Pathogenicity of aseptic Bursaphelenchus xylophilus

Pine wilt is a disease of pine (Pinus spp.) caused by the pine wood nematode (PWN), Bursaphelenchus xylophilus. However, the pathogenic mechanism of pine wilt disease (PWD) remains unclear. Although the PWN was thought to be the only pathogenic agent associated with this disease, a potential role for bacterial symbionts in the disease process was recently proposed. Studies have indicated that aseptic PWNs do not cause PWD in aseptic pine trees, while PWNs associated with bacteria cause wilting symptoms. To investigate the pathogenicity of the PWN and its associated bacteria, 3-month-old microcuttings derived from certain clones of Pinus densiflora Siebold & Zucc. produced in vitro were inoculated under aseptic conditions with aseptic PWNs, non-aseptic PWNs and bacteria isolated from the nematodes. Six-month-old aseptic P. densiflora microcuttings and 7-month-old P. massoniana seedlings were also inoculated under aseptic conditions with aseptic PWNs and non-aseptic PWNs. The results showed that the aseptic microcuttings and seedlings inoculated with aseptic PWNs or non-aseptic PWNs wilted, while those inoculated with bacterial isolates did not wilt. Nematodes were recovered from wilted microcuttings and seedlings inoculated with aseptic PWNs and non-aseptic PWNs, and the asepsis of nematodes recovered from aseptic PWN-inoculated microcuttings and seedlings was reconfirmed by culturing them in NB liquid medium at 30°C for more than 7 days. Taken together, the results indicate that the asepsis of PWN did not cause the loss of pathogenicity.     

化学通讯在松材线虫侵染和扩散中的作用

松材线虫为外来入侵种 ,由其引起的松材线虫病正在我国迅速扩散蔓延 ,造成我国部分地区松林资源的毁灭性破坏。松材线虫病的发生和流行与媒介天牛、寄主植物、共生真菌和细菌密切相关 ,松材线虫 -墨天牛 -松树 -共生微生物之间存在着广泛的化学联系 ,它们通过化学互作 ,调控松材线虫的行为 ,影响松材线虫的侵染和扩散     

Diversity of Bacteria Carried by Pinewood Nematode in USA and Phylogenetic Comparison with Isolates from Other Countries

Pine wilt disease (PWD) is native to North America and has spread to Asia and Europe. Lately, mutualistic relationship has been suggested between the pinewood nematode (PWN), Bursaphelenchus xylophilus the causal nematode agent of PWD, and bacteria. In countries where PWN occurs, nematodes from diseased trees were reported to carry bacteria from several genera. However no data exists for the United States. The objective of this study was to evaluate the diversity of the bacterial community carried by B. xylophilus, isolated from different Pinus spp. with PWD in Nebraska, United States. The bacteria carried by PWN belonged to Gammaproteobacteria (79.9%), Betaproteobacteria (11.7%), Bacilli (5.0%), Alphaproteobacteria (1.7%) and Flavobacteriia (1.7%). Strains from the genera Chryseobacterium and Pigmentiphaga were found associated with the nematode for the first time. These results were compared to results from similar studies conducted from other countries of three continents in order to assess the diversity of bacteria with associated with PWN. The isolates from the United States, Portugal and China belonged to 25 different genera and only strains from the genus Pseudomonas were found in nematodes from all countries. The strains from China were closely related to P. fluorescens and the strains isolated from Portugal and USA were phylogenetically related to P. mohnii and P. lutea. Nematodes from the different countries are associated with bacteria of different species, not supporting a relationship between PWN with a particular bacterial species. Moreover, the diversity of the bacteria carried by the pinewood nematode seems to be related to the geographic area and the Pinus species. The roles these bacteria play within the pine trees or when associated with the nematodes, might be independent of the presence of the nematode in the tree and only related on the bacteria''s relationship with the tree.     

Evolutionary change in a pine wilt system following the invasion of Japan by the pinewood nematode, <Emphasis Type="Italic">Bursaphelenchus</Emphasis><Emphasis Type="Italic">xylophilus</Emphasis>

Pinewood nematode (PWN), Bursaphelenchus xylophilus, is the causative agent of pine wilt disease (PWD) of pine trees and is transmitted by cerambycid beetles belonging to the genus Monochamus. PWN is believed to have been introduced into Japan from North America at the beginning of the 20th century. In this article, we first provide an outline of the PWD system and the range expansion of PWN in Japan and then review the literature, focusing on the virulence of PWN. Virulence is a heritable trait in PWN, with high virulence being closely related to a high rate of reproduction and within-tree dispersal. When two PWN isolates with different virulence levels are inoculated into pine seedlings, the more virulent nematodes always dominate in dead seedlings. In a laboratory setting, many more virulent nematodes board the insect vectors than avirulent ones. The age at which vectors transmit the most abundant PWNs to pine twigs changes during the course of a PWD epidemic. However, the relation between virulence and transmission of PWN remains as yet relatively unknown. Such information would enable ecologists to predict the evolution of the PWD system. In this review we also compare ecological traits between the PWN and the avirulent congener, B. mucronatus.     

