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1.  Pathogenesis in Pine Wilt Caused by Pinewood Nematode, Bursaphelenchus xylophilus 
Journal of Nematology  1988;20(2):236-244.
The progression of events in the development of pine wilt disease following the invasion by Bursaphelenchus xylophilus is reviewed from early migration through pine tissues until symptom development on foliage. Disease resistance in pines, especially the hypersensitive reaction that is successful in controlling many potential pests and pathogens, is explored. Pathologies resulting from the activities of pinewood nematode include cortical trails and cavities; formation of cambial gaps and traumatic resin cysts; browning and death of cortex, phloem, cambium, and ray tissues; granulation and shrinkage of cell cytoplasm in rays; and destruction of resin canal epithelial and ray parenchyma cells. Death of parenchyma, production of toxins, and leakage of oleoresins and other material into tracheids are typical of the hypersensitive reaction occurring in pines following migration of small numbers of pinewood nematodes. The hypothesis presented is that a spreading hypersensitive reaction results in some of the observed pathologies and symptoms and eventually causes pine death. The growth-differentiation balance hypothesis is used to help explain predisposition, oleoresin production and toxicity, susceptibility and resistance, and the effects of variation in climate on host pines as related to pinewilt disease.
PMCID: PMC2618809  PMID: 19290207
Bursaphelenchus xylophilus; pathogenesis; pathology; pine wilt disease; pinewood nematode
2.  Relative Susceptibility of Four Pine Species to Infection by Pinewood Nematode 
Journal of Nematology  1987;19(1):44-50.
Mature trees of eastern white, jack, Scotch, and shortleaf pines were inoculated with 25,000-34,000 pinewood nematodes, Bursaphelenchus xylophilus, isolated from infected Scotch pines in Missouri. Equal numbers of trees of each species inoculated with distilled water served as controls. Nine of fifteen Scotch pines died within 4 months of nematode infection or during the winter and early spring following infection. A single eastern white and shortleaf pine died. No jack pines died. A single Scotch pine control died, apparently the result of natural nematode infection. No other controls died. Mean oleoresin flow did not differ among nematode-inoculated jack and shortleaf pines and their respective controls. Oleoresin flow in nematode-inoculated eastern white and Scotch pines was significantly lower than in their controls. Oleoresin flow was temporarily reduced in mortality-resistant eastern white and Scotch pines following nematode infection. Thus a sublethal impact of nematode infection on mortality-resistant host trees was documented.
PMCID: PMC2618611  PMID: 19290105
Bursaphelenchus xylophilus; Monochamus carolinensis; mortality; oleoresin; pinewood nematode; Pinus banksiana; Pinus echinata; Pinus strobus; Pinus sylvestris; resistance
3.  A rapid decrease in temperature induces latewood formation in artificially reactivated cambium of conifer stems 
Annals of Botany  2012;110(4):875-885.
Background and Aims
Latewood formation in conifers occurs during the later part of the growing season, when the cell division activity of the cambium declines. Changes in temperature might be important for wood formation in trees. Therefore, the effects of a rapid decrease in temperature on cellular morphology of tracheids were investigated in localized heating-induced cambial reactivation in Cryptomeria japonica trees and in Abies firma seedlings.
Electric heating tape and heating ribbon were wrapped on the stems of C. japonica trees and A. firma seedlings. Heating was discontinued when 11 or 12 and eight or nine radial files of differentiating and differentiated tracheids had been produced in C. japonica and A. firma stems, respectively. Tracheid diameter, cell wall thickness, percentage of cell wall area and percentage of lumen area were determined by image analysis of transverse sections and scanning electron microscopy.
Key Results
Localized heating induced earlier cambial reactivation and xylem differentiation in stems of C. japonica and A. firma as compared with non-heated stems. One week after cessation of heating, there were no obvious changes in the dimensions of the differentiating tracheids in the samples from adult C. japonica. In contrast, tracheids with a smaller diameter were observed in A. firma seedlings after 1 week of cessation of heating. Two or three weeks after cessation of heating, tracheids with reduced diameters and thickened cell walls were found. The results showed that the rapid decrease in temperature produced slender tracheids with obvious thickening of cell walls that resembled latewood cells.
The results suggest that a localized decrease in temperature of stems induces changes in the diameter and cell wall thickness of differentiating tracheids, indicating that cambium and its derivatives can respond directly to changes in temperature.
PMCID: PMC3423807  PMID: 22843340
Cambial activity; conifers; latewood formation; morphology of tracheids; rapid decrease in temperature
4.  Pathogenicity of Bursaphelenchus xylophilus on Pines in Minnesota and Wisconsin 
Journal of Nematology  1986;18(1):44-49.
