Mycoviruses are viruses that infect fungi,and hypovirulence-associated mycoviruses have the potential to control fungal diseases.However,it is unclear how mycovirus-mediated hypovirulent strains live and survive in th...Mycoviruses are viruses that infect fungi,and hypovirulence-associated mycoviruses have the potential to control fungal diseases.However,it is unclear how mycovirus-mediated hypovirulent strains live and survive in the field,and no mycovirus has been applied for field crop protection.In this study,we found that a previously identified small DNA mycovirus(SsHADV-1)can convert its host,Sclerotinia sclerotiorum,from a typical necrotrophic pathogen to a beneficial endophytic fungus.SsHADV-1 downregulates the expression of key pathogenicity factor genes in S.sclerotiorum during infection.When growing in rapeseed,the SsHADV-1-infected strain DT-8 significantly regulates the expression of rapeseed genes involved in defense,hormone signaling,and circadian rhythm pathways.As a result,plant growth is promoted and disease resistance is enhanced.Field experiments showed that spraying DT-8 at the early flowering stage can reduce the disease severity of rapeseed stem rot by 67.6%and improve yield by 14.9%.Moreover,we discovered that SsHADV-1 could also infect other S.sclerotiorum strains on DT-8-inoculated plants and that DT-8 could be recovered from dead plants.These findings suggest that the mycoviruses may have the ability to shape the origin of endophytism.Our discoveries suggest that mycoviruses may influence the origin of endophytism and may also offer a novel strategy for disease control in which mycovirus-infected strains are used to improve crop health and release mycoviruses into the field.展开更多
: The genetic diversity and genetic structure of a population of isolates of Sclerotinia sclerotiorum (Lib.) de Bary from different regions and host plants were investigated using the random amplified polymorphic DNA ...: The genetic diversity and genetic structure of a population of isolates of Sclerotinia sclerotiorum (Lib.) de Bary from different regions and host plants were investigated using the random amplified polymorphic DNA (RAPD) method with 20 random decamer primer pairs in order to provide some information on the phylogenetic taxa and breeding for resistance to sclerotinia stem rot. A minimum of three and a maximum of 15 unambiguously amplified bands were generated, furnishing a total of 170 bands ranging in size from 100 to 3 200 bp, corresponding to an average of 8.5 bands per primer pair. One hundred and four of these 170 bands (61.2%) were polymorphic, the percentage of polymorphic bands for each primer pair ranging from 0.0% to 86.7%. The genetic relationships among the isolates, based on the results of RAPD analysis, were examined. The genetic similarity of all selected isolates was quite high. At the species level, the genetic diversity estimated by Nei's gene diversity (h) was 0.197 and Shannon's index of diversity (I) was 0.300. The unweighted pair-group mean analysis (UPGMA) cluster analysis showed that most isolates from the same regions were grouped in the same cluster or a close cluster. The population of isolates from Hefei (Anhui Province, China) was more uniform and relatively distant to other populations. The Canadian population collected from carrot (Daucus carota var. sativa DC.) was relatively close to the Polish population collected from oilseed rape (Brassica napus L.) plants. There was no relationship between isolates from the same host plants. An analysis of molecular variance (AMOVA) revealed that the percentage of variance attributable to variation among and within populations was 50.62% and 49.38%, respectively. When accessions from China, Europe, and Canada were treated as three separate groups, the variance components among groups, among populations within groups, and within populations were ?0.96%, 51.48%, and 49.47%, respectively. The genetic differentiations among and within population展开更多
