scholarly journals Combination of Suspension Array and Mycelial Growth Assay for Detecting Multiple-Fungicide Resistance in Botrytis cinerea in Hebei Province in China

Plant Disease ◽  
2019 ◽  
Vol 103 (6) ◽  
pp. 1213-1219
Author(s):  
Zehua Su ◽  
Xin Zhang ◽  
Jianjiang Zhao ◽  
Wenqiao Wang ◽  
Lei Shang ◽  
...  
Keyword(s):  
Botrytis Cinerea ◽  
High Throughput ◽  
Fungicide Resistance ◽  
Mycelial Growth ◽  
Accurate Method ◽  
Hebei Province ◽  
Combination Method ◽  
Major Type ◽  
Suspension Array ◽  
Growth Assay

To provide a high-throughput, efficient, and accurate method to monitor multiple-fungicide resistance of Botrytis cinerea in the field, we used the suspension array, sequencing, and mycelial growth assay in our research. Discriminating-dose bioassays for detecting carbendazim, diethofencarb, boscalid, and iprodione resistance (CarR, DieR, BosR, and IprR, respectively) were used to analyze 257 isolates collected from Hebei Province in China during 2016 and 2017. High resistance frequencies to carbendazim (100%), diethofencarb (92.08%), and iprodione (86.59%) were detected. BosR isolates accounted for 11.67% of the total. In addition, 103 isolates were randomly selected for phenotype and genotype detection. The high-throughput suspension array was utilized to detect eight genotypes simultaneously, including BenA-E198, BenA-198A, SdhB-H272, SdhB-272Y, BcOS1-I365, BcOS1-365S, erg27-F412, and erg27-412S, which were associated with resistance toward carbendazim or diethofencarb, boscalid, iprodione, and fenhexamid (FenR), respectively. Most of the benzimidazole-resistant isolates (81.55%) possessed the E198V mutation in the BenA gene. Ninety-three isolates with dual resistance to carbendazim and diethofencarb showed the E198V/K mutation. All BosR isolates carried the H272R mutation in the SdhB gene. The I365S and Q369P+N373S (66.99%) mutations in the BcOS1 gene were more frequently observed. No mutation was detected in the erg27 gene in Hebei isolates. There were 13 resistance profile phenotypes. Phenotypes with triple resistance were the most common (83.50%), and CarRDieRBosSIprRFenS was the major type. CarR isolates that carried E198V/K/A were all highly resistant (HR) and only one F200Y mutant was moderately resistant (MR) to carbendazim. Isolates that possessed E198V/K were MR or HR to diethofencarb. BosR isolates that possessed H272R mutation were lowly resistant (LR). IprR isolates were all LR or MR. The distribution of half maximal effective concentrations of CarR isolates with E198V/K mutations and IprR isolates with Q369P+N373S mutations significantly increased from 2016 to 2017. Combined with our observations, a combination method of the high-throughput suspension array and the mycelial growth assay was suggested to accurately monitor multiple resistance of B. cinerea in the field.

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

Plant Disease ◽  
2019 ◽  
Vol 103 (1) ◽  
pp. 59-64 ◽  
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.

scholarly journals Resistance to Thiophanate-Methyl in Botrytis cinerea Isolates From Californian Vineyards and Pistachio and Pomegranate Orchards

Plant Disease ◽  
2020 ◽  
Vol 104 (4) ◽  
pp. 1069-1075
Author(s):  
Hervé F. Avenot ◽  
David P. Morgan ◽  
Joel Quattrini ◽  
Themis J. Michailides
Keyword(s):  
Botrytis Cinerea ◽  
Mycelial Growth ◽  
Gray Mold ◽  
Fitness Components ◽  
Thiophanate Methyl ◽  
Significant Difference ◽  
Shoot Blight ◽  
Growth Assay ◽  
Free Media ◽  
Weak Resistance

