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.     

Cloning and expression of a toxin gene from Pseudomonas fluorescens GcM5-1A

Pseudomonas fluorescens GcM5-1A was isolated from the pine wood nematode (PWN), Bursaphelenchus xylophilus, obtained from wilted Japanese black pine, Pinus thumbergii, in China. In this paper, a genomic library of the GcM5-1A strain was constructed and a toxin–producing clone was isolated by bioassay. Nucleotide sequence analysis revealed an open reading frame of 1,290 bp encoding a protein of 429 amino acids with N-terminal putative signal peptide of 36 amino acids, which shared a similarity of 83, 82 and 80% identity with hypothetical protein PFLU2919 from P. fluorescens SBW25, Dyp-type peroxidase family protein from P. fluorescens Pf-5 and Tat-translocated enzyme from P. fluorescens Pf0-1, respectively. The gene encoding a full-length protein or without the putative signal peptide was cloned and expressed as a soluble protein in E. coli. The recombinant protein was purified to electrophoretic homogeneity by affinity chromatography using a Ni²⁺ matrix column. Its relative molecular weight was estimated to be 48.5 kDa by SDS-PAGE for full-length protein, and 45.0 kDa for the recombinant protein without putative signal peptide. Bioassay results showed that the recombinant protein with or without the putative signal peptide was toxic to both suspension cells and P. thunbergii seedlings. HPLC analysis demonstrated that components in branch extracts of P. thunbergii were significantly changed after addition of the recombinant full-length protein and hydrogen peroxide, which indicated that it is probably a peroxidase. This study offers information that can be used to determine the mechanism of pine wilt disease caused by the PWN.     

The chemiluminescence immunoassay for aflatoxin B1 based on functionalized magnetic nanoparticles with two strategies of antigen probe immobilization

A rapid and sensitive chemiluminescence immunoassay (CLIA) based on magnetic nanoparticles (MNPs) was developed to detect aflatoxin B1 (AFB1), which is a potent carcinogen in nature. We prepared monodisperse MNPs (300 nm in diameter) according to the solvothermal synthesis reaction and the MNPs were coated with silica by the Stöber method. Triethox was used as a one‐step carboxylation reagent, and 3‐aminopropyltriethoxysilane (APTES) an amination reagent, to modify the MNPs. We prepared two types of solid phase antigens using the above synthesized functionalized MNPs coupled with the later prepared AFB1‐oxime active ester and the purchased BSA–AFB1 respectively. 2′,6′‐dimethylcarbonylphenyl‐10‐sulfopropylacridinium‐9‐carboxylate 4′‐N‐hydroxysuccinimide (4′‐NHS) ester (NSP–DMAE–NHS), as a novel luminescent reagent, was used to label anti‐AFB1 antibodies. The two CLIA calibration curves based on the two types of solid phase antigens were obtained and compared. The acquired limit of detection (LOD) was about 0.001 ng/mL for the two functionalized MNPs‐based immunoassays, and the half maximal inhibitory concentration (IC₅₀) was 0.51 ng/mL for the MNPs–AFB1‐based method and 0.72 ng/mL for the MNPs–BSA–AFB1‐based method.     

Development of a highly sensitive ELISA for the determination of PBAN and its application to the analysis of hemolymph in Spodoptera littoralis

A highly sensitive enzyme linked immunosorbent assay (ELISA) for the determination of the pheromone biosynthesis activating neuropeptide (PBAN) has been developed. Six antisera have been obtained that recognize the carboxyl terminal side of this peptide. Two immunogens have been rationally designed and synthesized in order to direct antibody specificity, using as haptens PBAN or PBAN(20-33) with a Cys residue attached to their amino-terminal side. The Cys thiol group has been used to covalently bind the peptide to keyhole limpet hemocyanin (KLH) by using N-succinimidyl-4-(maleidimidomethyl) cyclohexane carboxylate (SMCC) as a convenient heterobifunctional cross-linker. Several usable competitive immunoassays have been obtained by synthesizing eight different coating antigens and screening the sera against all of them. The best assay was obtained with antibody 4 using Cys-Hez-PBAN(20-33) coupled to bovine serum albumin (BSA) through the Lys groups by using the homobifunctional cross-linker dimethylpimelidate dihydrochloride (DMP) as the coating antigen. The optimized assay allows to detect PBAN at concentrations as low as 1 fmol/well (l₅₀ = 2.5 fmol/well). An extraction procedure for the hemolymph has been developed that allows to perform PBAN measurements in this tissue even after a tenfold dilution. In these conditions matrix effect is negligible. Preliminary results on the presence of PBAN like immunoreactivity (PBAN-IR) in the hemolymph of Spodoptera littoralis females are reported.© 1995 Wiley-Liss, Inc.     

