Spread of the pinewood nematode vectored by the Japanese pine sawyer: modeling and analytical approaches

The pinewood nematode, Bursaphelenchus xylophilus, is the causative agent of pine wilt of Pinus thunbergii and P. densiflora in Japan. The nematode is vectored by cerambycid beetles of the genus Monochamus. It is inferred to have been introduced from North America early in the 1900s and then to have distributed in China, Korea, and Taiwan. Intensive and/or long-term studies of pine wilt systems have elucidated the pattern and mechanism of the nematode’s spread within a pine stand, dispersal of vector beetles, and spread pattern of pine wilt within a prefecture. The modeling of nematode spread over pine stands, which involves beetle reproduction within a pine stand, has been developing and should elucidate the factors influencing the rate at which the nematode range expands. In this review, we summarize the biologies of the nematode, beetle, and tree, and then characterize the spread of the nematode within a pine stand, locally over pine stands, and regionally over unit administrative districts. Local and regional spreading of the nematode is related primarily to long-distance dispersal by insect vectors and to the artificial transportation of pine logs infested with the nematode and its vector, respectively.     

携带与非携带松材线虫的松墨天牛miRNA表达谱比较分析

【目的】松墨天牛是松树的重要蛀干害虫,也是林业重大外来入侵种松材线虫的媒介昆虫。虽然松墨天牛和松材线虫互作的化学生态和分子进化机制受到人们的广泛关注,但miRNA等表观遗传因子在天牛—线虫互作中的作用未见报道。【方法】使用illumina HiSeq 2000平台进行miRNA高通量测序,得到4个携带线虫的天牛miRNA库和4个未携带线虫的天牛miRNA库。此外,对鉴定出的miRNA进行了差异表达分析,并对这些miRNA的靶基因进行了GO注释和KEGG通路富集分析。【结果】在携带线虫的天牛表皮、脂肪体、中肠和气管样本中分别鉴定出780、802、617和762个miRNA;在未携带松材线虫的天牛的不同组织样本中分别鉴定出784、723、713和837个miRNA。在携带松材线虫的松墨天牛中,某些已知miRNA表达量会显著升高,如miR-14、miR279和miR-312等。差异表达miRNA的功能大多指向代谢、免疫等方面。【结论】miRNA在松墨天牛和松材线虫互作中起着重要的调控作用。     

Application of harmonic radar to analyze dispersal behavior of the Japanese pine sawyer beetle,Monochamus alternatus (Coleoptera: Cerambycidae)

Monochamus alternatus (Hope) is a severe wood‐boring pest in coniferous forests and a major vector of pine wilt disease in East Asia. Harmonic radar is a powerful tool for studying the dispersal behavior of insects and it could be applied to control pine wilt disease. In this study, we validated the application of harmonic radar for analyzing the dispersal behavior of M. alternatus beetles in a field environment. We determined the wing capacities of the beetles and the effects of electronic tagging and marking on their movement, flight ability, survivorship, and food consumption in the laboratory to confirm the suitability of this technique. The detection rate and recovery rate for beetles were analyzed separately using radar on caged pine stands and in the field environment. The results showed that the minimum wing capacity of the Japanese pine sawyer was 24.9 mg, which was seven times the weight of the electronic tag (3.5 mg). Marking and tagging the beetles had no significant adverse effects on their movement, flight capacity, food consumption, and survivorship. The detection rate using the radar system and recovery rate based on visual observations of the beetles in caged pines were both 95.6%. However, in the field environment, the detection and recovery rates were only 55.6% and 37.8% after one week, respectively, and 33.3% and 7.8% after two weeks. Harmonic radar is a promising technique for studying the dispersal behavior of the Japanese pine sawyer, but its performance is not satisfactory and major improvements are required for both the radar system and electronic tags.     

History of Pine Wilt Disease in Japan

Pine wilt disease induced by the pinewood nematode, Bursaphelenchus xylophilus, is a great threat to pine forests in Japan. The first occurrence of the disease was reported in Nagasaki, Kyushu. During the 1930s the disease occurrence was extended in 12 prefectures, and in the 1940s the disease was found in 34 prefectures. The annual loss of pine trees increased from 30,000 m³ to 1.2 million m³ during these two decades. The enormous increase in timber loss in the 1970s resulted in 2.4 million m³ of annual loss in 1979. The affected area expanded into 45 prefectures of 47 prefectures in Japan. In cool areas the disease differs in epidemiology from that in heavily infested areas in the warm regions. A national project for controlling pine wilt disease lays special emphasis on the healthy pine forests predominating throughout cool areas in northern Japan.     

