Indian Journal of Experimental Biology Vol. 41, October 2003, pp. 1160-1164

Rhizobia as a biological control agent against soil borne plant pathogenic fungi

V K Deshwal l, P Pandey', S C Kang2 & D K Maheshwari I *

IDepartment of Botany & Microbiology, Gurukul Kangri University, Hardwar 249 404, India

2Department of Biotechnology, College of Engineering, Daegu University, Gyungsan City, Gyungbook 712-714, Korea

Rhizobia promote the growth of plants either directly through N2 fixation, supply of nutrients, synthesis of phytohormones and solubilization of minerals, or indirectly as a biocontrol agent by inhibiting the growth of pathogens. The biocontrol effect of rhizobia is due to the secretion of secondary metabolites such as antibiotics and HCN. Siderophore production in iron stress conditions provides rhizobia an added advantage, resulting in exclusion of pathogens due to iron starvation. : '

Keywords : Antibiotics, Biocontrol, HCN, Rhizobia, Siderophore

Widespread use of chemicals to control plant diseases has disturbed soil environment, contaminated underground water, resulted in the development of resistant races of pathogens, and caused health risks to humans. An alternate to these chemicals is the use of certain biocontrol agents which are inexpensive and eco-friendly, and have no harmful effects on human population. Rhizobia, which have been extensively studied as plant growth promoting rhizobacteria, have also been found to be important from the biocontrol point of view. These bacteria promote the growth of plants either directly through N2 fixation, supply of nutrients, synthesis of phytohormones and solubilization of minerals, or indirectly as a biocontrol agent by inhibiting the growth of pathogens.

Use of rhizobia in biocontrol Rhizobium has been shown to reduce root-rot of

soybeans caused by Phytophthora megasperma l•

Antoun et at. 2 studied the antagonism of 49 strains of Sino rhizobium meliloti towards Fusarium oxysporum and found that these strains showed inhibition regardless of their symbiotic effectiveness. The inhibition of the fungal growth by the S. meliloti strains varied from 5 to 50%. Chakraborty and Purkayastha3 reported that some rhizobitoxine­producing strains of Bradyrhizobium japonicum protected soybeans from the infection of Macrophomina phaseolina, the charcoal rot fungus of leguminous crops. Malajczuk et al.4 isolated rhizobia from the root nodules of Acacia pulchella and

*Correspondent author: E-mail: maheshwaridk@indiatimes.com

observed that these bacteria significantly reduced the survival of Phytophthora cinnamoni zoospores. Buonassisi et at. 5 inoculated the seeds of bean with strains of Rhizobium leguminosarum bv. phaseoli antagonistic to Fusarium solani f.sp. phaseoli. A significant reduction in root rot was observed in the bean plants grown in pasteurized soil artificially infested with the fungal pathogen. Inhibition of growth of seven pathogenic microorganisms of soybean by twenty B. japonicum strains was studied by Balasundaram and Sarbho/. Six rhizobial strains inhibited only the germination of brown sclerotia of Sclerotium rolfsii. The fast growing rhizobial strains were found to completely inhibit the growth of white sclerotia of S. rolfsii.

Cha07 tested six different Rhizobium strains for their antagonistic activity against 10 isolates of fungi and found that all the tested strains inhibited the growth of fungi. R. leguminosarum biovar phaseoli 6-3 showed the highest antagonIstic activity. Antagonistic rhizobia and bradyrhizobia used as seed dressing or as soil drench were observed to reduce the infection of M. phaseolina, Rhizoctonia solani and Fusarium spp. in both leguminous (soybean and mungbean) and non-leguminous (sunflower and okra) plants in field conditions8

. Perdomo et aI. 9 evaluated 64 strains of Rhizobium for their antifungal activity against M. phaseolina. These workers found that the expression of inhibition varied among strains and was dependent on growth media and screening methods. Nautiyal 'O observed that Rhizobium sp. NBRI9513 inhibited growth of F. oxysporum f.sp. ciceri, Rhizoctonia bataticola and Pythium sp. under in vitro

DESHWAL e/ al.: RHIZOBIA AS A CONTROL AGENT AGAINST SOIL BORNE PLANT PATHOGENIC FUNGI 1161

conditions. R. /eguminosarum bv. trifolii R39 strain showed in vitro antagonistic activity against the soil borne plant root pathogens, Fusarium spp., R. so/ani, Helminthosporium sativum and Gaeumanllomyces

graminisll. Several specIes of Rhizobium and Bradyrhizobium have been reported to restrict the growth of M. phaseolina I2

•13

• The rhizobia having biocontrol potential showed more competency in root

Fig. I ~ Microscopic examination of interaction between two pathogenic fungi and Bradyrhiz.ohiul11 (Arachis) sp. showing (A) Deforma­tion of hyphae of Rhizoctonia solani, (B) lysis of hyphae of R. solalli, (C) lysis of hyphae of Sclerotinia scleroriorum, and (D) degenera­tion of cytoplasm of S. sclerotiorul1l.

