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

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

Plant Disease ◽

10.1094/pdis-07-20-1561-re ◽

2020 ◽

Author(s):

Kim Lesniak ◽

Jingyu Peng ◽

Tyre J Proffer ◽

Cory Outwater ◽

Lauren Eldred ◽

...

Keyword(s):

Point Mutations ◽

Brown Rot ◽

Stone Fruit ◽

Monilinia Fructicola ◽

Eastern United States ◽

Sequence Element ◽

Wide Range ◽

Elevated Expression ◽

Demethylation Inhibitor ◽

Dmi Resistance

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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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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Discontinuance of tebuconazole in the field restores sensitivity of Monilinia fructicola in stone fruit orchards

Plant Pathology ◽

10.1111/ppa.13101 ◽

2019 ◽

Vol 69 (1) ◽

pp. 68-76 ◽

Cited By ~ 4

Author(s):

W. V. Pereira ◽

R. G. F. Morales ◽

A. I. G. Bauer ◽

K. Kudlawiec ◽

L. L. May‐De‐Mio

Keyword(s):

Stone Fruit ◽

Monilinia Fructicola ◽

Fruit Orchards

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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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Co-Infection with Three Mycoviruses Stimulates Growth of a Monilinia fructicola Isolate on Nutrient Medium, but Does Not Induce Hypervirulence in a Natural Host

Viruses ◽

10.3390/v11010089 ◽

2019 ◽

Vol 11 (1) ◽

pp. 89 ◽

Cited By ~ 4

Author(s):

Thao Tran ◽

Hua Li ◽

Duy Nguyen ◽

Michael Jones ◽

Stephen Wylie

Keyword(s):

Inhibitory Effect ◽

Fungal Species ◽

Natural Host ◽

Stone Fruit ◽

Monilinia Fructicola ◽

Isogenic Lines ◽

Nutrient Agar Medium ◽

Fruit Orchards ◽

Hyphal Tip

Monilinia fructicola and Monilinia laxa are the most destructive fungal species infecting stone fruit (Prunus species). High-throughput cDNA sequencing of M. laxa and M. fructicola isolates collected from stone fruit orchards revealed that 14% of isolates were infected with one or more of three mycoviruses: Sclerotinia sclerotiorum hypovirus 2 (SsHV2, genus Hypovirus), Fusarium poae virus 1 (FPV1, genus Betapartitivirus), and Botrytis virus F (BVF, genus Mycoflexivirus). Isolate M196 of M. fructicola was co-infected with all three viruses, and this isolate was studied further. Several methods were applied to cure M196 of one or more mycoviruses. Of these treatments, hyphal tip culture either alone or in combination with antibiotic treatment generated isogenic lines free of one or more mycoviruses. When isogenic fungal lines were cultured on nutrient agar medium in vitro, the triple mycovirus-infected parent isolate M196 grew 10% faster than any of the virus-cured isogenic lines. BVF had a slight inhibitory effect on growth, and FPV1 did not influence growth. Surprisingly, after inoculation to fruits of sweet cherry, there were no significance differences in disease progression between isogenic lines, suggesting that these mycoviruses did not influence the virulence of M. fructicola on a natural host.

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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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Baseline Sensitivity of Monilinia fructicola from China to the DMI Fungicide SYP-Z048 and Analysis of DMI-Resistant Mutants

Plant Disease ◽

10.1094/pdis-06-11-0495 ◽

2012 ◽

Vol 96 (3) ◽

pp. 416-422 ◽

Cited By ~ 41

Author(s):

F. P. Chen ◽

J. R. Fan ◽

T. Zhou ◽

X. L. Liu ◽

J. L. Liu ◽

...

Keyword(s):

Brown Rot ◽

Cross Resistance ◽

Monilinia Fructicola ◽

Lesion Area ◽

Site Mutation ◽

Baseline Sensitivity ◽

Dmi Fungicides ◽

Resistant Mutants ◽

Beijing China

Sterol 14α-demethylase inhibitors (DMIs) continue to be important in the management of brown rot of Monilinia spp. worldwide. In this study, the sensitivity of 100 Monilinia fructicola isolates from four unsprayed orchards and two packinghouses in Beijing, China, to the new DMI fungicide SYP-Z048 was evaluated and ranged from 0.003 to 0.039 and 0.016 to 0.047 μg/ml, respectively. Laboratory mutants resistant to SYP-Z048 were generated using UV irradiation but no mutants occurred spontaneously. Resistance was stable after 10 weekly consecutive transfers on fungicide-free medium. Three parameters, including growth rate, sporulation in vitro, and lesion area, were significantly different when sensitive isolates and resistant mutants were analyzed as groups. Mutants grew more slowly and developed significantly smaller lesions on detached fruit, and their sporulation ability in vitro was reduced. Cross resistance was found between SYP-Z048 and propiconazole (ρ = 0.82, P < 0.0001) but not between SYPZ048 and tridemorph, carbendazim, procymidone, azoxystrobin, or pyrimethanil. SYP-Z048 resistance in mutants exhibiting 50% mycelial growth inhibition values greater than 0.3 μg/ml was correlated with the presence of a mutation in the CYP51 gene that encodes the target protein for DMI fungicides. The mutation caused an amino acid change from tyrosine to phenylalanine at position 136 (Y136F). To our knowledge, this is the first baseline sensitivity of M. fructicola collected from China to a DMI fungicide. The inability of M. fructicola to generate spontaneous DMI-resistant mutants coupled with reduced fitness of Y136F mutants can explain why this target site mutation has not yet emerged as a DMI fungicide resistance determinant in M. fructicola field populations worldwide.

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