Multiple resistance to QoIs and other classes of fungicides in Botrytis cinerea populations from strawberry in Zhejiang Province, China

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1 Eur J Plant Pathol (2015) 141: DOI /s ORIGINAL RESEARCH Multiple resistance to QoIs and other classes of fungicides in Botrytis cinerea populations from strawberry in Zhejiang Province, China D. Yin & X. Chen & M. S. Hamada & M. Yu & Y. Yin & Z. Ma Accepted: 9 September 2014 /Published online: 28 September 2014 # Koninklijke Nederlandse Planteziektenkundige Vereniging 2014 Abstract Gray mold caused by Botrytis cinerea is a major fungal disease of strawberry in Zhejiang Province, which is considered as one of the most important strawberry-producing areas in China. Recently, effective management of this disease has become an urgent need for the farmers as a result of the failure of many fungicides in controlling this pathogen. A total of 486 B. cinerea isolates were collected from strawberry in nine locations of Zhejiang Province and screened for resistance to azoxystrobin, carbendazim, iprodione, pyrimethanil and fludioxonil. Among the 486 isolates, 353 (72.6 %) showed resistance to single or multiple fungicides, 254 (52.3 %) were simultaneously resistant to azoxystrobin, carbendazim, iprodione and pyrimethanil, but no fludioxonil-resistant (Flu R )isolates were found, indicating that fludioxonil has great potential for control of gray mold on strawberry. In mycelial growth assays, we determined the EC 50 values for 47 azoxystrobin-sensitive (Azo S ), 41 azoxystrobinresistant (Azo R ), 48 pyrimethanil-sensitive (Pyr S ), 44 pyrimethanil-resistant (Pyr R ) and 100 fludioxonilsensitive (Flu S ) isolates. The resistance factors (RF) of Azo R and Pyr R isolates ranged from 21.7 to 4,359.0 and D. Yin: X. Chen : M. S. Hamada : M. Yu : Y. Yin (*) : Z. Ma Institute of Biotechnology, Zhejiang University, Hangzhou , China ynyin@zju.edu.cn M. S. Hamada Pesticides department, Faculty of Agriculture, Mansoura University, Mansoura 35516, Egypt from 53.0 to 320.1, respectively. The EC 50 values for Flu S isolates ranged from to 0.04 (mean = 0.01) μg/ml. All 322 Azo R isolates harboured the G143A point mutation. B. cinerea populations were divided into two types according to the structure of the cyt b gene, with (12.2 %) or without (87.8 %) the third intron Bcbi- 143/144. The high frequency of isolates without the Bcbi-143/144 intron indicates that B. cinerea populations from strawberry have a high inherent risk for the development of resistance to QoIs. Keywords Botrytis cinerea. Gray mold. Multiple resistance. QoI fungicide. Cyt b. Strawberry Introduction In recent years, China has increased its strawberry production rapidly and become one of the major strawberry producers in the world. Zhejiang Province is located in China s southeastern coast, in the southern part of the Yangtze River Delta, and considered as one of the promising areas for strawberry production. The gray mold of strawberry caused by the fungus Botrytis cinerea Pers.:Fr. (teleomorph Botryotinia fuckeliana (de Bary) Whetzel) is a devastating disease and often causes serious losses in yield and quality of strawberry production particularly in cool wet conditions. Many studies worldwide revealed that the disease caused severe pre-harvest yield losses up to 35 % in untreated strawberry fields (Legard et al. 2001).