1162 INDIAN J EXP BlOL, OCTOBER 2003

hair infection in host plants as compared to non­biocontrol rhizobia I2

,13 ,

Rhizobia are capable of colonizing the roots of non-legumes 14 and hence are a potential biocontrol agent for the crops other than legumes. The strain Tal 629 of B. japonicum significantly increased the dry matter yield of radish. Significant shoot dry matter yield increases by inoculating maize, spring wheat and spring barley with R. leguminosarum bv.trifolii R39 strain have been reported in field experiments l5 . Wiehe and Hoflich 16 have demonstrated that strain R39 of R. leguminosarum bv,trifolii can multiply and survive under field conditions in the rhizosphere of com, rape, Brassica napus and wheat. Chabot et at. I7

have demonstrated that R. leguminosarum bv. phaseoli strains specifically selected for phosphate solubilization function as plant growth promoting rhizobacteria with lettuce and maize. Rhizobia have been reported to invade rice roots l8,19 and result in increased growth of lowland rice2o

.

Rhizobia, which lacked the tendency to inhibit plant pathogens, have been reported to show mutualistic behaviour with other biological control agents in rhizosphere. Inoculation with Pseudomonas fluorescens biocontrol strains did not affect the symbiosis between rhizobia and forage legumes2 1

.

Co-inoculation of antibiotic-forming bacteria (Bacillus and Streptomyces griseus) with Rhizobium or Bradyrhizobium resistant to the antibiotics has been reported to enhance nodulation of legumes22

.23

. Valdes et at. 24 have reported that rhizobia and V AM fungi when used together synergistically stimulate plant growth.

Rhizobia also have the potential as a biocontrol agent against the insect pests. Sk0t et al. 25

constructed transgenic R. leguminosarum bv. viciae and R. leguminosarum bv. trifolii strains in which the insecticidal crystal protein gene (crylIIA) from Bacillus thuringiensis subsp. tenebrionis was expressed and found that these rhizobia protected their host plant from damage by larvae of Sitona flavescens.

Mechanism of biocontrol effect of rhizobia

Biocontrol mechanisms of rhizobia may involve antibiotics, HCN and siderophores. Rhizobia also appear to influence the plant defence mechanism by stimulating the production of phytoalexins by plants. Microscopic examinations showed unfolding, abnormal intercalary swelling, tip deformation,

degeneration of cytoplasm and lysis of hyphae of fungal pathogens such as R. solani, F. oxysporum, Sclerotinia sclerotiorum and M. phaseolina during interaction with rhizobia (Fig 1, unpublished results) .

Antibiotics Antibiotics produced by rhizobia have been found

to play an important role in disease control. The secretion of peptide antibiotic trifolitoxin (TFX) by R. leguminosarum bv. trifolii T24 has been reported26. B. japonicum has been found to protect soybean crop against infection by M. phaseolina by the direct action of antibiotic rhizobitoxine3

. The inhibitory effect of Bradyrhizobium (Arachis) on M. phaseolina was due to rhizobitoxine production 13

.

HeN HCN, a secondary metabolite produced by several

microorganisms, has deleterious effect on the growth of some microbes27. Some rhizospheric microorganisms have been known to protect their host plants through the inhibition of growth of the pathogens by HCN production. Rhizobia are relatively less efficient in HCN production. Among rhizobia 12.5% and 3% strains were found to be HCN producers by Beauchamp et al.28 and Antoun et al. 14, respectively .

Siderophores Almost all facultative anaerobic organisms produce

extracellular siderophores to overcome iron limitation for their growth29

. Siderophores chelate and solubilize iron, and ferri-siderophore complexes are taken up by the ce1l30

. Rhizobia have been reported to produce a number of siderophores. These include rhizobactin (S. meliloti)31, citrate (E. japonicum)32, anthranilate (R. leguminosarum biovar viciae)33, catechol (R. leguminosarum34, Bradyrhizobium (cowpea)35, Bradyrhizobium (peanut)36, R. leguminosarum biovar trifoli/\ rhizobacti n 1021 (S. meliloti)38, vici bactin (R. leguminosarum biovar viciae)39. Siderophore production in iron stress conditions provides rhizobia an added advantage, resulting in exclusion of pathogens due to iron starvation.

Phytoalexins The involvement of 4-hydroxy-2,3,9-trimethoxy­

pterocarpan, a phytoalexin, in resistance of pea plants against infection by F.solani f.sp. pisi has been previously recorded40

,4I. Chakrabory and Chakrabort/2

reported that R. leguminosarum bv. viciae protected

DESHWAL et al.: RHIZOBIA AS A CONTROL AGENT AGAINST SOIL BORNE PLANT PATHOGENIC FUNGI 1163

pea roots against infection by F. solani f.sp. pisi. The protection appeared to be due to the increased production of 4-hydroxy-2,3,9-trimethoxypterocarpan by pea plants.

Future Research Prospects In nature, biocontrol by rhizobia appears to be very

rare. It is, therefore, essential to continue the screening process to obtain potential biocontrol rhizobial strains. Efforts should also be made to understand the biocontrol action of rhizobia. The survival and competitive ability of the rhizobial strains to be introduced as a biocontrol agent should be improved.

On the basis of their studies on the P. fluorescens CHAO strain, which produces ACC (I-aminocyclo­propane-I-carboxylic acid) dearninase enzyme, Wang et al.43 concluded that ACC dearninase may have a role in the biocontrol activity. The biocontrol potential of the rhizobial strains, natural or genetically engineered, having ACC deaminase activity should be investigated. Genetic engineering approach can also be used to introduce the genes coding for the synthesis of other antifungal metabolites into rhizobial strains selected for use in biocontrol.

Acknowledgement Financial assistance from TMOP-CSIR, New Delhi

is gratefully acknowledged to one of the authors (DKM).

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