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diff --git a/old/68670-0.txt b/old/68670-0.txt deleted file mode 100644 index 5fde5d6..0000000 --- a/old/68670-0.txt +++ /dev/null @@ -1,8223 +0,0 @@ -The Project Gutenberg eBook of The micro-organisms of the soil, by -Sir E. John Russell - -This eBook is for the use of anyone anywhere in the United States and -most other parts of the world at no cost and with almost no restrictions -whatsoever. You may copy it, give it away or re-use it under the terms -of the Project Gutenberg License included with this eBook or online at -www.gutenberg.org. If you are not located in the United States, you -will have to check the laws of the country where you are located before -using this eBook. - -Title: The micro-organisms of the soil - -Authors: Sir E. John Russell - Members of the biological staff of The Rothamsted Experimental Station - -Release Date: August 2, 2022 [eBook #68670] - -Language: English - -Produced by: Charlene Taylor, Harry Lamé and the Online Distributed - Proofreading Team at https://www.pgdp.net (This file was - produced from images generously made available by The - Internet Archive/Canadian Libraries) - -*** START OF THE PROJECT GUTENBERG EBOOK THE MICRO-ORGANISMS OF THE -SOIL *** - - - - Transcriber’s Notes - - Text printed in italics has been transcribed _between underscores_; - underlined text ~between tildes~. ^x and _{x} represent a superscript - and subscript x respectively. - - Uppercase letters between square brackets (such as [A]) refer to - footnotes (to be found directly underneath the paragraph or table), - numbers between square brackets (such as [1] or [1_a_] refer to - references at the end of the chapter(s) by the same author. - - More Transcriber’s Notes may be found at the end of this text. - - - - - _~THE ROTHAMSTED MONOGRAPHS ON - AGRICULTURAL SCIENCE~_ - - EDITED BY - SIR E. J. RUSSELL, D.Sc. (LOND.), F.R.S. - - - THE MICRO-ORGANISMS OF THE SOIL - - - - -THE ROTHAMSTED MONOGRAPHS ON AGRICULTURAL SCIENCE. - -EDITED BY SIR E. JOHN RUSSELL, D.Sc., F.R.S. - - -During the past ten years there have been marked developments in -knowledge of the relations between the soil and the growing plant. The -subject involves physical, biological, and chemical considerations, and -its ramifications are now so wide that they cannot be satisfactorily -dealt with in detail in any one book. These monographs collectively -cover the whole ground. In “Soil Conditions and Plant Growth” the -general outlines are presented: in the monographs the various divisions -are fully and critically dealt with by the Heads of the Departments -concerned at Rothamsted. A homogeneous treatment is thus secured that -will, it is hoped, much facilitate the use of the series. - - SOIL CONDITIONS AND PLANT GROWTH, Fourth Edition. By SIR E. JOHN - RUSSELL, F.R.S. 16_s._ net. - - The following volumes are in preparation:-- - - MANURING OF GRASS-LANDS - FOR HAY By WINIFRED E. BRENCHLEY, D.Sc., F.Z.S. - - THE MICRO-ORGANISMS OF THE - SOIL By Sir E. JOHN RUSSELL, F.R.S., and - Members of the Biological Staff of the - Rothamsted Experimental Station. - - SOIL PHYSICS By B. A. KEEN, B.Sc. - - SOIL PROTOZOA By D. W. CUTLER, M.A., and L. M. CRUMP, - M.Sc. - - SOIL BACTERIA By H. G. THORNTON, M.A. - - SOIL FUNGI AND ALGÆ By W. B. BRIERLEY, S. T. JEWSON, B.Sc., - and B. M. ROACH (Bristol), D.Sc. - - CHEMICAL CHANGES IN - THE SOIL By H. J. PAGE, B.Sc. - - - LONGMANS, GREEN AND CO., - LONDON, NEW YORK, TORONTO, BOMBAY, CALCUTTA, AND MADRAS. - - - - - THE MICRO-ORGANISMS - OF THE SOIL - - BY - - SIR E. JOHN RUSSELL, F.R.S. - - AND - - MEMBERS OF THE BIOLOGICAL STAFF OF THE - ROTHAMSTED EXPERIMENTAL STATION - - _WITH DIAGRAMS_ - - LONGMANS, GREEN AND CO. - 39 PATERNOSTER ROW, LONDON, E.C. 4 - NEW YORK, TORONTO - BOMBAY, CALCUTTA and MADRAS - 1923 - - -_Made in Great Britain_ - - - - -INTRODUCTION. - - -The purpose of this volume is to give the broad outlines of our present -knowledge of the relationships of the population of living organisms -in the soil to one another and to the surface vegetation. It is shown -that there is a close relationship with vegetation, the soil population -being dependent almost entirely on the growing plant for energy -material, while the plant is equally dependent on the activities of -the soil population for removing the residues of previous generations -of plants and for the continued production in the soil of simple -materials, such as nitrates, which are necessary to its growth. It is -also shown, however, that the soil population takes toll of the plant -nutrients and that some of its members may directly injure the growing -plant. - -The soil population is so complex that it manifestly cannot be dealt -with as a whole in any detail by any one person, and at the same -time it plays so important a part in the soil economy that it must -be seriously studied. Team work therefore becomes indispensable, and -fortunately this has been rendered possible at Rothamsted. - -Each group of organisms is here dealt with by the person primarily -responsible for that particular section of the work. The plan of the -book has been carefully discussed by all the authors, and the subject -matter has already been presented in a course of lectures given at -University College, London, under the auspices of the Botanical -Board of Studies of the London University. The interest shown in -these lectures leads us to hope that the subject may appeal to a -wider public, and above all to some of the younger investigators in -biological science. They will find it bristling with big scientific -problems, and those who pursue it have the satisfaction, which -increases as the years pass by, of knowing that their work is not only -of interest to themselves, but of great importance in ministering to -the intellectual and material needs of the whole community. - - - - -CONTENTS. - - - CHAP. PAGE - - I. DEVELOPMENT OF THE IDEA OF A SOIL POPULATION 1 - Sir E. JOHN RUSSELL, F.R.S., Director. - - II. OCCURRENCE OF BACTERIA IN SOIL--ACTIVITIES CONNECTED WITH - THE ACQUIREMENT OF ENERGY 20 - H. G. THORNTON, B.A., Head of the Department of - Bacteriology. - - III. CONDITIONS AFFECTING BACTERIAL ACTIVITIES IN THE - SOIL--ACTIVITIES CONNECTED WITH THE INTAKE OF PROTEIN - BUILDING MATERIALS 39 - H. G. THORNTON, B.A., Head of the Department of - Bacteriology. - - IV. PROTOZOA OF THE SOIL, I. 66 - D. W. CUTLER, M.A., Head of the Department of - Protozoology. - - V. PROTOZOA OF THE SOIL, II. 77 - D. W. CUTLER, M.A., Head of the Department of - Protozoology. - - VI. SOIL ALGÆ 99 - B. MURIEL BRISTOL, D.Sc., Algologist. - - VII. SOIL FUNGI--THE OCCURRENCE OF FUNGI IN THE SOIL 118 - W. B. BRIERLEY, D.Sc., Head of the Department of - Mycology. - - VIII. SOIL FUNGI--THE LIFE OF FUNGI IN THE SOIL 131 - W. B. BRIERLEY, D.Sc., Head of the Department of - Mycology. - - IX. THE INVERTEBRATE FAUNA OF THE SOIL (OTHER THAN PROTOZOA) 147 - A. D. IMMS, D.Sc., Head of the Department of Entomology. - - X. THE CHEMICAL ACTIVITIES OF THE SOIL POPULATION AND THEIR - RELATION TO THE GROWING PLANT 164 - Sir E. JOHN RUSSELL, F.R.S., Director. - - INDEX 181 - - - - -CHAPTER I. - -THE DEVELOPMENT OF THE IDEA OF A SOIL POPULATION. - - -From the earliest times agriculturists have been familiar with the idea -that decomposition of vegetable and animal matter takes place in the -soil, and that the process is intimately connected with soil fertility. - -By the middle of the nineteenth century three different ways were known -in which the decomposition occurred. One had been since early times -specially associated with soil fertility, in that it gave rise to -humus, the black sticky substance in farmyard manure or in soil--which -was supposed up to 1840 to be the special food of plants. No good -account of the process or of the conditions in which it occurred is, -however, given by the older writers. - -A second resulted in the formation of nitrates. This process became -known as nitrification: it was described by Georgius Agricola -(1494-1555) in his book “De Re Metallica,” and it was of great -importance in the seventeenth and eighteenth centuries, because it -was used for the manufacture of gunpowder in the great wars of that -period. The conditions for the making of successful nitre beds were so -thoroughly investigated that little fresh knowledge was added to that -of 1770[A] until quite recently. This process, however, was not usually -associated with soil fertility, although both Glauber (1656) and Mayow -(1674) had insisted on the connection. - - [A] See the remarkable collection of papers entitled - “Instructions sur l’établissement des nitrières,” publié par les - Régisseurs-généraux des Poudres et Salpêtre. Paris, 1777. - -A third type of decomposition was brought into prominence by Liebig -in 1840.[7][B] Reviewing the decomposition of organic matter in the -light of the newer chemistry, he concluded that the process was a -slow chemical oxidation, to which he gave the name “Eremacausis.” -He recognised that humus was formed, but he regarded it only as an -intermediate product, and emphatically denied its importance in -soil fertility. The true fertility agents, in his view, were the -final products--CO₂, potassium and other alkaline salts, phosphates, -silicates, etc. He went on to argue brilliantly that instead of -applying farmyard or similar manures to the soil it was altogether -quicker and better to apply these mineral compounds obtained from -other sources than to wait for the slow process of liberation as the -result of decomposition. For some reason, difficult to understand, he -overlooked nitrification and the part that nitrates might play in soil -fertility. Lawes and Gilbert[6] were much attracted by this new idea; -they showed that it was incomplete because it took no account of the -necessity for supplying nitrogen compounds to the crop. When ammonium -salts were added to Liebig’s ash constituents the resulting mixture had -almost as good a fertilising effect as farmyard manure. Lawes at once -saw the enormous practical importance of this discovery, and set up -a factory for the manufacture of artificial fertilisers. He did not, -however, follow it up more closely on the scientific side. - - [B] The numbers refer to the short bibliography on p. 18. - -Both Lawes and Gilbert were in constant touch with the idea of -decomposition in the soil, and they attached so much importance to -nitrogen compounds in plant nutrition that it is not easy to understand -how they missed the connection with nitrification. But they did so, and -like other English and German workers of the day, considered that plant -roots assimilated their nitrogen as ammonia. For the first ten years of -the history of Rothamsted only few experiments with nitrates were made, -and not till thirty-five years had elapsed were they systematically -studied. - -It was by Boussingault[2] and in France that the connection between -nitrification and soil fertility was first recognised. The news came -to England, but it was not accepted, although Way, one of the most -brilliant agricultural chemists of his time, showed that nitrates -were formed in soils to which nitrogenous fertilisers were added, and -that they were comparable in their fertiliser effects with ammonium -salts.[12] “The French chemists,” he wrote in 1856, “are going further, -several of them now advocating the view that it is in the form of -nitric acid that plants make use of compounds of nitrogen. With this -view I do not at present coincide, and it is sufficient here to admit -that nitric acid in the form of nitrates has at least a very high value -as manure.” Indeed, Kuhlmann went so far as to argue that the nitrates -found in the soil were there reduced to ammonia before assimilation -by plants could take place. The water-culture work of the plant -physiologists of the ’sixties finally showed the correctness of the -French view. - -Even when the importance of nitrification was realised its mechanism -was not understood: some thought it was chemical, some physical. -Again the explanation came from France. Pasteur in 1862 had expressed -the view that nitrification would probably be a biological action, -since purely chemical oxidation of organic matter was of very limited -occurrence. “Pénétrés de ces idées,” as Schloesing tells us, he and -Müntz in a memorable investigation cleared up the whole problem, and -in 1877 opened the way to a most fruitful field of research.[10] The -formal description is given in his papers in the “Comptes Rendus,” -but a more lively account is given in his lectures before the _École -d’application des Manufacteurs de l’état_, which, though not printed, -were collected and issued in script by his distinguished son, and a -copy of this work is among the treasures of the Rothamsted Library. - -He had been asked to study the purification of sewage, and he and Müntz -showed that it was bound up with nitrification. The process was slow in -starting, then it proceeded rapidly. Why, they asked, was the delay? -There should be none if the process were physical or chemical, and the -fact that it occurred strongly suggested biological action. The process -was stopped by chloroform vapour, but could be restarted after the -removal of the vapour by the addition of a little fresh soil. - -The importance of this work in connection with soil fertility -was immediately realised by Warington, who had recently come to -Rothamsted.[11] He quickly confirmed the result, and made the valuable -discovery that two stages were involved--the conversion of ammonia to -a nitrite by one organism, and of the nitrite to nitrate by another. -He made long and persistent attempts to isolate the organisms from the -soil, using the best technique of his time, but though he found many -bacteria none of them could nitrify ammonium salts; yet the soil did -it easily. For years he continued his efforts to find the nitrifying -organism, but always failed. His health was not good, his life at -Rothamsted was not happy owing to disagreements with Gilbert, and -although his other research work was succeeding, this investigation -on which he had set his heart was not coming out; bacterial technique -was not yet sufficiently far advanced. Ten bitter, disappointing years -passed, and the crown of disappointment came when Winogradsky, a young -bacteriologist in Paris, changed the technique and succeeded at once in -isolating both the nitrite and the nitrate-forming organisms.[13] - -The numerous bacteria found by Warington in the soil suggested the -presence of a soil population, and this idea was greatly strengthened -by another line of investigation which was being followed up in France. -Boussingault had shown that soils absorb oxygen and give out carbon -dioxide; Schloesing extended this discovery, as also did Wollny. It -was concluded that oxidation was the result of the activities of the -soil organisms in decomposing the organic matter of the soil, and thus -preparing the way for the nitrifying organisms. - -A third important function of soil bacteria was revealed by -Berthelot.[1] It was known that considerable loss of nitrogen from the -soil took place as the result of the conversion of nitrogen compounds -into nitrates, which were subsequently washed out in the drainage -water. It followed inevitably that the stock of nitrogen compounds in -the soil must long ago have become exhausted had there been no addition -of nitrogen compounds to the soil. Berthelot argued that there must be -fixation of atmospheric nitrogen, and, following the prevailing trend -of thought in France, he attributed it to bacteria. He confirmed the -anticipation by exposing soil to air in such conditions that dust, -rain, etc., were excluded, and he found an increase in the percentage -of nitrogen. - -Looking back over the work, it is difficult to understand the result. -The fixation of nitrogen is a process that absorbs energy, and -should have necessitated some source of energy, which apparently was -not supplied. But in spite of this drawback the investigation was -immediately fruitful in that it gave the key to another problem which -had long puzzled agriculturists. - -It had long been known that the growth of leguminous crops, unlike that -of others, enriched the ground,[C] and Lawes and Gilbert had shown -that this was due to an increase of soil nitrogen. But no explanation -could be found till Hellriegel and Wilfarth solved the problem.[4] In -studying the nitrogen nutrition of gramineous and leguminous crops, -they discovered that the gramineous plants died in absence of nitrate, -and in its presence made growth which increased regularly with nitrate -supply; while leguminous plants sometimes died and sometimes flourished -in absence of nitrate, and behaved equally erratically with increasing -nitrate supply. When the plants flourished nodules were invariably -present on the roots, but not otherwise. They concluded, therefore, -that the nitrogen nutrition of leguminous plants differed from that -of the gramineæ, and depended on some factor which sometimes came -into their experiments and sometimes did not, and, in any case, was -associated with the nodule. Knowing that the nodules on the roots of -leguminous plants contained bacteria-like bodies, and remembering -Berthelot’s results, they explored the possibility of bacterial -fixation. They sterilised the sand and found that peas invariably -failed to develop nodules and died, but after adding a little garden -soil nodules were found and vigorous growth was obtained. - - [C] “Of the leguminous plants the bean best reinvigorates the ground - ... because the plant is of loose growth and rots easily, wherefore - the people of Macedonia and Thessaly turn over the ground when it - is in flower” (i.e. dig it into the ground if the soil is poor). - Theophrastus, “Enquiry into Plants,” bk. viii. 2, and bk. ix. I. This - book is of profound interest to agriculturists and botanists. An - excellent translation by Sir Arthur Hort is now available. (Loeb’s - Classical Library.) - -Chemical analysis showed considerable fixation of gaseous nitrogen, -which Hellriegel associated with the nodule organism. This has proved -to be correct, and the fixation of nitrogen by bacteria is now a -well-recognised process, the conditions of which are being thoroughly -worked out. Two types of organisms are known--those associated with -leguminous plants, and those living in a free and independent state in -the soil. Of the latter the Clostridium, isolated by Winogradsky, is -anaerobic, and the Azotobacter of Beijerinck is aerobic. The essential -conditions are that a source of energy must be supplied--usually -given as sugar--that the medium must not be acid, and that sufficient -phosphate must be present. - -All this brilliant work had been accomplished in the short space of the -ten years 1880 to 1890. The inspiration had in each instance come from -France, and is traceable direct to Pasteur, although coming long after -his own work on bacteriology. It is impossible for us now to realise -the thrill of wonder and astonishment with which students, teachers, -and writers of those days learned that the nutrition of plants, and -therefore the growth of crops and the feeding of themselves, was -largely the result of the activity of bacteria in the dark recesses of -the soil. It is not surprising that the ideas were pushed somewhat too -far, that the soil population was regarded as solely bacterial, and -that important chemical and physical changes were sometimes overlooked. - -Gradually there came the inevitable reaction and a somewhat changed -outlook. Continued examination showed the presence in soil of almost -every kind of bacteria for which search was made. Some of them were -almost certainly in the resting condition as spores, and the new -generation of workers had an uneasy feeling that the case for the -overwhelming importance of bacteria in the economy of the soil was -not too well founded. It was shown that the decomposition of nitrogen -compounds to form ammonia would take place without micro-organisms if, -as presumably would happen, the plant enzymes continued to act after -they got into the soil. Even the oxidation of ammonia to nitrate--the -great stronghold of the biological school--was accomplished by chemical -agents. The fixation of nitrogen in soil conditions was beyond the -power of chemists to achieve, and here it was universally agreed that -bacteria were the active agents. And finally, chemists were themselves -bringing into prominence a set of bodies--the colloids--whose -remarkable properties seemed indefinitely expansible, and were in -addition sufficiently incomprehensible to the ordinary student to -attain much of the magnificence of the unknown. - -All the time, however, a faithful body of workers was busy exploring -the ground already won, improving the technique, making counts of the -numbers of bacteria in the soil, and trying to measure the amount of -bacterial activity. Much of the value of this work was limited by the -circumstance that the bacteria were regarded as more or less constant -in numbers and activities, so that a single determination was supposed -to characterise the position in a given soil. - -This was the condition of the subject when it was seriously taken up at -Rothamsted. Before turning to agriculture, the writer had been studying -the mechanism of certain slow chemical oxidations, and one of his first -experiments in agriculture was to examine the phenomena of oxidation -in soil. The results accorded with the biological explanation of -Schloesing: when the soil was completely sterilised oxidation almost -ceased. But the striking discovery was made, as the result of an -accident to an autoclave, that partial sterilisation increased the -rate of oxidation, and therefore presumably the bacterial activity. -This remarkable phenomenon had, however, already been observed, and -it had been shown that both bacterial numbers and soil fertility -were increased thereby. A full investigation was started in 1907 by -Dr. Hutchinson and the writer.[9] From the outset the phenomena were -recognised as dynamic and not static, and the rates of change were -always determined: thus the bacterial numbers, the nitrate and ammonia -present were estimated after the several periods. Close study of the -curves showed that the chemical and bacterial changes were sufficiently -alike to justify the view that bacteria were in the main the causes -of the production of ammonia and of nitrate; although non-biological -chemical action was not excluded, there was no evidence that it played -any great part. Thus the importance of micro-organisms in the soil was -demonstrated. - -The factor causing the increased bacterial numbers after partial -sterilisation was studied by finding out what agents would, and what -would not, allow the numbers to increase, e.g. it was found that the -bacterial increases became possible when soil had been heated at 56° -C., but not at 40° C. Again, it was shown that the high numbers in -partially sterilised soils rose for a time even higher if a little -fresh untreated soil were incorporated into the partially sterilised -soil, but afterwards they fell considerably. Putting all the results -together, it appeared that some biological cause was at work depressing -the numbers of bacteria in normal soils, but not--or not so much--in -the partially sterilised soils. Studied in detail, the data suggested -protozoa as the agent keeping down bacterial numbers, and they were -found in the untreated, but not in the treated, soils. The hypothesis -was therefore put forward that bacteria are not the only members of the -soil population, but that protozoa are also present keeping them in -check, and therefore adversely affecting the production of plant food. - -This conclusion aroused considerable controversy. It was maintained -that protozoa were not normal inhabitants of the soil, but only -occasional visitants, and, in any case, they were only there as cysts; -the soil conditions, it was urged, were not suitable to large organisms -like protozoa. The objection was not to be treated lightly, but, on -the other hand, the experiments seemed quite sound. As neither Dr. -Hutchinson nor the writer were protozoologists, Dr. T. Goodey and -(after he left) Mr. Kenneth R. Lewin were invited to try and find -out, quite independently of the partial sterilisation investigation, -whether protozoa are normal inhabitants of the soil, and if so, whether -they are in a trophic condition, and what is their mode of life and -their relation to soil bacteria. Had it turned out that protozoa had -nothing to do with the matter, search would have been made for some -other organism. Goodey showed that the ciliates were not particularly -important; Lewin soon demonstrated the existence of trophic amœbæ and -flagellates. Unfortunately he was killed in the war before he had got -far with the work. After the Armistice, Mr. Cutler accepted charge of -the work: he will himself relate in Chapters IV. and V. what he has -done. - -At first sight it might be thought comparatively easy to settle a -question of this kind by examining soil under a microscope or by -sterilising it and introducing successively bacteria and known types -of protozoa. Unfortunately neither method is simple in practice. It -is impossible to look into the soil with a microscope, and methods of -teasing-out small pieces of soil on a slide under the high, or even the -low power, give no information, because the particles of soil have the -remarkable power of attracting and firmly retaining protozoa, and no -doubt bacteria as well; indeed, for protozoa (which have been the more -fully investigated) there seems to be something not unlike a saturation -capacity (see Fig. 9, p. 78). Further, complete sterilisation of soil -cannot be effected without at the same time altering its chemical and -physical properties, and changing it as a habitat for micro-organisms. -Cutler has, however, overcome the difficulties and shown that the -introduction of protozoa into soils sterilised and then reinfected with -bacteria considerably reduces the numbers of these organisms. - -The method adopted, therefore, is to take a census of population and -of production. Counting methods are elaborated, and estimates as -accurate as possible are made of the numbers of the various organisms -in a natural field soil at stated intervals. Simultaneously, wherever -possible some measure is taken of the work done. The details of the -census are finally arranged in consultation with the Statistical -Department, to ensure that the data shall possess adequate statistical -value. From the results it is possible to adduce information of great -value as to the life of the population, the influence of external -conditions, etc. - -The most important investigation of this kind carried out at Rothamsted -was organised by Mr. Cutler.[3] A team of six workers was assembled, -and for 365 days without a break they counted every day the ciliates, -the amœbæ, the flagellates, and the bacteria in a plot of arable -ground, distinguishing no less than seventeen different kinds of -protozoa. The conclusions arrived at were carefully tested by the -Statistical Department. - -Of the protozoa the flagellates were found to be the most numerous, -the amœbæ came next, and the ciliates were by far the fewest. The -numbers of each organism varied from day to day in a way that -showed conclusively the essentially trophic nature of the protozoan -population. The numbers of amœbæ--especially _Dimastigamœba_ and of -a species called α--were sharply related to the numbers of bacteria: -when the amœbae were numerous the bacteria were few, and vice versa. -Detailed examination showed that the amœbæ were probably the cause -of the fluctuations in the bacterial numbers, but Mr. Cutler has not -yet been able to find why the amœbæ fluctuated; it does not appear -that temperature, moisture content, air supply or food supply were -determining causes. The flagellates and ciliates also showed large -fluctuations, amounting in one case--_Oicomonas_--to a definite -periodicity, apparently, however, not related to bacterial numbers, or, -so far as can be seen, to external conditions of moisture, temperature -and food supply, and showing no agreement with the fluctuations of the -amœbæ. However, one cannot be certain that lack of agreement between -curves expressing protozoan numbers and physical factors implies -absence of causal relationships: the observations (though the best that -can yet be made) are admittedly not complete. If we saw only the end of -the bough of a tree, and could see no connection with a trunk, we might -have much difficulty in finding relationships between its motion and -the wind; whatever the direction of the wind it would move backwards -and forwards in much the same way, and even when the wind was blowing -along the plane of its motion it would just as often move against the -wind as with it. - -Meanwhile evidence was obtained that the twenty-four hour interval -adopted by the protozoological staff was too long for bacteria, and -accordingly the Bacteriological Department, under Mr. Thornton, refined -the method still further. Bacterial counts were made every two hours, -day and night, for several periods of sixty or eighty hours without -a break. The shape of the curve suggests that two hours is probably -close enough, and for the present counts at shorter intervals are not -contemplated. But there is at least one maximum and one minimum in the -day, although the bacterial day does not apparently correspond with -ours, nor can any relationship be traced with the diurnal temperature -curve. - -The nitrate content of the soil was simultaneously determined by Mr. -Page and found to vary from hour to hour, but the variations did not -sharply correspond with the bacterial numbers; this, however, would not -necessarily be expected. The production of nitrate involves various -stages, and any lag would throw the nitrate and bacterial curves out -of agreement. There is a suggestion of a lag, but more counts are -necessary before it can be regarded as established. - -Examination of these and other nitrate curves obtained at Rothamsted -has brought out another remarkable phenomenon. No crop is growing on -these plots, and no rain fell during the eighty hours, yet nitrate -is disappearing for a considerable part of the time. Where is it -going to? At present the simplest explanation seems to be that it is -taken up by micro-organisms. A similar conclusion had to be drawn -from a study of the nitrogen exhaustion of the soil. The whole of the -nitrate theoretically obtainable from the organic matter of the soil -is not obtained in the course of hours or even days; in one of our -experiments at Rothamsted nitrification is still going on, and is far -from complete, even after a lapse of fifty-three years. The explanation -at present offered is that part of the nitrate is constantly being -absorbed by micro-organisms and regenerated later on. - -Now what organisms could be supposed to absorb nitrates from the soil? -Certain bacteria and fungi are known to utilise nitrates, and one -naturally thinks of algæ as possible agents also. Dr. Muriel Bristol -was therefore invited to study the algæ of the soil. Her account is -given in Chapter VI. She has found them not only on the surface, but -scattered throughout the body of the soil, even in the darkness of 4 -inches, 5 inches, or 6 inches depth, where no light can ever penetrate, -and where photosynthesis as we understand it could not possibly take -place. Some modification in their mode of life is clearly necessary, -and it may well happen that they are living saprophytically. Dr. -Bristol has not yet, however, been able to count the algæ in the soil -with any certainty, although she has made some estimates of the numbers. - -The quantitative work on the soil population indicates other -possibilities which are being investigated. There is not only a daily -fluctuation in the numbers, but so far as measurements have gone, -a seasonal one also. There seems to be some considerable uplift in -numbers of bacteria, protozoa, and possibly algæ and fungi in the -spring-time, followed by a fall in summer, a rise in autumn, and a -fall again in winter. At present we are unable to account for the -phenomenon, nor can we be sure that it is general until many more data -are accumulated. - -In the cases of the protozoa and the algæ, there was a definite reason -for seeking them in the soil. - -Another section of the population, the fungi, was simply found, and -at present we have only limited views as to their function. The older -workers considered that they predominated in acid soils, while bacteria -predominated in neutral soils. Present-day workers have shown that -fungi, including actinomycetes, are normal inhabitants of all soils. -The attempts at quantitative estimations are seriously complicated -by the fact that during the manipulations a single piece of mycelium -may break into fragments, each of which would count as one, while a -single cluster of spores might be counted as thousands. Little progress -has therefore been made on the quantitative lines which have been so -fruitful with protozoa. Dr. Brierley gives, in Chapters VII. and VIII., -a critical account of the work done on fungi. - -In addition to the organisms already considered there are others of -larger size. The nematodes are almost visible to the unaided eye, -most of them are free living and probably help in the disintegration -of plant residues, though a few are parasitic on living plants and do -much injury to clover, oats, and less frequently to onions, bulbs, and -potatoes. Further, there are insects, myriapods and others, the effects -of which in the soil are not fully known. Special importance attaches -to the earthworms, not only because they are the largest in size and -in aggregate weight of the soil population, but because of the great -part they play in aerating the soil, gradually turning it over and -bringing about an intimate admixture with dead plant residues, as first -demonstrated by Darwin. Earthworms are the great distributors of energy -material to the microscopic population. Systematic quantitative work -on these larger forms is only of recent date, and Dr. Imms, in Chapter -IX., discusses our present knowledge. - -TABLE I. - -SOIL POPULATION, ROTHAMSTED, 1922. - -(The figures for algæ and fungi are first approximations only, and have -considerably less value than those for bacteria and protozoa.) - - +----------------------+-----------+--------------------------------+ - | | |Approximate Weight per Acre of--| - | | +---------+----------+-----------+ - | | Numbers | Living |Dry Matter| Nitrogen | - | | per Gram | Organ- |in Organ- | in Organ- | - | | of Soil. | isms. | isms. | isms. | - +----------------------+-----------+---------+----------+-----------+ - |_Bacteria_-- | | lb. | lb. | lb. | - | High level |45,000,000 | 50} | 2 | 0·2 | - | Low level |22,500,000 | 25} | | | - |_Protozoa_-- | | | | | - | _Ciliates_-- | | | | | - | High level | 1,000 | -- | -- | -- | - | Low level | 100 | -- | -- | -- | - | _Amœbæ_-- | | | | | - | High level | 280,000 | 320} | 12 | 1·2 | - | Low level | 150,000 | 170} | | | - | _Flagellates_-- | | | | | - | High level | 770,000 | 190} | 7 | 0·7 | - | Low level | 350,000 | 85} | | | - | _Algæ_ | | | | | - | (not blue-green)| [100,000]| 125 | 6 | 0·6 | - | Blue-green | Not known.| | Say 6 | Say 0·6 | - | _Fungi_-- | | | | | - | High level |[1,500,000]| 1700} | 60 | 6·0 | - | Low level | [700,000]| 800} | | | - | | | +----------+-----------+ - | | | | 93 | 9·3 | - | | | |= 4 parts nitrogen per| - | | | |1,000,000 of soil. | - +----------------------+-----------+---------+----------------------+ - - +-------------------------------------- - | LARGER ORGANISMS. - +-----------------+-------------------+ - | | | - | | + - | | Numbers | - | | per Acre.[D] | - | +---------+---------+ - | | | Unma- | - | | Manured.| nured. | - +-----------------+---------+---------+ - |_Oligochaeta_ | | | - | (_Limicolae_)--| | | - | Nematoda, etc. |3,609,000| 794,000| - | Myriapoda |1,781,000| 879,000| - | Insects |7,727,000|2,475,000| - | Earthworms |1,010,000| 458,000| - +-----------------+---------+---------+ - | - +-------------------------------------- - - +-----------------------------------------------------------+ - | LARGER ORGANISMS. | - +-----------------+-----------------------------------------+ - | | Approximate Weight per Acre of-- | - | +-------------+-------------+-------------+ - | | Living | Dry Matter | Nitrogen in | - | | Organisms. |in Organisms.| Organisms. | - | +------+------+------+------+------+------+ - | | Ma- | Unma-| Ma- | Unma-| Ma- |Unma- | - | |nured.|nured.|nured.|nured.|nured.|nured.| - +-----------------+------+------+------+------+------+------+ - |_Oligochaeta_ | | | | | | | - | (_Limicolae_)--| lb. | lb. | lb. | lb. | lb. | lb. | - | Nematoda, etc. | 9 | 2 | 3 | 1 | -- | -- | - | Myriapoda | 203 | 99 | 85 | 42 | 4 | 2 | - | Insects | 34 | 16 | 14 | 6 | 1 | 1 | - | Earthworms | 472 | 217 | 108 | 50 | 10 | 5 | - +-----------------+------+------+------+------+------+------+ - | Total | 210 | 99 | 15 | 9 | - +-------------------------------+------+------+------+------+ - - Total organic matter (dry weight) in this soil = 126,000 lb. per acre. - - Total nitrogen = 5700 lb. per acre. (1 lb. nitrogen per acre = 0·4 - parts per 1,000,000 of soil.) - - [D] To a depth of 9 inches. The weight of soil is approximately - 1,000,000 kilos. - -Are there any other members of the soil population that are of -importance? As already shown, the method of investigating the soil -population in use at Rothamsted is to find by chemical methods the -changes going on in the soil; to find by biological methods what -organisms are capable of bringing about these changes; and then to -complete the chain of evidence by tracing the relationships between -the numbers or activities of these organisms and the amount of change -produced. The list as we know it to-day is given in Table I. - -The method, however, does not indicate whether the account is fairly -complete, or whether there are other organisms to be found. We might, -of course, trust to empirical hunting for organisms, or to chance -discoveries such as led Goodey to find the mysterious Proteomyxan -Rhizopods, which cannot yet be cultured with certainty, so that they -are rarely found by soil workers. It is possible that there are many -such organisms, and it is even conceivable that these unknown forms far -outnumber the known. The defect of the present method is that it always -leaves us in doubt as to the completeness of the list, and so we may -have to devise another. - -Reverting to Table I., it obviously serves no purpose to add the -numbers of all the organisms together. We can add up the weights of -living organisms, of their dry matter or nitrogen, so as to form some -idea of the proportion of living to non-living organic matter, and this -helps us to visualise the different groups and place them according -to their respective masses. But a much better basis for comparing the -activities of the different groups would be afforded by the respective -amounts of energy they transform, if these could be determined. It is -proposed to attempt such measurements at Rothamsted. The results when -added would give the sum of the energy changes effected by the soil -population as we know it: the figure could be compared with the total -energy change in the soil itself as determined in a calorimeter. If -the two figures are of the same order of magnitude, we shall know that -our list is fairly well complete; if they are widely different, search -must be made for the missing energy transformers. There are, of course, -serious experimental difficulties to be overcome, but we believe the -energy relationships will afford the best basis for further work on the -soil population. - -Finally, it is necessary to refer to the physical conditions obtaining -in the soil. These make it a much better habitat for organisms than one -might expect. At first sight one thinks of the soil as a purely mineral -mass. This view is entirely incorrect. Soil contains a considerable -amount of plant residues, rich in energy, and of air and water. The -usual method of stating the composition of the soil is by weight, but -this is misleading to the biologist because the mineral matter has a -density some two and a half times that of water and three times that -of the organic matter. For biological purposes composition by volume -is much more useful, and when stated in this way the figures are very -different from those ordinarily given. Table II. gives the results for -two Broadbalk arable plots, one unmanured and the other dunged; it -includes also a pasture soil. - -The first requirement of the soil population is a supply of energy, -without which it cannot live at all. All our evidence shows that the -magnitude of the population is limited by the quantity of energy -available. The percentage by weight of the organic matter is about -two to four or five, and the percentage by volume runs about four -to twelve. Not all of this, however, is of equal value as source of -energy. About one-half is fairly easily soluble in alkalis, and may -or may not be of special value, but about one-quarter is probably too -stable to be of use to soil organisms. - -A second requirement is water with which in this country the soil is -usually tolerably well provided. Even in prolonged dry weather the soil -is moist at a depth of 3 inches below the surface. It is not uncommon -to find 10 per cent. or 20 per cent. by volume of water present, spread -in a thin film over all the particles, and completely saturating the -soil atmosphere. - -TABLE II. - -VOLUME OF AIR, WATER AND ORGANIC MATTER IN 100 VOLUMES OF ROTHAMSTED -SOIL. - - +---+-----------------+-----------+--------------+ - | | | |In Pore Space.| - | | | |Values Common-| - | | Solid Matter. | | ly Obtained. | - | +--------+--------+ +-------+------+ - | |Mineral.|Organic.|Pore Space.| Water.| Air. | - +---+--------+--------+-----------+-------+------+ - |(1)| 62 | 4 | 34 | 23 | 11 | - |(2)| 51 | 11 | 38 | 30 | 8 | - |(3)| 41 | 12 | 47 | 40 | 7 | - +---+--------+--------+-----------+-------+------+ - - (1) Arable, no manure applied to soil. - - (2) Arable, dung applied to soil. - - (3) Pasture. - -The air supply is usually adequate owing to the rapidity with which -diffusion takes place. Except when the soil is water-logged, the -atmosphere differs but little from that of the one we breathe. There -is more CO₂, but only a little less oxygen.[8] The mean temperature is -higher than one would expect, being distinctly above that of the air, -while the fluctuations in temperature are less.