Review of Literature - Shodhganga

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of very nutritive and useful for milch cattle. It is also used as green ...... to control. Singh et al. (2008b) found in pot trial study a significant increase in seedling.
Review of Literature

Since millennia pulses are grown in India as legume food providing the nutritionally balanced food to the people. As defined over 1,000 years ago ‘balanced food’ consisted of pulses, besides cereals, vegetables and fruits, and milk products (Ayachit, 2002). The word pulse is derived from the Latin pulse, pultis, a thick soup. It is the broad term used to describe the dried, edible seeds of legumes. In our daily life pulses are very important as essential ingredient of the human diet. They are rich in protein and contain low fat, low sodium, high fiber and no cholesterol and a good source of protein. They are also a rich source of energy, minerals and certain vitamins of B-complex group. Further, the amino acid composition of pulse protein is such that a mixed diet of cereal and pulse has superior biological value than either of the component alone. Consequently, pulses help in checking the malnutrition among the children of our country. Chana (chickpea), mung, masur, tur and urad are the common pulses in India and consisted of most of the Indian families every day in daily diet (Nene, 2006). India is the most common and largest producer and consumer of pulses in the world. Although, India has the distinction of being the world’s single largest producer of pulses, the difference in production and population ratio is significant. The increase in population has pushed up demand of pulses, while the fall in availability has pushed up their prices. Although, a large area of approximately 20-22 million hectares is under different pulse crops, their production is more or less stagnant for the last four decades, which ranges between 11 and 13 million tonnes (Ali and Kumar, 2006). This fall in availability of pulses is attributed to many factors; pulses are mostly grown under rain-fed conditions where drought is a common feature. Other factors include their low harvest index, prolonged vegetative growth, low yield and their susceptibility to diseases. Madhya Pradesh is the leading state in producing pulses in India followed by Maharastra, Rajasthan and Andhra Pradesh. There has been decline in Characterization of endophytic rhizobacteria from Vigna mungo (L.) Hepper and their role in biocontrol of Macrophomina phaseolina (Tassi) Goid.

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annual production of pulses from 3488 million tonnes in 2003-04 to 2948 million tonnes in 2008-09 in Madhya Pradesh, and similarly in other states also. In 2003-04 pulses produced were 635 kg/ha in an area of 23.46 of million hectare that slightly decreased to 597 kg/ha grown in an area of 24.54 million hectare in 2008-09 as per report of Agriculture Ministry, Govt. of India (Goyal et al., 2010). There are numbers of variety of pulse crops grown in India and accepted as a major player in pulses contributing around 25-28% globally. A liberal trade regime has kept imports in this region around 25 lakh tonnes per annum, i.e. 20-25% of domestic production (Goyal et. al., 2010). To obtain high yields of pulses considerable improvement has been made in developing techniques, their production per hectare has remained the same for the last two centuries. In India, major 12 different pulse crops are grown such as: chickpea (Cicer arietinum), pigeonpea (Cajanus cajan), lentil (Lens culinaris), black gram (Vigna mungo), green gram or mung bean (Vigna radiata), lablab bean (Lablab purpureus), moth bean (Vigna aconitifolia), horse gram (Dolichos uniflorus), pea (Pisum sativum var. arvense), grass pea or khesari (Lathyrus sativus), and cowpea (Vigna unguiculata). Share of various pulses in total production of India is given in Fig. 1. Among total production of pigeon pea (arhar, tur, red gram) shares 15-16%, chickpeas (chana, Bengal gram) shares 40-45%, urad (black gram shares 12-16% and lentil (masoor) 912% (Fig. 1). Among these, Vigna mungo (L.) Hepper (= urad, mash bean, black gram) of the family Fabaceae (= Leguminoseae) is an important pulse crop grown throughout India in an area of about 3.2 million hectares (2007-08) as per report of Indian Institute of Pulses Research, Kanpur (UP). India has been universally accepted as the original home of the urad which has more or less confined to South Asia.There is a mention of urad in Vedic texts such as Kautilya's Arthasasthra' (Shamasastry, 1961) and 'Charak Samhita'. Sanskrit name of urad is mash parni written in the literature for Vigna dalzelliana. On Characterization of endophytic rhizobacteria from Vigna mungo (L.) Hepper and their role in biocontrol of Macrophomina phaseolina (Tassi) Goid.

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the basis of Sanskrit name urad is known as mash in Punjab and mash kalaya in West Bengal. The name of urad seems to have originated from the Tamil word ulundu. Masha (urad) has been talked about in the Brahadaranyaka (5500 BC), in the Mahabharata (2000 BC), in the Krishi Parashara (400 BC; Sadhale, 1996, 1999), and in the later literature (Achaya, 1994).

Fig. 1. Share of various pulses in total production of India (Source: NB research). From India it spread in many countries like Africa, Europe, America and Asia. It has become a popular pulse crop in Pakistan, India, Bangldesh, Burma, Ceylon, and most of the African countries (Achaya, 1998). In India, urad is very popularly grown in Andhra Pradesh, Bihar, Madhya Pradesh, Maharastra, Uttar Pradesh, West Bengal, Punjab, Haryana and Karnataka. Urad is mainly grown as a secondary mixed crop along with cotton, maize, jowar and other coarse cereals. Urad is referred as a kharif crop, but it is also grown in the rabi season also. It is sown in February, June-July, October depending on the cultivated area. In contrast to carnivorous species of mankind, purely vegetarian people obta8ined protein from pulses and milk. Urad is a rich protein food containing about 26% protein, which is almost three times more than that of cereals. Black gram supplies a major share of protein requirement of vegetarian population of the country. It is consumed in the form of split pulse as well as whole pulse, which is an essential supplement of cereal based diet. The combination of dal-chawal (pulse-rice) or dal-roti (pulse-wheat bread) is an important ingredient in the average Indian diet. The biological value improves greatly when wheat Characterization of endophytic rhizobacteria from Vigna mungo (L.) Hepper and their role in biocontrol of Macrophomina phaseolina (Tassi) Goid.

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or rice is combined with black gram because of the complementary relationship of the essential amino acids such as arginine, leucine, lysine, isoleucine, valine and phenylalanine, etc. (Goyal et al., 2010). Urad is a highly prized pulse, used for making several special South Indian dishes like idli, vada and dosa, etc. It is also used for making papad and dal. It is very rich in protein and richest in phosphoric acid among pulses. In addition, urad’s green fodder is of very nutritive and useful for milch cattle. It is also used as green manure. It is a cover crop and protects soil from erosion by its deep root system. Being a leguminous plant it has the capacity to fix atmospheric nitrogen and restore soil fertility (Goyal et al., 2010). In India urad is produced annually about 1.3 million tones, which is normally 10% of India's total pulse production, i.e. 12-15 million tonnes per annum. India imports urad around 1-2 lakh tonnes. This shows that annual production of urad is much lower than its consumption which leads to high price of urad. It has been reported that loss of production in pulse crop of urad is because of pests and pathogens that attack the crop. The yield of urad is low (504 kg/ha) due to several biotic and abiotic factors. Urad suffers from mildew (Cercospora leaf spot) under damp weather conditions and seedling blight, and root- and stem- rots of more than 500 cultivated and wild plant species including economically important crops. Soil supports the various microbial communities under the influence the root. There are vigorous microbial populations which bring to bear beneficial, neutral or harmful effects on plant growth. A large number of phytopathogens has been reported which have detrimental effects on urad crop. Among them root and stem rots are caused by one of the serious soil-borne sclerotial pathogen, Macrophomina phaseolina (Tassi) Goid., which belongs to the anamorphic Ascomycetes. Wheeler (1975) classified this fungus as : Division Eumycota, Sub-Division Deuteromycotina,

Class

Coelomycetes,

Order

Sphaeropsidales,

Family

Sphaeropsidaceae, Genus Macrophomina, Species phaseolina. Characterization of endophytic rhizobacteria from Vigna mungo (L.) Hepper and their role in biocontrol of Macrophomina phaseolina (Tassi) Goid.

