Bacillus subtilis. A bioagent in nematode management

Table of Contents

I. INTRODUCTION

II. Plant Growth Promoting Rhizobacteria in nematode management

III. Diversity of Bacillus species

IV. Biocontrol efficacy of Bacillus spp. on nematodes

V. Survey and isolation of PGPR bacteria

VI. Bacillus subtilis in nematode management

VII. Overview of the work

VIII. References

I. INTRODUCTION

Floriculture is a fast emerging venture throughout the world and is recognized as a lucrative profession with higher potential for returns per unit area. In India, 1,91,000ha are under cultivation of flower crops (2010- 2011) with the production of 7000 millions of cut flowers and 10.40 lakh metric tons of loose flowers and 7000 million cut flowers (NHB Database 2011).

Among the important ornamental bulbous plants grown in India, tuberose (Polianthes tuberosa L.) occupies a prime position due to its popularity and economic potential as a cut flower, loose flower and its potential in perfume industry. Tuberose belongs to the family Amaryllidaceae and is native of Mexico. Among the single flowered types, Calcutta single variety is extensively used as loose flower and for extraction of essential oil.Prajwal, a cross between Shringar and Mexican single cultivar is used as cut flower. Among the double flowered types, Calcutta double is preferred as cut flower, loose flower and also for oil extraction. Abiotic and biotic stress play a major role which affect the yield. Among abiotic factors, temperature is the most important one affecting growth, flower initiation and subsequently flower and bud development. Optimum temperature range for growth and development of tuberose is 20-30 °C (Sadhu and Bose, 1973).

Among the biotic stresses, nematodes are one of the major limiting factors reducing the yield of the crop both in quantity and quality. Most of the growers of tuberose are not aware of the menace since the nematodes are unseen enemies. The crop harbours many nematode pests, viz., root knot, spiral, reniform, lance and stunt nematodes. Monoculture of the crop increases the population of nematode fauna. The technologies such as cropping system approach, new fertilizer regime and irrigation schedule generally provide a congineal environment for the preponderance of the nematode species. Among the nematodes, root knot nematode, Meloidogyne incognita is the key nematode pest of the crop which causes serious damage and affects the flower yield of the crop. Root knot nematodes, viz. M. incognita, M. javanica and M. arenaria have been considered as the major limiting factors in the successful cultivation of tuberose in Tamil Nadu (Jayaraman et al., 1975). Affected plants exhibit the symptoms of yellowing, drying of leaves and retarded growth. In severely infested plants, emergence of the spike is also suppressed. At higher inoculum level of M. incognita, 65 per cent incidence was noticed in top growth of plant (Sundarababu and Vadivelu, 1988).

Nematode management with nematicides in farmer's field has limitations due to higher cost and difficulties in applying them in fields. Moreover, chemical applications cause hazards to the environment. Therefore, safe alternate methods for managing plant parasitic nematodes in field are critically needed for the development of sustainable cropping systems.

A promising alternative is the use of microbial antagonists against plant parasitic nematodes which are ecofriendly and economically feasible approaches and does not allow the nematodes to develop into new races or biotypes. In addition, they are amenable for the mass production, formulation and easy delivery in the field. In recent years, Plant Growth Promoting Rhizobacteria (PGPR), viz. Pseudomonas fluorescens and Bacillus subtilis (Oostendorp and Sikora, 1989) have been reported to be effective in boosting the plant vigour and also found to be deleterious to the plant pathogens and nematodes (Rodriguez Kabana et al., 1965; Singh et al., 1990).

An investigation was undertaken study the effect of liquid bioformulation of native antagonistic Bacillus spp. against the infestation of root knot nematode in tuberose with the objectives namely,

Survey and identify the level of infestation of root knot nematodes in tuberose grown in different areas of Tamil Nadu and to isolate native Bacillus.

Characterization of the Bacillus isolates and to study the in vitro efficacy of Bacillus isolates against the root knot nematode, M. incognita.

