Localized within‐ and between‐tree variation in nematode distribution during latent state of pine wilt disease makes the disease status cryptic

The pinewood nematode (PWN), Bursaphelenchus xylophilus, is the causal agent of pine wilt disease, an epidemic disease that has severely damaged pine forests in East Asia. The disease has spread to northern areas in Asia and parts of Europe. To prevent disease spread as the forefront of damage prevention, a better understanding of infection status is highly important. Not all infected trees show disease symptoms, and such asymptomatic PWN‐carrying trees are likely to be overlooked and can become a pathogen reservoir. To elucidate PWN infection status in asymptomatic trees, we performed PWN inspection of branches and trunks in 21 test trees in two different conditions: trees that had experienced PWN inoculation and those with suspected PWN infection that had experienced transient foliage discoloration. We detected PWNs in eight test trees (38%) and in 13 (1.5%) of a total of 843 samples. The difference in these percentages suggests that nematode inhabitation was highly localized within the trees, possibly owing to the restricted migration of PWNs. Our data demonstrated that trees that were once weakened but recovered their vigour can persist, as the asymptomatic carriers, in the forest. The implications for disease control are also discussed.     

Pine wilt disease: detection of the pinewood nematode (Bursaphelenchus xylophilus) as a tool for a pine breeding programme

The pinewood nematode (PWN), Bursaphelenchus xylophilus, is a serious quarantine pest first detected in Portugal and Europe in 1999. It is the causal agent of pine wilt disease (PWD). A resistance breeding programme has been initiated to contribute to control the evolution of the disease. Five hundred and four adult maritime pine, Pinus pinaster, trees were phenotypically selected as candidate trees for this programme from an area affected by PWD. To identify tolerance to the nematode, the selected trees were monitored monthly. Over the course of 1 year, 57 candidate trees died and were tested for the presence/absence of the PWN. As accuracy of detection is of major importance, an ITS‐PCR‐based method applied directly to wood from adult maritime pine trees was tested and compared with a standard morphological identification method. The results showed that the use of PCR to detect the pathogen provided more rapid and accurate results in comparison with the standard morphological identification. Thus, this method is suitable to be used in the survey of the breeding population for resistance/tolerance to PWD.     

Direct PCR‐based method for detecting Bursaphelenchus xylophilus,the pine wood nematode in wood tissue of Pinus massoniana

Pine wilt disease (PWD), caused by the pine wood nematode (PWN) Bursaphelenchus xylophilus, leads to serious losses to pine forestry around the world. Pinus massoniana, which is vulnerable to be attacked by the PWN, is the dominant species used in pine forestry in China. The objective of this study is to develop a direct PCR‐based method for detecting B. xylophilus in the wood of P. massoniana without a separate nematode extraction step. A simple procedure was first developed for isolating B. xylophilus DNA in 5 mg pine wood tissue samples harbouring PWN for detection by PCR amplification. A B. xylophilus‐specific amplicon of 403 bp (DQ855275) was generated by PCR from the infested wood tissue. The entire procedure can be completed within 5 h with one pair of primers. This assay can serve as a rapid, cheap and environmentally friendly method to detect B. xylophilus in samples of P. massoniana.     

An inoculation experiment of Japanese <Emphasis Type="Italic">Bursaphelenchus</Emphasis> nematodes on Japanese black and red pine, <Emphasis Type="Italic">Pinus thunbergii</Emphasis> and <Emphasis Type="Italic">P. densiflora</Emphasis>

The pinewood nematode (PWN) Bursaphelenchus xylophilus is an invasive pathogen that was introduced from North America to Asian countries and Portugal and is devastating native pine forests. Some native European and Asian Bursaphelenchus nematodes also have weak to moderate pathogenicity to native pine species. To evaluate the potential risk of native Bursaphelenchus species, we inoculated ten Japanese Bursaphelenchus species into native pine species (the dominant forest species) in Japan, and evaluated their pathogenicity using mortality and tracheal tissue damage as indices. Inoculation was conducted on August 3, 2007, and the symptoms were observed every 2 weeks until February 1, 2008. None of the inoculated trees, excluding the pathogenic PWN inoculated control, showed external disease symptoms; however, four species [a less pathogenic PWN isolate, B. luxuriosae, Bursaphelenchus sp. NK215 (undescribed), and NK224 (undescribed)] caused tracheal tissue damage in inoculated seedlings and showed weak pathogenicity. Therefore, we conclude that there are some potentially pathogenic native species of nematodes distributed in Japan. Interestingly, two of these weakly pathogenic species, B. luxuriosae and NK215, are not associated with Pinaceae trees, suggesting that nematode pathogenicity may be a pre-adaptive character. More experimental studies under different conditions are necessary to accurately evaluate the potential risk of these pathogens.     

