Validamycin A Enhances the Interaction Between Neutral Trehalase and 14-3-3 Protein Bmh1 in Fusarium graminearum.

In agriculture, Trehalase is considered the main target of the biological fungicide validamycin A, and the toxicology mechanism of validamycin A is unknown. 14-3-3 proteins, highly conserved proteins, participate in diverse cellular processes, including enzyme activation, protein localization, and acting as a molecular chaperone. In Saccharomyces cerevisiae, the 14-3-3 protein Bmh1could interact with Nth1 to respond to specific external stimuli. Here, we characterized FgNth, FgBmh1, and FgBmh2 in Fusarium graminearum. ΔFgNth, ΔFgBmh1, and ΔFgBmh2 displayed great growth defects and their peripheral tips hyphae generated more branches when compared with wild-type (WT) PH-1. When exposed to validamycin A as well as high osmotic and high temperature stresses, ΔFgNth, ΔFgBmh1, and ΔFgBmh2 showed more tolerance than WT. Both ΔFgNth and ΔFgBmh1 displayed reduced deoxynivalenol production but opposite for ΔFgBmh2, and all three deletion mutants showed reduced virulence on wheat coleoptiles. In addition, coimmunoprecipitation (Co-IP) experiments suggested that FgBmh1 and FgBmh2 both interact with FgNth, but no interaction was detected between FgBmh1 and FgBmh2 in our experiments. Further, validamycin A enhances the interaction between FgBmh1 and FgNth in a positive correlation under concentrations of 1 to 100 µg/ml. In addition, both high osmotic and high temperature stresses promote the interaction between FgBmh1 and FgNth. Co-IP assay also showed that neither FgBmh1 nor FgBmh2 could interact with FgPbs2, a MAPKK kinase in the high-osmolarity glycerol pathway. However, FgBmh2 but not FgBmh1 binds to the heat shock protein FgHsp70 in F. graminearum. Taken together, our results demonstrate that FgNth and FgBmh proteins are involved in growth and responses to external stresses and virulence; and validamycin enhanced the interaction between FgNth and FgBmh1in F. graminearum.

Pharmacological Characteristics and Efficacy of Fluazinam Against Corynespora cassiicola, Causing Cucumber Target Spot in Greenhouses.

Cucumber target spot, caused by Corynespora cassiicola, is a devastating fungal disease in greenhouses in China. Lack of resistant cultivars and unscientific use of fungicides aggravated the difficulty to manage this disease. In recent years, resistance of C. cassiicola to benzimidazoles, quinone outside inhibitors, and succinate dehydrogenase inhibitors has occurred in China. Here, we tested the fluazinam sensitivity distribution of 79 C. cassiicola isolates from different provinces in China based on mycelial growth inhibition. The EC50 values of fluazinam ranged from 0.1002 to 0.3129 µg/ml with a mean of 0.2136 ± 0.0495 µg/ml, and the sensitivity frequency was normally distributed (P = 0.2083, Shapiro-Wilk test). Meanwhile, the EC50 values for spore germination inhibition ranged from 0.0992 to 0.2278 µg/ml with a mean of 0.1499 ± 0.0504 µg/ml. This indicated that fluazinam exhibited an excellent in vitro fungicidal activity on both mycelial growth and spore germination. In addition, fluazinam also exhibited a good in planta control efficacy on detached cucumber leaves in the protective and curative assays. Moreover, the biological and physiological characteristics of C. cassiicola as affected by fluazinam were determined. Fluazinam not only significantly inhibited respiration and adenosine triphosphate production but also caused the increase of cell membrane permeability and the dysfunctions of cellular homeostasis. Interestingly, we found that fluazinam especially damaged vacuole structures, causing the redistribution of vacuole substances. Taken together, our findings provide not only essential references for resistance management of C. cassiicola but also interesting insights for further revealing the action mode of fluazinam against plant pathogens.

A ß2-tubulin dsRNA derived from Fusarium asiaticum confers plant resistance to multiple phytopathogens and reduces fungicide resistance.

