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III. The Rhizosphere Microflora in Relation to the Growth of Higher Plants

III. The Rhizosphere Microflora in Relation to the Growth of Higher Plants

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1. Influences on the Availability of Nutrient Elements

a. On the Availability of Combined Nitrogen. The amount of nitrogen available in soil is very frequently the limiting factor in crop growth :

consequently, the rate a t which it becomes mineralized from organically

combined, unavailable nitrogen, is a question of prime importance.

Numerous workers (Brown, 1912; King and Whitson, 1900; Lyon et al.,

1911, 1913, 1920, 1923, 1924, 1928; Wilson and Wilson, 1925) have noted

the greater accumulation of nitrate nitrogen after the growth of legumes

than after nonlegumes. This accumulation doubtless can be ascribed

to the higher nitrogen content of the legume residues, inasmuch as the

rate of mineralization of plant material in soil is dependent upon the

carbon/nitrogen ratio of the residues involved. It is generally believed

that nitrogen in the soil is transformed primarily by microbial activity,

and that claims for photonitrification processes have not as yet been adequately substantiated. The work of Broadbent and Norman (1947) has

suggested t*hatmicrobiological transformation of nitrogen in soil may be

limited a t times by the absence of available energy material to support

a vigorous microbial population.

Nitrogen-balance experiments on legume-cropped soils have been rendered impracticable by the fact that legumes in symbiosis with nodule

bacteria are able to fix atmospheric nitrogen. Several studies have been

made of the influence of the growth of nonlegumes on nitrogen mineralization in soil. Mineralization of nitrogen under such crops appears to

differ in rate from that in fallow soil. I n 1914 Russell reported that, at

the end of a growing season, land cropped to wheat and barley contained

less nitrate than fallow land, even though an accounting was made of

the nitrogen removed by the crop. Lyon et al. (1923), in experiments

with oats and maize, demonstrated that the accumulat*ionof nitrate under

these crops was less than in corresponding fallow soil. Allowance was

made for the nitrogen taken up by the crop. Prescott (1920) noted

depression of nitrification in similar experiments with maize and wheat.

It remained unsettled whether the rate in cropped soil was variable for

different crops or for differing growth stages of a single crop. Nor was

it determined whether there was an actual depression of mineralization

or an apparent one caused by denit,rification and loss of gaseous nitrogen

to the air. There have been occasional claims that cropping may result

in a loss of mineral nitrogen not entirely accounted for by that taken

up by the crop or by changes in the organic nitrogen content of the soil.

Pinck et al. (1945) suggested that there could be loss of nitrogen due to

metabolic processes occurring within growing plants.

Not all workers have agreed that crops depress mineralization.



Greaves et al. (1917) and Lohnis (1926) believed that plant growth

favored mineralization of nitrogen. Lyon e t al. (1923) believed that

maize might stimulate nitrogen mineralization during the earlier stages

of growth. Starkey (1931b) found that nitrates accumulated more

rapidly in soils taken from near the roots than in soils taken some distance away; he concluded that cropped soils have a greater nitrifying

capacity than do fallow soils. Soil taken from near the roots, however,

may contain more organic material than that taken some distance away,

and consequently the release of mineral nitrogen would be greater from

soil from within the root zone simply because such soil contains more

readily decomposable material. The results would not give true est.imates

of the nitrifying capacities of soils under growing crops. Starkey suggested that the influence of plant growth on nitrification may have been

due to the addition of organic matter with narrow carbon/nitrogen ratio

to the soil resulting in local liberation of ammonia on decomposition.

Goring and Clark (1949) have recently studied the mineralization of

nitrogen in soil under the influence of a number of growing crops, and

have reported nitrogen mineralized in soil cropped for 13 weeks to be

approximately half th at mineralized in aliquots of the soil maintained

fallow. I n fallow soil, nitrogen mineralized averaged 30 mg. per kg. of

soil; in cropped soil, with allowance for the nitrogen taken up by the

growing plant (tops and roots), 14 mg. of nitrogen per kg. were mineralized. Mineralizations determined for the several crops were as follows:

timothy, 20.4; wheat 19.4; brome grass, 18.6; tomato, 17.6; oats, 15.0;

tobacco, 11.6; rye, 7.6; and Sudan grass, 1.4 mg. per kg.

