松材线虫携带细菌产生抑制真菌的物质

研究发现松材线虫携带的两株荧光假单胞菌GcM5-1A、ZM4A和恶臭假单胞菌ZpB1-2A具有抑制真菌菌丝生长的作用.首先对供试的8个细菌菌株进行抑菌活性生物测定,从中筛选出3个具有抑菌活性的细菌菌株;接着对细菌培养液及滤液、死细菌及活细菌进行生物测定;最后对细菌代谢物的性质进行生物测定.实验证明抑制真菌菌丝生长的活性成分为松材线虫携带细菌的挥发性代谢产物.     

Pathogenicity of the pine wood nematode at different developmental stages

The ability of the pine wood nematode,Bursaphelenchus xylophilus, a pathogen that causes pine wilt disease, to kill cortical cells of Japanese black pine,Pinus thunbergii, during early development of the disease was conjectured to be a function of nematode developmental stage. A tangential segment of bark was removed from a 2-cm-long current-year stem. The cortex-exposed segments with cut cortical resin canals were designated as + RC-segments and those without them as − RC-segments. When a nematode population containing many older juveniles and adults (NL) was inoculated onto the cut surface, the − RC-segments were still alive 4 d after inoculation, as were non-inoculated control segments. When cortex-exposed segments were inoculated with either a nematode population containing many younger nematodes (NS) or with nematodes isolated from inoculated pine cuttings that also contained many younger juveniles, most tissue cells in − RC-segments died 4 d after inoculation, suggesting that younger juveniles killed pine cells directly, in contrast with older juveniles and adults. When nematodes were inoculated onto + RC-segments in which they could easily enter resin canals, both NL and NS killed the segment tissues. This suggests that NL is pathogenic to pine cells while living in resin canals. Such differences in the pathogenicity of NL and NS to pine parenchymatous cells were also demonstrated in a pathogenicity assay system using bark peelings, which allowed an estimate of direct attack on the cambial cells by nematodes. Based on these results, we hypothesize that younger juveniles are pathogenic to pine parenchymatous cells, while adults and older juveniles are not pathogenic. This work was supported in part by Grants-in-Aid for Scientific Research (No.01440012 and 06454088) and for Young Scientists (to K.I.) from Ministry of Education, Science, Culture, and Sports of Japan, and by a grant from PROBRAIN.     

Mutualistic symbiosis between Bursaphelenchus xylophilus and bacteria of the genus Pseudomonas

Interactions between the pine wood nematode (PWN), Bursaphelenchus xylophilus, and bacteria of the genus Pseudomonas were examined by cultivating axenic PWN and bacterial strains using callus of Pinus thunbergii. Ten (Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas cepacia and Pseudomonas spp.) of the 29 bacterial strains tested, significantly increased the reproduction of PWN. The rest of the bacteria (19 strains of 10 species) inhibited the reproduction of PWN completely. The growth of 18 of the 29 bacterial strains tested, including the 10 strains promoting PWN reproduction, was significantly increased by the presence of PWN. It indicated a mutualistic symbiotic relationship between PWN and the 10 bacterial strains in the genus Pseudomonas. The bacterial mutualistic symbionts are organisms, which may have co‐evolved with PWN rather than being accidentally associated. The finding provides further evidence for our hypothesis that pine wilt disease is complex, induced by both PWN and associated phytotoxin‐producing bacteria.     

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]     

Physiological process of the symptom development and resistance mechanism in pine wilt disease

Pathophysiological changes during the symptom development of pine wilt disease are reconsidered from recent investigations. The symptom development is divided into two stages: the early and the advanced stages. In the early stage, small number of nematodes migrate in cortex, then in xylem of the stem, and induce denaturation and necrosis of parenchyma cells. These changes in parenchyma are regarded as defense reactions of pines which result in terpene synthesis in xylem cells and embolism in tracheids. Such changes in the early stage can be induced in both susceptible and resistant pine species by either virulent or avirulent isolates of pinewood nematode (Bursaphelenchus xylophilus), or byB. mucronatus. No change occur in physiological status of leaves, and nematode reproduction is suppressed during this stage. Pine trees can survive if symptom does not progress from this stage. The symptoms of the advanced stage usually occur only in susceptible pines infected by virulent nematode isolates. At the beginning of the advanced stage, enhanced ethylene production by stem which coincides with cambial destruction occurs, and results in embolism of the outermost xylem in the portion. The embolism causes decrease in leaf water potential and cessation of photosynthesis. After cessation of photosynthesis, symptoms develop drastically with a burst of nematode population. There seems to be some unknown mechanism which suppress nematode reproduction and invasion to the cambial zone. This mechanism is thought to be photosynthesis-dependent, so that in photosynthesis-decrased conditions, even avirulent nematodes can multiply and invade cambium to induce tree death. Water stress in hot and dry summer should accelerates symptom development from the early to the advanced stage through such decrease of photosynthesis-dependent “cambial resistance”.     

