Multiple and multidrug resistance in Botrytis cinerea: molecular mechanisms of MLR/MDR strains in Greece and effects of co-existence of different resistance mechanisms on fungicide sensitivity

Abstract

Botrytis cinerea is a high-risk pathogen for fungicide resistance development. Within the fungal populations, strains have developed multiple mutations in different target genes leading to multiple resistance (MLR) or mutations associated with overexpression of efflux transporters leading to multidrug resistance (MDR). These types of resistance are a major threat, and their successful management is a major challenge. The current study was initiated to a) determine frequencies of MLR/MDR strains in populations originating from several crops, b) identify the types of MDR that occur in Greece, and c) determine interactions between MLR and MDR at the level of sensitivity to botryticides. The frequencies of MLR/MDR phenotypes were determined in 515 isolates subjected to bioassays using discriminatory concentrations of thiophanate-methyl, iprodione, cyprodinil, fenhexamid, boscalid, fluopyram, fludioxonil, pyraclostrobin, and tolnaftate. Interestingly, 7.8% and 31.3% of isolates from strawberry and rootstock seedlings were resistant to every single fungicide class, while MDR phenotypes from strawberries, rootstocks, and tomatoes accounted for 26%, 87%, and 13.4%, respectively. The MLR and MDR isolates were further molecularly analyzed regarding genes erg27, sdhB, Bcpos5, and Mrr1, responsible for resistance to fenhexamid, boscalid and fluopyram, cyprodinil, and MDR, respectively. The different mutations’ presence was determined along with a new mutation in Mrr1 leading to MDR. MDR isolates were characterized as MDR1 or MDR1h based on the presence of a 3-bp deletion in Mrr1. MDR1h was predominant in isolates from rootstocks and MDR1 from tomatoes and strawberries, whereas the most frequent target-site mutations were F412S (erg27), H272R (sdhB), and L412F (Bcpos5). To determine whether the accumulation of target-site mutations along with MDR mutations exhibits an additive effect concerning fungicide resistance, the sensitivity of isolates possessing the predominant target-site mutations was calculated in both the presence and the absence of MDR-associated mutations. EC50 in cyprodinil and boscalid increased to about twofold in the presence of MDR mutations, while there was no difference for fenhexamid. In conclusion, MLR/MDR frequencies are notably high in heavily treated crops in Greece, and the combination of MLR and MDR mutations leads to even higher fungicide resistance levels, highlighting the importance of resistance management.

Carboxylesterase and Cytochrome P450 Confer Metabolic Resistance Simultaneously to Azoxystrobin and Some Other Fungicides in Botrytis cinerea

“…42 Additionally, overexpression of efflux transporters also results in the development of resistance in fungi against several fungicides with different MOAs simultaneously. 18 This is evidenced by the four different MDR strains of B. cinerea reported, showing variations in both the range and level of resistance to fungicides and correlated with overexpression of efflux transporters encoded by atrB and mfsM2 genes. 43−45 Overexpression of efflux proteins typically leads to low to moderate levels of resistance.…”

“…Target site modifications are often associated with high levels of resistance, such as the G143A mutation in the CytB gene causing high-level resistance to QoI fungicides in various pathogens . Additionally, overexpression of efflux transporters also results in the development of resistance in fungi against several fungicides with different MOAs simultaneously . This is evidenced by the four different MDR strains of B.…”

“…cinerea has developed resistance to QoIs, aniline pyrimidine, phenylpyrrole, benzimidazole, dicarboximide, and various fungicides with distinct modes of action (MOAs). − Moreover, some strains have developed resistance to fungicides with different (MOAs) simultaneously, indicating the emergence of multiple fungicide resistance (MFR) and multidrug resistance (MDR). ,− The MFR typically arises from multiple mutations in different target genes. The mechanism of MDR in pathogenic fungi is primarily attributed to the overexpression of efflux transporters, − while metabolism-associated resistance or metabolic resistance have also been reported. , These types of resistance pose a significant threat to the successful management of Botrytis cinerea …”

“…16,17 These types of resistance pose a significant threat to the successful management of Botrytis cinerea. 18 Metabolic resistance is often due to the increased metabolism of pesticides within an organism, transforming these chemicals from parent compounds into functionally inactive structures. 19 Additionally, propesticides lacking inherent biological activities require conversion into active compounds after application.…”

A Conservative Mutant Version of the Mrr1 Transcription Factor Correlates with Reduced Sensitivity to Fludioxonil in Botrytis cinerea

“…The transcriptional regulation of atrB is convergent among various fungi and depends on the transcription factor Mrr1 [35]. Mutations in the mrr1 gene result in two main phenotypes: MDR1, conferring low resistance and characterized by different point mutations in the mrr1 gene [34,[36][37][38], and MDR1h, conferring moderate-to-high resistance to fludioxonil and characterized by a 3-bp deletion mutation in mrr1, leading to an amino acid deletion of L497 [38][39][40].…”