Comparative Phenotypic, Genomic, and Transcriptomic Analyses of Two Contrasting Strains of the Plant Beneficial Fungus Trichoderma virens.

Trichoderma virens is a beneficial fungus that helps plants fight pathogens and abiotic stresses and thereby enhances crop yields. Unlike other Trichoderma spp., there are two well-defined strains (P and Q) of T. virens, classified by secondary metabolites profiling, primarily the biosynthesis of the nonribosomal, strong antimicrobial agents gliotoxin (Q) and gliovirin (P). We have studied the phenotypic and biocontrol properties of two well-studied representative isolates (T. virens Gv29-8 and T. virens GvW/IMI304061) that represent a Q strain and a P strain of T. virens, respectively. We refined the genome assembly of the P strain using nanopore technology, and we compared it with the Q strain. The differences between the genomes include gene expansion in the Q strain. T. virens Gv29-8 is weaker than GvW as a mycoparasite on the broad host-range plant pathogen Sclerotium rolfsii, and it is ineffective as a biocontrol agent when applied to pathogen-infested soil. T. virens Gv29-8 proved to be phytotoxic to Arabidopsis seedlings, whereas the effect of T. virens GvW was not major. Both strains colonized the surface and outer cortex layer of tomato roots, with about 40% higher colonization by T. virens Gv29-8. T. virens Gv29-8 induced the expression of a larger set of tomato genes than did T. virens GvW, although some tomato genes were uniquely induced in response to T. virens GvW. We studied the comparative transcriptome response of T. virens Gv29-8 and T. virens GvW to S. rolfsii. A larger set of genes was regulated in T. virens GvW than in T. virens Gv29-8 in the presence of the plant pathogen. IMPORTANCE Trichoderma virens populations that were earlier classified into two strains (P and Q) based on secondary metabolites profiling are also phenotypically and genetically distinct, with the latter being ineffective in controlling the devastating, broad host range plant pathogen Sclerotium rolfsii. The two strains also provoke distinct as well as overlapping transcriptional responses to the presence of the plant and the pathogen. This study enriches our knowledge of Trichoderma-plant-pathogen interactions and identifies novel candidate genes for further research and deployment in agriculture.

Physiological responses of Pinus sylvestris var. mongolica seedlings to the interaction between Suillus luteus and Trichoderma virens.

The effects of the interaction between Suillus luteus (L.) Roussel and Trichoderma virens (J.H. Mill., Giddens & A.A. Foster) Arx on Pinus sylvestris var. mongolica Litv. were studied using plant physiology, mycorrhizal science, forest pathology, and biochemistry. Seedling growth and physiological parameters were determined, including the colonization rate of mycorrhizal fungi, biomass, root activity, photosynthetic pigment content, soluble protein content, antioxidant enzyme activities, rhizosphere soil enzyme activities, and protective enzyme activities. In addition, an optimal resistance system involving T. virens, mycorrhizal fungus (S. luteus), and P. sylvestris var. mongolica seedlings was constructed. Synergies between S. luteus and T. virens were observed, and most of the parameters of P. sylvestris var. mongolica seedlings inoculated with S. luteus 30 days + T. virens were higher than other treatments. After three months, when compared the control, the S. luteus 30 days + T. virens treatment gave increases in height (42.3 %); collar diameter (66.7 %); fresh weight (54 %); dry weight (50 %); soluble protein content (69.86 %); root activity (150 %); chlorophyll a (77.6 %); chlorophyll b (70.5 %); carotenoids (144 %); CAT activity (876.9 %); POD activity (268.3 %); SOD activity (66.18 %); ß-1,3-glucanase activity (125.8 %); chitinase activity (40 %); rhizosphere soil catalase activity (97.8 %); and phosphatase activity (266.7 %). These results indicate that there may be a stimulating factor between S. luteus and T. virens when they are inoculated together (S. luteus 30 days + T. virens).

Trichoderma-plant root colonization: escaping early plant defense responses and activation of the antioxidant machinery for saline stress tolerance.

