The process of decline of an endangered tree species,Pinus armandii Franch. var.amamiana (Koidz.) Hatusima, on the southern slope of Mt. Hasa-dake in Yaku-shima Island

The process of decline of an endangered tree species,Pinus armandii var.amamiana, was monitored on the southern slope of Mt. Hasa-dake in Yaku-shima Island from 1994 to 1998. There are 163 trees ofP. armandii var.amamiana over 1.3 m in height. They are distributed on steep ridges and rocks with a thin soil layer mostly consisting of friable granite. During the monitoring period, 21 trees ofP. armandii var.amamiana died and the mortality rate was 12.9%. Dead trees were categorized into three types: standing, uprooted and landslide. The uprooted- and landslide-typed dead trees were found only after a severe typhoon struck Yaku-shima Island. This suggests that the combination of fragile site conditions and severe typhoons play an important role in the process of decline ofP. armandii var.amamiana. The standing-typed dead trees were presumed to have been killed by pine wilt disease, accounting for 71.4% of the dead trees. However,Bursaphelenchus xylophilus, the cause of pine wilt disease, was not detected from any of the wood chips or branch samples from the standing-typed dead trees ofP. armandii var.amamiana. This might indicate that some factor(s) other than pine wilt disease could be responsible for the standing-typed death ofP. armandii var.amamiana in natural habitats. A part of this paper was presented at the 109th Meeting of the Japanese Forestry Society.     

Radial variations of wood properties of an endangered species, <Emphasis Type="Italic">Pinus armandii</Emphasis> var. <Emphasis Type="Italic">amamiana</Emphasis>

A dead tree of Pinus armandii Franch. var. amamiana (Koidz.) Hatusima (abbreviated to PAAm) was obtained from a natural habitat on Tanega-shima Island and various properties of its wood were investigated. Grain angle was measured and soft X-ray analysis was undertaken to obtain the density in each annual ring. Unit shrinkage and dynamic properties were measured by shrinkage, bending, and compression tests. Variations of wood properties in the radial direction, relationships of wood properties to density, and annual ring width were examined. Roughly speaking, variations in the radial direction of the grain angle, twist angle by drying, Young’s modulus and strength in static bending, absorbed energy in impact bending, compressive Young’s modulus, compressive strength, and compressive proportional limit corresponded to the variation of annual ring width. As a result, it was determined that if PAAm is afforested artificially for the purposes of lumber production and conservation, the annual rings of logs should not be too widely spaced. Wood properties of PAAm were similar to those of Japanese black pine (Pinus thunbergii Parl.), which is another representative pine on Tanegashima Island. This study was presented in part at the 56th Annual Meeting of the Japan Wood Research Society, Hiroshima, August 2007     

思茅松松材线虫病症状初步研究

思茅松是松材线虫在自然条件下感病的新寄主植物,对思茅松天然林区症状观察的同时进行了室内人工接种试验观察,结果表明,在思茅松上松材线虫病的症状表现与其他寄主上的有所不同,即松针当年陆续脱落和感病中期还有流脂现象。     

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”.     

Ectomycorrhizal development in a Pinus thunbergii stand in relation to location on a slope and effect on tree mortality from pine wilt disease

The relationship between ectomycorrhizal development and mortality from pine wilt disease was studied in an artificial Pinus thunbergii Parl. stand on a slope. The development of ectomycorrhizae and the survival of the trees showed the same tendency, which suggests a correlation between mycorrhizal development and resistance to pine wilt disease. The development of pine roots and mycorrhizae was greater in the upper part of the slope. The ratio of mycorrhizae to the total of mycorrhizae and fine taproots was also higher in the upper part of the slope. Tree mortality was clearly biased and more trees survived in the upper part of the slope than in the middle and the lower parts. There was no significant difference between the upper and the lower part of the slope in the number of feeding wounds made by the pine sawyer beetle, which demonstrates the opportunity of infection with this disease. There was no clear correlation between the development of mycorrhizae and the composition of the soil substrate such as total carbon, nitrogen, and phosphorus. The abundant mycorrhizae in the upper part of the slope, which mitigate drought stress, may also have decreased the rate of tree mortality.     

