Fusarium crown rot(FCR),predominantly caused by Fusarium pseudograminearum,has been listed as a Category Ⅱ disease in six provinces of China,posing a significant threat to wheat production.The phenylpyrrole fungicide...Fusarium crown rot(FCR),predominantly caused by Fusarium pseudograminearum,has been listed as a Category Ⅱ disease in six provinces of China,posing a significant threat to wheat production.The phenylpyrrole fungicide fludioxonil is a key agent for FCR control.Previous studies indicated that resistance to fludioxonil in F.pseudograminearum is primarily associated with altered expression levels of the FpOS1 gene,which encodes a hybrid histidine kinase.However,the roles of mutations in other FpOS genes and the molecular interactions between FpOS proteins and fludioxonil remain elusive.To address these gaps,we generated 16 fludioxonil-resistant mutants with heritable resistance traits by in vitro selection of four sensitive F.pseudograminearum isolates.These mutants exhibited high resistance levels,with resistance factors(RF)ranging from 633.73 to 8617.07.Compared to their parental isolates,the resistant mutants showed significantly reduced mycelial growth rate,sporulation capacity,and pathogenicity.They were also more sensitive to ionic,osmotic,and oxidative stresses and displayed compromised cell wall and membrane integrity.Fludioxonil demonstrated no cross-resistance with tebuconazole or pydiflumetofen;however,it exhibited weak positive crossresistance to pyraclostrobin and moderate positive cross-resistance to iprodione.Fludioxonil treatment significantly promoted glycerol synthesis and inhibited deoxynivalenol(DON)production in parental isolates,whereas these regulatory effects were markedly attenuated in the resistant mutants.Mutation analysis identified mutation sites in FpOS1,FpOS4,and FpOS5 genes,with a lower mutation frequency in FpOS1 and no mutations detected in FpOS2.Molecular docking indicated that amino acid substitutions in FpOS4 and FpOS5 significantly reduced the binding affinity of fludioxonil to these target proteins.In conclusion,F.pseudograminearum poses a moderate risk of resistance to fludioxonil.Point mutations in FpOS4 and FpOS5 genes emerge as key molecular drivers of resistance,likely by diminishing the binding affinity between the fungicide and its proteins.This study clarifies the molecular basis of fludioxonil resistance in F.pseudograminearum and provides a scientific rationale for the judicious use of this fungicide in managing FCR.展开更多
Wheat crown rot caused by Fusarium spp. is a common disease worldwide. Both Fusarium pseudograminearum and Fusarium graminearum infect wheat crown and produce mycotoxin leading to grain loss due to white head. F. pseu...Wheat crown rot caused by Fusarium spp. is a common disease worldwide. Both Fusarium pseudograminearum and Fusarium graminearum infect wheat crown and produce mycotoxin leading to grain loss due to white head. F. pseudograminearum （Fp） was reported in wheat from Henan Province of China a couple of years ago. The wheat crown rot （CR） caused by this new pathogen is as an emerging severe disease of wheat, which has recently expanded to several provinces in China and is, therefore, under rapid investigation. Colonization of wheat tissue by Fp is accomplished though the formation of a septated foot-shaped appressoria and generation of a penetration peg to break through the internal cells of leaf sheath. The molecular mechanism by which Fp regulates the pathogenesis on wheat host is unclear. Here, we report FpPDE1, a P-type ATPase-encoding predicted PDE1 orthologue gene of Magnaporthe oryzae, belonging to the DRS2 subfamily of aminophospholipid translocases. The gene deletion of FpPDE1 with the split-marker approach did not obviously affect hyphae growth and conidiation, but led to an attenuated virulence on wheat base stem and root. Our finding indicates that the putative aminophospholipid translocases is not essential for the infectious hyphae development in Fp.展开更多