三株芽胞杆菌菌株对松材线虫低温存活与繁殖的影响

为明确细菌在松材线虫生态适应性中的作用,本研究选择与致病相关的松材线虫伴生细菌GD1、马尾松内生细菌GD2以及具有杀松材线虫活性的湿地松内生细菌NJSZ-13为试验对象,测定经这3株芽胞杆菌菌株3个浓度低温驯化10、15和20 d后,在冷冻条件下松材线虫强毒虫株AMA3、中毒虫株AA3和弱毒虫株YW4的存活率和繁殖量。结果表明：低温驯化15 d和20 d后3株菌株对不同毒力线虫的活力影响较驯化10 d后的更显著。在低温驯化15 d、-20℃冷冻处理1 h后,5×10⁶ CFU/mL浓度菌株GD1处理下,虫株AMA3、AA3和YW4的存活率分别为77.22%、83.68%和84.26%,与对照差异显著;5×10⁵ CFU/mL浓度菌株GD1处理下,虫株AMA3、AA3和YW4的存活率分别为75.76%、80.67%和81.50%,与对照差异显著。5×10⁶ CFU/mL和5×10⁵ CFU/mL浓度菌株GD2处理下,与GD1处理组结果相似,菌株NJSZ-13处理组则与菌株GD1和GD2的结果相反。低温驯化15 d、-20℃冷冻处理1 h后,5×10⁶ CFU/mL浓度菌株GD1处理下,虫株AMA3、AA3和YW4的繁殖量分别为7 530、9 317和12 793条/皿,与对照（3 192、3 840和5 823条/皿）差异显著;5×10⁵ CFU/mL浓度菌株GD1处理时,3个虫株的繁殖量均与对照差异显著。而菌株GD2和NJSZ-13处理后,3个虫株的繁殖量均无显著变化。表明不同芽胞杆菌对松材线虫的低温适应性影响存在差异,松材线虫伴生细菌GD1和马尾松内生细菌GD2能增强其低温适应性,而湿地松内生细菌NJSZ-13菌株则相反。     

Temperature Effects on the Transmission of Bursaphelenchus xylophilus (Nemata: Aphelenchoididae) by Monochamus alternatus (Coleoptera: Cerambycidae)

The object of this research was to investigate the effects of ambient temperature on the transmission of the pinewood nematode, Bursaphelenchus xylophilus, by its vector, Monochamus alternatus. Ninety M. alternatus were reared individually at one of three constant temperatures (16, 20, and 25 °C). As the ambient temperature decreased from 25 to 16 °C, longevity of vectors decreased, nematode transmission efficiency decreased, and the peak period of nematode transmission was delayed and its peak height decreased. Low temperature may inhibit the transmission process, and this inhibition could be partially responsible for preventing pine wilt disease from devastating pine forests in cool regions.     

松材线虫galectin-1基因的克隆、原核表达和表达模式分析

【目的】galectin-1是凝集素的一种,广泛存在于各种生物体内,在生长发育、免疫调节方面起重要作用。本研究克隆和表达了松材线虫的galectin-1蛋白,并分析了各个龄期的表达量。【方法】设计引物,扩增松材线虫的galectin-1基因,使用双酶切的方法连接p ET-28a载体和目的基因,转化到大肠杆菌DH5α感受态细胞,筛选阳性克隆;在不同温度下,用不同浓度的异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,Western blot检验蛋白表达情况;采用RT-PCR技术检验松材线虫各个龄期galectin-1基因的表达情况。【结果】由SMART和Predict Protein软件分析可知,该蛋白有2个结构域,并且主要由无规卷曲和β折叠构成;生物学信息分析显示,松材线虫的galectin-1与小卷蛾斯氏线虫的相似性更高;与日本血吸虫相比,线虫能很好地聚集在一个分支上。Western blot检测纯化蛋白大小与查询所得蛋白分子质量一致。RT-q PCR结果显示,以繁殖型2龄松材线虫(L_2)为对照,galectin-1基因在繁殖型3龄(L_3)、繁殖型4龄(L_4)、扩散型3龄(L_(Ⅲ))和扩散型4龄(L_(Ⅳ))松材线虫中的表达量高,尤其是在LⅢ中的表达量最高;雌雄成虫没有显著性差异。【结论】松材线虫的galectin-1基因在p ET-28a原核表达系统中呈可溶性表达,在不同龄期的表达量有差异。本研究为进一步研究松材线虫的galectin-1基因奠定了基础,为松材线虫的防治提供了新的方向。     

