M. G. Zhou

Mutations in a β-tubulin confer resistance of Gibberella zeae to benzimidazole fungicides.

ABSTRACT 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.

Quantification of Fusarium graminearum in Harvested Grain by Real-Time Polymerase Chain Reaction to Assess Efficacies of Fungicides on Fusarium Head Blight, Deoxynivalenol Contamination, and Yield of Winter Wheat

We used a real time polymerase chain reaction-based assay and visual disease assessment to evaluate the efficacies of Js399-19, tebuconazole, a mixture of tebuconazole and thiram, azoxystrobin, carbendazim, and thiram on the development of Fusarium head blight (FHB) and deoxynivalenol (DON) contamination and on the yield of winter wheat (cv. Nannong no. 9918) after artificial inoculation under field conditions with Fusarium graminearum. The incidence of infected spikelets (IIS), amount of F. graminearum DNA (Tri5 DNA), total DON (containing DON, 3-acetyl-deoxynivalenol, and 15-acetyl-deoxynivalenol) concentration, and 1,000-grain weight (TGW) were quantified in 2006 and 2007. A strong positive correlation was found between IIS or Log10Tri5 DNA and total DON concentration in the harvested grain. The Js399-19, tebuconazole, and the mixture of tebuconazole and thiram significantly reduced IIS of FHB, amount of Tri5 DNA, and total DON within the grain and increased TGW. Although azoxystrobin, carbendazim, and thiram can increase TGW, they had no effect on the occurrence of F. graminearum compared with those of the untreated controls. Surprisingly, azoxystrobin and carbendazim significantly increased the total DON content in the harvested grain because they might have stimulated the amount of total DON production per Tri5 DNA. The fungicides Js399-19, tebuconazole, and the mixture of tebuconazole and thiram were the most effective in controlling FHB and reducing DON contamination of the wheat.

Sexual recombination of carbendazim resistance in Fusarium graminearum under field conditions

BACKGROUND Carbendazim has been the major fungicide for control of Fusarium head blight (FHB) caused by Fusarium graminearum Schwade in China. However, the effectiveness of carbendazim has been threatened by the emergence of resistant pathogen populations in the field. RESULTS Five isolates, representing three phenotypes of different carbendazim sensitivity levels (S, MR and HR), were randomly selected to study the inheritance of carbendazim resistance by three genetic crosses under field conditions. Each parent in all crosses was marked with a different class of nitrate non-utilizing (nit) mutation. The presence of sexual recombinants indicated that outcrossing had occurred in the crosses. Over 100 putative self-crossing or outcrossing perithecia for each cross were randomly sampled on the surface of the haulms of dead rice for each pair of parents. Results showed that 5.7-20.9% outcrossing frequency occurred in the three crosses and confirmed sexual recombination under field conditions. There were no significant differences in mycelial linear growth and pathogenicity between the selected recombinants and their parents, but they differed in sporulation ability and capacity to produce perithecia. Nevertheless, most of the sexual recombinants possessed fitness levels comparable with those of their parents. CONCLUSION Outcrossing between carbendazim-sensitive and -resistant isolates did occur under field conditions, and sexual recombination must play a role in the development of carbendazim resistance in the field.

Vegetative Compatibility of Fusarium graminearum Isolates and Genetic Study on Their Carbendazim-Resistance Recombination in China

ABSTRACT Monoconidial isolates of 33 carbendazim-sensitive isolates and 31 carbendazim-resistant isolates of Fusarium graminearum were selected from three regions of China for vegetative compatibility group (VCG) analysis. A total of 213 and 224 nit mutants were recovered from the 33 sensitive and the 31 resistant isolates, respectively. Of all the nit mutants, the frequency of the different phenotypes was 44.6, 46.5, 5.7, and 3.2% for nit1, nit3, nitM, and nitA, respectively. VCG analysis identified 30 different VCGs among the 33 sensitive- and the 31 carbendazim-resistant isolates, with VCG diversity 0.91 and 0.97, respectively. Both, a carbendazim-sensitive and a -resistant isolate from the same field belonged to the same VCG. In all then, a total of 59 VCGs were identified among the 64 isolates with an overall VCG diversity 0.92. Direct hyphal fusion was observed in six pairs of vegetatively compatible complements, which is evidence of heterokaryon formation. It was hypothesized that carbendazim resistance could not be transferred by hyphal fusion or there is a small chance to be transferred between two compatible isolates. Three stable sexual recombinants of F. graminearum were randomly chosen from each of the three genetic crosses to study their biological properties. There were no significant differences in mycelial linear growth and pathogenicity between recombinants and their parents, but they differ in sporulation ability and capacity to produce perithecia. We concluded that sexual recombination presumably played a role in the development of carbendazim resistance under field conditions.

A single amino acid substitution in the SdhB protein of succinate dehydrogenase determines resistance to amicarthiazol in Xanthomonas oryzae pv. oryzae.

BACKGROUND Xanthomonas oryzae pv. Oryzae Ishiyama, a causal agent of rice bacterial leaf blight, was found to be sensitive in vitro to the systemic fungicide amicarthiazol (2-amino-4-methylthiazole -5-carboxanilide), which is a potent inhibitor of succinate dehydrogenase (SDH, EC 1.3.99.1). This paper aimed to determine the molecular resistance mechanism of X. oryzae pv. oryzae to amicarthiazol. RESULTS UV-induced resistant mutants of X. oryzae pv. oryzae to amicarthiazol were isolated. The activity of SDH in wild-type X. oryzae pv. oryzae was strongly inhibited by amicarthiazol, while that in resistant mutants was insensitive, although their SDH activity was decreased compared with the wild-type sensitive strain without amicarthiazol. A mutation of Histidine(229) (CAC) to Tyrosine(229) (TAC) was identified in sdhB, which encoded the iron-sulfur protein subunit of SDH. The sdhB from the mutant was ligated into a cosmid, pUFR034, to generate pUFR034RAX, which conferred resistance to amicarthiazol when transformed into the wild-type sensitive strain. CONCLUSION A mutation of His(229) (CAC) to Tyr(229) (TAC) in SdhB was responsible for determining amicarthiazol resistance. .