The G143S mutation in cytochrome b confers high resistance to pyraclostrobin in Fusarium pseudograminearum.

BACKGROUND: Wheat crown rot (WCR), primarily caused by Fusarium pseudograminearum has become more and more prevalent in winter wheat areas in China. However, limited fungicides have been registered for the control of WCR in China so far. Pyraclostrobin is a representative quinone outside inhibitor (QoI) with excellent activity against Fusarium spp. There is currently limited research on the resistance risk and resistance mechanism of F. pseudograminearum to pyraclostrobin. RESULTS: Here, we determined the activity of pyraclostrobin against F. pseudograminearum. The EC50 values ranged from 0.022 to 0.172 µg mL-1 with an average EC50 value of 0.071 ± 0.030 µg mL-1. Four highly pyraclostrobin-resistant mutants were obtained from two sensitive strains by ultraviolet (UV) mutagenesis in the laboratory. The mutants showed decreased mycelial growth rate and virulence as compared with the corresponding wild-type strains, indicating that pyraclostrobin resistance suffered a fitness penalty in F. pseudograminearum. It was found that the high resistance of four mutants was caused by the G143S mutation in Cytb. Molecular docking analysis also further confirms that the G143S mutation in Cytb decreased the binding affinity between pyraclostrobin and Cytb. CONCLUSION: The resistance risk of F. pseudograminearum to pyraclostrobin could be low to medium. Although a mutation at the G143S position of Cytb could potentially occur, this mutation decreases the fitness of the mutant, which may reduce its survival in the environment. Therefore, the negative consequences of a possible mutation are lower. This makes pyraclostrobin a good candidate for controlling crown rot in wheat. © 2024 Society of Chemical Industry.

A new perspective: deciphering the aberrance and clinical implication of disulfidptosis signatures in clear cell renal cell carcinoma.

Recent research has discovered disulfidptosis as a form of programmed cell death characterized by disulfide stress. However, its significance in clear cell renal cell carcinoma (ccRCC) remains unclear. To investigate this, data from The Cancer Genome Atlas were collected and used to identify ccRCC subgroups. Unsupervised clustering was employed to determine ccRCC heterogeneity. The mutation landscape and immune microenvironment of the subgroups were analyzed. The Disulfidptosis-Related Score was calculated using the LASSO-penalized Cox regression algorithm. The E-MATB-1980 cohort was used to validate the signature. The role of SLC7A11 in ccRCC metastasis was explored using western blotting and Transwell assays. Disulfidptosis-related genes are commonly downregulated in cancers and are linked to hypermethylation and copy number variation. The study revealed that ccRCC is divided into two sub-clusters: the disulfidptosis-desert sub-cluster, which is associated with a poor prognosis, a higher mutation frequency, and an immunosuppressive microenvironment. A 14-gene prognostic model was developed using differentially expressed genes and was validated in the E-MATB-1980 cohort. The low-risk group demonstrated longer overall and disease-free survival and responded better to targeted immunotherapy. Results from in vitro experiments identified SLC7A11 as a key participant in ccRCC metastasis.

FgPfn participates in vegetative growth, sexual reproduction, pathogenicity, and fungicides sensitivity via affecting both microtubules and actin in the filamentous fungus Fusarium graminearum.

