Australasian Association of Nematologists

Regional News

NEWS FROM CANBERRA

Whilst in Canberra, many of you saw some of the Pratylenchus specimens in the nematode collection, and the sort of interactive key that can be built from such a collection. The support of GRDC is essential in building and maintaining the collection, and deserves the thanks of all the nematologists who will benefit in their research, as well as the grain growers who are the ultimate beneficiaries.

Latest additions to the collection include some several interesting specimens. We have been receiving quite a few stubby-root nematodes (Paratrichodorus), root-knot nematodes (Meloidogyne), a few root-lesion nematodes (Pratylenchus) and some more mermithids (Hexamermis). The stubby-root nematodes are quite widespread in sandier soils. Perhaps one to watch for the future.

The mermithids were taken from wingless grasshoppers and locusts and may have some role in regulation of the numbers of the insects, with high levels of parasitism in some areas. The root-lesion nematodes are a welcome addition to the collection of Pratylenchus which is continuing to add data on the species in Australia and their occurrence. The root-knot nematodes are also a welcome addition for a genus which is economically very important.

Many nematologists saw the sorts of interactive keys that can be built using data from an extensive collection. Conversely, the power of keys depends on the range of material available for study. Which brings me back to the collection, and the plea to keep sending us material. It is only through building the collection by including as much geographic, host crop and seasonal variation as possible, that the systematics and identification of nematodes can advance. Likewise it is the only way that we can recognise previously undiagnosed problems, and new threats identified. So when you are doing a study of any particular nematode problem, send us some specimens, so that we can add them to the collection where they will add to the data that will be the basis of future nematode systematics, identification, host and geographic records. As a specialist collection, we have the best possible curation equipment, expertise and a special purpose building for biological collections. We also have a separate unit creating specialist collection management software to ensure that the specimens are as accessible as possible. If you want to donate material, in whatever form (fixed or unfixed, mounted on slides or not, in pure or mixed culture), please contact me at the address below.

Mike Hodda

Leslie Research Centre, DPI, Toowoomba

It’s a hive of activity in Toowoomba. The field experiments are harvested and the glasshouse experiments are now ready to collect. We’ve been lucky enough to find a Pratylenchus neglectus site to complement the usual P. thornei experiments with wheat and chickpea.

Jason Sheedy impressed everyone at the 9^th Assembly of the Wheat Breeding Society of Australia with his talk - "Tolerance of Australian bread wheat (Triticum aestivum) varieties to the root-lesion nematode (Pratylenchus thornei) in a yellow spot (Pyrenophora tritici-repentis) year". He was awarded the prize for best oral presentation by a postgraduate student and was congratulated on his clear and logical presentation.

Nikki Seymour has disappeared for a short while on maternity leave. She had a girl on 5^th December, which was a little earlier than expected. She had intended to put the finishing touches on her PhD thesis before the baby arrived. Michelle O’Reilly is back helping with some of Nikki’s work.

Jason, Ros Reen and Rebecca Zwart are all currently at the Nematode Workshop in Adelaide. They were all looking forward to improving their nematode identification skills and re-visiting the Russian restaurant.

I’ve been awarded my PhD from Sydney University on "Resistance responses of grapevine to root-knot nematodes". (The abstract is included in this newsletter). I attended the Australasian Plant Pathology Conference and the Acquired Resistance workshop in Canberra in September (thankyou to RIRDC for the funds). At the workshop, Novartis, the suppliers of the systemic resistance activator, Bion, presented an optimistic future for the use of induced resistance for protecting plants. It was also a great opportunity to put a face to my fellow inducer of resistance against nematodes, Valerie Kempster. My new job here at the Leslie Research Centre is a GRDC project on "Cropping options for control of root-lesion nematodes". I’ve established high and low populations of P. neglectus and P. thornei at two field sites and will plant various summer crops to answer some questions that farmers have been asking for a while now about crop tolerance. Glasshouse experiments on resistance and ‘antagonisms’ to P. thornei in winter and summer crops are also well underway.

Kirsty Owen

NEWS FROM SOUTH AUSTRALIA

Andreas Hensel was successful in obtaining his Ph D from the University of Berlin. His research was conducted in Adelaide and resulted in his thesis entitled "Investigations of Rhabditis necromena (Rhabditidae, Nematoda), Ommatoiulus moreleti (Julidae, Diplopoda) and tripartite interactions with bacteria in South Australia". Also, Valerie Kempster plans to have submitted her PhD thesis "Induced resistance to clover cyst nematode" by the end of the year.

Kerrie Davies and Mike Hodda ran two well-received nematode identification workshops at the Waite Campus during December. The first workshop had about 15 people including three international participants, one each from PNG, Fiji and Vanuatu supported by ACIAR. The second course was designed specifically for quarantine plant pathologists having 9 participants. In addition to 24 people receiving quality nematode training, AAN recruited 7 new members.

In October, Astrid Schmidt commenced six months research on leaf gall Anguina spp. towards her Diploma thesis at the University of Bonn. Also, Prof. Bill Bowers from the Chemical Ecology Laboratory, University of Arizona is spending sabbatical at the Waite investigating the effects of plant defence chemicals on plant-parasitic nematode feeding and reproduction.

Wim Wouts, Landcare NZ, Auckland, spent two months collecting and extracting nematodes mostly in SA and WA, funded by GRDC, SARDI, Agwest and University of Adelaide. The prime aim being to examine species diversity in Pratylenchus and Radopholus in agricultural soils and nearby native vegetation in southern Australia. In addition to collecting a lot of prats, Wim managed to find what appear to be two or more previously unrecorded species of cyst nematodes and many other nematological treasures.

