Biochemical and enzymatic trait-based characterization of forest-derived Bacillus isolates from Uttara Kannada

Introduction

In the majority of growing nations across the world, agriculture sector is essential to the advancement of life in all its forms. Over time, the excessive and careless use of agrochemicals has unintentionally harmed the health of the soil (Bunemann et al., 2018) [6] . The biogeochemical cycles as well as the soil microbial communities are adversely affected by toxic substances (Rousk and Bengtson, 2014) [20] . Finding safe and efficient methods that increase agronomic productivity without endangering the soil's natural microflora is therefore essential (Bargaz et al., 2018) [4] . As carbon storage systems, forests often represent highly productive or regenerable environments. In these forests, carbon that is deposited in the rhizosphere and biomass leftovers decompose to generate organic matter in the soil. Different forest-dwelling fungi, bacteria, and archaea have high soil regeneration traits. According to Parray and Shameem (2020) [15] , maintaining healthy soil requires consideration of a number of factors, including microbial species diversity, water holding capacity, oxygen holding capacity, erosion reduction and carbon content. Chemical fertilisers promote plant development but have a negative effect on all of the aforementioned elements, reducing the fertility of the soil. In order to reduce or replace chemical fertilisers and increase soil fertility, plant growth-promoting rhizobacteria (PGPR) are applied in agriculture (Ramakrishna et al., 2019) [19] . Plants experience biotic and abiotic stress at various stages of their lifecycle. According to studies, using particular bacteria as fertilizers rather than chemical ones will benefit both plants and microbes (Ramakrishna et al., 2020) [18] . By producing various volatile organic chemicals, the plant growth-promoting bacterium Bacillus subtilis control its physicochemical processes. In comparison to other plant growth-promoting bacteria (PGPB) that do not create endospores, Bacillus spp. that produce endospores tend to survive longer in soil and stimulate plant growth more effectively (Yadav et al., 2020) [23] . Additionally, it was claimed that Bacillus spp. increases the photosynthetic ability of wheat plants because they create siderophores, which chelate iron and supply it to the photosynthetic machinery. In addition to protecting indole-3acetic acid (IAA) from oxidative damage, Bacillus spp. also ameliorate heavy metal induced oxidative stress and alleviate iron deficit in plants (Ferreira et al., 2019) [9] . It has been shown in the past that mycorrhizal fungi and siderophore-producing Bacillus spp. together improve the uptake of nutrients in wheat grain and root tissues (Yadav et al., 2021) [24]. These bacteria in the rhizosphere create siderophore, which by acidity solubilizes the inaccessible forms of several nutrients (Rajkumar et al., 2010) [17] . Bacillus spp. function as biostimulants by producing auxin, cytokinin, and expansin, which are phytohormones that support plant growth and development (Zubair et al., 2019) [25] . Bacillus spp. that contain ACC deaminase genes have the capacity to reduce ethylene levels in plants, which improves growth and drought tolerance in the treated plants (Gowtham et al., 2020) [10] . According to Radhakrishnan et al. (2017) [16] , Bacillus spp. have both direct and indirect effects on the disease management. The indirect method entails the development of biofilm, stimulation of plant growth, competition for nutrients and space, and ISR. B. subtilis produces a variety of lipopeptides, cell lytic enzymes, antioxidants and hormones that have an impact on a wide range of bacteria and fungi in the direct approach of disease prevention. The main lipopeptides produced are surfactin, iturin, and fengycin and they play a crucial role in controlling a wide range of phytopathogens. Therefore, the present investigation involved the isolation and characterization of Bacillus spp. from forests of Uttara Kannada with high regeneration.