The pinewood nematode, Bursaphelenchus xylophilus, was inoculated into established native jack and red pines (Pinus banksiana and P. resinosa) and exotic Austrian pine (P. nigra) in Minnesota and Wisconsin forests during summer 1981. The nematode isolates did not kill established nonstressed pine trees growing in the forest. However, the same nematode isolates killed pine seedlings under greenhouse conditions. Girdling the main stem of some trees to induce stress resulted in the death of the majority of inoculated and noninoculated branches of Austrian and jack pines, but no branch death was observed on red pine. Greater numbers of nematodes were extracted from branches of inoculated, girdled trees than from nongirdled trees. The mean number of nematodes extracted from branches of inoculated, nongirdled trees was 0.3 - 14 nematodes per gram of wood.
PMCID: PMC2618501  PMID: 19294138
pinewood nematode; Pinus banksiana; P. resinosa; P. nigra
5.  Effect of Local Heating and Cooling on Cambial Activity and Cell Differentiation in the Stem of Norway Spruce (Picea abies) 
Annals of Botany  2006;97(6):943-951.
• Background and Aims The effect of heating and cooling on cambial activity and cell differentiation in part of the stem of Norway spruce (Picea abies) was investigated.
• Methods A heating experiment (23–25 °C) was carried out in spring, before normal reactivation of the cambium, and cooling (9–11 °C) at the height of cambial activity in summer. The cambium, xylem and phloem were investigated by means of light- and transmission electron microscopy and UV-microspectrophotometry in tissues sampled from living trees.
• Key Results Localized heating for 10 d initiated cambial divisions on the phloem side and after 20 d also on the xylem side. In a control tree, regular cambial activity started after 30 d. In the heat-treated sample, up to 15 earlywood cells undergoing differentiation were found to be present. The response of the cambium to stem cooling was less pronounced, and no anatomical differences were detected between the control and cool-treated samples after 10 or 20 d. After 30 d, latewood started to form in the sample exposed to cooling. In addition, almost no radially expanding tracheids were observed and the cambium consisted of only five layers of cells. Low temperatures reduced cambial activity, as indicated by the decreased proportion of latewood. On the phloem side, no alterations were observed among cool-treated and non-treated samples.
• Conclusions Heating and cooling can influence cambial activity and cell differentiation in Norway spruce. However, at the ultrastructural and topochemical levels, no changes were observed in the pattern of secondary cell-wall formation and lignification or in lignin structure, respectively.
PMCID: PMC2803384  PMID: 16613904
Norway spruce; Picea abies; cambium; xylem; phloem; cell differentiation; heating; cooling; light microscopy; transmission electron microscopy; UV-microspectrophotometry
6.  Decline and Death of Pinus spp. in Delaware Caused by Bursaphelenchus xylophilus 
Journal of Nematology  1982;14(3):382-385.
Etiological studies to determine the cause of decline and death of Pinus spp. in Delaware were initiated in 1980. The pinewood nematode, Bursaphelenchus xylophilus, was found to be the major canse of mortality in Japanese black pine (Pinus thunbergii). When inoculated into healthy 5-yr-old Japanese black pines, B. xylophilus produced typical decline symptoms observed in the field. The xylophilous fungi most often associated with declining trees, Rhizosphaera pini, Fusarium spp., and Pestalotia funerea, were not pathogenic to Japanese black pine in greenhouse tests. Mineral analyses of soil and foliage showed no significant differences between healthy and infested trees. B. xylolyhilus was also found on loblolly pine (P. taeda), scrub pine (P. virginiana), Scots pine (P. sylvestris), red pine (P. resinosa), Eastern white pine (P. strobus), and pitch pine (P. rigida).
PMCID: PMC2618183  PMID: 19295726
pinewood nematode; Japanese black pine
7.  New Insights into the Phylogeny and Worldwide Dispersion of Two Closely Related Nematode Species, Bursaphelenchus xylophilus and Bursaphelenchus mucronatus 
PLoS ONE  2013;8(2):e56288.
The pinewood nematode, Bursaphelenchus xylophilus, is one of the greatest threats to coniferous forests worldwide, causing severe ecological damage and economic loss. The biology of B. xylophilus is similar to that of its closest relative, B. mucronatus, as both species share food resources and insect vectors, and have very similar morphological characteristics, although little pathogenicity to conifers has been associated with B. mucronatus. Using both nuclear and mitochondrial DNA markers, we show that B. xylophilus and B. mucronatus form distinct phylogenetic groups with contrasting phylogeographic patterns. B. xylophilus presents lower levels of intraspecific diversity than B. mucronatus, as expected for a species that evolved relatively recently through geographical or reproductive isolation. Genetic diversity was particularly low in recently colonised areas, such as in southwestern Europe. By contrast, B. mucronatus displays high levels of genetic diversity and two well-differentiated clades in both mitochondrial and nuclear DNA phylogenies. The lack of correlation between genetic and geographic distances in B. mucronatus suggests intense gene flow among distant regions, a phenomenon that may have remained unnoticed due to the reduced pathogenicity of the species. Overall, our findings suggest that B. xylophilus and B. mucronatus have different demographic histories despite their morphological resemblance and ecological overlap. These results suggest that Bursaphelenchus species are a valuable model for understanding the dispersion of invasive species and the risks posed to native biodiversity and ecosystems.
