Sensitivity of Sclerotinia sclerotiorum from oilseed crops to boscalid in Jiangsu Province of China

m Zh Accepted 26 June 2009 Disease management 20 i elin ctas d s �1 Bary is 400 s crops is the main production area, where oilseed rape is planted on about 700,000 ha every year. Because breeding programs for disease resistance have been hampered by limited gene sources (Lu, 2003), Sclerotinia stem rot imide fungicide, has also beenwidely used for the control of Sclero- tinia stem rot for more than 6 years in Jiangsu Province of China. However, it appears that repeated applications of dicarboximide fungicides have caused the emergence of resistant strains in several plant pathogens of Sclerotinia in other countries including S. homoeocarpa and S. minor (Smith et al., 1995; Jo et al., 2006). In a most recent study, the occurrence of insensitivity to dimetha- chlon in S. sclerotiorum was reported in Jiangsu Province of China (Ma et al., 2009). * Corresponding author. Tel./fax: þ86 025 8439 5641. E-mail address: [email protected] (M.-G. Zhou). 1 Contents lists availab Crop Pro .e l Crop Protection 28 (2009) 882–886 Joint first authorship. sunflower, canola and oilseed rape (Brassica napus L.) (Purdy, 1979). Infection can occur on leaves, stems and pods at different devel- opmental stages (Abawi and Grogan, 1979; Sylvester-Bradley and Donald, 1984; Dai et al., 2006). The disease is important in many countries including Canada (Bardin and Huang, 2001), the USA (Purdy, 1979; Bolton et al., 2007), Australia (Letham et al., 1976) and China (Zhou and Luo, 1994). For example, Sclerotinia stem rot caused by S. sclerotiorum in China leads to yield loss ranging from 10 to 80%, and oil quality declines as well (Anonymous, 1975). Oilseed rape is the major oilseed crop in China, with approximately 70� 106 ha in production (Zhao and Meng, 2003). Jiangsu province China, but wide spread control failures attributed to development of MBC resistance in S. sclerotiorumwere reported in 2001 and 2002 (Pan,1998; Shi et al., 2000; Zhang et al., 2003). Furthermore, a point mutation in the b-tubulin gene (E198A) in resistant isolates of S. sclerotiorum was characterized with practical resistance to MBC in the field (Li et al., 2003). Therefore, the use of MBC and related fungicides for controlling Sclerotinia stem rot in China was compromised after more than 30 years application of MBC in Jiangsu Province of China (Pan, 1998; Shi et al., 2000; Zhang et al., 2003). Dimethachlon [N-(3, 5-dichlorophenyl)-succinimide, Ohric, Wenzhou Pesticide Factory, Zhejiang Province China], a dicarbox- Fungicides Oilseed rape Carboxamides 1. Introduction Sclerotinia sclerotiorum (Lib.) de pathogen which attacks more than (Boland and Hall, 1994), including 0261-2194/$ – see front matter Crown Copyright � 2 doi:10.1016/j.cropro.2009.06.012 resistance between boscalid and MBC/dimethachlon already in commercial use. In field trials, the treatments boscalid (50% WG), at 125, 187.5 and 250 g ai ha�1 provided higher control efficacies of 65.2, 69.0 and 75.1%, respectively, than the treatments carbendazim (50% WP), at 750 g ai ha�1 and dime- thachlon (46% WP), at 690 g ai ha�1 with control efficacies of 18.9 and 44.6%, respectively. Crown Copyright � 2009 Published by Elsevier Ltd. All rights reserved. a cosmopolitan fungal pecies of higher plants such as soybean, bean, continues to impose serious limitations on oilseed rape production all over the world. In practice, application of fungicides is the principal tool in most oilseed rape crops for managing Sclerotinia stem rot. A benzimidazole fungicide, carbendazim (MBC), was widely used to control this disease routinely during the 1980s in Keywords: Sclerotinia stem rot dual resistance to both carbendazim and dimethachlon was similar, suggesting that there was no cross- unimodal curves with mean EC50 values of 0.17 � 0.09 mg ml for inhibition of mycelial growth. Inhi- bition of mycelial growth of isolates whether they had resistance to carbendazim or, dimethachlon or Sensitivity of Sclerotinia sclerotiorum fro in Jiangsu Province