Survey and genetic analysis of demethylation inhibitor fungicide resistance in Monilinia fructicola from Michigan orchards

Resistance to sterol demethylation inhibitor fungicides (DMIs) in Monilinia fructicola, causal agent of brown rot of stone fruit, has been reported in the southeastern and eastern United States and in Brazil. DMI resistance of some M. fructicola isolates, in particular those recovered from the southeastern U.S., is associated with a sequence element termed ‘Mona’ that causes overexpression of the cytochrome demethylase target gene MfCYP51. In this study, we conducted statewide surveys of Michigan stone fruit orchards from 2009-2011 and in 2019, and determined the sensitivity to propiconazole of a total of 813 isolates of M. fructicola. A total of 80.7% of Michigan isolates were characterized as resistant to propiconazole by relative growth assays but the ‘Mona’ insert was not uniformly detected, and was present in some isolates that were not characterized as DMI resistant. Gene expression assays indicated that elevated expression of MfCYP51 was only weakly correlated with DMI-resistance in M. fructicola isolates from Michigan, and there was no obvious correlation between the presence of the ‘Mona’ element and elevated expression of MfCYP51. However, sequence analysis of MfCYP51 from 25 DMI-resistant isolates did not reveal any point mutations that could be correlated with resistance. Amplification and sequencing upstream of MfCYP51 resulted in detection of DNA insertions in a wide range of isolates typed by DMI phenotype and the presence of ‘Mona’ or other unique sequences. The function of these unique sequences or their presence upstream of MfCYP51 cannot be correlated to a DMI-resistant genotype at this time. Our results indicate that DMI resistance was established in Michigan populations of M. fructicola by 2009 to 2011, and that relative resistance levels have continued to increase to the point that practical resistance is present in most orchards. In addition, the presence of the ‘Mona’ insert is not a marker for identifying DMI-resistant isolates of M. fructicola in Michigan.

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Identification and Characterization of Benzimidazole Resistance in Monilinia fructicola from Stone Fruit Orchards in California

Applied and Environmental Microbiology ◽

10.1128/aem.69.12.7145-7152.2003 ◽

2003 ◽

Vol 69 (12) ◽

pp. 7145-7152 ◽

Cited By ~ 127

Author(s):

Zhonghua Ma ◽

Michael A. Yoshimura ◽

Themis J. Michailides

Keyword(s):

Dna Sequences ◽

Point Mutations ◽

Brown Rot ◽

Stone Fruit ◽

Monilinia Fructicola ◽

Tubulin Gene ◽

Benzimidazole Fungicides ◽

Specific Pcr ◽

Allele Specific ◽

Fruit Orchards

ABSTRACT Low and high levels of resistance to the benzimidazole fungicides benomyl and thiophanate-methyl were observed in field isolates of Monilinia fructicola, which is the causative agent of brown rot of stone fruit. Isolates that had low levels of resistance (hereafter referred to as LR isolates) and high levels of resistance (hereafter referred to as HR isolates) were also cold and heat sensitive, respectively. Results from microsatellite DNA fingerprints showed that genetic identities among the populations of sensitive (S), LR, and HR isolates were very high (>0.96). Analysis of DNA sequences of theβ -tubulin gene showed that the LR isolates had a point mutation at codon 6, causing a replacement of the amino acid histidine by tyrosine. Codon 198, which encodes a glutamic acid in S and LR isolates, was converted to a codon for alanine in HR isolates. Based on these point mutations in the β-tubulin gene, allele-specific PCR assays were developed for rapid detection of benzimidazole-resistant isolates of M. fructicola from stone fruit.

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Characterizing Fenbuconazole and Propiconazole Sensitivity and Prevalence of ‘Mona’ in Isolates of Monilinia fructicola from New York

Plant Disease ◽

10.1094/pdis-12-10-0943 ◽

2011 ◽

Vol 95 (7) ◽

pp. 828-834 ◽

Cited By ~ 16

Author(s):

Sara M. Villani ◽

Kerik D. Cox

Keyword(s):