PMCID: PMC3568074  PMID: 23409167
8.  Changes in the localization and levels of starch and lipids in cambium and phloem during cambial reactivation by artificial heating of main stems of Cryptomeria japonica trees 
Annals of Botany  2010;106(6):885-895.
Background and Aims
Cambial reactivation in trees occurs from late winter to early spring when photosynthesis is minimal or almost non-existent. Reserve materials might be important for wood formation in trees. The localization and approximate levels of starch and lipids (as droplets) and number of starch granules in cambium and phloem were examined from cambial dormancy to the start of xylem differentiation in locally heated stems of Cryptomeria japonica trees in winter.
Electric heating tape was wrapped on one side of the stem of Cryptomeria japonica trees at breast height in winter. The localization and approximate levels of starch and lipids (as droplets) and number of starch granules were determined by image analysis of optical digital images obtained by confocal laser scanning microscopy.
Key Results
Localized heating induced earlier cambial reactivation and xylem differentiation in stems of Cryptomeria japonica, as compared with non-heated stems. There were clear changes in the respective localizations and levels of starch and lipids (as droplets) determined in terms of relative areas on images, from cambial dormancy to the start of xylem differentiation in heated stems. In heated stems, the levels and number of starch granules fell from cambial reactivation to the start of xylem differentiation. There was a significant decrease in the relative area occupied by lipid droplets in the cambium from cambial reactivation to the start of xylem differentiation in heated stems.
The results showed clearly that the levels and number of storage starch granules in cambium and phloem cells and levels of lipids (as droplets) in the cambium decreased from cambial reactivation to the start of xylem differentiation in heated stems during the winter. The observations suggest that starch and lipid droplets might be needed as sources of energy for the initiation of cambial cell division and the differentiation of xylem in Cryptomeria japonica.
PMCID: PMC2990657  PMID: 21037242
Cambial reactivation; confocal laser scanning microscopy; Cryptomeria japonica; lipid; starch; xylem differentiation
9.  Induction of Cambial Reactivation by Localized Heating in a Deciduous Hardwood Hybrid Poplar (Populus sieboldii × P. grandidentata) 
Annals of Botany  2007;100(3):439-447.
Background and Aims
The timing of cambial reactivation plays an important role in the control of both the quantity and the quality of wood. The effect of localized heating on cambial reactivation in the main stem of a deciduous hardwood hybrid poplar (Populus sieboldii × P. grandidentata) was investigated.
Electric heating tape (20–22 °C) was wrapped at one side of the main stem of cloned hybrid poplar trees at breast height in winter. Small blocks were collected from both heated and non-heated control portions of the stem for sequential observations of cambial activity and for studies of the localization of storage starch around the cambium from dormancy to reactivation by light microscopy.
Key Results
Cell division in phloem began earlier than cambial reactivation in locally heated portions of stems. Moreover, the cambial reactivation induced by localized heating occurred earlier than natural cambial reactivation. In heated stems, well-developed secondary xylem was produced that had almost the same structure as the natural xylem. When cambial reactivation was induced by heating, the buds of trees had not yet burst, indicating that there was no close temporal relationship between bud burst and cambial reactivation. In heated stems, the amount of storage starch decreased near the cambium upon reactivation of the cambium. After cambial reactivation, storage starch disappeared completely. Storage starch appeared again, near the cambium, during xylem differentiation in heated stems.
The results suggest that, in deciduous diffuse-porous hardwood poplar growing in a temperate zone, the temperature in the stem is a limiting factor for reactivation of phloem and cambium. An increase in temperature might induce the conversion of storage starch to sucrose for the activation of cambial cell division and secondary xylem. Localized heating in poplar stems provides a useful experimental system for studies of cambial biology.
PMCID: PMC2533603  PMID: 17621596
Populus sieboldii × Populus grandidentata; localized heating, cambial reactivation; model system; storage starch; xylem differentiation
10.  Molecular Characterization and Development of Real-Time PCR Assay for Pine-Wood Nematode Bursaphelenchus xylophilus (Nematoda: Parasitaphelenchidae) 
PLoS ONE  2013;8(11):e78804.