of China Jian-Xin Wang 1, Hui-Xia Ma 1, Yu Chen 1, Xiao-Fen Chang-Jun Chen, Ming-Guo Zhou* College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, China a r t i c l e i n f o Article history: Received 16 January 2009 Received in revised form 26 June 2009 a b s t r a c t Between 2006 and 2008, 1 characterized for the bas succinate ubiquinone redu 2006 and 2008 all showe journal homepage: www 009 Published by Elsevier Ltd. All oilseed crops to boscalid u, Wen-Yuan Yu, Zheng-He Tang, solates of Sclerotinia sclerotiorum from the Jiangsu Province of China were e sensitivity to boscalid, a new active ingredient that interferes with e in the electron transport chain of fungi. The isolates collected between imilar sensitivity to boscalid. Baseline sensitivities were distributed as le at ScienceDirect tection sevier .com/locate/cropro rights reserved. amended with dimethachlon to obtain final concentrations of 15, 30, 60, 120 and 240 mg ml�1 for the dimethachlon-resistant rotec One alternative to achieve and optimize disease control and minimize the risk of resistance development is to incorporate new fungicides with contrasting modes of action into a spray program (Staub,1991). Boscalid (trade name Cantus in China) is a new broad- spectrum fungicide belonging to the carboxamide group. It inhibits the enzyme succinate ubiquinone reductase (complex II), also known as succinate dehydrogenase (SDH), in the mitochondrial electron transport chain (Avenot and Michailides, 2007; Avenot et al., 2008). The enzyme SDH consists of four subunits: SdhA, SdhB, Sdhc and SdhD (Stammler and Speakeman, 2006). Boscalid is registered for use against Botrytis cinerea, Sclerotinia spp., Alternaria spp.,Monilinia spp., powdery mildews and other pathogens in fruit, vegetables and vines (Stammler and Speakeman, 2006) in other countries. However, presently, boscalid is only registered for controlling cucumber grey mould in China and provides an opportunity to study the baseline sensitivity of S. sclerotiorum to boscalid prior to its legal use. Moreover, the novel mode of action of boscalid makes it an excellent candidate for management of fungicide resistance in S. sclerotiorum in China. The objectives of this current study were to: (i) determine whether boscalid was effective against MBC-resistant and dime- thachlon-resistant isolates of S. sclerotiorum in vitro; (ii) establish the baseline sensitivity of S. sclerotiorum isolates to boscalid using the field isolates from Jiangsu Province of China; and (iii) test the efficacy of boscalid in controlling Sclerotinia stem rot in the field of Jiangsu Province where MBC was extensively used for decades and no longer controls the disease. 2. Materials and methods 2.1. Origin and collection of S. sclerotiorum isolates All isolates of S. sclerotiorum used in the study were collected from oilseed rape fields in Jiangsu Province of China between 2006 and 2008. The oilseed rape fields were separated from each other by more than 10 km. In each field several plants with symptoms of Sclerotinia stem rot were randomly collected from a designated 5.0 � 5.0 m2 area, air-dried, placed in paper envelopes, and stored at �4 �C. All isolates were derived from individual sclerotia collected from oilseed rape plants. The sclerotia were surface sterilized in 0.1% sodium hypochlorite for 5 min, rinsed in sterile distilled water for 30 s, bisected and one of the two halves were placed on potato dextrose agar (PDA, see details below) plates. The plates were incubated for 3 days at 25 �C in a growth chamber (12 h photoperiod). Pure cultures were obtained by transfer of a single sclerotium and maintained on PDA slants at 4 �C for 2–4 weeks (Christian et al., 2001; Li et al., 2003; Sarma et al., 2007). In all, a total of 120 isolates were collected throughout Jiangsu Province. Boscalid had never been used to control Sclerotinia stem rot before sampling. 