Sensitivity of Colletotrichum nymphaeae to Six Fungicides and Characterization of Fludioxonil-Resistant Isolates in China.

Colletotrichum nymphaeae is the dominant species causing anthracnose disease of peach in China. In this study, 140 isolates of C. nymphaeae were assessed for their sensitivity to six fungicides. It was found that C. nymphaeae was highly resistant to carbendazim, procymidone, and boscalid but sensitive to pyraclostrobin and prochloraz. For fludioxonil, the fungus exhibited differential sensitivities (i.e., approximately 14% of isolates were resistant to fludioxonil and the resistance was stable). Fludioxonil-resistant isolates had a mean EC50 value of 2.2380 µg/ml, whereas the mean EC50 value was 0.0194 µg/ml in fludioxonil-sensitive isolates. The mean EC50 values of C. nymphaeae for pyraclostrobin and prochloraz were 0.0083 µg/ml and 0.002 µg/ml, respectively. No cross-resistance was observed between fungicides from different groups. Mycelial growth rate, control efficacy, and osmotic stress responses were significantly different (P < 0.05) between fludioxonil-sensitive (FluS) and -resistant (FluR) isolates, but no significant difference was observed (P > 0.05) in virulence and sporulation between FluS and FluR isolates. No mutation was detected in coding regions of the CnOs-1, Cal, Hk1, Hog1, TPI, and Mrr1 genes. Interestingly, with fludioxonil treatment, the expression of ABC transporter gene atrB was significantly overexpressed in some resistant isolates. However, overexpression of the atrB gene was not detected in one moderately and one highly resistant isolate, indicating that other unknown mechanisms may be involved. Current findings uncovered several effective chemicals and provided the foundation for designing management strategies to practically control peach anthracnose with the most effective demethylation inhibitor fungicides and quinone outside inhibitor fungicides.

Sensitivity of Colletotrichum fructicola and Colletotrichum siamense of Peach in China to Multiple Classes of Fungicides and Characterization of Pyraclostrobin-Resistant Isolates.

Anthracnose, mainly caused by Colletotrichum gloeosporioides species complex including Colletotrichum fructicola and Colletotrichum siamense, is a devastating disease of peach. Chemical control has been widely used for years, but management failures have increased with the commonly used fungicides. Therefore, screening of sensitivity of Colletotrichum spp. to fungicides with different modes of action is needed to make proper management strategies for peach anthracnose. In this study, the sensitivity of 80 isolates of C. fructicola and C. siamense was screened for pyraclostrobin, procymidone, prochloraz, and fludioxonil based on mycelial growth inhibition at discriminatory doses. Results showed that C. fructicola and C. siamense isolates were highly resistant to procymidone and fludioxonil with 100% resistance frequencies to both fungicides, but sensitive to prochloraz, i.e., no resistant isolates were found. For pyraclostrobin, 74% of C. fructicola isolates showed high resistance, 26% showed low resistance, and all of the C. siamense isolates showed low resistance. No positive cross-resistance was observed between pyraclostrobin and azoxystrobin even when they are members of the same quinone outside inhibitor (QoI) fungicide group or between pyraclostrobin and non-QoIs. Resistant isolates to QoI fungicides were evaluated for the fitness penalty. Results showed that no significant differences except for the mycelial growth rates that were detected between high- and low-resistance isolates of C. fructicola. Molecular characterization of the Cyt b gene revealed that the G143A point mutation was the determinant of the high resistance in C. fructicola. This study demonstrated the resistance status of C. fructicola and C. siamense to different fungicides and briefly discussed implications of that resistance. Demethylation inhibitor fungicides were found to be the best option among the different chemicals studied here, to control peach anthracnose in China.

Effects of SHAM on the Sensitivity of Sclerotinia sclerotiorum and Botrytis cinerea to QoI Fungicides.

