Isolamento e identificação de fungos endofíticos da pupunha (Bactris gasípaes Kunth) e caracterização por marcadores moleculares.

Abstract

The tropical palm pupunha (Bactris gasipaes Kunth) was domesticated by Amerindians and is cultivated in several countries. In Brazil, the interest is in the agribusiness for heart-of-palm production, although the fruits nutritional value is also interesting. Endophytic microorganisms were isolated from fruit fragments of 18 plants using BDA and PDA (pupunha pulp-dextrose-agar) in the absence of light at 18ºC. In fragments of mesocarp, endocarp and endosperm the presence of endophytic bacteria, yeast and filamentous fungi was observed. The fungal isolates were identified as: Thielaviopsis paradoxa 36.4%, Ceratocystis paradoxa 17.1%, Phomopsis sp. 10.4%, Fusarium sp. 9.7% and Penicillium sp. 2,0%. The infection rate was highest in the mesocarp with 47.7%. More than one isolate was colonizing pupunha fruits in the same plant and in all plants investigated there were endophytes. C. paradoxa showed specificity for mesocarp while T. paradoxa for endocarp and endosperm. T. paradoxa was the most frequent isolate, and was chosen for molecular analysis. The DNA from 22 endophytes was extracted with CTAB and liquid nitrogen. A fragment with 1600 bp was amplified from rDNA by PCR and the purified amplification product was sequenced. The 22 samples had been submitted to the GenBank and all had presented greater similarity with access C. paradoxa (AF043607). The alignment among the 22 endophytes showed little variability among them. The samples were submitted to the TCS program, where gaps were considered as a fifth character, resulting in the identification of nine haplotypes. Among these only two were found in more than one individual. The greatest genetic variability was among the endosperm isolates. The PAUP 4.0 Program, utilizing the distance p method not corrected, was used to obtain a distance matrix and a parsimonious tree by neighbor-joining, with a length of 139 steps. The estimate of the transition/transvertion rate was 1.2, and the base proportions were 27.9% for adenine, 21.5% for cytosine, 21.5% for guanine and 29.1% for thymine. The proportion of invariable sites was 0.476 and the parameter gamma was 0.57; CI=0.897 and RI=0.976. There was little genetic divergence among isolates. The taxons topology was estimated by neighbor-joining, using a 1000 replication bootstrap. The tree was rooted with two outgroups, both of Chalara elegans (AF275509 e AF275482). Three major groups were formed, with one group containing C. paradoxa (AF043607) with all T. paradoxa isolates. No specific groups were formed according to the fragment sampled although some haplotypes from the endocarp and endosperm were grouped closely together. The molecular analysis confirmed the identification of T. paradoxa made from reproductive structures.