Changjun Chen

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.

Occurrence and Characterization of Dimethachlon Insensitivity in Sclerotinia sclerotiorum in Jiangsu Province of China

Sclerotinia stem rot, caused by Sclerotinia sclerotiorum, is the main fungal disease of oilseed rape (Brassica napus) in China. Numerous fungicide applications are required for control. Dimethachlon, one of the dicarboximide fungicides, has been the major fungicide for disease control after benzimidazole resistance became widespread. Fungal populations were collected throughout Jiangsu Province between 2006 and 2007 in order to determine their sensitivity to dimethachlon. A total of 1,066 single-sclerotium isolates of S. sclerotiorum were collected, and most of the isolates were considered sensitive to dimethachlon. Five isolates collected in Yancheng and Changzhou showed normal growth at 5 μg/ml dimethachlon with the resistance factor ≈10 (resistance factor was estimated as ratios between the EC50 values of resistant isolates and the average EC50 values of sensitive ones) compared to the sensitive isolates (EC50 is the concentration of fungicide causing 50% reduction in growth). Through in vitro selection for resistance to the fungicide, 25 dimethachlon-resistant mutants were derived from 10 wild-type isolates of S. sclerotiorum. The resistance factors for the isolates ranged from 198 to 484, and the isolates were considered highly resistant to dimethachlon. Therefore, at least two different mechanisms of resistance seem to be involved: one that may provide a moderate resistance (insensitivity) and a second that may give a high resistance level under laboratory conditions. There was positive cross-resistance between dimethachlon and other dicarboximide fungicides, such as iprodione and procymidone, in these S. sclerotiorum isolates. The field dimethachlon-insensitive and the laboratory-induced dimethachlon-resistant isolates appeared to have mycelial growth, sclerotial production, and pathogenicity comparable to their wild-type parental isolates. Also, results of osmotic tests showed that there were no significant difference in mycelial radial growth between the field dimethachlon-sensitive and field dimethachlon-insensitive isolates on potato dextrose agar plates amended with 2, 4, 6, or 8% (wt/vol) NaCl, but the laboratory-induced dimethachlon-resistant isolates grew significantly more slowly than their wild-type sensitive parents under all concentrations of NaCl. Because these studies yielded a high frequency of laboratory resistance in S. sclerotiorum, together with the occurrence of field insensitivity, appropriate precautions against resistance development in natural populations should be taken.

Localisation of the benzimidazole fungicide binding site of Gibberella zeae β2‐tubulin studied by site‐directed mutagenesis

BACKGROUND The efficacy of benzimidazole fungicides is often limited by resistance, and this is the case with the use of carbendazim for controlling Fusarium head blight caused by Gibberella zeae (Schwein.) Petch (anamorph Fusarium graminearum). Recent studies have shown that carbendazim resistance in field strains of G. zeae is associated with mutations in the β(2)-tubulin gene. The aims of the present study were to validate this mechanism and research the binding sites of carbendazim on β(2)-tubulin. RESULTS This work used site-directed mutagenesis followed by gene replacement to change the β(2)-tubulin gene of a carbendazim-sensitive field strain of G. zeae at residues 50, 167, 198 or 200. The transformants were confirmed and tested for their sensitivity to carbendazim. All the mutants were resistant to carbendazim, but the level of resistance differed depending on the mutation. Biological characteristics did not differ between the field strain and the site-directed mutants. A three-dimensional model of β(2)-tubulin was constructed, and the possible carbendazim binding site was analysed. CONCLUSION 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.

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.

Multiple Resistance of Botrytis cinerea from Vegetable Crops to Carbendazim, Diethofencarb, Procymidone, and Pyrimethanil in China

One hundred and eight isolates of Botrytis cinerea from greenhouse cucumber and tomato in two locations in Jiangsu Province (Nanjing and Huaiyin) and one location in Shandong Province were tested for their sensitivities to the four fungicides commonly used in China. Isolates with resistance to all four fungicides-carbendazim, diethofencarb, procymidone, and pyrimethanil (CarRDieRPrcRPyrR)-were found in all three regions in this study. High frequencies (52 and 53%) of resistance to all four fungicides were observed among the 62 isolates collected in Nanjing and the 36 isolates collected in Huaiyin in Jiangsu Province. The 10 isolates from Shandong Province were all resistant to the four fungicides. Pathogenicity and sporulation in vivo, and mycelial growth, sporulation, spore germination, and osmotic sensitivity to NaCl in vitro, were similar for the group of quadruple-resistant and wild-type isolates (P > 0.05). In the present study, the complete two-component histidine kinase gene (Bos1) was sequenced for 10 procymidone-resistant and 3 procymidone-sensitive B. cinerea isolates. Isolates representing four different procymidone-resistant phenotypes (CarRDieSPrcRPyrS, CarRDieRPrcRPyrS, CarRDieRPrcRPyrR, and CarRDieRPrcRPyrR) all had nucleic acid point mutations resulting in amino acid changes at position 369 (change from glutamine to proline) as well as at amino acid position 373 (asparagine to serine) in the Bos1 gene.

Integrated biological and chemical control of rice sheath blight by Bacillus subtilis NJ-18 and jinggangmycin.

