Bacillus species as versatile weapons for plant pathogens: a review

ABSTRACT Plant pathogens are the main threat for profitable agricultural productivity. Currently, chemical-based pesticides are thought to be an effective and reliable agricultural management measure for controlling pests. Chemical pesticides are highly effective and convenient to use but they are a potential threat for the environment and all kinds of life on earth. Therefore, the use of biological control agents for the management of plant pathogens is considered as a safer and sustainable strategy for safe and profitable agricultural productivity. Bacillus-based biocontrol agents play a fundamental role in the field of biopesticides. Many Bacillus species have proved to be effective against a broad range of plant pathogens. They have been reported as plant growth promoter, systemic resistance inducer, and used for production of a broad range of antimicrobial compounds (lipopeptides, antibiotics and enzymes) and competitors for growth factors (space and nutrients) with other pathogenic microorganisms through colonization. The aim of this article is to present the biocontrol potential of Bacillus species in relation with their antagonizing attributes against plant pathogens. These attributes include production of lipopeptides, antibiotics and enzymes as well as plant growth promotion and systemic induced resistance.


Introduction
It is of essence to control plant diseases for the production of quality and abundance of food, feed and fibre for maintaining a healthy and rapidly growing world population. Different kinds of plant disease management strategies may be adopted for the management or eradication of plant diseases. Most of the agriculturists in the world often depend on chemical-based pesticides and fertilizers for bounty agricultural output. Over the past centuries, agrochemicals have played a very important role for improving crop quality and quantity. However, during recent years, there is a change in attitude of people towards chemical-based fertilizer and pesticides due to the emerging hazards of environmental pollution and pesticide residual effects on human health and also on the earth ecosystem. As a result of these problems, stringent laws and regulations have been imposed for the safe use of agrochemicals. Consequently, scientists diverted their attention towards the alternative of synthetic agrochemicals which led to the development of biopesticides [1,2].
The earlier research work has repeatedly reported that numerous plant pathogenic diseases can be controlled by natural antagonistic microorganisms [3]. The relationship between antagonistic microbes and plant pathogens may be composite. It may involve the production of antipathogenic chemicals, competition for space and other necessities or triggering the host defensive mechanism and predation [4]. However, some strains of these microbes have abilities to overcome plant pathogenic diseases under various cropping environments and some of them have abilities to control a broad range of plant pathogens. Rigorous research activities have discovered several microorganisms for the development of biopesticides at commercial level.
Among the bacterial antagonists, many belong to the genus Bacillus and there are some other important genera but are of lesser applied importance than Bacillus [5]. The use and number of antagonistically important Bacillus species is increasing very rapidly. Bacillus species have a unique ability to replicate rapidly, resistant to adverse environmental conditions as well as they have broad spectrum of biocontrol ability. Volatile compounds produced by B. subtilis also play an important role in plant growth promotion and activation of plant defence mechanism by triggering the induced systemic resistance (ISR) in plants [6]. According to Denner and Gillanders [7], the US Food and Drug Administration (US FDA) declared B. subtilis as GRAS (generally recognized as safe) organisms for its use in food processing industries. Endosporic and enzymatic products of B. subtilis were found highly active against many fungal pathogens.
CONTACT Mingshan Ji jimingshan@163.com; jimingshan@syau.edu.cn Several microorganisms have been described as possible biocontrol agents such as Hypericum gramineum, Pseudomonas flurescens and some species of Streptomyces [8]. In addition, Ulocladium atrum and Trichoderma have the ability to control various bacterial and fungal diseases. They generally show control effectiveness ranging from 30% to 50%. Bacillus species have become attractive biological control agents due to their ability to produce hard, resistant endospores and antibiotics which control a broad range of plant pathogens [9]. This article seeks to show the biocontrol aptitudes of Bacillus, their antimicrobial compounds, mode of action and spectrum of these compounds and also indicate the missing aspects that should be considered for future research.