It is a common practice to add salicylhydroxamic acid (SHAM) into artificial medium in the in vitro sensitivity assay of fungal phytopathogens to the quinone outside inhibitor (QoI) fungicides. The rationale for adding SHAM is to inhibit fungal alternative oxidase, which is presumed to be inhibited by secondary metabolites of plants. Therefore, the ideal characteristics of SHAM should be almost nontoxic to phytopathogens and have no significant effect on control efficacy of fungicides. However, this study showed that the average effective concentration for 50% inhibition (EC50) of mycelial growth values of SHAM were 97.5 and 401.4 µg/ml for Sclerotinia sclerotiorum and Botrytis cinerea, respectively. EC50 values of the three QoI fungicides azoxystrobin, kresoxim-methyl, and trifloxystrobin in the presence of SHAM at 20 and 80 µg/ml for S. sclerotiorum and B. cinerea, respectively, declined by 52.7 to 78.1% compared with those without SHAM. For the dicarboximide fungicide dimethachlone, the average EC50 values in the presence of SHAM declined by 18.2% (P = 0.008) for S. sclerotiorum and 35.9% (P = 0.012) for B. cinerea. Pot experiments showed that SHAM increased control efficacy of the three QoI fungicides against the two pathogens by 43 to 83%. For dimethachlone, SHAM increased control efficacy by 134% for S. sclerotiorum and 86% for B. cinerea. Biochemical studies showed that SHAM significantly inhibited peroxidase activity (P = 0.024) of B. cinerea and esterase activity (P = 0.015) of S. sclerotiorum. The strong inhibitions of SHAM per se on mycelial growth of B. cinerea and S. sclerotiorum and significant influences on the sensitivity of the two pathogens to both the QoI fungicides and dimethachlone as well as inhibitions on peroxidase and esterase indicate that SHAM should not be added in the in vitro assay of sensitivity to the QoI fungicides.

Exploring mechanisms of resistance to dimethachlone in Sclerotinia sclerotiorum.

BACKGROUND: The dicarboximide fungicide dimethachlone has been widely used in China for more than 12 years to control the Sclerotinia stem rot caused by Sclerotinia sclerotiorum disease. First signs of resistance in the field are reported at low frequency. In this study, four resistant isolate/mutants were used to explore still unknown mechanisms leading to dimethachlone resistance. RESULTS: The resistant isolate/mutants had significantly higher EC50 values compared with the sensitive control isolates. Cross-resistance was confirmed between dimethachlone and procymidone, iprodione and fludioxonil. The resistant isolate/mutants revealed a decreased mycelial growth rate, were less pathogenic on leaves of oilseed rape, were more sensitive to osmotic pressure and oxidative stress and released more electrolytes compared with the sensitive isolates. Only in one lab mutant did we find a point mutation (V238A) in the SsOs1 gene of the high-osmolarity glycerol (HOG) signalling pathway. The expression of this gene was lost in the field resistant isolate HN456-1-JBJ and decreased in mycelium that was subjected to either high osmotic pressure or dimethachlone; however, another key gene in the HOG pathway, SsHog1, could be induced in the resistant isolate and mutants with NaCl treatment. CONCLUSION: This study demonstrates that resistance to dicarboximide fungicide dimethachlone in S. sclerotiorum is emerging in China. Several fitness parameters, including mycelial growth rate, sclerotia formed in vitro, aggressiveness on leaves and osmotic and H2 O2 sensitivity, indicate that the resistant strains may not effectively compete with sensitive isolates in the field in the absence of selection pressure. Lost expression or the V238A point mutation in the SsOs1 gene may confer resistance to dicarboximide fungicide dimethachlone in S. sclerotiorum, but this study illustrates that other, yet unknown mechanisms also exist.

Hormetic Effects of Trifloxystrobin on Aggressiveness of Sclerotinia sclerotiorum.