Zhongshuang9, a new semi-winter Brassica napus variety with high resistance to Sclerotinia sclerotiorum and lodging, high-yield, double-low quality and extensive adaptability, was bred by multiple crossing and microsp...Zhongshuang9, a new semi-winter Brassica napus variety with high resistance to Sclerotinia sclerotiorum and lodging, high-yield, double-low quality and extensive adaptability, was bred by multiple crossing and microspore culture technique. It was registered and released in China in 2002. In regional trial of Hubei Province in China, Zhongshuang9 yielded 2 482. 2 kg ha-1 averagely in 2000 - 2002, 15. 33% higher than the control variety Zhongyou821. Erucic acid, glucosinolates and oil contents of Zhongshuang9 were 0.23%, 22.69 μmol g-1(in meal)and 42%, respectively. In field assessment of resistance to S. Sclerotiorum , the disease incidence and disease index of Zhongshuang9 averaged 13.31 % and 6.47, respectively, which were lower than those of Zhongyou821 by 28% and 36%, respectively. After inoculation of detached leaves with mycelia, the lesion size of Zhongshuang9 was 4. 709 cm2, which was significantly smaller than that of the mid-resistant variety Zhongyou821(5. 933 cm2). The stem lesion length of Zhongshuang9 after match-stick inoculation was 1.275 cm, which was significantly lower than that of Zhongyou821(1.943 cm). The possible mechanism of resistance to S. sclerotiorum was studied through comparing the activities of phenylalanine ammonia lyase(PAL), exo-chitinase, β-1, 3-glucanase, peroxidase(POD)and polyphenoloxidase(PPO)in Zhongshuang9 with those in other resistant, mid-resistant and susceptible cultivars.展开更多
The fungal pathogen Sclerotinia sclerotiorum Libert causes rot diseases on many crops worldwide and large economic losses occur frequently because of a lack of resistant varieties. The pathogenesis of S. sclerotiorum ...The fungal pathogen Sclerotinia sclerotiorum Libert causes rot diseases on many crops worldwide and large economic losses occur frequently because of a lack of resistant varieties. The pathogenesis of S. sclerotiorum and the molecular basis of plant responses to the pathogen are poorly understood. In the present investigation, the process of S. sclerotiorum infection in Arabidopsis thaliana L., a plant that is highly susceptible to this fungus, was analysed. In addition, the defense activation in the host was investigated. A convenient inoculation method using millet grain was developed for S. sclerotiorum in Arabidopsis. The fungus rapidly infected the plants, probably through ball- or cushion-like infection structures. Visible symptoms developed within 24 h and plants were killed 72 h after inoculation. Cellulase, the main enzyme that caused host tissues to rot, was secreted by S. sclerotiorum in a pH-dependent manner. Oxalic acid, another pathogenic factor secreted by the fungus, induced necrotic lesions on the leaves, infection with S. sclerotiorum strongly induced the production of the pathogenesis-related （PR） proteins β-1,3-glucanase and chitinase in Arabidopsis. Furthermore, the PR gene PDF. 1 was induced, but not PR1, indicating that the pathogen activated basal defense of jasmonic acid/ethylene dependence, which is consistent with its necrotrophic characteristics. This pathosystem for Arabidopsis-S. sclerotiorum could provide an approach for the analysis of the interactions between S. sclerotiorum and other crops, thereby facilitating genetic manipulation techniques for controlling this pathogen.展开更多
The cause of virulence decline of hypovirulent isolate Ep-1PN of Sclerotinia sclerotiorum (Lib.) de Bary has been studied through transmitting test and analysing its offsprings derived from single ascospore. When the ...The cause of virulence decline of hypovirulent isolate Ep-1PN of Sclerotinia sclerotiorum (Lib.) de Bary has been studied through transmitting test and analysing its offsprings derived from single ascospore. When the colony of a normal strain Ep-1PNA 183 intermingled with the colony of Ep-1PN, its margin became abnormal, just like Ep-1PN. Cultures isolated from top-tip of the colony of Ep-1PNA 183 showed the hypovirulence and its associated properties of Ep-1PN. Moreover, a series of cultures which have characteristics between Ep-1PN and normal strain could be isolated from stipes of Ep-1PN. More than 200 offsprings derived from single ascospore of Ep-1PN were normal strains. According to those results, there exists a transmissible cytoplasmic hypovirulent element in hypovirulent isolate Ep-1PN of S. sclerotiorum.展开更多
Sclerotinia sclerotiorum is one of the most devastating necrotrophic phytopathogens. Virulence of the hyphae of this fungus at different ages varies significantly. Molecular mechanisms underlying this functional disti...Sclerotinia sclerotiorum is one of the most devastating necrotrophic phytopathogens. Virulence of the hyphae of this fungus at different ages varies significantly. Molecular mechanisms underlying this functional distinction are largely unknown. In this study, we confirmed the effect of mycelial culture time/age on virulence in two host plants and elucidated its molecular and morphological basis. The virulence of the S. sclerotiorum mycelia in plants dramatically decreases along with the increase of the mycelial age. Three-day-old mycelia lost the virulence in plants. Comparative proteomics analyses revealed