Lipopeptides-and antibiotics-based weapons
Bacillus species synthesize various types of lipopeptides based on secondary metabolites with specific activities against plant pathogens which give them a unique importance in agriculture, biotechnology and pharmaceutical industries. About 2428 antimicrobial peptides have been identified from various organisms such as bacteria (237), archaea (2), protists (7), fungi (12), plants (310), and animals (1819). Among them, 756 of such peptides have various degrees of antifungal properties [10]. The mechanisms by which microorganisms lead fungi to death include blockage, distraction and holes formation in the cell wall and cell membranes of the fungi. Also, some peptides are involved in the disintegration of fungal intracellular organs such as nucleic acid and mitochondria [11]. In addition to the production of different surfactant materials, Bacillus species synthesize many potent amphiphilic and surfactant lipopeptides comprising bacillomycins, iturins and mycosubtilin; with the modification of culturing conditions, these bacteria also produce fengycins or plipastatin [12]. Characterization based on the existence of L-and D-amino acids and adjustable hydrophobic studies revealed that all these fall into two categories: cyclic peptides (iturinics) and macrolactones (plipastatins, fengycins and surfactins). The iturinics cause cell leakage by inserting their hydrophobic tails in to cytoplasmic membrane and through autoaggregation to create pores in cellular membrane [13].
B. subtilis and B. cereus produce plipastatins to prevent phospholipase A 2 which plays an important role in different cytological process (inflammation and acute hypersensitivity) [14,15]. B. subtilis synthesize some other lipopeptides such as plipastatins indicated as isoformic mixture of lipopeptides that vary according to the composition of amino acids and hydrophobic tails [16]. The Bacillus-based lipopeptides and antibiotics are summarized in Table 1.
Haemolytic surfactant synthesized by B. subtilis was found to be very active against mosquitoes. Gong and his coworkers [22] studied the structure and composition of metabolites for the determination of inhibitory substances against Aspergillus flavus synthesized by Bacillus subtilis fmbJ. Four substances were found to be effective against target fungus after separation and purification of metabolic liquid produced by the bacteria. Bacillomycin D caused severe injury to both cell wall and cell membrane of fungal spores and hypha. Scanning and transmission electron microscopy showed the formation of holes by exudation of cytoplasm and cellular organelles. The purified bacillomycin significantly inhibited the spore germination (96.63%) and sporulation (98.10%) [22]. Morphological and ultrastructure studies of lipopeptides produced by B. subtilis strains on Podosphaera fusca revealed the reduction in germination of powdery mildew conidia by arresting the lipopeptides of P. fusca and various cellular changes [53].

Plant growth promotion as an indirect weapon
Quite apart from the antagonistic mechanism of Bacillus species, these microbes also have an important role in plant growth promotion by enhancing the biosynthesis of plant hormones (gibberellic acid (GA3) and indole-3acetic acid (IAA)) that have a close relation with plant nutrient availability [54,55]. Bacillus species play an important role in plant growth promotion. When spore or cell suspensions of B. subtilis OTPB1 were applied to tomato seeds on a plastic pot, it considerably enhanced the shoot and root growth, seedling vigour and leaf area of the tomato plant. Higher level of plant growth-promoting hormones (GA3 and IAA) and defence-related enzymes (peroxidase (PO), polyphenol oxidase (PPO) and superoxide dismutase) were detected in treated plants compared with non-treated plants [56].
Many scientists have discussed that B. subtilis had the ability of promoting plant growth and yield by enhancing nutrient uptake by increasing the production of plant harmones and decreasing ethylene production which facilitate the bacteria in root colonization [54,55,[57][58][59][60]. IAA has an important role in origination and emergence of adventitious roots. It also enhances shoot development by influencing cell expression, division and differentiation [61]. The GA3 plays it role in combination with auxin for elongation of plant and leaf bud formation [62]. These plant growth-promoting hormones enhance the nutrients uptake ability of plants and help the plant to defend against various biotic and abiotic stresses [63,64]. It was demonstrated that diluted cultural filtrate B. vallismortis

MT39
Antibacterial and fungal activity Sonorensin L. monocytogenes and S. aureus [51] of B. amyloliquefaciens significantly enhanced the growth of maize seedlings. Chemical analysis of supernatant of FZB42 showed the presence of substances capable of reacting with IAA-specific antibodies [57].