2 170 Eur J Plant Pathol (2015) 141: The application of synthetic fungicides at blossom time until harvest is the essential measure to reduce the losses possibly resulting from infection (Weber and Entrop 2011). Protecting flowers or foliage from infection and reducing sporulation of the pathogen on infected plant material are the main targets of chemical control strategies against Botrytis fruit rot (Wilcox and Seem 1994). Referring to fungicide resistance development, this pathogen is classified as a high-risk pathogen due to its short life cycle and prolific reproduction (Brent and Hollomon 1998). Fungicide resistance of B. cinerea often compromises the control efficacy and results in the failure of disease control (Leroux 2007). During the last decade, carbendazim, iprodione and pyrimethanil fungicides have been used extensively to control gray mold disease in strawberry in China (Li et al. 2007; Jiang et al. 2009). Resistance of B. cinerea to benzimidazole and dicarboximide fungicides has been reported by many authors from China and the world (Zhou et al. 1994;Kang et al. 2000; Yourman et al. 2000; Banno et al. 2008). QoI fungicides such as azoxystrobin and pyraclostrobin were introduced for gray mold control in recent years in China and used as alternatives or complements to a wide range of specific, single-site fungicides such as benzimidazoles or dicarboximides. Previous studies with plant- pathogenic fungi reported that point mutations G143A, F129L and G137R in the cyt b gene were related to different levels of resistance to QoI fungicides (Fernández-Ortuño et al. 2008). Usually, field resistance to QoIs in B. cinerea was caused by a G143A point mutation (Leroch et al. 2013). The cyt b gene in B. cinerea had six types of structure with various intron-deletion patterns and the presence of the third intron (Bcbi-143/144 intron) was related to the risk of development of resistance to QoIs (Yin et al. 2012). With the sustained application of QoIs and other fungicides to control gray mold of strawberry, we hypothesize that the frequency of multiple resistance in B. cinerea populations will largely increase. Meanwhile, we are interested in the molecular mechanism of the QoI-resistant isolates, and the occurrence frequency of the third intron of cyt b associated with the resistant risk. Therefore, the objectives of the present study were to: (a) screen for resistance of B. cinerea populations from strawberry to azoxystrobin, carbendazim, iprodione, pyrimethanil and fludioxonil and assess the resistance frequencies of these fungicides; (b) determine the sensitivity to azoxystrobin, pyrimethanil and fludioxonil for a subset of the B. cinerea isolates; (c) detect point mutations associated with QoI resistance for all azoxystrobin-resistant (Azo R ) isolates; and (d) identify the structure of the cyt b gene in B. cinerea populations from strawberry. Materials and methods Collection of isolates of B. cinerea To detect fungicide resistance of B. cinerea in strawberry, decayed fruits were sampled in Feburary and March 2012 and 2013 from nine locations in Zhejiang Province. In the eight locations Lin an, Gouzhuang, Jiande, Nanxun, Shaoxing, Ningbo-1, Ningbo-2 and Taizhou-1, carbendazim, iprodione and pyrimethanil had been used for gray mold control for more than 10 years and QoI fungicides had been used during the last 4 years. In Taizhou-2, none of these four fungicides were used for control of gray mold (Table 1). Fludioxonil has never been used in these nine locations. In each location, 80 decayed fruits were hand picked from four greenhouses. To isolate B. cinerea from decayed fruit, small pieces of decayed tissue were placed on potato dextrose agar (PDA) containing streptomycin sulphate (100 μg/ml), and incubated at 25 C for 3 5 days. Fungal colonies similar to those of B. cinerea on isolation plates were transferred onto fresh PDA containing the same antibiotic. Pure cultures were then grown on PDA and incubated at 20 C under 12-h light/12-h dark to