Different development of pine wilt disease in artificially infectedPinus thunbergii seedlings potted together with different tree species

To evaluate the effect of adjacent tree species on the susceptibility of Japanese black pine (Pinus thunbergii) to pine wilt disease, an inoculation experiment was conducted usingP. thunbergii seedlings potted with seedlings of six tree species,i. e. Alnus sieboldiana, Eurya japonica, Lespedeza bicolor formacutifolia, Pinus thumbergii, Robinia pseudo-acacia andSarothamus scoparius. About ten months after planting, they were inoculated with the pinewood nematode (Bursaphelenchus xylophilus) in early July 1992. After that, the proportion of pine seedlings with completely discolored foliage increased more quickly when the seedlings were potted withR. pseudo-acacia, S. scoparius orA. sieboldiana than when potted withP. thunbergii, L. bicolor orE. japonica. At the end of the study period, 17 weeks after inoculation, it reached 90.6%, 90.0%, 87.5%, 72.7%, 63.3%, and 59.4% when the pine seedlings were potted withR. pseudo-acacia, S. scoparius, A. sieboldiana, P. thunbergii, L. bicolor andE. japonica, respectively. This indicated that the susceptibility ofP. thunbergii seedlings to pine wilt disease was influence by the species of adjacent trees.     

Comparison of stand dynamics after dieback caused by pine wilt disease among pine forests with different management regimes in western Japan

In pine forests damaged by pine wilt disease, in western Japan, the effect of protection regimes of pine trees on the stand dynamics were examined in the following four stands: (1) lightly damaged stand (age 30–40 years) with no procedure in operation for protecting pine trees; (2) severely damaged stand (age 30–40 years) with no procedure in place for protecting pine trees; (3) severely damaged stand (age 50 years) with a selective cutting of infected trees; (4) severely damaged stand (age 30–40 years) with a selective cutting of infected trees. All the stands had been abandoned before the pine wilt disease damage. The understory structure of the severely damaged stand with no protection procedure was similar to that of the lightly damaged stand. Frequent invasion by tree species and acceleration in the growth of understory trees occurred after the dieback in the selective cutting stand. These results suggest that a deficiency in the canopy layer caused by the dieback resulted in low disturbance intensity in the early stages after the dieback, but the selective cutting increased the intensity by the reduction in the understory as well as the canopy layer. The intensity of the disturbance in the selective cutting stands was larger in the younger stand because it had a higher density of selectively cut pine trees. The different stand structure of pine forests occurred after the dieback because the intensity of the disturbance varied as a result of the selective cutting operation and the stand age.     

Management of pine wilt disease in Korea through preventative silvicultural control

Pine wilt disease (PWD) represents a major threat to forest ecosystems worldwide. Although PWD is now better understood, effective control measures for this disease have still not been devised. Here, we report several years of field studies on preventative silvicultural control of PWD. Silvicultural control through preventative clear-cutting and the manual removal of logs was implemented between 2005 and 2009 in 16 Korean districts that had newly PWD-infected stands. Preventative clear-cutting of neighboring asymptomatic pine trees (within a 10-50-m radius of wilt trees) and the removal of felled logs or branches suppressed spread of PWD. Occurrences of PWD wilt pines in districts (city or county) subjected to this silvicultural control method were significantly reduced compared with those in districts using conventional controls (physical or chemical treatment of wilt pine trees). Through silvicultural control, PWD was successfully suppressed in 11 of 16 districts investigated. In contrast, successful control was achieved in only 1 among 18 districts subjected to conventional control. Our results will be of considerable interest to those engaged in the very difficult battle against the global spread of PWD.     

Inhibiting factors of symptom development in several Japanese red pine (<Emphasis Type="Italic">Pinus densiflora</Emphasis>) families selected as resistant to pine wilt

Pinus densiflora and P. thunbergii, native to Japan, are highly susceptible to pine wilt disease caused by infection with a pine wood nematode (Bursaphelenchus xylophilus). Trees of these susceptible species have occasionally been found surviving in forests that are extensively damaged by this disease. Seedlings from a part of surviving trees that were selected as resistant families indicate lower mortality rates after the infection. The factors that prevent the symptoms from developing in resistant families of a susceptible species, P. densiflora, as based on the analysis of the pathogens behavior in the tree tissue and the anatomy of the resistant families, are presented in this paper. Nematode populations remained lower in the stems of seedlings from resistant families of P. densiflora than in the stems of non-resistant families. Areas dysfunctional in water transport developed in the stems of resistant families, but did not reach a size large enough to seriously block the ascent of sap. These results suggest that there are systems within the seedlings that prevent nematode migration and reproduction. The 2-year-old seedlings from resistant families of P. densiflora, however, did not suppress the pathogen activity. Numerous branches are a visible characteristic in the seedlings of some resistant families. The arrangement of the resin canals, the only channels in the tree to the pathogen migration, was disoriented at the joints between the branches and the main stem. Such a structure may be effective as a barrier to nematode dispersal.     

