In vivo study of trichoderma-pathogen-plant interactions, using constitutive and inducible green fluorescent protein reporter systems

Plant tissue colonization by Trichoderma atroviride plays a critical role in the reduction of diseases caused by phytopathogenic fungi, but this process has not been thoroughly studied in situ. We monitored in situ interactions between gfp-tagged biocontrol strains of T. atroviride and soilborne plant pathogens that were grown in cocultures and on cucumber seeds by confocal scanning laser microscopy and fluorescence stereomicroscopy. Spores of T. atroviride adhered to Pythium ultimum mycelia in coculture experiments. In mycoparasitic interactions of T. atroviride with P. ultimum or Rhizoctonia solani, the mycoparasitic hyphae grew alongside the pathogen mycelia, and this was followed by coiling and formation of specialized structures similar to hooks, appressoria, and papillae. The morphological changes observed depended on the pathogen tested. Branching of T. atroviride mycelium appeared to be an active response to the presence of the pathogenic host. Mycoparasitism of P. ultimum by T. atroviride occurred on cucumber seed surfaces while the seeds were germinating. The interaction of these fungi on the cucumber seeds was similar to the interaction observed in coculture experiments. Green fluorescent protein expression under the control of host-inducible promoters was also studied. The induction of specific Trichoderma genes was monitored visually in cocultures, on plant surfaces, and in soil in the presence of colloidal chitin or Rhizoctonia by confocal microscopy and fluorescence stereomicroscopy. These tools allowed initiation of the mycoparasitic gene expression cascade to be monitored in vivo.     

The Putative Protein Methyltransferase LAE1 of Trichoderma atroviride Is a Key Regulator of Asexual Development and Mycoparasitism

In Ascomycota the protein methyltransferase LaeA is a global regulator that affects the expression of secondary metabolite gene clusters, and controls sexual and asexual development. The common mycoparasitic fungus Trichoderma atroviride is one of the most widely studied agents of biological control of plant-pathogenic fungi that also serves as a model for the research on regulation of asexual sporulation (conidiation) by environmental stimuli such as light and/or mechanical injury. In order to learn the possible involvement of LAE1 in these two traits, we assessed the effect of deletion and overexpression of lae1 gene on conidiation and mycoparasitic interaction. In the presence of light, conidiation was 50% decreased in a Δ lae1 and 30–50% increased in lae1-overexpressing (OElae1) strains. In darkness, Δ lae1 strains did not sporulate, and the OElae1 strains produced as much spores as the parent strain. Loss-of-function of lae1 also abolished sporulation triggered by mechanical injury of the mycelia. Deletion of lae1 also increased the sensitivity of T. atroviride to oxidative stress, abolished its ability to defend against other fungi and led to a loss of mycoparasitic behaviour, whereas the OElae1 strains displayed enhanced mycoparasitic vigor. The loss of mycoparasitic activity in the Δ lae1 strain correlated with a significant underexpressionn of several genes normally upregulated during mycoparasitic interaction (proteases, GH16 ß-glucanases, polyketide synthases and small cystein-rich secreted proteins), which in turn was reflected in the partial reduction of formation of fungicidal water soluble metabolites and volatile compounds. Our study shows T. atroviride LAE1 is essential for asexual reproduction in the dark and for defense and parasitism on other fungi.     

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.     

Signal Transduction by Tga3, a Novel G Protein α Subunit of Trichoderma atroviride

Trichoderma species are used commercially as biocontrol agents against a number of phytopathogenic fungi due to their mycoparasitic characterisitics. The mycoparasitic response is induced when Trichoderma specifically recognizes the presence of the host fungus and transduces the host-derived signals to their respective regulatory targets. We made deletion mutants of the tga3 gene of Trichoderma atroviride, which encodes a novel G protein α subunit that belongs to subgroup III of fungal Gα proteins. Δtga3 mutants had changes in vegetative growth, conidiation, and conidial germination and reduced intracellular cyclic AMP levels. These mutants were avirulent in direct confrontation assays with Rhizoctonia solani or Botrytis cinerea, and mycoparasitism-related infection structures were not formed. When induced with colloidal chitin or N-acetylglucosamine in liquid culture, the mutants had reduced extracellular chitinase activity even though the chitinase-encoding genes ech42 and nag1 were transcribed at a significantly higher rate than they were in the wild type. Addition of exogenous cyclic AMP did not suppress the altered phenotype or restore mycoparasitic overgrowth, although it did restore the ability to produce the infection structures. Thus, T. atroviride Tga3 has a general role in vegetative growth and can alter mycoparasitism-related characteristics, such as infection structure formation and chitinase gene expression.     

