Antimycotic Compounds from the Plant Pathogen Rhizoctonia solani and its Antagonist Trichoderma harzianum

The soilborne rhizosphere-competent fungal biocontrol agent Trichoderma harzianum isolate Th008 secreted trichodermin (MW = 292) and a small peptide (MW = 876) in culture. These compounds were antagonistic in culture to the mycelial growth of the soilborne fungal pathogen Rhizoctonia solani isolate 2B-12, which is highly virulent to soybean ( Glycine max )seedlings. When 100mg of dried autoclaved mycelial mat of R. solani was added to 200 ml liquid cultures of T. harzianum , the quantity of antimycotic compounds secreted by the latter was 3.5 times greater than that of the antagonist alone. R. solani secreted a coumarin derivative (MW = 313) in liquid culture, which inhibited the mycelial growth of T. harzianum ; however, inhibition of the growth of the antagonist required a greater concentration than that for the antimycotic compounds produced by the antagonist against the pathogen. The inclusion of 100 mg of dried autoclaved mycelial mat of T. harzianum in a 200 ml liquid culture of R. solani did not affect the quantity of the antimycotic compound produced by the pathogen.     

Use of monoclonal antibodies to quantify the dynamics of alpha-galactosidase and endo-1,4-beta-glucanase production by Trichoderma hamatum during saprotrophic growth and sporulation in peat

Trichoderma species are ubiquitous soil and peat-borne saprotrophs that have received enormous scientific interest as biocontrol agents of plant diseases caused by destructive root pathogens. Mechanisms of biocontrol such as antibiosis and hyperparasitism are well documented and the biochemistry and molecular genetics of these processes defined. An aspect of biocontrol that has received little attention is the ability of Trichoderma species to compete for nutrients in their natural environments. Trichoderma species are efficient producers of polysaccharide-degrading enzymes that enable them to colonize organic matter thereby preventing the saprotrophic spread of plant pathogens. This study details the use of monoclonal antibodies (mAbs) to quantify the production of two enzymes implicated in the saprotrophic growth of Trichoderma species in peat. Using mAbs specific to the hemicellulase enzyme alpha-galactosidase (AGL) and the cellulase enzyme endo-1,4-beta-glucanase (EG), the relationship between the saprotrophic growth dynamics of a biocontrol strain of Trichoderma hamatum and the concomitant production of these enzymes in peat-based microcosms was studied. Enzyme activity assays and enzyme protein concentrations derived by enzyme-linked immunosorbent assay (ELISA) established the precision and sensitivity of mAb-based assays in quantifying enzyme production during active growth of the fungus. Trends in enzyme activities and protein concentrations were similar for both enzymes, during a 21-day sampling period in which active growth and sporulation of the fungus in peat was quantified using an independent mAb-based assay. There was a sharp increase in active biomass of T. hamatum 3 days after inoculation of microcosms with phialoconidia. After 3 days there was a rapid decline in active biomass which coincided with sporulation of the fungus. A similar trend was witnessed with EG activities and concentrations. This showed that EG production related directly to active growth of the fungus. The trend was not found, however, with AGL. There was a rapid increase in enzyme activities and protein concentrations on day 3, after which they remained static. The reason for the maintenance of elevated AGL probably resulted from secretion of the enzyme from conidia and chlamydospores. ELISA, immunofluoresence and immunogold electron microscopy studies of these cells showed that the enzyme is localized within the cytoplasm and is secreted extracellularly into the surrounding environment. It is postulated that release of oligosaccharides from polymeric hemicellulose by the constitutive spore-bound enzyme leads to AGL induction and could act as an environmental cue for spore germination.     

Rhizoctonia solani, an elicitor of 6-pentyl-α-pyrone production by Trichoderma harzianum in a two liquid phases, extractive fermentation system

6-pentyl-alpha-pyrone (6PP) production by Trichoderma harzianum, in an extractive fermentation system, was elicitated by Rhizoctonia solani. The extent of 6PP elicitation was related to the state of Rhizoctonia and to the Trichoderma inoculum type. The use of non-viable Rhizoctonia solani mycelium in mycelium-inoculated Trichoderma harzianum culture, yielded the maximal 6PP production (474 mg l(-1)) compared to control cultures (147 mg l(-1)) and decreased the process time from 192 to 96 h.     