Trichoderma spp. are versatile opportunistic plant symbionts which can colonize the apoplast of plant roots. Microarrays analysis of Arabidopsis thaliana roots inoculated with Trichoderma asperelloides T203, coupled with qPCR analysis of 137 stress responsive genes and transcription factors, revealed wide gene transcript reprogramming, proceeded by a transient repression of the plant immune responses supposedly to allow root colonization. Enhancement in the expression of WRKY18 and WRKY40, which stimulate JA-signaling via suppression of JAZ repressors and negatively regulate the expression of the defense genes FMO1, PAD3 and CYP71A13, was detected in Arabidopsis roots upon Trichoderma colonization. Reduced root colonization was observed in the wrky18/wrky40 double mutant line, while partial phenotypic complementation was achieved by over-expressing WRKY40 in the wrky18 wrky40 background. On the other hand increased colonization rate was found in roots of the FMO1 knockout mutant. Trichoderma spp. stimulate plant growth and resistance to a wide range of adverse environmental conditions. Arabidopsis and cucumber (Cucumis sativus L.) plants treated with Trichoderma prior to salt stress imposition show significantly improved seed germination. In addition, Trichoderma treatment affects the expression of several genes related to osmo-protection and general oxidative stress in roots of both plants. The MDAR gene coding for monodehydroascorbate reductase is significantly up-regulated and, accordingly, the pool of reduced ascorbic acid was found to be increased in Trichoderma treated plants. 1-Aminocyclopropane-1-carboxylate (ACC)-deaminase silenced Trichoderma mutants were less effective in providing tolerance to salt stress, suggesting that Trichoderma, similarly to ACC deaminase producing bacteria, can ameliorate plant growth under conditions of abiotic stress, by lowering ameliorating increases in ethylene levels as well as promoting an elevated antioxidative capacity.

The LysM receptor-like kinase LysM RLK1 is required to activate defense and abiotic-stress responses induced by overexpression of fungal chitinases in Arabidopsis plants.

Application of crab shell chitin or pentamer chitin oligosaccharide to Arabidopsis seedlings increased tolerance to salinity in wild-type but not in knockout mutants of the LysM Receptor-Like Kinase1 (CERK1/LysM RLK1) gene, known to play a critical role in signaling defense responses induced by exogenous chitin. Arabidopsis plants overexpressing the endochitinase chit36 and hexoaminidase excy1 genes from the fungus Trichoderma asperelleoides T203 showed increased tolerance to salinity, heavy-metal stresses, and Botrytis cinerea infection. Resistant lines, overexpressing fungal chitinases at different levels, were outcrossed to lysm rlk1 mutants. Independent homozygous hybrids lost resistance to biotic and abiotic stresses, despite enhanced chitinase activity. Expression analysis of 270 stress-related genes, including those induced by reactive oxygen species (ROS) and chitin, revealed constant up-regulation (at least twofold) of 10 genes in the chitinase-overexpressing line and an additional 76 salt-induced genes whose expression was not elevated in the lysm rlk1 knockout mutant or the hybrids harboring the mutation. These findings elucidate that chitin-induced signaling mediated by LysM RLK1 receptor is not limited to biotic stress response but also encompasses abiotic-stress signaling and can be conveyed by ectopic expression of chitinases in plants.

Plant-beneficial effects of Trichoderma and of its genes.

Trichoderma (teleomorph Hypocrea) is a fungal genus found in many ecosystems. Trichoderma spp. can reduce the severity of plant diseases by inhibiting plant pathogens in the soil through their highly potent antagonistic and mycoparasitic activity. Moreover, as revealed by research in recent decades, some Trichoderma strains can interact directly with roots, increasing plant growth potential, resistance to disease and tolerance to abiotic stresses. This mini-review summarizes the main findings concerning the Trichoderma-plant interaction, the molecular dialogue between the two organisms, and the dramatic changes induced by the beneficial fungus in the plant. Efforts to enhance plant resistance and tolerance to a broad range of stresses by expressing Trichoderma genes in the plant genome are also addressed.