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.     

Differential Inactivation of Seed Exudate Stimulation of Pythium ultimum Sporangium Germination by Enterobacter cloacae Influences Biological Control Efficacy on Different Plant Species

This study was initiated to understand whether differential biological control efficacy of Enterobacter cloacae on various plant species is due to differences in the ability of E. cloacae to inactivate the stimulatory activity of seed exudates to Pythium ultimum sporangium germination. In biological control assays, E. cloacae was effective in controlling Pythium damping-off when placed on the seeds of carrot, cotton, cucumber, lettuce, radish, tomato, and wheat but failed to protect corn and pea from damping-off. Seeds from plants such as corn and pea had high rates of exudation, whereas cotton and cucumber seeds had much lower rates of exudation. Patterns of seed exudation and the release of P. ultimum sporangium germination stimulants varied among the plants tested. Seed exudates of plants such as carrot, corn, lettuce, pea, radish, and wheat were generally more stimulatory to P. ultimum than were the exudates of cotton, cucumber, sunflower, and tomato. However, this was not directly related to the ability of E. cloacae to inactivate the stimulatory activity of the exudate and reduce P. ultimum sporangium germination. In the spermosphere, E. cloacae readily reduced the stimulatory activity of seed exudates from all plant species except corn and pea. Our data have shown that the inability of E. cloacae to protect corn and pea seeds from Pythium damping-off is directly related to its ability to inactivate the stimulatory activity of seed exudates. On all other plants tested, E. cloacae was effective in suppressing damping-off and inactivating the stimulatory activity of seed exudates.     

A Whole Cell Bioreporter Approach to Assess Transport and Bioavailability of Organic Contaminants in Water Unsaturated Systems

Bioavailability of contaminants is a prerequisite for their effective biodegradation in soil. The average bulk concentration of a contaminant, however, is not an appropriate measure for its availability; bioavailability rather depends on the dynamic interplay of potential mass transfer (flux) of a compound to a microbial cell and the capacity of the latter to degrade the compound. In water-unsaturated parts of the soil, mycelia have been shown to overcome bioavailability limitations by actively transporting and mobilizing organic compounds over the range of centimeters. Whereas the extent of mycelia-based transport can be quantified easily by chemical means, verification of the contaminant-bioavailability to bacterial cells requires a biological method. Addressing this constraint, we chose the PAH fluorene (FLU) as a model compound and developed a water unsaturated model microcosm linking a spatially separated FLU point source and the FLU degrading bioreporter bacterium Burkholderia sartisoli RP037-mChe by a mycelial network of Pythium ultimum. Since the bioreporter expresses eGFP in response of the PAH flux to the cell, bacterial FLU exposure and degradation could be monitored directly in the microcosms via confocal laser scanning microscopy (CLSM). CLSM and image analyses revealed a significant increase of the eGFP expression in the presence of P. ultimum compared to controls without mycelia or FLU thus indicating FLU bioavailability to bacteria after mycelia-mediated transport. CLSM results were supported by chemical analyses in identical microcosms. The developed microcosm proved suitable to investigate contaminant bioavailability and to concomitantly visualize the involved bacteria-mycelial interactions.     

Ability of Nonpathogenic Fusarium oxysporum Strain Fo47 To Induce Resistance against Pythium ultimum Infection in Cucumber