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

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

Biochemical and metabolic profiles of Trichoderma strains isolated from common bean crops in the Brazilian Cerrado, and potential antagonism against Sclerotinia sclerotiorum

Some species of Trichoderma have successfully been used in the commercial biological control of fungal pathogens, e.g., Sclerotinia sclerotiorum, an economically important pathogen of common beans (Phaseolus vulgaris L.). The objectives of the present study were (1) to provide molecular characterization of Trichoderma strains isolated from the Brazilian Cerrado; (2) to assess the metabolic profile of each strain by means of Biolog FF Microplates; and (3) to evaluate the ability of each strain to antagonize S. sclerotiorum via the production of cell wall-degrading enzymes (CWDEs), volatile antibiotics, and dual-culture tests. Among 21 isolates, we identified 42.86% as Trichoderma asperellum, 33.33% as Trichoderma harzianum, 14.29% as Trichoderma tomentosum, 4.76% as Trichoderma koningiopsis, and 4.76% as Trichoderma erinaceum. Trichoderma asperellum showed the highest CWDE activity. However, no species secreted a specific group of CWDEs. Trichoderma asperellum 364/01, T. asperellum 483/02, and T. asperellum 356/02 exhibited high and medium specific activities for key enzymes in the mycoparasitic process, but a low capacity for antagonism. We observed no significant correlation between CWDE and antagonism, or between metabolic profile and antagonism. The diversity of Trichoderma species, and in particular of T. harzianum, was clearly reflected in their metabolic profiles. Our findings indicate that the selection of Trichoderma candidates for biological control should be based primarily on the environmental fitness of competitive isolates and the target pathogen.     

Amendment with peony root bark improves the biocontrol efficacy of Trichoderma harzianum against Rhizoctonia solani

We tested Trichoderma harzianum as a biocontrol agent for Rhizoctonia solani AG2-1, using six natural antifungal materials to improve its efficacy. Among the six materials tested, peony (Paeonia suffruticosa) root bark (PRB) showed the strongest antifungal activity against R. solani AG2-1, and was not antagonistic to T. harzianum. Scanning electron microscopy showed that treatment with PRB extract resulted in shortened and deformed R. solani AG2-1 hyphal cells. The control of radish damping-off caused by R. solani AG2-1 was greatly increased by combined treatments of T. harzianum and PRB, as compared with either of the two treatments alone, with the control effect increased from 42.3-51.5% to 71.4-87.6%. The antifungal compound in PRB, which was isolated in chloroform and identified as paeonol by mass spectrometry, 1H NMR, and 13C NMR analyses, inhibited the growth of R. solani AG2-1 but not that of T. harzianum. Thus, PRB powder or extract may be used as a safe additive to T. harzianum to improve the control of the soil borne diseases caused by R. solani AG2-1.     

Self-fusion of protoplasts enhances chitinase production and biocontrol activity in Trichoderma harzianum

Protoplasts were isolated from Trichoderma harzianum strain PTh18 using lysing enzymes and self-fusion of T. harzianum protoplasts was carried out using polyethylene glycol in STC buffer. The fused protoplasts of T. harzianum were regenerated and 15 self-fusants were selected to study the chitinase production and biocontrol activity. High chitinase activity was measured in the culture filtrates of most of the self-fusants (87%) than the parent. Among the fusants, the strain SFTh8 produced maximum chitinase with a two-fold increase as compared to the parent strain. All the self-fusants exhibited increased antagonistic activity against Rhizoctonia solani than the parent. The crude chitinase preparation of SFTh8 lysed the mycelia of T. harzianum, Trichoderma viride and Trichoderma reesei and released the protoplasts in higher number than the crude chitinase preparation of parent strain PTh18.     

