Trichoderma harzianum及其近缘种的分子系统学研究

Thichoderma harzianum是木霉属内最常见的一个“集合种”。本研究对来源不同的T.harzianum及其相似种的46个菌株进行了ITS序列测定,将其ITSl—5．8S—ITS2序列与来自EMBL的参考菌株的序列进行比较,并进行系统发育分析,此外对其中的18个菌株进行了RAPD多态性分析,试图明确T.harzianum的多样性以及与其相似种之间的关系。ITS结果表明,T.harzianum及其相似种可分成2个群(A、B)：A群由T.hamatum、T.asperellum、T.at-roviride、T.koningii和T.viride组成,并形成2个分支,表明T.viride和T.koningii、T.atroviride的亲缘关系较近,而与T.hamatum、T.asperellum较远;B群由T.spirale、T.hamatum、T.inhamatum、T.harzianum和T.anam。Hypocrea vinosa组成,并形成6个分支。T.inhamatum可分成2个群(Ti1、Ti2)、T.harzianum至少可分成5个群(Thl、Th2、Th4、Th5、Th6)。结果还表明T.hamatum的遗传差异较大,T.hamatum的模式菌株归属于A群,而其他的T.hamatum的菌株归属于B群。RAPD结果与ITS的结果基本一致。     

四川省雅江松茸菌的分离与系统发育

对外生菌根真菌-松茸的纯培养条件进行了探讨,并从采集自四川省雅江县的松茸子实体中获得了10株松茸菌的纯培养物;分别以NS1和NS6 ,NS1和NS8,ITS4和ITS5为引物,对分离获得的松茸菌进行了18S r DNA PCR- RFL P和ITS PCR-RFL P分析,结果显示,用Alu I,H ae III,H inf I和Msp I四种限制性内切酶,这些松茸菌株的18S r DNA、ITS片段的酶切图谱完全相同;代表菌E7的ITS序列分析结果表明,本研究分离的松茸菌与Tricholoma matsutake的菌株在系统发育上高度同源,在分类上应属于同一个种     

Jasmonic acid signalling mediates resistance of the wild tobacco Nicotiana attenuata to its native Fusarium,but not Alternaria,fungal pathogens

We recently characterized a highly dynamic fungal disease outbreak in native populations of Nicotiana attenuata in the southwestern United States. Here, we explore how phytohormone signalling contributes to the observed disease dynamics. Single inoculation with three native Fusarium and Alternaria fungal pathogens, isolated from diseased plants growing in native populations, resulted in disease symptoms characteristic for each pathogen species. While Alternaria sp.‐infected plants displayed fewer symptoms and recovered, Fusarium spp.‐infected plants became chlorotic and frequently spontaneously wilted. Jasmonic acid (JA) and salicylic acid (SA) levels were differentially induced after Fusarium or Alternaria infection. Transgenic N. attenuata lines silenced in JA production or JA conjugation to isoleucine (JA‐Ile), but not in JA perception, were highly susceptible to infection by F. brachygibbosum Utah 4, indicating that products derived from the JA‐Ile biosynthetic pathway, but not their perception, is associated with increased Fusarium resistance. Infection assays using ov‐nahG plants which were silenced in pathogen‐induced SA accumulations revealed that SA may increase N. attenuata's resistance to Fusarium infection but not to Alternaria. Taken together, we propose that the dynamics of fungal disease symptoms among plants in native populations may be explained by a complex interplay of phytohormone responses to attack by multiple pathogens.     

Morphological and molecular phylogenetic analysis of two Saprolegnia sp. (Oomycetes) isolated from silver crucian carp and zebra fish