Fusarium head blight (FHB), caused by Fusarium graminearum species complexes (FGSG), is an epidemic disease in wheat and poses a serious threat to wheat production and security worldwide. Profilins are a class of actin-binding proteins that participate in actin depolymerization. However, the roles of profilins in plant fungal pathogens remain largely unexplored. Here, we identified FgPfn, a homolog to profilins in F. graminearum, and the deletion of FgPfn resulted in severe defects in mycelial growth, conidia production, and pathogenicity, accompanied by marked disruptions in toxisomes formation and deoxynivalenol (DON) transport, while sexual development was aborted. Additionally, FgPfn interacted with Fgα1 and Fgß2, the significant components of microtubules. The organization of microtubules in the ΔFgPfn was strongly inhibited under the treatment of 0.4 µg/mL carbendazim, a well-known group of tubulin interferers, resulting in increased sensitivity to carbendazim. Moreover, FgPfn interacted with both myosin-5 (FgMyo5) and actin (FgAct), the targets of the fungicide phenamacril, and these interactions were reduced after phenamacril treatment. The deletion of FgPfn disrupted the normal organization of FgMyo5 and FgAct cytoskeleton, weakened the interaction between FgMyo5 and FgAct, and resulting in increased sensitivity to phenamacril. The core region of the interaction between FgPfn and FgAct was investigated, revealing that the integrity of both proteins was necessary for their interaction. Furthermore, mutations in R72, R77, R86, G91, I101, A112, G113, and D124 caused the non-interaction between FgPfn and FgAct. The R86K, I101E, and D124E mutants in FgPfn resulted in severe defects in actin organization, development, and pathogenicity. Taken together, this study revealed the role of FgPfn-dependent cytoskeleton in development, DON production and transport, fungicides sensitivity in F. graminearum.

Risk assessment and molecular mechanism of Sclerotium rolfsii resistance to boscalid.

Boscalid, a widely used SDHI fungicide, has been employed in plant disease control for over two decades. However, there is currently no available information regarding its antifungal activity against Sclerotium rolfsii and the potential risk of resistance development in this pathogen. In this study, we evaluated the sensitivity of 100 S. rolfsii strains collected from five different regions in China during 2018-2019 to boscalid using mycelial growth inhibition method and assessed the risk of resistance development. The EC50 values for boscalid ranged from 0.2994 µg/mL to 1.0766 µg/mL against the tested strains, with an average EC50 value of 0.7052 ± 0.1473 µg/mL. Notably, a single peak sensitivity baseline was curved, indicating the absence of any detected resistant strains. Furtherly, 10 randomly selected strains of S. rolfsii were subjected to chemical taming to evaluate its resistance risk to boscalid, resulting in the successful generation of six stable and inheritable resistant mutants. These mutants exhibited significantly reduced mycelial growth, sclerotia production, and virulence compared to their respective parental strains. Cross-resistance tests revealed a correlation between boscalid and flutolanil, benzovindiflupyr, pydiflumetofen, fluindapyr, and thifluzamide; however, no cross-resistance was observed between boscalid and azoxystrobin. Thus, we conclude that the development risk of resistance in S. rolfsii to boscalid is low. Boscalid can be used as an alternative fungicide for controlling peanut sclerotium blight when combined with other fungicides that have different mechanisms of action. Finally, the target genes SDHB, SDHC, and SDHD in S. rolfsii were initially identified, cloned and sequenced to elucidate the mechanism of S. rolfsii resistance to boscalid. Two mutation genotypes were found in the mutants: SDHD-D111H and SDHD-H121Y. The mutants carrying SDHD-H121Y exhibited moderate resistance, while the mutants with SDHD-D111H showed low resistance. These findings contribute to our comprehensive understanding of molecular mechanisms underlying plant pathogens resistance to SDHI fungicides.

An Integrated Machine Learning Framework Identifies Prognostic Gene Pair Biomarkers Associated with Programmed Cell Death Modalities in Clear Cell Renal Cell Carcinoma.