New York ◽

No Reduction ◽

Quantitative Resistance ◽

Brown Rot ◽

Resistance Determinant ◽

Monilinia Fructicola ◽

Eastern United States ◽

Resistance Response ◽

Dmi Fungicides ◽

Dmi Resistance

Demethylation inhibitor (DMI) resistant populations of Monilinia fructicola, the causal agent of brown rot of stone fruit, and the presence of the genetic DMI resistance determinant ‘Mona’ have been reported throughout the eastern United States. In this study, we endeavored to conduct a comprehensive investigation of DMI sensitivity, the prevalence of ‘Mona’, and implications of DMI use for M. fructicola populations from New York and Pennsylvania. Of the 18 orchards surveyed, only 9 were primarily composed of isolates with either resistance or reduced sensitivity to fenbuconazole and propiconazole. The DMI resistance determinant ‘Mona’ was only found in 5 orchards, present in isolates with a range of sensitivity phenotypes, and not always present in resistant isolates. These results suggested that ‘Mona’ only contributes to a portion of the quantitative resistance response to DMI fungicides. On detached blossoms and fruit, protective applications of fenbuconazole (Indar 2F) against isolates with resistance or reduced sensitivity resulted in significantly (P < 0.05) lower brown rot incidence compared to applications of propiconazole (Orbit 3.6EC) and water controls. By comparison, therapeutic applications of fenbuconazole and propiconazole against isolates with resistance or reduced sensitivity provided little to no reduction in brown rot incidence on blossoms and fruit.

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Cross-Resistance Among Demethylation Inhibitor Fungicides With Brazilian Monilinia fructicola Isolates as a Foundation to Discuss Brown Rot Control in Stone Fruit

Plant Disease ◽

10.1094/pdis-04-20-0714-re ◽

2020 ◽

Vol 104 (11) ◽

pp. 2843-2850

Author(s):

Pamela Suellen Salvador Dutra ◽

Paulo S. F. Lichtemberg ◽

Maria Bernat Martinez ◽

Themis J. Michailides ◽

Louise Larissa May De Mio

Keyword(s):

Brown Rot ◽

Lesion Size ◽

Stone Fruit ◽

Cross Resistance ◽

Monilinia Fructicola ◽

Wild Type ◽

Specific Test ◽

Allele Specific ◽

Fruit Orchards ◽

Demethylation Inhibitor

Despite the resistance problems in Monilinia fructicola, demethylation inhibitor fungicides (DMIs) are still effective for the disease management of brown rot in commercial stone fruit orchards in Brazil. This study aims to investigate the sensitivity of M. fructicola isolates and efficiency of DMIs to reduce brown rot. A set of 93 isolates collected from Brazilian commercial orchards were tested for their sensitivities to tebuconazole, propiconazole, prothioconazole, and myclobutanil. The isolates were analyzed separately according to the presence or absence of the G461S mutation in MfCYP51 gene, determined by allele-specific test. The mean EC50 values for G461S mutants and wild-type isolates were respectively 8.443 and 1.13 µg/ml for myclobutanil, 0.236 and 0.026 µg/ml for propiconazole, 0.115 and 0.002 µg/ml for prothioconazole, and 1.482 and 0.096 µg/ml for tebuconazole. The density distribution curves of DMI sensitivity for both genotypes showed that myclobutanil and prothioconazole curves were mostly shifted toward resistance and sensitivity, respectively. Incomplete cross-resistance was detected among propiconazole and tebuconazole in both wild-type (r = 0.45) and G461S (r = 0.38) populations. No cross-sensitivity was observed among wild-type isolates to prothioconazole and the others DMIs tested. Fungicide treatments on detached fruit inoculated with M. fructicola genotypes showed significant DMI efficacy differences when fruit were inoculated with wild-type and G461S isolates. Protective applications with prothioconazole were more effective for control of both G461S and wild-type isolates compared with tebuconazole. Curative applications with tebuconazole were most effective in reducing the incidence and lesion size of G461S isolates. Sporulation occurred only for G461S isolates treated with tebuconazole under curative and preventative treatments. The differences found among the performance of triazoles against M. fructicola isolates will form the basis for recommendations of rational DMI usage to control brown rot in Brazil.

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The Point Mutation G461S in the MfCYP51 Gene is Associated with Tebuconazole Resistance in Monilinia fructicola Populations in Brazil

Phytopathology ◽

10.1094/phyto-02-17-0050-r ◽

2017 ◽

Vol 107 (12) ◽

pp. 1507-1514 ◽

Cited By ~ 17

Author(s):

Paulo S. F. Lichtemberg ◽

Yong Luo ◽

Rafael G. Morales ◽

Juliana M. Muehlmann-Fischer ◽

Themis J. Michailides ◽

...

