Antifungal Activity and Mechanism of Physcion against Sclerotium rolfsii, the Causal Agent of Peanut Southern Blight.

Peanut southern blight, caused by the soil-borne pathogen Sclerotium rolfsii, is a widespread and devastating epidemic. Frequently, it is laborious to effectively control by labor-intensive foliar sprays of agrochemicals due to untimely find. In the present study, seed treatment with physcion (PHY) at doses of 0.08, 0.16, and 0.32 g AI kg-1 seed significantly improved the growth and photosynthetic activity of peanuts. Furthermore, PHY seed treatment resulted in an elevated enzymatic activity of key enzymes in peanut roots, including peroxidase, superoxide dismutase, polyphenol oxidase, catalase, lipoxygenase, and phenylalanine ammonia-lyase, as well as an increase in callus accumulation and lignin synthesis at the infection site, ultimately enhancing the root activity. This study revealed that PHY seed treatment could promote the accumulation of reactive oxygen species, salicylic acid (SA), and jasmonic acid (JA)/ethylene (ET) in peanut roots, while also decreasing the content of malondialdehyde levels in response to S. rolfsii infection. The results were further confirmed by transcriptome data and metabolomics. These findings suggest that PHY seed treatment activates the plant defense pathways mediated by SA and JA/ET in peanut roots, enhancing the resistance of peanut plants to S. rolfsii. In short, PHY is expected to be developed into a new plant-derived immunostimulant or fungicide to increase the options and means for peanut disease control.

Design, synthesis and bioactivity of a new class of antifungal amino acid-directed phthalide compounds.

BACKGROUND: Peanut southern blight disease, caused by Sclerotium rolfsii, is a destructive soil-borne fungal disease. The current control measures, which mainly employ succinate dehydrogenase inhibitors, are prone to resistance and toxicity to non-target organisms. As a result, it is necessary to explore the potential of eco-friendly fungicides for this disease. RESULTS: Fourteen novel phthalide compounds incorporating amino acid moieties were designed and synthesized. The in vitro activity of analog A1 [half maximal effective concentration (EC50) = 332.21 mg L-1] was slightly lower than that of polyoxin (EC50 = 284.32 mg L-1). It was observed that on the seventh day, the curative activity of A1 at a concentration of 600.00 mg L-1 was 57.75%, while the curative activity of polyoxin at a concentration of 300.00 mg L-1 was 42.55%. These results suggested that our compound exhibited in vivo activity. Peanut plants treated with A1 showed significant agronomic improvements compared to the untreated control. Several compounds in this series exhibited superior root absorption and conduction in comparison to the endothermic fungicide thifluzamide. The growth promotion and absorption-conduction experiments demonstrated the reason for the superior in vivo activity of the target compound. Cytotoxic assays have demonstrated that this series of targeted compounds exhibit low toxicity levels toward human lo2 liver cells. CONCLUSION: Our results provide a new strategy for the design and synthesis of novel green compounds. Furthermore, the target compound A1 can serve as a lead for further development of green fungicides. © 2024 Society of Chemical Industry.

Bioactivity and Resistance Risk of Fluxapyroxad, a Novel SDHI Fungicide, in Didymella bryoniae.

Gummy stem blight, caused by Didymella bryoniae, is an important disease in watermelon in China. Fluxapyroxad, a new succinate dehydrogenase inhibitor fungicide, shows strong inhibition of the mycelia growth of D. bryoniae. However, its resistance risk in D. bryoniae is unclear. In this research, the sensitivities of 60 D. bryoniae strains to fluxapyroxad were investigated. The average EC50 value and MIC values of 60 D. bryoniae strains against fluxapyroxad were 0.022 ± 0.003 µg/ml and ≤0.1 µg/ml for mycelial growth, respectively. Eight fluxapyroxad-resistant mutants with medium resistance levels were acquired from three wild-type parental strains. The mycelial growth and dry weight of mycelia of most mutants were significantly lower than those of their parental strains. However, four resistant mutants showed a similar phenotype in pathogenicity compared with their parental strains. The above results demonstrated that there was a medium resistance risk for fluxapyroxad in D. bryoniae. The cross-resistance assay showed that there was positive cross-resistance between fluxapyroxad and pydiflumetofen, thifluzamide, and boscalid, but there was no cross-resistance between fluxapyroxad and tebuconazole and mepronil. These results will contribute to evaluating the resistance risk of fluxapyroxad for managing diseases caused by D. bryoniae and further increase our understanding about the mode of action of fluxapyroxad.