BACKGROUND: Clear cell renal cell carcinoma (ccRCC) is a common and lethal urological malignancy for which there are no effective personalized therapeutic strategies. Programmed cell death (PCD) patterns have emerged as critical determinants of clinical prognosis and immunotherapy responses. However, the actual clinical relevance of PCD processes in ccRCC is still poorly understood. METHODS: We screened for PCD-related gene pairs through single-sample gene set enrichment analysis (ssGSEA), consensus cluster analysis, and univariate Cox regression analysis. A novel machine learning framework incorporating 12 algorithms and 113 unique combinations were used to develop the cell death-related gene pair score (CDRGPS). Additionally, a radiomic score (Rad_Score) derived from computed tomography (CT) image features was used to classify the CDRGPS status as high or low. Finally, we conclusively verified the function of PRSS23 in ccRCC. RESULTS: The CDRGPS was developed through an integrated machine learning approach that leveraged 113 algorithm combinations. CDRGPS represents an independent prognostic biomarker for overall survival and demonstrated consistent performance between training and external validation cohorts. Moreover, CDRGPS showed better prognostic accuracy compared to seven previously published cell death-related signatures. In addition, patients classified as high-risk by CDRGPS exhibited increased responsiveness to tyrosine kinase inhibitors (TKIs), mammalian Target of Rapamycin (mTOR) inhibitors, and immunotherapy. The Rad_Score demonstrated excellent discrimination for predicting high versus low CDRGPS status, with an area under the curve (AUC) value of 0.813 in the Cancer Imaging Archive (TCIA) database. PRSS23 was identified as a significant factor in the metastasis and immune response of ccRCC, thereby validating experimental in vitro results. CONCLUSIONS: CDRGPS is a robust and non-invasive tool that has the potential to improve clinical outcomes and enable personalized medicine in ccRCC patients.

Resistance Risk and Molecular Mechanism of Tomato Wilt Pathogen Fusarium oxysporum f. sp. lycopersici to Pyraclostrobin.

Tomato wilt disease caused by Fusarium oxysporum f. sp. lycopersici (Fol) results in a decrease in tomato yield and quality. Pyraclostrobin, a typical quinone outside inhibitor (QoI), inhibits the cytochrome bc1 complex to block energy transfer. However, there is currently limited research on the effectiveness of pyraclostrobin against Fol. In this study, we determined the activity of pyraclostrobin against Fol and found the EC50 values for pyraclostrobin against 100 Fol strains (which have never been exposed to QoIs before). The average EC50 value is 0.3739 ± 0.2413 µg/mL, indicating a strong antifungal activity of pyraclostrobin against Fol, as shown by unimodal curves of the EC50 values. Furthermore, we generated five resistant mutants through chemical taming and identified four mutants with high-level resistance due to the Cytb-G143S mutation and one mutant with medium-level resistance due to the Cytb-G137R mutation. The molecular docking results indicate that the Cytb-G143S or Cytb-G137R mutations of Fol lead to a change in the binding mode of Cytb to pyraclostrobin, resulting in a decrease in affinity. The resistant mutants exhibit reduced fitness in terms of mycelial growth (25 and 30 °C), virulence, and sporulation. Moreover, the mutants carrying the Cytb-G143S mutation suffer a more severe fitness penalty compared to those carrying the Cytb-G137R mutation. There is a positive correlation observed among azoxystrobin, picoxystrobin, fluoxastrobin, and pyraclostrobin for resistant mutants; however, no cross-resistance was detected between pyraclostrobin and pydiflumetofen, prochloraz, or cyazofamid. Thus, we conclude that the potential risk of resistance development in Fol toward pyraclostrobin can be categorized as ranging from low to moderate.

Mitochondria-Associated Protein FgNdk1 Regulates the Development, Pathogenicity, and SDHI Fungicide Sensitivity of Fusarium graminearum by Interacting with Succinate Dehydrogenase.

Nucleoside diphosphate kinase (NDK) plays an important role in many cellular processes in all organisms. In this study, we functionally characterized a nucleoside diphosphate kinase (FgNdk1) in Fusarium graminearum, a causal agent of Fusarium head blight (FHB). FgNdk1 was involved in the generation of energy in the electron-transfer chain by interacting with succinate dehydrogenase (FgSdhA, FgSdhC1, and FgSdhC2). Deletion of FgNdk1 not only resulted in abnormal mitochondrial morphology, decreased ATP content, defective fungal development, and impairment in the formation of the toxisome but also led to the suppressed expression level of DON biosynthesis enzymes, decreased DON biosynthesis, and declined pathogenicity as well. Furthermore, deletion of FgNdk1 caused increasing transcriptional levels of FgSdhC1 and FgdhC2, in the presence of pydiflumetofen, related to the decreased sensitivity to SDHI fungicides. Overall, this study identified a new regulatory mechanism of FgNdk1 in the pathogenicity and SDHI fungicide sensitivity of Fusarium graminearum.