Preparation of effective Bacillus isolates in aqueous formulation and to study its effectiveness against the root knot nematode in tuberose under pot culture and field conditions and to visualize the histopathological changes in tuberose roots due to nematode -Bacillus interaction and the induction of defence mechanism with the application of biocontrol agent.

II. Plant Growth Promoting Rhizobacteria in nematode management

Soil borne diseases incited by nematode, fungi and bacteria cause extensive damage to crop plants and adversely affect the agricultural production. Nematodes are major pathogens in their own right but their interactions with other disease causing agents make it difficult to measure their impact on yield accurately.

More than 100 bioagents belonging to fungi, bacteria, viruses, nematodes, protozoans have been reported world over in the last five decades. Of these bioagents, few are predacious, some are parasitic and many are opportunistic. In the recent past, biological control of plant parasitic nematodes has been focused mainly on use of parasites and predators (Mankau, 1981). Opportunistic bioagents which are capable of deriving nutrition from organic source are easy to mass-produce and survive better in the nature. They have now emerged as promising bioagents against endoparasitic and semi-endoparasitic nematodes.

Most of the approaches in the management of phytonematodes exclusively based on the application of chemical pesticides. Environmental risks with nematicides warrant the development of safe options as many of these chemicals are proven to be carcinogens, build up residues in the plants and infiltrate into ground water (Zukerman and Esnard, 1994). Some of these chemicals are equally hazardous to livestock, and to the beneficial fauna and flora of the soil. Biocontrol approaches help to develop an ecofriendly sustainable management strategy for controlling the nematodes.

Antagonists most likely to be receptive to the management for the biological control of nematodes are Plant Growth Promoting Rhizobacteria (PGPR), antagonistic bacteria and predacious or trapping fungi. Among the antagonistic microorganisms, PGPR have been considered to be an important in sustainable agriculture for the management of nematodes due to their plant growth promotional ability as well as biocontrol potential.

Bacteria that colonize roots are termed Rhizobacteria (Schroth and Hancock, 1982). Root colonization is the process where bacteria survive on seeds, multiply in spermosphere in response to seed exudates rich in carbohydrates and amino acids (Kloepper et al., 1989) attach on to the root surfaces (Suslow, 1982) and colonize the developing root system. PGPR are free-living bacteria and some of them invade the tissues of living plants and cause unapparent and symptomatic infections (Sturz and Nowak, 2000) when applied to seeds or crops, enhance the growth of the plant or reduce the damage from soil-borne plant pathogens (Kloepper et al., 1980).

When the effect of PGPR on plants was demonstrated, the concept of PGPR began to gain importance. A large number of bacterial strains have been isolated, screened (Chanway and Holl, 1993; Bertrand et al., 2001) and were evaluated for plant growth promotion (Lifshtiz et al., 1987; Chanway et al., 1989; Glick et al., 1997; Bashan and Holguin, 1998; Bent et al., 2001). Several research groups have randomly screened rhizosphere bacteria for antagonistic activity against nematodes (Becker et al., 1988; Oostendorp and Sikora, 1990 and Spiegel et al., 1991). Isolates of Agrobacterium radiobacter, B. subtilis and Pseudomonas spp. that are familiar as antagonists of soil borne bacterial and fungal diseases of plants also have potential as biological control agents for nematodes.

Somers et al. (2004) classified PGPR as biofertilizers (increasing the availability of nutrients to plant) phytostimulators (plant growth promoting, usually by the production of phytohormones) rhizoremediators (degrading organic pollutants) and biopesticides (controlling diseases, mainly by the production of antibiotics and antifungal metabolites).

PGPR colonized the plant roots resulting in enhanced plant growth and soil suppressiveness against M. incognita (Marleny et al., 2008). They enhance plant growth by wide mechanisms like phosphate solubilization, siderophore production, biological nitrogen fixation, rhizosphere engineering, phytohormone production, exhibiting antifungal activity, production of volatile organic compounds (VOCs), induction of systemic resistance, promoting beneficial plant-microbe symbiosis, interference with pathogen toxin production etc. The potentiality of PGPR in agriculture is steadily increased as it offers an attractive way to replace the use of chemical fertilizers, pesticides and other supplements.

