Isolation of Antagonistic bacteria and Alternaria solani causing Early Blight in Tomato
The sampling was conductedduring the months of September to November 2019 in 20 locations of Tamil Nadu known for growing tomato crop in large acreages. Tomato growing fields were selected for sampling soil for the isolation antagonistic bacteria against early blight pathogen, Alternaria solani. Healthy tomato plants were uprooted carefully without disturbing the roots. Soil adhering to the roots was shaken and removed into poly Ziplock bags and immediately kept on ice until further processing.
Isolation of putative antagonistic Bacillus isolates
Soil samples collected from various tomato fields were removed from the cold storage and thawed to room temperature. A soil suspension was prepared using 10g of each sample and 90 ml of sterile water. The solution was agitated using a rotary shaker at 180 rev min− 1 for 30 minutes. The suspensions thus prepared using all the soil samples were heated to 60°C for one hour and cooled immediately by placing in an ice box giving a heat shock reaction for the formation of endospores. The suspensions were serially diluted up to 104 and spread plated on nutrient agar medium, followed by incubation at 30°C for 2 days in an incubator. Single colonies isolated were maintained as purified cultures on nutrient agar slants [24]. Purified putative antagonistic Bacillus isolates were stored at − 80°C in 60% glycerol stocks.
Screening of putative antagonistic Bacillus isolates against various plant pathogens
Putative antagonistic Bacillus isolates were subjected to screening through dual culture technique[25] against major pathogens of crops namely Fusarium oxysporum f.sp.lycopersici, Alternaria solani, Rhizoctonia solani, Macrophomina phaseolina and Pyricularia oryzae. All the phytopathogens were supplied by the Microbial Type Culture Collection culture (MTCC) bank, Chandigarh, India. Briefly, 5mm mycelial plug was removed from an actively growing plate of each phytopathogen and placed at one edge of the plate. Putative Bacillus isolates were streaked on other side of the mycelial plug near the edges of the plate followed by incubation at 28°C in an incubator for 7–10 days. Bacillus isolates which showed antagonistic activity through zone of inhibitions were selected. The percentage growth inhibition was calculated as per the below formula adapted from [26].
Inhibition (%) = [(Control-Treatment)/Control] x 100
Where Control is the colony diameter in Control plates and Treatment is the colony diameter in treatment plates. The experiment was performed in triplicates.
Characterization And Identification Of Putative Antagonistic Bacillus Isolates
The putative antagonistic Bacillus isolate (NKMV-3) that was shortlisted through dual plate screening was characterized according to Bergey’s manual of systematic Bacteriology [27] and using Himedia’sHiBacillus™ identification Kit. In order to identify the putative antagonistic Bacillus isolate, a loop full culture of NKMV-3 growing on Nutrient Agar was used for DNA extraction. Quick DNA- Fungal/Bacterial Miniprep Kit ofZymo Research was used for extracting DNA from the putative antagonistic Bacillus isolate (NKMV-3). 16S rRNA sequence of the extracted DNA was amplified throughPolymerase Chain Reaction (PCR) with two bacterial universal primers namely 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′)[28]. A 14µLPCR reaction mixture consisting of 8µL Taq Master mix, 2 µL of each forward and reverse primers, 2 µL of DNA template and 2 µL of molecular grade water was used for amplifying the DNA. PCR was performed in an Eppendorf Mastercycler X50s. The PCR conditions were adapted from Zhu et al., 2020 with slight modifications as described:1 minute of Initial denaturation at 95℃, followed by 35 cycles of denaturation at 95℃ for 30 seconds, 1 minute of annealing at 52℃followed by extension at 72℃ for 2 minutes and 30 seconds. A final extension of 72℃ for 10 minutes completed the PCR reaction. The PCR products were purified and sequenced by external sequencing facility.The sequencing results were compared with known bacterial NCBI Genbank sequences using BLAST and the identity was confirmed. The identified bacterial sequence was submitted with NCBI and an accession number was obtained. Mega 11 software was used for constructing a phylogenetic tree using the neighbor-joining method with 100 bootstrap replicates[30].
