Prochloraz Resistance Research Articles

Characterization, Molecular Mechanism of Prochloraz-Resistance in Fusarium fujikuroi and Development of Loop-Mediated Isothermal Amplification Rapid Detection Technique Based on the S312T Genotype of Resistances.

Rice bakanae disease (RBD) is a typical seed-borne fungal disease caused by Fusarium fujikuroi. Prochloraz is a sterol demethylation inhibitor, which is among the most important classes of active ingredients for the management of RBD. In 2022, the total resistance frequency of F. fujikuroi to prochloraz in Zhejiang Province was 62.67%. The fitness of the prochloraz-resistant population was lower than that of the susceptible population, but its pathogenicity was slightly stronger. The S312T and F511S double mutations of Ffcyp51b were detected in the resistant isolates. Loop-mediated isothermal amplification (LAMP) technology based on S312T was established to rapidly determine prochloraz resistance in F. fujikuroi. LAMP primer mismatch design was performed based on the cyp51b gene, and 100-300 bp sequences containing a mutation at codon 312 were amplified. In a 25 µL reaction tube, 1 pg/µL DNA of F. fujikuroi could be detected. The detection limit for the frequency of prochloraz resistance was 0.498% using this method. We performed LAMP detection on rice seedlings inoculated with prochloraz-sensitive and -resistant isolates and treated them with prochloraz. Prochloraz demonstrated good control in rice seedlings. A chromogenic reaction was observed in seedlings treated with prochloraz-resistant isolates, and the results were verified using electrophoresis. It has been demonstrated that LAMP technology based on the S312T genotype can quickly and specifically detect prochloraz-resistant isolates in rice seedlings.

Genotypic and phenotypic characterization of resistance to fenhexamid, carboxin, and, prochloraz, in Botrytis cinerea isolates collected from cut roses in Colombia.

Gray mold, caused by Botrytis sp., is a significant disease in Colombian rose crops and its control depends primarily on the intensive use of chemically synthesized fungicides. Despite the importance of this pathogen, there is limited information in Colombian floriculture about molecular taxonomy of species, fungicide resistance of populations and their genetic mechanism of resistance. In this study, we analyze 12 isolates of this fungus collected from rose-producing crops in the Department of Cundinamarca and conducted phylogenetic analysis using HSP60, G3PDH, and RPB2 gene sequences. Additionally, we realize phenotypic and genotypic characterization of resistance to the fungicides fenhexamid, carboxin, and prochloraz, evaluating the in vitro EC50 and presence of mutations of target genes of each isolate. All isolates were characterized as Botrytis cinerea in the phylogenetic analysis and presents different levels of resistance to each fungicide. These levels are related to mutations in target genes, with predominancy of L195F and L400F in the ERG27 gene to fenhexamid resistance, H272R/Y in the SDHB gene for carboxin resistance, and Y136F in the CYP51 gene for prochloraz resistance. Finally, these mutations were not related to morphological changes. Collectively, this knowledge, presented for the first time to the Colombian floriculture, contribute to a better understanding of the genetic diversity and population of B. cinerea from rose-producing crops in the department of Cundinamarca, and serve as a valuable tool for making informed decisions regarding disease management, future research, and improving crop management and sustainability in the Colombian floriculture industry.

Transcriptome Analysis of mfs2-Defective Penicillium digitatum Mutant to Reveal Importance of Pdmfs2 in Developing Fungal Prochloraz Resistance.