Growth promoting substances are likely to be produced in large quantities by these rhizosphere microorganisms that influence indirectly on the overall morphology of the plants. Diversity of PGPR in the rhizosphere along with their colonization ability and mechanism of action could facilitate their application as a reliable component in the management of sustainable agricultural system (Bhattacharyya and Jha, 2012).

They were also found to increase chlorophyll contents in the plant PGPR isolates viz., Azospirillum lipoferum, Azotobacter chroococcum, Pseudomonas fluorescens and Bacillus megaterium significantly increased germination rate, vigour index chlorophyll content and nutrient content of the plant. (Lenin and Jayanthi, 2012).

Although a large number of bacteria have shown antagonistic effects against nematodes but the most important genera include Rhizobium (R. leguminosorum), Bradyrhizobium japonicum, Mesorhizobium sp., Azorhizobium sp., Pseudomonas (P. fluorescens and P. aeruginosa) and Bacillus (B. subtilis). Application of some of these bacteria has accorded promising results.

There are several reports in the literature indicating that PGPR could be proved a boon in sustainable agriculture. Their beneficial events could be biological control of diseases and nematodes, plant growth promotion, increase in crop yields and quality improvement that can take place simultaneously and sequentially (Jonathan et al., 2005; Ambreen et al., 2012).

Mode of action of PGPR

Biological control is achieved through mechanisms such as parasitism, competition and antibiosis which adversely affect the fitness, survival and reproduction of nematodes. Antagonism is an umbrella term for parasites, predators, pathogens, competitors and other organisms that repel, inhibit or kill plant parasitic nematodes. There are many species of soil bacteria which are reported to promote the plant growth by producing growth regulators, inducing root exudation and enhancing the availability of nutrients to plants besides controlling the pathogens (Sinclair, 1989). PGPR induce plant growth promotion by direct or indirect modes of action (Kloepper et al., 1988; Kloepper et al., 1989; Beauchamp, 1993; Kloepper, 1993; Liu et al., 1995 and Lazarovits and Nowak, 1997). The mechanism involved in PGPR-mediated plant growth promotion is directly by production of plant growth regulators (auxins, cytokinins, gibberellins) and facilitation of the uptake of nutrients (nitrogen fixation, solubilization of phosphorus). The indirect promotion of plant growth occurs by preventing the deleterious effects of plant pathogens on plants by the production of inhibitory substances (antibiotics, antifungal metabolites, iron-chelating siderophores, cell wall-degrading enzymes and competition for sites on roots) or by increasing the natural resistance of the host (induced systemic resistance).

Although the mode of action of the bacteria differs, direct effects on egg hatching and nematode mobility and indirect effects such as alternation of root exudates and induced resistance, make roots less attractive to nematodes (Oostendorp and Sikora, 1990; Sikora and Hoffmann Hergarten, 1992).

Endophytic bacteria such as Bacillus, Pseudomonas have been shown to reduce root knot nematode infection (Mahdy et al., 2000, Munif et al., 2000) and induce systemic resistance in tomato (Munif et al., 2001).Application of P. fluorescens to the plants inhibit early root penetration of phytonematodes by alternating of specific root exudates such as polysaccharides and amino acids which modify nematode behaviour (Oostendrop and Sikora, 1990; Sikora, 1992; Sikora and Hoffman Hergarten, 1992).

III. Diversity of Bacillus species

The genus Bacillus consists of a heterogenic group of gram positive rods, able to form endospores that allow them to survive for extended periods under adverse environmental conditions.

Endospore formation is the dominant feature in the characterization of Bacillus. There is a boundary that separates this genus from other genera in which endospores are produced. The genus, Clostridium is distinguished from Bacillus by inability to grow on the surface of agar media if growth does occur under these conditions, it is slight and does not lead to sporulation. There is also little or no catalase activity (Gibson and Gordon, 1974).

