Antifungal Trait and Plant Growth Promotion Potential of Bacillus Spp. from Rhizosphere Soils of Black Aromatic Rice, ‘ Chakhao

Manipuri black aromatic rice, ‘ Chakhao ’, is a nutrient rich unique local rice cultivar of Manipur, India. Rhizosphere bacteria may have potential as antifungal agent and seedling growth promoter. Hence, the current investigation was aimed at screening, for the above traits, using bacteria, Bacillus spp., isolated from the soil of Rhizosphere of six Chakhao rice cultivars. Altogether, 323 bacterial isolates were obtained from the rhizospheric soils of 6 different cultivars of Chakhao , namely Chakhao Amubi (CA), Chakhao Poireiton (CP), Chakhao Sempak (CS), Chakhao Angoubi (CAng), Chakhao Angangbi (CR) and Chakhao Wairi (CW). All the rhizobacterial isolates were screened for antifungal activity against 5 rice fungal pathogens viz. Rhizoctonia solani (RS), Fusarium oxysporum (FO), Curvularia oryzae (CO), Pyricularia oryzae (PO) and Aspergillus niger (AN). The isolates were also subjected to PGP (plant growth promotion) assays such as ammonia, indole acetic acid (IAA), siderophore and 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase production, and phosphate solubilization. Of 323 isolates obtained, 64 were found to exhibit antifungal activity while 69 showed PGP traits. Twenty-five (25) isolates were found to be positive for both antifungal and PGP traits. Of 25 bioactive isolates, 4 (CR12, CW11, CA2 and CP2) potent isolates were shortlisted for further studies. The shortlisted potent isolates were subjected to quantitative estimation of PGP activities like phosphate solubilization, IAA and siderophore production. The strains could produce significant amount of IAA and siderophore and solubilize phosphate. Molecular characterization by 16S rDNA sequence analyses revealed the identity of the isolates: CR12:


Introduction
The scented rice of Manipur, Chakhao, meaning delicious rice in Manipuri language, possess high socio-cultural values and is in close relationship with spiritual cultural practices of Meiteis.Chakhao has a unique flavour and aroma.It is served as a special item in cultural and religious ceremonies such as Usob (death ceremonies), Kang-pali (religious festival), Chak-umba (first rice-eating ceremony). 1Chakhao, an indigenous rice variety of Manipur, is nutrient-rich and contains essential amino acids, minerals, and carotenoids.3] Chakhao is grown at certain parts of Imphal valley in Manipur.Due to its poor yield, Chakhao is grown in very limited acreage by farmers in Manipur for ceremonial and cultural purposes.There is a pressing need to increase its output because it is becoming more and more popular due to its nutraceutical characteristics and export demand. 4 modern agriculture, synthetic agrochemicals are used for enhancing crop production.The dependence on agrochemicals for improving crop production pose serious risks on ecological and human health. 5Rhizobacteria are associated with the soils near the roots and intimately interact with the exudates of the roots. 6Plant growth promoting rhizobacteria (PGPR) are among the beneficial soil microorganisms found in the rhizospheric region of the plant. 7Intensive research on plant growth promoting bacteria (PGPB) is underway worldwide for developing biofertilizers and biocontrol agents (BCAs) as better alternatives to agrochemicals, as the latter harm the environment and human health besides possess the burden of high costs to poor farmers. 8Biological control is slow but can be long lasting, inexpensive, and harmless to living organisms and the ecosystem; it neither eliminates the pathogen nor the disease, but brings them into natural balance. 9As most PGPBs show inconsistent performance in the field conditions, there is urgent need for survey of indigenous strains suited to local conditions.The use of bacteria having biocontrol and PGP activities is urgently warranted to increase Chakhao growth. 10rtain bacterial taxa e.g., actinomycetes and Bacillus spp have been shown to play a significant function in the plant rhizosphere by secreting a variety of antimicrobial substances that inhibit the growth of common root diseases.][13][14][15][16] Rhizospheric isolates associated with Chakhao varieties may have potential to produce unique metabolites, which can be exploited for application in agricultural, pharmaceutical and other industries.The present study aims at investigating the antifungal and biofertilizing potential of the rhizospheric isolates from 6 different Chakhao cultivars found in Manipur. 17
1 g of the soil sample was added to 99 ml of sterile distilled water (SDW).The sample was thoroughly vortexed (150 rpm, 30°C, 10 min).Serial dilutions of the soil suspensions were then performed (10 -2 to 10 -6 ).The aliquots of various dilutions from 10 -2 to 10 -6 were spread plated on agar plates.The isolation plates were incubated for 3-4 days at 30°C.Morphologically distinct bacterial colonies were subcultured till pure cultures were obtained. 18tifungal Assay Antifungal assay of the rhizospheric bacteria were carried out by dual culture method.[19][20] The inhibition zones are due to diffusible compounds by the bacterial isolate in the antifungal assay.After the full growth of fungal mycelia on the control plates, the inhibition in the remaining plates was measured.The percentage of mycelial growth inhibitions was calculated using the formula: Where, R represents the radial growth of the test pathogen in the control plates (measured in mm), and r is the radial growth of the test pathogen in the test plates.