Jiang et al. [65] screen out one-hundred bacterial strains for their antagonistic and growth-promotion activities and found out that B. amyloliquefaciens strain 54 significantly increased plant growth by enhancing the NPK and chlorophyll contents of plants. It also enhanced the level of resistance in plants against bacterial fruit blotch of cucurbitaceae crops by eliciting the defence-related gene PR1 and H 2 O 2 accumulation in plant tissues [65].
Zinc solubilization, crop growth, soil biology, and zinc mobilization aptitude of B. subtilis strains MDSR7, MDSR11 and MDSR14 were evaluated under lab conditions. Higher soluble zinc contents and an enhancement in total organic acids were observed where MDSR7 and MDSR14 were inoculated. MDSR7 and MDSR14 cause a significant reduction in soil pH and enhanced microbial respiration as well as b-glycosidase, dehydrogenase, auxin production and microbial biomass-C in the rhizosphere of wheat and soybean. All the strains significantly enhanced the availability of zinc which enhances the plant growth and assimilation of zinc in seeds by wheat and soybean plants [66].
The Bacillus strain SH1RP8 showed 8% salinity tolerance when applied to sand dune plant Peucedanum japonicum. It enhanced 10.9% shoot growth and 51.7% dry weight when grown in general soil [67]. Lin et al. [68] isolated 33 strains of bacteria from vinegar waste. After screening through self-developed screening method, he found out that, all the Bacillus strains were highly antagonistic to eight fungal pathogens and had the ability to produce IAA, while five strains exhibited plant growthpromotion abilities. Wei et al. [69] evaluated 38 Bacillus strains for anti Aspergillus parasiticus ability by tip culture method. Twleve strains were selected for their anti-fungal, growth promotion and phytohormones production ability.
Nain et al. [70] studied RM-2 strain of Bacillus species and found that the bacterial strain had many valuable features including acetyl-CoA carboxylase (ACC) deaminization activity, P solubilization ability, fungicidal, IAA production and ammonia production activity. The stains significantly enhanced seed germination, fresh and dry weight, leaf area, root and shoot length and also seed, pods and grain yield compared with untreated control.
Valdez et al. [71] treated sunflower seeds with B. subtilis for investigating its effect on plant growth, soil properties and greenhouse gases emission while using urea as positive control for comparison. The results showed that after one month, root length and root-shoot fresh and dry weight were considerably higher than ureatreated plants. No significant difference was observed for CO 2 production, while N 2 O production rate was higher in bacterial-treated plants compared to ureaembedded plants.

Systemically induced disease resistance
When a pathogen attacks a plant, the non-infected plant tissues acquire an ability to resist the subsequent attack; this type of broad spectrum and long-term ability of plants is known as systemic acquired resistance (SAR) [72]. Moreover, the beneficial nonpathogenic microorganisms can also trigger resistance in plants. This resistancetriggering mechanism was observed when colonization of roots by some nonpathogenic bacteria protects the above-ground plant parts from attack of various pathogenic organisms; this type of resistance response is called ISR [73]. Like SAR, nonpathogenic microbes trigger ISR which has also been shown in various plants and is effective against a broad range of diseases [74]. Park and his coworkers [75] evaluated five strains of Bacillus species against Ralstonia solanacearum. The bacterial strain EXTN-1 was proved to be effective and they also showed that reduction of disease was not due to direct antagonism but as a result of elicitation of host plant resistance genes. The main components of systemic-induced resistance are phenolic compounds, genetic and structural modifications, plant resistance activators, and activation of enzymatic weapons.