Bacteria of the genus Bacillus Cohn are widely distributed in nature, easy to multiply, have a long shelf life when sporulated and are nonpathogenic. The numbers of Bacillus are varying from 106 in cooler regions to 107 or more per gram in warmer latitudes (Alexander, 1961). B. subtilis, B. mycoides, B. pumihis, B. megaterium, B. thuringiensis and B. firmus are wide range of Bacillus present in rhizosphere soil (Wipat and Harwood, 1999; Garbeva et al., 2003) which produce cytotoxin (From et al., 2005).

Bacillus spp. being industrially important organism produces a wide variety of extra-cellular enzymes including proteases. Bacillus spp isolated from local soil samples collected from Bangalore were found to produce protease enzyme (Soundra et al., 2012).

Mannitol (1%) and soytone (1%) were found to be the best carbon and nitrogen sources, respectively, for use in antibiotic production of the Bacillus. On the basis of spectral data, including proton nuclear magnetic resonance (1H NMR), carbon nuclear magnetic resonance (13C NMR), and mass analyses, the chemical structure of antibiotic produced by Bacillus isolate, C9 was established as a stereoisomer of acetylbutanediol. Potential of compounds extracted from B. subtilis as a biocontrol agent and plant growth promoter with trigger induced systemic resistance of plants has been documented in the research conducted by Rezuanul et al. (2012).

Bacillus strains, MPB04 and MPB93 showed remarkable nematicidal activity which killed M. incognita within 2 h and completely destroyed tested nematode within 12 h. Nematicidal activity displayed by Bacillus strains is related with their proteolytic activity. The same Bacillus strains in pot culture trials reduced the root population of M. incognita by 60.95 and 35.28 per cent, respectively over control (Yap Chin Ann, 2013).

Chitin degrading B. subtilis and Bacillus atrophaeus were isolated from crustacean shells. (Anuradha and Revathi, 2013) and these bacteria can be effectively utilised for the nematode management.

IV. Biocontrol efficacy of Bacillus spp. on nematodes

Bacillus is the large group of bacteria that have shown diversified effects on plant-parasitic nematodes. Bacillus spp. that demonstrated nematicidal effects include B. subtilis, B. pumilis (Gokte and Swarup, 1988), B. cereus (Gokte and Swarup, 1988; Kempster et al., 2001) and B. licheniformis (Siddiqui and Mahmood, 1992). The non-cellular extract of Bacillus species exhibited high larvicidal properties. The first line of evidence of broad spectrum activity of antibiotics by PGPR was derived from culture filtrates or purified antibiotics (Nakayama et al., 1999).

In another in vitro studies, bacterial supernatant and whole culture of B. thuringiensis var. hrasiliensis and B. laterosporus was found to reduce the juvenile population within 24-48 hours of exposure whereas treatments with B. thuringiensis var. aizawai, B. thuringiensis var. morrisoni and B. circulans caused only immobilization of nematodes. Although the chemical nature of the supernatants was not determined, the results suggested the presence of extra cellular toxins with strong nematicidal activity (Regina et al., 1998).

Clover plants treated with P. fluorescens and B. subtilis had fewer galls and large root (Becker et al., 1988). Application of Bacillus licheniformis (Siddiqui and Mahmood, 1992) and B. subtilis (Khan et al., 2001) caused greater reduction in nematode multiplication on chickpea. Combined application of P. fluorescens, Bacillus spp. and VAM could be used as successful biocontrol agents for the management of M. incognita and Tylenchulus semipenetrans in the crops like citrus, tomato, potato and chilli (Rajendran et al., 2001).

