Wheat head blight caused by Gibberella zeae (anamorph: Fusarium graminearum) is a threat to food safety in China because of mycotoxin contamination of the harvested grain, the frequent occurrence of the disease, and the failure of chemical control in some areas due to benzimidazole resistance in the pathogen population. The molecular resistance mechanism, however, of G. zeae to benzimidazole fungicides (especially carbendazim; active ingredient: methyl benzimidazol-2-yl carbamate [MBC]) is poorly understood. DNA sequences of a beta-tubulin gene (beta(2)tub) (GenBank access number FG06611.1) in G. zeae were analyzed. Mutations in beta(2)tub in moderately resistant strains (MBC(MR)) included TTT (Phe)-->TAT (Tyr) at codon 167 or TTC (Phe)-->TAC (Tyr) at codon 200. A highly resistant strain (MBC(HR)) had two point mutations, one at codon 73, CAG (Gln)-->CGG (Arg), and the other at codon 198, GAG (Glu)-->CTG (Leu). To confirm that mutations in the beta(2)tub confer resistance to benzimidazole fungicides, the entire beta(2)tub locus was deleted from MBC(MR) and MBC(HR) strains of G. zeae. The resulting Deltabeta(2)tub mutants from both MBC(MR) and MBC(HR) strains grew normally on MBC-free potato dextrose agar medium and were supersensitive to MBC. Complementation of the Deltabeta(2)tub mutants by transformation with a copy of the intact beta(2)tub locus from their parent strains exhibited less resistance than the original strains, and complementation of the Deltabeta(2)tub mutants by transformation with a copy of the intact beta(2)tub locus from sensitive strains restored MBC sensitivity. The results indicated that the mutations in the beta(2)tub gene conferred resistance of G. zeae to benzimidazole fungicides and this gene can be used as a genetic marker in G. zeae.

Fusarium graminearum (teleomorph, Gibberella zeae) causes head blight of cereals and contaminates grains with trichothecene mycotoxins that are harmful to humans and domesticated animals. Control of Fusarium head blight relies on carbendazim (MBC) in China, but resistance to MBC in F. graminearum is now widespread. Sixty-seven strains were evaluated for trichothecene production in shake culture or in the field. The strains included 60 wild-type strains (30 MBC-resistant and 30 MBC-sensitive), three MBC-resistant site-directed mutants at codon 167 in beta(2)-tubulin, three MBC-sensitive site-directed mutants at codon 240 in beta(2)-tubulin, and their MBC-sensitive wild-type progenitor strain ZF21. The incidence of infected spikelets and the amount of F. graminearum DNA in field grain (AFgDNA) also were evaluated for all strains. MBC resistance increased trichothecene production in shake culture or in the field. Although MBC resistance did not change the incidence of infected spikelets, it did increase AFgDNA. Tri5 gene expression increased in MBC-resistant strains grown in shake culture. We found a significant exponential relationship between trichothecene production and Tri5 gene expression in shake culture and a linear relationship between the incidence of infected spikelets or AFgDNA and trichothecene production in field grain.

Mutations at codons 50, 167, 198 and 200 of G. zeae β(2)tub could cause resistance to carbendazim, and these codons may form a binding pocket.

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Summary The biotrophic fungal pathogen Ustilaginoidea virens causes rice false smut, a newly emerging plant disease that has become epidemic worldwide in recent years. The U. virens genome encodes many putative effector proteins that, based on the study of other pathosystems, could play an essential role in fungal virulence. However, few studies have been reported on virulence functions of individual U. virens effectors. Here, we report our identification and characterization of the secreted cysteine‐rich protein SCRE1, which is an essential virulence effector in U. virens. When SCRE1 was heterologously expressed in Magnaporthe oryzae, the protein was secreted and translocated into plant cells during infection. SCRE1 suppresses the immunity‐associated hypersensitive response in the nonhost plant Nicotiana benthamiana. Induced expression of SCRE1 in rice also inhibits pattern‐triggered immunity and enhances disease susceptibility to rice bacterial and fungal pathogens. The immunosuppressive activity is localized to a small peptide region that contains an important ‘cysteine‐proline‐alanine‐arginine‐serine’ motif. Furthermore, the scre1 knockout mutant generated using the CRISPR/Cas9 system is attenuated in U. virens virulence to rice, which is greatly complemented by the full‐length SCRE1 gene. Collectively, this study indicates that the effector SCRE1 is able to inhibit host immunity and is required for full virulence of U. virens.

