Endophytic Fungi Isolated from Vochysia divergens ... 115of fungicide residues in fruits (Schreiber et al. 2012). Therefore, we explored
the endophytic community in the medicinal plant V. divergens to identify
strains that could be used to control CBS disease caused by the fungus
Phyllosticta citricarpa (Hokama, 2012).
Three strains were selected for an exploration of their capacity to control
Phyllosticta citricarpa based in previous results from paired cultures
(Hokama, 2012). Strains LGMF1133 and LGMF1121, both belonging to the
genus Nigrospora, and LBMF1119, belonging to family Xylariaceae,
completely inhibit growth of the phytopathogen Phyllosticta citricarpa (Table
2 and Figure 13). In addition, these strains were examined for their production
of volatile compounds and activity of less than 20% was observed (Table 2),
suggesting that the activity against P. citricarpa was due to the production of
non-volatile metabolites. For the extraction of secondary metabolites, the three
strains were cultivated in PD liquid medium. The liquid phases of cultures
were separated from mycelia by filtration, and both were subjected to
extraction by ethyl acetate. The solvent was evaporated, and metabolites were
weighed and diluted to a final concentration of 10 mg/mL. The extract from
strains LGMF1133, LGMF1121, and LBMF1119 showed strong inhibition of
mycelial growth (>80%), except for the extract from strain LGMF1121 (Table
3 and Figure 14).
We also examined inhibition of Phyllosticta citricarpa pycnidia formation
by these extracts. High inhibitory activity (65.3%) was observed in the extract
from the liquid phase of strain LGMF1121 (Table 3 and Figure 15); however,
all extracts inhibited pycnidia formation by more than 15%. Minimization of
pycnidia in plants is important in terms of disrupting the CBS disease cycle,
because the asexual stage of fungal growth aggravates disease in the plant and
surrounding areas (Perryman et al. 2014). Therefore, strategies to minimize the
formation of P. citricarpa pycnidia in citrus plants are of fundamental
importance and may offer a breakthrough in disease control research. To
confirm the biological control activities of these strains, studies of interactions
in plants as well as chemical identification of the secondary metabolites will
be needed. Strains belonging to the genus Nigrospora and family Xylariaceae
are promising as disease control agents, because these taxa are associated with
the production of large numbers of secondary metabolites with diverse
biological activities (Buchanan et al. 1995, Pongcharoen et al. 2008; Zhan et
al. 2009; Zhang et al. 2016).