Triggering of phenolic compounds
Many experimental results have shown that Bacillus strains induced a broad spectrum of resistance against various bacterial and fungal plant pathogens. Park and coworkers [76] reported that B. vallismortis strain EXTN-1 induced SIR that was effective against a variety of bacterial and fungal diseases. Akram and Anjum [77] investigated the resistance elicitation ability of B. fortis 162 and B. subtilus 174 against Fusarium wilt of tomato and showed that both strains induced systemic resistance in tomato plants. They also observed the increased level of phenyl ammonia lyase (PAL), PPO and PO in bacterial-treated tomato plants and both strains significantly reduced tomato Fusarium wilt. Gupta and his coworkers [78] reported that cultural filtrate of B. subtilis strain FZB-G has the ability to trigger phytohormones precursor which plays an important role in signal transduction and activation of defence gene that results in the production of defence-related compounds [78]. Jayara and coworkers [79] investigated B. subtilis strain AUBS1 for systemic resistance induction against sheath blight of rice by its foliar application under greenhouse conditions and found a significantly increased level of PO and phenylalanine ammonia lyase (PAL). B. subtilis-treated leaves were found to have accumulation of PR proteins. They also found that the increased levels of thaumatin and b-1-3-glucanases with 17 and 30 kDa mass, respectively, play an important role for induction of resistance in rice plant.
Kloepper et al. [60] investigated the elicitation abilities of B. subtilis, B. cereus, B. amyloliquefaciens, B. pasteurii, B. sphaericus, B. pumilus and B. mycoides for induced resistance in tomato, muskmelon, bell pepper, sugar beet, watermelon, tobacco, Arabidopsis sp., loblolly pine, cucumber, and two tropical crops (green kuang futsoi and long cayenne pepper) against various pathogenic diseases under green house and field conditions. Significant reduction was observed in bacterial and spotting fungal pathogens, systemic viruses, crown-rotting, rootknot nematodes, and a stem-blight as well as blue mold, late bight and damping-off. Wang et al. [80] concluded that treatment with B. cereus AR156 enhances the defence related activities such as PAL, chitinase, b-1,3glucanase, PO and PPO, and stimulated amassing of H 2 O 2 .

Structural and genetic activation of host plants
Triggering of systemic resistance mechanism of plants by bacterial microbes is the main cause of disease suppression by crop plants [81]. Many studies suggested that the biochemical and structural modifications in plants are key sources of disease reduction by defending pathogen attack. Structural biochemical variation can reduce the spread of pathogens in the host plant [82]. Cytological studies showed that, root colonization of pea by B. pumilus strain SE34 restricted Fusarium oxysporum f. sp. pisi to the epidermis and outer cortex by strengthening the cell wall and epidermal cells. In addition, the newly formed barriers contained callose and phenolic compounds which were beyond the site of infection during electron microscopy [83]. In another study, it was observed that, treatment by strain SE34 reduced fungal colonization by changing the host physiology elicited by enhancing the host cell wall density, polymorphic deposition on potential susceptible sites, obstruction of intercellular gaps and epidermal cells with amorphous and osmophilic compounds [84].
Lee and coworkers [85] screened out 78 Bacilli for the stimulation of ISR and found that B. amyloliquefaciens strain HK34 showed disease reduction up to 99.1% against Phytophthora cactorum when applied to root, and similar results were perceived on leaf application under field evaluation. An enhancement in expression of genes PgPR10, PgPR5, and PgCAT in plant leaves was also observed after treatment with HK34. These outcomes showed the ISR-eliciting potential of strain HK34. A mixture of PGPR strains belonging to Bacillus spp. were tested for elicitation of resistance against Lycopersicon esculentum, Sclerotium rolfsii, Capsicum annuum var. acuminatum, Colletotrichum gloeosporioides and Cucumis sativus. Results showed that the mixture of PGPR significantly enhanced the resistance in plant for all of the above-listed plant pathogens [86].