Energetic benefits in coordinated circular swimming motion of two swimmers.

The coordinated movement of multiple swimmers is a crucial component of fish schools. Fish swimming in different formations, such as tandem, side-by-side, diamond, and phalanx, can achieve significant energetic advantages. However, the energetic benefits of nonstraight swimming behaviors, such as the collective motion of a milling pattern, are not well understood. To fill in this gap, we consider two swimmers in circular tracks, controlled by a PID approach to reach stable configurations. Our study finds that the optimal phase is affected by circumferential effects, and that substantial energy savings can result from both propulsion and turning. We also explore the radial effect in terms of energetic benefits. In a milling pattern, the inner swimmers can easily gain a certain energetic benefit (-8%), while their peers on the outside must be close enough to the inner swimmer with a proper phase to gain the energetic benefit (-14%). When the radial spacing becomes larger or is in an unmatched phase, the swimming of the outer swimmers becomes more laborious (+16%). Our results indicate that swimmers who maintain a matched phase and minimum radial effect obtain the highest energetic benefits (-26%). These findings highlight the energetic benefits of swimmers, even in a milling pattern, where the position difference dominates the extent of benefit.

Crucial Role of the Ca2+/CN Signaling Pathway in the Antifungal Activity of Seselin against Botrytis cinerea.

Botrytis cinerea causes gray mold in many fruit and vegetable crops. We previously found that Seselin (SL) displayed antifungal activity against B. cinerea (EC50 = 6.1 µg·mL-1), and this study investigated the effects of Ca2+ and the Ca2+/CN signaling pathway on its antifungal activity against B. cinerea. The results indicated that exogenous Ca2+, Cyclosporine A, and Verapamil reduced the sensitivity of SL against B. cinerea; SL significantly reduced the intracellular Ca2+ concentration in the hyphae; the sensitivity of strains ΔbcCCH1 and ΔbcMID1 to SL were significantly increased; and the expressions of CCH1, MID1, CNA, PMC1, and PMR1 genes of the Ca2+/CN signaling pathway were significantly downregulated by SL treatment. Hence, SL is a potential compound for developing fungicides against B. cinerea. SL dramatically reduces intracellular Ca2+ concentration and disturbs Ca2+ homeostasis, leading to cell death. The Ca2+/CN signaling pathway plays an important role in the antifungal activity of SL against B. cinerea.

FgMet3 and FgMet14 related to cysteine and methionine biosynthesis regulate vegetative growth, sexual reproduction, pathogenicity, and sensitivity to fungicides in Fusarium graminearum.

Fusarium graminearum is a destructive filamentous fungus, which widely exists in wheat and other cereal crops. Cysteine and Methionine are unique sulfur-containing amino acids that play an essential role in protein synthesis and cell life, but their functions and regulation in F. graminearum remain largely unknown. Here we identified two proteins, FgMet3 and FgMet14 in F. graminearum, which are related to the synthesis of cysteine and methionine. We found FgMet3 and FgMet14 were localized to the cytoplasm and there was an interaction between them. FgMet3 or FgMet14 deletion mutants (ΔFgMet3 and ΔFgMet14) were deficient in vegetative growth, pigment formation, sexual development, penetrability and pathogenicity. With exogenous addition of cysteine and methionine, the vegetative growth and penetrability could be completely restored in ΔFgMet3 and ΔFgMet14, while sexual reproduction could be fully restored in ΔFgMet3 and partially restored in ΔFgMet14. ΔFgMet3 and ΔFgMet14 exhibited decreased sensitivity to Congo red stress and increased sensitivity to SDS, NaCl, KCl, Sorbitol, Menadione, and Zn ion stresses. Moreover, FgMet3 and FgMet14 nonspecifically regulate the sensitivity of F. graminearum to fungicides. In conclusion, FgMet3 and FgMet14 interacted to jointly regulate the development, pathogenicity, pigment formation, sensitivity to fungicides and stress factors in F. graminearum.