[5] - -The reaction in normal soils is neutral to faintly alkaline; _p_H -values of nearly 8 are not uncommon. Results from certain English soils -are shown on p. 18. - -The soil reaction is not easily altered. A considerable amount of acid -must accumulate before any marked increase in intensity of _p_H value -occurs; in other words, the soil is well buffered. The same can be said -of temperature, of water, and of energy supply. Like the reaction, they -alter but slowly, so that organisms have considerable time in which to -adapt themselves to the change. - -HYDROGEN ION CONCENTRATION AND SOIL FERTILITY. - - _p_H - Alkaline 10 --+-- Sterile: Alkali soil. - ↑ | - | 9 --+-- - | | - | 8 --+-- Fertile: Arable. - | | - Neutral 7 --+-- - | | - | 6 --+-- - | | - | 5 --+-- Potato Scab fails. - | | Nitrification hindered. - | 4 --+-- Barley fails. - ↓ | - Acid 3 --+-- Sterile: Peat. - - -A SELECTED BIBLIOGRAPHY. - - [1] Berthelot, Marcellin, “Fixation directe de l’azote atmosphérique - libre par certains terrains argileux,” Compt. Rend., 1885, ci., - 775-84. - - [2] Boussingault, J. B., and Léwy, “Sur la composition de l’air - confiné dans la terre végétale,” Ann. Chim. Phys., 1853, xxxvii., - 5-50. - - [3] Cutler, D. W., Crump, L. M., and Sandon, H., “A Quantitative - Investigation of the Bacterial and Protozoan Population of the Soil, - with an Account of the Protozoan Fauna,” Phil. Trans. Roy. Soc., - Series B, 1922, ccxi., 317-50. - - [4] Hellriegel, H., and Wilfarth, H., “Untersuchungen über die - Stickstoffnahrung der Gramineen und Leguminosen,” Zeitsch. des - Vereins f. d. Rübenzucker-Industrie, 1888. - - [5] Keen, B. A., and Russell, E. J., “The Factors determining Soil - Temperature,” Journ. Agric. Sci., 1921, xi., 211-37. - - [6] Lawes, J. B., and Gilbert, J. H., “On Agricultural Chemistry, - Especially in Relation to the Mineral Theory of Baron Liebig,” Journ. - Roy. Agric. Soc., 1851, xii., 1-40. - - [7] Liebig, Justus, “Chemistry in its Application to Agriculture - and Physiology,” 1st and 2nd editions (1840 and 1841), 3rd and 4th - editions (1843 and 1847); “Natural Laws of Husbandry,” 1863. - - [8] Russell, E. J., and Appleyard, A., “The Composition of the Soil - Atmosphere,” Journ. Agric. Sci., 1915, vii., 1-48; 1917, viii., - 385-417. - - [9] Russell, E. J., and Hutchinson, H. B., “The Effect of Partial - Sterilisation of Soil on the Production of Plant Food,” Journ. Agric. - Sci., 1909, iii., 111-14; Part II., Journ. Agric. Sci., 1913, v., - 152-221. - - [10] Schloesing, Th., and Müntz, A., “Sur la Nitrification par les - ferments organisés,” Compt. Rend., 1877, lxxxiv., 301-3; 1877, - lxxxv., 1018-20; and 1878, lxxxvi., 892-5. “Leçons de chimie - agricole,” 1883. - - [11] Warington, R., “On Nitrification,” Part I., Journ. Chem. Soc., - 1878, xxxiii., 44-51; Part II, Journ. Chem. Soc., 1879, xxxv., - 429-56; Part III., Journ. Chem. Soc., 1884, xlv., 637-72; Part IV., - Journ. Chem. Soc., 1891, lix., 484-529. - - [12] Way, J. T., “On the Composition of the Waters of Land Drainage - and of Rain,” Journ. Roy. Agric. Soc., 1856, xvii., 123-62. - - [13] Winogradsky, S., “Recherches sur les organismes de la - nitrification,” Ann. de l’Inst. Pasteur, 1890, iv., 1^e Mémoire, - 213-31; 2^e Mémoire, 257-75; 3^e Mémoire, 760-71. - - “Recherches sur l’assimilation de l’azote libre de l’atmosphère par - les microbes.” Arch. des Sci. Biolog. St. Petersburg, 1895, iii, - 297-352. - - For further details and fuller bibliography, see E. J. Russell, “Soil - Conditions and Plant Growth,” Longmans, Green & Co. - - - - -CHAPTER II. - -SOIL BACTERIA. - - -_A._ OCCURRENCE AND METHODS OF STUDY. - -To understand the development of our knowledge of soil bacteria, it -must be remembered that bacteriology is under the disadvantage that -it started as an applied science. Although bacteria were first seen -by Leeuwenhoeck about the middle of the seventeenth century, and -some of their forms described by microscopists of the eighteenth and -early nineteenth centuries, it was only with the work of Pasteur on -fermentation, and of Duvaine, Pasteur, and their contemporaries on -disease bacteria, that bacteriology may be said to have started. -From the outset, therefore, attention has been directed mainly to -the bacteria in their specialised relationship to disease or to -fermentation and similar processes. As a result, little research was -done on the pure biology of the bacteria, so that even now many of the -most fundamental and elementary problems concerning them are quite -unsolved. - -In their work on fermentations and disease bacteria, the earlier -workers were assisted by the fact that under both sets of conditions -the causative bacteria exist, as a rule, either in practically pure -culture, or else in preponderating numbers. The study and elucidation -of such a mixed micro-population as exists in the soil, became possible -only when methods had been devised for isolating the different kinds -of bacteria, and thus studying them apart from each other. It was the -development of the gelatine plate method of isolating pure cultures by -Koch[36] in 1881 that made the study of the soil bacteria practicable. -The plating method opened up two lines of research. In the first -place, it provided a simple means of isolating organisms from the mixed -population of the soil, and thus enabled a qualitative study to be made -of each organism in pure culture, and, in the second place, from it was -developed a counting technique for estimating differences in bacterial -numbers between samples of soil, from which has sprung much of our -knowledge of the quantitative side. - -The earliest studies of the soil bacteria consisted of such estimations -of numbers, and showed that the soil contained a very numerous -population of bacteria. About 20,000,000 bacteria per gram of soil -is now considered a fair average number. The number and variety of -bacteria existing in the soil is so enormous that the method of -separating out all the different forms, and of discovering their -characters and functions, has proved impracticable. In practice, -therefore, the problem has been approached from the biochemical -standpoint. That is to say, the special chemical changes that the -bacteria produce in the soil have first been investigated, and this -has been followed by the isolation and study of the various groups of -bacteria that bring about the changes under investigation. - -The method commonly employed in isolating the organisms that produce a -given chemical change in the soil is called the “elective” method. The -soil is inoculated into a culture medium that will especially favour -the group of bacteria to be isolated, to the exclusion of others. -For example, if it is desired to isolate the organisms that attack -cellulose, a medium is made up containing no other organic carbon -compounds except cellulose. Such a selective medium encourages the -growth of the group of organisms to be investigated, so that after -several transfers to fresh medium a culture is obtained containing -only two or three different types of organisms. These are separated by -plating and pure cultures obtained. - -Another difficulty which has not yet been completely overcome is -that of adequately describing an organism when it is isolated. The -morphology of bacteria is not the constant thing that is seen in -the more stable higher organisms. In many cases the appearance of a -single strain is entirely different on different media, and may be -quite altered by such conditions as changes in acidity of the medium -or temperature of incubation. Even on a single medium remarkable -changes in morphology occur, at any rate, in some bacteria. This is -well seen in a cresol-decomposing organism under investigation at -Rothamsted. In cultures a few days old this organism develops as bent -and branching rods; these rods then break up into chains of cocci and -short rods, which separate, and in old cultures all the organisms -may be in the coccoid form (Fig. 1). It is claimed by Löhnis[47_b_] -that the possession of a complex life-cycle of changing forms is a -universal character in the bacteria. The instability of shape in many -bacteria makes it necessary to standardise very carefully the cultural -conditions under which they are kept when their appearance is described. - -[Illustration: - -Culture 15 hours old. - -Culture 3 days old. - -FIG. 1.--Change in appearance, in culture, of a cresol decomposing -bacterium.] - -The inadequacy of mere morphology as a basis for describing bacteria -led to the search for diagnostic characters, based on the biochemical -changes that they produced in their culture media, and the appearance -of their growth in the mass on various media. These characters -unfortunately have also proved to be very much influenced by the exact -composition of the medium and other conditions of culture. Recently -an attempt has been made by the American Society of Bacteriologists to -standardise the diagnostic characters used in describing bacteria, and -also the media and cultural conditions under which they are grown for -the purpose of description. The need for such precautions, however, -was not sufficiently realised by the early workers, many of whose -descriptions cannot now be referred to any definite organism. - -The large number of organisms found in the soil, and the difficulty -and labour of adequately describing them, is such that even now we -have no comprehensive description of the common soil bacteria that -appear on gelatine platings. A careful study based on modern methods of -characterisation has been made of certain selected groups of bacteria, -and it is hoped that the laborious systematic work of describing the -common forms will gradually be completed. - -Several attempts have been made to classify the bacteria that appear -commonly on gelatine platings. This work was commenced by Hiltner and -Stormer in Germany, and continued by Chester, Harding, and Conn in -America. Conn[10],[14] found that the common organisms fell into the -following main groups:-- - -(1) Large spore-forming bacteria, related to _Bacillus subtilis_, which -form about 5-10 per cent. of the numbers. He adduced evidence[12],[13] -that these organisms exist in the soil mainly as spores, so that they -may not form an important part of the active soil population. - -(2) Short non-sporing organisms, related to _Pseudomonas fluorescens_, -that are rapid gelatine liquefiers. These form another 10 per cent. of -the numbers. - -(3) Short rod forms that liquefy gelatine slowly or not at all, and -develop colonies very slowly. These form 40-75 per cent. of the -numbers, and may therefore be of considerable importance in the soil. - -(4) A few micrococci also occur. - -These groups comprise the larger portion of the bacterial flora of the -soil, but, in addition to these organisms, that develop on the media -commonly used for plating, there are special and important groups -that appear only on special media, either owing to their being unable -to grow on ordinary media or because they get swamped by other forms. -Examples of such groups are the ammonia and nitrite oxidising bacteria, -the nitrogen fixing groups, the cellulose decomposing organisms, and -the sulphur bacteria. - -In order that we may apply the results of the study of a definite -organism to other localities, a knowledge of the geographical -distribution of the soil bacteria is clearly needed. We have, -unfortunately, very little knowledge of the distribution of soil -organisms. The common spore-forming groups appear to be universally -distributed. Thus Barthel, in a study of the bacterial flora of soils -from Greenland and the island of Disko, obtained soil organisms -belonging to the groups of _Bacillus subtilis_, _B. amylobacter_, _B. -fluorescens_, _B. caudatus_, and _B. Zopfii_, which are common groups -in European soil, indicating that the general constitution of the -bacterial flora of the soil in arctic regions is not widely different -from that of Western Europe. Bredemann, who made an extensive study of -the _Bacillus amylobacter_ group, obtained soil samples from widely -scattered localities, and found these organisms in soil from Germany, -Holstein, Norway, Italy, Morocco, Teneriffe, Russia, Japan, China, -the East Indies, Samoa, Illinois, Arizona, German East Africa, and -the Cameroons. Some soil organisms, on the other hand, are apparently -absent from certain districts. This may be due to the conditions, -such as climatic environment, being unfavourable to them. A study has -recently been made at Rothamsted of the distribution over Great Britain -of a group of bacteria that are capable of decomposing phenol and -cresol. One of these organisms, apparently related to the acid-fast -_B. phlœi_, has an interesting distribution. It has been found in 50 -per cent. of the soils samples examined from the drier region, where -the annual rainfall is less than 30 inches, but in only 20 per cent. -of the samples in the wetter parts of Britain. Another example of -limited distribution is found in the case of _Bacillus radicicola_, -the organism that produces tubercles on the roots of leguminous plants. -The distribution of the varieties of this organism follows that of -the host plants with which they are associated, so that when a new -leguminous crop is introduced into a country, nodules may not appear -on the roots unless the soil be specially inoculated with the right -variety of organism. In cases where a group of soil organisms is widely -distributed over the globe, it may yet be absent from many soils -owing to the soil conditions not suiting it. Thus, phenol decomposing -bacteria, though abundant in the neighbourhood of Rothamsted, are yet -absent from field plots that have been unmanured for a considerable -period. The occurrence of the nitrifying organisms and the nitrogen -fixing _Azotobacter_ is also very dependent on the soil conditions. - -Owing to the method by which our knowledge of soil bacteria has been -acquired, by studying first the chemical changes in the soil and then -the bacteria that produce them, it is natural for us to divide them -into physiological groups according to the chemical changes that they -bring about. This grouping is the more reasonable since so little is -known as to the true relationships of the different groups of bacteria -that a classification based on morphology is well-nigh impossible. In -considering the activities of bacteria in the soil, it is convenient to -group the changes which they bring about into the two divisions into -which they naturally fall in the economy of the organisms. - -In the first place, there are the changes that result in a release of -energy, which the bacteria utilise for their vital processes. - -In the second place, there are the processes by which the bacteria -build up the material of their bodies. These building up processes -involve an intake of energy for their accomplishment. - -It will be convenient to deal first with the release of energy for -their own use by bacteria, and its consequences. - - -_B._ ACTIVITIES CONNECTED WITH THE ACQUIREMENT OF ENERGY. - -Unlike the green plants, most bacteria are unable to obtain the energy -that is required for their metabolism from sunlight. They must, -therefore, make use of such chemical changes as will involve the -release of energy. - -As an example of the acquirement of energy in this way may be taken the -oxidation of methane by _B. methanicus_. This organism, described by -Söhngen, obtains its energy supply by the conversion of methane into -CO₂ and H₂O. - - CH₄ + 2O₂ = CO₂ + 2H₂O 220 Cal. - -A further example is the acetic organism that obtains its energy -through the oxidation of alcohol to acetic acid. - - C₂H₆O + O₂ = C₂H₄O₂ + H₂O 115 Cal. - -The decomposition processes brought about by micro-organisms in -obtaining energy are usually oxidations, but this is not necessarily -so, as can be seen in case of the fermentation of sugar into alcohol.[E] - - C₆H₁₂O₆ = 2C₂H₆O + 2CO₂ 50 Cal. - - [E] These examples are from Orla-Jensen (Centralblatt f. Bakt., II., - Bd. 22, p. 305). - -By far the greater part of the decomposition of organic matter is -brought about by bacteria in the process of acquiring energy. In the -soil, nearly the whole of the material utilised by bacteria as a source -of energy is derived ultimately from green plants. The energy materials -left in the soil by the plant fall into two groups, the non-nitrogenous -compounds, which are mainly carbohydrates and their derivatives, and -the nitrogenous compounds, principally derived from proteins. - - -(1) _Decomposition of Non-nitrogenous Compounds._ - -The simpler carbohydrates and starches are attacked and decomposed by -a large variety of bacteria. The addition of such substances to soil -causes a rapid increase in bacterial numbers. In nature the sugars are -in all probability among the first plant constituents to be destroyed -during the decay processes. - -A large proportion of plant tissues consist of cellulose and its -derivatives. These compounds are consequently of great importance -in the soil. Unfortunately our knowledge of the processes by which -cellulose is broken down in the soil is very inadequate. The early -experimental study of cellulose decomposition, such as that of -Tappeiner[60] and Hoppe-Seyler,[33] was mostly carried out under -conditions of inadequate aeration, and the products of decomposition -were found to include methane and CO₂, and sometimes fatty acids and -hydrogen. The bacteriology of this anaerobic decomposition was studied -by Omelianski,[54] who described two spore-bearing organisms, one of -which attacked cellulose with the production of hydrogen, and the other -with the production of methane. Both species also produce fatty acids -and CO₂. It is probable that these organisms operate in the soil under -conditions of inadequate aeration. In swamp soils, in which rice is -grown, it has been shown that methane, hydrogen, and CO₂ are evolved -in the lower layers. In these soils, however, the methane and hydrogen -are oxidised when they reach the surface layers. This oxidation is -also effected by micro-organisms. Bacteria capable of deriving energy -by the oxidation of hydrogen gas have been isolated and studied by -Kaserer,[37] and by Nabokich and Lebedeff,[52] while Söhngen[57] has -isolated an organism which he named _Bacillus methanicus_, that was -capable of oxidising methane. - -Under normal conditions in cultivated soils, however, the decomposition -of cellulose takes place in the presence of an adequate air supply, -and so follows a different course from that studied by Omelianski. Our -knowledge of this aerobic decomposition is very scanty. A number of -bacteria, capable of decomposing cellulose aerobically, are known. A -remarkable organism was investigated by Hutchinson and Clayton,[30] -who named it _Spirochæta cytophaga_. This organism, which they isolated -from Rothamsted soil, though placed among the _Spirochætoidea_, is -of doubtful affinities. During the active condition it exists for -the most part as thin flexible rods tapered at the extremities. This -form passes into a spherical cyst-like stage, at first thought to be -a distinct organism (Fig. 2). _Spirochæta cytophaga_ is very aerobic, -working actively, only at the surface of the culture medium. It is -very selective in its action. It appears unable to derive energy from -any carbohydrate other than cellulose. Indeed, many of the simple -carbohydrates, especially the reducing sugars, are toxic to the -organism in pure culture. An extensive study of aerobic cellulose -decomposition by bacteria was made by McBeth and Scales,[50] who -isolated fifteen bacteria having this power. Five of these were -spore-forming organisms. Unlike _Spirochæta cytophaga_, they are all -able to develop on ordinary media such as beef agar or gelatine, and -are thus not nearly so selective in their food requirements. - -[Illustration: FIG. 2.--_Spirochæta cytophaga._ Changes occurring in -culture. (After HUTCHINSON and CLAYTON.)] - -We are at present ignorant as to which organisms are most effective -in decomposing cellulose in the soil under field conditions, or what -are the conditions best suited to their activity. It is possible that -fungi also help in the decomposition of cellulose to a great extent. -This subject of the decomposition of cellulose offers one of the most -promising fields of research in soil bacteriology. The difficulty -of the subject is further increased by our present ignorance of the -chemical aspect of cellulose decomposition. It has been supposed that -the early decomposition products are simpler sugars, but these are -not found under conditions in which cellulose is being decomposed by -pure cultures of the bacteria mentioned above. Hutchinson and Clayton -found that their organism produced volatile acids, mucilage, and a -carotin-like pigment. The organisms isolated by McBeth and Scales also -produce acids, and in some cases yellow pigments. It is known, however, -that the decomposition products of cellulose can be utilised as energy -supply for other organisms, such as nitrogen fixing bacteria. - -When plant remains decompose in the soil there are ultimately produced -brown colloidal bodies collectively known as humus. The processes by -which this humus is produced are not yet properly understood. Humus is -of great importance in the soil, in rendering the soil suitable for -the growth of crops. It affects the physical properties of the soil -to a great extent. In the first place, it improves the texture of the -soil, making heavy clay soils more friable, and loose sandy soils more -coherent. Secondly, it has great water-retaining powers, so that soils -rich in organic matter suffer comparatively little during periods of -drought. And lastly, it exerts a strong buffering effect against soil -acids. Now, it is one of the problems of present-day farming that -soil is becoming depleted of its humus. This is due to the increasing -scarcity of farmyard manure in many districts, and the consequent use -of mineral fertilisers to supply nitrogen, potash, and phosphate to -the crop. A need has therefore arisen for a substitute for farmyard -manure, by means of which the humus content of soils may be kept up in -districts where natural manure is scarce. - -[Illustration: FIG. 3.--Cellulose decomposed by _S. cytophaga_ in media -with increasing amounts of nitrogen. (After HUTCHINSON and CLAYTON.) - - X-axis: Milligrams of nitrogen supplied as sodium-ammonium phosphate. - - Y-axis: Milligrams of cellulose decomposed in 21 days.] - -It is well known that if fresh unrotted manure or straw be added to -the soil, it often produces harmful effects on the succeeding crop. -The problem, therefore, was to develop a method by which fresh straw, -before application to the soil, could be made to rot down to a mixture -of humus compounds such as occur in well-rotted farmyard manure. The -solution of this problem came as a result of an investigation by -Hutchinson and Richards,[30_b_] at Rothamsted, into food requirements -of the cellulose decomposing bacteria. They realised that since more -than 10 per cent. of the dry weight of bacteria consists of nitrogen, -it would be necessary to supply the cellulose decomposing bacteria with -a supply of nitrogen, in order that they should attain their greatest -activity. Experiments with cultures of _Spirochæta cytophaga_ showed -that the amount of cellulose decomposed depended upon an adequate -supply of nitrogen for the organism (Fig. 3). Similarly, materials such -as straw will scarcely decompose at all if wetted with pure water. An -adequate supply of nitrogen compounds is needed to enable decomposition -to take place. Hutchinson and Richards tested the effect of ammonium -sulphate, and discovered experimentally the proportion of ammonia to -straw that produced the most rapid decomposition. They found that if -a straw heap was treated with the correct proportion of ammonia, it -decomposed into a brown substance having the appearance of well-rotted -manure. This has resulted in the development of a commercial process -for making synthetic farmyard manure from straw. The method of -manufacture is as follows: A straw stack is made and thoroughly wetted -with water. The correct amount of ammonium sulphate is then sprinkled -on the top and wetted, so that the solution percolates through the -straw. The cellulose bacteria attack the straw, breaking it down -and assimilating the ammonia. This ammonia is not wasted, as it is -converted into bacterial protoplasm that eventually decays in the soil. -Field trials of this synthetic manure show that it produces an effect -closely similar to that of natural farmyard manure. - -While cellulose and related carbohydrates are by far the most -important non-nitrogenous compounds left in the soil by plants, there -are other compounds whose destruction by bacteria is of special -interest. Such, for example, is the case of phenol. This compound is -produced by bacterial action as a decomposition product of certain -amino-acids. It occurs in appreciable amounts in cow urine. It is -probable that it forms a common decomposition product in soil and -also in farmyard manure. If this phenol were to persist in the soil, -it would eventually reach a concentration harmful to plant growth. It -does not, however, accumulate in the soil; indeed, if pure phenol or -cresol be added to ordinary arable soil, a rapid disappearance occurs. -This disappearance is of some practical importance, since it limits -the commercial use of these compounds as soil sterilising agents. -The cause of the disappearance has been to some extent elucidated at -Rothamsted,[58] where it was found to be in part a purely chemical -reaction with certain soil constituents, and partly due to the activity -of bacteria capable of decomposing it. A large number of soil bacteria -have now been isolated that can decompose phenol, meta-, para-, and -ortho-cresol, and are able to use these substances as the sole sources -of energy for their life processes. These organisms have a wide -distribution, having been found in soil samples taken from all over -Great Britain, from Norway, the Tyrol, Gough Island, Tristan da Cunha -and South Georgia. Soil bacteria have also been isolated that are able -to decompose and derive their energy from naphthalene and from toluene. -The ability of the bacteria to break up the naphthalene is very -remarkable, and all the more so since they can hardly have come across -this compound in the state of nature. The naphthalene organisms have a -distribution as world-wide as the phenol group. - - -(2) _Ammonia Production._ - -The second main group of products left in the soil by higher plants -are the nitrogen-containing compounds, such as the proteins and -amino-acids. Plant remains are not the only source of organic nitrogen -compounds available to soil bacteria. There are, in addition, the dead -bodies of other soil organisms, such as protozoa and algæ. The relative -importance of these sources of nitrogen is not known, but almost -certainly varies greatly with the state of activity of the various -groups of the soil population. Bacteria are able to utilise organic -nitrogen compounds as energy sources, as can be exemplified in the -oxidation of a simple amino-acid:-- - - H O - | // - H--C--C + 3O = 2CO₂ + H₂O + NH₃ + 152 Cal. - | \ - NH₂ OH - -It will be seen that, in the acquirement of energy from such a -compound, ammonia is released as a by-product. It is not certainly -known what is the exact course of the reactions brought about by -bacteria in soil during the breaking-down of organic nitrogen -compounds, but they result in the splitting off of most of the nitrogen -as ammonia. Herein lies the great importance of the process, for the -production of ammonia is an essential stage in the formation of nitrate -in the soil, and on the supply of nitrate the growth of most crops -largely depends. - -[Illustration: FIG. 4.--Quantities of ammonia produced by pure cultures -from 5 grams of casein in the presence of varying quantities of -dextrose. (After DORYLAND.) - - X-axis: Percentage of dextrose added. - - Y-axis: Milligrams of NH₃ produced.] - -It is very important to note that the production of this ammonia is -only a by-product in the economy of the bacteria, the benefit that they -derive from the reactions being due to the release of energy involved -in the decomposition. The common ammonia-producing bacteria in the -soil have been found equally capable of deriving their energy by the -oxidation of sugars and similar non-nitrogenous compounds. Fig. 4 -shows an experiment by Doryland,[17] in which cultures of common soil -bacteria were grown in peptone solution, to which increasing quantities -of sugar were added. One can see that, as the amount of sugar is -increased, the production of ammonia is lowered, since the bacteria -are obtaining energy from the sugar instead of from the nitrogen -compound, peptone. Consequently, if soil contains a quantity of easily -decomposible carbohydrate material, bacteria will derive their energy -from this source, and the production of ammonia and nitrate will be -lowered. Thus the addition of sugar or unrotted straw to the soil often -lowers the nitrate production, and consequently reduces the crop yield. -If the soil is sufficiently rich in carbohydrate material, the bacteria -may multiply until the supply of organic nitrogen is used up, and -then will actually assimilate some of the ammonia and nitrate already -existing. There is thus a balance of conditions in the soil due to -varying proportions of nitrogenous and non-nitrogenous energy material. -When nitrogen compounds are the predominant energy source, the bacteria -utilise them, and ammonia is released. When a non-nitrogenous energy -source predominates, this is utilised and little or no ammonia is -released, and in extreme cases ammonia may be assimilated. - -Although a large number of the common organisms in the soil produce -ammonia in culture media containing peptone, the relative importance -of these in the soil has yet to be decided. It was supposed that the -spore-forming organisms related to _Bacillus mycoides_ were of chief -importance. This supposition dates from the work of Marchal,[49] who -studied the production of ammonia by an organism of this group in -culture solution, and found it to be a very active ammonifier. As -already mentioned, however, there is some doubt as to whether the large -spore-forming organisms are very active under soil conditions.[12],[13] -The existence of rapid fluctuations in nitrate content, found to exist -in soil, may in the future indicate which are the most active of the -common bacteria in the soil itself by enabling us to observe which -types increase during periods of rapid ammonia and nitrate formation. - - -(3) _Nitrate Production._ - -The ammonia produced in the soil under normal field conditions is -rapidly oxidised successively to nitrite and to nitrate, a process -known as nitrification. The process of nitrification is more rapid than -that of ammonia production, with the consequence that no more than -traces of ammonia are able to accumulate. The rate at which nitrate is -formed in the soil is consequently set by the slower process of ammonia -production. - -The work of Schloesing and of Warington showed that the oxidation -of ammonia was the work of living organisms. It is, however, to -Winogradsky’s isolation and study of the causative organisms that we -owe our present knowledge of the biology of the process. By a new -and ingenious technique, he isolated from soil two remarkable groups -of bacteria that bring about nitrification. The first group oxidises -ammonium carbonate to nitrite, and was divided by Winogradsky into -the two genera, _Nitrosomonas_, a very short rod-like organism bearing -a single flagellum, and _Nitrosococcus_, a non-motile form found in -South America. The second group oxidises nitrites to nitrates. They are -minute pear-shaped rods to which he gave the name _Nitrobacter_. - -Winogradsky found that the first, or nitrite-producing group, would -live in a culture solution containing:-- - - 2·25 grams ammonium sulphate, - 2·0 „ sodium chloride, - 1·0 „ magnesium carbonate, - to the litre of well water. - -Nitrobacter would grow in a similar medium containing sodium nitrite -instead of ammonium sulphate. There being no organic carbon in these -media, the organisms had no source of carbon for their nutrition, -except the CO₂ of the air, or possibly that of bicarbonate in solution. -It therefore followed that the organisms must obtain their carbon -supply from one of these sources. Unlike green plants, the nitrous and -nitric organisms are able to carry on this carbon assimilation in the -dark, and must therefore obtain the energy needed for the process from -some chemical reaction. The only sources of energy in Winogradsky’s -solutions were the nitrogen compounds, and it consequently followed -that the organisms must derive their energy supply by the oxidation -of ammonia and nitrite respectively. The release of energy obtained -by these two reactions has been calculated by Orla-Jensen to be as -follows:-- - - (NH₄)₂CO₃ + 3O₂ = 2HNO₂ + CO₂ + 3H₂O + 148 Cals. - - KNO₂ + O = KNO₃ + 22 Cals. - -The exact process by which ammonium carbonate is converted into nitrite -is not at present known. The two groups of organisms are extremely -selective in their source of energy. The nitrous organisms can derive -their energy only by the oxidation of ammonia to nitrite, and the -nitric organisms only by the oxidation of nitrite to nitrate. In -culture media they are, indeed, inhibited by soluble organic compounds -such as sugars. Under natural conditions, however, they appear to -be less sensitive, since ammonium carbonate is readily nitrified -in substrata rich in organic matter. The rapid nitrification that -takes place during the purification of sewage is an example of this. -The conditions in culture, with regard to aeration and the removal -of metabolic products from the neighbourhood of the organisms, are -very different from those in the soil, and perhaps account for the -discrepancies found. - -The oxidation of ammonium carbonate by nitrosomonas results in the -formation of nitrous acid. The organisms are very sensitive to acidity, -and can only operate if the nitrous acid produced is neutralised by an -available base. In normal soils calcium carbonate supplies this base, -and in acid soils the formation of nitrite is, as a rule, increased -by the addition of lime, or of calcium or magnesium carbonate. There -is evidence that in the absence of calcium carbonate, other compounds -can be used as a base. It was found by Hopkins and Whiting[32] that -in culture solution the nitrifying organisms could use insoluble rock -phosphate as a base, producing therefrom the soluble acid phosphate. -There is evidence, however, that in ordinary soil containing calcium -carbonate very little solution of phosphate takes place in this way. -The further oxidation of nitrite to nitrate by _Nitrobacter_ does not -produce acid, and requires no further neutralising base. - -The nitrate produced in this way is the main source of nitrogen supply -to plants under normal conditions. Experiments have shown that a number -of plants are capable of utilising ammonia as a source of nitrogen, and -Hesselmann[34] has found forest soils in Sweden where no nitrification -was proceeding, and where, therefore, plants would presumably obtain -their nitrogen in this way, but such cases must be regarded as -exceptional. - -Another group of bacteria capable of deriving their energy from -an inorganic source exists in the soil. This comprises the sulphur -bacteria, which are able to derive energy by the oxidation of sulphur, -sulphides, or thiosulphates to sulphuric acid:-- - - S + 3O + H₂O = H₂SO₄ + 141 Cals. - -One organism studied by Waksman and Joffe[63] is able to live in -inorganic solution, deriving its carbon from carbon dioxide. The -sulphur bacteria have recently come into prominence in America owing to -their faculty for producing acid. Thus Thiospirillum will increase the -acidity of its medium to a reaction of P_{H} 1·0 before growth ceases. -The potato scab disease in America is now treated by composting with -sulphur. This treatment depends on the production of sulphuric acid by -the sulphur oxidising bacteria, which renders the soil too acid for the -parasite. There is some evidence also that acid thus produced can be -used to render insoluble phosphatic manures more available in the soil. - -Analogous to the sulphur organisms are certain bacteria isolated from -sheep dig tanks in South Africa by Green,[28_b_] which can derive -energy by the oxidation of sodium arsenite to arsenate. - - -(4) _Anaerobic Respiration._ - -As is seen in the examples mentioned, energy is commonly obtained by -bacteria through an oxidation process in which free oxygen is utilised. -In water-logged soil, however, or in soil overloaded with organic -matter, anaerobic bacteria may develop, which obtain their oxygen -from oxidised compounds. Thus there are soil organisms described by -Beijerinck[2] and others which can obtain oxygen by reducing sulphates -to sulphides. - -A more important source of oxygen under these conditions is nitrate, -which can supply oxygen to a larger number of bacteria. The stage to -which the reduction can be carried varies according to the organism. -A very large number of bacteria are capable of reducing nitrates to -nitrites. Many can reduce nitrate to ammonia, and some can produce -an evolution of nitrogen gas from nitrate. The effects of nitrate -reduction, therefore, appear under water-logged conditions in soils. -For example, in swamp soils in which rice is grown, it has been found -by Nagaoka,[53] in Japan, that treatment with nitrate of soda depresses -the yield, probably owing to the formation of poisonous nitrites by -reduction. - -Under normal conditions of well aerated soil, however, it is unlikely -that the reduction of nitrate is of great importance. In such soils the -activities through which bacteria acquire their energy are, as we have -seen, of vital importance to the plant, resulting in the disintegration -of plant tissues, with the ultimate formation of humus, and in the -production of nitrate. - -In their activities connected with the building up of their protoplasm, -bacteria may, on the other hand, compete with the plant. These -activities and their consequences will be reviewed in the following -chapter. - - - - -CHAPTER III. - -SOIL BACTERIA. - - -_C._ ACTIVITIES CONNECTED WITH THE BUILDING-UP OF BACTERIAL PROTOPLASM. - - -(1) _Composition of Bacteria._ - -The activities of the soil bacteria that we have yet to consider are -those connected with the building-up from simpler materials of the -protoplasm of the bacterial cell. It is important to bear in mind that -this process is one requiring an expenditure of energy on the part of -the organism. The sources of energy we have already considered. - -The bodies of bacteria contain the same elements common to other -living matter. Analyses of various bacteria have been made by a number -of workers. About 85 per cent. of their weight is made up of water. -This analysis of Pfeiffer’s Bacillus by Cramer[15] shows the typical -percentages of carbon, nitrogen, hydrogen, and ash in the dry matter:-- - -_Composition of Pfeiffer’s Bacillus (Cramer)._ - - C 50 per cent. - N 12·3 „ - H 6·6 „ - Ash 9·1 „ - -About 65-70 per cent. of the dry matter of bacteria consists of protein. - - -(2) _Sources of Carbon._ - -The biggest constituent of the dry matter of bacteria is therefore -carbon. In the soil, bacteria find an abundance of organic matter from -which they may derive their carbon supply. A special case, however, -is furnished by the nitrifying organisms, certain sulphur oxidising -bacteria, and others that derive their carbon from the CO₂ of the soil -atmosphere. The sources from which these special groups obtain the -necessary energy to accomplish this, we have already considered. - - -(3) _Assimilation of Nitrogen Compounds._ - -Of chief importance in its consequences are the means adopted by -bacteria to obtain their nitrogen supply. - -There is some reason to believe that soil bacteria do not take up -protein and peptones as such, but must first break down these bodies -into simpler compounds. When a sufficient amount of easily decomposable -organic nitrogen is present in the soil, the ammonifying bacteria use -such compounds as sources of energy, and in this case have a nitrogen -supply exceeding their requirements. - -But where there is an excess of carbohydrate or other non-nitrogenous -source of energy available in the soil, the case is different. Here -the organisms have a supply of energy which enables them to multiply -rapidly until the organic nitrogen is insufficient for their needs. -Hence they turn to the ammonia and nitrate present in the soil, and -build up their proteins from this source. Doryland[17] has shown that -many common soil ammonifiers assimilate ammonia and nitrate when -supplied with carbohydrate. There may thus be a temporary loss of -nitrate from soil when sugar, starch, straw, or such materials are -added to it. - - -(4) _Fixation of Free Nitrogen._ - -The bacteria that we have so far considered take up their nitrogen -directly from compounds containing this element. There remain, however, -a comparatively small but very important group of bacteria possessing -the power of causing elemental nitrogen to combine, and of building it -up into their proteins. This fixation of nitrogen by micro-organisms -is a vital step in the economy of nature. Losses of nitrogen from the -land are continually occurring through the washing-out of nitrates -by rain, and through the evolution of gaseous nitrogen during the -processes of decay. To maintain the supply of combined nitrogen -which is essential to living organisms, there must therefore be a -compensating process by which the supply of nitrogen compounds in the -soil is kept up. - -It was discovered in the middle of the nineteenth century that if soil -were kept moist and exposed to the air, there was an increase in the -amount of nitrogen compounds present. Berthelot, in 1893, studied the -nitrogen relationships of soil, and recognised that this fixation of -nitrogen in soil was the work of micro-organisms. - -Winogradsky followed up his work and isolated from soil a large -anaerobic spore-forming organism, capable of fixing nitrogen, to which -he gave the name _Clostridium pasteurianum_. In 1901 the investigations -of Beyerinck, in Holland, led to the important discovery of a group of -large aerobic organisms, which he named _Azotobacter_. These were found -to be very active in fixing free nitrogen. More recently, a number of -other nitrogen-fixing bacteria have been described, and the property -has been found to exist to a small extent in several previously -well-known organisms. - -It becomes important to determine which are the groups of bacteria -whose nitrogen-fixing powers are of chief importance in the soil. - -On account of its energetic fixation of nitrogen in culture media, -_Azotobacter_ has attracted the greatest attention of workers. The -evidence seems to be consistent with the view that _Azotobacter_ is of -importance in the soil. Thus the distribution of _Azotobacter_ would -appear to be world-wide. It is found all over Western Europe and the -United States. Lipman and Burgess[45] found it in soils collected from -Italy and Spain, Smyrna, Cairo, the Fayum, the Deccan in India, Tahiti, -Hawaii, Mexico, Guatemala, and Canada. C. M. Hutchinson[29] found it -to be distributed throughout India. It was found by Omelianski[55] -to be widely distributed in European and Asiatic Russia, and by -Groenewege[28] in Java. Ashby[1] at Rothamsted, isolated it from soils -from the Transvaal, East Africa, and Egypt. Also, an association has -sometimes been found between the ability of a soil to fix nitrogen and -the occurrence and vigour of its _Azotobacter_ flora. Thus Lipman and -Waynick[46] found that if soil from Kansas were removed to California, -its power to produce a growth of _Azotobacter_, when inoculated into a -suitable medium, was lost, and, at the same time, its nitrogen-fixing -power was greatly reduced. Moreover, it is known that conditions -favourable to the fixation of nitrogen by _Azotobacter_ in cultures on -the whole favour nitrogen fixation in soils. The conditions that favour -other aerobic nitrogen-fixing bacteria are, however, not sufficiently -distinct to make such evidence of great value. - -It is usually found that nitrogen fixation is most active in -well-aerated soil. Thus Ashby,[1] at Rothamsted, found the -nitrogen-fixing power of a soil to decrease rapidly with depth. Similar -results were obtained in Utah by Greaves. This suggests, at first -sight, that anaerobic nitrogen fixers are unimportant under normal -soil conditions. It is, however, quite possible that they may assume -an importance when acting in conjunction with aerobic organisms. -Thus Omelianski and Salunskov[55] found that beneficial association, -or symbiosis, could occur between _Azotobacter_ and _Clostridium -pasteurianum_, the former absorbing oxygen from the surroundings, and -thus creating a suitable anaerobic environment for the _Clostridium_. - -The question of symbiosis of nitrogen-fixing bacteria with each other -and with other organisms offers an inviting field for research. -There is evidence that this factor may have considerable importance. -Beijerinck and Van Delden[3] early recognised that _Azotobacter_ -in mixed cultures fixed more nitrogen than in pure cultures. -_Granulobacter_, an organism which they found to be commonly associated -with _Azotobacter_ in crude cultures, appears to increase its -nitrogen-fixing powers (Krzeminiewski).[41] It was also found by -Hanzawa[31] that a greater fixation of nitrogen was obtained when two -strains of _Azotobacter_ were grown together. A symbiosis between -_Azotobacter_ and green algæ has been described, and will be further -discussed by Dr. Bristol. It is likely that this association may be of -importance under suitable conditions on the soil surface where the algæ -are exposed to light. - -The combination of elemental nitrogen is an endothermic process which -requires a very considerable amount of energy for its accomplishment. -This fact is well illustrated by the various commercial processes -in use for fixation of atmospheric nitrogen. The nitrogen-fixing -bacteria obtain this energy from the carbon compounds in the soil. -A number of compounds were compared as sources of energy by Löhnis -and Pillai,[47] who tested their effect on the amounts of nitrogen -fixed by _Azotobacter_ in culture. It was found that mannitol and the -simpler sugars give the best results as sources of energy, but that -other organic compounds can also be used. Mockeridge[51] has adduced -evidence that ethylene glycol, methyl-, ethyl-, and propyl-alcohol, -lactic, malic, succinic, and glycocollic acids could also be utilised. -Since so large a part of the organic matter added to soil is in the -form of celluloses, it is of great importance to ascertain how far -these compounds and their decomposition products can be utilised in -nitrogen fixation. Stubble, corn-stalks and roots, oak leaves, lupine -and lucerne tops, maple leaves, and pine needles may all serve as -useful sources of energy to nitrogen-fixing organisms in the soil. Pure -cellulose cannot apparently be used as a source of energy, but when -acted upon by cellulose decomposing organisms, it becomes available as -a source of energy. Hutchinson and Clayton, at Rothamsted, found that -a fixation of nitrogen could be brought about by mixed cultures of -_Azotobacter_, and of the cellulose attacking _Spirochæta cytophaga_, -when grown in cultures containing pure cellulose. It is not known -how far cellulose decomposition must proceed to produce an effective -source of energy, nor what are the substances thus produced that are -utilised. This point will not be decided until something more is known -of the course of changes in the breaking-down of cellulose in the soil. - -The amount of nitrogen fixed per unit of energy material decomposed -varies greatly, according to the organism and the conditions. -Winogradsky found that his _Clostridium_ assimilated 2-3 mgs. of -nitrogen per gram of sugar consumed. Lipman found that _Azotobacter_ -fixed 15-20 mgs. of nitrogen per gram of mannite consumed. - -[Illustration: FIG. 5. - - Caption: Azotobacter. Decrease in efficiency in N fixation with age - of culture. (Koch & Seydel.) - - X-axis: Days. - - Y-axis: Milligrams of Nitrogen fixed per gram of dextrose consumed.] - -It is found, however, that in liquid culture, the ratio of nitrogen -fixed to carbohydrates oxidised varies according to the age of the -culture, falling off rapidly as the age increases[42] (Fig. 5). This -decreasing efficiency in cultures may be due to the accumulation of -metabolic products such as would not occur under soil conditions. -Indeed, the efficiency of _Azotobacter_ in a sand culture has been -found by Krainskii[39] to be considerably greater than in solution. It -is thus probable that in soil the nitrogen-fixing organisms are less -wasteful of energy material than under the usual laboratory conditions. -It is to be hoped that future research will indicate what are the -conditions that produce the greatest economy of energy material in -nitrogen fixation. - -The fixation of nitrogen in soil is depressed by the presence of -considerable amounts of nitrates. This is, in all probability, due -to the fact that nitrogen-fixing organisms are able to utilise -compounds of nitrogen where these are available. The energy needed -to build up amino-acids and proteins from nitrate or ammonia is, of -course, far less than that required to build up these substances -from elemental nitrogen. It is, therefore, not surprising that where -nitrate is available, _Azotobacter_ will use it in preference to fixing -atmospheric nitrogen.