Isolation and characterization of bacterial strains for biocontrol activity in chilli against Fusarium sps.

Introduction

Chillies are one of the most widely cultivated and consumed spice crops globally. They belong to the Capsicum genus and are a crucial part of various cuisines, particularly in Asian and Latin American cultures. Chillies originated in the Americas over 6,000 years ago and were introduced to Asia by Portuguese traders in the 16th century. They quickly became an integral part of Indian, Chinese, and Southeast Asian cuisine. Microorganisms play a vital role in biocontrol activity, helping to manage chilli losses caused by diseases and pests. Screening and biocontrol potential of rhizobacteria native to plains and hilly regions to induce systemic resistance and promote plant growth in chilli against bacterial wilt disease by (Kashyap et al., 2021) [4] in vitro antagonist activity of rhizobacteria against chilli wilt. Out of the 63 isolates of rhizobacteria isolated from rhizospheric soil of chilli plants, 38.09% rhizobacteria were found to have antagonistic activity against R. solanacearum. A marked inhibition in the growth of R. solanacearum culture was observed in the presence of rhizobacterial isolates. The maximum inhibition zone (22.4 mm diameter) of R. solanacearum was found by the isolate PDS1 isolated from the Middle Gangetic Plain region, followed by KA9 isolate (20.06 mm diameter The use of glucose as a carbon source was found in 29.42% of rhizobacteria. Isolation and characterization of native antagonistic rhizobacteria against Fusarium wilt of chilli to promote plant growth by (shiva et al., 2024) [6] F. oxysporum f. sp. capsici, a soil borne pathogen, was found in wilted chilli plant samples during a survey. The fungus showed colonies with varying colors and velvety surfaces, changing color upon spore generation. Its hyphae were filaments, septate, hyaline, and branched at acute or right angles. The isolate's pathogenicity was evaluated in vitro using seedlings, causing symptoms like collapse and wilting of petiole, rachis, and leaflets 15 days post-inoculation. The signs of plant wilting in infected protrays resembled those in the main field.

Materials and Methods

Collection and Isolation of microbial isolates

Soil, seed, aerial parts of chilli samples were taken from warangal district of Telangana. Total 43 isolates were obtained from chilli samples using Nutrient Agar (NA) medium. These obtained isolates were purified by pure culture technique for ongoing further activities

Siderophore Production

Production of siderophores estimated qualitatively on aqueous ferric chloride solution for siderophores detection. Orange color zone around the colonies observed and recorded and modified by Alexander and Zuberer (1991) [1] . Siderophore production confirmed by the qualitative CAS agar test. The log-phase bacterial culture spotted on nutrient agar plates amended with the CAS solution. The plates incubated at 28°C under dark conditions for 5 days. The appearance of orange halo zones confirms siderophore production.

Hydrogen cyanide (HCN) production

Qualitative assay (Bakker and schippers, 1987) [2] for pure isolates tested by inoculating on agar plates supplemented with 4.4g glycine L-1 using alkaline picric acid as a reagent, incubated for 4 days and change to brown colour of the filter paper indicates positive for HCN production.

In vitro antagonist activity of the bacterial isolates

For in vitro antagonist activity both bacterial isolates and Fusarium sps pathogen were cocultured on Potato dextrose agar (PDA) medium according to the method described by Sijam and Dikin (2005) [7] . The bacteria which inhibiting the mycelial growth of fungi pathogen taken into consideration for biocontrol activity. The percentage reduction in growth was calculated following the formula. Where, C-diameter of fungal growth in control plate, T-diameter of fungal growth in test plate Percentage of inhibition (%) =

Molecular characterization of potential bacterial isolates

Genomic DNA extraction done for potential and unique characters of bacterial isolates by genomic DNA isolation kit (ZR Fungal/Bacterial DNA MiniPrepTM). Genomic DNA subjected to PCR (polymerase chain reaction) for amplification of 16s rRNA gene with universal primers (Forward & Reverse) and sequencing done through outsourcing. Identification of bacterial isolates based on 16s rRNA gene sequence by BLASTn in NCBI database.

Pot culture experiment

The experiment conducted for the evaluation of potential strains against wilt of chilli under pot culture in the Dept. of Food Safety and Quality Assurance, College of Food Science & Technology, Rudrur with seven treatments in chilli involving T 1 (Absolute Control), T 2 (100% RDF (Negative Control)), T 3 (100% RDF + Seed treatment with consortia), T 4 (100%RDF + Soil application with consortium), T 5 (100% RDF + Seed treatment + Soil application with consortium), T 6 (100% RDF + Application of fungicide Mancozeb @ 2.5g/ lit), T 7 (100% RDF + Seed treatment with consortium + Fungicidal spray).

