PCR-RFLP for Detection of Fusarium graminearum Genotypes with Resistance to Phenamacril.

Phenamacril is a cyanoacrylate fungicide that provides excellent control of Fusarium head blight (FHB) or wheat scab, which is caused predominantly by Fusarium graminearum and F. asiaticum. Previous studies revealed that codon mutations of the myosin-5 gene of Fusarium spp. conferred resistance to phenamacril in in vitro lab experiments. In this study, PCR restriction fragment length polymorphism (RFLP) was developed to detect three common mutations (A135T, GCC to ACC at codon 135; S217L, TCA to TTA at codon 217; and E420K, GAA to AAA at codon 420) in F. graminearum induced by fungicide domestication in vitro. PCR products of 841 bp (for mutation of A135T), 802 bp (for mutation of S217L), or 1,649 bp (for mutation of E420K) in the myosin-5 gene were amplified by appropriate primer pairs. Restriction enzyme KpnI, TasI, or DraI was used to distinguish phenamacril-sensitive and -resistant strains with mutation genotypes of A135T, S217L, and E420K, respectively. KpnI digested the 841-bp PCR products of phenamacril-resistant strains with codon mutation A135T into two fragments of 256 and 585 bp. In contrast, KpnI did not digest the PCR products of sensitive strains. TasI digested the 802-bp PCR products of phenamacril-resistant strains with codon mutation S217L into three fragments of 461, 287, and 54 bp. In contrast, TasI digestion of the 802-bp PCR products of phenamacril-sensitive strains resulted in only two fragments of 515 and 287 bp. DraI digested the 1,649-bp PCR products of phenamacril-resistant strains with codon mutation E420K into two fragments of 932 and 717 bp, while the PCR products of phenamacril-sensitive strains was not digested. The three genotypes of resistance mutations were determined by analyzing electrophoresis patterns of the digestion fragments of PCR products. The PCR-RFLP method was evaluated on 48 phenamacril-resistant strains induced by fungicide domestication in vitro and compared with the conventional method (mycelial growth on fungicide-amended agar). The accuracy of the PCR-RFLP method for detecting the three mutation genotypes of F. graminearum resistant to phenamacril was 95.12% compared with conventional method. Bioinformatics analysis revealed that the PCR-RFLP method could also be used to detect the codon mutations of A135T and E420K in F. asiaticum.

The ASK1 gene regulates the sensitivity of Fusarium graminearum to carbendazim, conidiation and sexual production by combining with ß2-tubulin.

ß-tubulin, a component of microtubules, is involved in a wide variety of roles in cell shape, motility, intracellular trafficking and regulating intracellular metabolism. It has been an important fungicide target to control plant pathogen, for example, Fusarium. However, the regulation of fungicide sensitivity by ß-tubulin-interacting proteins is still unclear. Here, ASK1 was identified as a ß-tubulin interacting protein. The ASK1 regulated the sensitivity of Fusarium to carbendazim (a benzimidazole carbamate fungicide), and multiple cellular processes, such as chromatin separation, conidiation and sexual production. Further, we found the point mutations at 50th and 198th of ß2-tubulin which caused carbendazim resistance decreased the binding between ß2-tubulin and ASK1, resulting in the deactivation of ASK1. ASK1, on the other hand, competed with carbendazim to bind to ß2-tubulin. The point mutation F167Y in ß2-tubulin broke the intermolecular H-bonds and salt bridges between ß2-tubulin and ASK1, which reduced the competitive effect of ASK1 to carbendazim and resulted in the similar carbendazim sensitivities in F167Y-ΔASK1 and F167Y. These findings have powerful implications for efforts to understand the interaction among ß2-tubulin, its interacting proteins and fungicide, as well as to discover and develop new fungicide against Fusarium.

Ethylenediaminetetraacetic Acid Disodium Salt Acts as an Antifungal Candidate Molecule against Fusarium graminearum by Inhibiting DON Biosynthesis and Chitin Synthase Activity.

