Fungicide Resistance Profiling in Botrytis cinerea Populations from Blueberry in California and Washington and Their Impact on Control of Gray Mold

Gray mold caused by Botrytis cinerea is a major postharvest disease of blueberry grown in the Central Valley of California and western Washington State. Sensitivities to boscalid, cyprodinil, fenhexamid, fludioxonil, and pyraclostrobin, representing five different fungicide classes, were examined for 249 (California) and 106 (Washington) B. cinerea isolates recovered from decayed blueberry fruit or flowers. In California and Washington, 7 and 17 fungicide-resistant phenotypes, respectively, were detected: 66 and 49% of the isolates were resistant to boscalid, 20 and 29% were moderately resistant to cyprodinil, 29 and 29% were resistant to fenhexamid, and 66 and 55% were resistant to pyraclostrobin. All isolates from California were sensitive to fludioxonil, whereas 70% of the isolates from Washington showed reduced sensitivity to fludioxonil. In California, 26 and 30% of the isolates were resistant to two and three classes of fungicides, respectively. In Washington, 31, 14, 16, and 9% of the isolates were resistant to two, three, four, and five classes of fungicides, respectively. Inherent risk of the development of resistance to quinone outside inhibitor (QoI) fungicides was assessed by detecting the presence of the Bcbi-143/144 intron in gene cytb. The intron was detected in 11.8 and 40% of the isolates in California and Washington, respectively, suggesting that the risk of QoI resistance is higher in California than in Washington. On detached blueberry fruit inoculated with 11 isolates exhibiting different fungicide-resistant phenotypes, most fungicides failed to control gray mold on fruit inoculated with the respective resistant phenotypes but the mixture of cyprodinil and fludioxonil was effective against all fungicide-resistant phenotypes tested. Our findings would be useful in designing and implementing fungicide resistance management spray programs for control of gray mold in blueberry.

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Fungicide resistance characterised across seven chemical classes in a Botrytis cinerea population isolated from Australian vineyards

10.1101/2021.03.31.437981 ◽

2021 ◽

Author(s):

Lincoln A. Harper ◽

Scott Paton ◽

Barbara Hall ◽

Suzanne McKay ◽

Richard P. Oliver ◽

...

Keyword(s):

Adverse Effect ◽

Botrytis Cinerea ◽

Fungicide Resistance ◽

Chemical Control ◽

Management Strategies ◽

Resistance Management ◽

Gray Mold ◽

Chemical Groups ◽

Important Disease ◽

Selection Of

AbstractGray mold, caused by Botrytis cinerea, is an economically important disease of grapes in Australia and across grape growing regions worldwide. Control of this disease relies heavily on canopy management and the application of single site fungicides. Fungicide application can lead to the selection of fungicide resistant B. cinerea populations, which has an adverse effect on the chemical control of the disease. Characterising the distribution and severity of resistant B. cinerea populations is needed to inform resistance management strategies. In this study, 725 isolates were sampled from 75 Australian vineyards during 2013 – 2016 and were screened against seven fungicides with different MOAs. The resistance frequencies for azoxystrobin, boscalid, fenhexamid, fludioxonil, iprodione, pyrimethanil and tebuconazole were 5, 2.8, 2.1, 6.2, 11.6, 7.7 and 2.9% respectively. Nearly half of the resistant isolates (43.7%) were resistant to more than one of the fungicides tested. The frequency of vineyards with at least one isolate simultaneously resistant to 1, 2, 3, 4 or 5 fungicides was 19.5, 7.8, 6.5, 10.4 and 2.6%.Resistance was associated with previously published genotypes in CytB (G143A), SdhB (H272R/Y), Erg27 (F412S), Mrr1 (D354Y), Os1 (I365S, N373S + Q369P, I365S + D757N) and Pos5 (P319A, L412F). Expression analysis was used to characterise fludioxonil resistant isolates exhibiting overexpression (6.3 - 9.6-fold) of the ABC transporter encoded by AtrB (MDR1 phenotype). Novel genotypes were also described in Mrr1 (S611N, D616G) and Cyp51 (P357S). Resistance frequencies were lower when compared to most previously published surveys of both grape and non-grape B. cinerea resistance. Nonetheless, continued monitoring of critical chemical groups used in Australian vineyards is recommended.