In this study, a mycelial growth assay was used to evaluate the sensitivity to thiophanate-methyl of 144 Botrytis cinerea isolates (collection A) from Californian vineyards and pistachio and pomegranate orchards. Based on the effective concentration that inhibits 50% of growth (EC50) values for mycelial growth inhibition on fungicide-amended media, 3, 28, 10, and 58% of the isolates showed sensitivity (SS; EC50 < 1 µg/ml), low resistance (LR; 1 < EC50 < 10 µg/ml), weak resistance (WR; 10 < EC50 < 50 µg/ml), and high resistance (HR; EC50 > 100 µg/ml) toward thiophanate-methyl, respectively. The LR and HR phenotypes were observed in pistachio and pomegranate orchards, even though pomegranate was not sprayed with thiophanate-methyl. Sensitivity to thiophanate-methyl of a historical collection of 257 B. cinerea isolates (collection B) isolated from pistachio orchards in 1992, 2005, and 2006 was assessed on potato dextrose agar amended with thiophanate-methyl at the discriminatory concentration of 10 µg/ml. Average percentages of thiophanate-methyl–resistant isolates were 50, 72, and 64% in the orchards in 1992, 2005, and 2006, respectively. A study of fitness components of selected thiophanate-methyl–resistant (LR, WR, and HR) and –sensitive (SS) isolates from collection A did not reveal any significant difference between them with respect to mycelial growth on fungicide-free media and pathogenicity on cultivar Crimson Seedless berries. Comparison of β-tubulin sequences from resistant and sensitive phenotypes revealed that a glutamic acid at position 198 was changed to alanine in all HR isolates and three LR isolates. The occurrence of thiophanate-methyl resistance in B. cinerea populations should be considered when designing spray programs against blossom and shoot blight of pistachio and gray mold of grape.

scholarly journals Resistance to the SDHI Fungicides Boscalid, Fluopyram, Fluxapyroxad, and Penthiopyrad in Botrytis cinerea from Commercial Strawberry Fields in Spain

Plant Disease ◽  
2017 ◽  
Vol 101 (7) ◽  
pp. 1306-1313 ◽  
Author(s):  
Dolores Fernández-Ortuño ◽  
Alejandro Pérez-García ◽  
Manuel Chamorro ◽  
Eduardo de la Peña ◽  
Antonio de Vicente ◽  
...  
Keyword(s):  
Amino Acid ◽  
Botrytis Cinerea ◽  
Fungicide Resistance ◽  
Mycelial Growth ◽  
Growing Season ◽  
Gray Mold ◽  
Effective Concentration ◽  
Site Specific ◽  
Resistance Patterns ◽  
Succinate Dehydrogenase Inhibitors

Gray mold, caused by the necrotrophic fungus Botrytis cinerea., is one of the most economically important diseases of strawberry. Gray mold control involves the application of fungicides throughout the strawberry growing season; however, B. cinerea isolates resistant to multiple classes of site-specific fungicides have been recently reported in the Spanish gray mold population. Succinate dehydrogenase inhibitors (SDHI) constitute a relatively novel class of fungicides registered for gray mold control representing new alternatives for strawberry growers. In the present study, 37 B. cinerea isolates previously characterized for their sensitivity to boscalid and amino acid changes in the SdhB protein were used to determine the effective concentration that reduces mycelial growth by 50% (EC50) to fluopyram, fluxapyroxad, and penthiopyrad. The present study was also conducted to obtain discriminatory doses to monitor SDHI fungicide resistance in 580 B. cinerea isolates collected from 27 commercial fields in Spain during 2014, 2015, and 2016. The EC50 values ranged from 0.01 to >100 μg/ml for fluopyram, <0.01 to 4.19 μg/ml for fluxapyroxad, and, finally, <0.01 to 59.65 μg/ml for penthiopyrad. Based on these results, as well as findings from a previous publication, the discriminatory doses chosen to examine sensitivities to boscalid, fluopyram, fluxapyroxad, and penthiopyrad were 100, 15, 1, and 6 μg/ml, respectively. Over the course of the 3-year monitoring period, the overall frequencies of resistance to the four SDHI were 56.9, 6.9, 12.9, and 24.6%, respectively. The frequency of boscalid-resistant isolates decreased from 73 to 41% over the years; however, the fluopyram-resistant isolates increased from 5 to 10% after 1 year of registration. Four SDHI resistance patterns were observed in our population, which included patterns I (30%; resistance to boscalid), II (13.8%; resistance to boscalid and penthiopyrad), III (5.7%; boscalid, fluxapyroxad, and penthiopyrad), and IV (7.9%; resistance to boscalid, fluopyram, fluxapyroxad, and penthiopyrad). Patterns I and II were associated with the amino acid substitutions H272R and H272Y; pattern III was associated only with the H272Y mutation; and, finally, pattern IV was associated with the N230I allele in the SdhB subunit. For gray mold management, it is suggested that the simultaneous use of boscalid and penthiopyrad should be limited to one application per season; however, fluxapyroxad and, especially, fluopyram could be used as valid SDHI alternatives for gray mold control, although they should be applied with caution.