The influence exerted by nonpathogenic Fusarium oxysporum strain Fo47 in triggering cucumber protection against infection by Pythium ultimum was investigated ultrastructurally. Macroscopic and microscopic observations of the pathogen colony in dual cultures revealed that reduction of Pythium growth was associated with marked disorders, including generalized disorganization of the host cytoplasm, retraction of the plasmalemma, and complete loss of the protoplasm. Cytochemical labeling of cellulose with an exoglucanase-gold complex showed that the cellulose component of the host cell walls was structurally preserved at a time when the host cytoplasm had undergone complete disorganization. A similar antagonistic process was observed at the root cell surface. Most striking and interesting was the finding that mycoparasitism, as evidenced by the frequent occurrence of Fo47 hyphae within nearly empty cells of the pathogen, occurred not only at the root surface but also within the invaded root tissues. The specific labeling pattern obtained with the exoglucanase-gold complex confirmed that Fo47 successfully penetrated cells of the pathogen, both in the rhizosphere and inside the root tissues. Pythium cells that could evade the first defensive line in the rhizosphere could penetrate the root epidermis, but their growth was restricted to the outermost tissues. Positive correlations between Fo47 treatment and induced resistance to infection by P. ultimum in cucumber were confirmed by (i) the reduction of pathogen viability; (ii) the elaboration of newly formed barriers, a phenomenon which was not seen in Fo47-free plants, where the pathogen proliferated in all root tissues within a few days; and (iii) the occlusion of intercellular spaces with a dense material likely enriched in phenolics. Taken together, our observations provide the first convincing evidence that Fo47 exerts a direct inhibitory effect on P. ultimum through a combination of antibiosis and mycoparasitism, in addition to being a strong inducer of plant defense reactions.     

Strong preference for the integration of transforming DNA via homologous recombination in Trichoderma atroviride

Trichoderma species play important roles in nature as plant growth promotors and antagonists of phytopathogenic fungi, and are used as models to study photomorphogenesis. Molecular tools have been implemented to manipulate and improve these fungi. However, instability of transformants or very low frequency of homologous recombination has been reported. Here, we report the fate of transforming DNA, demonstrating that it can follow two different fates. When a vector contains sequences also present in the Trichoderma atroviride genome, it mainly integrates by homologous recombination generating stable recombinant strains. In contrast, vectors with no sequence homology to the T. atroviride genome generate unstable transformants, losing the transforming DNA in the first generation of conidia produced without selection where, surprisingly, the vector behaves as autoreplicative. Integration by homologous recombination was demonstrated when transformants were generated with a truncated version of the blr2 gene, resulting in insertional mutants with phenotypes identical to those of knockout mutants. Our results indicate that T. atroviride is highly efficient in integrating DNA by homologous recombination and that plasmid vectors with no sequence homology to the genome are maintained for several generations in T. atroviride if kept under selective pressure even though they lacked fungal autonomous replication sequences.     

Biological control of southern corn leaf blight by Trichoderma atroviride SG3403

Trichoderma atroviride SG3403 showed high biocontrol activity against southern corn leaf blight (SCLB; pathogen: Cochliobolus heterostrophus). T. atroviride SG3403 could cause death of C. heterostrophus race O hypha on plates. Spraying T. atroviride SG3403 conidia suspension over maize seedling leaves protected the corn from SCLB infection. Biocontrol effect lasted for 30 days in the field. Trichoderma strain was able to induce resistance response in corn leaves against pathogen infection. In corn leaves treated with T. atroviride SG3403, the enzyme activities of phenylalanine ammonia lyase (PAL) and superoxide dismutase (SOD) reached the highest at 24 h, enzyme activity of catalase (CAT) reached the highest at 36 h after inoculation of pathogen C. heterostrophus race O. RNA expression levels of Pal, Sod and Cat (which synthesis enzyme PAL, SOD and CAT) were also upregulated and corresponded to the enzyme activity at the same time point. Enzyme activities and corresponding genes expression induced by Trichoderma SG3403 was more obvious than that induced by pathogen only, which implies that T. atroviride SG3403 induced corn defense gene expression against pathogen infection. Thus, induced resistance mechanism was possibly involved in the biocontrol of SCLB by T. atroviride SG3403.     

Isolation of Pythium Acanthicum,P. oligandrum,and P. periplocum from Soil and Evaluation of Their Mycoparasitic Activity and Biocontrol Efficacy Against Selected Phytopathogenic Pythium Species

