Peer review of the pesticide risk assessment of the active substance fludioxonil.

The conclusions of the European Food Safety Authority (EFSA) following the peer review of the initial risk assessments carried out by the competent authorities of the rapporteur Member State France and co-rapporteur Member State Spain for the pesticide active substance fludioxonil are reported. The context of the peer review was that required by Commission Implementing Regulation (EU) No 844/2012, as amended by Commission Implementing Regulation (EU) No 2018/1659. The conclusions were reached on the basis of the evaluation of the representative use of fludioxonil as a fungicide on wheat, oats, grapes, pome fruit and strawberry. The reliable end points, appropriate for use in regulatory risk assessment are presented. Missing information identified as being required by the regulatory framework is listed. Concerns are reported where identified.

A Novel Point Mutation M460I in Histidine Kinase FgOs1 Confers High Resistance to Fludioxonil in Fusarium graminearum.

Fusarium head blight (FHB), caused by Fusarium graminearum, severely impacts global wheat production, reducing both the yield and quality. In China, fludioxonil, a phenylpyrrole fungicide, is used for managing FHB. This study assessed fludioxonil activity against 120 F. graminearum strains collected from Hubei, Zhejiang, and Jiangsu in 2024, revealing an average EC50 value of 0.0273 ± 0.0062 µg/mL. We obtained two resistant mutants through chemical taming and discovered a novel point mutation of FgOs1-M460I. Site-directed mutagenesis confirmed that the FgOs1-M460I mutation greatly reduced fludioxonil sensitivity, with an EC50 value greater than 100 µg/mL. These mutants also displayed reduced sexual and asexual reproduction and lower virulence and accumulated less glycerol under fludioxonil and osmotic stress compared to sensitive strain. The resistant mutants showed no cross-resistance with carbendazim, tebuconazole, phenamacril, pyraclostrobin, or pydiflumetofen. Thus, we conclude that the FgOs1-M460I substitution regulates fludioxonil resistance and plays a role in asexual reproduction, sexual reproduction, and pathogenicity.

GABA-Decorated Nanocarrier for Smart Delivery of Fludioxonil for Targeted Control of Banana Wilt Disease.

Developing a targeted nanopesticide to control the vascular disease of banana in agriculture is crucial to improve pesticide utilization. In this study, according to the degree of functionalization, three γ-aminobutyric acid (GABA)-decorated nanocarriers (PSI-GABA8, PSI-GABA18, and PSI-GABA28) were constructed for smart delivery of nonsystemic fungicide in banana phloem tissues. Fludioxonil (Flu) was loaded in nanocarriers to form Flu@PSI-GABA nanoparticles with a core/shell structure for control of banana wilt disease. Results demonstrated that the delivery dosage of Flu was up to 1.6 mg/L in castor phloem sap using PSI-GABA28 nanocarriers. In vitro results showed that the EC50 of Flu@PSI-GABA28 was 0.0116 mg/L, and the inhibitory activity was about 8.8 times higher than that of technical-grade (TC) Flu. Flu@PSI-GABA28 could be transported for long distances and accumulated to the rhizome of banana by foliar application, and the control effectiveness was about 20 times that of the conventional Flu (50% WP) for the banana wilt. This study provides a distinctive guidance for effective control of vascular diseases in precision agriculture application.

Control of Botrytis cinerea from Chilean grapevines by pydiflumetofen: baseline and carboxamide-mutant sensitivity.