Fusarium fungi are the cause of an array of devastating diseases affecting yield losses and accumulating mycotoxins. Fungicides can be exploited against Fusarium and deoxynivalenol (DON) production. However, Fusarium resistance to common chemicals has become a therapeutic challenge worldwide, which indicates that new control agents carrying different mechanisms of action are desperately needed. Here, we found that a nonantibiotic drug, ethylenediaminetetraacetic acid disodium salt (EDTANa2), exhibited various antifungal activities against Fusarium species and DON biosynthesis. The infection of wheat seeding caused by F. graminearum was suppressed over 90% at 4 mM EDTANa2. A similar control effect was observed in field tests. Mycotoxin production assays showed DON production was significantly inhibited, 47% lower than the control, by 0.4 mM EDTANa2. In vitro experiments revealed a timely inhibition of H2O2 production as quickly as 4 h after amending cultures with EDTANa2 and the expression of several TRI genes significantly decreased. Chitin synthases of Fusarium were Mn2+-containing enzymes that were strongly inhibited by Mn2+ deficiency. EDTANa2 inhibited chitin synthesis and destroyed the cell wall and cytomembrane integrity of Fusarium, mainly via the chelation of Mn2+ by EDTANa2, and thus led to Mn deficiency in Fusarium cells. Taken together, these findings uncover the potential of EDTANa2 as a fungicide candidate to manage Fusarium head blight (FHB) and DON in agricultural production.

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.

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.

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.

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.

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.

An antibody that confers plant disease resistance targets a membrane-bound glyoxal oxidase in Fusarium.

Plant germplasm resources with natural resistance against globally important toxigenic Fusarium are inadequate. CWP2, a Fusarium genus-specific antibody, confers durable resistance to different Fusarium pathogens that infect cereals and other crops, producing mycotoxins. However, the nature of the CWP2 target is not known. Thus, investigation of the gene coding for the CWP2 antibody target will likely provide critical insights into the mechanism underlying the resistance mediated by this disease-resistance antibody. Immunoblots and mass spectrometry analysis of two-dimensional electrophoresis gels containing cell wall proteins from Fusarium graminearum (Fg) revealed that a glyoxal oxidase (GLX) is the CWP2 antigen. Cellular localization studies showed that GLX is localized to the plasma membrane. This GLX efficiently catalyzes hydrogen peroxide production; this enzymatic activity was specifically inhibited by the CWP2 antibody. GLX-deletion strains of Fg, F. verticillioides (Fv) and F. oxysporum had significantly reduced virulence on plants. The GLX-deletion Fg and Fv strains had markedly reduced mycotoxin accumulation, and the expression of key genes in mycotoxin metabolism was downregulated. This study reveals a single gene-encoded and highly conserved cellular surface antigen that is specifically recognized by the disease-resistance antibody CWP2 and regulates both virulence and mycotoxin biosynthesis in Fusarium species.

Host-induced gene silencing of an essential chitin synthase gene confers durable resistance to Fusarium head blight and seedling blight in wheat.

Fusarium head blight (FHB) and Fusarium seedling blight (FSB) of wheat, caused by Fusarium pathogens, are devastating diseases worldwide. We report the expression of RNA interference (RNAi) sequences derived from an essential Fusarium graminearum (Fg) virulence gene, chitin synthase (Chs) 3b, as a method to enhance resistance of wheat plants to fungal pathogens. Deletion of Chs3b was lethal to Fg; disruption of the other Chs gene family members generated knockout mutants with diverse impacts on Fg. Comparative expression analyses revealed that among the Chs gene family members, Chs3b had the highest expression levels during Fg colonization of wheat. Three hairpin RNAi constructs corresponding to the different regions of Chs3b were found to silence Chs3b in transgenic Fg strains. Co-expression of these three RNAi constructs in two independent elite wheat cultivar transgenic lines conferred high levels of stable, consistent resistance (combined type I and II resistance) to both FHB and FSB throughout the T3 to T5 generations. Confocal microscopy revealed profoundly restricted mycelia in Fg-infected transgenic wheat plants. Presence of the three specific short interfering RNAs in transgenic wheat plants was confirmed by Northern blotting, and these RNAs efficiently down-regulated Chs3b in the colonizing Fusarium pathogens on wheat seedlings and spikes. Our results demonstrate that host-induced gene silencing of an essential fungal chitin synthase gene is an effective strategy for enhancing resistance in crop plants under field test conditions.

Antagonistic mechanism of iturin A and plipastatin A from Bacillus amyloliquefaciens S76-3 from wheat spikes against Fusarium graminearum.