Biological control of phytopathogenic fungi through lytic action of Trichoderma species

Abstract The antagonistic effect of Trichoderma reesei and Trichoderma harzianum was studied towards a range of phytopathogenic fungi; Alternia solani, Botrytis fabae, Cladosporium cucumerinum, Fusarium oxysporum, Fusarium tricintum . Coculture of the phytopathogens and Trichodema under laboratory conditions clearly showed dominance of the Trichoderma species. In all cases, Trichoderma overgrew the phytopathogens and subsequently developed a conidial lawn over the surface. In these studies the lytic action of the pathogen was clearly apparent and the inhibition of growth appears directly related to its ability to hydrolyze the cell walls of the tested microorganisms rather than through the inhibitory action of antibiotics or toxins. Proteinase, mannanase, laminarinase and chitinase activities were determined in the extracellular fluid of Trichoderma . Glucose and laminaribiose were detected after the in vitro hydrolysis of the cell walls of the phytopathogens. The data imply that the nature of T. reesei and T. harzianum antagonism is based on mycoparasitism (lysis) and appears to optimalize with contact between the mycelia.     

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.     

芦竹内生真菌F0238对植物病原菌的拮抗作用

从黄海海岸低盐药用植物芦竹(Arundo donax L．)中分离得到木霉属的内生真菌F0238,对其进行拮抗植物病原菌活性及作用机制的试验研究。结果表明F0238发酵液对Botrytis cinerea,Sclerotium rolfsii等8种植物病原菌有强的抑菌活性及抑制孢子萌发作用,且这种作用在发酵原液被稀释了160倍后仍然存在;对峙培养试验表明F0238菌对植物病原菌有很强的拮抗作用,拮抗机制主要为重寄生作用、营养竞争作用及抗生作用等。     

Improved antifungal activity of a mutant of Trichoderma harzianum CECT 2413 which produces more extracellular proteins

Trichoderma harzianum is a well-known biological control agent against fungal plant diseases. In order to select improved biocontrol strains from Trichoderma harzianum CECT 2413, a mutant has been isolated for its ability to produce wider haloes than the wild type, when hydrolysing pustulan, a polymer of beta-1,6-glucan. The mutant possesses between two and four times more chitinase, beta-1,3- and beta-1,6-glucanase activities than the wild type, produces about three times more extracellular proteins and secretes higher amounts of a yellow pigment (alpha-pyrone). This mutant performed better than the wild type during in vitro experiments, overgrowing and sporulating on Rhizoctonia solani earlier, killing this pathogen faster and exerting better protection on grapes against Botrytis cinerea.     

Possible role of lectins in mycoparasitism.

Lectin activity in a host-mycoparasite relationship was demonstrated with Rhizoctonia solani and Trichoderma harzianum. Attachment of O but not A and B erythrocytes to hyphae occurred on R. solani but not on its mycoparasite. This phenomenon, which was Ca2+ and Mn2+ dependent, was prevented by galactose, present in T. harzianum cell walls, and by fucose.     

Major secondary metabolites produced by two commercial Trichoderma strains active against different phytopathogens

AIMS: Trichoderma harzianum strains T22 and T39 are two micro-organisms used as active agents in a variety of commercial biopesticides and biofertilizers and widely applied amongst field and greenhouse crops. The production, isolation, biological and chemical characterization of the main secondary metabolites produced by these strains are investigated. METHODS AND RESULTS: Of the three major compounds produced by strain T22, one is a new azaphilone that shows marked in vitro inhibition of Rhizoctonia solani, Pythium ultimum and Gaeumannomyces graminis var. tritici. In turn, filtrates from strain T39 were demonstrated to contain two compounds previously isolated from other T. harzianum strains and a new butenolide. The production of the isolated metabolites was also monitored by liquid chromatography/mass spectrometry during in vitro interaction with R. solani. CONCLUSIONS: This paper reports the isolation and characterization of the main secondary metabolites obtained from culture filtrates of two T. harzianum strains and their production during antagonistic interaction with the pathogen R. solani. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first work on secondary metabolites produced by the commercially applied strains T22 and T39. Our results provide a better understanding of the metabolism of these fungi, which are both widely used as biopesticides and/or biofertilizers in biocontrol.     

