Shinpei Banno

A Point Mutation in the Two-Component Histidine Kinase BcOS-1 Gene Confers Dicarboximide Resistance in Field Isolates of Botrytis cinerea

ABSTRACT Partial DNA fragments of Botrytis cinerea field isolates encoding the putative osmosensor histidine kinase gene (BcOS1) were cloned by polymerase chain reaction amplification and the predicted amino acid sequences were compared between dicarboximide-sensitive and resistant field isolates. The predicted BcOS1p is highly homologous to osmosensor histidine kinase OS1p from Neurospora crassa including the N-terminal six tandem repeats of approximately 90 amino acids. Four dicarboximide-resistant isolates of B. cinerea (Bc-19, Bc-45, Bc-682, and Bc-RKR) contained a single base pair mutation in their BcOS1 gene that resulted in an amino acid substitution in the predicted protein. In these resistant isolates, codon 86 of the second repeat, which encodes an isoleucine residue in sensitive strains, was converted to a codon for serine. The mutation of Botrytis field resistant isolates was located on the second unit of tandem amino acid repeats of BcOS1p, whereas the point mutations of the fifth repeat of OS1p confer resistance to both dicarboximides and phenylpyrroles and also osmotic sensitivity in Neurospora crassa. These results suggest that an amino acid substitution within the second repeat of BcOS1p is responsible for phenotypes of field resistant isolates (resistant to dicarboximides but sensitive to phenylpyrroles, and normal osmotic sensitivity) in B. cinerea.

Roles of putative His-to-Asp signaling modules HPT-1 and RRG-2, on viability and sensitivity to osmotic and oxidative stresses in Neurospora crassa

Neurospora crassa has a putative histidine phosphotransfer protein (HPT-1) that transfers signals from 11 histidine kinases to two putative response regulators (RRG-1 and RRG-2) in its histidine-to-aspartate phosphorelay system. The hpt-1 gene was successfully disrupted in the os-2 (MAP kinase gene) mutant, but not in the wild-type strain in this study. Crossing the resultant hpt-1; os-2 mutants with the wild-type or os-1 (histidine kinase gene) mutant strains produced no progeny with hpt-1 or os-1;hpt-1 mutation, strongly suggesting that hpt-1 is essential for growth unless downstream OS-2 is inactivated. hpt-1 mutation partially recovered the osmotic sensitivity of os-2 mutants, implying the involvement of yeast Skn7-like RRG-2 in osmoregulation. However, the rrg-2 disruption did not change the osmotic sensitivity of the wild-type strain and the os-2 mutant, suggesting that rrg-2 did not participate in the osmoregulation. Both rrg-2 and os-2 single mutation slightly increased sensitivity to t-butyl hydroperoxide, and rrg-2 and hpt-1 mutations increased the os-2 mutant’s sensitivity. Although OS-1 is considered as a positive regulator of OS-2 MAP kinase, our results suggested that HPT-1 negatively regulated downstream MAP kinase cascade, and that OS-2 and RRG-2 probably participate independently in the oxidative stress response in N. crassa.

Genotyping of Benzimidazole-Resistant and Dicarboximide-Resistant Mutations in Botrytis cinerea Using Real-Time Polymerase Chain Reaction Assays

Botrytis cinerea, an economically important gray mold pathogen, frequently exhibits multiple fungicide resistance. A fluorescence resonance energy transfer-based real-time polymerase chain reaction assay has been developed to detect benzimidazole- and dicarboximide-resistant mutations. Three benzimidazole-resistant mutations-(198)Glu to Ala (E198A), F200Y, and E198K-in beta-tubulin BenA were detected using a single set of fluorescence-labeled sensor and anchor probes by melting curve analysis. Similarly, three dicarboximide-resistant mutations-I365S, V368F plus Q369H, and Q369P-in the histidine kinase BcOS1 were successfully distinguished. Unassigned melting profiles in BenA genotyping assay resulted in the identification of a new benzimidazole-resistant BenA E198V mutation. This mutation conferred resistance to carbendazim as do E198A and E198K mutations. The isolates with BenA E198V mutation showed a negative cross-resistance to diethofencarb, but to a lesser extent than the E198A mutants. A survey of 210 B. cinerea field isolates revealed that most of benzimidazole-resistant isolates possessed the E198V or E198A mutation in the BenA gene, and the I365S mutation in the BcOS1 gene was also frequently observed in Japanese isolates. However, benzimidazole-resistant isolates with BenA F200Y or E198K mutations, which confer the diethofencarb-insensitive phenotype, were rare. Our BenA and BcOS1 genotyping is a rapid and reliable method that is suitable for monitoring the fungicide-resistant field population.

