中国西南地区木霉属分类研究

从西南四省区(云、贵、川、藏)的土壤和其它样品中分离木霉301株,鉴定出9个木霉集合种(species aggregates) :哈茨木霉(Trichoderma harzianum Rifai),拟康氏木霉(T.pseudokoningii Rifai),长枝木霉(T.longibrachiatum Rifai),深绿木霉(T.atrovirideBissett),桔绿木霉(T.citrinoviride Bissett),绿色木霉(T.viride Pers.ex S.F.Gray),钩状木霉(T.hamatum(Bon)Bain),康氏木霉(T.koningii Oud.)以及黄绿木霉(T.aureoviride Rifai)。从各个样点收集的42种不同作物和其它植被土样中都分离到木霉;土样pH值为4—8.5,海拔300—5400m。以哈茨木霉和拟康氏木霉出现频率最高,分别为28.5%和21.1%,似为西南地区木霉优势种群,而绿色木霉、钩状木霉和深绿木霉很少分离到。这样的种群结构可能与西南地区气候和采样季节有关。     

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。     

云南大围山自然保护区木霉菌多样性与RAPD分析

描述了从云南省大围山自然保护区土壤样品中分离鉴定的 6个木霉集合种 (speciesaggregates) :康氏木霉(Trichoderma .koningiiOud) ,哈茨木霉 (T .harzianumRifai) ,绿色木霉 (T .viridePersexS .F .Gray) ,长枝木霉(T .longibrachiatumRifai) ,桔绿木霉 (T .citrinovirideBissett) ,钩状木霉 (T .hamatum (Bon)Bain)。对 6种木霉分别进行拮抗活性测定和随机扩增多态性DNA(RAPD) ;其结果 ,6种木霉对 4种植物病原菌均有不同程度的拮抗性 ;6种木霉DNA扩增谱带差异明显 ,遗传相似性聚类分析结果按一定遗传距离可分 6群 ;与形态分类结果一致 ,可作为木霉分类鉴定的依据。     

作物根围土壤木霉菌物种多样性及其体外拮抗病原菌效应

对采自我国西北地区青海、宁夏、甘肃、陕西和新疆农作物根围土壤中的木霉菌进行分离纯化后,采用形态与分子生物学技术进行菌种鉴定,获得14个木霉种属346个菌株。其中哈茨木霉(Trichoderma harzianum)和棘孢木霉(Trichoderma asperellum)为优势菌种,分别占总数的35%和30%;其次为长枝木霉(Trichoderma longibrachiatum)占12%;绿木霉(Trichoderma virens)、深绿木霉(Trichoderma atroviride)、拟康宁木霉(Trichoderma koningiopsis)和平菇木霉(Trichoderma pleurotum)分别占8%,5%,2%和1%;绿色木霉(Trichoderma viride)/钩状木霉(Trichoderma hamatum)和短密木霉(Trichoderma brevicompactum)/矩孢木霉(Trichoderma oblongisporum)分别皆占2%和1%;渐绿木霉(Trichoderma viridescens)、侧耳木霉(Trichoderma pleuroticola)和康宁木霉(Trichoderma koningii)为我国西北地区农作物根围土壤中最小的木霉种群,各仅占0.3%。将纯化的菌株分别与5个靶标病原真菌在马铃薯葡萄糖培养基(PDA)平板上进行拮抗测试,研究结果表明各木霉菌株对病原菌的拮抗效果有明显差异,且其平均拮抗指数均60%。将2株短密木霉(TR1294TR1295)进行液体发酵培养,并用稀释5倍的发酵液配制PDA平板,对靶标病原真菌进行抑菌测试。结果显示,这5个病原真菌在含有TR1294和TR1295发酵液PDA平板上的生长抑制率可达72%,表明TR1294和TR1295在液体发酵过程中能产生抑菌次级代谢物。     

4种木霉菌对棉花黄萎病菌抑制作用的测定

以绿色木霉(Trichoderma viride)、康氏木霉(Trichoderma koningii)和二株未知木霉菌株(Trichoderma spp.)为供试木霉菌株,采用对峙培养法测定了不同温度处理下对棉花黄萎病菌(Verticilliam dahliae)的拮抗作用。结果表明,木霉在不同温度下对棉花黄萎病菌的抑制作用不一,其中以30℃和25℃黑暗条件下木霉对棉花黄萎病菌菌丝生长的抑制作用最强,对峙培养5d后,绿色木霉、康氏木霉和二株未知木霉菌在30℃条件下对棉花黄萎病菌的抑制率分别达到67.3%、65.9%、56.8%、65.9%,25℃条件下的抑制率分别为65.9%、72.9%、65.9%、78.8%;20℃下木霉菌株对棉花黄萎病菌的抑制作用次之,10℃、15℃和35℃条件下木霉菌丝扩散速度较慢,且孢子产生量少,不能有效地抑制棉花黄萎病菌菌丝的扩展,表明环境温度过高或过低对木霉菌丝的生长及分生孢子的产生均有较大影响。该研究为筛选棉花黄萎病菌更为有效的生防木霉菌株提供理论依据。     