2.2. Fungicides and media Boscalid technical grade (96.2%) was provided by BASF Co., China and was dissolved in acetone to 10 mg ml�1 for the stock solution. Dimethachlon technical grade (90.0%) was provided by Wenzhou Pesticide Factory (Zhejiang Province, China) and dis- solved in methanol (>99.5%) to 10 mg ml�1 for the stock solu- tion. Carbendazim (MBC) technical grade (98%) was provided by Shenyang Academy of Chemistry and Industry (Shengyang, China) and was dissolved in 0.1 mol l�1 hydrochloric acid (HCl) at 10 mg ml�1 as stock solution. Boscalid (50% Cantus, WG), carbendazim (50% Duojunling, WP) and dimethachlon (46% Junhejing or Ohric, WP) were purchased in Nanjing (the capital J.-X. Wang et al. / Crop P city of Jiangsu Province, China). mutants and 0, 0.125, 0.25, 0.5, 1.0 and 2.0 mg ml�1 for the selected wild-type dimethachlon-sensitive isolates. The EC50 values of dimethachlon-resistant mutants and the selected wild-type dimethachlon-sensitive isolates were determined as described above. The wild-type sensitive isolates, MBC-resistant isolates, the dimethachlon-resistant isolates and the isolates with dual resis- tance to both MBC and dimethachlon, chosen arbitrarily, were also compared regarding their sensitivity to boscalid to determine cross-resistance between boscalid and these two fungicides which were extensively used for control of Sclerotinia stem rot in Jiangsu PDA, which was a nutrient-rich medium, was made from 200 g potato, 20 g agar and 20 g dextrose l�1 of distilled water. Two poorer nutrient media, Czapek medium, consisted of 3 g NaNO3, 1 g K2HPO4, 0.5 g MgSO4, 0.5 g KCl, 0.01 g FeSO4, 30 g sucrose and 20 g agar l�1 of distilled water, and a synthetic complex medium, according to a previous study with modifications, was made of: 12 g glucose, 2 g asparagine, 5 g KH2PO4, 0.4 g MgSO4 and 10 g yeast extract l�1 of distilled water (Perez et al., 1992). These three media were used for routine culture and determination of sensi- tivity of S. sclerotiorum isolates to fungicides. 2.3. Analysis of cross-resistance of boscalid with MBC and dimethachlon Sensitivity of all 120 isolates to MBC was determined through a discriminatory dose test according to a previous study (Li et al., 2003). Briefly, an inverted mycelial plug (5 mm in diameter), cut from the edge of a 3 day-old colony was transferred to a 9 cm Petri dish containing the amended or unamended PDA. After 2 days of incubation in a growth chamber at 25 �C and 12 h photoperiod, mycelial growth was measured. Isolates that showed normal growth at 5 mg ml�1 MBC were considered resistant (scored as growth), whereas the completely inhibited ones were considered sensitive (scored as no growth). A series of tests was made to find the concentration of the fungicide causing a 50% reduction in the growth rate compared to an unamended control (EC50). In the EC50 assay, PDA medium was amended with six concentrations of MBC: 0, 0.1, 1, 10, and 100 mg ml�1 for the MBC-resistant isolates and 0, 0.125, 0.25, 0.5, 1.0 and 2.0 mg ml�1 of MBC for wild-type MBC- sensitive isolates. An inverted mycelial plug (5 mm in diameter), cut from the edge of a 3 day-old colony was transferred to 9 cm Petri dishes containing the amended media. Three replicates per concentration were used and all the tests were repeated twice. The mycelial growthwasmeasured after 2 days of incubation at 25 �C in a growth chamber (12 h photoperiod). Two perpendicular diame- ters of each fungal colony were measured and averaged (the diameter of the plug was subtracted). EC50 values were determined by probit analysis (Alberoni et al., 2005). Little information on the field resistance of S. sclerotiorum to dimethachlon was available in oilseed rape fields in Jiangsu Prov- ince (Ma et al., 2009), therefore, the dimethachlon resistance was obtained through selection. To induce dimethachlon resistance through selection, fresh mycelial plugs (5 mm in diameter) from colony margins of the wild-type dimethachlon-sensitive isolates were transferred to PDA plates amended with 5.0 mg ml�1 dime- thachlon and incubated in the growth chamber for 3 days. Any fast- growing sectors from the otherwise restricted colonies were selected and transferred after 2 days to PDA plates amended with 5.0 mg ml�1 dimethachlon. Single sclerotium isolates were estab- lished from dimethachlon-resistant mutants. Autoclaved PDA was tion 28 (2009) 882–886 883 Province of China. 