The gray mold (Botrytis cinerea; Botrytis) is the main disease affecting grapevines production in Chile. Succinate Dehydrogenase Inhibitors (SDHI) belonging to the carboxamides fungicide family are a key tool for the control of Botrytis in grapevines from Chilean Central Valley. This study aimed to determine the sensitivity of Chilean Botrytis population to the new generation carboxamide pydiflumetofen. Conidial germination (CG) and germ-tube elongation (GTE) sensitivity assays were conducted on 200 single-spore isolates collected during the 2016-2017 season. The mean effective concentration that inhibited 50% (EC50) of CG in the Botrytis population was 0.0545 µg/mL, with mean values of 0.066 µg/mL and 0.042 µg/mL, for table and wine grapes, respectively. The mean EC50 value of GTE was 0.000245 µg/mL, 0.0003 µg/mL, and 0.0019 µg/mL for the total, table grape, and wine grape populations, respectively. The comparison between pydiflumetofen and fludioxonil, a highly-efficient fungicide carrying a different mode of action, showed the 87.5% and 97.5% of Botrytis control with an EC50 threshold of 0.1 µg/mL, in table grape, and wine grape populations, respectively. No cross-resistance between pydiflumetofen and fludioxonil was detected. For nine isolates with reduced pydiflumetofen sensitivity, we evaluated SdhB mutations using a qPCR-HRM diagnostic system. Two isolates carried the sdhBP225/H272R genotype and two the sdhBP225/H272Y. Additional analysis of SdhB mutant isolates determined that pydiflumetofen controls wild-type as well as sdhBP225/H272R and sdhBP225H/H272 mutants. Pydiflumetofen does not control CG in the sdhBP225/H272Y mutant but is effective in the GTE control. Pydiflumetofen significantly controls Botrytis independently of the SdhB genotype in wounded berry assays. This condition resembles the berry cracking due to heavy rainfall right before harvest, as seen in recent years in the Chilean Central Valley. The findings demonstrate that pydiflumetofen effectively controls the grapevine Botrytis population, suggest a moderate risk of pydiflumetofen resistance, and highlight the significance of incorporating genetic data into the design of control programs.

A Fungicide, Fludioxonil, Formed the Polyploid Giant Cancer Cells and Induced Metastasis and Stemness in MDA-MB-231 Triple-Negative Breast Cancer Cells.

Fludioxonil, an antifungal agent used as a pesticide, leaves a measurable residue in fruits and vegetables. It has been identified to cause endocrine disruption, interrupt normal development, and cause various diseases such as cancers. In this study, fludioxonil was examined for its effects on the development and metastasis of breast cancer cells. On fludioxonil exposure (10-5 M) for 72 h, mutant p53 (mutp53) MDA-MB-231 triple-negative breast cancer (TNBC) cells significantly inhibited cell viability and developed into polyploid giant cancer cells (PGCCs), with an increase in the number of nuclei and expansion in the cell body size. Fludioxonil exposure disrupted the normal cell cycle phase ratio, resulting in a new peak. In addition, PGCCs showed greater motility than the control and were resistant to anticancer drugs, i.e., doxorubicin, cisplatin, and 5-fluorouracil. Cyclin E1, nuclear factor kappa B (NF-κB), and p53 expressions were remarkably increased, and the expression of cell cycle-, epithelial-mesenchymal-transition (EMT)-, and cancer stemness-related proteins were increased in the PGCCs. The daughter cells obtained from PGCCs had the single nucleus but maintained their enlarged cell size and showed greater cell migration ability and resistance to the anticancer agents. Consequently, fludioxonil accumulated Cyclin E1 and promoted the inflammatory cytokine-enriched microenvironment through the up-regulation of TNF and NF-κB which led to the transformation to PGCCs via abnormal cell cycles such as mitotic delay and mitotic slippage in mutp53 TNBC MDA-MB-231 cells. PGCCs and their daughter cells exhibited significant migration ability, chemo-resistance, and cancer stemness. These results strongly suggest that fludioxonil, as an inducer of potential genotoxicity, may induce the formation of PGCCs, leading to the formation of metastatic and stem cell-like breast cancer cells.

Fungicide-Loaded Liposomes for the Treatment of Fungal Diseases in Agriculture: An Assessment of Botrytis cinerea.

