d(GC) - Association of Biotechnology and Pharmacy

Current Trends in Biotechnology and Pharmacy
Vol. 6 (2) 173-182 April 2012, ISSN 0973-8916 (Print), 2230-7303 (Online)
understand the rice-Magnaporthe grisea
pathosystem, knowledge of the pathogen’s
genetic diversity and the mechanisms that lead
to the development of new virulent genotypes are
very much required. Population studies of the rice
blast pathogen have been studied across world
for their phenotypic and genotypic variations (4,
5, 6, 7, 8, 9, 10, 11, 12, 13). The genetic diversity
of Magnaporthe grisea and its correlation with
pathotypes have been studied by various
methods, including use of repetitive DNA
sequences i.e., Magnaporthe grisea repeats
(MGR), which have core repetitive sequence of
1,860 bp, with an estimated average of 46 copies
per genome (14, 15). Retrotransposon repetitive
elements like grh (16) and MAGGY (17) have
also been exploited for diversity analysis. But,
assessment of polymorphism at these repetitive
element regions has relied on restricted fragment
length polymorphism (RFLP) technique, which
is often costly and labor intensive. Kachroo et
al., (18) described a method that revealed the
polymorphism of Pot2, (a unique repetitive
element found in the M. grisea genome) based
on PCR with primers flanking the core repetitive
sequence but, the Pot2 marker also targets the
repetitive element which is shared by the isolates
which infects rice and other hosts. At present,
simple sequence repeats (SSR) which are
relatively abundant in genomes of eukaryotes
have become a method of choice for diversity
studies (19, 20, 21). To develop, microsatellite
markers, (AG)n microsatellite-enriched genomic
DNA library varying from fivefold to 60 repeat
motifs depending on the isolates was
constructed for Magnaporthe grisea and a set of
24 SSR markers were designed out of which
three markers were validated (22). In India, a
huge diversity of blast pathogen existing since
rice is grown in different agro climatic regions
(23), but the analysis of diversity was carried out
using random and repeat element based markers
(24). Therefore development of new and
alternative tools is required to analyze the
diversity and to monitor the dynamics of fungal
Madhan Mohan et al
174
populations which will help in designing strategies
for disease control in a more advanced and
precise manner. Admittedly a PCR based system
is much simple, inexpensive, convenient and
more accurate than a fingerprint based system.
Hence, the present study was aimed to determine
the population diversity and the relationship of
the blast pathogen races in the blast hotspot
regions of India using Magnaporthe grisea
genome specific microsatellite markers.
Materials and Methods
Isolation and maintenance of M. grisea
cultures: Thirty four isolates of M. grisea were
collected from blast infected rice leaves or
panicles from different blast hot spot regions of
India (Table 1). The leaf bits having a single, non
coalescing spot were selected for fungal isolation.
The leaf bits were surface sterilized with 0.1 %
mercuric chloride followed by 4 to 5 times
repeated washes with sterile distilled water. The
infected leaf bits were cultured on leaf extract
agar (Leaf decoction 100 g -l , sucrose 20 g -l and
agar 20 g -l ) and incubated at 27 o C for 5 days in
dark and 3 days in light for mycelial growth. From
the mycelia, spore suspension was prepared in
sterile distilled water and plated on to 1 % water
agar and incubated for 12 h at 27 o C for conidia
germination. Single germinating conidium was
observed under microscope and subsequently
transferred to test tubes containing sterile rice
bran agar for culture establishment. The
established cultures were kept at 4 o C for storage
and further investigations.
DNA extraction: Seven day old pre-inoculated
M. grisea agar block was transferred into sterile
2% Yeast Extract Glucose (YEG) broth and
incubated at 28 o C for 7 days for mass production
of the fungal mycelium. The obtained mycelium
(~250 mg) was lyophilized using liquid nitrogen
and used for DNA extraction. The DNA was
extracted by following CTAB method (25). A
working DNA solution was made by diluting DNA
stock to approximately 10 – 20 ngµl -1 .