松材线虫及其关键传媒墨天牛的研究进展

松材线虫Bursaphelenchusxylophilus (Steiner&Buhere,1 93 4Nickle,1 981 )是一种世界多国公认的重大外来生物。其原产地在美国 ,但松材线虫并不严重危害该国松林 ;日本受害最重 ,经过几十年的研究与防治 ,现已基本能控制松材线虫病大发生。自 1 982年我国南京中山陵首次发现松材线虫以来 ,其在我国的扩散趋势越来越明显 ,现已成为我国一种重要入侵病原生物。它不但对我国南方数百万公顷松林构成毁灭性威胁 ,影响我国经济和社会可持续发展 ,而且将破坏我国一些著名风景名胜区、文化遗产地。同时 ,松材线虫正成为一项技术壁垒 ,严重影响我国的进出口贸易。该文简要从松材线虫及其关键传煤墨天牛Monochamusspp .的生物学特性、分布状况 ,松材线虫病的蔓延条件及影响因素等方面概述国内外的研究动态 ,以期为我国对松材线虫的研究与防治工作提供参考     

Hazard ratings of pine forests to a pine wilt disease at two spatial scales (individual trees and stands) using self-organizing map and random forest

Pine wilt disease (PWD) caused by the pine wood nematode is the most serious global threat to pine forests. Hazard ratings of trees and forests to pest attacks provide important information to efficiently identify current or future hazardous conditions. However, in spite of the importance of hazard ratings for managing PWD, there are few studies on hazard ratings in this system. In this study, we evaluated the hazard ratings of pine trees and pine stands to PWD by considering environmental factors at the level of the stand and the individual tree. Our results showed that trees with larger diameter at breast height (DBH) showed a higher risk rate than those with smaller DBH, indicating that large trees have an increased probability of exposure to vector beetles because they are tall and have a large crown volume. We also found that reduced tree vigour could be related to susceptibility to PWD. In pine stands, geographical factors showed a high correlation with the occurrence of PWD. PWD occurrence was rare at high altitudes, but was more common on steep and south-facing slopes. These patterns were consistently observed in the results from 2 computational approaches: self-organizing map (SOM) and random forest models. The combination of SOM and random forest was effective to extract ecological information from the dataset. The SOM efficiently characterized relations among variables, and the random forest model was effective at predicting ecological variables, including the hazard rating of trees to disturbances.     

The developmental process of xylem embolisms in pine wilt disease monitored by multipoint imaging using compact magnetic resonance imaging

In pine wilt disease (PWD), embolized tracheids arise after virulent pine wood nematodes (PWN), Bursaphelenchus xylophilus, invade the resin canal of pine tree; infected pine trees finally die from significant loss of xylem water conduction. We used a compact magnetic resonance imaging system with a U-shaped radio frequency (rf) probe coil to reveal the developmental process of the xylem dysfunction in PWD. Multiple cross-sectional slices along the stem axis were acquired to periodically monitor the total water distribution in each 1-year-old main stem of two 3-year-old Japanese black pines (Pinus thunbergii) after inoculation of PWN. During the development of PWD, a mass of embolized tracheids around the inoculation site rapidly enlarged in all directions. This phenomenon occurred before the significant decrease of water potential. Some patch-like embolisms were observed at all monitoring positions during the experimental period. Patchy embolisms in a cross-section did not expand, but the number of patches increased as time passed. When the significant decrease of water potential occurred, the xylem dysfunctional rate near the inoculation point exceeded 70%. Finally, almost the whole area of xylem was abruptly embolized in all cross-sections along the stem. This phenomenon occurred just after water conduction was mostly blocked in one of the cross-sections. Thus, it appears that the simultaneous expansion of embolized conduit clusters may be required to induce a large-scale embolism across the functional xylem. Consequently, xylem dysfunction in infected trees may be closely related to both the distribution and the number of PWN in the pine stem.     

Effects of spring-killed pine trees on the epidemics of pine wilt disease

Pine wilt disease (PWD) is caused by the pinewood nematode (PWN), Bursaphelenchus xylophilus (Steiner et Buhrer) Nickle, and is transmitted by cerambycid beetles. In some pine trees infected with the PWNs in Japan, foliage changes from green to brown in summer to autumn of a nematode infection year (summer- autumn-killed trees) and the others in the following spring of a nematode infection year (spring-killed trees). The vector beetles require 1 or 2?years for development in cool summer areas and 1?year in warm summer areas. To evaluate the effects of the spring-killed trees and vectors with a long developmental time on the PWD epidemics, we presented simple mechanistic mathematical models. The models showed that it was possible for spring-killed trees to cause PWD epidemics when the transmission rate was high, and the efficacy of spring-killed trees as infection source was similar to that of summer?C autumn-killed trees. Spring-killed trees and vector beetles with a developmental time of 2?years harbored in summer- autumn-killed trees delayed epidemic timing by 3?C10?years or actually suppressed epidemics.     

