Determination Of Antagonistic Activity Of Volatiles From Bacillus Spp. Against Several Soilborne Pathogens And Components Analysis

Soilborne pathogens, such as fungi, bacteria, actinomycete and nematode, are the great cause that influence the plant health, crop production and ecosystem stability. Microbial volatile metabolites play significant roles in the control of soilborne plant diseases. In this study, the volatiles produced by soil Bacillus spp. strains and their bioactivities against several soil pathogenic fungi and nematode were studied. Besides, the determination of toxic action modes, the analysis of active components and biocontrol application in greenhouse was also carried out. The above work could provide scientific references to the exploit and application of bacterial volatile metabolites and to provide potential novel bioactive compounds for the control of plant diseases.The result of culture medium screening in sealed plates indicated that, compared to LB, NB and TSB, the test five Bacillus strains cultured on TSB-YE produced more bioactive volatiles. The result of bioactivity determination indicated that, the inhibition percentage of these volatiles against test fungi, including Sclerolinia sclerotirum, Botrytis cinerea, Alternaria brassicae, Alternaria solani, Ascochyta citrullina, Fusarium oxysporum, Cercospora kikuchii Chupp, Rhizoctonia solani, Cecospora arachidicola, Verticillium dahiae and Fusarium graminerum was from 20% to 100%, and had strong inhibition and nematicide effection against potato stem nematode (Ditylenchus detructor Thorne). The action of volatile-treated nematodes was gradually decreased and their mortality rate achieved 100%. All these results demonstrated the potential of volatiles from Bacillus in the control of soilborne pathogens.The determination of toxic action modes in sealed plates indicated that, these volatiles had obvious inhibition activity against the mycelium growth, spore germination, pigment production, sclerotia production and germination. The mycelium growth of S. sclerotirum exposed to bacterial volatiles for 7 d was completely inhibited, and the formed mycelium was freakish-contorted, swelled, protoplasm agglomerated and browned. After exposure to these volatiles for 24 h, most spores became cracked and browned, and the germination decreased (Inhibition percentage 70%-80%). An unexpected phenomenon was found that these volatiles strongly inhibited the production of pigment in test pathogenic fungi. The weight of sclerotia yielded in each treatment plates was decreased by 20%-40%. These sclerotia were oddly shaped, not plump and very spongy when touched, and their second germination rate decreased.Such effective antifungal VOCs were extracted using SPME and further determined by GC/MS technique. In total, 21-33 compounds were mainly detected from each bacterial TSB-YE cultures, which covered a wide range of alkyl, aldehyde, alcohol, ketone, fatty acid, amine, oxime, phenol and heterocyclic compounds. Besides, there were some overlap and particularity between VOCs detected from different strains.The bioactivity of 20 candidate compounds was further confirmed with commercially available pure compounds. The assay for antifungal activity against S. sclerotirum in sealed plates confirmed that, furfural, benzaldehyde, 1-octanol, 1-octen-3-ol, 3, 7-dimethyl-1, 6-octadien-3-ol, 2-ethyl-1-hexanol exhibited complete inhibition against S. sclerotiorum at a lowest treatment dosage of 0.01 g, but 2-furanmethanol, 1-dodecanol, n, n-dimethyl-1- hexadecanamine, azulene and tetradecane had no obvious growth inhibition.Other nine candidates treated between 0.01 g to 0.20 g also achieved complete inhibition effects (The minimal inhibitory concentration is about 0.94×10-5 to 26.15×10-5 g/cm3). The assay for nematicide activity against potato stem nematode confirmed that, 2-furanmethanol, 1-octanol, 1-octen-3-ol, benzothiazole, 3, 7-dimethyl-1, 6-octadien-3-ol, isovaleric acid, 2-ethyl-1- hexanol and cyclohexanol exhibited complete nematicidal effect at a lowest treatment dosage of 0.01 g. But xylene, (R)-3, 7-dimethyl-6-octen-1-ol, 1-dodecanol, n, n-dimethyl-1-hexadecanamine, azulene and tetradecane exhibited no obvious nematicidal activity. Other six candidates treated between 0.01 g to 0.05 g also achieved complete nematicidal effect (The minimum lethal concentration is about 7.65×10-5 to 53.23×10-5 g/cm3). These results indicated the important role of microbial compounds in soil antagonistic phenomenon and the potential to be applied as possible soil sterilants.In greenhouse, the soil fumigation against S. sclerrotium indicated that, the treatments thoroughly mixing the autoclaved soil with volatile-producing bacteria caused a higher control effect than those layer treatments and bacterial volatiles had no effect on the emergence rate of seedlings. Besides, an obvious growth promotion of cucumber seedlings was shown. When applied as direct fumigation for 7 d, these volatiles exhibited slight inhibition against the occurrence of powdery mildew (21.43%-45.10%). And obvious visible rot could easily come with this disease occurrence.