Isolation, purification, and identification of antifungal protein produced by Bacillus subtilis SL-44 and anti-fungal resistance in apple

Abstract

Abstract Apple anthracnose is a fruit fungal disease that is currently recognized as one of the most serious threats to apples worldwide. In this study, antifungal protein from B. subtilis SL-44 was isolated, purified, identified, and applied for apple anthracnose control. The antagonistic experiment showed that SL-44 had an excellent broad spectrum against plant pathogenic fungi. Initial pH, inoculum volume, and rotation speed were evaluated by single-factor experiments and Box-Behnken Design and which were optimized to be 7, 2%, and 180 rpm, respectively. Under these conditions, the yield of antifungal protein increased by 45.83% compared to that before optimization. The crude protein from SL-44 was isolated and purified by (NH4)2SO4 precipitation, DEAE-Sepharose fast flow column, and Sephadex G-100 column. TEM revealed that antifungal protein could severely disrupt the mycelial of C. gloeosporioides, resulting in the loss of its intracellular contents. LC-MS analyzed that antifungal protein was likely to be an uncharacterized protein YefB with a molecular weight of 35 kDa. Additionally, antifungal protein significantly alleviated the size of the lesion to more than 70% in the apple infection protection test. In conclusion, B. subtilis SL-44 and its main antimicrobial components have remarkable potential in the development of fungicides for biological control of apple anthracnose.