Pine Wood Nematode Bursaphelenchus Xylophilus Research Articles

Nematicidal Activity of 3-Acyltetramic Acid Analogues Against Pine Wood Nematode, Bursaphelenchus xylophilus.

Among 98 3-acyltetramic acid analogues, compounds 1c, 2c, 2f and 2g, showed >90% nematicidal activity against the pine wood nematode Bursaphelenchus xylophilus at a 10 μg/mL concentration. The nematicidal activities of compounds 1d, 1h, and 2k were a little lower at 88.0%, 85.8%, and 57.2% at a 10 μg/mL concentration, respectively. The nematicidal activity of emamection benzoate, widely used in Korea for the prevention of pine wilt disease, was 32.3% at a 10 μg/mL concentration. Other 3-acyltetramic acid analogues showed less than 30% nematicidal activity. A structure-activity relationship study indicated that the chain length of the C-acyl substituent was very important for high nematicidal activity. All active compounds had C13H27 or C11H23 acyl substituents, in two closely related groups with the common physicochemical properties of a polar surface area 57.6A2, PSA (polar surface area) 7.8–8.6% and ClogP (calculated partition coefficient) 5.1–5.9 and a polar surface area 75–84A2, PSA 11.1–11.6% and ClogP 4.7–5.1, respectively. Our study indicates that active 3-acyltetramic acid analogues could have potential as lead compounds for developing novel pine wood nematode control agents.

The Microbiome of Endophytic, Wood Colonizing Bacteria from Pine Trees as Affected by Pine Wilt Disease

Pine wilt disease (PWD) is a devastating forest disease present worldwide. In this study we analyzed the effects of the invasion of the pinewood nematode Bursaphelenchus xylophilus, the major pathogen causing PWD, on the endophytic microbiome of adult P. pinaster trees. Wood samples from trees with different degrees of PWD disease were collected at two sites (A and M) in Portugal. Endophytic bacteria were characterized based on directly extracted DNA by fingerprinting and barcoding using the 16S rRNA gene as marker. Furthermore, cultivation-based approaches were used to obtain isolates of the major taxa to study their ecophysiology. The endophytic microbiome from P. pinaster trees differed significantly between the two sampling sites. Main bacterial OTUs belonged to the Proteobacteria (39% (site M) - 97% (site A)), and Firmicutes (0.70% (site A) - 44% (site M)). However, consequences of the invasion with the pathogen were comparable. Interestingly diversity of wood endophytic bacteria increased with the severity of the diseases, with highest diversity levels observed in in the most affected trees. Our results suggest that in the first stages of the disease, the defence mechanisms of plants are repressed by the pathogen, resulting in a colonization of the wood interior by soil microorganisms.

Esteya vermicola Controls the Pinewood Nematode, Bursaphelenchus xylophilus, in Pine Seedlings.

Esteya vermicola (Ophiostomataceae) is an endoparasitic fungus that has great potential as a biological control agent against the pinewood nematode Bursaphelenchus xylophilus which causes pine wilt disease. We tested E. vermicola for control of pine wilt disease by spraying E. vermicola conidia on artificial wounds on pine seedlings, and the optimum E. vermicola treatment density and application time were also investigated in the greenhouse. The wounds were similar to those made by sawyer beetles (Coleoptera: Cerambycidae). Esteya vermicola treatments significantly increased the survival rate of pine seedlings that were infected by pinewood nematodes. Wounded plants sprayed with 107 CFU/ml E. vermicola had a 73.0% greater survival rate than nonwounded pine seedlings treated similarly. The treatment of pine seedlings with 107 CFU/ml E. vermicola 14 d before nematode infection increased their survival rate by 90.0%. The number of pinewood nematodes isolated from dead pine seedlings sprayed with E. vermicola was 76% less than the number of pinewood nematodes in the controls. Moreover, infected nematodes and the hyphae of E. vermicola were detected in the dead or wilting pine seedlings. Therefore, spraying E. vermicola on the wounds of pine seedlings made by sawyer beetles provides good control of the pine wilt disease that is caused by pinewood nematodes.