facilitate sporulation. B. cinerea isolates were then single-spore cultured and stored at 4 C. In total, 486 single-spore isolates of B. cinerea were obtained from these nine locations (Table 1). Monitoring of fungicide resistance in strawberry Isolates were characterized as resistant or sensitive using the discriminatory concentration (a concentration that fully inhibits mycelial growth of the sensitive strains). According to previous studies, the discriminatory concentrations were 10, 10, 5, 1 and 0.1 μg/ml for azoxystrobin, carbendazim (Martinez et al. 2005; Bardas et al. 2010), iprodione (Bardas et al. 2010), pyrimethanil (Topolovec-Pintaric 2009) and fludioxonil (Myresiotis et al. 2007), respectively. Technical grade azoxystrobin (96 % active ingredient [a.i.]; Syngenta Crop Protection, NC, USA) was dissolved in 100 % acetone, adjusted to a concentration of

3 Eur J Plant Pathol (2015) 141: Table 1 Information about 486 Botrytis cinerea isolates collected from strawberry in Zhejiang Province of China during 2012 and 2013 Location Year of collection No. of isolates No. of PCR assay with allelespecific primer pair b The history of fungicide application Azo Sa Azo Ra Azo S Azo R Lin an Carbendazim, iprodione and pyrimethanil Gouzhuang have been used during the last decade; Jiande azoxystrobin has been used during the last 4 years; and fludioxonil has never Nanxun been used Shaoxing Ningbo Ningbo Taizhou Taizhou None of the fungicides mentioned above was used Total / / / / a Azo S = sensitive to azoxystrobin and Azo R = resistant to azoxystrobin b +and indicate the presence and absence of the expected DNA fragment amplified by the allele-specific primer pair BcAR-F + BcAR-R (Jiang et al. 2009) 100 mg/ml, and added to PDA after sterilization to produce a final concentration of 10 μg/ml. The plates were amended with 100 μg/ml salicylhydroxamic acid (SHAM) (99 % a.i.; Sigma-Aldrich, Saint Louis, MO) to inhibit the alternative respiratory pathway. Technicalgrade carbendazim (98 % a.i.; Shanghai Shennong Pesticide Co., Ltd., Shanghai, China) was dissolved in 0.1 M hydrochloric acid (HCl), adjusted to a concentration of 5 mg/ml, and added to PDA to produce a final concentration of 10 μg/ml. Technical-grade iprodione (96.5 % a.i.; Heyi Agricultural Chemical Co. Ltd, Zhejiang, China) was dissolved in 100 % acetone, adjusted to a concentration of 100 mg/ml, and added to PDA to produce a final concentration of 5 μg/ml. Technicalgrade pyrimethanil (95 % a.i.; provided kindly by the Institute of the Control of Agrochemicals, Ministry of Agriculture, China) was dissolved in 100 % acetone at the concentration of 10 mg/ml, and added to ASP-agar medium (Hilber and Schuepp 1996) to produce a final concentration of 0.1 μg/ml. Technical-grade fludioxonil (93 % a.i.; Syngenta Inc., Switzerland) was dissolved in 100 % methanol, adjusted to a concentration of 10 mg/ml, and added to PDA to produce a final concentration of 1 μg/ml. For each fungicide, isolates that were able to grow on the amended PDA or ASP-agar were designated as resistant isolates; isolates that were unable to grow on the amended PDA or ASP-agar were designated as sensitive isolates. The experiment was conducted twice for all isolates and one additional test was performed for resistant isolates. Determination of sensitivity to azoxystrobin, pyrimethanil and fludioxonil The effective concentration that inhibits mycelial growth by 50 % relative to the control (EC 50 )values of azoxystrobin was determined for 47 azoxystrobinsensitive (Azo S ) and 41 Azo R isolates. The EC 50 values of pyrimethanil were determined for 48 pyrimethanilsensitive (Pyr S ) and 44 pyrimethanil-resistant (Pyr R ) isolates. The EC 50 values of fludioxonil were tested for 100 fludioxonil-sensitive (Flu S ) isolates. All the isolates used for the determination of EC 50 values were randomly selected. Azoxystrobin solution (100 mg/ml) was added to PDA to produce final concentrations of 0, 0.005, 0.01, 0.05, 0.1, 0.3, 0.6, and 1 μg/ml for Azo S isolates and 0, 1, 10, 100, 150 and 200 μg/ml for Azo R isolates. The plates were amended with 100 μg/ml SHAM to inhibit the alternative respiratory pathway. Pyrimethanil solution (10 mg/ml) was added to ASP-agar medium to produce final concentrations of 0, 0.08, 0.1, 0.14, 0.18, 0.2, 0.4, 0.8, 1, 1.2 and 1.4 μg/ml for Pyr S isolates and 10, 17.5, 25, 37.5, 50, 75 and 100 μg/ml for Pyr R isolates. Fludioxonil solution at the concentration of