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Charcoal rot caused by Macrophomina phaseolina [imperfect state Rhizoctonia bataticola (Taub.)] is of prime importance in reducing crop yield. Macrophomina is mostly known in two anamorphic forms belonging to M. phaseolina (Tassi) Goid. (= Tiarosporella phaseolina (Tassi) Van der Aa) and Rhizoctonia bataticola. It produces both pycnidia and sclerotia in host tissues and culture media. The sclerotial state is called Rhizoctonia bataticola and the pycnidial state as Macrophoma phaseolina. In 1947 Goidanich proposed Macrophomina phaseolina, since then it is written as Macrophomina phaseolina (Tassi) Goid. (Dubey and Upadhyay, 2001). Thus the fungus exists in two forms, one saprophytic (named R. bataticola) where the fungus mainly produces microsclerotia and another pathogenic (M. phaseolina) where the pathogen mainly produces pycnidia. In the pathogenic stage the fungus is a non- specific pathogen and attacks a broad spectrum of economically important crops such as common beans, maize, soybean, mungbean, uradbean, sesame, etc. (Dhingra and Sinclair, 1978). It survives in soil by sclerotia produced during parasitic phase in host tissues for about 20 years (Short et al., 1080; Dubey and Upadhyay, 2001; Baird et al., 2003).

M. phaseolina is a heterogeneous species that cannot be divided into subspecies groups based on pathogenicity and by pycnidium production. It shows a great morphological, physiological, pathogenic and genetic variability which increases its adaptability to diverse environmental condition. Disease symptoms appear from the time of seedling emergence and can be observed at various stages of plant development. However, plant withering can be observed from seedling to maturing stage. After seedling emergence, symptoms on cotyledons show brown to dark spots but cotyledons remain on the plant for only a few days. The margins of the cotyledons become bright red, and finally brown to black. Often, they are covered with a grayish mycelial pad bearing scattered sclerotia. Mycelia can also be observed inside the colonized cotyledons. The typical symptoms are pinhead-size, charcoal-coloured spots which are mostly restricted to the hypocotyl section of the Characterization of endophytic rhizobacteria from Vigna mungo (L.) Hepper and their role in biocontrol of Macrophomina phaseolina (Tassi) Goid. 10

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stem, including its underground part. The most common disease symptom is the sudden wilting and drying of the whole plant, remaining most of the leaves green. Then the stem and branches are covered with black bodies and give the charcoal or ashy appearance of dead plants. Infected spots may spread and develop into large necrotic lesions, resulting in death of the plant. M. phaseolina can also infect roots which show necrotic lesions (Adam, 1986). Infected plants die as the result of necrosis of roots and stems, and mechanical plugging of xylem vessels by microsclerotia, but also by toxin production and enzymatic action (Jones and Wang, 1997). Dhar et al. (1982) first isolated and elucidated the structure of a phytotoxic metabolite, phaseolinone 1, from the culture filtrate of M. phaseolina. Phaseolinone is a nonspecific exotoxin which inhibits seed germination of a large number of plants. The concentration required for complete inhibition of seed growth of Phaseolus mungo (black gram) has been found as 25 g/ml (Bhattacharya, 1987). It also causes wilting of seedlings and leaf necrosis in several plants. These symptoms were similar to those produced by the fungus itself, thus the toxin play a key role in pathogenesis. Bhattacharya et al. (1992) described an enzyme immunoassay procedure for the determination of phaseolinone levels in M. phaseolina-infected Seeds. They observed 50% inhibition in seed germination at a toxin concentration of 0.60 g/g of wet tissue. As a result of infection caused by charcoal rot yield losses are difficult to assess in quantitative value as the effects of disease caused by this fungus can be quite subtle and may not be noticed. In some crops, the yield losses caused by M. phaseolina may result from plant death or lodging. There are several information sources on soybean losses due to charcoal rot disease. Annual losses of 30-50 % in soybeans caused by M. phaseolina have been reported (Senthilkumar et al., 2009). Charcoal rot causes the greatest or second greatest economic loss for soybean producers (Wrather et al., 2003). Abiotic stress condition causes changes in the quality and quantity of the microflora of the rhizosphere. Also the stress condition adversely affects the growth and

Characterization of endophytic rhizobacteria from Vigna mungo (L.) Hepper and their role in biocontrol of Macrophomina phaseolina (Tassi) Goid. 11

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nodulation. Possibility of damage to the crops due to M. phaseolina increases under stress conditions (Atlas and Bartha, 1998). Rhizobia are a paraphyletic group that fall into two classes of the proteobacteria— the alpha- and beta-proteobacteria. As shown below, most belong to the order Rhizobiales, but several rhizobia occur in distinct bacterial orders of the proteobacteria. The alpha-proteobacteria comprises of mostly phototrophic genera but also several genera metabolising C1-compounds, symbionts of plants and animals, and a group of pathogens. Now rhizobia are divided into Rhizobium, Bradyrhizobium, Mesorhizobium, Sinorhizobium and Azorhizobium; they are Gram-negative, nitrogen-fixing bacteria that form nodules on host plants. They also have symbiotic relationships with legume plants, which can't live without the essential nitrogen-fixing processes of these bacteria. The species of Bradyrhizobium falls under the family Bradyrhizobiaceae. However, root nodules of urad are formed by species of Bradyrhizobium. It is Gram-negative bacillus (rod shaped), motile with a single sub-polar or polar flagellum. They are a common soil dwelling microorganism that can form symbiotic relationships with leguminous plants where they fix nitrogen in exchange for carbohydrates from the plant. They are slow growing in contrast to Rhizobium species, which are considered fast growing rhizobia. In a liquid medium, Bradyrhizobium species takes 3-5 days to create moderate turbidity, and 6-8 hours to double in population size. They tend to grow best with pentoses as a carbon source. The average G-C content of the genome is 64.1 mol % (Holt et al., 1994; Saharan et al., 2011). Appunu et al. (2009) have reported that Vigna mungo, V. radiata and V. unguiculata plants sampled in different agronomical-ecological climatic regions of India are nodulated by Bradyrhizobium yuanmingense. They made a core collection of 76 slowgrowing isolates from root nodules of V. mungo, V. radiata and V. unguiculata plants grown at different sites. The genetic diversity of the bacterial collection was assessed by restriction fragment length polymorphism (RFLP) analysis of PCR-amplified DNA fragments of the 16S-23S rDNA intergenic spacer (IGS) region, and the symbiotic

Characterization of endophytic rhizobacteria from Vigna mungo (L.) Hepper and their role in biocontrol of Macrophomina phaseolina (Tassi) Goid. 12

