Species Diversity,Distribution, and Genetic Structure of Endophytic and Epiphytic <Emphasis Type="Italic">Trichoderma</Emphasis> Associated with Banana Roots

Selective isolation, molecular identification and AFLP were used to investigate the distribution of the various species of endophytic and epiphytic Trichoderma associated with banana roots and to compare and contrast their genetic structure. Three specific groups of Trichoderma were observed in the roots of banana. Group one, which made up the largest population, comprised T. asperellum, T. virens, and Hypocrea lixii, which were isolated from both inside and on the surface of the banana roots, while group two, made up of T. atroviride and T. koningiopsis, existed on the surface only. Group three, comprising only T. brevicompactum was isolated from the inside of the roots. The AFLP analysis revealed Nei’s diversity indices of 0.15 and 0.26 for epiphytic T. asperellum and T. virens, respectively. The index values of 0.11 and 0.11 were obtained for endophytic T. asperellum and T. virens, respectively. The genetic diversity within endophytic T. asperellum and T. virens was lower than that within the epiphytes. This suggests that endophytic Trichoderma has a higher genetic conservation and is compatible with the relatively stable microenvironments inside roots.     

Evaluation of β-1,3-glucanase and β-1,4-glucanase enzymes production in some Trichoderma species

Trichoderma species have become the important means of biological control for fungal diseases. This research was carried on to access the high β-1,3-glucanase and β-1,4-glucanase enzyme producer of Trichoderma species isolates using two different carbon sources for finding a method to obtain more concentrate culture filtrates. Therefore, 14 Trichoderma isolates belonging to species: Trichoderma ceramicum, T. virens, T. pseudokoningii, T. koningii, T. koningiosis, T. atroviridae, T. viridescens, T. asperellum, T. harzianum1, T. orientalis, T. harzianum2, T. brevicompactum, T. viride and T. spirale were cultured in Wiendling’s liquid medium plus 0.5% glycerol or 0.5% Phytophthora sojae-hyphe as the carbon source in shaking and non-shaking (stagnant) statuses. Enzyme activity rate and total protein were evaluated in raw, acetony and lyophilized concentrated culture filtrates and the specific enzyme activity of β-1,3-glucanase and β-1,4-glucanase were measured by milligramme glucose equivalent released per minute per milligramme total protein in culture filtrates. The results showed that using Phytophthora – hyphe in medium increased the enzyme activities as compared to glycerol at all Trichoderma species which suggested that these substrates can also act as inducer for synthesis of lytic enzymes, in addition the most enzymes activity was observed in the lyophilised concentrated culture filtrate. The most successful species in β-1,3-glucanase and β-1,4-glucanase enzymes activities were T. brevicompactum and T. virens and these species can be used for mass production of these enzymes which are supposed to be used in commercial formulation and also will be able to control P. sojae directly.     

In vitro Antagonism of Phytophthora cinnamomi and P. citricola by Isolates of Trichoderma spp. and Gliocladium virens

Three fungi, isolated from soil from which Phytophthora was not obtained, were evaluated for antagonism of Phytophthora spp. shown to cause root rot of chestnut in South Australia. Trichoderma hamatum and T. pseudokoningii appeared to inhibit P. cinnamomi by mycoparasitism. with evidence of parallel growth and coiling, and both Trichoderma spp. and Gliocladium virens grew over P. cinnamomi in vitro, preventing further growth of this pathogen. Antibiotics produced by young T. hamatum cultures and G. virens in culture filtrate experiments inhibited growth of P. cinnamomi and P. citricola. with filtrate from 4-day-old cultures of G. virens showing the greatest potential for biocontrol. All three antagonists prevented P. cinnamomi and P. citricola from causing infection symptoms on micropropagated shoots of chestnut cvs Goldsworthy and Buffalo Queen in an in vitro excised shoot bioassay for biocontrol.     

Generation,annotation, and analysis of ESTs from four different <Emphasis Type="Italic">Trichoderma</Emphasis> strains grown under conditions related to biocontrol

The functional genomics project “TrichoEST” was developed focused on different taxonomic groups of Trichoderma with biocontrol potential. Four cDNA libraries were constructed, using similar growth conditions, from four different Trichoderma strains: Trichoderma longibrachiatum T52, Trichoderma asperellum T53, Trichoderma virens T59, and Trichoderma sp. T78. In this study, we present the analysis of the 8,160 expressed sequence tags (ESTs) generated. Each EST library was independently assembled and 1,000–1,300 unique sequences were identified in each strain. First, we queried our collection of ESTs against the NCBI nonredundant database using the BLASTX algorithm. Moreover, using the Gene Ontology hierarchy, we performed the annotation of 40.9% of the unique sequences. Later, based on the EST abundance, we examined the highly expressed genes in the four strains. A hydrophobin was found as the gene expressed at the highest level in two of the strains, but we also found that other unique sequences similar to the HEX1, QID3, and NMT1 proteins were highly represented in at least two of the Trichoderma strains. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Two new species of <Emphasis Type="Italic">Trichoderma</Emphasis> from Yunnan,China