Stand characteristics and downed woody debris accumulations associated with a mountain pine beetle (Dendroctonus ponderosae Hopkins) outbreak in Colorado

Lodgepole pine (Pinus contorta Dougl. ex Loud.)-dominated ecosystems in north-central Colorado are undergoing rapid and drastic changes associated with overstory tree mortality from a current mountain pine beetle (Dendroctonus ponderosae Hopkins) outbreak. To characterize stand characteristics and downed woody debris loads during the first 7 years of the outbreak, 221 plots (0.02 ha) were randomly established in infested and uninfested stands distributed across the Arapaho National Forest, Colorado. Mountain pine beetle initially attacked stands with higher lodgepole pine basal area, and lower density and basal area of Engelmann spruce (Picea engelmannii [Parry]), and subalpine fir (Abies lasiocarpa (Hook.) Nutt. var. lasiocarpa) compared to uninfested plots. Mountain pine beetle-affected stands had reduced total and lodgepole pine stocking and quadratic mean diameter. The density and basal area of live overstory lodgepole declined by 62% and 71% in infested plots, respectively. The mean diameter of live lodgepole pine was 53% lower than pre-outbreak in infested plots. Downed woody debris loads did not differ between uninfested plots and plots currently infested at the time of sampling to 3 or 4–7 years after initial infestation, but the projected downed coarse wood accumulations when 80% of the mountain pine beetle-killed trees fall indicated a fourfold increase. Depth of the litter layer and maximum height of grass and herbaceous vegetation were greater 4–7 years after initial infestation compared to uninfested plots, though understory plant percent cover was not different. Seedling and sapling density of all species combined was higher in uninfested plots but there was no difference between infested and uninfested plots for lodgepole pine alone. For trees ≥2.5 cm in diameter at breast height, the density of live lodgepole pine trees in mountain pine beetle-affected stands was higher than Engelmann spruce, subalpine fir, and aspen, (Populus tremuloides Michx.), in diameter classes comprised of trees from 2.5 cm to 30 cm in diameter, suggesting that lodgepole pine will remain as a dominant overstory tree after the bark beetle outbreak.     

日本松材线虫病治理技术考察

日本是世界上松材线虫发生最早、受害损失最严重的国家，也是对松材线虫研究最深入、控制经验最丰富、治理技术最先进的国家。通过对该国多个林木育种中心、森林综合研究所及基层森林经营管理机构、生产者等的访问交流和实地考察，较系统地介绍了松材线虫病在日本的发生危害、研究现状和实用控制技术，提出了有效预防、治理松材线虫病的措施和方法。     

Suppression of ectomycorrhizal development in young Pinus thunbergii trees inoculated with Bursaphelenchus xylophilus

In order to study the changes in ectomycorrhizal development during symptom expression of pine wilt disease, root window observations were conducted concurrent with measurements of leaf water potential as well as photosynthetic and transpiration rates of 5‐year‐old Pinus thunbergii trees that were inoculated with the pinewood nematode (PWN) Bursaphelenchus xylophilus. Infected trees were compared with girdled and uninfected control trees. Ectomycorrhizas developed constantly during the experimental period in control trees but did not develop in the girdled trees. Ectomycorrhizal development ceased within 2 weeks in those trees that finally died after PWN infection. In the trees that survived PWN infection, ectomycorrhizal development ceased within 1–4 weeks of inoculation but was resumed thereafter within 3–6 weeks. Ectomycorrhizal development ceased prior to a decrease in both photosynthetic rate and leaf water potential in the inoculated trees.     