The depression of mineralization was positively correlated with total

weight of roots in the cropped soil lots and also with total number of

bacteria contributed by the roots of the crop grown, but negatively

correlated with the nitrogen content of the harvested roots. These correlations were interpreted to indicate an immobilization of nitrogen by the

rhizosphere microflora. This int,erpretation was supported by a subsequent observation that during 16 weeks of incubation in the laboratory,

nitrogen mineralization in soil lots previously cropped exceeded th a t in

soil lots previously fallowed. I n fact, for the majority of crops, the

accelerated mineralization following cropping approximated the previously observed depression of mineralization. With longer incubation, it

is possible that the few soil lots for which exception must be made would

likewise have made up their previous deficits. It was also observed that

following cropping, the rates of nitrogen mineralization were surprisingly

uniform for the several soil lots previously cropped, regardless of the

crop grown.

The numbers of nitrifying bacteria were found unaffected by crop



growth. Other investigators have also failed t o find differences in numbers of nitrifying bacteria in rhizosphere soil and the surrounding soil.

Starkey (1932a), Graf (1930) , and Katznelson (1946) noted no appreciable influence of crop growth on numbers of ammonia-oxidizing and

nitrite-oxidizing bacteria. Numerous investigators have noted that in

the rhizosphere there are increased numbers of bacteria which under

suitable conditions are capable of bringing about denitrification, but

there has been no direct evidence that nitrogen loss to the atmosphere

does occur.

To the possibility that qualitative shifts in the microflora may be

sufficient to alter the rate of nitrogen mineralization must also be added

the possibility that with crop growth differences may be established in

aeration or in moisture that in turn affect nitrogen transformations.

Willis and Green (1949) have reported a more fairorable total nitrogen

balance in flooded soil cropped to rice than in flooded soil maintained


I n summary, mineralization of nitrogen appears depressed during crop

growth. At present no adequate explanation can be given for the data

recorded in the literature. It does appear plausible, however, that there

is a microbiological immobilization of nitrogen during crop growth, and

that this factor may account for the apparent difference in mineralization.

Undoubtedly, nitrogen transformations in soil present a fertile field for

the application of isotopic techniques.

b. On the Fixation of Free Nitrogen. Following Beijerinck’s discovery (1901) of the Azotobacter group, Hiltner (1904) stressed that these

nitrogen-fixing bacberia were stimulated by growing roots. Beij erinck

and Van Delden (1902) found Azotobacter in the vicinity of roots especially.in conjunction with B . radiobacter. Greaves (1918) reported Azotobacter more abundant in cropped than in virgin soils. Several Russian

workers (Rokitzkaya, 1932; Sheloumova and Menkina, 1936; Sidorenko,

1940) have noted favorable response by Azotobacter to roots of certain

plants. Starkey (1931a) cited scattered references that Azotobacter is

especially favored by cruciferous plants, but he himself did not find

appreciable stimulation of that microorganism by plant roote. Poschenreider (1929) examined the roots of crucifers and found Azotobacter

present; he then extended his work and found this bacterium associated

with the roots of a wide variety of plants (1930). His observations have

been cited in support of the contention that Azotobacter is particularly

adapted to the rhizosphere. Careful reading of Poschenreider’s papers,

however, shows that he used enrichment cultures only, and therefore his

results can hardly be considered as quantitative. I n fact, a t certain

seasons of the year, he could not find Azotobacter in the closer rhizo-



sphere, even though the organism was present in the adjacent soil. Truffaut and Vladykov (1930) reported Azotobacter generally present in the

rhizosphere of wheat, but again, the criticism may be made that their

observations are of doubtful value, as they did little more than record

the presence of some easily cultured and easily recognized soil organisms.

Several of the bacteria they named in addition to Azotobacter can hardly

he considered as rhizophilic organisms.