[5] - -TABLE III.--ASSIMILATION OF NITRATES. - -BY AZOTOBACTER IN PURE CULTURE--(_Bonazzi_). - - +---------------------------+--------+-----------+------+ - | | Nitrate| Organic |Total | - | | and | Nitrogen |Fixed | - | | Nitrite|and Ammonia| or | - | |Present.| Present. |Lost. | - +---------------------------+--------+-----------+------+ - | | mgs. | mgs. | mgs. | - |_Culture with nitrate_-- | | | | - | At beginning | 8·55 | 0·76 | -- | - | After growth | 0·2 | 8·71 | -0·4 | - |_Culture without nitrate_--| | | | - | At beginning | -- | 0·76 | -- | - | After growth | -- | 4·50 | +3.74| - +---------------------------+--------+-----------+------+ - - (Growth period--24 days at 25° C.) - -The chemical process by which nitrogen is fixed is quite unknown, -although a number of speculative suggestions have been made. The -appearance of considerable amounts of amino acids in young cultures of -_Azotobacter_ suggests that these may be a step in the process, but at -present the data are too inconclusive to form a basis for theorising. - -_Azotobacter_ is very rich in phosphorus, an analysis of the surface -growth in _Azotobacter_ cultures, made by Stoklasa, giving about 60 -per cent. of phosphoric acid in the ash. In cultures it has been found -that a considerable amount of phosphate is needed to produce full -development. As would be expected, therefore, nitrogen fixation in soil -is often greatly stimulated by the addition of phosphates. Christensen -has, indeed, found soils where lack of phosphate was the limiting -factor for _Azotobacter_ growth. - -_Azotobacter_ is very intolerant of an acid medium, and is very -dependent on the presence of an available base. In cultures this -is usually provided in the form of calcium or magnesium carbonate. -Gainey[21] found that _Azotobacter_ occurred in soils having an acidity -not greater than P_{H} 6·0, and Christensen,[7],[9] in Denmark, has -found a close association between the occurrence of _Azotobacter_ in -soils and the presence of an adequate supply of calcium carbonate. So -close was this association that he devised a technique based on this -fact for detecting a deficiency of lime in a soil sample. - -In addition to the groups already discussed, there is a remarkable and -important group of nitrogen-fixing bacteria that inhabit and can carry -on their functions within the root tissues of higher plants. It has -been known at least from classical times that certain leguminous plants -would, under suitable conditions, render the soil more productive. On -the roots of leguminosæ small tubercles are commonly found. These were -noted and figured by Malpighi in the seventeenth century, and for a -long time were regarded as root-galls. As was described in Chapter I., -the true nature of these tubercles was finally elucidated by Hellriegel -and Wilfarth in 1886. As the result of a series of pot experiments, -they made the very brilliant deduction that the ability to fix -nitrogen, possessed by the legumes, was due to bacteria associated with -them in the tubercles. - -These bacteria were finally isolated and studied in pure culture -by Beijerinck. Since then a very great deal of literature has -accumulated on the subject of the nodule-producing bacteria, which -it is impossible to deal with in a small space. The nodule organism, -_Bacillus radicicola_, when grown on suitable media, passes through a -number of different changes in morphology. The most connected account -of these changes is given in a paper by Bewley and Hutchinson.[4] -In a vigorous culture the commonest type is a rod-shaped bacillus -which may or may not be motile. As these get older they often become -branched, or irregular in shape, the formation of these branched forms -being perhaps due to conditions in the medium. These irregular forms, -known as “bacteroids,” are a characteristic type in the nodules. Their -production in culture media has been found to be stimulated by sugars -and organic acids such as would occur in their environment within the -host plant. In the older rods and bacteroids the staining material -becomes condensed into granules, and finally the rods disintegrate or -break up into coccoid forms. By suitable culture conditions, Bewley and -Hutchinson obtained cultures consisting almost entirely of this stage. -If such a culture be inoculated into a fresh medium rich in sugar, -the swarmer stage appears in great numbers. These swarmers are very -minute coccoid rods, ·9 × ·18 in size, that are actively motile. They -apparently develop later into the rod stage. - -[Illustration: FIG. 6.--_Bacillus radicicola._ Stages in the life -cycle. (After HUTCHINSON and BEWLEY.) - - Motile Rods - - Vacuolated Stage - - “Swarmers” - - “Bacteroids” - - “Pre-swarmers”] - -Very little is known as to the life of the organism in the soil. -It is able, however, to fix nitrogen in cultures, and it has been -claimed[35],[48] that it can do so in the soil outside the plant, so -that it is possible that we must take it into consideration in this -connection. More knowledge is needed as to the optimum conditions for -the growth of the organism in the soil. It seems to be more tolerant of -acid soil conditions than _Azotobacter_. The limiting degree of acidity -has been found to vary among different varieties of the organism from -P_{H} 3·15 to P_{H} 4·9. - -A long controversy has been held as to whether the nodule organisms -found in different host-plants all belong to one species, or whether -there are a number of separate species, each capable of infecting a -small group of host-plants. As the term “species” has at present no -exact meaning when applied to bacteria, the discussion in this form -is unlikely to reach a conclusion. The evidence seems to show that -the nodule organisms form a group that is in a state of divergent -specialisation to life in different host-plants, and that this -specialisation has reached different degrees with different hosts. -Thus the organisms from the nodule of the pea (_Pisum sativum_) will -also produce nodules on vicia, Lathyrus, and Lens, but seem to have -lost the ability normally to infect other legumes. On the other hand, -the bacteria from the nodules of the Soy Bean (_Glycine hispida_) -have become so specialised that they do not infect any other genus -of host-plant, and soy beans are resistant to infection by other -varieties of the nodule organism. Burrill and Hansen,[6] after an -extensive study, divided the nodule bacteria into eleven groups, within -each of which the host-plants are interchangeable. The existence of -different groups of nodule organisms has been confirmed by the separate -evidence of serological tests (Zipfel, Klimmer, and Kruger).[40] The -results of cross-inoculation tests have sometimes been conflicting. It -seems, indeed, that the host-plant has a variable power of resisting -infection, so that when its resistance is lowered it may be capable of -infection by a strange variety of the nodule organism. The question -that has thus arisen of the ability of the legume to resist infection -is of fundamental importance, and its elucidation should throw light on -the relation of plants to bacterial infection as a whole. - -The stage of the organism that infects the plant is not at present -known. It may be supposed that it is the motile “swarmer.” The entry is -normally effected through the root-hairs. The hair is attacked close -to the tip, and an enzyme is apparently produced which causes the tip -to bend over in a characteristic manner. The organisms multiply within -the root hair and pass down it, producing a characteristic gelatinous -thread filled with bacteria, in the rod form. This “infection thread” -passes down into the cells of the root tissue, where it branches -profusely. In young stages of nodule formation the branches can be seen -penetrating cells in the pericycle layer. Rapid cell division of these -root cells is induced. In the course of this cell division abnormal -mitotic figures are sometimes found, such as occur in pathological -growths. The cells push outward the root cortical layer, and so form a -nodule. - -Certain of the cells in the centre of the nodule become greatly -enlarged, and in the fully grown nodule are seen to be filled with -bacteria. Differences have been described in the morphology of the -organisms in different parts of the nodule.[62] Whether the different -stages of the organism are equally capable of fixing nitrogen, or what -is the significance of these stages within the nodule, is not certainly -known. It has been held that it is the irregular bacteroid forms that -are chiefly concerned with nitrogen fixation. In older nodules the -organisms become irregular and stain faintly, and the bacteroidal -tissue breaks down, the nodule finally decaying. In the fixation of -nitrogen that occurs in the nodules, the bacteria without doubt derive -the necessary energy from the carbohydrates of the host-plant. There -is evidence that the plant assists the process of fixation by removing -soluble metabolic products from the neighbourhood of the bacteria. -Golding[22] was able to obtain a greatly increased fixation of nitrogen -in artificial cultures by arranging a filtering device so as to remove -the products of metabolism. - -The great practical importance of leguminous crops in agriculture has -led to numerous attempts being made to increase their growth, and the -fixation of nitrogen in them, by inoculating the seed or the soil with -suitable nodule-bacteria. This inoculation can be effected either with -soil in which the host-plant has been successfully grown, and which -should consequently contain the organism in fair numbers, or else pure -cultures of the organisms isolated from nodules may be used. Very -varying results have been obtained with inoculation trials. - -In farm practice a leguminous crop has often been introduced into a -new area where it has never previously grown. In such soil it is very -probable that varieties of the nodule organism capable of infecting the -roots may not exist. In such cases inoculation with the right organism -or with infected soil often produces good results. - -The more difficult case, however, is that in which the legume crop -has been grown for a long time in the locality, and where the soil -is already infected with right organisms. This, the more fundamental -problem, applies especially to this country. Here it would seem that -inoculation with a culture of the organism will benefit the plant only -(1) if the naturally occurring organisms are present in very small -numbers; or (2) if the organisms in the culture added are more virulent -than those already in the soil. The problem of successful inoculation -would therefore seem to be bound up with that of grading up the -infective virulence of the organism to a higher level. - -Successful nodule development in a legume crop is also dependent to a -large degree on the soil conditions. The effects of soil conditions on -nodule development have been studied by numerous workers. Moisture has -been found very greatly to affect the nodule development. Certain salts -have a very definite effect on nodule formation.[64] Their effect on -the number of nodules developing has been studied, but the reason for -this effect is unusually difficult to decide. The action is usually a -complex one. Thus phosphates are known to stimulate nodule formation. -They probably act in several ways. In the first place, they may cause -the nodule organisms to multiply in the soil; in the second place, they -produce a greater root development in the plant, thus increasing the -chances of infection; and in the third place, Bewley and Hutchinson[4] -have found that phosphates cause the appearance of the motile stage -of the organism in cultures. A real understanding of the influence of -environment on nodule production will produce great improvements in our -methods of legume cropping. - - -_D._ THE RELATION OF BACTERIAL ACTIVITIES TO SOIL FERTILITY. - -The various activities of the soil bacteria have a vital importance to -the growth of higher plants, which are dependent for their existence -on certain of these processes. In the first place, as we have seen, -bacteria decompose the tissues of higher plants and produce humus -materials, which are essential to the maintenance of good physical -properties in the soil. Then the nitrate supply on which most higher -plants depend is produced by the decomposition of organic nitrogen -compounds by bacteria in their search for energy. The depletion of the -total nitrogen content of the soil through rain and through the removal -of nitrogen in the crops, is to some extent compensated by the fixation -of atmospheric nitrogen by certain bacteria. On the other hand, in the -assimilation of nitrogen compounds to build up protein, the bacteria -are competing with higher plants for one of their essential food -constituents, and their action may, under certain conditions, cause a -temporary nitrogen starvation. One must remember, however, that large -quantities of nitrate are lost from field soils by washing-out through -rain action, especially in winter. The assimilation of nitrate and -ammonia by micro-organisms keeps some of this nitrogen in the soil, and -at certain periods may thus be beneficial. - -There is another important respect in which soil bacteria influence -plant growth. Their activities result in the release of inorganic -salts, such as potash and phosphates, in a form available for the use -of plants. The release of phosphorus and potassium compounds takes -place in two ways. In the first place, organic matter containing -phosphorus and potassium, in an insoluble form, is attacked by -bacteria, resulting in these elements being set free as inorganic -salts available to the higher plant. Secondly, much of the phosphorus -supplied to the soil from rock minerals is present as insoluble -phosphates, such as apatite and iron phosphate. Much of the potassium, -too, is derived from insoluble silicate minerals. In both cases the -conversion of the insoluble minerals into soluble phosphates and -potassium compounds is brought about to a large extent by solution in -water containing carbonic and other acids. These acids are largely -produced by micro-organisms, which, in addition to carbonic acid, -produce organic acids, and in specialised cases, sulphuric and nitrous -acids. It has been found, for example, that in a compost of soil -with sulphur and insoluble phosphate, sufficient sulphuric acid may -be produced by the oxidation of the sulphur by bacteria to convert -an appreciable amount of phosphate into a soluble form. When we -consider the functions performed by soil bacteria, therefore, it is -not surprising to find that high bacterial activity in the soil is -associated, as a rule, with fertility. - - -_E._ CHANGES IN BACTERIAL NUMBERS AND ACTIVITIES, AND THEIR RELATION TO -EXTERNAL FACTORS. - -The object of soil bacteriologists is to discover means of favouring -the activity of soil bacteria, especially those activities that are -useful to the higher plant. Knowledge is therefore needed of the -changes in numbers and activities of the soil bacteria, and of the -influence of soil conditions on them. The necessity of studying these -changes has required the development of a quantitative technique by -which the numbers of bacteria in the soil and their activities can be -estimated. - -The method commonly used in counting bacteria in soil is a modification -of the plating method of Koch. In counting bacteria two difficulties -have to be overcome--their immense numbers and their small size. The -numbers of bacteria in soil are so large that the bacterial population -of a gram of soil could not, of course, be counted directly. The method -adopted, therefore, is to make a suspension of soil in sterile salt -solution, and to dilute this suspension to a convenient and known -extent, which will depend on the numbers of bacteria expected. In -ordinary field soils it is found convenient, for example, to dilute the -soil suspension so that one cubic centimeter of the diluted suspension -will contain 1/250,000th of a gram of soil. Such a volume will commonly -contain a number of bacteria sufficiently small to count. The second -difficulty is that the organisms are microscopic, and yet cannot be -readily counted under the microscope owing to the presence of soil -particles in the suspension. Hence recourse is had to plating. One -cubic centimeter of diluted suspension is placed in a petri dish and -mixed with a suitable nutrient agar medium, melted, and cooled to about -40° C. The medium sets, and after a few days’ incubation the organisms -multiply and produce colonies visible to the naked eye. By counting -these colonies we obtain an estimate of the number of bacteria in -the one cubic centimeter of suspension, it being assumed that every -organism has developed into one colony, and by multiplying this number -by the degree of dilution we obtain the numbers per gram of soil. -In practice a number of parallel platings are made from one cubic -centimeter portions of the diluted suspension and the mean number of -colonies per plate is taken. By this means the error due to the random -distribution of bacteria in the suspension is reduced, because of the -greater number of organisms counted. - -In drawing conclusions from bacterial count data, it is necessary to -distinguish between the indication which the method gives of the -absolute numbers of bacteria in the soil and the accuracy with which -it enables the numbers of two soil samples to be compared. The method -cannot be used for the former purpose at present. We do not know how -far the figures obtained by this counting method fall short of the -actual number of bacteria in the soil. One reason for this is the -difficulty of effecting a complete separation of the clumps of bacteria -into discrete individuals in the suspension. Then again, there is no -known medium upon which all the physiological groups of bacteria will -develop and produce colonies. And even on a suitable medium some of the -individuals may fail to multiply. - -In comparing the bacterial numbers in two soil samples, however, -the case is different. Within each bacterial group investigated the -plate method should give counts proportional to the bacterial numbers -in the soil. Thus, by the method one should be able to tell whether -the bacterial numbers are increasing or decreasing over a period of -time, or whether a certain soil treatment produces an increase or -a decrease. With this end in view the technique of the method has -been improved by recent workers. It was found that, when carefully -standardised, the process of dilution of the soil could be carried -out without significant variation in result (Table IV.), and that the -accuracy of the method is limited mainly by the variation in colony -numbers on parallel platings, due in part to random distribution of -bacteria throughout the final suspension, and partly to the uneven -development of colonies on the medium. The question of the medium was -therefore taken up with a view to improving the uniformity of results -obtained with it. Lipman, Conn, and others effected an improvement by -using chemical compounds as nutrient ingredients, thus making their -media more closely reproducible. On most agar media, an important -disturbing factor is the growth of spreading colonies, which prevent -the development of some of the other colonies. A medium has been -devised at Rothamsted on which these spreading organisms are largely -restricted.[61] A statistical examination[19] has shown that on this -medium errors due to the uneven development of colonies, except in -special cases, are prevented, so that in fact the variation in colony -numbers between parallel plates is found to be that produced merely by -random distribution of bacteria in the diluted suspension (see Table -IV.). In this case the accuracy of the counts of the bacteria in the -diluted suspension depend directly on the number of colonies counted, -and can be known with precision. - -TABLE IV.--BACTERIAL COUNTS OF A SOIL SAMPLE. - -PARALLEL PLATE COUNTS FROM FOUR SETS OF DILUTIONS MADE BY DIFFERENT -WORKERS. - - +------------------------------------------+ - | Counts of Colonies on each Plate. | - +------+--------+--------+--------+--------+ - |Plate.| Set I. | Set II.|Set III.| Set IV.| - +------+--------+--------+--------+--------+ - | 1 | 72 | 74 | 78 | 69 | - | 2 | 69 | 72 | 74 | 67 | - | 3 | 63 | 70 | 70 | 66 | - | 4 | 59 | 69 | 58 | 64 | - | 5 | 59 | 66 | 58 | 62 | - | 6 | 53 | 58 | 56 | 58 | - | 7 | 51 | 52 | 56 | 54 | - +------+--------+--------+--------+--------+ - | Mean | 60·86 | 65·86 | 64·28 | 62·86 | - +------+--------+--------+--------+--------+ - - Standard deviation between the four sets = 5·62. - - Standard deviation between plates within the sets = 7·76. - -The knowledge obtained from counts of soil bacteria is subject to -another serious limitation. We do not know which of the bacteria -counted are the most effective in bringing about the various changes -that take place in the soil. It is not even known which of them are -active in the soil and which are in a resting condition. It is thus -possible to have two soils containing equal numbers of bacteria but -showing widely different biochemical activity, if one soil contains -organisms of a higher efficiency. Moreover, as has been pointed -out, many important groups of soil bacteria do not develop on the -plating media, and so are not counted. These considerations led to -the development of supplementary methods by which it was hoped to -estimate the actual biochemical activity of the soil microflora. The -first of these methods was developed by Remy, who attempted to study -the biochemical activity of a soil by placing weighed amounts into -sterile solutions of suitable and known composition, keeping them -under standard conditions for a definite time and then estimating the -amount of the chemical change that was being studied. Thus, to test the -activity of the organisms that produce ammonia from organic nitrogen -compounds, he inoculated soil into 1 per cent. peptone solution and -measured the amount of ammonia produced in a given time. By similar -methods the power of a soil to oxidise ammonia to nitrate, to reduce -nitrate, or to fix atmospheric nitrogen, is tested. This method has -been extensively used and developed by more recent workers. It suffers, -however, from the same serious disadvantage that it was designed to -avoid, for we cannot be certain that those bacteria that develop in -the nutrient solution are the types that are active in the soil, and, -moreover, even where the same types do function in the two conditions, -we do not know that the degree of their activity is the same in soil -and in solution cultures. For instance, _Nitrosomonas_ appears to show -very different degrees of activity in soil and in culture. - -Another method, therefore, of studying the activity of soil -micro-organisms is the obvious one of estimating the chemical changes -that they produce in the soil itself. This method has obvious -advantages over the unnatural methods developed from Remy’s, but it has -a number of limitations that make its actual application difficult. -In the first place, we cannot always tell whether changes found to -occur in soil are due to the activity of micro-organisms, or are -purely chemical reactions unassisted by biological agencies. Then, if -we succeed in showing that the changes are due to micro-organisms, it -is very difficult to determine which organisms are effecting them. -This cannot be definitely tested by isolating suspected organisms and -testing their activity in sterile soil, because in sterilising soil its -nature and composition is altered. In spite of these difficulties, -however, the study of the chemical changes that take place in the soil -has produced valuable knowledge, when it has been combined with a -study of the changes in the number and variety of the micro-organisms -that accompany these reactions. This method of investigation is well -illustrated by the work of Russell and Hutchinson on the effects of -heat and volatile antiseptics on soil, where a study of the chemical -changes such as ammonia production, that occurred in these treated -soils, combined with a study of the changes in bacterial numbers, led -to the realisation that the soil micro-population was a complex one, -containing active protozoa. - -A great difficulty in applying quantitative methods to bacteria in the -field is the great variation in the density of the bacterial population -over a plot of field soil, which may be so great that a bacterial count -from a single sample is quite valueless. For example, the distribution -of bacterial numbers over a plot of arable soil near Northampton was -studied by taking sixteen samples distributed over an area about -12 feet square. The result showed that in some cases the bacterial -numbers in samples taken 6 inches apart differed by nearly 100 per -cent. Fortunately, under favourable conditions, a remarkably uniform -distribution of bacterial numbers over a plot of soil can be found. - -On such a plot it is possible to investigate the rapidity with which -the numbers of the soil micro-organisms alter in point of time. For -example, on the dunged plot of Barnfield, Rothamsted, which has been -cropped with mangolds for forty-seven successive years, the area -distribution of bacteria has been found to be so uniform that if a -number of samples of soil are taken from the plot at the same time, the -difference in bacterial numbers between the samples cannot be detected -by means of the counting technique (see Table V.). The work of Cutler, -Crump, and Sandon[16] on this plot showed that the bacterial numbers -vary very greatly from one day to the next, and that these fluctuations -took place over the whole plot, since two series of samples, taken in -two rows 6 feet apart, showed similar fluctuations (see Fig. 7). The -discovery of these big daily fluctuations in numbers led to an inquiry -as to how quickly bacterial numbers change, and samples from Barnfield, -taken at two-hourly intervals, showed that significant changes in -numbers took place even at such short intervals. - -TABLE V.--BACTERIAL COUNTS OF FOUR SOIL SAMPLES. - -FROM BARNFIELD, TAKEN SIMULTANEOUSLY. - - +------------------------------------------------------+ - | Counts of Colonies on each Plate. | - +------+-----------+-----------+-----------+-----------+ - |Plate.| Sample I. | Sample II.|Sample III.| Sample IV.| - +------+-----------+-----------+-----------+-----------+ - | 1 | 38 | 45 | 43 | 27 | - | 2 | 32 | 40 | 34 | 41 | - | 3 | 52 | 45 | 52 | 35 | - | 4 | 32 | 31 | 55 | 36 | - | 5 | 40 | 43 | 38 | 45 | - |Mean | 38·8 | 40·8 | 44·4 | 36·8 | - +------+-----------+-----------+-----------+-----------+ - - Standard deviation between the four samples = 7·25. - - Standard deviation between parallel plates within the sets = 7·55. - -[Illustration: FIG. 7. - - X-axis (top): Days. - - Y-axis (left): (Series A) Bacteria--millions per gramme of soil. - - Y-axis (right): (Series B) Bacteria--millions per gramme. - - Caption: Daily changes in bacterial numbers in field soil. - - Counts from two series of soil samples taken 6 feet apart. - - (After Cutler.)] - -Since the bacteria involved in this fluctuation are of great importance -to the crops, being for the most part ammonia producing types, further -knowledge as to the cause of this fluctuation and of its effect on the -ammonia and nitrate in the soil is of fundamental importance. There is -evidence, which will be discussed later, that the cause is connected -with the changing activities of certain soil protozoa, since the daily -changes in the numbers of active amœbæ in the soil have been found -to be in the reverse direction to those of the bacterial numbers. It -appears, therefore, that we are dealing with an equilibrium between -the various members of the soil population, the point of equilibrium -changing at frequent intervals. - -In addition to daily changes, it is possible to detect changes in the -numbers and activity of the soil population related to the season. -There is a well-marked increase in the spring and autumn (see Figs. -15, 16, pp. 89, 90). This is well seen when the fortnightly averages -of the daily bacterial and protozoal counts from Barnfield soil -are plotted. These spring and autumn increases comprise both the -bacterial and the protozoal population, and therefore differ from the -short time fluctuations in being due, not to a disturbance of the -bacteria-protozoa equilibrium, but to a general rise in activity of -both groups of organisms. - -When we consider the action of external conditions on the soil -bacteria, the existence of a complex soil population and the -interdependence of its members must be borne in mind. Changes in -external conditions may affect the different components of the -population in different ways or to different degrees, thus upsetting -the equilibrium between the various groups. For example, the addition -of a mild aromatic antiseptic to the soil apparently affects the -protozoa in such a way as to disturb the bacteria-protozoa equilibrium -in favour of the bacteria, while in some cases the aromatic compound -affords a food supply to special bacteria, causing these to increase, -upsetting the equilibrium between the different bacterial groups. When -our knowledge of the effect of external factors on the soil population -becomes sufficient, it will probably be found that in nearly all -cases a change in the soil conditions produces some disturbance in -the equilibrium between the components of the soil population, though -at present there are only certain examples where this disturbance is a -probable explanation of the facts. - -Since bacteria are dependent on adequate supplies of energy and food, -it is to be expected that additions of organic matter or of inorganic -food materials will greatly benefit their activities. The effect of -added farmyard manure in increasing bacterial activities has been much -studied.[27] Some of the increased bacterial numbers and activities -in this case may be due to the addition of bacteria with the manure, -but it is thought that this factor is of less importance than the -added energy and food supply which the general soil flora obtain from -it. Nutritive salts such as phosphates and salts of potassium usually -increase the bacterial activities. - -The effect of alkali salts on soil bacteria has been especially studied -in the Western United States, where the existence of alkali in the -soil is a serious problem.[23] Soil bacteria are usually stimulated by -small doses of alkali salts that are toxic in higher concentration. -As a rule, chlorides are the most toxic salts, the electronegative -ion playing an important part in the effect of the salt. Salts -affect bacteria both owing to the changes in osmotic pressure which -they produce, and through their specific action on the bacterial -protoplasm.[26] When equal weights of various salts are added to soil, -their toxic action on bacteria shows so little association with their -respective osmotic pressures that we must conclude that this factor is -the less important. There is reason to suppose that the toxic action -of salts on bacteria is often connected with an effect of the specific -ions on the permeability of the bacterial cell-wall. This conclusion -is based on the changes in electrical conductivity of bacterial -suspensions in the presence of various salts.[59] - -A definite antagonism between various salts has been found to exist. -It is possible that future work in this line may indicate what are -the proportions of common electrolytes which will produce a properly -“balanced” soil solution so that the harmful excess of one salt may be -antagonised. - -Certain salts, such as those of arsenic[24] and manganese, seem to -exercise a stimulating action on bacterial activities; the causes of -this action are not at present understood. - -The acidity of the soil has an important effect on the bacterial -processes. The acidity of soils may increase to such a point that the -decomposition of plant tissues by bacteria is hindered, a peat layer -being thus produced. The degree of acidity that is toxic varies very -greatly with different soil bacteria, some of them, like Azotobacter -and Nitrosomonas being very intolerant of acidity. - -The conditions of aeration, water content, and temperature are -inter-related in field soil. Ammonifying organisms are not greatly -dependent on aeration, but this factor is sometimes a limiting one in -the case of the very aerobic nitrifying bacteria. Hence efficient soil -cultivation is beneficial to nitrification. - -Many attempts have been made to correlate the temperature and moisture -of field soils with the bacterial numbers and activities. These -attempts have given very discordant results. It is generally agreed -that a plentiful moisture supply is beneficial. Thus Greaves, in Utah, -found the optimum water content for ammonia and nitrate production -to be about 60 per cent. of the water-holding capacity. On the other -hand, Prescott[56] found that the summer desiccation of soil in -Egypt was followed by increased bacterial activities. Fabricius and -Feilitzen,[18] using moor soil, found a direct relationship between -soil temperature and bacterial numbers, showing that temperature can -be a limiting factor under certain conditions. With normal arable -soils, however, no such direct effect of temperature or moisture can -be found[16] (see Fig. 8). It has even been found by Conn[11] that -freezing of the soil may cause a marked increase in bacterial numbers. -The erratic effects of temperature and moisture on the soil bacteria -probably afford instances of a disturbance of the equilibrium between -the bacteria and other components of the soil micro-population. Thus -desiccation and freezing, though they harmfully affect the bacteria, -may inhibit other micro-organisms to a greater degree, thus freeing -the bacteria from competition. It is in the investigation of this -equilibrium, and of the factors that can control it to our benefit, -that the great advances in soil biology in the future are to be -expected. - -[Illustration: FIG. 8.--Effect of frost on the bacterial numbers in the -soil. (After CONN.) - - X-axis: Nov.-May - - Y-axis (bottom): Temperature--Degrees C. - - Y-axis (top): Bacteria--Millions per Gramme of Soil.] - - -REFERENCES TO CHAPTERS II. AND III. - - [1] Ashby, S. F., Journ. Agric. Sci., 1907, vol. ii., p. 35. - - [2] Beijerinck, M. W., Centr. f. Bakt., 1900, Abt. II., Bd. 6, p. 1. - - [3] Beijerinck, M. W., and Van Delden, A., Centr. f. Bakt., 1902, - Abt. II., Bd. 9, p. 3. - - [4] Bewley, W. F., and Hutchinson, H. B., Journ. Agric. Sci., 1920, - vol. x., p. 144. - - [5] Bonazzi, E., Journ. Bact., 1921, vol. vi., p. 331. - - [6] Burrill, T. J., and Hansen, R., Illin. Exp. Sta., 1917, Bulletin - 202. - - [7] Christensen, H. R., Centralblatt. f. Bakt., 1915, Abt. II., Bd. - 43, p. 1. - - [8] Christensen, H. R., Centralblatt. f. Bakt., 1907, Abt. II., Bd. - 17, pp. 109, 161. - - [9] Christensen, H. R., and Larsen, O. H., Centralblatt. f. Bakt., - 1911, Abt. II., Bd. 29, p. 347. - - [10] Conn, H. J., Centralblatt. f. Bakt., 1910, Abt. II., Bd. 28, p. - 422. - - [11] Conn, H. J., Centralblatt. f. Bakt., 1914, Abt. II., Bd. 42, p. - 510. - - [12] Conn, H. J., Journ. Bact., 1916, vol. i., p. 187. - - [13] Conn, H. J., Journ. Bact., 1917, vol. ii., p. 137. - - [14] Conn, H. J., Journ. Bact., 1917, vol. ii., p. 35. - - [15] Cramer, E., Arch. f. Hyg., 1893, Bd. 16, p. 151. - - [16] Cutler, W., Crump, L. M., and Sandon, H., Phil. Trans. Roy. - Soc., 1923, Series B, vol. ccxi., p. 317. - - [17] Doryland, C. J. T., N. Dakota Agr. Exp. Sta., 1916, Bulletin 116. - - [18] Fabricius, O., and Feilitzen, H., Centr. f. Bakt., 1905, Abt. - II., Bd. 14, p. 161. - - [19] Fisher, R. A., Thornton, H. G., and Mackenzie, W. A., Ann. Appl. - Biol., 1922, vol. ix., p. 325. - - [20] Fred, E. B., and Hart, E. B., Wisconsin Agr. Exp. Sta. Research, - 1915, Bulletin 35. - - [21] Gainey, P. L., Journ. Agric. Research, 1918, vol. xiv., p. 265. - - [22] Golding, J., Journ. Agric. Sci., 1905, vol. i., p. 59. - - [23] Greaves, J. E., Soil Sci., 1916, vol. ii., p. 443. - - [24] Greaves, J. E., Journ. Agric. Res., 1916, vol. vi, p. 389. - - [25] Greaves, J. E., Soil Sci., 1920, vol. x., p. 77. - - [26] Greaves, J. E., and Lund, Y., Soil Sci., 1921, vol. xii., p. 163. - - [27] Greaves, J. E., and Carter, E. G., Journ. Agric. Research, 1916, - vol. vi., p. 889. - - [28] Groenewege, J., Arch. Suikerindust., 1913, Bd. 21, p. 790. - - [28_b_] Green, H. H., Union of S. Africa Dept. Agr., Rept. of - Director Vet. Res., 1918, p. 592. - - [29] Hutchinson, C. M., Rept. Agr. Res. Inst. and Col. of Pusa, 1912, - p. 85. - - [30] Hutchinson, H. B., and Clayton, J., Journ. Agric. Sci., 1919, - vol. ix., p. 143. - - [30_b_] Hutchinson, H. B., and Richards, H. H., Journ. Min. Agric., - 1921, vol. xxviii., p. 398. - - [31] Hanzawa, J., Centr. f. Bakt., 1914, Abt. II., Bd. 41, p. 573. - - [32] Hopkins, C. G., and Whiting, A. L., Ill. Agr. Exp. Sta., 1916, - Bulletin 190, p. 395. - - [33] Hoppe-Seyler, G., Ztschr. Phys. Chem., 1886, vol. x, pp. 201, - 401; 1887, vol. xi., p. 561. - - [34] Hesselmann, H., Skogsvårdsför. Tidskr., 1917, No. 4, p. 321. - - [35] Joshi, N. V., Mem. Dept. Agr. in India, Bact. Ser., 1920, vol. - i., No. 9. - - [36] Koch, R., Mitt. Kais. Gesundh., 1881, vol. i., p. 1. - - [37] Kaserer, H., Centr. f. Bakt., 1906, Abt. II., Bd. 16, p. 681. - - [38] Kaserer, H., Centr. f. Bakt., 1905, Abt. II., Bd. 15, p. 573. - - [39] Krainskii, A. V., Centr. f. Bakt., 1910, Abt. II., Bd. 26, p. - 231. - - [40] Klimmer, M., and Kruger, R., Centr. f. Bakt., 1914, Abt. II., - Bd. 40, p. 257. - - [41] Krzeminiewski, S., Centr. f. Bakt., 1909, Abt. II., Bd. 23, p. - 161. - - [42] Koch, A., and Seydel, S., Centr. f. Bakt., 1912, Abt. II., Bd. - 31, P. 570. - - [43] Lipman, C. B., Bot. Gaz., 1909, vol. xlviii., p. 106. - - [44] Lipman, C. B., and Burgess, P. S., Centr. f. Bakt., 1914, Abt. - II., Bd. 41, p. 430. - - [45] Lipman, C. B., and Burgess, P. S., Centr. f. Bakt., 1915, Abt. - II., Bd. 44, p. 481. - - [46] Lipman, C. B., and Waynick, D. O., Soil Sci., 1916, vol. i., p. - 5. - - [47] Löhnis, F., and Pillai, N. K., Centr. f. Bakt., 1908, Abt. II., - Bd. 20, p. 781. - - [47_b_] Löhnis, F., and Smith, T., Journ. Agric. Res., 1914, vol. - vi., p. 675. - - [48] Mackenna, J., Rept. Prog. Agric., India, 1917, p. 101. - - [49] Marchal, E., Bull. Acad. Roy. Belgique, 1893, vol. xxv., p. 727. - - [50] McBeth, I. G., and Scales, F. M., U.S. Dept. Ag., Bureau Plant - Indus., 1913, Bulletin 266. - - [51] Mockeridge, J., Biochem. Journ., 1915, vol. ix., p. 272. - - [52] Nabokich, A. J., and Lebedeff, A. F., Centr. f. Bakt., 1906, - Abt. II., Bd. 17, p. 350. - - [53] Nagaoka, M., Bull. Coll. Agr., Tokyo, 1900, vol. vi., No. 3. - - [54] Omelianski, W. L., Comptes Rendus Acad. Sci., 1895, vol. cxxi., - p. 653; 1897, vol. cxxv., pp. 907, 1131; Arch. Sci. Bio., (St. - Petersburg), 1899, vol. vii., p. 411. - - [55] Omelianski, W. L., and Sohmskov, M., Arch. Sci. Biol., Publ. - Inst. Imp. Med. Exp. (Petrograd), 1916, vol. xviii., pp. 327, 338, - 459; vol. xix., p. 162. - - [56] Prescott, J. A., Journ. Agr. Sci., 1920, vol. x., p. 177. - - [57] Söhngen, N. L., Centr. f. Bakt., 1905, Abt. II., Bd. 15, p. 513. - - [58] Sen Gupta, N., Journ. Agr. Sci., 1921, vol. xi., p. 136. - - [59] Shearer, C., Journ. Hyg., 1919, vol. xviii., p. 337. - - [60] Tappeiner, Ber. Deut. Chem. Gesell., 1883, vol. xvi., p. 1734; - Zeitsch. Biol., 1884, vol. xx., p. 52. - - [61] Thornton, H. G., Ann. Appl. Biol., 1922, vol. ix., p. 241. - - [62] Wallin, I. E., Journ. Bact., 1922, vol. vii., p. 471. - - [63] Waksman, S. A., and Joffe, J. S., Journ. Bact., 1922, vol. vii., - p. 239. - - [64] Wilson, J. K., Cornell Agric. Exp. Sta., 1917, Bulletin 386. - - - - -CHAPTER IV. - -PROTOZOA OF THE SOIL, I. - - -That protozoa could be isolated from the soil was a matter of common -knowledge to the biologists of the nineteenth century, but not until -the early part of the present century was it suggested that these -organisms might be playing some part in the general economy of the soil -micro-population. Of recent years a great deal of our knowledge of the -cytology of the different groups of protozoa, especially the Amœbæ, has -been obtained from the study of representatives normally living in the -soil; but unfortunately little or no knowledge has been gained of the -biology of these animals in their natural habitat. - -The view that the presence of these organisms in excessive numbers may -lead to “soil sickness” was first put forward by Russell and Hutchinson -in 1909, and elaborated in their further papers dealing with “Partial -Sterilisation of the Soil.” - -It is unnecessary to discuss in detail this important branch of -agriculture, but to obtain a clear idea of the development of -the study of soil protozoa it is necessary to give as briefly as -possible the conclusions deduced by Russell and Hutchinson from their -extensive experiments on soils treated with steam and various volatile -antiseptics[21],[22]:-- - -“(1) Partial sterilisation of the soil causes first a fall, then a -rise, in bacterial numbers, which goes on till the numbers considerably -exceed those present in the original soil. - -“(2) Simultaneously there is a marked increase in the rate of -accumulation of ammonia which is formed from organic nitrogen -compounds. - -“(3) The increase in bacterial numbers is the result of improvement in -the soil as a medium for bacterial growth, and not an improvement in -the bacterial flora. - -“(4) The improvement in the soil brought about by partial sterilisation -is permanent, the high bacterial numbers being kept up even for 200 -days or more. It is evident from (3) and (4) that the factor limiting -bacterial numbers in ordinary soil is not bacterial, nor is it any -product of bacterial activity, nor does it arise spontaneously in soils. - -“(5) But if some of the untreated soil is introduced into partially -sterilised soil, the bacterial numbers, after the initial rise, begin -to fall. Thus the limiting factor can be reintroduced from untreated -soils. - -“(6) Evidence of the limiting factor in untreated soils is obtained -by studying the effect of temperature on bacterial numbers. Untreated -soils were maintained at 10°, 20°, 30° C. in a well-moistened aerated -condition, and periodical counts were made of the numbers of bacteria -per gram. Rise in temperature rarely caused any increase in bacterial -numbers. But after the soil was partially sterilised the bacterial -numbers showed the normal increase with increasing temperatures. - -TABLE VI. - - +--------+------------------------+------------------------+ - | | | Soil Treated | - | | Untreated Soil. | with Toluene. | - |Tempera-+------+-----+-----+-----+------+-----+-----+-----+ - | ture of| |After|After|After| |After|After|After| - |Storage.| At | 13 | 25 | 70 | At | 13 | 25 | 70 | - | °C. |Start.|Days.|Days.|Days.|Start.|Days.|Days.|Days.| - +--------+------+-----+-----+-----+------+-----+-----+-----+ - | 5°-12° | 65 | 63 | 41 | 32 | 8·5 | 73 | 101 | 137 | - | 20° | 65 | 41 | 22 | 23 | 8·5 | 187 | 128 | 182 | - | 30° | 65 | 27 | 50 | 16 | 8·5 | 197 | 145 | 51 | - | 40° | 65 | 14 | 9 | 33 | 8·5 | 148 | 52 | 100 | - +--------+------+-----+-----+-----+------+-----+-----+-----+ - -“(7) It is evident, therefore, that the limiting factor in the -untreated soils is not the lack of anything, but the presence of -something active. The properties of the limiting factor are:-- - - “(_a_) It is active and not a lack of something. - - “(_b_) It is not bacterial. - - “(_c_) It is extinguished by heat or poisons. - - “(_d_) It can be re-introduced into soils from which it has been - extinguished by the addition of a little untreated soil. - - “(_e_) It develops more slowly than bacteria. - - “(_f_) It is favoured by conditions favourable to trophic life in the - soil, and finally becomes so active that the bacteria become unduly - depressed. - -“It is difficult to see what agent other than a living organism can -fulfil these conditions. Search was therefore made for a larger -organism capable of destroying bacteria, and considerable numbers of -protozoa were found. The ciliates and amœbæ are killed by partial -sterilisation. Whenever they are killed the detrimental factor is found -to be put out of action; the bacterial numbers rise and maintain a high -level. Whenever the detrimental factor is not put out of action, the -protozoa are not killed. To these rules we have found no exception.” - -From such premises as the above Russell and Hutchinson founded the -“protozoa theory of partial sterilisation,” and at Rothamsted there was -commenced the serious study of these soil organisms. - -Goodey was one of the early workers on this new subject, and added -considerably to our knowledge of the species living in normal soils, -and of the chemical constitution of the cyst wall of ciliates. He -also made investigations on the effects of various chemicals on the -micro-population of soils, but was unable to draw very definite -conclusions.