PCR-based genotyping of epidemic and preepidemic Trichoderma isolates associated with green mold of Agaricus bisporus.

We used randomly amplified polymorphic DNA (RAPD)-PCR to estimate genetic variation among isolates of Trichoderma associated with green mold on the cultivated mushroom Agaricus bisporus. Of 83 isolates examined, 66 were sampled during the recent green mold epidemic, while the remaining 17 isolates were collected just prior to the epidemic and date back to the 1950s. Trichoderma harzianum biotype 4 was identified by RAPD analysis as the cause of almost 90% of the epidemic-related episodes of green mold occurring in the major commercial mushroom-growing region in North America. Biotype 4 was more closely allied to T. harzianum biotype 2, the predominant pathogenic genotype in Europe, than to the less pathogenic biotype 1 and Trichoderma atroviride (formerly T. harzianum biotype 3). No variation in the RAPD patterns was observed among the isolates within biotype 2 or 4, suggesting that the two pathogenic biotypes were populations containing single clones. Considerable genetic variation, however, was noted among isolates of biotype 1 and T. atroviride from Europe. Biotype 4 was not represented by the preepidemic isolates of Trichoderma as determined by RAPD markers and PCR amplification of an arbitrary DNA sequence unique to the genomes of biotypes 2 and 4. Our findings suggest that the onset of the green mold epidemic in North America resulted from the recent introduction of a highly virulent genotype of the pathogen into cultivated mushrooms.     

A polymerase chain reaction-based test for the identification of Trichoderma harzianum biotypes 2 and 4, responsible for the worldwide green mold epidemic in cultivated Agaricus bisporus

We describe a polymerase chain reaction (PCR)-based test that is specific for the pathogenic European biotype 2 (Th2) and North American biotype 4 (Th4) of Trichoderma harzianum, responsible for the green mold epidemic in the cultivated mushroom, Agaricus bisporus. A PCR primer pair was designed that targets a 444-bp arbitrary sequence in the genome of Th4. The primers also amplified the same product with Th2, but showed no reactivity with other biotypes of T. harzianum, several biocontrol Trichoderma, or with 31 other genera and species of fungi. The PCR-based test should have application in disease management programs, and in the evaluation of biocontrol Trichoderma for potential pathogenicity on mushrooms. Received: 23 November 1998 / Received revision: 19 February 1999 / Accepted: 5 March 1999     

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.     

Genetic and metabolic biodiversity of Trichoderma from Colombia and adjacent neotropic regions

The genus Trichoderma has been studied for production of enzymes and other metabolites, as well as for exploitation as effective biological control agents. The biodiversity of Trichoderma has seen relatively limited study over much of the neotropical region. In the current study we assess the biodiversity of 183 isolates from Mexico, Guatemala, Panama, Ecuador, Peru, Brazil and Colombia, using morphological, metabolic and genetic approaches. A comparatively high diversity of species was found, comprising 29 taxa: Trichoderma asperellum (60 isolates), Trichoderma atroviride (3), Trichoderma brevicompactum (5), Trichoderma crassum (3), Trichoderma erinaceum (3), Trichoderma gamsii (2), Trichoderma hamatum (2), Trichoderma harzianum (49), Trichoderma koningiopsis (6), Trichoderma longibrachiatum (3), Trichoderma ovalisporum (1), Trichoderma pubescens (2), Trichoderma rossicum (4), Trichoderma spirale (1), Trichoderma tomentosum (3), Trichoderma virens (8), Trichoderma viridescens (7) and Hypocrea jecorina (3) (anamorph: Trichoderma reesei), along with 11 currently undescribed species. T. asperellum was the prevalent species and was represented by two distinct genotypes with different metabolic profiles and habitat preferences. The second predominant species, T. harzianum, was represented by three distinct genotypes. The addition of 11 currently undescribed species is evidence of the considerable unresolved biodiversity of Trichoderma in neotropical regions. Sequencing of the internal transcribed spacer regions (ITS) of the ribosomal repeat could not differentiate some species, and taken alone gave several misidentifications in part due to the presence of nonorthologous copies of the ITS in some isolates.     