Transcript and metabolite analysis of the Trichoderma-induced systemic resistance response to Pseudomonas syringae in Arabidopsis thaliana.

In the present study we have assessed, by transcriptional and metabolic profiling, the systemic defence response of Arabidopsis thaliana plants to the leaf pathogen Pseudomonas syringae pv. tomato DC3000 (Pst) induced by the beneficial fungus Trichoderma asperelloides T203. Expression analysis (qPCR) of a set of 137 Arabidopsis genes related to Pst defence responses showed that T203 root colonization is not associated with major detectable transcriptomic changes in leaves. However, plants challenged with the bacterial pathogen showed quantitative differences in gene expression when pre-inoculated with T203, supporting priming of the plant by this beneficial fungus. Among the defence-related genes affected by T203, lipid transfer protein (LTP)4, which encodes a member of the lipid transfer pathogenesis-related family, is upregulated, whereas the WRKY40 transcription factor, known to contribute to Arabidopsis susceptibility to bacterial infection, shows reduced expression. On the other hand, root colonization by this beneficial fungus substantially alters the plant metabolic profile, including significant changes in amino acids, polyamines, sugars and citric acid cycle intermediates. This may in part reflect an increased energy supply required for the activation of plant defences and growth promotion effects mediated by Trichoderma species.

Characterization of ACC deaminase from the biocontrol and plant growth-promoting agent Trichoderma asperellum T203.

1-aminocyclopropane-1-carboxylate (ACC) deaminase activity was evaluated in the biocontrol and plant growth-promoting fungus Trichoderma asperellum T203. Fungal cultures grown with ACC as the sole nitrogen source showed high enzymatic activity. The enzyme encoding gene (Tas-acdS) was isolated, and an average 3.5-fold induction of the gene by 3 mM ACC was detected by real-time PCR. Escherichia coli bacteria carrying the intron-free cDNA of Tas-acdS cloned into the vector pAlter-EX1 under the control of the tac promoter revealed specific ACC deaminase (ACCD) activity and the ability to promote canola (Brassica napus) root elongation in pouch assays. RNAi silencing of the ACCD gene in T. asperellum showed decreased ability of the mutants to promote root elongation of canola seedlings. These data suggest a role for ACCD in the plant root growth-promotion effect by T. asperellum.

Synthetic ultrashort cationic lipopeptides induce systemic plant defense responses against bacterial and fungal pathogens.

A new family of synthetic, membrane-active, ultrashort lipopeptides composed of only four amino acids linked to fatty acids was tested for the ability to induce systemic resistance and defense responses in plants. We found that two peptides wherein the third residue is a d-enantiomer (italic), C16-KKKK and C16-KLLK, can induce medium alkalinization of tobacco suspension-cultured cells and expression of defense-related genes in cucumber and Arabidopsis seedlings. Moreover, these compounds can prime systemic induction of antimicrobial compounds in cucumber leaves similarly to the plant-beneficial fungus Trichoderma asperellum T203 and provide systemic protection against the phytopathogens Botrytis cinerea B05, Pseudomonas syringae pv. lachrimans, and P. syringae pv. tomato DC3000. Thus, short cationic lipopeptides are a new category of compounds with potentially high utility in the induction of systemic resistance in plants.

Role of swollenin, an expansin-like protein from Trichoderma, in plant root colonization.

Swollenin, a protein first characterized in the saprophytic fungus Trichoderma reesei, contains an N-terminal carbohydrate-binding module family 1 domain (CBD) with cellulose-binding function and a C-terminal expansin-like domain. This protein was identified by liquid chromatography-mass spectrometry among many other cellulolytic proteins secreted in the coculture hydroponics medium of cucumber (Cucumis sativus) seedlings and Trichoderma asperellum, a well-known biocontrol agent and inducer of plant defense responses. The swollenin gene was isolated and its coding region was overexpressed in the same strain under the control of the constitutive pki1 promoter. Trichoderma transformants showed a remarkably increased ability to colonize cucumber roots within 6 h after inoculation. On the other hand, overexpressors of a truncated swollenin sequence bearing a 36-amino acid deletion of the CBD did not differ from the wild type, showing in vivo that this domain is necessary for full protein activity. Root colonization rates were reduced in transformants silenced in swollenin gene expression. A synthetic 36-mer swollenin CBD peptide was shown to be capable of stimulating local defense responses in cucumber roots and leaves and to afford local protection toward Botrytis cinerea and Pseudomonas syringae pv lachrymans infection. This indicates that the CBD domain might be recognized by the plant as a microbe-associated molecular pattern in the Trichoderma-plant interaction.