4 172 Eur J Plant Pathol (2015) 141: mg/ml was added to PDA to produce final concentrations of 0, 0.001, , 0.005, 0.01, 0.025, 0.05, and 0.1 μg/ml for Flu S isolates. A 5-mm mycelial plug was taken from the edge of a 3-day-old colony and placed at the centre of PDA or ASP-agar plates amended with the fungicides at each of the above concentrations. For each isolate, three replicates per concentration were used. The experiment was conducted twice for all isolates. After 3 days of incubation at 25 C, colony diameter in each plate was measured in two perpendicular directions with the original mycelial plug diameter (5 mm) subtracted from this measurement. The average EC 50 values from two experiments for each isolate were used in the data analysis since there was no significant difference (P>0.05) between the two experiments. Calculations were performed using SAS (JMP; SAS Institute, Cary, NC). Molecular detection of the G143A point mutation To extract DNA, approximately 100 mg of fresh mycelia of each isolate were harvested by gently scraping the surface of a culture on PDA plates with a sterilized loop. DNA from fungal mycelia was extracted using a previously published protocol (Saitoh et al. 2006). To detect whether the Azo R B. cinerea isolates had the G143A point mutation, a pair of allele-specific PCR primers, BcAR-F + BcAR-R, developed in our lab was used (Jiang et al. 2009). PCR products were examined by electrophoresis in a 1.5 % agarose gel in 1 Trisacetate-EDTA (TAE) buffer. Gene structure of cyt b According to previous studies, the cyt b gene in B. cinerea has various intron-deletion patterns (Banno et al. 2009; Yin et al. 2012). To detect the structure of the cyt b gene in B. cinerea populations from strawberry in Zhejiang Province, two primer pairs, 137S-F (5 -CTTG TGCTTTGCTTTGCTTTG-3 ) + 137S-R (5 -AACCAT CTCCATCCACCATAC-3 ) and1int-f(5 -ATGATC GATTCACCACAACCA-3 ) + 1 int-r (5 -CATCCA TTGTTAACATCACGC-3 ), were used (Yin et al. 2012). Primers 137S-F and 137S-R are located in the second and fourth introns, respectively, so they can simultaneously detect the presence of the second, third and fourth introns based on the size of PCR fragments. Amplifying with this primer pair, isolates giving a 687- bp band were identified as containing the second and fourth introns, isolates giving a 1,892-bp band were identified as containing the second, third and fourth introns, and isolates giving no PCR product were identified as lacking the second, fourth, or both introns (Yin et al. 2012). Primers 1 int-f + 1 int-r both distributed in exons only can detect the presence of the first intron. A total of 129 Azo R and 100 Azo S isolates randomly selected from the nine locations were tested for the structure of cyt b. PCR amplifications were conducted using the published assays (Yin et al. 2012). PCR products were examined by electrophoresis in a 1.5 % agarose gel in 1 TAE buffer. Results Resistance phenotypes of B. cinerea isolates from strawberry Monitoring of fungicide resistance among the 486 isolates based on discriminatory concentrations of azoxystrobin, carbendazim, iprodione, pyrimethanil and fludioxonil identified 11 resistance phenotypes. Among the 486 isolates, 133 (27.4 %) were sensitive to all fungicides tested, 7 (1.4 %) were resistant only to carbendazim, two (0.4 %) were resistant only to iprodione, two (0.4 %) were resistant to both iprodione and pyrimethanil, one (0.2 %) was resistant to azoxystrobin and pyrimethanil, four (0.8 %) were resistant to carbendazim and azoxystrobin, 14 (2.8 %) were resistant