Demethylation inhibitors (DMIs), including prochloraz, are popular fungicides to control citrus postharvest pathogens such as Penicillium digitatum (green mold). However, many P. digitatum strains have developed prochloraz resistance, which decreases drug efficacy. Specific major facilitator superfamily (MFS) transporter gene mfs2, encoding drug-efflux pump protein MFS2, has been identified in P. digitatum strain F6 (PdF6) to confer fungal strain prochloraz resistance. However, except for the drug-efflux pump function of MFS2, other mechanisms relating to the Pdmfs2 are not fully clear. The present study reported a transcriptome investigation on the mfs2-defective P. digitatum strain. Comparing to the wild-type strain, the mfs2-defective strain showed 717 differentially expressed genes (DEGs) without prochloraz induction, and 1221 DEGs with prochloraz induction. The obtained DEGs included multiple isoforms of MFS transporter-encoding genes, ATP-binding cassette (ABC) transporter-encoding genes, and multidrug and toxic compound extrusion (MATE) family protein-encoding genes. Many of these putative drug-efflux pump protein-encoding genes had significantly lower transcript abundances in the mfs2-defective P. digitatum strain at prochloraz induction, as compared to the wild-type strain, including twenty-two MFS transporter-encoding genes (MFS1 to MFS22), two ABC transporter-encoding genes (ABC1 and ABC2), and three MATE protein-encoding genes (MATE1 to MATE3). The prochloraz induction on special drug-efflux pump protein genes in the wild-type strain was not observed in the mfs2-defective strain, including MFS21, MFS22, ABC2, MATE1, MATE2, and MATE3. On the other hand, the up-regulation of other drug-efflux pump protein genes in the mfs2-defective strain cannot recover the fungal prochloraz resistance, including MFS23, MFS26, MFS27, MFS31, MFS33, and ABC3 to ABC8. The functional enrichment of DEGs based on Kyoto Encyclopedia of Genes and Genomes (KEGG), Clusters of Orthologous Groups (COG), and euKaryotic Orthologous Groups (KOG) database resources suggested some essential contributors to the mfs2-relating prochloraz resistance, including ribosome biosynthesis-related genes, oxidative phosphorylation genes, steroid biosynthesis-related genes, fatty acid and lipid metabolism-related genes, and carbon- and nitrogen-metabolism-related genes. The results indicated that the MFS2 transporter might be involved in the regulation of multiple drug-efflux pump protein gene expressions and multiple metabolism-related gene expressions, thus playing an important role in developing P. digitatum prochloraz resistance.

Biochemical and Molecular Characterization of Prochloraz Resistance in Lasiodiplodia theobromae Field Isolates.

Stem-end rot (SER), caused by Lasiodiplodia theobromae, is one of the most critical diseases of mango in China. The demethylation inhibitor fungicide prochloraz has been widely used in China to control mango diseases. Isolates (n = 139) of L. theobromae were collected in 2019 from six mango-producing regions in Hainan Province, China. The fungicide sensitivity of L. theobromae isolates to prochloraz revealed that the EC50 (50% effective concentration) values ranged from 0.0006 to 16.4131 µg/ml. In total, 21 of the 139 isolates were categorized as resistant to prochloraz. The resistant isolates sprayed with prochloraz could not be effectively controlled in detached fruit. The mycelial growth, conidia germination, and ability to grow at temperatures ranging from 12 to 35°C of resistant isolates decreased, suggesting fitness penalties. The experiment showed that, after treatment with prochloraz at 10 µg/ml, the content of ergosterol in the mycelia of the sensitive isolate decreased by 80.23%, whereas the resistant strain decreased by only 57.52%. The damage to membranes in the sensitive isolates was more serious than for resistant isolates. The target gene CYP51 and the ATP-binding cassette (ABC) subfamily ABCG gene were cloned but no mutation was found. When treated with prochloraz, the expression of CYP51 and ABCG in resistant isolates was significantly higher than in the sensitive isolates. Thus, induced expression of its target gene combined with the induction of expression drug efflux transporters appeared to mediate the prochloraz resistance of L. theobromae.

Targeted deletion of three CYP51s in Fusarium fujikuroi and their different roles in determining sensitivity to 14α-demethylase inhibitor fungicides.