In this work, liposomes loaded with the fungicide, Fludioxonil (FLUD), for the containment of fungal diseases in agriculture were developed. Three types of vesicles with different compositions were compared: (I) plain vesicles, composed of soy phosphatidylcholine and cholesterol; (II) PEG-coated vesicles, with an additional polyethylene glycol coating; and (III) cationic vesicles, containing didodecyldimethylammonium bromide. Nanometric-sized vesicles were obtained both by the micelle-to-vesicle transition method and by the extrusion technique, and encapsulation efficiency, drug loading content, and Zeta potential were determined for all the samples. The extruded and PEGylated liposomes were the most stable over time and together with the cationic ones showed a significant prolonged FLUD release capacity. The liposomes' biological activity was evaluated on conidial germination, germ tube elongation and colony radial growth of the ascomycete Botrytis cinerea, a phytopathogenic fungus affecting worldwide many important agricultural crops in the field as well as in the postharvest phase. The extruded and PEGylated liposomes showed greater effectiveness in inhibiting germ tube elongation and colony radial growth of the fungal pathogen, even at 0.01 µg·mL-1, the lowest concentration assessed.

A Conservative Mutant Version of the Mrr1 Transcription Factor Correlates with Reduced Sensitivity to Fludioxonil in Botrytis cinerea.

Fludioxonil is a highly effective phenylpyrrole fungicide for controlling Botrytis cinerea. Although the field efficacy of fludioxonil remains high, Botrytis cinerea isolates with reduced sensitivity have been reported globally. The molecular target of fludioxonil still remains unknown; however, a mechanism of reduced sensitivity to fludioxonil underlies the overexpression of the ATP binding cassette (ABC) transporter AtrB in a dependent pathway of the Mrr1 transcription factor. Fludioxonil is a key player in controlling B. cinerea infection in table grapes in Chile. However, some isolates with a reduced sensitivity to fludioxonil were detected. This study observed endogenous atrB overexpression in Chilean isolates with reduced sensitivity to fludioxonil (n = 22) compared to the sensitive isolates (n = 10). All isolates increased the expression of atrB in a growth medium supplemented with fludioxonil (0.05 µg/mL). However, sensitive isolates showed lower atrB expression than those with reduced fludioxonil sensitivity. Remarkably, a mutant version of the transcription factor Mrr1 carrying 21 amino acid modifications was identified in all isolates with reduced sensitivity to fludioxonil. These changes alter the protein's transcription factor domain and the C-terminal portion of the protein but not the Zn (2)-C6 fungal-type DNA-binding domain. These results suggest a direct relationship between the conserved and divergent mutant version of mrr1 and sensitivity to fludioxonil. This study provides a new target for developing molecular diagnostic strategies to monitor B. cinerea's sensitivity to fludioxonil in the field.

Molecular mechanism of reduced biological fitness of fludioxonil-resistant strains of Botrytis cinerea based on transcriptome analysis.

BACKGROUND: Fludioxonil is a fungicide used to control gray mold. However, the frequency of resistance in the field is low, and highly resistant strains are rarely isolated. The biological fitness of the resistant strain is lower than that of the wild strain. Therefore, the molecular mechanism underlying the decrease in the fitness of the fludioxonil-resistant strain of Botrytis cinerea was explored to provide a theoretical basis for resistance monitoring and management. RESULTS: Transcriptome analysis was performed on five different-point mutant resistant strains of fludioxonil, focusing on mining and screening candidate genes that lead to reduced fitness of the resistant strains and the functional verification of these genes. The differentially expressed genes (DEGs) of the five point-mutation resistant strains intersected with 1869 DEGs. Enrichment analysis showed that three downregulated genes (Bcin05g07030, Bcgad1, and Bcin03g05840) were enriched in multiple metabolic pathways and were downregulated in both domesticated strains. Bcin05g07030 and Bcin03g05840 were involved in mycelial growth and development, pathogenicity, and conidial yield, and negatively regulated oxidative stress and cell wall synthesis. Bcgad1 was involved in mycelial growth and development, conidial yield, oxidative stress, and cell wall synthesis. Furthermore, Bcin05g07030 was involved in osmotic stress and spore germination, whereas Bcin03g05840 and Bcgad1 negatively regulated osmotic stress and cell wall integrity. CONCLUSION: These results enable us to further understand the molecular mechanism underlying the decrease in the biological fitness of B. cinerea fludioxonil-resistant strains. © 2024 Society of Chemical Industry.