to carbendazim and iprodione, four (0.8 %) were resistant to azoxystrobin, iprodione and pyrimethanil, 42 (8.6 %) were resistant to carbendazim, azoxystrobin and pyrimethanil, 17 (3.5 %) were resistant to carbendazim, azoxystrobin and iprodione, six (1.2 %) were resistant to carbendazim, iprodione and pyrimethanil, and 254 (52.3 %) were resistant to carbendazim, azoxystrobin, iprodione and pyrimethanil (Fig. 1). The frequency distribution of fungicide resistance among nine locations Among the eight locations Lin an (n=50), Gouzhuang (n=52), Jiande (n=52), Nanxun (n=54), Shaoxing (n= 56), Ningbo-1 (n=59), Ningbo-2 (n=34) and Taizhou-1 (n=62) in Zhejiang Province, the frequency of Azo R isolates ranged from 46.0 to 90.7 %, the frequency of carbendazim-resistant (Car R ) isolates ranged from 54.0

5 Eur J Plant Pathol (2015) 141: Fig. 1 Frequencies of resistance phenotypes observed in Botrytis cinerea populations across nine strawberry locations in Zhejiang Province of China in 2012 and Azo R,Car R,Ipr R and Pyr R indicate resistant to azoxystrobin, carbendazim, iprodione and pyrimethanil, respectively. Sensitive = sensitive to the four fungicides to 94.2 %, the frequency of iprodione-resistant (Ipr R ) isolates ranged from 46.0 to 94.2 %, and the frequency of Pyr R isolates ranged from 44.0 to 90.7 % (Fig. 2). However, in location Taizhou-2 (n=67), the frequency of resistance to azoxystrobin, carbendzim, iprodione and pyrimethanil was 3.0, 3.0, 0 and 3.0 %, respectively. Of the total nine locations, no fludioxonil-resistant (Flu R ) isolates were found among the 486 isolates tested. Sensitivity of B. cinerea to azoxystrobin, pyrimethanil and fludioxonil Among 486 isolates of B. cinerea obtained in the study, 47 Azo S,48Pyr S and 100 Flu S isolates randomly selected were used to determine the EC 50 for azoxystrobin, pyrimethanil and fludioxonil, respectively, using mycelial growth inhibition assays. The EC 50 values of azoxystrobin, pyrimethanil and fludioxonil ranged from 0.02 to 2.5 μg/ml with a mean of 0.3 μg/ml (Fig. 3a), from 0.1 to 0.6 μg/ml with a mean of 0.2 μg/ml (Fig. 3c), and from to 0.04 μg/ml with a mean of 0.01 μg/ml (Fig. 3e), respectively. A total of 41 Azo R isolates and 44 Pry R isolates were tested for EC 50 values of azoxystrobin and pyrimethanil, respectively. The EC 50 values for 41 Azo R isolates ranged from 6.1 to 1,233.6 μg/ml, with a mean of μg/ml (Fig. 3b). The EC 50 values for 44 Pyr R isolates ranged from 9.4 to 57.0 μg/ml, with a mean of 33.0 μg/ml (Fig. 3d). The Azo R and Pyr R isolates showed a high level of resistance with RF (folds of EC 50 value for a resistant isolate relative to the mean EC 50 value for sensitive isolates) ranging from 21.7 to 4,359.0 and from 53.0 to 320.1, respectively. Molecular detection of the G143A point mutation and gene structure of cyt b All 322 Azo R isolates produced a 260-bp fragment amplifying with the allele-specific primers BcAR-F + BcAR-R (Table 1), indicating that they all harboured the G143A point mutation. After detecting the presence of four introns located in the cyt b gene, two types (A and B) of cyt b were found (Fig. 4). Among 229 B. cinerea isolates tested (129 Azo R and 100 Azo S ), 28 (12.2 %) had the type A cyt b gene containing all four introns (Bcbi-67/68, Bcbi- 131/132, Bcbi-143/144, and Bcbi- 164) and 201 (87.8 %) had the type B cyt b gene containing three introns (Bcbi-67/68, Bcbi- 131/132, and Bcbi-164). All the 129 Azo R isolates tested did not carry the Bcbi-143/144 intron in the cyt b gene. Among 100 Azo S isolates, only 28 contained the Bcbi- 143/144 intron in cyt b, while 72 did not contain this intron. Discussion To test our hypothesis that the continuous use of QoIs and other fungicides for the control of gray mold will lead to a high frequency of multiple resistance in B. cinerea populations, we analyzed the fungicide resistance of 486 B. cinerea isolates from nine strawberry locations and found that 52.3 % were simultaneously resistant to carbendazim, pyrimethanil, iprodione and