Fusarium fujikuroi is the pathogenic agent of rice bakanae disease and has developed serious resistance to prochloraz, a 14α-demethylase inhibitor (DMI). Prochloraz resistance in F. fujikuroi is caused by cooperation between FfCyp51B with Cyp51A and shows cross-resistance only to prothioconazole but not to tebuconazole, difenoconazole, propiconazole, metconazole, hexaconazole, and triadimefon. This study aimed to analyze the functions of the three Cyp51s in F. fujikuroi, especially their role in determining sensitivity to DMIs. The respective deletion of FfCyp51A, Cyp51B, and Cyp51C had no obvious effect on morphology, conidium germination, or pathogenicity. The involvement of growth, growth and ergosterol biosynthesis, and conidium production and ergosterol biosynthesis was observed for FfCyp51A, Cyp51B, and Cyp51C, respectively. Compared with the sensitive isolate of F. fujikuroi, the effect on sensitivity to the tested DMIs was divided into four groups: (i) both of Cyp51A and Cyp51B positively regulate the sensitivity to prochloraz and prothioconazole; (ii) Cyp51B positively regulate the sensitivity to tebuconazole and metconazole, but negatively regulate the sensitivity to difenoconazole; (iii) Cyp51A and Cyp51B play opposite roles in the sensitivity to triadimefon. Therefore, deletion of Cyp51A in F. fujikuroi confers a higher sensitivity to triadimefon, while deletion of Cyp51B results in a triadimefon-resistant mutant isolate; (iv) deletion of Cyp51B yielded a mutant isolate that was more resistant to propiconazole and hexaconazole. Sophisticated interactions exist within the three Cyp51 genes to DMIs fungicides sensitivity in F. fujikuroi, and Cyp51B probably plays a more critical role than Cyp51A and Cyp51C. © 2022 Society of Chemical Industry.

Synergistic effect of amino acid substitutions in CYP51B for prochloraz resistance in Fusarium fujikuroi

Prochloraz has been used to control Fusarium fujikuroi, the causative pathogen of rice bakanae disease. Linkage analysis of FfCYP51 genes in the progenies obtained from crossing prochloraz moderately resistant and sensitive strains suggested that the FfCYP51B gene is involved in prochloraz resistance. Sequence comparison revealed that the prochloraz-resistant strain had an F511S or S312T/F511S substitution in FfCYP51B compared with the sensitive strains. The contribution of the S312T and F511S substitutions in FfCYP51B to prochloraz resistance was investigated by creating S/F-, T/F-, or T/S- types at 312/511 codons from the S/S-type, which is a natural moderately resistant strain, using a gene-editing technique. T/S exhibited the highest prochloraz resistance, followed by S/S-, T/F-, and S/F-types. These results indicated that the S312T and F511S substitutions in FfCYP51B had a synergistic effect on prochloraz resistance in F. fujikuroi.

Monitoring and characterization of prochloraz resistance in Fusarium fujikuroi in China

Rice bakanae disease, caused by Fusarium fujikuroi, is a destructive seed-borne disease throughout the world. Prochloraz, a DMI (C-14α-demethylase inhibitor) fungicide, has been registered in China for >20 years. Prochloraz resistance in F. fujikuroi was severe in China with resistance frequencies of 34.56%, 45.33%, and 48.45% from 2019 to 2021. The fitness of prochloraz-resistant population was lower than that of sensitive population, with an average CFI of 2.86 × 106 and 4.56 × 106, respectively. No cross-resistance was detected between prochloraz and tebuconazole or hexaconazole, and the prochloraz-resistant isolates were still sensitive to fludioxonil, phenamacril, and pydiflumetofen. S312T mutation in Ffcyp51b or overexpression of Ffcyp51a and Ffcyp51b was detected in the highly resistant isolates. AS-PCR primers were designed to detect the prochloraz-resistant isolates with S312T mutation in the field. Resistant isolates carrying S312T mutation were the dominant group in prochloraz-resistant population with frequencies of 43.26%, 23.59%, and 71.20% from 2019 to 2021, which indicated that more attention should be paid to this genotype when monitoring and managing the prochloraz resistance in F. fujikuroi.

Characterization of Prochloraz Resistance in Fusarium fujikuroi from Heilongjiang Province in China.