Resistance mechanism of Phomopsis longicolla to fludioxonil is associated with modifications in PlOS1, PlOS4 and PlOS5.

Phomopsis longicolla, a causal agent of soybean root rot, stem blight, seed decay, pod and stem canker, which seriously affects the yield and quality of soybean production worldwide. The phenylpyrrole fungicide fludioxonil exhibits a broad spectrum and high activity against phytopathogenic fungi. In this study, the baseline sensitivity of 100 P. longicolla isolates collected from the main soybean production areas of China to fludioxonil were determined. The result showed that the EC50 values of all the P. longicolla isolates ranged from 0.013 to 0.035 µg/ml. Furthermore, 12 fludioxonil-resistance (FluR) mutants of P. longicolla were generated from 6 fludioxonil-sensitive (FluS) isolates. and the resistance factors (RF) of 12 FluR mutants were >3500. Sequence alignment showed that multiple mutation types were found in PlOS1, PlOS4 or/and PlOS5 of FluR mutants. All the FluR mutants exhibited fitness penalty in mycelial growth, conidiation, virulence and osmo-adaptation. Under fludioxonil or NaCl treatment condition, the glycerol accumulation was significantly increased in FluS isolates, but was slightly increased in FluR mutants, and the phosphorylation level of most FluR mutants was significantly decreased when compared to the FluS isolates. Additionally, positive cross-resistance was observed between fludioxonil and procymidone but not fludioxonil and pydiflumetofen, pyraclostrobin or fluazinam. This is first reported that the baseline sensitivity of P. longicolla to fludioxonil, as well as the biological and molecular characterizations of P. longicolla FluR mutants to fludioxonil. These results can provide scientific directions for controlling soybean diseases caused by P. longicolla using fludioxonil.

Binding Mode and Molecular Mechanism of the Two-Component Histidine Kinase Bos1 of Botrytis cinerea to Fludioxonil and Iprodione.

Gray mold caused by Botrytis cinerea is among the 10 most serious fungal diseases worldwide. Fludioxonil is widely used to prevent and control gray mold due to its low toxicity and high efficiency; however, resistance caused by long-term use has become increasingly prominent. Therefore, exploring the resistance mechanism of fungicides provides a theoretical basis for delaying the occurrence of diseases and controlling gray mold. In this study, fludioxonil-resistant strains were obtained through indoor drug domestication, and the mutation sites were determined by sequencing. Strains obtained by site-directed mutagenesis were subjected to biological analysis, and the binding modes of fludioxonil and iprodione to Botrytis cinerea Bos1 BcBos1 were predicted by molecular docking. The results showed that F127S, I365S/N, F127S + I365N, and I376M mutations on the Bos1 protein led to a decrease in the binding energy between the drug and BcBos1. The A1259T mutation did not lead to a decrease in the binding energy, which was not the cause of drug resistance. The biological fitness of the fludioxonil- and point mutation-resistant strains decreased, and their growth rate, sporulation rate, and pathogenicity decreased significantly. The glycerol content of the sensitive strains was significantly lower than that of the resistant strains and increased significantly after treatment with 0.1 µg/ml of fludioxonil, whereas that of the resistant strains decreased. The osmotic sensitivity of the resistant strains was significantly lower than that of the sensitive strains. Positive cross-resistance was observed between fludioxonil and iprodione. These results will help to understand the resistance mechanism of fludioxonil in Botrytis cinerea more deeply.

Characterization of the fludioxonil and phenamacril dual resistant mutants of Fusarium graminearum.

