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Development and use of in vitro culture of plant parasitic nematodes to study host feeding cell formation and function

Hutangura, Pokkwan (1999) Development and use of in vitro culture of plant parasitic nematodes to study host feeding cell formation and function. PhD thesis, Murdoch University.

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Root-knot nematodes (Meloidogyne spp.) are the most important obligate root parasites of horticultural and broad acre crops worldwide, particularly in tropical and subtropical regions. They invade roots of most economically important crops, causing yield losses and reduced quality of produce. The interaction between the plant host and the nematode is specific and includes alterations in host plant anatomy and physiology, which need to be investigated in detail. The information from such studies will be useful to develop new methods of nematode control.

In vitro culture of nematodes on host roots is a useful system to study various aspects of nematode-host interactions without interference from other pathogens. An optimised culture medium has been established in this work for in vitro infection of tomato with M. javanica. This consists of a quarterstrength Murashige and Skoog (MS) medium, 0.5% sucrose, pH 6.4 and 0.6% phytagel. On infection of cultured roots, M. javanica can complete its life cycle in 5-6 weeks, with production of eggs. These eggs can be hatched in drops of sterile distilled water (24-48 h) to generate sterile juveniles for further experiments. The established medium was also used to grow M. javanica on Arabidopsis and white clover roots in vitro, and for infection of Arabidopsis and tomato with Heterodera schachtii.

The optimised in vitro culture system has been used to study various aspects of plant-nematode interaction, including monitoring changes in glucuronidase (GUS) gene expression of four reporter gene constructs in transgenic Arabidopsis and white clover plants {haemoglobin\GUS, Adh’.GUS an alcohol dehydrogenase promoter, GH3:GUS an auxin responsive promoter and CHS.GUS chalcone synthase promoters) on infection with nematodes, and to study solute uptake at nematode feeding sites.

The expression patterns of the GUS reporter gene driven by two hypoxia inducible promoters {haemoglobin and Adh) in transgenic Arabidopsis roots has been studied during root-knot induced giant cell formation and gall development (2 to 45 days post infection; p.i.). Similar patterns of GUS expression from these two sets of transgenic plants indicate that the two genes are normally up-regulated only in mature giant cells (21-30 days p.i.). The results from this study support the hypothesis that giant cells are metabolically active and that low oxygen tension during later stages of infection may occur when giant cells are most active metabolically. This stage correlates with egglaying female nematodes. Both of these promoters could be used to develop a late giant cell specific promoter system for engineered plant resistance.

The role of the plant hormone, auxin, in giant cell initiation has been studied using a soybean auxin responsive promoter, GH3, linked to the GUS reporter gene in transgenic white clover {Trifolium repens) plants. High levels of GH3\GUS expression were detected 24-96 h after infection of roots with juveniles in cortical cells of gall tissue and in a ring of dividing pericycle cells but not in enlarging giant cells themselves. This result indicates that auxin is needed for initiation of giant cell formation, and as a trigger for mitotic division and enlargement of surrounding cells, but not for the subsequent enlargement of giant cells themselves.

The expression of the GUS gene driven by three chalcone synthase promoters {CHS1, CHS2 and CHS3), derived from different genes for the first enzyme of the flavonoid pathway in white clover, has been studied in relation to regulation of auxin levels during giant cell and gall development (24 to 120 h p.i.)- Because the CHS.GUS expression patterns precede GH3:GUS expression during early stages of giant cell/gall initiation, this suggests that auxin levels in initiating giant cells and developing galls are regulated by flavonoids either via an effect on IAA oxidase (IAA degradation enzyme) or inhibition of polar auxin transport in infected roots.

In order to learn more about off-loading of solutes at nematode feeding sites, experiments were carried out to study off-loading of a fluorescent tracer by epifluorescence and confocal laser scanning (CLSM) microscopy. 5(6)- carboxyfluorescein diacetate (CFDA), a non fluorescent dye precursor, was loaded into abraded Arabidopsis leaves and converted to carboxyfluorescein (CF), a non toxic fluorescent probe which is translocated in phloem sieve elements. CF transport was studied in intact cultured seedlings of Arabidopsis plants infected with M. javanica and H. schachtii at different stages of development in closed Petri dishes. Epifluorescence and CLSM microscopy showed that both giant cells and syncytia take up CF from sieve elements and that there is enhanced off-loading of CF at nematode feeding cells. A new concept which involves ATP-binding cassette transporters (ABC transporters) is suggested to explain solute unloading into feeding cells, since suggested symplastic transport through plasmodesmata neglects the previous ultrastructural studies of feeding cells which indicate that there are not enough plasmodesmata for symplastic transport. Although CF accumulated in giant cells and syncytia, it did not accumulate in any associated root-knot nematode females or in most of the equivalent female cyst nematodes. This indicates that there is ultrafiltration of solutes between feeding cell contents and the associated nematode, and that this effect is more pronounced for root-knot nematodes. The size exclusion limit of root-knot feeding tubes is therefore probably smaller than that for cyst nematodes.

The results presented in this thesis provide additional information on specific aspects of formation and function of nematode feeding cells. Such information will be useful in developing strategies to engineer host plant resistance to these economically important plant pathogens.

Item Type: Thesis (PhD)
Murdoch Affiliation(s): School of Biological Sciences and Biotechnology
Western Australian State Agricultural Biotechnology Centre
Division of Science and Engineering
Notes: Note to the author: If you would like to make your thesis openly available on Murdoch University Library's Research Repository, please contact: Thank you.
Supervisor(s): Jones, Michael
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