In order to optimize polymerase chain reaction（PCR） amplification of the internal transcribed spacer（ITS） rDNA region from fungi found in black soil in North China,an orthogonal experimental design [L16（45）] was...In order to optimize polymerase chain reaction（PCR） amplification of the internal transcribed spacer（ITS） rDNA region from fungi found in black soil in North China,an orthogonal experimental design [L16（45）] was used to evaluate five factors（template,Mg2＋,dNTP,Taq DNA polymerase,and primer） from four levels.Subsequently,the optimal annealing temperature,annealing time,extension time and cycle numbers were evaluated.The results showed that the optimized PCR solution for amplification of ITS region comprised 5 μL 10× buffer,30 ng soil DNA template,3.0 mmol·L-1 Mg2＋,0.2 mmol·L-1 dNTPs,0.1 μmol·L-1 each forward and reverse primer,and 2.0 U Taq enzyme in 50 μL reaction volume.The optimal thermal cycling protocol consisted of initial melting at 94℃ for 5 min,followed by 35 cycles at 94℃ for 30 s,56℃ for 30 s,72℃ for 90 s,and a final extension of 72℃ for 10 min.展开更多
While Ramularia leaf blight of cotton caused by Ramularia areola is of top most importance for Brazil, information is lacking regarding the survival mechanism of this pathogen during the cotton-free period. The teleom...While Ramularia leaf blight of cotton caused by Ramularia areola is of top most importance for Brazil, information is lacking regarding the survival mechanism of this pathogen during the cotton-free period. The teleomorph of R. areola is expected to belong to the genus Mycosphaerella. In the present study attempts were made to verify occurrence of this teleomorph in the State of Mato Grosso, Brazil. Decaying cotton leaves were collected two months after harvest of 2014 from 44 commercial and experimental fields where aerial fungicidal applications were made or not during the crop cycle to control the Ramularia leaf blight. Examination of the decaying cotton leaves revealed presence of abundant sclerotia, spermagonia and ascoma of Mycosphaerella sp. intermingled with each other during the cotton-free period in most of the leaf samples. Mono-ascospore isolations were obtained from the ascoma and considering their cultural, morphological, pathological and DNA sequence analysis they were identified as Mycosphaerella areola. M. areola and R. areola isolates produced similar symptoms under glasshouse inoculations. Reisolation of the pathogen from the symptoms produced by M. areola isolates yielded R. areola. Some cotton leaves showing such symptoms were kept on the soil surface on plastic trays for two months under natural field condition. After this period the decaying leaves showed abundant perithecia identical to their original M. areola. ITS rDNA sequence analyses revealed identical sequences from M. areola and R. areola isolates. Occurrence and the viability of the perfect stage M. areola during the cotton-free period on the left-over stubble from one season to another were interpreted as the survival mechanism of the pathogen and were considered responsible for the Ramularia blight epidemics in the State of Mato Grosso. Disease management practices for the State of Mato Grosso are discussed. This is the first report about the occurrence of the M. areola in Brazil.展开更多
The unmanaged Norway spruce montane forests in the Bohemian Forest National Park(Czech Republic)suffered from repeated large-scale bark beetle outbreaks in last decade.In this study,the diversity of culturable foliar ...The unmanaged Norway spruce montane forests in the Bohemian Forest National Park(Czech Republic)suffered from repeated large-scale bark beetle outbreaks in last decade.In this study,the diversity of culturable foliar endophyte microfungi in needles originating from eleven recently wind-fallen trees in this area was surveyed.Our aims were to describe their diversity and to determine the relative host and organ specificities of isolated endophyte species to estimate the species pool and abundance of foliar endophytes before the forest degradation.Microfungi were isolated from surface-sterilized needles,and the outgrowing strains were identified based on morphological and molecular characteristics(analyses of ITS1,ITS2 and partial 28S rDNA).Fungal communities in the needles were diverse,with ascomycetes(mostly anamorphs of Helotiales)dominating basidiomycetes.The most frequent species(Phacidiopycnis sp.,Cistella acuum,Sirococcus sp.and two species of Chalara)did not correspond with those recorded in previous studies of Picea spp.For example,the widely distributed Lophodermium piceae was rarely recorded in this study.This pattern may be caused by different methods of sterilization and cultivation or by physiological characteristics of the needles,or it may reflect the species distribution in the studied area.Members of the Helotiales,along with sequences from GenBank,showed substantial overlap in host affinities,most prominently between Pinus and Picea,and also among species from distant plant lineages.展开更多
基金Supported by National Natural Science Foundation of China (30571264)Nation Key Technology Program (2006BAD07B03)
文摘In order to optimize polymerase chain reaction（PCR） amplification of the internal transcribed spacer（ITS） rDNA region from fungi found in black soil in North China,an orthogonal experimental design [L16（45）] was used to evaluate five factors（template,Mg2＋,dNTP,Taq DNA polymerase,and primer） from four levels.Subsequently,the optimal annealing temperature,annealing time,extension time and cycle numbers were evaluated.The results showed that the optimized PCR solution for amplification of ITS region comprised 5 μL 10× buffer,30 ng soil DNA template,3.0 mmol·L-1 Mg2＋,0.2 mmol·L-1 dNTPs,0.1 μmol·L-1 each forward and reverse primer,and 2.0 U Taq enzyme in 50 μL reaction volume.The optimal thermal cycling protocol consisted of initial melting at 94℃ for 5 min,followed by 35 cycles at 94℃ for 30 s,56℃ for 30 s,72℃ for 90 s,and a final extension of 72℃ for 10 min.