FfCOX17 is Involved in Fumonisins Production, Growth, Asexual Reproduction, and Fungicide Sensitivity in Fusarium fujikuroi.

Fusarium fujikuroi, a causal agent of Rice Bakanae Disease, produces secondary metabolites such as gibberellin, pigments bikaverin, and mycotoxins fumonisins. Fumonisins produced by F. fujikuroi pose a severe threat to human and animal health. The copper chaperone protein plays a critical role in different growth stages of plants, fungi, and yeasts, but their functions and regulation in fumonisin biosynthesis are still unclear. Here, a copper chaperone protein, FfCOX17, was identified in F. fujikuroi. The FfCOX17 deletion mutant (∆FfCOX17) exhibited decreased vegetative growth and asexual reproduction. The transcriptional level of the FfFUM2 gene was significantly induced in ∆FfCOX17, and the fumonisin production in ∆FfCOX17 mutants was significantly increased compared to wild-type F. fujikuroi, but the pathogenicity of ∆FfCOX17 mutants was unaffected, which may be caused by the no significantly changed gibberellin content. ∆FfCOX17 showed decreased sensitivity to oxidative stress, osmotic stress, and increased sensitivity to cell wall stress, heat shock stress, and high concentration glucose. In addition, ∆FfCOX17 also showed increased sensitivity to fungicide fluazinam and fludioxonil, and decreased sensitivity to phenamacril and prochloraz. Taken together, this study suggested that FfCOX17 is critical for fumonisin production, vegetative growth, asexual reproduction, and fungicide sensitivity, but is not required for the virulence function of F. fujikuroi on rice.

Crucial role of Ca2+ /CN signalling pathway in the antifungal activity of disenecioyl-cis-khellactone against Botrytis cinerea.

BACKGROUND: Botrytis cinerea causes grey mould and is one of the most destructive fungal pathogens affecting important fruit and vegetable crops. In preliminary studies, we found that disenecioyl-cis-khellactone (DK) had strong antifungal activity against several fungi species including B. cinerea [half maximal effective concentration (EC50 ) = 11.0 µg mL-1 ]. In this study, we aimed to further evaluate the antifungal activity of DK against B. cinerea and determine the role of calcium ion/calcineurin (Ca2+ /CN) signalling pathway on its antifungal effect. RESULTS: DK was effective against B. cinerea in both in vitro and in vivo assays. Exogenous Ca2+ reduced the antifungal activity of DK. The combination of DK and cyclosporine A (CsA) did not exhibit an additive effect against B. cinerea. In contrast to CsA, DK reduced the intracellular Ca2+ concentration in B. cinerea. DK bound to calcineurin A (cnA) and up-regulated the expression of PMC1 and PMR1 genes. Moreover, DK sensitivity of △bccnA significantly decreased compared with that of Bc05.10 strain. CONCLUSION: DK is a promising lead compound for developing fungicides against B. cinerea. The Ca2+ /CN signalling pathway plays a crucial role in the DK antifungal activity, and cnA is one of the targets of DK against B. cinerea. DK directly reacts with cnA, which up-regulates the transcription of Ca2+ /CN-dependent target genes PMC1 and PMR1, decreasing the intracellular Ca2+ concentration and disturbing the intracellular Ca2+ balance, leading to cell death. © 2022 Society of Chemical Industry.

Diversity, prevalence, and expression of cyanase genes (cynS) in planktonic marine microorganisms.

Cyanate is utilized by many microbes as an organic nitrogen source. The key enzyme for cyanate metabolism is cyanase, converting cyanate to ammonium and carbon dioxide. Although the cyanase gene cynS has been identified in many species, the diversity, prevalence, and expression of cynS in marine microbial communities remains poorly understood. Here, based on the full-length cDNA sequence of a dinoflagellate cynS and 260 homologs across the tree of life, we extend the conserved nature of cyanases by the identification of additional ultra-conserved residues as part of the modeled holoenzyme structure. Our phylogenetic analysis showed that horizontal gene transfer of cynS appears to be more prominent than previously reported for bacteria, archaea, chlorophytes, and metazoans. Quantitative analyses of marine planktonic metagenomes revealed that cynS is as prevalent as ureC (urease subunit alpha), suggesting that cyanate plays an important role in nitrogen metabolism of marine microbes. Highly abundant cynS transcripts from phytoplankton and nitrite-oxidizing bacteria identified in global ocean metatranscriptomes indicate that cyanases potentially occupy a key position in the marine nitrogen cycle by facilitating photosynthetic assimilation of organic N and its remineralisation to NO3 by the activity of nitrifying bacteria.

