plant surface microbiology.pdf

comprises dipping samples in 96 % ethanol for 1 min, then in 65 % commercial
Chlorox (final concentration 3.25 % aqueous sodium hypochlorite)
for 10 min and finally in 96 % ethanol for 30 s. Malt extract agar is considered
the most suitable media for the growth and sporulation of endophytic
fungi (Bills and Polishook 1992; Bills 1996). Amendment of medium with
streptomycin sulfate is practised to prevent bacterial contamination. To prevent
the fast growing fungi overgrowing the plate, a growth inhibitor, Ross
Bengal is added to the agar.
Surface-sterilized plant tissues are plated in an appropriate medium
amended with antibiotics and Rose Bengal and incubated at room temperature
with periodic light and darkness. Incubated plates are checked after
1 week of incubation at regular intervals for fungal development. If the colony
is very small and there is a risk of engulfment by other colonies, it needs to be
subcultured. Subcultured isolates are generally maintained at room temperature
for many weeks to study morphological and other characteristics. Some
isolates may fail to produce reproductive structures even after several
months. Subculture of these isolates onto medium with autoclaved host tissue
strips can promote sporulation (Matsushima 1971). In general, sterile isolates
should be checked regularly for fruiting bodies over a period of 3–4 months
and the isolates failing to produce fruiting body are referred to as sterile
“morphotypes” depending on the characteristics of culture.
Other methods to promote sporulation of morphospecies should also be
tried. Guo et al. (1998) used twigs in conical flasks over a 3-month period to
promote sporulation of endophytes. Other methods can be designed, but
should try to mimic the situation in nature as closely as possible.
6 Molecular Characterization of Endophytes
17 Fungal Endophytes 285
Molecular approaches have been used to resolve the problems in fungal taxonomy
and in the identification of fungi (Rollo et al. 1995; Ma et al. 1997;
Zhang et al. 1997; Ranghoo et al. 1999). The use of molecular techniques for
the direct detection and identification of fungi within natural habitats has
been reviewed by Liew et al. (1998). Molecular techniques have mainly been
used in the detection and identification of mycorrhizal fungi and phytopathogenic
fungi directly from within plant tissues (Mills et al. 1992; Johanson and
Jeger 1993; Beck and Ligon 1995; Bonito et al. 1995; Abbas et al. 1996; Chambers
et al. 1998). Similarly, molecular techniques have been employed to detect
and identify fungi from the grass clothing of Iceman, from bamboo leaves and
glacial ice strata (Rollo et al. 1995; Ma et al. 1997; Zhang et al. 1997).
A most frequently encountered problem in endophyte study is the presence
of mycelia sterilia, making their identification difficult (Guo et al. 2000).Variable
proportions of mycelia sterilia have been reported ranging from 11 % of
isolates from palm (Trachycarpus fortunei) in China, 13 % of endophytes