Crops are attacked by a large number of pathogens which are responsible for an approximately 30% loss in global crop production at pre- and post-harvest levels. In light of the continuing emergence of fungicide resistance, the needs for new agricultural drugs turn out to be much more critical. Here we demonstrated a Faß2Tub-3 dsRNA derived from Fusarium asiaticum had broad-spectrum antifungal activity against Fusarium spp., Botrytis cinerea, Magnaporthe oryzae and Colletotrichum truncatum, with an additional function of reducing the dosage of carbendazim (MBC) fungicide. RNAi molecules derived from different regions of ß2-tubulin gene had different effects on mycelial growth, asexual reproduction and virulence. Faß2Tub-3 (one of ß2-tubulin segments) exhibited a strong silencing efficacy both on ß1-tubulin and ß2-tubulin genes in F. asiaticum. Faß2Tub-3 sequence was found to be highly conserved among Fusarium spp., Botrytis cinerea, Magnaporthe oryzae and Colletotrichum truncatum. The Faß2Tub-3 dsRNA demonstrated a broad-spectrum antifungal activity against these fungi in vitro and on living plant. More importantly, Faß2Tub-3 dsRNA increased the fungal sensitivity to MBC, while MBC increased the duration of Faß2Tub-3 dsRNA. Our findings suggest a new anti-fungal agent (Faß2Tub-3 dsRNA) for plant protection against diverse pathogens and for fungicide reduction.

A myosin5 dsRNA that reduces the fungicide resistance and pathogenicity of Fusarium asiaticum.

Fungal resistance to fungicides is a serious challenge in crop protection. Although strategies have been found to prevent the development of fungicide resistance, rare strategy has been found to quickly reduce such resistance once it has occurred. We demonstrate that the application of dsRNAs, which inhibit the expression of the phenamacril (fungicide JS399-19) target gene-Myosin 5 (Myo5) in Fusarium, decreased F. asiaticum resistance to phenamacril and infection. RNAi molecules derived from different regions of Myo5 gene had different effects on phenamacril-resistance. Myo5-8 (one of Myo5 segments) exhibited great and stable effect on phenamacril-resistant reduction both in vivo and in vitro. Myo5 mRNA and protein were both reduced when mycelium was treated with Myo5-8 dsRNA. After a mixture of Myo5-8 dsRNA and phenamacril treatment, plants can highly control the infection of phenamacril-resistant strain. The antifungal activity of Myo5-8 dsRNA plus phenamacril effected longer than a single Myo5-8 dsRNA. In addition, no off-target sequences were found in wheat and/or other plant and animal species for Myo5-8 dsRNA sequence. Our findings suggest a new strategy for fungicide resistant reduction and for designing new fungicides to control pathogens which easily develop fungicide resistance.

Simultaneous Detection of Multiple Benzimidazole-Resistant ß-Tubulin Variants of Botrytis cinerea using Loop-Mediated Isothermal Amplification.

Optimal disease management depends on the ability to monitor the development of fungicide resistance in plant pathogen populations. Benzimidazole resistance is caused by the point mutations of the ß-tubulin gene in Botrytis cinerea, and three mutations (E198A, E198K, and E198V) at codon 198 account for more than 98% of all resistant strains. Although traditional methods remain a cornerstone in monitoring fungicide resistance, molecular methods that do not require the isolation of pathogens can detect resistance alleles present at low frequencies, and require less time and labor than traditional methods. In this study, we present an efficient, rapid, and highly specific method for detecting highly benzimidazole-resistant B. cinerea isolates based on loop-mediated isothermal amplification (LAMP). By using specific primers, we could simultaneously detect all three resistance-conferring mutations at codon 198. The LAMP reaction components and conditions were optimized, and the best reaction temperatures and times were 60 to 62°C and 45 min, respectively. When B. cinerea field isolates were assessed for benzimidazole resistance, similar results were obtained with LAMP, minimal inhibition concentration, and sequencing. The LAMP assay developed in the current study was highly suitable for detection of highly benzimidazole-resistant field isolates of B. cinerea.

Secondary amplification of siRNA machinery limits the application of spray-induced gene silencing.

Spray-induced gene silencing (SIGS) is an innovative strategy for crop protection. However, the mechanism of SIGS is not known. Here, we first demonstrate that secondary small interfering RNA (siRNA) amplification limits the application of SIGS. A myosin5 gene (Myo5) was chosen as the target of SIGS in an agronomically important pathogen-Fusarium asiaticum. Five segments corresponding to the different regions of the Myo5 gene were found to efficiently silence Myo5, resulting in cell wall defects, life cycle disruption and virulence reduction. Myo5-8 (one of the Myo5 segments) induced sequence-specific RNA interference (RNAi) activity in F. asiaticum, F. graminearum, F. tricinctum and F. oxysporum, but not in other fungi, in vitro. Remarkably, the silencing of Myo5 lasted for only 9 h unless the double-stranded RNA (dsRNA) was continuously supplied, because F. asiaticum is unable to maintain siRNA amplification. After spraying on plants, dsRNAs were more efficiently taken up via the wounded surface. The antifungal activity of dsRNAs taken up by plant cells was higher and longer lasting than that dried onto the plant surface. In contrast with dsRNAs in fungi, dsRNAs in plant cells could efficiently turn into substantial siRNAs via secondary amplification machinery. Our findings provide new implications to develop SIGS as a mainstream disease control strategy against Fusarium and other fungi.