Inhibition of fungal and bacterial plant pathogens in vitro and in planta with ultrashort cationic lipopeptides.

Plant diseases constitute an emerging threat to global food security. Many of the currently available antimicrobial agents for agriculture are highly toxic and nonbiodegradable and cause extended environmental pollution. Moreover, an increasing number of phytopathogens develop resistance to them. Recently, we have reported on a new family of ultrashort antimicrobial lipopeptides which are composed of only four amino acids linked to fatty acids (A. Makovitzki, D. Avrahami, and Y. Shai, Proc. Natl. Acad. Sci. USA 103:15997-16002, 2006). Here, we investigated the activities in vitro and in planta and the modes of action of these short lipopeptides against plant-pathogenic bacteria and fungi. They act rapidly, at low micromolar concentrations, on the membranes of the microorganisms via a lytic mechanism. In vitro microscopic analysis revealed wide-scale damage to the microorganism's membrane, in addition to inhibition of pathogen growth. In planta potent antifungal activity was demonstrated on cucumber fruits and leaves infected with the pathogen Botrytis cinerea as well as on corn leaves infected with Cochliobolus heterostrophus. Similarly, treatment with the lipopeptides of Arabidopsis leaves infected with the bacterial leaf pathogen Pseudomonas syringae efficiently and rapidly reduced the number of bacteria. Importantly, in contrast to what occurred with many native lipopeptides, no toxicity was observed on the plant tissues. These data suggest that the ultrashort lipopeptides could serve as native-like antimicrobial agents economically feasible for use in plant protection.

The 18mer peptaibols from Trichoderma virens elicit plant defence responses.

SUMMARY Peptaibols, the products of non-ribosomal peptide synthetases (NRPS), are linear peptide antibiotics produced by Trichoderma and other fungal genera. Trichoderma virens strain Gv29-8, a well-known biocontrol agent and inducer of plant defence responses, produces three lengths of peptaibols, 11, 14 and 18 residues long, with several isoforms of each. Disruption of the NRPS gene, tex1, encoded by a 62.8-kb uninterrupted open reading frame, results in the loss of production of all forms of 18-residue peptaibols. Tex1 is expressed during all Trichoderma developmental stages (germinating conidia, sporulating and non-sporulating mycelia) examined on solid media. Expression analysis by reverse transcriptase PCR shows that in Gv29-8 wild-type the abundance of tex1 transcript is greater during co-cultivation with cucumber seedling roots than when grown alone. Cucumber plants co-cultivated with T. virens strains disrupted in tex1 show a significantly reduced systemic resistance response against the leaf pathogen Pseudomonas syringae pv. lachrymans, and reduced ability to produce phenolic compounds with inhibitory activity to the bacteria as compared with plants grown in the presence of wild-type. Two synthetic 18-amino-acid peptaibol isoforms (TvBI and TvBII) from Gv29-8 when applied to cucumber seedlings through the transpiration stream can alone induce systemic protection to the leaf pathogenic bacteria, induce antimicrobial compounds in cucumber cotyledons and up-regulate hydroxyperoxide lyase (hpl), phenylalanine ammonia lyase (pal1) and peroxidase (prx) gene expression. These data strongly suggest that the 18mer peptaibols are critical in the chemical communication between Trichoderma and plants as triggers of non-cultivar-specific defence responses.

Characterization of a mitogen-activated protein kinase gene from cucumber required for trichoderma-conferred plant resistance.