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genes nifH and nodC. These results reflect a long history of co-evolution between B. yuanmingense and Vigna spp. The genus Bradyrhizobium with a single species, B. japonicum, was proposed for symbionts of soybean (Jordan, 1982). Later, Hollis et al. (1981) separated B. japonicum into three DNA homology groups with species B. elkanii for one group and B. liaoningense for another group comprising extra slow growing Glycine isolates, retaining the name B. japonicum for slow-growing isolates of G. max. A major factor complicating the evaluation of the taxonomic status and interrelationships of bradyrhizobia is the high similarity of 16S rDNA gene sequences. Many strains have 16S rDNA sequence divergences of 0.1–2.0%. Only sequences for B. elkanii and related strains differ by up to 4% from those of other bradyrhizobia (Willems et al., 2001). On the basis of 16S rDNA similarities and total DNA homology values, B. elkanii is considered distinct from B. liaoningense and could represent a separate genus. B. liaoningense is phylogenetically closer to B. japonicum which is closer to genera Afipia, Agromonas, Blastobacter, Nitrobacter and Rhodopseudomonas. The Bradyrhizobium genus was described by Jordan in 1982. Currently, it consists of 9 rhizobia species as given in Table 1. Table 1. Bradyrhizobium species. Species

Host

Reference

B. japonicum

Glycine max

Jordan (1982)

B. elkanii

Glycine max

Kuykendall et al. (1992)

B. liaoningense

Glycine max

Xu et al. (1995)

B. yuanmingense

Lespedeza

Yao et al. (2002)

B. denitrificans

Aeschynomene indica

van Berkum & Eardly (2002)

B. betae

from the roots of Beta

Rivas et al. (2004)

vulgaris afflicted with tumorCharacterization of endophytic rhizobacteria from Vigna mungo (L.) Hepper and their role in biocontrol of Macrophomina phaseolina (Tassi) Goid. 13

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like deformations from genistoid legumes from

B. canariense

Vinuesa et al. (2005).

the Canary Islands B. jicamae

Pachyrhizus erosus

Ramírez-Bahena et al. (2009)

B. pachyrhizi

Pachyrhizus erosus

Ramírez-Bahena et al. (2009)

The other bacteria are also associated with root nodules. Bradyrhizobium is an important member of PGPR which shows several plant growth promoting activities. These bacteria carry out nitrogen fixation and provide several direct and indirect effects such as phytohormone production, iron-chelation, phosphorous solubilization, hormone production, HCN production, chitinase production, etc. (Deshwal et al., 2003). Obviously, bradyrhizobia are known to increase nodulation and nodule weight in legumes along with increase of host plant growth and development but Bradyrhizobium-bacterised seeds are known to reduce M. phaseolina infection (Gupta et al., 2002; Deshwal et al; 2003). Use of bradyrhizobia has dual advantage as compared to that of fluorescent pseudomonads as the former assimilates atmospheric nitrogen besides killing the deleterious phytopathogens (Siddiqui et al., 2001) and exhibit antagonistic effects towards many plant pathogenic fungi. The diverse endophytic bacteria (such as Pantoea agglomerans, Enterobacter kobei, Enterobacter

cloacae,

Leclercia

adecarboxylata,

Escherichia

vulneris,

and

Pseudomonas sp. belong to Gamma Proteobacteria) have been isolated from root nodules of Hedysarum Yacine et al. (2004). Wang et al. (2006) isolated diverse endophytic bacteria from a leguminous tree, Conzattia multiflora. Nine different groups were defined by PCR-based RFLP, which were classified as Pantoea, Erwinia, Salmonella, Enterobacter, Citrobacter and Klebsiella by the phylogenetic analysis of 16S rRNA genes.

Characterization of endophytic rhizobacteria from Vigna mungo (L.) Hepper and their role in biocontrol of Macrophomina phaseolina (Tassi) Goid. 14

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Similarly, 34 endophytic bacterial isolates associated from legumes root nodules have been characterized.

Phylogenetically, these isolates belong to the branches

containing the genera Inquilinus, Bosea, Rhodopseudomonas, Paracraurococcus, Phyllobacterium, Ochrobactrum, Starkeya, Sphingomonas, Pseudomonas, Agromyces, Microbacterium, Ornithinicoccus, Bacillus, and Paenibacillus. These strains did not induce any nodule formation when inoculated on the wide host spectrum legume species Microbacterium atropurpureum (Siratro) and no nodA gene could be amplified by PCR. However, nifH sequences, most similar to those of Sinorhizobium meliloti, were detected within strains related to the genera Microbacterium, Agromyces, Starkeya and Phyllobacterium (Zakhia et al., 2006). Dubey et al. (2010) isolated 8 strains of endophytic root nodule rhizobia from pigeon pea (Cajanus cajan) and identified as Ensifer sinorhizobium based on their physiological and biochemical characteristics; therefore, these strains were named as Ensifer spp. KCC1 to KCC4. KCC5 is placed in Ensifer fredii clade. Out of these, two isolates (KCC2 and KCC5) produced siderophore and showed strong antagonistic effect against F. udum. Besides Bradyrhizobium, Bacillus and Pseudomonas are the most common endophytes. These bacteria competitively colonize the roots of plant and can act as biofertilizers and/or antagonists (biopesticides) or simultaneously both. Bacillus is Gram-positive, rod shaped, motile and spore forming bacterium. Due to spore forming ability and adaptation it has been exploited for commercial formulation and field application (Liu and Sinclair, 1993). Physiological traits, such as multilayered cell wall, stress resistant endospore formation, and secretion of peptide antibiotics, peptide signal molecules, and extracellular enzymes, are ubiquitous to these bacilli and contribute to their survival under adverse environmental conditions for extended periods of time (Kumar et al., 2011a). The principal mechanisms of growth promotion include production of growth stimulating phytohormones, solubilization and mobilization of phosphate, siderophore production, antibiosis (i.e., production of antibiotics), ethylene Characterization of endophytic rhizobacteria from Vigna mungo (L.) Hepper and their role in biocontrol of Macrophomina phaseolina (Tassi) Goid. 15

Review of Literature

synthesis, and induction of plant systemic resistance to pathogens (Gutierrez-Manero et al., 2001; Whipps 2001; Idris et al., 2007; Richardson et al., 2009). Bacillus subtilis is commonly known for the positive effects on the plant growth, vitality and capacity of the plant to deal with phytopathogens which leads to great yield and productivity. Kumar et al. (2012b) have discussed that Bacillus has ability for nitrogen fixation, antibiotic production, degradation of cellulose, starch, pectin and protein and good plant growth promoting activities. Liquid, powder and granular formulations of sporeforming strains of bacilli have an advantage over the non-spore forming strains such as Pseudomonas (formulated as vegetative cells). Spores are more robust and resistant to the elevated temperature and high concentrations of chemicals. Moreover, the shelf-life of biological products based on bacterial spores can be up to 1-3 years. A disadvantage of the use of spores is that after application they need time to return to the metabolic active stage of a vegetative cell. Pseudomonas is one of the most important endophytic bacteria which are rodshaped, Gram-negative aerobes (some strains also have anaerobic respiration with nitrate as a terminal electron acceptor and for arginine fermentation), high genomic GC (59.68%) content (Holt et al., 1994) and motile in nature with several polar flagella. Pseudomonads are the most common genera of PGPR (Kloepper, 1993), which control pathogens by production of antibiotics (Gutterson et al., 1988), HCN (Defago et al., 1990), siderophores (Kloepper et al., 1980), etc. and competition for space and nutrients (Elad et al., 1987). P. aeruginosa isolated from potato rhizosphere displayed the strong antagonistic activity against important fungal pathogens viz., Macrophomina phaseolina and Fusarium oxysporum (Gupta et al., 1999) Boiero et al. (2007) evaluated phytohormone biosynthesis, siderophore production, and phosphate solubilization in Brdy. japonicum, most commonly used for inoculation of soybean and non-legumes. These strains did not produce siderophore and also did not solubilize phosphate in selective culture conditions. IAA, zeatin, and GA3 were found in Characterization of endophytic rhizobacteria from Vigna mungo (L.) Hepper and their role in biocontrol of Macrophomina phaseolina (Tassi) Goid. 16

