Agricultural productivity is hampered by various biotic and abiotic stresses under varied crop ecosystems worldwide. Plant growth promoting rhizobacteria (PGPR) have emerged as effective tools for holistic crop health management. Bacteria are the most abundant microorganisms in soils compared to fungi and other microbes. Among PGPR, Bacillus species have been well recognized for its effectiveness against biotic and abiotic stresses, since it is most common bacteria found to easily colonize plants. They have been reported as plant growth promoter, inducer of systemic resistance, and used for production of a wide range of antimicrobial compounds (lipopeptides, antibiotics and enzymes) and competitors for growth factors (space and nutrients) with other phytopathogenic microorganisms through colonization. The prime aim of this chapter is to focus on niche areas in PGPR research with respect to Bacillus species, mechanism of action and their potential role in alleviation of biotic and abiotic stresses and growth promotion of crops.
Keywords: Plant growth promoting rhizobacteria, crop health, systemic resistance inducer, antimicrobial compounds, Bacillus, biotic and abiotic stresses
Climate change poses serious threats due to abiotic and biotic stresses in plants which result in huge economic loss for farmers besides spoiling the food through production of toxins during storage. The conscious urge of farmers to combat the stresses lead to the development of a range of agrochemicals and their application which culminates in soil and groundwater contamination that ultimately endangers animal and human health. In order to get rid of these problems, biocontrol strategy for alleviation of plant diseases and environmental stresses assumed greater significance (Pal and Gardener 2006; Barr and Soila 1960).
The rhizosphere is densely inhabited with loads of microorganisms that are competing for space and nutrients (Walker et al. 2003). The soil microbiome is dynamic and affected by plant roots, soil management and other factors. Roots secrete primary and secondary metabolites, macromolecules and even cells into the rhizosphere that support nutrient acquisition and also shape the local microbiota (Driouich et al. 2013). Root exudates contain organic compounds that can serve as attractants for microorganisms which move towards roots using chemotaxis and may bring benefits to the plant (Ryan, Delhaize and Jones 2001). Exudates may also dissuade pathogens giving plants the possibility to affect the composition of the local soil microbiota (Baetz and Martinoia 2014). Further, certain microorganisms secrete compounds that favor their growth and association with plant roots.
Plant growth promoting rhizobacteria (PGPR) represent a wide group of bacteria that can colonize plant roots and support plant growth by mechanisms such as synthesis of phytohormones and increased nutrient uptake (Lugtenberg and Kamilova 2009). Many PGPR also have the capability to inhibit phytopathogens by releasing antibiotics (Ongena and Jacques 2008; Perez-Garcia et al. 2011) or by triggering (priming) innate immunity of plants referred to as induced systemic resistance (ISR) (Van Loon et al. 1998). It has been demonstrated that certain PGPR stimulate plant growth without being in physical contact with roots through release of volatile compounds (Ryu et al. 2003, 2004; Farag et al. 2006). Further, certain PGPR can restrict fungal growth by emission of volatile organic compounds (VOC) (Ryu et al. 2004; Ortiz- Castro et al. 2008). Bacterial VOC have been shown to serve various roles such as signal compounds for inter- and intra-species as well as cell-to-cell communication, stimulate or inhibit plant growth as well as affect phytopathogens (Wenke et al. 2010).
The use of agricultural chemical inputs can be reduced by soil microbes such as Bacillus sp. These beneficial bacterial strains are able to solubilize the immobile P in soil, which is then taken up by the plant roots (Ramani and Patel 2011; Tallapragada and Seshachala 2012). The biological health of soil remains poor if it has small amounts or nearly no microorganisms. It is considered as non-active soil and does not support healthy plant growth. Generally, the type and count of bacterial cells that are present in diverse soils are affected by soil environmental conditions such as pH, temperature, presence of various salts, heavy metals, moisture, and other inorganic and organic chemicals as well as by the types and number of flora and fauna found in that soils (Garbeva et al. 2004). The varied communities of aerobic endospore-forming beneficial bacteria (AEFB), i.e., Bacillus sp., commonly occur in all types of farming fields, with different types of plants, and play a significant role in enhancing crop productivity by its direct or indirect functions (Grayston et al. 1998). Other physiological properties, viz., multilayered cell walls, endospore formation which are stress resistant, and excretion of varied peptide antibiotics, signal peptide molecules, and extra cellular enzymes, are omnipresent in case of these various Bacillus sp., and these traits help in the proliferation and survival of bacterial cells under various adverse climatic conditions for very long duration (Pirttijärvi et al. 2000). Many species of