Activation of plant-resistance activators
Van and coworkers [87] determined that the phenotypic response of resistance induced by rhizobacteria is similar to that of pathogenically induced systemic resistance. Rhizobacterial-elicited resistance was verified against bacteria, fungi and viruses under conditions in which the rhizobacteria and pathogen were kept separately. The level of induced resistance was different for different strains of bacteria and also for different plant species. The determinants of rhizobacterial-elicited systemic-induced resistance were siderophores, lipopolysaccharides, jasmonic acid, ethylene and salicylic acid (SA) perception. Under challenging conditions, the level of resistance was higher than in non-challenging conditions; therefore, ISR is highly favourable and recommended for biological control of plant pathogenic diseases. B. sphaericus strain B43 was investigated for the induced resistance against Globodera pallida in potato roots by split root system. It was observed that, both living and heat-killed bacterial cell effectively reduced the incidence of G. pallida in potato roots [88]. It was reported that B. pumilus strain SE34 increased the level of SA in tobacco plant compared to non-treated plants [89].
Recently, it was demonstrated that microbial-originated volatiles have a role in elicitation of plant defence mechanism. The role of bacterial volatile for ISR elicitation was investigated by culturing bacteria and Arabidopsis seedlings on isolated edges of divided Petri dishes. Arabidopsis seedlings were continuously exposed to volatile compounds of Bacillus species for 4 days and a significant reduction in disease severity of soft rot was observed [90]. The chemical analysis of volatiles produced by the growth-promoting B. subtilis GB03 and B. amyloliquefaciens IN937a showed that their compositions are different from the non-growth-promoting bacterial strains DH5a. It was observed that the strains GB03 and IN937a released 2,3-butanediol and 3-hydroxy-2butanone (acetoin) abundantly which were not observed for DH5a [91]. It was also shown that B. subtilis GB03-originate volatile compounds that regulate ethylene biosynthesis enzymes, as well as ethylene biosynthesisrelated genes (ERF1, GST2, and CHIB). It also regulated jasminic acid-and SA-mediated defence mechanism.

Enzymatic weapons
Besides production of antibiotics and elicitation of systemic resistance in plant against a variety of plant pathogenic diseases, Bacillus species are also capable of producing enzymes like chitinase and b-1,3-glucanase having a very strong lytic activity. These lytic enzymes synthesized by Bacillus species have proved to be very active in degrading fungal cell wall [92]. Studies conducted have shown that Bacillus species also produced many defence-related oxidative enzymes such as PPO, PO and PAL [93]. These oxidative enzymes induced lignin and oxidative phenolic compounds which play a role in contraction defence-related obstacle by producing structural changes in the cellular defence system against plant pathogens [94]. Podile and Laxmi [95] proved that PAL has an important role in the formation of lignin and flavonoid production. It also plays a key role in phenylpropanoid biosynthetic pathway. The defence-related activities of the enzymes have been proved in various plant species and against various plant pathogens [94].
An increase in PAL formation and activity was observed when it is exposed to the bacterial strain in comparison with untreated control [96]. Kang and Buchenauer [97] observed an enhancement in the phenolic activity in wheat crop induced by the resistance inducers against the powdery mildew. Phenolic compounds are highly effective against fungal plant pathogens, and hence play an important role in plant protection. Hahlbrock and Scheel [98] reported that PAL has a crucial role for phenolic compounds formation in plants through phenylpropanoid pathway. PPO also has a key role in plant defence elicitation against various plant pathogens. Li and Steffens [99] reported that the genetically modified tomato plants for enhanced production of PPO are found to be highly resistant against various pathogenic diseases. Thipyapong and Steffens [100] observed that PPO acts as catalyst in oxidative reactions of phenolic compounds. Many members of Bacillus species were found to be highly active in these types of defence-eliciting activities.