Numerous Bacillus strains have been found to express activities that suppress pests and pathogens including nematodes (Siddiqui and Mahmood, 1999). The most thoroughly studied Bacillus includes B. subtilis and B. thuringiensis (Crickmore et al., 1998; Krebs et al., 1998; Siddiqui and Mahmood 1999). B. thuringiensis (Bt) produces one or more parasporal crystal inclusions (Cry or 6-endotoxins). These toxins are known to be toxic to a wide range of insect species (Feitelson et al., 1992). Some Cry proteins are also toxic to nematodes (Feitelson et al., 1992). To date, five Cry proteins (Cry5B, Cry6A, Cry13, Cryl4A, Cry21A) known to be toxic to juveniles of number of free-living or parasitic nematodes (Crickmore et al., 1998; Wei et al., 2003).

B. thuringiensis (Bt) is a potential biopesticide of large number of insect pests. Its toxic effects to eggs and juveniles of M. javanica in vitro (Prasad et al., 1972) and under natural conditions (Prasad and Tilak, 1972) were reported. They have nematicidal effects against plant-parasitic nematodes (Zuckerman et al., 1993) including Meloidogyne spp. (Rai and Rana, 1979), M. hapla (Chen et al., 2000) and lethality to eggs and second-stage juveniles of root knot nematode under laboratory conditions (Al Banna and Khyami-Horani, 2004). Cell free filtrates of Bt killed the juveniles of M. incognita completely 24 h of exposure in standard filtrates (S) and S/10 dilution (Dhawan et al., 2004).

B. thuringiensis var. kurstaki 113 exhibited effectiveness against the population ofR. similis infesting banana under field condition (Meena et al., 1997) and it reduced the tomato root galling by 51-59 per cent when M. javanica was inoculated (Khyami Horani and Al Banna, 2006). P. fluorescens and B. thuringiensis showed nematicidal activity against juveniles and adults of M. incognita infecting tomato plants. The mortality levels of M. incognita increased with increase in the concentration of bacterial cells (5 x 108 cfu/ml) (Hanna et al., 1999). Jonathan et al. 2000 reported that rhizobacteria, viz. P. fluorescens and Bacillus spp. induced profuse root development in banana, tomato and betelvine and reduced M. incognita population. In green house, B. subtilis exhibited strong nematicidal activity against M. javanica juveniles to varying degrees (Li Bin et al., 2005).

Microbial proteases have been proposed as virulence factors in the pathogenesis against nematodes. The most compelling evidences to support microbial proteases as virulence factors have come from the studies of protease-deficient mutants (Ahman et al., 2002; Siddiqui et al., 2005; Tian et al., 2006). The nematotoxic bacteria, B. laterosporus lost 57 per cent of its nematicidal activity because of the deletion of the extracellular alkaline protease, BLG4 (Tian et al., 2006). Siddiqui et al. (2005) also demonstrated that the deletion of a major extracellular protease from P. fluorescens CHA reduced bacterial activity against the root-knot nematode M. incognita. These researches suggested extracellular proteases might play an important pathogenic role in suppressing nematodes in the soil (Ambreen et al., 2012).

Jonathan and Umamaheswari (2006) conducted a pot culture study to assess the biocontrol potential of endophytic bacterial isolates of B. subtilis (EPB 5, 22, 31 and EPC 16) which was prepared in talc-based formulation against the nematodes of banana, viz., M. incognita, Pratylenchus coffeae, Radopholus similis and Helicotylenchus multicinctus on tissue cultured banana cv. Robusta (Musa AAA). Significant increase in shoot height and weight, root length and weight, pseudostem girth and number of leaves coupled with reduction in nematode population was observed in the combined treatment of EPB 5 and EPB 31.

Combined application of P. fluorescens (Pfbv 22 ) and B. subtilis (Bbv 57) as seed treatment each at 5g/kg of seeds and soil application at 1.25 kg/ha significantly reduced the R. similis and M. incognita infestation in soil and root of tomato. The microbial consortium treatment also significantly enhanced the plant growth parameters such as plant height, shoot weight, root length, root weight and fruit yield (Jonathan et al., 2009).