Bacillus and Paenibacillus are very well recognized to augment plant development and growth. The chief means and approach for growth encouragement includes the manufacture of growth energizing phyto-hormones, solubilization and mobilization of insoluble phosphate present in soil, production of proteinaceous components such as siderophore, and demonstration of the phenomenon of antibiosis, i.e., antibiotics production. Besides these beneficial properties, several Bacilli are also involved in inhibition of plant ethylene production and stimulation of plant systemic resistance against several plant pathogens (Gutiérrez‐Mañero et al. 2008; Idris et al. 2004; 2007a, b; Richardson et al. 2009). The disease-inciting microbes negatively affect the plant growth and health and, therefore, are a major challenge to the production of food. Conventional approaches such as rotation of crops, breeding resistant plant varieties, and applying chemical pesticides seem to be inadequate for controlling plant root diseases of significant crops (Johri et al. 2003). Additionally, it seems unavoidable that lesser amounts of chemical pesticides will be employed and that more and more dependence will be rested on novel bioagents based microbial technology, which predominantly includes the application of antagonistic beneficial microbes as potential biopesticides. The research on diversity, characterization and applications of novel bioagents has increased recently, partly due to reform in public thought towards chemical residue free farming (Bale et al. 2008). There is also an urgent need to find suitable substitutes for harmful chemicals employed in plant disease control. There are several reports about the Bacillus and Paenibacillus species expressing antagonistic behaviors by the process of suppressing pathogens under in vivo and in vitro conditions (Govindasamy et al. 2010). Bacillus subtilis was first isolated in 1872 by Ferdinand Cohn which is a rod-shaped filament bacterium. Bacillus sp. are mainly focused on the frame of biological control due to their cosmopolitan distribution in diverse ecosystems, safety, combating ability against adverse environment (Earl et al. 2008; Nakkeeran et al. 2004, 2005; Montesinos and Bonaterra 2009). In the past few years, investigations have demonstrated that several Bacillus sp. like Bacillus subtilis GB03, B. amyloliquefaciens IN937a and Paenibacillus polymyxa E681 (Lee et al. 2012) secrete volatiles that stimulated growth of Arabidopsis thaliana. Bacillus subtilis GB03 emitted more than 25 volatiles that activated transcripts in A. thaliana involved in e.g. modification of cell walls, metabolism, hormone regulation and protein synthesis (Zhang et al. 2007). Additionally, B. subtilis GB03 volatiles regulated processes such as cell expansion, photosynthetic efficiency and seed set (Zhang et al. 2007; Xie, Zhang and Pare 2009). Several PGPR strains produce the volatiles 2R, 3R-butanediol and acetoin that trigger ISR as demonstrated for e.g. B. subtilis GB03 and B. amyloliquefaciens IN937a against Erwinia carotovora in A. thaliana (Ryu et al. 2004) and Pseudomonas chlororaphis O6 against E. carotovora in tobacco (Han et al. 2006). Bacterial VOC can have many different chemical structures where compounds such as amines, benzaldehyde, benzothiazole, decanal, cyclohexanol, dimethyl trisulfide, 2-ethyl-1-hexanol and nonanal have been identified as fungicidal molecules (Kai et al. 2009).
Minerals are naturally occurring inorganic chemical compound as a solid material and among 17 nutrients reported, Phosphorous plays a critical role in the plant growth through photosynthesis, energy transfer, transformation of sugars and starches and transformation of genetic materials from one generation to another. Acquisition of plant nutrients was enhanced by beneficial soil microorganisms. Insoluble forms of phosphatic fertilizer like tricalcium phosphate (Ca3PO4)2, aluminium phosphate (Al3PO4) and iron phosphate (Fe3PO4) were converted into available forms by microorganisms (Gupta et al. 2007; Song et al. 2008; Khan et al. 2013; Sharma et al. 2013). Wide range of biological process involved in the transformation of insoluble nutrients into soluble nutrients (Babalola and Glick, 2012). Two types of phosphate utilization were observed like direct application of phosphate fertilizer and microbial solubilization. In the soil, artificial application of phosphatic fertilizers leads to little amount of absorption by plants, and the remaining will be converted into insoluble complexes. These will be solubilized by microorganisms in higher-level conversion (Mckenzie and Roberts, 1990) mediated by the enzymes released by the soil microbes called phosphatases (Yadav and Tarafdar 2003; Tarafdar et al. 1988; Aseri et al. 2009) and phytases (Maougal et al. 2014). There are several reports of phosphate solubilisation by Bacillus sp. Kang et al. (2014) studied the beneficial aspects of B. megaterium strain mj1212 inoculated with mustard plants. It was observed that application of Bacillus sp. enhanced shoot and root length as well as plant fresh weight. The biochemical analysis showed that chlorophyll, fructose, glucose, sucrose, and many amino acids’ contents were higher in B. megaterium strain mj1212 inoculated plants, as compared to the uninoculated