Hyphal fusion(anastomosis)is a common process serving many important functions at various developmental stages in the life cycle of ascomycetous fungi.However,the biological roles and molecular mechanisms in plant pat...Hyphal fusion(anastomosis)is a common process serving many important functions at various developmental stages in the life cycle of ascomycetous fungi.However,the biological roles and molecular mechanisms in plant pathogenic fungi were widely unknown.In this study,a hyphal fusion protein FpHam-2 was screened from a T-DNA insertion mutant library of Fusarium pseudograminearum,and FpHam-2 interacts with another 2 hyphal fusion protein homologues FpHam-3 and FpHam-4.Each of these 3 genes deletion mutant revealed in similar defective phenotypes compared with the WT and complemented strains,including reduction in growth rate,defects in hyphal fusion and conidiation,more sensitive for cell membrane,cell wall and oxidative stress responses,and decreased in virulence.The yeast two-hybrid assay was used to identify that FpHam-2 interacts with 3 autophagy-related proteins,including FpAtg3,FpAtg28 and FpAtg33.Furthermore,FpHam-2-deletion mutant showed decreased accumulation of autophagic bodies in hypha.In conclusion,FpHam-2,FpHam-3 and FpHam-4 have an essential role for hyphal fusion and regulating the growth,conidiation and virulence in F.pseudograminearum.展开更多
Fusarium pseudograminearum is a devastating pathogen that causes Fusarium crown rot(FCR)in wheat and poses a significant threat to wheat production in terms of grain yield and quality.However,the mechanism by which F....Fusarium pseudograminearum is a devastating pathogen that causes Fusarium crown rot(FCR)in wheat and poses a significant threat to wheat production in terms of grain yield and quality.However,the mechanism by which F.pseudograminearum infects wheat remains unclear.In this study,we aimed to elucidate these mechanisms by constructing a T-DNA insertion mutant library for the highly virulent strain WZ-8A of F.pseudograminearum.By screening this mutant library,we identified nine independent mutants that displayed impaired pathogenesis in barley leaves.Among these mutants,one possessed a disruption in the gene FpRCO1 that is an ortholog of Saccharomyces cerevisiae RCO1,encoding essential component of the Rpd3S histone deacetylase complex in F.pseudograminearum.To further investigate the role of FpRCO1 in F.pseudograminearum,we employed a split-marker approach to knock out FpRCO1 in F.pseudograminearum WZ-8A.FpRCO1 deletion mutants exhibit reduced vegetative growth,conidium production,and virulence in wheat coleoptiles and barley leaves,whereas the complementary strain restores these phenotypes.Moreover,under stress conditions,the FpRCO1 deletion mutants exhibited increased sensitivity to NaCl,sorbitol,and SDS,but possessed reduced sensitivity to H_(2)O_(2)compared to these characteristics in the wild-type strain.RNA-seq analysis revealed that deletion of FpRCO1 affected gene expression(particularly the downregulation of TRI gene expression),thus resulting in significantly reduced deoxynivalenol(DON)production.In summary,our findings highlight the pivotal role of FpRCO1 in regulating vegetative growth and development,asexual reproduction,DON production,and pathogenicity of F.pseudograminearum.This study provides valuable insights into the molecular mechanisms underlying F.pseudograminearum infection in wheat and may pave the way for the development of novel strategies to combat this devastating disease.展开更多
Fusarium crown rot,mainly caused by Fusarium pseudograminearum,is a destructive disease in wheat production.To establish a rapid and reliable detection method for F.peasudeograminearum,the specific PCR primer pair(Fpg...Fusarium crown rot,mainly caused by Fusarium pseudograminearum,is a destructive disease in wheat production.To establish a rapid and reliable detection method for F.peasudeograminearum,the specific PCR primer pair(Fpg-F1;R2)was designed based on the RPB sequence,and real-time fluorescence quantitative PCR(qPCR)was used to validate the efficiency of the primer.The results showed that the primer pair had high specificity and sensitivity of 100 pg of DNA.Furthermore,the qPCR system for early and rapid detection of F.peasudeograminearum had an amplification efficiency of 87.5%and correlation coefficient of 0.99,and the pathologic threshold of F.pseudograminearum in soil was determined by using this detection system.It was found that F.pseudograminearum could cause Fusarium crown rot when the DNA concentration of F.pseudograminearum in field soil exceeded 213 pg·g^(-1).Hence,the qPCR-based method we developed for F.pseudograminearum detection has the advantages of high specificity and sensitivity,and can be used for rapid and early detection of F.pseudograminearum even in field soils.展开更多
