Agricultural Microbiology

Unit 2

Soil Microorganisms

B.Sc Agriculture II

Composition of the Soil

The soil represents a medium or substrate in whichnumerous microorganisms Hve and bring about agreat variety of processes which are responsible forcontinuation of the cycle of life in nature.

A normal soil is made up of solid, liquid, and gaseousconstituents.

These can be broadly divided into five groups:

1. Mineral Particles. These vary greatly in size and inthe degree of their mechanical and chemicaldisintegration. They include particles ranging fromlarge pebbles to fine sand, clay, and silt.

2. Plant and Animal Residues. These comprise thefreshly fallen leaves and other plant stubble and deadremnants of a variety of insects and other animalforms.

Some of the materials are largely undecomposed; stillothers are partly or thoroughly decomposed, so thatthe original structure can no longer be recognized. Inthe last state they are spoken of as humus or humifiedmaterials.

3. Living Systems. These include the living roots ofhigher plants; the great number of living animal forms,which range from protozoa, insects, and earthwormsto rodents, as well as the numerous algae, fungi,actinomycetes, and bacteria.

4. Water. The liquid phase of the soil, comprising bothfree and hygroscopic water, contains in solutionvarying concentrations of inorganic salts and certainorganic compounds.

5. Gases. The soil atmosphere consists of carbondioxide, oxygen, nitrogen, and a number of other gasesin more limited concentrations.

At first, bacteria were considered to comprise the all-important group of microorganisms; the variousprocesses which influence soil fertility and for whichmicrobes were known or were believed to beresponsible were associated with the occurrence andabundance of bacteria.

When it was recognized that various other groups ofmicroorganisms must receive attention, and theirimportance in numerous soil processes could nolonger be ignored, there came a change in the generalconcept of the population and its importance.

The following major groups make up the soilmicrobiological population, or its flora and fauna:

1. Bacteria. These include spore-forming and non-spore-forming rods, cocci, vibrios, and spirilla.

They vary considerably in size, shape, oxygenrequirements (aerobic and anaerobic), energyutilization (autotrophic and heterotrophic), slimeformation, and relation to plants and animals(saprophytic and parasitic).

2. Actinomycetes. Three of the genera of actinomycetesare well represented in the soil. Species of Nocardia areclosely related to some of the bacteria, especially themycobacteria and corynebacteria.

Species belonging to the genera Strepfomyces andMicromonospora are more closely related to the truefungi.

Actinomycetes vary greatly in their biochemicalproperties, in their relation to higher plants andanimals (saprophytic vs. parasitic), and in their effectupon soil bacteria ( associative and antagonisticinterrelations )

3. Fungi. These include large groups of organisms,known as Phycomycetes, Ascomycetes, Hyphomycetesor Fungi Imperfecti, and Basidiomycetes.

They produce extensive mycelium and spores in soilsand in composts. Their growth throughout the soilmay be so extensive as to hold the mass of particlestogether by means of a very fine microscopic networkof mycelium and its excretion prod-nets.

Fungi vary greatly in their relation to higher forms oflife, notably plants (saprophytic vs. parasitic), to soilbacteria (formation of antibiotic substances), and toother members of the soil population.

4. Algae. These organisms comprise the grass-greenChlorophyceae, the blue-green Cyanophyccae, and theDiatomaceae. Their al)ilit\ to produce chloropln 11makes their life in the soil, especially oil its surface,independent of the presence of organic matter.

5. Protozoa. These comprise amoebae, flagellates, andciliates. The \egetati\e \s. cyst condition of theprotozoa in the soil has attracted considerableattention.

This is true also of their relation to the bacteria, sinceit was suggested at one time that protozoa function inthe soil as the natural enemies of the bacteria.

By feeding upon bacteria, protozoa exert, it wasbelieved, a controlling effect upon the abundance ofbacteria, thus affecting adversely a variety of soilprocesses.

6. Higher Animal Forms. These include nematodes,rotifers, earthworms, and lar\ae of insects. Theseorganisms have a variety of functions in the soil.

The ability of some of the soil-inhabiting insects toattack certain higher plants frequently makes them ofgreat economic importance.