Materials and Methods

The soil samples from rhizosphere were collected from different forest types like evergreen, deciduous and degraded patches of natural forests of Haliyal, Dandeli, Gutti, Joida, Mirjan-Ramnagar road regions of Karnataka. Later, the top litter layer of the soil (two cm) was removed and then the soil samples were collected, packed in fresh polythene covers, labeled and then stored at 4 o C to maintain the viability of organisms.The Bacillus spp. were isolated from forest soils by using the method given by Al-Humam (2016) [1] . Four grams of soil sample was suspended in 96 ml sterile distilled water and shaken vigorously for 2 min. The samples were heated at 60 ° C for 60 min in a water bath. Then, the suspension was kept on a bench surface at room temperature for two hours for soil particles to settle. The serial dilution plate count technique was followed. The plates were incubated at 28-30 ° C for 24-40 h for colony development. Colonies were purified by four-way streaking and pure cultures were transferred to nutrient agar slants and maintained for further experiments. Morphological characteristics of 30 Bacillus spp. isolates were done based on cell shape, colour and Gram reaction (Graham and Parker, 1964) [11] . The biochemical characteristics of the Bacillus spp. isolates were done according to the standard protocols. Starch hydrolysis test, indole production (MacWilliams, 2009) [14] , catalase test, oxidase test (Cappuccino & Sherman, 1996) [7] , gelatin liquefaction test (Bradbury, 1970) [5] , methyl red test, Voges prausker's test, citrate utilization test (Seeley & Vandemark, 1981) [21 ] were done. Screening for cellulase (Crabbe et al., 1994) [8] , lipase (Jaeger and Kouker, 1987) [12] , amylase (Amoozegar et al., 2003) [2] , urease (Aneja, 2006) [3] and solubilization of mineral phosphate (Aneja, 2006) [3] were done according to standard protocols.

Results

The soil samples were collected from different forests types like evergreen, deciduous and degraded patches of natural forest of Uttar Kannada in polyethylene cover. The details of the sources were recorded using the GPS systems and presented in Table 1. A total of 30 Bacillus spp. were isolated, purified and stored for further studies. These 30 isolates were designated by the following isolate codes which are depicted in Table 1. The Bacillus isolates were morphologically characterized based on the colony shape, cell shape, colour and Gram reaction. The details of morphological studies are presented in Table 2. All the isolates on nutrient agar medium plates were observed and colony shape was found to be circular, translucent, white or cream in colour and Gram positive. Out of 30 Bacillus isolates, 19 isolates were cream in colour and 11 isolates were white in colour. The cell shape of all the isolates was found to be rod. Later, these 30 isolates were biochemically characterized by conducting biochemical tests. The results indicated that all 30 isolates showed positive results for the catalase test, starch hydrolysis and gelatin hydrolysis, whereas only 29 isolates showed positive results for the VP test, gelatin hydrolysis; 28 isolates were positive for citrate utilization test, 26 were positive to oxidase test, 20 were positive to indole test and all 30 isolates showed negative result for MR test (Table 3). These 30 isolates were further characterized for the production of extracellular enzymes like cellulase, lipase, amylase, urease and mineral phosphate solubilization activity. All 30 isolates of Bacillus showed positive results for the production of cellulase, lipase and amylase. Whereas only three isolates (DBI-15, JBI-1 and JBI-2) showed positive results for urease production and two isolates (JBI-4 and MBI-18) showed positive results for mineral phosphate solubilisation (Table 4).

Discussion

The results indicated that these Bacillus isolates not only possessed metabolic potentials of these isolates but also exhibited an ability to mineralize plant nutrients. Similar results have been documented by Kumar et al. (2021) [13] on the identification and analysis of 120 bacterial isolates for their morphology, biochemistry and capacity to stimulate plant growth. Bacillus sp. was isolated from various soil samples and characterised by Shambhavi et al. (2020) [22] using morphological and biochemical assays such as the Gram staining, methyl red test, Vogesproskeur test, starch hydrolysis, gelatin hydrolysis and indole production. They reported that the isolate was Gram positive and produced white, slimy colonies on nutrient agar media.

Conclusion

The data from present study revealed that Bacillus isolates DBI-13 and MBI-18 have the ability to improve soil health as well as promote plant growth by producing various enzymes. These isolates should be further screened for their regenerative traits by conducting pot and field experiments.

References

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