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Characterization of Botrytis cinerea From Commercial Cut Flower Roses

Plant Disease ◽

10.1094/pdis-09-18-1623-re ◽

2019 ◽

Vol 103 (7) ◽

pp. 1577-1583 ◽

Cited By ~ 7

Author(s):

M. Muñoz ◽

J. E. Faust ◽

G. Schnabel

Keyword(s):

Botrytis Cinerea ◽

Fungicide Resistance ◽

Management Practices ◽

Cultural Practices ◽

Disease Incidence ◽

Resistance Management ◽

Gray Mold ◽

Economic Losses ◽

Cut Flower ◽

Aspergillus Brasiliensis

Botrytis cinerea Pers. infects cut flower roses (Rosa × hybrida L.) during greenhouse production and gray mold symptoms are often expressed in the postharvest environment, resulting in significant economic losses. Disease management is based on cultural practices and preventative chemical treatments; however, gray mold outbreaks continue to occur. Rose tissues from six commercial shipments from two greenhouses in Colombia were evaluated to determine the Botrytis species composition as well as identify other pathogens present, gray mold incidence and severity, and fungicide resistance profiles. Botrytis isolates (49 total) were grouped into six morphological phenotypes, and all were identified to be B. cinerea sensu stricto. Disease incidence was higher in the petals than in the stem, stamen, ovary, sepal, or leaf tissues. Other fungi were isolated infrequently and included Alternaria alternata, Cladosporium cladosporioides, Epicoccum nigrum, Penicillium citrinum, Aspergillus brasiliensis, and Diplodia sp. Fungicide resistance profiles were determined using previously established discriminatory doses. Isolates resistant to thiophanate-methyl, iprodione, boscalid, and cyprodinil were found frequently in all shipments and in both greenhouses. The frequency of resistance to penthiopyrad, fenhexamid, fluopyram, isofetamid, and fludioxonil varied between shipments and greenhouses. No resistance to pydiflumetofen was observed at the discriminatory doses tested. Isolates with resistance to multiple chemical classes were commonly found. These results indicate that fungicide resistance management practices may improve preharvest and postharvest gray mold control of cut flower roses.

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Resistance to Fludioxonil in Botrytis cinerea Isolates from Blackberry and Strawberry

Phytopathology ◽

10.1094/phyto-11-13-0308-r ◽

2014 ◽

Vol 104 (7) ◽

pp. 724-732 ◽

Cited By ~ 36

Author(s):

Xingpeng Li ◽

Dolores Fernández-Ortuño ◽

Anja Grabke ◽

Guido Schnabel

Keyword(s):

South Carolina ◽

Botrytis Cinerea ◽

Resistance Mechanism ◽

Gray Mold ◽

Development Of Resistance ◽

Atp Binding Cassette Transporter ◽

Glycerol Synthesis ◽

Increased Sensitivity ◽

Moderately Resistant ◽

Gene Alterations

Site-specific fungicides, including the phenylpyrrole fludioxonil, are frequently used for gray mold control but are at risk for the development of resistance. In this study, field isolates that were low-resistant (LR) and moderately resistant (MR) to fludioxonil from blackberry and strawberry fields of North Carolina, South Carolina, and Virginia were characterized. Genes involved in osmoregulation, including bcsak1, BcOS4, bos5, and BRRG-1, were cloned and sequenced to detect potential target gene alterations; however, none were found. A previously described mutation (R632I) in transcription factor Mrr1, which is known to increase the expression of ATP-binding cassette transporter AtrB, was found in MR but not in sensitive (S) or LR isolates. Expression of atrB in MR isolates was ≈200-fold increased compared with an S isolate; however, 30- to 100-fold overexpression was also detected in LR isolates. Both MR isolates exhibited increased sensitivity to salt stress in the form of mycelial growth inhibition at 4% NaCl, indicating a disruption of osmoregulatory processes in those strains. However, the glycerol content was indistinguishable between S, LR, and MR isolates with and without exposure to fludioxonil, suggesting that the glycerol synthesis pathway may not be a part of the resistance mechanism in LR or MR strains. An investigation into the origin of LR and MR isolates from blackberry revealed two insertions in the mrr1 gene consistent with those found in the Botrytis clade group S. The emergence of strains overexpressing atrB in European and now in North American strawberry fields underscores the importance of this resistance mechanism for development of resistance to fludioxonil in Botrytis cinerea.