that metabolism pathways were comprehensively reprogrammed to suppress the oxalic acid（OA） accumulation in old mycelia. The oxaloacetate acetylhydrolase（OAH）, which catalyzes OA biosynthesis, was identified in the S. sclerotiorum genome. Both gene expression and protein accumulation of OAH in old mycelia were strongly repressed. Moreover, in planta OA accumulation was strikingly reduced in old mycelia-inoculated plants compared with young vegetative mycelia-inoculated plants. Furthermore, supply with 10 mmol L^（-1） OA enabled the old mycelia infect the host plants, demonstrating that loss of virulence of old mycelia is mainly caused by being unable to accumulate OA. Additionally, aerial mycelia started to develop from 0.5-day-old vegetative mycelia and dominated over 1-day-old mycelia grown on potato dextrose agar plates. They were much smaller in hypha diameter and grew significantly slower than young vegetative mycelia when subcultured, which did not maintain to progenies. Collectively, our results reveal that S. sclerotiorum aerial hyphae-dominant old mycelia fail to accumulate OA and thereby lose the virulence in host plants.展开更多
With the development of tourism at home and abroad,Rapeseed(Brassica napus)has become an important ornamental plant.However,its ornamental value at the inflorescence stage is greatly reduced by Sclerotinia sclerotioru...With the development of tourism at home and abroad,Rapeseed(Brassica napus)has become an important ornamental plant.However,its ornamental value at the inflorescence stage is greatly reduced by Sclerotinia sclerotiorum.Identification of important genes in the defense responses is critical for molecular breeding,which is an important strategy for controlling the disease.In this study,we isolated a B.napus WRKY transcription factor gene,BnaWRKY75.BnaWRKY75 was found to encode a nucleus-localized protein and exhibited relatively high expression in the stems.Arabidopsis thaliana transgenic plants expressing BnaWRKY75 showed enhanced resistance to S.sclerotiorum,and both ProBnaWRKY75:GUS and gene expression analyses showed that BnaWRKY75 was highly responsive to S.sclerotiorum infection,indicating the involvement of BnaWRKY75 in response to this infection.Furthermore,overexpression(OE)of BnaWRKY75 in B.napus significantly enhanced the resistance to S.sclerotiorum,whereas the resistance was reduced in RNAi transgenic B.napus plants.Moreover,the BnaWRKY75-OE B.napus plants exhibited constitutive activation of salicylic acid-,jasmonic acid-,and ethylene-mediated defense responses and the inhibition of both H_(2)O_(2)and O_(2)·^(-)accumulation in response to this pathogen.By contrast,BnaWRKY75-RNAi plants showed a reverse pattern,suggesting that BnaWRKY75 is involved in hormonal signaling pathways and in the control of reactive oxygen species accumulation.In conclusion,these data indicate that BnaWRKY75,a regulator of multiple defense responses,positively regulates resistance against S.sclerotiorum,which may guide the improvement of resistance in rapeseed.展开更多
基金the National Science Foundation of China(31430070)the National Key R&D Program(2017YFD0200602)the China Agriculture Research System(CARS-12).
文摘Mycoviruses are viruses that infect fungi,and hypovirulence-associated mycoviruses have the potential to control fungal diseases.However,it is unclear how mycovirus-mediated hypovirulent strains live and survive in the field,and no mycovirus has been applied for field crop protection.In this study,we found that a previously identified small DNA mycovirus(SsHADV-1)can convert its host,Sclerotinia sclerotiorum,from a typical necrotrophic pathogen to a beneficial endophytic fungus.SsHADV-1 downregulates the expression of key pathogenicity factor genes in S.sclerotiorum during infection.When growing in rapeseed,the SsHADV-1-infected strain DT-8 significantly regulates the expression of rapeseed genes involved in defense,hormone signaling,and circadian rhythm pathways.As a result,plant growth is promoted and disease resistance is enhanced.Field experiments showed that spraying DT-8 at the early flowering stage can reduce the disease severity of rapeseed stem rot by 67.6%and improve yield by 14.9%.Moreover,we discovered that SsHADV-1 could also infect other S.sclerotiorum strains on DT-8-inoculated plants and that DT-8 could be recovered from dead plants.These findings suggest that the mycoviruses may have the ability to shape the origin of endophytism.Our discoveries suggest that mycoviruses may influence the origin of endophytism and may also offer a novel strategy for disease control in which mycovirus-infected strains are used to improve crop health and release mycoviruses into the field.