Prochloraz is widely used to control rice bakanae disease caused by Fusarium fujikuroi. The current study was aimed at monitoring the development of F. fujikuroi resistance to prochloraz in the Heilongjiang Province and analyzing the fitness of F. fujikuroi strains with different resistance levels. The results indicated that most of the 89 F. fujikuroi strains collected from the Heilongjiang Province were resistant to prochloraz, with resistance frequency reaching 92.1%. To assess the field resistance risk of prochloraz, 21 F. fujikuroi strains with different resistance levels were selected to investigate their biological characteristics and assess their fitness. Mycelial growth, sporulation, and germination rates were significantly different among the tested strains. However, when grouped into two subpopulations, no significant difference was tested between prochloraz-resistant and prochloraz-sensitive strains. Pathogenicity assays revealed that the disease severity index of prochloraz-resistant strains was higher than that of prochloraz-sensitive strains. Cross-resistance assays showed no cross-resistance between prochloraz and five other fungicides, namely phenamacril, ipconazole, tebuconazole, carbendazim, and fluopyram. Ffcyp51A gene overexpression was observed in the prochloraz-resistant F. fujikuroi strains after exposure to prochloraz. Collectively, these results indicated that F. fujikuroi resistance against prochloraz was severe. Furthermore, prochloraz-resistant strains were highly fit and could potentially become a dominant population in rice fields, consequently resulting in yield loss.

Monitoring and Characterization of Prochloraz Resistance in Fusarium Fujikuroi in China

Monitoring and Characterization of Prochloraz Resistance in Fusarium Fujikuroi in China

The FgCYP51B Y123H Mutation Confers Reduced Sensitivity to Prochloraz and Is Important for Conidiation and Ascospore Development in Fusarium graminearum.

Fusarium graminearum is one of the most important causal agents of Fusarium head blight disease and is controlled mainly by chemicals such as demethylation inhibitor (DMI) fungicides. FgCYP51B is one of the DMI targets in F. graminearum, and Tyrosine123 (Y123) is an important amino acid in F. graminearum CYP51B, located in one of predicted substrate binding pockets based on the binding mode between DMIs and CYP51B. Previous studies suggest that resistance to DMI fungicides is attributed primarily to point mutations in the CYP51 gene and that the Y123H mutation in F. verticillioides CYP51 confers prochloraz resistance in the laboratory. To investigate the function of FgCYP51B Y123 residue in the growth and development, pathogenicity, and DMI resistance, we generated and analyzed the FgCYP51B Y123H mutant. Results revealed that the Y123H mutation led to reduced conidial sporulation and affected ascospore development; moreover, the mutation conferred reduced sensitivity to prochloraz. Quantitative PCR and molecular docking were performed to investigate the resistance mechanism. Results indicated that Y123H mutation changed the target gene expression and decreased the binding affinity of FgCYP51 to prochloraz. These results will attract more attention to the potential DMI-resistant mutation of F. graminearum and increase our understanding of the DMI resistance mechanism.

Reconstruction of a Context-Specific Model Based on Genome-Scale Metabolic Simulation for Identification of Prochloraz Resistance Mechanisms in Penicillium digitatum.

Penicillium digitatum is the most destructive postharvest pathogen of citrus fruits, causing substantial economic losses. Prochloraz-resistant strains have emerged due to overuse of imidazole fungicides in agriculture. To study the prochloraz resistance mechanisms at the system level, a genome-scale metabolic model (GEM, iPD1512) of P. digitatum was reconstructed and constrained based on context-specific transcriptome data of the prochloraz-resistant strain, PdF6, from our previous work, a newly sequenced, context-specific transcriptome result of the major facilitator superfamily transporter-encoding gene mfs2 knockout mutant PdF6Δmfs2, and experimentally derived growth rate data. Through the model, iPD1512, the processes of prochloraz resistance in P. digitatum were well simulated. In detail, the growth rates of both wild-type and mutant P. digitatum under different prochloraz concentrations were simulated using constraint-based reconstruction and analysis. The growth rates of the mutant strains (sterol regulatory element-binding protein-encoding gene sreA knockout mutant PdF6ΔsreA and PdF6Δmfs2) were calculated and confirmed to be consistent with the simulation results. Furthermore, correlations between genes and prochloraz resistance were predicted and showed a great difference when compared with correlation results based on p-values from the hypothesis testing used by comparative transcriptomics. To sum up, in contrast to traditional transcriptome analysis, the GEM provides a systemic and dynamic drug resistance mechanism, which might help to detect some key upstream regulatory genes, but with small expression changes, and might provide more efficient targets to control prochloraz-resistant P. digitatum.