The action of earthworms as "soil cultivators“ placesthem in an important category. The fact that some ofthe injurious insects spend part of their life cycle in thesoil suggests certain methods of control.

7. Filterable Organisms. These include phages andother viruses. Although our knowledge of theimportance of these forms in soil processes is still verylimited, their ability to attack both lower and higherforms of life, ranging from the bacteria andactinomycetes to cultivated and wild plants andanimals, makes them of great potential importance.

8. Higher Plant Forms. In addition to the microscopicand ultramicroscopic organisms, the soil also harborsthe root systems of higher plants.

The activities of these are frequently dovetailed withthose of the microorganisms.

Although the soil-inhabiting organisms form only avery small part of the total soil mass, they areresponsible for the major changes that take place inthe soil.

These organisms are disti-ibuted throughout the soil,primarily in the upper layers, where the plants senddown their roots and where they obtain theirnecessary nutrients.

When the roots die, they are rapidly decomposed bythe fungi, bacteria, and other groups of organisms.

The constituent chemical elements, notably thecarbon, nitrogen, and phosphorus, are therebyreturned to circulation and again made available forthe growth of new roots and new plants.

In these processes of decomposition, microorganismsbuild up extensive cell substance, which contributes tothe organic matter of the soil or the soil humus. Themicrobial cells not only serve as a reservoir for thefurther activities of microorganisms, but also act asbinders for the soil particles.

Many of the groups of microorganisms found in thesoil are cosmopolitan in nature, whereas others are ofonly limited occurrence.

Some are found in a number of soil types, and othersonly in certain soils and under specific environmentalor cultural conditions.

Among the bacteria, the Bacillus subtilis and the B.mijcoides groups are cosmopolitan in nature, whereasthe Rhizobium legwninosarum is limited largely tosoils in which specific legumes are growing.

Azotobacter chroococcwn is found only in soils thathave a pH above 6.0, whereas A. indicum canwithstand much more acid reactions.

Fungi

are more abundant in acid soils, and actinomycetes inalkaline. Many organisms are controlled by the natureand abundance of the organic matter, by climaticconditions, aeration, and reaction, and by the specificvegetation.

The mycorrhiza fungi and the various plantpathogenicfungi and bacteria are particularly influenced byvegetation.

The associative and antagonistic effects amongmicroorganisms are often believed to exert acontrolling influence upon the specific nature of thesoil microbiological population.

The inhibition of many bacteria, notably of the spore-forming rods and cocci, by antibiotic substancesproduced by fungi; the feeding of certain fungi uponnematodes and protozoa; the feeding of manyprotozoa upon bacteria; the attack of many bacteriaand fungi upon insect larvae; the ability of variousphages to attack bacteria—all contribute to themodification of the soil population.

The methods most commonly used for theenumeration of microorganisms found in the soil arecommonly divided into several groups:

I. Microscopic methods.

1. Staining of soil and diuxt niicroscoi)ic examination.

2. Contact slide method.

3. Direct examination of unstained .soil.

II. Culture methods.

1. Plate culture methods.

2. Electi\e cultiu-e methods.

3. Soil enrichment methods.

The numbers of microorganisms in the soil vary withthe season of year, being highest in spring and fall andlowest in summer and winter.

The abundance of the individual constituent groups ofbacteria may also \ary with the season of year. Hiltnerand Stormer reported that actinomycetes make up 20per cent of the microbial population de\eloping on theplate in spring, 30 per cent in autumn, and 13 per centin winter.

Conn found larger numbers of bacteria in winter, e\enin frozen soil, than in summer; he explained this bythe existence of two types of bacteria, "winter" and"summer."

In addition to seasonal variations, there are also short-term variations among the microorganisms in the soil.These are believed to arise from different competitivefactors among the microorganisms.

The long-term fluctuations reflect the seasonalchanges in climatic conditions, as affecting the supplyof energy for microorganisms provided by plantmaterials.

These variations offer a more logical explanation thanthe "inherent urge" concept suggested by some soilinvestigators.