基金Basic Research and Development Plan of China，国家科技攻关项目，the Sino-Polish Scientific and Technological Cooperation Project for 2004-2006，中国科学院资助项目
文摘: The genetic diversity and genetic structure of a population of isolates of Sclerotinia sclerotiorum (Lib.) de Bary from different regions and host plants were investigated using the random amplified polymorphic DNA (RAPD) method with 20 random decamer primer pairs in order to provide some information on the phylogenetic taxa and breeding for resistance to sclerotinia stem rot. A minimum of three and a maximum of 15 unambiguously amplified bands were generated, furnishing a total of 170 bands ranging in size from 100 to 3 200 bp, corresponding to an average of 8.5 bands per primer pair. One hundred and four of these 170 bands (61.2%) were polymorphic, the percentage of polymorphic bands for each primer pair ranging from 0.0% to 86.7%. The genetic relationships among the isolates, based on the results of RAPD analysis, were examined. The genetic similarity of all selected isolates was quite high. At the species level, the genetic diversity estimated by Nei's gene diversity (h) was 0.197 and Shannon's index of diversity (I) was 0.300. The unweighted pair-group mean analysis (UPGMA) cluster analysis showed that most isolates from the same regions were grouped in the same cluster or a close cluster. The population of isolates from Hefei (Anhui Province, China) was more uniform and relatively distant to other populations. The Canadian population collected from carrot (Daucus carota var. sativa DC.) was relatively close to the Polish population collected from oilseed rape (Brassica napus L.) plants. There was no relationship between isolates from the same host plants. An analysis of molecular variance (AMOVA) revealed that the percentage of variance attributable to variation among and within populations was 50.62% and 49.38%, respectively. When accessions from China, Europe, and Canada were treated as three separate groups, the variance components among groups, among populations within groups, and within populations were ?0.96%, 51.48%, and 49.47%, respectively. The genetic differentiations among and within population
文摘Zhongshuang9, a new semi-winter Brassica napus variety with high resistance to Sclerotinia sclerotiorum and lodging, high-yield, double-low quality and extensive adaptability, was bred by multiple crossing and microspore culture technique. It was registered and released in China in 2002. In regional trial of Hubei Province in China, Zhongshuang9 yielded 2 482. 2 kg ha-1 averagely in 2000 - 2002, 15. 33% higher than the control variety Zhongyou821. Erucic acid, glucosinolates and oil contents of Zhongshuang9 were 0.23%, 22.69 μmol g-1(in meal)and 42%, respectively. In field assessment of resistance to S. Sclerotiorum , the disease incidence and disease index of Zhongshuang9 averaged 13.31 % and 6.47, respectively, which were lower than those of Zhongyou821 by 28% and 36%, respectively. After inoculation of detached leaves with mycelia, the lesion size of Zhongshuang9 was 4. 709 cm2, which was significantly smaller than that of the mid-resistant variety Zhongyou821(5. 933 cm2). The stem lesion length of Zhongshuang9 after match-stick inoculation was 1.275 cm, which was significantly lower than that of Zhongyou821(1.943 cm). The possible mechanism of resistance to S. sclerotiorum was studied through comparing the activities of phenylalanine ammonia lyase(PAL), exo-chitinase, β-1, 3-glucanase, peroxidase(POD)and polyphenoloxidase(PPO)in Zhongshuang9 with those in other resistant, mid-resistant and susceptible cultivars.
基金Supported by the National Natural Science Foundation of China （30125030） and the Knowledge Innovation Program of the Chinese Academy of Sciences （KSCX2-SW-301-02）.
文摘The fungal pathogen Sclerotinia sclerotiorum Libert causes rot diseases on many crops worldwide and large economic losses occur frequently because of a lack of resistant varieties. The pathogenesis of S. sclerotiorum and the molecular basis of plant responses to the pathogen are poorly understood. In the present investigation, the process of S. sclerotiorum infection in Arabidopsis thaliana L., a plant that is highly susceptible to this fungus, was analysed. In addition, the defense activation in the host was investigated. A convenient inoculation method using millet grain was developed for S. sclerotiorum in Arabidopsis. The fungus rapidly infected the plants, probably through ball- or cushion-like infection structures. Visible symptoms developed within 24 h and plants were killed 72 h after inoculation. Cellulase, the main enzyme that caused host tissues to rot, was secreted by S. sclerotiorum in a pH-dependent manner. Oxalic acid, another pathogenic factor secreted by the fungus, induced necrotic lesions on the leaves, infection with S. sclerotiorum strongly induced the production of the pathogenesis-related （PR） proteins β-1,3-glucanase and chitinase in Arabidopsis. Furthermore, the PR gene PDF. 1 was induced, but not PR1, indicating that the pathogen activated basal defense of jasmonic acid/ethylene dependence, which is consistent with its necrotrophic characteristics. This pathosystem for Arabidopsis-S. sclerotiorum could provide an approach for the analysis of the interactions between S. sclerotiorum and other crops, thereby facilitating genetic manipulation techniques for controlling this pathogen.