2.4. Determination of baseline EC50 values of boscalid against S. sclerotiorum To establish the baseline sensitivity to bocalid, the 120 S. scle- rotiorum isolates which were collected from the field of Jiangsu Province during 2006 to 2008 were tested to determine baseline sensitivity to boscalid. Autoclaved PDAwas amended with boscalid to obtain final concentrations of 0, 0.025, 0.05, 0.1, 0.2, 0.4 and 0.8 mg ml�1 and the EC50 values of these isolates were determined as described above. Czapek and the synthetic complex medium, were used to compare the sensitivity of the 120 S. sclerotiorum isolates to boscalid on different media. The concentrations of boscalid were the same, and the diameters of the colony were measured after 8 and 4 days on the Czapek medium and the synthetic complex medium, respectively. The EC50 values were determined as described above. 2.5. Field trial for controlling Sclerotinia stem rot of the stem; 7 ¼ lesions on 31–50% of the stem; 9 ¼ lesions on >50% of the stem (Anonymous, 2000). 2.6. Statistical analysis Data were processed with the analysis of variance (ANOVA) using the SAS GLM (SAS Institute, Inc., Cary, NC). When the ANOVA was significant (P ¼ 0.05), means were separated with Fisher’s protected least significant difference (PLSD). 3. Results 3.1. Cross-resistance between boscalid and MBC/dimethachlon There was significant difference (P ¼ 0.05) in sensitivity to boscalid between isolates (Table 1). However, there was no corre- lation between this difference in sensitivity to boscalid and sensitivity to MBC or dimethachlon. This suggested there was no cross-resistance between boscalid and MBC/dimethachlon already rotiorum isolates collected between 2006 and 2008 to boscalid and Cross-resistance between boscalid and carbendazim and dimethachlon in Sclerotinia scle daz 0.406 a 0.186 a 166.40 f 0.115 a J.-X. Wang et al. / Crop Protection 28 (2009) 882–886884 Isolatesa Resistance phenotypesb The EC50 of carben YZ112 MBCS dimethachlonS 0.106 a YZ114 MBCS dimethachlonS 0.134 a YZ116 MBCS dimethachlonS 0.137 a SZ47 MBCS dimethachlonS 0.177 a SZ62 MBCS dimethachlonS 0.186 a ZJ288 MBCR dimethachlonS 0.147 a CZ57 MBCR dimethachlonS 0.168 a ZJ44 MBCR dimethachlonS >100 b YC35 MBCR dimethachlonS >100 b ZJ47 MBCR dimethachlonS >100 b TZ83 MBCR dimethachlonS >100 b SZ48 MBCR dimethachlonS >100 b ZJ288-1 MBCR dimethachlonR >100 b ZJ288-2 MBCR dimethachlonR >100 b ZJ288-3 MBCR dimethachlonR >100 b CZ57-1 MBCR dimethachlonR >100 b CZ57-2 MBCR dimethachlonR >100 b CZ57-3 MBCR dimethachlonR >100 b a An oilseed rape field known to be naturally infested with S. sclerotiorumwas selected in 2008. The field, which was located in Jiangyan of Jiangsu Province, was divided into 21 plots; each plot was 5 � 6 m2. A completely randomized design was used with seven treatments (all sprays) and three replicates. The treatments were: (1) a control consisting of water, 750 L ha�1; (2) carbendazim (50%WP), 750 g ai ha�1; (3) dimethachlon (46%WP), 690 g ai ha�1; (4) boscalid (50% WG), 80 g ai ha�1; (5) boscalid, 125 g ai ha�1; (6) boscalid, 187.5 g ai ha�1; and (7) boscalid, 250 g ai ha�1. Fungicides were applied when 95% of the major stems were flowering. The sprayer was a Jacto Heavy-Duty HD400 (Agrolex, Singapore). No other fungicides were applied to the experimental plots. All other treatments, such as herbicides and fertilizers, were used in accordance with standard farm practices. The experiment was repeated twice in two different oilseed rape fields in Jiangyan. Disease incidence was determined at the end of the growing season, about 1 week before harvest. Two hundred randomly selected plants in each plot were rated for the presence of disease. Disease incidence for each plot was calculated as the percentage of plants with symptoms of Sclerotinia stem rot. Disease incidence for the control plot was high up to 25% in 2008. Severity was assessed using the following scale: 0 ¼ no lesions; 1 ¼ lesions on alid rotection 28 (2009) 882–886 885 data could be used as a baseline for monitoring the shift of sensi- tivity in S. sclerotiorum populations to boscalid. 