Keyword(s):

Gene Expression ◽

Point Mutation ◽

Molecular Mechanisms ◽

Brown Rot ◽

Polymerase Chain Reaction Method ◽

Stone Fruit ◽

Monilinia Fructicola ◽

Nucleotide Position ◽

Gene Encoding ◽

Elevated Expression

The ascomycete Monilinia fructicola is the causal agent of brown rot of stone fruit in Brazil, causing major pre- and postharvest losses. For many years, the demethylation inhibitor (DMI) fungicide tebuconazole has been used as the most effective active ingredient for controlling brown rot and, as a result, strains of M. fructicola resistant to this ingredient have emerged in many Brazilian states producing stone fruit. The aim of this study was to investigate the mechanisms associated with the resistance of M. fructicola to DMI tebuconazole. By sequencing the M. fructicola CYP51 (MfCYP51) gene, encoding the azole target sterol 14α-demethylase, a mutation was identified at the nucleotide position 1,492, causing the amino acid substitution from glycine to serine at the codon position 461, associated with reduced tebuconazole sensitivity. In addition, it was observed that MfCYP51 gene expression could play a secondary role in DMI fungicide resistance of M. fructicola strains in Brazil. However, for the specific isolate found to exhibit elevated expression levels of MfCYP51, no insertions that would trigger gene expression were found. Based on the point mutation associated with tebuconazole resistance, an allele-specific polymerase chain reaction method was developed to quickly identify resistant genotypes within the Brazilian population. This is the first report determining molecular mechanisms for DMI resistance identification for M. fructicola isolates from Brazil. This information provides an important advancement for risk assessment of DMI fungicides used to manage brown rot of stone fruit.

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Detection of Latent Monilinia Infections in Nectarine Flowers and Fruit by qPCR

Plant Disease ◽

10.1094/pdis-11-16-1682-re ◽

2017 ◽

Vol 101 (6) ◽

pp. 1002-1008 ◽

Cited By ~ 3

Author(s):

C. Garcia-Benitez ◽

P. Melgarejo ◽

A. De Cal

Keyword(s):

Prunus Persica ◽

Brown Rot ◽

Stone Fruit ◽

Monilinia Fructicola ◽

Latent Infections ◽

Established Method ◽

Latently Infected ◽

Dna Amounts

Most stone fruit with a latent brown rot infection caused by Monilinia do not develop visible signs of disease until the arrival of fruit at the markets or the consumer’s homes. The overnight freezing-incubation technique (ONFIT) is a well-established method for detecting latent brown rot infections, but it takes between 7 to 9 days. In this report, we inform on the advantages of applying a qPCR-based method to (i) detect a latent brown rot infection in the blossoms and fruit of nectarine trees (Prunus persica var. nucipersica) and (ii) distinguish between the Monilinia spp. in them. For applying this qPCR-based method, artificial latent infections were established in nectarine flowers and fruit using 10 Monilinia fructicola isolates, 8 M. fructigena isolates, and 10 M. laxa isolates. We detected greater amounts of M. fructicola DNA than M. laxa and M. fructigena DNA in latently infected flowers using qPCR. However, greater DNA amounts of M. laxa than M. fructicola were detected in the mesocarp of latently infected nectarines. We found that the qPCR-based method is more sensitive, reliable, and quicker than ONFIT for detecting a latent brown rot infection, and could be very useful in those countries where Monilinia spp. are classified as quarantine pathogens.

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Field Strains of Monilinia fructicola Resistant to Both MBC and DMI Fungicides Isolated from Stone Fruit Orchards in the Eastern United States

Plant Disease ◽

10.1094/pdis-12-12-1177-re ◽

2013 ◽

Vol 97 (8) ◽

pp. 1063-1068 ◽

Cited By ~ 41

Author(s):

F. Chen ◽

X. Liu ◽

G. Schnabel

Keyword(s):