A Nondestructive Method of Determining Bursaphelenchus xylophilus Infestation of Monochamus spp. Vectors

Pine wilt is caused by the nematode Bursaphelenchus xylophilus, which is transported to host trees in the trachea of Monochamus spp. (Coleoptera: Cerambycidae). The study of the relationship between the nematode and its beetle vectors has been hampered by the inability to estimate nematode presence or density within live beetles. This report describes a rapid method for estimating nematode load within live M. carolinensis and M. alternatus by visual examination of the atrium of the first abdominal spiracle. Visual estimates of nematode numbers correlated highly with actual nematode numbers. This method is a timesaving technique for determining relative numbers of B. xylophilus in pine wilt research.     

Spatial pattern of pine wilt disease caused byBursaphelenchus xylophilus (Nematoda: Aphelenchoididae) within aPinus thunbergii stand

To understand the mechanism of spread of pine wilt disease caused by the pinewood nematode, Bursaphelenchus xylophilus, which is vectored by a cerambycid, Monochamus alternatus, the spatial distribution of trees weakened by the nematode was examined within a Pinus thunbergii stand from June to October for 4 years. The weakened trees were distributed in a clumped pattern in 1980 and 1981, at an early stage of infestation. In many cases, they showed a double-clumped pattern. The degree of aggregation was higher in June or July than after August. They were uniformly distributed in June or July 1982 and in June 1983 whereas they showed a double-clumped pattern after August. The trees were frequently weakened in June or July when they were near the trees weakened during the previous year. At quadrat sizes of more than 25 m², spatial overlapping was pronounced between trees weakened during June–July of the current year and those weakened in the previous year. The seasonal changes in spatial distribution of weakened trees were explained by the interaction among M. alternatus, B. xylophilus and Pinus trees.     

植物根际益生细菌代表性菌株贝莱斯芽孢杆菌FZB42对松材线虫的抑杀性

[目的]由松材线虫导致的松树萎蔫病是松树的毁灭性病害,也是我国最主要的林业病害之一.本研究测评了在农业上广泛使用的、我国微生物肥料行业主要菌种资源之一——贝莱斯芽孢杆菌,对松材线虫的潜在抑杀性能.[方法]选用贝莱斯芽孢杆菌的代表性菌株FZB42为材料,测定对不同条件下的菌液上清、不同菌株的菌液上清、细菌素plantaz...     

Inhibitory Effect of Bursaphelenchus mucronatus (Nematoda: Aphelenchoididae) on B. xylophilus Boarding Adult Monochamus alternatus (Coleoptera: Cerambycidae)

Inhibitory effects of Bursaphelenchus mucronatus on the number of B. xylophilus carried by an adult Monochamus alternatus were investigated using artificial pupal chambers. When pupal chambers were infested with either B. xylophilus or B. mucronatus, the load of B. xylophilus onto the beetle was greater (P < 0.001) than that of B. mucronatus. However, within the pupal chamber there was no difference in the abundance of the third-stage dispersal juveniles, which would molt to the fourth-stage dispersal juveniles to board beetles. The nematode load on beetles that emerged from pupal chambers infested with both Bursaphelenchus species was smaller (P = 0.015) than that of beetles with B. xylophilus alone but greater (P < 0.001) than that of beetles with B. mucronatus alone, suggesting an inhibitory effect of B. mucronatus. As a result of this study, the rate of inhibition of B. mucronatus on molting of third-stage dispersal juveniles of B. xylophilus to fourth-stage dispersal juveniles was 0.65, which resulted in great inhibition on boarding beetles at a rate of 0.7.     