[11] - -One of the first criticisms raised against the protozoa theory of -partial sterilisation was that the protozoa were not normal inhabitants -of the soil, and were present only in small numbers, all of them in the -cystic, quiescent condition. It was further held that these cysts were -carried by the wind from dried-up ponds and streams. It is difficult to -trace the origin of this view, since early observers, viz., Ehrenberg -and Dujardin, in 1841, were of the opinion that the protozoa were -living in the trophic active condition in the soil, and it was not -until 1878 that Stein showed that free living protozoa can encyst. To -Martin and Lewin, however, must be ascribed the distinction of first -proving that the soil possesses an active protozoan population, for -by a series of ingenious experiments these observers isolated several -flagellates and amœbæ in a trophic condition from certain of the -Rothamsted soils.[18] The more recent work in this country has been -in the direction of devising new quantitative methods of research, -since by this means alone is it possible to elucidate many fundamental -questions. - -In America and elsewhere experiments have been devised for testing the -conclusions of Russell and Hutchinson. Cunningham and Löhnis,[2] in -America, Truffaut and Bezssonoff,[24] in France, supply evidence in -favour of the theory, but most of the American work is in opposition to -it. - -Sherman[23] is perhaps the most prominent in opposing the phagocytic -action of protozoa on soil bacteria in spite of the fact that certain -of his experimental results apparently show enormous decreases in -bacterial numbers in the presence of protozoa. In many of his soil -inoculation experiments, however, it was not demonstrated that his -active cultures remained alive after entering the soil. - -The experimental difficulties of dealing with soil protozoa are -considerable, and without a thoroughly sound technique investigators -may easily go astray. - - -CLASSIFICATION. - -The animal kingdom is divided into two main groups or sub-kingdoms--the -Protozoa and the Metozoa. In the latter the characteristic feature -is that the body is composed of several units, called cells, and -consequently such animals are often spoken of as multicellular. The -Protozoa, on the other hand, are usually designated as uni-cellular, -since their bodies are regarded as being homologous to a single unit or -cell of the metozoan body. For various reasons exception has been taken -by Dobell[9] and others to the use of the term uni-cellular, for, as -Dobell says, “If we regard the whole organism as an individual unit, -then the whole protozoan is strictly comparable with a whole metozoon, -and not with a part of it. But the body of a protozoan, though it shows -great complexity of structure, is not differentiated internally into -cells, like the body of a metozoon. Consequently it differs from the -latter not in the number of its cellular constituents, but in lacking -these altogether. We therefore define the sub-kingdom of the protozoa -as the group which contains _all non-cellular animals_.” - -It should be pointed out that this view does not find favour with many -zoologists, but it is useful in bringing into prominence the fact -that each protozoan is comparable as regards its functions with the -multi-cellular animals. - -The protozoa are again further divided into four main classes:-- - - I. Rhizopoda. - II. Mastigophora. - III. Ciliophora. - IV. Sporozoa. - -Of the above classes, representatives of each of the first three are -found living in the soil, but up to the present there is no evidence -that any sporozoon is capable of living an active life in the soil, -though the cysts of such organisms may be present. - -The class _RHIZOPODA_ consists of those protozoa whose organs of -locomotion and food capture are _pseudopodia_, that is, temporary -extensions of the living protoplasm. The body is typically naked, that -is to say, without any cuticular membrane, though in some forms, ex. -_Amœbæ terricola_, the external layer of protoplasm is thickened to -form a pellicle. A skeleton or shell may be present. - -The class is further sub-divided into various sub-classes, only -two of which concern the soil protozoologist, viz., the _Amœbæ_ -and the _Mycetozoa_, of which the most important representative is -_Plasmodiophora brassicæ_, which attacks the roots of many cruciferous -plants, causing the disease familiarly known as “Fingers and Toes.” - -The _Amœbæ_ are again divided into two orders:-- - - (_a_) _Nuda_, without shell or skeleton; - - (_b_) _Testacea_, with shells often termed _Thecamœbæ_. - -Representatives of the “naked” amœbæ commonly found in soils are -_Nægleria (Dimastigamœba) gruberi_, _Amœba diploidea_ (possessing -two nuclei) and _A. terricola_, the last two forms possessing a -comparatively thick skin or pellicles. _Trinema enchelys_, _Difflugia -constricta_ and _Chlamydophrys stercorea_ are examples of soil -Thecamœbæ. - -The class _MASTIGOPHORA_ consists of those protozoa whose typical modes -of progression are by means of flagella, whip-like filaments which, by -their continual lashing motion, cause movement of the animal. - -The body may be naked or corticate. The only organisms which concern -the soil biologist belong to the _Flagellata_ order. - -The Flagellates differ considerably among themselves, both as regards -their mode of feeding, and the number of flagella, thus making their -classification difficult and outside the scope of this book. Suffice -it to say that in the soil such organisms occur possessing one, two, -three or four flagella, ex. _Oicomonas termo_, _Heteromita globosus_, -_Dallengeria_ and _Tetramitus spiralis_. Further, their mode of feeding -may be _saprophytic_ in which nourishment is absorbed by diffusion -through the body surface in the form of soluble organic substances, -_holozoic_ where solid food particles are taken in, or _holophytic_ in -which food is synthesised by the energy of sunlight. This last group -is commonly spoken of as the _Phyto flagellates_, which are to all -intents and purposes unicellular algæ, and as such will be dealt with -in Chapter VI. - -The class _CILIOPHORA_ consists of those protozoa whose typical organs -of locomotion are threads or cilia. These organisms can in one sense -be regarded as the highest of the protozoa, since in no other division -does the body attain so great a complexity of structure. Moreover, they -are typically characterised by a complicated nuclear apparatus with -the vegetative and generative portions separated into distinct bodies, -the macro-nucleus and the micro-nucleus. Their mode of nutrition is -_holozoic_, though recently Peters has brought forward evidence that -certain species can obtain their nourishment saprophytically. - -The sub-class Ciliata comprises four orders, all of which are -represented in the soil. - -I. _Holotricha._ The cilia are equal in length and uniformly -distributed over the whole body in the primitive forms, though -restricted to special regions in the specialised forms. Typical soil -forms are _Colpoda cucullus_, _Colpidium colpoda_. - -II. _Heterotricha._ There is a uniform covering of cilia, and a -conspicuous spiral zone of larger cilia forming a vibratile membrane -and leading to the mouth. - -III. _Hypotricha._ The body is flattened dorso-ventrally and the cilia -are often fused to form larger appendages or cirri confined to the -ventral surface. Movement is typically a creeping one. Typical soil -forms are _Pleurotricha_, _Gastrostylis_, _Oxytricha_. - -IV. _Peritricha._ Typically of a sedentary habit and the cilia are -reduced to a zone round the adoral region of the body. A typical soil -form is _Vorticella microstomum_. - -The above classification is far from complete, but should be sufficient -to give an idea of the general grouping of the organisms. For a more -detailed account reference must be made to the numerous text books on -protozoa. - - -LIFE HISTORIES. - -The life history of each species has its own characteristic features as -regards nuclear division, etc., and in many forms, notably the amœbæ, -it is impossible to identify them with certainty unless the chief -stages of the life history are known. In general, however, the soil -protozoa pass through very similar phases and develop in a perfectly -straightforward way. Broadly speaking, there are two main phases of -the life history--a period of activity often mistermed vegetative, and -a period of rest. In the former the animal moves, feeds and reproduces, -while in the latter there is secreted round the body a thick wall, -capable of resisting adverse external influences. This condition is -termed the cystic stage, and by means of it the animals are distributed -from place to place by air, water, etc. Indeed, so resistant are the -cysts that many of them are capable of withstanding the action of the -digestive juices of the intestines of animals, through which they pass -to be deposited by the fæces on fresh ground. - -This cystic stage of the life history is found in practically all -free-living protozoa, though it is not formed in exactly the same -manner in every case. In the majority of instances the cyst is the -product of a single organism, round which is formed a delicate -gelatinous substance which soon hardens and gradually acquires the -peculiar characters of the wall. Concerning the chemical nature of this -wall there is little known, but Goodey,[11] working on the cysts of -_Colpoda cucullus_, found it to be formed of a carbohydrate, different -from all carbohydrates previously described, to which the name “Cytose” -was given. When in this state the animals are able to remain dormant -for considerable periods until favourable conditions once more obtain -when the wall is ruptured and the animal again resumes the active phase -of its life history. This simple process is characteristic of such -species as _Heteromita globosus_, _Cercomonas spp._, and many others. -It will be noted that no increase of numbers, i.e. reproduction, -occurs. A more complex condition is, however, sometimes found, as, for -example, in the ciliate _Colpoda steinii_, where actual reproduction -into small animals takes place within the cyst. - -Finally there is the less common type of cyst formation, such as is -found in the flagellate _Oicomonas termo_ described by Martin.[19] This -flagellate, in common with all other forms, reproduces by dividing into -two; the division of the nucleus initiating the process. At certain -undetermined periods of the life history, however, conjugation occurs -between two similar animals forming a large biflagellate body known as -the zygote. After swimming about for varying periods of time, during -which the size increases and a large vacuole appears, the zygote -secretes a thick wall, loses its flagella, and becomes a cyst. While in -this condition the two gamete nuclei fuse to form one, and eventually a -single _Oicomonas_ emerges from its cyst. - -Similarly in _A. diploidea_ the cysts are formed after two individuals -have come together. In the young cysts two amœbæ are found in close -association, and according to Hartmann and Nägler[12] a sexual process -occurs inside the cyst involving a “reductive” division of the nuclei. -This requires confirmation, but it is certain that only one individual -comes out of the cysts, which originally contained two amœbæ. - -Such cysts have been termed by some writers “reproductive,” evidently a -misleading term, since no increase in numbers, but rather a decrease, -results from the process. A better term is, perhaps, conjugation cyst. - -In soil protozoa, then, three different modes of cyst formation obtain, -and failure to make the distinction inevitably leads to confusion. - -Before leaving the question of life histories, reference must be made -to a peculiar and characteristic feature of _Nægleria gruberi_. This -amœba under certain circumstances assumes a free-swimming biflagellate -stage. After variable periods of time the flagella are lost and the -ordinary amœboid condition resumed. What are the factors concerned in -the production of flagellates is unknown, but flooding the coverslips -with distilled water is an effective method for causing their -appearance. - - -DISTRIBUTION OF SOIL PROTOZOA. - -For both the bacteria and algæ observations have been made regarding -their distribution through successive depths of the soil; little can -be said, however, about the protozoa in this connection. It is certain -that they occur throughout the first six inches of the Rothamsted -soils, though their relative frequencies in the successive inches has -not been determined, but probably they are most abundant in the 2nd to -the 4th inch. - -In this country experiments have not been made to determine whether -sub-soil normally contains protozoa; but from some South African -soil, taken under sterile conditions 4 ft. down and examined in this -laboratory, large numbers of protozoa were cultivated. - -This soil, however, could not, for various reasons, be regarded as a -typical sub-soil. - -Kofoid records the presence of _Nægleria gruberi_ in clay and rock -talus taken from the sides of excavations of over 20 ft. depth, but the -possibility of external infection does not appear to have been excluded. - -The presence of protozoa is not peculiar to British soil since they -have been found by various workers in Germany, France, the United -States, and elsewhere. In view of their probable importance in the soil -economy there has been instituted a survey of the protozoan species of -soil from all parts of the world. - -This work is in charge of Mr. Sandon, to whom I am indebted for the -following summary of his as yet unpublished research. - -“The majority of soil protozoa (like the fresh-water forms) appear to -be quite cosmopolitan, for the species found in such widely separated -localities as England, Spitsbergen, Africa, West Indies, Gough Island -(in the South Atlantic) and Nauru (in the Pacific) are, with few -exceptions, identical. This distribution indicates an ability to -withstand an extremely wide range of conditions, for the same species -occurring in Arctic soils, which are frozen for the greater part of -the year, are found also in soils exposed to the direct rays of the -tropical sun. Even sand from the Egyptian desert contains protozoa, -though it seems probable that in such cases they must be present only -in the encysted condition for the greater part of the time. - -“Not every sample of soil, however, contains all the species capable -of living in soil, but the local conditions determining the presence -or absence of any species are at present unknown. In general the -numbers, both of species and of individuals present, follow the number -of bacteria. They are consequently most numerous in rich moist soils. -The statement sometimes made that protozoa are most numerous in peaty -soils is based solely on the number of Rhizopod shells found in such -localities; but as most of these shells are empty, their abundance is -probably due simply to the slowness with which they disintegrate in -these soils where bacterial activity is low, they do not indicate a -great protozoal activity. Active protozoa do occur even in extremely -acid soils, but their numbers in such cases are low. The common soil -protozoa, in fact, appear to be as tolerant of differences in soil -acidity as they are of differences in climate, for many of the same -forms which occur in acid soils are found also in soils containing -high percentages of chalk. It is possible that some of the less common -species may be confined within closer limits of external conditions but -the information available on this point is inadequate. All the species, -however, which in Rothamsted soils occur in the highest numbers (e.g. -_Oicomonas termo_, _Heteromita spp._, _Cercomonas crassicauda_, -_Nægleria gruberi_, _Colpoda cucullus_, _C. steinii_) occur in -practically every soil which is capable of supporting vegetation, -though, of course, in very varying numbers.” - -It is evident, therefore, that the protozoa must be regarded as -constituting part of the normal micro-organic population of soils, -and as such are probably playing an important rôle. Unfortunately our -knowledge of the physiology of these organisms is extremely scant, and -much of future research must be directed towards elucidating their -functions and their responses to varying environmental conditions. - - - - -CHAPTER V. - -PROTOZOA OF THE SOIL, II. - - -In the preceding chapter an outline has been given of the development -of the study of soil protozoa, with especial reference to its -qualitative aspects. - -Here it is proposed to deal with the quantitative methods which have -been devised for studying these organisms and the results obtained. - -From the beginning great difficulty has been encountered in finding -means for counting protozoa; and most of the early results have been -obtained by the use of one of the following methods: (1) direct counts -in a known volume of soil suspension by means of a microscope; (2) -dilution method as used for counting bacteria, and suggested by Rahn, -who made dilutions of the soil and determined, by examination at -periodic intervals, the one above which protozoa did not grow; (3) Agar -plating as used by Killer; (4) counting per standard loop of suspension -as devised by Müller. Of these the two last have been little used, -and for various reasons are now discarded by most workers. Direct -methods have been used extensively in the United States by Koch[13] -and others,[16] who claim to have got satisfactory results; they are, -however, highly inaccurate and should be discontinued. The present -writer[3] has shown that there exists a surface energy relationship -between the soil particles and the protozoa, so that the two are always -in intimate contact; thus rendering it impossible to count under the -microscope the number of organisms in a given weight of soil suspension -(Fig. 9). Further, in a clay soil, such as is found at Rothamsted, the -clay particles alone make it very difficult to use such methods. - -The demonstration of this surface energy relationship affords an -effective rejoinder to the criticism made against Russell and -Hutchinson’s hypothesis, viz., that soil protozoa must be very few in -numbers, since it was impossible to see them on examining soil under -the microscope. - -[Illustration: FIG. 9.--Showing the number of amœbæ and flagellates -withdrawn from suspensions of varying strengths by different types of -solid matter. A = clay: B = partially sterilized soil: C = ignited -soil: D = fine sand: E = waste sand. Since complete withdrawal occurs -when the numbers of organisms added are less than the capacity of the -solid matter, the first part of each of the above curves is coincident -with the ordinate. The numbers of organisms are given in thousands. -(From Journ. Agric. Soc., vol. ix.) - - X-axis: Number of Organisms per c.c. left in Solution. - - Y-axis: Number of Organisms per c.c. taken up by Solid Matter.] - -The second or dilution method is the one, therefore, that has been most -extensively developed. - -Cunningham obtained concordant results in this way, and his method, -modified by L. M. Crump, was as follows: 10 grams of soil were added -to 125 c.c. of sterile tap-water and shaken for three minutes. This -gives a 1 in 12·5 dilution. From it further dilutions were made -until a sufficiently high one was obtained. Petri dishes, containing -nutrient agar, were inoculated with 1 c.c. of each of the dilutions and -incubated. At intervals covering 28 days the plates were examined and -the presence or absence of protozoa on each recorded. In this way the -approximate number of organisms per gram of soil could be found. - -By methods essentially similar to this numerous counts have been made -of the bacteria and protozoa in field soil and in partially sterilized -soils. They were, however, inconclusive; thus, on the one hand, -Goodey and several American observers, found no correlation between -the numbers of protozoa and bacteria, while Miss Crump and Cunningham -obtained evidence pointing to the reverse conclusion. - -Such divergence of opinion was probably mainly due to two causes: -firstly, that the time elapsing between the successive counts was too -long, for it has been shown recently that the number of bacteria and -protozoa fluctuate very rapidly; and secondly, the method was not -completely satisfactory since only the total numbers of protozoa were -considered, no means having been found of differentiating between the -cystic and active forms. This was a particularly serious source of -error for it is possible for soil to contain large numbers of bacteria -and protozoa, of which a high percentage of the latter are in the form -of cysts. A count made on such a soil would give results apparently -opposed to the theory that protozoa act as depressors of bacteria. - -This difficulty has, however, been overcome by a further modification -of the dilution method, and it is now possible in any soil sample -to count both the numbers of cysts and active forms. Also a further -advance in technique has made it possible to recognise and enumerate -the common species of protozoa, instead of simply grouping them as -Ciliates, Flagellates, and Amœbæ, as was done in the past.[7] - -Briefly the method consists in dividing the soil sample into equal -portions (usually 10 grams each) one of which is counted, thus giving -the total numbers of protozoa (active + cystic) present. The second -portion is treated over-night with 2 per cent. hydrochloric acid, the -HCl used being B.P. pure 31·8 per cent. Previous experiments have shown -that such acid kills all the active protozoa, leaving viable the cysts. -The number of cysts is therefore found by counting this treated sample, -and the number obtained subtracted from the total gives the active -number.[F] - - [F] The proof of the accuracy of this method will be found in the - following papers:-- - - (1) Cutler, D. W. (1920), Journ. Agric. Sci., vol. x., 136-143. - - (2) Cutler, D. W., and Crump, L. M. (1920), Ann. App. Biol., vol. - vii., 11-24. - -The discovery of this method at once puts into the hands of the -investigator a much more efficient instrument for studying the -activities of the soil micro-population, especially since at a slightly -later date Thornton’s method for counting bacteria was devised. - -Early in 1920 Cutler and Crump[6] decided to make a preliminary survey -of the protozoon and bacterial populations of one of the Rothamsted -field soils (Broadbalk dunged plot). The investigation was continued -for 28 days, daily soil samples being taken. The results so obtained -showed that an extended investigation of the micro-population of field -soil would yield interesting and important results, especially as -it was evident that certain views held by soil biologists required -modification. - -In July of the same year, therefore, it was decided to start an -extended investigation of the soil protozoa and bacteria. The method -adopted was to make counts of the numbers of bacteria and of six[G] -species of protozoa in soil samples taken daily direct from the field -(Barnfield dunged plot) and by statistical methods to correlate these -counts one with another and with the data for external conditions. -Observations at shorter periods than 24 hours could not be made, but it -was found possible to continue the research for 365 days.[7] - - [G] Actual counts were made of six species, though, as stated on p. - 10, observations were made on seventeen. - -[Illustration: FIG. 10.--Daily numbers of active amœbæ (Dimastigamœba -and Species α) and bacteria in 1 gram of field soil, from August 29 to -October 8, 1920. (From Phil. Trans. Roy. Soc., vol. ccxi.) - - X-axis: August September October - - Y-axis (left): Amoebae Active numbers per gramme of soil - - Y-axis (right): Bacteria in millions per gramme of soil - - Legend: Dimastigamoeba - - Species α - - Bacteria] - -The number of all the organisms showed large fluctuations of two kinds, -daily and seasonal. The size of the changes that took place within -so short a period as 24 hours was, perhaps, the most surprising fact -that the experiment revealed. Thus three consecutive samples gave -58·0, 14·25 and 26·25 millions of bacteria per gram respectively; and -the changes exhibited by any of the species of protozoa were at times -even larger. This fact is of extreme importance, since in the past it -has always been assumed that the number of bacteria remained fairly -constant from day to day, and investigators have not hesitated to -separate the taking of soil samples by long periods. It is now obvious -that such a procedure is of little use for comparative purposes (Fig. -10). - -It has usually been assumed that the changes in the external conditions -markedly affect the density of the soil population. To test this the -environmental conditions--temperature, moisture content and rainfall -were examined; but contrary to all expectation no connection could be -traced between any of these and the daily changes in numbers of any -of the organisms investigated, and moreover the species of protozoa -appeared in the main to be living independently of one another. - -It is difficult to believe that external conditions are as inoperative -as appears from the above; and in view of the known complexity of -the soil it is possible that further research will show that certain -combinations of external conditions are important agents in effecting -the changes. - -[Illustration: FIG. 11.--Numbers of active amœbæ (Dimastigamœba and -Species α) and bacteria to 1 gram of field soil for typical periods in -February and April, 1921. (From Phil. Trans. Roy. Soc., vol. ccxi.) - - X-axis: Feby. Feby. April - - Y-axis (left): Amoebae Active numbers per gramme of soil - - Y-axis (right): Bacteria millions - - Legend: Dimastigamoeba - - Species α - - Bacteria] - -In the case of the bacteria, however, the agent causing the -fluctuations is mainly the active amœbæ. This was well shown during the -year’s count, for with only 14 per cent. of exceptions, 10 per cent. -of which can be explained as due to rapid excystation or encystation, -a definite inverse relationship was established between the active -numbers of amœbæ and the number of bacteria (Figs. 11 and 12). Thus a -rise from one day to the next in the amœbic population was correlated -with a fall in the numbers of bacteria and vice versa. It must not be -supposed that the flagellates are of no account in this process; some -species, known to eat bacteria, undoubtedly induce slight depressions, -but, owing to their small size, any effect is masked by the greater -one of the amœbæ. - -[Illustration: FIG. 12.--Numbers of active amœbæ (Dimastigamœba and -Species α) and bacteria in 1 gram of field soil for typical periods in -September, October, and November, 1920. - - X-axis: August September October - - Y-axis (left): Amoebae, thousands - - Y-axis (right): Millions, Bacteria - - Legend: Dimastigamoeba - - Species α - - Bacteria] - -These experiments seem to admit of no doubt that in field soil the -active protozoa are instrumental in keeping down, below the level -they might otherwise have attained, the numbers of bacteria; but a -further proof of this contention ought to be obtained by inoculation -experiments. It should be possible, by inoculating sterile soil -with bacteria alone and with bacteria plus protozoa, to demonstrate -fluctuations in bacterial numbers in the latter, while those of the -former remained constant. This admittedly crucial test has often been -tried, but owing to difficulties in technique, etc., has always failed. -Recently, however, by using new methods confirmatory results have been -obtained.[5] - -Ordinary field soil was sterilised by heat at 100° C. for 1 hour on -four successive days; it was then divided into equal portions, one of -which was inoculated with three known species of bacteria, and the -other inoculated with the same number of bacteria plus the cysts of the -common soil amœba _Nægleria gruberi_. The numbers of bacteria in each -soil were counted daily for the first eight days and then daily from -the 15th to the 21st day after the experiment started. The results are -given in Table VII. and Fig. 13. - -TABLE VII. - - +------------+---------+--------+ - | Numbers of | Control | Control| - | Days after |(Bacteria|Bacteria| - |Inoculation.| alone).|+ Amœbæ.| - +------------+---------+--------+ - | 0 | 13·0 | 12·2 | - | 1 | 48·6 | 35·4 | - | 2 | 97·6 | 117·2 | - | 3 | 127·0 | 178·4 | - | 4 | 154·8 | 154·4 | - | 5 | 196·8 | 177·0 | - | 6 | 214·4 | 151·8 | - | 7 | 193·4 | 75·6 | - | 8 | 165·2 | 65·8 | - | 15 | 169·2 | 72·8 | - | 16 | 174·8 | 30·2 | - | 17 | 175·6 | 53·2 | - | 18 | 168·4 | 82·8 | - | 19 | 160·4 | 43·8 | - | 20 | 171·2 | 70·8 | - | 21 | 176·2 | 28·2 | - +------------+---------+--------+ - - The numbers of bacteria are given in millions per gram of soil. - -[Illustration: FIG. 13.--Numbers of bacteria counted daily in soils -containing - - A. Bacteria alone. - B. Same Bacteria as in A + Amœbæ. - C. Same Bacteria as in A + Flagellates. - -(From Ann. Appl. Biol., vol. x.)] - -It will be noted that the numbers of bacteria in each soil rose -steadily until a maximum was reached 6-8 days after inoculation. This -is in accordance with expectation, since the reproductive rate of -bacteria is much greater than that of the amœbæ, which, until their -active forms are numerous, will not exert any appreciable influence on -the bacterial population. Further, since the protozoa were inoculated -as cysts an appreciable time would elapse before excystation took -place. The last seven days of the experiment are of particular -interest. During this period the amœbæ were known to be active in the -soil, and were depressing the bacterial numbers, for in the control -(protozoa-free) soil the variation in numbers was within experimental -error, while in the other soil the variations were considerable and -well outside experimental error. In fact the variations were comparable -with those found from day to day in untreated field soils. Finally, -the experiment shows that the bacteria in protozoa-free soil are able -to maintain high numbers for a longer period than those living in -association with protozoa. - - -SEASONAL CHANGES. - -Superimposed on the daily variations in numbers there are seasonal -changes, as is clearly shown when fourteen day averages are made of -the numbers for each species. Bacteria have long been known to show -autumn and spring rises, but recent research has demonstrated that the -protozoan population also rises to a maximum at the end of November, -with a less marked spring rise at the end of March and beginning of -April (Figs. 14 and 15). - -It has sometimes been claimed that the numbers of soil organisms are -closely linked with the soil moisture, but no support for this view -was found during the course of the experiment. Similarly, as in the -case of the daily variations, no connection could be traced between the -seasonal changes and any of the external conditions considered. - -It is interesting to note, however, that the seasonal variations in the -numbers of soil organisms is very similar to those recorded for many -aquatic organisms. Miss Delf,[8] for instance, found that in ponds at -Hampstead the algæ are most numerous in spring and again in the autumn, -and like changes are recorded in British lakes by West and West[25] and -in the Illinois river by Kofoid.[14] - -[Illustration: FIG. 14.--Fortnightly averages of total numbers of -Oicomonas, Species γ, and Species α, and of bacteria, moisture, and -temperature. (From Phil. Trans. Roy. Soc., vol. ccxi.) - - X-axis: Fortnight beginning 1920. July. Aug. Sep^t. Oct. Nov. Dec. - Jan 1921. Feb^y. Mch. April. May. June. - - Y-axis (bottom left): Percentage of moisture - - Y-axis (top left): Logarithms of numbers of active protozoa per - gramme of soil - - Y-axis (bottom right): Temperature F - - Y-axis (top right): Bacteria in millions per gramme - - Legend: Oicomonas - - Species γ - - Species α - - Bacteria - - Temperature - - Moisture] - -It is difficult to resist the conclusion that these annual variations -are produced by similar causes, from which it follows that the increase -in the numbers of protozoa in the soil is not wholly conditioned by an -increased food supply--the bacteria--for the algæ are not dependent on -such a form of nourishment. This is substantiated by the fact that the -numbers of protozoa, except those of _Oicomonas_, rose during March, -whereas the corresponding increase in the bacteria was delayed till the -early part of April. - -[Illustration: FIG. 15.--Fortnightly averages of total numbers of -Heteromita, Cercomonas, and Dimastigamœba and of bacteria, moisture, -and temperature. (From Phil. Trans. Roy. Soc., vol. ccxi.) - - X-axis: Fortnight beginning July 1920. Aug. Sep^t. Oct. Nov. Dec. - Jan. 1921. Feb. Mar. April May June - - Y-axis (bottom left): Percentage of moisture. - - Y-axis (top left): Logarithms of numbers of active protozoa per - gramme of soil. - - Y-axis (bottom right): Temperature F - - Y-axis (top right): Bacteria in millions per gramme - - Legend: Heteromita - - Cercomonas - - Dimastigamoeba - - Bacteria - - Temperature - - Moisture] - -Owing to the variations in the numbers of both protozoa and bacteria, -little reliance can be placed on figures obtained from an isolated -count, since on one day the total numbers of flagellates may be nearly -2,000,000 per gram and drop by more than half this figure in 24 days. -It is certain, however, that the numbers recorded in the past are much -too low, since the total flagellate and amœbæ species were lumped -together in two groups. Some idea of the size of the soil population -can be obtained, nevertheless, by using the fourteen-day averages -mentioned above. In Table VIII. are tabulated the average total numbers -of flagellates, and amœbæ for the two periods of the year when the -population was at its maximum and minimum respectively. An endeavour -has also been made to strike a rough balance sheet as to the amount of -protoplasm represented by protozoa and bacteria in a ton of soil. For -this purpose it has been assumed that the organisms have a specific -gravity of 1·0 and are spheres of diameters, 6µ for the flagellates, -10µ for the amœbæ, and 1µ for the bacteria; and that they are uniformly -distributed through the top nine inches of soil. The top nine inches of -soil is taken as weighing 1000 tons. - -TABLE VIII. - - +-----------+---------------------------+---------------------------+ - | | Maximum Period. | Minimum Period. | - | + ____________/\___________ + ____________/\___________ + - | |( )|( )| - | | No. | Weight | Weight| No. per | Weight | Weight| - | | per |in Gram |in Tons| Gram. |in Gram |in Tons| - | | Gram. | per | per | | per | per | - | | | Gram. | Acre. | | Gram. | Acre. | - +-----------+----------+--------+-------+----------+--------+-------+ - |Flagellates| 770,000|0·000087| 0·087 | 350,000|0·000039| 0·039 | - |Amœbæ | 280,000|0·000147| 0·147 | 150,000|0·000078| 0·078 | - |Bacteria |40,000,000|0·000020| 0·02 |22,500,000|0·000012| 0·012 | - +-----------+----------+--------+-------+----------+--------+-------+ - -It must be remembered that the above figures are minimum ones, as many -species of bacteria and protozoa, known to occur in the soil, are not -included in the statement owing to their not appearing on the media -used for counting purposes. - -[Illustration: FIG. 16.--Daily variations in the numbers of active -individuals of a species of flagellate, _Oicomonas termo_ (Ehrenb.) -during March, 1921. (From Phil. Trans. Roy. Soc., vol. ccxi.) - - X-axis: March - - Y-axis: Active numbers per gramme of soil] - -Before leaving the discussion of daily variations in numbers of -protozoa, reference must be made to the flagellate species. As already -mentioned, their active numbers fluctuate rapidly, and for the most -part entirely irregularly. One species, however, _Oicomonas termo_, is -characterised by possessing a periodic change; high active numbers on -one day being succeeded by low, which are again followed by high on the -third day. This rhythm was maintained, with few exceptions, for 365 -days (Fig. 16), and has been shown to take place in artificial culture -kept under controlled laboratory conditions (Fig. 17). - -[Illustration: FIG. 17.--Daily variations in the numbers of active -individuals of _Oicomonas termo_ (Ehrenb.) in artificial culture media -kept at a constant temperature of 20° C. A, in hay infusion; B, in egg -albumen. - - X-axis: Days - - Y-axis: Thousands] - -It was thought that an explanation of this phenomenon might be found -in alternate excystation and encystation, since the latter is a -constituent part of the animals’ life history (see p. 73). This, -however, does not hold, for the cyst curve is not the inverse of that -of the active; and, moreover, statistical treatment demonstrated that -cyst formation is wholly unperiodic in character. - -An explanation must therefore be sought in the changes in the -organisms during the active period of their life, and the deduction -can be drawn that, increased active numbers tend to be followed by -death, conjugation, or both, while decreases in the active numbers -are followed by rises in total numbers, i.e., reproduction, and this -rhythmically. - -This somewhat surprising conclusion appears to hold, in a lesser -degree, for other soil protozoa, and is of sufficient importance -to warrant further research. The direction in which this is being -pursued is by a study of the reproductive rates of pure cultures of -certain ciliates and flagellates under varying external conditions. -Space does not admit of adequate discussion of this problem, but the -results already obtained justify the view that such lines of work will -elucidate some of the baffling problems of soil micro-biology. - - -SOIL REACTION. - -The development of the artificial fertiliser industry has in many -ways revolutionised farm practice, with the inevitable result that -new problems have arisen, not the least of which are biological in -character. - -If, as seems to be indubitable, the micro-organisms of the soil are of -importance to soil fertility, it is necessary for us to know in what -way this population is affected by the application of fertilisers, -and a start has been made by investigating the effects of hydrogen -ion concentration on soil protozoa. Much has already been written -concerning this question, but almost entirely on results obtained -in artificial cultures. It is always dangerous to argue from the -artificial to the natural environment of organisms and particularly -so in respect to the soil. Also, as Collett has shown, the toxic -effects of acids are probably not entirely a function of the hydrogen -ion concentration, but that the molecules of certain acids are in -themselves toxic, an action which can, however, be diminished by the -antagonistic powers of many substances such as NaCl. - -In this laboratory S. M. Nasir, by unpublished work, has shown that -the limiting value on the acid side for _Colpoda cucullus_ was P_{H} -3·3; for a flagellate (_Heteromita globosus_), 3·5; and for an amœba -(_Nægleria gruberi_), 3·9. - -Also Mlle. Perey, investigating the numbers of protozoa in one of the -Rothamsted grass plots of P_{H} 3·65, found a total of 13,600 protozoa, -of which 90 per cent. were active. - -The tolerance, therefore, of these organisms to varying external -conditions is greater than has formerly been supposed, a conclusion -which is becoming more evident from the researches mentioned in Chapter -IV. on soils from different parts of the world. - - -PROTOZOA AND THE NITROGEN CYCLE. - -In partially-sterilised soil from which protozoa were absent Russell -and Hutchinson obtained an increased ammonia production, a result -also obtained by Cunningham. Hill, on the other hand, concluded that -protozoa have no effect on ammonification, but his technique is open to -criticism. - -Lipman, Blair, Owen and McLean’s work[17] contains many figures -obtained by adding dried blood, tankage, soluble blood flour, -cottonseed meal, soy-bean meal, wheat flour, corn meal, etc., to soil. -It is difficult to understand how accurate results could be expected -when, to an already little understood complex substance, such as soil, -is added a series of substances whose effects are practically unknown. - -Free nitrogen-fixation in soils is an important process, more -especially in soils of a light sandy nature, from which crops are -taken year after year without any application of manure. The effect of -protozoa on the organisms causing this process has in the past received -little attention. Recently, however, Nasir[20] has studied the -influence of protozoa on Azotobacter, both in artificial culture and in -sand. From a total of 36 experiments done in duplicate or triplicate, -31 showed a decided gain in nitrogen fixation over the control, while -only 5 gave negative results. - -[Illustration: FIG. 18.--Showing the highest fixations of nitrogen -above the control recorded for Azotobacter in the presence of different -species of Protozoa. (From Ann. Appl. Biol., vol. ii.) - - X-axis (left): Artificial Media C A F AF AC ACF - - X-axis (right): Sand Cultures C A AF AC - - Legend: C represents CILIATES. - - A -do.- AMOEBAE. - - F -do.