Molecular Characterization of Boscalid Resistance in Field Isolates of Botrytis cinerea from Apple

Abstract

Botrytis cinerea isolates obtained from apple orchards were screened for resistance to boscalid. Boscalid-resistant (BosR) isolates were classified into four phenotypes based on the levels of the concentration that inhibited fungal growth by 50% relative to control. Of the 220 isolates tested, 42 were resistant to boscalid, with resistant phenotypes ranging from low to very high resistance. There was cross resistance between boscalid and carboxin. Analysis of partial sequences of the iron-sulfur subunit of succinate dehydrogenase gene in B. cinerea (BcSdhB) from 13 BosR and 9 boscalid-sensitive (BosS) isolates showed that point mutations in BcSdhB leading to amino acid substitutions at the codon position 272 from histidine to either tyrosine (H272Y) or arginine (H272R) were correlated with boscalid resistance. Allele-specific polymerase chain reaction (PCR) analysis of 66 BosR isolates (including 24 additional isolates obtained from decayed apple fruit) showed that 19 carried the point mutation H272Y and 46 had the point mutation H272R, but 1 BosR isolate gave no amplification product. Analysis of the BcSdhB sequence of this isolate revealed a different point mutation at codon 225, resulting in a substitution of proline (P) by phenylalanine (F) (P225F). The results indicated that H272R/Y in BcSdhB were the dominant genotypes of mutants in field BosR isolates from apple. A multiplex allele-specific PCR assay was developed to detect point mutations H272R/Y in a single PCR amplification. Levels of boscalid resistance ranged from low to very high within isolates carrying either the H272R or H272Y mutation, indicating that, among BosR isolates, different BosR phenotypes (levels of resistance) were not associated with particular types of point mutations (H272R versus H272Y) in BcSdhB. Analysis of genetic relationships between 39 BosR and 56 BosS isolates based on three microsatellite markers showed that 39 BosR isolates and 30 BosS isolates were clustered into two groups, and the third group consisted of only BosS isolates, suggesting that the development of resistance to boscalid in B. cinerea likely is not totally random, and resistant populations may come from specific genetic groups.

Frequency of fungicide-resistant Botrytis cinerea populations isolated from ‘Thompson Seedless’ table grapes in the Central Valley of Chile

“…Our results showed that H272R was the predominant mutation associated with the randomly selected Bos R B. cinerea isolates; 60% (n=18) possessed the H272R substitution and 40% (n=12) possessed the H272Y substitution, in agreement with previous studies conducted on grapevines where sdhB H272Y and sdhB H272R were the most frequent mutations in B. cinerea populations treated with boscalid (Esterio, 2014;Veloukas et al, 2011;Yin et al, 2011). In Champagne vineyards (Walker et al 2013), due to the availability of other classes of botryticides and the limited use of SDHIs, boscalid resistance frequency has not become widespread, and the emergence of the moderately resistant mutants sdhB H272Y and sdhB H272R has been favored over more resistant mutants.…”

Detection of Botrytis cinerea field isolates with multiple fungicide resistance from table grape in Sicily

“…Currently, field isolates of B. cinerea resistant to boscalid have been reported in a limited number of hosts Bardas et al, 2010;Fern andez-Ortuño et al, 2014;Veloukas et al, 2011;Yin et al, 2011) including grape in Germany (Wine Road region), France (Champagne region) and, more recently, in Italy (Apulia region) (De Miccolis Angelini et al, 2014;Leroch et al, 2011;Leroux et al, 2010). The low frequency of boscalid-resistant genotypes of B. cinerea detected in Sicilian vineyards and conferred by the SdhB H272R/Y mutation, could be due both to its relatively recent introduction (2006 in Italy) and the fact that after the product launch farmers did not use the fungicide frequently, performing a maximum of one application per growing season in recent years.…”

Baseline sensitivity and resistance risk assessmemt of Rhizoctonia cerealis to thifluzamide, a succinate dehydrogenase inhibitor

“…Although laboratory and field mutants with resistance to the SDHIs have been reported in eleven plant pathogens, including Ustilago maydis [17], Mycosphaerella graminicola [18][19][20][21][22], Botrytis cinerea [23][24][25][26], Botrytis elliptica from lilies [22], Alternaria alternata from pistachio [27][28][29], Corynespora cassiicola from cucurbits [30,31], Didymella bryoniae from cucurbits [32], Podosphaera xanthii from cucurbits (information from meeting by member of FRAC SDHI working group), Sclerotinia sclerotiorum from oilseed rape [16,21] and Stemphylium botryose from asparagus (information from meeting by member of FRAC SDHI working group), and in phytopathogenic bacteria, including Xanthomanas campestris pv. citri isolates from citrus (Citus reticulata) [33] and Xanthomonas oryzae pv.…”