Review of Literature

all three strains. This is the first report of IAA, GA3, zeatin, ethylene, and ABA production by B. japonicum in pure cultures using quantitative physicochemical methodology. The three strains have differential capability to produce the five major phytohormones and this fact may have an important technological implication for inoculant formulation. Boiero et al. (2007) have reported phytohormone biosynthesis, siderophores production and phosphate solubilization in three strains (E109, USDA110, and SEMIA5080) of Bradyrhizobium japonicum, most commonly used for inoculation of soybean and nonlegumes in USA, Canada, and South America. This is the first report of IAA, gibberellic acid (GA3), zeatin, ethylene, and abscisic acid (ABA) production by B. japonicum in pure cultures, using quantitative physicochemical methodology. The three strains have differential capability to produce the five major phytohormones and this fact may have an important technological implication for inoculant formulation. Direct growth promotiom mechanism involved various effects of PGPR on the plants such as phytohormons production; IAA (indole-3- acetic acid) is the most common phytohormone which positively affects the plant growth. It is known to stimulate both rapid and long term responses of plants. Endophytic bacteria such as Rhizobium, Bradyrhizobium, Bacillus and Pseudomonas produced IAA in the presence of tryptophan (precursor) via several pathways. Besides, they also produce auxins, cytokinins and gibberellins. They all are useful for plant growth which led increased of crop yield. Rhizospheric bacteria have been found to improve the availability of nutrients and showed detrimental effect on plant pathogens by producing hormones e.g. auxins. IAA produced by bacteria positively affected the plant growth and nodulation in green gram (V. radiata) and black gram (V. mungo) (Jangu et al., 2011). Mutants of Pseudomonas strain MPS 90 capable of producing IAA resulted in different IAA production. As compared to parent strain 3.33% mutants produced higher amount of IAA and low

Characterization of endophytic rhizobacteria from Vigna mungo (L.) Hepper and their role in biocontrol of Macrophomina phaseolina (Tassi) Goid. 17

Review of Literature

amount of IAA in 35.14% mutants. In black gram, majority of the Pseudomonas mutants increased the root growth of seedling (Jangu et al., 2011). The influence of endogenous root nodule’s phenolic acids (protocatechuic acid, 4hydroxybenzaldehyde and p-coumaric acid) on indoleacetic acid (IAA) production by its symbiont (Rhizobium) was examined by Mandal et al. (2007a). They reported that the phenolic acids present in the nodule might serve as a stimulator for IAA production by the symbiont (Rhizobium). Next to nitrogen, phosphorous is an essential nutrient for plant which can take from soil only in soluble form. Most of the soil phosphorous is in unavailable form; average percentage of phosphorous in soil is about 0.05% (w/w), however, only 0.1% of this is available to plants (Scheffer and Schachtschabel, 1992; Illmer and Schinner, 1995). There are number of endophytes which have ability to convert the insoluble inorganic phosphate into soluble and simple form. Pseudomonas, Bacillus, Rhizobium, Bradyrhizobium, etc. are the phosphate solubilising microorganisms (PSM). This improves and enhances the growth of both leguminous and non-leguminous plants (Barea et al., 2005; Sridevi and Mallaiah, 2009). Thus PSM is good inoculants for various crops in India. Iron is one of the most important elements essential for the growth of all microorganisms. As nitrogen and phosphorous, iron is also found in nature copiously but not easily available to the organisms for direct assimilation because ferric iron (Fe III) which is in nature in the majority is soluble and too low in concentration to support microbial growth. Insoluble ferric (Fe3+) state of iron exists only in oxidative environments and at physiological pH (Guerinot, 1994). Hence, to survive in such type of environment organisms secretes Fe-binding ligands called ‘siderophores’. Siderophores are ferric ion-specific ligands with high affinity for iron that are taken into cells via specific membrane receptors. Siderophores are the iron-chelators having high affinity to sequester iron from the environment. It form complex with iron and made them readily available to plant root surfaces. Competition exists in soil among the Characterization of endophytic rhizobacteria from Vigna mungo (L.) Hepper and their role in biocontrol of Macrophomina phaseolina (Tassi) Goid. 18

Review of Literature

microorganisns for iron uptake; under iron-limiting conditions some bacteria secrete ferric iron-specific siderophores to aid in sequestering and transport of iron. Besides iron uptake, proliferation of phytopathogens is also prevented, thereby facilitating plant growth (Kloepper et al., 1980). Under iron-limiting conditions, many bacteria secrete siderophores to aid in the sequestering and transport of iron. Gupta et al. (2000) have reported the production of siderophores by Bradyrhizobium sp. (Vigna). Ability of fluorescent Pseudomonas PS1 and PS2 for producing siderophores, indole acetic acid, hydrocyanic acid, and phosphate solubilization under normal growth conditions has also been reported by Bhatia et al. (2008). Some endophytic rhizobacteria (Rhizobium, Bacillus, Pseudomonas etc.), which play role in biological control of phytopathogens, produced HCN (hydrocyanic acid). According to Voisard et al. (1989), cyanide produced by Pseudomonas fluorescence strain CHAO showed antagonistic activity against Thielaviopsis basicola (causing black root rot of tobacco). Jian-Gang et al. (2008) isolated 353 strains from rhizosphere of eggplant among those chitinase-secreting strain was selected on chitin–Ayers (CA) medium and named as strain CH2. On the basis of several biochemical and physiological characteristics and 16S rDNA sequence alignment the strain was identified as Bacillus cereus. Estimation of its activity showed it to be a 15.0-KD chitinase. Germination of the fungal spores was effectively suppressed by the bacterial suspension, supernatant from the suspension, and 0.005% solution of chitinase extracted from the strain CH2. A plant growth promotion mechanism includes ACC deaminase activity. Jacobson et al. (1994) demonstrated that the Pseudomonas putida GR12-2 (promotes growth in canola seedling root) contains an enzyme, 1-aminocyclopropane-1-carboxylate (ACC) deaminase which hydrolysis the ACC. Mutant of P. putida GR12-2 lacked this enzyme resulting in growth promotion of roots of canola seedlings.