Synergistic attraction of pine sawyer Monochamus saltuarius (Coleoptera: Cerambycidae) to monochamol and α‐pinene

AbstractMonochamus (Coleoptera: Cerambycidae) species are longhorn pine sawyers that serve as insect vectors of the pinewood nematode Bursaphelenchus xylophilus (Nematoda: Parasitaphelenchidae), which are responsible for debilitating pine wilt disease. An aggregation pheromone, 2‐(1‐undecyloxy)‐1‐ethanol (hereafter referred to as monochamol), was shown to be effective at attracting Monochamus species. However, attraction of the pine sawyers to aggregation pheromones varied depending on semiochemicals, including host plant volatiles and kairomones. In this study, we investigated the abilities of monochamol and the host‐plant volatiles α‐pinene and ethanol to attract M. saltuarius in a pine forest in Cheongsong, Gyeongsangbuk‐do, Korea. A total of 91 M. saltuarius (28 males and 63 females) were captured. The combination of monochamol (700 mg) with α‐pinene and ethanol exhibited a synergistic effect on attracting M. saltuarius (11.0 beetles per trap), whereas monochamol alone and a mixture of α‐pinene and ethanol resulted in the capture of 3.2 beetles and 3.6 beetles per trap, respectively. Our results suggest that multi‐funnel traps baited with a blend of monochamol, α‐pinene and ethanol are highly effective for monitoring M. saltuarius and M. alternatus in pine forests.

Physiological development and dispersal ability of newly emerged Monochamus galloprovincialis

Novel associations between exotic pathogens and native insects may result in major ecological and economical losses. In Europe, Monochamus galloprovincialis (Olivier) (Coleoptera: Cerambycidae) is the only known vector of the exotic pine wood nematode Bursaphelenchus xylophilus (Steiner & Buhrer) Nickle (Nematoda: Aphelenchoididae), the causal agent of pine wilt disease (PWD). Transportation of goods containing nematode-infested beetles is the main pathway for the spread of the disease. In this scenario, management actions involving early detection and eradication are critical to stop further spread by the vector. Although dispersal of mature M. galloprovincialis has been successfully tracked using commercial baits and traps, dispersal ability of immature individuals is poorly understood. Sexual maturation and other physiological traits related to dispersal were studied for newly emerged M. galloprovincialis after different shoot-feeding spans. Sexual maturation was complete after on average 12 (males) or 13 (females) feeding days. Monochamus galloprovincialis adults emerged with an average of 10% lipid and 23.8% (males) or 29.9% (females) pterothorax muscle content, and these percentages did not change significantly during shoot feeding. Microtomography images of wing muscle structures at different maturation stages confirmed these results. Emerged adults that were kept unfed survived an average of 12 days in the lab. The ability of unfed immature insects to fly through hostless terrain was studied by marking and releasing newly emerged insects from a crop area located up to 3 km away from two small pine stands. The longest flown distance recorded was 2 km. Fitted regressions indicated that immature insects could have travelled up to 3 109 m to reach the experimental stands. We found that M. galloprovincialis emerge with well-developed thoracic muscles and energy reserves that enable them to sustain long flights over non-forested areas. These findings should aid managers and policy makers in devising sound procedures in areas where the risk of introducing PWD is high.

Understanding pine wilt disease: roles of the pine endophytic bacteria and of the bacteria carried by the disease-causing pinewood nematode.