Fusarium graminearum is an important fungal pathogen causing Fusarium head blight (FHB) in wheat and other cereal crops worldwide. Due to lack of resistant wheat cultivars, FHB control mainly relies on application of chemical fungicides. Both fludioxonil (a phenylpyrrole compound) and phenamacril (a cyanoacrylate fungicide) have been registered for controlling FHB in China, however, fludioxonil-resistant isolates of F. graminearum have been detected in field. To evaluate the potential risk of dual resistance of F. graminearum to both compounds, fludioxonil and phenamacril dual resistant (DR) mutants of F. graminearum were obtained via fungicide domestication in laboratory. Result showed that resistance of the DR mutants to both fludioxonil and phenamacril were genetically stable after sub-cultured for ten generations or stored at 4 °C for 30 days on fungicide-free PDA. Cross-resistance assay showed that the DR mutants remain sensitive to other groups of fungicides, including carbendazim, tebuconazole, pydiflumetofen, and fluazinam. In addition, the DR mutants exhibited defects in mycelia growth, conidiation, mycotoxin deoxynivalenol (DON) production, and virulence Moreover, the DR mutants displayed increased sensitivity to osmotic stress. Sequencing results showed that amino acid point mutations S217L/T in the myosin I protein is responsible for phenamacril resistance in the DR mutants. Our results indicate that mutations leading to fludioxonil and phenamacril dual resistance could result in fitness cost for F. graminearum. Our results also suggest that the potential risk of F. graminearum developing resistance to both fludioxonil and phenamacril in field could be rather low, which provides scientific guidance in controlling FHB with fludioxonil and phenamacril.

Fludioxonil induces cardiotoxicity via mitochondrial dysfunction and oxidative stress in two cardiomyocyte models.

Fludioxonil (Flu) is a phenylpyrrole fungicide and is currently used in over 900 agricultural products globally. Flu possesses endocrine-disrupting chemical-like properties and has been shown to mediate various physiological and pathological changes, such as apoptosis and differentiation, in diverse cell lines. However, the effects of Flu on cardiomyocytes have not been studied so far. The present study investigated the effects of Flu on mitochondria in AC16 human cardiomyocytes and H9c2 rat cardiomyoblasts. Flu decreased cell viability in a water-soluble tetrazolium assay and mediated morphological changes suggestive of apoptosis in AC16 and H9c2 cells. We confirmed that annexin V positive cells were increased by Flu through annexin V/propidium iodide staining. This suggests that the decrease in cell viability due to Flu may be associated with increased apoptotic changes. Flu consistently increased the expression of pro-apoptotic markers such as Bcl-2-associated X protein (Bax) and cleaved-caspase 3. Further, Flu reduced the oxygen consumption rate (OCR) in AC16 and H9c2 cells, which is associated with decreased mitochondrial membrane potential (MMP) as observed through JC-1 staining. In addition, Flu augmented the production of mitochondrial reactive oxygen species, which can trigger oxidative stress in cardiomyocytes. Taken together, these results indicate that Flu induces mitochondrial dysregulation in cardiomyocytes via the downregulation of the OCR and MMP and upregulation of the oxidative stress, consequently resulting in the apoptosis of cardiomyocytes. This study provides evidence of the risk of Flu toxicity on cardiomyocytes leading to the development of cardiovascular diseases and suggests that the use of Flu in agriculture should be done with caution and awareness of the probable health consequences of exposure to Flu.

Chilean Botrytis cinerea Isolates with Reduced Sensitivity to Fludioxonil Exhibit Low to Null Fitness Penalties.

The main phytosanitary problem for table grape production in Chile is gray mold caused by the fungus Botrytis cinerea. To manage this issue, the primary method utilized is chemical control. Fludioxonil, a phenylpyrrole, is highly effective in controlling B. cinerea and other plant pathogens. Consistently, there have been no field reports of reduced efficacy of fludioxonil; however, subpopulations with reduced sensitivity to fludioxonil are on the rise globally, as per increasing reports. Our study involved a large-scale evaluation of B. cinerea's sensitivity to fludioxonil in the Central Valley of Chile's primary table grape production area during the growing seasons from 2015 to 2018. Out of 2,207 isolates, only 1.04% of the isolates (n = 23) exceeded the sensitivity threshold value of 1 µg/ml. Remarkably, 95.7% are concentrated in a geographic region (Valparaíso Region). Isolates with reduced sensitivity to fludioxonil showed growth comparable with sensitive isolates and even more robust growth under nutritional deficit, temperature, or osmotic stress, suggesting greater environmental adaptation. When table grape detached berries were stored at 0°C, isolates less sensitive to fludioxonil caused larger lesions than sensitive isolates (2.82 mm compared with 1.48 mm). However, the lesions generated by both types of isolates were equivalent at room temperature. This study found no cross-resistance between fludioxonil and fenhexamid, an essential fungicide integrated with fludioxonil in Chilean B. cinerea control programs. All the Chilean isolates with reduced sensitivity to fludioxonil were controlled by the fludioxonil/cyprodinil mixture, a commonly employed form of fludioxonil. The cyprodinil sensitivity in the isolates with reduced sensitivity to fludioxonil explains their low field frequency despite their null fitness penalties. However, the emergence of fludioxonil-resistant isolates inside the Chilean B. cinerea population demands a comprehensive analysis of their genetic bases, accompanied by monitoring tools that allow the permanence of field fludioxonil efficacy.

