TABLE CONTENTS

Real Time SYBR GREEN1 Based Detection Technique for Globodera
rostochiensis of Potato Cyst Nematodes
Quader, M. & L. Nambiar
Nematology, Bio-Protection, Department of Primary Industries, Knoxfield Centre, 621 Burwood highway,
Knoxfield 3180, Victoria, Australia
A new DNA primer (ITS1H) was designed based on broader sequence information to
distinguish Globodera rostochiensis (Gr) from Globodera pallida (Gp). This primer (ITS1H)
along with universal primer (ITS5) will amplify a short piece of DNA (174 bp) for Gr in both
conventional and real time PCR. This amplified DNA product effectively differentiated Gr
from Gp in both types of PCRs. The melting peak for Gr with ITS1H primer was 85 O C
against 85.7 O C for Gp with primer ITSp4. The primer ITS1H has been produced consistent
melting peaks compared to inconsistent peaks with published primer (ITSr3) during
validation of new primer across a number of isolates of Gr from different locations of
Victoria, Australia. The new primer gave significant positive relationships between threshold
cycles and DNA concentrations in quantitative real time PCR. The method described in this
article could be an efficient tool in identification and quantification of DNA from Gr.
Molecular Confirmation of a Xiphinema americanum Complex (Virusvectoring
Nematode) from New Zealand
Shah, F.A. (1), N. Bell (2) & S. Bulman (1)
(1) Crop & Food Research, Private Bag 4707, Christchurch New Zealand; (2) AgResearch Limited, Ruakura
Research Centre, Private Bag 3123, Hamilton, New Zealand.
Species in the Xiphinema americanum complex have been subject to much study, as they
transmit at least four plant pathogenic nepoviruses. The identity, taxonomy and worldwide
distribution of nematodes in this species complex remain much debated due to their close
morphological similarity. Virus vectoring nematodes pose biosecurity and a market access
threats for New Zealand. At least three X. americanum species have been described
previously from New Zealand, but no molecular techniques have been used in these
identifications. We describe the first use of DNA barcoding to identify X. brevicollum, a
nematode from the X. americanum complex, in New Zealand. We used the prepGEM enzyme
system for DNA extraction from nematode isolated from soil and DNA fragments were
amplified with the LSUD3B/LSUD2A primer pairs. DNA sequences obtained from the New
Zealand nematode were used for comparison against GenBank sequences. BLASTN
comparison indicated that the LSU fragment was most similar to sequences from X.
brevicollum. Phylogenetic trees inferred from the collected nematode sequences show this
close relationship as well as the clustering of the X. brevicollum with X. diffusum and X.
taylori sequences. The prepGEM enzyme system for DNA extraction and the barcoding
techniques were simple to apply and gave excellent species identification of longidorid
nematodes in our sample.
5 th International Congress of Nematology, 2008 332