Pine wilt disease (PWD) is one of the most destructive diseases in trees of the genus Pinus and is responsible for environmental and economic losses around the world. The only known causal agent of the disease is the pinewood nematode (PWN) Bursaphelenchus xylophilus. Despite that, bacteria belonging to several different genera have been found associated with PWN and their roles in the development of PWD have been suggested. Molecular methodologies and the new era of genomics have revealed different perspectives to the problem, recognizing the manifold interactions between different organisms involved in the disease. Here, we reviewed the possible roles of nematode‐carried bacteria in PWD, what could be the definition of this group of microorganisms and questioned their origin as possible endophytes, discussing their relation within the endophytic community of pine trees. The diversity of the nematode‐carried bacteria and the diversity of pine tree endophytes, reported until now, is revised in detail in this review. What could signify a synergetic effect with PWN harming the plant, or what could equip bacteria with functions to control the presence of nematodes inside the tree, is outlined as two possible roles of the microbial community in the etiology of this disease. An emphasis is put on the potential revealed by the genomic data of isolated organisms in their potential activities as effective tools in PWD management.

Versatile glycoside hydrolase family 18 chitinases for fungi ingestion and reproduction in the pinewood nematode Bursaphelenchus xylophilus

The glycoside hydrolase family 18 (GH18) of chitinases is a gene family widely expressed in archaes, prokaryotes and eukaryotes, and hydrolyzes the β-1,4-linkages in chitin. The pinewood nematode Bursaphelenchus xylophilus is one of the organisms that produces GH18 chitinases. Notably, B. xylophilus has a higher number of GH18 chitinases compared with the obligate plant-parasitic nematodes Meloidogyne incognita and Meloidogyne hapla. In this study, seven GH18 chitinases were identified and cloned from B. xylophilus based on genomic analyses. The deduced amino acid sequences of all these genes contained an N-terminal signal peptide and a GH18 catalytic domain. Phylogenetic analysis showed that the origin of B. xylophilus GH18 chitinases was independent of those from fungi and bacteria. Real-time quantitative reverse transcription PCR analysis indicated that GH18 chitinase genes had discrete expression patterns, representing almost all the life stages of B. xylophilus. In situ hybridisation showed that the mRNA of GH18 chitinase genes of B. xylophilus were detected mainly in the spermatheca, esophageal gland cells, seminal vesicle and eggs. RNA interference (RNAi) results revealed different roles of GH18 chitinase genes in B. xylophilus. Bx-chi-1, Bx-chi-2 and Bx-chi-7 were associated with reproduction, fungal cell-wall degradation and egg hatching, respectively. Bx-chi-5 and Bx-chi-6 may be involved in sperm metabolism. In conclusion, this study demonstrates that GH18 chitinases have multiple functions in the life cycle of B. xylophilus.

Using an evapo-transpiration model (ETpN) to predict the risk and expression of symptoms of pine wilt disease (PWD) across Europe

The pine wood nematode (PWN) Bursaphelenchus xylophilus is the causal agent of pine wilt disease (PWD), a xylem restricting disease of pine trees. PWN, a native of North America where it very rarely kills native pine trees, has spread internationally killing host trees in China, Japan, Korea, Taiwan and Portugal, with isolated incursions into Spain. Based on the locations where tree mortality has been recorded, it appears that pine trees growing in hot, dry conditions are more susceptible to pine wilt disease. This paper describes the ETpN model, an evapo-transpiration model (previously developed by Forest Research), which has been modified to incorporate the presence of PWN inside a tree and which predicts the regions of Europe that are likely to succumb to PWD. ETpN acts independently of the vector beetle (Monochamus spp.), predicting the likelihood of PWD on the assumption that a tree in a particular region has already been infested by the pine wood nematode. Different regions across Europe are included to investigate and demonstrate how different climates affect PWD incidence significantly. Simplified, “lite” and latency models have been developed to allow a non-specialist user to determine respectively the risk of PWD at a particular location and the likelihood of delays (latency) in expression of wilt symptoms.

