Bacillus valezensis: A New Plant Growth Promoting Rhizobacterium for Plant Growth Promotion and Inhibition of Rhizoctonia bataticola for the Management of Dry Root Rot of Chickpea

Dry root rot in chickpea caused by Rhizoctoniabataticola is an economically important disease and results in more yield losses wherever the crop is grown across the globe as well as in India in recent years. All the states wherever chickpea is grown are affected by the disease and it is particularly severe in Karnataka, Maharashtra, Gujarat, Tamil Nadu, Madhya Pradesh, Tellangana and Andhra Pradesh. The disease is more prevalent between flowering and podding stages and causes yield loss up to 100 per cent in susceptible varieties of chickpea under favorable conditions (Gupta and Sharma, 2015).
       
At present, the disease management strategy relies heavily on the use of fungicides, which are not eco-friendly and affordable to many farmers under organic farming of chickpea. Additionally, the use of plant growth-promoting rhizobacteria (PGPR) is potentially advantageous for improving crop productivity, food quality and security in more sustainable and eco-friendly manner (Etesami, 2020).
       
Hence, keeping the above facts in view, use of plant growth promoting rhizobacteria.  Bacillus spp. is a rod-shaped, gram positive bacterium, frequently found in soil and reported to produce endospores to resist dry environments and high temperatures. The aim of present investigation was to evaluate the antagonistic potential, plant growth promoting traits of indigenous Bacillus sp. strains against Rhizoctonia bataticola causing dry root rot of chickpea and to identify them at molecular level using 16S rDNA.

Isolationand maintenance of the pathogen
 
Plants showing typical dry root rot symptoms were collected from chickpea fields of UAS, Raichur during rabi 2020-21 cropping season and the pathogen was isolated from the infected portions and cultured on potato dextrose agar (PDA) medium using hyphal tip isolation method. Pure cultures of the pathogen R. bataticola were maintained on PDA slants.
 
Collection and isolation of native Bacillus PGPR strains
 
Soil samples from healthy chickpea rhizosphere were collected during rabi, 2020 for isolation PGPR. Nineteen plant growth promoting rhizobacteria, Bacillus sp. strains were obtained by serial dilution technique using Hichrome Bacillus agar medium (Waksman, 1922) from different regions of Karnataka (Table 1). The Bacillus sp. strains were kept under observation daily for the appearance of green colonies.
 

 
Antagonistic potential of PGPR strains against R. bataticola
 
Thirty PGPR strains of Bacillus sp. were screened for antagonistic potential against R. bataticola by dual culture technique (Xu and Kim, 2014). The degree of antagonism was determined by measuring the radial growth of pathogen with bacterial culture and control. The per cent inhibition over the control was calculated by using the formula (Vincent, 1927).

                                                   
                                             
Where;
I = Per cent inhibition of mycelium.
C = Growth of fungal mycelium in control.
T = Growth of fungal mycelium in treatment.
       
The efficient PGPR strains of indigenous Bacillus sp. which showed more than 50 per cent mycelial inhibition of pathogen were selected to study plant growth promoting traits.
 
Plant growth promoting traits of potential PGPR
 
Indole acetic acid (IAA) production
 
It was measured by the method described by Patten and Glick (2002) and the detection of IAA was determined by the development of pink color.
 
Ammonium production
 
The test was carried out as explained by Cappuccino and Sherman (1992). Development of brown to yellow colour indicated positive test for ammonium production.
 
Phosphate solubilization
 
The phosphate solubilization study was conducted as per protocol given by Nautiyal (1999). Test PGPR strain was stabbed on plate using sterile cork borer. The observation on halo zone was measured after 3 days of incubation at 28°C.
 
HCN production
 
Production of HCN was determined in slants containing NA supplemented with 4.4 g/l of glycine (Lorck, 1948) and the rhizobacteria were streaked and plates were inverted. Later, a piece of Whatman filter paper no. 1 impregnated with 0.5% picric acid and 2% of sodium carbonate was placed on the lid. Petri plates were sealed with parafilm and incubated for and incubated at 28-30°C for 96 h. Change in colour of the filter paper from orange to brown was considered as production of HCN. Change in colour of inserted filter paper to brown or reddish-brown was recorded as positive reaction.
 
