Thirty-eight isolates of Alternaria alternata from pistachio orchards with a history of Pristine (pyraclostrobin + boscalid) applications and displaying high levels of resistance to boscalid fungicide (mean EC(50) values >500 microg/ml) were identified following mycelial growth tests. A cross-resistance study revealed that the same isolates were also resistant to carboxin, a known inhibitor of succinate dehydrogenase (Sdh). To determine the genetic basis of boscalid resistance in A. alternata the entire iron sulphur gene (AaSdhB) was isolated from a fungicide-sensitive isolate. The deduced amino-acid sequence showed high similarity with iron sulphur proteins (Ip) from other organisms. Comparison of AaSdhB full sequences from sensitive and resistant isolates revealed that a highly conserved histidine residue (codon CAC in sensitive isolates) was converted to either tyrosine (codon TAC, type I mutants) or arginine (codon CGC, type II mutants) at position 277. In other fungal species this residue is involved in carboxamide resistance. In this study, 10 and 5 mutants were of type I and type II respectively, while 23 other resistant isolates (type III mutants) had no mutation in the histidine codon. The point mutation detected in type I mutants was used to design a pair of allele-specific polymerase chain reaction (PCR) primers to facilitate rapid detection. A PCR-restriction fragment length polymorphism (RFLP) assay in which amplified gene fragments were digested with AciI was successfully employed for the diagnosis of type II mutants. The relevance of these modifications in A. alternata AaSdhB sequence in conferring boscalid resistance is discussed.

During February 2005, 55 single-spore isolates of Botrytis cinerea were collected at the end of the season from vegetable crops grown in 18 greenhouses on the island of Crete, Greece. They were tested for sensitivity to the anilinopyrimidine fungicides pyrimethanil and cyprodinil, the hydroxyanilide fungicide fenhexamid, the phenylpyrrole fungicide fludioxonil, the dicarboximide fungicide iprodione, and the benzimidazole fungicide carbendazim. Results of the study showed the existence of benzimidazole- and dicarboximide-resistant strains at frequencies of 61.8 and 18%, respectively. Moreover, for first time, the development of resistance to anilinopyrimidine fungicides by B. cinerea was detected in greenhouse vegetable crops on the island of Crete. High resistance frequencies of 49.1 and 57.4% were observed for pyrimethanil and cyprodinil, respectively. In addition, one isolate was found to be resistant to the hydroxyanilide fungicide fenhexamid, while no strains resistant to the phenylpyrrole fungicide were detected. Among the 55 isolates tested, 13 were resistant only to carbendazim, 6 were resistant only to anilinopyrimidines, 3 were resistant to both benzimidazoles and dicarboximides, 17 were resistant to both benzimidazoles and anilinopyrimidines, 6 were resistant to both dicarboximides and anilinopyrimidines, 1 was simultaneously resistant to benzimidazoles, dicarboximides, and anilinopyrimidines, 1 was resistant to both anilinopyrimidines and hydroxyanilides, and 8 were sensitive to all fungicides tested. A strong cross-resistance relationship was found between the two anilinopyrimidine fungicides tested when log transformed EC50 values of the isolates were subjected to a linear regression analysis (r = 0.71). Despite the detection of several phenotypes with simultaneous resistance to chemically unrelated active ingredients, in none of the remaining possible fungicide pairs was there observed any kind of cross-resistance relationship.

This is the first report of B. cinerea field isolates with resistance to both boscalid and pyraclostrobin, and it strongly suggests that there may be a major problem in controlling this important pathogen on kiwifruit.

Botrytis cinerea isolates (n = 122) were collected from strawberry fields located in northern Greece during a 3-year period (2008–10) and tested for their sensitivity to the succinate dehydrogenase inhibitor boscalid. Sensitivity measurements showed three distinct phenotypes consisting of isolates highly sensitive (fungicide concentration causing inhibition of germ tube growth by 50% [EC50 values] of 0.05 to 0.21 μg ml–1), moderately resistant (EC50 values of 1.37 to 7.79 μg ml–1), or highly resistant (EC50 values of >50 μg ml–1) to boscalid. Sequence analysis of the sdhB gene revealed five mutations leading to amino acid substitutions in the SdhB subunit in isolates moderately resistant and highly resistant to boscalid. Three moderately resistant isolates showed a nucleotide change from A to T at codon 230, resulting in an asparagine to isoleucine (N230I) substitution. Several moderately resistant isolates showed a nucleotide change from C to T at codon 272, resulting in a substitution from histidine to arginine (H272R) whereas, in another set of isolates, a nucleotide change from A to G was found at the same codon, leading to a substitution from histidine to tyrosine (H272Y). One highly resistant isolate had a nucleotide change from A to T at codon 272, leading to a substitution from histidine to leucine (H272L), whereas, in three other highly resistant isolates, a double nucleotide change from CC to TT was observed at codon 225, resulting in a substitution from proline to phenylalanine (P225F). To facilitate rapid detection of these mutations associated with resistance to boscalid, a primer-introduced restriction analysis polymerase chain reaction was developed. The method was successfully applied to the moderately and highly resistant subpopulations and showed that the H272R mutation was predominant with relative frequencies of 28.5, 37.5, and 30% during 2008, 2009, and 2010, respectively. In contrast, the H272L mutation was detected at a frequency of 2.5% only in the 2009 population, whereas the P225F mutation was detected at a frequency of 7.5% only in the 2010 population.

Succinate dehydrogenase inhibiting (SDHI) fungicides constitute a relatively novel fungicide group used for gray mold control caused mainly by Botrytis cinerea. Shortly after registration, resistance was observed in fungal populations that correlated with several mutations in the succinate dehydrogenase complex (complex II). In the current study, 30 B. cinerea isolates possessing five different mutations at three different codons of SdhB (P225F, N230I, and H272L/R/Y) were characterized for their sensitivities to eight SDHI fungicides. The results show different sensitivities and cross-resistance patterns between structurally different SDHIs. P225F mutants were resistant in vitro to all SDHIs tested. Similarly, isolates possessing the H272L mutation were highly resistant to boscalid but showed low to moderate levels of resistance to other SDHIs. The N230I mutants were moderately resistant to boscalid, fluopyram, and fluxapyroxad and showed low resistance levels to isopyrazam, bixafen, fenfuram, benodanil, and carboxin. The H272R mutants showed moderate levels of resistance to boscalid and low resistance levels to isopyrazam, fenfuram, and carboxin but remained sensitive to fluopyram, bixafen, fluxapyroxad, and benodanil. Similarly, the H272Y showed moderate levels of resistance to boscalid and very low resistance levels to isopyrazam, bixafen, fenfuram, and carboxin but showed increased sensitivity to benodanil and fluopyram. Boscalid provided moderate to high control of H272R/Y and N230I mutants in detached fruit assays but provided little control against the H272L and P225F mutants. In contrast, fluopyram controlled H272R/Y mutants and provided moderate levels of control toward H272L, N230I, and P225F mutants. Our findings suggest that sensitivity to SDHIs may vary greatly, dependent on the point mutation in the sdhb subunit.