plants. Phosphate content was also higher in inoculated plants than control plants. In another study conducted by Swain et al. (2012), Bacillus subtilis thermotolerant strains (<50 °C) were isolated from cow dung. This strain also solubilized tricalcium phosphate, and this solubilization was associated with enzyme phosphatase production, especially the acid phosphatase (AcP). The inoculation of this bacterium with cowpea (Vigna unguiculata L.) resulted in increased root length, shoot height, and plant biomass as compared to the control plants. Wang et al. (2014) isolated phosphate-solubilizing bacteria Bacillus thuringiensis strain B1 from an acidic soil in China. Inoculation of B1 increased available P and peanut growth under acidic soil condition. Inoculation by strain B1 also considerably enhanced shoot length, branch number, hundred seed weight, and crude protein contents. Similar results on groundnut have also been reported by Maheswar and Sathiyavani (2012). As an endophytic bacterium, Bacillus sp. has been reported to solubilize P and enhance growth of banana plants (Matos et al. 2017). Kibrom et al. (2017) reported on the isolation and characterization of phosphate-solubilizing Bacillus sp. from various agroclimatic regions of Tigray, Ethiopia. The isolated bacteria enhanced P uptake as well as root and shoot length. Mohamed et al. (2018) isolated phosphate-solubilizing Bacillus subtilis and Serratia marcescens from the tomato plant rhizosphere. Inoculation of both bacteria in tomato plants enhanced phosphate uptake. Similarly, Turan et al. (2007) reported about the influence of Bacillus strain FS3 on growth and development of tomato (Lycopersicon esculentum L.) plants and enhanced phosphate content. The inoculation of FS3 increased plant height and root length. In another study conducted by Tahir et al. (2013), three phosphate-solubilizing bacterial strains, viz., Azospirillum, Bacillus, and Enterobacter, were isolated and characterized. Later these strains were identified based on 16SrRNA sequence analysis. Inoculation of these three strains improved wheat (Triticum aestivum L.) growth and phosphate uptake in grains. Jadhav (2016) studied the phosphate solubilization and biocontrol aspects of Bacillus licheniformis isolated from the pigeon pea (Cajanus cajan L.) rhizosphere. It was found that Bacillus licheniformis solubilized a good amount of phosphate under laboratory conditions. Owing to its P-solubilizing attribute, this isolate may be utilized as a potential P-mobilizing biofertilizer. Ahmad et al. (2018) studied the effect of phosphate-solubilizing Bacillus subtilis Q3 and Paenibacillus sp. Q6 for enhancing cotton plant growth under alkaline soil conditions. The strains Q3 and Q6 enhanced plant growth and P uptake. From a unique rhizosphere of an aromatic plant, Tallapragada and Seshachala (2012) isolated and characterized phosphate-solubilizing microbes from the rhizospheres of Piper betel. The isolated Bacillus sp. exhibited good amount of phosphate-solubilizing potential. Under semiarid conditions, the effect of salt-tolerant and phosphate solubilizers Bacillus sphaericus and Burkholderia cepacia were evaluated by Ramani and Patel (2011) on food and fodder crops. Both the bacterial isolates exhibited noteworthy effect under pot culture and field conditions. Bahadir et al. (2018) discovered potential bioinoculants 440 Bacillus isolates from various sources. These were investigated qualitatively for P solubilization, and affirmative isolates were further tested for quantitative determination of P solubilization and production of organic acid. The six best phosphate solubilizers were again tested for production of phytohormone (IAA), seed germination under in vitro conditions, and pot experiments. All six best Bacillus strains produced a good amount of IAA, meaningfully improved root and shoot length, and noticeably enhanced plant growth and development. In another study, the effect of P-solubilizing B. pumilus was studied on cauliflower. Bacillus sp. not only improved P uptake but also enhanced cauliflower size and weight as compared to the control (Dipta et al. 2017). The phosphate-solubilizing Bacillus sp. employed several mechanisms for P solubilization.
Potassium (K) is considered as a major constituent and essential element in all living cells. Naturally, soils contain K in larger amounts than any other nutrients; however, most of the K is unavailable for plant uptake. Depending on soil type, 90–98% of potassium in the soil is in the unavailable form (Sparks and Huang, 1985). This can be converted to soluble forms by potassium-solubilizing bacteria for the plant uptake (Etesami et al. 2017) and mostly belong to the genera Bacillus sp. include B. pumilus, B. mucilaginous, B. amyloliquefaciens, B. firmus, B. megaterium, B. subtilis, B. licheniformis, Paenibacillus macerans (Glick 2012) having the capacity to solubilize K minerals like feldspar, muscovite, biotite, orthoclase, illite and mica. This can be made possible by various processes in conversion of silicate minerals through the process like acidolysis, complexolysis, chelation and exchange reaction. Upon artificial inoculation of phosphate-solubilizing bacteria may lead to improve plant growth by increasing seed emergence, plant weight and yield.