Screening of Plant Growth Promoting (PGP) Traits
Indole Acetic Acid (IAA) Production Test IAA production was determined by inoculating bacterial isolates in NB medium containing 2 mg/ ml of L-tryptophan, (150 rpm, 30ºC, 5 d). 21The fully grown culture broth was centrifuged (10,000 rpm, 10 min) and 1 ml of the supernatant was mixed with 2 ml of Salkowski's reagent.A pink colour indicates a positive test for IAA production.
For the quantitative assay, the bacterial isolates were inoculated in NB containing 2 mg/mL of Tryptophan under shaking conditions (150 rpm, 30ºC).5 mL aliquot was withdrawn periodically from each culture flask at 24 hours intervals and centrifuged at 10,000 rpm for 10 min. 211 mL of the supernatant was mixed with 2 mL of Salkowski's reagent and incubated at 30ºC for 20 min.The absorbance was measured at 530 nm and the amount of IAA produced was calculated by comparing it with the standard IAA curve.

Phosphate Solubilization Test
Spot inoculation on NBRIP-BP (National Botanical Research Institute's Phosphate Growth Medium) was carried out to determine phosphate solubilization by bacterial isolates.A halo zone around the bacterial colony after 4 days of incubation at 30 o C indicated a positive test. 22antitative estimation of phosphate solubilization was done by inoculating in 100 mL of NBRIP-BP medium (pH 7).It was then incubated in a shaker incubator (150 rpm, 30°C).5 mL aliquot withdrawn periodically at 24 hours intervals was centrifuged (10,000 rpm, 10 min) and the supernatant was analysed for pH and Phosphate concentration. 23H 2 PO 4 was used as the standard. 24derophore Production Test SCNA (without iron) amended with CAS-substrate (Chrome Azurol S) were inoculated with 6 mm agar plugs of bacterial isolate and incubated at 30°C for 7 days.The formation of an orange colour halo surrounding the colony was considered a positive result for siderophore production. 257] 5 mL aliquots were withdrawn periodically at 24 hours intervals and centrifuged (10,000 rpm, 10 min).An equal volume of CAS reagent was added to the supernatant.Absorbance at 630 nm was noted.A reference made of 1 mL uninoculated broth and 1 mL CAS reagent was used.The amount of siderophore produced (percentage siderophore units) was calculated by using the formula: Percentage siderophore units = (Ar − As)/Ar× 100 Where, Ar represents the absorbance of reference and As represents the absorbance of the sample at 630 nm.

Ammonia Production Test
The bacterial isolates were inoculated in 10ml of peptone water and incubated in a shaker incubator (150 rpm, 30°C) for 4 days.To each test tube, 0.5 ml of Nessler reagent was then added.A colour change of brown to yellow indicated ammonia production by the bacterial isolates. 28

1-Aminocyclopropane-1-Carboxylic Acid (ACC) Deaminase Production Test
] Isolates were inoculated on the media and then incubated at 30°C for 4 days.Bacterial growth in the plates indicated a positive test for ACC deaminase production.