Ustilaginoidea virens is the causal agent of rice false smut disease resulting in quantitative and qualitative losses in rice. To gain insights into the pathogenic mechanisms of U. virens, we established a T-DNA insertion mutant library of U. virens through Agrobacterium tumefaciens-mediated transformation and selected an enhanced pathogenicity mutant (i.e., B3277). We analyzed the biological characteristics of the wild-type P1 and B3277. The growth rate and sporulation of B3277 were decreased compared with those of P1; the ferrous iron could be utilized by B3277, but inhibited the growth of P1. Southern blot analysis was performed to verify the copy number of the foreign gene inserted in the genomic DNA and only one copy of the T-DNA was found. The combined hiTAIL-PCR with RACE-PCR analysis showed the successful cloning of full length of the T-DNA flanking gene associated with pathogenicity, named Uvt3277. Gene expression was analyzed using real-time PCR. Results revealed that Uvt3277 was expressed at lower levels in B3277 than in P1. This gene was then subjected to bioinformatics analysis. The encoded protein of Uvt3277 exhibited high homology with low-affinity iron transporter proteins in some fungi. Transformation of the RNAi vector by constructing the hairpin RNA of the target gene was confirmed as successful. The pathogenicity of the transformant also increased. These results suggested that Uvt3277 may have an important function associated with the pathogenesis of U. virens. This study provides insights into the pathogenic mechanism of U. virens and a molecular target of disease control.

Lodging is an important limiting factor in wheat because it affects growth, yield and grain quality. Plant growth regulators (PGRs) are often used to restrain elongation of internodes, improve lodging traits, and protect yield potentials. An experiment was set up in the greenhouse at the Ottawa Research and Development Centre (ORDC) to study the effect of the selected PGRs (Manipulator, the active ingredient of which is chlormequat; and Palisade, the active ingredient of which is trinexapac-ethyl) on yield, stem height and morphological traits in six spring wheat cultivars (AC Carberry, AAC Scotia, Hoffman, Fuzion, FL62R1, and AW725). Both PGRs reduced plant height and caused a 6% to 48% reduction in the length of the second basal internode. The mixture of the two PGRs had a synergistic affect and made the stem shorter. The application of PGRs significantly reduced lodging, increased stem diameter, thickness, filling degree, and stem strength, and increased leaf relative chlorophyll content. However, application of PGRs significantly reduced grain yield, and the combination of the two PGRs (Manipulator and Palisade) had a synergistic effect and lowered the yield. In general, the effect of Palisade was more evident than that of Manipulator.

Ascospores of Villosiclava virens are a primary infection source of rice false smut. This phytopathogenic fungus exists in heterothallic form, and mating compatibility is regulated by mating-type locus 1 (MAT1). However, the MAT1 locus structure remains unknown. The MAT1-1 and MAT1-2 idiomorphs of V. virens were characterized and annotated on the basis of cDNA sequencing. A multiplex polymerase chain reaction (PCR) method was developed to identify the mating types of hyphae and sclerotia. The MAT1-1 locus of V. virens contains three mating-type genes: MAT1-1-1, MAT1-1-2 and MAT1-1-3, and a pseudogene similar to MAT1-2-1. The MAT1-2 locus harbors the MAT1-2-1 gene and a new mating-type gene MAT1-2-8. The mRNA of MAT1-1-1, MAT1-1-3 and MAT1-2-1, but not MAT1-1-2, was detectible by reverse transcription PCR in vegetative mycelia. However, the mRNA of MAT1-1-2 was detectible in the stroma, which is a sexual reproduction structure of V. virens. A multiplex PCR detection method was developed for the identification of the MAT1-1 and MAT1-2 idiomorphs. All 20 wild-type strains harbored either the MAT1-1 or MAT1-2 idiomorphs. Sclerotia that harbored both the MAT1-1 and MAT1-2 idiomorphs had potential to form fertile stromata, whereas those that harbored only the MAT1-1 idiomorph could not form mature stromata.