Marine Fungal Diversity and Bioprospecting 2.5 Collection, Isolation, and Identification of Fungi 19
Part A | 2.5
duction of appendages that provide buoyancy in water,
entrapment, and adherence to substrates, as reported
in mangrove wood [2.63] and driftwood [2.64]. On
the contrary, temperatures below 10
ı C support the
growth of wood fungi, Digitatispora marina, but when
the temperature reaches 10
ı C and above, the fungi
stop fruiting on the wood [2.65]. Likewise, a few marine fungi have been recovered from Antarctic waters
such as Thraustochytrium antarticum, Leucosporidium
anatartica, and Spathulospora antartica, which leads
us to the conclusion that the low temperature of the seawater and the availability of suitable substrata in the
Antarctic region are responsible for the lower numbers
of marine fungi [2.66].
There is clear evidence that temperature is responsible for sporulation, deposition, and germination of
endophytic fungi. It has been demonstrated that endophytes enhance the thermotolerance of temperate
plants, augmenting their potential to colonize extreme
environments [2.67]. Further, temperature appears to
be a major variable affecting the fluctuation of endophyte frequency in plant tissue [2.68]. Recent studies
have provided evidence that the population density of
leaves is influenced by summer temperatures; however, the number species of endophytic fungi increases
with increasing air temperature [2.69]. It is interesting
to note that seven isolates of Corollospora maritime
have been investigated on the 18S gene [2.70], and it
was found that five isolates from temperate localities
grouped together; those from subtropical collections
formed a separate group, while the strain from Aldabra
separated from both groups.
2.5 Collection, Isolation, and Identification of Fungi
The water, sediments, substrates, and any solid material
from the habitat can be collected in sterile polythene
bags for fungal examination. The samples are transferred to the laboratory for further analysis. Isolation
of fungi is performed by using a pour plate method
with different fungal agar medium plates. The media
is weighed out and prepared according to the manufacturer’s specifications, with respect to the given
instructions and directions. The isolated fungal species
are identified up to species level by referring standard
mycological manuals and books [2.4, 71–73]. After
identification, the fungal species are maintained on
a Sabouraud chloromphenicol agar (SCA) medium at
27
ı C and they are subcultured at regular intervals using a sterile cork borer. Mycelial disks are cut at random
and used for further studies.
2.5.1 Techniques for Sample Collection
Direct examination method. Fungi are directly examined under a dissection microscope for the presence of
ascocarps, basidiocarps, or pycnidia. Such fruit bodies
are transferred with a needle to a microscopic slide,
torn apart in a drop of water to expose the spores, and
carefully squeezed under a cover glass. To allow the
development of fruit bodies, the litter samples can be
incubated in a sterile moist chamber/petridish/polybag
at room temperature. The incubated samples are examined under a microscope for fungal fruit bodies.
Another method is called the wood baiting technique, where terrestrial wooden logs measuring 6
3 2 cm are immersed in mangrove waters for certain
period and then collected to examine the fungal colonization. To determine the fungal biota in any habitat,
grab samples are satisfactory. At the chosen sites, samples are obtained scooping the water, mud, sand, or soil
to be examined into the sample container. Plastic vials,
soil cans, or other containers may be used to collect the
samples. The container should be clean and the sample
should be handled aseptically. It is not possible to obtain samples from the bottom of a body of water without
water; to obtain samples from the bottom of a body of
water deeper than elbow depth any of the available sampling devices can be used. The container is closed with
its proper cover, excess sludge, dirt, etc., is wiped or
rinsed off after which it is returned to the laboratory.
The container should not be completely airtight.
