SOIL M O I S T U R E C O N T E N T A N D R O O T E X U D A T E S I N R E L A T I O N TO R H I Z O S P H E R E E F F E C T IN

L E G U M I N O U S WEEDS 1

By SHANKER BHAT SULLIA ~

(Department of Botany, Bonaras Hindu University, Varanasi-5)

Received May 17, 1973

(Communicated by Prof. T. S. Sadasivan, F.^.sc.)

ABSTRACT

In leguminous weeds, Cassia torn L. and Crotalaria medicaginea Lamk., the ageing of the plants was accompanied by a quantitative increase in the rhizosphere fungal population which reached the maximum level at the senescent stage of the plants when the moisture content of the soil had fallen significantly. Concomitant with these phenomena was the decrease in the amino acids and sugars contained in the root exudates. Th role of root exudates, moribund root cells and the moisture corltent of the soil in bringing about these changes in the mycoflora are discussed.

INTRODUCTION

It is well known that the rhizosphere, apart from differing from the rest of the soil in its microflora, exhibits a pattern of fluctuation depending on fac- tors such as plant age and environmental factors. Earlier workers have visualised the rhizosphere effect as something closely connected with the active growth of the plant. It was also shown that the rhizosphere eitect increased with the age of the plant fill the peak period of vegetative growth, .e., the flowering stage and then deoreased (Timonin, 1940; Contois, 1953;

Ivarson and Katznelson, 1960; Rao, 1962; Vidal artd Visorta, 1965). Some workers have, however, observed a decrease in the fungal population of the rhizosphere with plant age (Subba-Rao et al., 1961). These changes in the microflora have been mainly attributed to root exudates which were shown to contain several nutrients such as amino acids, sugars, organic acids, vita- mins and some hormones (Andal et al., 1956; Rovira, 1956; Sulochana, 1962 a, b; Vancura and Hovadik, 1965). However, previous workers have studied the root exudates only at the seedling stage due to technical difficul- ties, although a few have attempted studies at two stages of growth of the

1 Part of Doctoral Thesis, Banaras Hindu University. 2 Present address: Department of Bot~my, Bangalore University, Bangalore~l.

264

Soil Moisture Content and Root Exudates 265

plants (Katznelson et aL, 1955; Vancura and Hovadik, 1965). The present study was aimed at finding out the nature of the mycofloral changes in cer- tain leguminous weeds growing in their own ecological niches, since it was felt that patterns of mycopopulation changes in these plants might differ from those encountered in the cultivated plants grown under artificial condi- tions. Three important phases of plant growth, viz., seedling, flowering and fruiting stages, were studied in order to obtain a picture of root exudates in relation to the ageing of the plants. Cassia tora L. and Crotalaria medi- caginea Lamk. were selected for the study in view of their abundance in the area and wide range of distribution. The dynamics of the rhizosphere myeo. flora of these plants were studied in relation to the quantitative changes in the chemical composition of the soil, soil reaction, soil moisture content and root exudates.

MATERIALS AND METHODS

Samplings were made at monthly intervals from three plots situated at a distance of about one kilometer from one another, near the University Campus. The plots were fenced to avoid biotic disturbance. From each plot, three plants were picked and their rhizosphere soil was bulked. The non-rhizosphere soil was sampled from a distance of about 60 cm from the base of the plants. The number of fungal propagules per gram of soil was estimated by dilution plate method using Martin's medium (Martin, 1950).

The moisture content of the soil was determined from a representative 100 g sample from each plot after drying at 100 ° C to constant weight.

Root exudates were collected at three stages of plant growth, viz., seed- ling, flowering and fruiting stages. Seeds were scarified with carborundum to ensure uniform germination, surface-sterilized with 0-1 per cent mercuric chloride and aseptically transferred to 250 ml flasks containing acid-washed sand wetted with sterile distilled water. Fifteen seeds were transferred to each flask and 6 replicates were maintained for each plant species. After 2 weeks under light, root washings were collected freeing the roots carefully from the sand, the washings from the sand also being collected. All the washings were bulked and concentrated in a water bath at 60 ° C, and this sample was used for chromatographic analysis.

