洞庭湖湿地木霉多样性及生防活性

【目的】了解湖南省洞庭湖湿地木霉种类及分布,丰富我国的木霉种质资源,为功能菌株筛选应用奠定基础。【方法】利用ITS序列比对分析结合形态学特征对分离到的木霉菌株进行种类鉴定,构建系统发育进化树分析其亲缘关系。通过菌丝生长速率法测定菌株的平板抑菌能力,根据水解带大小检测菌株的水解酶活性,利用灰色关联度分析筛选综合生防效果较好的木霉菌株。【结果】从52个土样和18个水样中分离得到114株木霉菌株,经鉴定分属16个木霉种类:哈茨木霉、绿木霉、刺孢木霉、土星孢木霉、钩状木霉、拟康宁木霉、短密木霉、深绿木霉、猥木霉、毛细木霉(中国新记录种)、长枝木霉、卵孢木霉、侧耳木霉、加纳木霉、厚木霉及一个疑似新种;哈茨木霉为洞庭湖湿地中的优势种,占总菌株数量的19.30%;16种木霉在系统发育树中归于7个进化支:Harzianum Clade、Virens Clade、Longibrachiatum Clade、Lutea Clade、Viride Clade、Hamatum Clade、Unknown Clade。灰色关联度分析表明,菌株TW21990、QT22040和QT22094的灰色关联度较高,分别为0.849 5、0.798 6和0.732 6,综合生防性状较好。【结论】洞庭湖湿地木霉具有种类多样性和分布多样性,发现了一个中国新记录种毛细木霉和一个疑似新种,哈茨木霉TW21990、长枝木霉QT22040和卵孢木霉QT22094是潜在的优良生防菌株。     

木霉属真菌分离培养的研究进展

木霉属真菌（Trichoderma spp.）种类众多、分布广泛,在农业、工业和环境修复领域应用广泛。因此,木霉属真菌的分离培养具有重要的研究价值。本文通过详尽的文献查找和分析对木霉属真菌的分离培养研究进展予以综述,从生存环境、不同分离基质和分离方法的样品前处理方法等总结了木霉属真菌的分离基质及预处理方法;详细回顾了木霉属真菌分离培养基研究的发展历程和主要原理;简要介绍了目前木霉属真菌的纯化与保存方法;并从开展多种抗菌物质和表面活性剂探索角度对未来木霉属真菌分离培养进行了展望,以期为木霉属真菌资源开发提供参考。     

设施番茄根围土样中木霉菌多样性及功能活性分析

为探索设施栽培条件下蔬菜根围土壤中木霉种群多样性特征及其抗常见植物病原真菌及有机磷农药降解活性,从连续20多年种植番茄的设施大棚根围土样中以及番茄根系内部分离木霉菌,并以tef1-α序列和系统发育分析进行种类鉴定,估算各菌株的相对种群密度;对其生防和农残降解活性进行了测定。结果共分离不同木霉菌株27株,结合形态学特征和tef1-α序列分析,鉴定出分属6个不同的木霉种,即绿色木霉(Trichoderma virens)、深褐木霉(T.atrobrunneum)、西蒙斯木霉(T.simmonsii)、深绿木霉(T.atroviride)、厚木霉(T.crassum)和1个疑似木霉新种(T.sp.nov.);木霉各菌株产孢簇常呈鲜绿色到暗绿色,分布不均匀,常形成同心圆结构。对峙条件下这些木霉对土壤中常见土传病原菌立枯丝核菌(Rhizoctonia solani)、大丽轮枝菌(Verticillium dahliae)、尖孢镰孢菌(Fusarium oxysporum)均有普遍的不同程度的抑菌活性;除深褐木霉两个菌株对毒死蜱的降解效果不明显外,其它种类的木霉均有菌株能有效降解毒死蜱,木霉菌株在根围土壤中的相对种群密度仅为104 cfu/g,且菌株间差异较大。研究结果表明设施农业土壤中木霉菌具有丰富的种质资源多样性,在形态特征及功能活性方面表现明显的菌株差异;木霉对3种常见植物病原真菌有普遍的抑菌作用,少数菌株对常用有机磷农药降解作用明显,可以发挥生防和农化残留降解方面的协同效应。该结果对于研究设施农业土壤中木霉的多样性,指导功能菌株筛选及应用,更好挖掘利用木霉菌资源有着较好的借鉴意义。     