Protective formula and preservation conditions for the endoparasitic fungus, Esteya vermicola

The endoparasitic nematophagous fungus, Esteya vermicola, is a bio-control agent with demonstrated ability to attack pinewood nematode (Bursaphelenchus xylophilus). An optimized solution for the protection and preservation of E. vermicola conidia is needed in order to ensure their survival during transportation, preservation, and application. Five protectants, kaolin, arabinose, sorbitol, PEG8000, and Span 80, were selected from 34 agents. These were incorporated into calcium alginate gel capsules at the following concentrations: 10% kaolin, 0.1% Span 80, 1% arabinose, 5% sorbitol, and 5% PEG8000. The improved diffluent formula contained 69.9% soluble starch, 14% wheat flour, 5% PEG8000, 0.1% span 80, 1% arabinose and 10% skim milk. The viability of E. vermicola conidia preserved in the protectant (5% sorbitol and 20% PEG8000) at six temperatures,–70,–20, 4, 26, 37°C, and room temperature (uncontrolled), was also assessed. The highest viability after storage for one month was achieved at–70°C.

Effect of family, crown position, number of winter buds, fresh weight and the length of needle on rooting ability of Pinus thunbergii Parl. cuttings

Abstract: As a measure for contrasting pine wilt disease, which caused serious damage in Japanese black pine (Pinus thunbergii Parl.) by the pine wood nematode (Bursaphelenchus xylophilus (Steiner et Buhrer) Nickle), resistant trees have been widely planted in Japan. The propagation of resistant trees using cuttings obtained from healthy stock plants and inoculated with pine wood nematode is expected to further increase in the next future. To improve the cutting propagation of Japanese black pine trees resistant to pine wilt disease, the factors associated with rooting and root volume were investigated. The type of cutting and the crown position of stock plants from which cuttings were taken, were markedly associated with rooting. The crown position did not show significant interactions with any other investigated factor, while fresh weight of cuttings and their number of winter buds did not affect rooting. The rooting percentage was markedly higher for cuttings taken from the lower crown than for those from the upper crown, as already reported for other coniferous tree species. The length of the longest needle was significantly correlated with the root volume of cuttings, and showed significantly interactions with crown position and fresh weight of cuttings. Such correlation suggests that the growth of needles can be considered a useful predictor in the assessment of the root volume of cuttings during the propagation period, allowing growers to transplant rooted cuttings at the appropriate time without excavating or uprooting. These findings may contribute to the improvement of cutting propagation of Japanese black pine.

The genome and genetics of a high oxidative stress tolerant Serratia sp. LCN16 isolated from the plant parasitic nematode Bursaphelenchus xylophilus.

BackgroundPine wilt disease (PWD) is a worldwide threat to pine forests, and is caused by the pine wood nematode (PWN) Bursaphelenchus xylophilus. Bacteria are known to be associated with PWN and may have an important role in PWD. Serratia sp. LCN16 is a PWN-associated bacterium, highly resistant to oxidative stress in vitro, and which beneficially contributes to the PWN survival under these conditions. Oxidative stress is generated as a part of the basal defense mechanism used by plants to combat pathogenic invasion. Here, we studied the biology of Serratia sp. LCN16 through genome analyses, and further investigated, using reverse genetics, the role of two genes directly involved in the neutralization of H2O2, namely the H2O2 transcriptional factor oxyR; and the H2O2-targeting enzyme, catalase katA.ResultsSerratia sp. LCN16 is phylogenetically most closely related to the phytosphere group of Serratia, which includes S. proteamaculans, S. grimessi and S. liquefaciens. Likewise, Serratia sp. LCN16 shares many features with endophytes (plant-associated bacteria), such as genes coding for plant polymer degrading enzymes, iron uptake/transport, siderophore and phytohormone synthesis, aromatic compound degradation and detoxification enzymes. OxyR and KatA are directly involved in the high tolerance to H2O2 of Serratia sp. LCN16. Under oxidative stress, Serratia sp. LCN16 expresses katA independently of OxyR in contrast with katG which is under positive regulation of OxyR. Serratia sp. LCN16 mutants for oxyR (oxyR::int(614)) and katA (katA::int(808)) were sensitive to H2O2 in relation with wild-type, and both failed to protect the PWN from H2O2-stress exposure. Moreover, both mutants showed different phenotypes in terms of biofilm production and swimming/swarming behaviors.ConclusionsThis study provides new insights into the biology of PWN-associated bacteria Serratia sp. LCN16 and its extreme resistance to oxidative stress conditions, encouraging further research on the potential role of this bacterium in interaction with PWN in planta environment.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2626-1) contains supplementary material, which is available to authorized users.