The soil microbiological population has been dividedby Winogradsky into two broad groups:

(a) the autochthonous or native microbes, which arecharacteristic of the particular soil and which may beexpected always to be found there;

{h) the zymogenic microbes, or those which developunder the influence of specific soil treatments, asaddition of organic matter, fertilization, or aeration. Tothese two groups, another may be added,

(c) the transient microbes, comprising organisms thatare introduced into the soil intentionally, as by legumeinoculation, or unintentionally, as in the case of agentsproducing animal and plant diseases; these may dieout rapidly or may sur\i\e in the soil for \'aryingperiods, especially in the presence of plant or animalhosts.

For the classification of soil bacteria, the Bergeysystem is now almost universally used. It will also beadopted here, with certain slight modifications. Thefollowing five orders are now recognized:

I. Simple and undifferentiated forms, not producingany tlireads and not branching under normalconditions of culture Eubacteriales.

II. Rod-shaped, clubbed, or filamentous cells, withdecided tendency to true branching Actinomycetales.

III. Filamentous, largely aquatic forms, some showingfalse branching. Chlamydobacteriales.

IV. Cells enclosed in a slimy mass, forming apseudoplasmodium-hke aggregation before passinginto a cyst-producing resting stage .... Myxobacteriales.

V. Cells slender, spiral, flexuous Spirochaetales.

Another system of classification of bacteria based upontheir physiological activities has frequently been employedin soil studies.

A. Autotrophic and facultative autotrophic bacteria, deri\ing their carbon primarily from the COj of the atmosphereand their energy from the oxidation of inorganicsubstances or simple compounds of carbon.

I. Bacteria using simple nitrogen comiDounds, ammoniaand nitrite, as sources of energy.

II. Bacteria using sulfur and simple inorganic sulfurcompounds as sources of energy.

III. Bacteria using iron (and manganese) compounds assources of energy.

IV. Bacteria using hydrogen as a source of energy.

V. Bacteria using simple carbon compounds (CO, CH4) assources of energy.

Heterotrophic bacteria deriving their carbon and energyfrom organic compounds.

I. Nitrogen-fixing bacteria, deriving their nitrogen from theatmosi^here as gaseous atmospheric nitrogen.

1. Nonsymbiotic nitrogen-fixing bacteria.

a. Anaerobic, but>'ric acid organism.

b. Aerobic Azotohacter, Radiohacter, Aerobacter, etc.

2. Symbiotic nitrogen-fixing, or root-nodule, bacteria.

II. Bacteria requiring combined nitrogen.

1. Aerobic bacteria.

a. Spore-forming bacteria.

h. Non-spore-forming bacteria:

( 1 ) Gram-positi\'e bacteria.

(2) Gram-negative bacteria.

2. Anaerobic bacteria, requiring combined nitrogen.

Autotrophic Bacteria

Autotrophic hactcria arc characterizect by certainphysiological properties that differentiate them sharplyfrom all the other bacteria.

The principles originally laid down by Winogradsky for thegrowth of these bacteria still hold today with only slightmodifications.

The characteristic properties of these organisms can besummarized as Follows

1. Their development in nature takes place in stronglyelective mineral media, which contain specific oxidizableinorganic substances.

2. Their existence is connected with the presence of suchinorganic elements or simple compounds, which undergooxidation as a result of the life activities of the organisms.

3. The oxidation of the inorganic substances suppliesthe only source of energy for the growth of theseorganisms.

4. They do not need any organic nutrients either forcell synthesis or as a source of energy.

5. They are almost incapable of decomposing organicsubstances and may even be checked in theirdevelopment by certain compounds.

6. They use carbon dioxide as an exclusive source ofcarbon, which is assimilated chemosynthetically.

Nitrifying Bacteria. Among the autotrophic bacteria,the nitrifying organisms have received the greatestconsideration, because of the importance of theprocess of nitrification in the soil, in composts, insewage-disposal systems, and in fresh and salt waters.

Sexeral typos of nitrite-forming organisms are foundin various soils. These bacteria were classified byWinogradsky into four groups:

1. Nitrosonionas. Free, motile forms, present in the soilas cocci or as rods with rounded ends. Optimimireaction is at pH 8.6-8.8; some strains may ha\e theiroptimum at /jH 9.1-9.2, and others at pH 7.5-7.8;growth ceases at pH 6.0.

