Mutations and Overexpression of CYP51 Associated with DMI-Resistance in Colletotrichum gloeosporioides from Chili

Abstract

Chili anthracnose caused by Colletotrichum spp. is an annual production concern for growers in China. Sterol C14-demethylation inhibitors (DMIs, such as tebuconazole) have been widely used to control this disease for more than three decades. In the current study, of 48 isolates collected from commercial chili farms in Jiangsu Province of China during 2018 and 2019, 8 single-spore isolates were identified as Colletotrichum gloeosporioides and the rest were identified as C. acutatum. To determine whether the DMI resistance of isolates develops in the field, mycelial growth of the 48 isolates was measured in culture medium with and without tebuconazole. In all, 6 of the 8 C. gloeosporioides isolates were resistant to tebuconazole, but all 40 of the C. acutatum isolates were sensitive to tebuconazole. The fitness cost of resistance was low based on a comparison of fitness parameters between the sensitive and resistant isolates of C. gloeosporioides. Positive cross-resistance was observed between tebuconazole and difenconazole or propiconazole, but not prochloraz. Alignment results of the CgCYP51 amino acid sequences from the sensitive and resistant isolates indicated that mutations can be divided into three genotypes. Genotype I possessed four substitutions (V18F, L58V, S175P, and P341A) at the CgCYP51A gene but no substitutions at CgCYP51B, while genotype II had five substitutions (L58V, S175P, A340S, T379A, and N476T) at CgCYP51A, concomitant with three substitutions (D121N, T132A, and F391Y) at CgCYP51B. In addition, genotype III contained two substitutions (L58V and S175P) at CgCYP51A, concomitant with one substitution (T262A) at CgCYP51B. Molecular docking models illustrated that the affinity of tebuconazole to the binding site of the CgCYP51 protein from the resistant isolates was decreased when compared with binding site affinity of the sensitive isolates. Our findings provide not only novel insights into understanding the resistance mechanism to DMIs, but also some important references for resistance management of C. gloeosporioides on chili.

Resistance risk assessment for a novel succinate dehydrogenase inhibitor pydiflumetofen in Fusarium asiaticum

“…Expressions of FaSdh genes from resistant mutants was analyzed according to a previous study. 32 Briefly, five mycelial discs of each strain/mutant were incubated in six flasks containing 100 mL YBA liquid medium, respectively. After 2 days of incubation at 25°C on a shaker, pydiflumetofen was added into the three flasks to obtained the final concentrations of half maximal effective concentration (EC 50 ) values of the corresponding strain/mutants, respectively, and the other three flasks without fungicide were served as control.…”

Mutations at sterol 14α‐demethylases (CYP51A&B) confer the DMI resistance in Colletotrichum gloeosporioides from grape

“…All isolates were separated from infected fruits that were collected from commercial grape vineyards in Jurong, Zhenjiang, Jiangsu Province in eastern China. Isolates were purified by single conidium and identified by morphology and DNA sequencing of the internal transcribed spacer (ITS) region and β ‐tubulin 29 . Sixty‐six single‐conidium isolates were obtained and kept on potato dextrose agar (PDA) slants at 4 °C.…”

“…The first prochloraz-resistance case in C. gloeosporioides from mango was reported in 2003, 46 and mutations and overexpression of CYP51s of C. gloeosporioides from chili orchards are associated with DMI resistance. 29 In the two other Colletotrichum spp. from peach orchards, however, C. truncatum resistant to flutriafol, fenbuconazole, tebuconazole and metconazole, and C. nymphaeae resistant to flutriafol and fenbuconazole were reported in 2016.…”

“…Isolates were purified by single conidium and identified by morphology and DNA sequencing of the internal transcribed spacer (ITS) region and ⊎-tubulin. 29 Sixty-six single-conidium isolates were obtained and kept on potato dextrose agar (PDA) slants at 4°C. Six azole fungicides, including difenoconazole (96.3%), propiconazole (98%), prochloraz (97%), triadimefon (98%), prothioconazole (97%), and tebuconazole (95%), were used to test sensitivity.…”

Analysis of Colletotrichum musae populations from Brazil reveals the presence of isolates with high competitive ability and reduced sensitivity to postharvest fungicides

“…They act by inhibiting the cytochrome P450 sterol 14α‐demethylase, which is important in the ergosterol biosynthesis pathway (Ziogas and Malandrakis, 2015). Among the mechanisms of resistance to DMIs, point mutation in the CYP51 gene that encodes this enzyme is the most common and has been reported for several plant pathogens (Luo et al ., 2008; Narayanasamy, 2008; Cools et al ., 2010; Mair et al ., 2016), including in the genus Colletotrichum (Xu et al ., 2014; Zhang et al ., 2017; Wei et al ., 2020).…”