In Vitro Seed Germination (Vigor Index)
In Vitro rice seed germination by the isolates were carried out according to Tamreihao et al. (2016). 31he bacterial isolates were inoculated in NB and incubated for 3 days to get fully grown cultures.It was then centrifuged (10,000 rpm, 10 min).The pellets were collected and washed thrice with SDW (Sterile Distilled Water) and culture suspensions were prepared using SDW.Black rice (Variety: Chakhao Amubi) seeds were surface sterilized with 70% ethanol for 5 min, followed by 0.2% sodium hypochlorite for 5 min, and rinsed four times with SDW.Surface sterilized seeds were soaked in the cell suspensions and kept under shaking conditions (150 rpm, 30°C) for 2 hours.The seeds were then transferred to sterile plates containing wetted filter papers (10 seeds per plate).Untreated sterile seeds soaked in SDW were used as a control.The plates were then incubated at 28°C for 5 days.The number of germinated seeds, roots and shoot lengths were measured and vigor indices were calculated using the following formula 32 ;

Vigor index= Percentage germination × Seedling length
Where,

Statistical Analysis
All the data were subjected to one-way analysis of variance (ANOVA) followed by independent t-test (p≤0.05) using the SPSS software.

Strain Identification
Genomic DNA extraction and PCR amplification of 16S rDNA sequences were performed. 33The PCR products obtained were sent to AgriGenome Labs Pvt. Ltd. (Kerala, India) for sequencing.The bacterial sequences were subjected to BLAST alignment analysis using the NCBI GenBank database to obtain the accession number. 34

Isolation
Altogether, 323 morphologically distinct isolates were obtained from rhizospheric soils of six (6)  different cultivars of Chakhao.These isolates were preserved in 30% glycerol stock at − 20°C for further studies.

Antifungal Assay
All the 323 isolates were screened for antifungal assays against the test fungal pathogens, of which 64 showed antifungal activities against one or more fungal pathogens.Four (4) isolates (CR12, CW11, CA2 and CP2) exhibited potent antifungal activity (Figs.1-2 and Table 1).

Plant growth promoting (PGP) Activities
All the isolates were then subjected to screening for PGP traits such as IAA, siderophore, ammonia and ACC deaminase production, and phosphate solubilization.69 isolates showed positive results for one or more PGP traits (Table 2).

Shortlisting of Promising Isolates
Based on the screening of antifungal and PGP activities, 4 most potent isolates (CR12, CW11, CA2 and CP2) were shortlisted for molecular characterization and seedling vigor studies.

Quantitative Estimation of PGP Activities
The shortlisted potent strains were subjected to quantitative estimation of PGP activities like phosphate solubilization, IAA and siderophore production.The strain CR12 produced the highest amount of IAA (73 μg/mL) followed by CW11 (71.4 μg/mL), CP2 (70.5 μg/mL) and CA2 (62.91 μg/ mL) respectively after 10 d of incubation.The strain CA2 could solubilize the highest amount of inorganic P ( up to 200 μg/mL) after 3 d of incubation with concomitant decrease in medium pH (from 5.5 to 2.69) followed by CR12, CW11 and CP2 ( up to 156.006, 113.5 and 105.7 μg/mL respectively).CA2 showed maximum siderophore production (74.37% siderophore units in SCNB) followed by CR12, CP2 and CW11 (73.15% in NB, 67.80% in SCNB and 65.50% in NB respectively) after 6 d of incubation.