2. Nitrosocystis. Masses of cocci surrounded by amembrane. Optimum pH 7.4-7.8.

3. Nitrosospira. Spiral-shaped forms.

4. Nitrosogloca. Zoogloea-producing organisms.

Sulfur Bacteria. The sulfur bacteria, or those bacteriawhich are capable of obtaining the energy necessaryfor their growth from the oxidation of sulfur or itscompounds, should be distinguished from otherbacteria taking part in the sulfur cycle, such as thoseliberating hydrogen sulfide in the hydrolysis ofproteins or in the reduction of sulfates.

At least eleven species of TJiiobacilliis are found in theliterature, and twelve others have been described but notnamed. There is considerable overlapping among thevarious forms, many of them having been onlyincompletely described.

Some, like TJi. tliiooxidans, also oxidize sulfiu and areobligate autotrophic. The thiosulfate- oxidizing bacteriahave been separated into the strictly autotrophic (Th.thioparus), facultative autotrophic (TJi. noveUus), andheterotrophic (Pseudomonas fluorescens) forms: the firsttwo groups increase the acidity of the medium, and thethird group decreases its acidity.

Other Autotrophic Bacteria. Among the other autotrophicbacteria should be mentioned those that oxidize hydrogen,carbon monoxide, and ferrous iron; not all of these bacteriaare strictly soil inhabitants, although certain conditionsmake some of them abundant in the soil.

Heterotrophic Bacteria Heterotrophic bacteriacomprise the great majority of soil organisms. Theydepend on organic materials for then energy sources,and are primarily concerned with the decompositionof cellulose and hemicelluloses, starches and sugars,proteins and other nitrogenous materials, fats andwaxes.

These bacteria vary greatly in structure andphysiology, in abundance, and in importance. Someare aerobic; others are anaerobic.

Some are spore-forming; others are non-spore-forming. Some are Gram-positive; others areGramnegative.

Some are able to fix atmospheric nitrogen; othersdepend upon fixed forms of organic or inorganicnitrogen.

Spore-Forming Bacteria. The soil harbors a largenumber of spore-forming bacteria. The three mostcommon forms can be readily recognized by thegelatin plate method.

Bacillus mycoides is a rapidly liquefying form; itproduces large filamentous to rhizoid colonies.Bacillus cereus liquefies gelatin almost as rapidly andusually forms round colonies with entire edges; thesurface membrane contains granules that tend to bearranged concentrically.

Non spore forming bacteria Thermophilic Bacteria. Miquelwas the first to isolate, in 1879, bacteria capable ofdeveloping at 72°C.

These organisms were found in river mud, sewage, animalexcreta, dust, and soil. It was soon established that variousbacteria capable of growing at 50-70°C, but not at roomtemperature, are found in the soil. Organisms capable ofgrowing at temperatures up to 79.5 °C were also found instable manure.

Myxobacteria.

Myxobacteria occur abundantly in manure and in soil. Thetotal number of these organisms depends upon the natureof the soil.

Some are found only in alkaline, neutral, or faintly acid(pH 8.0-6.0) soils, others in very acid soils (pH 3.7-5.9), andstill others are independent of the reaction.

Denitrifying Bacteria.

A large number of microorganisms are able to reducenitrates to nitrites or to ammonia. Only specificbacteria, however, can reduce, under certainconditions, the nitrate to elementary nitrogen and tooxides of nitrogen, which can thus escape into theatmosphere.

Under anaerobic conditions, the nitrate may serve as asource of oxygen for these bacteria, with organiccarbon compounds as sources of energy.

This process is usually referred to as completedenitiification, and the bacteria are spoken of asdenitrifying bacteria.

Sulfate-Reducing Bacteria. Several organisms capable ofreducing sulfate to hydrogen sulfide have been described.

Vibrio desulfuricans was isolated from soil and othersubstrates. It is a strictly anaerobic, Gram-negative form,growing at 30-55 °C, and able to use salts of organic acids assources of energy.