Two new species of the fungal genus Trichoderma, Trichoderma compactum and Trichoderma yunnanense, isolated from rhizosphere of tobacco in Yunnan Province, China are described based on morphological characters and phylogenetic analyses of nucleotide sequences. Our DNA sequences included the internal transcribed spacer (ITS) regions of the rDNA cluster (ITS1 and ITS2), and partial sequences of the translation elongation factor 1-alpha (tef1) and a fragment of the gene coding for endochitinase 42 (ech42). The analyses show that T. compactum belongs to the Harzianum clade, and T. yunnanense belongs to the Hamatum clade.     

Trichoderma species for biocontrol of soil-borne plant pathogens of pasture species

Soil-borne plant pathogens such as Rhizoctonia solani (Kuhn), Pythium ultimum (Trow) and Sclerotinia trifoliorum (Eriks) can reduce grass and forage legume establishment. The potential for biocontrol of these pathogens by Trichoderma fungi was evaluated. Following dual culture assays, nine Trichoderma isolates (five of Trichoderma atroviride and one each of Trichoderma hamatum, Trichoderma koningiopsis, Trichoderma viride and Trichoderma virens) were chosen for assessment in pot experiments. In the presence of R. solani, perennial ryegrass (Lolium perenne L.) emergence was increased by 60–150% by two isolates of T. atroviride and by 35–212% by the isolate of T. virens, with the increase depending on growing medium and amount of pathogen inoculum. Red clover (Trifolium pratense L.) emergence in the presence of S. trifoliorum was significantly increased by two T. atroviride isolates and the T. hamatum isolate. In the presence of P. ultimum, white clover (Trifolium repens L.) emergence was increased by 25–42% by one isolate of T. atroviride and the T. hamatum isolate. However, for all three pasture species, some Trichoderma isolates reduced seedling emergence. Seedling growth (shoot and root fresh weight/plant) of the three pasture species was significantly increased by one or more T. atroviride isolates. On the basis of these results for both disease reduction and growth promotion, four T. atroviride isolates were selected for field assessment as biocontrol agents of soil-borne pathogens of pasture species.     

Biocontrol potential of Trichoderma species against mango malformation pathogens

Malformation disease of Mango (Mangifera indica L.) caused by Fusarium moniliforme var. subglutinans is one of the most destructive diseases, which is a major production constraint in the mango-growing regions of India. In this study, The bioagents Trichoderma viride (Tr1), Trichoderma virens (Tr2) and Trichoderma harzianum (Tr3) were evaluated in culture with the pathogens to monitor the antagonistic effect and their volatile compound and culture filtrates (non-volatile compound). It was found that all the three isolates of bioagents significantly checked the growth of F. moniliforme var. subglutinans. In dual culture, the best result was obtained with T. harzianum followed by T. virens and T. viride. A similar result was also observed in the case of culture filtrates ofTrichoderma spp. The results clearly showed that inhibition of the growth of the fusaria isolates by T. harzianum was significantly superior to T. viride andT.virens. In case of antifungal activity of volatile compounds released by Trichoderma isolates, it was also observed that T. virens was more superior to T.harzianum and T. viride.     

Effects of temperature,pH and water potential on growth of four fungi with disease biocontrol potential

Effects of temperature, pH and water potential on blomass production or hyphal extension of Gliocladium virens (G20) and three Trichoderma isolates were determined in vitro. Optimum blomass production occurred between 20 and 30°C and at pH ranges between 4.6 and 6.8. Two isolates of T. viride grew at 5°C and G. virens grew at 35°C but no isolates grew at 40°C. Hyphal extension rates and conidial germination of all fungi declined with decreasing water potential over the range -0.7 to -14.0 MPa. In general, growth rates for each isolate were lower on potato/dextrose agar with water potential adjusted with polyethylene glycol than when adjusted with NaCl or glycerol. No mycelial growth or spore germination occurred on agar at-14.0 MPa.The authors are with the Microbiology and Crop Protection Department, Horticulture Research International, Littlehampton, West Sussex BN17 6LP, UK. J.M. Whipps is the corresponding author     