木霉属2个中国新记录种

【背景】木霉菌现存的Stromaticum进化支为Samuels等2012年定义,包括9个木霉种;国内目前仅报道子座木霉(Trichoderma stromaticum)、蠕状毛木霉(T.vermipilum)和絮状木霉(T.floccosum)3个种。【目的】报道2个木霉属中国新记录种。【方法】采用THSM选择性培养基,从北京和山东两地土壤中分离木霉菌株,通过形态学特征、TEF1-α和RPB2序列对菌株进行鉴定。【结果】通过对TEF1-α和RPB2的系统发育分析,2个菌株分别与T.ivoriense(科特迪瓦木霉)和T.barbatum(毛簇木霉)相近;且形态学特征上存在差异。综合鉴定2个菌株分别为科特迪瓦木霉(T.ivoriense)和毛簇木霉(T.barbatum)或其近缘种。【结论】在国内新发现科特迪瓦木霉(T.ivoriense)和毛簇木霉(T.barbatum)两个木霉种,它们属于Stromaticum进化支,该进化支国内木霉种类增加到5个。     

检疫性昆虫DNA条形码检测技术的研发与应用实例

【目的】DNA条形码技术是近年来生物分类鉴定的研究热点之一,已成为植物检疫性昆虫鉴定的有力工具。为快速、准确地鉴定口岸截获的昆虫种类,实现"检得出、检得准、检得快"的要求,我们研发了昆虫DNA条形码试剂盒检测技术(Insect DNA barcoding identification kit)。【方法】该检测技术针对出入境植物检疫性及危险性昆虫的主要类群,选择合适的基因片段、设计引物、对目标基因进行扩增测序,找出基因片段上区分每个物种的多态位点规律,作为该物种的鉴定特征并建立数据库,应用于植物检疫性及危险性昆虫的物种鉴定。【结果】以检疫性昆虫木蠹象属Pissodes为例,确定了木蠹象属5种昆虫的多态位点规律(鉴定特征),构建了用于物种鉴定的数据库。通过比对数据库里的鉴定特征,将未知样品鉴定为榛梢木蠹象P.terminalis(相似度100%),与形态鉴定结果一致。本文介绍了检测技术的原理、方法、技术流程及应用实例,并展望了其在有害生物检测中的推广应用前景。【结论】昆虫DNA条形码试剂盒检测技术为建立标准化,准确性高的物种鉴定平台打下基础,有着良好的推广应用前景。     

木霉属2个中国新记录种（英文）

【背景】木霉菌现存的Stromaticum进化支为Samuels等2012年定义,包括9个木霉种;国内目前仅报道子座木霉(Trichoderma stromaticum)、蠕状毛木霉(T.vermipilum)和絮状木霉(T.floccosum)3个种。【目的】报道2个木霉属中国新记录种。【方法】采用THSM选择性培养基,从北京和山东两地土壤中分离木霉菌株,通过形态学特征、TEF1-α和RPB2序列对菌株进行鉴定。【结果】通过对TEF1-α和RPB2的系统发育分析,2个菌株分别与T.ivoriense(科特迪瓦木霉)和T.barbatum(毛簇木霉)相近;且形态学特征上存在差异。综合鉴定2个菌株分别为科特迪瓦木霉(T.ivoriense)和毛簇木霉(T.barbatum)或其近缘种。【结论】在国内新发现科特迪瓦木霉(T.ivoriense)和毛簇木霉(T.barbatum)两个木霉种,它们属于Stromaticum进化支,该进化支国内木霉种类增加到5个。     