6 174 Eur J Plant Pathol (2015) 141: Fig. 2 Frequency distribution of different resistant populations of Botrytis cinerea collected from nine strawberry locations in Zhejiang Province of China. The number of isolates from Lin an, Gouzhuang, Jiande, Nanxun, Shaoxing, Ningbo-1, Ningbo-2, azoxystrobin, 20.3 % were resistant to one, two or three of these four fungicides, and only 27.4 % were sensitive to these four fungicides. The development of multiple resistances to chemically unrelated fungicides has been reported in different populations of B. cinerea (Yourman et al. 2000; Baroffio et al. 2003; Weber 2011; Amiri et al. 2013; Leroch et al. 2013). Some B. cinerea isolates from German strawberry-growing regions even possess resistance to six fungicides (Leroch et al. 2013). Because carbendazim, iprodione and pyrimethanil have been used to control strawberry gray mold during the last decade, it is not surprising to find the high frequency of triple-resistant isolates of B. cinerea. It was surprising that the frequency of resistance to azoxystrobin was up to 66.2 % in this study even though QoI fungicides were recently introduced to control gray Taizhou-1 and Taizhou-2 was 50, 52, 52, 54, 56, 59, 34, 62 and 67, respectively. Azo R,Car R,Ipr R and Pyr R indicate resistance to azoxystrobin, carbendazim, iprodione and pyrimethanil, respectively mold in China. In a previous study, only two out of 65 B. cinerea isolates collected from strawberry in Zhejiang Province showed resistance to azoxystrobin (Jiang et al. 2009). Within 4 years the frequency of Azo R isolates from the same Province has increased approximately 21 times, indicating that the risk for development of azoxystrobin resistance in B. cinerea is high. The study on pyraclostrobin resistance in B. cinerea populations from apple in Washington State supported our conclusion (Yin et al. 2012). The present work was initiated to assess the fungicide resistance in B. cinerea populations from strawberry and to find a proper strategy to control the populations with multiple resistance to a group of fungicides with different modes of action. The most important finding of this work is that no Flu R isolates were detected among all

7 Eur J Plant Pathol (2015) 141: Fig. 3 Frequency distribution of the effective concentration resulting in 50 % mycelial growth inhibition (EC 50 ) for (a) 47 azoxystrobin-sensitive (Azo S ) isolates, (b) 41 azoxystrobinresistant (Azo R ) isolates, (c) 48 pyrimethanil-sensitive isolates (Pyr S ), (d) 44 pyrimethanil-sensitive isolates (Pyr R )and(e)100 fludioxonil-sensitive (Flu S ) isolates, of Botrytis cinerea collected from strawberry 486 isolates tested, indicating that fluidioxonil has great potential in controlling gray mold and could be an alternative to carbendazim, iprodione, pyrimethanil and azoxystrobin. Similar results were found by Myresiotis et al. (2007), who detected resistance of B. cinerea isolates collected from vegetable crops to carbendazim, iprodione and pyrimethanil, and did not find any isolates resistant to fludioxonil. Moreover, Fernández-Ortuño et al. (2013b) also did not observe any B. cinerea isolates resistant to fludioxonil in strawberry fields treated with this fungicide. Among nine locations in Zhejiang Province, eight had a high frequency (from 44.0 to 94.2 %) of resistance to azoxystrobin, carbendazim, iprodione or pyrimethanil, and only location Taizhou-2 had a very low frequency (3.0 %) of resistance to azoxystrobin,