Sclerotinia sclerotiorum is a devastating ascomycete plant pathogen with an extremely wide host range. Fungicides are still the mainstay for control of this pathogen, and stimulations to mycelial growth and aggressiveness by subtoxic doses of fungicides carbendazim and dimethachlon have been reported. The present study assessed hormetic effects of the quinone outside inhibitor (QoI) fungicide trifloxystrobin on aggressiveness of S. sclerotiorum. Trifloxystrobin at 0.0001, 0.0005, and 0.001 µg/ml exerted significant stimulatory effects on aggressiveness to potted rapeseed plants, and the highest percent stimulation were 20.5 and 24.2% for isolates HB15 and SX11, respectively. At 18 h postinoculation (HPI), initial necrotic lesions were visible to the naked eye on leaves treated with trifloxystrobin, whereas no obvious disease symptoms were discerned for the nontreated control. At 24, 36, and 48 HPI, aggressiveness stimulation was more obvious than at 18 HPI. Scanning electron microscopic observations demonstrated that no mycelia were detected on the nontreated leaves at 4 HPI; by contrast, mycelia were observed on leaves treated with trifloxystrobin at 0.0001 µg/ml. At 8 and 12 HPI, there were more mycelia and infecting hyphae on the treated leaves than on the nontreated control. These results indicated that fungal stimulation had occurred in the first 4 and 8 HPI, suggesting that direct stimulation was likely to be the underlying mechanism for hormetic actions of trifloxystrobin. Pretreatment with trifloxystrobin did not significantly affect subsequent mycelial growth on PDA or aggressiveness to detached rapeseed leaves in the absence of trifloxystrobin. However, in the presence of trifloxystrobin, mycelial growth and aggressiveness were significantly (P < 0.05) greater for the pretreatment with trifloxystrobin at 0.003 and 0.03 µg/ml compared with the nonpretreatment control, indicating that a prior exposure to the fungicide may undermine its subsequent effectiveness. These studies will raise our awareness of fungicide hormesis and have important implications for judicious application of fungicides.

Time Course of Carbendazim Stimulation on Pathogenicity of Sclerotinia sclerotiorum Indicates a Direct Stimulation Mechanism.

Previous studies have demonstrated that subtoxic doses of carbendazim have a stimulatory effect on pathogenicity of Sclerotinia sclerotiorum on rapeseed plants. The present study focused on the time-course profile of the stimulatory effect and its relevance to stimulation mechanisms. At 12 h postinoculation (HPI), initial necrotic lesions were visible only for rapeseed leaves treated with carbendazim at 0.2 and 1 µg/ml, whereas no disease symptoms were observed for the nontreated control. At 18 HPI, carbendazim stimulation on pathogenicity was more obvious than at 12 HPI. Study with scanning electron microscopy demonstrated that no discernable differences in the development of disease symptoms could be detected at 8 HPI. However, at 12 HPI, necrotic symptoms of the epidermal cells were apparent only for leaves sprayed with carbendazim. These results indicated that stimulations on pathogenicity occurred in the first 12 h, implying that direct stimulation rather than overcompensation to the disruption of homeostasis was likely to be the underlying mechanism for pathogenicity stimulation. Greenhouse experiments showed that spraying carbendazim at 400 µg/ml on potted rapeseed plants had statistically significant (P < 0.05) stimulations on pathogenicity for inoculations at 1, 3, 5, and 7 days after application (DAA). The stimulation action eventually disappeared for inoculations at 14 DAA. Mycelia grown on potato dextrose agar (PDA) supplemented with carbendazim at 400 µg/ml were more pathogenic than the nontreated control. However, after additional growth of the mycelia on fungicide-free PDA for 2 days, the stimulatory effect disappeared completely, indicating that carbendazim was indispensable for pathogenicity stimulations. Studies on biochemical mechanisms indicated that cell-wall-degrading enzymes such as cellulase, pectinase, and polygalacturonase were not involved in pathogenicity stimulations. These results will advance our understanding of the nature and mechanisms of fungicide stimulation on fungal pathogenicity and, thus, are valuable for judicious applications of fungicides.

