Diversidade e potencial biotecnológico de fungos endofíticos de cacau (Theobroma cacao L.) / Diversity and biotechnological potential of the cacao (Theobroma cacao L.) endophytic fungi




Endophytes are all microorganisms, culturable or not, that inhabit the interior of plant tissues, causing no harm to the host, and that do not develop external structures, excluding in this way, nodulating bacteria and mycorrhizal fungi. Theobroma cacao L. is important to food and cosmetic industry due the fact that it is used for production of cacao butter. Considering the importance of cacao and associated endophytic fungal community studies, the objectives of this work were to: i) study the diversity on endophytic community of cacao; ii) evaluate the potential of this community to biological control of Crinipellis perniciosa, causal agent of witches’ broom disease and other phytopathogens and iii) study the interaction endophyte-pathogen and endophyte-host. The work began with isolation of the cacao endophytic fungi that was characterizated based on morphological and molecelar characters. The diversity was evaluated by rDNA (ITS1, ITS2 e 5.8 subunit regions) analyses (ARDRA and sequencing). The ARDRA analysis generated 64 haplotypes, which included at least 16 species. Following, antagonistic endophytic fungi from cacao were selected in vitro against C. perniciosa. It was observed that in 145 evaluated isolates, 38.6% was classified as antagonic, while 13.1% were classified as parasitic. After the selection, isolates were used for secondary metabolites extraction and further characterization. Also, detection of δ-(L-α-aminoadipil)-L-cisteinil-D-valin (ACV) gene, which is the precursor of β-lactans, antibiotics like penicilins and cephalosporins, was carried out. The amplification of this gene generated a fragment of 600pb in 9.1% of the isolates evaluated. Some isolates were further evaluated against phytophathogenic fungi (Colletotrichum falcatum, C. sublineorum, Erythricium salmonicolor, Rhizoctonia solani, Ceratocystis paradoxa, Phytophthora sp., P. parasitica, P. palmivora and Fusarium moniliforme). Only C. sublineorum of sorghum and Ceratocystis paradoxa of sugarcane were in vitro inhibited by the endophytes 42.3 and 2, respectively. The investigation of the penetration and colonization ability of endophytic fungus 42.3 on susceptible cacao comum was carried out, using RAPD and SEM techniques. The penetration of the fungus was initiated between 3 and 6 hours after inoculation. The interaction between cacao endophyte 42.3 with C. perniciosa 281 (pathogenic), was evaluated by SDSPAGE, aiming the detection of expressed proteins by pathogen and endophyte. The SDS-PAGE allowed the visualization of bands expressed in the interaction, when the microorganisms were grown together. In the presence of the endophyte growth substrate, C. perniciosa do not express visible proteins or, at least, distinguishable in SDS-PAGE, but the co-cultive of these fungi generated non observed bands in previously analysis.


cacau endophytic fungal cacao fungo endofítico biodiversidade biodiversity

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