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Independent Emergence of Resistance to Seven Chemical Classes of Fungicides in Botrytis cinerea

Phytopathology ◽

10.1094/phyto-06-14-0161-r ◽

2015 ◽

Vol 105 (4) ◽

pp. 424-432 ◽

Cited By ~ 63

Author(s):

Dolores Fernández-Ortuño ◽

Anja Grabke ◽

Xingpeng Li ◽

Guido Schnabel

Keyword(s):

United States ◽

Botrytis Cinerea ◽

Target Genes ◽

Management Strategies ◽

Resistance Management ◽

Monitoring Program ◽

Gene Mutations ◽

The United States ◽

Gray Mold ◽

Development Of Resistance

Gray mold, caused by the fungal pathogen Botrytis cinerea, is one of the most destructive diseases of small fruit crops and control is largely dependent on the application of fungicides. As part of a region-wide resistance-monitoring program that investigated 1,890 B. cinerea isolates from 189 fields in 10 states of the United States, we identified seven isolates (0.4%) from five locations in four different states with unprecedented resistance to all seven Fungicide Resistance Action Committee (FRAC) codes with single-site modes of action including FRAC 1, 2, 7, 9, 11, 12, and 17 registered in the United States for gray mold control. Resistance to thiophanate-methyl, iprodione, boscalid, pyraclostrobin, and fenhexamid was based on target gene mutations that conferred E198A and F200Y in β-tubulin, I365N/S in Bos1, H272R/Y in SdhB, G143A in Cytb, and T63I and F412S in Erg27. Isolates were grouped into MDR1 and MDR1h phenotypes based on sensitivity to fludioxonil and variations in transcription factor mrr1. MDR1h isolates had a previously described 3-bp deletion at position 497 in mrr1. Expression of ABC transporter atrB was increased in MDR1 isolates but highest in MDR1h isolates. None of the isolates with seven single resistances (SR) had identical nucleotide variations in target genes, indicating that they emerged independently. Multifungicide resistance phenotypes did not exhibit significant fitness penalties for the parameters used in this study, but MDR1h isolates produced more sclerotia at low temperatures and exhibited increased sensitivity to salt stress. In this study we show that current resistance management strategies have not been able to prevent the geographically independent development of resistance to all seven site-specific fungicides currently registered for gray mold control in the United States and document the presence of MDR1h in North America.

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Advances in understanding fungicide resistance in Botrytis cinerea in China

Phytopathology ◽

10.1094/phyto-07-20-0313-ia ◽

2020 ◽

Author(s):

Wenyong Shao ◽

Youfu Zhao ◽

Zhonghua Ma

Keyword(s):

Botrytis Cinerea ◽

Fungicide Resistance ◽

Target Genes ◽

Agricultural Practices ◽

Resistance Mechanisms ◽

Gray Mold ◽

Yield Losses ◽

Quinone Outside Inhibitor ◽

Significant Yield ◽

Fungicide Target

Gray mold, caused by Botrytis cinerea, is a devastating disease causing significant yield losses in various economic plants. Fungicide application is one of the main strategies for management of gray mold; however, B. cinerea has developed resistance to various groups of fungicide. In China, benzimidazole-, dicarboximide-, and quinone outside inhibitor (QoI)-resistant populations of B. cinerea have become dominant. Substitute mutations in fungicide target genes are responsible for resistance in B. cinerea. Based on known resistance mechanisms, molecular methods including loop-mediated isothermal amplification (LAMP) have been developed for rapid detection of resistant isolates of B. cinerea. Since B. cinerea is able to quickly develop resistance to various fungicides, various integrated strategies have been developed, including biological and agricultural practices, to manage fungicide resistance in B. cinerea in the last decade.