Mycoparasitic Pythium species with spiny oogonia were surveyed in 50 Palestinian agricultural fields subject to different cropping practices using the Sclerotia Bait Technique (SBT) and the Surface-Soil-Dilution-Plate method (SSDP) with the selective VP3 medium. The mycoparasitic Pythium species were obtained from 21 (42%) soils using the SSDP method and from 37 (74%) soils using SBT. Pythium acanthicum and P. oligandrum were isolated by both methods, whereas P. periplocum was isolated only by the SBT. Using a newly modified dual plate culture method (MDPCM), the three mycoparasites showed varying antagonistic performance against several Pythium host species under a range of in vitro conditions. However, P. periplocum and P. oligandrum were found to be active biocontrol agents against P. ultimum, the damping-off organism of cucumber. This pathogen was antagonized, on thin films of water agar, by the three mycoparasites, and was moderately susceptible to P. periplocum while slightly susceptible to P. acanthicum and P. oligandrum. In direct application method in which antagonistic mycoparasites were incorporated into peat/sand mixture artificially infested with P. ultimum under growthroom conditions, Pythium oligandrum and P. periplocum (at 500 CFUg⁻¹) significantly improved seedling emergence and protected seedlings from damping-off. In the seed coating method, biocontrol by two types of seed dressing (homogenate- or oospore coated seeds), was comparable to that achieved by direct application. This revised version was published online in June 2006 with corrections to the Cover Date.     

Mycoparasitic actions against fungi and oomycetes by a strain (CCFEE 5003) of the fungusLecanicillium muscarium isolated in Continental Antarctica

A strain (CCFEE 5003) ofLecanicillium muscarium, isolated in Continental Antarctica,showed mycoparasitism in agar cocultures, at 5 and 25 °C, againstMucor mucedo, Botrytis cinerea, Pythium aphanidermatum andPhytophthora palmivora. Different sequential steps were observed in the process leading to parasitism and resulting in a complete host disruption. Parasitism against fungi was characterised by diffused penetration into the host mycelium; with oomycetes, penetration was less evident and the contact between the two organisms was more intimate. Production of glucanolytic and chitinolytic enzymes appeared related to the mycoparasitic process.     

Trichoderma-induced plant immunity likely involves both hormonal- and camalexin-dependent mechanisms in Arabidopsis thaliana and confers resistance against necrotrophic fungus Botrytis cinerea

Filamentous fungi belonging to the genus Trichoderma have long been recognized as agents for the biocontrol of plant diseases. In this work, we investigated the mechanisms involved in the defense responses of Arabidopsis thaliana seedlings elicited by co-culture with Trichoderma virens and Trichoderma atroviride. Interaction of plant roots with fungal mycelium induced growth and defense responses, indicating that both processes are not inherently antagonist. Expression studies of the pathogenesis-related reporter markers pPr1a:uidA and pLox2:uidA in response to T. virens or T. atroviride provided evidence that the defense signaling pathway activated by these fungi involves salicylic acid (SA) and/or jasmonic acid (JA) depending on the amount of conidia inoculated. Moreover, we found that Arabidopsis seedlings colonized by Trichoderma accumulated hydrogen peroxide and camalexin in leaves. When grown under axenic conditions, T. virens produced indole-3-carboxaldehyde (ICAld) a tryptophan-derived compound with activity in plant development. In Arabidopsis seedlings whose roots are in contact with T. virens or T. atroviride, and challenged with Botrytis cinerea in leaves, disease severity was significantly reduced compared with axenically grown seedlings. Our results indicate that the defense responses elicited by Trichoderma in Arabidopsis are complex and involve the canonical defense hormones SA and JA as well as camalexin, which may be important factors in boosting plant immunity.Key words: Arabidopsis, Trichoderma, phytostimulation, defense responses, jasmonic acid, salicylic acid, camalexin     

An N-acetyl-β-d-glucosaminidase gene, cr-nag1, from the biocontrol agent Clonostachys rosea is up-regulated in antagonistic interactions with Fusarium culmorum