松兰山景区松材线虫病综合治理研究

研究分析了浙江宁波市象山松兰山景区松材线虫病发生规律与防治效果。结果表明：只采取“封锁”措施，会使松材线虫病迅速爆发毁灭全林。从1992年到1998年，松兰山枯死松树从6株增到5893l株，增长了9820．8倍；采用皆伐松林防治松材线虫病并改造成板栗、杨梅的方法，会导致逆向演替，使松林最后成为以疏花野青茅为主的荒山。而采取综合防治措施就可有效地控制松材线虫病的危害。从1998年到2002年，松兰山枯死松树下降到137株，缩小了430倍，使松兰山仍然保持着葱郁的松林群落。     

Retardation of pine wilt disease symptom development in Japanese black pine seedlings exposed to simulated acid rain and inoculated withBursaphelenchus xylophilus

Three- or 4-year-old Japanese black pine seedlings were exposed to simulated acid rain (SAR) at pH 3 for two months, then inoculated with a virulent isolate (S 10) of pinewood nematodes. The experiments were repeated three times in 1996–1998. The exposure to SAR killed no seedlings, and retarded the development of disease symptoms in the seedlings inoculated with nematodes. In the experiments in 1996 and 1997, however, cessation of resin exudation, and primary decrease in the xylem conductivity occurred earlier in the seedlings exposed to SAR than in those exposed to tap water as a control. These results imply that acid rain at pH 3 influenced an increase in some resistance of Japanese black pine seedlings to pinewood nematode, and that this improved resistance could potentially overcome damage caused by acid rain. The exposure to SAR did not have any significant effect on the water relations of the seedlings itself, suggesting that retardation of the symptom development after the exposure to SAR could not be attributed to the improvement in the water relations. This work was supported in part by a Grants-in-Aid for Scientific Research from the Japan Forest Technical Association (JAFTA).     

重灾区松材线虫病的治理试验

松材线虫病近距离传播是由松褐天牛自然扩散完成的。通过人工清理病死木结合化学防治,在中心疫区保住了1块松林。实践证明,人工清理后通过有效保护措施,能够控制松材线虫病。     

Role of asymptomatic carrier trees in epidemic spread of pine wilt disease

To determine why pine wilt disease caused by the pinewood nematode (Bursaphelenchus xylophilus) recurs in the same pine stand even after thorough eradication of dead pine trees, the amount of oleoresin exudation from artificial wounds was measured from 72 Pinus koraiensis trees, highly susceptible to this disease, for 4 years. The amount of exuded oleoresin was rated from 0 to 4. All values obtained for each tree were summed at each measurement; thereby a cumulative curve was drawn to monitor the physiological condition of each test tree. Cumulative curves suggest that some pine trees that died had already been infected in the previous year or earlier and then had survived without any visible symptoms. If cessation of oleoresin exudation delays, and overlaps with activity of Monochamus alternatus, the vector beetle of pinewood nematodes, in the following season, such trees can be referred to as latent carriers or asymptomatic carriers. They could play a significant role as attractants for M. alternatus that could then transmit B. xylophilus to neighboring trees. Behavior of M. alternatus caged with several pine seedlings, only one of which acted as an asymptomatic carrier of B. xylophilus, confirmed this idea. The presence of asymptomatic pine trees, which harbored B. xylophilus nematodes or had reduced annual elongation, near stumps of newly dead trees in the study stand also substantiated this hypothesis.     

抗松材线虫病赤松组培再生植株田间生长表现

为了评价从日本引进的抗松材线虫病赤松通过无性快繁获得的再生植株在我国的生长状况.本研究对田间试种的抗病赤松6个家系12个无性系共688株再生植株进行跟踪监测.结果表明,抗病赤松0.5年生再生植株试种成活率明显低于2.5年、2年、1.5年及1年生再生植株;不同无性系间抗病赤松再生植株生长状况存在明显差异.其中,1#-A和5#-5无性系再生植株生长较好.菌根化造林试验表明,两种外生菌根菌美味牛肝菌(Be)和黄色须腹菌(R1)对抗病赤松1#-A5代、8代再生植株具有明显促生效果.此研究为今后抗病赤松再生植株造林育种提供了参考依据.     

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.     

