Suggestions on management measures of pine forest ecosystems invaded by <Emphasis Type="Italic">Bursaphelenchus xylophilus</Emphasis>

Pine wood nematode （PWN）, Bursaphelenchus xylophilus （Steiner et Buhrer） Nickle is an important invasive alien species in forests of China and has become one of the most destructive forest diseases. In order to improve the resistance and resilience of pine forest ecosystems against B. xylophilus invasion and make the pine forest ecosystem more timely responsive to PWN invasion, we made some recommendations based on five years of intensive observations. We advocate a set of management measures with the theme “Prevention is priority, but integrated with curative techniques and ecological resilience” on the pine forest ecosystem invaded by B. xylophilus; details of accurate measures are proposed. The aim is to discover the underlying problems of present pine forest ecosys-tems and to take, correspondingly, administrative measures and strategies, which will encourage the pine forest ecosystem to develop in a benign way.     

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.     

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.     

First isolation of pine wood nematode from Pinus tabuliformis forests in China

Previous risk prediction studies have mostly shown that suitable areas for pine wood nematode, Bursaphelenchus xylophilus (PWN), in China are mainly concentrated in eastern and southern China, with pest reaching no further northern than the Yangtze River basin. The Chinese pine (Pinus tabuliformis, Chinese pine or Chinese red pine) is the main, native, afforestation species in areas to the north of the Yangtze River basin. To our knowledge, there is no report on PWN infecting Chinese pines in the north of the Yangtze. In this study, we used Baermann funnel method and several PCR primers to detect the presence of PWN in Chinese pine forest fields in 2010 in Shaanxi, China, which is far to the north of the Yangtze River and a cause for concern. Pinus koraiensis (Korean pine) may also be at risk and should also be monitored to prevent invasion by PWN.     

Comparison of histological responses and tissue damage expansion between resistant and susceptible Pinus thunbergii infected with pine wood nematode Bursaphelenchus xylophilus

Pine wilt disease caused by the pine wood nematode (PWN), Bursaphelenchus xylophilus, has been epidemic and has had disastrous impacts on pine forests and forest ecosystems in eastern Asia. Many pine species in this area are susceptible to this disease. Pinus thunbergii is particularly susceptible. In Japan, tree breeders have selected surviving trees from severely damaged forests as resistant candidates, and have finally established several resistant varieties of P. thunbergii. However, this breeding procedure requires much time and effort due to the lack of physiological and phenotypical information about resistance. To investigate the resistance mechanisms of selected P. thunbergii, we compared histochemical responses, tissue damage expansion, and PWN distribution in resistant and susceptible clones of P. thunbergii after PWN inoculation. The results suggested that the mechanisms of resistance are as follows: damage expansion in the cortex, cambium, and xylem axial resin canals are retarded in resistant trees soon after inoculation, probably due to the induction of wall protein-based defenses. Suppression of PWN reproduction was particularly caused by inhibition of damage expansion in the cambium. The slow expansion of damage in each tissue provides time for the host to complete the biosynthesis of lignin in the walls of cells that surround the damaged regions. This lignification of cell walls is assumed to effectively inhibit the migration and reproduction of the PWNs. The mechanism of initial damage retardation is presumed to be a key for resistance.     

Comparison of the gene expression profiles of resistant and non‐resistant Japanese black pine inoculated with pine wood nematode using a modified LongSAGE technique