The plasma membrane H+ -ATPase FgPMA1 regulates the development, pathogenicity, and phenamacril sensitivity of Fusarium graminearum by interacting with FgMyo-5 and FgBmh2.

Fusarium graminearum, as the causal agent of Fusarium head blight (FHB), not only causes yield loss, but also contaminates the quality of wheat by producing mycotoxins, such as deoxynivalenol (DON). The plasma membrane H+ -ATPases play important roles in many growth stages in plants and yeasts, but their functions and regulation in phytopathogenic fungi remain largely unknown. Here we characterized two plasma membrane H+ -ATPases: FgPMA1 and FgPMA2 in F. graminearum. The FgPMA1 deletion mutant (∆FgPMA1), but not FgPMA2 deletion mutant (∆FgPMA2), was impaired in vegetative growth, pathogenicity, and sexual and asexual development. FgPMA1 was localized to the plasma membrane, and ∆FgPMA1 displayed reduced integrity of plasma membrane. ∆FgPMA1 not only impaired the formation of the toxisome, which is a compartment where DON is produced, but also suppressed the expression level of DON biosynthetic enzymes, decreased DON production, and decreased the amount of mycelial invasion, leading to impaired pathogenicity by exclusively developing disease on inoculation sites of wheat ears and coleoptiles. ∆FgPMA1 exhibited decreased sensitivity to some osmotic stresses, a cell wall-damaging agent (Congo red), a cell membrane-damaging agent (sodium dodecyl sulphate), and heat shock stress. FgMyo-5 is the target of phenamacril used for controlling FHB. We found FgPMA1 interacted with FgMyo-5, and ∆FgPMA1 showed an increased expression level of FgMyo-5, resulting in increased sensitivity to phenamacril, but not to other fungicides. Furthermore, co-immunoprecipitation confirmed that FgPMA1, FgMyo-5, and FgBmh2 (a 14-3-3 protein) form a complex to regulate the sensitivity to phenamacril and biological functions. Collectively, this study identified a novel regulating mechanism of FgPMA1 in pathogenicity and phenamacril sensitivity of F. graminearum.

Automated Identification of Skull Fractures With Deep Learning: A Comparison Between Object Detection and Segmentation Approach.

Objective: Skull fractures caused by head trauma can lead to life-threatening complications. Hence, timely and accurate identification of fractures is of great importance. Therefore, this study aims to develop a deep learning system for automated identification of skull fractures from cranial computed tomography (CT) scans. Method: This study retrospectively analyzed CT scans of 4,782 patients (median age, 54 years; 2,583 males, 2,199 females; development set: n = 4,168, test set: n = 614) diagnosed with skull fractures between September 2016 and September 2020. Additional data of 7,856 healthy people were included in the analysis to reduce the probability of false detection. Skull fractures in all the scans were manually labeled by seven experienced neurologists. Two deep learning approaches were developed and tested for the identification of skull fractures. In the first approach, the fracture identification task was treated as an object detected problem, and a YOLOv3 network was trained to identify all the instances of skull fracture. In the second approach, the task was treated as a segmentation problem and a modified attention U-net was trained to segment all the voxels representing skull fracture. The developed models were tested using an external test set of 235 patients (93 with, and 142 without skull fracture). Results: On the test set, the YOLOv3 achieved average fracture detection sensitivity and specificity of 80.64, and 85.92%, respectively. On the same dataset, the modified attention U-Net achieved a fracture detection sensitivity and specificity of 82.80, and 88.73%, respectively. Conclusion: Deep learning methods can identify skull fractures with good sensitivity. The segmentation approach to fracture identification may achieve better results.

Potential surgical therapies for drug-resistant focal epilepsy.