Bursaphelenchus xylophilus, the pine-wood nematode (PWN), is the causal agent of pine wilt disease, one of the most damaging emerging pest problems to forests around the world. It is native to North America where it causes relatively minor damage to native conifers but is labeled an EPPO-A-2 pest and a quarantine nematode for many countries outside of the United States because of its potential for destruction to their native conifers. Exports of wood logs and commodities involving softwood packaging materials now require a lab test for the presence/absence of this regulated nematode species. We characterized the DNA sequences on the ribosomal DNA small subunit, large subunit D2/D3, internal transcribed spacer (ITS) and mitochondrial DNA cytochrome oxidase subunit one on the aphelenchid species and described the development of a real-time-PCR method for rapid and accurate identification of PWN targeting the ITS-1. A total of 97 nematode populations were used to evaluate the specificity and sensitivity of this assay, including 45 populations of B. xylophilus and 36 populations of 21 other species of Bursaphelenchus which belong to the abietinus, cocophilus, eggersi, fungivorus, hofmanni, kevini, leoni, sexdentati, and xylophilus groups and one unassigned group from a total of 13 groups in the genus Bursaphelenchus; 15 populations of Aphelenchoides besseyi, A. fragariae, Aphelenchoides species and Aphelenchus avenae; and one population of mixed nematode species from a soil sample. This assay proved to be specific to B. xylophilus only and was sensitive to a single nematode specimen regardless of the life stages present. This approach provides rapid species identification necessary to comply with the zero-tolerance export regulations.
PMCID: PMC3823978  PMID: 24244367
11.  Immunolocalization indicates plasmodesmal trafficking of storage proteins during cambial reactivation in Populus nigra 
Annals of Botany  2010;106(3):385-394.
Background and Aims
Cambium reactivation after dormancy and budbreak in deciduous trees requires a supply of mobilized reserve materials. The pathway and mode of transfer of these materials are poorly understood.
Transport of reserve materials during cambium reactivation in Populus nigra was investigated by conventional and immunocytochemical TEM analyses, SDS–PAGE, western blotting and intracellular microinjection of fluorescent dyes.
Key Results
Proteinaceous compounds stored in vacuoles and protein bodies of vascular cells and ray cells disappeared within 3 weeks after cambial reactivation and budbreak. Some of these proteins (32 kDa, 30 kDa and 15 kDa) were labelled by lectin antibodies in SDS–PAGE. The same antibodies were localized to plasmodesmata (PDs) between phloem parenchyma, ray cells and fusiform cambial cells. In addition, proteinaceous particles were localized inside the cytoplasmic sleeves of these PDs during budbreak. During this period, the functional diameter of PDs was about 2·2 nm which corresponds approximately to the Stokes' radius of the detected 15-kDa protein.
Lectin-like reserve proteins or their degradation products seem to be transferred through PDs of phloem parenchyma and rays during cambial reactivation and budbreak. PD transfer of storage proteins is a novelty which supports the concept of symplasmic nutrient supply to the cambial region.
PMCID: PMC2924828  PMID: 20584737
Cambial region; lectins; plasmodesmal trafficking; Populus nigra ‘italica’; size exclusion limit; storage proteins; vascular tissues
12.  Searching for resistance genes to Bursaphelenchus xylophilus using high throughput screening 
BMC Genomics  2012;13:599.
Pine wilt disease (PWD), caused by the pinewood nematode (PWN; Bursaphelenchus xylophilus), damages and kills pine trees and is causing serious economic damage worldwide. Although the ecological mechanism of infestation is well described, the plant’s molecular response to the pathogen is not well known. This is due mainly to the lack of genomic information and the complexity of the disease. High throughput sequencing is now an efficient approach for detecting the expression of genes in non-model organisms, thus providing valuable information in spite of the lack of the genome sequence. In an attempt to unravel genes potentially involved in the pine defense against the pathogen, we hereby report the high throughput comparative sequence analysis of infested and non-infested stems of Pinus pinaster (very susceptible to PWN) and Pinus pinea (less susceptible to PWN).
Four cDNA libraries from infested and non-infested stems of P. pinaster and P. pinea were sequenced in a full 454 GS FLX run, producing a total of 2,083,698 reads. The putative amino acid sequences encoded by the assembled transcripts were annotated according to Gene Ontology, to assign Pinus contigs into Biological Processes, Cellular Components and Molecular Functions categories. Most of the annotated transcripts corresponded to Picea genes-25.4-39.7%, whereas a smaller percentage, matched Pinus genes, 1.8-12.8%, probably a consequence of more public genomic information available for Picea than for Pinus. The comparative transcriptome analysis showed that when P. pinaster was infested with PWN, the genes malate dehydrogenase, ABA, water deficit stress related genes and PAR1 were highly expressed, while in PWN-infested P. pinea, the highly expressed genes were ricin B-related lectin, and genes belonging to the SNARE and high mobility group families. Quantitative PCR experiments confirmed the differential gene expression between the two pine species.