2.5.2 Media Preparation for Isolation
of Fungi
Various culture media are used for the isolation of fungi
from the samples. The most commonly used media
are Sabouraud dextrose agar or potato dextrose agar,
along with chloramphenicol to avoid bacterial contamination. The chemical should be added in proper
proportion along with agar (for solidification), autoclaved at 121
ı C for 15 min, and plated in a sterile Petri
dish that is free from contamination. Media used in plating the samples may be prepared the day before the
samples are collected. The most convenient method of
storage is in 10 ml lots in culture tubes in culture tube
Part A | 2.5
duction of appendages that provide buoyancy in water,
entrapment, and adherence to substrates, as reported
in mangrove wood [2.63] and driftwood [2.64]. On
the contrary, temperatures below 10
ı C support the
growth of wood fungi, Digitatispora marina, but when
the temperature reaches 10
ı C and above, the fungi
stop fruiting on the wood [2.65]. Likewise, a few marine fungi have been recovered from Antarctic waters
such as Thraustochytrium antarticum, Leucosporidium
anatartica, and Spathulospora antartica, which leads
us to the conclusion that the low temperature of the seawater and the availability of suitable substrata in the
Antarctic region are responsible for the lower numbers
of marine fungi [2.66].
There is clear evidence that temperature is responsible for sporulation, deposition, and germination of
endophytic fungi. It has been demonstrated that endophytes enhance the thermotolerance of temperate
plants, augmenting their potential to colonize extreme
environments [2.67]. Further, temperature appears to
be a major variable affecting the fluctuation of endophyte frequency in plant tissue [2.68]. Recent studies
have provided evidence that the population density of
leaves is influenced by summer temperatures; however, the number species of endophytic fungi increases
with increasing air temperature [2.69]. It is interesting
to note that seven isolates of Corollospora maritime
have been investigated on the 18S gene [2.70], and it
was found that five isolates from temperate localities
grouped together; those from subtropical collections
formed a separate group, while the strain from Aldabra
separated from both groups.
2.5 Collection, Isolation, and Identification of Fungi
The water, sediments, substrates, and any solid material
from the habitat can be collected in sterile polythene
bags for fungal examination. The samples are transferred to the laboratory for further analysis. Isolation
of fungi is performed by using a pour plate method
with different fungal agar medium plates. The media
is weighed out and prepared according to the manufacturer’s specifications, with respect to the given
instructions and directions. The isolated fungal species
are identified up to species level by referring standard
mycological manuals and books [2.4, 71–73]. After
identification, the fungal species are maintained on
a Sabouraud chloromphenicol agar (SCA) medium at
27
ı C and they are subcultured at regular intervals using a sterile cork borer. Mycelial disks are cut at random
and used for further studies.
2.5.1 Techniques for Sample Collection
Direct examination method. Fungi are directly examined under a dissection microscope for the presence of
ascocarps, basidiocarps, or pycnidia. Such fruit bodies
are transferred with a needle to a microscopic slide,
torn apart in a drop of water to expose the spores, and
carefully squeezed under a cover glass. To allow the
development of fruit bodies, the litter samples can be
incubated in a sterile moist chamber/petridish/polybag
at room temperature. The incubated samples are examined under a microscope for fungal fruit bodies.
Another method is called the wood baiting technique, where terrestrial wooden logs measuring 6
3 2 cm are immersed in mangrove waters for certain
period and then collected to examine the fungal colonization. To determine the fungal biota in any habitat,
grab samples are satisfactory. At the chosen sites, samples are obtained scooping the water, mud, sand, or soil
to be examined into the sample container. Plastic vials,
soil cans, or other containers may be used to collect the
samples. The container should be clean and the sample
should be handled aseptically. It is not possible to obtain samples from the bottom of a body of water without
water; to obtain samples from the bottom of a body of
water deeper than elbow depth any of the available sampling devices can be used. The container is closed with
its proper cover, excess sludge, dirt, etc., is wiped or
rinsed off after which it is returned to the laboratory.
The container should not be completely airtight.
2.5.2 Media Preparation for Isolation
of Fungi
Various culture media are used for the isolation of fungi
from the samples. The most commonly used media
are Sabouraud dextrose agar or potato dextrose agar,
along with chloramphenicol to avoid bacterial contamination. The chemical should be added in proper
proportion along with agar (for solidification), autoclaved at 121
ı C for 15 min, and plated in a sterile Petri
dish that is free from contamination. Media used in plating the samples may be prepared the day before the
samples are collected. The most convenient method of
storage is in 10 ml lots in culture tubes in culture tube