ALLEVIATION OF CROP STRESSES
Bacillus sp. is recognized as effective bioagent as well as plant growth promoting bacteria, and its potential has been proved during the last 20 years (Fiddaman and Rossall 1995; Sharga 1997; Campbell 1989). These Bacillus sp. mostly include B. subtilis (Mishagi and Donndelinger 1990), B. insolitus, B. pumilus (McInroy and Kloepper 1995), Paenibacillus polymyxa (Shishido et al. 1999), B. amyloliquefaciens (Reva et al. 2002), B. cereus (Pleban et al. 1997), B. megaterium (McInroy and Kloepper 1995), B. licheniformis and B. endophyticus from the inner tissues of cotton plant. Besides, the endospore-forming potential of the Bacillus sp. enhances their survival capability under diverse environmental conditions. Some strains of Bacillus species have the ability to intrude into the innermost plant tissues and play an essential role in plant growth promotion and protection against biotic and abiotic stresses. Bacillus sp. has been reported to produce diverse antibiotics with multifaceted mode of action that aids in the control of various phytopathogens (Kumar 1999; Asaka and Shoda 1996). The antibiotic compounds are of diverse types and structures, viz. aliphatic hydrocarbons, fatty acids, phenolics, and lipopeptides (Silo-suh et al. 1994; Sathyaprabha et al. 2010; Mihailovi et al. 2011; Sadashiva et al. 2010; Musthafa et al. 2012; Mora et al. 2011). Among the antibiotic compounds, antimicrobial peptides of short-chained amino acids namely bacillomycin, subtilin, iturins, mersacidin, surfactins, bacilysin, and fengycins exhibit a noteworthy performance in plant disease management (Mora et al. 2011; Chung et al. 2008; Rajesh Kumar et al. 2014; Ramarathnam et al. 2007; Ongena and Jacques 2007; Vinodkumar and Nakkeeran 2015). Major antimicrobial peptides produced by Bacillus sp. can be grouped into three categories, viz., iturins, surfactins, and fengycins/plipastatins. Iturin produced by B. subtilis, was effective against wide spectrum of fungal phytopathogens. A significant decrease in seed mycoflora was noticed in the seed loads that were tested with iturin A with concentrations of 50–100 ppm (Asaka and Shoda 1996). Treatment of corn seeds with iturin A @ 5 and 20 g/100 Kg showed a significant reduction in total microbial count was observed. B. subtilis strain RB14 that was capable of producing iturin B and surfactin that aided in suppressing damping-off disease of tomato (Asaka and Shoda 1996). In addition to disease control, B. subtilis strain BACT-O also promoted the plant growth and yield of cucumber (Utkhede et al. 1999). Liang et al. (2007) reported that seed priming with Bacillus polymixa increased the seedling height of safflower. Two types of chitinase enzymes are synthesized by B. amyloliquefaciens and can inhibit F. oxysporum growth (Wang et al. 2004). Chitinase enzymes with high chitinitic character are also produced by B. subtilis against various fungal pathogens (Chang et al. 2009). A chitinolytic bacterial strain (YC300), isolated from a compost sample from Republic of Korea, and produced an iturin-like compound. Later, this strain identified as Paenibacillus koreensis also provided a fair to good antifungal activities against Colletotrichum lagenarium, F. oxysporum and S. sclerotiorum, B. cinerea and R. solani (Chung et al. 2000). In an independent research, iturin A isolated from bacteria showed much stronger performance than surfactin against phytopathogens (Asaka and Shoda 1996). Three strains, B. cereus (L-07-01), B. subtilis (H-08-02), and Bacillus mycoides (S-07-01), exhibited significant antifungal activity against F. graminearum (Fernando et al. 2005; Ramarathnam et al. 2007). Soil application of Bacillus sp. was highly effective in the management of Fusarium wilt of carnation (Rajesh Kumar, 2014). B. subtilis, B. amyloliquefaciens, B. licheniformis, and B. cereus resulted in inhibiting the soil, air, and post-harvest plant diseases (Yoshida et al. 2002). Zwittermicin A is an aminopolyol compound produced by B. cereus and is also known to possess good inhibitory action against pathogenic fungi including oomycetes group of pathogens (Silo-Suh et al. 1998; Fernando et al. 2005). Delivering of talc-based consortia formulation comprising of B. subtilis (S2BC-2) + Burkholderia cepacia (TEPF-Sungal) reduced vascular wilt and corm rot of gladiolus under protected cultivation. Besides, increase in cormel and corm production and flowering was