3.3. Efficacy of boscalid in controlling Sclerotinia stem rot The incidence of Sclerotinia stem rot was correlated with moisture periods before and during flowering. The symptoms of stem rot were severe and the disease incidence for control plots was 25% in 2008 because the weather was favourable for an epidemic of Sclerotinia stem rot. The treatments boscalid at 125, 187.5 and 250 g ai ha�1 provided the highest control efficacies of 65.2, 69.0 and 75.1%, respectively, whereas MBC failed to control Fig. 1. Frequency distribution of sensitivity of 120 Sclerotinia sclerotiorum isolates to bosc J.-X. Wang et al. / Crop P Sclerotinia stem rot with a control efficacy of only 16.0% (Table 2). However, another extensively used fungicide for the control of Sclerotinia stem rot, dimethachlon exhibited a control efficacy of 48.7%, which was similar to the control efficacy of the treatment boscalid at 80 g ai ha�1 (49.7%), the lowest dosage in this field trial (Table 2). 4. Discussion This study suggested that there was no cross-resistance between boscalid and two commonly used fungicides (MBC and dimethachlon) for the control of Sclerotinia stem rot. This could be explained by the difference in mode of action between boscalid and other fungicides (MBC and dimethachlon) extensively used in practice. Meanwhile, boscalid showed strong antifungal activity in inhibiting mycelial growth (Zhang et al., 2007). This, together with the wide spread occurrence of MBC resistance and dimethachlon insensitivity in S. sclerotiorum in the oilseed rape crops of Jiangsu Province (Ma et al., 2009), makes boscalid an excellent candidate for the control of Sclerotinia stem rot. For inhibition of growth, EC50 values showed a range of distri- bution from 0.028 to 0.398 mg ml�1 for the most and least sensitive isolates, respectively. Therefore, 0.17 (�0.09) mgml�1 boscalid could be used as a baseline for monitoring the shift of sensitivity in S. sclerotiorum populations to boscalid for mycelial growth. More- over, it is very important to monitor the sensitivity of S. sclerotiorum populations to boscalid in the future as resistance to this fungicide has already occurred in Alternaria alternata causing alternaria late blight of pistachios in California (Avenot and Michailides, 2007). Based on our knowledge, this is the first report on the baseline sensitivity of S. sclerotiorum isolates to boscalid in China. A previous study also reported that a mycelial growth test in tissue culture plates with liquid YBA medium was developed for a rapid and reliable sensitivity determination of S. sclerotiorum to boscalid (Stammler et al., 2007). The results from that study showed that EC50 values in inhibiting mycelial growth of S. scle- rotiorum ranged from 0.001 to 0.056 mg ml�1, with the mean EC50 values less than 0.012 mg ml�1. It was also argued that a rich medium should not be used to assay the sensitivity of boscalid in on potato dextrose agar (a), Czapek medium (b) and the synthetic complex medium (c). S. sclerotiorum (Stammler et al., 2007). However, in our study, the EC50 values were 0.028–0.398 mg ml �1, higher than that in the previous study. Moreover, the average EC50 values were similar for the nutrient-rich PDA medium and the nutrient-poor Czapek and the synthetic complex media, and based on the EC50 values, these isolates were still very sensitive to boscalid. The difference between the previous study and our results might be caused by the isolates, whichwere fromdifferent countries and the differentmedia, which were used for the determination of sensitivity to boscalid (in that previous study, liquid YBAwas used, while agar mediawere used in Table 2 Control of Sclerotinia stem rot of rape in Taizhou (two different sites) by boscalid. Treatments Dosage per ha (g.ai) Disease control (%)a 50% carbendazim (WP) 750 18.9 db 46% dimethachlon (WP) 690 44.6 c 50% boscalid (WG) 