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.     

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.     

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.     

The effect of lyophilization and storage time on the survival rate and hydrolytic activity of <Emphasis Type="Italic">Trichoderma</Emphasis> strains

The study evaluates the survivability and storage stability of seven Trichoderma strains belonging to the species: T. harzianum (1), T. atroviride (4), and T. virens (2) after the lyophilization of their solid state cultures on wheat straw. Biomass of Trichoderma strains was freeze-dried with and without the addition of maltodextrin. Furthermore, in order to determine the ability of tested Trichoderma strains to preserve selected technological features, the biosynthesis of extracellular hydrolases (cellulases, xylanases, and polygalacturonases) after a 3-month storage of lyophilizates was investigated. Strains of T. atroviride (except TRS40) and T. harzianum TRS85 showed the highest viability after lyophilization process (up to 100%). After 3 months of storage, T. atroviride TRS14 exhibited the highest stability (95.23%); however, the number of active conidia remained at high level of 10⁶–10⁷ cfu/g for all tested T. atroviride strains and T. harzianum TRS85. Interestingly, after a 3-month storage of lyophilized formulations, most of the tested Trichoderma strains exhibited higher cellulolytic and xylanolytic activities compared to the control, i.e., before freeze-drying process. The highest activities of these enzymes exhibited the following: T. atroviride TRS14–2.37 U/g and T. atroviride TRS25–21.47 U/g, respectively, whereas pectinolytic activity was weak for all tested strains, with the highest value of 0.64 U/g registered for T. virens TRS109.     

Enzyme Diffusion from Trichoderma atroviride (= T. harzianum P1) to Rhizoctonia solani Is a Prerequisite for Triggering of Trichoderma ech42 Gene Expression before Mycoparasitic Contact

A plate confrontation experiment is commonly used to study the mechanism by which Trichoderma spp. antagonize and parasitize other fungi. Previous work with chitinase gene expression (ech42) during the precontact period of this process in which cellophane and dialysis membranes separated Trichoderma harzianum and its host Rhizoctonia solani resulted in essentially opposite results. Here, we show that cellophane membranes are permeable to proteins up to at least 90 kDa in size but that dialysis membranes are not. ech42 was expressed during the precontact stage of the confrontation between Trichoderma atroviride and its host only if the cellophane was placed between the two fungi. These results are consistent with enzyme diffusion from T. atroviride to R. solani generating the trigger of ech42 gene expression.     

Bioactivity of endophytic Trichoderma fungal species from the plant family Cupressaceae

Trichoderma fungal species are universal soil residents that are also isolated from decaying wood, vegetables, infected mushroom and immunocompromised patients. Trichoderma species usually biosynthesize a plethora of secondary metabolites. In an attempt to explore endophytic fungi from healthy foliar tissues of the plant family Cuppressaceae, we explored Cupressus arizonica, C. sempervirens var. cereiformis, C. sempervirens var. fastigiata, C. sempervirens var. horizontalis, Juniperus excelsa, Juniperus sp. and Thuja orientalis plants and recovered several endophytic Trichoderma fungal strains from Trichoderma atroviride and Trichoderma koningii species. We found that the host plant species and biogeographical location of sampling affected the biodiversity and bioactivity of endophytic Trichoderma species. Furthermore, the bioactivity of Trichoderma isolates and the methanol extracts of their intra- and extra-cellular metabolites were assessed against a panel of pathogenic fungi and bacteria. Fungal growth inhibition, conidial cytotoxicity, minimum inhibitory concentration and minimum bactericidal concentration were evaluated and analyzed by statistical methods. Our data showed that both intra- and extracellular secondary metabolites from all endophytic isolates had significant cytotoxic and antifungal effects against the model target fungus Pyricularia oryzae and the cypress fungal phytopathogens Diplodia seriata, Phaeobotryon cupressi and Spencermartinsia viticola. Further research indicated their significant antimicrobial bioactivity against the model phytopathogenic bacteria Pseudomonas syringae, Erwinia amylovora and Bacillus sp., as well. Altogether, the above findings show for the first time the presence of T. atroviride and T. koningii as endophytic fungi in Cupressaceae plants and more importantly, the Trichoderma isolates demonstrate significant bioactivity that could be used in future for agrochemical/drug discovery and pathogen biocontrol.     