The Japanese black pine (Pinus thunbergii) is highly susceptible to pine wilt disease caused by the pine wood nematode (PWN; Bursaphelenchus xylophilus). To cope with this disease, researchers and tree breeders selected PWN‐resistant individuals in a previous breeding program. In an attempt to understand the mechanisms of resistance in the Japanese black pine, we created four LongSAGE (serial analysis of gene expression) libraries. A total of 20 818 tags were studied, including 5194 tags from a PWN inoculated resistant pine, 5218 a non‐inoculated resistant pine, 5194 an inoculated non‐resistant pine, and 5212 a non‐inoculated non‐resistant pine. The analysis of the libraries indicated that 14 tag species were significantly up‐regulated (e.g., pathogenesis‐related proteins 2 and 4, osmotin, lipoxygenase, and chalcone synthase), and nine were down‐regulated (eukaryotic translation initiation factor SUI1, translationally controlled tumor protein, and xyloglucan endotransglycosylase) by the PWN inoculation in both the resistant and non‐resistant pines. On the other hand, 38 tag species were significantly expressed at a higher level only in the resistant pine (catalase, dienelactone hydrolase family protein) and 25 were expressed at a higher level in the non‐resistant pine (pathogenesis‐related proteins 1, 2, and 3, and leucoanthocyanidin dioxygenase). These differentially expressed genes are presumed to reflect some of the differences between the resistant and non‐resistant pines. Our results provide valuable information on the complex responses induced in the resistant and non‐resistant pine trees in response to PWN invasion.     

Impact of the invasion of pinewood nematode and the following different removal disturbance intensities on the plant diversity of Masson pine community

Reported in this paper are our findings of the study on changes in species diversity of different Masson pine communities after the invasion of Bursaphelenchus xylophilus (Steiner and Buhren) Nickle and subsequent and different removal disturbance management approaches. Based on the results, the species diversity indices for the arbor layers of the different communities can be arranged in the following order (from high to low): 1) broad-leaved stand after the removal of all infected pine trees in the pure pine stand in Fuyang; 2) lightly infected Masson pine and Schima superba mixed stand in Fuyang; 3) uninfected stand mixture of Masson pine and Castanopsis fargesii as a control; 4) lightly infested pure Masson pine stand in Fuyang; 5) Quercus variables stand formed following the selective removal of infected pine trees from a mixed Masson pine and Q. variables stand in Zhoushan Islands; 6) pure young Masson pine stand formed following the removal of all infected pine trees from a pure Masson pine stand; 7) pure Liquidambar formosana stand after the removal of infected pine trees from a pure pine stand in Zhoushan Islands; 8) a mixed stand consisting of Pinus thunbergii and the Masson pine in Zhoushan Island; and 9) moderately infected Masson pine stand in Zhoushan Islands. All the three diversity indices (R ₀ = richness, H′ = Shannon-Wiener index, and E = evenness) for the shrub layer did not show any significant differences among the various communities except for the pure pine stand in Zhoushan Island, which had the lowest diversity. The three indices for the herb layer of the pure young Masson pine, Q. variables stand, and L. formosana stand were higher than that of other stands. The integrated analysis showed that the Masson pine forest in different geographical situations and extent of damage had distinct disparity, just as in different disturbance degrees and restoring manners. We created the “index of disturbing intensity of stump and fallen woods” (IDISFW) to represent the degree of disturbance of tree removal to plant diversity. We found that the relationship between the species diversity index and the IDISFW shows both a lower and higher degree of removal disturbance that follows the “mid-altitude bulge” theory. Specifically, both excessive and insufficient removal of infected trees will cause a decline of plant species diversity to a certain degree. Covariance analysis indicated that different IDISFW had no significant effects on the species diversity of the arbor layer, but had different impacts on that of the shrub and herb layers, and therefore can be used to assess changes in species diversity of different Masson pine communities after the invasion of pine wood nematode. __________ Translated from Chinese Journal of Applied Ecology, 2006, 17(7): 1,157–1,163 [译自: 应用生态学报]     

Parasitoids associated with Monochamus galloprovincialis (Oliv.) (Coleoptera: Cerambycidae) within the pine wilt nematode-affected zone in Portugal

Until recently, the pine sawyer Monochamus galloprovincialis (Olivier) (Coleoptera; Cerambycidae) was considered a secondary forest pest in Portugal. Its pest status changed when it was found to be the main vector of the recently introduced pine wood nematode (PWN) Bursaphelenchus xylophilus (Steiner and Bührer) Nickle (Nematoda; Aphelenchoididae). A survey in three locations within the PWN-affected zone was conducted in order to identify parasitoids associated with immature stages of the beetle and to evaluate their importance. No parasitoid was found associated with the insect eggs whereas 10% of the larvae were found parasitised by three Braconidae. Cyanopterus flavator Fabricius was the most numerous species; Iphiaulax impostor (Scopoli) and Coeloides sordidator Ratzeburg were the other species present. A review of the parasitoids associated with Monochamus in Europe, eastern Asia and North America was made, and the possibility of using the local species in future biological control programmes in Portugal is briefly discussed.     