80 49.7 c 50% boscalid (WG) 125 65.2 b 50% boscalid (WG) 187.5 69.0 b 50% boscalid (WG) 250 75.1 a a Percentage of disease control (¼disease control relative to the water-treated control) was calculated as follows: percentage of disease index ¼ [n (1) þ n (3) þ n (5) þ n (7) þ n (9)]/number of leaves scored � 100 (where n ¼ number of leaves showing scores 1, 3, 5, 7 and 9); percentage of disease control ¼ (disease index in the water control � disease index in the treatment)/disease index in the water control � 100. b Values in columns followed by similar letters were not significantly different according to Fisher’s protected LSD test (P ¼ 0.05). our study). The authors also suggested that it was easy, convenient and not time-consuming to evaluate the sensitivity of S. scle- rotiorum isolates to boacalid on PDA, Czapek and the synthetic complex medium. Management of benzimidazole resistance relies on reducing selection pressure by limiting exposure to fungicides with the same mode of action, and using tank mixtures or replacement of fungi- cides with different biochemical mechanisms of action. Boscalid did exhibit strong fungicidal activity against S. sclerotiorum in vitro and had the expected effect on limiting Sclerotinia stem rot in field trials. Furthermore, it also showed excellent efficacy in controlling Sclerotinia stem rot in the field where MBC was extensive used for decades and the control failure of MBC occurred. To our knowledge, this is also the first report on the excellent efficacy of boscalid in controlling Sclerotinia stem rot in China. A lack of any cross-resistance between boscalid and MBC/ Bardin, S.D., Huang, H.C., 2001. Research on biology and control of Sclerotinia diseases in Canada. Can. J. Plant Pathol 23, 88–98. Boland, G.J., Hall, R., 1994. Index of plant hosts of Sclerotinia sclerotiorum. Can. J. Plant Pathol 16, 93–108. Bolton, D.M., Thomma, P.H.J.B., Nelson, D.B., 2007. Sclerotinia sclerotiorum (Lib.) de Bary: biology and molecular traits of a cosmopolitan pathogen. Mol. Plant Pathol 7, 1–16. Christian, T., Christopher, J.W., Jolanta, J.B., Rikard, H., Berndt, G., 2001. Suppression of Sclerotinia sclerotiorum apothecial by the soil bacterium Serratia plymuthica: identification of a chlorinated macrolide as one of the causal agents. Soil Biol. Biochem 33, 1817–1826. Dai, F.M., Xu, T., Wolf, G.A., He, Z.H., 2006. Physiological and molecular features of the pathosystem Arabidopsis thaliana L. and Sclerotinia sclerotiorum Libert. J. Integr. Plant Biol. 48, 44–52. Jo, Y.-K., Niver, A.L., Rimelspach, J.W., Boehm, M.J., 2006. Fungicide sensitivity of Sclerotinia homoeocarpa from golf courses in Ohio. Plant Dis 90, 807–813. Letham, D.B., Huett, D.O., Trimboli, D.S., 1976. Biology and control of Sclerotinia sclerotiorum in cauliflower and tomato crops in coastal New South Wales. Plant Dis. Rep 60, 286–289. Li, H.X., Lu, Y.J., Zhou, M.G., Wang, X.F., 2003. Mutation in b-tubulin of Sclerotinia sclerotiorum conferring resistance to carbendazim in rapeseed field isolate. Chin. J. Oil Crop Sci 25, 56–60. J.-X. Wang et al. / Crop Protection 28 (2009) 882–886886 dimethachlon suggests that the unique mode of action of boscalid makes an excellent candidate for the control of Sclerotinia stem rot and the management of the field MBC resistance, although boscalid is not registered for the control of Sclerotinia stem rot in China. The results in this study suggest that combining or alternating appli- cation of boscalid with other fungicides will be a valuable tool to limit fungicide resistance development, and wewill make efforts to popularize this new fungicide for the control of Sclerotinia stem rot in China. Acknowledgements This study was sponsored by the Science and Technology Support Programs from Jiangsu Province and the Ministry of Science and Technology (Nos. 2006CB101907, 3-21000004, 2006BAD08A03 2006AA10A211 and 2008AA102414). References Abawi, G.S., Grogan, R.G., 1979. Epidemiology of diseases caused by Sclerotinia species. Phytopathology 69, 899–904. Alberoni, G., Collina, M., Pancaldi, D., Brunelli, A., 2005. Resistance to dicarboximide fungicides in Stemphylium vesicarium of Italian pear orchards. Eur.. J. Plant Pathol 113, 211–219. Anonymous, 1975. Sclerotinia Disease of Oilseed Crops. Oilcrop Research Institute, Chinese Academy of Sciences. Agriculture Press, Beijing, China. Anonymous, 2000. Pesticide d Guidelines for the Field Efficacy Trials (I), National StandardsofChina. StandardsPress ofChina,pp.144–147 (GB/T17980. 