As a main causal agent of wheat crown rot,Fusarium pseudograminearum secrets numerous proteins into the host during the infection process to regulate host immune responses and contribute to the virulence of F.pseudogr...As a main causal agent of wheat crown rot,Fusarium pseudograminearum secrets numerous proteins into the host during the infection process to regulate host immune responses and contribute to the virulence of F.pseudograminearum.In this study,the secreted protein Fp00392 from F.pseudograminearum was found to trigger cell death in Nicotiana benthamiana.Purified Fp00392 protein could activate the ROS burst,callose deposition,and the upregulation of defense-related genes in N.benthamiana.Moreover,the VIGS assay in N.benthamiana showed that Fp00392-triggered cell death is independent of BAK1 and SOBIR1.Furthermore,the transcript level of Fp00392 was significantly induced during F.pseudograminearum infection.Knockout of Fp00392 significantly attenuated the pathogenicity of F.pseudograminearum on wheat coleoptiles.Deletion of Fp00392 affected the sensitivity of F.pseudograminearum to H_(2)O_(2)and Congo Red.Overall,these results indicate that Fp00392 can not only induce plant immune response as a PAMP,but it can also promote F.pseudograminearum infection as a virulence factor.展开更多
The fungicide metconazole,which acts as a sterol 14α-demethylation inhibitor(DMI),can exhibit strong inhibitory effects on Fusarium pseudograminearum.However,the resistance mechanism as well as the risk that F.pseudo...The fungicide metconazole,which acts as a sterol 14α-demethylation inhibitor(DMI),can exhibit strong inhibitory effects on Fusarium pseudograminearum.However,the resistance mechanism as well as the risk that F.pseudograminearum develops resistance to metconazole is yet to be fully assessed.In this study,metconazole displayed a mean EC50 value of 0.0559μg/mL against 105 F.pseudograminearum isolates.Ten sensitive parental isolates were then subjected to fungicide adaptation to generate resistant mutants,with in vitro experiments subsequently highlighting the inferior fitness of the mutants.In addition,metconazole exhibited positive cross-resistance with both mefentrifluconazole and tebuconazole.Altogether,the results confirmed the low risk that F.pseudograminearum develops resistance to metconazole.Finally,a mutation genotype(M151T)was identified in FpCYP51B,with the mutants also overexpressing the FpCYP51 genes.Subsequent molecular docking and transformation-based experiments indicated that M151T substitution and overexpression in FpCYP51 genes conferred resistance to metconazole in F.pseudograminearum.Highlights The baseline sensitivity of Fusarium pseudograminearum to metconazole was established.F.pseudograminearum demonstrated a low resistance risk to metconazole.The increased expression of the CYP51 genes as well as M151T mutations in FpCYP51B could confer metconazole resistance.展开更多
C_(2)H_(2)zinc finger transcription factors such as FlbC and Msn2,have broad regulatory roles in fungal growth and conidiation.In the present study,we cloned and characterized a C_(2)H_(2)zinc finger transcription fac...C_(2)H_(2)zinc finger transcription factors such as FlbC and Msn2,have broad regulatory roles in fungal growth and conidiation.In the present study,we cloned and characterized a C_(2)H_(2)zinc finger transcription factor gene,FpCzf14,in the wheat pathogen Fusarium pseudograminearum.FpCzf14 was localized to the nuclei.The expression of FpCzf14 was significantly upregulated in conidia,suggesting that FpCzf14 might contribute to conidiation.Further analysis of the fpczf14-deleted mutant(Δfpczf14)demonstrated that it exhibited defect in conidiation,and this defect was restored in the complemented strainΔfpczf14-C expressing FpCzf14,demonstrating that FpCzf14 was essential for conidiation.Moreover,FpCzf14 was required for mycelial growth and pathogenicity of F.pseudograminearum.Microscopic observation results showed thatΔfpczf14 produced only very few penetration pegs and invasive hyphae inside host tissues compared with WT andΔfpczf14-C.Additionally,results of reverse transcription quantitative PCR(RT-qPCR)showed that FpCzf14 regulated expression of several conidiation-related genes in F.pseudograminearum.In conclusion,FpCzf14,as a core regulatory gene in conidiation,provides new insights into the mechanism of conidiation in F.pseudograminearum.展开更多
基金Supported by Funding from the Henan Provincial Scientific and Technological Breakthrough Project(No.242102111113).