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.     

Effect of pine wood nematode invasion on pine community functions in the Pinghu region, Zhejiang Province, eastern China

In order to investigate the effect of invastion by pine wood nematode （PWN）, this study analyzed severalfunctional indices, i.e., the increment in DBH and stand volume and biomass, in the damaqed stands with various mixedpercentages of Pinus massoniana and P. thunbergii and with different levels of damage. According to the results of rate of change in increment of DBH and stand volume, the forest ecosystem resistance against PWN increased with a reduct on n the m xed ratio of pine. The resistance was highest with a mixed percentage of 50%. The invasion of PWN hanged the corresponding relationship of increment between DBH and stand volume （pure stands 〉 7：3 conifer and roadleaf 〉 6：4 conifer and broadleaf 〉 5：5 conifer and broadleaf） among the P. thunbergii stands when there is no amage, but for P. massoniana stands this phenomenon did not occur. For the increment rate of DBH and stand volume, is significant change in P, thunbergii forest indicates that the resistance of pure P. thunbergii forest was higher than at of P. massoniana. The invasion of PWN accelerates the succession from pure stands to mixed stands and then tohe broadleaf evergreen stands.     

Community-based management of rural pine forests in a suburban village of Hiroshima prefecture, western Japan

Community-based management of a rural pine forest in a small suburban community, was examined and assessed. In particular, the study focused on theMatsutake project, which is an initiative seeking to help in the maintenance of a communal pine forest via the cultivation of theMatsutake mushroom by a local seniors’ group. From an ecological perspective, the maintenance work is found to be effective in the conservation and regeneration of the pine forest ecosystem, including its species diversity, especially in the herb layer. From a sociological perspective, theMatsutake project presented a valuable opportunity to strengthen connections not only within the seniors’ group, but also between senior citizens and other generations. However, subsidies for the project have tended to be gradually reduced, because it is difficult for non-involved community members to appreciate the benefits of the project. For the project to be sustainable, more widespread participation of the community is essential. A framework for wider analysis of local participatory forest management is also deemed necessary.     

Proactive intervention to sustain high-elevation pine ecosystems threatened by white pine blister rust

Only recently have efforts begun to address how management might prepare currently healthy forests to affect the outcome of invasion by established non-native pests. Cronartium ribicola, the fungus that causes the disease white pine blister rust (WPBR), is among the introductions into North America where containment and eradication have failed; the disease continues to spread. Ecosystem function is impaired by high rust-caused mortality in mature five-needle white pine forests. This paper evaluates five proactive management options to mitigate the development of impacts caused by white pine blister rust in threatened remote high-elevation five-needle pine ecosystems of western North America. They are: reducing pest populations; managing forest composition; improving host vigor; introducing resistant stock with artificial regeneration; and diversifying age class structure to affect the natural selection process for resistance. Proactive intervention to manage and facilitate evolutionary change in the host species may sustain host populations and ecosystem function during pathogen naturalization.     

中国松材线虫病研究

松材线虫作为一种外来入侵物种, 自其发现之日起就对中国的松林生态系统造成了巨大的破坏和冲击。松材线虫在中国有着相当广泛的适生区, 到目前为止已在江苏、浙江、安徽、福建、江西及台湾和香港等17个省(市)和地区发生危害。由于其致病机理尚不明确, 致使该病的防治和控制工作进展缓慢。文中指出目前该病的检验检疫与防治方法中存在的不足, 并提出松材线虫致病性的分子机制研究、松属树木抗病机制研究、松林系统对松材线虫病入侵的生态抵御机制等几个方面是将来研究的重点方向。     