Lipman and Starkey (1935) stated that the bulk of the available

evidence showed that Azotobacter does not appear more frequently in

the rhizosphere than in soil apart from roots. Most studies since 1935

have been in agreement with their summarizing statement. Neither

Clark (1940) nor Jensen (1940) found Azotobacter in the rhizosphere of

wheat in soils generally free of Azotobacter. Jensen and Swaby (1940)

failed to find Azotobacter stimulated by the roots of legumes. Krassilnikov (1934) reported that Azotobacter was depressed by the roots of

plants. Katznelson (1946) failed to note any positive response to mangels by Azotobacter, even t,hough other microbial groups were markedly

stimulated. Recently, Clark (1948a) has reported that Azotobacter introduced into the rhizosphere of tomatoes disappeared rapidly. The

disappearance of Azotobacter was found to be more rapid in cropped

than in uncropped soil. Addit,ion of tomato root fragments to soil did

not affect the Azotobacter content of Webster loam normally containing

Azotobacter. The Azotobacter flora of soil normally containing these

bacteria was no greater when such soil was cropped to tomatoes, to soybeans, to broccoli, or to mustard than when it was maintained fallow.

Paralleling the disagreement on the occurrence of Azotobacter in the

rhizosphere, there has been controversy concerning the value of crop

inoculation wit.h Azotobacter. Several early investigators claimed benefit

from inoculation of nonlegumes, but others failed to duplicate their successes, and prior to 1925 there was very little inoculation of nonlegumes

(Allison, 1947). Within the past two decades, extensive claims have

been made by a number of Soviet scientists that yields of nonleguminous

crops are superior following seed or seedling inoculation with Azotobacter.

Field inoculation with this bacterium in the U.S.S.R. reached 5 million

acres in 1942, and is estimated to have tripled since that time. Allison

(1947) has recently reviewed t,he Russian literature, and it is unnecessary

to duplicate his citations here,

Although few workers outside the U.S.S.R. believed that Azotobacter

could be used profitably for crop inoculation, the extent of the Russian

claims has prompted some furt,her investigation. Only negative results

have been reported. Allison and coworkers (1947) failed to obtain increased crop yields or increased nitrogen content in crops receiving



Azotobacter inoculations in greenhouse experiments. Gainey (1946) has

reported negative results from Kansas field experiments carried on for

20 years. Timonin (1949) has reported failure to obtain increased yields

in field experiments conducted in Canada under widely differing conditions of soil and climate.

Although anaerobes capable of fixing nitrogen in culture solution have

been found in association with the roots of rice plants (Sen, 1929), reports

concerning the incidence of anaerobic, free-nitrogen-fixing organisms in

the rhizosphere are too few and too incomplete in character either to

justify any summarizing statements concerning them or to ascribe to

them any importance in fixation of nitrogen in the rhizosphere. Nor does

there appear any satisfactory evidence that algae capable of fixing

atmospheric nitrogen are of any especial importance in the nitrogen

economy of cropped soils.

I n summary, there are a t present neither adequate microbiological

data, nor acceptable crop yield data, to warrant a positive st,atement that

there is any increased fixation of atmospheric nitrogen in the rhizosphere,

with or without attendant benefit to t,he growing plant, by either anaerobic or aerobic microorganisms other than rhizobia.

c. The Chemical Transformation of Elements Other Than Nitrogen.

In soil, microorganisms transform many elements in addition to nitrogen. I n general, microbial transformations affecting plant growth involve

either the oxidation or reduction of inorganic compounds, the decomposition of organic compounds, or the assimilation of materials into

microbial tissue. The availability of iron, sulfur, phosphorus, potassium,

and of other elements, as affected by microbial transformations apart

from t,he rhizosphere, has been discussed by Waksman and Starkey


It was established early that manganese could be rendered unavailable

to plants by the oxidizing action of soil bacteria. Beijerinck (1913) and

Sohngen (1914) demonstrated that both soil bacteria and fungi were

capable of converting available manganese into the unavailable form.

It was also noted that various plants grown under conditions of manganese deficiency developed characteristic diseases. The grey-speck

disease of oats is illustrative. The role of manganese in the etiology of

this disease has been discussed by Samuel and Piper (1929) and Gerretsen (1937).