Pathogenic cellulase assay of pine wilt disease and immunological localization

The pine wilt disease caused by Bursaphelenchus xylophilus (BX), also known as the pine wood nematode (PWN), is the most devastating disease of pine trees. In this work, a high molecular weight B. xylophilus cellulase antigen (BXCa) was purified from total homogenates of nematodes. BXCa was found to be able to hydrolyze carboxymethyl cellulose (CMC) efficiently (155.65 U/mg) and to have an approximate molecular mass of 58.9 kDa. We harvested anti-BXCa antibodies and performed immunocytochemical assays, which revealed the localization of cellulase pools in the esophageal gland cells of the PWN. It was also discovered that cellulase was secreted from the stylet and was used to hydrolyze cellulose to facilitate the PWN entering host cells. These results are consistent with other plant parasitical nematodes. Interestingly, strong fluorescence signals from cellulase staining were observed in tracheid cells in naturally infected pine wood, in addition to ray cells and the resin canal zone. These results strongly suggest that the cellulase released by the PWN is one of the pathogenic substances of pine wilt disease and is responsible for the development of the early symptoms of the disease.     

Pathogenic Cellulase Assay of Pine Wilt Disease and Immunological Localization

The pine wilt disease caused by Bursaphelenchus xylophilus (BX), also known as the pine wood nematode (PWN), is the most devastating disease of pine trees. In this work, a high molecular weight B. xylophilus cellulase antigen (BXCa) was purified from total homogenates of nematodes. BXCa was found to be able to hydrolyze carboxymethyl cellulose (CMC) efficiently (155.65 U/mg) and to have an approximate molecular mass of 58.9 kDa. We harvested anti-BXCa antibodies and performed immunocytochemical assays, which revealed the localization of cellulase pools in the esophageal gland cells of the PWN. It was also discovered that cellulase was secreted from the stylet and was used to hydrolyze cellulose to facilitate the PWN entering host cells. These results are consistent with other plant parasitical nematodes. Interestingly, strong fluorescence signals from cellulase staining were observed in tracheid cells in naturally infected pine wood, in addition to ray cells and the resin canal zone. These results strongly suggest that the cellulase released by the PWN is one of the pathogenic substances of pine wilt disease and is responsible for the development of the early symptoms of the disease.     

Host Deception: Predaceous Fungus,Esteya vermicola,Entices Pine Wood Nematode by Mimicking the Scent of Pine Tree for Nutrient

Background

A nematophagous fungus, Esteya vermicola, is recorded as the first endoparasitic fungus of pine wood nematode (PWN), Bursaphelenchus xylophilus, in last century. E. vermicola exhibited high infectivity toward PWN in the laboratory conditions and conidia spraying of this fungus on Japanese red pine, Pinus densiflora, seedlings in the field protected the pine trees from pine wilt disease to some extent, indicating that it is a potential bio-control agent against PWN. Previous research had demonstrated that the living fungal mycelia of E. vermicola continuously produced certain volatile organic compounds (VOCs), which were responsible for the PWN attraction. However, identity of these VOCs remains unknown.

Methodology/Principal Findings

In this study, we report the identification of α-pinene, β-pinene, and camphor produced by living mycelia of E. vermicola, the same volatile compounds emitted from PWN host pine tree, as the major VOCs for PWN attraction using gas chromatography-mass spectrometry (GC-MS). In addition, we also confirmed the host deception behavior of E. vermicola to PWN by using synthetic VOCs in a straightforward laboratory bioassay.

Conclusions/Significance

This research result has demonstrated that the endoparasitic nematophagous fungus, E. vermicola, mimics the scent of PWN host pine tree to entice PWN for the nutrient. The identification of the attractive VOCs emitted from the fungus E. vermicola is of significance in better understanding parasitic mechanism of the fungus and the co-evolution in the two organisms and will aid management of the pine wilt disease.     