Drug-resistant focal epilepsy (DRFE), defined by failure of two antiepileptic drugs, affects 30% of epileptic patients. Epilepsy surgeries are alternative options for this population. Preoperative evaluation is critical to include potential candidates, and to choose the most appropriate procedure to maximize efficacy and simultaneously minimize side effects. Traditional procedures involve open skull surgeries and epileptic focus resection. Alternatively, neuromodulation surgeries use peripheral nerve or deep brain stimulation to reduce the activities of epileptogenic focus. With the advanced improvement of laser-induced thermal therapy (LITT) technique and its utilization in neurosurgery, magnetic resonance-guided LITT (MRgLITT) emerges as a minimal invasive approach for drug-resistant focal epilepsy. In the present review, we first introduce drug-resistant focal epilepsy and summarize the indications, pros and cons of traditional surgical procedures and neuromodulation procedures. And then, focusing on MRgLITT, we thoroughly discuss its history, its technical details, its safety issues, and current evidence on its clinical applications. A case report on MRgLITT is also included to illustrate the preoperational evaluation. We believe that MRgLITT is a promising approach in selected patients with drug-resistant focal epilepsy, although large prospective studies are required to evaluate its efficacy and side effects, as well as to implement a standardized protocol for its application.

Temperature-Responded Biological Fitness of Carbendazim-Resistance Fusarium graminearum Mutants Conferring the F167Y, E198K, and E198L Substitutions.

Understanding the effects of temperature on Fusarium graminearum infection can provide theoretical guidance for chemical control of Fusarium head blight (FHB). Here, we evaluated the effects of various temperatures on biological fitness development of wild-type sensitive strain 2021 and carbendazim-resistance mutants conferring ß2-tubulin substitutions F167Y, E198K, and E198L. The results showed that mycelial growth and conidiation of four strains increased with the increase in temperature between 10 and 25°C. Conidia of F167Y displayed strong adaptability to low temperature. The virulence of the four strains was largely similar at the same temperature, showing an upward trend between 10 and 25°C. At 10°C, the hyphal growth of all strains was significantly inhibited, metabolism was slowed down, and the accumulation of secondary metabolites decreased. Subsequently, the production of deoxynivalenol (DON) and its intermediates pyruvate and aurofusarin decreased at low temperature, and the expression of DON biosynthesis-related genes Tri5, FgPK, and AUR decreased accordingly. At the same temperature, the aurofusarin production of the strains F167Y and E198K was higher than that of strains 2021 and E198L. The contents of DON and pyruvic acid in carbendazim-resistance mutants were higher than those in the wild-type strain 2021. The sensitivity of four strains to different fungicides changed at various temperatures. The sensitivity to most fungicides increased with decreasing temperature. The carbendazim-resistance mutants showed positive cross-resistance with other benzimidazoles. However, there was no cross-resistance to pyraclostrobin and azoles. These results would direct us to use fungicides preventing the infection of F. graminearum with changeable atmospheric temperature at the wheat flower stage.

Molecular and Biochemical Characterization of Pydiflumetofen-Resistant Mutants of Didymella bryoniae.

Gummy stem blight (GSB), caused by Didymella bryoniae, is a devastating disease on watermelon. Pydiflumetofen belongs to succinate dehydrogenase inhibitor (SDHI) fungicide, which is effective in controlling many plant diseases. The EC50 values of 69 D. bryoniae isolates to pydiflumetofen ranged from 0.0018 to 0.0071 µg/mL, and the minimal inhibitory concentration (MIC) value of all strains to pydiflumetofen was <0.05 µg/mL. Eight pydiflumetofen-resistant mutants were obtained, and the level of resistance was stable. The mycelial growth, dry weight of mycelia, hyphal morphology, and pathogenicity of most resistant mutants did not change significantly compared with their parental strains, which indicated that the resistance risk of D. bryoniae to pydiflumetofen would be medium to high. Sequencing alignment showed that five resistant mutants presented a mutation at codon 277 (H277Y) in the SdhB gene. The point mutants FgSdhBH248Y/R exhibited decreased sensitivity to pydiflumetofen in Fusarium graminearum, which indicated that the point mutants of SdhB could reduce sensitivity to pydiflumetofen. These results further increase our understanding about the mode of action and the resistance mechanism of pydiflumetofen.

