Exploration of the Potential of Bacillus spp. as an Antagonist and PGPR against Stem and Pod Rot of Groundnut.

Groundnut (Arachis hypogaea L.) is an important oilseed crop and in recent years, stem and pod rot caused by Sclerotium rolfsii Sacc. was found to be most devastating causing significant yield losses and results in huge losses. The groundnut growing farmers are facing serious problem of the disease. All states that grow groundnuts are affected by the stem and pod rot disease and it is particularly more in Maharashtra, Gujarat, Tamil Nadu and Karnataka. The yield loss caused by the pathogen is 25 per cent, but sometimes reaches 80-90 per cent (Grichar and Bosweel, 1987).
       
At present, a number of fungicides have been used for the management of disease. However, chemicals are harmful to human and animal health. Alternatives to fungicides include antagonistic microorganisms. The beneficial organisms known as bioagents reduce the harmful effects of plant diseases and encourage good plant responses. The Biological control agents are also known to promote the plant growth. is the process by which one or more organisms reduce the inoculums or disease-causing activities of a pathogen (Cook and Baker, 1983).
       
Hence, use of plant growth promoting rhizobacteria provides a useful alternative for growth promotion as well as plant disease management. 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 purpose of the current investigation was to study antagonistic potential, characterization and plant growth promoting traits of indigenous Bacillus spp. against Sclerotium rolfsii.

Isolation and maintenance of the pathogen
 
Groundnut plants showing typical symptoms of stem and pod rot were collected from groundnut fields of UAS, Raichur during Kharif, 2021 cropping season and the pathogen was isolated on Potato Dextrose Agar (PDA) medium by hyphal tip technique. Pure cultures of the pathogen S. rolfsii were maintained on PDA by periodical transfers.
 
Isolation of Bacillus spp.
 
Healthy rhizosphere soil from groundnut fields was collected by sampling the random soil sampling. The nineteen isolates of plant growth promoting rhizobacteria, Bacillus spp. Isolates were obtained by serial dilution technique using Hichrome Bacillus agar medium from different regions of Karnataka. The Bacillus spp isolates were kept under observation daily for the appearance of green colonies.
 
Antagonistic potential of Bacillus spp. isolates against Sclerotium rolfsii
 
Nineteen isolates of Bacillus spp. were screened for antagonistic potential against S. rolfsii by dual culture technique. 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 isolates of indigenous Bacillus spp. which showed more than 50 per cent mycelial inhibition of pathogen were selected for molecular, morphological as well as biochemical characterization and to study plant growth promoting traits.
 
Molecular characterization of efficient Bacillus spp.
 
The DNA isolation of five efficient isolates 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.
 
Morphological characterization of efficient Bacillus species
 
The characteristics of five efficient Bacillus species such as shape of cells was recorded under microspcope during Grams staining process, the colour and texture of colony by visual observation.
 
Biochemical characterization of efficient Bacillus species
 
Gram’s staining
 
The Gram staining of Bacillus spp. was carried out as per the standard protocol by using Grams staining kit and subjected for microscopic examination.
 
KOH test
 
A loopful of bacterial culture from well grown colony was taken and the test was carried out as per the procedure given by Sallam et al., (2013). The formation of strands of viscoid material represents the Gram negative bacteria and Gram positive bacteria do not produce viscoid thread.
 
Catalase test
 
A loopful of single well isolated colony was placed on a clean microscope slide and added with 3 per cent H₂O₂. A positive result of catalase production was characterized by the rapid evolution of O₂ which is evidenced by bubble formation (Yunting et al., 2013).
 
Starch hydrolysis
 
The test culture was spot inoculated on to the starch agar plates and kept for incubation. Later, the petri plates flooded with Lugol’s iodine and left few minutes. Formation of clear zone around the colony was taken as positive for the test (Eckford,1927).
 
Urease test
 
The young cultures were inoculated to the test tubes containing sterilized ureabroth and incubated. The development of pink color was taken as positive for the test.
 
Casein hydrolysis
 
Young cultures of Bacillus spp. were spotted on to the skimmed milk agar plates and incubated. The production of clear halo around the colony was taken as positive for the test (Seeley and Vandemark, 1970).
 
Gelatin liquefaction
 
The test cultures of Bacillus spp. were inoculated to the presterilized nutrient gelatin deep tubes and were incubated at 28±2°C for 24 h and kept in a refrigerator at 4°C for 30 minutes. The tubes with cultures that remained liquefied were taken as positive and those that solidified on refrigeration were taken as negative for the test (Blazevic and Ederer, 1975).
 
Indole production
 
The test cultures were inoculated to the pre-sterilized SIM agar tubes and incubated for 48 h. After incubation, each tube was added with 10 drops of Kovac’s reagent. The production of cherry red colour was taken as positive for test.
 