松材线虫伴生细菌的分离鉴定及其致病性

从松材线虫虫体上分离到两个细菌菌株:GD1和R,经鉴定,菌株GD1为坚强芽孢杆菌.利用水培马尾松离体松枝作接种材料,接种消毒后的松材线虫(Bx)、GD1、R、Bx GD1和Bx R.结果表明,松材线虫和坚强芽孢杆菌混合接种,松枝发病,松枝流脂减少至停止,蒸腾作用降低,单独接种松材线虫或坚强芽孢杆菌,松枝不发病.菌株R对松材线虫病的发生没有影响.提出松材线虫及其伴生细菌均是松材线虫病不可缺少的致病因素.     

Development of two reverse transcription‐PCR methods to detect living pinewood nematode,Bursaphelenchus xylophilus,in wood

Pinewood nematode, Bursaphelenchus xylophilus, is an inhabitant of native pine species of North America, where its presence in trees is non‐pathogenic. By contrast, the introduction of this nematode to forests overseas has devastated some pine stands and is recognized as a pest of phytosanitary concern by some countries' National Plant Protection Organizations. The ability to detect B. xylophilus in internationally traded wood products is crucial to reduce the spread of this organism. Current molecular techniques for the detection of B. xylophilus rely on the presence of genomic DNA and thus will detect both living and dead nematodes without differentiation. The detection of dead nematodes could lead to unnecessary trade disruption. Therefore, accurate techniques for the detection of and differentiation between live and dead B. xylophilus are critical. We have developed an endpoint RT‐PCR assay and a SYBR Green 1 real‐time RT‐PCR assay, both of which selectively identify living pinewood nematode by detecting the presence of Hsp70 mRNA as a viability marker. Both of these assays may help overcome or resolve disputes involving the detection of pinewood nematode at the port of entry and can also be used to evaluate the efficiency of wood treatment procedures.     

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]     

内寄生真菌Esteya vermicola对松材线虫侵染活力的测定

从不同培养基、不同测定方法、不同菌株、不同孢子浓度4个方面测定内寄生真菌Esteya vermicola孢子对松材线虫的侵染活力,结果表明:该真菌离体分生孢子对松材线虫有侵染活力;PDA固体培养比PDB液体培养所得孢子能更有效的粘附和杀死松材线虫;在WA平板上,采用浸渍法测定孢子对线虫的侵染活力比其他处理效果好;在供试的3个菌株(CUN 120806、CBS 10082和CBS 115803)中,CBS 115803菌株对松材线虫具有较高侵染活性;4个不同浓度(1×109、1×108、1×107、1×106个·mL-1)孢子对线虫的作用中,随着孢子浓度降低,孢子对线虫侵染能力也随之降低。     