Activity of a novel succinate dehydrogenase inhibitor fungicide pyraziflumid against Sclerotinia sclerotiorum.

Pyraziflumid is a novel member of succinate dehydrogenase inhibitor fungicides (SDHI). In this study, baseline sensitivity of Sclerotinia sclerotiorum (Lib.) de Bary to pyraziflumid was determined using 105 strains collected during 2015 and 2017 from different geographical regions in Jiangsu Province of China, and the average EC50 value was 0.0561 (±0.0263)µg/ml for mycelial growth. There was no cross-resistance between pyraziflumid and the widely used fungicides carbendazim, dimethachlon and the phenylpyrrole fungicide fludioxonil. After pyraziflumid treated, hyphae were contorted with offshoot of top increasing, cell membrane permeability increased markedly, oxalic acid content significantly decreased and mycelial respiration was strongly inhibited. But the number and dry weight of sclerotia did not change significantly. The protective and curative activity test of pyraziflumid suggested that pyraziflumid had great control efficiency against S. sclerotiorum on detached rapeseed leaves, and protective activity was better than curative activity. These results will contribute to us on evaluating the potential of the new SDHI fungicide pyraziflumid for management of diseases caused by S. sclerotiorum and understanding the mode of action of pyraziflumid against S. sclerotiorum.

A new point mutation in ß2-tubulin confers resistance to carbendazim in Fusarium asiaticum.

Resistance to benzimidazole fungicides in many phytopathogenic fungi is caused by specific point mutations in the ß-tubulin gene (ß-tubulin). However, the mutated locus and genotype of ß-tubulin differ among phytopathogenic fungi. To validate the point mutation in Fusarium asiaticum ß2-tubulin that confers resistance to carbendazim and to analyze the molecular interaction between carbendazim and F. asiaticum ß2-tubulin. In this study, a new point mutation (GAG→GCG, E198A) at codon 198 of ß2-tubulin in a wild-type F. asiaticum strain was constructed by site-directed mutagenesis followed by a split marker strategy. The site-directed mutants were verified and exhibited a high level of resistance to carbendazim. In the absence of fungicide treatment, the biological characteristics did not differ between the site-directed mutants and the wild-type strain. Molecular docking between carbendazim and ß2-tubulin was carried out using the Surflex-Dock program in Sybyl X-2.0 version and the results indicated that the E198A mutation altered the configuration of ß2-tubulin, resulting in the change of the bonding sites and docking scores. We concluded that the point mutation of F. asiaticum ß2-tubulin conferring carbendazim resistance may not always be the bonding site for carbendazim.

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.

Resistance mechanism of Fusarium fujikuroi to phenamacril in the field.

BACKGROUND: Rice bakanae disease, mainly caused by Fusarium fujikuroi, is an important disease of rice. Phenamacril has been used to control the disease for a few years in China. In 2016, nine phenamacril-resistant strains were found in the field in Zhejiang Province. The aim of the study was to clarify the mechanism of resistance of F. fujikuroi to phenamacril and the fitness of resistant strains. RESULTS: The nine F. fujikuroi strains examined were highly resistant to phenamacril. Eight of them had the point mutation TCA (Ser) → CCA (Pro) at codon 219 in the Myosin-5 protein, while the other had the point mutation TCA (Ser) → TTA (Leu) at codon 219. Myosin-5 replacement between resistant and sensitive strains confirmed that the point mutation in Myosin-5 caused the resistance of F. fujikuroi to phenamacril. Docking of phenamacril into the modeled binding pocket of Myosin-5 showed that the affinity between phenamacril and Myosin-5 decreased and a hydrogen bond could not be formed between phenamacril and the amino acid at codon 219 after it changed to Pro or Leu. There was no cross-resistance between phenamacril and other fungicides. The eight resistant strains containing the point mutation S219P had almost the same fitness as the sensitive strains, while the one resistant strain containing the point mutation S219 L showed decreased mycelial growth, sporulation and pathogenicity. CONCLUSION: In the field, the point mutation S219P or S219 L in Myosin-5 conferred high resistance to phenamacril in F. fujikuroi. The point mutation S219P did not affect the fitness of F. fujikuroi, while the point mutation S219 L decreased its fitness. © 2017 Society of Chemical Industry.