H₂S production
 
Pre-sterilized tubes containing SIM agar were stabbed with the test cultures all along the walls of the test tubes and incubated for 48 h at 28±2°C. After incubation, the development of black color along the line of the stab was positive for the test (Cappuccino and Sherman, 1992).
 
Endospore staining
 
The endospore staining was carried out as per the procedure given by Harley and Prescott (2002) by using Malachite green. Under a light microscope, green colour cells were endospores and pink coloured cells were vegetative cells.
 
Plant growth promoting traits of Bacillus species
 
HCN production
 
The rhizobacteria were streaked onto nutrient agar plates amended with Glycine (4.4 g/l), plates were inverted and 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 96 h. Change in colour of the filter paper from orange to brown was considered as production of HCN. A change of colour from yellow to light brown, brown or reddish-brown was recorded as weak (+), moderate (++) or strong (+++) reaction, respectively.
 
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.
 
Siderophore production
 
It was assayed by a plate method using the Chromoazurol sulphonate (CAS) agar method (Neilands and Schwayn, 1987).
 
Volatile compound production
 
In vitro volatile antimicrobial activity was assessed using paired plate method using NA medium for bacterium and PDA for pathogen along with control and kept for incubation. Later, colony diameter of the fungal pathogen was measured and compared with the control.

Isolation of pathogen
 
The results indicated that, the white mycelia growth of pathogen was observed within 3 to 4 days. Formation of sclerotial bodies was also after 8-10 days.
 
Isolation and maintenance of native isolates of Bacillus spp.
 
Nineteen isolates of Bacillus spp. were successfully isolated and designated as BS-1 to BS-19. Similarly, Bacillus spp. were isolated by using nutrient agar by serial dilution technique (Bharose et al., 2017).
 
Antagonistic potential of Bacillus spp. isolates against Sclerotiumrolfsii
 
The per cent inhibition of mycelial growth varied greatly among the nineteen isolates. The isolates BS-3 (58.51%), BS-2 (57.03%), BS-6 (56.29%), BS-10 (55.55%) and BS-7 (52.96%) showed more than 50% mycelial inhibition of pathogen (Table 1 and Fig 1). Rajkumar (2016) screened thirty B. subtilis isolates against S. rolfsii reported that the isolates showed different levels of inhibition of mycelial growth of S. rolfsii. Among them, BS16 inhibited maximum mycelial growth 64.04 per cent followed by BS 30 (62.20%) and minimum inhibition of mycelial growth was observed in case of BS17 (11.98%).
 

 

 
Molecular characterization of potential Bacillus spp. Isolates
 
Five efficient Bacillus spp. isolates, BS-2, BS-3, BS-6, BS-7 and BS-10 were amplified for 16S rDNA (Fig 2) and the species were identified as Bacillus tropicus (ON564730), Bacillus subtilis (ON564689), Bacillus pacificus (ON564610), Bacillus cereus (ON564773) and Bacillus tropicus (ON564907), respectively. The accession numbers were deposited in genebank. Phylogram of five Bacillus species was constructed to analyze the diversity among them (Fig 3). The results in the dendrogram showed two major clusters. Cluster-I comprised two sub groups. In sub-group I, B. pacificus shared 36 per cent similarity with reference organism B. cereus. Whereas, B.cereus shared 16 per cent similarity between B. pacificus and B. cereus. B. tropicus was out grouped from B. pacificus, B. cereus and two reference B. cereus strain. Sub-group II consists of B. tropicus which was out grouped from B. pacificus and B. tropicus reference strains. Cluster-II consists of B. subtilis shared 99 per cent similarity with B. subtilis strain (Fig 3). Hernandez et al., (2014) reported that B. subtilisand B. cereusare known to suppress growth of S. rolfsii.
 

 

 
Morphological characterization of potential isolates of Bacillus spp.
 
The results (Table 2 and Fig 4) indicated that all species were rod shaped and colony was rough in texture. The colour of the colony was white in B. pacificus and creamy inrest of the species. Rajkumar (2016) identified the cultures of B. subtilis based on characters such as shape, texture of colony, colony morphology and colour of colony. All the isolates were rod shaped, isolates BS-2, BS-3, BS-4, BS-5, BS-11, BS-12, BS-19 and BS-20 were cream and remaining isolates white in colour. Colony morphology was smooth in case of BS-3, BS-6, BS-9, BS-13, BS-17 and BS-18 and remaining isolates were rough. Biochemical characterization of all the isolates showed Gram positive and positive for endospore production, catalase, oxidase, gelatin hydrolysis and starch hydrolysis and negative for KOH test and indole test. Among the 30 different isolates of B. subtilis, the isolates BS-1, BS-5, BS-6, BS-7, BS-9, BS-13, BS-16, BS-20, BS-23, BS-24, BS-25, BS-27, BS-28 and BS-29 showed positive for HCN production, The isolates like, BS-5, BS8, BS-13, BS-20 and BS-20 were negative and all the isolates recorded positive for IAA production except BS-20, BS-23, BS-24, BS-25, BS-27 and BS-2. Wafula et al., (2014) reported the morphological characterization of B. subtilis like colour, form, shape and elevation of pure colonies. The colony morphology of the isolates ranged from flat and filamentous or branching. They were smooth or rough and the colour ranged from white to creamy.
 

