Australasian Association of Nematologists
SOIL MICROBES AS POTENTIAL CONTROL AGENTS FOR PLANT-PARASITIC NEMATODES IN PASTURE
University of Adelaide 1999
Summary of Valerie Kempster's PhD thesis
Induced systemic resistance (ISR) is widely manifest in the plant kingdom, but there are few reports of its occurrence against nematodes and, prior to this study, it had not been reported in white clover. It was decided to investigate the induction of resistance to the clover cyst nematode, Heterodera trifolii Goffart, an economic pest in white clover pastures that are a key to high milk yields in dairy cattle in Australia and New Zealand. This study aimed to explore the potential of soil and rhizosphere bacteria to induce systemic resistance in white clover, Trifolium repens L.
Salicylic acid (SA) and benzo (1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH) are known to induce resistance in both monocotyledonous and dicotyledonous plants against a wide variety of pathogens and pests. In a growth cabinet soil bioassay, both chemicals were applied separately as soil drenches to T. repens seedlings, which were subsequently inoculated with infective juveniles of H. trifolii. Both chemicals induced resistance to H. trifolii, manifest as a reduction in fecundity of the nematode and a higher proportion of abnormal cysts and fewer eggs per cyst, compared to controls treated with water. Resistance was induced in two cultivars of T. repens, 'Haifa', and 'Grasslands Huia'. The latter is considered to be very susceptible to H. trifolii.
Soil samples and white clover plants were collected from pastures known to be infested with H. trifolii in Victoria. These and soil samples from South Australia were examined for the presence of Bacillus and Pseudomonas spp., both known to be potential inducers of ISR in other plants. Bacillus strains were isolated on a medium selective for B. thuringiensis and B. cereus. Fluorescent Pseudomonas strains were isolated on King's B medium, further selected as to colony type on tetrazolium chloride agar, and then selected for pectinolysis on crystal violet pectate agar. Two pectinolytic Pseudomonas strains, P29 and P80, applied as nutrient broth (NB) cultures, induced resistance in white clover seedlings equivalent to that resulting from application of SA and BTH. Both live and dead cells of strain P29, resuspended in sterile distilled water and applied as a soil drench, had the same effect as the NB culture of the strain. This suggests that there is some plant 'recognition' of the bacterial cell walls which triggers the plant response. Cell-free culture filtrate of strain P29, applied to white clover seedlings, did not induce resistance to H. trifolii, suggesting that the bacterial metabolites did not act as inducing agents. The metabolites had little antagonistic effect against infective juveniles of H. trifolii in vitro.
The plant response to BTH and P29 as inducing agents was investigated by quantitative biochemical assays for lignin and callose. At 4 weeks after inoculation with infective juvenile nematodes, there was no difference in the concentrations of lignin or callose in the leaves of plants pre-treated with BTH, P29 or water. Similarly, there was no difference in the concentrations of lignin or callose in the roots of plants pre-treated with BTH, P29 or water, 4 weeks after inoculation with the nematodes.
Bacillus strain B1 was also found to induce resistance against H. trifolii in the growth cabinet soil-based bioassays, equivalent to that induced by P29 or BTH.
A greenhouse experiment was carried out to investigate whether resistance could be induced to the blue-green aphid, Acyrthosiphon kondoi, in white clover and a medic, Medicago truncatula. 'Grasslands Huia', and the medic cultivars 'Sephi 6297' (resistant to the blue-green aphid) and 'Jemalong' (susceptible) were treated with strain P29, BTH or water and 4-5 aphids were released onto the plants. Some resistance to the blue-green aphid was observed. Resistance was manifest as greater plant growth in treated than control plants. Tests of strain P29 as a potential growth-promoting bacterium had shown no increase in growth of white clover plants after 52 days compared to SDW-treated controls. No increase in callose or lignin was found in the leaves and stems of the treated white clover plants.
It is concluded that resistance to the clover cyst nematode can be induced in white clover with a soil drench of BTH, of Pseudomonas strains P29 and P80, or of Bacillus strain B1 isolated from soil. This is a first report of induced resistance in white clover, and to the clover cyst nematode. These bacterial strains are candidates for further evaluation as biocontrol agents against nematodes in white clover.