The root exudates at the flowering and fruiting stages were collected from 3 and 4 month-old plants grown in sterilized sand inside aseptically maintained glass chambers. The plants were transferred from sand to flasks containing 150 ml of 10-fold diluted Pfeffer's culture solution. Five plants

Acad.--B 4

266 SHANKER BHAT SULLIA

were kept in each flask supported by a plug of sterile cotton in such a way that the roots dipped in the culture solution and the shoots were exposed. The flasks were covered on the outside with black paper. After 7 days under diffused light, the solution with root exudates was collected and concentrated.

Circular Paper Chromatography with Whatman Paper No. 1 and butanol- acetic acid-water solvent was used for both amino acids and sugars. Nin- hydrin (0.1 per cello was used as spraying reagent for amino acids while aniline-diphenyl amine-phosphoric a~,id reagent was used for sugars (Buehan and Savage, 1952).

The carbon, total nitrogen, calcium and magnesium contents of the soil were estimated by the method of Piper (1944). The dilute acid-soluble phosphorus was estimated by the colorimetric method of Jackson (1958).

Statistical analysis of data was done by applying analysis of variance.

RESULTS AND DISCUSSION

Members of Deuteromycetes were the most abundant fungi in both rhizosphere and non-rhizosphere soils. Phycomycetes were more ill the rhizosphere than in the non-rhizosphere soil (Table I).

There was an increase in the number of fungal propagules in the rhizo- sphere with the age of the plants. The non-rhizosphere mycopopulation was relatively stable. In all the cases, the maximum number of fungal propa- gules were found at the senescent stage of the plants. The R/S ratios (No. of fungi irt rhizosphere/No, of fungi in non-rhizosphere) increased with plant age from July to December of two successive years of observation (Figs. 1 and 2). Statistically, the effect of plant age on the quantitative changes in the rhizo- sphere ftmgal population was significant. The quantitative differences in the mycopopulations of the rhizosphere and non-rhizosphere were also significant but the differences between the three plots and between the two plant species were insignificant. The dominant fungi in the rhizosphere of the two plant species are given in Table II. Only those fungal species that occurred in more than 50 per cent of the dilution plates were cortsidered as dominant.

It must be noted that there was only a quantitative increase in the rhizo- sphere mycopopulation with plant age and not a succession of fungi. The dominant fungal species present at the seedling stage persisted throughout without being replaced by other species.

The number of amino acids and sugars in root exudates decreased with plant age (Tables III and IV). In Cassia tora, root exudates contained 8 amino

Soil Moisture Content and Root Exudates 267

TABLE I

Number of species belonging to different major groups of fungi isolated from the rhizosphere and non-rhizosphere soils

Plant species Major groups of Rhizosphere fungi

Non-rhizosphere

Plots Plots

i ii iii i ii iii

Cassia tora

Phycomycetes 7 12 8 7 10 7

Ascomycetes 5 4 4 6 7 6

Basidiomycetes 1 . . . . . . . . . . .

Deuteromycetes 41 36 34 45 42 36

TOTAL 54 52 46 58 59 49

Crotalaria medicaginea

Phycomycetes 11 10 8 6 8 7

Ascomycetes 4 4 4 5 5 4

Basidiomycetes . . . . . . . . 1 ..

Deuteromyeetes 36 34 31 36 44 38

TOTAL 51 48 43 47 58 49

acids in the seedling stage, 7 in the flowering stage and 5 in the fruiting stage. Leucine, methionine, valine, glutamic acid and aspartic acid were present in all the stages. An unidentified amino acid U1 was present only in the seedling stage. A marked decrease in the number of sugars exuded by Cassia tora roots in the flowering and fruiting stages could be noted. Only glucose was present in the above two stages while in the seedling stage there were 4 different sugars (Table IV). A similar decrease in the number of amino acids and sugars in the root exudates was evident in the case of Crotalaria medicaginea (Tables III and IV).