Biocontrol of onion white rot by application of Trichoderma species formulated on wheat bran powder

In vitro, Trichoderma album, Trichoderma harzianum, Trichoderma koningii, Trichoderma viride and Trichoderma virens showed antagonistic effect against the most pathogenic isolate (Sc2) of Sclerotium cepivorum, the cause of onion white rot disease. Five Trichoderma preparations of each Trichoderma sp. were prepared on wheat bran powder to be used for controlling white rot disease of onion. Greenhouse and field experiments followed the same trend where T. harzianum and T. koningii were the most effective in reducing the incidence and severity of white rot disease compared with the control. Trichoderma species preparations caused promotion to vegetative parameters of onion plants in pots and increase bulb productivity in filed. In this regard, T. harzianum and T. koningii were the most effective. A positive correlation was found between the biocontrol activity of Trichoderma species preparations and enhancement of peroxidase, polyphenoloxidase and chitinase enzymes in onion plants to resist infection with S. cepivorum.     

Species diversity of <Emphasis Type="Italic">Trichoderma</Emphasis> in Poland

In the present study, we reinvestigate the diversity of Trichoderma in Poland utilizing a combination of morphological and molecular/phylogenetic methods. A total of 170 isolates were collected from six different substrata at 49 sites in Poland. These were divided among 14 taxa as follows: 110 of 170 Trichoderma isolates were identified to the species level by the analysis of their ITS1, ITS2 rDNA sequences as: T. harzianum (43 isolates), T. aggressivum (35), T. citrinoviride (11), T. hamatum (9), T. virens (6), T. longibrachiatum (4), T. polysporum (1), and T. tomentosum (1); 60 isolates belonging to the Viride clade were identified based on a fragment of the translation-elongation factor 1-alpha (tef1) gene as: T. atroviride (20 isolates), T. gamsii (2), T. koningii (17), T. viridescens (13), T. viride (7), and T. koningiopsis (1). Identifications were made using the BLAST interface in TrichOKEY and TrichoBLAST (). The most diverse substrata were soil (nine species per 22 isolates) and decaying wood (nine species per 75 isolates). The most abundant species (25%) isolated from all substrata was T. harzianum.     

Biotransformation of Trichoderma spp. and Their Tolerance to Aromatic Amines,a Major Class of Pollutants

Trichoderma spp. are cosmopolitan soil fungi that are highly resistant to many toxic compounds. Here, we show that Trichoderma virens and T. reesei are tolerant to aromatic amines (AA), a major class of pollutants including the highly toxic pesticide residue 3,4-dichloroaniline (3,4-DCA). In a previous study, we provided proof-of-concept remediation experiments in which another soil fungus, Podospora anserina, detoxifies 3,4-DCA through its arylamine N-acetyltransferase (NAT), a xenobiotic-metabolizing enzyme that enables acetyl coenzyme A-dependent detoxification of AA. To assess whether the N-acetylation pathway enables AA tolerance in Trichoderma spp., we cloned and characterized NATs from T. virens and T. reesei. We characterized recombinant enzymes by determining their catalytic efficiencies toward several toxic AA. Through a complementary approach, we also demonstrate that both Trichoderma species efficiently metabolize 3,4-DCA. Finally, we provide evidence that NAT-independent transformation is solely (in T. virens) or mainly (in T. reesei) responsible for the observed removal of 3,4-DCA. We conclude that T. virens and, to a lesser extent, T. reesei likely utilize another, unidentified, metabolic pathway for the detoxification of AA aside from acetylation. This is the first molecular and functional characterization of AA biotransformation in Trichoderma spp. Given the potential of Trichoderma for cleanup of contaminated soils, these results reveal new possibilities in the fungal remediation of AA-contaminated soil.     

Evaluation of Trichoderma spp. as biocontrol agents against avocado white root rot

Five isolates of Trichoderma atroviride and one isolate each of T. virens, T. harzianum and T. cerinum were tested for in vivo biological control of white root rot of avocado (Rosellinia necatrix). Five of these Trichoderma isolates were previously selected as possible biological control agents on the basis of their capacity to control the disease and high levels of colonization of the avocado rhizoplane. Combinations of the five selected isolates were evaluated on cellophane for compatibility with each other and T. virens CH 303 was eliminated because of a high incompatibility with other Trichoderma isolates. The four remaining isolates, all T. atroviride, were tested singly and in combination for their capacity to control avocado white root rot. Isolate CH 304.1 provided the highest levels of control when tested singly or in combination with isolate CH 101.     