Bursaphelenchus xylophilus: opportunities in comparative genomics and molecular host-parasite interactions

Most Bursaphelenchus species are fungal feeding nematodes that colonize dead or dying trees. However, Bursaphelenchus xylophilus, the pine wood nematode, is also a pathogen of trees and is the causal agent of pine wilt disease. B. xylophilus is native to North America and here it causes little damage to trees. Where it is introduced to new regions it causes huge damage. The most severely affected areas are found in the Far East but more recently B. xylophilus has been introduced into Portugal and the potential for damage here is also high. As incidence and severity of pine wilt disease are linked to temperature we suggest that climate change is likely to exacerbate the problems caused by B. xylophilus and, in addition, will extend (northwards in Europe) the range in which pine wilt disease can occur. Here we review what is currently known about the interactions of B. xylophilus with its hosts, including recent developments in our understanding of the molecular biology of pathogenicity in the nematode. We also examine the potential developments that could be made by more widespread use of genomics tools to understand interactions between B. xylophilus, bacterial pathogens that have been implicated in disease and host trees.     

Diversity of bacteria associated with Bursaphelenchus xylophilus and other nematodes isolated from Pinus pinaster trees with pine wilt disease

The pinewood nematode (PWN), Bursaphelenchus xylophilus, has been thought to be the only causal agent of pine wilt disease (PWD), however, since bacteria have been suggested to play a role in PWD, it is important to know the diversity of the microbial community associated to it. This study aimed to assess the microbial community associated with B. xylophilus and with other nematodes isolated from pine trees, Pinus pinaster, with PWD from three different affected forest areas in Portugal. One hundred and twenty three bacteria strains were isolated from PWN and other nematodes collected from 14 P. pinaster. The bacteria strains were identified by comparative analysis of the 16S rRNA gene partial sequence. All except one gram-positive strain (Actinobacteria) belonged to the gram-negative Beta and Gammaproteobacteria. Most isolates belonged to the genus Pseudomonas, Burkholderia and to the family Enterobacteriaceae. Species isolated in higher percentage were Pseudomonas lutea, Yersinia intermedia and Burkholderia tuberum. The major bacterial population associated to the nematodes differed according to the forest area and none of the isolated bacterial species was found in all different forest areas. For each of the sampled areas, 60 to 100% of the isolates produced siderophores and at least 40% produced lipases. The ability to produce siderophores and lipases by most isolates enables these bacteria to have a role in plant physiological response. This research showed a high diversity of the microbial community associated with B. xylophilus and other nematodes isolated from P. pinaster with PWD.     

Using the nematophagous fungus Esteya vermicola to control the disastrous pine wilt disease

The present study evaluated the protective effects of the nematophagous fungus Esteya vermicola on the large pine trees of Mt. Wora, Jinju, South Korea for six years. When pine trees were treated with E. vermicola 110 days before artificial normal pinewood nematode (PWN) infection, 30–50% of the trees survived for six years. When pine trees were treated with E. vermicola one week after artificial normal PWN infection, 40% of the trees were saved. In contrast, all of the control trees were killed by pine wilt disease in the first year. Although it has been more than six years since the beginning of this experiment, the existence of E. vermicola inside the treated pine trees was successfully detected using a PCR method with two pairs of specific primers for E. vermicola. These results suggest that E. vermicola possesses great potential as a biocontrol agent to combat the disastrous pine wilt disease. This is the first report of using nematophagous fungi to control pine wilt disease in the field for a duration of over five years.     

Fast R-CNN深度学习和无人机遥感相结合在松材线虫病监测中的初步应用研究

松材线虫病因其破坏性强、传播速度快和防治难度大等特点,严重威胁着我国的松林资源.及时发现、定位和清理病死松树是控制松材线虫病蔓延的有效手段.本研究利用小型无人机获得松材线虫病疫点的可见光和多光谱的航摄影像.根据松树针叶颜色变化,将松材线虫Bursaphelenchus xylophilus侵染的松树分为病树和枯死树两种类型.将无人机遥感正摄影像图切割成瓦片图,根据不同植被指数的特征差异,筛选出含病树和枯死树的瓦片图.训练Fast R-CNN深度学习框架形成最终模型,通过模型运算获得病枯死松树的分布地图及坐标点位置.研究结果显示Fast R-CNN深度学习和无人机遥感相结合能有效识别出病树和枯死树,正确率分别达到90％和82％,漏检率分别为23％和34％,可为大面积监测松材线虫病的发生现状和流行动态、评估防控效果和灾害损失提供技术支撑.     

松材线虫及其传媒松墨天牛的监测和防治现状

松材线虫起源于北美大陆。1 982年侵入中国。松材线虫能引起一种林业上最具危险性、毁灭性的病害“松材线虫病”。松材线虫病的病因复杂,传播途径多样,不同国家地区对其采取的监测和防治策略也有所差异。目前,该病局限分布于北美和东北亚,但在世界范围内其扩散趋势日益明显。该文简要从监测预警,物理、化学、生物等防治方法、以及监测防治中存在问题和发展趋势等方面概述了国内外松材线虫Bursaphelenchusxylophilus及其关键传媒松墨天牛MonochamusalternatusHope的研究现状,对制定适宜我国的松材线虫病监测和防治策略有借鉴和参考价值。