34d2000). Avenot, H.F., Michailides, T.J., 2007. Resistance to boscalid fungicide in Alternaria alternata isolates from pistachio in California. Plant Dis 91, 1345–1350. Avenot, H.F., Sellam, A., Karaoglanidis, G., Michailides, T.J., 2008. Characterization of mutations in the iron-sulphur subunit of succinate dehydrogenase correlating with boscalid resistance in Alternaria alternate from California pistachio. Phytopathology 98, 736–742. Lu, G., 2003. Engineering Sclerotinia sclerotiorum resistance in oilseed crops. Afr. J. Biotechnol 2, 509–516. Ma, H.X., Feng, X.J., Chen, Y., Chen, C.J., Zhou, M.G., 2009. Occurrence and charac- terization of dimethachlon insensitivity in Sclerotinia sclerotiorum in Jiangsu Province of China. Plant Dis 93, 36–42. Pan, Y.L., 1998. The resistance of Sclerotinia sclerotiorum of rape to carbendazim and its management. Jiangsu J. Agr. Sci. 14, 159–163. Perez, F., Riba, J.P., Strehaiano, P., 1992. Effect of yeast extract concentration on growth of Schizoccharomyces pombe. Biotechnol. Lett. 14, 123–126. Purdy, L.H., 1979. Sclerotinia sclerotiorum: history, disease, and symptomatology, host range, geographic distribution, and impact. Phytopathology 69, 875–880. Sarma, B.K., Ameer, B.S., Singh, D.P., Singh, U.P., 2007. Use of non-conventional chemicals as an alternative approach to protect chickpea (Cicer arietinum) from Sclerotinia stem rot. Crop Prot 26, 1042–1048. Shi, Z.Q., Zhou, M.G., Ye, Z.Y., 2000. Resistance of Sclerotinia Sclerotiorum to car- bendazim and dimethachlon. Chin. J. Oil Crop Sci. 22, 54–57. Smith, F.D., Phipps, P.M., Stipes, R.J., Brenneman, T.B., 1995. Significance of insen- sitivity of Sclerotinia minor to iprodione in control of Sclerotinia blight of peanut. Plant Dis 79, 517–523. Stammler, G., Speakeman, J., 2006. Microtiter method to test the sensitivity of Botrytis cinerea to boscalid. J. Phytopathol 154, 508–510. Stammler, G., Benzinger, G., Speakman, J., 2007. A rapid and reliable method for monitoring the sensitivity of Sclerotinia sclerotiorum to Boscalid. J. Phytopathol 155, 746–748. Staub, T., 1991. Fungicide resistance: practical experience with antiresistance strategies and the role of integrated use. Annu. Rev. Phytopathol 29, 421–442. Sylvester-Bradley, R., Donald, C.M., 1984. A code for stages of development in oilseed rape (Brassica napus L.) Aspect. Appl. Biol. 6, 399–419. Zhang, X.L., Sun, X.M., Zhang, G.F., 2003. Preliminary report on the monitoring of the resistance of Sclerotinia libertinia to carbendazim and its internal manage- ment. Chin. J. Pest. Sci. Adm 24, 18–22. Zhang, C.Q., Yuan, S.K., Sun, H.Y., Qi, Z.Q., Zhou, M.G., Zhu, G.N., 2007. Sensitivity of Botrytis cinerea from vegetable greenhouses to boscalid. Plant Pathol 56, 646–653. Zhao, J., Meng, J., 2003. Detection of loci controlling seed glucosinolate content and their association with Sclerotinia resistance in Brassica napus. Plant Breeding 122, 19–23. Zhou, B.W., Luo, Q., 1994. Rapeseed Diseases and Control. China Commerce Publishing Co, Beijing, China, pp. 346(in Chinese). Sensitivity of Sclerotinia sclerotiorum from oilseed crops to boscalid in Jiangsu Province of China Introduction Materials and methods Origin and collection of S. sclerotiorum isolates Fungicides and media Analysis of cross-resistance of boscalid with MBC and dimethachlon Determination of baseline EC50 values of boscalid against S. sclerotiorum Field trial for controlling Sclerotinia stem rot Statistical analysis Results Cross-resistance between boscalid and MBC/dimethachlon Baseline sensitivity to boscalid Efficacy of boscalid in controlling Sclerotinia stem rot Discussion Acknowledgements References