Antifungal Activity of Bacillus amyloliquefaciens NJN-6 Volatile Compounds against Fusarium oxysporum f. sp. cubense

Abstract

Bacillus amyloliquefaciens NJN-6 produces volatile compounds (VOCs) that inhibit the growth and spore germination of Fusarium oxysporum f. sp. cubense. Among the total of 36 volatile compounds detected, 11 compounds completely inhibited fungal growth. The antifungal activity of these compounds suggested that VOCs can play important roles over short and long distances in the suppression of Fusarium oxysporum. Microbial antagonist strains capable of producing both nonvolatile compounds and volatile compounds (VOCs), which exhibit strong inhibitory activity against plant pathogens, have received much attention (4, 14). These antagonists include bacteria, such as Pseudomonas spp. (5), and nonpathogenic fungi like Trichoderma spp. (11). The release of VOCs by soil microbes has been reported to promote plant growth (13), display nematicidal activity (7), and induce systemic resistance in crops (3). Previous researchers also found that VOCs produced by bacteria could inhibit the growth (6) and the spore germination of pathogenic fungi (10), suggesting that VOCs produced by bacteria could be a mechanism of biocontrol against some soilborne fungal diseases.Fusarium oxysporum is a well-known soilborne fungus, and some strains of F. oxysporum are pathogenic to plants and are difficult to control; however, biological methods may be a reliable alternative to chemical methods for controlling soilborne fungal growth. For applications in agriculture, the Bacillus species are considered important biological control agents. Bacillus amyloliquefaciens (NJN-6), isolated from the rhizosphere soil of healthy banana plants, acts as an efficient antagonist against F. oxysporum f. sp. cubense by producing several antibiotics (15,16). In this study, we characterized the volatile organic compounds produced by strain NJN-6. We used solid-phase microextraction (SPME) combined with gas chromatography-mass spectrometry (GC-MS) to extract and identify the VOCs. Finally, we identified antagonistic VOCs as those that reduced the growth and inhibited the spore germination of F. oxysporum.Microorganisms and culture conditions. The antagonistic strain NJN-6 was identified as B. amyloliquefaciens (CGMCC [China General Microbiology Culture Collection Center] accession no. 3183) by 16S rRNA sequencing (15). The fungal strain F. oxysporum f. sp. cubense, which exhibited high virulence in banana plants, was used as the target fungus.Antagonistic assay of VOCs against fungi. One compartment of the divided plates containing modified MS medium (with 1.5% [wt/vol] agar, 1.5% [wt/vol] sucrose, and 0.4% [wt/vol] TSA [3]) was inoculated with NJN-6, except for control plates. Another compartment containing PDA medium was used for F. oxysporum to test growth inhibition, or 100 l of spore solution (10 8 CFU/ ml) was spread evenly to test the ability of the VOCs to inhibit the spore germination of fungi, or 10 g of diseased banana field soil from Ledong, Hainan Province, was added to one compartment. The plates were incubated at 28°C for 3 days, and then the diame...

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