Recognizing that susceptibility of oats to grey-speck disease is varietal, Timonin (1947) compared the rhizosphere microfloras of resistant.

and susceptible varieties. It was found that the rhizosphere of the susceptible variety of oats harbored a denser population of manganeseoxidizing, casein-hydrolyzing, and denitrifying bacteria than did the



rhizosphere of a resistant, variety grown in the same soil under identical

conditions. Fungi on the other hand were most numerous in the rhizosphere of the resistant variety. With application of soil fumigants, the

bacteria capable of oxidizing manganese were greatly reduced or completely eradicated, and susceptible plants grown in such treated soil

showed lowered incidence of disease or were disease-free. There was a

positive correlation of 0.939 between severity of disease and number of

manganese-oxidizing and cellulose-decomposing organisms. Such a study

is of interest as an example of the microbiological factors involved in

manganese transformations. Fuj imoto and Sherman (1948b) have discussed chemical aspects of the manganese cycle in soil.

Activities of microorganisms in phosphorus transformations in soil

have been summarized by Pierre (1948) under the following headings:

( a ) mineralization of organic phosphorus; (b) immobilization of available phosphorus; and (c) effects on the solubi1it.y of organic and inorganic phosphorus compounds.

Organic phosphorus compounds, which make up a significant fraction

of the total phosphorus of soils, are readily subject to microbial attack.

Thompson and Black (1948) and Thompson et al. (1949) noted close

correlat,ion between the mineralization of phosphorus, nitrogen, and carbon in soils during incubation. Only a few scattered references exist

concerning the role of the rhizosphere flora in mineralization of phosphorus from organic compounds. Verona and Luchetti (1931) ascribed the

ability of cruciferous plants to use more phosphorus to a favorable microflora in their rhizosphere. Some Russian microbiologists have isolated

microorganisms which break down lecithin and nucleic acid in culture

and have claimed that seed inoculation with such types gives increased

crop yields (Menkin, 1946). Such inoculation claims have not been


Phosphorus immobilization by microbial populations in soil has been

shown by Thompson et al. (1949). The problem of phosphorus immobilization in the rhizosphere has not been investigated. Whether there

occurs immobilization commensurate with the size of the root system

and the magnitude of the microbial population in the rhizosphere has

not been shown. At least it can be said with certainty that if nitrogen

immobilization by the microbial population of the rhizosphere occurs,

then phosphorus immobilization also occurs, inasmuch as neither element

can be immobilized in microbial tissue independently of the other.

d. I n the Solution of Relatively Insoluble Minerals. I n the soil,

carbon dioxide may facilitate the release of nutrients from relatively

insoluble soil minerals. Therefore, i t is appropriate to preface a discussion of the effects of the rhizosphere flora upon the solubility of



minerals with a few remarks on the role of microorganisms in carbonic

acid production in soil.

Carbon dioxide in cropped soil is produced almost entirely from

biological activity, either directly from root respiration, or from microbial

attack upon soil organic matter and upon organic materials coming from

plant roots. Carbon dioxide production has been observed greater from

planted than from unplanted soil (Neller, 1922; Headdon, 1927) and

bicarbonates have been found in much greater concentration near plant

roots than in soil apart from roots (Metzger, 1928). St.arkey ( 1 9 2 9 ~ )

has shown that soil taken from near roots yielded more carbon dioxide

than did distant soil. Such an observation doubtless can be explained

by the greater organic matter content of the root-adjacent soil, together

with its sharply higher numbers of microorganisms.

Attempts have been made to evaluate the relative importance of

rhizosphere microorganisms in the formation of carbon dioxide about

plant roots. Following the experiments of Liebig (1858) and Sachs

(1860) , who demonstrated that marble and ostheolite were etched by

plant roots, Fred and Haas (1919) showed that the etching of marble

by roots of peas was markedly less in sterile cultures than in cultures

contaminated with bacteria. They concluded that microorganisms played

an important role in etchings obtained.

Data of Lundeglrdh (1924) indicate that nearly half of the carbon

dioxide arising from plant roots growing in unsterilized sand is produced

by their accompanying microorganisms. Root respiration in unsterilized

and in sterilized sand amounted to 5.57 mg. and 3.05 mg. carbon dioxide

per hour, respect.ively. Barker and Broyer (1942) noted that 63 per cent

of the carbon dioxide produced by squash roots in aerobic culture came

from bacterial respiration. Stille (1938) concluded that microorganisms

were responsible for roughly 35 per cent of the carbon dioxide evolved

from a sand and solution substrate in which mustard plants were being


Parker (1924, 1925) questioned whether carbon dioxide was of any

value in the feeding power of plants. He noted no correlation between

carbon dioxide in the soil and mineral content of plants grown. The

removal of carbon dioxide by continuous aeration did not influence plant

composition. Truog (1927), and McGeorge (1938) , however found that

carbon dioxide increased the availability and absorption of phosphorus

by plants, at least in calcareous soils.