South Carolina ◽

Sweet Cherry ◽

Management Strategies ◽

Stone Fruit ◽

Monilinia Fructicola ◽

Eastern United States ◽

Peach Fruit ◽

Thiophanate Methyl ◽

Dmi Fungicides ◽

Fruit Orchards

In 2012, significant brown rot disease was observed on stone fruit in Pennsylvania, Maryland, and South Carolina despite preharvest application of methyl benzimidazole carbamate (MBC) and demethylase inhibitor (DMI) fungicides. In total, 140 Monilinia fructicola isolates were collected from diseased orchards and examined for fungicide sensitivity. In addition to isolates resistant to either the DMI propiconazole or the MBC thiophanate-methyl, 22 isolates were discovered that were resistant to both fungicides, including 4 isolates from peach in South Carolina, 12 isolates from peach and sweet cherry in Maryland, and 6 isolates from sweet cherry in Pennsylvania. Analysis of MBC resistance revealed that dual-resistant isolates from South Carolina carried the β-tubulin E198A mutation, whereas isolates from Maryland and Pennsylvania carried E198 mutations not previously described in the Monilinia genus, E198Q or F200Y. The genetic element Mona, associated with DMI fungicide resistance in M. fructicola, was detected in the dual-resistant isolates from South Carolina but not in the isolates from the two more northern states. An investigation into the molecular mechanism of DMI resistance in the latter isolates revealed that resistance was not based on increased expression or mutation of MfCYP51, which encodes the target of DMI fungicides. Label rates of formulated propiconazole or thiophanate-methyl were unable to control dual-resistant isolates on detached peach fruit, confirming field relevance of dual resistance. The same isolates were not affected by fitness penalties based on mycelial growth rate, ability to sporulate, and virulence on detached peach fruit. The emergence of M. fructicola strains resistant to both DMI and MBC fungicides in multiple states and multiple stone fruit crops is a significant development and needs to be considered when designing resistance management strategies in stone fruit orchards.

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Significance of Thinned Fruit as a Source of the Secondary Inoculum of Monilinia fructicola in California Nectarine Orchards

Plant Disease ◽

10.1094/pdis.1997.81.5.519 ◽

1997 ◽

Vol 81 (5) ◽

pp. 519-524 ◽

Cited By ~ 68

Author(s):

Chuanxue Hong ◽

Brent A. Holtz ◽

David P. Morgan ◽

Themis J. Michailides

Keyword(s):

Brown Rot ◽

Weather Conditions ◽

San Joaquin Valley ◽

Stone Fruit ◽

Monilinia Fructicola ◽

Inoculum Source ◽

Secondary Infections ◽

Fruit Orchards ◽

Semi Arid

The significance of thinned fruit as a source of secondary inoculum in the spread of brown rot, caused by Monilinia fructicola, under semi-arid weather conditions of the San Joaquin Valley in California, was investigated in seven nectarine orchards in 1995 and 1996. Between 6 and 60% (depending on the orchard) of thinned fruit showed sporulation by M. fructicola. Brown rot was significantly less severe at preharvest (five orchards) and postharvest (one orchard) on fruit harvested from trees in plots from which thinned fruit were completely removed than on those in plots from which thinned fruit were not removed. M. fructicola sporulated more frequently on thinned fruit placed into irrigation trenches than on those left on the dry berms in tree rows. The incidence of preharvest fruit brown rot increased exponentially as the density of thinned fruit increased on the orchard floor. These results suggest that thinned fruit left on the floor of nectarine orchards can be a significant inoculum source of secondary infections. Removal or destruction of thinned fruit should reduce brown rot in nectarine and possibly other stone fruit orchards under semi-arid California conditions.

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First Report of Brown Rot of Stone Fruit Caused by Monilinia fructicola in Italy

Plant Disease ◽

10.1094/pdis-93-6-0668a ◽

2009 ◽

Vol 93 (6) ◽

pp. 668-668 ◽

Cited By ~ 39

Author(s):

C. Pellegrino ◽

M. L. Gullino ◽

A. Garibaldi ◽

D. Spadaro

Keyword(s):