First record of Ektaphelenchoides pini associated with Ips sexdentatus on Pinus nigra laricio in Italy

In February 2015, an unexpected windstorm downed five hectares of a European black pine Pinus nigra subsp. laricio forest formation located close to Vallombrosa, Florence (Central Italy). In the following spring, an extensive survey was conducted in the area. Felled trees, stumps and all the suitable plant material were screened for the presence of the pinewood nematode (PWN), Bursaphelenchus xylophilus, by sampling wood and bark. Bark beetles were then collected from the gallery systems on the inner side of bark samples and observed in the laboratory. The following bark beetles were morphologically identified: Ips sexdentatus, Orthotomicus erosus, O. laricis and Pityogenes bidentatusa. The dissection of Ips sexdentatus allowed the extraction of numerous nematodes that were morphologically and molecularly identified as Ektaphelenchoides pini. Conversely, only few nematode specimens were isolated from either pine bark or wood. These individuals could be only molecularly identified and belonged to an undescribed nematode taxon. Even though no PWN was recorded in the investigated sites, our survey allowed the detection of a new association between E. pini and I. sexdentatus on P. nigra.     

A new on‐site detection method for Bursaphelenchus xylophilus in infected pine trees

The pinewood nematode (PWN), Bursaphelenchus xylophilus, is the causal agent of pine wilt disease (PWD), which is a major problem in East Asia and West Europe. Quick identification of PWN is needed to prevent the dispersal of PWD to healthy forests. Various detection methods of PWN have been developed using anatomical characters and molecular markers. These methods are not suitable for rapid diagnosis because it is difficult to distinguish B. xylophilus from the non‐pathogenic species Bursaphelenchus mucronatus based on morphological characters without expertise in nematode taxonomy and most PCR or isothermal amplification detection methods require time‐consuming processes. In this study, we developed an on‐site PWN detection method using a recombinase polymerase amplification (RPA) assay with a novel extraction buffer (DAP buffer). This new PWN detection method is able to extract genomic DNA from PWN in pinewood by simple buffer consisting of sodium hydrate, polyethylene glycol 200 and dimethyl sulfoxide in 10 min without using the experimental devices and able to distinguish between B. xylophilus and other Bursaphelenchus spp. by amplifying the species‐specific 5S rDNA fragment of B. xylophilus in 10 min. Taken together, our protocol can obtain the result for the detection of PWN in pine tree samples within 30 min. This result suggests that RPA/DAP assay is much faster, easier and cheaper than the conventional methods for detecting PWN.     

Insect vectors of the pinewood nematode: a review of the biology and ecology of Monochamus species

Pine wilt disease (PWD), caused by the pinewood nematode (PWN), Bursaphelenchus xylophilus (Steiner and Buhrer) Nickle 1970 , is a serious threat to susceptible pine forests of the world. The PWN is primarily vectored by Monochamus species (Coleoptera: Cerambycidae). The first occurrence of PWD was reported from Japan in the early 1900s. Following this report, Japanese scientists documented the community of bark‐ and phloem‐inhabiting insects associated with the nematodes in dying trees to determine possible vectors of the nematode. Monochamus alternatus was reported to be the most effective vector in Japan. The primary vector in North America is Monochamus carolinensis, and in Europe, it is Monochamus galloprovincialis. Further studies have been expanded through the nematode‐invaded countries of Korea, Taiwan, China and Portugal. There is an interspecific association between the PWN and its insect vectors, and it is an obligatory component of the disease cycle. It is crucial to understand this relationship as well as the population ecology of the beetle to aid in monitoring and control of this worldwide threat to pine forests. Studies to date indicate a remarkable similarity among beetle species around the globe for a variety of life‐history traits, including lifespan, adult emergence numbers, flight capability, nematode transmission rates and attraction to pine volatiles. Wherever pines are found, there is a beetle species capable of transmitting the nematode. Although flight performance and range is generally poor for this group of beetle vectors, the cryptic nature of the species and the lack of interest in the beetles by countries in the absence of the nematode have led to the disease establishing a foothold in a variety of countries such as Portugal. In this paper, studies conducted in different countries on Monochamus vector species of the PWN are compared and discussed.     