Diversity of root-endophytic <Emphasis Type="Italic">Trichoderma</Emphasis> from Malaysian Borneo

Trichoderma species form endophytic associations with plant roots and may provide a range of benefits to their hosts. However, few studies have systematically examined the diversity of Trichoderma species associated with plant roots in tropical regions. During the evaluation of Trichoderma isolates for use as biocontrol agents, root samples were collected from more than 58 genera in 35 plant families from a range of habitats in Malaysian Borneo. Trichoderma species were isolated from surface-sterilised roots and identified following analysis of partial translation elongation factor-1α (tef1) sequences. Species present included Trichoderma afroharzianum, Trichoderma asperelloides, Trichoderma asperellum, Trichoderma guizhouense, Trichoderma reesei, Trichoderma strigosum and Trichoderma virens. Trichoderma asperellum/T. asperelloides, Trichoderma harzianum s.l. and T. virens were the most frequently isolated taxa. tef1 sequence data supported the recognition of undescribed species related to the T. harzianum complex. The results suggest that tropical plants may be a useful source of novel root-associated Trichoderma for biotechnological applications.     

Identification of novel gene clusters for secondary metabolism in <Emphasis Type="Italic">Trichoderma</Emphasis> genomes

Trichoderma species are widely used in agriculture as biofungicides. These fungi are rich source of secondary metabolites and the mycoparasitic species are enriched in genes for biosynthesis of secondary metabolites. Most often, genes for secondary metabolism are clustered in fungal genomes. Previously, no systematic study was undertaken to identify the secondary-metabolism related gene clusters in Trichoderma genomes. In the present study, a survey of the three Trichoderma genomes viz. T. reesei, T. atroviride and T. virens, was made to identify the putative gene clusters associated with secondary metabolism. In T. reesei genome, we identified one new NRPS and 6 new PKS clusters, which is much less than that found in T. atroviride (4 and 8) and T. virens (8 and 7). This work would pave the way for discovery of novel secondary metabolites and pathways in Trichoderma.     

Antagonistic potential of Gliocladium virens and Trichoderma longibrachiatum to phytopathogenic fungi

Three isolates of Gliocladium virens (G1, G2 and G3) and two of Trichoderma longibrachiatum (T1 and T2) were screened against isolates of three soilborne plant pathogens namely Rhizoctonia solani, Sclerotium rolfsii and Pythium aphanidermatum. G. virens exhibited stronger hyperparasitism and wider biological spectrum than T. longibrachiatum. Further, similarities as well as variation was observed in the ability of the various isolates to invade the test pathogens in dual culture. For the hyperparasites, acidic pH range (5.0 to 5.5) favoured both growth and spore germination. The hyperparasites made direct contact with the pathogens followed by varied modes of attack invariably leading to cell disruption. Antagonists, G1 and G3 revealed strong antibiosis while T2 showed moderate effect. All the isolates produced enhanced levels of lytic enzymes adaptively and there were marked differences among them. However, no correlation was observed between these attributes and the hyperparasitic potential of the various isolates in dual culture. The relevance and the role of enzymes and toxic metabolite(s) in the antagonism of G. virens and T. longibrachiatum to these pathogens are discussed.     

Expression and characterization of <Emphasis Type="Italic">Trichoderma virens</Emphasis> UKM-1 endochitinase in <Emphasis Type="Italic">Escherichia</Emphasis> <Emphasis Type="Italic">coli</Emphasis>

A gene encoding endochitinase from Trichoderma virens UKM-1 was cloned and expressed in E. coli BL21 (DE3). Both the endochitinase gene and its cDNA sequences were obtained. The endochitinase gene encodes 430 amino acids from an open reading frame comprising of 1,690 bp nucleotide sequence with three introns. The endochitinase was expressed as soluble and active enzyme at 20°C when induced with 1 mM IPTG. Maximum activity was observed at 4 h of post-induction time. SDS-PAGE showed that the purified endochitinase exhibited a single band with molecular weight of 42 kDa. Biochemical characterization of the enzyme displayed a near neutral pH characteristic with an optimum pH at 6.0 and optimum temperature at 50°C. The enzyme is stable between pH 3.0–7.0 and is able to retain its activity from 30 to 60°C. The presence of Mg²⁺ and Ca²⁺ ions increased the enzyme activity up to 20%. The purified enzyme has a strong affinity towards colloidal chitin and low effect on ethyl cellulose and D-cellubiose which are non-chitin related substrates. HPLC analysis from the chitin hydrolysis showed the release of (GlcNAc)₃, (GlcNAc)₂ and GlcNAc, in which (GlcNAc)₂ was the main product.     

