Resistance and its molecular mechanism of Phyllosticta citricarpa and Phyllosticta citriasiana to benzimidazole fungicide

In order to control citrus black spot (CBS, Phyllosticta citricarpa) and pummelo black spot (PBS, Phyllosticta citriasiana) effectively, we evaluated the resistance frequency, resistance level and their molecular mechanisms of these two pathogen populations collected from seven provinces of China to the fungicide carbendazim. The results showed that two carbendazim-resistant (Cab-R) isolates were found from the Jiangxi subpopulation of P. citricarpa out of 114 strains. The resistant frequency was 1.75%, and their coefficient of resistance were 5 333.6 and 379.2, respectively. One Cab-R strain with coefficient of resistance of 303.3 was found in the Sichuan subpopulation of P. citricarpa out of 32 strains, and the resistant frequency was 3.13%. However, none of the Cab-R isolates was detected from the Zhejiang (23 strains) and the Chongqing (3 strains) subpopulations. For P. citriasiana, one Cab-R strain, whose coefficient of resistance was 13 719.5, was obtained in the Guangdong subpopulation (54 strains in total), and the resistant frequency was 1.85%. None of the Cab-R strains was found in the Guangxi (34 strains) and the Fujian (34 strains) subpopulations. In addition, there was a positive cross-resistance between thiophanate-methyl and carbendazim in P. citricarpa and P. citriasiana, while there was no cross-resistance between azoxystrobin and carbendazim. Based on the results obtained, we suggest that the fungicide carbendazim can still be used in controlling CBS and PBS in the fields, but the rotation of the fungicides with different antifungal mechanisms is necessary given the existence of high-resistant strains. Finally, the two molecular mechanisms of carbendazim-resistance of P. citricarpa were found. One was a mutation at the amino acid (aa) 198 of β-tubulin gene, in which the glutamic acid changed to lysine (the strain with a coefficient of resistance of 5 333.6), and the other one was a mutation at the aa 200 of β-tubulin gene, in which the phenylalanine changed tyrosine (the strain with a coefficient of resistance of 379.2). However, the molecular mechanism of the carbendazim-resistance of P. citriasiana was a mutation at the aa 198 in the β-tubulin gene, in which the glutamic acid changed to alanine.