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.     

Effects of inoculum density of pinewood nematode on the development of pine wilt disease in Japanese black pine seedlings pretreated with simulated acid rain

Six‐month‐old Japanese black pine seedlings (Pinus thunbergii) were exposed to simulated acid rain (SAR) at pH 3 and 2 three times a week. After treatment for 2 months, the seedlings were inoculated with a virulent isolate (S10) of the pinewood nematode (Bursaphelenchusxylophilus), at three inoculum levels (P_i = 50, 160 or 500 nematodes per seedling). In seedlings inoculated with 500 nematodes, both population growth of nematodes and disease development were accelerated by pretreatment with SAR at pH 3 or 2. In seedlings inoculated with 50 nematodes, population growth of the nematodes was suppressed and more time was needed for seedlings to die when pretreated with pH 3 SAR. This suggests that exposure to pH 3 SAR increased not only the progress of mortality, but also simultaneously enhanced the tolerance limit of the seedlings to the pinewood nematode – the critical value of physiological burden (represented as a product of time and initial nematode population) necessary to kill a seedling. Exposure to pH 2 SAR accelerated nematode reproduction in seedlings and increased seedling mortality irrespective of the number of nematodes inoculated.     

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.     

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).     

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。     

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.     

Avirulent isolate of the pinewood nematode Bursaphelenchus xylophilus,survives 7 months in asymptomatic host seedlings

An avirulent isolate (C14‐5) of the pinewood nematode, Bursaphelenchus xylophilus, was inoculated onto 45 seedlings of the susceptible host Japanese black pine (Pinus thunbergii) and its viability was investigated. The nematode survived inside host seedlings for approximately 7 months even when the host seemed to overcome the infection based on lack of foliar wilting and the observation of normal oleoresin flow.     

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.     

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.     

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.     

松材线虫侵染对松树苯丙氨酸解氨酶及酚类物质的影响

以湿地松（Pinus elliottii Engelm）、日本黑松（Pinus thunbergii Parl）为材料,研究松材线虫[Bursaphelenchus xylophilus（Steiner＆Buhrer,1934）Nickle]感染对苯丙氨酸解氨酶和酚类物质的影响.两种松树的苯丙氨酸解氨酶的活性在发病过程中都有明显增加,并且伴随着酶活性的变化,各种苯丙烷次生代谢产物,如总酚、类黄酮的含量也发生明显的变化.但湿地松在苯丙氨酸解氨酶活力和酚类物质的积累上的变化比黑松要大些.结果表明,松材线虫病的发生、发展与寄主植物苯丙氨酸解氨酶活性和酚类物质含量变化有关.     

A comparison of needle retention on Japanese black pine and Japanese red pine

Needle retention (number of needle sets), needle density, height increment and radial increment were surveyed on seven Japanese black pines (Pinus thunbergii Parl.) and seven Japanese red pines (Pinus densiflora Sieb. et Zucc.) growing on the same site. Number of needle sets on branches with respect to whorl position was estimated visually. In 1999, maximum summer needle retention was observed on the fifth and sixth whorl from the top, with values of 3.4 needle sets for the black pines and 2.4 needle sets for the red pines, respectively. The needle trace method (NTM) was used to determine needle retention and needle density along the main stems retrospectively for the years 1968–1998. The long-term mean summer needle retention along the main stem was 3.7 needle sets for the black pines and 2.2 needle sets for the red pines. In both pine species, the number of needle sets varied from year to year. However, the long-term budget between newly born and annually shed needle sets was in equilibrium. The long-term average of needle density per cm of stem shoot was 9.4 needle pairs for the black pines and 7.4 needle pairs for the red pines. The results showed clear intra-specific and inter-specific similarities in needle retention, height increment and radial increment trends. The values for number of needle sets, height increment and radial increment positively increased with favorable growing conditions, whereas the value of needle density had an opposite tendency.     

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.     

油松受松毛虫危害后恢复能力的研究

油松经人工模拟失去一定的量的１年生针叶后对第２年高生长,针叶长度有明显的影响：失杏越多,影响越大,经２ａ后其后高生长虽仍有差异,但均可恢复到正常生长状态,而失去少量２－３年生针叶后的油松具有有补偿和超补偿效应,失去１年生针叶后油松不具有补偿和超补偿效应。     

Virulence of Bursaphelenchus mucronatus to pine seedlings and trees under field cownditions