Molecular characterization of highly potential PGPR
 
The rhizospheric PGPR which showed higher plant growth promoting traits were further selected for molecular characterization. The DNA isolation of four efficient strains was carried out by using standard CTAB method. The universal primers 16s rDNA F (GAG-TTT-GAT-CCT-GGC-TCA) and 16s rDNA R (AGA-AAG-GAG-GTG-ATC-CAG) were synthesized at Eurofins, Bangalore, India for characterization of isolates. Amplification was conducted in a thermal cycler (Veriti, Applied Biosystems, Singapore) and the PCR programming was done as per Yugander et al., (2017). Initial denaturation was set for 1 min at 94°C for 1 cycle. Whereas, denaturation time was set for 1 min at 96°C, annealing for 1.5 min at 58°C and extension for 1.5 min at 72°C for 35 cycles. The final extension was set for 8 min at 72°C for 1 cycle.

Isolation of pathogen
 
The results indicated that, the white mycelial growth of pathogen was observed within 3 to 4 days. Later, it turned to black colour showing sclerotial bodies after 8-10 days. The mycelium was brown in colour and branching was right angled under microscope showing specific character of the pathogen.
 
Isolation and maintenance of native PGPR strains of Bacillus sp.
 
Thirty strains of Bacillus sp.were successfully isolated by using Hichrome Bacillus agar medium (Bharose et al., 2017) and strains were designated as SBPGPM- 1to SBPGPM-30 (Table 2).
 

 
Antagonistic potential of rhizospheric PGPR strains against R. bataticola
 
The per cent inhibition of mycelial growth of pathogen varied greatly (2.59-83.89 mm) among the thirty isolates. Among them, nine strainsviz., SBPGPM-4, SBPGPM-7, SBPGPM-9, SBPGPM-15, SBPGPM-19, SBPGPM-21, SBPGPM-23, SBPGPM-28 and SBPGPM-30 recorded more than 50 per cent inhibition of pathogen (Table 1 and Fig 1). However, the minimum mycelial inhibition was observed in SBPGPM-22 with per cent inhibition of 2.59 (Table 2, Fig 1). Similarly, Pandey et al., (2016) studied the antagonistic action of fluorescent Pseudomonas which was isolated from native soil (Raipur), tested against three different isolates of F. oxysporum f. sp. ciceris by dual culture technique. The fluorescent Pseudomonas inhibited the growth of pathogen isolates such as F. oxysporum f. sp. ciceris 1, F. oxysporum f. sp. ciceris 2 and F. oxysporum f. sp. ciceris to the extent of 46.70, 46.83 and 46.03 per cent, respectively. Likewise, B. subtilis strain PRBS-1 and AP-3 inhibited five soybean seed pathogenic fungi, viz., Rhizoctonia solani, C. truncatum, S. sclerotium, M.  phaseolina and Phomopsis spp. under in vitro conditions (Araujo et al., 2005). In the current study also the rhizospheric PGPR strains able to inhibit the R. bataticola which indicated that rhizospheric bacteria can inhibit R. bataticola.
 

 
Plant growth promoting traits of potential PGPR strains
 
Plant growth promoting microorganisms significantly ameliorate plant growth by a number of mechanisms including increase of uptake of essential nutrients such as phosphate, ammonia and nitrogen, producing phytohormones such as indole-3-acetic acid and gibberellins. Hence, nine potential rhizospheric bacterial PGPMs, which were efficient in inhibition of pathogen in the dual culture assay were further screened for the production of plant growth promoting traits like IAA production, ammonia production, phosphate solubilization and HCN production (Table 3).
 

 
IAA production
 
Indole-3-acetic acid is the most common, naturally occurring plant hormone. IAA influences the process of forming plant tissues, namely growth, division and cell differentiation and protein synthesis. A diverse genus of microbes can augment significant amount of IAA. That being the case, in the present study,nine strains such as SBPGPM-4, SBPGPM-7, SBPGPM-9, SBPGPM-15, SBPGPM-19, SBPGPM-21, SBPGPM-23, SBPGPM-28 and SBPGPM-30exhibited pink color which indicated the production of indole-3-acetic acid as detected by the Salkowaski’s reagent (Table 3 and Fig 2a). The results indicated that PGPR strains have the ability to induce plant growth promotion of chickpea through the synthesis of IAA which helped in plant cell elongation, proliferation and also indirectly supported the growth of root and shoot. The present results are supported by Reetha et al., (2014) who undertook a study for isolation of P. fluorescens and B. subtilis from rhizosphere of onion and analysis of these bacteria for in vitro IAA acetic acid production. The results indicated that the two tested PGPR exhibited a pink to red color with a little variation in intensity.
 