8 176 Eur J Plant Pathol (2015) 141: Type A B. cinerea with intron bi-143/144 1 intron bi-67/68 Met 67 His intron bi-131/132 Ile Gly bp Tyr Gly ATG.CAT atggca.agtttg ATT.GGT 1366 bp TAT.GGT tataaa.attttg intron bi-143/ Ala 163 Glu 1205 bp tttttt...gccttg GCT..GAGT intron bi Leu Stop 1268 bp caaaca...atattg TTTTA.TAA Type B B. cinerea without intron bi-143/144 1 Met 67 His ATG.CAT intron bi-67/ bp atggca.agtttg Fig. 4 Structures of the cytochrome b gene (cyt b) in Botrytis cinerea from strawberry. Type A = cyt b containing four introns (Bcbi-67/68, Bcbi-131/132, Bcbi-143/144, and Bcbi-164); type B intron bi-131/ Ile Gly 132 intron bi-164 Tyr 143 Gly Ala 163 Glu Leu Stop 1366 bp 1268 bp ATT.GGT TAT.GGT GCT..GAGT TTTTA.TAA tataaa.attttg caaaca...atattg = cyt b containing three introns (Bcbi-67/68, Bcbi-131/132, and Bcbi-164). Boxes indicate exons; lines indicate introns. Lengths of exons and introns are not scaled carbendzim or pyrimethanil. The main difference between Taizhou-2 and the other eight locations is owing to the selection pressure of fungicides. These eight locations have been heavily treated with the four fungicides mentioned above in 2 5 spray applications during the whole blossom for several years, whereas strawberries in the location Taizhou-2 are never treated with these fungicides. Even within the eight locations, the resistance frequencies of the same fungicide showed varying levels. Similarly, Ishii et al. (2009) determined the sensitivity to azoxystrobin for B. cinerea isolates collected from strawberry fields in different regions of Japan and found that the resistance frequencies ranged from 78 to 100 %. In the sensitivity tests, all 41 Azo R isolates showed high resistance to azoxystrobin with RF ranging from 21.7 to 4, This result was supported by the study of Yin et al.(2012),who reported that B. cinerea isolates highly resistant to the QoI fungicide pyraclostrobin had RF higher than 900. The mean EC 50 value of 47 Azo S isolates was 0.3 μg/ml and fell within the range ( μg/ml) of EC 50 values for B. cinerea populations from vineyards in southern Italy (De Miccolis et al. 2012). Similarly, all the tested Pyr R isolates with EC 50 values from 9.4 to 57.0 μg/ml also showed high resistance to pyrimethanil (53.0 RF 320.1). The 48 Pyr S isolates with EC 50 values from 0.1 to 0.6 μg/ml had a similar range compared with Pyr S populations from vegetable crops in Greece (Myresiotis et al. 2007). To our knowledge, this is the first report about EC 50 values and RFs for Pyr R populations of B. cinerea, which is useful for the study of resistance levels probably relating to resistance mechanism. All 486 isolates obtained in this study were sensitive to fludioxonil using mycelial growth assays, and the EC 50 values of 100 Flu S isolates ranging from to 0.04 μg/ml were close to those of B. cinerea populations from vegetable crops (Myresiotis et al. 2007). More recently, moderately Flu R populations of B. cinerea from small fruit in Germany (Weber 2011) and from strawberry in Maryland and South Carolina (Fernández-Ortuño et al. 2014) were detected in conidial germination assays, while lowly Flu R populations from strawberry in Virginia were observed by Fernández-Ortuño et al. (2013a). In this study, all 322 Azo R isolates harboured point mutation G143A, which is in agreement with many previous studies (Leroux et al. 1999; Banno et al. 2009; Yin et al. 2012). None of the 129 Azo R isolates tested carried the Bcbi-143/144 intron, which is consistent with those of previous studies on B. cinerea from different crops (Leroux et al. 1999; Yin et al. 2012). Moreover, 87.8 % of the 229 isolates of B. cinerea tested from various strawberry-production locations in Zhejiang Province did not contain the Bcbi143/144 intron. According to the previous studies that the presence of this intron in cyt b prevents the occurrence of G143Amediated resistance (Grasso et al. 2006; Banno et al. 2009), our finding indicates that B. cinerea populations from strawberry in Zhejiang Province possess a high inherent risk for the development of resistance to QoI fungicides. Acknowledgments This research was supported by the Special Program for Agricultural Research ( ), the 973 Project (2013CB127802), and National Science Foundation ( ). References Amiri, A., Heath, S. M., & Peres, N. A. (2013). Phenotypic characterization of multifungicide resistance in Botrytis cinerea isolates from strawberry fields in Florida. Plant Disease, 97,

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