Peroxidase assay

Peroxidase activity was estimated quantitatively by the method described by Chen et al. (2000) [2] . 100  of the extract was taken into a spectrophotometer cuvette into which 7.5 1 of guaiacol of 50 mM concentration and 792 1 of 0.05 mM Tris HCl buffer of pH 6.0 was added. The reaction was started by adding 100 1 of 0.6 M Hydrogen peroxide. Optical density (OD) values were taken at 470 nm. A blank was prepared which comprises guaiacol, Tris HCl buffer and Hydrogen peroxide to set the spectrophotometer at 100% absorbance. One unit of the enzyme was defined as the change in absorbance (in units) min -1 g -1 fresh tissue.

Polyphenol oxidase assay

Chen et al. (2000) [2] have described the method for the estimation of PPO activity with slight modifications. The reaction mixture for PPO comprises 1.5 ml of 0.1 M citrate phosphate buffer of pH 6.0, 5 mg/ml of proline, 500 1 of 0.01 M of catechol and 500 1 of enzyme extract. Moreover, the reaction mixture was aerated before the addition of catechol, which serves as a substrate. Enzyme activity was analysed by taking the optical density value at 546 nm. One unit of the enzyme was defined as the change in absorbance (in units) min -1 g -1 fresh tissue.

2.9 Phenylalanine ammonia lyase assay PAL assay was determined quantitatively by the following method which was given by Ross and Sederoff (1992). The reaction mixture comprises 600 1 of L-phenylalanine in 500  of 50 mM Tris HCl of pH 8.8 and 100 1 of enzyme extract, which was incubated for 60 min at 40 C. The reaction was stopped by adding 1 ml of 2 N HCl and 1.5 ml of toluene, vortexed for 30 sec, centrifuged at 4,000 rpm for 5 min and the toluene fraction containing tans-cinnamic acid(lower face) was separated. The absorbance was measured at 290 nm, against the toluene blank.

Results and Discussion

Siderophore production

Production of siderophores estimated qualitatively on aqueous ferric chloride solution for siderophores detection. Orange color zone around the colonies observed and recorded (Fig. 1). Out of 43 isolates 26 isolates showed positive in which all epiphytic bacterial isolates showed positive. Among all the isolates MB 2 RC 4 (45mm) produced highest with a great diffference from other isolates.Whereas MB 1 RC 3 (7mm), MB 2 RC 1 (8mm), MB 2 RC 3 (5mm), MB 3 RC 2 (6mm), W 1 ENC 1 (8mm),W 2 ENC 4 (5mm), MB 1 ENC 3 (8mm), MB 3 ENC 3 (6mm), W 1 EPC 2 (5mm), W 2 EPC 3 (5mm),W 3 EPC 2 (3mm), MB 1 EPC 2 (8mm), MB 3 EPC 1 (6mm). Among all the isolates W 3 EPC 2 showed least siderophore production and rest all isolates were moderate as shown in (Table 1)

The study by Ghazy and El-Nahrawy (2021) [9] . found that B. subtilis MF497446 and P. koreensis MG209738 are the most effective PGPRs by qualitative and quantitative assays for siderophore production and individually or in combination inoculation condition, leading to induce the disease resistance of late wilt caused by C. maydis in maize plant and improvement of growth promotion and yield.

HCN production

Pure isolates tested qualitatively by inoculating on agar plates supplemented with 4.4g glycine L-1 using alkaline picric acid as a reagent, incubated for 4 days and change to orange brown colour of the filter paper indicated positive for HCN production (Fig. 2) All isolates showed positive for HCN production except MB 3 EPC 2. Based on amount of colour change intensity, classification of HCN producer isolates were done as strong, medium and slow producers. (Table 2) De Vrieze et al. (2020) [10] verified the involvement of this known particular biocontrol trait and quantified its relative contribution to the overall anti-oomycete activity of two cyanogenic Pseudomonas using in vitro and leaf disk experiments. Findings by Anand showed the indication of higher activity or altered phenotype observed in the cyanide deficient mutant of strain suggests that the presence of cyanogenic strains.

In vitro antagonist activity-Dual culture

With Fusarium sps W 1 RC 1, MB 2 RC 4, MB 1 ENC 1, MB 2 ENC 1, W 1 EPC 2, MB 1 EPC 1 showed positive for antagonistic activity in which all had very strong percent inhibition. Among them MB 1 ENC 1 (77%), MB 2 ENC 1 (79%) highest percentage than other isolates. (Fig. 3)

Peroxidase assay

This activity carrried by taking samples of root and shoot from all seven treatments. The results showed that peroxidase activity was in the increasing order of T 5 (100% RDF + Seed treatment + Soil application with consortium)> T 7 (100% RDF + Seed treatment with consortium + Fungicidal spray) >T 3 (100% RDF + Seed treatment with consortia) >T 4 (100%RDF + Soil application with consortium)> T 6 (100% RDF + Application of fungicide Mancozeb @ 2.5g/ lit) >T 1 (Absolute Control) > T 2 (100% RDF (Negative Control)) for Fusarium sps in both root and shoot extract (Fig 5)