Fitness is Recovered with the Decline of Dimethachlon Resistance in Laboratory-induced Mutants of Sclerotinia sclerotiorum after Long-term Cold Storage.

After four years of cold storage, dimethachlon resistance of two laboratory-induced resistant Sclerotinia sclerotiorum isolates SCG7 and LA50 declined by 99.5% and 98.9%, respectively, and cross resistance to iprodione and procymidone also declined dramatically. Along with the decline of fungicide resistance, osmotic sensitivity to sodium chloride and glucose decreased tremendously; mycelial growth rate, sclerotia number and weight per potato dextrose agar (PDA) plate increased on average by 118.6%, 85. 5% and 64.5%, respectively; and virulence to detached leaves of oilseed rape increased by 72.7% on average. Significant negative correlations were detected between dimethachlon resistance levels and mycelial growth rate on PDA (r = -0.980, P = 0.021), and between resistance levels and lesion diameters on detached leaves of oilseed rape plants (r = -0.997, P = 0.002). These results have profound implications for assessing the potential risk for resistance development to dicarboximide fungicides in S. sclerotiorum.

Pathogenicity Stimulation of Sclerotinia sclerotiorum by Subtoxic Doses of Carbendazim.

Sclerotinia sclerotiorum is a devastating ascomycete fungus capable of infecting more than 400 species of plants worldwide. Carbendazim has been a principal fungicide for control of this pathogen and high levels of carbendazim resistance have been reported in eastern China. In this study, stimulatory effect of subtoxic doses of carbendazim on pathogenicity of S. sclerotiorum was investigated. All seven field resistant isolates with EC50 values greater than 1,000 µg/ml exhibited stimulated pathogenicity toward detached leaves of rapeseed at subtoxic concentrations of carbendazim. Detailed studies on pathogenicity of two resistant isolates AH-17 and LJ-86 toward potted rapeseed plants and detached leaves demonstrated that carbendazim at 0.2 to 5 µg/ml could consistently stimulate significantly higher (P < 0.05) pathogenicity than the control. On potted rapeseed plants, the percent stimulations on pathogenicity ranged from 18.8 to 22.0% for isolate AH-17 and from 15.1 to 23.2% for isolate LJ-86. On detached leaves of rapeseed, the percent stimulations ranged from 18.7 to 31.29% for isolate AH-17 and from 16.7 to 24.3% for isolate LJ-86. Studies on stimulation mechanism indicated that secretion of oxalic acid and tolerance to oxidative stresses H2O2 and paraquat after exposed to subtoxic doses of carbendazim did not change significantly. These results have profound implications for judicious application of fungicides and sustainable management of fungicide resistance.

Baseline Sensitivity of Pyraclostrobin and Toxicity of SHAM to Sclerotinia sclerotiorum.

Sclerotinia sclerotiorum is a cosmopolitan plant pathogen notable for its wide host range. The quinone outside inhibitor (QoI) fungicide pyraclostrobin has not been registered for control of S. sclerotiorum in China. In this study, baseline sensitivity of pyraclostrobin was established based on effective concentration for 50% inhibition of mycelial growth (EC50) values of 153 isolates of S. sclerotiorum collected from five provinces of China and toxicity of alternative oxidase inhibitor salicylhydroxamic acid (SHAM) to S. sclerotiorum was determined. Results showed that the frequency distribution of EC50 values of the 153 isolates was unimodal but with a right-hand tail. The mean EC50 value was 0.1027 µg/ml and the range of EC50 values was 0.0124 to 0.6324 µg/ml. Applied as a preventive fungicide in pot experiments, pyraclostrobin at 5, 15, and 45 µg/ml provided control efficacies of 61, 77, and 100%, respectively. There was no positive cross-resistance between pyraclostrobin and carbendazim or dimethachlon. EC50 values for SHAM against four isolates of S. sclerotiorum were 44.4, 51.8, 54.4, and 68.7 µg/ml. SHAM at 20 µg/ml could significantly increase not only the inhibitory effect of pyraclostrobin on mycelial growth on potato dextrose agar media but also the control efficacy in planta. These results indicated that SHAM should not be added into artificial media in in vitro assay of S. sclerotiorum sensitivity to pyraclostrobin. This has broad implications for assay of sensitivity of fungal pathogen to QoI fungicides.

