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.

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.

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.