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Resistance to Pyraclostrobin and Boscalid in Populations of Botrytis cinerea from Stored Apples in Washington State

Plant Disease ◽

10.1094/pdis-94-5-0604 ◽

2010 ◽

Vol 94 (5) ◽

pp. 604-612 ◽

Cited By ~ 43

Author(s):

Y. K. Kim ◽

C. L. Xiao

Keyword(s):

Botrytis Cinerea ◽

Fungicide Resistance ◽

Mycelial Growth ◽

Gray Mold ◽

Apple Fruit ◽

Conidial Germination ◽

Postharvest Disease ◽

Cross Resistance ◽

Baseline Sensitivity

Gray mold caused by Botrytis cinerea is a major postharvest disease of apple. Pristine, a formulated mixture of pyraclostrobin and boscalid, was recently registered for use on apple. Pristine applied within 2 weeks before harvest is effective in controlling gray mold in stored apple fruit. To determine the baseline sensitivity of B. cinerea populations to these fungicides, 40 isolates from organic and 80 from conventional apple orchards where Pristine had not been used were tested for mycelial growth or conidial germination on fungicide-amended media. To monitor fungicide resistance, gray-mold-decayed apple fruit originating from orchards in which Pristine had been used were sampled from a fruit packinghouse. Isolates of B. cinerea recovered from the fruit were tested for resistance to the two fungicides. In the in vivo study in the orchards, Pristine was applied to fruit 1 day before harvest. Fruit were then harvested, wounded, and inoculated with isolates exhibiting different fungicide-resistance phenotypes. Fruit were stored at 0°C for 8 weeks for decay development. The effective concentration that inhibits mycelial growth by 50% relative to the control (EC50) values for sensitive isolates ranged from 0.008 to 0.132 μg/ml (mean = 0.043, n = 116) for pyraclostrobin and from 0.003 to 0.183 μg/ml (mean = 0.075, n = 117) for Pristine in a mycelial growth assay on potato dextrose agar. The EC50 values of boscalid for sensitive isolates ranged from 0.065 to 1.538 μg/ml (mean = 0.631, n = 29) in a conidial germination assay on water agar. Four isolates were resistant to pyraclostrobin, with resistance factors (RFs) ranging from 12 to 4,193. Of the four pyraclostrobin-resistant isolates, one also was resistant to boscalid (RF = 14) and Pristine (RF = 373), and two exhibited reduced sensitivity to Pristine (RF = 16 and 17). The minimum inhibitory concentration for conidial germination (for boscalid) or mycelial growth (for pyraclostrobin and Pristine) of sensitive isolates was 5 μg/ml, which is thus recommended as a discriminatory concentration for phenotyping isolates for resistance to these fungicides. Of the 56 isolates obtained from decayed apple fruit that had been exposed to Pristine, 11 (approximately 20%) were resistant to both pyraclostrobin and boscalid and 1 was resistant only to pyraclostrobin. Of the additional 43 isolates obtained from decayed apple fruit originating from an organic orchard, 3 were resistant only to pyraclostrobin, 2 were resistant only to boscalid, and 2 were resistant to both fungicides. It appeared that there was no cross resistance between pyraclostrobin and boscalid because of the existence of isolates resistant only to either pyraclostrobin or boscalid. Pristine applied at label rate in the orchard failed to control gray mold on apple fruit inoculated with the Pristine-resistant isolates. This is the first report of multiple resistance to pyraclostrobin, boscalid, and Pristine in field populations of B. cinerea. Our results suggest that the development of dual resistance to pyraclostrobin and boscalid in B. cinerea populations could result in the failure to control gray mold with Pristine.