Trichoderma species on Agaricus bisporus farms in Serbia and their biocontrol

Twenty Trichoderma isolates were collected on 13 Serbian Agaricus bisporus farms and one in Bosnia and Herzegovina during 2006–2010. Twelve isolates were classified into five species by standard mycological studies and ITS1/ITS4 sequence analyses, namely Trichoderma atroviride, Trichoderma koningii, Trichoderma virens, Trichoderma aggressivum f. europaeum and Trichoderma harzianum. Eight isolates were not identified to the species level but were shown to be related to T. harzianum. The isolates of T. harzianum exhibited the highest virulence to the harvested A. bisporus pilei and T. virens and T. aggressivum f. europaeum the lowest. Antifungal activity of two biofungicides based on Bacillus subtilis and tea tree oil and the fungicide prochloraz manganese were tested in vitro to all Trichoderma isolates. Prochloraz manganese and B. subtilis were highly toxic to all tested Trichoderma isolates, their ED₅₀ values were below 0.3 and 1.3 mg L^?1, respectively. Tea tree oil did not exhibit a significant antifungal activity (ED₅₀ = 11.9–370.8 mg L^?1). The effectiveness of biofungicides was evaluated against T. harzianum in a mushroom growing room, and they were applied alone or in combination with the fungicide at a respective proportion of 20:80%. Prochloraz manganese showed higher effectiveness than both tested biofungicides or their respective mixtures. The biofungicide based on B. subtilis demonstrated greater effectiveness in preventing disease symptoms than tea tree oil. B. subtilis combined with the fungicide revealed less antagonism in effectiveness against pathogen than tea tree oil.     

Native Trichoderma strains isolated from Bangladesh with broad spectrum antifungal action against fungal phytopathogens

Nineteen Trichoderma isolates, collected from different locations in Bangladesh, were characterised through phenotypic, biochemical and molecular means. Besides, they were assessed for their antifungal action in vitro. The isolates were divided into three groups: T. asperellum, T. virens and T. harzianum. A dual culture assay and a culture filtrate assay against 6 phytopathogens revealed that 9 of the 19 isolates showed significant antifungal activities. The isolate T. harzianum TR05 showed the highest inhibition against Fusarium oxysporum, Rhizoctonia solani, Fusarium circinatum and Phomopsis vexans, followed by T. asperellum TR08 and T. virens TR06. TR08 had the highest inhibition against Sclerotium rolfsii and Pythium aphanidermatum, followed by TR05 and TR06. These findings were in agreement with their activities of extracellular hydrolytic enzymes, including chitinase, β-1,3-glucanase, and proteinase. Our results suggest that isolates TR05, TR06 and TR08 have the potential to be effective biocontrol agents against the phytopathogenic fungi.     

Molecular cloning,sequence analysis,expression and characterization of the endochitinase gene from <Emphasis Type="Italic">Trichoderma</Emphasis> sp. in <Emphasis Type="Italic">Escherichia coli</Emphasis> BL21

The endochitinase DNA and cDNA from Trichoderma sp. were cloned, sequenced and expressed. The cloned DNA and cDNA sequences were 1,476 and 1,275 bp in length, respectively. There were three introns in DNA sequence in comparison with the cDNA sequence. The endochitinase protein contained three regions: the signal peptide, the prepro-region and the mature protein region. The gene fragment encoding the mature endochitinase was ligated into the expression vector pET-28a+, yielding pET-1. The plasmid pET-1 was transformed into the Escherichia coli BL21 (DE3). The clone bearing pET-1 was picked and cultured at 30°C for the expression of endochitinase. SDS-PAGE analysis showed that the endochitinase was expressed in the periplasmic space and the purified protein showed a single band. The activity of 70.2 U/mg was obtained from the cellular extract of the recombinant strain. The activity of endochitinase was 2.5-fold higher at 24 h than at 16 h in the periplasmic space. The optimal pH and temperature of the recombinant endochitinase were determined to be 7.0 and 35°C, respectively. It was relatively stable within the pH range of 5–8. Significant activity stimulation by 1 mM Mg²⁺ and 5 mM Fe²⁺ and inhibition by 5 mM Co²⁺ and 5 mM Hg²⁺ were observed. The kinetic constants K_m, V_max and K_cat for the hydrolysis of the colloidal chitin were 1.5 mM, 1.37 μmol min⁻¹ and 6.23 min⁻¹, respectively.