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IV. The Numbers of Microorganisms Associated with Plant Roots

IV. The Numbers of Microorganisms Associated with Plant Roots

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SOIL MICROORGANISMS A N D PLANT ROOT6



265



bers in successive washings of the root sample and (e) as numbers per

square centimeter of root surface. The two last named procedures have

not been widely used.

Starkey (1929-1931) preferred to use root surface scrapings for microbiological study of the rhizoplane. Clark (1940) and Mitchell et ul.

(1941) have occasionally employed root surface scrapings, but for the

most part this group (Clark, 1939-1948b; Clark and Thom, 1939;

Stumbo et al., 1942; Thom and Humfeld, 1932) has used samples containing whole roots with adhering soil. For fibrous rooted plants, such as

grasses, the whole root system, or a reasonable aliquot thereof, may be

employed; for larger tap-rooted and fleshy-rooted plants, only the

secondary or feeding roots are collected. The attempt is made t o secure

as much root surface as possible, and to avoid including in the sample

fleshy or bulky portions, which contain a large amount of material not

considered a part of the rhizoplane. When large or fleshy roots are being

studied, root surface scrapings certainly should be employed in preference

to the whole roots.

Samples of composition similar to the ones recommended have been

employed by workers in Canada (Kat,znelson, 1946; Lochhead et ul.,

1940; Timonin, 1940a, b), but whereas many American workers have

expressed microbial numbers per gram of gross sample, the Canadian

investigators generally have determined the soil fraction of each gross

sample, and have computed numbers of organisms per gram of oven-dry

“rhizosphere soil.” There are a few exceptions, for example, Tyner

(1948), also working a t Ottawa, has expressed microbial numbers per

gram of gross sample, oven dry basis. According to Katznelson (1946),

the “rhizosphere effect” is expressed by means of the rhizosphere-soil

ratio (R:S), that is, the number of organisms in the rhizosphere soil

divided by the number in the soil a t a distance from the root.

The desirability of expressing microbial populations on root surfaces

as numbers per gram of adhering soil, regardless of the amount of roots

included, is open to serious question. For soil taken near the roots, or

with but small amount of root material included, observations may well

be reported per gram of soil. For roots with very little soil adhering, it

appears reasonable to express the numbers per gram of sample material.

I t is generally conceded that the roots or materials coming therefrom are

responsible for the increased microbial populations observed in t.heir

vicinity. Clark (1948b) has recently shown that when both root weight

and soil weight in a series of rhizosphere samples from a number of

plants were determined by recovery procedures and oven-drying, there

was significant, correlation between magnitude of microbial population

and root, content of the sample, but no significant correlation between



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soil content and microbial population. Katznelson et al. (1948) have

suggested that for root samples from which as much superfluous soil as

possible has been removed, the recorded data include dry weights of

both roots and rhizosphere soil in the material studied. This suggestion

appears commendable.

9. Density of the Microbial Population Within the Rhizosphere

Plate count estimates of the soil population vary widely; some workers consistently obtain only a very few millions per gram, while others

obtain much higher figures. Thom (1938) has discussed the range of

findings recorded in the literature, and has preferred to accept a general

estimate of 50 millions per gram. Populations in excess of this estimate

are nearly always reported for rhizosphere samples. For the rhizoplane

of corn, mangel beets, and beans, Starkey (1931a) obtained populations

of 653, 427, and 199 millions, respectively, while the second year growth

of clover, mangel beets, and table beets yielded counts of 3,470, 583,

and 485 millions per gram. Considering all the plants together, there

were 24.8 times as many bacteria on root surfaces as in soils close to

roots. Exclusive of the legumes, bacteria were 12.1 times as numerous

on roots; for the legumes alone, they were 50.3 times as numerous as in

soil. Other workers generally have reported rhizosphere populat.ion/soil

population ratios of approximately this same order. Timonin (1940a)

reported increases in total microbial numbers of from 7 to 71 times for

roots of wheat, oats, alfalfa, and peas. Lochhead (1940) reported increases of from 2 to 45 times for a variety of crops; Clark (1940) from

2 to 24 times for cotton and wheat; Zukovskaya (1941), up to 100 times

for potatoes, flax, and clover; and Adati (1939), nearly 200 times for

peas, with lesser increases for other plants.

