RESUMO
Trichoderma species are known for their ability to produce lytic enzymes, such as exoglucanases, endoglucanases, chitinases, and proteases, which play important roles in cell wall degradation of phytopathogens. ß-glucanases play crucial roles in the morphogenetic-morphological process during the development and differentiation processes in Trichoderma species, which have ß-glucans as the primary components of their cell walls. Despite the importance of glucanases in the mycoparasitism of Trichoderma spp., only a few functional analysis studies have been conducted on glucanases. In the present study, we used a functional genomics approach to investigate the functional role of the gluc31 gene, which encodes an endo-ß-1,3-glucanase belonging to the GH16 family in Trichoderma harzianum ALL42. We demonstrated that the absence of the gluc31 gene did not affect the in vivo mycoparasitism ability of mutant T. harzianum ALL42; however, gluc31 evidently influenced cell wall organization. Polymer measurements and fluorescence microscopy analyses indicated that the lack of the gluc31 gene induced a compensatory response by increasing the production of chitin and glucan polymers on the cell walls of the mutant hyphae. The mutant strain became more resistant to the fungicide benomyl compared to the parental strain. Furthermore, qRT-PCR analysis showed that the absence of gluc31 in T. harzianum resulted in the differential expression of other glycosyl hydrolases belonging to the GH16 family, because of functional redundancy among the glucanases.
Assuntos
Antibiose/genética , Parede Celular/enzimologia , Parede Celular/metabolismo , Endo-1,3(4)-beta-Glucanase/metabolismo , Trichoderma/enzimologia , Trichoderma/metabolismo , Ascomicetos/metabolismo , Benomilo/farmacologia , Parede Celular/química , Parede Celular/efeitos dos fármacos , Quitina/metabolismo , Endo-1,3(4)-beta-Glucanase/genética , Fusarium/metabolismo , Regulação Fúngica da Expressão Gênica/genética , Genômica , Microscopia de Fluorescência , Filogenia , Rhizoctonia/metabolismo , Trichoderma/efeitos dos fármacos , Trichoderma/patogenicidade , beta-Glucanas/metabolismoRESUMO
The present work was carried out to study the potential of plant rhizosphere associated bacteria for the biocontrol of potato black scurf disease caused by Rhizoctonia solani Khun AG-3. A total of twenty-eight bacteria isolated from diseased and healthy potato plants grown in the soil of Naran and Faisalabad, Pakistan were evaluated for their antagonistic potential. Nine bacterial strains were found to be antagonistic in vitro, reduced the fungal growth and caused the lysis of sclerotia of R. solani in dual culture assay as well as in extracellular metabolite efficacy test. The selected antagonistic strains were further tested for the production and efficacy of volatile and diffusible antibiotics, lytic enzymes and siderophores against R. solani. Selected antagonistic bacteria were also characterized for growth promoting attributes i.e., phosphate solubilization, nitrogen fixation and indole acetic acid production. Biocontrol efficacy and percent yield increase by these antagonists was estimated in greenhouse experiment. Statistical analysis showed that two Pseudomonas spp. StT2 and StS3 were the most effective with 65.1 and 73.9 percent biocontrol efficacy, as well as 87.3 and 98.3 percent yield increase, respectively. Potential antagonistic bacterial strain StS3 showed maximum homology to Pseudomonas sp. as determined by 16S rRNA gene sequencing. These results suggest that bacterial isolates StS3 and StT2 have excellent potential to be used as effective biocontrol agents promoting plant growth with reduced disease incidence.
Assuntos
Antibacterianos , Sequência de Bases , Técnicas In Vitro , Controle Biológico de Vetores , Plantas Comestíveis , Pseudomonas/genética , Pseudomonas/metabolismo , Rhizobium/genética , Rhizobium/metabolismo , Rhizoctonia/genética , Rhizoctonia/metabolismo , Metabolismo , Métodos , Métodos , VirulênciaRESUMO
The genus Trichoderma is a potential biocontrol agent against several phytopathogenic fungi. One parameter for its successful use is an efficient coiling process followed by a substantial production of hydrolytic enzymes. The interaction between fifteen isolates of Trichoderma harzianum and the soil-borne plant pathogen, Rhizoctonia solani, was studied by light microscopy and transmission electron microscopy (TEM). Macroscopic observations of fungal growth in dual cultures revealed that growth inhibition of the pathogen occurred soon after contact with the antagonist. All T. harzianum isolates tested exhibited coiling around the hyphae of R. solani. The strains ALL23, ALL40, ALL41, ALL43 and ALL49 did not differ in coiling frequency and gave equal coiling performances. No correlation between coiling frequency and the production of cell wall-degrading chitinases, N-acetyl-beta-D-glucosaminidase and beta-1,3-glucanases, was found.