Effects of PGPR can occur via local antagonism to soil-borne pathogens and nematodes or by induction of systemic resistance against pathogens. Rhizobacteria belonging to the genera Pseudomonas and Bacillus are well known for their antagonistic effects and their ability to trigger ISR. Resistance-inducing and antagonistic rhizobacteria might be useful in formulating new inoculants with combinations of different mechanisms of action, leading to a more efficient use for biocontrol strategies to improve cropping systems (Beneduzi Aziz, 2013).

Kavitha et al. (2012) reported that the B. subtilis strains viz., BsN3, Bs5 and Bbv57 produced surfactin and iturin which suppressed the hatching of M. incognita and also killed the juveniles.

Low cost organic substrates such as rice grain, chickpea, wheat grains, rice husk, chickpea husk and wheat bran were used to culture B. subtilis and B. firmus. Vegetative cell counts of both bacterial species generally increased on majority of substrates in initial 5 days and declined in subsequent incubation. Spore counts also increased with time but declined at 10th day. The effectiveness of bacterial species grown on different organic substrates may vary but they controlled root-knot nematodes population in okra (Muhammad et al., 2013).

Bacillus alvei NRC-14 potentiality reduced nematode eggs and juveniles by production of hydrolytic enzymes which directly hydrolyze nematode eggs and juveniles (Abdel et al., 2013).

Combined application of Trichoderma koningii and Bacillus megaterium for the management of disease complex caused by a mixed population of root knot nematode(M. javanica and M.incognita) and the wilt fungus Fusarium oxysporum, on the growth of potato cv. Nicola, caused significant reduction of Fusarium wilt disease incidence and nematode infection on potato and improved plant growth components. Generally, the combination of the two bio control agents was more effective in controlling the plant disease and improving plant growth components than either of the two organisms used singly (Shennawy et al., 2012).

Plant growth promotion ability of Bacillus

De Freitas et al. (1997) assessed the potential use of phosphate solubilizing Bacilli and other rhizobacteria as biofertilizers for canola and reported that Bacillus thuringiensis isolate was that most effective inoculants which significantly increased the number and weight of pods and seed yield without rock phosphate.

Podile and Dube (1988) reported enhanced plant growth and yield for agricultural crops through seed bacterization with an antibiotic producing strain of B. subtilis AF1. In growth chamber, B. subtilis B2 significantly increased growth parameters of onion (Reddy and Rahe, 1989). B. subtilis increased yield of peanut upto 37 per cent and reduced plant disease in field conditions (Turner and Blackman, 1991). It also increased the dry and fresh weight of cucumber plants by 29 per cent, fruit yield by 14 per cent and fruit number by 50 per cent in greenhouse (Uthede et al., 1999).

Rajarathnam et al. (1995) found that B. megaterium and B. polymyxa to be the most efficient phosphate solubilizing bacteria (PSB) in Tamil Nadu (India). Chanway (1995) studied B. Polymyxa strain L6-16R and obtained significant increase in seedling emergence, height and biomass accumulation of western hemlock. The influence of inoculation of Pseudomonas striate, and B. polymyxa on sorghum significantly increased yield and nutrient uptake of sorghum as well as available phosphate content in soil (Jisha and Alagawadi, 1996). Increase in growth and yield of black gram under rice fallow conditions was reported by Sundari and Sureshkumar (2004) due to inoculation of B. megaterium var. phosphaticum.

Growth promotion activities of B. amyloliquefaciens has been studied by (Sharma et al., 2013) where he observed improved plant growth, nutrient assimilation and yield of soybean with the application of the isolate.

Application of P. fluorescens and B. subtilis were effective in improving seed quality such as seed germination, vigour index and nutritional quality such as protein content and carbohydrate content in sorghum. In addition, they increased the plant peroxidase activity (Prathibha and Siddalingeshwara, 2013).

Bacillus species have been reported to promote the growth of a wide range of plants (De Freitas et al., 1997; Kokalis-Burelle et al., 2002). Trials with rhizosphere associated plant growth promoting N2-fixing and phosphate solubilising Bacillus species indicated yield increase in sorghum (Broadbent et al., 1977), maize (Pal, 1998), rice (Sudha et al., 1999), (Sahin et al., 2004) and apples (Aslantas et al., 2007).

