Molecular characterization and identification of biocontrol isolates of Trichoderma spp

The most common biological control agents (BCAs) of the genus Trichoderma have been reported to be strains of Trichoderma virens, T. harzianum, and T. viride. Since Trichoderma BCAs use different mechanisms of biocontrol, it is very important to explore the synergistic effects expressed by different genotypes for their practical use in agriculture. Characterization of 16 biocontrol strains, previously identified as "Trichoderma harzianum" Rifai and one biocontrol strain recognized as T. viride, was carried out using several molecular techniques. A certain degree of polymorphism was detected in hybridizations using a probe of mitochondrial DNA. Sequencing of internal transcribed spacers 1 and 2 (ITS1 and ITS2) revealed three different ITS lengths and four different sequence types. Phylogenetic analysis based on ITS1 sequences, including type strains of different species, clustered the 17 biocontrol strains into four groups: T. harzianum-T. inhamatum complex, T. longibrachiatum, T. asperellum, and T. atroviride-T. koningii complex. ITS2 sequences were also useful for locating the biocontrol strains in T. atroviride within the complex T. atroviride-T. koningii. None of the biocontrol strains studied corresponded to biotypes Th2 or Th4 of T. harzianum, which cause mushroom green mold. Correlation between different genotypes and potential biocontrol activity was studied under dual culturing of 17 BCAs in the presence of the phytopathogenic fungi Phoma betae, Rosellinia necatrix, Botrytis cinerea, and Fusarium oxysporum f. sp. dianthi in three different media.     

Mitochondrial DNAs and plasmids as taxonomic characteristics in Trichoderma viride.

Mitochondrial DNA (mtDNA) was purified from 12 isolates of the Trichoderma viride aggregate and found to be, on the average, 32.7 kb in size. Plasmids were present in the mtDNA preparations from 8 of 12 strains of T. viride examined. Plasmids in four of the strains produced ladderlike banding patterns on gels, and these plasmids were studied in detail. The ladderlike patterns were produced by single molecules that were supercoiled to various degrees. Plasmids from two of the strains do not have homology with the mtDNA but do have a limited amount of homology with each other. No phenotype could be associated with the presence of a plasmid. Restriction endonuclease digestion of the mtDNAs produced patterns in which the presence or absence of certain fragments correlated with the classification of the strains into T. viride group I or II. Phenetic cluster analysis and parsimony analysis of the fragment patterns produced groups that corresponded to T. viride groups I and II. The fragment patterns were very diverse, with nearly all strains having a unique pattern. However, two strains of T. viride group I from widely different geographical locations did have identical restriction patterns for all the enzymes used in this study. This result indicates that it may not be possible to use mtDNA restriction patterns alone to identify Trichoderma strains.     

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.     

木霉(Trichoderma spp.)对三种引起大棚蔬菜病害病原菌的影响

通过木霉属(Trichoderma) 3菌株与双鸭山蔬菜大棚中的黄瓜枯萎病菌(FusariumoxysporumSchlecht.f.cucumerinum)、黄瓜果腐病菌(PhytophthoracapsiciLeonian)、菜豆叶枯病菌(Cladosporiumsp .)的对峙培养试验,结果表明:绿色木霉1(TrichodermaviridePers.exGray 1)可作为双鸭山蔬菜大棚中的黄瓜枯萎病、黄瓜果腐病、菜豆叶枯病3种病害的生物防治拮抗菌加以利用,该拮抗菌对菜豆叶枯病菌抑制效果最好;绿色木霉2 (Tricho dermaviride 2 )对黄瓜果腐病菌抑制效果最好;而哈茨木霉(TrichodermaharzianumRifai)对以上3种病原菌都有抑制效果,对菜豆叶枯病菌抑制效果最好。从试验结果还可看出,绿色木霉2对黄瓜枯萎病菌和菜豆叶枯病菌的生长有促进作用。     

