In vitro studies on the potential for biological control of Aspergillus flavus and Fusarium moniliforme by Trichoderma species

Culture filtrates of Trichoderma viride and Trichoderma harzianum were inhibitory of Fusarium moniliforme and, to a lesser extent, Aspergillus flavus. The degree of inhibition was, however, dependent on the carbon or nitrogen source incorporated into the medium. Scanning electron microscopy revealed the development of abnormal fruiting structures on exposure to some Trichoderma culture filtrate, while macroscopically, growth restriction and, in the case of A. flavus, altered colony colouration were observed. Based on the results of inverted colony culture, it would appear that some isolates of Trichoderma produce inhibitory volatile compounds. The production of possible antibiotics was also demonstrated. The aggressive behaviour (towards A. flavus and F. moniliforme) demonstrated by Trichoderma spp. may be partly explained by the liberation of extracellular enzymes by these fungi. An isolate of T. viride exhibited amylolytic, pectinolytic, proteolytic and cellulolytic activity. Based on the results of the present investigation, Trichoderma spp. are potential candidates for biocontrol of some mycotoxin-producing fungi, but there exists some doubt as to their osmotolerance within the air-dry seed. This revised version was published online in August 2006 with corrections to the Cover Date.     

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。     

Growth of Trichoderma harzianum and Myceliophthora thermophila in palm oil sludge

Uncentrifuged palm oil mill sludge (POS) diluted to about 50% (v/v in tap water) supports good mycelial growth of Myceliophthora thermophila and Trichoderma harzianum. Both of the selected fungi are non-toxic to mice. After 24 h M. thermophila grown in batch culture in POS yielded 28.6 g/l of mycelial biomass with biological oxygen demand (BOD) and chemical oxidation demand (COD) reductions of 72% and 74% respectively. T. harzianum yielded 24.4 g/l of mycelial biomass with BOD and COD reductions of 67% and 68% respectively. The crude protein of the mycelial biomass of M. thermophila and T. harzianum was twice that of untreated POS. T. harzianum showed amylolytic activity while M. thermophila was cellulolytic and lipolytic.     

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。     

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.     

Randomly amplified polymorphic DNA fingerprinting identifies subgroups of Trichoderma viride and other Trichoderma sp. capable of chestnut blight biocontrol

Abstract Eleven strains of Trichoderma viride , 2 strains of the putative teleomorph Hypocrea rufa and 9 of several other Trichoderma sp. were characterized by random polymorphic DNA amplification (RAPD) fingerprinting and screened for their ability to antagonize growth of European strains of the chestnut blight causing fungus Cryphonectria parasitica , using a dual-culture assay. The best strains were found in the species T. harzianum, T. parceramosum , a distinguishable subgroup of T. viride and a not named Trichoderma sp. The successful application of these strains against chestnut blight in vivo is demonstrated.     

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．     

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.     

An image analysis technique to estimate the cell density and biomass concentration of Trichoderma reesei

Aim:  The objective is to develop an automated image analysis protocol to quantify the cell volume fraction of filamentous fungi ( Trichoderma reesei ) and estimate the biomass concentration.
Methods and Results:  Both dry weight and image analyses were performed on samples collected periodically from 7-l stirred tank fermentations. Using the projected area of lactophenol blue-stained hyphae, the fraction occupied by the cells in a given volume was estimated. Combined with the biomass dry weight obtained by filtration, the method was used to estimate the density of filamentous fungi. Knowing the density of fungi, the algorithm was employed to quantitatively assess the biomass evolution during the course of fermentation even in the presence of solid particles.
Conclusions:  A density of 0.334 g dry weight cm⁻³ was found for T. reesei RUT C-30. The image analysis protocol allowed successful estimation of biomass concentration in the presence or absence of solid particles.
Significance and Impact of the Study:  Methods to quantify biomass during the industrial production of cellulase with T. reesei are often limited due to the presence of solid substrates. The image analysis protocol presented here offers a quick and easy way to estimate biomass concentration of filamentous micro-organisms in insoluble medium.     