工厂化生产海鲜菇菌包污染霉菌的鉴定及防治

对工厂化生产中海鲜菇菌包污染霉菌进行分离,根据霉菌的形态特征、培养性状及ITS序列分析,鉴定其为哈茨木霉、拟康氏木霉、脉孢霉、长枝木霉、黑曲霉、产红青霉和产黄青霉;在此基础上探讨了常用抑菌剂对霉菌的防治效果及对海鲜菇菌丝生长的影响。结果表明,质量浓度100 mg/L克霉灵对哈茨木霉、黑曲霉、产红青霉、产黄青霉、拟康氏木霉有强抑制作用,质量浓度100 mg/L多菌灵对长枝木霉、产红青霉、产黄青霉、拟康氏木霉有强抑制作用,二者对海鲜菇菌丝生长的抑制都比较弱。可为海鲜菇工厂化生产中污染霉菌的综合防治提供参考。     

我国农作物根际土壤木霉菌资源分布调查

通过对我国18个省市地区的农作物根际土壤进行采样,使用木霉菌选择性培养基(TSM)和马铃薯葡萄糖培养基(PDA),对采样土壤中的木霉菌(Trichoderma spp.)进行分离和纯化,再与同一地区的多种植物病原菌进行平板对峙测试,筛选和保存了1 300余株有拮抗功能的木霉菌株。经形态学和分子生物学(ITSTef1-α)鉴定,这些菌株分属9个木霉菌种。其中哈茨木霉(T.harzianum)和棘孢木霉(T.asperellum)为优势菌种,分别占29.07%和29.91%(总数:393和382株);其次是绿色木霉(T.viride),占筛选总数的14%(总数:185株);而渐绿木霉(T.viridescens)和康宁木霉(T.koningii)分别占7.53%和7.00%(总数:99和92株);深绿木霉(T.atroviride)和钩状木霉(T.harmatum)分别只占5%以上(总数:69和67株);长枝木霉(T.longibrachiatum)和橘绿木霉(T.citrinoviride)的群落在我国农作物根际土壤中的分布最少,分别只占1.5%和0.5%以上(总数:21和7株)。同时,为了解不同地区的木霉菌株对上海地区的草莓灰霉菌(Botrytis cinerea)的拮抗作用的差异性,选用14个不同地区的木霉菌株,通过对峙培养拮抗测定,筛选出6个木霉菌株,进一步做了抑制草莓灰霉病的室内生测试验。结果显示,来源于华东地区的木霉菌株TR78和TR85,在平板对峙试验中对草莓灰霉菌生长的抑菌率分别为85.49%和82.90%,室内生测对灰霉病的防治效果分别为80.19%和80.02%,显著高于(P0.05)其他菌株对灰霉病的防治效果。     

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。     

粮食上木霉菌的分离鉴定及其生防效果

【背景】粮食在生长和收储期极易受到病原真菌或产毒真菌的污染,造成严重的损失。众多实践证明木霉属(Trichoderma)可以有效防治植物病原真菌。【目的】鉴定和筛选能有效抑制粮食常见危害真菌的木霉生防菌株,开发生防菌剂,保障粮食生产安全。【方法】从粮食上分离筛选出35株木霉,通过多基因系统发育分析和形态学观察方法进行菌种鉴定,利用平板对峙试验筛选出对粮食常见危害真菌有抑制作用的菌株。【结果】35株木霉分属于8个种,分别为非洲哈茨木霉(Trichodermaafroharzianum)、类棘孢木霉(Trichodermaasperelloides)、 Trichoderma amoenum、近深绿木霉(Trichoderma paratroviride)、Trichoderma obovatum、长枝木霉(Trichoderma longibrachiatum)、东方木霉(Trichodermaorientale)和深绿木霉(Trichodermaatroviride)。对峙试验结果表明,这8种木霉对于粮食上分离到的10种危害真菌均具有较好的抑制效果。非洲哈茨木霉(T.afroharzi...     

