Identification of main postharvest pathogenic fungi in Hongdeng cherry and the anti- fungal spectrum of Bacillus velezensis G-1 natural product

Abstract:【Objective】Postharvest fungal disease is one of the important factors affecting storage of sweet cherry [Cerasus avium (L.) Moench], and mycotoxins produced by pathogenic fungi can contami- nate sweet cherry. In addition, chemical pesticides have been forbiddened to be used during postharvest storage in many contries. On the other hand, some new pathogens have been founded from many crops in recent years. Therefor, it is important to clarify whether there are new diseases in postharvest sweet cherry fruits for the management of pre-harvest diseases and the technological innovation of post-har- vest control. Our study aimed to explore the potential application of natural products from Bacillus velezensis G-1 to postharvest fungal diseases of sweet cherry in order to provided a new source for the development of broad-spectrum biocontrol agents for cherry postharvest diseases.【Methods】The fruits of Hongdeng sweet cherry infested, with postharvest diseases were used as materials to isolate the pathogenic fungi by the methods of tissue separation. The fruits were soaked in 75% ethanol for 30 s and rinsed three times by sterile distilled water. Then the fruits were soaked in 3% ethanol for 3 min and  rinsed three times by sterile distilled water. The fruits were cut to 3 mm × 3 mm in size and plated on potato dextrose agar (PDA). All isolates were purified by single spore isolation method. All pure fungal isolates were identified by morphology and ITS sequence analysis. The morphology of the colony, spores size, mycelium and spore microscopy on PDA medium were used by morphology analysis. The pathogenic ability of the pathogenic fungi was tested based on Robert Koch's rule. In addition, the TEF(translation elongation factor-1α) was used to identify strain YT-2 and YT-4. NCBI’s BLASTn tools were used to obtain highly homologous DNA sequence in GenBank and molecular phylogenetic tree was constructed by MEGA 5.0. The growth rate method and plate-to-plate method were used to evalu- ate the antifungal spectrum of lipopeptide (500 μg·mL-1)and 2,4-Di-tert-butylphenol (10 μg·μL-1) pro- duced by B. velezensis G-1 against the pathogenic fungi obtained as mentioned above【. Results】There were 8 types of pure fungi isolated from the disease symptomatic tissues, including YT-1 (Rhizopus ory- zae), YT-2 (Fusarium dlaminii), YT-4 (Fusarium fujikuroi), YT-5 and YT-7 (Alternaria sp.), YT-6 (Botrytis pelargonii), YT-8 (Mucor hiemalis f. hiemalis) and YT-9 (Penicillium crustosum). YT-1, YT-6 and YT-8 were the main pathogenic strains among the 8 strains, and had much higher pathogenic ability than other pathogenic fungi. TEF gene could be used to identify F. dlaminii and F. fujikuroi. The antifun- gal rate of lipopeptide (500 μg · mL- 1), produced by B. velezensis G- 1, against M. hiemalis f. hiemalisand P. crustosum were less than 60%. However, the antifungal rates of lipopeptide against other fungi species were over 80%. In addition, the volatile compound 2, 4-di-tert-butylphenol (10 μg·μL-1) had much lower antifungal activity against P. crustosum, and the inhibition rate was only 0.41%. But the an- tifungal rates of 2, 4-di-tert-butylphenol against other pathogenic fungi were all over 90%.【Conclu- sion】In this study, F. dlaminii and F. fujikuroi were found in Hongdeng cherry for the first time, indicat- ing that the postharvest diseases of Hongdeng cherry have changed in recent years. Toxin contamination from F. dlaminii and F. fujikuroi should be paid attention to during cherry storage. The lipopeptide and volatile 2,4-di-tert-butylphenol from B. velezensis G-1 could be potentially used to control postharvest diseases. But the final control effect of lipopeptide and 2, 4-di-tert-butylphenol still needs to verify in commercial storage conditions. This study would provide a theoretical basis for controling postharvest fungal disease and for preventing preharvest fungal disease of Hongdeng sweet cherry in the future.