Survey of mutations of a histidine kinase gene BcOS1 in dicarboximide-resistant field isolates of Botrytis cinerea

Previously, we cloned a putative osmosensing histidine kinase gene (BcOS1) and revealed that a single amino acid substitution, isoleucine to serine at codon 365, conferred dicarboximide resistance in field isolates of Botrytis cinerea. This point mutation (type I) occurred within the restriction enzyme TaqI site of the wild-type BcOS1 gene. Thus, a procedure was developed for detecting the type I mutation of the BcOS1 gene using a polymerase chain reaction (PCR) in combination with restriction fragment-length polymorphism (RFLP). Diagnosis by PCR-RFLP was conducted on the 105 isolates isolated from 26 fields in Japan. All dicarboximide-sensitive isolates (49 isolates) had the wild-type BcOS1 gene, and the 43 isolates with the type I mutation were resistant to dicarboximides without exception. These data indicate that dicarboximide-resistant isolates with type I mutation are widespread throughout Japan. However, other types of dicarboximide resistance were detected among isolates from Osaka; among the 24 resistant isolates from Osaka, 12 had the BcOS1 gene without the type I mutation. BcOS1 gene sequencing of these resistant isolates classified them into two groups, type II and type III. The type II isolates have three amino acid substitutions within BcOS1p (368Val to Phe, 369Gln to His, and 447Thr to Ser). The type III isolates have two amino acid substitutions within BcOS1p (369Gln to Pro and 373Asn to Ser). These amino acid changes are located on the amino acid repeat domain in BcOS1p. The three types of resistant isolates were all moderately resistant to dicarboximides without significant osmotic sensitivity, and their pathogenicity on cucumber leaves was also very similar to that of the wild-type isolate.

Quantitative nested real-time PCR detection of Verticillium longisporum and V. dahliae in the soil of cabbage fields

Verticillium longisporum and V. dahliae, causal agents of Verticillium wilt, are spreading through the cabbage fields of Gunma Prefecture. Using the V. longisporum-specific intron within the 18S rDNA and differences between ITS 5.8S rDNA sequences in Japanese isolates of V. longisporum and V. dahliae, we developed three quantitative nested real-time (QNRT) PCR assays. The QNRT-PCR quantification of V. longisporum or V. dahliae in cabbage field soil was consistent with the severity of Verticillium wilt disease in those fields. In field trials of resistant cultivar YR Ranpo grown for three seasons in soil infested with the pathogen, disease severity and pathogen density in the soil were significantly reduced in a field moderately contaminated by V. dahliae, but only slightly reduced in a highly contaminated field. These results suggest that continuous cultivation of a resistant cultivar is an effective way to reduce the pathogen population. QNRT-PCR assays provide a powerful analytical tool to evaluate the soil population dynamics of V. longisporum and V. dahliae for disease management.

Histidine Kinase Inhibitors

Dicarboximides and phenylpyrroles have been mainly used to control diseases caused by fungal strains that belong to the genera Botrytis, Sclerotinia, Monilinia, and Alternaria. Both types of fungicides overactivate Hog-like mitogen-activated protein kinases in the osmotic signal transduction pathway and result in cell death. Cross-resistance among dicarboximides, phenylpyrroles, and aromatic hydrocarbons has been observed in most laboratory Botrytis cinerea-resistant mutants, which are generally hyperosmotic sensitive. However, such resistant strains have rarely been isolated from the fields. All dicarboximide-resistant field isolates contained point mutations in a putative osmosensor histidine kinase BcOS1/Daf1, did not show cross-resistance to phenylpyrroles, and were insensitive to osmotic stress. In contrast, Alternaria field-resistant strains carried various mutations, including null mutations, in their osmosensor histidine kinase genes. The introduction of several new fungicides against B. cinerea, such as anilinopyrimidine fungicides, fenhexamid, QoIs, and succinate dehydrogenase inhibitors, reduced the use of dicarboximides, thereby reducing the populations of dicarboximide-resistant strains. However, several types of multidrug resistance strains, in which efflux pumps are activated, have emerged. Gain-of-function mutations of the transcription factor Mrr1, which leads to an overexpression of the ATP-binding cassette transporter AtrB, confers reduced sensitivities to some fungicides, including fludioxonil and cyprodinil. In addition, strains that overexpress the major facilitator superfamily transporter mfsM2 by promoter rearrangements lead to reduced sensitivities to iprodione, fenhexamid, and cyprodinil. Therefore, in addition to target modifications of BcOS1, multidrug resistance caused by the overexpression of drug transporters is another resistance mechanism in B. cinerea against dicarboximides and phenylpyrroles.

Characterization and distribution of two subtypes of Verticillium longisporum isolated from cabbage fields in Japan

Verticillium longisporum is an allodiploid hybrid fungus that consists of at least three groups, A1/D1, A1/D2, and A1/D3. PCR-based analysis of 18S rDNA, 5.8S rDNA-ITS region, mtSSU-rDNA, cytochrome b gene, and mating type gene, as well as RAPD analysis, revealed that Verticillium wilt of cabbage in Gunma Prefecture in Japan is caused by two groups of V. longisporum—A1/D1 and A1/D3—and by V. dahliae. A1/D1 and A1/D3 lineage strains were equally distributed in cabbage fields, whereas V. dahliae strains were most frequently isolated. The proportion of the three types of Verticillium had not changed in 1998 and 2008. Although both V. longisporum strains produced longer conidia than the V. dahliae strains, the A1/D3-type strains produced larger conidia than A1/D1-type strains. In addition, the A1/D3-type strains formed microsclerotia that were distinguishable from those of A1/D1-type of V. longisporum and V. dahliae. The pathogenicity of the A1/D3-type strains on cabbage was similar to that of the A1/D1-type strains. These results will contribute to understanding of genotypic diversity, distribution, and pathogenicity of Verticillium species pathogenic on cabbage.