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。     

Identification of Trichoderma strains from building materials by ITS1 ribotyping, UP-PCR fingerprinting and UP-PCR cross hybridization

To study the role of Trichoderma in sick building syndrome, it is essential to be able to accurately identify species. Forty-four strains of Trichoderma spp. isolated from Danish buildings damaged by water leaks were identified using ITS1 ribotyping and universally primed PCR, UP-PCR. Ribotyping allowed the assignment of the strains into three distinct groups. High similarity of UP-PCR banding profiles of the strains allowed species designation for almost all strains (43 out of 44) when compared with the UP-PCR banding profiles obtained from reference strains of T. atroviride, T. citrinoviride, T. harzianum, T. longibrachiatum and T. viride. However, cross hybridization of UP-PCR products showed that the latter strain had high DNA homology to the ex-type strain of T. hamatum. The combined approach is a convenient way for reliable identification of Trichoderma strains.     

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.     

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.     

绿色木霉代谢产物的植物毒性研究

通过人工固体培养和液体发酵研究发现绿色木霉的代谢产物对植物的幼苗生长有抑制作用,其代谢产物大量分泌到它所生长的环境中,在不同的营养基质中其代谢产物的抑制作用有差异．     

Proteolytic activity of Trichoderma viride in mixed culture with Sclerotium rolfsii in soil.

Cultures of Sclerotium rolfsii and Trichoderma viride together in autoclaved soil were assayed at intervals during 8 days of incubation for proteolytic activity (PA) of T. viride. Significant proteolytic activity was detected only in soil containing T. viride (i.e., T. viride alone or S. rolfsii + T. viride); greatest activity occurred between 3 and 4 days after infestation and declined rapidly thereafter. Maximal PA in the mixed-culture soil was accompanied by an increase in soil pH. optimal pH values for PA was 5.5-6.5 with a maximum at 6.0.     

Lipid accumulation in Trichoderma species

Abstract Two filamentous fungi, Trichoderma harzianum and Trichoderma viride , were compared for their ability to synthesize lipids on different carbon and nitrogen sources. Three culture media were selected for each strain after preliminary screening. All the test media were nitrogen-deficient (C/N = 60) so as to stimulate lipid accumulation. For both microorganisms the glucose-ammonium sulphate medium was the most conducive to lipid production: a lipid accumulation of 17% (w/w) of biomass dry weight was obtained for T. harzianum and of 32% (w/w) of biomass dry weight for T. viride . In sucrose-sodium nitrate medium T. harzianum was able to accumulate almost 25% (w/w) of its biomass in lipid form. However the small quantity of biomass produced (2 g dry weight/l) limited the quantity of lipid obtained. Neutral lipids, free fatty acids and phospholipids were monitored during 8 days of cultivation of the two fungi.     

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。     

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.     

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。     

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。     

Typing and selection of wild strains of Trichoderma spp. producers of extracellular laccase

Using the ITS region and the gene tef1, 23 strains of the genus Trichoderma were identified as belonging to the species T. harzianum (n = 14), T. olivascens (n = 1), T. trixiae (n = 1), T. viridialbum (n = 1), T. tomentosum (n = 2), T. koningii (n = 1), T. atroviride (n = 1), T. viride (n = 1), and T. gamsii (n = 1). Strains expressing extracellular laccase activity were selected by decolorization/oxidation assays in solid media, using azo, anthraquinone, indigoid, and triphenylmethane dyes, and the phenolic substances tannic acid and guaiacol. No strain decolorized Direct Blue 71 or Chicago Blue 6B, but all of them weakly oxidized guaiacol, decolorized Methyl Orange, and efficiently oxidized tannic acid. Based in decolorization/oxidation assays, strains CMU‐1 (T. harzianum), CMU‐8 (T. atroviride), CMU‐218 (T. viride), and CMU‐221 (T. tomentosum) were selected for evaluating their extracellular laccase activity in liquid media. Strain CMU‐8 showed no basal laccase activity, while strains CMU‐1, CMU‐218, and CMU‐221 had a basal laccase activity of 1,313.88 mU/mL, 763.88 mU/mL, and 799.53 mU/mL, respectively. Addition of sorghum straw inhibited laccase activity in strain CMU‐1 by 34%, relative to the basal culture, while strains CMU‐8, CMU‐21, and CMU‐221 increased their laccase activity by 1,321.5%, 64%, and 47%, respectively. These results show that assayed phenolic substrates are good tools for selecting laccase producer strains in Trichoderma. These same assays indicate the potential use of studied strains for bioremediation processes. Straw laccase induction suggests that analyzed strains have potential for straw delignification in biopulping and other biotechnological applications. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:787–798, 2016     