promoted upon the application (Shanmugam and Kanoujia, 2011). Kumar et al. (2014) reported the abiotic as well as biotic stress regulating potential of Bacillus sp. isolated from different rhizospheric zones of India. It was found that Bacillus sp. could control the growth of pathogenic fungal species such as Sclerotium rolfsii, Botrytis ricini, Macrophomina phaseolina, Fusarium oxysporum, and Rhizoctonia solani. On the other hand, this bacterium also demonstrated abiotic stress tolerance against salinity (NaCl 7%), higher temperature (50 °C), and drought (−1.2 MPa). Zwittermicin A is also popularly used as a broad spectrum of compound against different harmful microbes (Silo-suh et al. 1998). These groups of compounds also exhibit diverse biological activity against Oomycetous plant diseases as well as insecticidal activity (Emmert et al. 2004). Moreover, there are several reports which demonstrated the biological activity against different groups of plant pathogens by Bacillus species (Kloepper et al. 2004; Correa et al. 2009; Jogaiah et al. 2010). B. amyloliquefaciens with 23 diverse AMP genes effectively inhibited S. sclerotiorum which causes stem rot of carnation. Further, it significantly enhanced the plant growth and yield (Vinodkumar et al. 2015). In another study, the synergistic action of iturin and surfactin against Colletotrichum gloeosporioides was performed successfully (Kim et al. 2010). Surfactin like compounds isolated from B. subtilis (R14) and Bacillus circulans (Das et al. 2008; Fernandes et al. 2007) were found to suppress multidrug-resistant bacteria such as Escherichia coli, Alcaligenes faecalis, Pseudomonas aeruginosa, Staphylococcus aureus, Proteus vulgaris, and methicillin-resistant. The four strains of B. subtilis which were effective against cucurbit powdery mildew also exhibited highest antibacterial activity against Xanthomonas campestris pv. cucurbitae and Pectobacterium carotovorum sub-sp. Carotovorum (Romero et al. 2007). These strains produced lipopeptide antibiotics, viz. fengycins, surfactins, and iturins. Further, thin-layer chromatography studies and direct bioautography revealed that the antibacterial activity was correlated due to iturin compound. This result was further validated using defective mutants of lipopeptide, thereby elucidated the importance of AMPs in plant disease control. US EPA (Environmental Protection Agency) has also provided a catalogue of biopesticides in which commercial formulations of distinct strains of Bacillus that can be employed as biocontrol agents are prescribed (McSpadden Gardener, 2004). These products are accessible in distinct types of preparations such as a wettable powder, dry cakes, or liquid or suspension in a liquid, as it depends on the nature of relationship between the biocontrol strain and carrier molecule. Products such as Taegro, Sublilex, Companion, Serenade, and Kodiak are all dependent on the utilization of various strains of B. subtilis as a biocontrol agent. Kodiak is known for the eradication of root inhabiting disease-causing agents of soybean and cotton like Aspergillus sp., Alternaria sp., Fusarium sp., and R. solani. Moreover, Serenade (Agra Quest, Daius CA, USA) constituting B. subtilis strain QST713 is known to remediate the Cercospora leaf spot, early and late blight diseases related with different crop plants. Similarly, bacterial strain, Bacillus sp. AZ-11 was also found as effective against reduction in Ganoderma growth. Several species of the genus Bacillus have been reported as potential BCAs. For instance, the bacterium B. tequilensis is reported to manage root knot nematode when co-inoculated with Trichoderma harzianum (Tiwari et al., 2017). Thus, this bacterium can have dual advantage in irrigated pockets where several vegetables suffer from root knot nematode. Incidentally, B. tequilensis is also known to solubilize Zinc for increased yield of wheat and soybean (Khande et al., 2017). In arid region, one strain of B. firmus has also been reported as specific antagonist to Macrophomina phaseolina (Lodha et al. 2013) in earlier studies. Bajoria et al. (2008) isolated B. subtilis, B. cereus, B. pumilus and B. sphaericus from hot arid region having biocontrol potential against plant pathogenic fungi. Therefore, the isolated antagonistic bacterial strains will be potentially very useful and yield effective results in further field studies for management of this terrific pathogen.