scholarly journals Sensitivity of Botrytis cinerea Isolates from Conventional and Organic Strawberry Fields in Brazil to Azoxystrobin, Iprodione, Pyrimethanil, and Thiophanate-Methyl

Plant Disease ◽  
2018 ◽  
Vol 102 (9) ◽  
pp. 1803-1810 ◽  
Author(s):  
Juliana S. Baggio ◽  
Natalia A. Peres ◽  
Lilian Amorim
Keyword(s):  
Botrytis Cinerea ◽  
Fungicide Resistance ◽  
Mycelial Growth ◽  
Commercial Production ◽  
Effective Concentration ◽  
Fruit Rot ◽  
Strawberry Fruit ◽  
Molecular Analyses ◽  
Thiophanate Methyl ◽  
Control Disease

Botrytis fruit rot, caused by Botrytis cinerea, is one of the most important strawberry diseases worldwide, and fungicide applications are often used to manage the disease in commercial production. Isolates of B. cinerea were collected from conventional and organic strawberry fields in four Brazilian States from 2013 to 2015 and their sensitivity to the main single-site mode-of action fungicides used in Brazil was tested. Resistance to azoxystrobin, iprodione, pyrimethanil, and thiophanate-methyl was found and values for effective concentration that inhibited mycelial growth by 50% were higher than 71.9, 1.2, 5.0, and 688 µg/ml, respectively, regardless the production system. Resistance to these fungicides was observed in 87.5, 76.6, 23.4, and 92.2% of isolates from conventional fields and 31.4, 22.9, 14.3, and 51.4% of isolates from organic fields, respectively. Moreover, frequencies of isolates with multiple fungicide resistance to the four active ingredients were 20.6 and 2.8% whereas 6.3 and 27.8% were sensitive to the four fungicides for conventional and organic areas, respectively. Molecular analyses of the cytochrome b, β-tubulin, and bos1 genes revealed the presence of G143A; E198A; and I365 N/S, Q369P, or N373S mutations, respectively, in resistant isolates of B. cinerea. Field rates of fungicides sprayed preventively to inoculated strawberry fruit failed to control disease caused by the respective resistant isolates.

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

Plant Disease ◽  
2010 ◽  
Vol 94 (5) ◽  
pp. 604-612 ◽  
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.

scholarly journals Novel Fungitoxicity Assays for Inhibition of Germination-Associated Adhesion of Botrytis cinerea and Puccinia recondita Spores

2002 ◽  
Vol 68 (2) ◽  
pp. 597-601 ◽  
Author(s):  
Richard A. Slawecki ◽  
Eileen P. Ryan ◽  
David H. Young
Keyword(s):  
Botrytis Cinerea ◽  
High Throughput ◽  
Spore Germination ◽  
Mycelial Growth ◽  
Puccinia Recondita ◽  
Ergosterol Biosynthesis ◽  
Microtiter Plates ◽  
Sulforhodamine B ◽  
Ergosterol Biosynthesis Inhibitors ◽  
Biosynthesis Inhibitors