Urea-Decomposing Bacteria. Pasteur was the first torecognize, in 1860, that ammonia formation from urea isbrought about by a living organism, which he designated asTorula ammoniacale.

It was later established that organisms capable ofdecomposing urea are found in most families of bacteria,actinomycetes, and fungi, but that only certain specificbacteria, whose metabolism is closely connected with thetransformation of this substance, are termed "ureabacteria."

Anaerobic Bacteria. By selective culture methods,Diiggeli found the following numbers of anaerobicbacteria per gram of soil: 1,000-1,000.000 but>Tic acid,0-1,000 cellulose-decomposing, 100- 1,000,000nitrogen-fixing, 100-1,000,000 protein-decomposing,and 100-1,000,000 pectin-decomposing forms.

By the deep tube method, only between 19,000 and900,000 anaerobic bacteria were found per gram ofsoil. No single solid medium can be devised whichwould be fa\orable for the development of allanaerobic bacteria.

Cellulose-Decomposing Bacteria. Cellulosedecomposition in nature is carried out by numerousgroups of microorganisms.

Among these, bacteria occupy a prominent place. Theanaerobic bacteria were at first believed to be the mostimportant agents in the decomposition of cellulose.

It was later found, however, that aerobic bacteria andvarious fungi are far more important than theanaerobic bacteria.

A detailed systematic study of various aerobic cellulose-decomposing bacteria found in the soil has been made byWinogradsky, who divided these organisms into threegenera:

1. Cytophaga: slender, flexible filaments, 3-8 fi long, andpointed at each end; only cellulose can be used as a sourceof energy; the cellulose is changed into a colloidal gel,colored yellow, orange, rose, red.

2. Cellvibrio: slender, bent rods with rounded ends, 2-5 jxlong; actively motile, with one flagellum; cellulosedecomposition is not invariably specific; cream- to ocher-colored pigment, readily diffusing; very abundant,although only two species were described.

3. Cellfalcicula: spindle- or sickle-shaped cells, notexceeding 2 fi in length, with pointed ends; motile, withone flagellum; paper stained green and creani-coloied,ne\er distinctl)' yellow, red, or orange, as the first twogenera are; three species were described.

Other Bacteria. Many other groups of bacteria are foundabundantly in the soil. Among these are mycobacteria,corynebacteria, \arious anaerobic bacteria in addition to thoselisted above, and a host of other bacteria characterized byspecific physiological or morphological properties.

ACTINOMYCETES

Actinomycetes form, taxonomically, a link between the bacteria,tlirough the genera Mycobacterium and Conjnebacterhim, andthe true fungi. They are characterized by the formation of aunicellular mycelium, composed of hyphae, which show truebranching, similar to that of fungi.

The hyphae are rather long and are usually 0.5-0.8 fx in diameter.The mycelium develops either in the substrate or on the surfaceof the substrate as aerial growth.

The mycelium breaks up into short fragments, which may looklike bacterial rods and resemble true bacteria in theirprotoplasmic properties.

When examined directly under the microscope, the aerialmycelium is found to consist of ^'ery fine, characteristic, long orshort branching hyphae, with distinct spore-bearing hyphae.

The reproductive conidia, which are characteristic ofthe genus Streptomijces, are produced by asimultaneous division of the protoplasm in thesporogenoiis hyphae, progressing from the tip towardthe base.

The spores possess a somewhat greater power ofresistance to environmental factors than the vegetativehyphae. They resemble bacteria in size, shape, andstaining properties, are 0.5- 1.5 fx in diameter, 1-2 jxlong, oval to rod-shaped.

All actinomycetes, particularly in young preparations,are Grampositive. In stationary liquid media, theynever cause trn-bidity, but grow either on the surfaceof the medium or in the form of flakes or smallcolonies throughout the medium; they may sink to thebottom of the container or adhere to the glass.

The surface colonies may grow together to form asmooth or wrinkled surface membrane. The colonieson solid media are usually tough, leathery, smooth orwrinkled, often growing high above the surface of themedium, and are broken up only when appreciableeffort is applied.