- FLAGELLATES.] - -As might be expected, the fixation figures varied from culture to -culture, the highest recorded being 36·04 per cent. above the control -and this in a sand culture (Fig. 18). Reference to the details of the -experiments shows that the criticisms made against similar work done in -the past do not hold here, and one must conclude that Azotobacter is -capable of fixing more atmospheric nitrogen in the presence of protozoa -than in their absence. - -At present it is impossible to say how this occurs, but it is highly -improbable that the protozoa are themselves capable of fixing nitrogen. -A more likely explanation is that the protozoa, by consuming the -Azotobacter, kept down the numbers, and transfer the nitrogen to their -own bodies. This will tend to prevent the bacteria from reaching a -maximum density, and reproduction, involving high metabolism, will be -maintained for a longer period than would have otherwise occurred. This -and other possible explanations, are being tested. - -Little has been said regarding the application of protozoology to the -question of soil partial sterilisation. As already pointed out, in -the past much work has been done, but the results were conflicting. -In view, however, of our recently acquired knowledge of the life of -protozoa in ordinary field soil, most of the early experiments require -repeating. A beginning has already been made, but the work is not -sufficiently advanced to warrant discussion. - -What is urgently needed, however, is to increase our knowledge of the -general physiology of these unicellular animals. Until we know what -are the inter-relationships between the members of the micro-organic -population of normal soil it is almost impossible to hope that means -will be devised by which they can be controlled. - -At present we are almost entirely ignorant of the simplest of -physiological reactions, such as the exact effect of various inorganic -salts found in the soil. - -Also some experiments in Germany and the States indicate that amœbæ are -selective as regards the bacteria they ingest. If this is substantiated -it may prove of importance to economic biology. - -It has been shown that the flagellates occur in the soil in large -numbers, and many of them feed on bacteria. It is probable, however, -that certain of them feed saprophytically and must therefore exert some -influence on the soil solution, though what this may be is entirely -unknown. - -Finally, as Nasir has shown, the protozoa play a part in the -complicated nitrogen cycle, and work of this type needs extending. - -Such, then, are a few of the outstanding problems that confront the -soil protozoologist; but he must always remember that the organisms he -studies are but a small fraction of the total, and that any influence -affecting one part of the complex will be reflected in another. As -Prof. Arthur Thomson said in his Gifford Lectures, “No creature lives -or dies to itself, there is no insulation. Long nutritive chains often -bind a series of organisms together in the very fundamental relation -that one kind eats the others.” Such nutritive chains obtain in the -soil as markedly as in other haunts of living creatures. - - -SELECTED BIBLIOGRAPHY. - -* _Papers giving extensive bibliographies._ - - [1] Cunningham, A., Journ. Agric. Sci., 1915, vol. xvii., p. 49. - - [2] Cunningham, A., and Löhnis, F., Centrlb. f. Bakt. Abt. II., 1914, - vol. xxxix., p. 596. - - [3] Cutler, D. W., Journ. Agric. Sci., 1919, vol. ix., p. 430. - - [4] Cutler, D. W., Journ. Agric. Sci., 1920, vol. x., p. 136. - - [5] Cutler, D. W., Ann. App. Biol., 1923, vol. x., p. 137. - - [6] Cutler, D. W., and Crump, Ann. App. Biol., 1920, vol. vii., p. 11. - - [7] * Cutler, D. W., Crump and Sandon, Phil. Trans. Roy. Soc. B., - 1922, vol. ccxi., p. 317. - - [8] Delf, E. M., New Phytologist, 1915, vol. xiv., p. 63. - - [9] Dobell, C. C., Arch. f. Protisenk., 1911, vol. xxiii., p. 269. - - [10] * Goodey, T., Roy. Soc. Proc. B., 1916, vol. lxxxix., p. 279. - - [11] Goodey, T., Roy. Soc. Proc. B., 1913, vol. lxxxvi, p. 427. - - [12] Hartmann, M., and Nägler, K., Sitz-Ber. Gesellsch. Naturf. - Freunde, 1908, Berlin, No. 4. - - [13] Koch, G. P., Journ. Agric. Res., 1916, vol. li., p. 477. - - [14] Kofoid, C. A., Bull. Illinois State Lab. Nat. Hist., 1903 and - 1908. - - [15] * Kopeloff, N., Lint, H. C., and Coleman, D. A., Centrlb. f. - Bakt. Abt. II., 1916, vol. xlvi., p. 28. - - [16] Kopeloff, N., Lint, H. C., and Coleman, D. A., Centrlb. f. Bakt. - Abt. II., 1916, vol. xlv., p. 230. - - [17] Lipman, J. G., Blair, A. W., Owen, L. L., and McLean, H. C., - N.J. Agric. Exp. Sta. 1912, Bull., No. 248. - - [18] Martin, C. H. and Lewin, K. R., Journ. Agric. Soc., 1915, vol. - vii., p. 106. - - [19] Martin, C. H., Roy. Soc. Proc. B., 1912, vol. lxxxv., p. 393. - - [20] * Nasir, S. M., Ann. App. Biol., 1923, vol. x., p. 122. - - [21] Russell, E. J., Roy. Soc. Proc. B., 1915, vol. lxxxix., p. 76. - - [22] * Russell, E. J., “Soil Conditions and Plant Growth,” 1921, 4th - ed. - - [23] * Sherman, J. M., Journ. Bact., 1916, vol. i., p. 35, and vol. - ii., p. 165. - - [24] Truffaut, G., and Bezssonoff, H., Compt. Rend. Acad. Sci., 1920, - vol. clxx., p. 1278. - - [25] West, W., and West, G. S., Journ. Linn. Soc. Bot., 1912, vol. - xl., p. 395. - - - - -CHAPTER VI. - -ALGÆ. - - -I. GENERAL AND HISTORICAL INTRODUCTION. - -Speaking broadly, the organisms of the soil may be classified into -several distinct groups differing conspicuously in their general -characters and physiological functions and therefore in their economic -significance; among such groups may be mentioned the bacteria, -protozoa, algæ and fungi. It is found, however, that though typical -members of these groups are conspicuously different from one another, -yet there exist a number of unicellular forms which have characters -in common with more than one of these big groups, and the lines of -demarcation between them become difficult to define. It becomes -advisable, therefore, to depart a little from the systematist’s rigid -definitions and to adopt a somewhat more logical grouping of the soil -organisms based on their mode of life. - -To give but a single example: _Euglena viridis_ occurs quite commonly -in soil. Through its single flagellum, its lack of a definite cellulose -wall, its changeable shape and its ability to multiply by simple -fission in the motile state it definitely belongs systematically to the -group of protozoa. But its possession of chlorophyll, in enabling it to -synthesise complex organic substances from CO₂ and water in a manner -entirely typical of plants, connects it physiologically so closely with -the lower green algæ that in studying the biology of the soil it seems -best to include it and other nearly related forms with the algæ. - -On this physiological basis “soil-algæ” may be defined as those -micro-organisms of the soil which have the power, under suitable -conditions, to produce chlorophyll. Such a definition has the -advantage that it is wide enough to include the filamentous protonema -of mosses, which, though alga-like in form and in physiological -action, is nevertheless separated from the true algæ by a wide gulf. -A more accurate name for such a group of organisms would be the -“chlorophyll-bearing protophyta” of the soil; they may be classified -briefly as follows (Table IX.):-- - -TABLE IX. - - +----+------------------------------------+-----------+--------------+ - | | Group. | Colour. | Pigments. | - +----+------------------+-----------------+-----------+--------------+ - | I.|_Flagellatæ._ |Euglenaceæ. |Green. |_Chlorophyll._| - | | |Cryptomonadineæ. | | | - | | | | | | - | II.|_Algæ_-- | | | | - | | 1. Myxophyceæ. |Mostly filamen- |Blue-green |Phycocyanin. | - | | |tous, chiefly |to violet |_Chlorophyll._| - | | |Oscillatoriaceæ |or brown. |Carotin. | - | | |and Nostocaceæ. | | | - | | 2. Bacillariaceæ.|Mostly pennate, |Golden- |Carotin. | - | | |chiefly Navi- |brown. |Xanthophyll. | - | | |culoideæ. | |_Chlorophyll._| - | | 3. Chlorophyceæ. | (i) Protococ- |Green. |_Chlorophyll._| - | | | cales, Ulo- | | | - | | | trichales, | | | - | | | Conjugatæ, | | | - | | | etc. | | | - | | |(ii) Heterokontæ.|Yellow- |_Chlorophyll._| - | | | |green. |Xanthophyll. | - | | | | | | - |III.|_Bryophyta._ |Filamentous moss |Green. |_Chlorophyll._| - | | |protonema. | | | - +----+------------------+-----------------+-----------+--------------+ - -The importance of the lower algæ from a biological standpoint has -long been recognised, since their extremely primitive organisation, -coupled with their ability to synthesise organic compounds from simple -inorganic substances, singles them out as being not very distantly -removed from the group of organisms in which life originated upon the -earth. But the possibility of their having a very much wider economic -significance was completely overlooked until about a quarter of a -century ago, when Hensen demonstrated their importance in marine -plankton as the producers of the organic substance upon which the -whole of the animal life of the ocean is ultimately dependent. In -consequence, it has been generally assumed that the growth of algæ, -since they contain chlorophyll, is entirely dependent on the action of -light. Hence the recent idea of the existence of algæ which actually -inhabit the soil has been received with a certain amount of scepticism, -though the results of modern physiological research on a number of the -lower algæ show that there is very good reason to believe that such a -soil flora is entirely possible. - -In considering the alga-flora of a soil it is necessary to distinguish -between two very different sets of conditions under which the organisms -may be growing. In the first place, they may grow on the surface of the -soil, being subjected directly to insolation, rain, the deposition of -dew, the drying action of wind, relatively quick changes of temperature -and other effects of climate. Certain combinations of these conditions -present so favourable an environment for the growth of algæ that at -times there appears on the surface of the soil a conspicuous green -stratum, sometimes so dark in colour as to appear almost black. Strata -of this nature are well known, and in systematic works there are -constant references to species growing “on damp soil”; for instance, of -the 51 well-defined species of _Nostoc_ recognised by Forte, no less -than 31 are characterised as terrestrial. Such appearances, however, -seem to have been regarded as sporadic and more or less accidental, -rather than as the unusually luxuriant development of an endemic -population, and have been frequently attributed to an excessively moist -condition of the soil due to defective drainage. - -In the second place, the algæ may be living within the soil itself, -away from the action of sunlight and under somewhat more uniform -conditions of moisture and temperature. - -Up to the present time the greater number of the investigations carried -out in this subject have been of a systematic nature, and extremely -little direct evidence has been obtained which can throw any light on -the subject of the economic significance of the soil algæ. - -The earliest systematic work was carried out by Esmarch, in 1910-11, -who investigated by means of cultures the blue-green algæ of a number -of soils from the German African Colonies, the samples being taken from -the surface and also from the lower layers of the soil. He obtained a -considerable number of species and observed that in cultivated soils -they were not confined to the surface but occurred regularly to a depth -of 10-25 cms. and occasionally as low as 40-50 cms. He attributed -their existence in the lower layers to the presence of resting spores -carried down in the processes of cultivation, since his samples from -uncultivated soils were unproductive. - -Later, Esmarch extended his investigations to a far larger -number of samples, 395 in all, of soils of different types from -Schleswig-Holstein. He found that blue-green algæ were very widely -distributed in soils of certain types, though they occurred rarely -in uncultivated soils of low water-content, and he described no less -than 45 species of which 34 belonged to the _Oscillatoriaceæ_ and -_Nostocaceæ_. Certain of the commoner species were obtained from soils -of widely different types, as shown in Table X., while other forms -occurred only rarely and with a much more limited distribution. - -TABLE X.--FREQUENCY OF OCCURRENCE OF CERTAIN COMMON SPECIES IN -ESMARCH’S SOIL SAMPLES. - - +-------------------------+----------------------------------------+ - | | Percentage of Samples | - | | containing given Alga. | - | +--------------------+-------------------+ - | | Uncultivated | Cultivated | - | | Damp Sandy Soil. | Soils. | - | +------+------+------+------+-----+------+ - | |Shores|Shores| | | | | - | | of | of | Sea- | | |Marsh-| - | Species. | Elbe.|Lakes.|shore.|Sandy.|Clay.| land.| - +-------------------------+------+------+------+------+-----+------+ - |Anabæna variabilis | 46 | 43 | 9 | 10·3 | 60 | 46 | - |Anabæna torulosa | 31 | 14·3 | 63·6 | 27·6 | 34·3| 56·4 | - |Cylindrospermum muscicola| 23 | 28·6 | 0 | 24 | 48·6| 59 | - |Cylindrospermum majus | 0 | 14·3 | 0 | 38 | 40 | 33·3 | - |Nostoc Sp. III. | 7·7 | 0 | 0 | 38 | 37 | 48·7 | - +-------------------------+------+------+------+------+-----+------+ - -Taking the number of samples containing blue-green algæ as a rough -measure of their relative abundance, Esmarch obtained the following -interesting figures (Table XI.):-- - -TABLE XI. - - +------------------------------+----------------+---------+ - | | Percentage | | - | | of Samples |Number of| - | | Containing | Samples | - | Kind of Soil. |Blue-green Algæ.|Examined.| - +------------------------------+----------------+---------+ - | | | | - |Cultivated marshland | 95 | 40 | - |Cultivated clay soil | 94·6 | 37 | - |Uncultivated moist sandy soils| 88·6 | 35 | - |Cultivated sandy soil | 64·4 | 45 | - | {Woodland | 12·5 | 40 | - |Uncultivated{Sandy heathland | 9 | 34 | - | {Moorland | 0 | 35 | - +------------------------------+----------------+---------+ - -In noting that the soils fell into two groups, those relatively rich -and those poor in blue-green algæ, Esmarch concluded that the two -chief factors governing the distribution of the _Cyanophyceæ_ on the -surface of soils are, (1) the moisture content of the soil, (2) the -availability of mineral salts, cultivated soils being especially -favoured in both of these respects. He further distinguished between -cultivated land of two kinds, viz. arable land and grass land, and -found that on all types of soil grassland was richer in species than -was arable land. - -Esmarch examined, in addition, 129 samples taken from the lower layers -of the soil immediately beneath certain of his surface samples, 107 at -10-25 cms. and the rest at 30-50 cms. depth. - -In cultivated soils, whether grassland or arable land, he found that -blue-green algæ occurred almost invariably in the lower layers in those -places bearing algæ on the surface and that, with rare exceptions, the -algæ found in the lower layers corresponded exactly to those on the -surface, except that with increasing depth there was a progressive -reduction in the number of species. - -In uncultivated, moist, sandy soils the agreement was far less -complete, for though algæ were rarely absent from the lower layers -their vertical distribution was frequently disturbed by the action -of wind and rain. Other uncultivated soils not subject to periodic -disturbance were found to be uniformly lacking in algæ in the lower -layers, but as the limited number of samples examined came completely -from places where there were no algæ on the surface this means very -little. - -By direct microscopic examination of soil Esmarch claims to have -found living filaments of blue-green algæ at various depths below the -surface. He realised, however, that there was no indication of the -length of time that such filaments had been buried, and therefore -conducted a series of experiments from which he concluded that the -period during which the algæ investigated could continue vegetatively -in the soil after burial varied with different species from 5-12 weeks, -but that during the later part of the period the algæ gradually assumed -a yellowish-green colour. - -It is unfortunate that Esmarch’s investigations were directed only -towards the blue-green algæ since observations made in this country -indicate that such a series of records gives but a very incomplete -picture of the soil flora as a whole. - -Petersen, in his “Danske Aërofile Alger” (1915) added considerably to -our knowledge of soil algæ, especially of diatoms. Unfortunately he -confined his investigations of the green algæ to forms growing visibly -on the surface of the ground. He observed, however, that acid soils -possessed a different flora from that commonly found on alkaline or -neutral soils, the former being dominated by _Mesotænium violascens_, -_Zygnema ericetorum_, and 2 spp. of _Coccomyxa_, while the latter were -characterised by _Mesotænium macrococcum var._, _Hormidium_, 2 spp., -and _Vaucheria_, 3 spp. - -Of diatoms he obtained no less than 24 species and varieties from -arable and garden soils, and five characteristic of marshy soils, while -from forest soils and dry heathland they appeared to be often absent. -He omitted all reference to blue-green algæ. - -Meanwhile Robbins, examining a number of Colorado soils that contained -unprecedented quantities of nitrate, obtained from them 18 species of -blue-green algæ, 2 species of green algæ, and one diatom. Moore and -Karrer have demonstrated the existence of a subterranean alga-flora of -which _Protoderma viride_, the most constantly occurring species, was -shown to multiply when buried to a depth of one metre. - -In this country attention was first called to the subject by Goodey and -Hutchinson of Rothamsted who, in examining certain old stored soils -for protozoa, obtained also a number of blue-green forms which were -submitted to Professor West for identification. This ability of certain -algal spores to retain their vitality for a long resting period was so -very striking that an investigation was begun at Birmingham in 1915 to -ascertain whether other forms were equally resistant. The investigation -was carried out on a large number of freshly collected samples of -arable and garden soils which were first aseptically air-dried for at -least a month and then grown in culture. No less than 20 species or -varieties of diatoms, 24 species of blue-green and 20 species of green -algæ were obtained from these cultures (Table XII.). In the majority -of the samples there was found a central group of algæ, including -_Hantzschia amphioxys_, _Trochiscia aspera_, _Chlorococcum humicola_, -_Bumilleria exilis_ and rather less frequently _Ulothrix subtilis -var. variabilis_, while moss protonema was universally present. These -species were thought to form the basis of an extensive ecological plant -formation in which, by the inclusion of other typically terrestrial -but less widely distributed species smaller plant-associations were -recognised. - -In certain of the soils, associations consisting very largely of -diatoms were present, and it is to be noted that the majority of the -forms that have been described are of exceedingly small size. It is -doubtless this characteristic which enables them to withstand the -conditions of drought to which the organisms of the soil are liable to -be subjected, small organisms having been shown to be better able to -resist desiccation than are larger ones. Since the soil diatoms belong -to the pennate type, they are further adapted to their mode of life by -their power of locomotion, which enables them in times of drought to -retire to the moister layers of the soil. - -In the soils examined in this work blue-green algæ were less -universally present than were diatoms or green algæ, and the species -found appeared to be more local in occurrence. There seemed to be, -however, an association between the three species, _Phormidium tenue_, -_Ph. autumnale_, and _Plectonema Battersii_, at least two of the three -species having been found together in no less than 16 of the samples, -while all three occurred in 7 of them. - -TABLE XII.--ALGÆ IN DESICCATED ENGLISH SOILS. (BRISTOL.) - - +---------------+-----------+------------------------------+ - | | | Number of Species. | - | | Number of +-----------+-----------+------+ - | | Samples | Maximum | Average | | - | Group. |Productive.|per Sample.|per Sample.|Total.| - +---------------+-----------+-----------+-----------+------+ - | | per cent. | | | | - |Diatoms | 95·5 | 9 | 3·7 | 20 | - |Blue-green algæ| 77·3 | 7 | 2·5 | 24 | - |Green algæ | 100 | 7 | 4·3 | 20 | - |Moss protonema | 100 | -- | -- | -- | - +---------------+-----------+-----------+-----------+------+ - | Total | -- | 20 | 10·5 | -- | - +---------------+-----------+-----------+-----------+------+ - -It was generally noticeable that those soils found to be rich in -blue-green algæ contained only a few species of diatoms, and vice -versa. Diatoms appeared most frequently in soils from old gardens, -whereas blue-green algæ were more characteristic of arable soils. The -green algæ and moss protonema, on the other hand, were distributed -universally. - -The majority of green algæ typically found in soils are unicellular, -but a few filamentous forms occur. With the exception of _Vaucheria_ -spp. these are characterised, however, by an ability to break down -in certain circumstances into unicellular or few-celled fragments, in -which condition identification is often very difficult. - -It was also found by cultural examination of a number of old stored -soils from Rothamsted that germination of the resting forms of a -number of algæ could take place after an exceedingly long period of -quiescence. No less than nine species of blue-green algæ, four species -of green algæ, and one species of diatom were obtained from soils that -had been stored for periods of about forty years, the species with the -greatest power to retain their vitality being _Nostoc muscorum_ and -_Nodularia Harveyana_. - - -II. THE SOIL AS A SUITABLE MEDIUM FOR ALGAL GROWTH. - -Were it not for the recent advances that have been made in our -knowledge of the mode of nutrition of many of the lower algæ, it would -be very difficult to account for the widespread occurrence of algæ in -the soil, for it is undoubtedly true of some of the more highly evolved -algæ that their mode of nutrition is entirely typical of that of green -plants in general. The application of bacteriological technique to the -algæ, however, by Beijerinck, by Artari, and by Chodat and his pupils, -and the introduction of pure-culture methods have led to a study of -the physiology of some of the lower algæ, in the hope of getting to -understand some of the fundamental problems underlying the nutrition -of organisms containing chlorophyll. It is impossible here to do more -than mention the names of a few of the more important of those who -have worked along these lines, such as Chodat, Artari, Grintzesco, -Pringsheim, Kufferath, Nakano, Boresch, Magnus and Schindler, and to -condense into a few sentences some of their more important conclusions. - -It is now established that although in the light the algæ are able to -build up their substance from CO₂ and water containing dilute mineral -salts, yet in such conditions growth is sometimes very slow, and with -some species at any rate it is greatly accelerated by the addition of -a small quantity of certain organic compounds. The ability of the lower -algæ to use organic food materials varies specifically, quite closely -related forms often reacting very differently to the same substance, -but there have been shown to be a considerable number of forms which -can make use of organic compounds to such an extent that they can -grow entirely independently of light. In such cases the nutrition -of the organism becomes wholly saprophytic, and the chlorophyll may -be completely lost; it has frequently been observed, however, that -on suitable nutrient media, even in complete darkness, certain algæ -continue to grow and retain their green colour, provided that a -sufficient supply of a suitable nitrogenous compound is present. - -_Chlorella vulgaris_, an alga frequently found in soil, has been shown -to be extremely plastic in its relations to food substances. Given only -a dilute mineral-salts solution as food source, it absorbs CO₂ from -the air, and grows in sunlight with moderate rapidity. The addition -of glucose to the medium in the light greatly increases the rate and -amount of growth and the size of the cells, while in the dark the -colonies not only remain green but have been shown to develop more -vigorously than in full daylight. The organism is also able to use -peptone as a source of nitrogen in place of nitrates. - -_Stichococcus bacillaris_ and _Scenedesmus spp._, also occurring -in soils, have been shown to be almost equally adaptable, though -in these cases the organisms grow more slowly in the dark than on -the corresponding medium in the light. Liquefaction of gelatine by -the secretion of proteolytic enzymes has been shown to be a further -property of certain species, resulting in the formation of amino acids -such as glycocoll, phenylalanine, dipeptides, etc. This property is, -however, possessed by only a limited number of species and in varying -degree. - -Up to the present very little work of this kind has been done upon algæ -actually taken from the soil, and our knowledge is therefore very -scanty. Of the species so far examined all show considerable increase -in growth on the addition to the medium of glucose and other sugars, -and tend to be partially saprophytic; a few have been shown to liquefy -gelatine to some extent. - -Servettaz, Von Ubisch, and Robbins have also demonstrated that the -protonema of some mosses can make use of certain organic substances, -especially the sugars, and grow vigorously in the dark. It has been -shown, however, that light is essential for the development of the moss -plant. - -It was thought at Rothamsted that some light might be thrown upon the -activities of the soil-algæ by making counts of the numbers present -in samples of soil taken periodically within a circumscribed area. -A dilution method similar to that in use in the protozoological -laboratory was adopted and applied to samples of arable soil taken -from the surface, and at depths of 2, 4, 6 and 12 inches vertically -beneath. A considerable number of samples were examined in this way -from two plots on Broadbalk wheat-field, viz.: the unmanured plot and -that receiving a heavy annual dressing of farmyard manure. The numbers -in the unmanured soil were observed to fall far short of those in that -containing a large amount of organic matter, while in both plots the -numbers varied considerably at different times of the year. The chief -species in both plots were identical, and their vertical distribution -was fairly uniform, but it was observed that the numbers of individuals -varied according to the depth of the sample. The 6th and 12th inch -samples contained very few individuals of comparatively few species, -but the 4th inch samples yielded numbers that were not significantly -less than those in the top inch. The 2nd inch sample was usually much -poorer in individuals than either the top or the 4th inch. - -It is unfortunate that this method of counting is not really -satisfactory for the algæ, chiefly because it takes no account of the -blue-green forms. The gelatinous envelope which encloses the filaments -of these algæ prevents their breaking up into measurable units. -Assuming, as appears to be the case for the two plots investigated, -that the blue-green algæ are at least as numerous as the green forms, -the total numbers should probably be at least twice as great as those -calculated. Taking 100,000 as a rough estimate of the number of algæ -per gram of manured soil in a given sample, and assuming the cells to -be spherical and of average diameter 10µ, it has been calculated that -the volume of algal protoplasm present was at least 3 times that of -the bacteria though only one-third of that of the protozoa. This is -probably only a minimum figure for this sample. - -A soil population of this magnitude can not be without effect on the -fertility of the soil. When growing on the surface of the ground -exposed to sunlight the algæ must, by photosynthesis, add considerably -to the organic matter of the soil, but when they live within the soil -itself their nutrition must be wholly saprophytic, and they can be -adding nothing either to the energy or to the food-content of the soil. -How these organisms fit into the general scheme of life in the soil is -at present undetermined, and there is a wide field for research in this -direction. - - -III. RELATION OF ALGÆ TO THE NITROGEN CYCLE - -Probably the most important limiting factor in British agriculture is -the supply of nitrogen available for the growing crop, and it seems -likely that the soil-algæ are intimately connected with this question -in several ways. - -Periodic efforts have been made during the last half century to -establish the fact that a number of the lower organisms, including -the green algæ, have the power of fixing atmospheric nitrogen and -converting it into compounds which are then available for higher -plants. This property has been definitely established for certain -bacteria, and rather doubtfully for some of the fungi, but until -recently no authentic proof had been produced that algæ by themselves -could fix nitrogen. The subject is too wide to be discussed in much -detail here. - -Schramm in America, working with pure cultures of algæ, tried for -ten years to establish the fact of nitrogen fixation, and failed -completely; more recently Wann has extended Schramm’s work, and claims -to have proved indisputably that, given media containing nitrates as -a source of nitrogen and a small amount of glucose, the seven species -of algæ tested by him fixed atmospheric nitrogen to the extent of 4-54 -per cent. of the original nitrogen content of the medium. So important -a result needed corroboration, and Wann’s experiment, with some slight -improvements, was therefore repeated at Rothamsted last summer. - -This work has not yet been published, but in the whole series of -ninety-six cultures, with four different species, each growing on -six different media, there is no evidence that nitrogen fixation has -taken place; but there has been a total recovery at the end of the -experiment of 98·93 per cent. of the original nitrogen supplied. On -the other hand, a flaw has been detected in Wann’s method of analysing -those media containing nitrates, sufficiently great to account for the -differences he obtained between the initial and final nitrogen content -of his cultures. Hence, though one hesitates to say that the algæ are -unable, given suitable conditions, to fix atmospheric nitrogen, one -must admit that no one has yet proved that they can do so. - -It is far more likely, however, that the experiments of Kossowitsch -and others throw more light on the relation of soil algæ to -nitrogen fixation. They affirm that greater fixation of nitrogen is -effected by mixtures of bacteria and certain gelatinous algæ than -by nitrogen-fixing bacteria alone, and that the addition of algæ to -cultures of bacteria produces a stimulating effect only slightly less -than that of sugar. It is probable, therefore, that the algæ, in their -gelatinous sheaths, provide easily available carbohydrates from which -the bacteria derive the energy essential to their work, and that -nitrogen fixation in nature is due to the combined working of a number -of different organisms rather than to the individual action of single -species. - -Russell and Richards have shown that the rate of loss of nitrogen by -leaching from uncropped soils is far less than would be expected from -a purely chemical standpoint, and suggest that certain organisms are -present in the soil which, by absorbing nitrates and ammonium salts -as they are formed, remove them from the soil solution and so help to -conserve the nitrogen of the soil. It is probable that the soil algæ -act in this manner, though to what extent has not yet been determined. - - -IV. RELATION OF ALGÆ TO SOIL MOISTURE AND TO THE FORMATION OF HUMUS -SUBSTANCES. - -In warmer countries than our own, especially those with an adequate -rainfall, the significance of soil algæ is perhaps more obvious to -a casual observer. Treub states that after the complete destruction -of the island of Krakatoa by volcanic eruption in 1883, the first -colonists to take possession of the island were six species of -blue-green algæ, viz., _Tolypothrix_ sp., _Anabæna_ sp., _Symploca_ -sp., _Lyngbya_ 3 spp. Three years after the eruption these organisms -were observed to form an almost continuous gelatinous and hygroscopic -layer over the surface of the cinders and stones constituting the soil, -and by their death and decay they rapidly prepared it for the growth -of seeds brought to the island by visiting birds. Hence the new flora -which soon established itself upon the island can be said to have -had its origin in the alga-flora which preceded it. Fritsch has also -emphasised the importance of algæ in the colonisation of new ground in -Ceylon. - -Welwitsch ascribes the characteristic colour from which the “pedras -negras” in Angola derive their name to the growth of a thick stratum of -_Scytonema myochrous_, a blue-green alga, which gradually becomes black -and completely covers the soil. At the close of the rainy season this -gelatinous stratum dries up very slowly, enabling the underlying soil -to retain its moisture for a longer period than would otherwise be the -case. - -The gelatinous soil algæ are probably very important in this respect, -for their slow rate of loss of water is coupled with a capacity for -rapid absorption, and they are therefore able to take full advantage of -the dew that may be deposited upon them and increase the power of the -soil to retain moisture. - - -V. RELATION OF ALGÆ TO GASEOUS INTERCHANGES IN THE SOIL. - -In the cultivation of rice the algæ of the paddy field have been found -to be of extreme importance. Brizi in Italy has shown that although -rice is grown under swamp conditions yet the roots of the rice plant -are typical of those of ordinary terrestrial plants and have none -of the structural adaptations to aquatic life so characteristic of -ordinary marsh plants. Hence the plants are entirely dependent for -healthy growth upon an adequate supply of oxygen to their roots from -the medium in which they are growing. A serious disease of the rice -plant, characterised by the browning and dying off of the leaves, -which was thought at first to be due to the attacks of fungi, was -found to be the effect of the inadequate aeration of the roots, while -the entry of the fungi was shown to be subsequent to the appearance -of the physiological disease. The presence of algæ in the swamp water -was found to prevent the appearance of this disease, in that they -unite with other organisms to form a more or less continuous stratum -over the surface of the ground, and add to the gases which accumulate -there large quantities of oxygen evolved during photosynthesis. The -concentration of dissolved oxygen in the water percolating through the -soil is thereby raised to a maximum, and the healthy growth of the crop -ensured. - -This work has been corroborated by Harrison and Aiyer in India, and a -sufficient supply of algæ in the swamp water is now regarded as one of -the essentials for the production of a good rice crop. - -From what has been said, it appears that, although our knowledge of -the soil algæ is extremely limited, and our conception of the part -they play is largely based on speculation, yet the subject is one of -enormous interest and worthy of investigation in many directions. In -its present undeveloped state, it is a little difficult to foresee -which lines of study are likely to prove most profitable, but there -is little doubt that eventually the soil algæ will be shown to play a -significant part in the economy of the soil. - - -SELECTED BIBLIOGRAPHY. - -* _Papers giving extensive bibliographies._ - - -I. GENERAL. - - [1] Bristol, B. M., “On the Retention of Vitality by Algæ from Old - Stored Soils,” New Phyt., 1919, xviii., Nos. 3 and 4. - - [2] Bristol, B. M., “On the Alga-Flora of some Desiccated English - Soils: an Important Factor in Soil Biology,” Annals of Botany, 1920, - vol. xxxiv., No. 133. - - [3] Brizi, U., “Ricerche sulla Malattia del Riso detta ‘Brusone,’ - Sect. IV. Influenza che le alghe verdi esercitano in risaia,” - Annuario dell Instituzione Agraria Dott. A. Ponti, Milan, 1905, vol. - vi., pp. 84-89. - - [4] Esmarch, F., “Beitrag zur Cyanophyceen-Flora unserer Kolonien,” - Jahrb. der Hamburgischen wissensch. Anstalten, 1910, xxviii., 3. - Beiheft, S. 62-82. - - [5] Esmarch, F., “Untersuchungen über die Verbreitung der - Cyanophyceen auf und in verschiedenen Boden,” Hedwigia, 1914, Band - lv., Heft 4-5. - - [6] Fritsch, F. E., “The Rôle of Algal Growth in the Colonisation of - New Ground and in the Determination of Scenery,” Geog. Journal, 1907. - - [7] Harrison, W. H., and Aiyer, P. A. Subramania, “The Gases of - Swamp Rice Soils,” Mem. Dept. Agr. in India, Chem. Ser. (I.) “Their - Composition and Relationship to the Crop,” 1913, vol. iii., No. - 3; (II.) “Their Utilisation for the Aeration of the Roots of the - Crop,” 1914, vol. iv., No. 1; (IV.) “The Source of the Gaseous Soil - Nitrogen,” 1916, vol. v., No. 1. - - [8_a_] Hensen, V., “Ueber die Bestimmung des Planktons oder des im - Meere treibenden Materials am Pflanzen und Thieren.” Fünfter Ber. - Komm. wiss. Unters. deutschen Meere, 1887. - - [8] Moore, G. T., and Karrer, J. L., “A Subterranean Alga Flora,” - Ann. Miss. Bot. Gard., 1919, vi., pp. 281-307. - - [9] Nadson, G., “Die perforierenden (kalkbohrende) Algen und ihre - Bedeutung in der Natur,” Scripta bot. hort. Univ. Imp. Petrop., 1901, - Bd. 17. - - [10] Petersen, J. B., “Danske Aërofile Alger,” D. Kgl. Danske - Vidensk. Selsk. Skrifter, 7 Raekke, Naturv. og mathem., 1915, Bd. - xii., 7, Copenhagen. - - [11] Robbins, W. W., “Algæ in some Colorado Soils,” Agric. Exp. Sta., - Colorado, 1912, Bulletin 184. - - [12] Treub, “Notice sur la nouvelle Flora de Krakatau,” Ann. Jard. - Bot. Buitenzorg, 1888, vol. vii., pp. 221-223. - - -II. RELATION OF ALGÆ TO LIGHT AND CARBON. - - [13] Artari, A., “Zur Ernährungsphysiologie der grünen Algen,” Ber. - der D. bot. Ges., 1901, Bd. xix., S. 7. - - [14] Artari, A., “Zur Physiologie der Chlamydomonaden (Chlam. - Ehrenbergii);” (I.) Jahrb. f. Wiss. Bot., 1913, Bd. lii., S. 410; - (II.) _Ibid._, 1914, Bd. liii., S. 527. - - [15] Adjarof, M., “Recherches expérimentales sur la Physiologie de - quelques Algues vertes,” Université de Genève--Institut Botanique, - Prof. R. Chodat--1905, 6 serie, vii. fascicule, Genève. - - [16] Beijerinck, M. W., “Berichte über meine Kulturen niederer Algen - auf Nährgelatine,” Centr. f. Bakt. u. Paras., 1893, Abt. I., Bd. - xiii., S. 368, Jena. - - [17] Boresch, K., “Die Färbung von Cyanophyceen und Chlorophyceen in - ihrer Abhängigkeit vom Stickstoffgehalt des Substrates,” Jahrbücher - für Wiss. Botanik., 1913, lii., pp. 145-85. - - [18] Chodat, R., “Étude critique et expérimentale sur le - polymorphisme des Algues,” Genève, 1909. - - [19] Chodat, R., “La crésol-tyrosinase, réactif des peptides et - des polypeptides, des protéides et de la protéolyse,” Archiv. des - Sciences physiques et naturelles, 1912. - - [20] Chodat, R., “Monographie d’Algues en Culture pure: Matériaux - pour la Flore Cryptogamique Suisse,” 1913, vol. iv., fasc. 2, Berne. - - [21] Dangeard, P. A., “Observations sur une Algue cultivée à - l’obscurité depuis huit ans,” Compt. Rend. Acad. Sci. (Paris), 1921, - vol. clxxii., No. 5, pp. 254-60. - - [22] Étard et Bouilhac, “Sur la présence de la chlorophyll dans un - Nostoc cultivé à l’abri de la lumière,” Compt. Rend., t. cxxvii, 1898. - - [23] Grintzesco, J., “Recherches expérimentales sur la morphologie et - la physiologie expérimentale de _Scenedesmus acutus_,” Meyen. Bull. - herb. Boiss., 1902, Bd. ii., pp. 219-64 and 406-29. - - [24] Grintzesco, J., “Contribution à l’étude des Protococcoidées: - _Chlorella vulgaris_ Beyerinck,” Revue générale de Botanique, 1903, - xv., pp. 5-19, 67-82. - - [25] * Kufferath, H., “Contribution à la physiologie d’une - protococcacée nouvelle, _Chlorella luteo-viridis_ Chod. n. sp. var., - _lutescens_ Chod. n. var.,” Recueil de l’institut bot. Léo Errera, - 1913, t. ix, p. 113. - - [26] Kufferath, H., “Recherches physiologiques sur les algues vertes - cultivées en culture pure,” Bull. Soc. Roy. Bot. Belgique, 1921, - liv., pp. 49-77. - - [27] Magnus, W., and Schindler, B., “Ueber den Einflusz der Nährsalze - auf die Färbung der Oscillarien,” Ber. der D. Bot. Gesellschaft, - 1912-13, xxx., p. 314. - - [28] * Nakano, H., “Untersuchungen über die Entwicklungs- und - Ernährungsphysiologie einiger Chlorophyceen,” Journ. College of Sci. - Imp. Univ. Tokyo, 1917, vol. xl., Art. 2. - - [29] Pringsheim, E., “Kulturversuche mit chlorophyll-führenden - Mikroorganismen,” Cohns Beiträge Z. Biol. d. Pflanzen. (I.) - Die Kultur von Algen in Agar, 1912, Bd. xi., S. 249; (II.) Zur - Physiologie der _Euglena gracilis_, 1913, Bd. xii., S. 1.; (III.) Zur - Physiologie der Schizophyceen, 1913, Bd. xii., S. 99. - - [30] Radais, “Sur la culture pure d’une algue verte; formation de - chlorophylle à l’obscurité,” Comptes Rendus, 1900, cxxx., p. 793. - - [31] Richter, O., “Zur Physiologie der Diatomeen.” (I.) Sitzber. d. - kais. Akad. d. W. in Wien, math, naturw. Kl., 1906, Bd. cxv., Abt. - I., S. 27; (II.) Denkschrift d. math. naturw. Kl. d. kais. Akad. - d. W. in Wien, 1909, Bd. lxxxiv., S. 666; (III.) Sitzber. d. Kais. - Akad., etc., 1909, Bd. cxviii., Abt. I., S. 1337. - - [32] Richter, O., “Ernährung der Algen,” 1911. - - [33] Robbins, W. J., “Direct Assimilation of Organic Carbon by - _Ceratodon purpureus_,” Bot. Gaz., 1918, lxv., pp. 543-51. - - [34] Schindler, B., “Ueber den Farbenwechsel der Oscillarien,” - Zeitsch. f. Bot., 1913, v., pp. 497-575. - - [35] Ternetz, Charlotte, “Beiträge zur Morphologie und Physiologie - der _Euglena gracilis_,” Jahrb. f. Wiss. Bot., 1912, Bd. 51, S. 435. - - -III. RELATION OF ALGÆ TO NITROGEN. - - [36] Berthelot, “Recherches nouvelles sur les microorganismes - fixateurs de l’azote,” Comptes Rend., 1893, cxvi., pp. 842-49. - - [37] Bouilhac, R., “Sur la fixation de l’azote atmosphérique par - l’association des algues et des bactéries,” Comptes Rend., 1896, - cxxiii., pp. 828-30. - - [38] Bouilhac and Giustiniani, “Sur une culture de sarrasin en - présence d’un mélange d’algues et de bactéries,” Comptes Rendus, - 1903, cxxxvii., pp. 1274-76. - - [39] Charpentier, P. G., “Alimentation azotée d’une algue: Le - Cystococcus humicola,” Ann. Inst. Pasteur, 1903, 17, pp. 321-34. - - [40] Fischer, Hugo, “Über Symbiose von Azotobacter mit Oscillarien,” - Centr. f. Bakt., 1904, xii. - - [41] Frank, B., “Uber den experimentellen Nachweis der Assimilation - freien Stickstoffs durch Erdbewohnende Algen,” Ber. der D. Bot. - Gesellsch., 1889, vol. vii., pp. 34-42. - - [42] Frank, B., “Ueber den gegenwärtigen Stand unserer Kenntnisse der - Assimilation elementaren Stickstoffs durch die Pflanze,” Ber. der. D. - Bot. Ges., 1889, vii., 234-47. - - [43] Frank, B., and Otto, R., “Untersuchungen über Stickstoff - Assimilation in der Pflanze,” Ber. der D. Bot. Ges., 1890, viii., - 331-342. - - [44] Gautier and Drouin, “Recherches sur la fixation de l’azote par - le sol et les végétaux,” Compt. Rend., 1888, cvi., pp. 1174-76; - General Conclusions, p. 1232. - - [45] Kossowitsch, P., “Untersuchungen über die Frage, ob die Algen - freien Stickstoff fixiren,” Bot. Zeit., 1894, Heft 5, S. 98-116. - - [46] Krüger, W., und Schneidewind, “Sind niedere chlorophyllgrüne - Algen imstande, den freien Stickstoff der Atmosphäre zu assimilieren - und Boden an Stickstoff zu bereichern?” Landwirtschaftliche Jahrb., - 1900, Bd. 29, S. 771-804. - - [47] Moore, Benjamin, and T. Arthur Webster, “Studies of the - photosynthesis in f.w.a.” (I.) “The fixation of both C and N from - atmosphere to form organic tissue by green plant cell”; (II.) - “Nutrition and growth produced by high gaseous dilutions of simple - organic compounds, such as formaldehyde and methylic alcohol”; (III.) - “Nutrition and growth by means of high dilution of CO₂ and oxides of - N without access to atmosphere,” Proc. Roy. Soc., London, 1920, B. - xci., pp. 201-15. - - [47_a_] Moore, B., Whiteley, Webster, T. A., Proc. Roy. Soc., London, - B., 1921; xcii., pp. 51-60. - - [48] Reinke, J., “Symbiose von Volvox und Azotobacter,” Ber. der d. - Bot. Ges., 1903, Bd. xxi., S. 481. - - [49] Russell, E. J., and Richards, E. H., “The washing out of - Nitrates by Drainage Water from Uncropped and Unmanured Land,” Journ. - Agric. Sci., 1920, vol. x., Part I. - - [50] Schloesing, fils, and Laurent, E., “Recherches sur la fixation - de l’azote libre par les plantes,” Ann. de l’Institut Pasteur, 1892, - vi., pp. 65-115. - - [51] Schramm, J. R., “The Relation of Certain Grass Green Algæ to - Elementary Nitrogen,” Ann. Mo. Bot. Gard., 1914, i., No. 2. - - [52] Wann, F. B., “The Fixation of Nitrogen by Green Plants,” Amer. - Journ. Bot., 1921, viii., pp. 1-29. - - - - -CHAPTER VII. - -THE OCCURRENCE OF FUNGI IN THE SOIL. - - - NOTE.