Bioactivity and Resistance Risk of Fluxapyroxad, a Novel SDHI Fungicide, in Didymella bryoniae.

Gummy stem blight, caused by Didymella bryoniae, is an important disease in watermelon in China. Fluxapyroxad, a new succinate dehydrogenase inhibitor fungicide, shows strong inhibition of the mycelia growth of D. bryoniae. However, its resistance risk in D. bryoniae is unclear. In this research, the sensitivities of 60 D. bryoniae strains to fluxapyroxad were investigated. The average EC50 value and MIC values of 60 D. bryoniae strains against fluxapyroxad were 0.022 ± 0.003 µg/ml and ≤0.1 µg/ml for mycelial growth, respectively. Eight fluxapyroxad-resistant mutants with medium resistance levels were acquired from three wild-type parental strains. The mycelial growth and dry weight of mycelia of most mutants were significantly lower than those of their parental strains. However, four resistant mutants showed a similar phenotype in pathogenicity compared with their parental strains. The above results demonstrated that there was a medium resistance risk for fluxapyroxad in D. bryoniae. The cross-resistance assay showed that there was positive cross-resistance between fluxapyroxad and pydiflumetofen, thifluzamide, and boscalid, but there was no cross-resistance between fluxapyroxad and tebuconazole and mepronil. These results will contribute to evaluating the resistance risk of fluxapyroxad for managing diseases caused by D. bryoniae and further increase our understanding about the mode of action of fluxapyroxad.

The intrinsic resistance of Fusarium solani to the Fusarium-specific fungicide phenamacril is attributed to the natural variation of both T218S and K376M in myosin5.

Fusarium solani is responsible for causing root rot in various crops, resulting in wilting and eventual demise. Phenamacril, a specific inhibitor of myosin5 protein, has gained recognition as an effective fungicide against a broad spectrum of Fusarium species. It has been officially registered for controlling Fusarium diseases through spray application, root irrigation, and seed dipping. In this study, phenamacril was observed to exhibit negligible inhibitory effects on F. solani causing crop root rot, despite the absence of prior exposure to phenamacril. Considering the high selectivity of phenamacril, this phenomenon was attributed to intrinsic resistance and further investigated for its underlying mechanism. Sequence alignment analysis of myosin5 proteins across different Fusarium species revealed significant differences at positions 218 and 376. Subsequent homology modeling and molecular docking results indicated that substitutions T218S, K376M, and T218S&K376M impaired the binding affinity between phenamacril and myosin5 in F. solani. Mutants carrying these substitutions were generated via site-directed mutagenesis. A phenamacril-sensitivity test showed that the EC50 values of mutants carrying T218S, K376M, and T218S&K376M were reduced by at least 6.13-fold, 9.66-fold, and 761.90-fold respectively compared to the wild-type strain. Fitness testing indicated that mutants carrying K376M or T218S&K376M had reduced sporulation compared to the wild-type strain. Additionally, mutants carrying T218S exhibited an enhanced virulence compared to the wild-type strain. However, there were no significant differences observed in mycelial growth rates between the mutants and the wild-type strain. Thus, the intrinsic differences observed at positions 218 and 376 in myosin5 between F. solani and other Fusarium species are specifically associated with phenamacril resistance. The identification of these resistance-associated positions in myosin5 of F. solani has significantly contributed to the understanding of phenamacril resistance mechanisms, thereby discouraging the use of phenamacril for controlling F. solani.