 
Ammonium production
 
Ammonium plays a key role in the plant growth by providing the nitrogen to plants.All nine strains (SBPGPM–4, SBPGPM-7, SBPGPM-9, SBPGPM- 15, SBPGPM-19, SBPGPM-21, SBPGPM- 23, SBPGPM-28 and SBPGPM-30) were found positive for production of ammonium by exhibiting yellow color (Table 3 and Fig 2b). The results also indicated that the PGPMs which could produce the ammonia can supply the crops with a sufficient amount of ammonium required for root and shoot elongation and consequently promote plant growth.
       
There is a supporting report given by Hassan (2017) that the rhizobacteria such as B. cereus and B. subtilis and fungal PGPMs, P. chrysogenum and P. crustosum also produced indole acetic acid, ammonium and phosphatein Teucrium polium.
 
Phosphate solubilization
 
Phosphorus is one of the macronutrients required for plant growth promotion. In most cases, phosphorus is present in the soil as insoluble inorganic forms. But, different rhizospheric and endophytic PGPM strains have the efficacy to convert it from an unavailable to available source for plant uptake. In the present study, the phosphate solubilizing activity for all nine isolates was assessed on Pikoviskaya medium supplemented with tri-calcium phosphate as an inorganic phosphate source. Out of nine, five isolates such as SBPGPM-9, SBPGPM-19, SBPGPM-21, SBPGPM- 23, SBPGPM-28 were positive for the reaction (Table 3 and Fig 2c).
 
HCN production
 
HCN is recognized as a biocontrol agent, based on its ascribed toxicity against plant pathogens. Among the nine strains tested, five strains viz., SBPGPM-19, SBPGPM-21, SBPGPM-23, SBPGPM-28, SBPGPM-30 produced light reddish brown color on the filter paper which indicated the production of HCN (Table 3 and Fig 2d).Similar type of results was obtained with Ramyabharathi and Raguchander (2014), who reported that, B. subtilis was found to be positive for hydrogencyanide production.
       
There is a previous evidence similar to present investigation in which one hundred and fifty strains (endophytic and rhizospheric PGPMs) isolated from Canola were characterized for plant growth promoting traits. Among them, hundred isolates produced indole-3-acetic acid, seventeen isolates solubilized phosphate, forty four isolates produced siderophores, thirty four produced 1-aminocyclopropane-1-carboxylate deaminase and five produced hydrocyanic acid (Etesami et al., 2014). In the present investigation also the native rhizospheric PGPR strains could produce similar PGP traits.
       
It is clear from the present investigation that out of nine potential PGPR strains, only four PGPR strains namely SBPGPM-19, SBPGPM-21, SBPGPM- 23 and SBPGPM-28 successful in exhibiting more PGP traits tested. Hence, all the four native PGPRstrains were further carried for molecular characterization.
 
Molecular characterization of highly potential PGPR strains
 
Four highly potential rhizospheric bacterial strains such as SBPGPM-19, SBPGPM-21 SBPGPM- 23 and SBPGPM- 28 were amplified for 16S rRNA gene (Table 4, Fig 3). The results indicated the size of PCR amplified products of all four strains was 1500 bp. Four strains such as SBPGPM-19, SBPGPM-21 SBPGPM-23 and SBPGPM-28 were identified as B. cereus (Acc. No. ON567448), B. velezensis (ON568504), B. subtilis (ON566236), B.subtilissub sp. subtilis (ON566124), respectively and the accession numbers were deposited in genebank. Further, diversity analysis among them was studied by phylogenetic analysis. The phylogeny results  recorded homology with B. cereus (100 %), Bvelezensis (100%), B. subtilis (100%), B. subtilis sub sp. subtilis (99.34%) (Fig 4). Among four highly potential rhizosphaeric PGPR, Bacillus velezensis is new report for antagonistic potential and plant growth promotion in case of chickpea crop.
 

 

The present investigation put forward about the exploitation of plant growth promoting microorganisms for the pathogen suppression. In another terms, a healthy plant with its associated microorganisms can fight back against pathogens. As an evidence, in the current study, Bacillus valezensis, isolated from healthy chickpea rhizosphere could inhibit Rhizoctonia bataticola which causes major threat to its host. Additionally, it could exhibit plant growthpromoting activities. It is the first report that Bacillus valezensis could inhibit the pathogen.

The work has been undertaken as part of the of doctoral research programme at department of plant pathology, Agricultural college, UAS Raichur. The first author is chairmen and second author being a research scholar,isextremely thankful to the University for providing financial assistance and laboratory facilities for conducting the work.

None.