--I am indebted to my late colleague Miss Sibyl S. Jewson, - M.Sc., for permission to include unpublished data from our - investigations on the soil fungi. - -In 1886 Adametz,[1] investigating the biochemical changes occurring -in soils, isolated several species of fungi. It was, however, only -with the work of Oudemans and Koning,[17] in 1902 when forty-five -species were isolated and described, the majority as new to science, -that the real study of the fungus flora of the soil commenced. There -is now no doubt that fungi form a large and very important section -of the permanent soil population, and certain forms are found only -in the soil. Indeed, Takahashi[22] has reversed the earlier ideas by -suggesting that fungus spores in the air are derived from soil forms. -The majority of investigations on this subject fall, perhaps, into -one or more of three classes: (_a_) purely systematic studies such as -those of Oudemans and Koning,[17] Dale,[5] Jensen,[9] Waksman,[25a] -Hagem,[8c] Lendner,[12] and others, which consist in the isolation -and identification of species from various soils: (_b_) physiological -researches, such as those of Hagem[8c] on the Mucorineæ of Norway, or -the many investigations on the biochemical changes in soils produced by -fungi, such as those of Muntz and Coudon,[15] McLean and Wilson,[15] -Kopeloff,[11] Goddard,[7] McBeth and Scales,[14] and others: (_c_) -quantitative studies, such as those of Remy,[20] Fischer,[6] -Ramann,[18] Waksman,[25c] and Takahashi,[22] which involve numerical -estimates of the fungus flora in soils. - - -QUALITATIVE STUDY. - -With very rare exceptions soil fungi cannot be examined in situ, and -the necessary basis of any qualitative research is the isolation of -the organisms in pure culture. Most soil forms belong to the _Fungi -imperfecti_, and often show considerable plasticity on artificial -media. This makes it very difficult to determine them by comparison -with type herbarium specimens or published morphological diagnoses. -In consequence many soil fungi have not infrequently been given new -specific names, as _humicola_, _terricola_, and so forth, which is -very unsatisfactory, and means that the determinations have little -significance. - -Furthermore, most artificial media are slight variations on a few -common and simple themes, and are very selective, permitting the growth -of a moiety only of the fungi present. In addition, many fungi grow -so slowly that they are overwhelmed by the more rapidly germinating -or spreading forms, or on the other hand, they may be eliminated by -the metabolic products of different adjacent colonies. The extremely -selective nature of the technique commonly used is shown if one -tabulates systematically all the fungi which have been recorded or -described in soil investigations. Of _Phycomycetes_ there are fifty-six -species of eleven genera; of _Ascomycetes_ twelve species of eight -genera; and of _Fungi imperfecti_, including _Actinomycetes_ but not -sterile _Mycelia_, 197 species of sixty-two genera. Rusts and Smuts -one might not expect, but that of the multitudes of _Basidiomycetes_ -growing in wood and meadow not one should have been recorded is indeed -startling. It was at first thought that many imperfect fungi might -be conidial stages of _Basidiomycetes_, but much search among forms -isolated at Rothamsted has, up to the present, failed to reveal clamp -connections in the hyphæ. - -Since various species of soil fungi have different optimum temperature, -humidity and other conditions[3] one would not expect to find an even -geographic distribution. Very little is yet known of this aspect, but -_Rhizopus nigricans_, _Mucor racemosus_, _Zygorrhynchus vuilleminii_, -_Aspergillus niger_, _Trichoderma koningi_, _Cladosporium herbarum_, -and many species of _Aspergillus_, _Penicillium_, _Fusarium_, -_Alternaria_, and _Cephalosporium_ have been commonly found throughout -North America and Europe wherever soils have been examined. Species of -_Aspergillus_, however, would appear to be more common in the soils -of south temperate regions and species of _Penicillium_, _Mucor_, -_Trichoderma_, and _Fusarium_ more abundant in northern soils. - -It is well known that in many plant and animal communities there -occurs a definite rhythm, various species following each other in a -regular sequence as dominants in the population. Although it is not yet -possible to make any definite statement there would seem indications -that this may also be true of the soil fungi. - -Much work has been done on the distribution of species at different -depths in the soil, but the results are still confusing. Thus, -examining eighteen species, Goddard[7] found no difference in relative -distribution down to 5½ inches. Werkenthin[26] found identical species -from 1-4 inches, and then an absence of fungi from 5-7 inches, which -latter was the greatest depth he examined. Waksman[25] found little -difference in the first six inches, but very few species below 8 inches -except _Zygorrhynchus vuilleminii_, which extended down to 30 inches -and was often the only species occurring below 12 inches. Taylor[23] -has reported species of _Fusarium_ at practically every depth to 24 -inches. Rathbun[19] found _Aspergillus niger_, _Rhizopus nigricans_, -and species of Fusarium and Mucor down to 34 inches, and _Oospora -lactis_, _Trichoderma koningi_, _Zygorrhynchus vuilleminii_ and species -of _Penicillium_, _Spicaria_ and _Saccharomyces_ as deep as 44 inches. -Eleven species were isolated from the alimentary canal of grubs and -worms, and Rathbun concluded that soil fungi may be spread by these -organisms. - -On an unmanured grass plot at Rothamsted twenty species were isolated -from a depth of 1 inch, nineteen from 6 inches, and eleven from -12 inches, whereas on the unmanured plot of Broadbalk wheat field -twenty-six species were obtained from 1 inch, seven from 6 inches, and -five from 12 inches. There appeared to be no conspicuous differences -between the floras of the two plots save that in the Broadbalk plot -there were fewer Mucorales, and _Zygorrhynchus mœlleri_ and _Absidia -cylindrospora_ were absent. In the grass plot samples about one-half -the forms occurring at the lower levels were isolated also from the -upper levels, but in the Broadbalk sample the five forms isolated from -12 inches, and five out of seven of those at 6 inches occurred only at -those levels, i.e. each of the three levels appeared to have a specific -flora. The difference in depth distribution in these two cases may -be due to the fact that in the Broadbalk plot the stiff clay subsoil -occurs at 5-7 inches, whereas in the grass plot the depth of soil is -greater than 12 inches. Much further work needs to be done on this -aspect before any definite conclusion can be reached. - -Much scattered information is available concerning the effect of -soil type, manuring, treatment, cropping, and so forth upon the -fungus content, but no clear issue as yet emerges from the results. -Hagem[8] found that cultivated soils vary greatly from forest soils -in the species of _Mucor_ present, and that certain species seem -to be associated in similar environments. Thus in pinewoods _Mucor -ramannianus_ is usually found, together with _M. strictus_, _M. -flavus_, and _M. sylvaticus_, and with this “_M. Ramannianus Society_,” -_M. racemosus_, _M. hiemalis_, and _Absidia orchidis_, are frequently -associated. The differences found by Hagem between the species of -_Mucor_ from forest and cultivated land could not, however, be -confirmed by Werkenthin.[26] - -Dale,[5] examining sandy, chalky, peaty and black earth soils, found -specific differences, although many of the species were common to all. -A soil which had been manured continuously for thirty-eight years -with ammonium sulphate alone, contained twenty-two species, whereas -the same soil with the addition of lime only had thirteen species. -Both Goddard[7] and Werkenthin,[26] in their investigations, found -a constant and characteristic fungus flora regardless of soil type, -tillage, or manuring. Waksman’s[25] studies of forest soils showed -few species of _Mucor_ but many of _Penicillium_ and _Trichoderma_[2]; -orchard soil contained no species of _Trichoderma_, very few of -_Penicillium_, but a large number of species of _Mucor_; species of -_Trichoderma_ were common in acid soils, whilst cultivated garden -soil contained all forms. The examination of very differently manured -plots on the Broadbalk wheat field at Rothamsted has not shown any -striking differences in the fungus flora, all the more important groups -of species being represented in every plot, but significant minor -differences are present. Thus, plot 13, manured with double ammonium -salts, superphosphate and sulphate of potash, is especially rich in -“species” of Trichoderma, whereas the unmanured plot contains large -numbers of species of green _Penicillium_, _Trichoderma_, and a species -of _Botrytis_ (pyramidalis?). - -The effect of the crop upon the fungus flora is seen in cases where -the same crop is grown year after year as in certain flax areas, where -species of _Fusarium_ accumulate in the soil and tend to produce “flax -sickness.”[13] - - -QUANTITATIVE STUDY. - -As it is not possible to count the soil fungi _in situ_, any estimation -of the numbers present in a soil must be arrived at by indirect means. -The method adopted is to make as fine a suspension as possible of -a known quantity of soil sample in a known amount of water, dilute -this to 1/5000, 1/10000, and so forth by regular gradations, incubate -cubic centimetres of the final dilution on artificial media in petri -dishes, and count the colonies of fungi developing in each plate. -Using the average figures from a series of duplicate plates, the -number of “individual” fungi in a gram of the original soil sample may -then be calculated. The very few students who have made quantitative -estimations have obtained very unsatisfactory results. In bacterial or -protozoal estimations, the shaking of the soil suspension separates the -unicellular individuals, so that in the final platings each individual -from the soil theoretically gives rise to one colony on the medium. -In the case of fungi, the organisms may be in the form of unicellular -or multicellular spores or larger or smaller masses of unicellular or -multicellular mycelium differing for each particular species or phase -of development within the single species. The organisms may be sterile -in the soil or form fruiting bodies, consisting of few or myriads -of locally or widely distributed spores. In the process of shaking -the soil-suspension fungi of different organisation or of differing -developmental stages may be broken up and moieties fragmented in -totally different ways or to very different degrees. With protozoa and -bacteria the relation of soil individual to plate colony is direct; -with fungi we do not know what is the soil “individual” nor whether it -is the same for different fungi; nor can we yet profitably discuss any -significant numerical relationship of plate colonies to soil organisms. -Thus Conn[4] has pointed out that the plate count of a fungus indicates -only the ability to produce reproductive bodies and found that the -spores of one colony of _Aspergillus_, if distributed evenly through -a kilogram of soil, could produce the average plate counts obtained -by Waksman. Abundant vegetative growth may, in some species, reduce -or inhibit spore formation, so that of two species the one giving a -lower count might really be much the more important and plentiful -in the soil. Further, the colonies developing in the final plates -represent only a selected few of the fungi present in the soil sample, -the _Basidiomycetes_, and no doubt many other forms, being absent. -In addition, different media differ among themselves in the average -number of colonies developing on the plates, each medium giving, -as it were, its own point of view. Thus, in one experiment carried -out at Rothamsted by Miss Jewson, using the same soil suspension, -twenty plates of Coon’s Agar gave 357 colonies, of Cook’s Agar 246, -of Czapek’s Agar 215, and of Prune Agar 366. Thus if one only used -Coon’s Agar and Prune Agar one would obtain a total of 723 colonies, -whereas the same suspension on Cook’s Agar and Czapek’s Agar would -give only 461, and the calculated numbers of fungi per gram of soil -would be totally different. Further, if a single medium be taken, it -is found that slight alterations in the degree of acidity may make -very considerable differences in the final numbers. Thus Coon’s Agar -acidified to a hydrogen ion concentration of 5·0 gave as the results of -four series the following average numbers of colonies per plate, 17, -23·75, 18, 23. When, however, the medium was acidified to a PH of 4·0 -to 4·3, corresponding averages from three series were 38, 46·3, and -44·8; i.e. the final estimations of numbers of fungi in the soil was -about twice as great. Again, the degree of dilution of soil suspension -used in plating may also be a very serious factor. Thus, if a series of -dilutions be made of 1/80,000, 1/40,000, 1/20,000, 1/10,000, 1/5,000 -and 1/2,500, the average plate numbers should be in the proportions -of 1, 2, 4, 8, 16, and 32 respectively. In an actual experiment, the -following average plate numbers were obtained, 15·4, 32·8, 59·1, 104·0, -150, 224·5, which show a very decided reduction in the higher numbers. -If, however, dilutions of a suspension of spores of a single species be -made, this reduction does not occur. - -These are but three of the very numerous factors involved in the -technique of quantitative estimation, and every single factor may be -the source of errors of similar magnitude, minute fluctuations in the -operations leading to the final platings having very considerable -effect upon the numbers of colonies that develop. - -By critically evaluating each particular factor in the method, and -making statistical correction, it has, however, been found possible to -obtain series of duplicate plates comparing very favourably and thus to -extract certain figures which, whilst not possessing any final value, -have yet a certain general and comparative worth. Thus, 20·0, 18·2, -and 16·8 were obtained as the averages of six plates each, of a soil -suspension divided into three parts, and the individual plate numbers -in all three series were within the range of normal distribution. The -meaning of these numerical estimates in relation to fungi per gram -of soil sample is, however, entirely hypothetical, and to have value -quantitative comparison should only be made between single species -or groups of species closely related physiologically, and where the -technique is standardised. - -[Illustration: FIG. 19.--Monthly Counts of Numbers of Fungi per gramme -of Dry Soil. Broadbalk Plot 2 (Farmyard Manure), Rothamsted. - - X-axis: ~Apr.~ 1921 May Jun. ~Jul.~ Aug. Sep. ~Oct.~ Nov. ~Dec.~ Jan. - 1922 ~Feb.~ Mar. Apr. May ~Jun.~ Jul. Aug. ~Sep.~ ~Oct.~ - - Y-axis: 10.000 per Gramme of Soil] - -No comparative estimations have been made of the number of fungi in -the soils of different regions. There are, however, certain figures -which show that decided seasonal differences exist. Thus, correcting -and averaging certain of Waksman’s results[25] the following numbers -of fungi per gram of soil at 4 inches deep are obtained; September, -768,000; October, 522,000; November, 310,000; January, 182,000. At -Rothamsted results have been obtained which would appear to mark -a clear seasonal rhythm, corresponding in the time of its maxima -in Autumn and Spring with the periodicities known for many other -ecological communities (Fig. 19). - -The numbers of fungi at various depths in the soil show very clearly -marked differences. The distribution in the top 4-6 inches depending -probably upon the depth of soil, is more or less equal, but there is a -very rapid falling off in numbers, especially between 5-9 inches, until -at 20-30 inches fungi are either very few in number or absent. Thus -Takahashi[22] found 590,000 fungi per gram at a depth of 2 cms. and -only 160,000 at 8 cms. - -TABLE XIII.--INFLUENCE OF SOIL TREATMENT UPON THE NUMBERS OF FUNGI AS -DETERMINED BY THE PLATE METHOD--(AFTER WAKSMAN). - - +--------------------------+---------+-----------------+ - | | |Numbers of Fungi | - | Soil Fertilisation. |Reaction.|per Gram of Soil.| - +--------------------------+---------+-----------------+ - | | P.H. | | - |Minerals only | 5·6 | 37,300 | - |Heavily manured | 5·8 | 73,000 | - |Sodium nitrate | 5·8 | 46,000 | - |Ammonium sulphate | 4·0 | 110,000 | - |Minerals and lime | 6·6 | 26,200 | - |Ammonium sulphate and lime| 6·2 | 39,100 | - +--------------------------+---------+-----------------+ - -The type of soil and its treatment exercise a great influence over -the number of fungi present. Fischer[6] found that farmyard manure -increased the number of fungi in uncultivated “Hochmoor,” cultivated -“Grunlandmoor,” and a clay soil by two, three, and five times -respectively. Waksman’s results[25] indicate that the more fertile -soils contain more fungi, both in number and species, than the less -fertile ones, and if one averages his results, the following figures -are obtained: garden soil, 525,000 per gram; orchard soil, 250,000; -meadow soil, 750,000; and forest soil, 151,000. Recently Waksman[25_e_] -has found that manure and acid fertilisers increase the numbers of -fungi in the soil, whereas the addition of lime decreases them (Table -XIII.). - -Jones and Murdock[10] examined surface and sub-surface samples of -forty-six soils representing seventeen soil types in eastern Ontario. -Molds were fairly uniform in numbers in all soils except a sandy clay -loam and sandy clay shale, in which they were absent. - -It has also frequently been pointed out that acid and water-logged -soils are richer in fungus content than normal agricultural soils. -On the other hand, Brown and Halversen[2] found, examining six plots -receiving different treatment and studied through a complete year, that -the numbers of fungi were unaffected by moisture, temperature, or soil -treatment. Against this, however, must be set the work of Coleman[3] -who studied the activities of fungi in sterile soils and found such -factors as temperature, aeration and food supply to exercise a deciding -control. - -Investigations at Rothamsted show that Broadbalk plot 13, receiving -double ammonium salts, superphosphate and sulphate of potash and -yielding 31 bushels per acre, and plot 2, receiving farmyard manure and -yielding 35·2 bushels, contain approximately equal numbers of fungi. -This figure is about half as high again as that for plot 3, which -is unmanured and yields 12·6 bushels, plot 10, with double ammonium -salts alone and yielding 20 bushels, and plot 11, with double ammonium -salts and superphosphate and yielding 22·9 bushels per acre. A primary -factor, however, in all considerations such as these is the equality -of distribution of fungi laterally in any particular soil. There are -probably few soils so homogeneous as the Broadbalk plots at Rothamsted, -and on plot 2 (farmyard manure since 1852) samples taken from the lower -and upper ends and the middle region gave average numbers of colonies -per plate of 24, 23, and 25 respectively. On the other hand, soil -samples taken only a few yards apart in the middle region of the plot -gave average plate counts of 33·7 and 56·8. - - -CONCLUSION. - -Surveying generally the field covered in this chapter, one can only -be impressed with the fragmentary character of our knowledge and with -the fact that, owing to the selective nature of the technique, the -data we possess, if assumed to be representative, give an entirely -partial and erroneous picture of the soil fungi. From the qualitative -aspect, the chief impediment is the impossibility of obtaining reliable -specific determinations of very many of the soil fungi. Lists of -doubtfully-named forms from particular soils or geographic regions -are useless or a positive evil, and there is imperative need for the -systematising of selected genera by physiological criteria, such as has -been partially done for _Penicillium_, _Fusarium_, and _Aspergillus_. -Furthermore, until a standardised and non-selective technique has been -devised, or a number of standardised selective methods for particular -groups, comparative investigations into specific distribution can -give little of value. This latter criticism is also very applicable -if regard be paid to the quantitative aspect of soil work, for -progress here largely depends upon the elaboration of a standardised -fractionation technique. Every single factor in these methods needs -exact analysis, for each gives opportunity for great error, and each -error is magnified many thousand times in the final results. Much -has been done in this direction at Rothamsted, but more remains to -do. Finally, working with single species in sterilised soil under -standardised conditions, there is fundamental work to be done on the -relation of plate colony to soil “individual.” - - [1] Adametz, I., “Untersuchungen über die niederen Pilze der - Ackerkrume,” Inaug. Diss., Leipzig, 1886. - - [2] Brown, P. E., and Halversen, W. V., “Effect of Seasonal - Conditions and Soil Treatment on Bacteria and Molds in Soil,” Iowa - Agric. Expt. Sta. 1921, Res. Bull., 56. - - [3] Coleman, D. A., “Environmental Factors Influencing the Activity - of Soil Fungi,” Soil Sci., 1916, v., 2. - - [4] Conn, H. J., “The Microscopic Study of Bacteria and Fungi in - Soil,” N.Y. Agric. Expt. Sta., 1918, Bull. 64. - - [5] Dale, E., (_a_) “On the Fungi of the Soil,” Ann. Mycol., 1912, - 10; (_b_) “On the Fungi of the Soil,” Ann. Mycol., 1914, 12. - - [6] Fischer, H., “Bakteriologisch-chemische Untersuchungen; - Bakteriologischen Teil,” Landw. Jahrb., 1909, 38. - - [7] Goddard, H. M., “Can Fungi living in Agricultural Soil Assimilate - Free Nitrogen?” Bot. Gaz., 1913, 56. - - [8] Hagem, O., (_a_) “Untersuchungen über Norwegische Mucorineen - I., Vidensk. Selsk, I.,” Math. Naturw. Klasse, 1907, 7; (_b_) - “Untersuchungen über Norwegische Mucorineen II., Vidensk. Selsk. I.,” - Math. Naturw. Klasse, 1910, 10. - - [9] Jensen, C. N., “Fungus Flora of the Soil,” N.Y. (Cornell) Agric. - Expt. Sta., 1912, Bull. 315. - - [10] Jones, D. H., and Murdock, F. G., “Quantitative and Qualitative - Bacterial Analysis of Soil Samples taken in Fall of 1918,” Soil Sci., - 1919, 8. - - [11] Kopeloff, N., “The Effect of Soil Reaction on Ammonification by - Certain Soil Fungi,” Soil Sci., 1916, 1. - - [12] Lendner, A., “Les Mucorinées de la Suisse,” 1908. - - [13] Manns, S. F., “Fungi of Flax-sick Soil and Flax Seed,” Thesis, - N. Dak. Agric. Expt. Sta., 1903. - - [14] McBeth, I. G., and Scales, F. M., “The Destruction of Cellulose - by Bacteria and Filamentous Fungi,” U.S. Dept. Agric. Bur. Plant - Indust., 1913, Bull. 266. - - [15] McLean, H. C., and Wilson, G. W., “Ammonification Studies with - Soil Fungi,” N.J. Agric. Expt. Sta., 1914, Bull. 270. - - [16] Muntz, A., and Coudon, H., “La fermentation ammoniaque de la - terre,” Compt. Rend. Acad. Sci. (Paris), 1893, 116. - - [17] Oudemans, A. C., and Koning, C.J., “Prodrome d’une flore - mycologique, obtenue par la culture sur gelatin préparée de la terre - humeuse du Spanderswoud, près de Bussum,” Arch. Néerland. Sci. Exact - et Nat., 1902, s. ii., 7. - - [18] Ramann, E., “Bodenkunde,” Berlin, 1905. - - [19] Rathbun, A. E., “The Fungus Flora of Pine Seed Beds,” - Phytopath., 1918, 8. - - [20] Remy, T., “Bodenbakteriologischen Studien,” Centr. f. Bakt., - 1902, ii., 8. - - [21] Sherbakoff, C. D., “Fusaria of Potatoes,” N.Y. (Cornell) Agric. - Expt. Sta., 1915, Mem. 6. - - [22] Takahashi, T., “On the Fungus Flora of the Soil,” Anns. - Phytopath. Soc., Japan, 1919, 1. - - [23] Taylor, M. W., “The Vertical Distribution of _Fusarium_,” - Phytopath., 1917, 7. - - [24] Thom, Ch., “Cultural Studies of Species of Penicillium,” U.S. - Dept. Agric. Bur. Animal Indus., 1910, Bull. 118. - - [25] Waksman, S. A., (_a_) “Soil Fungi and their Activities,” Soil - Sci., 1916, 2; (_b_) “Do Fungi Actually Live in the Soil and Produce - Mycelium?” Science, 1916, 44; (_c_) “Is there any Fungus Flora of - the Soil?” Soil Sci., 1917, 3; (_d_) “The Importance of Mold Action - in the Soil,” Soil Sci., 1918, 6; (_e_) “The Growth of Fungi in the - Soil,” Soil Sci., 1922, xiv. - - [26] Werkenthin, F. C., “Fungus Flora of Texas Soils,” Phytopath., - 1916, 6. - - - - -CHAPTER VIII. - -THE LIFE OF FUNGI IN THE SOIL. - - -In the last chapter fungi were considered as so many specific but -functionless units in the soil. Unless, however, they are regarded -merely as inert spore contaminations from the air, a view which is -now no longer tenable, their very presence implies the existence -of innumerable vital relationships between the organisms and their -environment. From this point of view the studies treated in the -previous chapter are but the necessary first steps to an understanding -of the relation of soil fungi to living plants and of the part played -by them in the soil economy. - - -RELATION OF SOIL FUNGI TO LIVING PLANTS. - -Older classifications of fungi frequently divided these organisms -into four categories--parasites, saprophytes, facultative parasites, -and facultative saprophytes, but the further mycological studies are -carried the more clearly it is seen that these groups are entirely -artificial. There are probably few fungi that cannot, under particular -conditions, invade living tissues, and it only seems a question of -time before at all events the vast majority of fungi will be grown on -synthetic media in the laboratory. From our present point of view the -importance of this lies in the fact that fungi living saprophytically -in the soil may, given the right conditions or the presence of some -particular host plant, become parasites or symbionts, and conversely -well-known pathogens may live a saprophytic existence. Thus Cucumber -Leaf Spot is caused by _Colletotrichum oligochætum_, and Bewley[3] has -repeatedly isolated this fungus from glasshouse manure and refuse of -various kinds. In his early studies, Butler[13] isolated many parasitic -species of _Pythium_ from Indian soils, and the presence of _P. de -Baryanum_ as a soil saprophyte has been confirmed by Bussey, Peters, -and Ulrich.[11] De Bruyn[17] has recently found that most species of -_Phytophthora_, including _P. erythroseptica_ and _P. infestans_ may -live as saprophytes in the soil, whilst Pratt[53] has isolated from -virgin lands and desert soils various fungi, which cause disease in -potatoes. In 1912 Jensen[29] gave a list of twenty-three “facultative -parasites” isolated from soil, and these are but a moiety of those -which could be listed to-day. - -Furthermore, it was shown by Frank[24] many decades ago that forest -humus is not merely a mass of the remains of animals and plants, -but that a considerable part of its organic substance is made up of -fungus hyphæ, which ramify and penetrate in all directions. Evidence -is rapidly accumulating that this is also true of most other soils -containing organic matter. It is well known that many of the higher -plants live in symbiotic or commensal relationship with these humus -fungi, which are present in the host tissues as mycorrhiza, and further -studies only serve to show the widespread and fundamental nature -of this relationship. Thus many _Basidiomycetes_[50] (species of -_Tricholoma_, _Russula_, _Cortinarius_, _Boletus_, _Elaphomyces_, etc.) -possess a mycorrhizal relationship with various broad leaved trees, -such as beech, hazel, and birch[57] and with various conifers and -certain Ericales. Other Ericales show this relationship with species of -the genus _Phoma_,[62] many orchids, with species of _Rhizoctonia_[2] -(or _Orcheomyces_[10]), whilst _Gastrodia elata_ contains _Armillaria -mellea_.[36] Certain species of _Pteridophyta_ and _Bryophyta_ are -also known to certain mycorrhizal fungi. Of the numerous fungi taking -part in these mycorrhizal relationships, only a small number have yet -been identified, but there is little doubt that perhaps the majority -of these organisms must be regarded as true soil forms.[14],[45] The -mycological flora of the soil thus plays an important part in the life -of many higher forms of vegetation, and this relationship is a very -fruitful field for study. - - -RELATION OF FUNGI TO SOIL PROCESSES. - -The great cycle of changes occurring in the soil whereby organic matter -is gradually transformed and again made available as plant food is -entirely dependent upon micro-organisms. Until a decade ago it was -thought that bacteria were by far the most important group concerned -in the bringing about of these changes, but recent studies have shown -that, in at all events certain arcs of this great organic cycle, -the fungi have, perhaps, an equal part to play. The life of fungi -in the soil may, for our purposes, be considered from three points -of view--their part in the decomposition of carbon compounds, their -nitrogen relationships, and their work in the mineral transformations -of the soil. - - -CARBON RELATIONSHIPS. - -Of primary importance in the carbon relationships of soil fungi is -the part played in the decomposition of the celluloses, which compose -almost all the structural remains of plant tissues. Our first real -knowledge of this subject was given by Van Iterson[28] in 1904 when he -showed the wide extent of cellulose destruction by fungi, and devised -methods whereby fifteen cellulose-decomposing forms, many of which have -since proved to be common soil fungi, were isolated. Three years later -Appel[1] published his account of the genus _Fusarium_, and showed -that many of the species could destroy filter paper. A difficulty was -introduced in 1908 by Schellenberg,[60] who, working with common soil -forms, found that only hemicelluloses and not pure cellulose were -destroyed. This has recently been supported by Otto,[48] but from the -practical point of view the discussion is academic for the amount of -pure cellulose in plants is insignificant. - -In 1913 McBeth and Scales[43] showed that a considerable number -of common soil fungi were most active cellulose destroyers, pure -precipitated cellulose and cotton being readily attacked. This was -supported by McBeth in 1916,[42] whilst Scales[59] has found that -most species of _Penicillium_ and _Aspergillus_ decompose cellulose, -especially where ammonium sulphate is the source of nitrogen. -Waksman[65] tested twenty-two soil fungi and found that eleven -decomposed cellulose rapidly and four slowly, whilst Dascewska,[16] -Waksman,[66],[67] and others have concluded that soil fungi play -a more important part in the decomposition of cellulose and in -“humification” than soil bacteria. Schmitz[61] has recently shown that -cellulose-destroying bacteria play no important part in the decay of -wood under natural conditions. - -In addition to the celluloses, practically all simple and complex -organic carbon compounds are attacked by soil fungi, and in many cases -the decomposition is very rapid.[26] Many _Actinomycetes_, _Aspergilli_ -and _Penicillia_ are active starch splitters, and it is of interest to -note that some of the strongest cellulose decomposers (_Melanconium -sp._, _Trichoderma sp._, and _Fusaria_) secrete little diastase.[66] -The _Mucorales_ apparently do not attack cellulose, but can only -utilise pectin bodies, monosaccharides, and partly disaccharides.[26] -Dox and Neidig[19] have shown that various species of _Aspergillus_ -and _Penicillium_ are able to attack the soil pentosans. Roussy,[58] -Kohshi,[24] Verkade and Söhngen,[64] and many other workers have found -that fats and fatty acids are readily used as food by soil fungi, and -Koch and Oelsner[33] have recently shown that tannins are readily -assimilated. Klöcker,[32] Ritter,[56] and others have shown that the -utilisation of many carbon compounds is to a large extent determined by -the source of nitrogen and its concentration in the pabulum. - -There would seem, therefore, no doubt that the decomposition of -celluloses and other carbon compounds is of primary importance in the -life-activities of soil fungi. - - -NITROGEN RELATIONSHIPS. - -In this section we shall consider the problems of nitrogen fixation and -nitrification, of ammonification, and of the utilisation of nitrogenous -compounds by soil fungi. - -As soil fungi form so large a part of the soil population, the question -of whether they can make use of the free nitrogen of the air is of -primary importance. During the last two decades many investigators have -attempted to solve the problem, often studying allied or identical -species; but if one consults some thirty researches published during -this period, opinion is found to be about equally divided. Even, -however, in those studies where nitrogen fixation has been recorded the -amounts are very slight, usually being below 5 mgrms. per 50 c.c. of -solution, and often being obviously within the limits of experimental -error. Latham,[37] however, working on _Aspergillus niger_, recorded -variations ranging from a nitrogen loss of 42·5 mgrms. to a nitrogen -fixation of 205·1 mgrms. per 50 c.c. of medium. Ternetz[63] found -that different strains of _Phoma radicis_ may fix from 2·5 mgrms. of -nitrogen in the lowest case, to 15·7 mgrms. in the highest per 50 c.c. -of nutrient solution. Duggar and Davis[20] report that _Phoma betæ_ -may fix nitrogen in quantities of 7·75 mgrms. per 50 c.c. of medium. -The latter authors, in a very able critique of the problem, indicate -certain possible sources of error in previous work, and if one examines -the studies in which nitrogen fixation has been recorded in the light -of these criticisms, it is difficult not to think that, with the -exception of the genus _Phoma_, good evidence for nitrogen fixation -by fungi is lacking. _Phoma betæ_ is a common pathogen attacking -beets, whilst _P. radicis_ is a mycorrhizal form inhabiting various -Ericales. Apart from these exact quantitative studies, which have -given a negative verdict, there is a considerable amount of positive -but indirect evidence for nitrogen fixation by mycorrhizal fungi,[55] -and it is very unfortunate that more of these forms have not been -investigated quantitatively. As the evidence stands to-day, one must -conclude that the fungus flora does not play any part in the direct -nitrogen enrichment of the soil. - -Equally obscure is the question of nitrification and denitrification -by soil fungi, but this is the result of a lack of study rather than -of a plethora of indeterminate researches. Direct nitrification or -denitrification has not been established, but the work of Laurent[38] -and a few other workers appears to show that soil fungi can reduce -nitrates to nitrites. - -The second primary nitrogen relationship that we have to consider is -the process of ammonification. The ammonifying power of soil fungi was -first demonstrated by Muntz and Coudon,[46] and by Marchal[40] in 1893, -the former showing that _Mucor racemosus_ and _Fusarium Muntzii_ gave a -larger accumulation of ammonia in soil than any of the bacteria tested; -and the latter that _Aspergillus terricola_, _Cephalothecium roseum_ -and other soil fungi were active ammonifiers, especially in acid -soils. Shibata,[62] Perotti,[49] Hagem,[26] Kappen,[31] Löhnis,[39] -and others, have observed that urea, dicyanamide and cyanamide are -decomposed with the liberation of ammonia; and Hagem[26] has recorded -the same process for peptones, amino acids, and other organic nitrogen -compounds in plant and animal remains in the soil. The latter author -considers soil fungi more important ammonifying agents in the soil than -bacteria, a conclusion in which McLean and Wilson,[44] and perhaps most -later workers concur. McLean and Wilson[44] found large differences in -the ammonifying powers of various soil fungi, the _Moniliaceæ_ being -the strongest ammonifiers, the _Aspergillaceæ_ the weakest. Generic and -specific differences have been confirmed by Coleman,[15] Waksman,[67] -and other authors. Waksman and Cook[70] suggested that such variations -may be due, not to innate differences in the metabolic activities of -the several organisms, but to differences in reproductive times, and -that there might be some relationship between sporogeny and the ability -to accumulate nitrogen. Kopeloff[35] has carried out experiments on -the inoculation of sterilised soil with known quantities of spores -and found that, although the amount of ammonia accumulated increased -with the number of spores the proportion was not direct but modified -by the food supply. After the first five days’ growth, the rate of -ammonia production varied markedly in a two-day rhythm which seemed -to be due to the metabolism of the fungus rather than to recurrent -stages of spore formation and germination in the life history. The -amount of ammonia liberated has been shown by recent work[66] to depend -upon the available sources of carbon and nitrogen. In the absence of -a carbohydrate supply the protein is attacked both for carbon and -nitrogen, and since more of the former is required much ammonia is -liberated. In addition, however, to the carbon and nitrogen control, -the process of ammonification by soil fungi is intimately related to -physical conditions. Working with pure cultures, McLean and Wilson,[44] -Coleman,[15] Kopeloff,[35] Waksman and Cook,[70] and other students, -have shown that the amount of ammonia accumulated depends upon such -factors as the presence of phosphates, the period of incubation of the -fungi, aeration, the moisture in the soil, the temperature, the degree -of soil acidity, the type of soil, and so forth. - -That fungi take a very important place as ammonifying agents in the -soil can no longer be doubted, but the question yet remains to be -considered of the balance of profit or loss resulting from their -activities. It has usually been considered that a part of the ammonia -freed is used by the fungi themselves, but that the greater part -is liberated, and so rendered available to nitrifying organisms. -Both Neller[47] and Potter and Snyder[51] found that typical soil -fungi inoculated into sterile soil grew with a vigour approximately -equal to the growth induced by an inoculation of the entire soil -flora. This is largely to be accounted for by the fact that when -soils are sterilised by heat or by certain chemicals, breaking-down -changes occur, and substances are liberated which are peculiarly -favourable to fungus growth. This fact must be borne in mind when -interpreting ammonification and other studies where the method is that -of inoculation of fungi into sterilised soil. In many cases it tends -to nullify any application of the results to normal soils, whilst in -others the conclusions must be accepted with some reserve. In all -cases Potter and Snyder[51] found that fungi caused a diminution in -the amount of nitrates, that the ammonia was not much changed in -amount, and that there was a decrease in the quantities of soluble -non-protein nitrogen. The range of organic and inorganic nitrogenous -compounds utilisable by soil fungi is very great. Ritter[56] has shown -that certain forms can use the nitrogen of “free” nitric acid in the -medium; Ritter,[56] Hagem,[26] and others, that soil fungi can use -ammonia nitrogen equally with nitrate nitrogen, and Ehrenberg[21] -concluded that soil fungi play a more important part in the building -of albuminoids from ammonia than bacteria do. Ehrlich[22] has shown -that various heterocyclic nitrogen compounds and alkaloids can serve as -sources of nitrogen to soil fungi, whilst Ehrlich and Jacobsen[23] have -found that soil fungi can form oxy-acids from amino-acids. Hagem,[26] -Povah,[52] Bokorny,[6],[8] and others, state that for many soil forms -organic nitrogen sources are better than inorganic sources, and that -peptones, amino-acids, urea, and uric acids, etc., are very quickly -utilised by species of _Mucor_, yeasts, and so forth. Butkevitch,[12] -and Dox[18] have recently found that it depends on circumstances which -compounds of protein molecule can be utilised by particular fungi, and -that soil fungi can utilise both amino and amido complexes for the -formation of ammonia. In 1919 Boas[4] showed for _Aspergillus niger_ -that if a number of nitrogenous compounds are available the fungus -absorbs the most highly dissociated. - -In the welter of scattered observations on the utilisation of -nitrogenous compounds, it is difficult to trace any clear issue. That -proteins, amino-acids, and other complex organic compounds are readily -broken down to ammonia by soil fungi is clear, and, on the other hand, -it is also clear that soil fungi utilise extensively ammonia and -nitrates as sources of nitrogen. On which side the balance lies it is -yet impossible to say. - - -MINERAL RELATIONSHIPS. - -Heinze[27] and Hagem[26] have stated that soil fungi make the insoluble -calcium, phosphorus, and magnesium compounds in soil soluble and -available for plant food; and Butkevitch[12] has used _Aspergillus -niger_ in determining the availability of the mineral constituents, -but practically no work has yet been carried out on these problems. -A further matter on which sound evidence is greatly to be desired is -the part played by soil fungi in the oxidation processes of iron and -sulphur. - -A point which may be mentioned here, as it is of some considerable -practical importance, is the large quantity of oxalic, citric, and -other acids formed by certain common soil fungi. Acid formation -is partly dependent upon the species of fungus--even more the -physiological race within the species--and partly upon the substratum, -particularly the source of carbon.[5],[54] It is interesting that as -a group _Actinomycetes_ do not form acids from the carbon source but -alkaline substances from the nitrogen sources.[69] - - -CONTROL OF SOIL FUNGI. - -In the preceding sections an attempt has been made to sketch rapidly -the chief outlines of the widespread relationships of soil fungi and -of the fundamental part that they play in the biochemical changes -occurring in the soil. It will be evident, even from this survey, -that their occurrence is of the utmost agricultural importance, both -when helpful as in mycorrhizal relationships or as agents in making -complex organic materials available as plant food, or when harmful -as when causal agents of disease in plants. It is clear that could -the soil fungi be controlled to human ends by the encouragement of -the useful forms and the elimination of the harmful, a valuable power -would be placed in the hands of the grower of plants. Certain aspects -of this control, the cruder and more destructive perhaps, are already -practicable, whilst the finer and more constructive aspects remain -possibilities of to-morrow. - -Theoretically, the technique of control is selective in that it aims -to determine one or more particular fungi, leaving the remaining flora -untouched. Its highest expression is seen, perhaps, in the utilisation -of pure cultures of mycorrhizal fungi for horticultural purposes, such -as orchid cultivation, but there is no reason why this should not be -done for other purposes on a field scale similar to the way in which -cultures of special strains of the root nodule organisms of legumes -are employed. A second aspect is the direct encouragement of special -components of the fungus flora for particular purposes by selective -feeding. Thus, in a laboratory experiment, McBeth and Scales[43] record -an increase of 2000 times in cellulose-destroying and other soil fungi -by this method. It has been pointed out that soil fungus activities -such as ammonification, proteolysis and carbohydrate decomposition are -controlled by factorial equilibria, and for special purposes it would -seem feasible to weight the balance so that particular activities may -be favoured. A further step in this direction is the controlling of -particular physical conditions so that the activities of certain fungi -may be restricted. Professor L. R. Jones[30] and his colleagues at -Madison have shown the primary importance of the control of the soil -temperature in certain parasitic relationships; the work of Gillespie -and Hurst[25] and later workers has demonstrated that the parasitism -of certain species and strains of _Actinomyces_ upon the potato -is conditioned by definite ranges of soil acidity; and many other -relationships of similar nature are known. Data along such lines are -rapidly accumulating, and in certain cases are already susceptible of -practical application. In other cases, particular soil fungi are less -open to persuasive influences, and more drastic treatment needs to be -adopted. Certain chemicals mixed intimately with the soil increase or -diminish the numbers of particular fungi or groups of fungi; whilst -these organisms may be totally eliminated from the soil by wet or -dry heat for definite periods or by treatment with potent fungicides -such as formaldehyde. Although soil sterilisation and crude treatment -in other ways has been practised for decades, the possibility of a -more delicate control of soil fungi is only now being realised. Its -concrete expression will depend upon the progress that is made in exact -knowledge of the activities of soil fungi under natural and controlled -conditions, of the balance of factors in the environment which controls -any particular function and of the genetic nature of the soil fungi -which occur. Each of these aspects is a fruitful field of study. - - -RELATION TO SOIL FERTILITY. - -From a general survey of the researches that have been carried out on -soil fungi during the past two decades certain issues emerge. It would -seem clear that fungi occupy, perhaps, a primary place as factors in -the decomposition of celluloses, and thus may be the chief agents in -the transformation of plant remains to humus and to soluble compounds -which can be used as food by the nitrogen-fixing bacteria. Furthermore, -soil fungi are very important ammonifiers, but whether the balance -of ammonia freed is utilised by the fungi themselves, or whether it -is made available to nitrifying bacteria is not yet clear. If the -latter is the case, soil fungi play a valuable indirect rôle in the -accumulation of available plant food in the soil. On the other hand, by -utilising nitrates as sources of nitrogen, fungi may play an important -part in the depletion of the nitrogenous food in the soil available to -crop plants. Thirdly, soil fungi apparently take no part in the direct -nitrogen enrichment of the soil. Thus, soil fungi would seem to be the -most important factor in the first half of that great cycle whereby -organic remains become again available as organic food. - -The impression left on one’s mind by the study of the life of fungi -in the soil is of an infinitely complex series of moving equilibria, -the living activities being determined by both biological and -physico-chemical conditions. All these factors play an integral part in -the life of the soil fungi and must be considered if a true picture is -to be drawn. The principal factors may be classified into the following -groups: Most evident, perhaps, are the natures and specificities of -the fungi and the relative composition of the fungus flora. Equally -important, however, are the quantity and quality of the foods available -and the non-biological environment which results from the complex -series of physical and chemical changes occurring in the soil causally -independent of the organisms present, which interacts with the equally -vast series of changes resulting from fungus activities. Finally, one -must consider the interacting biological environment of surface animals -and plants and the microscopic fauna and flora. The complexities are -such that only the application of Baconian principles can unravel -them. A beginning has been made in the study of pure cultures of soil -fungi on synthetic media, and much valuable data have accrued, but -it is obviously not possible to apply directly to soil the results -obtained in such work. They remain possibilities; in certain cases -probabilities, but nothing more. A further step, one already taken -and of great promise, is the investigation of the changes occurring -in sterilised soils inoculated with known quantities of one or more -pure cultures of particular soil fungi. Such intensive study of single -factors in a standardised natural or artificial soil, to which has been -added a pedigreed fungus, is, perhaps, the most fruitful avenue of -progress. In all such work, however, one must bear acutely in mind the -fact that a sterilised soil and, still more, an artificial soil, is a -very different complex from a normal soil, and that results obtained -from the inoculation of such soils are not applicable directly in the -elucidation of ordinary soil processes. At present there is no method -known of completely sterilising a soil which does not destroy the -original physico-chemical balance. It is evident that the complexities -are such that chemist, physicist, and biologist must all co-operate -if the significance of the processes is to be understood, and a solid -foundation laid for future