Identification of Pathogenic Fungi and Screening for Biocontrol Bacteria during Fermentation of Yuxi Cigars

In order to clarify the pathogenic fungi involved in the fermentation process of Yuxi cigar tobacco, a tissue isolation method was used to isolate the pathogenic fungi from moldy tobacco leaves. The pathogenic species were identified based on the Koch's postulates, combined with morphological and ITS gene identification. The pathogenic fungi were used as indicator fungi to screen for biocontrol bacteria. The anti-mildew effect and the effect of aroma components were verified by indoor fermentation. The Koch's test results showed that the original isolated pathogenic fungus XJ-a and XJ-b re-isolated from symptomatic tobacco leaves were highly similar in morphology, microscopy, and disease symptoms. The ITS sequences of both strains of pathogenic fungi clustered with multiple strains of Aspergillus flavus, with a sequence similarity of more than 99%. Based on the comprehensive morphological and molecular biological characteristics, it was determined that the pathogenic fungus responsible for the mold on cigar tobacco leaves was A. flavus. Three biocontrol bacteria with strong inhibition activities were obtained through the agar diffusion method. Among them, strain CC-3 had an inhibition bandwidth of 9.04 mm against A. flavus. It was identified as B. cereus. Adding CC-3 during fermentation was able to significantly reduce the infection rate of A. flavus, with infection rates decreasing by 64.44% and 52.66% after 15 and 30 days of fermentation, respectively, and the total amount of aroma substances increasing by 28.96%. The results of this study indicated that A. flavus is the main mold fungus responsible for the fermentation of Yuxi cigar tobacco. Adding the biocontrol bacteria CC-3 during fermentation can reduce the infection of A. flavus and increase the aroma component content.