Defense-related genes triggered by nematode infestation were detected in both P. pinaster and P. pinea transcriptomes utilizing 454 pyrosequencing technology. P. pinaster showed higher abundance of genes related to transcriptional regulation, terpenoid secondary metabolism (including some with nematicidal activity) and pathogen attack. P. pinea showed higher abundance of genes related to oxidative stress and higher levels of expression in general of stress responsive genes. This study provides essential information about the molecular defense mechanisms utilized by P. pinaster and P. pinea against PWN infestation and contributes to a better understanding of PWD.
PMCID: PMC3542250  PMID: 23134679
13.  Change in Water Status of Pinus thunbergii Parl. Inoculated with Species of Bursaphelenchus 
Journal of Nematology  1990;22(1):132-135.
Maximum and minimum xylem pressure potentials of needles were measured to evaluate water status of Pinus thunbergii Parl. after inoculation with the virulent or avirulent populations of Bursaphelenchus xylophilus or B. mucronatus. In virulent B. xylophilus-inoculated pines, the water status changed abruptly and needle chlorosis occurred by day 29 after inoculation. Similar changes were not seen in B. mucronatus-inoculated and uninoculated control pines. Oleoresin flow ceased in virulent B. xylophilus-inoculated pines. Avirulent B. xylophilus-inoculated pines responded very little to nematode invasion by a slight decrease in oleoresin flow. Oleoresin flow did not vary in B. mucronatus-inoculated and uninoculated control pines. A decrease in soil water potential below field capacity seemed to accelerate the development of pine wilt disease.
PMCID: PMC2619013  PMID: 19287700
avirulent population; Bursaphelenchus mucronatus; B. xylophilus; oleoresin flow; pine wilt disease; pinewood nematode; Pinus thunbergii; soil water potential; virulent population; water status; xylem pressure potential
14.  Ribosomal DNA haplotype distribution of Bursaphelenchus xylophilus in Kyushu and Okinawa islands, Japan 
Journal of Nematology  2009;41(3):194-202.
Ribosomal DNA region sequences (partial 18S, 28S and complete ITS1, 5.8S, and ITS2) of the pinewood nematode (Bursaphelenchus xylophilus) were obtained from DNA extracted directly from wood pieces collected from wilted pine trees throughout the Kyushu and Okinawa islands, Japan. Either a 2569bp or 2573bp sequence was obtained from 88 of 143 samples. Together with the 45 rDNA sequences of pinewood nematode isolates previously reported, there were eight single nucleotide polymorphisms and two indels of two bases. Based on these mutations, nine haplotypes were estimated. The haplotype frequencies differed among regions in Kyushu island (northwest, northeast and center, southeast, and southwest), and the distribution was consistent with the invasion and spreading routes of the pinewood nematode previously estimated from past records of pine wilt and wood importation. There was no significant difference in haplotype frequencies among the collection sites on Okinawa island.
PMCID: PMC3380500  PMID: 22736814
Bursaphelenchus xylophilus; haplotype; invasive species; pine wilt disease; pinewood nematode; rDNA; spread route
15.  Survival and Infectivity of Bursaphelenchus xylophilus in Wood Chip-Soil Mixtures 
Journal of Nematology  1992;24(4):495-503.
To determine the effect of soil environment on the life stages and total numbers of Bursaphelenchus xylophilus, nematode-infested wood chips alone and mixed with soil were incubated at 12 and 20 C. Nematodes were extracted at 2-week intervals for 12 weeks. Numbers of nematodes and percentage of third-stage dispersal larvae were greater at 12 C and in chips without soil. Percentage of juveniles of the propagative cycle was greater at 20 C and in chips with soil. Although B. xylophilus survived in chips with soil for 12 weeks, nematode numbers and life stage percentages changed little over time. To determine if B. xylophilus was capable of infecting wounded roots, infested and uninfested chips were mixed with soil in pots with white and Scots pine seedlings. Trees were maintained at 20 and 30 C and harvested at mortality or after 12 weeks. Only seedlings treated with infested chips contained nematodes. In field experiments, planted seedlings were mulched with infested chips to determine if nematodes would invade basal stem wounds. Among these trees, Scots pine was more susceptible than white or red pines to infection and mortality.
PMCID: PMC2619303  PMID: 19283027
Bursaphelenchus xylophilus; nematode; pine; pinewood nematode; Pinus strobus; P. sylvestris; root; soil; temperature; wood chip
16.  Scanning Electron Microscopy of Pine Seedling Wood Tissue Sections Inoculated with the Pinewood Nematode Bursaphelenchus xylophilus Previously Prepared for Light Microscopy 
Journal of Nematology  2012;44(3):255-259.
