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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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