Activity of the Succinate Dehydrogenase Inhibitor Fungicide Penthiopyrad Against Sclerotinia sclerotiorum.

In present study, the morphological and physiological characteristics of Sclerotinia sclerotiorum (Lib.) de Bary to a novel succinate dehydrogenase inhibitor (SDHI) fungicide penthiopyrad has been reported. The baseline sensitivity of S. sclerotiorum to penthiopyrad was determined using 119 strains by inhibition of mycelial growth. The median effective concentration (EC50) values for penthiopyrad ranged from 0.0096 to 0.2606 µg/ml, and the mean value was 0.0578 (±0.0626) µg/ml. After 1 µg/ml penthiopyrad treatment, mycelia of S. sclerotiorum strains showed increased apical branching and were denser compared with control, and cell membrane permeability significantly increased. In addition, glycerol content, oxalic acid (OA), and exopolysaccharide (EPS) content decreased markedly and mycelial respiration was distinctly inhibited. The number and dry weight of sclerotia significantly decreased after being treated with 2 µg/ml penthiopyrad. Penthiopyrad exhibited both protective and curative activity on the detached rapeseed leaves. Importantly, the above results will provide us more information on penthiopyrad for management of diseases caused by S. sclerotiorum and increase our understanding of action of penthiopyrad against S. sclerotiorum.

Impact of fluazinam on morphological and physiological characteristics of Sclerotinia sclerotiorum.

Sclerotinia sclerotiorum is a necrotrophic and filamentous fungus with a broad host range. Fluazinam is a pyridinamine fungicide with a broad spectrum of antifungal activity and had a strong inhibition effect on mycelial growth of S. sclerotiorum populations. But the impact of fluazinam on morphological and physiological characteristics of S. sclerotiorum is little known. In this study, the EC50 values of fluazinam to three strains of S. sclerotiorum (CZ17S, YZ55S and SA42S) were 0.0084, 0.007, 0.0065 µg/ml respectively. After fluazinam treatment, hyphae of S. sclerotiorum became thinner, hyphal offshoot of top increased, the distance between one septum and another became shorter, cell membrane permeability increased markedly, exopolysaccharide (EPS) content and oxalic acid content decreased significantly, peroxidase (POD) activity increased significantly and mycelial respiration was inhibited. While the number and dry weight of sclerotia, glycerol content in the mycelia did not significantly change. In protective activity assay on detached rapeseed leaves, application of fluazinam at 40 µg/ml and 80 µg/ml, the control efficacy reached to 41.4% and 100%, respectively. In curative activity assay, application of fluazinam at 100 µg/ml, the control efficacy reached to 61.09%. In the same concentration, protective activity of fluazinam against S. sclerotiorum was higher than curative activity. These results will contribute to us on evaluating the potential of the fungicide fluazinam for management of Sclerotinia stem rot and understanding the mode of action of fluazinam against S. sclerotiorum.

Activity of the dinitroaniline fungicide fluazinam against Bipolaris maydis.

Fluazinam is a dinitroaniline fungicide with broad-spectrum activities. However, the activity of fluazinam against Bipolaris maydis which is the causal agent of southern corn leaf blight is unknown yet. In this study, baseline sensitivity of B. maydis to fluazinam was determined using 92 isolates collected during 2015 and 2016 from different geographical regions in Jiangsu Province of China, and the EC50 values ranged from 0.0396 to 0.9808 µg/ml with average value of 0.3853 ±â€¯0.2297 µg/ml, and 0.079 to 0.7832 µg/ml with average value of 0.3065 ±â€¯0.1384 µg/ml for mycelial growth and conidium germination respectively. Fluazinam did not affect the distribution of cell nucleus and the formation of septum of B. maydis. However, fluazinam could make mycelium of B. maydis contorted and the mycelial branches increased and inhibit the development of conidia. The result of transmission electron microscope showed that fluazinam damaged cell wall and cell membrane of mycelium, and make organelles in mycelial cell dissolved and vacuolated, and the cell almost broke up, which caused the intracellular plasma leakage increase. The protective activity test of fluazinam suggested that fluazinam had great control efficiency against B. maydis on detached corn leaves. Application of fluazinam at 10 µg/ml and 20 µg/ml, the control efficacy reached to 87.70% and 98.25% respectively. However, fluazinam had no curative activity against B. maydis on detached corn leaves. These results will contribute to us on evaluating the potential of the dinitroaniline fungicide fluazinam for management of diseases caused by B. maydis and understanding the mode of action of fluazinam against B. maydis.