Characterization of two novel mycoviruses from Penicillium digitatum and the related fungicide resistance analysis

Pathogenic fungi including Penicillium digitatum and Penicillium italicum are the main destructive pathogens in the citrus industry, causing great losses during postharvest process. To our knowledge, only one mycovirus from P. digitatum has been reported, and the prevalence of such mycoviruses against citrus postharvest pathogenic fungi and their genotyping were still under investigation. In the present study, we showed that 39 of 152 Penicillium isolates from main citrus-growing areas in China were infected with various mycoviruses belonging to polymycoviruses, Narna-like viruses, and families Totiviridae, Partitivirdae and Chrysoviridae. The next generation sequencing (NGS) towards virus genome library and the following molecular analysis revealed two novel mycoviruses Penicillium digitatum polymycovirus 1 (PdPmV1) and Penicillium digitatum Narna-like virus 1 (PdNLV1), coexisting in P. digitatum strain HS-RH2. The fungicide-resistant P. digitatum strains HS-F6 and HS-E9 coinfected by PdPmV1 and PdNLV1 exhibited obvious reduction in triazole drug prochloraz resistance by mycelial growth analysis on both PDA plates and citrus fruit epidermis with given prochloraz concentration. This report at the first time characterized two novel mycoviruses from P. digitatum and revealed the mycovirus-induced reduction of fungicide resistance.

A novel major facilitator superfamily transporter in Penicillium digitatum (PdMFS2) is required for prochloraz resistance, conidiation and full virulence.

To clone a novel major facilitator superfamily (MFS, a large protein family with diverse physiological functions in all kingdoms) transporter gene, Pdmfs2, and characterize its function in Penicillium digitatum. A novel MFS transporter gene, Pdmfs2, was isolated from P. digitatum. The full-length DNA of Pdmfs2 had a 1590bp ORF encoding a full-size MFS transporter with 529 amino acids. In a prochloraz-resistant strain (PdHS-F6), Pdmfs2 transcript level was up-regulated compared with the prochloraz-sensitive strain (PdHS-E3) and could be induced by 7μg prochloraz/ml. The deletion of Pdmfs2 (ΔPdmfs2) in PdHS-F6 led to increased susceptibility to prochloraz and lower EC50 value (the concentration of prochloraz producing 50% growth inhibition) compared with the PdHS-F6 or complementation strain (COPdmfs2). The ΔPdmfs2 strain was defective in conidia yield and virulence towards citrus fruits, while the complementation of Pdmfs2 could restore the phenotypic features to a large extent. Pdmfs2 is the second MFS transporter gene in P. digitatum and is required for prochloraz resistance, conidiation and full virulence.

Whole transcriptome analysis of Penicillium digitatum strains treatmented with prochloraz reveals their drug-resistant mechanisms.

BackgroundPenicillium digitatum is one of the most destructive postharvest pathogen of citrus fruits, causing fruit decay and economic loss. The emergence of fungicide-resistant strains made the control of P. digitatum more difficult. While the genome of P. digitatum is available, there has been few reports about its resistant mechanism from the transcriptome perspective and there has been no large-scale functional annotation of the genome using expressed genes derived from transcriptomes.MethodsTotal RNA of P. digitatum strain HS-F6 (prochloraz-resistant strain) and HS-E3 (prochloraz-susceptible strain) before and after prochloraz-treatment were extracted and sequenced on an Illumina Hiseq 2000 platform. The transcriptome data of four samples were compared and analyzed using differential expression analysis, novel transcripts prediction and alternative splicing analysis, SNP analysis and quantitative real-time PCR. ResultsWe present a large scale analysis about the transcriptome data of P. digitatum. The whole RNA was extracted from a prochloraz-resistant strain (HS-F6) and a prochloraz-susceptible strain (HS-E3) before and after prochloraz-treatment and sequenced by Illumina technology. A total of more than 100 million reads were generated and de novo assembled into 9760 transcripts that contained annotated genes after quality control and sequence assembling. 6625 single nucleotide variations (SNVs) were identified from the sequences aligned against the reference genome. Gene expression profiling analysis was performed upon prochloraz treatment in HS-F6 and HS-E3, and differential expression analysis was used to identify genes related to prochloraz-response and drug-resistance: there are 224 differentially expressed genes in HS-E3 and 1100 differentially expressed genes in HS-F6 after prochloraz-treatment. Moreover, gene expression profile in prochloraz-resistant strain HS-F6 is quite different from that in HS-E3 before prochloraz-treatment, 1520 differential expression genes were identified between the two strains. Gene ontology (GO) term enrichment and KEGG enrichment were then performed to classify the differential expression genes. Among these genes, there are a lot of transporter encoding genes including 14 MFS (Major Facilitator Superfamily) transporters, 8 ABC (ATP-binding cassette transporter) and 3 MATE (multidrug and toxic compound extrusion family) transporters. Meanwhile, the roles of typical MFS, ABC and MATE proteins in prochloraz resistance were investigated using real-time quantitative PCR.ConclusionsThe sequencing-based transcriptome data of P. digitatum demonstrate differences between prochloraz-resistant and prochloraz-susceptible strains with prochloraz-treatment. The differences existed in expressed transcripts, splice isoforms and GO categories, which would contribute to our knowledge on the molecular mechanisms involved in drug resistance of P. digitatum.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2043-x) contains supplementary material, which is available to authorized users.

