Resistance of Mulberry Fruit Sclerotiniosis Pathogens to Thiophanate-Methyl and Boscalid

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Fruit sclerotiniosis (FS) is becoming the most important disease in recently transformed mulberry fruit gardens (TFMGs), where traditional production of mulberry leaves for sericulture takes place; FS has a long history as a secondary disease in Zhejiang province, China. Thiophanate-methyl and boscalid are the two main fungicides adopted for the management of FS in these gardens. A decrease in efficacy has been observed by growers and local technicians. For this new situation regarding TFMGs, however, the resistance status of them has not yet been investigated and reported. In the present study, pathogens were isolated from diseased fruits and identified through a combination of morphological characteristics with ITS sequence analysis. Results showed that the pathogens included Scleromitrula shiraiana and Sclerotinia sclerotiorum. All isolates of S. shiraiana (n = 12) and S. sclerotiorum (n = 12) were sensitive to boscalid, and no resistance was detected. The S. shiraiana sub-population was sensitive to thiophanate-methyl, whereas the S. sclerotiorum sub-population developed resistance with a frequency of 33.3%. Thiophanate-methyl-resistant (ThR) S. sclerotiorum grew faster at a low temperature (15 °C) than sensitive ones. These ThR exhibited negative cross-resistance to diethofencarb, as previously observed in Botrytis cinerea, and showed no cross-resistance to procymidone or boscalid. Further studies indicated that resistance to thiophanate-methyl is caused by a novel double mutation (E198V+V349I) in the β-tubulin of S. sclerotiorum. This E198V+V349I mutation produced structural alterations in the β-tubulin protein, the action target of thiophanate-methyl, leading to reorientation of the substrate binding site and conformational change in the active pocket. In conclusion, avoiding the sole use of thiophanate-methyl on TFMGs is necessary. Application of boscalid in combination or rotation with other fungicides without cross-resistance is recommended for the management of FS in TFMG practices.