Plant parasitic nematodes infect almost all crop plants and annually cause losses of millions of dollars worldwide. The relationship between these pathogens and their hosts is still poorly understood in spite of several decades of research. In this research project, I attempted to investigate different aspects of this host-parasite relationship with respect to root-parasitic nematodes, and the effect these nematodes have on growth and development of the affected plants. I studied the host-parasite interactions using two different host plants, Arabidopsis thaliana and Solanum lycopersicum, and two nematodes which contrasted in their modes of infection, life cycle and pathogenicity: the root-knot nematode, Meloidogyne javanica, and the root lesion nematode, Pratylenchus thornei. Studies on A. thaliana were conducted in the laboratory under aseptic conditions in Petri dishes, and studies on tomato were conducted in the greenhouse as pot culture experiments. In my first investigation I examined the changes occurring in the cytoskeleton of A. thaliana root cells due to infection by M. javanica and P. thornei. This experiment was conducted over a period of one year from March 2003 to March 2004 at the Research School of Biological Sciences, Australian National University, Canberra, ACT in the laboratory of Drs Geoff Wasteneys and David Collings. The plant cytoskeleton plays a vital role in almost all cellular functions including cell signalling, cell division and wound response. I used three different investigative techniques to compare the effects of plant parasitic nematodes on the host root cytoskeleton. These techniques were whole root double immunolabelling, double immunolabelling of BMM sections and use of GFP-hTalin-transformed A. thaliana. Samples were observed using confocal laser scanning microscopy. I found all three techniques to be effective in studying cytoskeletal changes in infected roots. Whole root immunolabelling and GFP-hTalin-transformed A. Thaliana were most useful for studying the early stages of infection. However, immunolabelling of BMM sections was most effective in the later stages of infection to study large and thick gall tissue, as the transmission of light through thick tissue was not adequate to obtain high quality images by the other two techniques. Whole root immunolabelling showed that both M. javanica and P. thornei initially (even within the first 3 h) caused similar changes in A. thaliana. All stages of M. javanica were observed in A. thaliana roots; their migration and development was observed for 14 d. However, observations on P. thornei could not be conducted beyond the first 24 h after inoculation due to bacterial contamination. While P. thornei damaged A. thaliana roots, they were not observed within roots at any stage during this observation period. This was probably because the small diameter of the roots made them unattractive for entry by this larger nematode species. Material labelled for actin was observed to accumulate in a rounded or disc-shaped plug on the root surface and appeared to line the wound surface on the inside of the cell within the first 24 h after inoculation. Microtubules maintained their orientation, giving support to the cell wall. Increased actin and microtubule labelling was detected in nematode-infected root tissue with all three techniques used. In A. thaliana roots inoculated with M. javanica, this was especially prominent during initial entry and feeding site formation but was not as noticeable during nematode migration. This may have been due to a change in permeability of the cell walls resulting from nematode infection. A diffuse fluorescence was observed whenever actin fixation was not adequate. Once the M. javanica nematode entered the root, it followed a path (probably associated with physiological or chemical signals derived from the different cell types) during different stages of its migration to reach its ultimate feeding site. Microtubules in host cells close to the nematode body in its migration path were in a wavy, rather than a taut, arrangement. In nematode feeding sites, giant cells were formed around the head of the nematode and numerous small cells were observed towards the posterior of the nematode, forming the gall. Unusual divisions were observed in nematode-infected root tissues, with abnormal spindles and incomplete phragmoplasts in a region where cell divisions do not normally occur. Using immunolabelled BMM sections I observed well-defined microtubules and actin bundles in an infected cell. Giant cells appeared to have lost their growth polarity and anisotropy. These giant cells were spindle-shaped, with two tapering ends and with an enlarged middle section, indicating that there may be significant differences between the end (cross) walls of cells and the walls parallel to the longitudinal axis of the root. I subsequently conducted a temporal investigation in tomato, as an adjunct to a pot trial, to determine the similarities and differences in the way in which the two nematode species entered, migrated and caused damage, as well as their development within roots. My observations of roots were made using light microscopy and staining with acid fuchsin. This study showed, in detail, the entry and migration for both nematode species and, for M. javanica, feeding site formation and developmental life stages. This enabled longer time for observations of P. Thornei than was the case with the A. thaliana study. The initial stages of infection were consistent with the Arabidopsis results. However, I was able to observe the later stages of infection, which were markedly different between the two nematode species, although at a lower magnification than in the confocal study. To investigate the effects of the nematode-plant interactions that I had observed in my laboratory studies on plant growth and development, I conducted a pot trial using tomato seedlings, which ran over a period of 11 weeks, using the two previously studied nematode species. Treatments involved inoculation of pots with 5000 larvae. Different pots were infested weekly, from the date of planting to one week before harvest. At harvest (11 weeks), I recorded the length of aerial shoots, the number of green leaves, dry weight of shoots and roots, the combined number of flowers and flower buds and the number, fresh weight and diameter of fruits. M. Javanica significantly (p < 0.05) reduced shoot length, the diameter of the largest fruit, and dry weight of shoots and roots, while P. thornei significantly (p < 0.05) reduced shoot length, number of green leaves, the combined number of flowers and buds, and the dry weight of shoots and roots. While fruit from plants infected with M. javanica had similar moisture content to those in the untreated control, fruit from plants infected with P. thornei had lower moisture content than comparative control plants. These results indicate that the differences between the modes of infection of these two nematode species that I observed in the laboratory studies are manifested in host plant growth and development.Texture, together with pore size and sufficient moisture, is reported to be the primary soil factor influencing plant root-attacking nematode movement towards host roots for infection. I investigated the movement of M. javanica and P. thornei through soils towards the roots of tomato, S. lycopersicum, in a pot trial. For this I used three soils with different textures; namely sand, sandy clay loam and clay. While only a small proportion (< 19% in M. javanica and < 11.5% in P. thornei) of the original inoculum of 300 nematodes were recorded in the tomato roots, this is consistent with previously published studies. However, contrary to previous reports, there were many fewer nematodes present in roots of plants in sandy clay loam soil than in clay soils. The likely explanation is that the sandy clay loam soil had substantially high organic matter, electrical conductivity and silt. This indicates that soil chemistry and, probably, soil biology can play an even more important role than texture in determining nematode movement to and their damage of plant roots. I conclude that M. javanica and P. thornei differ in the way they interact with root tissues during the later stages of infection, although the host cytoskeletal reaction is similar during initial entry by both nematodes. These effects are manifested in the subsequent growth and development of host plants. A combination of soil physical, chemical and biological factors influence the movement of nematodes through soil to roots of host plants, and, in the case of P. thornei, may also influence length of time they spend within roots. The results of my study provide opportunities for further in-depth examination of the infection process, to understand the host-parasite interaction between nematodes and their host plants. A better understanding of the nature of these interactions will assist in formulating improved control strategies for these important nematode pests.
| Date of Award | 2009 |
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| Original language | English |
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