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.     

Evaluation of antifungal,phosphate solubilisation,and siderophore and chitinase release activities of endophytic fungi from <Emphasis Type="Italic">Pistacia vera</Emphasis>

Fungal endophytes use different strategies to protect host plants from abiotic and biotic stress. In this study, we isolated endophytic fungi from Pistacia vera and characterised their antifungal activity against Aspergillus flavus, Rhizoctonia solani and Sclerotinia sclerotiorum, and their release of some factors that can alter plant growth capability. Trichoderma harzianum TH 5-1-2, T. harzianum TH 10-2-2 and T. atroviride TA 2-2-1 exhibited the highest growth inhibition percentages in dual culture assays against A. flavus, R. solani and S. sclerotiorum, respectively. Among the fungal endophyte cultures, ethyl acetate extracts of T. harzianum TH 10-2-2, T. harzianum TH 5-1-2 and T. atroviride TA 2-2-1 exhibited the highest growth inhibition of S. sclerotiorum, R. solani and A. flavus, respectively. Phosphate solubilisation was induced by Byssochlamys nivea BN 1-1-1 in culture. Large amounts of siderophore production were observed with Quambalaria cyanescens QC 11-3-2 and Epicoccum nigrum EN1, but Trichoderma spp. also produced siderophore in lower amounts. Trichoderma harzianum TH 5-1-2 produced the highest chitinase activity (2.92 U/mL). In general, among the endophytes isolated, Trichoderma spp. appear to have the most promise for promoting healthy growth of P. vera.     

Colonization of Onions by Endophytic Fungi and Their Impacts on the Biology of Thrips tabaci

Endophytic fungi, which live within host plant tissues without causing any visible symptom of infection, are important mutualists that mediate plant–herbivore interactions. Thrips tabaci (Lindeman) is one of the key pests of onion, Allium cepa L., an economically important agricultural crop cultivated worldwide. However, information on endophyte colonization of onions, and their impacts on the biology of thrips feeding on them, is lacking. We tested the colonization of onion plants by selected fungal endophyte isolates using two inoculation methods. The effects of inoculated endophytes on T. tabaci infesting onion were also examined. Seven fungal endophytes used in our study were able to colonize onion plants either by the seed or seedling inoculation methods. Seed inoculation resulted in 1.47 times higher mean percentage post-inoculation recovery of all the endophytes tested as compared to seedling inoculation. Fewer thrips were observed on plants inoculated with Clonostachys rosea ICIPE 707, Trichoderma asperellum M2RT4, Trichoderma atroviride ICIPE 710, Trichoderma harzianum 709, Hypocrea lixii F3ST1 and Fusarium sp. ICIPE 712 isolates as compared to those inoculated with Fusarium sp. ICIPE 717 and the control treatments. Onion plants colonized by C. rosea ICIPE 707, T. asperellum M2RT4, T. atroviride ICIPE 710 and H. lixii F3ST1 had significantly lower feeding punctures as compared to the other treatments. Among the isolates tested, the lowest numbers of eggs were laid by T. tabaci on H. lixii F3ST1 and C. rosea ICIPE 707 inoculated plants. These results extend the knowledge on colonization of onions by fungal endophytes and their effects on Thrips tabaci.     

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.     