Correlation of seedling size and branch number with disease resistance of Pinus thunbergii seedlings to Bursaphelenchus xylophilus

Japanese black pine (Pinus thunbergii) seedlings resistant to pine wood nematode (PWN; Bursaphelenchus xylophilus) are routinely selected in Japanese field inoculation trials. Correlations between morphological factors (such as height, stem diameter at ground level and number of branches on seedlings) and disease resistance were examined to improve the production efficiency of 1‐year‐old black pine seedlings for inoculation. Family relatedness and environmental conditions strongly affected seedling resistance; accordingly, logistic regression analysis was used to separate effects of these two variables. Height and stem diameter at ground level significantly correlated with disease resistance in seedlings inoculated with PWN. Because (a) interactions between stem diameter at ground level and environmental condition were significant and (b) height did not interact with any other factor, it was concluded that height of 1‐year‐old Japanese black pine seedlings independently correlated with PWN resistance. Thus, field inoculation tests should use tall seedlings to achieve enhanced survival rates.     

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.     

Niche of insect borers within Pinus massoniana infected by pine wood nematode

In November 2003 and June 2004, the insect borers and their spatial distribution within Pinus massoniana were investigated in Zhoushan City, in East China’s Zhejiang Province, where pine wood nematode (Bursaphelenchus xylophilus) are typically found. The niche width, proportional similarity of niche and the niche overlap of dominant species of dying trees were computed. Results show that five insect species infect and damage Pinus massoniana, which had been infected by pine wood nematodes, among which four are wood boring beetles and one termite. Species within host trees vary from winter to summer and all the species have their own niche width, proportional similarity of niche and the niche overlap. They can achieve competitive equilibrium and coexistence according to their biological characteristics and life habits. __________ Translated from Journal of Beijing Forestry University, 2005, 27(6): 108–111 [译自: 北京林业大学学报, 2005, 27(6): 108–111]     

CO2 fluxes of a Scots pine forest growing in the warm and dry southern upper Rhine plain, SW Germany

The effects of the warm and dry weather in the southern upper Rhine plain in the southwest of Germany on the carbon balance of the Scots pine forest at the permanent forest meteorological experimental site Hartheim were analysed over a 14-month period. The investigation of the net ecosystem exchange of carbon dioxide (F _NEE) of the Scots pine forest started in the extraordinary hot and dry August 2003. Carbon dioxide fluxes were measured continuously using an eddy covariance system and analysed by use of the EDDYSOFT software package. After determining the temperature dependence of the forest ecosystem respiration and the daytime light dependence of the CO₂ exchange, monthly and annual carbon balances of the Scots pine forest were calculated. Mean peak daytime F _NEE rates observed in August and September 2003 (−6.5±3.6 μmol m⁻² s⁻¹) were drastically lower than in August and September 2004 (−11.8±5.2 μmol m⁻² s⁻¹), which did not show pronounced deviations from the mean long-term (1978–2002) climatic conditions. In August 2003, the Hartheim Scots pine forest was a distinct CO₂ source (35 g C m⁻²). The estimates of the annual carbon sink strength of the Scots pine forest ranged between −132 g C m⁻² (August 2003–July 2004) and −211 g C m⁻² (October 2003–September 2004). The main uncertainty in the determination of the carbon balance of the Hartheim Scots pine forest was introduced by the frequently low turbulence levels, i.e. the friction velocity corrected night-time F _NEE fluxes.     

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.     

马尾松不同种源氨基酸含量与抗松材线虫病的关系

在选出和验证抗病马尾松种源的基础上,研究了马尾松不同抗病性种源的树体内氨基酸含量的差异。结果表明,抗病种源在接种松材线虫前后均比较感种源含有较少的组氨酸、苏氨酸、甘氨酸、异亮氨酸、胱氨酸、甲硫氨酸、亮氨酸、酪氨酸和精氨酸等９种游离氨基酸。它们与感病指数均呈正相关,这说明游离氨基酸含量低的马尾松种源抗松材线虫病。１１种固态氨基酸的含量与感病指数有负相关性,说明其含量较高的种源具较强抗病性能。