The fungal biocontrol agent Trichoderma asperellum has been recently shown to induce systemic resistance in plants through a mechanism that employs jasmonic acid and ethylene signal transduction pathways. Mitogen-activated protein kinase (MAPK) proteins have been implicated in the signal transduction of a wide variety of plant stress responses. Here we report the identification and characterization of a Trichoderma-induced MAPK (TIPK) gene function in cucumber (Cucumis sativus). Similar to its homologs, wound-induced protein kinase, MPK3, and MPK3a, TIPK is also induced by wounding. Normally, preinoculation of roots with Trichoderma activates plant defense mechanisms, which result in resistance to the leaf pathogen Pseudomonas syringae pv lachrymans. We used a unique attenuated virus vector, Zucchini yellow mosaic virus (ZYMV-AGII), to overexpress TIPK protein and antisense (AS) RNA. Plants overexpressing TIPK were more resistant to pathogenic bacterial attack than control plants, even in the absence of Trichoderma preinoculation. On the other hand, plants expressing TIPK-AS revealed increased sensitivity to pathogen attack. Moreover, Trichoderma preinoculation could not protect these AS plants against subsequent pathogen attack. We therefore demonstrate that Trichoderma exerts its protective effect on plants through activation of the TIPK gene, a MAPK that is involved in signal transduction pathways of defense responses.

TasHyd1, a new hydrophobin gene from the biocontrol agent Trichoderma asperellum, is involved in plant root colonization.

SUMMARY A hydrophobin-like clone (TasHyd1) was isolated during a PCR differential mRNA display analysis conducted on Trichoderma asperellum mycelia interacting with plant roots. The open reading frame encodes a 145-amino-acid protein showing similarity to Pbhyd1, a Class I hydrophobin from the dimorphic fungus Paracoccidioides brasiliensis. TasHyd1 expression was detected in planta up to 5 days after Trichoderma root inoculation. TasHyd1 is constitutively expressed at low levels in mycelia in young cultures but gene expression is not detected in sporulating hyphae or in non-germinating spores. Carbon limitation stimulates expression of TasHyd1 whereas nitrogen or phosphate starvation down-regulate expression. TasHyd1 fused to an HA tag was over-expressed in Trichoderma and the protein was detected with an anti-HA antibody in the trifluoroacetic-acid-soluble fraction of mycelial cell walls. Over-expressor mutants were not affected in their mycoparasitic activity when tested in vitro against the plant pathogen Rhizoctonia solani and retained root colonization capacity comparable with that of the wild-type. TasHyd1 deletion mutants had no significant reduction in in vitro mycoparasitic activity but were altered in their wettability and were severely impaired in root attachment and colonization. These phenotypes were recovered by complementation of TasHyd1, indicating that the protein is a new hydrophobin that contributes to Trichoderma interaction with the plant.

Trichoderma mitogen-activated protein kinase signaling is involved in induction of plant systemic resistance.

The role of a mitogen-activated protein kinase (MAPK) TmkA in inducing systemic resistance in cucumber against the bacterial pathogen Pseudomonas syringae pv. lacrymans was investigated by using tmkA loss-of-function mutants of Trichoderma virens. In an assay where Trichoderma spores were germinated in proximity to cucumber roots, the mutants were able to colonize the plant roots as effectively as the wild-type strain but failed to induce full systemic resistance against the leaf pathogen. Interactions with the plant roots enhanced the level of tmkA transcript in T. virens and its homologue in Trichoderma asperellum. At the protein level, we could detect the activation of two forms reacting to the phospho-p44/42 MAPK antibody. Biocontrol experiments demonstrated that the tmkA mutants retain their biocontrol potential against Rhizoctonia solani in soil but are not effective against Sclerotium rolfsii in reducing disease incidence. Our results show that, unlike in many plant-pathogen interactions, Trichoderma TmkA MAPK is not involved in limited root colonization. Trichoderma, however, needs MAPK signaling in order to induce full systemic resistance in the plant.