A new point mutation (D1158N) in histidine kinase Bos1 confers high-level resistance to fludioxonil in field gray mold disease.

Gray mold, caused by the fungus Botrytis cinerea, is one of the most important plant diseases worldwide that is prone to developing resistance to fungicides. Currently, the phenylpyrrole fungicide fludioxonil exhibits excellent efficacy in the control of gray mold in China. In this study, we detected the fludioxonil resistance of gray mold disease in Shouguang City of Shandong Province, where we first found fludioxonil-resistant isolates of B. cinerea in 2014. A total of 87 single spore isolates of B. cinerea were obtained from cucumbers in greenhouse, and 3 of which could grow on PDA plates amended with 50 µg/mL fludioxonil that was defined as high-level resistance, with a resistance frequency of 3.4%. Furthermore, the 3 fludioxonil-resistant isolates also showed high-level resistance to the dicarboximide fungicides iprodione and procymidone. Sequencing comparison revealed that all the 3 fludioxonil-resistant isolates had a point mutation at codon 1158, GAC (Asp) â†’ AAC (Asn) in the histidine kinase Bos1, which was proved to be the reason for fludioxonil resistance. In addition, the fludioxonil-resistant isolates possessed an impaired biological fitness compared to the sensitive isolates based on the results of mycelial growth, conidiation, virulence, and osmotic stress tolerance determination. Taken together, our results indicate that the high-level resistance to fludioxonil caused by the Bos1 point mutation (D1158N) has emerged in the field gray mold disease, and the resistance risk is relatively high, and fludioxonil should be used sparingly.

Resistance to Seven Site-Specific Fungicides in Botrytis cinerea from Greenhouse-Grown Ornamentals.

The fungal pathogen Botrytis cinerea is a notorious problem on many floriculture greenhouse hosts including petunia, geranium, and poinsettia; these key crops contribute to the $6.43 billion U.S. ornamental industry. While growers use cultural strategies to reduce relative humidity and free moisture to limit Botrytis blight, fungicides remain a primary component of control programs. Isolates (n = 386) of B. cinerea sampled from symptomatic petunia, geranium, and poinsettia in Michigan greenhouses from 2018 to 2021 were screened for resistance to eight fungicides belonging to seven Fungicide Resistance Action Committee (FRAC) groups. Single-spored isolates were subjected to a germination-based assay using previously defined discriminatory doses of each fungicide. Resistance was detected to thiophanate-methyl (FRAC 1; 94%), pyraclostrobin (FRAC 11; 80%), boscalid (FRAC 7; 67%), iprodione (FRAC 2; 65%), fenhexamid (FRAC 17; 38%), cyprodinil (FRAC 9; 38%), fludioxonil (FRAC 12; 21%), and fluopyram (FRAC 7; 13%). Most isolates (63.5%) were resistant to at least four FRAC groups, with 8.7% of all isolates demonstrating resistance to all seven FRAC groups tested. Resistance frequencies for each fungicide were similar among crops, production regions, and growing cycles but varied significantly for each greenhouse. Phenotypic diversity was high, as indicated by the 48 different fungicide resistance profiles observed. High frequencies of resistance to multiple fungicides in B. cinerea populations from floriculture hosts highlight the importance of sustainable and alternative disease management practices for greenhouse growers.