PLANT GROWTH PROMOTION
Bacillus sp. utilizes various means (direct as well as indirect) to promote plant growth. Bacillus sp. transforms the intricate or difficult form of indispensable nutrients, such as N and P, into very simple and bioavailable forms that can be taken up by the plant root system (Kuan et al. 2016; Shafi et al. 2017). The biological available form of Nitrogen in soils which is an essential constituent in nucleic acids, proteins, and other organic components, is partial or inadequate, which slows down plant growth in its natural ecosystem (Barker et al. 1974; De-Willigen 1986). There are some species of Bacillus sp. which release NH3 from nitrogenous organic matter (Hayat et al. 2010). Oliveira et al. (1993) reported that some of the Bacillus sp. possess the nif gene and demonstrate nitrogenase enzyme activity. This enzyme helps in the fixation of atmospheric Nitrogen and delivers it to the plants to augment plant development, growth, and yield by fulfilling N requirements and delaying the process of senescence (Seldin et al. 1984; Ding et al. 2005). The Bacillus sp. also excretes the proteinaceous compound known as a siderophore, which helps in iron chelation from the rhizospheric zone of plant (Wilson et al. 2006). The iron-chelating compound siderophores bind Fe+++ in complex substances and reduce the ferric form into ferrous form, which can easily enter plant root system (Dertz et al. 2006). Kumar et al. (2012) isolated seven soil bacterial isolates from bean rhizosphere in the Uttarakhand Himalayan region. These demonstrated excellent plant growth-promoting and biocontrol activities. On the bases of 16S rRNA gene sequence, the soil isolate was identified as Bacillus sp. and named BPR7. The rhizospheric strain BPR7 released phytohormone IAA, siderophores, enzyme phytase, organic acids, cyanogens, and ACC deaminase and was also able to solubilize numerous types of inorganic and organic phosphatic material as well as Zn and K. Gutiérrez-Mañero et al. (2008), reported that, B. pumilus and B. licheniformis isolated from alder (Alnus glutinosa [L.] Gaertn.) rhizosphere exhibited strong plant growth-promoting potential. Therefore, both the Bacillus bacteria demonstrated the good potential of promoting plant growth. The bioassay facts displayed that the dwarf phenotype produced in alder seedlings by a chemical paclobutrazol (which inhibits biosynthesis of gibberellin or GA) was reversed effectively by applying bacterial extracts from media and by exogenous treatment of GA3.
Calvo et al. (2010) isolated 63 Bacillus strains from the native potato rhizospheric regions growing in the Andean highlands of Peru. The strains demonstrated strong antagonism against Rhizoctonia solani and Fusarium solani. The antagonistic Bacillus strains were further verified for additional plant growth promotional aspects. It was observed that 81% of the isolates produced a certain amount of phytohormone indole-3-acetic acid (IAA) and 58% could solubilize tricalcium phosphate (TCP). The phylogenetic assessment revealed that the majority of isolates belonged to B. amyloliquefaciens sp., while other three potential strains may be novel putative Bacillus sp. Therefore, these Bacillus sp. may be used as potential potato growth promoters. Shakeel et al. (2015) obtained 234 rhizospheric isolates from basmati super rice and basmati-385 varieties cultivated in clay loam and saline soils from various locations of Punjab province in Pakistan. Out of the total 234 rice rhizospheric isolates, 27 isolates solubilized Zinc (Zn) from zinc oxide, zinc carbonate, and zinc phosphate. The soil isolate SH10 exhibited maximum zone of Zn solubilization (24 mm) using zinc phosphate, and soil isolate SH17 showed maximum zone of Zn solubilization (14–15 mm) using zinc oxide and zinc carbonate. Strains SH10 and SH17 also solubilized P (38–46 mm) and K (47–55 mm) under laboratory conditions. Both the strains also exhibited bio control activities against root rot (Fusarium moniliforme) and blast (Pyricularia oryzae) inciting plant pathogens by 22–29% and manufactured many biological control factors in vitro conditions. Bacterial strains SH10 and SH17 also enhanced Zinc uptake and translocation into the grains and augmented plant yield of super basmati rice and basmati-385 varieties by 18–47% and 22–49%, correspondingly. Using 16S rRNA gene analysis, both the SH10 and SH17 bacterial strains were identified as Bacillus sp. and Bacillus cereus. Idris et al. (2002) isolated many Bacillus strains belonging to B. subtilis or B. amyloliquefaciens from plant pathogen-contaminated soils and demonstrated PGPR properties. Soil isolate B. amyloliquefaciens could biodegrade extracellular compound phytate (myoinositol hexakisphosphate). The maximum phytase activity was found in isolate FZB45 (B. amyloliquefaciens), and the diluted bacterial cultural filtrate of this isolate encouraged maize seedling growth in phytate presence and P limitation. Lucas Garcia et al. (2004) studied the effect of inoculation of B. licheniformis on development and growth of tomato and pepper in three experiments. In the first experimental condition, the bacterium meaningfully enhanced plant height and the leaf surface area in both cultivars, and the effect was more on pepper as compared to tomato. In a second experiment, the plant seedlings were grown in sand, and hydroponic systems were tested. Interestingly, the size and number of tomato fruit increased after application of bacterial inoculation in sand and in the hydroponic system. Moreover, the inoculated cultivars exhibited less disease infestation as compared to untreated plants. In the third experiment, the pepper yield was greater in inoculated plants as compared to the uninoculated plants. This Bacillus strain had appreciable colonization and survival potential, and it could be employed as a biofertilizer as well as biocontrol agent. Kayasth et al. (2013) isolated Bacillus strain and designated as ML3 from a soil sample, and it produced the phytohormone and IAA and NH3 in the range