Controlling toxigenic Fusarium graminearum (FG) is challenging. A bacterial strain (S76-3, identified as Bacillus amyloliquefaciens) that was isolated from diseased wheat spikes in the field displayed strong antifungal activity against FG. Reverse-phase high performance liquid chromatography and electrospray ionization mass spectrometry analyses revealed that S76-3 produced three classes of cyclic lipopeptides including iturin, plipastatin and surfactin. Each class consisted of several different molecules. The iturin and plipastatin fractions strongly inhibited FG; the surfactin fractions did not. The most abundant compound that had antagonistic activity from the iturin fraction was iturin A (m/z 1043.35); the most abundant active compound from the plipastatin fraction was plipastatin A (m/z 1463.90). These compounds were analyzed with collision-induced dissociation mass spectrometry. The two purified compounds displayed strong fungicidal activity, completely killing conidial spores at the minimal inhibitory concentration range of 50 µg/ml (iturin A) and 100 µg/ml (plipastatin A). Optical and fluorescence microscopy analyses revealed severe morphological changes in conidia and substantial distortions in FG hyphae treated with iturin A or plipastatin A. Iturin A caused leakage and/or inactivation of FG cellular contents and plipastatin A caused vacuolation. Time-lapse imaging of dynamic antagonistic processes illustrated that iturin A caused distortion and conglobation along hyphae and inhibited branch formation and growth, while plipastatin A caused conglobation in young hyphae and branch tips. Transmission electron microscopy analyses demonstrated that the cell walls of conidia and hyphae of iturin A and plipastatin A treated FG had large gaps and that their plasma membranes were severely damaged and separated from cell walls.

Trehalose 6-phosphate phosphatase is required for development, virulence and mycotoxin biosynthesis apart from trehalose biosynthesis in Fusarium graminearum.

Trehalose 6-phosphate synthase (TPS1) and trehalose 6-phosphate phosphatase (TPS2) are required for trehalose biosynthesis in yeast and filamentous fungi, including Fusarium graminearum. Three null mutants Δtps1, Δtps2 and Δtps1-Δtps2, each carrying either a single deletion of TPS1 or TPS2 or a double deletion of TPS1-TPS2, were generated from a toxigenic F. graminearum strain and were not able to synthesize trehalose. In contrast to its reported function in yeasts and filamentous fungi, TPS1 appeared dispensable for development and virulence. However, deletion of TPS2 abolished sporulation and sexual reproduction; it also altered cell polarity and ultrastructure of the cell wall in association with reduced chitin biosynthesis. The cell polarity alteration was exhibited as reduced apical growth and increased lateral growth and branching with increased hyphal and cell wall widths. Moreover, the TPS2-deficient strain displayed abnormal septum development and nucleus distribution in its conidia and vegetative hyphae. The Δtps2 mutant also had 62% lower mycelial growth on potato dextrose agar and 99% lower virulence on wheat compared with the wild-type. The Δtps1, Δtps2 and Δtps1-Δtps2 mutants synthesized over 3.08-, 7.09- and 2.47-fold less mycotoxins, respectively, on rice culture compared with the wild-type. Comparative transcriptome analysis revealed that the Δtps1, Δtps2 and Δtps1-Δtps2 mutants had 486, 1885 and 146 genotype-specific genes, respectively, with significantly changed expression profiles compared with the wild-type. Further dissection of this pathway will provide new insights into regulation of fungal development, virulence and trichothecene biosynthesis.

Type II myosin gene in Fusarium graminearum is required for septation, development, mycotoxin biosynthesis and pathogenicity.

Type II myosin is required for cytokinesis/septation in yeast and filamentous fungi, including Fusarium graminearum, a prevalent cause of Fusarium head blight in China. A type II myosin gene from the Chinese F. graminearum strain 5035, isolated from infected wheat spikes, was identified by screening a mutant library generated by restriction enzyme-mediated integration. Disruption of the Myo2 gene reduced mycelial growth by 50% and conidiation by 76-fold, and abolished sexual reproduction on wheat kernels. The Δmyo2 mutants also had a 97% decrease in their pathogenicity on wheat, and mycotoxin production fell to just 3.4% of the normal level. The distribution of nuclei and septa was abnormal in the mutants, and the septal ultrastructure appeared disorganized. Time-lapse imaging of septation provided direct evidence that Myo2 is required for septum initiation and formation, and revealed the dynamic behavior of GFP-tagged Myo2 during hyphal and macroconidia development, particularly in the delimiting septum of phialides and macroconidial spores. Microarray analysis identified many genes with altered expression profiles in the Δmyo2 mutant, indicating that Myo2 is required for several F. graminearum developmental processes and biological activities.