Antifungal Activity of Bacillus Species Against Fusarium and Analysis of the Potential Mechanisms Used in Biocontrol

Abstract

Fusarium is a complex genus of ascomycete fungi that consists of plant pathogens of agricultural relevance. Controlling Fusarium infection in crops that leads to substantial yield losses is challenging. These economic losses along with environmental and human health concerns over the usage of chemicals in attaining disease control are shifting focus toward the use of biocontrol agents for effective control of phytopathogenic Fusarium spp. In the present study, an analysis of the plant-growth promoting (PGP) and biocontrol attributes of four bacilli (Bacillus simplex 30N-5, B. simplex 11, B. simplex 237, and B. subtilis 30VD-1) has been conducted. The production of cellulase, xylanase, pectinase, and chitinase in functional assays was studied, followed by in silico gene analysis of the PGP-related and biocontrol-associated genes. Of all the bacilli included in this study, B. subtilis 30VD-1 (30VD-1) demonstrated the most effective antagonism against Fusarium spp. under in vitro conditions. Additionally, 100 μg/ml of the crude 1-butanol extract of 30VD-1's cell-free culture filtrate caused about 40% inhibition in radial growth of Fusarium spp. Pea seed bacterization with 30VD-1 led to considerable reduction in wilt severity in plants with about 35% increase in dry plant biomass over uninoculated plants growing in Fusarium-infested soil. Phase contrast microscopy demonstrated distortions and abnormal swellings in F. oxysporum hyphae on co-culturing with 30VD-1. The results suggest a multivariate mode of antagonism of 30VD-1 against phytopathogenic Fusarium spp., by producing chitinase, volatiles, and other antifungal molecules, the characterization of which is underway.

Keywords: Bacillus; Fusarium; biocontrol bacteria; hydrolytic enzymes; plant growth promoting bacteria.

FIGURE 1

Average of the measurements of fungal radial growth toward the bacterial strain after 7 days of incubation compared to the control. All four Bacillus strains (B. subtilis 30VD-1, B. simplex 30N-5, B. simplex 11, and B. simplex 237) inhibited the growth of Fusarium spp. (A) F. oxysporum f. sp. conglutinans (FOC), (B) F. oxysporum f. sp. matthioli (FOM), (C) F. solani (FS). Values presented as mean ± standard error. The experiment was repeated 6 times with 10 replicates in each trial.

FIGURE 2

Homologs of a reference set of genes likely to be involved in inhibiting fungal growth through hydrolytic enzyme production. Top row, IMG gene ID number; second row, general categories of biocontrol; third row, genes identified in each Bacillus strain (left column); B. simplex strains (orange); B. cereus/megaterium group (purple); and typical PGP bacilli (blue). The percentages of gene identity are included within the individual cells. Sequence alignments were based on blastp searches (% values in cells). Varying shades of orange represent percent identities to B. simplex 30N-5 (set to 100%): ≥90%, dark orange; 71–89%, medium orange; 60–70%, light orange; 50–59%, pale orange. The turquoise-blue cells represent sequence identities of ≤49%. Genes not detected, n.d.

FIGURE 3

Photographs of chitosan-degrading activity of (A) Paenibacillus tundrae 47 (positive control) and (D) Kocuria rosea 245 (negative control). The experimentals are (B) B. subtilis 30VD-1; (C) B. simplex 30N-5; (E) B. simplex 237; (F) B. simplex 11. Bar, 10 μm.

FIGURE 4

(A) Disease severity in pea under in vivo conditions. The presented treatments are uninoculated control (C), inoculation with B. subtilis 30VD-1 (30VD-1), inoculation with F. oxysporum f. sp. matthioli (FOM), and co-inoculation of B. subtilis 30VD-1 and FOM (30VD-1+FOM). (B) Graphical representation of dry biomass of pea plants in response to different treatments in C, 30VD-1, FOM, and 30VD-1+FOM. The asterisks indicate that the co-inoculation of B. subtilis 30VD-1 and Fusarium had a positive effect on growth. ANOVA univariate analyses were performed and a post hoc LSD test was used to identify inoculation treatments with means significantly different from the control at P ≤ 0.05.