TABLE CONTENTS

From Microscope to Molecule: Initial Work at Developing Diagnostic
Tools for Plant Parasitic Nematodes of Sugarcane
Berry, S.D. (1), M. Fargette (2), S. Morand (3), V.W. Spaull (1) & P. Cadet (2)
(1) South African Sugarcane Research Institute, Private Bag X02, Mount Edgecombe, 4300, South Africa; (2)
IRD-UMR 1062 - CBGP, Centre de Biologie et de Gestion des Populations, Campus International de
Baillarguet, CS 30 016, 34988, Montferrier sur Lez Cedex, France; (3) CNRS, Institut des Sciences de
l'Evolution, CC064, Universite Montpellier 2, 34095 Montpellier cedex 05, France.
Many crops are attacked simultaneously by a combination of a number of nematode species
and genera. Conventionally their identification has been by means of morphological
characteristics. However, recent advances in molecular biological techniques enable the
development of newer, potentially more powerful, means of identification. This work reports
on: (1) the use of the rDNA internal transcribed spacer 1 (ITS1) region to discriminate
between nematodes commonly found associated with sugarcane. The ITS1 amplification
fragments varied in size, from 400 bp (Meloidogyne javanica) to 1200 bp (Longidorus pisi,
Paratrichodorus minor, Xiphinema elongatum, Xiphinema zulu). Discrimination between
certain species was possible by size differentiation of the ITS1 amplification products.
However this type of discrimination also has potential pitfalls, such as when different genera
(Longidorus and Xiphinema) and different species (Scutellonema africanum, S. brachyurus
and S. truncatum) have similar size fragments. Discrimination with nematode universal
primers also has limitations when multiple taxa compete for reagents and primers in the same
PCR reaction; (2) the use of sequence data to study the genetic diversity of these nematodes.
It was found that variation between specimens belonging to the same species was low for
Helicotylenchus dihystera, P. lobatus, P. sacchari, S. brachyurus and X. elongatum.
Conversely, specimens of M. javanica and Pratylenchus zeae exhibited greater sequence
variation. Variation was usually greater between species than within species and consistent
specific clusters were identified; some variation between individuals belonging to the same
population was observed; (3) the use of sequence data to design species-specific reverse
primers that could be used in conventional- and quantitative PCR assays. Real-time PCR with
SYBR Green I dye enabled discrimination between M. javanica and P. zeae or X. elongatum
by melting peak analyses. Due to competition between amplicons, multiplex reactions were
not feasible. Real-time PCR enabled quantification of the numbers of these three species in
nematode samples extracted from the field, with a significant positive correlation between
real-time PCR and counts performed with microscopy. Altogether, these results provide a
good base for further development of molecular tools for diagnostic and ecological studies.
5 th International Congress of Nematology, 2008 174