The ATP Synthase Subunits FfATPh, FfATP5, and FfATPb Regulate the Development, Pathogenicity, and Fungicide Sensitivity of Fusarium fujikuroi.

ATP synthase catalyzes the synthesis of ATP by consuming the proton electrochemical gradient, which is essential for maintaining the life activity of organisms. The peripheral stalk belongs to ATP synthase and plays an important supporting role in the structure of ATP synthase, but their regulation in filamentous fungi are not yet known. Here, we characterized the subunits of the peripheral stalk, FfATPh, FfATP5, and FfATPb, and explored their functions on development and pathogenicity of Fusarium Fujikuroi. The FfATPh, FfATP5, and FfATPb deletion mutations (∆FfATPh, ∆FfATP5, and ∆FfATPb) presented deficiencies in vegetative growth, sporulation, and pathogenicity. The sensitivity of ∆FfATPh, ∆FfATP5, and ∆FfATPb to fludioxonil, phenamacril, pyraclostrobine, and fluazinam decreased. In addition, ∆FfATPh exhibited decreased sensitivity to ionic stress and osmotic stress, and ∆FfATPb and ∆FfATP5 were more sensitive to oxidative stress. FfATPh, FfATP5, and FfATPb were located on the mitochondria, and ∆FfATPh, ∆FfATPb, and ∆FfATP5 disrupted mitochondrial location. Furthermore, we demonstrated the interaction among FfATPh, FfATP5, and FfATPb by Bimolecular Fluorescent Complimentary (BiFC) analysis. In conclusion, FfATPh, FfATP5, and FfATPb participated in regulating development, pathogenicity, and sensitivity to fungicides and stress factors in F. fujikuroi.

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.

Activity and cell toxicology of fluazinam on Fusarium graminearum.

Fusarium graminearum is an important plant pathogen and the causal agent of Fusarium head blight (FHB). At present, the principal method of controlling FHB is through fungicides. Fluazinam is an agent with strong broad-spectrum antifungal activity and has been used to control many diseases. However, there are no reported uses of fluazinam for controlling FHB. This study reports the activity and cell toxicology mechanisms of fluazinam on the filamentous fungus F. graminearum and its effect on fungal growth and development. The activity of fluazinam was tested for 95 wild-type field strains of F. graminearum. The EC50 values (the 50% effective concentration) of fluazinam for inhibition of mycelial growth and spore germination ranged from 0.037 µg/ml to 0.179 µg/ml and from 0.039 µg/ml to 0.506 µg/ml, respectively. The fluazinam sensitivity of these strains varied in 4.9 and 13.0 folds, implying that the target of the fungicide remained unchanged. After treatment with 0.3 µg/ml (≈EC90) fluazinam, the production of conidia was reduced, and the cell wall and cell membrane had shrunked; the cell nucleus and septum morphology, cell membrane permeability, and sexual development were not affected. When treated with 0.1 µg/ml (≈EC50) or 0.3 µg/ml fluazinam, the mycelial respiration and deoxynivalenol (DON) synthesis of F. graminearum were decreased. Confocal images showed that the formation of toxisomes was disturbed after fluazinam treatment, suggesting that fluazinam reduces DON synthesis by inhibiting toxisome formation. Infection of wheat coleoptiles revealed that fluazinam had a strong protective activity against F. graminearum. At 250 µg/ml fluazinam the control efficacy of protective treatments reached 100% and controlled strains resistant to carbendazim. These results contribute to the understanding of the mode of action of fluazinam and its application.

FgMet3 and FgMet14 related to cysteine and methionine biosynthesis regulate vegetative growth, sexual reproduction, pathogenicity, and sensitivity to fungicides in Fusarium graminearum.