ABSTRACT Botrytis cinerea and Puccinia recondita spores adhere strongly to polystyrene microtiter plates coincident with germination. We developed assays for inhibition of spore adhesion in 96-well microtiter plates by using sulforhodamine B staining to quantify the adherent spores. In both organisms, fungicides that inhibited germination strongly inhibited spore adhesion, with 50% effective concentrations (EC50s) comparable to those for inhibition of germination. In contrast, fungicides that acted after germination in B. cinerea inhibited spore adhesion to microtiter plates only at concentrations much higher than their EC50s for inhibition of mycelial growth. Similarly, in P. recondita the ergosterol biosynthesis inhibitors myclobutanil and fenbuconazole acted after germination and did not inhibit spore adhesion. The assays provide a rapid, high-throughput alternative to traditional spore germination assays and may be applicable to other fungi.

scholarly journals A synthetic agar assay for determining sensitivity of Venturia inaequalis to anilinopyrimidine fungicides in New Zealand apple orchards

2013 ◽  
Vol 66 ◽  
pp. 293-302 ◽  
Author(s):  
N.J. Larsen ◽  
R.M. Beresford ◽  
P.N. Wood ◽  
P.J. Wright ◽  
B.M. Fisher
Keyword(s):  
New Zealand ◽  
Disease Control ◽  
Botrytis Cinerea ◽  
Mycelial Growth ◽  
Venturia Inaequalis ◽  
Black Spot ◽  
Apple Trees ◽  
Colony Diameter ◽  
Growth Assay ◽  
Agar Method

A synthetic agar mycelial growth assay adapted from a Botrytis cinerea method was used to determine the sensitivity of Venturia inaequalis isolates (the cause of apple black spot) to anilinopyrimidine (AP) fungicides Nineteen singleconidium isolates were classified as sensitive (S) or resistant (R) to cyprodinil or pyrimethanil after 21 days growth according to concentrations that inhibited colony diameter by 50 (EC50) Isolate classification on agar was compared with leaf disease control by AP fungicides on potted apple trees inoculated with S or R isolates Three S isolates were completely inhibited by AP fungicides on plants Three R isolates were partially inhibited on fungicidetreated leaves One isolate classified as R for both fungicides on agar was inhibited by cyprodinil but not pyrimethanil on plants The agar method will be used for screening V inaequalis populations for AP resistance in orchard surveys with plant tests on some isolates to determine implications for orchard disease control

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

Plant Disease ◽  
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.

scholarly journals The Role of Saprotrophy and Virulence in the Population Dynamics of Botrytis cinerea in Vineyards

2005 ◽  
Vol 95 (6) ◽  
pp. 692-700 ◽  
Author(s):  
Fabian Martinez ◽  
Bernadette Dubos ◽  
Marc Fermaud
Keyword(s):  
Botrytis Cinerea ◽  
Fungicide Resistance ◽  
Mycelial Growth ◽  
Phenological Stages ◽  
Grape Berries ◽  
Genetic Types ◽  
Group I ◽  
Pathogenicity Tests ◽  
Group Ii

Change in relative frequencies of the three main genetic types of Botrytis cinerea (Group I, Group II vacuma, and Group II transposa) were monitored over time from 1998 to 2000 in three Bordeaux vineyards not treated with fungicides. During 2000, Group I isolates, detected mostly at flowering comprised only 2.5% of the entire population. Within Group II, the complementary frequencies of vacuma and transposa isolates differed significantly depending on grapevine phenological stages and organs. Every year and at all sites, including one noble rot site, transposa isolates dominated at every stage, particularly on overwintering canes and at harvest (greater than 86.7% on berries). The complementary frequency of vacuma isolates reached a maximum on senescing floral caps (between 23.5 and 71.4%) and then decreased significantly until harvest on leaves and berries. In pathogenicity tests on grape berries, transposa isolates were significantly more virulent than were vacuma isolates. Mycelial growth rate was negatively correlated with virulence, notably on leaves in transposa and with double resistance to the fungicides carbendazim and vinclozolin. In vacuma, this double resistance was positively correlated with virulence on leaves. Change in the vacuma and transposa frequencies was most likely caused by differences in saprotrophic and pathogenic fitness. Possible interactions between fungicide resistance profiles and fitness are discussed.

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