Two Saprolegnia isolates, JY isolated from silver crucian carp (Carassius auratus gibelio Bloch) and BMY isolated from zebra fish (Brachydanio rerio Hamilton) came from infections occurring concurrently in different locations in China. To confirm whether the two isolates were from the same Saprolegnia clone, comparative studies have been carried out based on their morphological, physiological and molecular characteristics. Observations showed that morphologically (both asexual and sexual organs) the two isolates were broadly similar and both isolates underwent repeated zoospore emergence. Comparing 704 base pairs of internal transcribed spacer (ITS) region and the 5.8S rDNA, we found isolates JY and BMY shared an identical ITS sequence with a minor variation (99.6 % similarity). Forty available sequences for representatives Saprolegnia spp. belonged to four phylogenetically separate clades. The two studied isolates fell within clade I that comprised a group of isolates which showed almost an identical ITS sequence but had been identified as a number of different morphological species. Our findings suggest that isolates JY and BMY appear to belong to the S. ferax clade and this clade (I) contains a number of closely related phylogenetic species. This is distinct from the more common fish pathogenic isolates, which belong to the S. parasitica clade (III) and are characterized by having cysts decorated by bundles of long hooked hairs and two further clades (II and IV) containing largely saprotrophic or soil born species.     

Molecular identification and phylogenetic analysis of economically important acaroid mites (Acari: Astigmata: Acaroidea) in Korea

Molecular diagnosis is highly valuable for the species identification of microscopic mites. Here, we collected some economically important mites of the superfamily Acaroidea from various stored products in Korea. Those nucleotide sequences of ribosomal second internal transcribed spacer (ITS2) and the mitochondrial cytochrome oxidase subunit I (COI) regions were determined by PCR using universial primers. In nucleotide sequence comparison at GenBank database, seven species including Rhizoglyphus robini, R. echinopus, Sancassania sp. Acarus siro, Tyrophagus putrescentiae, T. similis in Acaridae and Suidasia medanensis in Suidasiidae were identified. Particularly, COI sequences of R. robini, R. echinopus, Sancassania sp. and T. similis were firstly determined. Our results suggest that the phylogenetic relationships inferred from the ITS2 region, rather than COI region, were similar to those derived based on their morphological classification. Our study provides molecular information for the identification and phylogenetic relationship of acaroid mites in Korea.     

Epigenetic regulation of development and pathogenesis in fungal plant pathogens

Evidently, epigenetics is at forefront in explaining the mechanisms underlying the success of human pathogens and in the identification of pathogen‐induced modifications within host plants. However, there is a lack of studies highlighting the role of epigenetics in the modulation of the growth and pathogenicity of fungal plant pathogens. In this review, we attempt to highlight and discuss the role of epigenetics in the regulation of the growth and pathogenicity of fungal phytopathogens using Magnaporthe oryzae, a devastating fungal plant pathogen, as a model system. With the perspective of wide application in the understanding of the development, pathogenesis and control of other fungal pathogens, we attempt to provide a synthesized view of the epigenetic studies conducted on M. oryzae to date. First, we discuss the mechanisms of epigenetic modifications in M. oryzae and their impact on fungal development and pathogenicity. Second, we highlight the unexplored epigenetic mechanisms and areas of research that should be considered in the near future to construct a holistic view of epigenetic functioning in M. oryzae and other fungal plant pathogens. Importantly, the development of a complete understanding of the modulation of epigenetic regulation in fungal pathogens can help in the identification of target points to combat fungal pathogenesis.     

Comparison of phenotypical and genetic identification of Aeromonas strains isolated from diseased fish

Phenotypicaly identified Aeromonas strains (n=119) recovered mainly from diseased fish were genetically re-identified and the concordance between the results was analysed. Molecular characterization based on the GCAT genus specific gene showed that only 90 (75.6%) strains belonged to the genus Aeromonas. The 16S rDNA-RFLP method identified correctly most of the strains with the exception of a few that belonged to A. bestiarum, A. salmonicida or A. piscicola. Separation of these 3 species was correctly assessed with the rpoD gene sequences, which revealed that 5 strains with the RFLP pattern of A. salmonicida belonged to A. piscicola, as did 1 strain with the pattern of A. bestiarum. Correct phenotypic identification occurred in only 32 (35.5%) of the 90 strains. Only 14 (21.8%) of the 64 phenotypically identified A. hydrophila strains belonged to this species. However, coincident results were obtained in 88% (15/17) of the genetically identified A. salmonicida strains. Phenotypic tests were re-evaluated on the 90 genetically characterized Aeromonas strains and there were contradictions in the species A. sobria for a number of previously published species-specific traits. After genetic identification, the prevailing species were A. sobria, A. salmonicida, A. bestiarum, A. hydrophila, A. piscicola and A. media but we could also identify a new isolate of the recently described species A. tecta. This work emphasizes the need to rely on the 16S rDNA-RFLP method and sequencing of housekeeping genes such as rpoD for the correct identification of Aeromonas strains.     