文摘While Ramularia leaf blight of cotton caused by Ramularia areola is of top most importance for Brazil, information is lacking regarding the survival mechanism of this pathogen during the cotton-free period. The teleomorph of R. areola is expected to belong to the genus Mycosphaerella. In the present study attempts were made to verify occurrence of this teleomorph in the State of Mato Grosso, Brazil. Decaying cotton leaves were collected two months after harvest of 2014 from 44 commercial and experimental fields where aerial fungicidal applications were made or not during the crop cycle to control the Ramularia leaf blight. Examination of the decaying cotton leaves revealed presence of abundant sclerotia, spermagonia and ascoma of Mycosphaerella sp. intermingled with each other during the cotton-free period in most of the leaf samples. Mono-ascospore isolations were obtained from the ascoma and considering their cultural, morphological, pathological and DNA sequence analysis they were identified as Mycosphaerella areola. M. areola and R. areola isolates produced similar symptoms under glasshouse inoculations. Reisolation of the pathogen from the symptoms produced by M. areola isolates yielded R. areola. Some cotton leaves showing such symptoms were kept on the soil surface on plastic trays for two months under natural field condition. After this period the decaying leaves showed abundant perithecia identical to their original M. areola. ITS rDNA sequence analyses revealed identical sequences from M. areola and R. areola isolates. Occurrence and the viability of the perfect stage M. areola during the cotton-free period on the left-over stubble from one season to another were interpreted as the survival mechanism of the pathogen and were considered responsible for the Ramularia blight epidemics in the State of Mato Grosso. Disease management practices for the State of Mato Grosso are discussed. This is the first report about the occurrence of the M. areola in Brazil.
基金supported by the Grant Agency of the Czech Republic(project Nr.526/08/0751)the Ministry of Education,Youth and Sports of the Czech Republic(project No.MSM0021620828)the Institutional Research Concept of the Institute of Microbiology of the ASCR,v.v.i.(AV0Z50200510).
文摘The unmanaged Norway spruce montane forests in the Bohemian Forest National Park(Czech Republic)suffered from repeated large-scale bark beetle outbreaks in last decade.In this study,the diversity of culturable foliar endophyte microfungi in needles originating from eleven recently wind-fallen trees in this area was surveyed.Our aims were to describe their diversity and to determine the relative host and organ specificities of isolated endophyte species to estimate the species pool and abundance of foliar endophytes before the forest degradation.Microfungi were isolated from surface-sterilized needles,and the outgrowing strains were identified based on morphological and molecular characteristics(analyses of ITS1,ITS2 and partial 28S rDNA).Fungal communities in the needles were diverse,with ascomycetes(mostly anamorphs of Helotiales)dominating basidiomycetes.The most frequent species(Phacidiopycnis sp.,Cistella acuum,Sirococcus sp.and two species of Chalara)did not correspond with those recorded in previous studies of Picea spp.For example,the widely distributed Lophodermium piceae was rarely recorded in this study.This pattern may be caused by different methods of sterilization and cultivation or by physiological characteristics of the needles,or it may reflect the species distribution in the studied area.Members of the Helotiales,along with sequences from GenBank,showed substantial overlap in host affinities,most prominently between Pinus and Picea,and also among species from distant plant lineages.