Seed dispersal of the Korean pine,Pinus koraiensis,by the red squirrel,Sciurus vulgaris

Seed hoarding behavior of the red squirrel,Sciurus vulgaris, was studied in relation to the amount of dispersed seeds of the Korean pine,Pinus koraiensis, and the distribution of its seedlings. After removing a cone from a tree, squirrels sat on the ground and ripped off its cone scales before transporting it. A mean of 3.2 seeds were scatter-hoarded per hole. Of 7.7×10⁴ mature seeds produced in a 0.21 ha planted Korean pine forest, 22% were estimated to be directly eaten by four squirrels, 9% were hoarded by them in the pine forest and 65% were cached outside the forest. Squirrels rediscovered hoarded seeds frequently, until the ground was covered with snow, during the period from snow fall until seed germination the next spring, few hoarded seeds were utilized. Korean pine seedlings were found up to 600 m from their mother trees. Scatter-hoarding by squirrels extensively contributes to seed dispersal to places suitable for the regeneration of the Korean pine. The large size of the cone, the absciss-layer at the cone penduncle, the infrequent dehiscence of cone scales, the large and wingless seeds, and the thick seed-coats have probably all been specialized to facilitate utilization by GenusSciurus.     

Fungi associated with the southern pine beetle: avoidance of induced defense response in loblolly pine

Summary Loblolly pines (Pinus taeda) are rapidly killed by colonizing southern pine beetle (Dendroctonus frontalis). The female beetles carry two species of fungi (Ceratocystis minor var. barrasii and an unnamed basidiomycete) within a mycangium. The insects are also frequently associated with a blue-staining form of C. minor. These fungi are inoculated into the tree during colonization. The tree has an induced defensive response that involves resin soaking and necrosis of affected tissue isolating the invading organlsms. The blue-staining fungus stimulates formation of this response in the tree, but the two mycangial fungi do not. These results suggest that the beetles are closely associated with two highly pathogenic fungi that do not stimulate one of the critical components of tree defense.     

Successful spread of a biocontrol agent reveals a biosecurity failure: elucidating long distance invasion pathways for <Emphasis Type="Italic">Gonatocerus ashmeadi</Emphasis> in French Polynesia

Invasive species are generally detected in new ecosystems long after their first arrival, making it difficult to elucidate pathways leading to successful invasion. In this study, the dispersal of a classical biological control agent, the mymarid egg parasitoid Gonatocerus ashmeadi, was monitored across ten islands in three major island groups in French Polynesia from the exact moment of its introduction into Tahiti to combat the invasive pest Homalodisca vitripennis. Within 10 months, the parasitoid spread quickly from Tahiti to widely separated islands (up to 1,400 km from Tahiti); presumably through the transportation of plant material containing parasitized H. vitripennis eggs. Gonatocerus ashmeadi thus functioned as a “biomarker”, providing an informal audit of the effectiveness of inter-island quarantine measures designed to curb the accidental spread of noxious organisms. Survey results suggest that invasive organisms, like deliberately released biological control agents, can be unintentionally and rapidly transmitted across vast distances by humans. Furthermore, even remote islands appear to experience relentless pressure from invasive propagules associated with human travel. Implications of survey work documenting the spread and impact of G. ashmeadi are discussed within the context of biological control programs, non-target impacts, and biosecurity initiatives. Handling Editor: Dirk Babendreier.     

Host acceptance behaviour of the jack pine tip beetle, Conophthorus anksianae and the red pine cone beetle, C. resinosae

The jack pine tip beetle, Conophthorus banksianae McPherson (Coleoptera: Scolytidae) and the red pine cone beetle, C. resinosae Hopkins, are doubtful sibling species. However, it is possible that these two taxa are valid species that maintain reproductive isolation because they accept different hosts. In a series of laboratory and field cage experiments, the host acceptance behaviours of these two species under choice and no choice conditions were compared. The field experiments demonstrated that the two species have a similar propensity to accept jack pine cones and shoots for feeding, but differ consistently in their acceptance of red pine cones, and variably in their acceptance of red pine shoots. However, the laboratory experiments did not indicate a difference between the two species in their propensity to accept red pine cones for feeding. In field cages, C. resinosae accepted significantly more red pine cones for oviposition than C. banksianae; the situation was reversed for jack pine shoots. In comparison to C. banksianae, C. resinosae is a more generalist feeder. The results from this study suggest that host acceptance behaviour is a permeable barrier unable to ensure reproductive isolation between the two species. Although there are differences in the host acceptance behaviours between C. banksianae and C. resinosae, we conclude that these differences do not necessarily support their designation as distinct species.     

