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”.     

Mechanism of cavitation development in the pine wilt disease

Volatile terpenes increase in xylem tissue after infection of Pinus thunbergii with the pine wood nematode (Bursaphelenchus xylophilus). The role of these terpenes in traeheid cavitation, which blocks xylem-sap ascent and leads to water deficit in pine trees, was assessed. Volatile terpene concentration increased long before initiation of tracheid cavitation. After the volatile terpenes reached the highest concentration, severe cavitation developed. Direct injection of α-pinene into healthy pine trunks formed artificial cavitation in xylem. These observations support the hypothesis that excessively produced volatiles, which are hydrophobic and have lower surface tension than water can promote tracheid cavitation in pine wilt disease.     

Influence of water stress on development of pine wilting disease caused by Bursaphelenchus lignicolus

In the development of pine wilting disease caused by Bursaphelenchus lignicolus, water status in pines plays an important role in the pinenematode relationship. Levelling off in transpiration causes a population growth of nematodes in the wood. Such pines invariably wilt several weeks after the reduction of transpiration. The paper contains details of oleoresin exudation, transpiration and xylem water potential as well as the nematode population in the wood in connection with disease development.     

松材线虫病致病机理的研究进展

该文对松材线虫病的致病机制的研究进行了概述。目前对松材线虫病的病原有两种看法，一种认为松材线虫是惟一病原，另一种认为病原为松材线虫和细菌两种生物。对于松材线虫的致病机理，目前存在3种观点。第1种观点认为松材线虫的酶使松树薄壁细胞的细胞壁和细胞膜遭到破坏，树脂不正常地从树脂道中渗漏并扩散到相邻的管胞中，使水分输导受阻，导致萎蔫。第2种观点认为松树感染了松材线虫后，木质部内挥发性萜烯类物质的含量增加，这些物质进入管胞在管胞中形成空洞，致使水分输导受阻。第3种观点认为松树感染松材线虫后，体内产生有毒物质，这些物质使松树萎蔫。     

越南松树枯死病症状及原因初步研究

为查明越南松树枯死的原因,对越南南部的林同省,中部的承天-顺化省,北部的广宁省的思茅松、马尾松和南亚松枯死症状和原因进行了调查,分别从枯死、濒死、健康松树木质部中抽取52份样品,用于线虫分离镜检。调查发现松树在感病后,树冠部分针叶失去光泽,逐渐褪绿变黄,进而褐红干枯,最后整个树冠变为褐色或红褐色,全株枯死,针叶下垂且当年不落;随着病程的发展,松脂分泌逐渐减少,直至停止;树干上有天牛产卵刻槽,死亡的松树上有天牛的羽化孔。在抽取的松树样品中分离鉴定到了泰国伞滑刃线虫(Bursaphelenchus thailandae BrasschBrassch-Bidasak,2002),为病原的确定和防治工作提供初步的依据。     

松材线虫在我国的分布

松材线虫[Bursaphelenchus xylophilus(Steiner＆Buhrer,1934)Nickle,1970]引起的松树萎蔫病(Pine Wilt Disease)是一种毁灭性病害。松树感染此线虫后可很快死亡。作者在温室用松材线虫接种三年生黑松苗,35d后76％的松苗即枯死了。据记载,在日本由于松材线虫为害引起的木材年损失量最高可达240万m~3 。自1981年以来,在我国湖南、贵州、云南、浙江、江苏等省都有松树枯死现象。1982年首次在南京发现松材线虫危害。以后,病情逐年加剧,到1987年已累计病死松树60万株,成为我国突出的、至今尚无防治良策的一大森林病害。     

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。     

松材线虫病的潜伏侵染及松墨天牛传播新途径

在南京用松材线虫接种 7种松树 ,于第 2a在无任何症状的松树的接种点上方 10cm处取样 ,分离线虫。结果表明松材线虫病的潜伏侵染现象比较普遍。在不同的松树品种上潜伏侵染现象有所差异。感病黑松和赤松很快死亡 ,只有在接种量小的情况下才有潜伏侵染现象发生。抗病性强的火炬松和湿地松感病后潜伏侵染现象比较普遍 ,并且样品中的线虫量也较高。而抗病性中等的马尾松和刚松感病后潜伏侵染现象的普遍性及样品中的线虫量均处于中等。短针松接种松材线虫后既不枯死 ,也无潜伏侵染现象。松墨天牛传播松材线虫的试验结果表明 ,未携带松材线虫的松墨天牛成虫在松材线虫病松枝上取食后 ,再到健康松枝上取食 ,有传播松材线虫的可能。这些结果表明在松材线虫病新病区清理病死木时 ,应采取早期诊断技术 ,把未表现症状的松树一起清除 ,以达到防治目的。     