 

 
Biochemical characterization of efficient isolates of Bacillus spp.
 
Gram’s staining
 
All the Bacillus species for gram staining indicated that, they were Gram positive showing blue coloured cells by taking primary stain (Fig 5).
 

 
KOH test
 
All five Bacillus species showed negative for KOH test without forming any mucoid string (Fig 5).
 
Catalase test
 
All five Bacillus species showed positive for the catalase test by forming gas bubbles (Fig 5).
 
Strach hydrolysis
 
The clear zone around the colony indicates positive for test for all five Bacillus species (Fig 5).
 
Urease test
 
All five Bacillus species developed pink colour indicating positive urease test (Fig 5).
 
Casein hydrolysis
 
The formation of halo zone indicated positive test for casein hydrolysis in all the five Bacillus species (Fig 5).
 
Gelatin liquefaction
 
In this study, all the five Bacillus species have shown positive for the test indicated by the tubes remained liquefied even after refrigeration (Fig 5).
 
Indole test
 
All five Bacillus species showed negative for the indole test wherein there was no formation of cherry red colour (Fig 5).
 
H₂S production
 
Formation of black colour along the line of stab indicated H₂S production by all five Bacillus species (Fig 5).
 
Endospore staining
 
All the tested Bacillus species produced endospore when stained with malachite green stain (Fig 5).
       
Rajkumar (2016) reported that all the tested isolates were Gram positive and positive for endospore production, catalase, oxidase, gelatin hydrolysis and starch hydrolysis and negative for KOH test and indole test. Among the 30 different isolates of B. subtilis, 25 isolates showed positive for HCN production and all the isolates recorded positive for IAA production. Venant et al., (2013) reported that isolates of B. subtilis were indole and methyl red negative and positive for citrate, catalase, urease, oxidase, starch hydrolysis, KOH test and gelatin hydrolysis.
 
Plant growth promoting traits efficient Bacillus species

HCN production
 
Bacillustropicus and B. subtilis showed higher level of HCN production and recorded as strong (+++). B. pacificus and B. cereus recorded moderate scoring (++) and B. tropicus weak (+) (Fig 6). Similar type of results was obtained with Ramya bharathi and Raguchander (2014), who reported that, B. subtilis was found to be positive for hydrogencyanide production.
 

 
IAA production
 
Five Bacillus species were showed positive for the production of IAA (Fig 6). Bharose et al., (2017) also observed the production of IAA by the isolate JND-KHGn-29-B of B. subtilis. Sivasakthi et al., (2013) tested 10 isolates of B. subtilis for the production of IAA, results revealed that, the isolate BS8 produced maximum amount of IAA i.e., 26 μg/ml. (Dinesh Singh et al., 2012) studied on nine isolates of B. subtilis which were isolated from rhizospheric soil of tomato plants. Among them, BS9 exhibited maximum indole acetic acid production (0.139 μg/ml) and ammonia production under in vitro conditions.  
 
Siderophore production
 
All five species of Bacillus produced siderophore where the  B. tropicus (BS-2) and B. pacificus produced higher amount of siderophore (4 mm orange colour zone) and B. subtilis and B. cereus recorded moderate siderophore production (2-4 mm) and B. tropicus (BS-10) produced less siderophore (2 mm) (Fig 6). Sivasakthi et al., (2013) tested10 isolates of B. subtilis for the production of siderophore, results revealed that, the isolate BS8 produced maximum amount of siderophore i.e., 9 μg/ml. The bacterial isolateJND-KHGn-29-A was found to have siderophore activity (Bharose et al., 2017).
 
Volatile compounds production
 
The results revealed that the species produced considerable amount of volatile metabolites which varied with the species. The B. subtilis showed maximum per cent inhibition (47.03%) followed by B. tropicus (BS-2) (44.07%) and B. pacificus (42.22%) (Fig 6). The results obtained in this study are in line with Ramyabharathi and Raguchander (2014) they reported that, B. subtilis EPCO16 strain was effectively inhibited the growth of F. oxysporum f. sp. lycopersici to an extent of 60.78 per cent invitro through the production of volatile compound. The volatile compounds produced bya strain of B. subtilis found to be antifungal to R. solani and Pythium ultimum (Fiddaman and Stephen, 1993).