of 174.72 and 0.66 μg ml−1, correspondingly. This strain also produced siderophores and effectively solubilized tricalcium phosphate (TCP). The main functional nitrogenase gene nif H was also found in this strain. Based on morphological, biochemical, and partial 16S rRNA gene sequencing, this strain ML3 was identified as B. licheniformis. This strain has all properties to be utilized as an efficient plant growth promoter. Agarwal and Agarwal (2013) isolated 28 bacterial strains from dissimilar tomato rhizospheric soils from Dehradun area of Uttarakhand, India. All of the soil isolates were characterized biochemically and tested for plant growth-promoting abilities such as indole acetic acid production (IAA), Phosphate (P) solubilization, HCN production, siderophore excretion, and catalase activity. Out of 28 soil isolates, only 5 Bacillus isolates exhibited potential plant growth-promoting abilities. Inoculation of tomato plants with selected Bacillus sp. resulted in increment in shoot and root length of tomato seedlings as compared to the uninoculated plants. Bacillus sp. also enhanced the seed germination percentage as well as seed vigor index. Ramírez and Kloepper (2010) have studied the consequence of inoculum concentration and soil phosphate-associated properties on plant growth encouragement by B. amyloliquefaciens strain FZB45, which produced phytase. A noteworthy synergy between soil phosphate and bacterial application was observed. B. amyloliquefaciens strain FZB45 encouraged plant growth and phosphate uptake, which confirms the role of enzyme phytase and less P uptake in uninoculated plants. This strain also produced IAA under laboratory conditions, but its role was not determined. Sharma et al. (2013) isolated a bacterium from the rhizosphere of soybean plants grown at Directorate of Soybean Research, Indore, Madhya Pradesh, India, and this bacterium was identified as Bacillus sp. on the basis of morphological and biochemical tests as well as FAME profile. Studies on 16S rRNA gene showed 98.7% homology to B. amyloliquefaciens, and thereafter, it was designated as strain sks-bnj-1 (AY 932823). This strain owned manifold plant growth-promoting attributes such as IAA production; siderophore production; ACC deaminase activity; enzymes like phosphatases, phytases, and cellulases; Zinc solubilization; and HCN production. This bacterium also exhibited biocontrol properties. Interaction of this strain with soybean increased the shoot root biomass as well as nutrient uptake as compared to the uninoculated control plants. In another study on Bacillus sp. by Cruz-Martín et al. (2015) conducted on banana plant in Cuba, it was observed that strain B. pumilus could fix atmospheric nitrogen and was able to grow in nitrogen-free culture media and produced IAA (28.9 μg ml−1). Moreover, strain B. pumilus significantly enhanced the plant height and thickness of the stem, altered root architecture, and improved fresh and dry plant weight. In the recent past, a novel bacterium, B. firmus was isolated from naturally heated cruciferous residue amended soil that showed antagonistic activity against M. phaseolina. In dual culture tests, it produced a scarlet pigmentation only against M. phaseolina (Lodha et al., 2013). Separate dual culture tests were also performed to ascertain the activity of antagonistic bacterium against prevalent soil fungi, Fusarium oxysporum f. sp. cumini and a bio-agent T. harzianum. However, B. firmus could not inhibit the growth of any of these fungi nor any scarlet pigmentation was observed during interaction. Bacillus sp. is particularly suited for studies on biological control due to its omnipresence in soils, high thermal tolerance, rapid growth in broth culture and ready formation of resistant spores. They are uniformly distributed throughout the soil rather than being concentrated in the plant rhizosphere. The main characteristics of B. firmus includes its thermal tolerance (45°C), phosphate solubilizing nature, compatibility with T. harzianum, increased nodulation, plant growth promotion and potential to colonize roots.
BIO FORMULATIONS FOR DISEASE SUPPRESSION
Many locally available on-farm wastes were evaluated to select food substrates that can improve the shelf life of B. firmus for a reasonable period. Our efforts successfully culminated in identifying a suitable food substrate, which was combined with carrier to retain adequate moisture in the bio-formulated product so that bacterium could survive for a period of 180 days. The product designated as Maru Sena 3 was made available for distribution to the farmers. The technology was validated after developing bio-formulated products and their efficacy was demonstrated at growers’ field to disseminate this eco-friendly and easy management strategy in the region (Mawar et al., 2018). Field demonstrations were carried out at adopted villages of the Institute’s Krishi Vigyan Kendra (Farm Science Centre dedicated for extension activities). Effectiveness of seed coating with bio-formulated product of B. firmus on incidence of dry root rot and seed yield of cluster bean, moth bean and sesame were demonstrated at grower’s field during last ten years. The percent increase in seed yield just by seed treatment ranged from 13.3-23.5% in all legumes and oil seed crops in different demonstrations. These products are made available for the farmers through Agricultural Technology Information Center (ATIC: Single window delivery mechanism) of the institute. In the last five years, hundreds of hectares have been sown with bacterium coated seeds. Farmers are getting positive response in checking incidence of dry root rot in rainfed crops of arid region namely cluster bean, cowpea, green gram, mothbean, sesame, etc. The process of developing bio-formulated product of these bio-pesticides has been patented in order to put these products for commercialization for wider adoption among growers. A patent “Bioformulation of a biopesticide and a process for preparing the same” was granted in 2019 with patent no. 309385.