Dimethachlon Resistance in Sclerotinia sclerotiorum in China.

The dicarboximide fungicide dimethachlon has been widely used for controlling Sclerotinia sclerotiorum in China for more than a decade. To assess the current status of dimethachlon resistance in S. sclerotiorum in China, 2,424 isolates were collected from disease-infected oilseed rape and soybean plants in five provinces of China in 2011 and 2012, and dimethachlon resistance was monitored by mycelial growth inhibition method on potato dextrose agar (PDA) media. Dimethachlon at 5 µg/ml was used as a discriminatory dose to detect resistance in all isolates, and 50% effective concentration values were determined for all dimethachlon-resistant isolates and some sensitive isolates. No dimethachlon resistance was detected in isolates from Anhui province (eastern China), Gansu province (northwestern China), and Qinghai province (western China). In Hunan province (central China), 3 of 268 (1.12%) isolates collected from oilseed rape plants in 2012 were resistant to dimethachlon, and the resistance ratios for the three resistant isolates were 4.56, 32.70, and 105.53, respectively. In Heilongjiang province (northeastern China), 8 of 243 (3.29%) isolates collected from soybean plants in 2011 were resistant to dimethachlon, with resistance ratios of 5.57 to 94.80; 11 of 409 (2.69%) isolates collected in 2012 were resistant to dimethachlon, with resistance ratios of 3.21 to 9.69. Cross-resistance studies showed that there was positive cross-resistance between dimethachlon and iprodione, procymidone, and the N-phenyl carbamate fungicide diethofencarb. No cross-resistance was found between dimethachlon and carbendazim, tebuconazole, kresoxim-methyl, thiram, and boscalid. Compared with the sensitive isolates of S. sclerotiorum, the field-dimethachlon-resistant isolates were more sensitive to osmotic pressure, grew more slowly on PDA media, and were less pathogenic on leaves of oilseed rape.

Stimulatory Effects of Sublethal Doses of Dimethachlon on Sclerotinia sclerotiorum.

Growth and virulence stimulations of sublethal doses of fungicides on plant-pathogenic fungi and oomycetes have been reported and the stimulatory effects are potentially relevant to plant disease management. Sclerotinia sclerotiorum is one of the most devastating and economically important necrotrophic fungal phytopathogens, capable of infecting more than 400 species of plants worldwide. In order to study stimulatory effects of sublethal doses of fungicides on S. sclerotiorum, 55 dimethachlon-sensitive isolates and 3 dimethachlon-resistant isolates of S. sclerotiorum were assayed to determine effects of sublethal doses of dimethachlon on mycelial growth rate on potato dextrose agar (PDA) media and virulence on oilseed rape plants. Results showed that all 3 dimethachlon-resistant isolates and 13 of the 55 sensitive isolates exhibited stimulatory responses to sublethal doses of dimethachlon. Dimethachlon-resistant isolates grew significantly (P < 0.05) faster on PDA media amended with dimethachlon at 0.5 to 4 µg/ml than on fungicide-free PDA media. As for virulence on detached leaves of oilseed rape plants, lesion diameters of dimethachlon-resistant isolates after growth on PDA media amended with dimethachlon at 0.5 to 2 µg/ml were significantly larger (P < 0.05) than the control. The maximum stimulatory effects were 42.40 to 59.80%. In pot experiments, for both dimethachlon-sensitive and -resistant isolates, significant (P < 0.05) virulence stimulations were observed after spraying with dimethachlon at a concentration of 2 µg/ml. After growing on dimethachlon-amended PDA media, H2O2 sensitivity of S. sclerotiorum decreased significantly (P < 0.05) compared with the nonamended PDA control.