Monochamus galloprovincialis’in Tunus’taki Halep çamı ormanlarında yayılışı

The pine wilt disease, caused by the pine wood nematode (PWN) Bursaphelenchus xylophilus, was detected in Europe in 1999 in Portugal and the longhorn beetle Monochamus galloprovincialis reported as the only vector since 2001. Although not present in northern Africa, it is feared that the PWN may cause significant damage if introduced into the Maghreb region, where several susceptible pine species which can serve as hosts are found, along with insects of the Monochamus genus which can act as vectors. In order to assess the risk of propagation of the wilt disease, we surveyed for the presence of possible vectors of the Monochamus genus in Tunisia, characterizing the distribution and emergence pattern. Studies were carried in nine locations with Aleppo pine (Pinus halepensis) forests. Sampling for insects was based on the trap tree technique, allowing beetles to lay eggs in the field and subsequently emerging. We confirmed the presence of Monochamus beetles in Tunisia, with only one species detected, M. galloprovincialis, which was widespread in the Aleppo pine forests. Our results show that this specie can develop and emerge from the basal, median and the upper part of the Aleppo pines with similar success. The larval development took nearly one year and adult emergences occurred from May to August during 2012. Results are discussed in view of similar biological studies conducted in other Mediterranean countries and the implications for the risk assessment of pine wilt disease in Tunisia. Keywords: Pine sawyer, Pinus halepensis, Emergence, Xylophagous beetle, Pine wilt disease

Protein markers of Bursaphelenchus xylophilus Steiner & Buhrer, 1934 (Nickle, 1970) populations using quantitative proteomics and character compatibility.

The Pine Wood Nematode (PWN) Bursaphelenchus xylophilus is a severe forest pathogen in countries where it has been introduced and is considered a worldwide quarantine organism. In this study, protein markers for differentiating populations of this nematode were identified by studying differences among four selected Iberian and one American population. These populations were compared by quantitative proteomics (iTRAQ). From a total of 2860 proteins identified using the public database from the B. xylophilus genome project, 216 were unambiguous and significantly differentially regulated in the studied populations. Comparisons of their pairwise ratio were statistically treated and supported in order to convert them into discrete character states, suggesting that 141 proteins were not informative as population specific markers. Application of the Character Compatibility methodology on the remaining 75 proteins (belonging to families with different biological functions) excludes 27 which are incompatible among them. Considering only the compatible proteins, the method selects a subset of 30 specific unique protein markers which allowed the compared classification of the Iberian isolates. This approach makes it easier search for diagnostic tools and phylogenetic inference within species and populations of a pathogen exhibiting a high level of genetic diversity.

Identification of Genes Relevant to Pesticides and Biology from Global Transcriptome Data of Monochamus alternatus Hope (Coleoptera: Cerambycidae) Larvae.

Monochamus alternatus Hope is the main vector in China of the Pine Wilt Disease caused by the pine wood nematode Bursaphelenchus xylophilus. Although chemical control is traditionally used to prevent pine wilt disease, new strategies based in biological control are promising ways for the management of the disease. However, there is no deep sequence analysis of Monochamus alternatus Hope that describes the transcriptome and no information is available about gene function of this insect vector. We used next generation sequencing technology to sequence the whole fourth instar larva transcriptome of Monochamus alternatus Hope and successfully built a Monochamus alternatus Hope transcriptome database. In total, 105,612 unigenes were assigned for Gene Ontology (GO) terms, information for 16,730 classified unigenes was obtained in the Clusters of Orthologous Groups (COGs) database, and 13,024 unigenes matched with 224 predicted pathways in the Kyoto Encyclopedia of Genes and Genome (KEGG). In addition, genes related to putative insecticide resistance-related genes, RNAi, the Bt receptor, intestinal digestive enzymes, possible future insect control targets and immune-related molecules are described. This study provides valuable basic information that can be used as a gateway to develop new molecular tools for Monochamus alternatus Hope control strategies.