Specific PCR Assays for the Detection and Quantification of DNA from the Biocontrol Strain Trichoderma harzianum 2413 in Soil

Strain identification in situ is an important factor in the monitoring of microorganisms used in the field. In this study, we demonstrated the use of sequence-characterized amplified region (SCAR) markers to detect genomic DNA from Trichoderma harzianum 2413 from soil. Two primers (SCAR A1/SCAR A1c) were tested against DNA of 27 isolates of Trichoderma spp. and amplified a 990-bp fragment from T. atroviride 11 and a 1.5-kb fragment from T. harzianum 2413, using an annealing temperature of 68°C. These fragments showed no significant homology to any sequence deposited in the databases. The primer pair, BR1 and BR2, was designed to the 1.5-kb fragment amplified from T. harzianum 2413, generating a SCAR marker. To test the specificity of these primers, experiments were conducted using the DNA from 27 Trichoderma spp. strains and 22 field soil samples obtained from four different countries. PCR results showed that BR1 and BR2 amplified an 837-bp fragment unique to T. harzianum 2413. Assays in which total DNA was extracted from sterile and nonsterile soil samples, inoculated with spore or mycelium combinations of Trichoderma spp. strains, indicated that the BR1 and BR2 primers could specifically detect T. harzianum 2413 in a pool of mixed DNA. No other soil-microorganisms containing these sequences were amplified using these primers. To test whether the 837-bp SCAR marker of T. harzianum 2413 could be used in real-time PCR experiments, new primers (Q2413f and Q2413r) conjugated with a TaqMan fluorogenic probe were designed. Real-time PCR assays were applied using DNA from sterile and nonsterile soil samples inoculated with a known quantity of spores of Trichoderma spp. strains.     

Genomic Analysis of the Mycoparasite Pestalotiopsis sp. PG52

Pestalotiopsis sp. is a mycoparasite of the plant pathogen Aecidium wenshanense. To further understand the mycoparasitism mechanism of Pestalotiopsis sp., we assembled and analyzed its genome. The genome of Pestalotiopsis sp. strain PG52 was assembled into 335 scaffolds and had a size of 58.01 Mb. A total of 20,023 predicted genes and proteins were annotated. This study compared PG52 with the mycoparasites Trichoderma harzianum, Trichoderma atroviride, and Trichoderma virens. This study reveals the entirely different mycoparasitism mechanism of Pestalotiopsis compared to Trichoderma and reveals this mycoparasite’s strong ability to produce secondary metabolites.     

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.     

Mycoparasitism-related genes expression of Trichoderma harzianum isolates to evaluate their efficacy as biological control agent

The selection of new isolates of Trichoderma harzianum with high suppressive activity against Fusarium oxysporum is a suitable strategy to avoid the increase of chemical pesticides. In this study, 31 isolates of Trichoderma sp. were analyzed by RAPD-PCR and five isolates of T. harzianum (T-30, T-31, T-32, T-57 and T-78) were selected. The expression of genes encoding for NAGases (exc1 and exc2), chitinases (chit42 and chit33), proteases (prb1) and β-glucanases (bgn13.1) activities and their respective in vitro enzymatic activities were measured. Dual plate confrontation assays of the isolates against F. oxysporum were also tested. Different profiles of gene expression between the different T. harzianum isolates were related to enzymatic activities values and dual plate confrontation. In this work, the T. harzianum isolates T-30 and T-78 showed the greatest mycoparasitic potential against F. oxysporum, which could lead to improved biocontrol of this phytopathogen.     

Geographic range, impact, and parasitism of lepidopteran species associated with the invasive weed Lantana camara in South Africa

Six isolates of Trichoderma were screened for antagonism to Armillaria in tea stem sections buried in the soil. The inability of Armillaria to invade Trichoderma-colonized stem sections and the reduction of its viability in the plant materials following invasion of these by Trichoderma were used as indicators of antagonism. Four isolates of the species Trichoderma harzianum significantly (P<0.001) reduced the incidence of the pathogen in the plant materials. Isolate T4 completely eliminated the pathogen from plant materials in sterile soil and also antagonized two different isolates of the pathogen in nonsterile soil. Application of this T. harzianum isolate to the soil as a wheat bran culture significantly (P<0.001) reduced viability of Armillaria in woody blocks of inoculum. Soil amendment with coffee pulp also reduced the inoculum viability but did not affect the incidence of Trichoderma in the blocks of inoculum. We conclude that the direct application of wheat bran-formulated T. harzianum into soil surrounding woody Armillaria inoculum sources can suppress the pathogen. Further, no organic amendment is needed to enhance development of the antagonist in the soil as a pre-requisite to suppressing the pathogen.