Discussion
According to a survey of Chakhao rice cultivation in some selected villages of Manipur by Borah  et al. (2018), it was found that the yield of Chakhao is generally lower (~1.3-1.8 tons/hectare) as compared to hybrid and traditional rice varieties (~2.0-5.5 tons/ hectare). 35Farmers generally do not apply chemical fertilizers and farmyard manure in the cultivation of Chakhao rice as application of excess inorganic and organic fertilizer increases lodging and sterility. 1o, there is an urgent need to search for organic fertilizers that can enhance Chakhao rice growth.In our study, plant growth promoting rhizobacteria (PGPR) were isolated from the rhizospheric soils of 6 Chakhao rice cultivars cultivated at different locations of Manipur, India.Even though there are many reports on the nutritional value of Chakhao rice and its importance to the people of Manipur, the application of PGPRs in Chakhao cultivation is still an underexplored area.
In the present study, 4 multi-trait PGP rhizospheric strains viz.CR12 (Bacillus subtilis strain CR12), CW11 (Bacillus paralicheniformis strain CW11), CA2 (Bacillus sp.CCMB1014 strain CA2) and CP2 (Bacillus licheniformis strain CP2) were chosen as potent biocontrol and biofertilizing agents as they can inhibit all the 5 fungal pathogens tested and were positive for all the PGP traits.The shortlisted potent strains exhibited antifungal activities against the 5 test fungal pathogens and showed significant mycelial growth inhibition against the fungal pathogens.Bacillus amyloquefaciens strain Bk7, a PGPR isolated from rice rhizosphere could inhibit the mycelial growth of Magnaporthe oryzae, Rhizoctonia solani, Botrytis cinerea and Fusarium graminearum. 36Bacillus spp.strains B-1, B-2, B-3 and B-4 isolated from rice rhizospheric soils of Odisha, India showed strong antifungal activity (upto 90%) against the phytopathogens like Rhizoctonia solani, Sclerotium rolfsii and Sclerotium oryzae invitro. 37A recent report showed a rice rhizospheric antifungal Bacillus sp.KM5 synthesized prominent antibiotics such as Bacillomycin F, Bacillomycin D, Bacillopeptin A, etc. 38 Significant inhibition of the five (5) fungal pathogens by the potent shortlisted strains can also be noted.Therefore they may hold potential to be developed as antifungal agents for rice cultivation.
Bacillus spp.strains K3 and N4 isolated from rice rhizospheric soil samples from different villages of Bardoli, Gujarat, India were reported to exhibit PGP activities like phosphate solubilization, IAA and siderophore production39.Phosphate solubilizing rhizobacteria (PSRB) such as Pseudomonas aeruginosa, Bacillus subtilis strain 1, Bacillus subtilis strain 2 and Bacillus subtilis strain 3, isolated from rice rhizosphere have been reported as promising agents for rice cultivation in soils deficient in phosphorus. 40A rhizobacterial isolate UPMB19, belonging to the genera Lysinibacillus obtained from rice rhizosphere has been reported as a PGPR showing IAA and siderophore production and phosphate solubilization which can promote rice seed growth significantly. 41ACC deaminase producing Bacillus sp.strain SB1-ACC3 isolated from the coastal rice field soils of West Bengal, India promoted rice seed germination. 42Streptomyces corchorusii strain UCR3-16, obtained from rice rhizospheric soils showed antifungal activities, found positive for plant growth promoting traits such as IAA, ammonia, siderophore, ACC deaminase production and phosphate solubilization and the strain showed significant increase in growth and grain yield of rice plants. 277] Strain CA2 could produce siderophore in significant amount (74.37%) in SCNB.A common bean rhizospheric Bacillus sp.BPR7 could produce siderophore upto 38 µg/mL in 72 hrs. 43 Chakhao rice seedling germination by CR12, CW11, CA2 and CP2 were performed and exhibited higher vigor indices over the control.

Conclusion
The best approach for sustainable Chakhao rice agriculture will be the use of microbes especially rhizospheric bacteria associated with the host plants.
In the present study, 4 most promising rhizospheric bacteria from 6 different cultivars of Chakhao with PGP potential were found.All 4 shortlisted strains showed good inhibition against major rice fungal pathogens and so they can be used as promising antifungal agents.Moreover, these isolates also showed significant PGP activities.Then shortlisted strains showed notable higher seedling vigor indices over the control under In Vitro conditions.Further studies are underway to investigate and confirm their growth promotion potency and biocontrol efficacy under pot trial and limited field conditions.

Fig. 1 :
Fig. 1: Antifungal activities of the 4 best strains against the fungal pathogens (a-y)

Table 3 : Vigor indices of the shortlisted best strains against control
*Values with different alphabet within a column are significant at P≤0.05 51gor indices of CR12 (668.00),CW11(478.91),CA2(618.66)andCP2 (510.92) were found to be higher than vigor index of control (164.48).The PGPR strains UPMB10 (Bacillus sphaericus), Rhizobium strains (SB16, UPMR1006 and UPMR1102) reported byMia  et al. (2012)showed significant increase in seedling vigor and overall root growth of lowland rice variety MR21950.Bacillus subtilis strain MBI600, a PGPR obtained from Department of Entomology and Plant Pathology, Auburn University, AL, USA enhanced seedling vigor index of rice (13192) relative to the control (5028).51