Molecular and biological characterization of Sclerotinia sclerotiorum resistant to the anilinopyrimidine fungicide cyprodinil.

Cyprodinil belongs to the chemical class of anilinopyrimidines fungicides. In this study, baseline sensitivity of Sclerotinia sclerotiorum (Lib.) de Bary to cyprodinil was determined using 100 strains collected from the fields in Jiangsu Province of China. The EC50 (50% effective concentration) values ranged from 0.0636-0.8163 µg/ml with a mean value of 0.1869 (±0.1118) ug/ml for mycelial growth. Nine cyprodinil-resistant mutants (Range of resistance factor: 20.22-271.59) were obtained from sensitive strains exposed on PDA medium amended with cyprodinil and the resistance was stable after their ten transfers on PDA without the fungicide or stored at 4 °C for two months. There was positive cross-resistance between cyprodinil and pyrimethanil but not to fludioxonil, dimetachlone, procymidone, carbendazim and boscalid in S. sclerotiorum. Compared with the parental strains, all of the nine cyprodinil-resistant mutants decreased in sclerotial production. The dry weight of mycelia, pathogenicity and cell membrane permeability of most resistant mutants decreased. The mycelial growth, oxalic acid content, and the response to various stress for resistant mutants were almost the same as the sensitive parental strains. Sequencing alignment results showed that there was no alteration of amino acid in cystathionine γ-synthase (MetB) and cystathionine ß-lyase (MetC) between cyprodinil-resistant mutants and their sensitive parental strains, which indicated that MetB or MetC was not the molecular target of cyprodinil in S. sclerotiorum. The addition of amino acids L-methionine, L-cystine or L-cysteine decreased the inhibition of cyprodinil against mycelial growth of S. sclerotiorum, which indicated that cyprodinil could not only inhibited methionine biosynthesis but also suppressed cystine and cysteine biosynthesis. These results will contribute to evaluating the resistance risk of cyprodinil for management of the plant diseases of Sclerotinia stem rot caused by S. sclerotiorum and further increase our understanding about the mode of action of cyprodinil.

Resistance risk assessment for fluazinam in Sclerotinia sclerotiorum.

In the current study, sensitivity distribution of Sclerotinia sclerotiorum populations to fluazinam was determined using 103 strains collected from the fields of Jiangsu Province of China in 2016-2017 and the resistance risk of fluazinam was assessed. The average EC50 (50% effective concentration) values and MIC (minimum inhibitory concentration) values of 103 S. sclerotiorum strains against fluazinam were 0.0073±0.0045µg/ml and <0.3µg/ml for mycelial growth, respectively. Nine mutants with low resistance level were obtained from wild type sensitive strains exposed on PDA medium amended with fluazinam and the resistance was stable after their ten transfers on PDA without the fungicide. Compared with the parental strains, the nine fluazinam-resistant mutants decreased in mycelial growth, sclerotial production, pathogenicity and were more sensitive to 0.7M NaCl. In addition, cell membrane permeability of resistant mutants was higher than that of their parental strains. Cross resistance assay showed that there was no cross-resistance between fluazinam and fludioxonil, dimetachlone, prochloraz, tebuconazole, azoxystrobin, or procymidone in S. sclerotiorum. The above results indicated that there was a low resistance risk for fluazinam in S. sclerotiorum. However, the sensitivity of all fluazinam-resistant mutants to fludioxonil decreased. Sequencing alignment results showed that there were no mutations in the two-component histidine kinase gene (Shk1) of the resistant mutants and the expression levels of Shk1 of three resistant mutants were significantly up-regulated while others were almost the same as their parental strains. These results will contribute to evaluating the resistance risk of fluazinam for management of diseases caused by S. sclerotiorum and further increase our understanding about the mode of action of fluazinam.