卵巢线虫(Contortylenchus genitalicola)在中国松褐天牛上的分布与作用研究

[目的]日本学者Kosaka与Ogura发现松褐天牛成虫除携带松材线虫外,雌成虫卵巢内还携带有另一种线虫,他们将其命名为卵巢线虫,并认为这种线虫是松褐天牛成虫的寄生性线虫。我国对该种线虫的研究迄今未见报道。为了证明这种线虫在我国是否存在和分布,开展了本项研究。[方法]分期分批捕获刚羽化的松褐天牛雌雄成虫,采用解剖松褐天牛成虫松树木质部及感病的松褐天牛幼虫等方法,调查卵巢线虫的存在与分布。[结果]通过调查研究,发现我国的松褐天牛成虫体内有该卵巢线虫存在,分布于松褐天牛成虫、幼虫体内和松树木质部3个部位;在松褐天牛成虫体内的卵巢线虫通过松褐天牛雌成虫产卵而接种、进入寄主树木木质部中。同时,木质部的卵巢线虫也有一部分进入松褐天牛幼虫体内寄生,另一部分仍在木质部生活,当松褐天牛幼虫再次发育为成虫时,在木质部中的卵巢线虫和已被寄生的松褐天牛幼虫体内的卵巢线虫再次进入松褐天牛成虫体内,完成循环。卵巢线虫在松褐天牛雌、雄虫体内均有分布,携带率为44.4%,其中松褐天牛雌成虫携带率为43.8%,松褐天牛雄成虫携带率为45.0%,两者间无显著差异;每头松褐天牛成虫平均携带卵巢线虫574条,其中雌成虫平均携带816条,雄成虫平均携带308条,具显著差异。初步研究表明,卵巢线虫在松褐天牛成虫体内只能完成产卵到1~4龄的幼虫阶段,不能完成一个完整的世代;在松褐天牛幼虫体内寄生和在木质部生活的卵巢线虫能完成一个完整世代,但具体过程尚不清楚;调查中未观察到卵巢线虫对松褐天牛成虫有寄生致病或致死的现象,但对松褐天牛幼虫有寄生致死的能力;卵巢线虫常与松材线虫同时存在,而且侵入松树及离开松树的方式与松材线虫相同。[结论]我国松褐天牛体内也发现有卵巢线虫存在;目前尚不能证明该线虫对松褐天牛成虫具有寄生致死性,但对松褐天牛幼虫具有一定的寄生致死性;卵巢线虫的生活史与松材线虫相似,是否与松材线虫一样对松树具有危害性以及其病理作用还有待于进一步研究。     

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]     

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。     

松材线虫(Bursaphelenchus xylophilus)耐久型幼虫(LIV)携带的香茅醇假单胞杆菌(Pseudomonas citronellolis)分离与鉴定

[目的]目前有关松材线虫与伴生细菌的关系及伴生细菌的病原作用是松树枯萎病研究的重点。为了揭示松材线虫与伴生细菌之间存在的密切关系,作者对松材线虫L_IV幼虫携带的细菌进行了分离鉴定。[方法]根据培养性状和16S rDNA序列同源性以及系统发育学等方面进行分析鉴定。[结果]确定L_IV幼虫携带的是香茅醇假单胞杆菌（Pseudomonas citronellolis）,携带率为100%;每条L_IV幼虫携带量在1.4×10⁵~4.5×10⁵。L_IV幼虫生活在松褐天牛体内,是引起松材线虫病侵染流行的唯一虫态;新发现的香茅醇假单胞杆菌能分解纤维素及降解或合成萜烯和酚类化合物。[结论]L_IV幼虫携带香茅醇假单胞杆菌的发现,揭示了松树、松褐天牛、松材线虫、细菌同为一体的紧密关系,并为揭示松树枯萎病机制提供了一种新病原和重要的研究思路。     

水合霉素对松材线虫病的影响

松材线虫病是松树的一种毁灭性病害。为进一步弄清细菌在病害中的作用，本文筛选了几种对细菌菌株GD1有抑菌活性的抗菌剂，在马尾松(Pinu massomiama水培1a～2a生离体松枝上测定了其对病害发生的影响。结果表明，红霉素和环丙沙星对菌株GD1均有较强的抑菌活性，水合霉素的抑菌活性较弱，叶枯宁、菌毒清、农用链霉素、青霉素钠和氯霉素均没有抑菌活性；离体松枝上的试验显示，水合霉素能减轻混合接种松材线虫(Bursaphelenchus xylophilus)和菌株GD1松枝的病情，而红霉素和环丙沙星无此现象，从而再次证明了细菌在松材线虫病中的重要作用。据此提出了从细菌和松材线虫两方面入手的该病防治策略。     

Effect of damaged pine needles on growth and development of pine caterpillar larvae