Fusarium graminearum is a destructive filamentous fungus, which widely exists in wheat and other cereal crops. Cysteine and Methionine are unique sulfur-containing amino acids that play an essential role in protein synthesis and cell life, but their functions and regulation in F. graminearum remain largely unknown. Here we identified two proteins, FgMet3 and FgMet14 in F. graminearum, which are related to the synthesis of cysteine and methionine. We found FgMet3 and FgMet14 were localized to the cytoplasm and there was an interaction between them. FgMet3 or FgMet14 deletion mutants (ΔFgMet3 and ΔFgMet14) were deficient in vegetative growth, pigment formation, sexual development, penetrability and pathogenicity. With exogenous addition of cysteine and methionine, the vegetative growth and penetrability could be completely restored in ΔFgMet3 and ΔFgMet14, while sexual reproduction could be fully restored in ΔFgMet3 and partially restored in ΔFgMet14. ΔFgMet3 and ΔFgMet14 exhibited decreased sensitivity to Congo red stress and increased sensitivity to SDS, NaCl, KCl, Sorbitol, Menadione, and Zn ion stresses. Moreover, FgMet3 and FgMet14 nonspecifically regulate the sensitivity of F. graminearum to fungicides. In conclusion, FgMet3 and FgMet14 interacted to jointly regulate the development, pathogenicity, pigment formation, sensitivity to fungicides and stress factors in F. graminearum.

Evolution of Benzimidazole Resistance Caused by Multiple Double Mutations of ß-Tubulin in Corynespora cassiicola.

Cucumber target leaf spot caused by Corynespora cassiicola has devastated greenhouse cucumber production. In our previous study, the resistance monitoring of C. cassiicola to carbendazim was carried out, and a large number of resistant populations carrying various mutations (M163I&E198A, F167Y&E198A, F200S&E198A, or E198A) in ß-tubulin were detected. However, the single-point mutations M163I, F167Y, and F200S have remained undetected. To investigate the evolutionary mechanism of double mutations in ß-tubulin of C. cassiicola resistance to benzimidazoles, site-directed mutagenesis was used to construct alleles with corresponding mutation genotypes in ß-tubulin. Through PEG-mediated protoplast transformation, all the mutants except for the M163I mutation were obtained and conferred resistance to benzimidazoles. It was found that the mutants conferring the E198A or double-point mutations showed high resistance to carbendazim and benomyl, but the mutants conferring the F167Y or F200S mutations showed moderate resistance. Except, the F200S mutants showed low resistance, the resistance level of the other mutants to thiabendazole seemed no difference. In addition, compared to the other mutants, the F167Y and F200S mutants suffered a more severe fitness penalty in mycelial growth, sporulation, and virulence. Thus, combined with the resistance level, fitness, and molecular docking results, we concluded that the field double mutations (F167Y&E198A and F200S&E198A) evolved from the single mutations F167Y and F200S, respectively.

The G143A/S substitution of mitochondrially encoded cytochrome b (Cytb) in Magnaporthe oryzae confers resistance to quinone outside inhibitors.

BACKGROUND: Rice blast, caused by Magnaporthe oryzae, is a destructive disease threatening the production of staple foods worldwide. Quinone outside inhibitors (QoIs) are a group of chemicals exhibiting excellent activity against a majority of plant pathogens, with the disadvantage that pathogens can easily develop resistance to QoIs. RESULTS: Here, we investigated the activity of picoxystrobin against M. oryzae, which showed a great inhibitory effect on 100 strains of M. oryzae with half-maximal effective concentrations (EC50 ) ranging from 0.0251 to 0.1337 µg ml-1 . The EC50 values showed a continuous unimodal distribution that was identical to the normal distribution, suggesting the potency of our study to represent baseline sensitivity. In addition, nine resistant mutants were obtained by exposing M. oryzae to a high dosage of picoxystrobin in the laboratory; all of them showed cross-resistance to the other five QoI fungicides. Although some mutants showed a decreased resistance factor after ten successive cultures on fungicide-free medium, the resistance to picoxystrobin was still inheritable. Amino acid substitution of G143S was detected in eight of nine picoxystrobin-resistant mutants, and G143A was detected in only one of nine mutants. A fitness penalty was found in the mutants carrying G143S rather than G143A. CONCLUSION: Our findings suggested that M. oryzae had a mid to high risk of resistance to picoxystrobin. Considering this, we should be vigilant to the resistance risk and apply picoxystrobin sensibly in the field. © 2022 Society of Chemical Industry.