我国河北、浙江、云南及西藏木霉种记述

对从中国河北、浙江、云南及西藏分离的72个木霉菌株进行了鉴定和分类学研究。采纳Gams&Bissett(1998)及Kullnig—Gradinger et al.(2002)的分类观点,鉴定出木霉属的12个种, 其中包括8个已知种:深绿木霉T.atroviride,桔绿木霉T.citrinoviride,哈茨木霉T.harzianum, 康宁木霉T.koningii,长枝木霉T.longibrachiatum,中国木霉T.sinensis,绿木霉T.virens和绿色木霉T.viride;4个我国新记录种是:木霉组(Trichoderma section)的棘孢木霉T.asperellum及粗梗组(Pachybasium section)的淡黄木霉T.cerinum,螺旋木霉T.spirale和茸状木霉T.velutinum。     

根癌农杆菌介导的木霉遗传转化及应用进展

木霉作为土传植物病原菌的生防真菌,研究其功能基因具有重要的意义。根癌农杆菌介导的遗传转化(ATMT)为木霉功能基因的研究提供了一个强有力的工具。对根癌农杆菌介导木霉遗传转化的机理、特点、方法及其在木霉中的应用进行了综述。     

河北安国药用植物根区土壤木霉物种多样性

为探明安国药用植物根区土壤木霉Trichoderma spp.群落组成和物种分布,2017年7月在河北省安国市中药材种植基地选取麦冬Ophiopogon japonicus、半枝莲Scutellaria barbata和亚麻Linum usitatissimum等26种药用植物,采集根区0～30 cm土层土壤样品,采用形态特征结合r DNA ITS序列分析方法对木霉种类进行分类鉴定,探讨木霉属真菌群落组成、物种多样性以及土壤因子的生态功能。共分离鉴定出10种木霉,包括绿色木霉Trichoderma viride、深绿木霉Trichoderma atroviride、哈茨木霉Trichoderma harzianum、短密木霉Trichoderma brevicompactum、长枝木霉Trichoderma longibrachiatum、突里巴木霉Trichoderma turrialbense、贵州木霉Trichoderma guizhouense、猬木霉Trichoderma erinaceus、康宁木霉Trichoderma koningii和黄绿木霉Trichoderma aureoviride,其中优势种为绿色木霉、深绿木霉和哈茨木霉,分离频率分别为38%、18%和17%。金银花Lonicera japonica根区土壤木霉物种分离频率最高(15%),分离到绿色木霉、深绿木霉、哈茨木霉、贵州木霉和康宁木霉,其香农维纳指数最高(1.359),均匀度指数较小(0.845);甘草Glycyrrhiza uralensis分离频率次之(9%),分离到绿色木霉、哈茨木霉、长枝木霉和突里巴木霉,其香农维纳指数为1.330,均匀度指数为0.959;其他植物均分离到绿色木霉或深绿木霉,多样性指数较低,表明不同药用植物根区木霉群落组成差异显著。主成分分析发现,土壤磷酸酶、有机碳和速效氮是影响河北安国样地土壤理化性质的主要变量。深绿木霉与土壤脲酶和酸性磷酸酶显著正相关,哈茨木霉与土壤有机碳显著正相关,与速效磷显著负相关;短密木霉与植物种类和p H显著负相关,长枝木霉与速效氮正相关,表明土壤理化性质和植物对木霉分布有显著影响。方差分解表明木霉多样性主要受植物种类的影响。     

木霉菌及其系统分类学研究回顾

从木霉菌(Trichodermaspp.)的分类历史、主要分类系统、分子生物学在木霉菌分类研究中的应用、我国木霉菌分类研究状况等几个方面,对木霉菌的分类研究进展进行了简要回顾。近些年来国际上对木霉菌的分类研究取得了显著进展,陆续发表了许多新种,目前木霉属已报道有3组60种和2个变型。另外,对木霉分类研究中存在的问题也进行了讨论。     

Exploring the species diversity of Trichoderma in Norwegian drinking water systems by DNA barcoding

