Evolution of Life Cycles of Nematodes Parasitizing Woody Plants As a Result of Ecological and Phylogenetic Co-Adaptations with Hosts and Vectors.

Fundamental aspects in the evolution of nematodes parasitizing woody plants are reviewed. (1) Nematode faunal lists of natural refugia are useful to predict the risks of opportunistic pathogens becoming true pathogens in the forest and park communities. (2) Nematode composition in natural refugia gives a chance to identify nematode antagonists of insect vectors of dangerous fungal and nematode infections, which can be potentially used as the biological agents for woody plants' protection. (3) Dauers in the ancestors of wood-inhabiting nematodes played a role as a survival stage in the detritus decomposition succession, and they later acquired the functions of dispersal and adaptations for transmission using insect vectors. (4) When inspecting wilted trees, it is necessary to use dauers for diagnostics, as sexually mature nematodes may be absent in tree tissues. (5) Plant parasitic nematodes originated from members of the detritus food web and retained a detritivorous phase in the life cycle as a part of the propagative generation. (6) Vectors in the life cycles of plant parasitic nematodes are inherited from the ancestral detritivorous nematode associations, rather than inserted in the dixenic life cycle of the 'nematode-fungus-plant' association. (7) Despite the significant difference in the duration of the nematode-tree and nematode-vector phases of the life cycle, the actual parasitic nematode specificity is dual: firstly to the vector and secondly to the natural host plant (as demonstrated in phytotests excluding a vector).

The simplest "field" methods for extractin of nematodes from plants, wood, insects and soil, with additional description how to keep extracted nematodes alive for a long time.

The simplest modification of the dynamic extraction method using cottonwool filter based on the Baermann funnel principle, is described. This modification excludes the funnel because a great share of Sticky worms attach to sloping walls of a funnel and thus do not reach the collector Eppendorf tube. But the main principle of the Baermann funnel is used, I. e. sinking down of actively moving heavy narrow bodies via wide holes of filter and thus separating the active worms from passive non-Brownian moving substrate particles, which do not pass the filter and remain above it. This principle is illustrated because it has never been described before. In the proposed modification any sloping walls in the extraction paths are excluded and thus the probability to attach sticky nemotodes to walls is also excluded; only cylindrical equipment with abrupt vertical walls is used; procedures are extremely simplified to be user-friendly for beginners: only filter (cotton pads), Eppendorf tubes, plastic glasses and narrow PVC tubing are applied. The new simplified modification allows one to collect nematodes by non-professional workers, e. g. in Polar expeditions without microscopic study of results. As an addition, an efficient method to maintain extracted nematodes alive is proposed, using the "effect of water film" in foam rubber inside the Eppendorf tube. To maintain nematodes alive during several months it is recommended to suppress bacteria via addition of 0.2-0.4% formaldehyde solution and then keep the tube with nematodes in a refrigerator.

Pine Wilt Disease and Possible Causes of Its Incidence in Russia.

Surveys of forests and stockpiled timber of pine, spruce, larch, and silver fir in 14 administrative subjects of the Russian Federation revealed widespread occurrence of the coniferous wood parasitic nematode Bursaphelenchus mucronatus. Twenty species of bacteria belonging to 13 genera have been detected in 25 B. mucronatus isolates, and their identity has been determined by direct sequencing of the 16S- RNA gene. The most frequently occurring were bacteria from the genera Pseudomonas, -Stenotrophomonas, Pantoea, Bacillus, Burkholderia, and Serratia. Prevalence of Pseudomonas brenneri and P. fluorescence, which were also found in the nematode dauer larva (Liv) isolated from the fir sawyer beetle Monochamus urussovi, have also been assessed. Two nematode B. xylophilus isolates from Portugal and one isolate from the United States have been examined, and 10 symbiotic bacteria species have been isolated, including Agrobacterium tumefacience, P.fluorescens, P. brenneri, Rahnella aquatilis, Stenotrophomonas maltophilia, S. rhizophila, and Yersinia mollaretfi.