Resistance monitoring of Colletotrichum spp. population to carbendazim in grape vineyards

Abstract:【Objective】Grapevine anthracnose caused by Colletotrichum spp. species complex popula-tion (GG) has been the main disease during the grapevine spike stage in the hilly area of Jiangsu province. In recent years, the controlling failure of common chemicals to the disease has caused serious yield loss. Therefore, it has become an important research target to clarify the evolution, epidemic dy-namics and molecular mechanism of resistance of GG to benzimidazole fungicide, and to provide deci-sion-making basis for resistance control.【Methods】Employing the methods of discriminative dose (a concentration that fully inhibits mycelial growth of the sensitive isolates) and effective inhibition medi-um concentration (inhibits mycelia growth by 50% relative to the control, EC50), the EC50 values were identified to distinguish sensitivity and sensitive baseline migration to carbendazim. According to previ-ous studies, the discriminatory concentration of carbendazim was 10 mg · L^-1. Then EC50 values of car-bendazim were determined from 98 isolates and 34 isolates in 2013 and 2017, respectively. Carbendaz-im solution was added to PDA to produce final active ingredient concentrations of 0, 0.04, 0.12, 0.37,1.11, 3.33, 10.00 and 30.00 mg·L^-1. For each isolate, three replicates per concentration were used. The conidia suspension of sensitive, resistant and mixed isolates (the resistant isolate and sensitive isolate with the same conidia concentration were mixed in equal volume) was inoculated during young berry  growing stage after spray with carbendazim. The concentration of conidia suspension was 1×104 sporesper mL. The concentration of carbendazim was 1000 mg·L-1. The controlling efficacy was investigated during berry ripening stage, and the sensitivity of the back separative isolates to the carbendazim was detected to evaluate the resistant evolution of GG. This field control test was repeated twice in 2017 and 2018, respectively. Furthermore, the molecular mechanisms of carbendazim were determined by the se-quence analysis of target gene (TUB2). First, DNA from fungal mycelia was extracted using a DNA kit. One primer pairs, TubF1 (5’-ACTTCGTCTTCGGCCAGTCTG-3’) and TubR1 (5’-TTCTGGACGTT-GCGCATCTG-3’) was used. PCR products were examined by electrophoresis in a 1.2 % agarose gel in1×TAE buffer. DNASTAR software was used to assemble and align the nucleotide and amino acid se-quences. All nucleotide sequences were compared to previously reported sequences using BLAST.【Re-sults】The mean EC50 values of GG to carbendazim were 0.528 5 mg · L^-1 and 7.787 8 mg · L^-1 in 2013 and 2017, respectively. The sensitivity of GG shifted from the baseline significantly. The resistant fre- quencies of GG to carbendazim in different sampling areas in the same year and in different sampling years in the same region were different. However, when the whole city was taken as a sampling unit, the resistant frequencies of GG to carbendazim within five years (2013—2017) increased year by year.The resistant frequencies of GG to carbendazim increased from 2.34% in 2013 to 32.21% in 2017. Field test of carbendazim at twice the recommended field dose (a.i. 1000 mg · L^-1) against resistant isolates showed that the controlling efficacy of carbendazim was very low, only 0.64% and 4.18% in 2017 and 2018, respectively. The resistance frequencies of back separative isolates were 65.00% and 68.18% in 2017 and 2018, respectively, which was significantly higher than the initial ratio of inoculated resistant isolates (50% ). All the resistant isolates harbored the E198A (glutamate substituted by alanine) or F200Y (phenylalanine substituted by arginine) point mutation in TUB2.【Conclusion】The resistance frequencies of GG to carbendazim in the hilly area of Jiangsu province fluctuated from year to year, but the resistance frequencies of the population showed an overall upward trend within five years, and resis- tant epidemic formed. The results also showed that the current resistance control measures had poor ef- fect on the resistance management of GG. We suggested stopping carbendazim application completely. Carbendazim should be replaced by fungicides with different mechanisms. The selection pressure caused by the continuous application of carbendazim was the main driving factor for the emergence and prevalence of resistance to GG in the field. The genotype of resistance to carbendazim of GG belonged to the point mutation of single base of target gene, which resulted in the substitution of amino acids, i.e. E198A or F200Y, and no other genotypes were found in this research.