Such figures show the range of values reported from several different

laboratories. These values are of course all subject to the many errors

of the standard plate count (Harmsen, 1940; James and Sutherland,

1939). Accepting Thom’s estimate of 50 millions microorganisms per

gram of soil, and accepting a rhizosphere/soil microbial population ratio

of 10 as representative of reports in the literature, then roughly 500

millions of microorganisms are to be expected per gram of rhizosphere

sample. This value merely provides a perspective-it will be pointed

out shortly that many factors, such as type of plant, stage or condition

of plant growth, and environmental influences, produce extreme fluctuations in the actual value to be determined.

At this time brief comparison may be made of the magnitude of

microbial counts on roots and those of other plant materials occurring

in nature. Humfeld and Smith (1932) reported plate counts of from



SOIL MJCROORGANTSMS AND PLANT ROOTS



267



5 to 8 billions for the decomposing layer of a green manure in soil.

Clark et al. (1948) reported counts as high as five billion, primarily of a

single species, for late season cotton fibers in bolls prematurely opened

by frost. Starkey (1931a) reported a maximum population of approximately 3.5 billions per gram for roots of sweet clover; the majority of the

plants which he studied yielded microbial populations for the rhizoplane

of approximately one-half billion. For the root system generally, although a t times the populations thereon may approach those encountered

in decaying materials in the field, the microbial populations encountered

ordinarily do not equal those on plant residues subject to immediate

and complete decay in soil. The fact that roots can support comparatively dense populations without undergoing destruction and decay attests

to the continuing supply of organic material sloughed or excreted by the

roots. Some writers consider that the magnitude of the root population

is such that it is indicative of secretory or excretory products being

supplied by the roots in considerable quantity, as the sloughing of dead

parts alone appears inadequate as a source of stimulation.

The question may be raised concerning the proportion of the soil that

consists of microbial tissue and the relative contribution of the root

microflora to the total microbial mass. It is probable that the total

microbial tissue in a fertile soil comprises no more than 0.3 per cent of

the soil weight. This estimate may be reached from data given for the

total number of microorganisms in soil, as revealed by direct microscopic

count.

Using a fluorescence microscope for direct examination of soil, and

employing acridine orange staining to differentiate living and dead organic matter, Strugger (1948) recently has reported from 1,038 millions

to 8,640 millions bacteria per gram of soil on the dry basis. Kendall

(1928) has placed the volume of an average bacterial cell a t 15.7 x

mm.3, its density a t 1.04 and its moist weight a t 16.3 x 10-lo mg. If the

mg., then

dry weight of a single bacterium is taken as 3.26 times

according t o the data cited from Strugger (1948), the total bacterial

mass in soil ranges from 0.03 to 0.28 per cent.

This estimate appears plausible in the light of certain observations

on the relative organic phosphorus contents of soil and of microorganisms.

Converting the estimate just reached from a percentage to a p.p.m. basis,

bacterial tissue contributes roughly 300 t o 3000 p.p.m. to the soil mass.

If microbial tissue contains approximately 3 per cent phosphorus (Porter,

1947), then such tissue may account for from 9 to 90 p.p.m. of phosphorus

in soil. The observation that nucleic acid and microbial tissue show a

similar course of mineralization (Pearson et al. 1942; Thompson and

Black, 1948) has led the latter authors to conclude that the nucleic acid



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fraction of phosphorus in soil and the microbial fraction roughly are

identical. Bower (1949) places the fraction of organic phosphorus occurring a5 nucleic acid as approximately 50 per cent. The values for

microbial phosphorus given above, therefore,, indicate an organic phosphorus content in the soils of from 18 to 180 p.p.m. These values appear

reasonable in view of current concepts of organic phosphorus in soils

(Pierre, 1948).

Krassilnikov (1944) has estimated the microbial content of noncropped soil a t 0.03 to 0.06 per cent, and of soil cropped to legumes, a t

0.18 to 0.27 per cent by weight. His estimates agree rather closely with

those calculated from direct microscopic counts. They also indicate that

the total microbial mass in soil is markedly higher in the presence of

plant roots than in their absence.