安徽部分湖泊养殖鱼类暴发性出血病的病原分离与鉴定

目的确定引起安徽省部分湖泊养殖鱼类暴发性出血病的病原,为防治该病提供理论依据。方法随机取濒死期鲫鱼和白鲢的肌肉组织分别进行细菌和病毒分离培养,联合采用细菌表型鉴定法和16S rRNA基因序列分析法鉴定分离菌株,并使用分离菌株进行人工感染实验。结果从5尾患病鱼的肌肉组织中分离获得5株细菌,综合分离菌株的形态特征、理化特性和16S rRNA基因序列与系统发育学分析的结果,确定L1菌株为温和气单胞菌、L2菌株为维氏气单胞菌、J1、J2和L3菌株为嗜水气单胞菌。人工感染实验表明这5个气单胞菌分离株均具有较强的致病性。结论气单胞菌是该次鱼类暴发性出血病的病原,水温剧变、水质恶化和缺氧是疾病暴发的诱因,应采取改善环境、加强饲养管理、提高鱼体抵抗病力和及时杀灭病原体的综合措施防控该病。     

Molecular identification and phylogenetic analysis of nuclear rDNA sequences among three opisthorchid liver fluke species (Opisthorchiidae: Trematoda)

In this study, we describe the development of a fast and accurate molecular identification system for human-associated liver fluke species (Opisthorchis viverrini, Opisthorchis felineus, and Clonorchis sinensis) using the PCR-RFLP analysis of the 18S-ITS1-5.8S nuclear ribosomal DNA region. Based on sequence variation in the target rDNA region, we selected three species-specific restriction enzymes within the ITS1 regions, generating different restriction profiles among the species: MunI for O. viverrini, NheI for O. felineus, and XhoI for C. sinensis, respectively. Each restriction enzyme generated different-sized fragments specific to the species examined, but no intraspecific polymorphism or cross-reaction between the species was detected in their restriction pattern. These results indicate that PCR-linked restriction analysis of the ITS1 region allows for the rapid and reliable molecular identification among these opisthorchid taxa. In addition, phylogenetic analysis of rDNA sequences using different methods (MP, ML, NJ, and Bayesian inference) displayed O. viverrini and O. felineus as a sister group, but this relationship was not strongly supported. The failure of recovering a robust phylogeny may be due to the relatively small number of synapomorphic characters shared among the species, yielding weak phylogenetic signal. Alternatively, rapid speciation within a very short period time could be another explanation for the relatively poorly resolved relationships among these species. Our data are insufficient for discriminating between sudden cladogenesis and other potential causes of poor resolution. Further information from independent loci might help resolve this phylogeny.     

分子系统发育分析揭示中国云南省间座壳属两个新种

间座壳属真菌在多种经济上重要的农作物、观赏植物和森林树木上常作为植物病原菌、内生菌或腐生菌被人熟知.本研究调查我国云南省患病植物叶片上的间座壳属真菌,通过核糖体内部转录间隔区、β微管蛋白基因、翻译延伸因子基因、钙调蛋白基因和组蛋白基因的DNA序列数据比较和形态学特征描述了 2个新种.它们是分别采集自密花胡颓子和荔枝患病...     

煤附生真菌产漆酶菌株的分离鉴定及产酶特性研究

从煤炭样品中筛选到一株产漆酶活性菌株,经菌体形态观察和ITS序列分析,鉴定为Trichoderma asperellum W03。菌株所产漆酶的最适反应pH为3.5-4.5,最适反应温度45℃,类似于白腐真菌漆酶。液态发酵条件的均匀设计实验表明,适宜的发酵培养基组成为:土豆200.00g/L、葡萄糖9.36g/L、米糠粉37.44g/L、硝酸钾4.00g/L、KH2PO43.20g/L、MgSO4·7H2O2.00g/L、CuSO4·5H2O0.005g/L、初始pH8.0;在33℃、180r/min、50mL/250mL的摇瓶培养条件下,棘孢木霉W03在孢子接种培养后48h、84h产酶量较高,分别处在菌体的快速生长期和衰亡期;菌体产酶受Cu2+、联苯胺诱导,而受1-萘酚、愈创木酚和2,4-D抑制。     