文摘Fusarium crown rot(FCR),predominantly caused by Fusarium pseudograminearum,has been listed as a Category Ⅱ disease in six provinces of China,posing a significant threat to wheat production.The phenylpyrrole fungicide fludioxonil is a key agent for FCR control.Previous studies indicated that resistance to fludioxonil in F.pseudograminearum is primarily associated with altered expression levels of the FpOS1 gene,which encodes a hybrid histidine kinase.However,the roles of mutations in other FpOS genes and the molecular interactions between FpOS proteins and fludioxonil remain elusive.To address these gaps,we generated 16 fludioxonil-resistant mutants with heritable resistance traits by in vitro selection of four sensitive F.pseudograminearum isolates.These mutants exhibited high resistance levels,with resistance factors(RF)ranging from 633.73 to 8617.07.Compared to their parental isolates,the resistant mutants showed significantly reduced mycelial growth rate,sporulation capacity,and pathogenicity.They were also more sensitive to ionic,osmotic,and oxidative stresses and displayed compromised cell wall and membrane integrity.Fludioxonil demonstrated no cross-resistance with tebuconazole or pydiflumetofen;however,it exhibited weak positive crossresistance to pyraclostrobin and moderate positive cross-resistance to iprodione.Fludioxonil treatment significantly promoted glycerol synthesis and inhibited deoxynivalenol(DON)production in parental isolates,whereas these regulatory effects were markedly attenuated in the resistant mutants.Mutation analysis identified mutation sites in FpOS1,FpOS4,and FpOS5 genes,with a lower mutation frequency in FpOS1 and no mutations detected in FpOS2.Molecular docking indicated that amino acid substitutions in FpOS4 and FpOS5 significantly reduced the binding affinity of fludioxonil to these target proteins.In conclusion,F.pseudograminearum poses a moderate risk of resistance to fludioxonil.Point mutations in FpOS4 and FpOS5 genes emerge as key molecular drivers of resistance,likely by diminishing the binding affinity between the fungicide and its proteins.This study clarifies the molecular basis of fludioxonil resistance in F.pseudograminearum and provides a scientific rationale for the judicious use of this fungicide in managing FCR.
基金financially supported by the National Special Fund for Agro-scientific Research in the Public Interest of China(201503112)the Basic and Advance Technology Research Program in Henan Province,China(152300410073)the Talent Project of Henan Agricultural University,China(3600861)
文摘Wheat crown rot caused by Fusarium spp. is a common disease worldwide. Both Fusarium pseudograminearum and Fusarium graminearum infect wheat crown and produce mycotoxin leading to grain loss due to white head. F. pseudograminearum （Fp） was reported in wheat from Henan Province of China a couple of years ago. The wheat crown rot （CR） caused by this new pathogen is as an emerging severe disease of wheat, which has recently expanded to several provinces in China and is, therefore, under rapid investigation. Colonization of wheat tissue by Fp is accomplished though the formation of a septated foot-shaped appressoria and generation of a penetration peg to break through the internal cells of leaf sheath. The molecular mechanism by which Fp regulates the pathogenesis on wheat host is unclear. Here, we report FpPDE1, a P-type ATPase-encoding predicted PDE1 orthologue gene of Magnaporthe oryzae, belonging to the DRS2 subfamily of aminophospholipid translocases. The gene deletion of FpPDE1 with the split-marker approach did not obviously affect hyphae growth and conidiation, but led to an attenuated virulence on wheat base stem and root. Our finding indicates that the putative aminophospholipid translocases is not essential for the infectious hyphae development in Fp.