A novel sterol regulatory element-binding protein gene (sreA) identified in penicillium digitatum is required for prochloraz resistance, full virulence and erg11 (cyp51) regulation.

Penicillium digitatum is the most destructive postharvest pathogen of citrus fruits, causing fruit decay and economic loss. Additionally, control of the disease is further complicated by the emergence of drug-resistant strains due to the extensive use of triazole antifungal drugs. In this work, an orthologus gene encoding a putative sterol regulatory element-binding protein (SREBP) was identified in the genome of P. digitatum and named sreA. The putative SreA protein contains a conserved domain of unknown function (DUF2014) at its carboxyl terminus and a helix-loop-helix (HLH) leucine zipper DNA binding domain at its amino terminus, domains that are functionally associated with SREBP transcription factors. The deletion of sreA (ΔsreA) in a prochloraz-resistant strain (PdHS-F6) by Agrobacterium tumefaciens-mediated transformation led to increased susceptibility to prochloraz and a significantly lower EC50 value compared with the HS-F6 wild-type or complementation strain (COsreA). A virulence assay showed that the ΔsreA strain was defective in virulence towards citrus fruits, while the complementation of sreA could restore the virulence to a large extent. Further analysis by quantitative real-time PCR demonstrated that prochloraz-induced expression of cyp51A and cyp51B in PdHS-F6 was completely abolished in the ΔsreA strain. These results demonstrate that sreA is a critical transcription factor gene required for prochloraz resistance and full virulence in P. digitatum and is involved in the regulation of cyp51 expression.

Novel mutations in CYP51B from Penicillium digitatum involved in prochloraz resistance.

Green mold caused by Penicillium digitatum is one of the most serious postharvest diseases of citrus fruit, and it is ubiquitous in all citrus growing regions in the world. Sterol 14α-demethylase (CYP51) is one of the key enzymes of sterol biosynthesis in the biological kingdom and a prime target of antifungal drugs. Mutations in CYP51s have been found to be correlated with resistance to azole fungicides in many fungal species. To investigate the mechanism of resistance to prochloraz (PRC) in P. digitatum, the PRC sensitivity was determined in vitro in this study to assess the sensitivity of 78 P. digitatum isolates collected in Hubei province. The results showed that 25 isolates were prochloraz-resistant (PRC-R), including six high-resistant (HR) strains, twelve medium-resistant (MR) and seven low-resistant (LR) strains. A sequence analysis showed no consistent point mutations of PdCYP51A in the PRC-R strains, but four substitutions of CYP51B were found, Q309H in LR strains, Y136H and Q309H in HR strains, and G459S and F506I in MR strains, which corresponded to the four sensitivity levels. Based on the sequence alignment analysis and homology modeling followed by the molecular docking of the PdCYP51B protein, the potential correlation between the mutations and PRC resistance is proposed.

