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R.
Buruchara, G. Mahuku, S. Mayanja, J. Mukalazi &
A. Levesque
Pythium root rot
caused by a number of Pythium spp is the most destructive
soilborne disease of beans (Phaseolus vulgaris L) in East and
Central Africa and can result in complete crop failures in some seasons.
Identification of species belonging to the Pythium genus is both
slow and difficult using morphological or pathogenic characteristics.
Rapid and accurate detection assays are required because correct species
identification is important as a basis for identification and
development of host resistance and other management strategies. The
objective of this study is to adapt a relatively fast and accurate
diagnostic method known as “Reverse Dot Blot Hybridization” (RDBH) for
species detection and identification. RDBH is based on species-specific
oligonucleotides designed and blotted onto a membrane array which
currently only includes species occurring in temperate regions. The
first step for the application of the RDBH in tropical regions of Africa
requires validation, through extensive characterization, so that
Pythium species prevalent in Africa are represented on the membrane
array. DNA from over 400 Pythium isolates, obtained from soil and
diseased bean plant samples in Uganda, Kenya and Rwanda, were amplified
with Pythium group–specific primers that target the internally
transcribed spacer (ITS) region of the ribosomal genes. Direct
sequencing of the PCR fragments was done for over 150 isolates using
primers that annealed inside of the first fragment. The sequences were
analyzed by comparing them to sequences of known Pythium species
from a Pythium database managed by Dr A. Levesque. Sequence
analysis of the isolates identified 12 different species with P.
ultimum Var. ultimum being the most prevalent. Other species
identified included P. irregulare, P.
dissotocum, P. indigoferae, P. oligandrum, P. salpingophorum, P.
spinosum, P. torulosum P. vexans, P. nodusum, P. deliense/aphanidatum,
and P. pachycuale. Interestingly,
P. oligandrum, a known biocontrol agent, was among the Pythium
species isolated. Four new putative species found in association with
beans were among the neo (type) strains, implying they could be novel.
A number of isolates were identified as Mortierella spp, a common
saprophyte that can be isolated on Pythium-specific media and
which is culturally and morphologically similar to Pythium spp.
Implications for resistance breeding imposed by species diversity and
distribution are discussed.
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