The basic goal of releasing hydrolase is to utilize the nutrient stored in substrate by converting it from unavailable form to available form. Moreover, bacteria release extracellular enzymes in combination with some other compounds to overcome the competition with other microbial agents. Most soils have sufficient amount of plant nutrients but present in insoluble form due to which plants are unable to uptake these nutrients for their normal growth [101]. b-1,3-Glucanases and chitinases actively contributed in defence of plant against a variety of plant pathogens while PO and PAL actively involved in phenylpropanoid breakdown in plant tissues [102].
Ramyabharathi et al. [103] studied the B. subtilis EPCO16 for the control of F. oxysporum f. sp. lycopersici. It was reported that the liquid and telic formulation of strain EPCO16 considerably elicited the defence-related enzymes and proteins (phenolics lyase, catalase and phenylalanine ammonia) in infected tomato plants. Liquid formulation significantly enhanced enzymatic activities in the tomato plant as well as in seed, seedling and soil treated with EPCO16. Spectrophotometric analysis of plants showed that the defensive proteins (phenolics, PAL and catalase) reached maximum levels at the seventh day of pathogen inoculation.
Chitinases and glucanases (laminarinase) fungal cell wall hydrolytic enzymes are produced by some hyperparasitic Bacillus species. Many strains of B. subtilis have been reported for their chitinolytic activities [104]. Podile and Prakash [105] reported the chitinolytic mechanism of B. subtilis. Chitin holds second position among the polysaccharides according to its abundance in nature. It is a b-1!4-linked homopolymer of N-acetyl-d-glucosamine which is very important fungal cell wall strengthening element that offers rigidity to cell wall by hydrogen bonding between adjacent polymers. Thus, glycosidic bonds of constituting polysaccharides are the base for fungal cell wall integrity. Interference in these bonds deteriorates the cell wall and hence cell leakage. Bacillus species produced cell wall hydrolyzing enzymes (chitinases, glucanases and chitosanases) that efficiently hydrolyzed the cell wall of fungal pathogens. Therefore, these hydrolytic enzymes hold a great potential for the management of fungal pathogenic plant disease. As the plant cells lack chitin, chitinase application is more effective than glucanase [104]. Chitinase-producing bacteria supplemented with a chitinase substrate and 1% colloidal chitin considerably reduced the groundnut leaf spot disease [106,107].
B. amyloliquefaciens V656 synthesized two types of chitinases enzymes and both significantly inhibited F. oxysporum growth [108]. B. subtilis produced an antifungal chitinase enzyme with strong chitinitic activity against various pathogenic fungi [109]. B. thuringiensis inhibited S. rolfsii and various other pathogenic fungal diseases of soybean. Liu et al. showed that the chitinase from B. thuringiensis subsp. colmeri prevented the germination of fungal spores [110]. Bargabus et al. [111] investigated in their research that B. mycoides strain BacJ elicites systemic resistance in sugar beet which was related with increased activity of PO and also enhanced the production of two ribozymes of b-1,3-glucanase and one isozyme of chitinase.
In nature, there is a diverse range of chitins presented with a diverse structure, and hence bacteria synthesize a variety of chitinases. According to amino acid sequence, chitinases are classified into 18, 19, and 20 glycosyl hydrolases. Family 18 of chitinolytic enzymes is multifarious and comprises chitinolytic enzymes from fungi, bacteria, viruses and some plants and animals. Type 19 chitinolytic enzymes comprise some Streptomyces and plant chitinases I, II, and IV [112] while type 20 consists of human, Streptomycetes and bacterial b-N-acetylhexosaminidases. Based on catalytic domains stature, bacterial chitinases are further classified in to A, B, and C groups [113]. Bacillus licheniformis synthesizes five chitinolytic enzymes (66,62,53,49, and 42 kDa) while B. circulans synthesizes six different chitinases but only chitinase A1 is mainly involved in degradation of chitin. Chitin-binding protein (Cbp21) has a synergistic effect on b-chitin disruption [114].