Scanning electron microscopy (SEM) was applied to paraffin-embedded wood sections to study the histopathology of pine seedlings inoculated with the pinewood nematode (PWN), Bursaphelenchus xylophilus. The sections, which had been previously prepared and observed by light microscopy (LM) on glass slides, were originally obtained from experiments in which pine seedlings had been inoculated with PWN. The cover glass was removed by soaking the glass slide in xylene for 3 to 5 days. The glass slides were cut into small pieces so that each piece contained one wood section. Each piece of the glass slide was attached with double adhesive tape to an aluminum stub. The specimens were sputter-coated with gold and examined with a scanning electron microscope (JEOL-JSM 5200). Compared to LM (as documented in previous reports) SEM provided greater depth of focus and resolution of the damaged wood tissues, nematodes and associated bacteria. SEM made it possible to observe the relationship between bacterial distribution and nematode distribution in wood tissues. SEM observations also suggested the possibility of documenting the death of ray cells and other parenchyma cells in relation to disease development. Finally, the current study of PWN in pine seedlings demonstrated that glass slides prepared for LM observations more than 25 years earlier could be successfully processed for examination by SEM.
PMCID: PMC3547340  PMID: 23481606
bacterial mass; inoculation experiments; Pinus sp.; scanning electron microscopy; technique
17.  Cambial Activity and Intra-annual Xylem Formation in Roots and Stems of Abies balsamea and Picea mariana 
Annals of Botany  2008;102(5):667-674.
Background and Aims
Studies on xylogenesis focus essentially on the stem, whereas there is basically no information about the intra-annual growth of other parts of the tree. As roots strongly influence carbon allocation and tree development, knowledge of the dynamics of xylem production and maturation in roots at a short time scale is required for a better understanding of the phenomenon of tree growth. This study compared cambial activity and xylem formation in stem and roots in two conifers of the boreal forest in Canada.
Wood microcores were collected weekly in stem and roots of ten Abies balsamea and ten Picea mariana during the 2004–2006 growing seasons. Cross-sections were cut using a rotary microtome, stained with cresyl violet acetate and observed under visible and polarized light. The number of cells in the cambial zone and in differentiation, plus the number of mature cells, was counted along the developing xylem.
Key Results
Xylem formation lasted from the end of May to the end of September, with no difference between stem and roots in 2004–2005. On the contrary, in 2006 a 1-week earlier beginning of cell differentiation was observed in the stem, with cell wall thickening and lignification in roots ending up to 22 d later than in the stem. Cell production in the stem was concentrated early in the season, in June, while most cell divisions in roots occurred 1 month later.
The intra-annual dynamics of growth observed in stem and roots could be related to the different amount of cells produced by the cambium and the patterns of air and soil temperature occurring in spring.
PMCID: PMC2712372  PMID: 18708643
Abies balsamea; boreal forest; cambium; cell differentiation; cell wall thickening; lignification; Picea mariana; root; stem; xylem
18.  Pinewood Nematode, Bursaphelenchus xylophilus, Associated with Red Pine, Pinus resinosa, in Western Maryland 
Journal of Nematology  1986;18(4):575-580.
Red pines Pinus resinosa in Garrett and Allegany counties, Maryland, were examined during 1982-84 to determine distribution of the pinewood nematode, Bursaphelenchus xylophilus, within and among trees. Approximately 25-year-old (younger) and 47-year-old (older) trees were subdivided into the following categories: 1) trees with mostly green needles; 2) trees with mostly reddish-brown needles; 3) trees lacking needles but with bark intact; 4) trees lacking both needles and bark; and 5) trees with chlorotic, bleached-green needles. Bursaphelenchus xylophilus was found infecting 68% of younger red pines and 77% of older red pines. Nematodes were not evenly distributed in trees within any given tree decadence category or in trees of the same age. Nematodes were recovered from 20% of wood samples from trunks and primary and secondary branches in younger pines and from 15 % of older red pines. On the basis of tree decadence category, the highest incidence of infection in younger trees (31%) was in bleached-green needled trees (category 5), whereas in older trees the highest infection (25%) occurred in green needled trees (category 1). At both sites trunks were infected more often than branches.
PMCID: PMC2618579  PMID: 19294229
Bursaphelenchus xylophilus; pinewood nematode; pinewih disease; Pinus resinosa; red pine
19.  First Insights into the Genetic Diversity of the Pinewood Nematode in Its Native Area Using New Polymorphic Microsatellite Loci 
PLoS ONE  2013;8(3):e59165.