Response of pine forest to disturbance of pine wood nematode with interpretative structural model

Pine wood nematode (PWN, Bursaphelenchus xylophilus), originating from North America, causes destructive pine wilt disease. Different pine forest ecosystems have different resistances to B. xylophilus, and after its invasion, the resilience and restoration direction of different ecosystems also varies. In this study, an interpretative structural model was applied for analyzing the response of pine forest ecosystem to PWN disturbance. The result showed that a five-degree multi-stage hierarchical system affected the response of the pine forest ecosystem to PWN disturbance, in which direct affecting factors are resistance and resilience. Furthermore, the analysis to the 2nd, 3rd and 4th degree factors showed that not only does distribution pattern of plant species and pine’s ecological features affect the resistance of pine forests’ ecosystem, but removal of attacked trees and other measures also influence the resistance through indirectly affecting the damage degree of Monochamus alternatus and distribution pattern of plant species. As for resilience, it is influenced directly by soil factors, hydrology, surrounding species provenance and biological characteristics of the second and jointly dominant species, and the climate factors can also have a direct or indirect effect on it by affecting the above factors. Among the fifth elements, the elevation, gradient and slope direction, topographical factors, diversity of geographical location and improvement of prevention technology all influence the response of pine forest ecosystem to PWN disturbance. __________ Translated from Scientia Silvae Sinicae, 2007, 43(8): 85–90 [译自: 林业科学]     

Heartwood/sapwood variation of western redcedar as influenced by cultural treatments and position in tree

We studied heartwood and sapwood variation in western redcedar (Thuja plicata) at three sites, including a 95-year-old naturally regenerated, unmanaged stand, a 35-year-old planted spacing trial, and a 30-year-old naturally regenerated stand to which thinning and fertilization treatments had been applied. In the 95-year-old stand, we studied within-tree variation in heartwood and sapwood. In the thinning/fertilization trial and the planted spacing trial, we studied effects of cultural practices and growth rate on heartwood and sapwood. In the trees that we studied, sapwood width was generally fairly narrow, rarely exceeding 3.5 cm. Heartwood formation in western redcedar appeared to begin at a relatively small stem diameter (7 cm) and at a young age, probably 10–15 years. The amount and proportion of heartwood increased with distance downward from the top of the tree, with the implication that older trees will contain a greater proportion of heartwood than younger trees. For any given age, it appears that cultural treatments that favor rapid growth will result in stems with greater amounts of both sapwood and heartwood, and a greater proportion of heartwood.     

Identification and genetic analysis of the pinewood nematode Bursaphelenchus xylophilus from Pinus yunnanensis

In China, pinewood nematode (PWN), Bursaphelenchus xylophilus, was first discovered from Pinus thunbergii in 1982. Thus far, 14 species in the genus Pinus have been reported to be infected by PWN under natural conditions. Pinus yunnanensis, a pine species native to south‐western China, is considered a pioneer tree for barren hill afforestation in areas undergoing rocky desertification. In this study, we detected PWN in dead P. yunnanensis trees in Anlong County, Guizhou Province, China, using both morphological and molecular methods. To our knowledge, this is the first report of PWN from P. yunnanensis in China. To investigate the possible origin of this new outbreak, mitochondrial cytochrome oxidase gene subunit I and cellulase gene sequences were used to evaluate genetic relationships among worldwide PWN isolates. Phylogenetic tree and haplotype networks revealed that the Anlong isolate (BxChQAL008) sequence was identical to those of seven Chinese isolates collected from Sichuan, Chongqing, Zhejiang, Anhui and Shandong (372–1500 km from Anlong County), but different from the isolate BxChQZY030 collected from the same province (330 km from Anlong County). It is suggested, therefore, that more than one introduction of PWN into Guizhou Province has taken place. The Anlong isolate was likely introduced from neighbouring or more distant provinces rather than from outside China. Moreover, the absence of a correlation between geographic and genetic distance was observed using Mantel test analysis, providing evidence that human‐induced dispersal plays a fundamental role in the spread of the PWN in this region.     