Compatibility of Bacillus species with other biocontrol agents

The antagonostic effect of naturally occurring rhizobacteria on plant parasitic nematodes has been reported by many workers. P. fluorescens and B. subtilis applied as seed dressing reduced Globodera spp. in root system of potato and sugar beet in greenhouse studies (Oostendorp and Sikora, 1989; Racke and Sikora, 1992; Hackenberg and Sikora, 1994). Combined application of P. fluorescens and B. thuringiensis in tomato reduced juveniles and adults of M. incognita infesting the plants. Mortality of M. incognita increased with increase in the concentration of bacterial cells (5x108 cfu/ml) (Hanna et al., 1999). Application of the same was found to increase the yield of chick pea with reduced the infestation of M. incognita (Khan et al., 2001).

Bacillus sp. in association with P. fluorescens and Vesicular Arbuscular Mycorrhiza (VAM) tested against M. incognita and Tylenchulus semipenetrans in tomato and citrus, effectively reduced the nematode population in the plants (Rajendran et al., 2001). They also reduced the population of R. similis, P. coffeae and Helicotylenchus multicinctus in banana cv. Robusta (Shanthi et al., 2002). Combined treatment of P. fluorescens isolate, Pfbv 22 and B. subtilis isolate, Bbv 57 recorded the highest plant growth parameters in betelvine, significantly reduced nematode (M. incognita) and wilt (Phytophthora capsici) incidence (Jonathan et al., 2006).

Liquid formulation of Bacillus - Advantages

An important area of microbial research with regard to biocontrol is the development of formulations that would preserve microbial activity forlong enough to enable delivery of an effective product for field application. First report on liquid inoculants for seed isolation of Rhizobium was published from Holland (Van Schrevan et al., 1953).

Rice and Olsen (1992) suggested liquid inoculants as a better method than seed treatments with carrier inoculants. Soybean nodule numbers in a tropical soil was found to be increased with increasing the rates of Rhizobia applied in a liquid form (Smith, 1992). Inoculation of liquid inoculants with cell number of 2.7 x108 cfu/ml resulted in increased growth and yield of tomato (Terry et al., 2000).

Sumathy (2001) reported that survival of sporulated cultures of Bacillus with rice gruel was optimum upto 12 h of inoculation. Inoculation of sporulated cultures through seedling root dipping showed better establishment and also found that establishment of Bacillus was more when inoculated through carrier based spores than vegetative cells.

Formulation of the yeast, Rhodotorula minuta (109 cfu/ml) with the addition of glycerol (20 %) and xanthane (5g/l) avoided contamination and yeast sedimentation and it preserved the cells upto 107 cfu/ml upto 6 months at the temperature of 40 °C (Patino-vera et al., 2005).

Liquid formulation have the advantages over other formulations in terms of high cell count, zero contamination, longer shelf life, greater protection against environmental stresses, increased field efficacy and convenience of handling (Hedge, 2002; Tamizhvendan and Thangaraju, 2007).

Viability of the plant growth promoting bacteria A. diazotrophicus L1 andH. seropedicae J24 stored as liquid formulation was evaluated by studying the effect of growth medium, preservation medium and temperature. (Patil Nita et al., 2012).The studies revealed that, A. diazotrophicus L1 and H. seropedicae J24 cells preserved in gum arabica (5% w/v) and PEG 300 (5% w/v), respectively for 8-9 months of storage period at 4°C. Bacterial cultures of different ages in these liquid formulations exhibited different plant growth promoting traits up to 240 days.

Cell protectants in enhancing the viability of microbial cells

Addition of cell protectant and certain chemicals (glycerol, polyvinylpyrrolidone (PVP), trehalose, Fe EDTA etc.) are known to keep the cells viable for long period under liquid culture.