The pinewood nematode, Bursaphelenchus xylophilus, native to North America, is the causative agent of pine wilt disease and among the most important invasive forest pests in the East-Asian countries, such as Japan and China. Since 1999, it has been found in Europe in the Iberian Peninsula, where it also causes significant damage. In a previous study, 94 pairs of microsatellite primers have been identified in silico in the pinewood nematode genome. In the present study, specific PCR amplifications and polymorphism tests to validate these loci were performed and 17 microsatellite loci that were suitable for routine analysis of B. xylophilus genetic diversity were selected. The polymorphism of these markers was evaluated on nematodes from four field origins and one laboratory collection strain, all originate from the native area. The number of alleles and the expected heterozygosity varied between 2 and 11 and between 0.039 and 0.777, respectively. First insights into the population genetic structure of B. xylophilus were obtained using clustering and multivariate methods on the genotypes obtained from the field samples. The results showed that the pinewood nematode genetic diversity is spatially structured at the scale of the pine tree and probably at larger scales. The role of dispersal by the insect vector versus human activities in shaping this structure is discussed.
PMCID: PMC3598798  PMID: 23554990
20.  Intra-annual dynamics of stem CO2 efflux in relation to cambial activity and xylem development in Pinus cembra 
Tree physiology  2009;29(5):641-649.
The relationship between stem CO2 efflux (ES), cambial activity and xylem production in Pinus cembra was determined at the timberline (1950 m a.s.l.) of the Central Austrian Alps, throughout one year. ES was measured continuously from June 2006 to August 2007 using an infrared gas-analysis system. Cambial activity and xylem production was determined by repeated microcore sampling of the developing tree ring and radial increment was monitored using automated point dendrometers. Aside of temperature, the number of living tracheids and cambial cells was predominantly responsible for ES: ES normalized to 10°C (ES10) was significantly correlated to number of living cells throughout the year (r2 = 0,574; p < 0,001). However, elevated ES and missing correlation between ES10 and xylem production was detected during cambial reactivation in April and during transition from active phase to rest, which occurred in August and lasted until early September. Results of this study indicate that (i) during seasonal variations in cambial activity non-linearity between ES and xylem production occurs and (ii) elevated metabolic activity during transition stages in the cambial activity-dormancy cycle influence the carbon budget of Pinus cembra. Daily radial stem increment was primarily influenced by the number of enlarging cells and was not correlated to ES.
PMCID: PMC3013296  PMID: 19203979
cambial reactivation; dormancy; Pinus cembra; radial stem growth; sap flow; stem CO2 efflux; stem respiration; xylem production
21.  Nemtaode-Vector Relationships in the Pine Wilt Disease System 
Journal of Nematology  1988;20(2):227-235.
Pinewood nematode, Bursaphelenchus xylophilus, is the causal agent of pine wilt disease in North America and Japan. Dispersal stage dauer larvae are transported to new host trees on the body surface and within the tracheal system of several beetle species. Worldwide, 21 species of Cerambycidae, 1 genus of Buprestidae, and 2 species of Curculionidae are known to carry pinewood nematode dauer larvae upon emerging from nematode-infested trees. Five species of cerambycids in the genus Monochamus are known to transmit dauer larvae to new host trees, four North American species and one Japanese species. Primary transmission to healthy trees occurs through beetle feeding wounds on young branches. Secondary transmission to stressed trees or recently cut logs occurs through Monochamus oviposition sites.
PMCID: PMC2618795  PMID: 19290206
Bursaphelenchus xylophilus; Cerambycidae; insect vector; Monochamus alternatus; Monochamus carolinensis; Monochamus scutellatus; Monochamus titillator; pine sawyer; pine wilt disease; pinewood nematode; transmission
22.  Effect of Copper Sulfate and Lead Acetate on Infection of Pines with Bursaphelenchus xylophilus 
Journal of Nematology  1989;21(1):1-9.
Treatment of 3-year-old Scots, white, and Austrian pine seedlings with copper sulfate or lead acetate significantly affected energy homeostasis and oleoresin production in the seedlings but did not induce wilting of the seedlings. Inoculation of copper sulfate-treated or lead acetate-treated white, Scots, and Austrian pine seedlings with the white pine specific pathotype of Bursaphelenchus xylophilus, VPSt-1, caused a significant increase in oleoresin production, stressed energy homeostasis, and induced rapid wilting of the seedlings. Scots pine lost tolerance and Austrian pine lost resistance to VPSt-1 after the seedlings were treated with either copper sulfate or lead acetate. These results suggest that environmental pollution may significantly affect susceptibility of pines to B. xylophilus and may have a role in establishment of this nematode in uninfested areas.
PMCID: PMC2618893  PMID: 19287570
Bursaphelenchus xylophilus; copper; heavy metal; lead; pinewood nematode; pine wilt; Pinus nigra; P. strobus; P. sylvestris; resistance; susceptibility
23.  Transmission of the Pinewood Nematode, Bursaphelenchus xylophilus, to Slash Pine Trees and Log Bolts by a Cerambycid Beetle, Monochamus titillator, in Florida 
Journal of Nematology  1984;16(1):37-40.