Activities of these bacterial enzymes have been improved by the use of modern biotechnology. Homological-based enzymatic modelling played an important role in enhancing the activities of bacterial enzymes. Activity of b-galactosidase has been improved sixty six times while substrate activity was enhanced one-thousand times by DNA shuffling and screening [115]. Without any reduction in catalytic activity of phospholipase A1, its melting point increased by 7-10 C by error-prone PCR and screening [116]. Thermal stability of Kanamycin nucleotidyl transferase increased up to 20 C by DNA shuffling and screening/selection [117]. PCR-based sitedirected mutagenesis was used to detect half-life of raw starch-digesting amylase (RSDA) by 20 folds [118]. Cellulose consumption of cellulase was increased three-fold through metabolic engineering [119].
The three-dimensional structure of catalytic domain showed that the tryptophan residues (W122 and W134) played a very important role in hydrolysis of chitin. Mutational studies represented that any change in these residues significantly reduced the catalytic activity of enzyme [120]. Bacillus-based chitinases vary with environmental conditions. Studies on macular diversity of bacterial-based chitinases showed that it is more diversified in arable soils. Phylogenetic data and multidimensional scaling (MDS) analysis of T-RFLP profiles revealed that the composition was considerably affected by the type of soil and pH [121]. Principle eliciting factors of systemic resistance and target pathogen are summarized in Table 2.

Colonization
Interspecies competition causes reduction in growth, productiveness and other activities of the competing organisms. Biological control arises when pathogenic and nonpathogenic organisms compete for space and nutrients around the host plant [1]. Plant and soil surface has a limited amount of nutrients. Therefore, for effective colonization of plant surface, pathogenic and nonpathogenic microbes must compete to fulfil their nutrient requirements. Plant exudates, senesced tissue or leachates are the main sources of nutrient supply on host plant surface. It is difficult to demonstrate the direct role of competition in plant disease control but there are numerous indirect studies that have proved that pathogenic and nonpathogenic microbes compete for nutrients and space and thus play a very crucial role in pathogen severity and incidence reduction. In general, the soil-borne plant diseases (e.g. Fusarium and Pythium species) are more exposed to competition because they only infect through mycelial contact in comparison with those pathogenic diseases whose causal organisms directly germinate on plant surface and infect through infection peg and appressoria. Nonpathogenic microbes associated with photosphere protect plants by fast colonization and exhausting the developmental necessities thus makes them unavailable for pathogenic microbes. Biocontrol can be established by competing for essential micronutrients, for example iron in rhizosphere, is extremely limited and its availability is highly dependent on soil. Iron is mostly in ferric form in oxidized and aerated soils which are water-insoluble at pH 7.4 and its concentration may be as low as 10 ¡8 mol/L. This concentration is insufficient to support the growth and development of microorganisms. For survival under such situations, the microorganisms develop siderophores which enable the microbes to fulfil their iron requirement from the microenvironment. Kloepper and his coworkers [60] found out the role of siderophores for the biological management of Erwinia carotovora by several PGPR bacteria. The enhanced ability of beneficial nonpathogenic commercial microbes for utilization was suggested to be an important element for rapid colonization of rhizosphere and helpful for the shift of pathogenic microbes from the probable site of infection. Rapid colonization of tomato tissues by Bacillus species significantly reduced Fusarium oxysporium infection as well as the wilting index. For successful biological management of plant disease, colonization of plant tissues is considered as principle element, while for soilborne diseases, root colonization by biological control agents has direct contribution [128]. A significant increase in colonization ability of Bacillus species was observed when pathogen (Fusarium cf. incarnatum) and biocontrol agent (Bacillus Isolate B2-5) were inoculated simultaneously on the ginseng tissues in comparison with separate inoculation [129]. B. subtilis strain HJ5 was tagged with a green fluorescent protein-encoding gene for easy investigation of its colonization ability. HJ5 successfully colonized the differentiating and elongating root tissues and bacterial population reached to its highest point (10 7 ) after three days of inoculation. Strain HJ5 significantly reduced the Verticillium wilt of cotton and apparent mechanism of this disease reduction was rapid colonization ability of strain HJ5 [130]. Zhang and coworkers [131] investigated the colonization of banana root by B. subtilis N11 under sand, hydroponic and natural soil systems and the results showed that the strain N11 efficiently colonized the banana roots under all of three culturing systems.