Clonostachys rosea is a widely distributed fungus that often acts as a parasite on other soil fungi. This fungus has also been reported as a potential parasite against nematodes and insects. The antagonistic activity is thought to be correlated with the secretion of cell wall-degrading enzymes, including chitinases. In this work, we identified and characterized an N-acetyl-β-d-glucosaminidase-encoding gene, cr-nag1, belonging to glycosyl hydrolase family 20, from the C. rosea strain IK726 using a degenerated primer strategy designed from conserved motifs. The complete gene, including its promoter region, was obtained by genomic walking. Southern analysis showed that cr-nag1 is present as a single copy gene in C. rosea. Phylogenetically, cr-nag1 showed the highest similarity to N-acetyl-β-d-glucosaminidase genes from other mycoparasitic fungi. Enzymatic assays and RT-PCR showed that the NAGase activity of C. rosea is specifically repressed in medium containing a high glucose content and is expressed in media containing chitin or Fusarium culmorum cell walls as sole carbon sources. Macroscopic and microscopic observations indicated that the mycelial growth of F. culmorum and Pythium ultimum were inhibited during interactions with C. rosea. High expression of cr-nag1 was found in interactions between C. rosea and F. culmorum, whereas the expression of cr-nag1 in interactions between C. rosea and P. ultimum was similar to the control. This indicates that although C. rosea secretes chitin-hydrolysing agents in order to target the cell wall of F. culmorum, it seems to use another strategy for controlling the development of the oomycete P. ultimum.     

Structural and Phylogenetic Analysis of Laccases from Trichoderma: A Bioinformatic Approach

The genus Trichoderma includes species of great biotechnological value, both for their mycoparasitic activities and for their ability to produce extracellular hydrolytic enzymes. Although activity of extracellular laccase has previously been reported in Trichoderma spp., the possible number of isoenzymes is still unknown, as are the structural and functional characteristics of both the genes and the putative proteins. In this study, the system of laccases sensu stricto in the Trichoderma species, the genomes of which are publicly available, were analyzed using bioinformatic tools. The intron/exon structure of the genes and the identification of specific motifs in the sequence of amino acids of the proteins generated in silico allow for clear differentiation between extracellular and intracellular enzymes. Phylogenetic analysis suggests that the common ancestor of the genus possessed a functional gene for each one of these enzymes, which is a characteristic preserved in T. atroviride and T. virens. This analysis also reveals that T. harzianum and T. reesei only retained the intracellular activity, whereas T. asperellum added an extracellular isoenzyme acquired through horizontal gene transfer during the mycoparasitic process. The evolutionary analysis shows that in general, extracellular laccases are subjected to purifying selection, and intracellular laccases show neutral evolution. The data provided by the present study will enable the generation of experimental approximations to better understand the physiological role of laccases in the genus Trichoderma and to increase their biotechnological potential.     

青蒿内生真菌分离、分子鉴定及深绿木霉对青蒿的促生作用研究

为从青蒿(Artemisia annua L.)内生真菌中筛选有促生作用的菌株,用组织分离法从青蒿侧枝中分离内生真菌并进行分子鉴定,对其中1株真菌深绿木霉对青蒿幼苗的促生作用进行了研究。结果表明,从青蒿侧枝中获得23株内生真菌,鉴定出16种,以炭疽菌属占优势。深绿木霉发酵液对青蒿种子发芽和幼苗生长具有显著抑制作用;深绿木霉液体发酵菌丝体在4-5月对青蒿幼苗生长具有显著的促进作用,但7月以后没有明显促进作用。因此,深绿木霉可作为青蒿苗肥,与化肥配合可施用于成株。     

Moths that Vector a Plant Pathogen also Transport Endophytic Fungi and Mycoparasitic Antagonists

Claviceps paspali, a common fungal pathogen of Paspalum grasses, attracts moth vectors by producing sugary exudates in the grass florets it infects. These exudates also support mycoparasitic Fusarium species that may negatively influence C. paspali fitness. We examined the potential for moths on which C. paspali depends to also transmit mycoparasitic Fusarium and fungal endophytes, which inhabit asymptomatic plant tissue and may influence host susceptibility to pathogens. We quantified infections by C. paspali, Fusarium spp., and endophytic fungi associated with Paspalum spp. at focal sites in the southeastern USA and used data from the nuclear internal transcribed spacer (ITS rDNA) to compare communities of plant-associated and moth-borne fungi. ITS sequences of moth-borne fungi were identical to reference sequences of mycoparasitic Fusarium heterosporum and to three distinct endophytic fungi isolated from Paspalum species. Our results demonstrate an unexpected overlap of fungal communities between disparate locations and among plant species and plant tissues, and suggest an unexpected role of moths, which vector a plant pathogen, to transmit other guilds of fungi. In turn, the potential for insects to transmit plant pathogens as well as mycoparasites and endophytic fungi suggests complex interactions underlying a commonly observed grass–pathogen system. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.