Gerretsen (1948) recently has published interesting data showing an

effect on plant nutrition by microorganisms in the rhizosphere. Oats,

mustard, sunflower, and rape were grown under sterile and nonsterile

conditions in pot culture in order to determine the influence of micro-



organisms on the top growth and the phosphate intake of plants. Data

in Table I1 are illustrative of the order of increases in yield and in

phosphorus content for plants grown with microorganisms present in their


Gerretsen pointed out that even though the presence of bacteria considerably increased the quantity of phosphorus in the plants grown,

nevertheless the plants with sterile rhizospheres also absorbed notable

amounts of phosphorus from tricalcium phosphate and bone meal. I n

view of t,he negligible solubility of phosphorus in such compounds, he

assumed that plant roots are able to mobilize some phosphorus independently of microbial activity, and he considered it possible that carbon

dioxide production by microorganisms was the effective mechanism in

the increased phosphate intake observed. Whatever the explanation, his

evidence supports the view that the rhizosphere flora increases the phosphate intake by plants.

Gerretsen’s experiments were performed with basic phosphates. I n

acid soils, in which the inorganic phosphates of iron and aluminum tend

to accumulate, it is possible that dissimilar results might be obtained.

His observation that in an iron-deficient substrate, the increased phosphorus brought into solution when bacteria were present caused precipitation of the small amounts of available iron, and that as a result, much

poorer plant growth was obtained in the presence of bacteria than in

their absence. These aspects of nutrient intake certainly warrant further

investigation. The solubility of elements other than phosphorus may be

affected by microbial activity in the rhizosphere. Calcium, magnesium,

potassium, and iron may be influenced by carbonic acid of microbial

origin or by other acids arising as products of nitrification, sulfur oxidation, and fermentation.

e. Mycorrhizae and Bacteriorrhizae i n Relation to Plant Nutrition.

Although Frank (1885) early suggested that mycorrhizal-forming fungi

are beneficial to the plants on which they occur, there was for many

years controversy concerning the mutualistic nature of the association

(Schmidt, 1947). Within the past dozen or so years, several workers

have presented convincing data that mycorrhizae benefit the higher plant.

Hatch (1936,1937) compared pine seedlings in seedbeds with and without

mycorrhizal fungi, and found that the mycorrhizal plants absorbed 86

per cent more nitrogen, 75 per cent more potassium, and 234 per cent

more phosphorus than did non-mycorrhizal plants. Young (1936) obtained similarly positive results, as did Mitchell et al. (1937). McComb

(1938, 1943) and McComb and Griffith (1946) noted increased phosphorus intake with mycorrhizae present on pine seedlings. Rosendahl

(1942) found that mycorrhizae increased intake of potassium from a


Influence of Microorganisms on the Yield and Phosphate Intake of Plantsa


Total dry weight

of plants

Increase in yield with

microbes present, compared with sterile



PsO, absorbed

by plants


Increase in P,06 absorbed

with microbes present,

compared with sterile


Oats, Avena sativa

Blank, without





Algerian phosphate












































75 2








Mustard, Sinapk albo







Sunflower, Helicrnthus annuus





After Gerretsen (1948).





Mean values only from original tables.



sand orthoclase medium. Rout,ien and Dawson (1943) reported that

mycorrhizae enabled pine roots to absorb Ca, Fe, K, Mg, and P a t lower

levels of base saturation of the clay substrate than was possible in their


As such reports indicate, most of the recent workers on mycorrhizae

suggest that such formations are of importance in the intake of nutrient

salts, but do not claim that mycorrhizae enhance the nitrogen nutrit.ion

of the plants on which they occur. Hatch (1937) concluded that mycotrophy is not a special adaptation for acquiring nitrogen, as mycorrhizal

organisms neit,her make available combined organic nitrogen, nor do they

fix free nitrogen. Allison et al. (1934) failed to find mycorrhizal fungi

capable of fixing appreciable quantities of free nitrogen.

