Molecular and biochemical characterization of field boscalid-resistant isolates of Alternaria alternata from broccoli and peanut

Leaf spots and blights, caused by filamentous fungus Alternaria alternata, are common diseases of numerous economic crops in China. Boscalid, a succinate dehydrogenase inhibitor, is widely used to control Alternaria leaf blights in China for decades. In current study, among the 114 isolates of A. alternata separated from broccoli and peanut grown in Yancheng City of Jiangsu province, the resistance frequency of boscalid is 4.39%, however, for the 54 isolates from Zoucheng City of Shandong province, the resistance frequency of boscalid is 5.56%. Compared to boscalid-sensitivity isolates, some boscalid-resistant isolates exhibited defects on fitness defined as mycelial growth, sporulation, spore germination and virulence. There was no cross-resistance between boscalid and thifluzamide, azoxystrobin, tebuconazole, fludioxonil or procymidone in the isolates of A. alternata from broccoli and peanut. Sequence alignment results of AaSDHB, AaSDHC or AaSDHD coding genes from eight field boscalid-resistant isolates showed that five broccoli-originated isolates possessed six types of amino acid substitutions in AaSDHB and AaSDHC (I21V, A28V, I52V, I62T, A118V and V169F), other three peanut-originated isolates possessed two types of amino acid substitutions in AaSDHD (V17A and A47T). Under the boscalid treatment the gene expressions of AaSDHB, AaSDHC or AaSDHD in boscalid-sensitivity isolate but not in boscalid-resistant isolates are increased, moreover, the succinate dehydrogenase (SDH) activity of the boscalid-sensitive isolate was lower than that of resistant isolates. Compared to the boscalid‑free condition, boscalid inhibited melanin synthesis in A. alternata, with down‑regulated expression of melanin synthesis genes. These results provide directions for controlling of leaf spots and blights in broccoli and peanut via scientifically using fungicide, and promote research development in fungicide resistance and fungi secondary metabolism.