BACKGROUND Sheath blight caused by Rhizoctonia solani Kühn is a major disease of rice that greatly reduces yield and grain quality and jinggangmycin is the most widely used fungicide to control this disease in China. Bacillus subtilis NJ-18 has broad antimicrobial activity to many phytopathogenic bacteria and fungi; it is especially effective against Rhizoctonia solani. Laboratory, greenhouse and field tests were conducted to determine the effect of combining the biological control agent Bacillus subtilis NJ-18 with the fungicide jinggangmycin for control of rice sheath blight. RESULTS Growth of NJ-18 in vitro was not affected by jinggangmycin. In a greenhouse experiment, disease control was greater with a mixture of NJ-18 and jinggangmycin than with either alone; a mixture of NJ-18 at 10(8)  cfu mL(-1) and jinggangmycin at 50 or 100 mg L(-1) reduced lesion length by 35% and 20%, respectively, and the combinations showed a synergistic action. In three field trials, disease control was significantly greater with a mixture of NJ-18 at 10(8)  cfu mL(-1) and jinggangmycin at 75 or 150 g a.i. ha(-1) than with either component alone. CONCLUSION The results of the study indicate that, when Bacillus subtilis NJ-18 strain was combined with jinggangmycin, there was an increased suppression of rice sheath blight, and thus could provide an alternative disease control option.

Functional analysis of the β2‐tubulin gene of Fusarium graminearum and the β‐tubulin gene of Botrytis cinerea by homologous replacement

BACKGROUND Resistance to carbendazim and other benzimidazole fungicides in Botrytis cinerea and most other fungi is usually conferred by one or several allelic mutations in the β-tubulin. Carbendazim resistance in Fusarium graminearum, however, differs from that in B. cinerea and other fungi in that F. graminearum has two β-tubulins (Fgtub1 and Fgtub2) rather than one, and the resistance is conferred by mutations in the β2 -tubulin. In a previous study, the β1 -tubulin of F. graminearum (Fgtub1) was replaced with the β-tubulin of B. cinerea conferring carbendazim resistance (BctubE198A). The transformants were sensitive to carbendazim. RESULTS BctubE198A was successfully transferred into the β2 -tubulin locus of F. graminearum (Fgtub2) via homologous replacement. The mutants were still sensitive to carbendazim. Furthermore, Fgtub2 of the mutant 20C1 (Fgtub1 had been replaced with BctubE198A) and Fgtub1 of the mutant 20D10 (Fgtub2 had been replaced with BctubE198A) were deleted. Surprisingly, the mutants were also sensitive to carbendazim. Meanwhile, the biological characteristics of all the mutants were determined. CONCLUSION The B. cinerea β-tubulin (Bctub) could complement the function of the two F. graminearum β-tubulins even when both were deleted. Expression of the β-tubulin conferring carbendazim resistance differs between pathogenic fungi.

Transfer of the β-tubulin gene of Botrytis cinerea with resistance to carbendazim into Fusarium graminearum

BACKGROUND Resistance to carbendazim and other benzimidazole fungicides in Botrytis cinerea (Pers. ex Fr.) and most other fungi is usually conferred by mutation(s) in a single chromosomal beta-tubulin gene, often with several allelic mutations. In Fusarium graminearum Schwade, however, carbendazim resistance is not associated with a mutation in the corresponding beta-tubulin gene. RESULTS The beta-tubulin gene conferring carbendazim resistance in B. cinerea was cloned and connected with two homologous arms of the beta-tubulin gene of F. graminearum by using a double-joint polymerase chain reaction (PCR). This fragment was transferred into F. graminearum via homologous double crossover at the site where the beta-tubulin gene of F. graminearum is normally located (the beta-tubulin gene of F. graminearum had been deleted). The transformants were confirmed and tested for their sensitivity to carbendazim. CONCLUSION The beta-tubulin gene conferring carbendazim resistance in B. cinerea could not express this resistance in F. graminearum, as transformants were still very sensitive to carbendazim.

Activity of Azoxystrobin and SHAM to Four Phytopathogens

Abstract The study was conducted to make clear the activity of azoxystrobin to 4 plant pathogens and the synergistic effects of salicylhydroxamic acid (SHAM), which acted on the alternative oxidase. It was also conducted to be aware of the mechanism of azoxystrobin in inhibition on mycelial respiration and the influence of SHAM. The activity test of azoxystrobin and SHAM was carried out with a mycelial linear growth test and spore germination test. Other related biological properties were also observed. Inhibition of azoxystrobin and SHAM on 4 pathogens was determined by using SP-II oxygraph system. Azoxystrobin inhibited mycelial growth in Colletotrichum capsici, Botrytis cinerea, Rhizoctonia solani, and Magnaporthe grisea, respectively; it also inhibited conidia germination, and conidia production in C. capsici, B. cinerea M. grisea, and sclerotia formation in R. solani. Moreover, it created stayed pigment biosynthesis in C. capsici and M. grisea somehow. Salicylhydroxamic acid enhanced inhibition by azoxystrobin. An oxygen consuming test of the mycelia showed that azoxystrobin inhibited all the 4 fungis respiration in the early stages. With the concentration rising up, the effectiveness increased. However, as time went on, the respiration of the mycelia treated with fungicides recovered and SHAM could not inhibit the oxygen consuming. This reaction between the mycelia and the fungicides appeared not to initiate alternative respiration but rather the other mechanism created a lack of efficacy.

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.