A total of 123 Trichoderma strains were isolated from Norwegian surface-sourced drinking water. The water samples included raw water, treated water, and water from private homes and hospital installations. Trichoderma species are difficult to differentiate morphologically, but recent molecular identification tools, including DNA barcoding, successfully distinguish between closely related species. The diversity of Trichoderma spp. was explored by DNA sequencing of internal transcribed spacer (ITS) and translation elongation factor 1 alpha (TEF-1α). Sequence identification was performed in the TrichOKEY version 2.0 barcode program and in the multilocus similarity search database TrichoBLAST, combined with traditional blast searches in the EMBL/GenBank. A total of 11 known Trichoderma/Hypocrea species were identified. In addition, one group of unidentified Trichoderma strains was found to represent a separate, strongly supported subclade within the Pachybasium'A'/Hamatum clade, based on their TEF-1α haplotypes. Trichoderma viride comprised 49% of the identified strains, and was represented by four and eight slightly different ITS and TEF-1α haplotypes, respectively. Approximately 22% of the surface-derived water samples were positive for T. viride, and the species was frequently isolated throughout the surface-sourced drinking water distribution system. The results indicate that a broad range of Trichoderma species are present in Norwegian surface-sourced drinking. Water treatment has minor effect in removing Trichoderma from raw water, and active growth in the water distribution system is likely to occur.     

SCAR-based real time PCR to identify a biocontrol strain (T1) of Trichoderma atroviride and study its population dynamics in soils

Strains of Trichoderma spp. are known for their antagonistic properties against plant pathogens, some are already on the market, others are under development. In order to launch a strain on the market its perfect identification at the species and strain levels is needed. The aim of this study is to (i) design a SCAR marker for specific identification of strain T1 of Trichoderma atroviride and (ii) monitor population dynamics of this strain in soil by real time PCR. A primer pair targeting a 141-bp fragment enabled specific detection of this strain without cross detection of autochthonous populations of Trichoderma in several field soils. In two soils, population dynamics assessed by real time PCR and the soil plate technique gave similar results. The molecular tools developed in this study satisfy the requirement for specific identification of the biocontrol strain and for detection and quantification of T. atroviride T1 population in complex environments.     

Identification of Trichoderma strains by image analysis of HPLC chromatograms

Forty-four Trichoderma strains from water-damaged building materials or indoor dust were classified with chromatographic image analysis on full chromatographic matrices obtained by high performance liquid chromatography with UV detection of culture extracts. The classes were compared with morphological identification and rDNA sequence data, and for each class all strains were of the same identity. With all three techniques each strain--except one--was identified as the same species. These strains belonged to Trichoderma atroviride (nine strains), Trichoderma viride (three strains), Trichoderma harzianum (10 strains), Trichoderma citrinoviride (12 strains), and Trichoderma longibrachiatum (nine strains). The odd strain was identified as Trichoderma hamatum by morphology and rDNA sequencing, but not by image analysis as no reference strains of this species were included. It is concluded that the secondary metabolite profile contains sufficient information for classification and species identification.     

M13-microsatellite PCR and rDNA sequence markers for identification of Trichoderma (Hypocreaceae) species in Saudi Arabian soil

Seven fungal isolates were identified as pan-global Hypocrea/Trichoderma species, from section Trichoderma, on the basis of their morphology. These species were H. lixii/T. harzianum and H. orientalis/T. longibrachiatum. PCR-based markers with primer M13 (core sequence of phage M13) and internal-transcribed spacer sequences of ribosomal DNA were used to confirm the identity of the two Trichoderma species. Sequence identification was performed using the TrichOKEY version 2.0 barcode program and the multilocus similarity search database TrichoBLAST. Sequences from the ribosomal DNA internal-transcribed spacer regions showed limited variation among the Trichoderma species. This analysis divided the isolates into two main groups. Grouping the isolates based on cluster analysis of their DNA profiles matched the grouping based on morphological taxonomy. Molecular data obtained from analyses of gene sequences are essential to distinguish phonetically cryptic species in this group and to establish phylogenetic relationships.     