Characterization of endophytic rhizobacteria from Vigna mungo (L.) Hepper and their role in biocontrol of Macrophomina phaseolina (Tassi) Goid. 19

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Dubey et al. (2012a) evaluated six isolates of Bradyrhizobium sp. (VR1-VR6) from Vigna mungo for their plant growth promoting (PGP) attributes and antifungal properties in vitro. All the isolates produced IAA but none of them produced HCN. Isolates VR1 and VR2 produced siderophore, and enzymes chitinase and ACC deaminase besides phosphate solubilisation and antagonism against M. phaseolina. There are number of abiotic factors such as temperature, pH, salt concentration, etc. that affects growth and survival of bacteria. Endophytic bacteria vary in their tolerance to these stresses. Temperature is a one of the limiting factors for legumeBradyrhizobium spp. symbiosis and other endophytes (Bacillus, Pseudomonas, etc.). They are sensitive to temperature and other environmental factors such as pH, salt tolerance, etc. which can modify the plant growth or height and bacteria associated with plant survival. Twelve strains of Bradyrhizobium spp. (pigeon pea and cow pea nodulating bacteria) were tested for temperature tolerance (20ºC/10 ºC, 30 ºC/20 ºC and 38 ºC/25ºC) and their temperature tolerating ability were observed on the basis of their growth. Only five strain of Bradyrhizobium were most temperature tolerant strains viz., USDA 3278, USDA 3362, USDA 3364, USDA 3458 and USDA3472. Growth of both crop were mainly dependent on the temperature variation not on the Bradyrhizobium strain, they are independent of Bradyrhizobium strain. It was recorded that at lowest temperature height of plants were the shortest and nitrogen fixation was inhibited (of pigeon pea). The optimum temperature was 30 ºC/20 ºC (Marsh Lurline E. et al., 2006). Temperature affects the legume-Bradyrhizobium symbiosis either directly, by limiting the growth of the microsymbiont and/or indirectly, by regulating the growth of the acrosymbiont (Hashem et al., 1998; Kuykendall et al., 2000). Kumar (2010) tested the temperature effect for number of Rhizobium strains among those only 7 strains could grow at maximum temperature (55ºC) and grew at 5ºC. The optimum temperature for all the isolates of Rhizobium spp. was 28ºC. The observed optimum temperature regime for endophytes (Rhizobium, Bacillus and Pseudomonas) Characterization of endophytic rhizobacteria from Vigna mungo (L.) Hepper and their role in biocontrol of Macrophomina phaseolina (Tassi) Goid. 20

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was 28ºC. Minimum and maximum tolerated temperature regimes for isolates were 5ºC and 55ºC, respectively (Kumar, 2012). Maximum temperature for survival of B. japonicum was reported from 33.7 ºC to 48.7 ºC (Kulkurni et al., 2000). In addition to temperature, fluctuation in pH also affects the survival of bacteria. Acidic pH is a significant reason for limited survival of bacteria and reduced nodulation. Low pH levels also affect the production and excretion of Nod metabolites (O’ Hara and Glenn, 1994). The optimum pH for rhizobial population is neutral to slightly acidic. Study have shown that in acidic soil the rhizobial population is often small and ineffective (Taurian et al., 1998). High pH may also have negative effects on survival of endophytic rhizobacteria (such as Bradyrhizobium, Bacillus, Pseudomonas, etc.). Salt concentration is one of the abiotic factors whose fluctuation can negatively affect the growth of endophytic rhizobacteria. Salinity is a hazardous to agriculture in arid and semiarid regions (Rao and Sharma 1995). Around 40% of the land surface of the world has potential salinity problems (Cordovilla et al., 1994). Optimum salt concentration for the isolates strains of Rhizobium, Bacillus and Pseudomonas was 23%, while the maximum and minimum salt concentration tolerated were 0.5% and 6% (Kumar, 2012). Antagonism is a balancing wheel of nature. It operates via three facets viz., amensalism (antibiosis and lysis), competition (for nutrient and space) and parasitism (between two microorganisms). Antibiotic molecules include antibiotics, bacteriocins, HCN, and several other extracellular metabolites; they pose inhibitory or cidal effects individually or in combination. Culture filtrates of bacteria grown for different time consist of many primary and secondary metabolites including proteins, amino acids, antibiotic, toxins, etc. Cha (1990) demonstrated that mycelial dry weight of fungal isolates was reduced when treated with culture filtrates of R. leguminosarum in vitro. In an antifungal activity test fifteen Bradyrhizobium strains had been found to inhibit the mycelia growth, reduction Characterization of endophytic rhizobacteria from Vigna mungo (L.) Hepper and their role in biocontrol of Macrophomina phaseolina (Tassi) Goid. 21

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of sclerotial formation and inhibition of sclerotial germination of Rhizoctonia solani AG-1. In contrast, Bradyrhizobium or their cell-free culture filtrate (CFCF) negatively affected the mycelial growth, sclerotial formation and germination of Rhizoctonia solani Kuhn AG-1 (Kelemu et al., 1995). It has been proved that endophytic bacterial strains of Bradyrhizobium sp. as well as its culture filtrate have inhibitory properties which help to act as potential biocontrol agent of M. phaseolina. Complete inhibition in mycelial dry weight and sclerotia germination of pathogen was caused by culture filtrates of strain VR2. Moreover, patterns of sclerotia germination varied with concentration of culture filtrates of VR1 and VR2. The number of hyphae produced per sclerotium was more in control than the culture filtrate-amended plates. The number of sclerotia producing less hyphae got increased with increasing the concentration of culture filtrate of strains VR1 than VR2 (Dubey et al., 2012a). Chakraborty and Purkayastha (1984) found that R. japonicum inhibited the growth of M. phaseolina on both liquid and solid media. Replacement of nutrient medium with culture filtrate of R. japonicum significantly reduced mycelial growth of M. phaseolina. Whole culture extracts of R. japonicum yielded a toxic substance which was identified as rhizobitoxin after chromatographic, ultraviolet, and infrared spectrophotometric analyses. This compound was also detected in the roots of soybean inoculated with either R. japonicum alone or in combination of R. japonicum and M. phaseolina. Dosage-response curves with rhizobitoxin showed it to be antifungal. Endophytic bacteria produce antibiotic which is an effective mechanism for prevention of pathogens. Bacillus has commonly shown antagonism against many pathogens. The potential of several strains have been checked on a number of plants for control of several pathogens. Members of pseudomonas have also capacity for control of soil-borne pathogens. During the last three decades fluorescence pseudomonads appeared as potentially most promising group of PGPR for biocontrol of plant diseases. Bacillus strain BPR7 strongly inhibited the growth of several phytopathogens such as Characterization of endophytic rhizobacteria from Vigna mungo (L.) Hepper and their role in biocontrol of Macrophomina phaseolina (Tassi) Goid. 22

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Macrophomina phaseolina, Fusarium oxysporum, F. solani, Sclerotinia sclerotiorum, Rhizoctonia solani and Colletotricum sp. in vitro. Cell-free culture filtrate of strain BPR7 also caused colony growth inhibition of all test pathogens (Kumar et al., 2012b). Ahmed et al. (2009) studied the antagonism effect of eight Bacillus isolates against Fusarium oxysporum f. sp. cucumerinum in vitro and in glasshouse. It was showed that Bacillus subtilis No.2, Bacillus spp. No.2 and Bacillus Subtilis No.1 caused the highest inhibition zone (35.7, 34.0 and 30.37 mm, respectively); hence they were best antagonistic bacteria against F. oxysporum f. sp. cucumerinum. Besides this Bacillus culture filtrates also affected the spore germination of F. oxysporum at different concentration (10-50%). As concentration increased it caused decrease in spore germination. Culture filtrates of Bacillus subtilis No.2 and Bacillus spp. No.2 also were more effective for reduction of mycelial growth and reducing the spore germination of F. oxysporum by 80.74 and 80.37 %, respectively. In comparison to Bacillus subtilis No.2 and Bacillus subtilis spp. 2, Bacillus megtla was the best and effective isolate for completely prevention of disease severity by 93.33% and 91.67%, respectively (Ahmed et al., 2009). Singh et al. (2008b) reported that cell-free culture filtrate of B. subtilis BN1 also prevented the growth of M. phaseolina. Some lytic enzymes, chitinase and β-1,3glucanase are also produced by B. subtilis BN1, which are cause hyphal degradation and cell wall digestion of M. phaseolina. Thus B. subtilis BN1 proved as effective biocontrol agent. Siddiqui et al. (2002) found that after inoculation Pseudomonas fluorescens strains CHA0 and IE-6S+ inhibited in vitro growth of Bradyrhizobium japonicum 569Smr, while IE-6S+ suppressed CHA0. Unlike antagonism most of the bacteria show synergistic effects; some of the rhizobia isolates has synergistic interaction with Pseudomonas fluorescence and potential antagonistic activity against pathogen. Samavat et al. (2011) applied singly or in combination with the culture filtrate of five rhizobia isolates to

