Development and application of loop-mediated isothermal amplification for detection of the F167Y mutation of carbendazim-resistant isolates in Fusarium graminearum.

Resistance of Fusarium graminearum to carbendazim is caused by point mutations in the ß2-tubulin gene. The point mutation at codon 167 (TTT → TAT, F167Y) occurs in more than 90% of field resistant isolates in China. To establish a suitable method for rapid detection of the F167Y mutation in F. graminearum, an efficient and simple method with high specificity was developed based on loop-mediated isothermal amplification (LAMP). A set of four primers was designed and optimized to specially distinguish the F167Y mutation genotype. The LAMP reaction was optimal at 63 °C for 60 min. When hydroxynaphthol blue dye (HNB) was added prior to amplification, samples with DNA of the F167Y mutation developed a characteristic sky blue color after the reaction but those without DNA or with different DNA did not. Results of HNB staining method were reconfirmed by gel electrophoresis. The developed LAMP had good specificity, stability and repeatability and was suitable for monitoring carbendazim-resistance populations of F. graminearum in agricultural production.

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.

Biological characteristics and resistance analysis of the novel fungicide SYP-1620 against Botrytis cinerea.

SYP-1620, a quinone-outside-inhibitor (QoI), is a novel broad-spectrum fungicide. In this study, 108 isolates of Botrytis cinerea from different geographical regions in Jiangsu Province of China were characterized for baseline sensitivity to SYP-1620. The curves of baseline sensitivity were unimodal with a mean EC50 value of 0.0130±0.0109 µg/mL for mycelial growth, 0.01147±0.0062 µg/mL for spore germination, respectively. The biological characterization of SYP-1620 against B. cinerea was determined in vitro. The results indicated that SYP-1620 has a strong inhibiting effect on spore germination, mycelial growth, and respiration. The protective and curative test of SYP-1620 suggested that protective effect was better than curative either on strawberry leaves or on cucumber leaves in vivo. In addition, the biological characterization of SYP-1620-resistant mutants of B. cinerea was investigated. SYP-1620 has no cross-resistance with other types of fungicide. Compared to the sensitive isolates, the resistant mutants had lower mycelial growth and virulence but not differ in mycelial dry weight. Sequencing indicated that SYP-1620 resistance was associated with a single point mutation (G143A) in the cytochrome b gene.

A two-component histidine kinase Shk1 controls stress response, sclerotial formation and fungicide resistance in Sclerotinia sclerotiorum.

Fungal histidine kinases (HKs) are involved in osmotic and oxidative stress responses, hyphal development, fungicide sensitivity and virulence. Members of HK class III are known to signal through the high-osmolarity glycerol mitogen-activated protein kinase (HOG MAPK). In this study, we characterized the Shk1 gene (SS1G_12694.3), which encodes a putative class III HK, from the plant pathogen Sclerotinia sclerotiorum. Disruption of Shk1 resulted in resistance to phenylpyrrole and dicarboximide fungicides and increased sensitivity to hyperosmotic stress and H2 O2 -induced oxidative stress. The Shk1 mutant showed a significant reduction in vegetative hyphal growth and was unable to produce sclerotia. Quantitative real-time polymerase chain reaction (qRT-PCR and glycerol determination assays showed that the expression of SsHOG1 (the last kinase of the Hog pathway) and glycerol accumulation were regulated by the Shk1 gene, but PAK (p21-activated kinase) was not. In addition, the Shk1 mutant showed no change in virulence. All the defects were restored by genetic complementation of the Shk1 deletion mutant with the wild-type Shk1 gene. These findings indicate that Shk1 is involved in vegetative differentiation, sclerotial formation, glycerol accumulation and adaption to hyperosmotic and oxidative stresses, and to fungicides, in S. sclerotiorum. Taken together, our results demonstrate, for the first time, the role of two-component HKs in Sclerotinia.

Functional analysis of the ß2 -tubulin gene of Fusarium graminearum and the ß-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 ß-tubulin. Carbendazim resistance in Fusarium graminearum, however, differs from that in B. cinerea and other fungi in that F. graminearum has two ß-tubulins (Fgtub1 and Fgtub2) rather than one, and the resistance is conferred by mutations in the ß2 -tubulin. In a previous study, the ß1 -tubulin of F. graminearum (Fgtub1) was replaced with the ß-tubulin of B. cinerea conferring carbendazim resistance (BctubE198A). The transformants were sensitive to carbendazim. RESULTS: BctubE198A was successfully transferred into the ß2 -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 ß-tubulin (Bctub) could complement the function of the two F. graminearum ß-tubulins even when both were deleted. Expression of the ß-tubulin conferring carbendazim resistance differs between pathogenic fungi.