辽宁木霉属(Trichoderma)真菌的形态分类研究

对采自辽宁省内14个地方的173份土样和植物组织材料进行分离,获得了54株Trichoderma菌株,采用形态学分类方法鉴定出12种木霉菌,分别是拟康氏木霉(Trichoderma pseudokoningii)、长枝木霉(T.longi-brachiatum)、粘绿木霉(T.virens)、卷曲木霉(T.spirale)、顶孢木霉(T.fertile)、粗壮木霉(T.strigosum)、长孢木霉(T.longipile)、钩状木霉(T.hamatum)、绿色木霉(T.viride)、康氏木霉(T.koningii)、深绿木霉(T.atroviri-de)和哈茨木霉(T.harzianum)。其中长孢木霉为中国新记录种,粗壮木霉和卷曲木霉为东北地区首次报道。文中列有辽宁省木霉属真菌的分种检索表,并附有各木霉菌的生境和分布。     

An oligonucleotide barcode for species identification in Trichoderma and Hypocrea

One of the biggest obstructions to studies on Trichoderma has been the incorrect and confused application of species names to isolates used in industry, biocontrol of plant pathogens and ecological surveys, thereby making the comparison of results questionable. Here we provide a convenient, on-line method for the quick molecular identification of Hypocrea/Trichoderma at the genus and species levels based on an oligonucleotide barcode: a diagnostic combination of several oligonucleotides (hallmarks) specifically allocated within the internal transcribed spacer 1 and 2 (ITS1 and 2) sequences of the rDNA repeat. The barcode was developed on the basis of 979 sequences of 88 vouchered species which displayed in total 135 ITS1 and 2 haplotypes. Oligonucleotide sequences which are constant in all known ITS1 and 2 of Hypocrea/Trichoderma but different in closely related fungal genera, were used to define genus-specific hallmarks. The library of species-, clade- and genus-specific hallmarks is stored in the MySQL database and integrated in the TrichOKey v. 1.0 - barcode sequence identification program with the web interface located on . TrichOKey v. 1.0 identifies 75 single species, 5 species pairs and 1 species triplet. Verification of the DNA-barcode was done by a blind test on 53 unknown isolates of Trichoderma, collected in Central and South America. The obtained results were in a total agreement with phylogenetic identification based on tef1 (large intron), NCBI BLAST of vouchered records and postum morphological analysis. We conclude that oligonucleotide barcode is a powerful tool for the routine identification of Hypocrea/Trichoderma species and should be useful as a complement to traditional methods.     

The first 100 Trichoderma species characterized by molecular data

Trichoderma species are generally abundant on decaying wood and in soil because of their success in various heterotrophic interactions, including decomposition, parasitism, and even opportunistic endophytism. Many Trichoderma species or, precisely, many individual Trichoderma strains, have various important applications in industry and human life, which led to the inclusion of Hypocrea jecorina (Trichoderma reesei), the well-known producer of industrial enzymes, in the list of organisms whose genomes have been sequenced. Trichoderma species also have been adopted as agents of biological control of plant pathogenic fungi and as antibiotic producers. Trichoderma longibrachiatum is known as an opportunistic pathogen of immunocompromised mammals, including humans, and some species are common indoor contaminants. Given these properties, correct identification at the species level is highly desirable. However, within the past decade, the number of recognized Trichoderma species has tripled, reaching 100. Therefore, Trichoderma taxonomy and species identification is a difficult issue. The abundant homoplasy in phenetic characters is likely the reason, given that the number of morphologically distinct species is significantly lower than the number of phylogenetically distinct species recognized using methods of gene sequence analysis. In this review, we introduce to the scientific community the development of modern tools for Trichoderma species identification: the oligonucleotide barcode program TrichOKEY version 1.0, and TrichoBLAST, the multilocus database of vouchered sequences powered by a similarity search tool. We also discuss the application of the Genealogic Concordance Phylogenetic Species Recognition approach. In combination, these advances make it possible to identify all known Trichoderma species based on sequence analysis.     