Involvement of Jasmonic Acid/Ethylene Signaling Pathway in the Systemic Resistance Induced in Cucumber by Trichoderma asperellum T203.

ABSTRACT Trichoderma spp. are effective biocontrol agents for a number of soilborne plant pathogens, and some are also known for their ability to enhance plant growth. It was recently suggested that Trichoderma also affects induced systemic resistance (ISR) mechanism in plants. Analysis of signal molecules involved in defense mechanisms and application of specific inhibitors indicated the involvement of jasmonic acid and ethylene in the protective effect conferred by Trichoderma spp. against the leaf pathogen Pseudomonas syringae pv. lachrymans. Moreover, examination of local and systemic gene expression by real-time reverse transcription-polymerase chain reaction analysis revealed that T. asperellum (T203) modulates the expression of genes involved in the jasmonate/ethylene signaling pathways of ISR (Lox1, Pal1, ETR1, and CTR1) in cucumber plants. We further showed that a subsequent challenge of Trichoderma-preinoculated plants with the leaf pathogen P. syringae pv. lachrymans resulted in higher systemic expression of the pathogenesisrelated genes encoding for chitinase 1, beta-1,3-glucanase, and peroxidase relative to noninoculated, challenged plants. This indicates that Trichoderma induced a potentiated state in the plant enabling it to be more resistant to subsequent pathogen infection.

Isolation of two aspartyl proteases from Trichoderma asperellum expressed during colonization of cucumber roots.

Trichoderma asperellum and cucumber seedlings were used as a model to study the modulation of Trichoderma gene expression during plant root colonization. Seedlings were grown in an aseptic hydroponics medium and inoculated with Trichoderma spore suspension. Proteins differentially secreted into the medium were isolated. Three major proteins of fungal origin were identified: two arabinofuranosidases (Abf1 and Abf2) and an aspartyl protease. Differential mRNA display was conducted on Trichoderma mycelia interacting and non-interacting, with the plant roots. Among the differentially regulated clones another aspartyl protease was identified. Sequencing of the genes revealed that the first aspartyl protease is a close homologue of PapA from T. harzianum and the other, of AP1 from Botryotinia fuckeliana. RT-PCR analysis confirms that the proteases are induced in response to plant roots attachment and are expressed in planta. papA, but not papB, is also induced in plate confrontation assays with the plant pathogen Rhizoctonia solani. These data suggest that the identified proteases play a role in Trichoderma both as a mycoparasite and as a plant opportunistic symbiont.

The global regulator genes from biocontrol strain Serratia plymuthica IC1270: cloning, sequencing, and functional studies.

The biocontrol activity of various fluorescent pseudomonads towards plant-pathogenic fungi is dependent upon the GacA/GacS-type two-component system of global regulators and the RpoS transcription sigma factor. In particular, these components are required for the production of antifungal antibiotics and exoenzymes. To investigate the effects of these global regulators on the expression of biocontrol factors by plant-associated bacteria other than Pseudomonas spp., gacA/gacS and rpoS homologues were cloned from biocontrol strain IC1270 of Serratia plymuthica, which produces a set of antifungal compounds, including chitinolytic enzymes and the antibiotic pyrrolnitrin. The nucleotide and deduced protein sequence alignments of the cloned gacA/gacS-like genes-tentatively designated grrA (global response regulation activator) and grrS (global response regulation sensor) and of the cloned rpoS gene revealed 64 to 93% identity with matching genes and proteins of the enteric bacteria Escherichia coli, Pectobacterium carotovora subsp. carotovora, and Serratia marcescens. grrA, grrS, and rpoS gene replacement mutants of strain IC1270 were deficient in the production of pyrrolnitrin, an exoprotease, and N-acylhomoserine lactone quorum-sensing signal molecules. However, neither mutant appeared to differ from the parental strain in the production of siderophores, and only grrA and grrS mutants were deficient in the production of a 58-kDa endochitinase, representing the involvement of other sigma factors in the regulation of strain IC1270's chitinolytic activity. Compared to the parental strain, the grrA, grrS, and rpoS mutants were markedly less capable of suppressing Rhizoctonia solani and Pythium aphanidermatum under greenhouse conditions, indicating the dependence of strain IC1270's biocontrol property on the GrrA/GrrS and RpoS global regulators.