Glycerol

Glycerol is a carbon source which has high water binding capacity and protects cells from the effects of dessication by slowing the drying rate. Lorda and Balatti (1996) described the growth characteristics of B. japonicumin glycerol based liquid media under various environmental conditions. More rapid growth was observed when 10 m1 of glycerol was substituted for mannitol and populations in the glycerol medium at times reached densities in excess of 1 x 1010 cells/ ml.

In terms of viability and virulence, Verticillium lecani and glycerol 2% and tween 80 1% and arachnid oil (0.5%) emerged as the best combinations (Chavan and Kadam, 2009).

Polyvinylpyrrolidone (PVP)

PVP has high water binding capacity and slows down the drying of the inoculants. Singleton et al. (2002) reported that PVP appear to enhance survival of B. japonicum and the promotion is concentration dependent. Increasing amounts of PVP in the media increased survival on the seed by 100 fold at 48 h after inoculation. The author also found that after 180 days of storage, the number of viable cells remained nearly constant in the medium with 20g PVP/l.

Trehalose

Leslie et al. (1995) reported that the protective action of trehalose has been ascribed to its ability to replace water in proteins and membrane structures. Trehalose was observed to be either hydrolyzed or presumably acting as a carbon source and is likely to function as a membrane stabilizer and protectant (Majara et al., 1996).

Torres et al. (2003) reported that trehalose at (1%) was the best protective agent for Candida sake and showed viability of 72 per cent after four months storage. Accumulation of trehalose was enhanced by supplying the disaccharide in culture media and it was found that trehalose loaded Bradyrhizobium cells survived significantly better during subsequent desiccation stress (Streeter, 2003).

Induced systemic resistance in plans due to the application of Bacillus

i. Peroxidase (PO)

Peroxidases have been found to play a major role in the regulation of plant cell elongation, phenol oxidation; polysaccharide cross-linking, IAA oxidation, cross linking of extension monomers, oxidation of hydroxyl-cinnamyl alcohols into free radical intermediates and wound healing (Vidhyasekaran et al., 1997). Bharathi (2001) reported that chilli plants treated with mixtures of strains of PGPR viz., Pf 1, B. subtilis, Neem and chitin showed enhanced PO activity against cucumber mosaic virus.

Kavitha et al. (2007) tested the efficacy of P. fluorescens B. subtilis and T. viride against M. incognita in tropical suger beet cv. indus they observed significant increased in plant growth parameters and decrees nematode infestation and observed sig increased in enzyme activity peroxidases, polyphenol oxidase, phenylalanine ammonia lyase.

Jonathan and Umamaheswari (2006) accessed the biocontrol potential of entophytic bacterial isolates of B. subtilis (EPB 5, 22, 31 and EPC 16) prepared in talc base against nematodes of banana cv. robusta significant increase plant growth coupled with reduction in nematode population was observed in the combined treatment of EPB 5 + 31. The treatment also enhanced the activity of defence enzyme responsible for induction of systemic resisitance such as peroxidases, polyphenol oxidase, phenylalanine ammonia lyase etc.

ii. Polyphenol oxidase (PPO)

Phenolics play a major role in plant disease where necrosis is involved (Wallace, 1961). PPO accumulated upon wounding in plants and it involved jasmonic acid as an intermediate signal and culminated in the production of proteins such as PPO and proteinase inhibitors (Schaller and Ryan, 1995). PPO usually accumulates upon wounding in plants. PPO can be induced viz., octadecanoid defence signal pathway (Constabel et al., 1995). PGPR treated sugarcane after pathogen inoculation showed comparatively enhanced induction of PPO isoforms than the untreated control plants (Viswanathan, 1999).

iii. Phenylalanine ammonia lyase (PAL)

PAL is the first enzyme involved in phenyl propanoid pathway and plays a key role in the biosynthesis of phenolics and phytoalexins (Bashan et al., 1985). Induction of enzymes such as PAL and PO leading to the accumulation of phenolics and lignin can occur in response to insect and pathogen attack, exposure to oxidizing pollutants, mechanical stimulation. They are thought to function in enhancing the resistance of plants against the damage by these biotic and abiotic stresses.

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