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sand. After three months, plant shoots were
removed, and soil and roots were collected, airdried
and stored in a refrigerator (4-10ºC) until
use for spore extractions and identification.
Isolation of VAM Spores
The researchers use sieving pan
method for the isolation process (Remy et
al. 1994). The pans were arranged with the
least degree of filtration on top and the pan of
highest degree of filtration at the bottom. Then,
a 600-gram soil sample was mixed with a 5-liter
pail of water and was left for 10 minutes until
the soil settled at the bottom of the pail. After
the soil settled at the bottom of the pail, fungal
spores are already floating on the water.
To isolate these spores, the mixture of
soil and water were poured on the arranged
sieving pans while letting the remaining
soil particles remain in the pail. The spores
remained on the sieving pans afterwhich
they were transferred by gradually washing
the sieving pans with tap water using a wash
bottle while simultaneously letting the water
flow into the 100ml glass bottle with a funnel.
The researchers repeated the same procedure
with the other sieving pans using different
glass bottles. The water drains slowly through
the lower sieve; hence, the 38 um sieve was
continuously checked by separating the two
sieves and visually looking at the height of the
water. If the water does overflow the lower sieve,
spores are lost. The glass bottles were labeled
according to the pans used and the plantations
where the soil sample came from. The previous
procedures were repeated for the other soil
samples. The top sieve will concentrate most of
the soil particles: so only the fine soil particles
along with the AMF spores will collect on the
bottom sieve.
VAM Spores Identification
Spores were extracted and mounted
on PVLG and Melzer’s reagent. Species
identification was done according to Schenck
and Pérez (2001) and by comparison with
J.I Yago, et.al
reference culture information available in the
web page (http://invam.caf.wvu.edu/fungi/
taxonomy/speciesID.htm) of the International
Culture Collection of (Vesicular) Arbuscular
Mycorrhizal Fungi (INVAM).
VAM Diversity
Analysis of the following diversity
parameters were computed based from the
methods used by Miller et al. (1987).
Species Richness. After identification,
the total number of species recovered (T) and
AM fungal species richness (R= average of
species number per sample) were determined.
Species richness was computed by the
following formula:
where:
S = species richness
n = total number of species present in
sample population
k = number of “unique” species (of
which only one organism was
found in sample population)
Diversity Index. Diversity index was
computed by the following formula:
where:
D = diversity index
N = Total number of organisms of all
species found
n = number of individuals of a
particular species
A high D value suggests a stable and
ancient site, while a low D value could suggest a
polluted site, recent colonization or agricultural
management.
Repetition Index.
Repetition index
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