Ultrasonic phytosanitation of pinewood nematode infected wood

Pinewood nematode (Bursaphelenchus xylophilus) mortality was investigated after ultrasonic treatment at 20 and 40 kHz frequency. Experiments were conducted with infected small wood specimens that were ultrasonically treated for 1, 3, 5 and 7 hours and two variable temperature conditions, namely, a gradually increasing from ambient to a maximum of 70oC and a decreasing from ambient to a minimum of 5oC. The results revealed that the ultrasonic treatment itself had no significant effect on the nematode mortality at the 5oC level, while at the 70oC level, considerable nematode mortality was observed in short time periods and at 7 hours of sonic exposure it reached 100%. Therefore, certain combinations of timing and frequency of ultrasonic waves and produced heat can be effective in killing pinewood nematodes thus resulting in phytosanitized wood.

Functional analysis of the venom allergen-like protein gene from pine wood nematode Bursaphelenchus xylophilus using a baculovirus expression system

The pine wood nematode, Bursaphelenchus xylophilus, infects pine trees, leading to fatal pine wilt disease. Here, recombinant venom allergen-like protein (VAP) was obtained by expressing Bx-vap-1 in insect cells. Three-year-old Pinus massoniana were inoculated with recombinant VAP, simulating B. xylophilus esophageal gland secretions. Recombinant VAP up-regulated α-pinene synthase gene expression, the trees showed disease symptoms 15 d after inoculation and the xylem pith revealed brown tissue discoloration, indicating that recombinant VAP could damage P. massoniana cells. Recombinant VAP did not, however, lead to cavitation, indicating that the VAP secreted from B. xylophilus acts as a defense response elicitor.

집합페로몬 2-(Undecyloxy)ethanol을 이용한 솔수염하늘소 유인 실험

The pinewood nematode Bursaphelenchus xylophilus (Nematoda: Parasitaphelenchidae) poses a serious threat to pine forests in Europe and East Asia, leading to a debilitating pine wilt disease. Infected pine trees in Korea are generally fumigated or crushed to small wood chips after felling. Although pine wilt disease often recurs in pest management sites, there are no adequate means to monitor the effectiveness of pest control measures in those sites. Recently, a male-produced aggregation pheromone, 2-(undecyloxy)ethanol, was shown to be useful for attracting several Monochamus species, which are vectors for the pinewood nematodes. In this study, we investigated the abilities of 2-(undecyloxy)ethanol at three different doses (175, 350, and 700 mg), as well as host plant volatiles (α-pinene and ethanol), to attract M. alternatus (Coleoptera: Cerambycidae) at a pine forest in Pohang, Korea where infected pine trees had been cut down and fumigated. Twenty-seven M. alternatus were captured in cross-vane panel traps made of polyethylene terephthalate bottles and acrylic sheets. The results indicate that a high dose of 2-(undecyloxy)ethanol (700 mg per trap) is the most effective for attracting M. alternatus. The aggregation pheromone could be used to monitor the effectiveness of pest control measures as well as M. alternatus populations.