基金supported by the grants from the National Natural Science Foundation of China(U2004140)the Henan Provincial Science and Technology Major Project,China(221100110100)。
文摘Hyphal fusion(anastomosis)is a common process serving many important functions at various developmental stages in the life cycle of ascomycetous fungi.However,the biological roles and molecular mechanisms in plant pathogenic fungi were widely unknown.In this study,a hyphal fusion protein FpHam-2 was screened from a T-DNA insertion mutant library of Fusarium pseudograminearum,and FpHam-2 interacts with another 2 hyphal fusion protein homologues FpHam-3 and FpHam-4.Each of these 3 genes deletion mutant revealed in similar defective phenotypes compared with the WT and complemented strains,including reduction in growth rate,defects in hyphal fusion and conidiation,more sensitive for cell membrane,cell wall and oxidative stress responses,and decreased in virulence.The yeast two-hybrid assay was used to identify that FpHam-2 interacts with 3 autophagy-related proteins,including FpAtg3,FpAtg28 and FpAtg33.Furthermore,FpHam-2-deletion mutant showed decreased accumulation of autophagic bodies in hypha.In conclusion,FpHam-2,FpHam-3 and FpHam-4 have an essential role for hyphal fusion and regulating the growth,conidiation and virulence in F.pseudograminearum.
基金supported by grants from the National Natural Science Foundation of China(31901835)the Science and Technology Planning Project of Henan Province of China(212102110145)the International(Regional)Cooperation and Exchange Program of the National Natural Science Foundation of China(31961143018).
文摘Fusarium pseudograminearum is a devastating pathogen that causes Fusarium crown rot(FCR)in wheat and poses a significant threat to wheat production in terms of grain yield and quality.However,the mechanism by which F.pseudograminearum infects wheat remains unclear.In this study,we aimed to elucidate these mechanisms by constructing a T-DNA insertion mutant library for the highly virulent strain WZ-8A of F.pseudograminearum.By screening this mutant library,we identified nine independent mutants that displayed impaired pathogenesis in barley leaves.Among these mutants,one possessed a disruption in the gene FpRCO1 that is an ortholog of Saccharomyces cerevisiae RCO1,encoding essential component of the Rpd3S histone deacetylase complex in F.pseudograminearum.To further investigate the role of FpRCO1 in F.pseudograminearum,we employed a split-marker approach to knock out FpRCO1 in F.pseudograminearum WZ-8A.FpRCO1 deletion mutants exhibit reduced vegetative growth,conidium production,and virulence in wheat coleoptiles and barley leaves,whereas the complementary strain restores these phenotypes.Moreover,under stress conditions,the FpRCO1 deletion mutants exhibited increased sensitivity to NaCl,sorbitol,and SDS,but possessed reduced sensitivity to H_(2)O_(2)compared to these characteristics in the wild-type strain.RNA-seq analysis revealed that deletion of FpRCO1 affected gene expression(particularly the downregulation of TRI gene expression),thus resulting in significantly reduced deoxynivalenol(DON)production.In summary,our findings highlight the pivotal role of FpRCO1 in regulating vegetative growth and development,asexual reproduction,DON production,and pathogenicity of F.pseudograminearum.This study provides valuable insights into the molecular mechanisms underlying F.pseudograminearum infection in wheat and may pave the way for the development of novel strategies to combat this devastating disease.
基金Yong Science and Technology Talent of AAAS(QNYC-201911)。
文摘Fusarium crown rot,mainly caused by Fusarium pseudograminearum,is a destructive disease in wheat production.To establish a rapid and reliable detection method for F.peasudeograminearum,the specific PCR primer pair(Fpg-F1;R2)was designed based on the RPB sequence,and real-time fluorescence quantitative PCR(qPCR)was used to validate the efficiency of the primer.The results showed that the primer pair had high specificity and sensitivity of 100 pg of DNA.Furthermore,the qPCR system for early and rapid detection of F.peasudeograminearum had an amplification efficiency of 87.5%and correlation coefficient of 0.99,and the pathologic threshold of F.pseudograminearum in soil was determined by using this detection system.It was found that F.pseudograminearum could cause Fusarium crown rot when the DNA concentration of F.pseudograminearum in field soil exceeded 213 pg·g^(-1).Hence,the qPCR-based method we developed for F.pseudograminearum detection has the advantages of high specificity and sensitivity,and can be used for rapid and early detection of F.pseudograminearum even in field soils.
