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.
The obtained biological activity, baseline sensitivity and cross-resistance relationship data suggest that fluopyram could play a key role in grey mould management in the near future and encourage its introduction into spray programmes.
Respiration inhibitors such as the succinate dehydrogenase inhibitors (SDHIs) and the quinone outside inhibitors (QoIs) are fungicide classes with increasing relevance in gray mold control. However, recent studies have shown that dual resistance to both fungicide classes is a common trait in Botrytis cinerea populations from several hosts throughout the world. Resistance of B. cinerea to SDHIs is associated with several mutations in the sdhB, sdhC, and sdhD genes, while resistance to QoIs, in most cases, is associated with the G143A mutation in the cytb gene. The objective of the current study was to investigate the fitness and the competitive ability of B. cinerea field strains possessing one of the H272Y/R/L, N230I, or P225F sdhB substitutions and the G143A mutation of cytb. Fitness parameters measured were (i) mycelial growth and conidia germination in vitro, (ii) aggressiveness and sporulation capacity in vivo, (iii) sclerotia production in vitro and sclerotia viability under different storage conditions, and (iv) sensitivity to oxidative stress imposed by diquat treatments. The competitive ability of the resistant isolates was measured in the absence and presence of the SDHI fungicides boscalid and fluopyram selection pressure. The measurements of individual fitness components showed that the H272R/G143A isolates had the lower differences compared with the sensitive isolates. In contrast, the groups of H272Y/L/G143A, N230I/G143A, and P225F/G143A isolates showed reduced fitness values compared with the sensitive isolates. Isolates possessing only the cytb G143A substitution did not show any fitness cost. The competition experiments showed that, in the absence of fungicide selection pressure, after four disease cycles on apple fruit, the sensitive isolates dominated in the population in all the mixtures tested. In contrast, when the competition experiment was conducted under the selection pressure of boscalid, a gradual decrease in the frequency of sensitive isolates was observed, whereas the frequency of H272L and P225F isolates was increased. When the competition experiment was conducted in the presence of fluopyram, the sensitive isolates were eliminated even after the first disease cycle and the P225F mutants dominated in the population. Such results suggest that the sdhB mutations may have adverse effects on the mutants. The observed dominance of sensitive isolates in the competition experiments conducted in the absence of fungicides suggest that the application of SDHIs in alternation schemes may delay the selection or reduce the frequency of SDHI-resistant mutants.
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