B. amyloliquefaciens strain C06 synthesized g-polyglutamic acid and studies suggested that it played very important role in colonization of stain C06 and any interruption in the synthesis of g-polyglutamic acid caused significant reduction in colonization ability of strain C06 [132]. Table 3 shows the important Bacillus strains in relation to colonization, their target pathogens and host plant.

Conclusion
Bacillus-based biological control agent delivers attractive, efficient and friendly method for the management of plant pests. The matters that demand considerations for the practical adoption of biological control agents comprise the exposition of their mode of action, stability under field application, development of such formulation that can be used with other pesticides with synergetic effect and perfect demonstration of cost-benefit ratio for effective commercialization.
Bacillus species produce a variety of compounds that can be used for the management of a broad range of plant pests. Stable growth and development of biological agent under field conditions is still a problem due to adverse environmental conditions. Formulation of active product with perfect stabilizer that can optimize its activity under field conditions is an alternative and more effective strategy for the management of plant pest instead of using living bacteria directly. The bacterial-based formulated products, when used with other synthetic chemical pesticides, can be harmful for the living bacteria. The formulation that can enhance the shelf-life of bacterial product during storage, transportation and also during field application is also important. Proper knowledge and understanding of the bacterial active compound is necessary for a stable and efficient formulation.
Lipopeptides belong to a group of microbial-based peptides that enable the plant activation of defence mechanism. For biological control of plant pathogens Bacillus-originated lipopeptides play a very important role. Their unique physiochemical properties are mainly responsible for their effectiveness against a broad range of plant pathogens. Agitation of lipid bilayers, decline in surface tension and many other alterations in surface properties are the main qualities of these lipopeptides. Mass spectrometry is a rapid and efficient method for selection of effective strains of biological control of plant pathogens. Although recent advances have provided us some useful understanding of mechanism of lipopeptides with other interacting pathogens, studies mostly based on physiochemical activities and a broader view of the mechanisms are still missing.
Combined use of Bacillus species with different mechanism of action can enhance their effectiveness under greenhouse conditions. While combining the different groups, care must be taken so that each of them should have equal opportunity of colonization without any antagonistic effect among the biological control agents. Spore-forming ability and genetically engineered plants with Bacillus genes have been provided as an effective solution of many plant pathogenic diseases. The sporeforming property of Bacillus species gives them a prime importance in the field of biological control. This is because the spores produced by Bacillus species have the ability to endure the extreme environmental conditions. Therefore, attention should be put on the development of cost-effective and stable spores based products. Diversity in Bacillus strains provides protection to plants in a variety of ways. Systemic-induced resistance and plant growth promotion play a principle role in biological plant protection. A large number of Bacillus strains have been developed for an effective control of plant pest and diseases. Most of the scientific investigations are made on signal transduction pathways in Bacillus species-treated plants. Biologically active microbes synthesize a variety of antimicrobial secondary metabolites but the exact measurement of active compound is difficult because of its low quantity in comparison with other ineffective compounds present on plant surface. The Bacillus species have the ability to produce compounds that belong to multiple classes of antibiotics which can be used for control of a broad range of plant pathogenic diseases.
For successful application of these biological agents, their ecological study is necessary. Definitely safety and worth of biological agents will be determined by its ecological success under field conditions. Practical understanding about the mode of action, diversity, ecological distribution and interacting environment into which the biological agents have to work will be valuable for successful adaptation of biological control for sustainable agricultural productivity.
Inaugurating the occurrence and functionality of biologically important microbes in a particular environment is the first step for understanding their nature and mode of action for controlling the pathogens under that environment. Due to the complexity of field conditions, extensive studies are required for fully understanding and characterizing the mode of action of these biological control agents.

Disclosure statement
No potential conflict of interest was reported by the authors.