文摘The cause of virulence decline of hypovirulent isolate Ep-1PN of Sclerotinia sclerotiorum (Lib.) de Bary has been studied through transmitting test and analysing its offsprings derived from single ascospore. When the colony of a normal strain Ep-1PNA 183 intermingled with the colony of Ep-1PN, its margin became abnormal, just like Ep-1PN. Cultures isolated from top-tip of the colony of Ep-1PNA 183 showed the hypovirulence and its associated properties of Ep-1PN. Moreover, a series of cultures which have characteristics between Ep-1PN and normal strain could be isolated from stipes of Ep-1PN. More than 200 offsprings derived from single ascospore of Ep-1PN were normal strains. According to those results, there exists a transmissible cytoplasmic hypovirulent element in hypovirulent isolate Ep-1PN of S. sclerotiorum.
基金supported by grants from the Special Fund for Agro-Scientific Research in the Public Interest, China (201103016)the Specialized Research Fund for the Doctoral Program of Higher Education, China (SRFDP) (20110101110092)+2 种基金the National Natural Science Foundation of China (31371892)the Program for New Century Excellent Talents in University (NCET-08-0485)the Program for New Century 151 Talents of Zhejiang Province, China
文摘Sclerotinia sclerotiorum is one of the most devastating necrotrophic phytopathogens. Virulence of the hyphae of this fungus at different ages varies significantly. Molecular mechanisms underlying this functional distinction are largely unknown. In this study, we confirmed the effect of mycelial culture time/age on virulence in two host plants and elucidated its molecular and morphological basis. The virulence of the S. sclerotiorum mycelia in plants dramatically decreases along with the increase of the mycelial age. Three-day-old mycelia lost the virulence in plants. Comparative proteomics analyses revealed that metabolism pathways were comprehensively reprogrammed to suppress the oxalic acid（OA） accumulation in old mycelia. The oxaloacetate acetylhydrolase（OAH）, which catalyzes OA biosynthesis, was identified in the S. sclerotiorum genome. Both gene expression and protein accumulation of OAH in old mycelia were strongly repressed. Moreover, in planta OA accumulation was strikingly reduced in old mycelia-inoculated plants compared with young vegetative mycelia-inoculated plants. Furthermore, supply with 10 mmol L^（-1） OA enabled the old mycelia infect the host plants, demonstrating that loss of virulence of old mycelia is mainly caused by being unable to accumulate OA. Additionally, aerial mycelia started to develop from 0.5-day-old vegetative mycelia and dominated over 1-day-old mycelia grown on potato dextrose agar plates. They were much smaller in hypha diameter and grew significantly slower than young vegetative mycelia when subcultured, which did not maintain to progenies. Collectively, our results reveal that S. sclerotiorum aerial hyphae-dominant old mycelia fail to accumulate OA and thereby lose the virulence in host plants.
基金supported by the National Natural Science Foundation of China(Grant No.31771836)the National Key Research and Development Program of China(Grant No.2018YFD0201003).
文摘With the development of tourism at home and abroad,Rapeseed(Brassica napus)has become an important ornamental plant.However,its ornamental value at the inflorescence stage is greatly reduced by Sclerotinia sclerotiorum.Identification of important genes in the defense responses is critical for molecular breeding,which is an important strategy for controlling the disease.In this study,we isolated a B.napus WRKY transcription factor gene,BnaWRKY75.BnaWRKY75 was found to encode a nucleus-localized protein and exhibited relatively high expression in the stems.Arabidopsis thaliana transgenic plants expressing BnaWRKY75 showed enhanced resistance to S.sclerotiorum,and both ProBnaWRKY75:GUS and gene expression analyses showed that BnaWRKY75 was highly responsive to S.sclerotiorum infection,indicating the involvement of BnaWRKY75 in response to this infection.Furthermore,overexpression(OE)of BnaWRKY75 in B.napus significantly enhanced the resistance to S.sclerotiorum,whereas the resistance was reduced in RNAi transgenic B.napus plants.Moreover,the BnaWRKY75-OE B.napus plants exhibited constitutive activation of salicylic acid-,jasmonic acid-,and ethylene-mediated defense responses and the inhibition of both H_(2)O_(2)and O_(2)·^(-)accumulation in response to this pathogen.By contrast,BnaWRKY75-RNAi plants showed a reverse pattern,suggesting that BnaWRKY75 is involved in hormonal signaling pathways and in the control of reactive oxygen species accumulation.In conclusion,these data indicate that BnaWRKY75,a regulator of multiple defense responses,positively regulates resistance against S.sclerotiorum,which may guide the improvement of resistance in rapeseed.