Although on a dry basis, microorganisms may account for only a

fractional per cent of the soil mass, in their living state in soil, because

of their high water content, and relatively low density in comparison

to the mineral fraction of the soil, microorganisms may comprise a much

higher percent,age of the soil volume.

3. Some Factors Affecting Rhizosphere Populations

Determined Culturally



a. Influence of Soil Moisture Content a t Sampling. I n the course of

rhixosphere studies with flax grown a t two differing soil moisture contents, Timonin (1940b) noted higher microbial populat,ions in the rhizosphere of plants in soil maintained at 30 per cent moisture holding

capacity than a t 60 per cent. Similarly, Clark (1940) noted increased

bacterial numbers in the rhizoplane of wheat as soil moisture content a t

sampling was decreased from 24.5 to 12 per cent. Thus in both laboratories, higher populations were encountered a t the lower moisture contents employed.

A dissimilar observation commonly is made in microbiological studies

on field soils apart from the rhixosphere. For such samples, decreasing

soils moisture content within the range of maximum water retention to

the permanent wilting percentage is accompanied by decreasing bacterial

populations.

I n a recent study of the effect,of moisture on the rhixosphere population of soybeans grown in the greenhouse, Clark (1948b) again noted that

microbial numbers were higher for roots taken from drier than from

wetter soils. The greater weights of the gross root samples collected

from t.he wetter soils, the macroscopic appearance of such samples, and

the smaller percentage of root material determined as present in them,

make it appear plausible that the microbiological differences encountered



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269



resulted largely from the adhesion of more soil to those roots taken from

the wetter soils. The further observation that when paired containers

in which plants were being grown were brought to low moisture simultaneously, one container then being sampled while dry, the other after

wetting to near saturation just prior to collection of root, material, reduced microbial numbers were found for roots from the wetter soil, also

provided evidence of a mechanical or sampling influence. There is little

reason to believe that there would be an act.ual reduction in bacterial

numbers with heavy watering; such a treatment certainly is not bacteriocidal, a t least not within a short interval of t,ime.

b. Influence of Type and Stage of Plant Growth. Environmental and

sampling effects are not responsible for many differences in microbial

populations reported for plant roots. Starkey (1929b) noted t.hat rhizosphere populations of individual crops differed a t successive stages of

plant growth. H e found relatively small numbers of organisms in the

early growth stages, increased numbers after the plants had reached considerable size, and decreased numbers after fruiting. Greatest numbers

were usually noted a t the stage of maximum vegetative growth and a t

fruiting. Graf (1930), Krassilnikov et al. (1936a, b) and Timonin

(1940a) also noted that rhizosphere populations differ during differing

stages of plant growth.

Rhizosphere populations also differ for different groups of plants.

Almost without exception, microbiological studies of random series of

plants, including both legumes and nonlegumes, show that the former

support higher rhizosphere populations. In due time, differing relative

effects may be determined for other plant groups or species, but until

procedures of sampling and cultural methods of study are refined, one

cannot attempt to arrange the common agricultural plants according to

their relative stimulation of microorganisms in the rhizosphere.

Different varieties of a single plant species may harbor dissimilar

rhizosphere populations. The occurrence of greater numbers of bacteria

in the rhizospheres of varieties of plants susceptible to soil-borne plant

pathogens, even when grown entirely free from disease, than in the

rhiaospheres of resistant varieties has been discussed in Section III-2-b.

c. Influence of the Region of the Rhizosphere Studied. The several

parts of the root system of a single plant harbor dissimilar rhizosphere

populations, a t least insofar as t.hose populations are revealed by cultural

studies. Some of the effects noted may be due to differences in the composition of the samples employed. Whatever the reason, greater rhizosphere populations commonly are noted for the central or crown porbion

of the root and for that portion of the root system in the upper soil

profile than for the more distal portions (Clark, 1939; Sabinin and



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Minina, 1932). Direct microscopic st.udies, however, show that even the

root hairs carry clusters of microorganisms upon them (Linford, 1942;

Rossi e t al. 1936; Starkey, 1939). Inasmuch as the upper horizons in

the soil profile normally contain higher numbers of bacteria and more

organic matter, it is of interest that with roots of wheat, the greatest

numbers of microorganisms were found associated with those roots growing in the uppermost 4 inches of soil, regardless of whether the greatest

soil population was established in the lst, 2nd, or 3rd four inches of soil

by means of heavy appIications of manure (Clark, 1939). Concentration

of bacteria in the upper portion of the rhizosphere, therefore, appears

independent of the normally occurring microbial concentration in the

upper portion of the soil profile. Further studies are needed concerning

t.he distribution of microorganisms in the individual root systems of

plants.