Effect of crude lipopeptides against Alternaria solani
The effect of the crude lipopeptides obtained from NKMV-3 was tested through a modified poison food technique[31]. The dried crude lipopeptide powder was used for preparing various concentrations starting from 1–5% in potato dextrose agar medium on W/V basis. Media containing various concentrations of the crude lipopeptides was autoclaved and dispensed in petri plates. After solidification, a 5mm disc from an actively growing A.solani plate was extracted using a sterile cork borer and placed at the centre of the plate. The PDA plates containing no crude lipopeptides served as control for the trial. The plates were incubated at 28°C for 7 days in an incubator. After 7 days, the plates were observed for the growth of A.solani. The inhibition rate of mycelial growth was calculated as follows.
Inhibition rate (%) = [Diameter of colony in Control (mm) – Diameter of colony in treatment (mm)/Diameter of colony in Control (mm)] x 100
The experiment was conducted in triplicates.
Molecular Detection Of Surfactin, Fengycin And Iturin Genes
A loop full of culture from an actively growing slant of NKMV-3 was used for DNA extraction. DNA was extracted as mentioned in the previous section of this article. Each PCR reaction was performed in an Eppendorf Mastercycler X50s thermal cycler using a 14 µL reaction mixture containing Taq Master mix (8µL), forward and reverse primers (2µL each), Molecular grade water (2µL). The primers were chosen from already available literature. The details of primers and expected amplicon sizes are provided in Table 1.The PCR conditions were adapted from [32, 33] with slight modifications. Briefly, SfrA gene was amplified using a 35-cycle reaction consisting of 4 minutes of initial denaturation at 95°C, followed by denaturation for 1 minute at 94°C. Annealing was performed at 52°C for 30 sec, followed by an extension at 70°C for 1 minute. A final extension was performed at 70°C for 5 minutes. fen B, fen D and Itu C genes were amplified using a 40-cycle reaction consisting of 3 minutes of initial denaturation at 94°C, followed by denaturation for 1 minute at 94°C. Annealing was performed at 59°C (fen B), 50°C (fen D &Itu C) for 1 minute, followed by an extension at 70°C for 1 minute. A final extension was performed at 72°C for 10 minutes. The PCR amplicons were analysed through gel electrophoresis on a 1% agarose run at constant voltage of 100V for 40 minutes followed visualization using a gel documentation system (make: Vilber Quantum).
Extraction Of Extracellular Metabolites Produced By Nkmv-3
NKMV-3 was grown in Nutrient brothfor 72 hoursat 37°Cin a rotatory shaker with constant shaking of130 rotations min− 1. The cells were harvested after 3 days by centrifugation at 6,000 xgfor 15 minutes followed by the reduction of pH of the cell free extract to 2.0 by the addition of 3N HCl and left for overnight precipitation at 4°C. The so precipitated crude lipopeptides was separated by centrifugation at 8,000 xg for 30 minutes at 4°C. The pellet was dissolved in methanol and extracted thrice and evaporated under vacuum using a rotatory evaporator at 50°C and 65 rpm [34, 35]. The resulting viscous liquid was left for drying at 50°C for 48hours in a hot air oven. The dried crude lipopeptide extract was scrapped and dissolved in Tris HCl pH 7.5 and stored until further use.
