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。     

松树抗松材线虫病机理的研究进展

松材线虫病 [Bursaphelenchusxylophilus(Steiner&Buhrer)Nickle]是一种毁灭性的森林病害。不同松种和同一松种不同种源的抗病性存在明显差异 ;松树通过固有或诱导的防卫系统抵抗松材线虫的扩散和繁殖。本文就国内外关于松树抗松材线虫病机理方面的研究进行综述     

湖南省松材线虫病的首次确认

通过病原形态学和分子生物学鉴定,在国内首次确认了湖南省松材线虫病疫情,并通过疫情普查准确掌握了病害在郴州市的苏仙区、北湖区,益阳市的资阳区、沅江市,常德市的汉寿县等3个市、5个县（市、区）的发生范围和危害程度。     

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.     

乐清市松材线虫病的发生情况与防治对策

论述了乐清市松材线虫病的发生情况,并提出分区治理、健全检疫检查、监测普查、清理除治、建设健康安全的森林生态、稳定高效的保障等6大体系的防治对策。     

松材线虫发病条件的研究概况

研究和综述了寄生松科植物的伞滑刃属(Bursaphelenchus)的线虫23种,松材线虫的寄主松科植物108种、携带昆虫40种及发病的环境条件。     

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.     

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.     

松材线虫病致死松树上的松褐天牛季节性分布规律

连续两年定期监测、调查采样和鉴定证明：每年６～１０月出现的松材线虫病Ｂｕｒｓａｐｈｅｌｅｎｃｈｕｓｘｙｌｏｐｈｉｌｕｓ致死松树上，松褐天牛Ｍｏｎｏｃｈａｍｕｓａｌｔｅｒｎａｔｕｓ虫株率为１００％，而１１月至次年５月间出现的病死株上，虫株率为零。对前者应集中砍除，并加以严格的除害处理；后者可经剥皮处理后在疫情发生区内加工利用，部分可留在林间作为天牛产卵的饵木     

Possible altitude and temperature limits on pine wilt disease: the reproduction of vector sawyer beetles (Monochamus alternatus), survival of causal nematode (Bursaphelenchus xylophilus), and occurrence of damage caused by the disease

Pine wilt disease is caused by the pine wood nematode [Bursaphelenchus xylophilus (Steiner et Buhrer) Nickle]. In East Asia, an important vector of the nematode is Monochamus alternatus Hope. We determined the tolerance and reproductive ability of sawyer beetles and the nematode to altitude and temperature at elevations between 850 and 1,450 m on Mt. Fuji in Japan. The number of emergent adults decreased markedly along the altitudinal gradient, but the beetle could still reproduce at 1,050 m (8.2 °C annual mean temperature). Beetles with a 2-year life cycle increased rapidly in number with increasing altitude. The pine wood nematode survived through winter at all altitudes tested (850–1,450 m). The beetle population decreased between 950 (9.1 °C) and 1,150 m (8.3 °C). Therefore, the beetle population seems to be stable at 850 m (10.2 °C) and lower altitudes (higher temperatures) but cannot be maintained from 950 (9.1 °C) to 1,150 m (8.3 °C) without constant immigration of beetles from lower altitudes. The beetles could not reproduce at altitudes above 1,150 m (lower than 8.2 °C). From the mean and effective cumulative temperatures, we concluded that the beetle (and its population) can endure temperatures lower than those previously reported. Pine wilt disease also occurred at lower temperatures and higher altitudes than expected. We have summarized the principal strategies for controlling the disease at high altitudes based on these results.     

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.     

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.     

马尾松抗松材线虫病候选无性系的抗病性测定

为筛选抗松材线虫病强的马尾松无性系,以前期筛选获得的抗松材线虫病强的马尾松实生苗为接穗,使用湿地松作为砧木培育嫁接苗,开展无性系2次接种松材线虫抗病性测定。结果表明:使用湿地松作为砧木培育马尾松抗性无性系苗木,经过对马尾松无性系嫁接苗2次接种松材线虫测定,获得健全率较火炬松高的无性系。抗病性第1次、第2次测定的健全率超过火炬松的合格无性系分别为77.7%、66.2%,平均健全率分别为81.4%、94.5%,对照树种火炬松平均健全率分别为61.3%和82.1%,获得的无性系抗病性显著提高。为了减小误差,应该选用嫁接成活苗数量相对多的无性系间进行抗病性强弱次序评价。采用了10株以上无性系间抗病性强弱次序评价进行对照,减少了抗性强弱无性系两极分化,抗性能力中等的无性系相应增多,在选拔植株营建抗性种子园过程中应给予重视,尽量选择接种测定数量较多的高抗无性系。     