2 6 8 SHANKER BHAT SULLIA

TABLE I I

Dominant fungal specie~ in the rhizosphere of C a s s i a t o r a L. and C r o t a l a r i a

m e d i c a g i n e a Lamk.

F u n g a l s p e c i e s

Cassia tora Crotalaria medicaginea

P l o t s P l o t s

i ii iii i ii iii

Rhizopus nigricans E h r e n b e r g . . + - - + + + - -

Mucor luteus L i r m e m a n n . . + - - + - - + - -

M. hiemalis W e h m e r . . - - + - - -J- + - -

Cunnighamella echinulata T h a x t e r . . + - - - - + + - -

A~pergillns nidulans ( E i d a m ) W i n t e r . . + - - - - + + - -

Thielavia terricola ( G i l m a n a n d A b b o t )

E m m o n s . . - - + - - + - - +

Phoma glomerata ( C o r d a ) W o l l e n w e b e r

a n d H o c h a p f e l . . - - - - + - - - - +

Aspergillus'terreus T h o r n . . + + + + + +

.4. flavus L i n k . . + + + + + +

A. niger v a n T i e g h a m . . + + + + + +

A. candidus L i n k . . + - - - - + + +

A. luchuemis I n u i . . - - + - - - - - - +

A. sydowi ( B a i n e r a n d S a r t o r y ) T h o r n

a n d C h u r c h . . - - - - + + + - -

Penieillium humicoIa O u d e m a l l s . . - - - - -~ - - + +

P. herquei B a i n e r a n d S a r t o r y . . - - + - - - - - - +

Paeeilomy¢exfusisporus S a k s e n a . . + - - + + - - - -

Cladosporium herbarum ( P e r s o o n ) L i n k + + + + + +

C. epiphyllum P e r s o o n . . + - - - - - - + - -

Curvularia lunata ( W a l k e r ) B o e d i j n . . + + + + + - -

Trichoderma lignorum ( T o d e ) H a r z . . - - + - - - - + - -

Fusarium udum (Be rke l ey ) W o l l e n w e b e r - - - - - - + + +

Fusarium s p . -- + + + + + +

Ster i le w h i t e m y c e l i u m . . - - - - + - - + - -

+ D o m i n a n t (Colonies in more than 50 per cent of dilution plates).

- - Not dominan t (Colonies always in less than 50 per cent of the dilution plates).

,So ii Moisture Content and Root Exudates

TABLE III

Amino acids from root exudates o f Crotalaria medicaginea Lamk. and Cassia tora L. at different stages of plant growth

269

Amino acid

Seedling Flowering Fruiting stage stage stage

CR CA CR CA CR CA

1. Leucine

2. Methionine

3. Valine

4. Tryptophan

5. Tyosine

6. Proline

7. Alanine

8. Glutamic acid

9. Aspartic acid

10. Asparagine

11. Unidentified (U1)

12. Cystine

• °

Q 9

Q 6

Q

g O

O Q

+ + + + + +

+ + - + - +

+ + - + - +

- - + + + + - -

+ + + + + +

+ + + + + +

C R - Crotalaria medicag#lea C A - - Cassia torn ÷ - - P r e s e n t

A b s e n t

The pH of the soil ranged from 7.1 to 7-4. It was never acidic. The quantitative changes in the carbon, nitrogen, phosphorus, calcium artd magnesium content of soil were statistically insignificant and since no corre- lation could be shown between the above factors and the mycopopulation changes in the rhizosphere, the chemical analysis data are not presented in this paper.

A c a d ' - - B 5

270 S H A N K E R B H A T S U L L I A

TABLE IV

Sugars in the root exudates of Crotalaria medicaginea Lamk. and Cassia tora L at different stages of plant growth

Seedling Flowering Fruiting Sugar stage stage stage

CR CA CR CA CR CA

1. Rhamnose . . -- -- + -- -- --

2. Fructose .. 4- + + -- -- --

3. Glucose .o + + 4- 4- + +

4. Sucrose .. + 4- + + + --

5. Lactose .. 4- . . . . .

6. Raffinose .. -- 4- . . . .

CR -- Crotalada medicaginea

C A -- Cassia tara + -- Prosent

Absen t

The moisture content of the soil showed a decrease from July to December in all the plots (Figs. 1 and 2).