progress and for practical application. - - [1] Appel, O., “Untersuchungen über die Gattung _Fusarium_,” Mitt. - Biol. Reichanst. Land- u. Forstw., 1907, 4. - - [2] Bernard, N., “L’évolution dans la symbiose. Les Orchidées et - leurs Champignons commensaux,” Ann. Sci. Nat. (Bot.), Ser. 9, 1909, 9. - - [3] Bewley, W. F., “Anthracnose of the cucumber under glass,” Journ. - Min. Agric., 1922, xxix. - - [4] Boas, F., “Die Bildung löslicher Stärke im elektiven - Stickstoff-Stoffwechsel,” Ber. deut. bot. Ges., 1919, 37. - - [5] Boas, F., und Leberle, H., “Untersuchungen über Säurenbildung bei - Pilzen und Hefen II.,” Biochem. Ztschr., 1918, 92. - - [6] Bokorny, T., “Benzene derivatives as sources of nourishment,” - Zentr. Physiol., 1917, 32. - - [7] Bokorny, T., “Sugar fermentation and assimilation,” Allg. Brau. - Hopfen Zeit., 1917, 57. - - [8] Bokorny, T., “Verhaltung einiger organischer Verbindungen in der - lebenden Zelle,” Pflügers Archiv., 1917, 168. - - [9] Brown, P. E., “Mould action in soils,” Science, 1917, 46. - - [10] Burgeff, H., “Die Wurzelpilze der Orchideen,” Jena. 1909. - - [11] Bussey, W., Peters, L., and Ulrich, P., “Ueber das Vorkommen - von Wurzelbranderregern im Boden,” Arb. Kais. Biol. Anst. Land- u. - Forstw., 1911, 8. - - [12] Butkevitch, V. S., “Ammonia as a product of protein - transformations caused by mould fungi, and the conditions of its - formation,” Recueil d’articles dedié au Prof. C. Timiriazeff, 1916. - - [13] Butler, E. J., “An account of the genus _Pythium_ and some - _Chytridiaceæ_,” Mem. Dept. Agr. India, 1907, Bot. Ser. 5, 1. - - [14] Christoph, H., “Untersuchungen über die mykotrophen Verhältnisse - der Ericales und die Keimung von Pirolaceen,” Beihefte Bot. Centr., - 1921, 28. - - [15] Coleman, D. A., “Environmental factors influencing the activity - of soil fungi,” Soil Sci., 1916, 2. - - [16] Dascewska, W., “Étude sur la désagrégation de la cellulose dans - la terre de bruyère et la tourbe,” Univ. Genève, Inst. Bot., 1913, S. - 8. - - [17] De Bruyn, H. L. G., “The saprophytic life of _Phytophthora_ in - the soil,” Meded. v. d. Landbouwhoogeschool Wageningen, 1922, xxiv. - - [18] Dox, A. W., “Amino acids and micro-organisms,” Proc. Iowa Acad. - Sci., 1917, 24. - - [19] Dox, A. W., and Neidig, R. E., “Pentosans in lower fungi,” - Journ. Biol. Chem., 1911, 9. - - [20] Duggar, B. M., and Davis, A. R., “Studies in the physiology of - the fungi. (I.) Nitrogen fixation,” Ann. Mo. Bot. Gard., 1916, 3. - - [21] Ehrenberg, P., “Die Bewegung des Ammoniakstickstoffs in der - Natur,” Mitt. Landw. Inst., Breslau, 1907, 4. - - [22] Ehrlich, F., “Yeasts, moulds, and heterocyclic nitrogen - compounds and alkaloids,” Biochem. Ztschr., 1917, 79. - - [23] Ehrlich, F., and Jacobsen, K. A., “Über die Umwandlung von - Aminosäuren in Oxysäuren durch Schimmelpilze,” Ber. Deut. Chem. - Gesell., 1911, 44. - - [24] Frank, B., “Ueber die auf Wurzelsymbiose beruhende Ernährung - gewisser Bäume durch unterirdische Pilze,” Ber. d. Deut. Bot. - Gesell., 1885, 3. - - [25] Gillespie, L. J., and Hurst, L. A., “Hydrogen-ion - concentration--soil type--common potato scab,” Soil Sci., 1918, 6. - - [26] Hagem, O., “Untersuchungen über Norwegische Mucorineen,” - Vidensk. Selsk. I., Math. Naturw. Klasse, 1910, 7. - - [27] Heinze, B. H., “Sind Pilze imstande den elementaren Stickstoff - der Luft zu verarbeiten und den Boden an Gesamtstickstoff - anzureichen,” Ann. Mycol., 1906, 4. - - [28] Van Iterson, C., “Die Zersetzung von Cellulose durch Aërobe - Mikroorganismen,” Centr. f. Bakt., 1904, ii, 11. - - [29] Jensen, C. N., “Fungous flora of the soil,” Agric. Expt. Sta. - Cornell, Bull. 1912, 315. - - [30] Jones, L. R., “Experimental work on the relation of soil - temperature to disease in plants,” Trans. Wisc. Acad. Sci., 1922, 20. - - [31] Kappen, H., “Ueber die Zersetzung des Cyanamids durch Pilze,” - Centr. f. Bakt., 1910, ii, 26. - - [32] Klöcker, A., “Contribution à la connaissance de la faculté - assimilatrice de douze espèces de levure vis-à-vis de quatre Sucres,” - Compt. Rend. Trav. Lab., Carlsberg, 1919, 14. - - [33] Koch, A., und Oelsner, A., “Einfluss von Fichtenharz und Tannin - auf den Stickstoffhaushalt des Bodens und seiner physikalischen - Eigenschaften,” Centr. f. Bakt., 1916, ii, 45. - - [34] Kohshi, O., “Ueber die fettzehrenden Wirkungen der Schimmelpilze - nebst dem Verhalten des Organfettes gegen Fäulnis,” Biochem. Ztschr., - 1911, 31. - - [35] Kopeloff, N., “The inoculation and incubation of soil fungi,” - Soil Sci., 1916, 1. - - [36] Kusano, S., “_Gastrodia elata_ and its symbiotic association - with _Armillaria mellea_,” Journ. Coll. Agric., Imp. Univ., Tokyo, - 1911, iv. - - [37] Latham, M. E., “Nitrogen assimilation of _Sterigmatocystis - niger_ and the effect of chemical stimulation,” Torrey Bot. Club, - Bull. 1909, 36. - - [38] Laurent, “Les reduction des nitrates en nitrites par les graines - et les tubercles,” Bull. Acad. Roy. Sci. Belg., 1890, 20. - - [39] Löhnis, F., “Ammonification of cyanamid,” Ztschr. f. - Gärungsphysiol., 1914, v. - - [40] Marchal, E., “Sur la production de l’ammoniaque dans le sol par - les microbes,” Bull. Acad. Roy. Sci. Belg., 1893, 25. - - [41] Mazé, P., Vila et Lemoigne, “Transformation de la cyanamide en - urée par les microbes du sol,” Compt. Rend. Acad. Sci., Paris, 1919, - 169. - - [42] McBeth, I. G., “Studies on the decomposition of cellulose in - soils,” Soil Sci., 1916, I. - - [43] McBeth, I. G., and Scales, F. M., “The destruction of cellulose - by bacteria and filamentous fungi,” U.S. Dept. Agric, Bur. Pl. Ind., - 1913, Bull. 266. - - [44] McLean, H. C, and Wilson, G. W., “Ammonification studies with - soil fungi,” New Jersey Agric. Expt. Sta., 1914, Bull. 270. - - [45] Melin, E., “Ueber die mykorrhizenpilze von _Pinus silvestris_ - (L.) und _Picea abies_ (L.), Karst.” Svensk. Botan. Tidskr., 1921, xv. - - [46] Muntz, A., and Coudon, H., “La fermentation ammoniaque de la - terre,” Compt. Rend. Acad. Sci., Paris, 1893, 116. - - [47] Neller, J. R., “Studies on the Correlation between the - production of carbon dioxide and the accumulation of ammonia by soil - organisms,” Soil Sci., 1918, 5. - - [48] Otto, H, “Untersuchungen über die Auflösung von Zellulosen und - Zellwänden durch Pilze,” Dissert., Berlin, 1916. - - [49] Perotti, B., “Uber das physiologische Verhalten des Dicyanamides - mit Rücksicht auf seinen Wert als Düngemittel,” Centr. f. Bakt., - 1907, ii, 18. - - [50] Peyronel, B., “Nuovi casa di rapporti micorizici tra - Basidiomiceti e Fanerogame arboree,” Bull. Soc. Bot. Ital., 1922. - - [51] Potter, R. S., and Snyder, R. S., “The production of carbon - dioxide by moulds inoculated into sterile soil,” Soil Sci., 1918, 5. - - [52] Povah, A. H. W., “A critical study of certain species of - _Mucor_,” Bull. Torrey Bot. Club, 1917, 44. - - [53] Pratt, O. A., “Soil fungi in relation to diseases of the Irish - potato in Southern Idaho,” Journ. Agric. Res., 1918, 13. - - [54] Raistrick, H., and Clark, A. B., “On the mechanism of oxalic - acid formation by _Aspergillus niger_,” Biochem. Journ., 1919, 13. - - [55] Rayner, M. C., “Nitrogen fixation in Ericaceae,” Bot. Gaz., - 1922, 73. - - [56] Ritter, G. E., “Contributions to the physiology of mould fungi,” - Voronege, 1916. - - [57] Rosseels, E., “L’influence des microorganismes sur la croissance - des végétaux supérieurs,” Bull. Soc. Centrale Forest. Belg., 1916, 23. - - [58] Roussy, A., “Sur la vie des champignons en milieux Gras,” Compt. - Rend. Acad. Sci., Paris, 1909, 149. - - [59] Scales, F. M., “The Enzymes of _Aspergillus terricola_,” Journ. - Biol. Chem., 1914, 19. - - [60] Schellenberg, H. C., “Untersuchungen über das Verhalten einiger - Pilze gegen Hemizellulosen,” Flora, 1908, 98. - - [61] Schmitz, H., “The relation of bacteria to cellulose fermentation - induced by fungi with special reference to the decay of wood,” Ann. - Mo. Bot. Gard., 1919, vi. - - [62] Shibata, K., “Uber das Vorkommen vom Amide spaltenden Enzymen - bei Pilzen,” Beitr. Chem. Physiol. u. Path., 1904, 5. - - [63] Ternetz, C., “Über die Assimilation des atmosphärischen - Stickstoffs durch Pilze,” Jahrb. f. wiss. Bot., 1907, 44. - - [64] Verkade, P. E., and Söhngen, N. L., “Attackability of cis- and - trans-isomeric unsaturated acids by moulds,” Centr. f. Bakt., 1920, - ii, 50. - - [65] Waksman, S. A., “Soil fungi and their activities,” Soil Sci., - 1916, 2. - - [66] Waksman, S. A., “The influence of available carbohydrate upon - ammonia accumulation by micro-organisms,” Journ. Amer. Chem. Soc., - 1917, 39. - - [67] Waksman, S. A., “Proteolytic enzymes of soil fungi and - _Actinomycetes_,” Journ. Bact., 1918, 3. - - [68] Waksman, S. A., “On the metabolism of _Actinomycetes_,” Proc. - Soc. Amer. Bact. Abstract Bact., 1919, 3. - - [69] Waksman, S. A., “The influence of soil reaction upon the growth - of _Actinomycetes_ causing potato scab,” Soil Sci., 1922, xiv. - - [70] Waksman, S. A., and Cook, R. C., “Incubation studies with soil - fungi,” Soil Sci., 1916, 1. - - - - -CHAPTER IX. - -THE INVERTEBRATE FAUNA OF THE SOIL (OTHER THAN PROTOZOA). - - -The micro-organisms of the soil have been fully discussed in the -preceding chapters of this volume. There now remains to be considered -the fauna of invertebrate animals, other than protozoa, which inhabit -that same medium. In the first place, it is necessary to define what -groups of invertebrate animals are to be regarded as coming under the -category of soil organisms. The latter expression has rather a wide -application and, for the present purpose, is held to mean any organism -of its kind which, in some stage or stages of its life-cycle, lives -on or below the surface of the soil. It will be obvious that, with so -comprehensive a definition, the intimacy of the association of these -animals with the soil will vary within very wide limits. Some animals -pass their whole life-cycle in the soil; others are only present during -a limited phase, and not necessarily in a trophic condition, but since -their occurrence is constant, they cannot be entirely omitted from -consideration. - -Unlike the groups of organisms which have been dealt with in the -foregoing pages, the invertebrates of the soil do not admit, as a rule, -of investigation in culture media. It is, in consequence, much more -difficult to achieve in the laboratory the same control over their -environmental conditions. This fact in itself largely explains why -the interpretations of field observations in animal ecology have not -usually been subjected to the test of laboratory experimentation. The -study of animal ecology, in so far as the denizens of the soil are -concerned, is of very recent birth. It has not, as yet, passed the -preliminary stage of cataloguing empirical data, and much spade work -will be necessary before the various factors controlling the phenomena -actually observed are understood. - -Owing to the paucity of information available, this chapter is -essentially based upon observations conducted at Rothamsted. Its object -is not so much to attempt to evaluate the invertebrate fauna of the -soil, as to suggest a line of ecological work demanding investigation -on land of many different types. - - -METHOD OF INVESTIGATING THE SOIL FAUNA. - -The method adopted at Rothamsted consists in taking weekly soil samples -from a given area for a period of twelve months. Each sample is a cube -of soil, with a side dimension of nine inches, and a total content -of 729 cubic inches. The samples are taken by means of an apparatus -consisting of four iron plates, which are driven into the ground down -to the required depth so as to form a kind of box, which encloses a -cube of soil (_vide_ Morris, 1922 A). The latter is then removed in -layers, each layer being transferred to a separate bag for the purpose. -When the complete sample has been extracted, there are five bags -containing layers of soil taken from the surface to a depth of 1″, from -1″ to 3″, from 3″ to 5″, from 5″ to 7″, and 7″ to 9″ respectively. -Below a depth of 9″ no samples have been taken. - -The sample obtained in this manner may be gradually worked into small -fragments by hand, and examined whenever necessary under a binocular -microscope for the smaller organisms present. This procedure, however, -is very tedious and has been replaced by the use of an apparatus -consisting of a series of three sieves, with meshes of decreasing size -(_vide_ Morris, 1922). The soil is washed through these sieves by means -of a stream of water, and the meshes of the final strainer are small -enough to retain all except the most minute organisms present, while at -the same time they allow the finest soil particles to be carried away. -When desirable, the effluent can be passed through a bag or sieve of -bolting silk, in order to collect such organisms that may have passed -through the third sieve. - -In addition to the actual taking and examination of the samples, a -botanical survey of the area under investigation is made; chemical -and mechanical analyses of the soil are also required. It is further -necessary to take soil temperature readings, to determine the moisture -content of the samples taken, and the amount of organic matter which -they contain. - - -GROUPS OF INVERTEBRATA REPRESENTED IN THE SOIL. - -The various groups of invertebrates represented in the soil may be -briefly referred to in zoological order. - -_Nematoda._--The Nematoda or thread-worms are chiefly animal parasites, -nevertheless they usually lead an independent existence in the soil -in certain stages of their development. The numerous small species -belonging to the family _Anguillididæ_, or eel-worms, form a definite -constituent of the soil fauna; they are generally free-living and -non-parasitic. Certain members of this family, however, are enemies of -cultivated plants. - -_Annelida._--Terrestrial Annelida are almost entirely confined to the -order _Oligochæta_, the majority of which are earthworms (_Terricolæ_), -whose whole life-cycle is passed within the confines of the soil. -The small white worms of the family _Enchytræidæ_ belong to the -aquatic section (_Limicolæ_) of the order, but they have various -representatives which are abundant in damp soil containing organic -matter. - -_Mollusca._--The terrestrial Mollusca are included in the sub-order -_Pulmonata_ of the _Gastropoda_. These organisms, which include the -snails (_Helicidæ_) and slugs (_Limacidæ_), regularly deposit their -eggs in moist earth. Slugs adopt the soil as a frequent habitat, only -leaving it for feeding purposes in the presence of sufficient moisture. -They are frequent consumers of vegetation, with the exception of -_Testacella_, which is carnivorous. - -_Crustacea._--The few species of Crustacea inhabiting the soil belong -to the order _Isopoda_, family _Oniscidæ_, which are popularly referred -to as “woodlice,” “slaters,” etc. - -_Myriapoda._--The _Diplopoda_ or millipedes include enemies of -various crops and are common denizens of the soil. The _Chilopoda_ or -centipedes are usually less abundant and are carnivorous. The minute -_Symphyla_ are often evident but are of minor importance. - -_Insecta._--Insects form the dominant element in the invertebrate -fauna. Phytophagous species devour the subterranean parts of plants, -and notable examples are afforded by the larvæ of _Melolontha_, -_Agriotes_ and _Tipula_. Saprophagous forms are abundantly represented -by the _Collembola_, and by numerous larval _Diptera_ and _Coleoptera_. -Predaceous species preying upon other members of the soil fauna -are exemplified by the _Carabidæ_ and many larval _Diptera_. -Parasitic species pass their larval stages on or within the bodies -of other organisms. The groups of _Hymenoptera_, and the dipterous -family _Tachinidæ_, which exhibit this habit, constitute, along -with predaceous forms, one of the most important natural agencies -controlling the multiplication of insect life. There are also insects -(ants, and other of the aculeate _Hymenoptera_) which utilize the -soil as a suitable medium wherein to construct their habitations or -brood chambers, without necessarily deriving their food from the soil. -Lastly, there are many insects, notably _Lepidoptera_, which only -resort to the soil for the purpose of undergoing pupation. The insect -fauna is, therefore, a closely inter-connected biological complex; for -a discussion and an enumeration of its representatives reference may be -made to papers by Cameron (1913, 1917), and Morris (1921, 1922 a). - -_Arachnida._--The two principal classes represented in the soil are the -_Areinida_, or spiders, and the _Acarina_, or mites, and ticks. The -_Areinida_, which are well-known to be carnivorous, are an unimportant -constituent of the fauna. _Acarina_, on the other hand, are abundant, -and exhibit a wide range of feeding habits; most of the soil forms are -probably carnivorous, and either free-living or parasitic. - - -NUMBER OF ORGANISMS PRESENT AND THEIR DISTRIBUTION IN DEPTH. - -In computing the number of invertebrates normally present in a given -type of soil, the method adopted consists of making individual counts -of all such organisms as occur in each sample of a series taken over -a period of twelve months. This method considerably reduces errors -due to season and to the possible deviation of one or more samples -from the average. If the total number of these organisms is known for -the samples taken, it becomes a simple procedure to arrive at their -approximate numbers per acre. - -TABLE XIV. - -(Based on Morris, 1922 A.) - - +------------------------------+---------------+-------------+ - | |Unmanured Plot.|Manured Plot.| - +------------------------------+---------------+-------------+ - |Insects | 2,474,700 | 7,727,300 | - +------------------------------+---------------+-------------+ - |Larger Nematoda and Oligochæta| | | - | Limicolæ | 794,600 | 3,600,400 | - +------------------------------+---------------+-------------+ - |Myriapoda-- | | | - | Diplopoda | 596,000 | 1,367,000 | - | Chilopoda | 215,400 | 208,700 | - | Symphyla | 64,000 | 215,500 | - | +---------------+-------------+ - | Total | 875,400 | 1,791,200 | - +------------------------------+---------------+-------------+ - |Oligochæta (Terricolæ) | 457,900 | 1,010,100 | - +------------------------------+---------------+-------------+ - |Arachnida-- | | | - | Acarina | 215,400 | 531,900 | - | Areinida | 20,200 | 20,200 | - | +---------------+-------------+ - | Total | 235,600 | 552,100 | - +------------------------------+---------------+-------------+ - |Crustacea (Isopoda) | 33,700 | 80,800 | - +------------------------------+---------------+-------------+ - |Mollusca (Pulmonata) | 13,500 | 33,700 | - +------------------------------+---------------+-------------+ - | Total Invertebrata | 4,885,400 | 14,795,600 | - +------------------------------+---------------+-------------+ - -[Illustration: FIG. 20.--Distribution in depth of the more important -groups of soil invertebrates in the manured and unmanured (or control) -plots at Rothamsted. (From Morris, “Annals of Applied Biology,” vol. -ix., nos. 3 and 4, Cambridge University Press.)] - -Table XIV. represents a numerical estimate of the invertebrate fauna of -two plots of arable land at Rothamsted. The soil is clay with flints -overlying chalk, and the land in question has been devoted for eighty -years to continuous cropping with wheat; one plot (No. 3) receives an -annual dressing of farmyard manure at the rate of 14 tons per acre, and -the other plot (No. 2) receives no natural or artificial fertilizer. -The significant feature in a comparison of the fauna of the two plots -is the great numerical increase in organisms due to the addition of -manure. From the point of view of distribution in depth, Fig. 20 -clearly demonstrates that the bulk of the fauna is concentrated in the -first three inches of the soil. With the exception of the _Acarina_ it -is evident that the limits of vertical distribution extend below the -depth of nine inches investigated, although the numbers of organisms -likely to be present are inconsiderable. The _Oligochæta_, or true -earthworms, occur in Rothamsted soil in numbers very much in excess of -the figures given by Darwin, who quoted observations by Hensen. The -latter authority calculated that there were 53,767 earthworms in an -acre of garden soil, and estimated that about half that number would -be present in an acre of corn field. In the Rothamsted investigations -their numbers exceeded Hensen’s estimate over 16 times in unmanured -land, and over 36 times in manured land. - -In an area of pasture-land in Cheshire few insects occurred below -a depth of 2 inches, and they reached the limit of their vertical -distribution at or near 6 inches. Their number (3,586,000 per acre) -is considerably in excess of that present in unmanured arable land at -Rothamsted. - -[Illustration: - - 1, _Collembola_; 2, _Thysanura_; 3, _Orthoptera_; 4, _Thysanoptera_; - 5, _Hemiptera_; 6, _Lepidoptera_; 7, _Coleoptera_; 8, _Diptera_; 9, - _Hymenoptera_. - -FIG. 21.--Number of individuals in the different orders of insects in -manured and unmanured arable land at Rothamsted. (From Morris, “Annals -of Applied Biology,” vol. ix., nos. 3 and 4, Cambridge University -Press.)] - - -DOMINANCE OF CERTAIN SPECIES AND GROUPS. - -In Fig. 21 a numerical analysis is given of the different orders -of insects represented in Rothamsted soil. The ascendency of the -_Hymenoptera_ and _Collembola_ is almost entirely due to the occurrence -of three species in large numbers, viz., the ant _Myrmica lævinodis_ -and the _Collembola_, _Onychiurus ambulans_ and _O. fimetarius_. In -the unmanured plot these two _Collembola_ constituted 13 per cent. of -the insects and the species of ant accounted for nearly 28 per cent. -In the manured plot they amounted respectively to 27 per cent. and 36 -per cent. of the insects present. Next in order of numerical ascendency -are larval _Diptera_, mainly belonging to the families _Cecidomyidæ_, -_Chironomidæ_, and _Mycetophilidæ_. The _Diptera_ are followed by -the _Coleoptera_, whose most abundant representatives are larval -_Elateridæ_ (wireworms). - -[Illustration: - - 1, _Collembola_; 2, _Thysanura_; 3, _Orthoptera_; 4, _Thysanoptera_; - 5, _Hemiptera_; 6, _Lepidoptera_; 7, _Coleoptera_; 8, _Diptera_; 9, - _Hymenoptera_; 10, _Diplopoda_; 11, _Chilopoda_; 12, _Areinida_; 13, - _Acarina_. - -FIG. 22.--Number of species of different orders of invertebrates -present in the manured and unmanured (or control) plots at Rothamsted. -(From Morris, “Annals of Applied Biology,” vol. ix., nos. 3 and 4, -Cambridge University Press.)] - -In point of view of number of species present (Fig. 22), _Coleoptera_ -take the front rank; in the unmanured plot they are very closely -approached by _Collembola_ and _Diptera_. - -Passing from the insects, the next assemblage of organisms in point of -number of individuals are the smaller worms. The difficulties attending -the specific identification of these organisms are great, and, in the -present survey, the _Nematodes_ and all the smaller _Oligochætes_ have -not been separated. - -The abundance of the _Myriapoda_ is mainly due to the prevalence of -_Diplopoda_, which are represented by four species. The _Chilopoda_ -almost entirely consist of a single species _Geophilus longicornis_. - -The dominant group of the _Arachnida_ is the _Acarine_ family -_Gamascidæ_, which are represented by about a dozen species. - - +---------+-------------+-------------+------------+--------------+ - | |Phytophagous.|Saprophagous.|Carnivorous.|Heterophagous.| - +---------+-------------+-------------+------------+--------------+ - |Unmanured| | | | | - |plot | 14 | 48 | 13 | 20 | - |Manured | | | | | - |plot | 13 | 58 | 9 | 20 | - +---------+-------------+-------------+------------+--------------+ - - -CLASSIFICATION OF SOIL INVERTEBRATES ACCORDING TO FEEDING HABITS. - -From the point of view of the fauna as a whole, the zoological -classification of the soil invertebrates is only significant when the -various groups are analysed according to the feeding habits of their -members. All animals are directly or indirectly dependent upon plant -life for their nutrition. For the present purpose they are divided -into four categories, and the position of each class of animals in the -scheme is based upon the habits of its chief representatives in the -soil. Definite information on this subject, however, is not always -forthcoming, and it is only possible to achieve approximate estimates. -In the table above the percentages in number of individuals present in -the two plots investigated at Rothamsted are given under each type of -feeding habit. - -It must be borne in mind that these estimates only apply to average -conditions; the outbreak of a plant pest in any one year must naturally -materially alter the proportions given. The phytophagous organisms are -represented by a certain number of the _Insecta_ together with the -pulmonate _Mollusca_. Carnivorous forms which are mainly beneficial -from the agricultural standpoint, include _Insecta_, together with -the _Chilopoda_, many _Acarina_ and the _Areinida_. Saprophagous -forms constitute the dominant element of the soil fauna. More than -30 per cent. of the _Insecta_ exhibit this habit, which is also the -dominant one in the _Oligochæta_, _Symphyla_, and in many of the soil -_Nematodes_. Heterophagous species include all those of somewhat -plastic habits; for the most part they are saprophagous, but, on the -other hand, a considerable proportion of the species attack growing -plants or exhibit both habits. Under this category are included a -certain number of the _Insecta_, the _Diplopoda_, _Isopoda_, and some -_Acarina_. - - -THE INFLUENCE OF ENVIRONMENTAL FACTORS UPON THE INVERTEBRATES OF THE -SOIL. - -Since animals are endowed with powers of independent locomotion: they -are not necessarily tied to their environment to the same extent -that plants are. The investigation of the influence of environmental -factors sooner or later involves a study of the tropisms of the -animals concerned. Until these are adequately understood it is -scarcely possible to arrive at any exact conclusions relative to their -behaviour in the soil. Insects, for example, respond to the stimuli of -various, and often apparently insignificant forces, acting upon their -sensory organs. Such responses are known as chemotropism, phototropism, -hydrotropism, thermotropism, and so forth according to the nature of -the stimuli. Tropisms are automatic and, so far as they distinguish -sensations, are independent of any choice, and consequently of psychic -phenomena. Animal automatism, however, does not present the rigidity -of mechanical automatism. Differential sensibility, vital rhythms, or -periodicity, etc., are other important aspects of animal behaviour. - -The environmental factors, affecting more especially the insect -population of the soil, have been discussed by Cameron (1917) and -Hamilton (1917), and certain broader aspects of animal ecology by -Adams (1915) and Shelford (1912). These factors are so numerous and -so inter-connected, that it is only possible to refer to them briefly -in the space available. As might be expected, soils that are of a -light and open texture are the ones most frequented by soil insects, -nutritional and other factors being equal. Furthermore, it has already -been shown that in arable land insects and other animals penetrate to -a greater depth than in pastures. This fact is primarily due to the -greater looseness of the soil occasioned by agricultural operations, -which ensure at the same time better drainage aeration, and greater -facilities for penetration. Hamilton found that soil insect larvæ are -very sensitive to evaporation, and especially so if the temperature -is 20° C. or over. In their natural habitat the relative humidity of -the air, in moist or wet soil, is not far below saturation, and the -temperature of the soil rarely goes above 20°-23°C., and then only in -exposed, dry, hard soil in which these larvæ do not occur. - -The significance of the rate of evaporation as an environmental -factor was first emphasised by Shelford. According to him the best -and more accurate index of the varying physical conditions affecting -land animals, wholly or in part exposed to the atmosphere, is the -evaporating power of air. By means of a porous cup-atmometer, as -devised by Livingston, Shelford has carried out an important series -of experiments on the reactions of various animals to atmospheres of -different evaporation capacities, and reference should be made to his -text-book. - -The importance of the organic matter present in the soil is well -illustrated in the table on p. 152. The great increase in the number of -insects and other animals is partly due to their direct introduction -along with the manure, and partly to their entry into the soil in -response to chemotropic stimuli exerted by fermentation. Organic matter -influences the fauna in other ways also; it increases the moisture -content of the soil, and it provides many species with an abundance of -food material. Also, the amount of carbon dioxide present in the soil -is partly dependent upon decaying organic matter. Hamilton conducted -experiments on the behaviour of certain soil insects in relation to -varying amounts of carbon dioxide. Although his work is of too limited -a nature to be accepted without reserve, it lends support to the -conclusions of Adams who says: “The animals which thrive in the soil -are likely to be those which tolerate a large amount of carbon dioxide, -and are able to use a relatively small amount of oxygen, at least for -considerable intervals, as when the soil is wet during prolonged rains. -The optimum soil habitat is therefore determined, to a very important -degree, by the proper ratio or balance between the amount of available -oxygen and the amount of carbon dioxide which can be endured without -injury.” - -Little is known concerning the occurrence of ammonia in the soil -atmosphere, but its presence in minute quantities is probably an -important chemotropic factor in relation to saprophagous organisms -which are the largest constituent of the fauna. A great increase in -Dipterous larvæ occurs on the addition of farmyard manure, and this -is noteworthy in the light of Richardson’s experiments (1916), which -indicate that ammonia exercises a marked attraction for _Diptera_, -which spend some part of their existence in animal excrement in some -form or another. - -The nature of the vegetation supported by the soil is of paramount -importance in relation to phytophagous organisms, and examples need -scarcely be instanced of certain species of soil insects being -dependent upon the presence of their specific food plants. - - -THE RELATION OF SOIL INVERTEBRATES TO AGRICULTURE. - -The relation of these organisms to agriculture may be considered from -three points of view: (_a_) their influence upon the soil itself; (_b_) -their relation to the nitrogen cycle; and (_c_), their direct influence -upon economic plants. - -(_a_) The behaviour of earthworms as a factor inducing soil fertility -is discussed by Darwin in his well-known work on the subject, and their -action may be briefly summarised as follows. In feeding habits they -are very largely saprophagous, and consume decaying vegetable matter -including humus, which they swallow, together with large quantities of -soil. Earthworms come to the surface to discharge their fæces (“worm -casts”), and in this process they are continually bringing up some of -the deeper soil to the air. Darwin estimated that earthworms annually -brought to the surface of the soil in their “casts” sufficient earth -to form a layer ·2 inch in depth, or 10 tons per acre. Their action, -along with the atmosphere, are the chief agencies which produce the -uniformity and looseness of texture of the surface soil. By means of -their burrows earthworms facilitate the penetration of air and water -into the soil, while their habit of dragging leaves and other vegetable -material into these burrows increases the organic matter present -below the surface. These facts are generally agreed upon, but it is a -disputed point whether earthworms, by devouring organic matter, aid the -conversion of the latter into plant food more rapidly than takes place -solely through the activities of micro-organisms. - -Soil insects and other arthropods, by their burrowing activities, -are also instrumental in loosening the soil texture and thereby -facilitating soil aeration and the percolation of water. The action of -termites in warmer countries is discussed by Drummond in his “Tropical -Africa,” who compares the rôle of subterranean termites to that of -earthworms. The great abundance of ants renders them also significant -in this same respect, and very few species are direct enemies of the -agriculturist. - -[Illustration: FIG. 23.--Diagram showing the Relation of the Soil -Invertebrata (other than Protozoa) to the Nitrogen Cycle.] - -(_b_) In their relation to the nitrogen cycle (_vide_ p. 174), the -activities of the soil invertebrates may be expressed diagrammatically, -as a side-chain in the process (Fig. 23). The proteins, elaborated by -plants, are utilised as nitrogenous food by the phytophagous animals -present. The waste products of the latter, which contain the nitrogen -not used for growth or the replacement of loss by wear and tear, are -returned to the soil. Here they disintegrate, and are ultimately -converted into ammonium salts, mainly by bacterial action. The dead -bodies of these animals are also broken down by various means, becoming -eventually chemically dissociated and available as plant food. Animal -(and plant) residues serve, however, as food for the large number of -saprophagous invertebrates present in the soil. In this event the -nitrogen contained in such residues becomes “locked up,” as it were, -for the time being in their bodies. Both saprophagous and phytophagous -animals are preyed upon by carnivorous species, but ultimately the -nitrogen is returned to the soil upon the death of those organisms. -The amount present in the bodies of the whole invertebrate fauna has -been calculated by Morris (1922) upon analyses furnished by chemists -at Rothamsted. It is estimated that the fauna of manured land contains -about 7349 grm., or 16·2 lb. of nitrogen per acre, and that of -untreated land, 3490 grm., or 7·5 lb. per acre. These amounts are equal -respectively to the nitrogen content of 103·6 lb. and 48 lb. of nitrate -of soda. - -The primary question affecting agriculture is, whether any notable -loss of nitrogen is occasioned by the presence of these organisms in -the soil. It has been mentioned that their nitrogenous waste material, -and their dead bodies, ultimately undergo disintegration; any loss, -if any, takes place during the latter process. With the more complex -compounds it probably consists in the production of amino-acids and -their subsequent hydrolysis or oxidation. During this process an -appreciable loss of nitrogen in the gaseous form occurs. This loss, -which is discussed on p. 173 would represent the net deficit occasioned -by the incidence of invertebrates in the soil. Against this loss must -be placed the beneficial action of such organisms as earthworms, which, -in all probability, more than counterbalances it. - -(_c_) Many soil insects, on account of their phytophagous habits, are -well-known to be some of the most serious enemies of agriculture. -Certain of these, and also other classes of invertebrates, which are -likewise directly injurious, have been instanced in the earlier pages -of this chapter. Detailed information on this subject will be found in -textbooks of economic zoology, notably the volume by Reh (1913). - - -LITERATURE REFERRED TO. - - ADAMS, C. C., “An Ecological Study of Prairie and Forest - Invertebrates,” Bull. Illin. St. Lab. Nat. Hist., 1915, xi. - - CAMERON, A. E., “General Survey of the Insect Fauna of the Soil,” - Journ. Econ. Biol., 1913, viii. “Insect Association of a Local - Environmental Complex in the District of Holmes Chapel, Cheshire,” - Trans. Roy. Soc. Edin., 1917, lii. - - DARWIN, C., “Vegetable Mould and Earthworms,” London, 1881. - - HAMILTON, C. C., “The Behaviour of some Soil Insects in Gradients of - Evaporating Power of Air, etc.,” Biol. Bull., 1917, xxxii. - - MORRIS, H. M., “Observations on the Insect Fauna of Permanent Pasture - in Cheshire,” Ann. App. Biol., 1921, vii. “On a Method of Separating - Insects and other Arthropods from Soil,” Bull. Entom. Res., 1922, - xiii. “The Insect and Other Invertebrate Fauna of Arable Land at - Rothamsted,” Ann. App. Biol., 1922 A, ix. - - REH, L., In Sorauer’s “Pflanzenkrankheiten,” 1913, iii. - - RICHARDSON, C. H., “The Attraction of Diptera to Ammonia,” Ann. Ent. - Soc. Amer., 1916, ix. - - RUSSELL, E. J., “The Effect of Earthworms on Soil Productiveness,” - Journ. Agric. Sci., 1910, iii. - - SHELFORD, V. E., “Animal Communities in Temperate America,” Chicago. - 1914, “The Importance of the Measure of Evaporation in Economic - Studies of Insects,” Journ. Econ. Entom., 1912, vii. - - - - -CHAPTER X. - -THE CHEMICAL ACTIVITIES OF THE SOIL POPULATION AND THEIR RELATION TO -THE GROWING PLANT. - - -In the preceding chapters it is shown that the soil is normally -inhabited by a very mixed population of organisms, varying in size from -the smallest bacteria up to nematodes and others just visible to the -unaided eye, on to larger animals, and finally earthworms, which can -be readily seen and handled. These organisms all live in the soil, and -therefore must find in it the conditions necessary for their growth. -We have dealt in the first chapter with the supplies of water, air, -and heat, without which life is clearly impossible. Equally necessary -is the source of energy, for the organism requires energy material as -surely as the motor engine requires petrol, and it ceases to function -unless an adequate supply is forthcoming. - -All the energy comes in the first instance from the sun, if we exclude -the unknown but probably small fraction coming from radio-active -elements. But this radiant energy is not utilisable by the soil -population, excepting surface algæ; it has to be transformed into -another kind. So far, chlorophyll is the only known transformer; -it fixes the energy of sunlight and stores it up in bodies like -hemicellulose, sugar, starch, protein, etc. The transformation is -imperfect; even the heaviest yielding crops grown under glass, in -conditions made as favourable as our knowledge permits, utilise only -about 4 per cent. of the total energy available during their period of -growth; in natural conditions not more than 0·4 per cent. is utilised. -Such as it is, however, the energy fixed in the plant represents all, -indeed more than all, that the soil organisms can obtain. - -In the state of Nature, vegetation dies and is left on the soil. Two -things may then happen. It may become drawn into the soil by earthworms -and other agents; the energy supply is thus distributed in the soil -to serve the needs of the varied soil population. This is the normal -case, associated with the normal soil population and the normal flora. -If, however, the mingling agents are absent, the dead vegetation lies -like a mat on the surface of the soil, only partially decomposing, -unsuitable for the growth of most seedlings, and effectually preventing -most of the vegetation below from pushing a way through: thus there -comes to be no vegetation at all, or only a very restricted and special -flora. The soil population becomes also specialised. Peats and acid -grassland afford examples. - -On the neutral grass plots at Rothamsted, the dead vegetation does not -accumulate on the surface but is rapidly decomposed or drawn into the -soil, leaving the surface of the earth bare and free for the growth -of seedlings. On the acid plots dead vegetation remains long on the -surface, blotting out all new growth excepting two or three grasses -which form underground runners capable of penetrating the mat, and -sorrel, the seedling roots of which seem to have the power of boring -through a fibrous layer of this sort. It is possible to remove the -mat entirely by bacterial action alone, if sufficient lime be added -periodically to make the reaction neutral, but failing these repeated -additions the mat persists. - -We shall confine ourselves to the normal case where earthworms bring -the source of energy into the soil. - -Directly the energy is available, it begins to be utilised. Two laws -govern the change. The first is well-known to biologists: it states -that the total energy of the system remains constant and can neither -be increased nor diminished except from outside; in other words, that -energy can be neither created nor destroyed. The second law is less -familiar: it is that energy once transformed to heat by one organism -cannot be used again by another. It is not destroyed; it remains -intact, but is useless to the organism. One cannot have an indefinite -chain of organisms living on each other’s excretory products; there was -a certain quantity of energy in the food eaten by the first, and no -more than this quantity can be got out whether one organism obtains the -whole or whether others share it. - -The outside value for the amount of energy fixed in the soil is -obtainable by combustion of the soil in a calorimeter, but much of -this is not available to the soil organisms. The normal sedimentary -soils of England still contain decomposition products of the débris -of plants and animals originally deposited with them, but in the long -course of ages much of the extractable energy has been utilised. The -soil population is thus dependent on recently grown vegetation, and it -is therefore largely confined to the layer, usually in this country -about 6 inches thick, through which the recently dead vegetation is -distributed. Below this level there may be sufficient air, water, -temperature, etc., but there is insufficient source of energy for any -large population. - -Unfortunately there is no ready means for distinguishing between the -total and the actually available quantity of energy in the soil. But -it is not difficult, by adopting the Rothamsted analytical method, to -ascertain the approximate amount of energy that has been transformed in -a given period. The Rothamsted plots are periodically analysed and a -balance sheet is drawn up showing how much of each constituent has been -added to and removed from the soil in the intervening period. For two -of the Broadbalk plots the results are shown in Tables XV., XVI. - -The dunged plot receives 14 tons farmyard manure per annum, a quantity -in excess of what would usually be given; the unmanured plot, on the -other hand, has received no manure for many years and is abnormally -poor. Normal soils lie somewhere between these limits, but tending -rather to the value for the dunged than for the unmanured plot. It will -be seen that each acre of the dunged land loses on an average 41,000 -calories per day, while each acre of the unmanured land loses on an -average 2700 calories per day. - -TABLE XV.--MATERIAL BALANCE SHEET: BROADBALK SOIL, ROTHAMSTED. - -(LB. PER ACRE PER ANNUM.) - - +-------------------------+-------------+---------------+ - | | Farmyard | No Manure | - | |Manure Added.| Added. | - | +------+------+-------+-------+ - | | C. | N. | C. | N. | - +-------------------------+------+------+-------+-------+ - |Added in farmyard manure| 3600 | 200 | nil | nil | - |Added in stubble | 300 | 3 | 100 | 1 | - +-------------------------+------+------+-------+-------+ - | Total added | 3900 | 203 | 100 | 1 | - |Taken from soil | nil | nil | 200 | nil | - |Stored in soil | 200 | 30 | nil | nil | - +-------------------------+------+------+-------+-------+ - | Lost from soil | 3700 | 170 | 300 | nil[H]| - | Per cent. | 95 | 84 | 100 | nil | - +-------------------------+------+------+-------+-------+ - - Initial C : N ratio in farmyard manure, 18 : 1 - - Final C : N ratio in soil, 10 : 1. - - [H] Gain of 6 lb. See p. 173. - -TABLE XVI.--ANNUAL ENERGY CHANGES IN SOIL: BROADBALK. APPROXIMATE -VALUES ONLY. - -MILLIONS OF KILO CALORIES PER ACRE PER ANNUM. - - +---------------------------------+-------------+---------+ - | | Farmyard |No Manure| - | |Manure Added.| Added. | - +---------------------------------+-------------+---------+ - |Added in manure | 14 | nil | - |Added in stubble | 2 | 0·3 | - | +-------------+---------+ - | Total added | 16 | 0·3 | - |Taken from soil | nil | 0·5-1 | - |Stored in soil | 0·5-1 | nil | - | +-------------+---------+ - | Dissipated per annum | 15 | 1 | - +---------------------------------+-------------+---------+ - |Per day: calories | 41,000 | 2700 | - |Equivalent to | 12 men. | ¾ man. | - |The human food grown provides for| 2 men. | ½ man. | - +---------------------------------+-------------+---------+ - -These numbers are interesting when we reflect that the human food -produced on the dunged land yields only 7000 calories per day, from -which it is clear that our agricultural efforts so far provide more -energy for the soil population, for which it was not intended, than for -ourselves. - -The account is not complete; we have omitted all reference to the -oxidation of ammonia and of elements other than carbon. Nature -seems to be in an unexpectedly economical mood in the soil, and all -compounds which can be oxidised with liberation of energy seem to -have corresponding organisms capable of utilising them. Even phenol, -benzene, hydrogen, and marsh gas can all be oxidised and utilised as -energy sources by some of the soil population. - -Even with this remarkable power the soil population has insufficient -energy to satisfy all its possibilities; our present knowledge -indicates that energy supply is, in this country at any rate, the -factor limiting the numbers of the population. Increases in the water -supply or the temperature of the soil produce no consistent effect on -the population, but directly the energy supply is increased the numbers -at once rise. - - -MATERIAL CHANGES. - -These transformations of energy involve transformations of matter. -The original plant residues may be divided roughly into substances -forming the structure of the plant, such as the hemicelluloses, the -pentosans, gums, and the contents of the cell--the protoplasm and the -storage products, protein; in addition, there are smaller quantities of -fats and waxes and other constituents. Some of the easily-decomposable -carbohydrates never reach the soil at all, being broken down by -intracellular respiration or attack of micro-organisms. But much of the -structure material--hemicelluloses, pentosans, etc.