哈茨木霉与绿色木霉对杉木种子萌发和幼苗生长的影响

以杉木种子为材料,研究不同浓度(0.003、0.03、0.3、3、30、300 mg·L^-1)哈茨木霉和绿色木霉溶液对杉木种子萌发和幼苗生长的影响.结果表明: 各浓度木霉溶液处理对杉木种子萌发和幼苗生长均有一定的促进作用,其促进效果随着处理浓度的增加均呈先升后降的趋势.与对照相比,0.03 mg·L^-1哈茨木霉和绿色木霉处理对提高杉木种子的发芽率、发芽势、发芽指数、活力指数、根长、苗高和鲜质量效果最佳,分别提高了57.6%、125.0%、51.0%、209.2%、114.3%、16.1%、24.6%和42.7%、76.7%、43.9%、185.4%、113.8%、8.6%、22.6%;0.3 mg·L^-1哈茨木霉和绿色木霉显著提高杉木幼苗超氧化物歧化酶和过氧化物酶活性,分别增加了157.6%、179.9%和127.5%、116.2%,而丙二醛含量降低了86.1%和72.4%. 0.03～0.3 mg·L^-1浓度的哈茨木霉和绿色木霉不仅能显著促进杉木种子的萌发和幼苗生长,而且能够提高其抗氧化酶活性,增强杉木幼苗的抗逆性.     

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。     

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。     

Influence of resource unit distribution and quality on the activity of soil fungi in a particulate medium

Experiments were set up to assess the relative impacts of grass lamina fragment density, size and quality on the activity and growth of four fungi in a particulate soil-like matrix. The fungi studied were Cladosporium cladosporioides , Fusarium lateritium , Phoma exigua and Trichoderma viride , all of which are common inhabitants of UK upland grassland soils. Resource quality was varied by using three contrasting grasses as sources of lamina fragments: Lolium perenne , Agrostis capillaris and Nardus stricta . All the fungi were able to forage effectively through the soil-like matrix (at a rate of 1–2 ml matrix d⁻¹) and colonize and partially decompose available lamina fragments (up to 40% d. wt loss). Foraging rates (ml matrix d⁻¹) were affected by lamina fragment species but not by fragment density or size. In general, F. lateritium and T. viride foraged at a faster rate than the other species. Mycelial activity in the soil-like matrix was directly proportional to total lamina fragment availability and was unaffected by fragment size. Biomass production on solid media was also directly related to substrate (carbohydrate) availability. The results indicate that these fungi can adjust their growth patterns in response to fragment density so as to maximize foraging efficiency (energy acquired per unit exploratory biomass production). Differences in lamina fragment species were responsible for up to 50% reduction in mycelial activity.     

木霉属中国新纪录种Trichoderma pleuroticola和T. pleurotum

【目的】对蔬菜大棚土壤中和阿魏菇腐烂的菌盖上分离的两株木霉菌进行分类鉴定。【方法】结合形态学分类特征和ITS序列分析的方法进行鉴定。【结果】从蔬菜大棚的土壤中和阿魏菇腐烂的菌盖上分离的两株木霉菌分别为Trichoderma pleuroticola和T.pleurotum。T.pleuroticola的形态特征与T.harzianum相似,但其分生孢子显著大于T.harzianum的分生孢子,且在PDA上产生黑褐色的色素以及黄色的结晶物。T.pleurotum典型特征是分生孢子梗单生,有时匍匐,分枝散生,初级分枝和分生孢子梗顶端聚生,类似粘帚霉。【结论】分离的两株木霉分别是T.pleuroticola和T.pleurotum,为木霉菌中国新纪录种。     

Biological control of Sclerotinia sclerotiorum attacking soybean plants. Degradation of the cell walls of this pathogen by Trichoderma harzianum (BAFC 742)

Two experiments of biological control of Sclerotinia sclerotiorum, one in the greenhouse and the other in the field, were carried out with soybean and Trichoderma harzianum as host and antagonist, respectively. Significant control of disease was achieved in both experiments, but there were no significant differences in plant growths. In the greenhouse, the application of T. harzianum as alginate capsules, increased the survival of soybean plants more than 100% with respect to the disease treatment. In the field, T. harzianum treated plants survived 40% more than those from the disease treatment, showing a similar survival level to control plants. Besides, a significant reduction (62.5%) in the number of germinated sclerotia was observed in the Trichoderma treated plot. Chitinase and 1,3-β- glucanase activities were detected when T. harzianum was grown in a medium containing Sclerotinia sclerotiorum cell walls as sole carbon source. In addition, electrophoretic profiles of proteins induced in T. harzianum showed quantitative differences between major bands obtained in the media induced by S. sclerotiorum cell walls and that containing glucose as a sole carbon source. This revised version was published online in June 2006 with corrections to the Cover Date.     

Changes in selected enzyme activities during growth of pure and mixed cultures of the white-rot decay fungus Trametes versicolor and the potential biocontrol fungus Trichoderma harzianum.