土壤中木霉的分离及其对毒死蜱降解特性研究

为挖掘保护地土壤中的能降解毒死蜱的木霉资源,从长期污染的土壤中共分离6个不同木霉菌株,通过形态学特征和ITS r DNA序列对各菌株进行鉴定,6个菌株分别为哈茨木霉(Trichoderma harzianum)、绿色木霉(T.viride)、康宁木霉(T.koningii)、拟康宁木霉(T.koningiopsis)、长枝木霉(T.longibrachiatum)和短密木霉(T.brevicompactum)。选择降解活性最高的长枝木霉TC5菌株,测定各种条件下对毒死蜱的降解特性。结果表明,木霉TC5在中性或偏碱性条件下对毒死蜱具有更好的降解活性;添加碳源或提高木霉的接种浓度,能提高对毒死蜱的降解活性;在50-300 mg/L范围内,随毒死蜱浓度升高,木霉的降解活性也明显提高。盆栽试验中木霉TC5对毒死蜱保持较强的降解活性,但在自然土壤中降解活性显著低于在灭菌土壤中。分离的木霉菌株在土壤农药污染修复方面具有应用开发潜力。     

Phylogeny of the genus trichoderma based on sequence analysis of the internal transcribed spacer region 1 of the rDNA cluster

Sequences of the internal transcribed spacer region 1 (ITS1) of the ribosomal DNA were used to determine the phylogenetic relationships of species of Trichoderma sect. Pachybasium. To this end, 85 strains-including all the available ex-type strains-were analyzed. Parsimony analysis demonstrated that the section is nonmonophyletic, distributing the 85 strains among three main groups that were supported by bootstrap values. Group A comprises two clades (A1 and A2), with A1 including T. polysporum, T. piluliferum, and T. minutisporum, while A2 included T. hamatum, T. pubescens, and T. strigosum in addition to species previously included in sect. Trichoderma (i.e., T. viride, T. atroviride, and T. koningii). The ex-type strain of T. fasciculatum formed a separate branch basal to clade A. Clade B contained the sect. Pachybasium members T. harzianum, T. fertile, T. croceum, T. longipile, T. strictipile, T. tomentosum, T. oblongisporum, T. flavofuscum, T. spirale, and the anamorphs of Hypocrea semiorbis and H. cf. gelatinosa. Sequence differences among clades A1, A2, and B were in the same order of magnitude as between each of them and T. longibrachiatum, which was used as an outgroup in these analyses. Sequence differences within clades A1, A2, and B were considerably smaller: in some cases (i.e., T. virens and T. flavofuscum; T. strictipile and H. cf. gelatinosa), the ITS1-sequences were identical, suggesting conspecifity. In other cases (e.g., T. crassum and T. longipile; T. harzianum, T. inhamatum, T. croceum, T. fertile, and H. semiorbis; T. hamatum and T. pubescens; and T. viride, T. atroviride, and T. koningii) differences were in the range of 1-3 nt only, suggesting a very close phylogenetic relationship. The sequence of a previously described aggressive mushroom competitor group of T. harzianum strains (Th2) was strikingly different from that of the ex-type strain of T. harzianum and closely related species and is likely to be a separate species. Copyright 1998 Academic Press.     

Peptaibiomics: Screening for polypeptide antibiotics (peptaibiotics) from plant-protective Trichoderma species

Eight strains of Trichoderma species (T. strigosum, T. erinaceus, T. pubescens, T. stromaticum, and T. spirale as well as T. cf. strigosum, T. cf. pubescens) were selected because of their antagonistic potential against Eutypa dieback and Esca which are fungal diseases of grapevine trunks. These isolates were screened for the production of a group of polypeptide antibiotics named peptaibiotics, including its subgroups peptaibols and lipopeptaibols. Fully-grown fungal cultures on potato-dextrose agar were extracted with CH(2)Cl(2)/MeOH, and these extracts were subjected to SPE using C(18) cartridges. The methanolic eluates were analyzed by on-line LC/ESI-MS(n) coupling--a method which is referred to as 'peptaibiomics'. New seven-, ten-, and eleven-residue lipopeptaibols, with N-terminal alkanoyl, and C-terminal leucinol or isoleucinol residues were found and named lipostrigocins and lipopubescins. Furthermore, new 18-residue peptaibols named trichostromaticins and 19-residue peptaibols named trichostrigocins were discovered. One peptaibiotic carrying a free C-terminal valine (or isovaline) named trichocompactin XII was also sequenced. These results corroborate the hypothesis that peptaibiotics might contribute to the plant-protective action of their fungal producers. The data also point out that comparison of peptaibiotic sequences is of limited relevance in order to establish chemotaxonomic relationships among species of the genus Trichoderma.     