基金supported by the National Natural Science Foundation of China(32102153)the Natural Science Foundation of Anhui Higher Education Institutions,China(2023AH040139)the Talent Research Project of Anhui Agricultural University,China(rc342218)。
文摘As a main causal agent of wheat crown rot,Fusarium pseudograminearum secrets numerous proteins into the host during the infection process to regulate host immune responses and contribute to the virulence of F.pseudograminearum.In this study,the secreted protein Fp00392 from F.pseudograminearum was found to trigger cell death in Nicotiana benthamiana.Purified Fp00392 protein could activate the ROS burst,callose deposition,and the upregulation of defense-related genes in N.benthamiana.Moreover,the VIGS assay in N.benthamiana showed that Fp00392-triggered cell death is independent of BAK1 and SOBIR1.Furthermore,the transcript level of Fp00392 was significantly induced during F.pseudograminearum infection.Knockout of Fp00392 significantly attenuated the pathogenicity of F.pseudograminearum on wheat coleoptiles.Deletion of Fp00392 affected the sensitivity of F.pseudograminearum to H_(2)O_(2)and Congo Red.Overall,these results indicate that Fp00392 can not only induce plant immune response as a PAMP,but it can also promote F.pseudograminearum infection as a virulence factor.
基金funded by National Key Research and Development Program of China(2022YFD1400900).
文摘The fungicide metconazole,which acts as a sterol 14α-demethylation inhibitor(DMI),can exhibit strong inhibitory effects on Fusarium pseudograminearum.However,the resistance mechanism as well as the risk that F.pseudograminearum develops resistance to metconazole is yet to be fully assessed.In this study,metconazole displayed a mean EC50 value of 0.0559μg/mL against 105 F.pseudograminearum isolates.Ten sensitive parental isolates were then subjected to fungicide adaptation to generate resistant mutants,with in vitro experiments subsequently highlighting the inferior fitness of the mutants.In addition,metconazole exhibited positive cross-resistance with both mefentrifluconazole and tebuconazole.Altogether,the results confirmed the low risk that F.pseudograminearum develops resistance to metconazole.Finally,a mutation genotype(M151T)was identified in FpCYP51B,with the mutants also overexpressing the FpCYP51 genes.Subsequent molecular docking and transformation-based experiments indicated that M151T substitution and overexpression in FpCYP51 genes conferred resistance to metconazole in F.pseudograminearum.Highlights The baseline sensitivity of Fusarium pseudograminearum to metconazole was established.F.pseudograminearum demonstrated a low resistance risk to metconazole.The increased expression of the CYP51 genes as well as M151T mutations in FpCYP51B could confer metconazole resistance.
基金supported by grants from the International(Regional)Cooperation and Exchange Program of National Natural Science Foundation of China(31961143018)the National Key R&D Plan of China(2017YFD0301104).
文摘C_(2)H_(2)zinc finger transcription factors such as FlbC and Msn2,have broad regulatory roles in fungal growth and conidiation.In the present study,we cloned and characterized a C_(2)H_(2)zinc finger transcription factor gene,FpCzf14,in the wheat pathogen Fusarium pseudograminearum.FpCzf14 was localized to the nuclei.The expression of FpCzf14 was significantly upregulated in conidia,suggesting that FpCzf14 might contribute to conidiation.Further analysis of the fpczf14-deleted mutant(Δfpczf14)demonstrated that it exhibited defect in conidiation,and this defect was restored in the complemented strainΔfpczf14-C expressing FpCzf14,demonstrating that FpCzf14 was essential for conidiation.Moreover,FpCzf14 was required for mycelial growth and pathogenicity of F.pseudograminearum.Microscopic observation results showed thatΔfpczf14 produced only very few penetration pegs and invasive hyphae inside host tissues compared with WT andΔfpczf14-C.Additionally,results of reverse transcription quantitative PCR(RT-qPCR)showed that FpCzf14 regulated expression of several conidiation-related genes in F.pseudograminearum.In conclusion,FpCzf14,as a core regulatory gene in conidiation,provides new insights into the mechanism of conidiation in F.pseudograminearum.
