Resistance characterization of the natural population and resistance mechanism to pyraclostrobin in Lasiodiplodia theobromae.

Lasiodiplodia theobromae is the main pathogen of mango stem-end rot disease, causing mango fruit decay and major economic loss. QoI resistance has been found in field populations of L. theobromae. The characterization and resistance mechanism of pyraclostrobin-resistant L. theobromae was investigated by using a combination of bioassays and biochemical and molecular methods. The pyraclostrobin resistance among the L. theobromae population samples from Hainan was 93.41%. The resistant isolates were stable after successive subculturing for 10 times on PDA. Cross-resistance was observed only between the Qols pyraclostrobin and azoxystrobin. The alternative oxidase (AOX) inhibitor SHAM notably decreased the EC50 values of pyraclostrobin for all tested L. theobromae isolates. Induction of AOX by pyraclostrobin was observed in mycelia cells of L. theobromae. After treatment with pyraclostrobin, the final ATP and AOX contents of all sensitive isolates were significantly lower than those of resistant isolates. The relevant mutation and high expression of the cytochrome b gene were not detected in resistant isolates. However, there were 4 mutations in the AOX gene, which were only observed in highly resistant isolates. Pretreatment with pyraclostrobin resulted in a significant upregulation of AOX gene expression, and the average expression level of the highly resistant isolates was 33-fold that of the control group. These results suggested that the AOX pathway is responsible for resistance to pyraclostrobin, and that the AOX-related resistance mechanism is common in field populations of L. theobromae in Hainan mango orchards.

Molecular characterization and overexpression of the difenoconazole resistance gene CYP51 in Lasiodiplodia theobromae field isolates.

Stem-end rot (SER) caused by Lasiodiplodia theobromae is an important disease of mango in China. Demethylation inhibitor (DMI) fungicides are widely used for disease control in mango orchards. The baseline sensitivity to difenoconazole of 138 L. theobromae isolates collected from mango in the field in 2019 was established by the mycelial growth rate method. The cross-resistance to six site-specific fungicides with different modes of action were investigated using 20 isolates randomly selected. The possible mechanism for L. theobromae resistance to difenoconazole was preliminarily determined through gene sequence alignment and quantitative real-time PCR analysis. The results showed that the EC50 values of 138 L. theobromae isolates to difenoconazole ranged from 0.01 to 13.72 µg/mL. The frequency of difenoconazole sensitivity formed a normal distribution curve when the outliers were excluded. Difenoconazole showed positive cross-resistance only with the DMI tebuconazole but not with non-DMI fungicides carbendazim, pyraclostrobin, fludioxonil, bromothalonil, or iprodione. Some multifungicide-resistant isolates of L. theobromae were found. Two amino acid substitutions (E209k and G207A) were found in the CYP51 protein, but they were unlikely to be related to the resistance phenotype. There was no alteration in the promoter region of the CYP51 gene. However, difenoconazole significantly increased the expression of the CYP51 gene in the resistant isolates compared to the susceptible isolates. These results are vital to develop effective mango disease management strategies to avoid the development of further resistance.