Evaluation of antifungal activities and structure-activity relationships of coumarin derivatives.

BACKGROUND: Osthol is a natural coumarin and lead compound that has been developed into commercial fungicides in China. Natural coumarins comprise five major subtypes: simple coumarins, linear furanocoumarins, angular furanocoumarins, linear pyranocoumarins and angular pyranocoumarins. Studies pertaining to the antifungal activities of linear pyranocoumarins are few, and no reports exist for the antifungal activities of angular pyranocoumarins. In order to discover more antifungal natural coumarins, we synthesised a series of simple natural coumarins and isolated several plant-based furanocoumarins and pyranocoumarins using previously described methods. The compounds were biologically evaluated against some plant fungal pathogens. RESULTS: Several of the 35 coumarins evaluated here exhibited strong activities against specific fungal species, including compound 25 (Pd-D-V, a linear pyranocoumarin), compound 26 (libanorin, an angular furanocoumarin) and compound 34 (disenecioyl khellactone, an angular pyranocoumarin). Compound 25 exhibited a high activity against Sclerotinia sclerotiorum (EC50 = 13.2 µg mL-1 ); compound 34 displayed a strong antifungal activity against Botrytis cinerea (EC50 = 11.0 µg mL-1 ). CONCLUSION: This study demonstrates that several natural coumarins (one linear pyranocoumarin and one angular pyranocoumarin in particular) exhibit strong antifungal activities. These results call for further studies, where these coumarins can be examined as potential lead compounds for developing novel antifungal agents. © 2016 Society of Chemical Industry.

Development and application of loop-mediated isothermal amplification for detecting the highly benzimidazole-resistant isolates in Sclerotinia sclerotiorum.

Resistance of benzimidazole fungicides is related to the point mutation of the ß-tubulin gene in Sclerotinia sclerotiorum. The point mutation at codon 198 (GAG → GCG, E198A) occurs in more than 90% of field resistant populations in China. Traditional detection methods of benzimidazole-resistant mutants of S. sclerotiorum are time-consuming, tedious and inefficient. To establish a suitable and rapid detection of benzimidazole-resistant mutants of S. sclerotiorum, an efficient and simple method with high specificity was developed based on loop-mediated isothermal amplification (LAMP). Eight sets of LAMP primers were designed and four sets were optimized to specially distinguish benzimidazole-resistant mutants of S. sclerotiorum. With the optimal LAMP primers, the concentration of LAMP components was optimized and the reaction conditions were set as 60-64 °C for 60 min. This method had a good specificity, sensitivity, stability and repeatability. In the 1491 sclerotia, 614 (41.18%) were positive by LAMP, and 629 (42.19%) positive by MIC. Therefore, the LAMP assay is more feasible to detect benzimidazole-resistant mutants of S. sclerotiorum than traditional detection methods.

Development and evaluation of a novel and rapid detection assay for Botrytis cinerea based on loop-mediated isothermal amplification.

Botrytis cinerea is a devastating plant pathogen that causes grey mould disease. In this study, we developed a visual detection method of B. cinerea based on the Bcos5 sequence using loop-mediated isothermal amplification (LAMP) with hydroxynaphthol blue dye (HNB). The LAMP reaction was optimal at 63 °C for 45 min. When HNB was added prior to amplification, samples with B. cinerea DNA developed a characteristic sky blue color after the reaction but those without DNA or with DNA of other plant pathogenic fungi did not. Results of HNB staining method were reconfirmed when LAMP products were subjected to gel electrophoresis. The detection limit of this LAMP assay for B. cinerea was 10(-3) ng µL(-1) of genomic DNA per reaction, which was 10-fold more sensitive than conventional PCR (10(-2) ng µL(-1)). Detection of the LAMP assay for inoculum of B. cinerea was possible in the inoculated tomato and strawberry petals. In the 191 diseased samples, 180 (94.2%) were confirmed as positive by LAMP, 172 (90.1%) positive by the tissue separation, while 147 (77.0%) positive by PCR. Because the LAMP assay performed well in aspects of sensitivity, specificity, repeatability, reliability, and visibility, it is suitable for rapid detection of B. cinerea in infected plant materials prior to storage and during transportation, such as cut flowers, fruits and vegetables.