Characterization of endophytic rhizobacteria from Vigna mungo (L.) Hepper and their role in biocontrol of Macrophomina phaseolina (Tassi) Goid. 23

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evaluate the potential of two isolates of P. fluorescence (UTPF 68 and UTPF 109) for biocontrol of Rhizoctonia solani causing damping-off of bean. Many species of rhizobia not only could fix the nitrogen for plant growth but also have antagonistic effect on soil-borne pathogens (Muthamilan and Jeyarajan, 1996; Deshwal et al., 2003; Bardin et al., 2004). It can cause inhibitory affect on some other pathogenic fungi such as Macrophomina phaseolina, Fusarium spp., Rhizoctonia spp. and Pythium spp. in both legumes and non-legumes (Hossain and Mohammed, 2002). Potential of biocontrol in B. subtilis BN1 against M. phaseolina was associated with root rot disease of the same plant (Singh et al., 2008b). Several other workers have also found the biocontrol activities of Bacillus against many common phytopathogens (Chung et al., 2008; Gajbhiye et al., 2010). Deshwal et al. (2003) isolated ten strains of Bradyrhizobium sp. (Arachis) in peanut. Among those only three Bradyrhizobium strains AHR-2amp+, AHR-5amp+ and AHR-6amp+ were produced siderophore, IAA and exhibited phosphate solubilization in vitro. They showed antagonistic activity against Macrophomina phaseolina. These results prove the antagonistic as well as plant growth-promotory properties of Bradyrhizobium strains. Bradyrhizobia has dual advantage and the more potential for biocontrol compared to that of fluorescent pseudomonads because bradyrhizobia assimilates atmospheric nitrogen besides killing deleterious phytopathogens. Dubey et al. (2012a) found that in dual culture the metabolites of Bradyrhizobium strains VR2 caused several deformities in hyphae and sclerotia of M. phaseolina such as fragmentation, shrinkage and lysis of hyphae, cytoplasm vacuolation, loss of mycelial pigment, and inability of sclerotia formation and germination as observed in SEM. Such deformities have also been reported in the hyphae of Sclerotinia sclerotiorum by Pseudomonas aeruginosa GRC1 (Gupta et al., 2006) and Pseudomonas fluorescens PS1 (Aeron et al., 2011), M. phaseolina by Bacillus subtilis BN1 (Singh et al., 2008b), Fusarium udum by root nodulating Sinorhizobium fredii KCC5 and P. fluorescens

Characterization of endophytic rhizobacteria from Vigna mungo (L.) Hepper and their role in biocontrol of Macrophomina phaseolina (Tassi) Goid. 24

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LPK2 (Kumar et al., 2010), and F. oxysporum by Ensifer meliloti and R. leguminosarum (Kumar et al., 2011b), etc. Gupta et al. (2006) studied antagonistic activity of Pseudomonas aeruginosa GRC1 against Sclerotinia sclerotiorum, in vitro and in vivo. Scanning electron microscopic (SEM) observation showed that P. aeruginosa GRC1 caused morphological deformities (damage, lysis and destruction) of hyphae of S. sclerotiorum due to the production of extracellular chitinase enzyme, the role of which was clearly demonstrated through Tn5 mutagenesis. From the rhizosphere of chirpine (Pinus roxburghii), number of bacterial isolates were isolated having antifungal and good plant growth-promoting trait. Among those, Bacillus subtilis BN1 showed strong antagonistic activity against phytopathogens such as Macrophomina phaseolina, Fusarium oxysporum and Rhizoctonia solani, in which it caused vacuolation, hyphal constriction, swelling, abnormal branching and lysis of mycelia (Singh et al., 2008b). Kumar et al. (2012b) monitored seven bacterial isolates from the rhizosphere of common bean which showed prospective plant growth promoting (PGP) and antagonistic activities. Bacillus sp. strain BPR7 produced IAA, siderophore, phytase, organic acid, ACC deaminase, cyanogens, lytic enzymes, oxalate oxidase, and solubilized various sources of organic and inorganic phosphates as well as potassium and zinc. Strain BPR7 has strongly antagonistic property resulting inhibited the growth of several phytopathogens (in vitro) such as Macrophomina phaseolina, Fusarium oxysporum, F. solani, Sclerotinia sclerotiorum, Rhizoctonia solani and Colletotricum sp. Efficacy of Cell-free culture filtrate of strain BPR7 also checked by its growth inhibition of colony of all test pathogens (Kumar et al., 2012b). Most of the control methods aim to reduce the number of sclerotia in soil or to minimize the contact of the inoculum and the host. Chemical fertilizers and fungicides have been recommended to enhance the yield and control the pathogen but it has resulted in degradation of soil health. Therefore, the alternative methods are being Characterization of endophytic rhizobacteria from Vigna mungo (L.) Hepper and their role in biocontrol of Macrophomina phaseolina (Tassi) Goid. 25

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imagined in an ecofriendly approach aimed at sustainable agriculture. Researchers have investigated the in vitro sensitivity of different isolates of M. phaseolina to fungicides and the efficacy of fungicide application to seed and soil to reduce fungal germination and infection. However, chemical control of M. phaseolina is difficult and neither profitable nor desirable (Alice et al., 1996). Soil solarization, addition of organic amendments, and maintenance of high soil moisture content (Dhingra and Sinclair, 1975) are the better alternative of hazardous chemical pesticides and have been suggested as possible methods to manage soil-borne pathogens. Solarization alone was not effective for controlling M. phaseolina in field (Mihail and Alcorn, 1984) soils. Soil moisture content greatly affects the sensitivity of resting structures to heat treatment (Lodha et al., 2003), and one summer irrigation was sufficient to reduce the population of M. phaseolina by 25–42 % (Lodha and Solanki, 1992; Lodha, 1995). Solarization of moistened soil further augmented this reduction in the top soil, but many propagules survived at lower depths (Lodha and Solanki, 1992, Dubey et al., 2009a). Hence, the combined effect of soil solarization and amendment of neem products (leaf, bark and oil cake powders and neem oil) was studied in detail on the survival of M. phaseolina sclerotia in soil. Propagules of M. phaseolina treated with different neem products gradually decreased with increase in duration of soil solarization. The effectiveness of solarization got potentiated upon addition of different neem products. The bacterial counts increased after addition of neem cake powder in solarized soil (Dubey et al., 2009b). Management strategies to control charcoal rot also include the use of biocontrol agents to prevent host infection or to suppress the growth of the pathogen (Siddiqui and Mahmood, 1993). In a broader sense, biological control (also called biocontrol) is the suppression of damaging activities of one organism by one or more other organisms, often referred to as natural enemies. More narrowly, biological control refers to the purposeful utilization Characterization of endophytic rhizobacteria from Vigna mungo (L.) Hepper and their role in biocontrol of Macrophomina phaseolina (Tassi) Goid. 26