V. THEKINDSOF MICROORGANISMS

FOUND

ON PLANT

ROOTS

1 . Relative Occurrence of the Major Groups of Soil Microorganisms



During the time of the initial investigations on types of organisms

present on plant roots, there was controversy concerning whether the general soil microflora was simply encouraged e n mmse by the presence of

plant roots, or whether certain types were especially favored. Gottheil

(1901) believed that the general soil flora const.ituted the bulk of the root

microflora; Lohnis (1910) was in agreement with this opinion. Various

other workers have contended that there exists a specialized microbial

population on plant roots. With the development of microbiological technique and the accumulation of a more extensive literature, it has become

increasingly apparent that not only are bacteria stimulated by roots to a

greater extent than are fungi and actinomycetes, but, also that particular

groups or species of bacteria are disproportionately encouraged or depressed.

Katznelson et aE. (1948) classify investigations on the qualitative

nature of the rhizosphere microflora along t.wo broad lines; (a) those in

which numbers of specific groups or species are determined on selective

media by plating or dilution methods, and (b) those in which a nonselective medium is used and all colonies on a plate or a representative

sector of a plate are picked and studied from the point of view of cult*ural,

morphological, and physiological behavior. They have also noted that

the study of types of bacteria adhering to buried slides has yielded

useful information. Inasmuch as the literature resulting from the application of such methods of study has become too extensive to permit

reviewing separately each contribution, only summary attention will be



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271



given to many observations, and some isolated and unconfirmed reports

will be ignored altogether.

The increased microbial flora within the rhizosphere is predominantly

bacterial. Starkey’s (1931a) data on sweet clover show that bacteria

are 200 times more numerous in the rhizoplane than in root-free soil, and

that fungi and actinomycetes are 10 times more numerous. Thom and

Humfeld (1932) found that the roots of alfalfa, rye, and vetch stimulated

bacteria, fungi, and actinomycetes in the order named. Adati (1939)

noted increases in these groups in the same relative order. Frequently,

t.he rhizosphere effect on fungi and actinomycetes is reported as negligible.

There is direct microscopic evidence that protozoa and microphagow

nematodes are more numerous within the root zone. By cultural means,

Katznelson (1946) has found a twenty-fold increase of protozoa on roots

of mangels grown in manured soil. Further attempt6 to determine

whether other plants exerted a favorable influence on protozoa were not

successful (Katznelson et al., 1948). The fact that protozoa quite frequently increase in numbers following increases in bacterial populations

makes i t probable that the accumulation of bacteria on roots is aceompanied by increased numbers of protozoa.

6. The Fungal Flora of the Rhizosphere



It remains unsettled whether certain species of fungi are preferentially

encouraged by plant roots. Timonin (1940a) failed to find significant

differences in the fungal flora of the rhizosphere of seedling wheat, oats,

alfalfa, and clover. Later, he (1941) reported that flax varieties susceptible to disease preferentially encouraged certain genera of fungi.

West and Hildebrand (1941) also noted qualitative differences in the

fungal flora of strawberry roots grown under different treatments for

root rot control, and Clark (1942) found that certain groups of fungi

responded t o cotton roots variously treated. Inasmuch as these observations are complicated by plant treatment and by the factor of disease,

there does not yet appear sufficient evidence to name individual species

of fungi as rhizophilic in habit. Even less is known about t,he response

of individual species of actinomycetes, although the direct microscopic

as well as the cultural evidence indicates some increased growth of this

group about plant roots.

3. T h e Bacterial Flora of the Rhizosphere

a. Physiological and Morphological Characterization. According to

Thom (1935), the microorganisms associated with root surfaces belong

to species active in the decomposition of fresh organic matter in soil, and

not to species associated with the breakdown of humus residues. The



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IV. The Numbers of Microorganisms Associated with Plant Roots

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