Screening of Tomato Seed Bacterial Endophytes for Antifungal Activity Reveals Lipopeptide Producing Bacillus siamensis Strain NKIT9 as a Potential Bio-Control Agent

Abstract

The current study investigates the diversity pattern and fungicidal potential of bacterial endophytes isolated from two different organic varieties of tomato plants (V1 and V2). A total of seventy-five bacterial isolates identified by 16S rRNA gene sequencing revealed a majority of genus as Bacillus and one Planococcus, which were grouped into eight different species. The Shannon diversity H' (1.56), Simpson's index of diversity (0.93), Magalef' index (2.23), Evenness (0.96), and Species richness (7) indicated the high endophytic bacterial diversity in the V1 variety of the tomato. Bacterial endophytes isolated from both of the varieties were screened for their antifungal activity against five economically critical fungal pathogens (viz., Botrytis cinerea, Rhizoctonia solani, Fusarium solani, Verticillium lateritium, and Alternaria solani) of tomato crop through dual culture assay. The data revealed B. siamensis strain NKIT9 as the most potent antagonist, significantly (p < 0.05) inhibiting the mycelial growth between 75 to 90% against selected fungal pathogens. High bioactivity of lipopeptide extract of strain NKIT9 was recorded against R. solani with minimum IC₅₀ value of 230 μg/ml. The Ultra Performance Liquid Chromatography-High Definition Mass Spectrometry (UPLC-HDMS) analysis of this lipopeptide extract revealed the presence of Surfactin and Bacillomycin D. Furthermore, in-vitro results showed that the selected bacterial strain significantly minimized the disease incidence in damping-off assay which makes this strain a promising antifungal bio-control agent. Moreover, in the pot experiment the NKIT9 increased the fruit yield by 59.2% compared with the untreated R. solani infested control.

Keywords: UPLC; antagonistic; bio-pesticide; crop protection; diversity indices; plant growth promotion.

FIGURE 1

Taxonomic profiles of the bacterial community in each variety at the representative species level with the relative abundance.

FIGURE 2

Evolutionary relationships of bacterial isolates investigated in this study: Phylogenetic tree was constructed using 16S rRNA gene sequences and the bootstrap values (100 replicates) are indicated (BS > 50) at the nodes. Closest neighbor of strain NKIT38, Planococcus ruber strain CW1 was used outgroup. The evolutionary history was inferred using the NJ method. The optimal tree with the sum of branch length = 0.07256030 is shown. The evolutionary distances were computed using the Maximum Composite Likelihood method and are in the units of the number of base substitutions per site. The analysis involved 55 nucleotide sequences. All positions containing gaps and missing data were eliminated. There were a total of 545 positions in the final dataset. Evolutionary analyses were conducted in MEGA7.

FIGURE 3

Venn diagram representing the shared species of isolated bacterial endophytes within the variety.

FIGURE 4

Antagonistic effect against five pathogenic test fungi. (A) Graph for the antifungal activity of bacterial endophytes isolated from V1 variety seeds. (B) Graph for the antifungal activity of bacterial endophytes isolated from V2 variety seeds. Three replicates were used for the assay. Bars labeled with the same letters are not significantly different according to Duncan Multiple Range Test at p = 0.05. Vertical lines represent the standard errors of the mean.

FIGURE 5

Antagonizing effect of strain NKIT9 against (A) Rhizoctonia solani, (B) Verticillium lateritium (C) Botrytis cinerea, (D) Alternaria solani, and (E) Fusarium solani after 6 days of inoculation (“E” represents endophytic bacterial strain whereas “P” represents Pathogenic fungi).

FIGURE 6

Antifungal bioassay of lipopeptide extracted from Bacillus siamensis strain NKIT9 at 5 different concentrations of 50, 100, 250, 500, and 1000 μg/ml against five test pathogenic fungi (R. solani, F. solani, B. cinerea, A. solani, and V. lateritium).

FIGURE 7

(A) UPLC chromatogram of lipopeptides extracted from Bacillus siamensis strain NKIT9; (B) HDMS accurate mass revealed the production of Surfactin and Bacillomycin D analogues.

FIGURE 8

General molecular structure of lipopeptides (A) Surfactin and (B) Bacillomycin isolated from B. siamensis strain NKIT9.

FIGURE 9

Effect of priming of tomato seeds with strain NKIT9 on the (A) shoot length of the seedling (F = 54.109; df = 53) and (B) seedling fresh weight (F = 70.928; df = 53). Evaluation was made after 15 days of planting of tomato seeds. Nine replicates were used for the assay with 10 seedlings in each replicate. Bars labeled with the same letters are not significantly different according to Duncan Multiple Range Test at p = 0.05. Vertical lines represent the standard errors of the mean.

FIGURE 10

Growth of tomato plants (V1) obtained after 45 days of sowing of tomato seeds of all the treatments: T1 (Surface-sterilized seeds only); T2 (Surface-sterilized seeds + fungal pathogen); T3 (Bio-primed seeds only); T4 (Bio-primed seeds + fungal pathogen); T5 (Bio-primed seeds + fungal pathogen + soil treatment of the bacterial strain NKIT9); T6 (Chemical treated seeds (Bavistin-Carbendazim) + fungal pathogen.