Genome-wide survey and characterization of the small heat shock protein gene family in Bursaphelenchus xylophilus

Temperatures directly influence the distribution and intensity of pine wilt disease, which is caused by the pine wood nematode Bursaphelenchus xylophilus. Small heat shock proteins (sHSPs) are molecular chaperones that contribute to nematode survival during the stress response to high temperatures. Seven B. xylophilus sHSPs (Bx-sHSPs) were identified and studied in a whole-genome shotgun project. The replacement of aromatic amino acids with aliphatic amino acids in motifs was the most significant difference between Bx-sHSPs and Caenorhabditis elegans sHSPs (Ce-sHSPs). In Bx-sHSPs, two motifs showed consensus sequences similar to the known palindromic nGAAn sequence or variants of this sequence. A phylogenetic tree of Bx-sHSPs and corresponding Ce-sHSPs suggests the existence of a one-to-one orthologous relationship for all sHSPs. Gene evolution patterns corresponding to both purifying selection and positive selection were found in orthologous pairs of Ce-sHSPs and Bx-sHSPs. The upregulation of Bx-sHSPs in response to heat stress (30°C) suggests that these proteins play a role in thermoregulation.

Nematicidal effect of Sparassis latifolia-derived sparassol and disodium sparassol against Bursaphelenchus xylophilus

The pine wood nematode (Bursaphelenchus xylophilus) causes pine wilt disease, which is a major problem affecting pine forests. This study aimed to identify metabolites from Sparassis latifolia that have nematicidal activity and to investigate the effect of water-soluble formulations of the active compounds for use as trunk-injectable control agents against the nematode B. xylophilus. When the submerged cultures of S. latifolia were bioassayed against B. xylophilus, strain KFRI 747 showed the most potent activity (52.8%), followed by KFRI 1080 (44.4%) and KFRI 645 (34.8%); their activities were correlated with the concentration of sparassol in their mycelia. Six fractions (SLE1-6) were obtained from ethyl acetate extracts of the liquid culture medium; nematicidal activity was found only in SLE4 (which included sparassol). Sparassol bioassayed against B. xylophilus and had LC50 and LC95 of 84.92 and 132.13ppm, respectively. Sparassol was modified to disodium sparassol using NaOH because sparassol was not water-soluble and thus was not useful as an agent for trunk injection. Disodium sparassol exhibited similar nematicidal activity to that found with sparassol. The trunk-injectable nematicides (abamectin and emamectin benzoate) were more effective than sparassol and disodium sparassol in terms of LC50 and LC95 values. However, sparassol has a more simple chemical structure than these compounds and could be more easily synthesized. To our knowledge, this is the first report indicating that disodium sparassol might be useful as nematicide for use against the pine wood nematode.

The EctoparasitoidScleroderma guani(Hymenoptera: Bethylidae) Uses Innate and Learned Chemical Cues to Locate Its Host, Larvae of the Pine SawyerMonochamus alternatus(Coleoptera: Cerambycidae)

In Asia, the pine sawyer beetle Monochamus alternatus Hope (Coleoptera: Cerambycidae) is the most important vector of the pinewood nematode Bursaphelenchus xylophilus Steiner and Buhrer (Aphelenchida: Parasitaphelenchidae), the causal agent of pine wilt disease, and the ectoparasitoid Scleroderma guani Xiao et Wu (Hymenoptera: Bethylidae) is the most important natural enemy of this pest. Efficient host location is critical to parasitoid fitness, and chemical cues are key factors guiding the host searching process. This study was conducted to elucidate the odor cues guiding host location in S. guani and the impact of previous host experience on this behavior. Tests were conducted in which S. guani oriented to different odor resources associated with M. alternatus and its habitat, and components of these volatiles were analyzed with gas chromatography/mass spectrometry. Orientation to various odors also was investigated using adult S. guani subjected to different adult experiences. Female S. guani could easily distinguish M. alternatus larvae from other odor resources. Hydrocarbons were the main components of volatiles derived from M. alternatus larvae and were absent in volatiles from wood and frass associated with M. alterna- tus larvae. Female S. guani oriented most strongly to 1st and 3rd instars of M. alternatus. Prior experience of host odor without oviposition decreased orientation of S. guani towards the host, and the host used for rearing had no impact on subsequent odor orientation by adult S. guani. Volatile hydrocarbons emanating from host larvae might be the key to host location by adult S. guani, and experience with hosts appears to reinforce behavioral responses. The present results may be useful references for improving augmentative biological control using S. guani against M. alternatus and other forest pests.