First Report of the β-Tubulin E198A Mutation Conferring Resistance to Methyl Benzimidazole Carbamates in European Isolates of Monilinia fructicola

Abstract

The causal agent of brown rot on stone and pome fruits, Monilinia fructicola (G. Wint.), is a quarantine pathogen in Europe. It has been detected in Austria (later eradicated), Spain, the Czech Republic, Italy, Germany, and Switzerland (1). In the United States and other countries, M. fructicola isolates were reported to show resistance to different classes of fungicides, including methyl benzimidazole carbamates (MBC) (2). Lichou et al. (2) reported the presence of isolates resistant to the MBC carbendazim in France, but the mechanisms inducing MBC resistance in these isolates were not studied. Ma et al. (3) in California, and more recently, Zhu et al. (4) in South Carolina, demonstrated that the molecular mechanisms accounting for low and high levels of resistance to MBC fungicides in M. fructicola isolates were the mutations H6Y and E198A, respectively, in the β-tubulin gene. Four M. fructicola isolates each from Italy, France, Spain, and Switzerland (16 isolates total), all having an unknown level of MBC resistance, were selected. In each isolate, the section of the β-tubulin gene containing the two potentially mutant codons was PCR-amplified with the primers TubA and TubR1 (3) and the amplicons were sequenced directly. Sequence analysis revealed the amino acid histidine (H) at codon 6 in all the isolates, which would not predict MBC resistance, while alanine (A) at codon 198 (the mutation predictive of a high level of MBC resistance) was found in all isolates from Spain and Switzerland and in three isolates each from France and Italy. A representative sequence of the four identical partial β-tubulin gene sequences from the Swiss isolates was submitted to GenBank under the Accession No. HQ709265. All isolates were tested in a potato dextrose agar (PDA) petri dish assay for resistance to the MBC fungicide thiophanate-methyl (Nippon Soda Co., Ltd., Tokyo, Japan) at the discriminatory dose of 50 μg/ml (4). All isolates with the E198A mutation were able to grow on the media, while the two isolates without the E198A mutation were not able to grow. The result indicated that most isolates had a high level of resistance to the MBC fungicide. To our knowledge, this is the first report of the presence of the E198A mutation conferring resistance to MBC fungicides in European isolates of M. fructicola. As the mutation appears to be widely distributed, we anticipate that MBC fungicides may be ineffective at controlling brown rot in countries with occurrence of M. fructicola. References: (1) M. Hilber-Bodmer et al. Plant Dis. 94:643, 2010. (2) J. Lichou et al. Phytoma 547:22, 2002. (3) Z. H. Ma et al. Appl. Environ. Microbiol. 69:7145, 2003. (4) F. X. Zhu et al. Plant Dis. 94:1511, 2010.

Sensitivity of Monilinia fructicola from Spanish peach orchards to thiophanate-methyl, iprodione, and cyproconazole: fitness analysis and competitiveness

“…In a recent study that included four M. fructicola isolates of our population, it was demonstrated that mutations leading to high levels of resistance to MBC fungicides were present in Europe (Weger et al 2011). Despite the fact that resistance to fungicides has been also detected in M. laxa populations in Europe (Thomidis et al 2009;Malandrakis et al 2012Malandrakis et al , 2013, we have no detected resistance to MBCs, DFCs or DMIs until 2006, in our M. laxa study population.…”

Analysis of genetic diversity in Monilinia fructicola from the Ebro Valley in Spain using ISSR and RAPD markers

“…Furthermore, rare alleles from the source population may become common in the new population if they are present in the founders of this new population (Milgroom et al 1992). Weger et al (2011) has proposed that these processes could explain the high levels of resistance against methyl benzimidazole carbamate (MBC) that has been found in some M. fructicola isolates from Italy, France, Spain, and Switzerland posing a significant risk for peach production in the Ebro Valley, Spain, where M. fructicola is now present. Disease management programs against M. fructicola should take this into account in order to slow down the development of fungicidal-resistant strains.…”

Epidemiología y control de la podredumbre parda del melocotón en postcosecha

“…However, fungicide-based methods are not sustainable in the long term because of the emergence and subsequent spread of M. fructicola and M. laxa strains with reduced sensitivity to fungicides (Egüen et al, 2015;Ma et al, 2005;Malandrakis et al, 2013;Thomidis et al, 2009;Weger et al, 2011;. Recent studies in the Ebro Valley have detected a M. fructicola population with isolates that were single resistant, double resistant, or even triple resistant to tiophanate-methyl, iprodione and tebuconazole (Egüen et al, 2015).…”

“…11). Furthermore, since 2014 M. (Weger et al, 2011). Due to the extended usage of molecular tools in disease diagnostic, it has been discovered that there are at least six species causing brown rot on Prunus spp., three more species than the initialy described (M. fructicola, M. fructigena, and M. laxa).…”