Field-collected adults of the southern pine sawyer, Monochamus titillator (F.) (Coleoptera: Cerambycidae), naturally infested with fourth-stage juveniles (dauerlarvae) of the pinewood nematode, Bursaphelenchus xylophilus (Steiner and Buhrer, 1934) Nickle, 1970, were maturation fed on excised shoots of typical slash pine, Pinus elliottii Engelm. var elliottii, for 21 days. During August 1981, a male and female adult beetle were held in a sleeve cage placed on the terminal of a side branch of each of seven replicate, healthy 10-year-old slash pine trees. All seven branch terminals showed evidence of beetle feeding on the bark after 1 week, and pinewood nematodes were present in wood samples taken near these feeding sites. Four of the seven trees showed wilt symptoms in 4-6 weeks and died about 9 weeks after beetle feeding. Pinewood nematodes were recovered from the roots and trunks of the dead trees. Each of seven replicate slash pine log bolts was enclosed in a jar with a pair of the same beetles used in the sleeve cages. After 1 week, wood underlying beetle oviposition sites in the bark of all replicate log bolts was infested with the pinewood nematode.
PMCID: PMC2618336  PMID: 19295871
Bursaphelenchus xylophilus; Pinus elliottii; Monochamus titillator; maturation-feeding; oviposition
24.  Pinewood nematode-associated bacteria contribute to oxidative stress resistance of Bursaphelenchus xylophilus 
BMC Microbiology  2013;13:299.
Pine wilt disease (PWD) caused by the pinewood nematode Bursaphelenchus xylophilus is one of the most serious forest diseases in the world. The role of B. xylophilus-associated bacteria in PWD and their interaction with the nematode, have recently been under substantial investigation. Several studies report a potential contribution of the bacteria for the PWD development, either as a helper to enhance the pathogenicity of the nematode or as a pathogenic agent expressing interesting traits related to lifestyle host-adaptation.
We investigated the nematode-bacteria interaction under a severe oxidative stress (OS) condition using a pro-oxidant hydrogen peroxide and explored the adhesion ability of these bacteria to the cuticle surface of the nematodes. Our results clearly demonstrated a beneficial effect of the Serratia spp. (isolates LCN-4, LCN-16 and PWN-146) to B. xylophilus under the OS condition. Serratia spp. was found to be extremely OS-resistant, and promote survival of B. xylophilus and down-regulate two B. xylophilus catalase genes (Bxy-ctl-1 and Bxy-ctl-2). In addition, we show that the virulent isolate (Ka4) of B. xylophilus survives better than the avirulent (C14-5) isolate under the OS condition. The bacterial effect was transverse for both B. xylophilus isolates. We could not observe a strong and specific adhesion of these bacteria on the B. xylophilus cuticle surface.
We report, for the first time, that B. xylophilus associated bacteria may assist the nematode opportunistically in the disease, and that a virulent B. xylophilus isolate displayed a higher tolerance towards the OS conditions than an avirulent isolate.
PMCID: PMC3880045  PMID: 24365493
Bursaphelenchus xylophilus; Catalase; Oxidative stress; Pine wilt disease
25.  Characterization of the Pinus massoniana Transcriptional Response to Bursaphelenchus xylophilus Infection Using Suppression Subtractive Hybridization 
Pine wilt disease (PWD) caused by pine wood nematode (PWN), Bursaphelenchus xylophilus, is the most destructive diseases of pine and poses a threat of serious economic losses worldwide. Although several of the mechanisms involved in disease progression have been discovered, the molecular response of Pinus massoniana to PWN infection has not been explored. We constructed four subtractive suppression hybridization cDNA libraries by taking time-course samples from PWN-inoculated Masson pine trees. One-hundred forty-four significantly differentially expressed sequence tags (ESTs) were identified, and 124 high-quality sequences with transcriptional features were selected for gene ontology (GO) and individual gene analyses. There were marked differences in the types of transcripts, as well as in the timing and levels of transcript expression in the pine trees following PWN inoculation. Genes involved in signal transduction, transcription and translation and secondary metabolism were highly expressed after 24 h and 72 h, while stress response genes were highly expressed only after 72 h. Certain transcripts responding to PWN infection were discriminative; pathogenesis and cell wall-related genes were more abundant, while detoxification or redox process-related genes were less abundant. This study provides new insights into the molecular mechanisms that control the biochemical and physiological responses of pine trees to PWN infection, particularly during the initial stage of infection.
PMCID: PMC3709736  PMID: 23759987
pine wilt disease; differentially expressed genes; suppression subtractive hybridization; Pinus massoniana

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