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.     

The effect of invasive plant management on the rate of removal of vertebrate-dispersed fruits

Chrysanthemoides monilifera ssp. rotundata is an invasive plant in eastern Australia. It produces large quantities of fleshy fruits, which are dispersed by birds and mammals, and has the potential to alter ecological processes in native plant communities in several ways. Seed dispersal of native plants growing amongst C. monilifera could be decreased (if C. monilifera is effective in competing for limited seed dispersers), increased (if C. monilifera-infested vegetation supports more seed dispersers), or be unchanged (if there is sufficient separation in fruiting time or dispersal agents between the species, or if dispersers are not limiting). We compared the rates of removal of fruits of native plants (Monotoca elliptica and Elaeocarpus spp.) from experimental stations in C. monilifera stands, in uninfested coastal vegetation, and in vegetation from which C. monilifera had been eliminated with herbicide. The rate of removal of fruits differed between plant species and locations, but not between any of the habitat treatments. This may have resulted from temporal separation of peak fruit availability between M. elliptica and C. monilifera, and differences in fruit morphology and disperser assemblages between Elaeocarpus spp. and C. monilifera. We also measured removal of C. monilifera fruits from stations in C. monilifera stands and from where C. monilifera had been eliminated with herbicide. In the herbicide-treated habitat, removal of C. monilifera fruits was reduced. This may assist control efforts and suggests that, to reduce seed dispersal, dense (core) populations should be targeted in control programs.     

Oviposition deterrence associated with larval frass of the Japanese pine sawyer,Monochamus alternatus (Coleoptera: Cerambycidae)

To determine the effect of larval frass of Monochamus alternatus on oviposition preference of the female adults, three tests were performed in the laboratory. Individual females were provided with a frass-coated, Pinus densiflora bolt and an untreated bolt simultaneously and were allowed to oviposit for 24 h. They deposited a significantly smaller number of eggs on frass-coated bolts. The females supplied with frass-coated bolts deposited no eggs on them, indicating that the larval frass deterred the females from oviposition. When individual females were provided simultaneously with a pine bolt applied with a methanol extract of larval frass and another bolt applied with methanol alone, they deposited a significantly smaller number of eggs on frass extract-applied bolts for 24 h. These results suggest the presence of a putative oviposition deterrent in larval frass of M. alternatus. Each test was replicated 19-20 times using different females.     

Newly Discovered Transmission Pathway of Bursaphelenchus xylophilus from Males of the Beetle Monochamus alternatus to Pinus densiflora Trees via Oviposition Wounds

The transmission of Bursaphelenchus xylophilus from Monochamus alternatus males to Pinus densiflora trees via oviposition wounds has been determined. Nematode-infested males, with mandibles fixed experimentally to prevent feeding, were placed for 48 hours with pine bolts containing oviposition wounds that had been made by nematode-free females. After removal of the nematode-infested males, the pine bolts were held for 1 month and then examined for the presence of nematodes. Reproducing nematode populations were recovered from pine bolts that were exposed to male beetles carrying a high number of nematodes. No reproducing nematode population could be recovered from pine bolts exposed to beetles with a small number of nematodes. Nematode reproduction in the pine bolts was not related to the number of oviposition wounds per bolt. Fourth-stage dispersal B. xylophilus juveniles, collected from beetle body surfaces, were inoculated on pine bolt bark 0, 5, 10, and 15 cm away from a single artificial, small hole. These dauer juveniles successfully entered some bolts. The probability of successful nematode reproduction decreased with increased distance between inoculation point and artificial hole. The results indicated that B. xylophilus can move a significant distance to oviposition wounds along the bark surface and enter a tree via the wounds. The new transmission pathway is considered important for the nematode to persist in pine forests such as in North America where pine wilt disease does not occur.