Chinese pine caterpillar (Dendrolimus tabulaeformis) larvae were fed with pine needles of different degrees of damage to evaluate the effects of pine needles on the growth and development of larvae. The results showed that the nutritional index of the larvae declines after feeding on the damaged pine needlings. The lowest amount of food ingested and voided feces, the lowest nutritional index, slowest development, lightest pupae and most mortality were found in those pine caterpillar larvae fed with pine needles which were 50% damaged. The damaged pine needles significantly affected the population dynamics of Chinese pine caterpillars. The nutritional indices of larvae fed with 25% and 75% damaged pine needles were similar. The nutritional index of the dark morphs was higher than that of the tinted morphs, however, their mortality was lower than that of the tinted morphs. This phenomenon was reversed at the later stage of development when the larvae were fed on 50% damaged pine needles. __________ Translated from Journal of Beijing Forestry University, 2005, 27(6): 83–88 [译自: 北京林业大学学报, 2005, 27(6): 83–88]     

Improvement of wood properties by urea-formaldehyde resin and nano-SiO2

In order to improve wood properties of triploid clones of Populus tomentosa, urea-formaldehyde (UF) resin was compounded with nano-SiO₂, coupling agents and flame retardants in different ways to prepare five kinds of modifiers. The poplar wood samples were impregnated with the modifiers and heated to prepare UF-SiO₂-wood composites. The antiswelling efficiency, resistance of water absorption, oxygen index and hardness of the composites were measured. Results show that all of the modifiers reduced water absorption of poplar wood and enhanced flame resistance and hardness. Nano-SiO₂ showed a marked effect in improving the hardness of wood. In addition, all of the modifiers, except UF-C-SiO₂-polymer, improved the dimensional stability of poplar wood. The UF resin and nano-SiO₂ compound improved general properties of poplar wood. __________ Translated from Journal of Beijing Forestry University, 2006, 28(2): 123–128 [译自: 北京林业大学学报]     

Organic acid exudation from the roots of Cunninghamia lanceolata and Pinus massoniana seedlings under low phosphorus stress

Organic acid exudation from the roots of Chinese fir (Cunninghamia lanceolata) and Masson pine (Pinus massoniana) seedlings under low phosphorus stress was studied using the solution culture method. The results revealed that organic acid exudation from the roots of Chinese fir and Masson pine seedlings under low phosphorus stress increased. Compared with P₃ (KH₂PO₄, 0.5 mmol/L), the average organic acid exudation from the root of Masson pine seedlings under P₀ (KH₂PO₄, 0 mmol/L), P₁ (KH₂PO₄, 0.03 mmol/L) and P₂ (KH₂PO₄, 0.09 mmol/L) increased by 328.6%, 267.9% and 126.4% respectively. Masson pine from Zhejiang Province in China had the highest organic acid exudation. Under low phosphorus stress, the increase in organic acid exudation from the different provinces of Chinese fir and Masson pine varied. Masson pine from Zhejiang Province mainly increased oxalic acid, tartaric acid, citric acid and malic acid exudation, that from Guangxi Province mainly increased oxalic acid and tartaric acid exudation, and that from Guizhou Province, China mainly increased oxalic acid, tartaric acid and malic acid exudation. Chinese fir mainly increased oxalic acid and tartaric acid exudation. __________ Translated from Journal of Nanjing Forestry University, 2007, 31(2): 9–12 [译自: 南京林业大学学报]     

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.     

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.     

伊氏杀线真菌与苏云金芽孢杆菌对松材线虫的联合毒力研究

[目的]探究伊氏杀线真菌与苏云金芽孢杆菌(Bacillus thuringiensis,Bt)对松材线虫的联合毒力。[方法]使用Esteya vermicola孢子悬浮液与Bt发酵液处理松材线虫,通过线虫的形态变化、死亡率及死亡速度3个方面测定E.vermicola与Bt联合对松材线虫的影响。[结果]经过E.vermicola孢子悬浮液处理过的线虫会出现内容物渗漏,体腔收缩、弯曲、断裂的现象;高浓度的E.vermicola孢子悬浮液与Bt发酵液联合处理松材线虫可以明显地提高线虫的死亡率,最高可以达到100%;联合处理的线虫死亡速度较快,随着处理时间的延长,死亡率会一直呈上升的趋势。[结论]应用这两种生防微生物在合适的浓度下混配能够在较短时间内提高松材线虫的死亡率。