无人机监测松材线虫病的精度比较

利用无人机搭载可见光相机进行遥感拍摄,在430 m和700 m两个飞行高度下采集异常枯死松树遥感影像,将野外GPS采集的位置信息同ENVI遥感数据处理软件处理与解读数据信息进行比对。人工甄别和NDVI值提取枯死松树分别为6株与7株,数据有效率提取为85.71%;对无人机遥感获取的6株枯死松树地理位置信息进行实地验证,水平误差在0.86 m^4.20 m之间。表明无人机遥感基本实现了松材线虫病致死松树的精准定位,对于松材线虫病监测和后期除治具有重要意义。     

松材线虫病的诊断方法探讨

控制松材线虫病的关键是及时诊断疑似疫木、疫点,既能采用有效、简单、易行的方法进行诊断,且能有的放矢地进行诊断。文章根据广东省松材线虫病疫区的特点,对林间枯死松树外观症状诊断法、林间枯死松树体内线虫的分离鉴定法和松褐天牛引诱剂辅助诊断法等3种松材线虫病的诊断方法及其适应范固进行探讨。     

Ethylene production during symptom development of pine-wilt disease

Ethylene release from Pinus thunbergii Parl. seedlings was examined after inoculation with virulent and avirulent isolates of Bursaphelenchus xylophilus. Enhanced ethylene release was observed only in seedlings inoculated with the virulent isolate. The avirulent isolate induced necrosis of xylem ray cells and embolism in some tracheids, however, the cambium and outermost xylem was intact where water conduction was maintained. The ethylene increase coincided with cambial death in seedlings inoculated with the virulent isolate.     

Haustorial morphology of Cronartium conigenum in naturally infected cones of three Pinus species from Guatemala1

Haustorial morphology of the cone-rust pathogen, C. conigenum, was investigated among naturally infected female strobili of three Guatemalan pines, P. maximinoi, P. pseudostrobus, and P. oocarpa. Among the three pine species, haustorial shapes and sizes were more variable in P. maximinoi and P. oocarpa than in P. pseudostrobus. The haustorial shapes and sizes were more variable in parenchyma cells of the cortex, xylem, and pith than in the cells in the phloem, xylem rays, and tracheids. The haustoria were also present in larger numbers in the parenchyma cells of the cortex, xylem, and pith than in phloem parenchyma cells and tracheids. In living cells, the tips of some haustoria were appressed to the host nuclei.     

Survival ofPinus densiflora andP. thunbergii forests in Ibaraki Prefecture severely damaged byBursaphelenchus xylophilus

The total area ofPinus densiflora andP. thunbergii forests in Ibaraki Prefecture in 1978 was 65,200 ha, which decreased to 30,300 ha by 1985 mainly due to pine wilt mortality caused byBursaphelenchus xylophilus. This damage has also continued thereafter. To estimate the survivability of pine trees in Ibaraki Prefecture, pine tree mortality has been studied in eight experimental forests for over 20 years, and ground surveys throughout the Prefecture were also conducted in 1995. Survival in the experimental forests corresponded well to the results of ground surveys. Pine forests remained as pure stands if control measures were undertaken or if they were located in cool areas. In warm areas where no control was undertaken, most of the pine forests disappeared and only a few pine trees remained in mixed forests, while on dry soils no mature or old pine trees survived. Since surviving pine forests are often cut for wood utilization,P. densiflora andP. thunbergii may decrease in area to become rare species in the future unless controls are applied and/or reforestation with resistant pines is carried out. A part of this paper was orally presented at the 108th Annual Meeting of the Japanese Forestry Society (1997).     

林内就地火烧病死木防治松材线虫病试验

林内就地火烧病死木防治松材线虫病使病死树砍伐与除害同步进行即现砍现浇。该法操作方便、成本低,效果好,文内具体提出了现砍现浇的操作方法,以避免诱发森林火灾,消除燃烧不彻底的隐患。     

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.     

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.     