The increase in the number of fungal propagules with plant age observed in the two plant species cannot be directly correlated to root exudates as the nutrient status of the root exudates goes down with ageing. Similar decrease in the number of certain organic compounds in root exudates has been noted by some earlier workers in other plants (Rovira, 1959, 1965; Vancura and Hovadik, 1965). It has also been shown by Christiansen and Thimann (1950) that even free amino acids in certain plant parts decrease with ageing. With the onset of flowering, the bulk of the amino acids and sugars being utilized, a decrease of the above compounds in the root exudates is to be expected. It is, therefore, difficult to understand how the maximum rhizosphere popula- tion recorded at this stage of plant growth by earlier workers could be correla- ted to root exudates. The explanation given by some (Rao, 1962; Rovira, 1965) that moribund root cells peeled off from drying roots are responsible

Soil Moisture Content and Root Exudates 271

for the increase in the mycopopulation during senescence while the earlier peak of l:hizosphere effect is due to root exudates, does not seem to hold good here. A shift in the composition of the fungal flora towards cellulose decom- posers consequent upon a shift in the nutritional status has rtot beert noted in this case.

t O PLOT- I l '

6 MOISTURE N • PLOT- 3 0

0 5 m t--- .< rY

ID 4 It: LJ P o 3 w ii ii LLt w 2

[1. m I 0 m

I ne

JUL.

FIG. 1.

g

2O

15 ~ l - Z w I-

I0

~ U ~ O [ 6

AUG. S E E OCT. NOV. D E C . M O N T H S

Rhizosphere effect in terms of R/S ratio (No. of fungi in rhizosphere/No, of fungi in soil), and s gil m)isture content in the three different plots of Cassia tora L.

It appears that the decrease in moisture content of the soil, as found under field conditions, influences the fungal flora markedly. It may have either a direct effect on the fungal population or an indirect one through the plants or both. The direct effect may be to induce the fungi to sporulate and leave behind their conidia, chlamydospores or other hybernating structures in large

272 SHANKER ]}HAT SULLIA

numbers. The quantitative estimation of the mycoflora by the dilution plate method is, essentially, a count of the total number of fungal propagules and an increased sporulation under drying conditions may show an increase in the mycopopulation, whereas, more of mycelial growth as under favourable condi- tions may give a low colony count under the method used. The accumulation of fungi more in the vicinity of roots as the non-rhizosphere soil begins to dly up, may also account for the large number of fungi under low moisture condi- tions. The indirect effect must be visualised as the release of some specific sporulation-stimulant by the drying roots, for which there is no evidence.

~ S RATIO 0 P L O T - I

6 MOISTURE% A P L O T - 2 30

• P L O T - 3 c-, O o X \

• i •i20~

\ • ~ • o z

o

0 • t~

JUL. AUG. SEP. OCT. N OV. D EC. M O N T I-I.~

F[3.2. Rhizosphere effect in terms of R/S ratio and soil moisture content in the three different plots of Crotalaria medicaginea.

The absence of a significant difference in the qualitative nature of the rhizosphere mycopopulafion of the two plant species s~ms surprising in the

Soil Moisture Content and Root Exudates 273

context of claims made by several workers for a specific rhizosphere myco- flora for each plant species and even different strains of a single plant species (Buxton, 1957 ; Bhuvaneshwari, 1960; Subba Rao and Bailey, 1961 ; Lacy and Horner, 1962). However, the more frequent occurrence of Fusarium udum in the rhizosphere of Crotalaria medicaginea may be attributed to the susceptibilty of this pllmt to fusarium-wilt.

What seems to be significant in the nature of mycopopulation changes in these weed plants as compared to cultivated plants, is the absence of the maximum rhizosphere effect at the period of flowering and a decrease there- after. There is no reason to believe that greater exudation of nutrients takes place through the roots at the time of initiation of flowering. On the contrary, seedling roots are shown to exude more nutrients. As Rovira (1965) stated, the fungi are, perhaps, less affected by root exudates than bacteria in general.