Bursaphelenchus mucronatus is a parasitic nematode of pine that is widely distributed in the natural pine forests of Asia and Europe. It has a very similar morphology and biology to that of Bursaphelenchus xylophilus, the causal agent of pine wilt disease, but has generally been considered to be non‐pathogenic to pine. However, in some provinces of China, B. mucronatus has been isolated from dead pine trees rather than B. xylophilus. Previous studies have shown that B. mucronatus can induce the death of pine seedlings under glasshouse conditions. To investigate the virulence of B. mucronatus, 2‐year‐old seedlings of Pinus massoniana and Pinus elliottii were inoculated with one of six isolates of B. mucronatus under field conditions in April 2014 and their condition was monitored over a year. The virulence of the six B. mucronatus isolates differed on the three host species: P. elliottii seedling mortality ranged from zero to six of the 18 inoculated seedlings, whereas P. massoniana seedling mortality ranged from four to 12 of the 18 inoculated seedlings. Three B. mucronatus isolates that appeared to cause different levels of mortality among the seedlings were used to inoculate 12‐year‐old Pinus thunbergii trees in August 2014. The trees were monitored for a year, during which time between 4 and 12 of the 18 inoculated trees in each treatment wilted and died. The average monthly temperature during the test period appeared to be similar to that of the historical average in the test areas; however, both study sites experienced above‐average rainfall. This study demonstrated that B. mucronatus has potential virulence on pine trees and provided experimental evidence that high temperatures or drought stress is not essential for the virulence of B. mucronatus.     

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.     

不同松树感染松材线虫后NO和核酸酶变化与其抗病性的关系

以黑松、马尾松和火炬松为研究对象,对其接种松材线虫后4,12,24,48,72,96和120 h的NO和核酸酶的活性进行测定.3种松树接种松材线虫后,针叶内NO平均含量的积累为火炬松＜马尾松＜黑松,这种差异在12 ～48 h尤为明显;48h后较其割伤处理(CK1)的增加幅度为火炬松＞马尾松＞黑松.同时3种松树接种后的核酸酶变化趋势存在差异,单链核酸酶活性在4～48 h为火炬松＞马尾松＞黑松,而火炬松双链核酸酶活性在4～96h时却一直低于马尾松和黑松,这表明不同松树体内单链或双链核酸酶的活性变化与其抗病性有关.且3种松树在接种松材线虫后,表现出症状的时间顺序为黑松最早,马尾松次之,火炬松最晚.这表明NO和核酸酶的变化与松树感染松材线虫后的病程发展密切相关.     

古油松衰弱衰老诊断的生理指标

选取颐和园不同树龄油松及其衰弱株的一年生针叶为试材,测量不同季节的针叶单位质量的长度、叶绿素含量、丙二醛(MDA)含量、超氧化物歧化酶(SOD)活性等指标。结果表明:与健康油松比较,衰弱油松树冠不完整,一年生针叶柔弱细长,针叶单位质量的长度值大;衰弱油松叶绿素含量低、光合能力弱,叶绿素a/b值低,光能利用效率低。在一年的生长季节中,油松MDA含量随针叶的发育逐渐升高;与健康油松比较,衰弱油松针叶MDA含量远高于健康油松,而针叶SOD总活性和SOD比活力均低于健康油松,其抗氧化能力弱。这些结果说明:针叶单位质量的长度、叶绿素含量、MDA含量、SOD活性等可以作为古油松衰弱衰老的诊断指标。     

Inoculation of several Bursaphelenchus xylophilus group nematodes into adult trees of Pinus thunbergii and their survival in the trees

To clarify the pathogenicity of Bursaphelenchus nematodes to adult pine trees, inoculation experiments using six species of B. xylophilus group nematodes and ca. 10‐year‐old trees of Pinus thunbergii were conducted. Trees inoculated with an avirulent isolate (C14‐5) of B. xylophilus did not die during the survey, but showed a decline in oleoresin exudation compared with the controls. Fifteen months after the inoculation, a small number of B. xylophilus survived in a tree inoculated with B. xylophilus C14‐5. Trees inoculated with B. mucronatus, B. doui, B. luxuriosae, B. conicaudatus and Bursaphelenchus sp. NK224 (undescribed) showed no decline in oleoresin exudation and no external symptoms of wilt. However, 9 months after the inoculation, a small number of B. luxuriosae survived in a tree inoculated with the nematodes, although four other nematode species were not isolated from trees inoculated with them. These results were approximately consistent with our previous results (Kanzaki, N.; Aikawa, T.; Maehara, N.; Ichihara, Y., 2010, J. For. Res.; in press), in which an avirulent isolate (OKD‐1) of B. xylophilus and B. luxuriosae caused water flow inhibition without external symptoms in 3‐year‐old seedlings. Therefore, to examine the pathogenicity of the nematodes to pines, it is useful to use 3‐year‐old seedlings in inoculation experiments when adult trees cannot be used.     

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.