When transferred to suitable media, the sporesgerminate readily. The older the mycelium, the morereduced is the germinating power of the individualfragments. In shaken cultures, they grow in the formof "clumps" or "colonics" throughout the medium.

This mass of growth can easily be removed byfiltration, leaving a clear fluid.

Soil Fungi

Although fungi are not represented in the soil by somany physiological groups as are the bacteria, manythousands of species find in the soil a temporary orpermanent habitat.

Of the various genera of fungi found in the soil, themost common, both in the number of species and inthe Ireciuenc) ot occurrence, are 7Aj<:,oihijiichus,Mucor, Rhizopus, PcnicilUum, Aspergillus,frichodenna, Fusarium, and Clodosporiuni.

Cellulose-Decomposing Fungi

The addition of cellulose to the soil brings about anextensive development of fungi, most of which possessvery strong cellulosedecomposing power.

These include various species of Penicillium,Aspergillus, Trichoderma, SporotricJuwi, Fusarium,Chaetomium, and other forms.

McBeth suggested that the fungi play a much moreimportant part in cellulose decomposition in moistsoils, particularly in humus soils, than in dry soils.

Mycorrhiza Fungi

The mycorrhiza fungi form a special group oforganisms. They are capable of attacking thesubterranean organs of plants, feeding upon theirorganic constituents.

The plant cells may recover, however, and in their turndigest the fungus mycelium. In this instance, thesubterranean part of the plant and the fungusmycelium form an association which is frequently ofbenefit to both, this union being known as mycorrhizaor fungus-root.

Frank divided the mycorrhiza into two groups:

(1) Ectotrophic mycorrhiza, in which the fungus producesan external investment of the root, in the form of a crownof hyphae, without penetrating into cells other than tlioseof the epidermis; there is an extensive intcrcelluhu‘ de\elopmcnt between the cortical cells of the roots which isespecially characteristic of forest trees.

(2) Endotrophic mycorrhiza, in which the hyphae of thefungus penetrate to the inner parts of the roots, intodefinite root layers, and into the cells, and have littleconnection with the mycelium in the soil.

This is true of plants belonging to the Orchidaceae,Ericaceae, and Eparidaceae, and is now known also formany other plants. Root hairs are frequently absent inectotrophic mycorrhiza and are replaced by hyphae offungi.

Algae

Algae are widely distributed in the soil. Although theyare largely confined to the surface layer and arecontrolled by the moisture content, they may also befound below the surface and even in fairly dry soils.

Since they depend on sunlight for their growth, thesubterranean forms must either lead heterotrophicexistence or remain there largely in an inactive state.

Protozoa

Protozoa are unicellular organisms, varying in sizefrom a few microns to 4-5 mm. Some protozoa are alsoable to form colonies which consist of numerousindividuals.

Other Animal Forms

Animal forms larger than protozoa also occurabundantly in the soil. They range from microscopicnematodes to large earthworms and insect larvae.

Some nematodes (Hetcrodera schachtii) and certaininsects are parasitic on plants; Viruses and PhagesCertain viruses and various phages exist independentlyin the soil.

The mosaic \'irus of wheat can be transmitted from thesoil. Heating the soil for 10 minutes inactivates thisvirus.

Use of Soil Inoculants

The methods of biological control of disease-producing organisms are still insufficiently studied.

Here belong the introduction of birds and otherhigher animals, as well as of certain insects feedingupon specific injurious insects and worms, or the useof predaceous nematodes against plant-pathogenicnematodes, or of entomogenous fungi and bacteriaparasitic upon insects. The phenomena of antagonismmay also be listed here.

Numerous attempts have been made to inoculate thesoil with antagonistic organisms for the purpose ofcontrolling plant diseases.

These efforts have proved to be, in most instances,complete failures, as pointed out previously.

This is largely because, unless the conditions of thesoil are modified by supplying more nutrients to theantagonists or by creating a favorable reaction, theantagonists will not develop.

The introduction of organic materials, such as greenmanures and stable manures, may correct such acondition, thus favoring development of theantagonists, which bring about, directly or indirectly,suppression of the disease-producing agent.

References:SoilMicrobiology by SELMAN A. WAKSMAN

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