Two filamentous fungi, the white-rot fungus Trametes versicolor and the soil fungus and potential biocontrol organism Trichoderma harzianum, have been grown in pure and mixed cultures on low-N (0.4 mM) and high-N (4 mM) defined synthetic media to determine the activities of selected wood-degrading enzymes such as cellobiase, cellulase, laccase, and peroxidases. Growth characteristics and enzyme activities were examined for potential correlations. Such correlations would allow the use of simple enzyme assays for measuring biomass development and would facilitate predictions about competitiveness of species in mixed fungal cultures. Our results show that while laccase and Poly Red-478 peroxidase activities indicate survival of the decay fungus, none of the monitored extracellular enzymes can serve as a quantitative indicator for biomass accumulation. As expected, the level of available nitrogen affected the production of the enzymes monitored: in low-N media, specific cellobiase, specific cellulase, and peroxidase activities were enhanced, while laccase activities were reduced. Most importantly, laccase activities of Trametes versicolor, and to a smaller extent, cellobiase activities of both fungi, were significantly induced in mixed cultures of Trametes versicolor and Trichoderma harzianum.     

Biomass production from Trichoderma viride in nonconventional oat medium

Oatmeal, an alternative, renewable, and low‐cost substrate, was used for the production of Trichoderma viride spores by submerged fermentation. The nonconventional oat medium was only supplemented with potato peptone, which is a green source of nitrogen for the microorganism. Because particles are suspended in the nonconventional oat medium, the characterization was based on viscosity, average particle diameter, size distribution, and porosity of the particles. Because of the complexity of the fungal biomass extraction, the dry weight and protein content were used as methods for quantifying the growth of T. viride. The inversion between the proportion of mycelia and spores was captured in the microscopic image analysis during the fermentation process. After 60 h, spores began to appear, accounting for most of the form present at 120 h of fermentation. The decrease in pH and the increase in glucose concentration during fermentation indicate that glucan hydrolysis occurs and that glucose is released into the medium. The potential for industrial applications of submerged fermentation with oats for biomass production of T. viride is noted in the results. This simple and easily controllable process has several advantages, including the use of low‐cost substrates for the propagation of a microorganism that is widely used in scientific and commercial settings. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

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.     

Screening of bioagents against root rot of mung bean caused by Rhizoctonia solani

A laboratory and green house experiment was carried out on the comparative antagonistic performance of four different bioagents (Aspergillus sp., Gliocladium virens, Trichoderma harzianum and T. viride) isolated from soil against Rhizoctonia solani. Under laboratory conditions, T. harzianum exhibited maximum (75.55%) mycelial growth inhibition of R. solani This was followed by T. viride, which showed 65.93 per cent mycelial growth inhibition of the pathogen. Gliocladium virens was also found to be effective antagonists, which exhibited 57.77 per cent mycelial growth inhibition. While Aspergillus sp exhibited minimum growth inhibition (45.74%) in comparison to other bioagents. Under green house conditions, T. harzianum gave maximum protection of the disease (72.72%) followed by T. viride, which exhibited 54.54 per cent disease control. However, G. virens and Aspergillus sp were found least effective in controlling root rot of mungbean.     

Screening Selected Fungi for Antagonism towards Pseudocercosporella herpotrichoides (Fron) Deighton,the Cause of Eyespot Disease of Cereals

In the laboratory, a dual culture primary screen using wheat straw agar (WSA) showed that 64 out of 152 selected fungi inhibited the in vitro growth of Pseudocercosporella herpotrichoides by 50% or more. An ‘asymmetric’ species of Penicillium isolated from wheat stubble was the most effective, inhibiting growth of the pathogen by 70%. The results were similar using both W‐type and R‐type isolates of P. herpotrichoides. Other fungi showing in vitro antagonism included: Trichoderma viride, T. koningii, T. harzianum, Chaetomium globosum, Acremonium persicum, Botryotrichum piluliferum, Sordaria alcina, Microdochium bolleyi, Rhizoctonia cerealis and Laetisaria arvalis.

In a glasshouse secondary screen, the 64 fungi showing antagonism in vitro were assessed further for their ability to reduce eyespot disease symptoms of wheat seedlings using a straw collar co‐inoculation technique. Of them, 13 fungi reduced disease symptoms significantly: a Chaetomium sp. from stubble, two Fusarium spp., a F. culmorum isolate T. viride, T. koningii, T. harzianum, B. piluliferum, M. bolleyi, L. arvalis, the ‘asymmetric’ Penicillium sp. and two unidentified fungi from stubble. A F. culmorum isolate was the most effective antagonist on average, giving disease reductions of 54 and 58% for the W‐type and R‐type of P. herpotrichoides respectively. M. bolleyi, T. harzianum and the F. culmorum isolate were the most effective antagonists of the W‐type, all three reducing disease by 54%. T. viride was most effective against the R‐type, reducing disease by 64%.