Strain-specific SCAR markers for the detection of Trichoderma harzianum AS12-2, a biological control agent against Rhizoctonia solani, the causal agent of rice sheath blight

In order to identify a specific marker for T. harzianum AS12-2, a strain capable of controlling rice sheath blight caused by Rhizoctonia solani, UP-PCR was performed using five universal primers (UP) both separately and in pairwise combinations. The application of two UP primers resulted in the amplification of unique fragments from the genomic DNA of T. harzianum AS12-2, clearly distinguishing it from other Trichoderma strains. The unique fragments had no significant sequence homology with any other known sequence available in databases. Based on the sequences of the unique fragments, 14 oligonucleotide primers were designed. Two primer sets amplified a fragment of expected size from the DNA of strain T. harzianum AS12-2 but not from any other examined strains belonging to T. harzianum, to other Trichoderma species assayed, or to other common fungi present in paddy fields of Mazandaran province, Iran. In conclusion, SCAR (sequence characterized amplified regions) markers were successfully identified and rapid, reliable tools were provided for the detection of an effective biocontrol Trichoderma strain, which can facilitate studies of its population dynamics and establishment after release into the natural environment.     

Chitinase gene expression during mycoparasitic interaction of Trichoderma harzianum with its host.

For monitoring chitinase expression during mycoparasitism of Trichoderma harzianum in situ, we constructed strains containing fusions of green fluorescent protein (GFP) to the 5'-regulatory sequences of the T. harzianum nag1 (N-acetyl-beta-d-glucosaminidase-encoding) and ech42 (42-kDa endochitinase-encoding) genes. Confronting these strains with Rhizoctonia solani led to induction of gene expression before (ech42) or after (nag1) physical contact. A 12-kDa cut-off membrane separating the two fungi abolished ech42 expression, indicating that macromolecules are involved in its precontact activation. No ech42 expression was triggered by culture filtrates of R. solani or by placing T. harzianum onto plates previously colonized by R. solani. Instead, high expression occurred upon incubation of T. harzianum with the supernatant of R. solani cell walls digested with culture filtrates or purified endochitinase 42 (CHIT42, encoded by ech42) from T. harzianum. The chitinase inhibitor allosamidin blocked ech42 expression and reduced inhibition of R. solani growth during confrontation. The results indicate that ech42 is expressed before contact of T. harzianum with R. solani and its induction is triggered by soluble chitooligosaccharides produced by constitutive activity of CHIT42 and/or other chitinolytic enzymes.     

Structure, production characteristics and fungal antagonism of tensin - a new antifungal cyclic lipopeptide from Pseudomonas fluorescens strain 96.578

AIM: To study the antagonistic activity by Pseudomonas fluorescens strain 96.578 on the plant pathogenic fungus Rhizoctonia solani. METHODS AND RESULTS: Strain 96.578 produced a new cyclic lipopeptide, tensin. High tensin production per cell was detected in liquid media with glucose, mannitol or glutamate as growth substrate while fructose, sucrose and asparagine supported low production. Tensin production was nearly constant in media with different initial C levels, while low initial N contents reduced production. When applied to sugar beet seeds, strain 96.578 produced tensin during seed germination. When challenged with strain 96.578 or purified tensin, Rhizoctonia solani reduced radial mycelium extension but increased branching and rosette formation. CONCLUSION: The antagonistic activity of strain 96.578 towards Rhizoctonia solani was caused by tensin. SIGNIFICANCE AND IMPACT OF THE STUDY: When coated onto sugar beet seeds, tensin production by strain 96.578 could be of significant importance for inhibition of mycelial growth and seed infection by Rhizoctonia solani.