ACKNOWLEDGEMENTS

Grateful thanks are due to Dr. R. Y. Roy for guidance and to Professor, R. Misra for laboratory facilities. This work was carried out during the tenure of a Junior Research Fellowship from the Council of Scientific and Industrial Research.

Andal, R., Bhuvaneshwari, K. and Subba-Rao, N. S.

Bhuvaneshwari, K.

Buchan, J. L. and Savage, R. I.

Buxton, E. W.

Christiansen, G. S. and Thimann, K. V.

REFERENCES

"Root exudates of paddy," Nature, Lend., 1956, 178, 1063.

.. "Rhizosphere and root diseases," Mem. Indian bot. Soc., 1960, 3, 152-55.

.. "Paper chromatography of some starch conversion products," Analyst, 1952, 77, 401--406.

.. "Some effects of pea root exudates on physiologic races of Fusarium oxysporum Ft. f pisi (Linf.) Snyder and Hansen ," Trans. Br. mycoL Soc., 1957, 40, 145-54.

•. "The metabolism of stem tissue during its growth and its inhibition. II. Respiration and ether soluble material ," Arch. Biochem. Biophys., 1950, 26, 248-59.

274 SHANKER BHAT SULLIA

Contois, D. E.

[varson, K. C. and Katznelson, H.

Jackson, M. L

Katznelson, H., Rouatt, J. W.

and Payne, T. M. B.

Lacy, M. L. and Hornet, C. E.

Martin, J. P.

Piper, C. S.

R a o , A. S.

Rovira, A. D.

Subba-Rao, N. S. and Bailey, L.

Sulochana, C. B.

.. " Microflora of the rhizosphere of pine apple plant," Soil Sci., 1953, 76, 259-72•

"Studies on the rhizosphere mycoflora of yellow birch seedlings," Plant and Soil, 1960, 12, 30--40.

. . Soil Chemical Analysis, 1958, Constable and Co. Ltd., London.

"The liberation of amino acids and reducing com- pounds from plant roots ", Plant and Soil, 1955,

7, 35-48.

•. "Reproduction of Verticillium in the rhizosphere and subsequent root infection of resistant and suscepti- ble mint species," Phytopathology, 1962, 52, 739.

. . " Use of acid, rose bengal and streptomycin in the plate method for estimating soil fungi ," Soil. Sci., 1950, 9, 26-31.

.. Soil and Plant Analysis, 1944, University of Adelaide.

.. "Fungal populations in the rbizosphere of peanut (Arachis hypogaea L.) ," Plant and Soil, 1962, 17, 260-66.

.. "Plant root excretions in relation to rhizosphere effect. I. The nature of root exudates from oats and peas", 1bid., 1956, 7, 178-94

•. "Plant root excretions in relation to rhizosphere effect. IV. Influence of plant species, age of plants, light, temperature and calcium nutrition on exudation" 1bid, 1959, 11, 5364.

.. "Interactions between plant roots and soil micro- organisms," A. Rev. Microbiol., 1965, 19, 241-266.

•. "Rhizosphere studies in relation to varietal resistance and susceptibility of tomato to Verticillium wilt," Can. J. Bot., 1961, 39, 1747-57•

.. "Amio acids in the root exudates of cotton," Plant and Soil, 1962 (a), 16, 312-326.

.. "B-vitamis in root exudates of cotton," Ibid.p 1962 (b), 16, 327-234.

Timonin, M. I.

Vancura, V. and Hovadik, A.

Vidal, G. and Visona, L.

Soil Moisture Content and Root Exudates

o . .

275

"The interaction of higher plants and soil micro- organisms. II. Study of the microbial population of the rhizosphere in relation to resistance of plants to soil-borne diseases," Can. J. Res., 1940, 18, 444-56.

"Root exudates of plants. 11. Composition of root exudates of some vegetables," Plant and Soil, 1965, 22, 21-32.

" Etude de la mycoflore de la rhizosphere de trois legumineuses," Annales de l'lnstitut Pasteur, 1965, 108, 535-40.