Lack of an Intron in Cytochrome b and Overexpression of Sterol 14α-Demethylase Indicate a Potential Risk for QoI and DMI Resistance Development in Neophysopella spp. on Grapes

Abstract

Asian grapevine leaf rust, caused by Neophysopella meliosmae-myrianthae and N. tropicalis, is often controlled by quinone outside inhibitor (QoI) and demethylation inhibitor (DMI) fungicides in Brazil. Here, we evaluated the sensitivity of 55 Neophysopella spp. isolates to pyraclostrobin (QoI) and tebuconazole (DMI). To elucidate the resistance mechanisms, we analyzed the sequences of the cytochrome b (CYTB) and cytochrome P450 sterol 14α-demethylase (CYP51) target proteins of QoI and DMI fungicides, respectively. The CYP51 expression levels were also determined in a selection of isolates. In leaf disc assays, the mean 50% effective concentration (EC50) value for pyraclostrobin was around 0.040 µg/ml for both species. CYTB sequences were identical among all 55 isolates, which did not contain an intron immediately after codon 143. No amino acid substitution was identified at codons 129, 137 and 143. The mean EC50 value for tebuconazole was 0.62 µg/ml for N. tropicalis and 0.46 µg/ml for N. meliosmae-myrianthae and no CYP51 sequence variation was identified among isolates of the same species. However, five N. meliosmae-myrianthae isolates grew on leaf discs treated at 10 µg/ml tebuconazole and these were further exposed to tebuconazole selection pressure. Tebuconazole-adapted laboratory isolates of N. meliosmae-myrianthae showed an eight- to 25-fold increase in resistance after four rounds of selection that was not associated with CYP51 target alterations. In comparison with sensitive isolates, CYP51 expression was induced in the presence of tebuconazole in three out of four tebuconazole-adapted isolates tested. These results suggest a potential risk for QoI and DMI resistance development in Neophysopella spp.

Molecular Characterization and Overexpression of CYP51 gene of Difenoconazole Resistance in Lasiodiplodia Theobromae Field Isolates

“…Our study showed that overexpression of LtCYP51 of resistant isolates was observed in response to difenoconazole. This is consistent with what has been found in Mycosphaerella graminicola of wheat, Blumeriella jaapii of cherry and Neophysopella meliosmae-myrianthae of grapevine 30,43,44 . Overexpression of CYP51 caused by promoter insertions or retrotransposons has only been described in a few phytopathogenic fungi so far.…”

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

“…Some studies reported mutations and overexpression of the CYP51 gene simultaneously in some DMI-resistant isolates of plant pathogens 17 , 20 , 45 . Furthermore, in L. theobromae of papaya, Mycosphaerella graminicola of wheat, Blumeriella jaapii of cherry and Neophysopella meliosmae-myrianthae of grapevine no CYP51 gene point mutation linked to DMI resistance has been identified, but CYP51 overexpression has been observed 18 , 30 , 46 , 47 . A similar pattern of results was obtained in this study.…”

Damage on grapevine cv. Niagara Rosada leaves caused by the combined effect of temperature and Asian grapevine leaf rust (Neophysopella tropicalis)

“…Asian grapevine leaf rust (AGLR), caused by Neophysopella tropicalis , promotes severe damage to grapevine production, especially in latitudes lower than 25° S (Santos, Amorim, et al, 2021), which include the largest grapevine‐producing regions in Brazil. Most cultivars of Vitis labrusca as well as V. vinifera and their interspecific hybrids are susceptible to rust, although the occurrence of the disease in the field is more frequent in V .…”