Extending the Host Range of Fusarium Poae Virus 1 from Fusarium poae to other Fusarium Species in the Field.

Fusarium poae virus 1 (FpV1, a betapartitivirus) is one of the mycoviruses which is discovered earlier. Due to the vegetative incompatibility barrier that often exists between different species or strains of filamentous fungi, FpV1 has been thought to be limited to its host, F. poae, as a non-hypovirulence mycovirus in the past 20 years in the field. Here, a novel strain of FpV1 (FpV1-Fa) with two dsRNA segments (2157-and 2080-nt) was consistently identified in F. asiaticum isolates in the field. FpV1-Fa induced abnormal morphology and hypovirulence of F. asiaticum, along with a high viral load. FpV1-Fa was detected only from the F. asiaticum and F. tricinctum strains at a FpV1-Fa sampling site (119.014289, 33.8261), while the other strains from other sites were not identified FpV1-Fa. A horizontal transmission experiment showed that FpV1-Fa can transfer from F. asiaticum to F. poae and F. tricinctum, but not to F. graminearum. The selection analysis of FpV1-Fa revealed RdRP and CP were under strong purifying selection, and the C-terminal side of RdRP was under positive selection. In these regions, 9 amino acid mutations in RdRP and 21 mutations in CP appeared to cause the variation of host range and virulence in FpV1-Fa.

Cytochrome bc1 Complex: Potential Breach to Improve the Activity of Phenazines on Xanthomonas.

The effects of the natural pesticides, phenazines, were reported to be limited by some tolerant metabolism processes within Xanthomonas. Our previous studies suggested that the functional cytochrome bc1 complex, the indispensable component of the respiration chain, might participate in tolerating phenazines in Xanthomonas. In this study, the cytochrome bc1 mutants of Xanthomonas campestris pv. campestris (Xcc) and Xanthomonas oryzae pv. oryzae (Xoo), which exhibit different tolerance abilities to phenazines, were constructed, and the cytochrome bc1 complex was proven to partake a critical and conserved role in tolerating phenazines in Xanthomonas. In addition, results of the cytochrome c mutants suggested the different functions of the various cytochrome c proteins in Xanthomonas and that the electron channeled by the cytochrome bc1 complex to cytochrome C4 is the key to reveal the tolerance mechanism. In conclusion, the study of the cytochrome bc1 complex provides a potential strategy to improve the activity of phenazines against Xanthomonas.

FfCOX17 is Involved in Fumonisins Production, Growth, Asexual Reproduction, and Fungicide Sensitivity in Fusarium fujikuroi.

Fusarium fujikuroi, a causal agent of Rice Bakanae Disease, produces secondary metabolites such as gibberellin, pigments bikaverin, and mycotoxins fumonisins. Fumonisins produced by F. fujikuroi pose a severe threat to human and animal health. The copper chaperone protein plays a critical role in different growth stages of plants, fungi, and yeasts, but their functions and regulation in fumonisin biosynthesis are still unclear. Here, a copper chaperone protein, FfCOX17, was identified in F. fujikuroi. The FfCOX17 deletion mutant (∆FfCOX17) exhibited decreased vegetative growth and asexual reproduction. The transcriptional level of the FfFUM2 gene was significantly induced in ∆FfCOX17, and the fumonisin production in ∆FfCOX17 mutants was significantly increased compared to wild-type F. fujikuroi, but the pathogenicity of ∆FfCOX17 mutants was unaffected, which may be caused by the no significantly changed gibberellin content. ∆FfCOX17 showed decreased sensitivity to oxidative stress, osmotic stress, and increased sensitivity to cell wall stress, heat shock stress, and high concentration glucose. In addition, ∆FfCOX17 also showed increased sensitivity to fungicide fluazinam and fludioxonil, and decreased sensitivity to phenamacril and prochloraz. Taken together, this study suggested that FfCOX17 is critical for fumonisin production, vegetative growth, asexual reproduction, and fungicide sensitivity, but is not required for the virulence function of F. fujikuroi on rice.

