Endophytic Fungi Isolated from Vochysia divergens ... 95variety of habitats and its hydrological cycle, with seasonal inundation
enduring for more than 200 days a year (Junk et al. 2006). Because of the
dynamism of the Pantanal, a limited number of plants are able to tolerate the
long periods of flooding. Among them is Vochysia divergens Pohl (Arieira et
al. 2006), a medicinal plant found in large communities in areas of the
Pantanal that are both periodically and permanently flooded (Arieira et al.
2006). This plant, used in folk medicine to treat respiratory and gastric
diseases (Pott et al. 1994) is a source of various substances, some with
antimicrobial potential (Honda et al. 1995) and others with pharmacological
importance (Hess et al. 1999).
This study aimed to assess the biodiversity of the endophytic community
in the medicinal plant V. divergens Pantanal/Brazil, as well as the capacity of
three isolates to control the phytopathogen Phyllosticta citricarpa, the
etiological agent of citrus black spot (CBS) disease, which depreciates the
commercial value of fruit and increases production cost considerably in citrus-
producing regions worldwide (Wulandari et al. 2009, Glienke et al. 2011).
MATERIAL AND METHODS
Sample Collection and Endophyte Isolation
Leaves of V. divergens Pohl (Cambará) were collected from Santa Emilia
Farm (Rio Negro Pantanal sub-region), Mato Grosso do Sul, Brazil (Figure 1).
Nine plants were collected between the years 2008 and 2010 (Table 1).
V. divergens leaves without any stains or bruises caused by insects,
mechanical damage, or pathogens were collected. All plant materials were
placed in sterile plastic bags and transported to laboratory under refrigeration
within 72 hours of collection. The leaves were submitted to a five-step
disinfection protocol to eliminate epiphytic microorganisms as described by
Petrini et al. (1993). Leaves were cut, aseptically, into 0.5-cm pieces and
seeded in petri dishes containing potato dextrose agar (PDA), pH 6.8, with
antibiotic tetracycline (100 μg/mL). Petri plates were incubated at 28°C with a
12 - h photoperiod for 30 days. Fungi growing from leaf segments were
subsequently transferred to fresh PDA plates.