Fungal Pathogens ◽

Brown Rot ◽

Stone Fruit ◽

Fruit Rot ◽

Brown Spot ◽

Morphological Identification ◽

Monilinia Fructicola ◽

Stone Fruits ◽

First Report ◽

Sequence Characterized Amplified Region

Monilinia fructicola, causal agent of brown rot, is one of the most important fungal pathogens of stone fruit. M. fructicola is a quarantined pathogen in Europe. During the summer of 2008 in 15 orchards located in Piedmont (northern Italy), 12,500 stone fruits (cherries, apricots, peaches, nectarines, and plums) were stored in cold chambers at 4 and 6°C and monitored for 8 weeks for the presence of Monilinia spp. M. fructicola was detected on 0.5% of nectarines (cvs. Sweet Red and Orion) that originated from two orchards in Lagnasco. Symptoms appeared on the fruit during storage, starting 3 weeks after harvest. Fruit rot lesions were brown, sunken, and covered with grayish tufts. The majority of infected fruit became dry and mummified. Brown rot symptoms were similar to those caused by endemic M. fructigena and M. laxa. Symptoms began with a small, circular, brown spot, and the rot spread rapidly. At the same time, numerous, small, grayish stromata developed. Finally, the whole surface of the fruit was covered by conidial tufts. Tissues were excised from diseased stone fruits and cultured on potato dextrose agar (PDA) amended with 25 μg of streptomycin per liter. The isolates produced abundant mycelium on PDA at 20 ± 2°C. Colonies were initially gray, but after sporulation, they became hazel, showing concentric rings (sporulation is sparse in M. laxa or M. fructigena). Conidia were one-celled, ellipsoid, hyaline, 15.2 × 10.1 μm, and produced in branched monilioid chains (2). Preliminary morphological identification of fungi resembling M. fructicola was confirmed by PCR using genomic DNA extracted from the mycelia of pure cultures. The DNA was amplified with a common reverse primer and three species-specific forward primers (3) obtained from a sequence characterized amplified region and a product of 535 bp, diagnostic for the species M. fructicola, was obtained. BLAST analysis of the amplified sequence (GenBank Accession No. FI569728) showed 96% similarity to the sequence of a M. fructicola isolated from Canada (GenBank Accession No. AF506700), 15% similarity to M. laxa ATCC11790 (GenBank Accession No. AF506702), and 35% similarity to a M. fructigena sequence isolated in Italy (GenBank Accession No. AF506701). Moreover, two sequences obtained through the amplification of ribosomal region ITS1-5.8S-ITS2, showing 100% similarity to the same ribosomal sequence of M. fructicola, were deposited in GenBank (Accession Nos. FJ411109 and FJ411110). The pathogen was detected on some mummified fruit from the same orchards in November of 2008. Pathogenicity was tested by spraying 103 conidia/ml on 10 surface-sterilized artificially wounded nectarines per strain of M. fructicola. After 5 days of incubation at 20 ± 2°C, typical, brown, rot symptoms developed on inoculated fruit. M. fructicola was reisolated from the inoculated fruit on PDA. Symptoms did not appear on control fruit. To our knowledge, this is the first report of M. fructicola in Italy. Its occurrence in Europe has been reported sporadically in Austria and France, and in 2006, it was detected in Hungary and Switzerland on peaches and nectarines imported from Italy and Spain (1,4). References: (1) E. Bosshard et al. Plant Dis. 90:1554, 2006. (2) R. J. W. Byrde and H. J. Willetts. The Brown Rot Fungi of Fruit: Their Biology and Control. Pergamon Press, Oxford, 1977. (3) M. J. Coté et al. Plant Dis. 88:1219, 2004. (4) M. Petròczy and L. Palkovics. Plant Dis. 90:375, 2006.

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Recovery Plan for Monilinia polystroma Causing Asiatic Brown Rot of Stone Fruit

Plant Health Progress ◽

10.1094/php-12-17-0080-rp ◽

2018 ◽

Vol 19 (2) ◽

pp. 107-124 ◽

Cited By ~ 1

Author(s):

K. D. Cox ◽

S. M. Villani ◽

Anna Poniatowska ◽

Guido Schnabel ◽

Imre Holb ◽

...

Keyword(s):

United States ◽

Crop Production ◽

Plant Disease ◽

Brown Rot ◽

The United States ◽

Stone Fruit ◽

Fruit Rot ◽

Eastern United States ◽

Recovery Plan ◽

The Impact

Stone fruit are an economically important group of specialty fruit crops in the United States. Species of the fungal genus Monilinia are some of the most important pathogens of stone fruit worldwide. These pathogens cause blossom blight, shoot blight, and brown fruit rot in temperate production regions. The most common species of Monilinia pathogenic on stone fruit include Monilinia fructicola, M. laxa, M. fructigena, and M. polystroma. Presently, neither M. polystroma, the causal agent of “Asiatic brown rot”, nor M. fructigena, one of the causal agents of “European brown rot”, have been reported in North America. Interestingly, both species can also cause brown rot of apple, which is densely planted in the eastern United States. This recovery plan was produced as part of the National Plant Disease Recovery System (NPDRS), called for in Homeland Security Presidential Directive Number 9 (HSPD-9) to ensure that the tools, infrastructure, communication networks, and capacity required to mitigate the impact of high-consequence plant disease outbreaks are such that a reasonable level of crop production is maintained. It is intended to provide a brief primer on the disease, assess the status of critical recovery components, and identify disease management research, extension, and education needs.

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