The possibility that mycorrhizae benefit the plant by affecting its

carbohydrate nutrition is likewise not well supported. Schmidt (1947)

has contrasted Bjorkman’s view that mycorrhizal fungi invade the plant

and benefit it by removing excess carbohydrate with the reports of Falck

(1923) , Francke (1934) and MacDougal and Dufrenoy (1944, 1946)

that mycorrhizal fungi make additional energy material available to the

plant. The suggestion of Perotti (1926) that bacteriorrhizae mobilize

carbohydrates for transportation within the plant has not been substantiated.

Both the experimental data on tree growth and composition, as cited

above, and the greatly increased absorptive area provided in mycotrophy

(mycorrhizal roots may expose several hundred times as much absorbing

surface as nonmycorrhizal roots) support the proposit,ion of Stahl (1900)

that mycorrhizal formations are of benefit because such organs increase

nutrient salt absorption from the soil. It is interesting to note that

essentially the same benefits are ascribed to the nonsymbiotic rhizosphere

microflora as to mycorrhizal fungi. Preceding sections have indicated

that combined nitrogen is not made availale more rapidly by microorganisms in the rhizosphere and also that there is no acceleration of

nitrogen fixation therein by nonsymbiotic nitrogen-fixing organisms, but

that there is good possibility that the rhizosphere flora increases the

availability of mineral salts to the plant.

Thus far, however, the experimental evidence concerning the role of

the nonsymbiotic root-surface bacterial flora in plant nutrition is far

more limited than that. for the mycorrhizal fungi, and it is only within

the last decade or so that mycotrophy has been accepted generally aB of

practical significance (Schmidt, 1947). I n rhizosphere microbiology,

further studies such as the one by Gerretsen (1948) on phosphorus intake

are clearly needed. Until factual data are a t hand, the role of the rhizosphere microflora in plant nutrition cannot accurately be assessed.



2. Some Influences on Plant Growth and Welfare

a. Production of Plant Growth Substances in the Rhizosphere. In

1914 Hoffman reported that growth of individual species of bacteria in

soil produced changes in the soil solution manifested by an increased or

decreased development of plant seedlings when grown in extracts made

from such soils. There soon followed a number of papers concerning the

occurrence, nature, and action of plant growth substances. It is now

generally considered that such substances are essential to the best growth

and development of green plants. General reviews on plant growth

hormones have been given by Boysen-Jensen (1936) and by Thimann

and Bonner (1938). A general review of the role of vitamins in plant

development has been prepared by Bonner (1937).

Soil microorganisms can produce effective quantities of growth stimulating substances. Bacterial synthesis of various growth substances used

by plants has been shown by McBurney et al. (1935), West and Wilson

(1938), West (1939), Roberts and Roberts (1939), and Thompson

(1942). That such syntheses by microorganisms are of more than

academic interest has also been indicated. Clark (1930) and Clark and

Roller (1931) found that sterile organic extracts added to nonsterile

medium accelerated the rate of reproduction in Lemna, but were without

influence when added to sterile medium. Thimann (1939) expressed the

opinion that growth substances produced by rhizobia are of significance

in nodule formation on legume roots. Wilson (1940) lists vit,amin B-1,

vitamin C, nicotinic acid and biotin as of probable importance in the

symbioses between leguminous plants and rhizobia. Isakova (1936,

1939, 1940) and Isakova and Smirnova (1937) concluded that bacteriorrhizal types are stimulatory to germinating seedlings, and that such

stimulation extends throughout plant development. Brown (1946) concluded that some seeds will not germinate in the absence of external

supplies of plant growth accelerators.

Berezova et al. (1938) believed that the success of seed or seedling

inoculation with Azotobacter was due to auxin production by the introduced organisms. Allison (1947) considered the most plausible explanation of the beneficial effect of Azotogen on crops to be the production of

growth accelerating substances, which promote plant growth a t the

maximum rate.

It is also possible that toxic as well as stimulatory factors may be

produced by rhizosphere bacteria. Schreiner and Shorey (1909) reported y’ears ago that decomposition products in the soil had harmful

effects upon plant growth. More recently, McCalla and Duley (1948)

have demonstrated that extracts of decaying sweet clover inhibit the

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