Endophytic Fungi Isolated from Vochysia divergens ... 99and subjected to electrophoresis on automated DNA sequencer MegaBACE
(Amersham Biosciences).
DNA sequences were compared with sequences available in the National
Center for Biotechnology Information database (http://blast.ncbi.
nlm.nih.gov/Blast.cgi) using the BLAST tool (Altschul, 2005). Sequences of
type strains were obtained from MycoBank (http://www.mycobank.org) and
GenBank (http://www.ncbi.nlm.nih.gov/genbank). Alignments of DNA
sequences were performed using the BioEdit version 7.2.5 (Hall, 2013) and
ClustalW (Thompson et al. 1994) in MEGA version 6 (Tamura et al. 2013).
Bayesian inference of the phylogeny was performed in MrBayes version 3.2.1
(Ronquist et al. 2011), with permutations allowed until a frequency of division
≤0.01 was reached. The general time-reversible (GTR) substitution model was
used. FigTree version 1.4.2 (Rambaut, 2012) was used to edit the phylogenetic
trees that were constructed. Sequences obtained in this study were deposited in
GenBank with the accession numbers listed in Table 1.
Antifungal Activity against the Phytopathogen
Phyllosticta citricarpa
Based on previous results, isolates LGMF1133, LGMF1121, and
LGMF119 were selected to evaluate their antifungal activity against the
phytopathogen Phyllosticta citricarpa. The fungal isolates were examined for
the production of non-volatile or volatile metabolites in a randomized design
with five replicates, using methods outlined by Morris et al. (2010). Each
sample was cultured in a petri dish and evaluated after 14, 21 and 28 days. The
negative control received only Phyllosticta citricarpa, and the positive control
received fungicide glyphosate (1 mg/mL). To determine the inhibition
percentage (IP), the diameters of colonies were measured, and the IP was
calculated according to the following formula: IP = mycelial growth in the
control − mycelial growth in the treated sample/mycelial growth in the control
× 100. The endophytes that produced non-volatile active metabolites were
selected for the production of extracts.
Extract Production
Extracts from the endophytes were obtained by fermenting three mycelial
discs (8 mm) in 250 mL of potato dextrose (PD) medium (200 g of potato, 20