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of introduced or resident living organisms, other than disease resistant host plants, to suppress the activities and populations of one or more plant pathogens. This may involve the use of microbial inoculants to suppress a single type or class of plant diseases. Or, this may involve managing soils to promote the combined activities of native soil- and plant-associated organisms that contribute to general suppression. Most narrowly, biological control refers to the suppression of a single pathogen (or pest), by a single antagonist, in a single cropping system (Pal and Gardener, 2006). Biological control may be defined as ‘the use of an organism or organisms to reduce disease caused by other organisms in crops. Biocontrol of plant pathogens includes management of resident populations of organisms and introductions of specific organisms to reduce diseases. Organisms used in biological controls of plant pathogens utilise various mechanisms; therefore they cannot classified into a single group. Since Macrophomina blight may inflict heavy losses to the crop in country and the present cultivars are susceptible to this disease, plant growth promoting rhizobacteria (PGPR) is the best substitute for control of soil borne pathogens. Several genera of bacteria have ability of promoting plant growth termed as plant growth promoting rhizobacteria (PGPR). Plant growth promoting rhizobacteria (PGPR) were first defined by Kloepper and Schroth (1978) as the soil bacteria that colonize the roots of plants by following inoculation onto seed and that enhance plant growth. There are various PGPR to be used as biocontrol agents such as Rhizobium, Bradyrhizobium, Bacillus, Pseudomonas, etc. PGPR enhance plant growth by direct and indirect means, but the specific mechanisms involved have not been well characterized (Kloepper, 1993; Glick, 1995). In nature different plant supports different community of endophytic bacteria which have ability to affect the plant positively. The surroundings of root where plentifully microorganisms present (called rhizosphere or area under the influence the root) is termed as ‘rhizobacteria’. There are number of microbes that have ability to colonize root internally without affecting the host plant negatively termed as ‘endophytes’. They

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share characteristics with PGPR and form nodules for nitrogen fixation in legumes and they have ability to colonize the root of non-legumes. In general, endophytes have been defined as bacteria that are able to colonize living plant tissues without harming the plant or gaining benefit other than securing residency (Kado 1992). Several studies have shown that the interaction between plants and some endophytic bacteria was associated with beneficial effects such as plant growth promotion and biocontrol potential against plant pathogens (Chen et al., 1995; Hallmann et al., 1995; Pleban et al., 1995).There are number of endophytic bacteria, such as Enterobacter, Pseudomonas, Rhizobium, Bradyrhizobium, Bacillus, Pantoea, etc.,

which prevent

infection or disease of soil-borne pathogen and positively affect the plant growth. Strains of Bradyrhizobium sp. and Rhizobium meliloti were reported to be antagonistic against M. phaseolina and to have plant growth promoting properties in urad (Dubey et al., 2012a) and groundnut (Arora et al., 2001; Deshwall et al., 2003). PGPR enhance plant growth by both direct and indirect method. Direct mechanisms of plant growth promotion by PGPR can be demonstrated in the absence of plant pathogens or other rhizosphere microorganism, while indirect mechanisms involve the ability of PGPR to reduce the deleterious effects of phytopathogens on crop yield. PGPR have been reported to directly enhacnc plant growth by a variety of mechanisms such as fixation of atomospheric nitrogen that is transferred to the plant (Kennedy et al., 2004), production of siderophore that chelate iron and make it available to the plant root (Gupta et al., 2001), solubilization of minerals such as phosphorus, zink, and potassium (Gupta et al., 2012) and synthesis of phytohormones such as indole acetic acid (Patten and Glick, 2002), abscisic acid (Dobbelaere et al., 2003), gibberellic acid (Mahmoud et al., 1984), cytokinins (Timmusk et al., 1999) and ethylene (Zahir et al., 2004). Direct enhancement of mineral uptake due to increase in specific ion fluxes at the root surface in the presence of PGPR has also been reported (Bertrand et al., 2000).

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Indirect enhancement of plant growth by PGPR via suppression of phytopathogens occurs by a variety of mechanisms, such as the ability to produce siderophores that chelate iron making it unavailable to pathogens (Pandey et al., 2005), the ability to synthesize antifungal metabolites like antibiotics (Kang et al., 2004), expression of fungal cell wall-lysing enzymes e.g. β-1, 3-glucanases (Ruiz Duenas and Martinez, 1996), β-1, 4-glucanases (Diby et al., 2005), cellulases (Chatterjee et al., 1995), chitinases (Gupta et al., 2006) and hydrogen cynide (Senthilkumar et al., 2009), which suppress the growth of fungal pathogens. Thus the PGPR successfully compete with pathogens for nutrients of specific niches on the roots and thereafter develop systemic resistance. Among these a very important bacterial enzyme like 1-aminocyclopropane1-carboxylate (ACC) deaminase plays a significant role in the regulation of a plant hormone, ethylene and enhance the growth and development of plants (Glick, 2005). Bacterial strains containing ACC deaminase can at least alleviate the stress-induced ethylene-mediated negative impact on plants (Glick, 2005). Remans et al. (2007) examined the potential of PGPR containing ACC-deaminase to enhance nodulation of common bean (P. vulgaris). Different types of functions performed by PGPR are shown in Fig. 2.

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Fig. 2. Different functions of plant growth-promoting rhizobacteria (PGPR) (sources: http://www.envitop.co.kr/10chumdan/07/sp1.htm)

Kumar and Dubey (2012) reviewed the plant growth promoting rhizobacteria for biocontrol of phytopathogens and yield enhancement of Phaseolus vulgaris with special reference to IAA production, phosphate solubilization, organic acid production, zinc solubilization, potassium solubilization, ACC deaminase production, HCN production, siderophore production, oxalate-oxidase enzyme production, lytic enzyme production, and nitrogen fixation. PGPR have the potential to contribute in sustainable agricultural systems by functioning in three different ways: (i) synthesizing particular compounds for the plants, (ii) facilitating the uptake of certain nutrients from the soil, and (iii) preventing the plants from diseases (Deshwal et al., 2003; Singh et al., 2008b, 2010). Siddiqui et al. (2002) studied the effect of Pseudomonas fluorescens CHA0, P. aeruginosa IE-6S+ and B. japonicum 569Smr singly and in combinations for biological control against multiple tomato pathogens such as M. phaseolina, Fusarium solani and Characterization of endophytic rhizobacteria from Vigna mungo (L.) Hepper and their role in biocontrol of Macrophomina phaseolina (Tassi) Goid. 30