Ian Riley

Murdoch University, Western Australia – Mike Jones and colleagues

Major areas of research are in the molecular basis of host-parasite relations of endoparasitic nematodes (root-knot and cyst-nematodes). These include:

1. Confocal Laser Scanning Microscopy of GFP expressing transgenic Arabidopsis with enhancer trap tagged cell lineages on infection with root-knot nematodes

In this work, transgenic Arabidopsis thaliana plants, in which specific cell lineages within the root have been tagged with the gene for Green Fluorescent Protein (GFP), have been used to study changes in gene expression in host root cells on infection with Meloidogyne javanica.

A culture system has been designed to allow high resolution study of individual host-parasite relationships for up to 5 weeks from infection, using confocal scanning laser microscopy to look at GFP expression in giant cells and other surrounding tissues. Twelve lines of transgenic Arabidopsis out of 122 available have been studied in detail. Both up- and down- regulation of GFP expression has been observed.

For example, in one line in which only endodermal cells fluoresced green with GFP, infection with M. javanica resulted in switching off GFP expression around giant cells, but nowhere else in the root. One interpretation of these results is that there is no functional endodermis between giant cells and the surrounding gall tissues.

In another line in which the GFP expression was limited to phloem cells in control roots, there was very strong up-regulation of GFP within giant cells.

This approach promises to provide new information on changes in patterns of gene expression on infection of roots with nematodes. Because the transgenic plants were produced by enhancer trapping, it should also be possible to isolate the control sequences responsible for the changes of GFP expression on infection with nematodes. We are looking for support to continue this line of work.

John Blinco, Rob Potter and Mike Jones

2. Use of transgenic plants to study gene expression in roots infected with M. javanica

(i) Adh-gus

Transgenic Arabidopsis plants containing the gus reporter gene and adh (alcohol dehydrogenase) promoter have been studied after infection with M. javanica. The results indicate strong up-regulation of gus expression during egg laying. This gene is also up-regulated in non-infected roots by hypoxia, and this result supports the view that the partial pressure of oxygen in giant cells is reduced when there is maximum demand for nutrients by the associated nematode. These reduced oxygen levels leads to up-regulation of gus expression in adh – gus plants.

(ii) Auxin responsive and chalcone synthase promoters

The expression of the auxin responsive promoter (GH3) fused to the gusA reporter gene in white clover, Trifolium repens cv. Haifa has been studied during the initiation of root galls by root-knot nematodes (Meloidogyne javanica) to investigate whether nematode infection affects auxin distribution in developing galls. In a search for a plant signal that would mediate changes in auxin location we studied the induction of the flavonoid pathway because flavonoids can act as auxin transport regulators. Three chalcone synthase (CHS1, CHS2 and CHS3) promoter:gusA fusions were examined in transgenic plants and flavonoids were detected using fluorescence microscopy. Within 24 hr post inoculation, CHS:gusA expression occurred around the invading nematode. At 48 hr post inoculation, CHS:gusA expression and flavonoids were detected throughout the infection site, followed by high GH3:gusA expression in the gall 48-72 hr post inoculation. Initially (48-72 hr post inoculation) high GH3:gusA expression in giant cell precursors was followed by low expression in the enlarging giant cells (96-120 hr post inoculation), suggesting that auxin is needed as a trigger for giant cell initiation but not for later enlargement. It is suggested that nematodes control auxin distribution in the root and that flavonoids could be responsible for controlling auxin accumulation.

Pokkwan Hutangua, Ulrike Mathesius, Mike Jones and Barry Rolfe (Murdoch/RSBS/ANU)

3. Use of fluorescent dyes to study changes in transport properties around giant cells.

The phloem mobile tracer, carboxymethyl fluorescein has been used to study changes in tracer accumulation on infection of intact Arabidopsis seedlings with M. javanica and Heterodera schachtii.

In intact seedlings, this fluorescent dye, introduced into cotyledons and leaves, moves as two fluorescent files down the phloem of the root. Without nematode infection, the fluorescence remains confined to sieve elements and is off loaded at the root tip, as determined by epifluorescence microscopy and confocal scanning laser microscopy. With nematode infection, the fluorescent tracer accumulates rapidly in the feeding cells, and can also enter the nematodes (this is usually the case for M. javanica, and occurred only rarely for H. schachtii).

The question posed here is whether there are sufficient plasmodesmata to allow symplastic off loading of the tracer at feeding sites, or whether the permeability of the cell membranes is altered at the infection sites such that the tracer can cross the membranes directly. The microscopic distribution of plasmodesmata suggests the latter explanation.

Pokkwan Hutangura and Mike Jones

4. Molecular studies on gene expression in giant cells.

(i) DD-RT PCR

Following Differential Display RT-PCR, comparing giant cell enriched and control root tissues, 12 up- or down- regulated transcripts have been identified. Their expression has been studied by Southern blotting and quantitative RT-PCR. One transcript found is of nematode origin, and this is being studied further. Up-regulated transcripts of plant origin have high homology with a cucumber basic blue protein and a gibberellic acid biosynthesis enzyme.

Audrey Ah Fong, Rob Potter and Mike Jones

(ii) Gene expression in giant cell cytoplasm.

A method has been developed to extract the cytoplasm directly from individual giant cells and to carry out DD-RT PCR from these cells to identify genes up- and down- regulated. A method has also been developed to carry out quantitative RT-PCR to look at relative levels of transcripts in giant cell cytoplasm compared with non-infected tissue. Actin mRNA is used to normalize mRNA levels for comparative results. This approach promises to provide new information on giant cell function because giant cell contents themselves are analyzed.

Zhao-Hui Wang, Rob Potter and Mike Jones