--remains. - -Once the plant residues pass through the earthworm bodies they become -completely disintegrated and lose all signs of structure. - -The only visible product so far known is humus, the black sticky -substance characteristic of soil and of manure. Two modes of formation -have been suggested. Carbohydrates, sugars, pentosans, etc., are -known to yield furfuraldehyde or hydroxymethylfurfuraldehyde on -decomposition, and it has been shown at Rothamsted that this readily -condenses to form a humus-like body, if not humus itself. In the -laboratory the reaction is effected in presence of acid, but even -amino-acids suffice. All the necessary conditions occur in the soil, -and humus formation may proceed in this way. - -Some of the structure material--the lignin--contains aromatic ring -groupings. Fischer and Schrader have shown that in alkaline conditions -these ring substances absorb oxygen and form something very like humus. -It is quite possible that humus formation also proceeds in the soil -in this way. Whether the two products are chemically identical is not -known. - -The scheme can be represented thus:-- - - Cell structure material - | - +----------------+----------------+ - | | - Aliphatic Aromatic - (Hemicelluloses, (Lignin, etc., - Pentosans, etc.) in presence of - | oxygen and under - +-------+------------+ aerobic conditions) - | | | - Fatty acids Furfuraldehyde | - | or | - | Hydroxymethylfurfuraldehyde | - | (in presence of acid) | - | · | - | · | - | · | - | · | - | · | - | · | - | · | - | · | - | · | - ↓ ↘ ↓ - Calcium carbonate. Humus. - -The disintegration of the cell and the first stages in the -decomposition of the structure material are almost certainly brought -about by micro-organisms. Whether they complete the process is not -known: purely chemical agencies could easily account for part. - -The decomposition of protein in the soil has not been studied in any -detail. From what is known of the acid hydrolysis and the putrefactive -decompositions, however, it is not difficult to draw up a scheme which, -at any rate, accords with the facts at present known. It is probable -that the protein gives rise to amino-acids, which then break down by -one of the known general reactions. - -Two types of non-nitrogenous products may be expected: The aliphatic -amino-acids give rise to ammonia and fatty acids; these form -calcium salts which break down to calcium carbonate. The aromatic -amino-acids--tyrosin, phenylalanine, etc.--which would account for -about 6 per cent. of the nitrogen of vegetable proteins, would be -expected to give ammonia and phenolic substances. Now phenols are -poisonous to plants and if no method existed for their removal the -accumulation would ultimately render the soil sterile. Matters would be -even worse on cultivated soils, since cows’ urine, which enters into -the composition of farmyard manure and is the chief constituent of -liquid manure, contains, according to Mooser, no less than 0·25 to 0·77 -grams of _p_-cresol per litre,[I] a quantity three to ten times that -present in human urine. Fortunately this contingency never arises, for -the soil contains a remarkable set of organisms capable of decomposing -the phenols and leaving the soil entirely suitable for plant growth. -This affords an interesting case of an organism--in this case the -plant--growing well in a medium in spite of some adverse condition, not -because it is specially adapted to meet this condition, but because -some wholly different agent removes it. - - [I] Mooser, Zeitschrift physiol. Chem., 1909, lxiii., 176. No phenol - was found. It is possible that the _p_-cresol is not entirely derived - from the protein, but that some comes from the glucosides in the - animals’ food. - -Other ring compounds, e.g. pyrrol, arise in smaller quantity in the -decomposition of protein, but their fate in the soil is not known. - -We may summarise the probable changes of the protein as follows:-- - - Protein. - +-------------------+------------------+ - | | | - Aliphatic Aromatic Other - amino-acids amino-acids compounds - | | (Pyrrol, etc.) - +--------+--------+ +--------+--------+ - | | | | - Fatty acids and Ammonia Phenolic - hydroxy acids | compounds - | Nitrite | - | | | - | | | - ↓ ↓ ↓ - Calcium Nitrate CO₂ - carbonate - -It must be admitted that the evidence is indirect. The rate of -oxidation of ammonia by bacteria in the soil is more rapid than the -rate of formation, so that ammonia is practically never found in the -soil in more than minimal amounts (1 or 2 parts per 1,000,000); indeed, -the only evidence of its formation was for a long time the fact that -no compound other than ammonia could be oxidised by the nitrifying -organism. It has, however, since been shown at Rothamsted that ammonia -accumulates in soils in which the nitrifying organism has been killed. - -Nothing is known of the mechanism of the oxidation of ammonia beyond -the fact that it is biological; the reaction is not easily effected -chemically at ordinary temperatures. Possibly the organism assimilates -ammonia at one end of a chain of metabolic processes and excretes -nitrates at the other. Or, the reaction may be simply a straight -oxidation for energy purposes, the ammonia changing to hydroxylamine -and then to nitrous and nitric acids. - -The nitrate does not remain long in the soil. Some is taken up by the -plant and some is washed out from the soil. Part, however, either of -the nitrate itself or of one of its precursors is converted into an -insoluble form: probably it is changed into protein by the action of -micro-organisms; it then goes through the whole process once more. - -These are the general outlines; they present no particular chemical -difficulties. When we come to details, however, there is much that -cannot be understood. - -First of all, there is the slow rate at which complex nitrogen -compounds disappear from the soil in comparison with the rate of -oxidation of the carbon. Thus, in the original plant residues, there -is some forty times as much carbon as nitrogen: before they have been -long in the soil there is only ten times as much carbon as nitrogen; -this seems to be the stable position. What is the reason for this -preferential oxidation of the carbon? No explanation can yet be given. - -[Illustration: FIG. 24. - - X-axis: 1887-8 1890-1 1900-1 1910-11 - - Y-axis: ℔ per acre] - -An equally difficult problem arises in connection with the length of -time the process will continue. Decomposition of the nitrogen compounds -never seems to be complete in the soil; it dribbles on interminably. -In the year 1870 Lawes and Gilbert cut off a block of soil from its -surroundings and undermined it so that the drainage water could be -collected and analysed. The soil has been kept free from vegetation -or addition of nitrogen compounds from that time till now; yet it has -never failed to yield nitrates, and the annual yield falls off only -very slowly (Fig. 24). This same peculiarity is seen in the yield of -crops on unmanured land: it decreases, but very gradually; even after -eighty years the process is far from complete, and there is no sign -that it will ever come to an end. - -TABLE XVII.--APPROXIMATE LOSS OF NITROGEN FROM CULTIVATED SOILS: -BROADBALK WHEAT FIELD, ROTHAMSTED, FORTY-NINE YEARS (1865-1914.) - - +------------------------+---------------------+---------------------+ - | | Rich Soil: Plot 2. | Poor Soil: Plot 3. | - | | Lb. per Acre. | Lb. per Acre. | - +------------------------+---------------------+---------------------+ - |Nitrogen in soil in 1865|·175 per cent. = 4340|·105 per cent. = 2720| - |Nitrogen added in | | | - |manure, rain (5 lb. per | | | - |annum), and seed (2 lb. | | | - |per annum) | 10,140 | 340 | - +------------------------+---------------------+---------------------+ - |Nitrogen expected in | | | - |1914 | 14,480 | 3060 | - |Nitrogen found in 1914 |·259 per cent. = 5950|·095 per cent. = 2590| - +------------------------+---------------------+---------------------+ - |Loss from soil | 8530 | 470 | - |Nitrogen accounted for | | | - |in crops | 2500 | 750 | - +------------------------+---------------------+---------------------+ - |Balance, being dead loss| 6030 | -280[J] | - |Annual dead loss | 123 | - 6[J] | - +------------------------+---------------------+---------------------+ - - [J] Gains. Possibly the result of bacterial action. - -A further remarkable fact connected with the decomposition of the -nitrogen compounds is that it seems invariably to be accompanied by -an evolution of gaseous nitrogen. Apparently there are two cases. -Under anaerobic conditions many of the soil organisms have the power -of obtaining their necessary oxygen from nitrates, thereby causing -a change in the molecule which leads in some cases to liberation of -gaseous nitrogen; but the same result seems to be attained in aerobic -conditions, especially when carbon is being rapidly oxidised. - -It is possible that the reaction is the same, and that in spite of the -general aerobic conditions there is locally an anaerobic atmosphere. -But it is also possible that some direct oxidation of protein or -amino-acids may yield gaseous nitrogen. However it is brought about -it affects a considerable proportion of the entire stock of nitrogen, -and it becomes more serious as cultivation is intensified. Thus, on -the Broadbalk plot receiving farmyard manure the loss is particularly -heavy; on the unmanured plot it cannot be detected. The nitrogen -balance-sheet is shown in Table XVII. - -The oxidation of carbonaceous matter, however, is not invariably -accompanied by a net loss of nitrogen; in other circumstances there is -a net gain. In natural conditions there seems always to have been some -leguminous vegetation growing; the gain may, therefore, be ascribed -to the activity of the nodule organism. In pot experiments, however, -it has been found possible, by adding sugar to the soil, to obtain -gains of nitrogen where there is no leguminous vegetation, and this is -attributed to the activity of Azotobacter. - -The nitrogen cycle as observed in the soil is as follows:-- - - +--------------→ Protein ←----------------+ - | · · | - | · · | - | · · | - By certain| · · | - organisms | | | |By Azotobacter, - and by | ↓ | |Clostridium, - growing | Ammonia | Mechanism |nodule organisms, - plants | | | uncertain |etc. - | ↓ | | - | Nitrite | | - | | ↓ | - | ↓ Gaseous | - +------- Nitrate ------→ Nitrogen --------+ - - By denitrifying organisms - -There has been but little study of the process of decomposition of the -other compounds in plants. Part, if not all, of the sulphur is known -to appear as sulphate, and some of the phosphorus as phosphate. It is -certain that the plant constituents decompose, for there is no sign of -their accumulation in the soil. They may exert transitory effects, but -there is nothing to show permanent continuance. The toxic conditions -which cause trouble in working with pure cultures of organisms in -specific cultures media do not, so far as is known, arise in the soil. -All attempts to find bacterio-toxins or plant toxins in normal soils -have failed. The product toxic to one organism seems to be a useful -nutrient to another, and so the mixed population keeps the soil healthy -for all its members. - -There is little precise knowledge as to the part played by the -different members of the soil population in bringing about these -changes. - -We know in a general way that earthworms effect the distribution of -the plant residues in the soil, and serve to disintegrate them; there -is no evidence, however, that they play any indispensable part in the -decomposition. Many root and other fragments do not go through this -process; observation shows that fungi can force a way in, and they may -be followed by nematodes which continue the disintegration. Possibly -some of the flagellates help, and certainly the bacteria do. After that -nothing is certain. We cannot, with certainty, assign any particular -reaction in the decomposition to any specific organism, with the -exception of the oxidation of the phenolic substances, the conversion -of ammonia to nitrite and nitrate, and the fixation of nitrogen. With -these exceptions many organisms seem capable of bringing about the -reactions, and indeed some of the reactions may be purely chemical and -independent of biological agencies. - -The relationships between the soil population and soil fertility are -readily stated in general outline, but they are by no means clear cut -when one comes to details; fertility is a complex property, and some -of its factors are independent of soil micro-organisms. - -The general relationship between plants and soil organisms is one of -complete mutual interdependence. The growing plant fixes the sun’s -energy and converts it into a form utilisable by the soil organisms; -without the plant they could not exist. The plant is equally dependent -on the soil organisms in at least two directions: their scavenging -action removes the dead vegetation which would, if accumulated on the -surface of the soil, effectively prevent most plants from growing. -Further, the plant is dependent on the soil population for supplies -of nitrates. Nothing is known about the relative efficiencies of the -various soil organisms as scavengers. Numerous fungi and bacteria are -effective producers of ammonia, the precursor of nitrates; it is not -known, however, whether flagellates and such higher forms as nematodes -act in this way. - -This widespread power of producing ammonia makes it impossible in our -present knowledge to regard any particular group of organisms as _par -excellence_ promoters of fertility. Indeed, it is safest not to attempt -to do so. The primary purpose of the activities of a soil organism -is to obtain energy and cell material for itself; any benefit to the -plant is purely incidental. For cell material it must have nitrogen -and phosphorus; here it competes with the plant. If it produces more -ammonia than it utilises--in other words, if it is driven to nitrogen -compounds for its energy, then the plant benefits. If, on the other -hand, it absorbs more ammonia than it produces, as happens when it -derives its energy from non-nitrogenous substances, the plant suffers. -Thus, addition of peptone to the soil or an increase in bacterial -numbers effected without addition of external energy (e.g. by partial -sterilisation) leads to increased ammonia supply, and, therefore, -to increased fertility. But addition of sugar to the soil causes so -great an increase of numbers of bacteria and other organisms that -considerable absorption of ammonia and nitrate occurs, and fertility -is for a time depressed. - -Both actions proceed in soils partially sterilised by organic -substances, such as phenol, which are utilised by some of the soil -organisms; there is first a great rise in numbers of these particular -organisms with a depression of ammonia and nitrate, then a drop to the -new level, higher than the old one, and an increased production of -ammonia and nitrate resulting from the partial sterilisation effects. - -We must then regard the soil population as concerned entirely to -maintain itself, and only incidently benefiting the plant, sometimes, -indeed, injuring it; always essential, yet always taking its toll, and -sometimes a heavy toll, of the plant nutrients it produces. - -This effect makes it difficult to deduce simple quantitative -relationships between bacterial activity and soil fertility, and the -difficulty is increased by the fact that bacteria and plants may both -be injured or benefited by the same causes, so that high bacterial -numbers in a fertile soil would not necessarily be the cause, but might -be simply the result of fertility. - -The circumstance that certain soil organisms--bacteria, algæ, and -fungi--themselves assimilate ammonia and nitrate may account for -the remarkable slowness of nitrate accumulation, to which reference -has already been made. The protein formed from the assimilated -nitrogen remains in the bodies of the organisms, living or dead, till -decomposition sets in. It is not difficult to picture a cycle of events -in which much of the nitrate formed is at once reabsorbed by other -organisms, and only little is actually thrown off into the soil. Such a -process might continue almost interminably so long as any carbonaceous -material remained. - -Finally, we come to the very interesting problem--is it possible to -control the population of the soil? - -The problem may seem superfluous in view of the difficulties just -mentioned. Some aspects of it, however, are fairly clearly defined. - -In the first instance, some organisms appear to be wholly harmful to -the plant; among them are parasitic eelworms and fungi, and bacteria -causing disease. - -Control of these organisms can be brought about by partial -sterilisation, and of all methods heat is the most effective, but it -is costly, and attempts are now being made to replace it by chemical -treatment. The results are promising, but the investigation is -laborious; the organisms show specific relationships, and in finding -a sufficiently potent and convenient poison it is necessary in each -case to make an investigation into the relationship between chemical -constitution and toxicity to the particular organism concerned. -Formaldehyde is usually potent against fungi, and the cresols, and -particularly their chlor- and chloronitro-derivatives, are potent -against animals (eelworms, etc.). - -One group of organisms is wholly beneficial, those associated with -leguminous plants. Attempts have been made to increase their activities -by inoculating the soil with more vigorous strains. The practical -difficulties still remain very considerable, but there is hope that -they may be overcome. - -It is also possible to shift the balance of the soil population in -certain directions. Special groups of soil organisms can be caused -to multiply temporarily, if not permanently, by satisfying their -particular requirements. Thus, when a soil has been heated above 100° -C. it becomes specially suited to the growth of fungi, and quite -unsuited to certain bacteria such as the nitrifying organisms and -others; if this heated soil is infected with a normal soil population -the fungi develop to a remarkable extent. The nodule organisms appear -to be stimulated by addition of farmyard manure and of phosphates, and -the phenol-destroying organisms by successive small additions of phenol. - -Finally, quite apart from the control of disease organisms, it is -possible to alter the soil population considerably by partial -sterilisation, using a temperature of only about 60° C., or a poison -like toluene that favours few of the soil organisms. This problem has -already been discussed in Chapter I. - -The control of the soil population is still only in its infancy, but -it already promises useful developments. It cannot, however, be too -strongly insisted that the only sure basis of control is knowledge, and -we cannot hope to push control further till we have learned much more -about the soil population than we know at present. - - - - -AUTHOR INDEX. - - - ADAMETZ, 118. - - Adams, 158. - - Aiyer, 113. - - Appel, 133. - - Artari, 107. - - Ashby, 42. - - - BARTHEL, 24. - - Beijerinck, 6, 37, 41, 42, 46, 107. - - Berthelot, 5, 6, 41. - - Bewley, 47, 51, 132. - - Bezssonoff, 69. - - Boas, 138. - - Bokorny, 138. - - Bonazzi, 45. - - Boresch, 107. - - Boussingault, 3. - - Bredemann, 24. - - Bristol, 106. - - Brizi, 113. - - Brown and Halversen, 127. - - Burgess, 41. - - Burrill, 48. - - Bussey, Peters and Ulrich, 132. - - Butkevitch, 138, 139. - - Butler, 132. - - - CAMERON, 150, 158. - - Chodat, 107. - - Christensen, 46. - - Clayton, 28, 43. - - Coleman, 127, 136. - - Conn, 23, 54, 61, 123. - - Cramer, 39. - - Crump, 57, 79, 80. - - Cunningham, 69. - - Cutler, 57, 58, 78, 80. - - - DALE, 118, 121. - - Darwin, 153. - - Dascewska, 134. - - De Bruyn, 132. - - van Delden, 42. - - Delf, 87. - - Doryland, 33, 40. - - Dox, 138. - - Dox and Neidig, 134. - - Drummond, 160. - - Duggar and Davis, 135. - - Duvaine, 20. - - - EHRENBERG, 138. - - Ehrlich and Jacobsen, 138. - - Esmarch, 102, 103. - - - FABRICIUS, 61. - - Feilitzen, 61. - - Fischer, 118, 126. - - Forte, 101. - - Frank, 132. - - Fritsch, 112. - - - GAINEY, 46. - - Gillespie and Hurst, 140. - - Goddard, 118, 120, 121. - - Golding, 49. - - Goodey, 68, 73, 79, 105. - - Greaves, 42, 61. - - Green, 37. - - Grintzesco, 107. - - Groenewege, 42. - - - HAGEM, 118, 121, 136, 138, 139. - - Hamilton, 158, 159. - - Hansen, 48. - - Hanzawa, 43. - - Harrison, 113. - - Heinze, 139. - - Hellriegel, 5, 6, 46. - - Hensen, 100. - - Hesselmann, 36. - - Hill, 94. - - Hiltner, 23. - - Hopkins, 36. - - Hutchinson, C. M., 42. - - Hutchinson, H. B., 27, 43, 47, 51, 57, 105. - - - VAN ITERSON, 133. - - - JENSEN, 118, 132. - - Jewson, 123. - - Joffe, 37. - - Jones, D. H. and Murdock, 127. - - Jones, L. R., 140. - - - KAPPEN, 136. - - Karrer, 105. - - Kaserer, 27. - - Klöcker, 134. - - Koch, A., 44, 134. - - Koch, R., 20, 53. - - Kofoid, 88. - - Kohshi, 134. - - Kopeloff, 118, 136. - - Kossowitsch, 111. - - Krainskii, 44. - - Krzeminiewski, 43. - - Kufferath, 107. - - - LATHAM, 135. - - Laurent, 136. - - Lawes and Gilbert, 5. - - Lebedeff, 27. - - Leeuwenhoeck, 20. - - Lendner, 118. - - Lipman, C. B., 41, 42, 44, 54. - - Lipman, J. G., Blair, Owen, and McLean, 94. - - Löhnis, 22, 43, 69, 136. - - - MAGNUS, 107. - - Malpighi, 46. - - Marchal, 34, 136. - - Martin, 73. - - Martin and Lewin, 69. - - McBeth, 28, 134. - - McBeth and Scales, 118, 140. - - McLean and Wilson, 118, 136. - - Mockeridge, 43. - - Moore, G. T., 105. - - Morris, 150, 151, 162. - - Muntz and Coudon, 118, 136. - - - NABOKICH, 27. - - Nagaoka, 38. - - Nakano, 107. - - Nasir, 94, 95. - - Neller, 137. - - - OMELIANSKI, 27, 42. - - Orla-Jensen, 26, 35. - - Otto, 133. - - Oudemans and Koning, 118. - - - PASTEUR, 3, 20. - - Perey, 94. - - Perotti, 136. - - Petersen, 104. - - Pillai, 43. - - Potter and Snyder, 137, 138. - - Povah, 138. - - Pratt, 132. - - Prescott, 61. - - Pringsheim, 107. - - - RAMANN, 118. - - Rathbun, 120. - - Reh, 162. - - Remy, 118. - - Richards, 112. - - Richardson, 159. - - Ritter, 138. - - Robbins, W. J., 109. - - Robbins, W. W., 105. - - Roussy, 134. - - Russell, 112. - - Russell and Hutchinson, 57, 66, 94. - - - SALUNSKOV, 42. - - Sandon, 57, 75. - - Scales, 28, 134. - - Schellenberg, 133. - - Schindler, 107. - - Schloesing, 3, 4, 34. - - Schmitz, 134. - - Schramm, 111. - - Servettaz, 109. - - Seydel, 44. - - Sherman, 69. - - Shibata, 136. - - Söhngen, 26, 27, 134. - - - TAKAHASHI, 118. - - Taylor, 120. - - Ternetz, 135. - - Treub, 112. - - Truffaut, 69. - - - VERKADE and Söhngen, 134. - - Von Ubisch, 109. - - - WAKSMAN, 37, 118, 120, 121, 123, 125, 126, 134, 136. - - Waksman and Cook, 136. - - Wann, 111. - - Warington, 4, 34. - - Waynick, 42. - - Welwitsch, 112. - - Werkenthin, 120, 121. - - West, 88, 105. - - Whiting, 36. - - Wilfarth, 46. - - Winogradsky, 4, 6, 34, 41, 44. - - - - -SUBJECT INDEX. - - - _Absidia_, 121. - - _Acarina_, 150, 151, 157. - - Acid formation by Fungi, 139. - - Acidity of soil, 17; effect on Actinomyces, 140; relation to - nitrification, 36. - - _Actinomycetes_, 119, 134, 139. - - Aeration of soil, effect on bacteria of, 61. - - _Agriotes_, 150. - - Air supply in soil, 17. - - Algæ, agents causing disappearance of nitrate from soil, 12; - associations of, in soil, 105, 106; blue green, 102 _sqq._ (see also - Cyanophyceæ and Myxophyceæ); colonisation of new ground by, 112; - conditions of growth for, 101, 104, 107, 108; distribution of, 102, - 104, 106, 109; economic significance of, 100, 102; filamentous, - 106; flora of soil, 101, 112; formation of humus substances, 112; - fragmentation of filaments, 107, 110; frequency of occurrence, 102 - _sqq._; glucose, effect of, on growth, 108, 109; green, 104 _sqq._ - (see also Chlorophyceæ); importance in cultivation of rice, 113; - numbers in soil of, 109, 110; nutrition of, 107, 108, 110; producers, - of organic substance, 100; pure cultures of, 107, 111; relation to - gaseous interchange in soil, 113; relation to soil moisture, 112; - seasonal changes in numbers of, 88; subterranean, 105. - - Alkaloids, as source of nitrogen for fungi, 138. - - _Alternaria_, 119. - - Amino-acids, formation of, by algæ, 108. - - Amino-compounds, decomposition of, by fungi, 136, 138. - - Ammonia, assimilation of, by bacteria, 33, 40, 45; effect of partial - sterilisation on soil content of, 66; formation in soil, 170; - formation in soil by bacteria, 32 _sqq._; formation in soil by fungi, - 135 _sqq._, 141; influence of physical conditions on formation of, - 137; property of attracting Diptera, 159; utilisation by higher - plants, 36. - - Ammonium sulphate, effect on fungi, 121, 126, 127. - - _Anabæna_, 102, 112. - - _Annelida_, 149. - - Antagonism of salts in soil, 60. - - Ants, 153. - - _Arachnida_, 150, 151. - - Arctic soil, bacterial flora of, 24. - - _Areinida_, 150, 151, 157. - - _Armillaria_, 132. - - _Ascomycetes_, 119. - - _Aspergillaceæ_, 136. - - _Aspergillus_, 119, 120, 135, 136, 138, 139. - - Azotobacter, 6, 41, 95, 96; assimilation of nitrates by, 45; - decreasing efficiency in liquid culture, 44; indicator of soil - acidity, 44. - - - BACILLARIACEÆ, 100 (see also Diatom). - - _Bacillus amylobacter_, distribution of, 24. - - _Bacillus radicicola_, 24, 46 _sqq._; inoculation of soil with, 50; - life cycle of, 47. - - Bacteria, association with algæ in nitrogen fixation, 111; anærobic - respiration of, 37; effect of arsenic on, 61; cellulose destroying, - 134; changes in morphology in culture, 22, 47; classification of main - groups, 23, 25; composition of cells of, 39; inverse relationship - with protozoa, 10, 79, 82 _sqq._; isolation from soil, 21; methods - of describing, 21; method of estimating numbers of, 53 _sqq._, 80; - nitrogen fixation by, 110, 111; numbers in relation to algæ, 110; - numbers in soil, 52 _sqq._; oxidation of hydrogen by, 27, 37; effect - of partial sterilisation on, 8, 9, 66, 67; part played in soil - fertility by, 7; pure cultures, isolation by plating, 20; seasonal - changes in numbers of, 59, 87 _sqq._; effect of salts on, 60; short - time changes in numbers of, 11, 57, 58; effect of temperature on, 67; - uneven distribution of, 57. - - _Basidiomycetes_, 119, 123, 132. - - Beets, attacked by _Phoma betæ_, 135. - - _Boletus_, 132. - - _Botrytis_, 122. - - Bryophyta, 100, 132. - - _Bumilleria_, 105. - - - CALCIUM compounds in soil and fungi, 139. - - _Carabidæ_, 150. - - Carbohydrates, decomposition by bacteria, 26 _sqq._; decomposition by - fungi, 140; decomposition in soil, 168; effect on ammonia production - in soil, 33; presence in algal sheath and bacteria, 111. - - Carbon, changes in amount in soil, 167; relationships of bacteria, - 27; relationships of fungi, 133; source of, for soil bacteria, 39; - sources of, for soil fungi, 139. - - Carbon dioxide, assimilation by algæ, 99, 107, 108; assimilation by - soil bacteria, 35, 36, 40. - - Carotin, in algæ, 100; formed by _Spirochæta cytophaga_, 29. - - _Cecidomyidæ_, 155. - - Cellulose, decomposition by bacteria, 27, _sqq._; decomposition by - fungi, 133, 134, 141; relation of nitrogen supply to decomposition - of, 30; decomposition in soil, 168; as source of energy for nitrogen - fixation, 43. - - Centipedes, see _Chilopoda_. - - _Cephalosporium_, 120. - - _Cephalothecium_, 136. - - _Chilopoda_, 157. - - _Chironomidæ_, 155. - - _Chlorella_, 108. - - _Chlorococcum_, 105. - - _Chlorophyceæ_, 100. - - Chlorophyll, loss of, from algæ, 108. - - Ciliates, classification of, 72; cyst wall of, 73. - - Citric acid, formation of, by fungi, 139. - - _Cladosporium_, 119. - - Clamp connections in fungi, 119. - - Classification, of algæ, 100; of bacteria, 23, 25; of fungi, 131; of - protozoa, 69 _sqq._ - - Climate, effect of, on algæ, 101. - - _Clostridium_, 41, 44; as fixer of nitrogen, 6. - - _Coccomyxa_, 104. - - _Coleoptera_, 150, 154, 155. - - _Collembola_, 150, 153, 154. - - _Colletotrichum_, 131. - - Commensals, 132. - - _Conjugatæ_, 100. - - _Cortinarius_, 132. - - Cotton, destroyed by fungi, 134. - - Counting, of algæ, 109; of bacteria, 53 _sqq._; of fungi, 122; of - protozoa, 77, 79, 80. - - Cresol, decomposition of, by bacteria, 22, 24, 31. - - Criteria, physiological, of fungi, 128. - - Crop growth, effect on fungi, 122. - - _Cryptomonadineæ_, 100. - - Cucumber leaf spot, 131. - - Cyanamide, decomposition of, by fungi, 136. - - _Cyanophyceæ_, 103 (see also _Myxophyceæ_ and blue-green algæ). - - _Cylindrospermum_, 102. - - Cysts, 68, 73, 74. - - - DENITRIFICATION, by bacteria, 37; by fungi, 136. - - Desiccation, resistance to, by algæ, 106. - - Dew, relation to algæ, 101, 113. - - Diatoms, 104 _sqq._ (see also _Bacillariaceæ_). - - Dicyanamide, decomposition of, by fungi, 136. - - Dipeptides, formation of, by algæ, 108. - - _Diplopoda_, 157. - - _Diptera_, 150, 154, 155, 159. - - Disaccharides and fungi, 134. - - - EARTHWORMS, abundance of, in soil, 153; effect of, in soil, 13, 160, - 175. - - Eel-worms, 149 (see also _Nematoda_). - - _Elaphomyces_, 132. - - _Enchytræidæ_, 149. - - Energy, laws of, 165; relationships of soils, 166; requirements of - soil organisms, 15, 16. - - Energy supply, relation of bacterial activities to, 25 _sqq._, 40, - 44; sources of, for soil bacteria, 26 _sqq._, 40, 43; supplies of, - for soil organisms, 111, 164, 167, 168. - - Environmental conditions in soil, 16. - - Eremacausis, 2. - - Ericales, 132, 135. - - _Euglena_, 99. - - _Euglenaceæ_, 100. - - Experimental error, in bacterial counts, 54; in fungal counts, 124. - - - FARMYARD manure, see Manure. - - Fats, used by fungi, 134. - - Fatty acids used by fungi, 134. - - Fertility of soil, views on, 2; effect of decomposition of plant - residues on, 1, 165; effect of organisms on, 175. - - Filter paper, destruction of, by fungi, 133; destruction of, by - _Spirochæta cytophaga_, 28. - - Fixation of nitrogen, discovery of, by Berthelot, 5; by bacteria, 40 - _sqq._; by algæ, 110, 111; by mixtures of bacteria and algæ, 111; by - fungi, 135 _sqq._ (see also Nitrogen Fixation). - - _Flagellatæ_, 100. - - Flax sickness and fungi, 122. - - Formaldehyde, as agent for destroying fungi, 141. - - Fungi, control of, in soil, 139 _sqq._; counting of, 122; - distribution of, in soil, 119 _sqq._, 127; fertilisers, effect of, on - numbers of in soil, 126; as facultative parasites, 131, 132; fruiting - bodies of, 123; destruction of hemicelluloses by, 133; individual, - 122, 123; action on monosaccharides of, 134; mineral relationships - of, 139; mycorrhizal, 132, 135, 139, 140; heterocyclic nitrogen - compounds and, 138; occurrence in soil, 118; qualitative study of, - 118; selective feeding of, 140; specific determination of, 119. - - _Fungi imperfecti_, 119. - - _Fusaria_, 134. - - _Fusarium_, 119, 120, 122, 128, 133, 136. - - - _Gamascidæ_, 156. - - Gases of swamp water (Paddy soils), 113. - - _Gastrodia_, 132. - - Gelatinous envelope of algæ, 109, 111. - - Geographical distribution of azotobacter, 41; of soil bacteria, 24; - of protozoa, 75, 76; of soil fungi, 119, 125. - - Germination, of algal spores, 107. - - Glucose, use of, by algae, 108, 109, 111; use of, by moss protonema, - 109. - - Glycocoll, formation of, by algæ, 108. - - _Granulobacter_, 42. - - Greenland, bacteria in soil from, 24. - - “Grunlandmoor,” fungi in, 126. - - - _Hantzschia_, 105. - - _Hemiptera_, 154. - - _Heterokontæ_, 100. - - “Hochmoor,” fungi in, 126. - - _Hormidium_, 104. - - Humus, the food of plants, 1; formation of, by fungi, 134, 141; - formation of, in soil, 168; forest, 132; fungal hyphæ as constituent - of forest humus, 132. - - Hydrogen ion concentration, in soil, 17; effect on fungi of, 124. - - _Hymenoptera_, 150, 154. - - - _Insecta_, 150, 157. - - Insects, numbers present in soil, 154. - - Invertebrata, definition of, 147; method of investigating, 148; - groups represented, 149; distribution in the soil, 151; dominant - species and groups, 153; environmental factors of, 157; feeding - habits, 156; relation to agriculture, 160; relation to nitrogen - cycle, 161. - - Iron compounds, oxidation by fungi, 139. - - _Isopoda_, 150, 151. - - - _Leguminosæ_, association with bacteria, 46 _sqq._; enrichment of - ground by, 5. - - _Lepidoptera_, 150, 154. - - Life cycles, of bacteria, 22, 47; of protozoa, 72 _sqq._ - - Lime, effect on fungi in soil, 121, 126. - - _Lyngbya_, 112. - - - MAGNESIUM compounds, effect on fungi, 139. - - Manganese compounds, effect on bacteria, 61. - - Manure, farmyard, effect on algæ, 109, 110; effect on numbers of - bacteria, 60; effect on numbers of fungi, 126; effect on numbers of - insects, 154, 155. - - Manure, Artificial, effect on fungi, 127. - - Manure, town stable, occurrence of disease organisms in, 132. - - _Mastigophora_, classification of, 71; species of, 71. - - Media, containing nitrates, chemical analysis of, 111; for counting - soil bacteria, 54; for counting protozoa, 79; for counting fungi, - 119, 123. - - _Melanconium_, 134. - - _Melolontha_, 150. - - Methane, oxidation of, by bacteria, 26, 27. - - Millipedes, see _Diplopoda_. - - Mites, see _Acarina_. - - _Mollusca_, 149, 157. - - _Moniliaceæ_, 136. - - _Mucor_, 120, 121, 136, 138. - - _Mucorales_, 121, 134. - - _Mucorineæ_, 118. - - _Mycetophilidæ_, 155. - - Mycorrhiza, 132, 135, 139, 140. - - _Myriapoda_, 150, 156. - - _Myxophyceæ_, 100 (see also _Cyanophyceæ_ and blue-green algæ). - - - NAPHTHALENE, decomposition of, by bacteria, 31. - - _Naviculoideæ_, 100. - - _Nematoda_, 149, 151, 157. - - Nitrate, assimilation by algæ, 105, 108, 111; assimilation by - bacteria, 33, 40, 44, 51; assimilation by fungi, 136, 138; removal - from soil, 12, 112, 171; variations in amount in soil, 11. - - Nitre-beds, 1. - - Nitrification, and bacteria, 34; chemical changes in, 171; and fungi, - 136; energy supply in, 35; mechanism of, 1, 3; and soil fertility, 1, - 3. - - Nitrites and fungi, 136; formation by bacteria, 34. - - _Nitrobacter_, 35. - - Nitrogen, changes in amount in soil, 167; cycle in soil, 161; - fixation by bacteria, 6, 40 _sqq._; fixation by fungi, 135, 136, 141; - fixation of, in clover plant, 5; increase by protozoa of fixation - of, 94, 95 (fig.); fixation sources of energy for, 43, 49; gain of, - in soil, 174; in invertebrates, 162; loss of, by leaching, 112; - loss of, from cultivated soils, 173; relationships of fungi, 135; - relationships of algæ, 110-112; relationships of bacteria, 32 _sqq._, - 40 _sqq._; relationships of insects, 162. - - _Nitrosococcus_, 35. - - _Nitrosomonas_, 35. - - Nodule Organism of the Leguminosæ, 6, 46 _sqq._ - - _Nostocaceæ_, 100, 101, 102, 107. - - - _Oligochæta_, 149, 151, 153, 157. - - _Oospora_, 120. - - _Orcheomyces_, 132. - - Orchid cultivation and fungi, 132, 140. - - _Orthoptera_, 154. - - _Oscillatoriaceæ_, 100, 102. - - Osmotic pressure, influencing effect of salts on bacteria, 50. - - Oxalic acid, formation of, by fungi, 139. - - Oxidations effected by soil organisms; by bacteria, 26 _et seq._; by - fungi, 139. - - Oxygen, absorption by soils, 4. - - - PARTIAL sterilisation of soil, 8, 66 _sqq._, 96, 178; influence of - organic antiseptics, 177; limiting factor in, 67, 68. - - Pectin, effect of, on fungi, 134. - - _Pedras negras_, 112. - - _Penicillia_, 134. - - Pentosans, effect of, on fungi, 134. - - Peptones, decomposition of, by fungi, 136, 138; source of nitrogen - for algæ, 108. - - Periodicity, of protozoa in soil, 90 _sqq._ (fig.), 92 (fig.), 93. - - Phenol, decomposition of, by bacteria, 24, 25, 31. - - Phenylalanine, formation of, by algæ, 108. - - _Phoma_, 132. - - _Phormidium_, 106. - - Phosphates, availability of, influenced by bacteria, 52; by fungi, - 139; effect on bacteria, 46, 51, 60. - - Photosynthesis, 99, 100, 107, 110, 113. - - Phycocyanin, 100. - - Physical conditions in soil, 16. - - Physiological criteria, of bacteria, 22; of fungi, 128. - - _Phycomycetes_, 119. - - _Phytophthora_, 132. - - Plant disease, and fungi, 139. - - Plant residues, decomposition of, in soil, 168; influence of soil - reaction on, 165; relation to soil fertility, 1, 165. - - Plasticity of fungi, 119. - - _Plectonema_, 106. - - Potassium salts, effect on bacteria, 60; influence of bacteria on the - availability of, 52. - - Protein, decomposition of, in soil, 169; decomposition by bacteria, - 32; decomposition by fungi, 138, 140. - - _Protococcales_, 100. - - _Protoderma viride_, 105. - - Protonema of mosses, 100, 105, 106, 109. - - Protophyta, chlorophyll-bearing, 100. - - Protozoa, inoculation into soil of, 85 _sqq._; isolation from soil, - 69; classification of, 69 _sqq._; life histories of, 72 _sqq._; - species of, in soil, 70 _sqq._; distribution of, in soil, 74 _sqq._; - retention of, by soil, 78 (fig.); size of, 90; reproductive rates, - 93; inverse relation with bacteria, 79 _sqq._; presence of trophic - forms in soil, 9; numbers of, in soil, 90, 96, 97; fluctuations in - numbers of, 10, 81 (fig.), 82; external conditions, effect on, 82; - seasonal changes, effect on, 87 _sqq._; weight of, 90. - - _Pteridophyta_, 132. - - _Pythium_, 132. - - - REACTION of soil, 17. - - Reaction of soil, effect on bacteria, 36, 37, 46, 48, 61; effect on - protozoa, 93, 94 (see also hydrogen ion concentration). - - Relationships of Fungi, commensal, 132; mycorrhizal, 132; symbiotic, - 132. - - _Rhizopoda_; classification of, 70, 71; species of, 70, 71. - - Rhythm, supposed in ammonification by fungi, 137. - - _Rhizoctonia_, 132. - - _Rhizopus_, 119, 120. - - Rice plant, aeration of roots, 113; physiological disease of, 113. - - Rock Phosphate as base for nitrifying organisms, 36. - - Rothamsted, Broadbalk plot 2 (Farmyard Manure) algæ, 109; fungi, 125, - 127; Insects, 152. - - Rothamsted, Broadbalk plot 3 (Unmanured) algæ, 109; fungi, 120, 122, - 127; Insects, 152. - - Rothamsted, Broadbalk Plots 10, 11, and 13; 122, 127. - - Rothamsted, Barnfield Plot 1-0 (Farmyard Manure), Protozoa, 80. - - Rothamsted, unmanured grass plot, 120. - - _Russula_, 132. - - Rusts, 119. - - - _Saccharomyces_, 120. - - Saprophytes, facultative, 131. - - Saprophytism and algæ, 108, 110. - - _Scenedesmus_, 108. - - Seasonal fluctuations in numbers of soil organisms, 12, 87 _et seq._, - 125. - - Selective media, use of, in isolation of soil bacteria, 21. - - Serological tests, separation of varieties of _B. radicicola_ by, 48. - - Slugs, see _Mollusca_. - - Smuts, 119. - - Snails, see _Mollusca_. - - Soil; comparison of, by volume, 17; effect of depth below surface - on algæ, 101, 104, 109, 110, 113; effect of depth below surface on - insects, 151; effect of depth below surface on fungi, 121, 126, 127; - effect of various treatments on fungi, 126, 127, 132; environmental - factors in, 16; inoculation of, for leguminous plants, 50; moisture - (see Water supply); population, control of, 177 _sqq._; population, - methods of investigation, 10, 15; sterilisation and fungi, 137, 138, - 141 (see Partial Sterilisation); stored, survival of algæ in, 107; - type and fungi, 121, 126, 127. - - Soil conditions, effect on bacteria, 33, 36, 37, 40, 46, 48, 50, 59 - _sqq._; effect on protozoa, 82. - - Soil fertility, see Fertility of soil. - - _Spicaria_, 120. - - Spiders, see _Areinida_. - - _Spirochæta cytophaga_, 28, 43. - - Spore forming bacteria in soil, 23, 34. - - Spore, fungus, inhibition of formation, 123; presence in air of, 118. - - Standardisation of cultural methods for soil bacteria, 54 _sqq._ - - Starch, decomposition of, by fungi, 134. - - _Stichococcus_, 108. - - Straw; effect on nitrate production in soil, 33; manure, 29; rotting - of, 30. - - Sulphur oxidation, by bacteria, 37; by fungi, 139. - - Symbiosis, of Azotobacter with other organisms, 42, 43, see also - Mycorrhiza and Nodule organism. - - _Symphyla_, 150, 151, 157. - - _Symploca_, 112. - - - _Tachinidæ_, 150. - - Tannins, used by fungi, 134. - - Temperature of soil and fungi, 127, 140. - - Termites, 160. - - _Testacella_, 149. - - _Thiospirillum_, 37. - - _Thysanura_, 154. - - _Thysanoptera_, 154. - - _Tipula_, 150. - - Toluene, decomposition by soil bacteria, 31. - - _Tolypothrix_, 112. - - _Trichoderma_, 119, 120, 122, 134. - - _Trochiscia_, 105. - - Tropisms, 157. - - - _Ulothrix_, 105. - - _Ulotrichales_, 100. - - Urea, by fungi, 136, 138. - - Uric acid, utilisation of, by fungi, 138. - - - _Vaucheria_, 104, 106. - - Vitality, retention of, by algæ and moss protonema, 105, 107. - - - WATER; supply in soil, 17; and algæ, 112; bacteria, 50, 61, 82; - fungi, 127; protozoa, 82. - - Wireworms, 155. - - Wood, decay of, 134. - - Woodlice, 150; (see also _Isopoda_). - - - YEASTS, 138. - - - _Zygnema_, 104. - - _Zygorrhynchus mœlleri_, 119, 120, 121. - - -PRINTED IN GREAT BRITAIN BY THE UNIVERSITY PRESS, ABERDEEN - - - - - Transcriber’s Notes - - - Inconsistent and archaic or unusual spelling, capitalisation, - italicisation, hyphenation, etc. have been retained, unless mentioned - below. The names and classifications of the organisms as used in the - book do not always conform to modern names and classifications; these - have not been changed. - - Depending on the hard- and software used and their settings, not all - elements may display as intended. - - Page 14, table, lower right hand cell: the data given add up to 8, - not to 9. - - Page 47, ·9 × ·18 in size: the source document does not include the - units; presumably the sizes are in microns. - - Page 118, endnote 8c (2×): this note does not exist. - - Subject Index, entry Zygorrhynchus mœlleri: also refers to - Zygorrhynchus vuilleminii. - - - Changes made: - - Footnotes, tables and illustrations have been moved out of text - paragraphs; some tables have been split or re-arranged. - - Several minor obvious typographical, punctuation and spelling errors - (including accents) have been corrected silently. In several cases - spelling differences (mainly of proper names) between the text and - the index and endnotes have been standardised. In the indexes and in - tables some ditto marks have been replaced with the dittoed - text. Some page references have been corrected to indicate the - correct page number. - - Indented text under illustrations is not present as such in the - source document, but has been transcribed from the illustration for - legibility and ease of reference. Some tables have been re-arranged - or split to fit the available width. - - Page 28, 29: MacBeth changed to McBeth as elsewhere (in the Author - Index the entries MacBeth and McBeth have been merged). - - Page 32, formula: 30 changed to 3O. - - Page 58: From Barnfeild, ... changed to From Barnfield, .... - - Page 85: closing bracket deleted after ... Table VII. and Fig. 13. - - Page 90, Table VIII, column 5: 350·000 and 150·000 changed to 350,000 - and 150,000. - - Page 97: No creature lies or dies to itself, ... changed to No - creature lives or dies to itself, ... - - Page 104: Danske Aerofile Alghe changed to Danske Aërofile Alger. - - Page 114: Recherche sulla Malattia del Riso ... changed to Ricerche - sulla Malattia del Riso .... - - Page 115: ... sur de polymorphisme ... changed to ... sur le - polymorphisme .... - - Page 116: literature notes 38 (Robbins) and 48 (Schindler) changed to - 33 and 34 respectively. - - Page 120: Zygorrhynchus vuillemini changed to Zygorrhynchus - vuilleminii as elsewhere. - - Page 126: references to Waksman[24] and [24_e_] changed to [25] and - [25_e_]. - - Page 129: ... preparée de la pres de Russum ... changed to ... - préparée de la terre humeuse du Spanderswoud, près de Bussum .... - - Page 134: reference to Kohshi[24] changed to [34]. - - Page 143: Sämenbildung changed to Säurenbildung (entry 5); - Wurzelbranderregern im Baden changed to Wurzelbranderregern im Boden - (entry 11). - - Page 144: ... Umwandlung von Aminosamen in Oxysämen ... changed to - ... Umwandlung von Aminosäuren in Oxysäuren ....; ... Wirkungen der - Schimmelze ... changed to ... Wirkungen der Schimmelpilze ....; - Hydrogen-iron concentration changed to Hydrogen-ion concentration. - - Page 145: Ztschr. f. Garungs. Physiol. changed to Ztschr. f. - Gärungsphysiol. - - Page 146: einige Pilze gegen Hemizellulosen changed to einiger Pilze - gegen Hemicellulosen. - - Page 157: Such responses are known chemotropism ... changed to Such - responses are known as chemotropism .... - - Page 170: ... alphatic amino-acids ... changed to ... aliphatic - amino-acids .... - -*** END OF THE PROJECT GUTENBERG EBOOK THE MICRO-ORGANISMS OF THE -SOIL *** - -Updated editions will replace the previous one--the old editions will -be renamed. - -Creating the works from print editions not protected by U.S. copyright -law means that no one owns a United States copyright in these works, -so the Foundation (and you!) can copy and distribute it in the -United States without permission and without paying copyright -royalties. 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