Relationship between the incidence of pine wilt disease and the drainage area

We examined the relationship between the incidence of pine wilt disease and moisture conditions in the stand level ofPinus densiflora Sieb. et Zucc. forests in the warm-temperate zone of the western part of Japan. For this analysis, pine trees killed by pine wilt disease were distinguished from the suppressed trees by their position in the layer of the pine forest stand. The drainage area, which is small in the upper part and large in the lower part of the slope, was adopted for representing the moisture conditions in the soil of the slope. The percentage of the pine trees killed by pine wilt disease increased as the size of the drainage area increased. This result suggested that the incidence of pine wilt disease tended to be high in areas with moist conditions. Pine trees attacked by the pinewood nematode die from extensive water deficit due to tracheid cavitations. Pine wilt disease mainly emerges in the summer when the soil water conditions become especially severe, and the radical water stress is thought to accelerate the disease. It was assumed that pine trees in the plots with the small drainage area resisted the influence of the attack of the pinewood nematode because pine trees in the plots with the small drainage area encountered long-term water stress and acquired drought tolerance. Pine trees in the plots with the large drainage area were presumed to be well established in the moist conditions and not to have acquired drought tolerance. The drought tolerance of pine trees was thought to be an important factor in resistance to pine wilt disease.     

Promotion of the population growth of pinewood nematode in 4-month-old Japanese black pine seedlings by pretreatment with simulated acid rain

Simulated acid rain (SAR) at three pH levels (pH 4, 3, 2) was applied to only the top or both the top and roots of 4-month-old Japanese black pine seedlings repeatedly for two months. Then the seedlings were inoculated with a virulent isolate (S10) of pinewood nematode,Bursaphelenchus xylophilus. The exposure to SAR at any level did not kill the seedlings. When inoculated with nematodes, however, the nematode population build-up was significantly higher in the seedlings pretreated with SAR at any level than in the control at the 7th and 17th day after inoculation, and subsequent disease symptom development was also significantly accelerated by the exposure to SAR at pH 2 and 3. This result indicates that even acid rain at pH 4 has the potential for promoting population growth of pinewood nematodes in 4-month-old Japanese black pine seedlings.     

Spatiotemporal analysis of pine wilt disease: Relationship between pinewood nematode distribution and defence response in Pinus thunbergii seedlings

Pine wilt disease is of major concern as it has destroyed pine forests in East Asia and Europe. Several studies have suggested that invasion by the pinewood nematode (PWN) Bursaphelenchus xylophilus, which causes this disease, evokes an excessive defence response in pine trees, resulting in tree death. However, few studies have quantitatively evaluated the correlation between PWN distribution and tree defence responses. Therefore, the present study aimed to quantify the number of PWNs and expression levels of putative pathogenesis‐related (PR) genes in different positions of Japanese black pine (Pinus thunbergii) seedlings over time. To quantify the number of PWNs in the seedlings, we used TaqMan quantitative real‐time PCR (qPCR) assay. During the early phase of infection, most PWNs were distributed around the inoculated sites, with only a small number being detected at distant sites, but the expression levels of PR genes were highly upregulated throughout the seedlings. Both the number of PWNs and expression levels of PR genes then increased drastically throughout the seedlings, all of which exhibited external symptoms. Thus, it appears that the rapid migration of PWNs induces a defence response throughout the seedling; however, this may not be effective in controlling these parasites, thereby ultimately leading to plant death.     

Spatial distribution pattern, scale and gap characteristics of Pinus armandii population in Qinling Mountains, China

Based on the data collected from 27 plots of the Pinus armandii community in Qinling Mountains, we studied the spatial distribution pattern, scale, and gap characteristics of the P. armandii population. The results showed that the population had a clumped distribution before age 50. At the age range from 15 to 25, though the population tended to be distributed randomly, the distribution was still clumped. The population distribution at the age range from 40 to 50 was at the transitional stage from clumped to random. After age 50, the population started to be senesced, the distribution pattern turning from clumped to random. The distribution pattern scale of P. armandii always changes with the development stage of the population, being 100 m² in general. The gap size of P. armandii population was similar to its distribution pattern scale, and the gaps of 80–130 m² occupied 59% of the total. Because of the better light and nutrient condition in the gap, P. armandii seedlings grew well, which helped the population keep its stability through “mobile mosaic circling”. __________ Translated from Chinese Journal of Ecology, 2006, 25(6): 652–656 [译自: 生态学杂志]     

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。