Risk assessment for resistance to fludioxonil in Corynespora cassiicola in Liaoning China.

Cucumber corynespora leaf spot, caused by Corynespora cassiicola, is the primary disease of cucumber leaves in greenhouses in China. Fludioxonil is a phenylpyrrole fungicide that inhibits C. cassiicola growth. We studied the sensitivity of 170 isolates of C. cassiicola to fludioxonil and evaluated resistance risk. All of the isolates were sensitive to fludioxonil. The EC50 values ranged from 0.082 to 0.539 µg/mL with a mean of 0.207 ± 0.0053 µg/mL. Laboratory-created mutants with a high resistance factor to fludioxonil were genetically stable after 10 transfers and showed positive cross-resistance to iprodione and procymidone but not to azoxystrobin, carbendazim, pydiflumetofen, and prochloraz. There was no significant difference in mycelial growth and temperature adaptation between the mutant s and the sensitive isolates, except for pathogenicity and sporulation. The resistant isolates accumulated less glycerol than their parental isolates and were more sensitive to osmotic stress. The histidine kinase activity of the sensitive isolates was significantly inhibited compared to that of the resistant mutants. Sequence alignment of the histidine kinase gene CCos revealed that the mutants RTL4, RXM5, and RFS102 had point mutations at different sites that resulted in amino acid changes at G934E, S739F, and A825P in the CCos protein. The mutant RFS102 had an alanine deletion at site 824. After fludioxonil treatment, CCos expression by RFS20 was significantly lower than that of the parental isolate. Our findings demonstrate that C. cassiicola exhibits moderate resistance to fludioxonil.

Distinct transcriptional responses to fludioxonil in Aspergillus fumigatus and its ΔtcsC and Δskn7 mutants reveal a crucial role for Skn7 in the cell wall reorganizations triggered by this antifungal.

BACKGROUND: Aspergillus fumigatus is a major fungal pathogen that causes severe problems due to its increasing resistance to many therapeutic agents. Fludioxonil is a compound that triggers a lethal activation of the fungal-specific High Osmolarity Glycerol pathway. Its pronounced antifungal activity against A. fumigatus and other pathogenic molds renders this agent an attractive lead substance for the development of new therapeutics. The group III hydride histidine kinase TcsC and its downstream target Skn7 are key elements of the multistep phosphorelay that represents the initial section of the High Osmolarity Glycerol pathway. Loss of tcsC results in resistance to fludioxonil, whereas a Δskn7 mutant is partially, but not completely resistant. RESULTS: In this study, we compared the fludioxonil-induced transcriptional responses in the ΔtcsC and Δskn7 mutant and their parental A. fumigatus strain. The number of differentially expressed genes correlates well with the susceptibility level of the individual strains. The wild type and, to a lesser extend also the Δskn7 mutant, showed a multi-faceted stress response involving genes linked to ribosomal and peroxisomal function, iron homeostasis and oxidative stress. A marked difference between the sensitive wild type and the largely resistant Δskn7 mutant was evident for many cell wall-related genes and in particular those involved in the biosynthesis of chitin. Biochemical data corroborate this differential gene expression that does not occur in response to hyperosmotic stress. CONCLUSIONS: Our data reveal that fludioxonil induces a strong and TcsC-dependent stress that affects many aspects of the cellular machinery. The data also demonstrate a link between Skn7 and the cell wall reorganizations that foster the characteristic ballooning and the subsequent lysis of fludioxonil-treated cells.

Sensitivity and resistance risk of Botryosphaeria dothidea causing Chinese hickory trunk canker to fludioxonil.