温度对松材线虫的生物效应

对松材线虫在不同温度下的存活及生长繁殖状况进行了观察。松材线虫在-70℃不能存活,在-20℃可存活约8d,在10℃~35℃的温度范围内,松材线虫的生长繁殖速度随温度上升而加快,生长繁殖数量随温度提高而增加,松材线虫在40℃可存活约7d,在45℃可存活约5d,在50℃以上不能存活。松材线虫与松树在不同温度下生长速度的差异提示松树在不同气候条件下发病轻重与此差异有关。     

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.     

Leaf-level and whole-plant gas exchange characteristics of shortleaf pine exposed to ozone and simulated acid rain

Field-grown shortleaf pine (Pinus echinata Mill.) seedlings were exposed to ozone (O(3)) and simulated acid rain (SAR) in open-top chambers over three growing seasons. Ranges of O(3) and SAR spanned ambient levels found in the southern USA. Effects of O(3) on leaf-level and whole-plant gas exchange were characterized for a single measurement period immediately before the third summer of exposure. Decreased photosynthesis rates were attributed to O(3), but not SAR. Stomatal conductance decreased in response to O(3) exposure, and either increased or was unaffected by SAR. Increased internal CO(2) concentration (c(i)) in response to O(3) treatment indicated a greater effect of O(3) on photosynthetic capacity than stomatal conductance. Whole-seedling gas exchange characteristics indicated that whole-seedling carbon assimilation was more severely affected by O(3) than was evident from leaf-level gas exchange characteristics. Seedlings exposed to O(3) retained fewer flushes than seedlings grown in charcoal-filtered air.     

松材线虫入侵的黑松内栖真菌区系初步研究

松材线虫（Bursaphelenchus xylophilus（Steiner ＆ Buhrer）Nickle）是毁灭性森林病害——松材线虫病的病原物,主要危害针叶树种,尤其是松属（Pinus spp．）树种。黑松（Pinus thunbergii Pad．）原产日本及朝鲜半岛东部沿海地区,因其耐海雾、抗海风、可在海滩上生长等特性,在我国辽宁、山东、河北、江苏、安徽、浙江、福建、湖北、广东等省大量栽植,总面积约159300hm^2。研究证明,黑松是松材线虫病的高度感病树种,一旦松材线虫入侵,后果是毁灭性的,将给森林造成严重的、不可挽回的损失。     

A rapid staining‐assisted wood sampling method for PCR‐based detection of pine wood nematode Bursaphelenchus xylophilus in Pinus massoniana wood tissue

For reasons of unequal distribution of more than one nematode species in wood, and limited availability of wood samples required for the PCR‐based method for detecting pinewood nematodes in wood tissue of Pinus massoniana, a rapid staining‐assisted wood sampling method aiding PCR‐based detection of the pine wood nematode Bursaphelenchus xylophilus (Bx) in small wood samples of P. massoniana was developed in this study. This comprised a series of new techniques: sampling, mass estimations of nematodes using staining techniques, and lowest limit Bx nematode mass determination for PCR detection. The procedure was undertaken on three adjoining 5‐mg wood cross‐sections, of 0.5 × 0.5 × 0.015 cm dimension, that were cut from a wood sample of 0.5 × 0.5 × 0.5 cm initially, then the larger wood sample was stained by acid fuchsin, from which two 5‐mg wood cross‐sections (that adjoined the three 5‐mg wood cross‐sections, mentioned above) were cut. Nematode‐staining‐spots (NSSs) in each of the two stained sections were counted under a microscope at 100× magnification. If there were eight or more NSSs present, the adjoining three sections were used for PCR assays. The B. xylophilus– specific amplicon of 403 bp ( DQ855275 ) was generated by PCR assay from 100.00% of 5‐mg wood cross‐sections that contained more than eight Bx NSSs by the PCR assay. The entire sampling procedure took only 10 min indicating that it is suitable for the fast estimation of nematode numbers in the wood of P. massonina as the prelimary sample selections for other more expensive Bx‐detection methods such as PCR assay.