The Y123H substitution perturbs FvCYP51B function and confers prochloraz resistance in laboratory mutants of Fusarium verticillioides

Fusarium verticillioides reduces corn yield and contaminates infected kernels with the toxin fumonisin, which is harmful to humans and animals. Previous research has demonstrated that F. verticillioides can be controlled by the azole fungicide prochloraz. Currently, prochloraz is used as a foliar spray to control maize disease in China, which will increase the risk of resistance. Although F. verticillioides resistance to prochloraz has not been reported in the field, possible resistance risk and mechanisms resulting in prochloraz resistance were explored in the laboratory. Four prochloraz‐resistant strains of F. verticillioides were generated by successive selection on fungicide‐amended media. The mycelial growth rates of the mutants were inversely related to the level of resistance. All four mutants were cross‐resistant to the triazole fungicides triadimefon, tebuconazole and difenoconazole, but not to the multisite fungicide chlorothalonil or to the MAP/histidine‐kinase inhibitor fungicide fludioxonil. Based on the Y123H mutation in FvCYP51B, the four resistant mutants were subdivided into two genotypes: PCZ‐R1 mutants with wildtype FvCYP51B and PCZ‐R2 mutants with substitution Y123H in FvCYP51B. Wildtype FvCYP51B complemented the function of native ScCYP51 in Saccharomyces cerevisiae YUG37::erg11, whereas Y123H‐mutated FvCYP51B did not. For the PCZ‐R1 mutants, induced expression of FvCYP51A increased resistance to prochloraz. For the PCZ‐R2 mutants, disruption of FvCYP51B function by the Y123H substitution caused constitutive up‐regulation of FvCYP51A expression and thus resistance to prochloraz.

Involvement of an efflux transporter in prochloraz resistance of Fusarium fujikuroi CF245 causing rice bakanae disease

Rice bakanae disease pathogens resistant to conazole fungicide prochloraz have been reported in Korea. Understanding fungal response associated with the resistant is required for successful control of the disease. Investigation of prochlorazresistant responses of the rice bakanae disease pathogen, Fusarium fujikuroi, showed significant growth inhibition of the resistant strain after sodium azide treatment suggested involvement of the ABC transporter in fungal prochloraz-resistant responses. Prochloraz degradation was accompanied by fungal growth, dependently on sodium azide treatment. Partial sequence analysis of the ABC transporter gene confirmed that the resistant strain possesses an efflux transporter with high sequence similarity to genes of conazole fungicide-resistant pathogens. These results suggest that the prochloraz-resistant responses of F. fujikuroi CF245 involve an efflux transporter.

Cloning of the CYP51 gene from the eyespot pathogen Tapesia yallundae indicates that resistance to the DMI fungicide prochloraz is not related to sequence changes in the gene encoding the target site enzyme

Resistance to sterol 14α-demethylase inhibitor (DMI) fungicides has been correlated with mutations in the CYP51 gene encoding the target enzyme eburicol 14α-demethylase. CYP51 was isolated from the eyespot pathogen Tapesia yallundae revealing a predicted 526-amino acid product exhibiting homology to other fungal CYP51s. CYP51 was sequenced from four field isolates sensitive or resistant to the DMI fungicide prochloraz and partially sequenced from two further isolates and eight progeny from a cross between prochloraz-sensitive and -resistant parents. Two alleles of the gene were detected termed CYP51-1 and CYP51-2. No correlation was found between sequence change and fungicide sensitivity. Therefore prochloraz resistance involved a mechanism other than mutation in the target site gene.

Cloning of the CYP51 gene from the eyespot pathogen Tapesia yallundae indicates that resistance to the DMI fungicide prochloraz is not related to sequence changes in the gene encoding the target site enzyme.

Resistance to sterol 14 alpha-demethylase inhibitor (DMI) fungicides has been correlated with mutations in the CYP51 gene encoding the target enzyme eburicol 14 alpha-demethylase. CYP51 was isolated from the eyespot pathogen Tapesia yallundae revealing a predicted 526-amino acid product exhibiting homology to other fungal CYP51s. CYP51 was sequenced from four field isolates sensitive or resistant to the DMI fungicide prochloraz and partially sequenced from two further isolates and eight progeny from a cross between prochloraz-sensitive and -resistant parents. Two alleles of the gene were detected termed CYP51-1 and CYP51-2. No correlation was found between sequence change and fungicide sensitivity. Therefore prochloraz resistance involved a mechanism other than mutation in the target site gene.