Molecular Mechanism of Sclerotinia sclerotiorum Resistance to Succinate Dehydrogenase Inhibitor Fungicides.

Succinate dehydrogenase inhibitor (SDHI) fungicides have a wide spectrum of fungicidal effects on a variety of fungi causing plant diseases, including Sclerotinia stem rot caused by Sclerotinia sclerotiorum. However, the consistent use of site-specific SDHI fungicides can result in the development of resistant isolates with mutations in the SDHB, SDHC, or SDHD subunit thereby leading to a rapid decline of fungicide performance. In this study, we found that SDHC was genetically evolved into two isotypes SDHC1 and SDHC2 in S. sclerotiorum but not involved in the sensitivity to SDHI fungicides. In addition, we demonstrated that the A11V substitution in SDHB was not involved in the resistance of S. sclerotiorum to boscalid, and this substitution widely emerged in the field populations. Meanwhile, the P226L substitution in SDHB was demonstrated to confer boscalid resistance in S. sclerotiorum. The result of cross-resistance showed that the SDHB-P226L substitution exhibited a positive cross-resistance between boscalid and carboxin, fluopyram, pydiflumetofen, flubeneteram, pyraziflumid, fluindapyr, or penthiopyrad. Taken together, our results indicated that the P226L substitution in SDHB resulted in the resistance of S. sclerotiorum to SDHI fungicides but suffered from fitness penalty, especially the homozygous mutants conferring the P226L substitution in SDHB.

Risk assessment and molecular mechanism of Fusarium incarnatum resistance to phenamacril.

BACKGROUND: Cucumber fruit rot (CFR) caused by Fusarium incarnatum is a devastating fungal disease in cucumber. In recent years, CFR has occurred frequently, resulting in serious yield and quality losses in China. Phenamacril exhibits a specific antifungal activity against Fusarium species. However, no data for phenamacril against F. incarnatum is available. RESULTS: The sensitivity of 80 F. incarnatum strains to phenamacril was determined. The half maximal effective concentration (EC50 ) values ranged from 0.1134 to 0.3261 µg mL-1 with a mean EC50 value of 0.2170 ± 0.0496 µg mL-1 . A total of seven resistant mutants were obtained from 450 mycelial plugs by phenamacril-taming on potato dextrose agar (PDA) plates with 10 µg mL-1 of phenamacril, and the resistant frequency was 1.56%. Phenamacril-resistant mutants showed decreased mycelial growth, conidiation and virulence as compared with the corresponding wild-type strains, indicating that phenamacril resistance suffered a fitness penalty in F. incarnatum. In addition, using sequence analysis, the point mutations of S217P or I424S were discovered in Fimyosin-5 (the target of phenamacril). The site-directed mutagenesis of the S217P, P217S, I424S and S424I substitutions were constructed to reveal the relationship between the point mutations and phenamacril resistance. The results strongly demonstrated that the mutations of S217P and I424S in Fimyosin-5 conferred phenamacril-resistance in F. incarnatum. CONCLUSION: Phenamacril-resistant mutants were easily induced and their resistance level was high. The S217P or I424S substitutions in Fimyosin-5 conferring phenamacril resistance were detected and futherly verified by transformation assay with site-directed mutagenesis. Thus, we proposed that the resistance development of F. incarnatum to phenamacril is high risk. © 2022 Society of Chemical Industry.