Various studies reported that certain biocontrol consortia were unable to show at least comparable effects on plants when compared to their individual applications. One of the major causes for such contrary results may be attributed to incompatibility of the microbes in the mixture with each other. The findings clearly advocate for screening of compatible microbes for development of microbial consortia. The basic objective of developing microbial consortium would fail if the microbes used in the consortium do not have any additive or synergistic effects on disease suppression.
Application of bioagents in consortium may improve efficacy, reliability and consistency of the microbes under diverse soil and environmental conditions (Berg et al., 2005). Use of different species of biocontrol occupy different niches in the root zone and thereby restrict competition among them. Diversity in biocontrol mechanisms offered by each microbial component may also help in enhancing disease suppressiveness. A number of microbial consortia have been developed by many scientists and were tested in different crops in our country. Therefore, in the same stream our consortia prepared with two organisms worked in a synergistic manner. B. firmus operates through antibiosis while T. harzianum kills the mycelium of the pathogen mainly through hyper parasitism. It was, therefore, thought worthwhile to develop a consortium of native bio-control agents in the form of a single bio-formulated product to improve pathogen control by way of operating different mechanisms of antagonism for plant disease suppression. The biocontrol consortium activates the antioxidant enzyme activities and the phenylpropanoid pathway leading to accumulation of total phenolics, proline and pathogenesis related (PR) proteins after the pathogen challenge. The impact of triple microbial consortia consisting of fluorescent Pseudomonas (PHU094), Trichoderma (THU0816) and Rhizobium (RL091) for alleviation of biotic stress in chickpea is through enhanced antioxidant and phenylpropanoid activities (Akanksha et al., 2012). Periodical oxidase and accumulation of total phenol content was higher when challenged with the pathogen compared to the single microbe and dual microbial consortia. Pseuomonas aeruginosa PJHU15, Tricoderma harzianum TNHU27 and Bacillus subtilis BHHU100 as a consortium has been used to assess suppression of soft-rot pathogen Sclerotina sclerotiorum (Jain et al., 2012). The triple-microbe consortium and single-microbe treatments showed 1.4-2.3 and 1.1-1.7 fold increment in defense parameters respectively when compared to untreated challenged control. The compatible microbial consortia triggered defense responses in an enhanced level in pea than when the microbes were alone and provided better protection against Sclerotinia rot. Trichoderma species and fluorescent Pseudomonas sp. have been reported to induce systemic resistance in plants. These biological control agents were tested as a single application and in combination for their abilities to elicit induced resistance in cucumber against Fusarium oxysporum f. sp. radices cucumerinum and in A. thaliana against Botrytis cinerea. The combination of Tr6 and Ps14 induced a significantly higher level of resistance in cucumber, which was associated with the primed expression of a set of defense-related genes upon challenge with Fusarium. In Arabidopsis, both Ps14 and Tr6 triggered ISR against B. cinerea but their combination did not show enhanced effects. In the induced systemic resistance-defective Arabidopsis mutant myb72, none of the treatments protected against B. cinerea, whereas in the SA-impaired mutant sid2, all treatments were effective. Taken together, these results indicate that in Arabidopsis Ps14 and Tr6 activate the same signaling pathway and thus have no enhanced effect in combination. The enhanced protection in cucumber by the combination is most likely due to activation of different signaling pathways by the two biocontrol agents.
CONCLUSION AND PERSPECTIVES
Diversity in Bacillus species produce multiple classes of antimicrobial compounds inducing systemic resistance in a variety of ways that can be used for the plant growth promotion and management of a broad range of plant stresses and ultimately sustaining crop health. The spore forming ability of Bacillus species gives them a key importance in the field of biological control. The research areas that demand considerations for the successful adoption of Bacillus sp. as bioagents comprise the exploration for biodiverse antagonistic and antibiotic strains, elucidation of their mode of action and signalling pathways, stability under field application, development of effective formulations that can be used with other bioinoculants with synergetic effect and perfect demonstration of cost–benefit ratio for effective commercialization.
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