In silico environmental sampling of emerging fungal pathogens via big data analysis

Yeast species in the Candida haemulonii complex (C. haemulonii, C. haemulonii var. vulnera, C. duobushaemulonii, C. pseudohaemulonii, and C. vulturna) and closely related species (C. auris, C. heveicola, and C. ruelliae) are of significant public health concern worldwide. Little is known about their natural habitat. To understand the worldwide emergence of new fungal pathogens, it is important to identify key environmental habitats. Showing the effectiveness of the primary fungal DNA barcode and leveraging big data archived in the Sequence Read Archive (SRA) database enabled the identification of novel reservoirs over a wide range of geographical areas for those yeasts. We identified 1209 datasets corresponding to species in the C. haemulonii complex and three closely related species. Our results imply that climate change is not the main driver for the emergence of pathogenic multidrug-resistant yeast species. This approach opens the door for further big data analysis using the accessible resources of such databases.     

Iturin A is the principal inhibitor in the biocontrol activity of Bacillus amyloliquefaciens PPCB004 against postharvest fungal pathogens

Aims: A Bacillus amyloliquefaciens strain, surviving epiphytically on the surface of fruit, was isolated while searching for naturally occurring biological control agents. This bacterial strain was characterized for its antifungal activity against seven selected fungal postharvest pathogens of citrus. Methods and Results: To understand the antifungal activity, seven postharvest fungal pathogens were screened for growth inhibition by B. amyloliquefaciens strain. Assays using B. amyloliquefaciens lipopeptide extracts showed a strong inhibitive activity. The inhibitory effect was observed in abnormal conidial germination and germ tube development when conidia were treated with different lipopeptide extract concentrations. Further analysis using PCR and chromatography confirmed the presence of fengycin, iturin and surfactine, of which iturin A showed the strongest and most common inhibitory effect. The results are supported by site‐directed mutagenesis analysis, targeted to suppress the biosynthesis of iturin A production. Fruit trials confirmed disease development inhibition when the antagonist was applied 1 day prior to or 1 day after fungal application. Conclusions: We conclude that the iturin family of lipopeptides are vital in the antagonism of B. amyloliquefaciens against the seven citrus postharvest pathogenic fungi tested. Significance and Impact of the Study: We elucidated the principal mechanism used by B. amyloliquefaciens PPCB004 to suppress postharvest disease development on stored fruits.     

Molecular identification and phylogenetic analysis of Aloe shadensis from Saudi Arabia based on matK,rbcL and ITS DNA barcode sequence

The Kingdom of Saudi Arabia thrives with great plant diversity, including rare plants of the family Asphodelaceae that have multiple benefits and are still being studied. Aloe shadensis is one of these plants that must be preserved and documented in its natural environment. The most appropriate molecular approach currently approved for documentation is the sequencing of some genomic markers. The current study is the first to use genomic markers to record this rare plant. In this study, the plastid genes matK (Maturase K), rbcL (Ribulose-bisphosphate carboxylase/oxygenase large subunit), and the nuclear region ITS (Internal transcribed spacer) were used to reveal their efficiency in identifying the plant under study. This study is the first to deal with this plant and document it using these genetic markers. The study showed a promising result concerning identifying the sequence of the matK gene and ITS region, while the rbcL gene did not give a good indicator through the used primers. The obtained sequences of the matK gene and the ITS region were determined through two different sets of primers in each case then deposited in GenBank. The evolutionary relatedness of Aloe shadensis was established with the different species of Aloe. The study showed that the closest species is Aloe vera with a similarity of more than 99 %. The study concludes with the possibility of using these genes to correctly identify, distinguish and document the species of Aloe shadensis.     

In vitro and in vivo antagonism of Streptomyces violaceusniger YCED9 against fungal pathogens of turfgrass

The biocontrol agent Streptomyces violaceusniger YCED9 inhibited in vitro growth of seven fungal pathogens of turfgrass. Three different antibiotics were produced by the actinomycete, including nigericin, geldanamycin and a complex of macrocyclic lactone antibiotics. Each had a different spectrum of antifungal activity. Only nigericin was detected in soil or in grass rhizospheres inoculated with YCED9. However, all three antibiotics were produced when YCED9 was grown in a mixture of moistened grass and thatch. In greenhouse experiments, a grass seedling disease caused by the Rhizoctonia solani and a crown-foliar disease caused by Sclerotinia homeocarpa were partially controlled with commercial spore formulations of YCED9.     