Migration of Bursaphelenchus xylophilus in cortical and xylem axial resin canals of resistant pines

Migration of the pine wood nematode (PWN), Bursaphelenchus xylophilus, in susceptible and resistant pines was investigated at the tissue level. PWN was inoculated onto the top cross‐cut surface of 20‐cm stem cuttings of susceptible Pinus thunbergii and resistant pines (P. strobus, P. rigida and P. thunbergii of a resistant family Namikata‐(t)‐73 (half‐sib)). PWNs were mainly distributed in cortical resin canals of susceptible P. thunbergii down to 15 cm from the inoculated surface by 6 h after inoculation (HAI) and all tissues (including cortical and xylem resin canals) down to the bottom at 192 HAI. In P. strobus, P. rigida and P. thunbergii family Namikata‐(t)‐73 (half‐sib), PWN was distributed in cortical resin canals down to 5 cm by 6 HAI and down to the base at 192 HAI. However, the distribution of PWN in xylem resin canals of the resistant pines was restricted near inoculated surfaces down to 5 cm, even at 192 HAI. These results demonstrated that migration of PWN in resistant pines was slowed in cortical resin canals and restricted in xylem axial resin canals, features which may be associated with the resistance.     

Pathogenicity of canadian isolates of the Bursaphelenchus xylophilus (pinewood nematode) to provenances of Pinus sylvestris and Pinus contorta as grown in Finland: A greenhouse study

Seedlings of Scots pine (Pinus sylvestris L.) and lodgepole pine (P. contorta Dougl.) provenances, as grown in Finland, were inoculated with “m”; and “r”; “forms”; of the pinewood nematode (Bursaphelenchus xylophilus) from Canada, an m form isolate from France and an r form isolate from Missouri, USA. Scots pine was highly susceptible to an Alberta r form and a British Columbia m form isolate and moderately susceptible to two Quebec m form isolates. Lodgepole pine was higly susceptible to the two r form (Alberta and Missouri) isolates and moderately susceptible to the British Columbia m form and the two Quebec m form isolates. Mortality of both pines after inoculation with the French isolate was inconsistent. Mortality of both pines occurred more rapidly following inoculation with r form than with m form nematodes. Large numbers of nematodes were generally found in the tissues of both pines. Our results with seedlings need to be corroborated by inoculating larger field‐grown trees.     

松材线虫病感病松树中致萎毒性物质的研究

本研究用我国自然感染松材线虫病的15年生以上黑松和马尾松以及人工感染松材线虫病的3年生黑松的木质组织进行萃以和粗提纯，方法为高压蒸煮法和氯仿碱法。生物测定结果显示，有致萎活性的毒性物质仅存在于自然感病未死的黑松和马尾松以及人工感病未死的黑松木质组织中。自然感病死后1年的黑松和马尾松以及健康松树不中含有这些毒性物质。通过气相色谱－质谱联用仪（GC／MS System）对上述木质组织萃取物作定性分析发现，自然感病未死的黑松、马尾松和人工感病未死的黑松有致萎活性的萃取物中均含苯乙酸和2－甲氧基肉桂酸，自然感病死后1年的松树和健康松树中则无。而在健康松树和感病松树中都发现有苯甲醛存在。表明毒性物质与苯乙酸和2－甲氧基肉桂酸有关，苯甲醛则是松树正常的代谢产物。苯乙酸和2－甲氧基肉桂酸均为酚类化合物，是植物体内重要的次生物质。说明感病松树中的毒性物质是松树组织由于松材线虫侵染而产生的异常代谢产物，应称之为致萎毒性物质。     

Mutual influences in growth and reproduction between pine wood nematode Bursaphelenchus xylophilus and bacteria it carries

The interactions between pine wood nematode and three bacterium strains isolated from the nematode, Bursaphelenchus xylophilus, which are two strong pathogenic bacterium strains, Pseudomonas fluorescens GcM5-1A and Pseudomonas putida ZpB1-2A and a weak-pathogenic bacterium strain, Pantoea sp. ZM2C, were studied. The result showed that the strong-pathogenic GcM5-1A strain and ZpB1-2A strain significantly increased fecundity, reproduction rate, and the body volume of the adult nematode. Meanwhile, pine wood nematodes significantly promoted reproduction of the two strong-pathogenic bacterium strains. However, the weak-pathogenic bacterium strain, ZM2C, completely inhibited reproduction of pine wood nematodes. Aseptic pine wood nematodes significantly inhibited reproduction of the strain ZM2C. The results indicated that mutualistic symbiosis exists between pine wood nematodes and the two pathogenic bacteria it carries. The phenomenon showed that the pathogenic bacteria carried by the nematode were not accidentally contaminated, but rather had existed as symbionts of the nematode with which it had coevoluted over a long period. The role of mutualistic symbiosis in the process of pine wilt disease was also discussed. __________ Translated from Journal of Nanjing Forestry University, 2005, 29(3): 1–4 [译自: 南京林业大学学报, 2005, 29(3): 1–4]