Long‐term survival and non‐vector spread of the pinewood nematode,Bursaphelenchus xylophilus,via wood chips

The pinewood nematode, Bursaphelenchus xylophilus, is the causal agent of pine wilt disease and is transmitted to new host trees by beetles of the genus Monochamus. The increasing interest in imported wood chips from North America for paper production and energy purposes and the corresponding phytosanitary risk of non‐vector transmission of B. xylophilus has been discussed since 1984, the year of the first interception of B. xylophilus in wood chips in the European Union. The long‐term survival of B. xylophilus in wood chips and its non‐vector spread from infested wood chips to non‐infested trees were studied. Pinus sylvestris logs were inoculated with a suspension of B. xylophilus to produce infested wood chips. During the long‐term storage test, B. xylophilus in P. sylvestris wood chips were examined. Four variants, including sealed and openly stored wood chips at both 15°C and 25°C, were studied. For the test of non‐vector spread, B. xylophilus ‐infested wood chips were placed on three‐ to four‐year‐old P. sylvestris saplings under different conditions. Bursaphelenchus xylophilus survived for more than 1 year at both temperatures in the sealed wood chips, which was significantly longer than for the openly stored variant at 25°C. Temperature, tree condition and wood chip location all influenced non‐vector spread through wood chips. Of the 480 trees that were in contact with infested wood chips and showed clear symptoms of pine wilt disease, B. xylophilus were extracted from 42 pines at 25°C and one pine at 15°C. The highest B. xylophilus infestation rates resulting in clear pine wilt disease symptoms (75%) were found in infested wood chips directly attached to stem‐wounded trees at 25°C. However, more variants exhibited B. xylophilus infestation at this temperature; trees with stem or root injuries plus direct contact with infested wood chips to the wounded part were primarily affected. Moreover, non‐vector spread was also detected in stem‐ and root‐injured pines without any direct contact with infested wood chips. Our results confirmed that B. xylophilus can survive for long periods in wood chips and can be transmitted from infested wood chips to damaged trees, but the likelihood of such PWN establishment should be low compared to spread through vectors. These findings must be considered in the pest risk analysis of B. xylophilus, and studies using outdoor trials should be carried out to complete this pest risk analysis.     

Examination of within-tree variations and the heights representing whole-tree values of derived wood properties for quasi-non-destructive breeding of Eucalyptus camaldulensis and Eucalyptus globulus as quality pulpwood

Within-tree variations of derived wood properties of Runkel ratio, Luces shape factor, slenderness ratio, and solids factor were examined for Eucalyptus camaldulensis and Eucalyptus globulus trees and the tendency difference in the within-tree variations between individuals and between species, in both radial and axial directions by statistical data analysis. These properties are important for quality breeding of pulpwood. In both species, within-tree variations were generally observed as higher values in the upper and outer parts compared with other parts of the trunk for Runkel ratio and Luces shape factor. In E. camaldulensis, within-tree variations were observed as higher values in the upper and outer parts compared with other parts of the trunk for slenderness ratio and solids factor. In E. globulus, within-tree variations were observed as higher values in the outer parts compared with other parts for slenderness ratio and solids factor. However, significant difference of tendency was observed in radial variation between individuals of E. globulus for Runkel ratio and in both radial and axial variations between species for solids factor. Furthermore, within-tree variations of derived wood properties were analyzed to determine a sampling height in the trunk which can be used to represent whole-tree values. Representative heights of derived wood properties from two trees were found to be 2.8m in E. camaldulensis (except for Runkel ratio and Luces shape factor) and 1.8m in E. globules (except for Runkel ratio), regardless of differences in tree height (growth rate) and in tendency of within-tree variation of derived wood properties.     

Pine wilt disease: a short review of worldwide research

This article summarizes the results of the research papers presented at the International Symposium on pine wilt disease (IUFRO Working Party Meeting 4.04.03) held in July 2009, at Nanjing, China. The general topics covered were on pine wilt disease (PWD), its causal organism, the pinewood nematode (PWN) Bursaphelenchus xylophilus, plus other PWN-associated microorganisms that play a significant role in PWD such as bacteria (e.g. Pseudomonas fluorescens). Most of the papers that are reviewed are based on work on PWD-PWN in East Asia and Russia. Specific topics covered include: 1) the fundamental conceptions of PWD development, 2) pathogenicity, 3) host-parasite relationships including the histopathology of diseased conifers and the role of toxins from bacteria-nematode ecto-symbionts, 4) PWN life cycle and transmission, 5) B. xylophilus dissemination models, 6) associations (with other nematodes), 7) diagnostics, 8) quarantine and control of the PWN and 9) biocontrol of the PWN.     