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Within-Season Shift in Fungicide Resistance Profiles of Botrytis cinerea in California Strawberry Fields

Plant Disease ◽

10.1094/pdis-03-18-0406-re ◽

2019 ◽

Vol 103 (1) ◽

pp. 59-64 ◽

Cited By ~ 8

Author(s):

Scott D. Cosseboom ◽

Kelly L. Ivors ◽

Guido Schnabel ◽

Patricia K. Bryson ◽

Gerald J. Holmes

Keyword(s):

Botrytis Cinerea ◽

Fungicide Resistance ◽

Mycelial Growth ◽

Target Genes ◽

Resistance Management ◽

Gray Mold ◽

Field Resistance ◽

Early Season ◽

Late Season ◽

Growth Assay

Sensitivity of Botrytis cinerea to seven fungicide chemical classes was determined for 888 isolates collected in 2016 from 47 California strawberry fields. Isolates were collected early season (minimum fungicide exposure) and late season (maximum fungicide exposure) from the same planting block in each field. Resistance was determined using a mycelial growth assay, and variable frequencies of resistance were observed to each fungicide at both sampling times (early season %, late season %): boscalid (12, 35), cyprodinil (12, 46), fenhexamid (53, 91), fludioxonil (1, 4), fluopyram (2, 7), iprodione (25, 8), isofetamid (0, 1), penthiopyrad (8, 25), pyraclostrobin (77, 98), and thiophanate-methyl (81, 96). Analysis of number of chemical class resistances (CCRs) revealed an increasing shift in CCR from the early to late season. Phenotypes of 40 isolates that were resistant or sensitive to different chemical classes were associated with presence or absence of mutations in target genes. Fungicide-resistance phenotypes determined in the mycelial growth assay closely matched (93.8%) the genotype observed. Previously described resistance-conferring mutations were found for each gene. A survey of fungicide use from 32 of the sampled fields revealed an average of 15 applications of gray mold–labeled fungicides per season at an average interval of 12 days. The most frequently applied fungicides (average number of applications during the 2016 season) were captan (7.3), pyraclostrobin (2.5), cyprodinil (2.3), fludioxonil (2.3), boscalid (1.8), and fenhexamid (1.4). Multifungicide resistance is widespread in California. Resistance management tactics that reduce selection pressure by limiting fungicide use, rotating among Fungicide Resistance Action Committee codes, and mixing/rotating site-specific fungicides with multisite fungicides need to be improved and implemented.

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Efficacy of Natamycin Against Gray Mold of Stored Mandarin Fruit Caused by Isolates of Botrytis cinerea With Multiple Fungicide Resistance

Plant Disease ◽

10.1094/pdis-04-19-0844-re ◽

2020 ◽

Vol 104 (3) ◽

pp. 787-792

Author(s):

S. Saito ◽

F. Wang ◽

C. L. Xiao

Keyword(s):

Botrytis Cinerea ◽

Fungicide Resistance ◽

Mycelial Growth ◽

Fungal Growth ◽

Gray Mold ◽

Conidial Germination ◽

Postharvest Disease ◽

Cross Resistance ◽

Postharvest Diseases ◽

Modes Of Action

Gray mold caused by Botrytis cinerea is an emerging postharvest disease of mandarin fruit in California. Management of postharvest diseases of mandarins relies on postharvest fungicides; however, multiple resistance to fungicides of different modes of action is common in B. cinerea populations from mandarin, leading to their failure to control decay. Natamycin is commonly used in the food industry as an additive, and it has been registered as a biofungicide for postharvest use on citrus and some other fruits. Sensitivity to natamycin of 64 isolates of B. cinerea from decayed mandarin fruit with known resistance phenotypes to other citrus postharvest fungicides (azoxystrobin, fludioxonil, pyrimethanil, and thiabendazole) was tested. Effective concentrations of natamycin to cause a 50% reduction relative to the control for conidial germination were from 0.324 to 0.567 µg/ml (mean of 0.444 µg/ml), and those for mycelial growth were 1.021 to 2.007 µg/ml (mean of 1.578 µg/ml). Minimum inhibitory concentrations where no fungal growth was present were 0.7 to 1.0 µg/ml for conidial germination and 5.0 to 10.0 µg/ml for mycelial growth. No cross-resistance between natamycin and other citrus postharvest fungicides was detected. Decay control efficacy tests with natamycin were conducted on mandarin fruit inoculated with B. cinerea isolates exhibiting five different fungicide resistance phenotypes, and natamycin significantly reduced incidence and lesion size of gray mold on fruit, regardless of fungicide resistance phenotypes. Natamycin has the potential to be an effective tool for integration into postharvest fungicide programs to control gray mold and manage B. cinerea isolates resistant to fungicides with other modes of action.