Hickory trunk canker (HTC), primarily caused by Botryosphaeria dothidea, is an aggravating disease that threatens an important regional economic tree species of Chinese hickory and few information is available in the control of this disease. Here, the sensitivity of 93 isolates to fludioxonil and the resistance risk were investigated. All the isolates tested were sensitive to fludioxonil and the EC50 ranged from 0.0028 to 0.0569 µg/mL. The tamed fludioxonil-resistant mutants remained highly resistant to fludioxonil even after 10 consecutive transfers to fludioxonil-free PDA plates. As for fitness penalty, the fludioxonil-resistant mutants demonstrated a reduction in conidia production and virulence as well as increased sensitivity to high osmotic stress. While, variations in mycelial growth and responses to SDS and H2O2 were not detected in all the resistant mutants. In addition, the resistant mutants demonstrated positive cross-resistance to iprodione but not to fungicides of other modes of action. Sequential analysis of BdNik1 showed that premature stop codon occurred in all the resistant mutants despite of point mutation (BD16-22R9 and BD16-22R20) or frameshift mutation (BD16-22R8, BD16-22R11 and BD16-22R18). Our study suggested that fludioxonil exhibited excellent inhibition activity on mycelial growth of B. dothidea in vitro, the resistance risk of B. dothidea to fludioxonil should be low to moderate and fludioxonil would be a nice candidate in controlling HTC caused by B. dothidea.

Overexpression of FgPtp3 Is Involved in Fludioxonil Resistance in Fusarium graminearum by Inhibiting the Phosphorylation of FgHog1.

Fusarium graminearum is the main causal agent of Fusarium head blight (FHB), a destructive disease in cereal crops worldwide. Resistance to fludioxonil has been reported in F. graminearum in the field, but its underlying mechanisms remain elusive. In this study, 152 fludioxonil-resistant (FR) mutants of F. graminearum were obtained by selection in vitro. The FR strains exhibited dramatically impaired fitness, but only 7 of the 13 analyzed strains possessed mutations in genes previously reported to underlie fludioxonil resistance. Comparison between the FR-132 strain and its parental strain PH-1 using whole genome sequencing revealed no mutations between them, but transcriptome analysis, after the strains were treated with 0.5 µg/mL fludioxonil, revealed 2778 differently expressed genes (DEGs) mapped to 96 KEGG pathways. Investigation of DEGs in the MAPK pathway showed that overexpression of the tyrosine protein phosphatase FgPtp3, but not FgPtp2, enhanced fludioxonil resistance. Further analysis found that FgPtp3 interacted directly with FgHog1 to regulate the phosphorylation of Hog1, and overexpressed FgPtp3 in PH-1 could significantly suppress the phosphorylation of FgHog1 and hinder signal transmission of the HOG-MAPK pathway. Overall, FgPtp3 plays a significant role in regulating fludioxonil resistance in F. graminearum.

Occurrence and Chemical Control Strategy of Wheat Brown Foot Rot Caused by Microdochium majus.

Wheat brown foot rot (WBFR), caused by a variety of phytopathogenic fungi, is an important soilborne and seedborne disease of wheat. WBFR causes wheat lodging and seedling dieback, which seriously affect the yield and quality of wheat. In this study, 64 isolates of WBFR were isolated from different wheat fields in Yancheng city, Jiangsu Province, China. The internal transcribed spacer, elongation factor 1α, and RNA polymerase II subunit were amplified and the sequencing results of the fragments were analyzed with BLAST in NCBI. Through morphological and molecular identification, all of the isolates were identified as Microdochium majus. Verification by Koch's postulates confirmed that M. majus was the pathogen causing WBFR. The antifungal activities of fludioxonil and prochloraz against 64 isolates of M. majus were determined based on mycelial growth inhibition method. The results showed that fludioxonil and prochloraz had good antifungal activity against M. majus. The mean 50% effective concentration values of fludioxonil and prochloraz against M. majus were 0.2956 ± 0.1285 µg/ml and 0.0422 ± 0.0157 µg/ml, respectively. Control efficacy for seed-coating treatments conducted in a greenhouse indicated that M. majus severely damaged the normal growth of wheat, while seed coating with fludioxonil or prochloraz significantly reduced the disease incidence and improved the seedling survival rates. At fludioxonil doses of 7.5 g per 100 kg and prochloraz doses of 15 g per 100 kg, the incidence was reduced by 22.26 and 25.33%, seedling survival rates increased by 25.37 and 22.66%, and control efficacy reached 70.02 and 72.30%, respectively. These findings provide vital information for the accurate diagnosis and effective management of WBFR.