Genetic and metabolic diversity of Trichoderma: a case study on South-East Asian isolates

We have used isolates of Trichoderma spp. collected in South-East Asia, including Taiwan and Western Indonesia, to assess the genetic and metabolic diversity of endemic species of Trichoderma. Ninety-six strains were isolated in total, and identified at the species level by analysis of morphological and biochemical characters (Biolog system), and by sequence analysis of their internal transcribed spacer regions 1 and 2 (ITS1 and 2) of the rDNA cluster, using ex-type strains and taxonomically established isolates of Trichoderma as reference. Seventy-eight isolates were positively identified as Trichoderma harzianum/Trichoderma inhamatum (37 strains) Trichoderma virens (16 strains), Trichoderma spirale (8 strains), Trichoderma koningii (3 strains), Trichoderma atroviride (3 strains), Trichoderma asperellum (4 strains), Hypocrea jecorina (anamorph: Trichoderma reesei; 2 strains), Trichoderma viride (2 strains), Trichoderma hamatum (1 strain), and Trichoderma ghanense (1 strain). Analysis of biochemical characters revealed that T. virens, T. spirale, T. asperellum, T. koningii, H. jecorina, and T. ghanense formed clearly defined clusters, thus exhibiting species-specific metabolic properties. In biochemical character analysis T. atroviride and T. viride formed partially overlapping clusters, indicating that these two species may share overlapping metabolic characteristics. This behavior was even more striking with T. harzianum/T. inhamatum where genotypes defined on the basis of ITS1 and 2 sequences overlapped significantly with adjacent genotypes in the biochemical character analysis, and four strains from the same location (Bali, Indonesia) even clustered with species from section Longibrachiatum. The data indicate that the T. harzianum/T. inhamatum group represents species with high metabolic diversity and partially unique metabolic characteristics. Nineteen strains yielded three different ITS1/2 sequence types which were not alignable with any known species. They were also uniquely characterized by morphological and biochemical characters and therefore represent three new taxa of Trichoderma.     

Trichoderma biodiversity in China

In the present study, we made further investigation into the diversity of Trichoderma in China than previous ones utilizing comprehensive approaches of morphological microscopic observation and phylogenetic analysis by detecting molecular markers. One thousand nine hundred ten Trichoderma strains were isolated from soil or other materials in China: East (Anhui, Fujian, Jiangsu, Jiangxi, Shandong, Zhejiang province and Shanghai municipality), South-West (Guizhou, Qinghai, Shanxi, Sichuan and Yunnan province, Tibet Autonomous Region and Chongqing municipality), South-East (Guangdong, Guangxi, Hainan province), and Middle China (Henan, Hubei and Hunan province). Representative isolates were verified at the species level by morphological characters and the oligonucleotide barcode program TrichoOKey v.10 and the custom BLAST server TrichoBLAST, using sequence of the ITS 1 and 2 region of the rDNA cluster and partial sequences of translation elongation factor 1-alpha(tef1-α). A total of 23 Trichoderma species were identified : T.asperellum, T.atrioviride, T.aureovriride, T.brevicompactum, T.citrioviride, T.erinaceum, T.gamsii, T.hamatum, T.harzianum (H.1ixii), T.intricatum, T.koningii (H.koningii), T.koningiopsis, T.longibranchiatum, T.pleuroticola, T.reeseii (H.jecorina), T.sinensis, T.spirale, T.stromaticum, T.tomentosum, T.velutinum, T.vermipilum, T.virens (H.virens), T.viride. Among them, 3 species: T.intricatum, T.stromaticum, T.vermipilum were first reported in China; T.harzianum (H,1ixii) was the most widely distributed species in China. This study further shows that, the highest biodiversity of Trichoderma population appeared in South-West China.     