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Population Structure, Fungicide Resistance Profile, and sdhB Mutation Frequency of Botrytis cinerea from Strawberry and Greenhouse-Grown Tomato in Greece

Plant Disease ◽

10.1094/pdis-04-14-0373-re ◽

2015 ◽

Vol 99 (2) ◽

pp. 240-248 ◽

Cited By ~ 30

Author(s):

Sotirios Konstantinou ◽

Thomas Veloukas ◽

Michaela Leroch ◽

George Menexes ◽

Matthias Hahn ◽

...

Keyword(s):

Botrytis Cinerea ◽

Fungicide Resistance ◽

Sensu Stricto ◽

Sdhb Mutation ◽

Resistance Development ◽

Low Frequencies ◽

Resistance Profile ◽

Development Of Resistance ◽

Crete Island ◽

Of Greece

Botrytis cinerea is a pathogen with high genetic variability that has also shown high risk for fungicide resistance development. In total, 1,169 isolates obtained from strawberry (n = 297) and tomato (n = 872) in five geographic regions of Greece were tested for their sensitivity to several botryticides. A high frequency of isolates with multiple resistance to carbendazim, cyprodinil, pyraclostrobin, and boscalid was found in isolates from strawberry. In the isolates from tomato, the predominant phenotype was that of dual resistance to carbendazim and cyprodinil in the Crete island, of single resistance to carbendazim in the region of Preveza, and of sensitive isolates in the region of Kyparissia. None of the tested isolates was found to be fludioxonil resistant. High frequencies of boscalid-resistant phenotypes were observed in the strawberry isolates, while boscalid-resistance frequency in the tomato isolates was lower. H272R was the predominant sdhB mutation, associated with resistance to boscalid, in all the sampled isolates, while other sdhB mutations were found at low frequencies. B. cinerea group S, identified by the presence of a 21-bp insertion in the transcription factor mrr1 gene, was predominant within the tomato isolates obtained from all three sampled regions, with frequencies ranging from 62 to 75% of the isolates; whereas, within strawberry isolates, B. cinerea was predominant, with frequencies of 75 to 95%. Correlations of isolate genotype and fungicide resistance profile showed that B. cinerea sensu stricto isolates were more prone to the development of resistance to boscalid compared with the Botrytis group S isolates, which may explain the observed predominance of B. cinerea sensu stricto in strawberry fields.

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Succinate dehydrogenase inhibitor (SDHI) fungicide resistance prevention strategy

New Zealand Plant Protection ◽

10.30843/nzpp.2011.64.5972 ◽

2011 ◽

Vol 64 ◽

pp. 119-124 ◽

Cited By ~ 4

Author(s):

A.H. McKay ◽

G.C. Hagerty ◽

G.B. Follas ◽

M.S. Moore ◽

M.S. Christie ◽

...

Keyword(s):

New Zealand ◽

High Risk ◽

Succinate Dehydrogenase ◽

Fungicide Resistance ◽

Fungal Pathogens ◽

Resistance Management ◽

Resistance Development ◽

Development Of Resistance ◽

Action Committee ◽

Succinate Dehydrogenase Inhibitor

Succinate dehydrogenase inhibitor (SDHI) fungicides are currently represented in New Zealand by eight active ingredients bixafen boscalid carboxin fluaxapyroxad fluopyram isopyrazam penthiopyrad and sedaxane They are either currently registered or undergoing development in New Zealand for use against a range of ascomycete and basiodiomycete pathogens in crops including cereals ryegrass seed apples pears grapes stonefruit cucurbits and kiwifruit These fungicides are considered to have medium to high risk of resistance development and resistance management is recommended by the Fungicide Resistance Action Committee (FRAC) in Europe Guidelines are presented for use of SDHI fungicides in New Zealand to help avoid or delay the development of resistance in the fungal pathogens that they target

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