Insect (Salebria sp.) infestation and impact on Vitellaria paradoxa C.F. Gaertn. fruit production in agroforestry parklands

Vitellaria paradoxa, known as Shea tree or karité, is one of the dominant trees of agroforestry parklands in Sahelian region with an immense commercial value. In this study, we examined infestation of fruit-bearing shoots and fruits on two sites in western Burkina Faso across different reproductive phenophases, crown heights and aspects. The number of infested and healthy shoots and fruits were counted on 60 reproductively active trees randomly selected from on-year shea trees in each site, using a metal frame (0.5 × 0.5 m) to mark a fixed area of the canopy for sampling. The shoot and fruit borer was identified as Salebria sp. (Lepidoptera:Pyralidae), which was not reported earlier. The proportion of infested trees and shoots varied significantly between sites and among reproductive phenophases (P < 0.0001). The site close to the natural forest had the highest infestation rate than distantly located site. Among reproductive phenophases, the proportions of infested trees and shoots were the highest during flowering and fruit set, respectively. Fruit infestation also varied significantly between sites, crown heights and aspects (P < 0.001). The site close to the natural forest and the crown with north-east aspect were heavily infested while the bottom part of the crown had the lowest infestation. As a whole, 49–80% of trees, 24–31% of shoots and 4–15% of fruits were found infested, depending on the site. These infestation levels combined with precocious flower and fruit abscissions would pose serious economic losses that justify a call for immediate pest control initiatives.     

A loop‐mediated isothermal amplification‐based method for detecting Bursaphelenchus xylophilus from Monochamus alternatus

The introduction of the pine wood nematode (PWN), Bursaphelenchus xylophilus, to new areas has impacted on the international economy. Therefore, accurate and reliable detection methods for PWN are essential for the control and management of this pest. A rapid and economic method for detecting PWN may be developed focusing on the PWN vector (Monochamus alternatus). This work standardized a loop‐mediated isothermal amplification (LAMP) method using newly designed primer sequences based on the syg‐2 gene, which encodes the synaptogenesis protein syg‐2. Loop‐mediated isothermal amplification was conducted at 63°C for 60 min using six sets of primers. The result was confirmed by visual observation. A positive reaction was confirmed by SYBR Green I fluorescence dye under light thermal cycling. The lower limit of DNA detection was 51.4 pg/μl in both LAMP and 51.4 ng/μl in PCR. Therefore, the LAMP was 1,000 times more sensitive in DNA detection than PCR. The LAMP is a relatively new, highly accurate and rapid molecular technique that can rapidly detect infectious agents in the field without requiring sophisticated instruments, giving a visually readable result. The method greatly improves detection, without requiring professional knowledge and expensive, sophisticated equipment. Therefore, this system is suitable for quarantine and field detection.     

Representative heights for assessing whole-tree values of cell-type proportions in Eucalyptus camaldulensis and E. globulus

Eucalyptus camaldulensis Dehnh. and E. globulus Labill. are economically important species for wood and pulpwood materials. Representative heights for assessing whole-tree values of cell-type proportions(vessels, fibers, ray and axial parenchyma percentages) using increment cores were examined by analysis of within-tree variations. Pattern differences were evaluated between trees and species in both radial and axial directions by statistical data analysis(Moses test). In E. camaldulensis,within-tree variation of vessel percentage was generally higher in the upper and outer regions of the trunk. In contrast, E. globulus within-tree variation was unclear. In both species, although no clear pattern of fiber percentages was observed, within-tree variations of ray and axial parenchyma levels were higher in the lower and inner regions.Significant differences in patterns were observed in the axial variation between species for vessel percentages and in the radial variation between trees of E. camaldulensis for ray parenchyma percentages. The representative heights for assessing whole-tree cell-type proportions were 0.8 mabove the ground for E. camaldulensis and 2.8 m for E.globulus, regardless of differences in tree height and pattern of within-tree variation of cell-type proportions.