An immunological approach to quantifying the saprotrophic growth dynamics of Trichoderma species during antagonistic interactions with Rhizoctonia solani in a soil-less mix

Studies of the saprotrophic growth dynamics of Trichoderma species and their fungal hosts during antagonistic interactions are severely hampered by the absence of methods that allow the unambiguous identification and quantification of individual genera in complex environments such as soil or compost containing mixed populations of fungi. Furthermore, methods are required that allow discrimination between active hyphal growth and other components of fungal biomass such as quiescent spores that are produced in large numbers by Trichoderma species. This study details the use of monoclonal antibodies to quantify the saprotrophic growth dynamics of the soil-borne plant pathogen Rhizoctonia solani and biological control strains of Trichoderma asperellum and Trichoderma harzianum during antagonistic interactions in peat-based microcosms. Quantification was based on the immunological detection of constitutive, extracellular antigens that are secreted from the growing tip of Rhizoctonia and Trichoderma mycelium and, in the case of Trichoderma harzianum, from quiescent phialoconidia also. The Trichoderma-specific monoclonal antibody (MF2) binds to a protein epitope of the enzyme glucoamylase, which was shown by immunofluorescence and immunogold electron gold microscopy studies of Trichoderma virens in vitro to be produced at the origin of germ tube emergence in phialoconidia and from the growing tip of germ tubes. In addition, a non-destructive immunoblotting technique showed that the enzyme was secreted during active growth of Trichoderma asperellum mycelium in peat. The Rhizoctonia solani-specific monoclonal antibody (EH2) similarly binds to a protein epitope of a glycoprotein that is secreted during active mycelial growth. Extracts derived from lyophilized mycelium were used as a quantifiable and repeatable source of antigens for construction of calibration curves. These curves were used to convert the absorbance values obtained in ELISA tests of peat extracts to biomass equivalents, which allowed comparisons of the saprotrophic growth dynamics of the pathogen and antagonists to be made in single or mixed species microcosms. Trichoderma species were able to compete successfully with R. solani for nutrients and to prevent saprotrophic growth of the pathogen. Specificity of the Trichoderma quantitative assay was tested in non-sterile soil-based microcosms artificially inoculated with T. asperellum. The assay was highly specific and only detected T. asperellum population dynamics. No cross-reactivity was found with extracts from soil samples containing contaminant fungi.     

A new cultivation independent approach to detect and monitor common Trichoderma species in soils

A set of primers was developed for the detection, identification and quantification of common Trichoderma species in soil samples. Based on a broad range master alignment primers were derived to amplify an approximate 540 bp fragment comprising the internal transcribed spacer region 1 (ITS 1), 5.8S rDNA and internal transcribed spacer region 2 (ITS 2) from all taxonomic Clades of the genus Trichoderma. The primer set was applied to test strains as well as community DNA isolated from arable and forest soil. For all tested isolates the corresponding internal transcribed spacer regions of Trichoderma spp. strains were amplified, but none of non-Trichoderma origin. PCR with community DNA from soil yielded products of the expected size. Analysis of a clone library established for an arable site showed that all amplified sequences originated exclusively from Trichoderma species mainly being representatives of the Clades Hamatum, Harzianum and Pachybasioides and comprising most of the species known for biocontrol ability. In a realtime PCR approach the primer set uTf/uTr also proved to be a suitable system to quantify DNA of Trichoderma spp. in soils.     

木霉属补充DNA条形码筛选

木霉属真菌是一类重要的生物资源,在工农业、环境保护等方面具有较高经济价值,对其进行快速、准确的物种鉴定兼具理论意义和应用前景。以木霉属35个概念清晰的种为材料,选择ITS、rpb2和 tef1作为候选基因序列,利用TaxonGap对231个序列片段进行分析,将种内与种间序列差异以及序列获取难易程度作为评价指标,筛选该属的补充条形码片段。结果表明,rpb2具有适宜的种内与种间序列差异,其最小的种间差异（2.48%）,大于最大种内差异（1.8%）,种内、种间遗传距离存在明显的间隔区,并且该基因序列具有较高的PCR扩增与测序成功率（94.4%）;ITS和tef1基因序列的种内与种间序列差异之间存在交叉重叠。因此建议rpb2作为木霉属的补充DNA条形码序列。