Soils of a Mediterranean hot spot of biodiversity and endemism (Sardinia, Tyrrhenian Islands) are inhabited by pan-European, invasive species of Hypocrea/Trichoderma

We have used a Mediterranean hot spot of biodiversity (the Island of Sardinia) to investigate the impact of abiotic factors on the distribution of species of the common soil fungus Trichoderma . To this end, we isolated 482 strains of Hypocrea / Trichoderma from 15 soils comprising undisturbed and disturbed environments (forest, shrub lands and undisturbed or extensively grazed grass steppes respectively). Isolates were identified at the species level by the oligonucleotide BarCode for Hypocrea / Trichoderma ( TrichO KEY), sequence similarity analysis ( Tricho blast ) and phylogenetic inferences. The majority of the isolates were positively identified as pan-European and/or pan-global Hypocrea / Trichoderma species from sections Trichoderma and Pachybasium , comprising H. lixii/T. harzianum , T. gamsii , T. spirale , T. velutinum , T. hamatum , H. koningii/T. koningii , H. virens/T. virens , T. tomentosum , H. semiorbis , H. viridescens/T. viridescens , H. atroviridis/T. atroviride , T. asperellum , H. koningiopsis/T. koningiopsis and Trichoderma sp. Vd2. Only one isolate represented a new, undescribed species belonging to the Harzianum–Catoptron Clade. Internal transcribed spacer sequence analysis revealed only one potentially endemic internal transcribed spacer 1 allele of T. hamatum . All other species exhibited genotypes that were already found in Eurasia or in other continents. Only few cases of correlation of species occurrence with abiotic factors were recorded. The data suggest a strong reduction of native Hypocrea / Trichoderma diversity, which was replaced by extensive invasion of species from Eurasia, Africa and the Pacific Basin.     

Genetic relatedness of Trichoderma sect. Pachybasium species based on molecular approaches

Molecular approaches including internal transcribed spacer (ITS) sequences of ribosomal DNA, universal primer polymerase chain reaction (UP-PCR) fingerprinting, and DNA-DNA hybridization were used to study the genetic relatedness of species within Trichoderma sect. Pachybasium. In the analysis of ITS and 5.8S sequences of ribosomal DNA, parsimony analysis demonstrated that forty-one strains were distributed into five main groups supported by high bootstrap values. The species of Trichoderma sect. Pachybasium were clustered into groups I, II, and IV, with the strains of Trichoderma fasculatum and Trichoderma strictipile forming a separate branch, an independent group V. Some species within each group showed nearly identical sequence differences (fewer than 1-3 bp). UP-PCR and DNA-DNA hybridization were further used to clarify the genetic relatedness of these species with highly similar ITS sequences. Highly similar or identical UP-PCR profiles and high values of DNA complementarity (>70%) were observed among some species, Trichoderma hamatum and Trichoderma pubescens; Trichoderma croceum, Trichoderma polysporum and Trichoderma album, Trichoderma crassum and Trichoderma flavofuscum; and Trichoderma strictipile and Trichoderma fasciculatum. Although every species can be differentiated morphologically, the species showed highly similar molecular characteristics in the above cases, indicating that they could be conspecific. However, in some cases (Trithoderma longipile, T. crassum and T. flavofuscum; Trichoderma fertile and Trichoderma minutisporum; Trichoderma tomentosum, Trichoderma inhamatum and Trichoderma harzianum) there were discriminative patterns of UP-PCR and (or) low levels (<50%) of DNA-DNA hybridization; even their ITS sequences were similar, suggesting a closely phylogenetic relationship.     

耐热真菌的分类鉴定及rDNA-ITS系统发育分析

分别从山东、山西、河北、陕西、河南、云南、江西、海南、新疆、内蒙古、甘肃、广州等地实地采集堆肥、培养料、草堆等标本600多份,分离鉴定出耐热真菌28种,其中包括2个中国新记录种,沙生梭孢壳Thielavia arenaria和丰孢木霉Trichoderma saturnisporum,其余26个为中国已知种.在形态分类...     

木霉属Trichoderma组和Pachybasium组的分子系统学研究

对来源不同的木霉及其有性型Longibrachiatum组、Trichoderma 组和Pachybasium组的81个菌株进行了ITS序列测定,并对ITS1序列用PHYLIP程序包中的DNAPARS程序进行系统发育分析。结果表明Trichoderma 组和Pachybasium组的所有菌株可分成两个群(A,B),B群进一步分为4个分支(B1,B2,B3,B4);A群由Trichoderma 组的H. aureoviridis和未鉴定到种的3个Hypocrea菌株构成;B1,B2,B4群均由Pachybasium组菌株构成;B3群由Pachybasium组的T. hamatum、T. strigosum和Trichoderma 组的T. asperellum、T. atroviride、T. koningii、T. viride和Hypocrea菌株T261构成。2个组相互交叉,组间没有明确的区分,进一步证明Pachybasium组是多系的。建议将Trichoderma 组中的T. viride aggr.、T. atroviride、T. koningii归并入Pachybasium组,对Trichoderma 组重新定义。