Further investigations of the population dynamics and pathogenicity of the pinewood nematode, Bursaphelenchus xylophilus (Steiner and Buhrer 1934) Nickle 1970, and its non-vector transmission to Pinus sylvestris

Abstract

The pinewood nematode, Bursaphelenchus xylophilus (Steiner and Buhrer) Nickle 1970, is the causal agent of the so-called pine wilt disease in susceptible conifer species, mainly Pinus spp., outside its natural range (North America) and in non-native pine species. Since its introduction to Asia and Europe, it has become the most harmful plant parasitic nematode of trees. Diseased trees wilt and die. Currently affected European countries are Portugal and Spain. Because of the observed threat in infested countries, B. xylophilus is listed as a quarantine pest in the European Union. In this PhD thesis, which is part of a European Union research project of the European Union Commission, investigations were conducted to support the pest risk analysis, management strategies and contingency planning of European Union Plant Health policy.

The transmission of B. xylophilus to new host trees is commonly by vector beetles of the genus Monochamus. However, B. xylophilus have been complained in wood chips with origin North America imported to the European Union, and the demand for wood chip imports from North America is increasing. Therefore, the phytosanitary risk of non-vector transmission of B. xylophilus by wood chips is of interest.

Moreover, Pinus sylvestris, a widespread tree species in Germany and northeastern Europe, was found to be highly susceptible to B. xylophilus in greenhouse trials using saplings. In Europe, mature trees of this species have not yet been tested.

The third part of this thesis was an evaluation of potentially tolerant or resistant host tree provenances as an option for management of pine wilt disease in affected countries.

The long-term survival of B. xylophilus in wood chips and its non-vector spread from infested wood chips to non-infested trees were investigated. B. xylophilus-infested wood chips were produced by inoculating a nematode-tap water suspension into P. sylvestris logs. During the long-term storage test, the survival of B. xylophilus was studied in sealed and openly stored P. sylvestris wood chips at 15 °C and 25 °C. For the investigation of non-vector spread, B. xylophilus-infested wood chips were placed on P. sylvestris saplings under different conditions.

Investigations using seven- to eight-year-old trees were conducted to examine the significance of sapling-based analyses of the population dynamics and pathogenicity of the pest for mature P. sylvestris trees. The trees were artificially inoculated with B. xylophilus using a nematode-tap water suspension. For nematode extraction during the population dynamics investigation, the pines were divided into 48 segments. Physiological changes and the development of wilt symptoms were recorded until tree death.

The pathogenicity of B. xylophilus towards different German pine provenances (according to the German Legal Ordinance on Regions of Provenance) was studied. For this purpose, P. sylvestris saplings were artificially inoculated with B. xylophilus using a nematode-tap water suspension.

In the sealed wood chips, B. xylophilus was found for more than 1 year at 15 °C and 25 °C. This was significantly longer than the duration observed for the variant openly stored at 25 °C. Furthermore, non-vector spread through wood chips was influenced by temperature, tree condition and wood chip location. Trees with stem or root injuries plus direct contact of the wounded part with infested wood chips at 25 °C were primarily B. xylophilus-infested and showed clear symptoms of pine wilt disease. Moreover, for stem- and root-injured pines, direct contact with infested wood chips was not always necessary for non-vector spread. At 15 °C, one B. xylophilus-infested pine exhibited clear symptoms of pine wilt disease.

At the start of the population dynamics investigation, B. xylophilus was located at the inoculation site and in adjacent segments. Before any external wilt symptoms developed, B. xylophilus was located in the entire stem, adjacent branch segments, root collar and roots. Finally, B. xylophilus was detected in all wood and root segments in combination with an increase in pine wilt disease and high nematode densities. Shortly before tree death, the treetop was partly nematode-free, and the subjacent tree segments were highly nematode-infested. During the pathogenicity investigation, all B. xylophilus-inoculated pines died and exhibited a significant but variable decline in the water potential in the needles compared to a drought-stressed variant.

All tested P. sylvestris provenances showed a mortality of 100 %. However, significant differences in the time course of disease development were found for a few provenances.

In conclusion, long-term survival in wood chips and non-vector transmission from infested wood chips to damaged trees were clearly shown, although such establishment should be less likely than spread via vectors. These findings should be tested in outdoor trials.

P. sylvestris saplings are good indicator trees for investigations of B. xylophilus population dynamics and pathogenicity because the results in seven- to eight-year-old P. sylvestris trees were comparable to those in saplings, although delayed in reaching a population peak and developing wilt symptoms.

The phenomena of delayed symptom development and delayed tree death of some pine provenances should be more closely examined with respect to potential defence traits. An ongoing search for tolerant or resistant provenances or individuals for cross-breeding purposes is suggested as part of a long-term phytosanitary strategy against B. xylophilus.

Overall, the threat based on infested wood chips and the high risk for P. sylvestris forests must be considered in pest risk analysis, management strategies and contingency planning.