Metabolism of Linoleic Acid or Mevalonate and 6-Pentyl-alpha-Pyrone Biosynthesis by Trichoderma Species

The understanding of the biosynthetic pathway of 6-pentyl-alpha-pyrone in Trichoderma species was achieved by using labelled linoleic acid or mevalonate as a tracer. Incubation of growing cultures of Trichoderma harzianum and T. viride with [U-C]linoleic acid or [5-C]sodium mevalonate revealed that both fungal strains were able to incorporate these labelled compounds (50 and 15%, respectively). Most intracellular radioactivity was found in the neutral lipid fraction. At the initial time of incubation, the radioactivity from [C]linoleic acid was incorporated into 6-pentyl-alpha-pyrone more rapidly than that from [C]mevalonate. No radioactivity incorporation was detected in 6-pentyl-alpha-pyrone when fungal cultures were incubated with [1-C]linoleic acid. These results suggested that beta-oxidation of linoleic acid was a probable main step in the biosynthetic pathway of 6-pentyl-alpha-pyrone in Trichoderma species.     

Isolation and characterization of a trypsin-like protease from Trichoderma viride.

A serine endopeptidase with a molecular mass of 25 kDa has been purified from the culture filtrate of Trichoderma viride to electrophoretic homogeneity. The isoelectric point was determined at 7.3. Two carboxyl sites at Arg22 and Lys29 of the oxidized insulin B-chain were cleaved, and peptidyl-p-nitroanilide substrates with Lys or Arg at the P1 position were also hydrolyzed by the enzyme. These results suggest that the specificity of T. viride protease is similar to that of trypsin. However, the hydrolytic activity toward casein of T. viride protease was less than that of porcine trypsin. The amino-terminal sequence of the enzyme protein is similar to that of bovine trypsin. It seems that the trypsin of T. viride is a protease which is promising for the substitution of animal trypsin in the food industry and in medicine at this stage.     

Comparison of four purified extracellular 1,4-beta-D-glucan cellobiohydrolase enzymes from Trichoderma viride.

Four electrophoretically distinct 1,4-beta-D-glucan cellobiohydrolase enzymes (exo-cellobiohydrolase, EC 3.2.1.91) from Trichoderma viride have been purified to homogeneity. Three enzymes (A, B, and C) were from a commercial T. viride preparation whereas the other (D) was from T. viride QM 9123 grown on cellulose in submerged culture. The enzymes were similar with respect to ultraviolet light absorption, amino acid and amino sugar composition, heat stability, molecular weight, specific activity, and carboxyterminal residues, indicating very nearly identical polypeptide portions. The enzymes also exhibited immunological cross-reactivity. The enzymes differed most in the content and composition of covalently bound neutral carbohydrate.     

New Mycotoxin, Trichotoxin A, from Trichoderma viride Isolated from Southern Leaf Blight-Infected Corn

A new mycotoxin, trichotoxin A, was found in a solvent extract of the mycelium of Trichoderma viride isolated from corn infected with southern leaf blight. Trichotoxin A is a cyclic peptide with the following amino acid composition: (gluN)(2), (glu)(1), (pro)(2), (gly)(1), (ala)(3), (leu)(3), (2-methyl alanine)(1).