Trichoderma species--opportunistic, avirulent plant symbionts.

Trichoderma spp. are free-living fungi that are common in soil and root ecosystems. Recent discoveries show that they are opportunistic, avirulent plant symbionts, as well as being parasites of other fungi. At least some strains establish robust and long-lasting colonizations of root surfaces and penetrate into the epidermis and a few cells below this level. They produce or release a variety of compounds that induce localized or systemic resistance responses, and this explains their lack of pathogenicity to plants. These root-microorganism associations cause substantial changes to the plant proteome and metabolism. Plants are protected from numerous classes of plant pathogen by responses that are similar to systemic acquired resistance and rhizobacteria-induced systemic resistance. Root colonization by Trichoderma spp. also frequently enhances root growth and development, crop productivity, resistance to abiotic stresses and the uptake and use of nutrients.

Regulation of two homodimer hexosaminidases in the mycoparasitic fungus Trichoderma asperellum by glucosamine.

Trichoderma asperellum is a mycoparasitic fungus which is used as a biocontrol agent against plant pathogens. Its hydrolytic enzymes take part in its parasitic interaction, degrading the pathogen cell wall and thereby helping to control disease. One of those enzymes, beta- N-acetyl- d-glucosaminidase (GlcNAcase), degrades chitin, which is a major component of the cell wall of many plant-pathogenic fungi. Two GlcNAcases of T. asperellum T203, designated EXC1Y and EXC2Y, were purified, their genes and their promoters were sequenced, and their regulation was studied. The enzymes share homology (59% identity) but are easily distinguished by PAGE assay. Biochemical characterization, Edman degradation, and mass spectrometry demonstrated that EXC1Y and EXC2Y are both active as homodimers. Both genes are up-regulated by glucosamine (GlcN), in contrast to two endochitinases of this fungus. GlcN induces the secretion of several proteins (including a beta-glucosidase), among which EXC1Y is the most abundant. An exc2y knockout was constructed, to study the regulation of EXC1Y expression and secretion. The fungus has the ability to store a high amount of this enzyme in an active form and secrete it into the medium later.

Concomitant induction of systemic resistance to Pseudomonas syringae pv. lachrymans in cucumber by Trichoderma asperellum (T-203) and accumulation of phytoalexins.

Most studies on the reduction of disease incidence in soil treated with Trichoderma asperellum have focused on microbial interactions rather than on plant responses. This study presents conclusive evidence for the induction of a systemic response against angular leaf spot of cucumber (Pseudomonas syringae pv. lachrymans) following application of T. asperellum to the root system. To ascertain that T. asperellum was the only microorganism present in the root milieu, plants were grown in an aseptic hydroponic growth system. Disease symptoms were reduced by as much as 80%, corresponding to a reduction of 2 orders of magnitude in bacterial cell densities in leaves of plants pretreated with T. asperellum. As revealed by electron microscopy, bacterial cell proliferation in these plants was halted. The protection afforded by the biocontrol agent was associated with the accumulation of mRNA of two defense genes: the phenylpropanoid pathway gene encoding phenylalanine ammonia lyase (PAL) and the lipoxygenase pathway gene encoding hydroxyperoxide lyase (HPL). This was further supported by the accumulation of secondary metabolites of a phenolic nature that showed an increase of up to sixfold in inhibition capacity of bacterial growth in vitro. The bulk of the antimicrobial activity was found in the acid-hydrolyzed extract containing the phenolics in their aglycone form. High-performance liquid chromatography analysis of phenolic compounds showed a marked change in their profile in the challenged, preelicited plants relative to that in challenged controls. The results suggest that similar to beneficial rhizobacteria, T. asperellum may activate separate metabolic pathways in cucumber that are involved in plant signaling and biosynthesis, eventually leading to the systemic accumulation of phytoalexins.