Identity and virulence properties of Aeromonas isolates from diseased fish, healthy controls and water environment in China

Aims: To investigate the species distribution in Aeromonas isolates from diseased fish, healthy controls and water environment in China; to evaluate the frequency of the aerolysin (aer), cytotonic enterotoxin (alt), cytotoxic enterotoxin (act), temperature‐sensitive protease (eprCAI) and serine protease (ahp) genes in Aeromonas isolates; and to determine the potential pathogenicity of these isolates. Methods and Results: Two hundred and two Aeromonas isolates from diseased fish (n = 42), healthy fish (n = 120) and water environment (n = 40) in China were identified to species levels based on sequencing of the housekeeping gene gyrB, while the distribution of five virulence factors, including aer, alt, act, eprCAI and ahp, was investigated by PCR. Aeromonas veronii (25/42; 60%) and Aeromonas hydrophila (14/42; 33%) were the species most commonly isolated from diseased fish, while Aer. veronii was the most common species in healthy fish (90/120; 75%) and water samples (25/40; 62·5%). All the five virulence genes were present in 9% (19/202), among which 10 strains were from diseased fish and nine were identified as Aer. hydrophila. For the strains carrying five virulence genes, the average 50% lethal doses (LD_50s) of strains from diseased fish were lower when compared with the strains from healthy fish and water environment. Conclusions: Aeromonas veronii is the most common species, but no significant difference exists in the isolates obtained from diseased fish and from healthy fish. However, Aer. hydrophila isolates were significantly more frequent from diseased fish than from healthy fish. aer⁺alt⁺act⁺eprCAI⁺ahp⁺ was more frequent virulence genotype in Aeromonas isolates from diseased fish than from healthy fish and water environment, and the aer⁺alt⁺act⁺eprCAI⁺ahp⁺ isolates were more virulent to zebrafish comparing to the other genetic profiles. Significant and Impact of the Study: Aeromonas species in aquatic environments are various and have considerable virulence potential, and therefore, there is a need for more careful and intensive epidemiology studies.     

Genetic analysis of the br gene in halophilic archaea isolated from Xinjiang region, China

Some novel members of extremely halophilic archaea, strains AJ 11, AJ 12 and AJ 13, were isolated from the Aularz Lake located in the Altun Mountain National Nature Reserve of Xinjiang, Uygur Autonomous Region in China. Partial DNA fragments encoding a bacteriorho-dopsin (BR), as well as for 16S rRNA of isolated strains, were amplified by PCR and their DNA sequences were determined subsequently. On the basis of homology and phylogenetic analysis of the 16S rDNA, we thought that the isolated strains forming a microbiological population are the members of the genus Natrinema. The results of genetic analysis, such as GC content, transition/transver-sion (Ti/Tv) rate ratios and synonymous substitution rates (Ks) indicate that the br fragments, with a high level of genetic divergence, are faced with both purifying selection and bias mutation pressure. The study provides the basis for use of species and BR proteins resources.     

安徽省土壤绿僵菌分离株的分子鉴定

目的进一步验证ITS序列的系统发育分析可为绿僵菌属种的鉴定提供重要的参考依据。方法对分离自安徽土壤的13株绿僵菌菌株的内转录间隔区（ITS）片段进行PCR扩增和序列测定,采用Blast方法将测序结果在GenBank中进行同源搜索,依据邻接法构建获得与其相关菌株的ITS序列系统发育树。结果供试菌株分别位于系统发育树的3个分支上,分支I包括8个菌株和金龟子绿僵菌小孢变种,1个菌株和金龟子绿僵菌鳞鳃金龟变种形成分支III,另外4个菌株和黄绿绿僵菌棉蚜变种聚为分支X。结论结合同源比较的数据,将这8个、4个和1个绿僵菌菌株分别鉴定为金龟子绿僵菌小孢变种、黄绿绿僵菌棉蚜变种和金龟子绿僵菌鳞鳃金龟变种。