Adaptation to pyrrolnitrin in Botrytis cinerea and cost of resistance

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Abstract
The objective of this work was to estimate the risk of a decrease in the efficacy of biocontrol as a result of selection pressure exerted by biocontrol agents on Botrytis cinerea , focusing on pyrrolnitrin, an antibiotic identified in diverse biocontrol agents having an effect on B. cinerea . To evaluate a possible decrease in sensitivity to pyrrolnitrin, 10 successive generations of five isolates of B. cinerea were produced in vitro in the presence of a sublethal dose (10 μg L −1 ) of the antibiotic. For one isolate, a significant reduction in the sensitivity to pyrrolnitrin at the fifth generation was observed with a resistance factor of c . 11. The production of 10 additional generations for four of these isolates, with increasing doses of pyrrolnitrin (100–4000 μg L −1 ), resulted in the development of variants of B. cinerea with high levels of resistance to the antibiotic (RF > 1000) and a reduced sensitivity in vitro to a pyrrolnitrin‐producing bacterium. Reverse adaptation of resistant variants after 10 additional generations in the absence of selection pressure was not observed, suggesting stability of the resistance. Comparison of the pyrrolnitrin‐resistant generations and their sensitive parental isolates for mycelial growth, sporulation and aggressiveness on plant tissues revealed that the high level of resistance to pyrrolnitrin resulted in a high fitness cost. Mycelial growth was reduced between 1·7 to 3·6 times and sporulation reduced 3·8 to 6·6 times that of sensitive parental isolates. Similarly, aggressiveness was 7 to 10 and 3 to 10 times lower for resistant isolates on tomato and apple, respectively. This study provides evidence that a fungal plant pathogen is able to gradually build up resistance to an antibiotic produced by a biocontrol agent.

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