Table 1
PCR Details for amplification of AMP genes
S.No | Gene | Primer | Primer sequence | Expected base pairs (bp) |
1 | SfrA | SRFA-F1/SRFA-R1 | 5′-AGAGCACATTGAGCGTTACAAA-3’ 5′-CAGCATCTCGTTCAACTTTCAC-3’ | 670 |
2 | fen B | FEN B-F1/ FEN B-R1 | 5'-CCTGGAGAAAGAATATACCGTACCY-3' 5'-GCTGGTTCAGTTKGATCACAT-3' | 670 |
3 | fen D | FEN D-F1/ FEN D-R1 | 5'-GGCCCGTTCTCTAAATCCAT-3' 5'-GTCATGCTGACGAGAGCAAA-3' | 670 |
4 | Itu C | ITU C - F1/ ITU C - R1 | 5'-CCCCCTCGGTCAAGTGAATA-3' 5'-TTGGTTAAGCCCTGATGCTC-3' | 594 |
Identification and quantification of Iturin, Surfactin and Fengycin in crude Lipopeptides using Liquid Chromatography
All solvents used were of HPLC grade (Merck) and standards of Iturin, Surfactin and Fengycin were purchased from Sigma, USA. Crude lipopeptides were quantified by Reverse-Phase High performance liquid chromatography using a chromatograph (Waters, USA) equipped with a quartinary pump and diode array detector. Analytical scale Purospher® RP-C18 (250 × 4.6 mm, 5 µm particle size) column was used. 1mg/ml of crude lipopeptide was prepared and filtered and filled into HPLC vials for injection. A volume of 2µl of sample was injected into the column. The mobile phase and chromatographic conditions were adapted from[36]. This method provided a single protocol for the detection and quantification of lipopeptides in a combined method rather than the conventional method of separate chromatographic runs for the identification and quantification of individual lipopeptides. The mobile phase consisted ofMilli-Q Water (Solvent A) and 0.1% HPLC grade trifluoroacetic acid dissolved in Acetonitrile(Solvent B). The elution of lipopeptide homologues was monitored at 210 nm. The conditions of the chromatography are provided in Table 2.
Table 2
Details for HPLC gradient conditions for the isolation and quantification of lipopeptides
Time (min) | Solvent A (%) | Solvent B (%) | Flow rate (mL min 1) |
0–4 | 60 | 40 | 2 |
4–11 | 55 | 45 | 2 |
11–17 | 40 | 60 | 0.8 |
17–22 | 30 | 70 | 0.4 |
22–30 | 15 | 85 | 1.5 |
30–33 | 5 | 95 | 1.0 |
33–35 | 60 | 40 | 2 |
Analysis of crude Lipopeptides through Fourier Transformation Infra-Red (FTIR) spectroscopy
The crude methanolic lipopeptide extract was subjected to a FTIR analysis (make:Perkin Elmer) to elucidate the structural groups of the crude lipopeptides.100 mg of KBr and an mg of crude lipopeptide extractof NKMV-3were ground using a pestle and pressed with load for 30 s to obtain translucent pellets.These pellets were subjected to FTIR between a frequency range from 4000 to 400 cm − 1 [37].
Detached leaf bioassays of crude lipopeptides against A.solani in Tomato
Detached leaf bioassays were conducted with slight modifications as described by [38]. Briefly, Tomato (Variety: PKM − 1) leaves from 45 days old potted plants were obtained. The leaves were surface sterilized using 1% sodium hypochlorite solution, followed by two washes with sterile water. The leaves were left for air drying inside the Laminar air flow chamber. Various concentrations of crude lipopeptides were prepared from 1–5% on W/V basis in sterile water and methanol in the ratio of 9:1. Using a handheld atomizer, the leaves were sprayed on both sides with the test solution. Untreated controls were maintained which were sprayed with only sterile water and methanol in the ratios as mentioned above. Control leaves with sterile water spray alone were also maintained. All the leaves were left to air dry inside the laminar air flow chamber. Upon drying, a 5mm disc from an actively growing 7 days old A.solani plate was cut and place in the centre of each leaf, except for the untreated control leaves. All the leaves were placed on wet cotton inside petri dishes followed by incubation for 7 days at 28°C in an incubator. After 7 days the leaves were examined for lesions and the extent of lesion formation was measured in centimetres. The inhibition rate was calculated as follows
Inhibition rate (%) = [Diameter of lesion in Control (cm) – Diameter of lesion in treatment (cm)/Diameter of lesion in Control (cm)] x 100
The experiment was conducted in triplicates and statistically analysed.
Statistical analysis
All data was analysed statistically using WASP - Web Agri Stat Package 2.0 and Microsoft excel (2016) to assess statistically significant differences among the various treatments.