Assuntos
Biotecnologia/métodos , Rhizoctonia/metabolismo , Trichoderma/metabolismo , Parede Celular/metabolismo , Quitinases/química , Quitinases/metabolismo , Enzimas/química , Hidrólise , Microscopia Eletrônica de Transmissão , Modelos Biológicos , Controle Biológico de Vetores , Doenças das Plantas , Extratos Vegetais/metabolismoRESUMO
Trichoderma harzianum 650 (Th650) and Paenebacillus lentimorbus 629 (Pl629) selected earlier for their ability to control Rhizoctonia solani, Fusarium solani and F. oxysporum in vitro, were applied alone or combined with solarization (summer assay) and/or with methyl bromide (MeBr) (summer and winter assays) to a soil with a high inoculum level, for the control of tomato root rot caused by the complex F. oxysporum f. sp. lycopersici - Pyrenochaeta lycopersici - Rhizoctonia solani. Evaluations were also performed independently for root damage caused by P. lycopersici, and also for R. solani in the summer assay. MeBr decreased tomato root damage caused by the complex from 88.7 percent to 21.2 percent and from 78.4 percent to 35.7 percent in the summer and in the winter assay, respectively. None of the bio-controllers could replace MeBr in the winter assay, but Th650 and Pl629 reduced root damage caused by this complex in the summer assay. Treatments with bio-controllers were improved by their combination with solarization in this season. Independent evaluations showed that the positive control of Th650 towards R. solani and the lack of effect on P. lycopersici correlates well with the endochitinase pattern expressed by Th650 in response to these phytopathogens. Root damage caused by R. solani can be controlled at a similar level as it does MeBr in summer assays, thus representing an alternative to the use of this chemical fungicide for the control of this phytopathogen.
Assuntos
Antifúngicos/metabolismo , Controle Biológico de Vetores/métodos , Fusarium/metabolismo , Solanum lycopersicum/microbiologia , Rhizoctonia/metabolismo , Bacillus/metabolismo , Estufas para Plantas , Hidrocarbonetos Bromados , Fungos Mitospóricos/metabolismo , Modelos Biológicos , Raízes de Plantas/microbiologia , Estações do Ano , Microbiologia do Solo , Luz Solar , Trichoderma/metabolismoRESUMO
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.
Assuntos
Técnicas de Cultura de Células/métodos , Técnicas de Cocultura/métodos , Pironas/isolamento & purificação , Pironas/metabolismo , Rhizoctonia/metabolismo , Trichoderma/metabolismoRESUMO
AIMS: A bacterial strain producing antifungal compounds active against the plant pathogenic fungi Fusarium, Rhizoctonia and Sclerotinia has been characterized and shown to control Rhizoctonia root rot of soya bean. METHODS AND RESULTS: The metabolites excreted by Bacillus BNM 122 remained active after autoclaving, were resistant over a wide pH range and to hydrolytic enzymes. By (1)H-NMR and thin-layer chromatography analyses surfactin and iturin-like compounds were partially identified. Moreover, soya bean seeds bacterization with BNM 122 in a compost-based formulation was as effective controlling Rhizoctonia solani as pentachloronitrobenzene. According to its 16S rDNA sequence BNM 122 was closely related to Bacillus amyloliquefaciens and Bacillus subtilis. PCR analysis of the 16S-23S rRNA intergenic spacer region and repetitive sequence-based PCR (rep-PCR) genomic fingerprinting revealed a close genetic relationship to B. amyloliquefaciens. However, by physiological characterization using API tests, this strain resembled more B. subtilis. CONCLUSIONS: This is the first report describing the co-production of surfactin and iturin-like compounds by a putative strain of B. amyloliquefaciens. The synergistic effect of both lipopetides is a remarkable trait for a candidate biocontrol agent. SIGNIFICANCE AND IMPACT OF THE STUDY: This kind of research has relevance in order to minimize the use of synthetic fungicides and surfactants, contributing to the preservation of the environment.
Assuntos
Bacillus/genética , Inibidores Enzimáticos/metabolismo , Fungicidas Industriais/metabolismo , Peptídeos Cíclicos/metabolismo , Ascomicetos/metabolismo , Bacillus/metabolismo , Sequência de Bases/genética , Cromatografia em Camada Fina/métodos , DNA Bacteriano/genética , DNA Ribossômico/genética , Fusarium/metabolismo , Germinação/fisiologia , Lipopeptídeos , Espectroscopia de Ressonância Magnética/métodos , Dados de Sequência Molecular , Controle Biológico de Vetores/métodos , Rhizoctonia/metabolismo , Sementes/microbiologia , Glycine max/microbiologia , Esporos Fúngicos/metabolismoRESUMO
Trichoderma asperellum produces at least two extracellular beta-1,3-glucanases upon induction with cell walls from Rhizoctonia solani. A beta-1,3-glucanase was purified by gel filtration and ion exchange chromatography. A typical procedure provided 35.7-fold purification with 9.5% yield. The molecular mass of the purified exo-beta-1,3-glucanases was 83.1 kDa as estimated using a 12% (w/v) SDS-electrophoresis slab gel. The enzyme was only active toward glucans containing beta-1,3-linkages and hydrolyzed laminarin in an exo-like fashion to form glucose. The K(m) and V(max) values for exo-beta-1,3-glucanase, using laminarin as substrate, were 0.087 mg ml(-1) and 0.246 U min(-1), respectively. The pH optimum for the enzyme was pH 5.1 and maximum activity was obtained at 55 degrees C. Hg(2+) strongly inhibited the purified enzyme.