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Rhizoctonia solani AG 8, and root-knot nematodes (e.g. Meloidogyne javanica). When using an iron chelator to create iron deficiency in the soil, the biocontrol efficacy of the bacteria against F. solani and R. solani was enhanced. P. aeruginosa increased growth of plant and nodulation in urad plants. For reduction of M. phaseolina, P. aeruginosa was used with or without Bradyrhizobium. Because of root knot nematode (Meloidogyne spp.), root infecting fungi viz., M. Phaseolina, R. solani and Fusarium spp. causes various diseases in urad resulting serious losses in crop (Ethteshamul-Haque, 1994; Ghaffar, 1995). Rhizobia termed as the root nodulating bacteria are also known to reduce the soil-borne root infecting fungi (EthteshamulHaque and Ghaffar, 1993; Siddiqui et al., 1998). It has shown the potential of coinoculation of P. aeruginosa and Bradyrhizobium to control the root rot disease (M. phaseolina, R. solani and F. solani) on urad. Nitrogen is an essential plant nutrient and its average content is up to 80% in atmosphere. It is deficient in soils; causative plants are unable to use this atmosphere nitrogen which leads to reduced agricultural yields. Biological nitrogen fixation system possibly is a good alternative. It makes available the nitrogen supply to the plants directly (as fixed nitrogen) without any loss. Treatment with nitrogen fixing microorganisms shows a considerable increase in growth. Biological nitrogen fixation process by bacteria fix around 65% of the nitrogen currently utilized in agriculture, and will continue to be important in future sustainable crop production systems (Matiru and Dakora, 2004). In response to symbiotic association with rhizobia, flavonoid molecules are released as signals by the leguminous plant, which leads to induce the expression of nod genes (for nodulation) in rhizobia, which in turn produce lipo-chitooligosaccharide (LCO) signals that generate mitotic cell division in roots leading to nodule formation (Dakora, 2003). In legumes N2 fixation also benefits to linked non-legumes through transfer of fixed nitrogen to cereals growing with legume crops (Snapp et al., 1998) or to the following crops rotated with symbiotic legumes (Deshwal et al., 2006; Hayat et al., 2008). Characterization of endophytic rhizobacteria from Vigna mungo (L.) Hepper and their role in biocontrol of Macrophomina phaseolina (Tassi) Goid. 31

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Bradyrhizobium fix more nitrogen that the plant can use. The excess nitrogen is left in the soil and available for other plants or later crops. Intercropping with a legume has the potential to decrease the need for applied fertilizer. Co-inoculation of PGPR and Rhizobium spp. have been shown to increase root and shoot dry weight, plant vigour, nodulation, and nitrogen fixation in various legumes. In the root surroundings presence of PGPR improve ability of rhizobia to compete with indigenous populations for nodulation. Chakraborty and Purkayastha (1984) reported that bacterization of soybean seeds or roots with R. japonicum significantly reduced charcoal rot disease caused by M. phaseolina. Possibly rhizobitoxine may play a role in protecting soybean roots from infection by M. phaseolina. Co-inoculation of Bradyrhizobium with P. striata has also been observed to enhance biological nitrogen fixation in soybean (Dubey, 1996). Bacillus sp. co-inoculated with Rhizobium etli has been found to enhance nodulation in common bean (Srinivasan et al., 1997). Bai et al. (2003) have found the increased soybean growth and nodulation upon inoculation of Bradyrhizobium with Bacillus. Bacillus is frequently isolated from rhizosphere, some species were also common plant endophyte. B. mucilaginous has been observed for its capability in solubilizing potassium (Wu et al., 2005) and phosphate (Idriss et al., 2002). It has also been reported that wheat yield increased up to 30% with Azotobacter inoculation and up to 43% with Bacillus inoculation due to some growth hormones such as indole acetic acid (IAA) (Kloepper et al., 1991). Deshwal et al. (2003) reported that seeds bacterized with Bradyrhizobium strains were significant by improved seed germination, seedling biomass, nodule number, and nodule fresh weight, average nodule weight compared to un-inoculated and uninfected controls. Gupta et al. (2006) have found that neomycin resistant Pseudomonas aeruginosa GRC1neo+ bacterium which was habitually isolated from rhizosphere of peanut plants was a good root colonizer and a potential biocontrol agent against S. sclerotiorum. Peanut seeds bacterization with strain GRC1 led to increased seed Characterization of endophytic rhizobacteria from Vigna mungo (L.) Hepper and their role in biocontrol of Macrophomina phaseolina (Tassi) Goid. 32

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germination and reduced stem-rot by 97%. Other vegetative and yield plant parameters such as nodules per plant, pods and grain yield per plant were enhanced in comparison to control. Singh et al. (2008b) found in pot trial study a significant increase in seedling biomass besides reduction in root rot symptoms in chir-pine seedlings. Consequently, root and shoot dry weights got increased by 43.6% and 93.54%, respectively as compared to control. Deshwal et al. (2006) found that peanut seeds coated with Bradyrhizobium strains enhanced seed germination, seedling biomass, nodule number, nodule fresh weight, and average nodule weight as compared to uninoculated and uninfected controls. The potential of several nodule inducing bacteria was tested by Antoun Hani et al. (1998) by using radish as a model plant. Among 266 strains tested, three percent were found to be cyanogens, 83% of strains produced siderophores. 58% produced indole 3acetic acid (IAA) and 54% solubilized phosphorus. Some of the bacterial species have deleterious effect, while the others were neutral or displayed a stimulatory effect on radishes. B. japonicum strain Soy 213 was found to have the highest stimulatory effect (60%), and an arctic strain (N44) was the most deleterious, causing 44% reduction in dry matter yield of radish. A second plant inoculation test, performed in growth cabinets, revealed that only strain Tal 629 of B. japonicum significantly increased (15%) the dry matter yield of radish. This indicates that specific bradyrhizobia have the potential to be used as PGPR on non-legumes. Javaid (2009) investigated the effect of EM application and two strains of nitrogen fixing Bradyrhizobium japonicum (TAL- 102 and MN-S) on plant growth, nodulation and yield of black gram. They recorded a marked increase in nodule biomass due to B. japonicum inoculation in two types of soils. Grain yield was significantly increased by 46% due to either of the two B. japonicum strains in NPK-amended soil. In greenhouse experiments, suspension of the cells of Bacillus cereus CH2 strain reduced the severity of Verticillium wilt on eggplant by 69.69%, its supernatant by 54.04%, and the enzyme diluted to 0.01% strength by 53.13% in 14 days. Strain CH2 Characterization of endophytic rhizobacteria from Vigna mungo (L.) Hepper and their role in biocontrol of Macrophomina phaseolina (Tassi) Goid. 33

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and its chitinase have good commercial potential in controlling Verticillium wilt (JianGang et al., 2008). Bakshi et al. (2006) studied survival, nodulation and N2 fixing ability of root nodule bacteria under different environmental conditions. They found a better survival ability of all slow growing strains of Bradyrhizobium than the fast growing strains. Siddiqui et al. (2002) reported better rhizosphere colonization by Pseudomonas fluorescens strain IE-6S+ than CHA0 and Bradyrhizobium japonicum 569Smr. Populations of P. fluorescens strain CHA0 declined in rhizosphere when the bacterium was used with either IE-6S+ and/or 569Smr, while populations of P. fluorescens IE-6S+ in the rhizosphere were enhanced when used in combination with CHA0 and/or 569Smr. IE-6S+ was the only bacterium that colonized inner root tissues of tomato plants. Singh et al. (2008b) reported a continuous increase in population of Bacillus subtilis strain B1 was 1.5  104 c.f.u. g-1 root after one month, which increased to 4.5  104 c.f.u. g-1 root in three months. Positive root colonization capability of B. subtilis BN1 proved it as a potential biocontrol agent. Bhatia et al. (2008) reported that the population of fluorescent Pseudomonas strains PS1 and PS2 increased due to aggressive root colonization in the rhizosphere in the first 15 days which constantly increased up to 60 days. Singh et al. (2010) evaluated P. aeruginosa strain PN1rif+strep+ for colonization of chir-pine roots. The strain successfully colonized chir-pine roots both alone and in combination with M. phaseolina, and increased its population in the chir-pine rhizosphere.

Characterization of endophytic rhizobacteria from Vigna mungo (L.) Hepper and their role in biocontrol of Macrophomina phaseolina (Tassi) Goid. 34