高亲和力莱克多巴胺单克隆抗体的研制及ciELISA检测方法的建立

  用混合酸酐法（MA）将莱克多巴胺（RAC）偶联于牛血清白蛋白（BSA）,合成人工抗原BSA-RAC,用UV和SDS-PAGE鉴定;用BSARAC免疫Balb/c小鼠,细胞融合技术建立高亲和力RAC单克隆抗体（mAb）杂交瘤细胞株,体内诱生腹水法制备RAC mAb;应用RAC mAb研制RAC残留快速检测ciELISA试剂盒（RAC-Kit）,并测定其性能。结果表明,BSA-RAC偶联成功,分子结合比为24.5∶1;筛选出3株杂交瘤细胞,其中最好的4D8株亲和常数（Ka）为1.65×1010L/mol; RAC-Kit的检测限为0.5ng/ml,检测范围为0.5～151ng/ml,饲料样、猪尿样的平均添加回收率为87.2%,89.4%,平均批内和批间变异系数小于15%,与多巴酚丁胺的交叉反应率（CR%）为9.7%,与其它化合物无CR,RAC-Kit在4 ℃保存期为180d。     

Determination of the reactivities and cross-reactivities of monoclonal antibodies against staphylococcal enterotoxin A by indirect ELISA and immunoblot including a semiautomated electrophoresis system

Eight murine monoclonal antibodies (Mabs) to staphylococcal enterotoxin A (SEA) were produced using standard hybridoma techniques. We studied reactivities and cross-reactivities by indirect ELISA and immunoblotting. Two of these Mabs (A5 and A7) reacted with five serovars (A-E) of SE in both systems. Only Mab A1 reacted specifically with the homologous toxin, while four Mabs reacted with SEA and SEE. Mabs A5 and A7 could be used to detect all five serovars of SEs in a single assay.     

Evidence of century-scale environmental changes: Trace element in tree-ring from Fuling Mausoleum Shenyang,China

In order to detect the response of trees to environmental pollution in Shenyang during the last century, tree rings of two 100+ year old Chinese pine (Pinus tabulaeformis Carr.) were analyzed for nine trace element concentrations (K, Ca, Mg, Mn, Cu, Zn, Al, Pb and Cd) and compared to local environmental change. The concentrations of Cu, Zn and Al had significant (p < 0.01) increasing trends after 1940s and correlated significantly (p < 0.01), with increasing production of nonferrous metal from local industrial processing. Concentrations of nutrient element (K, Ca and Mg) showed significant (p < 0.01) increasing trend from heartwood to sapwood with declines in the last three decades. Significant correlation between climatic factors and element concentration in tree rings were also found, in which the temperature (November to May) and relative humidity in April and May showed significant positive correlation with Ca, Zn and Cu (p < 0.05) as well as Al (p < 0.01) concentrations in tree rings. April rainfall showed significant positive correlation with K and Zn (p < 0.05) as well as Al (p < 0.01) concentration in tree rings. The results suggest that tree rings from Chinese pine in Shenyang are sensitive to environmental change and have the potential to be used as a biomonitor for the environmental pollution. The results also demonstrate the feasibility of applying dendrochemical techniques to areas with limited samples.     

玉米赤霉烯酮单克隆抗体和免疫酶技术研究

采用蛋白质连接技术合成玉米赤霉烯酮抗原,免疫Balb/c鼠,通过淋巴细胞杂交瘤技术建立六株分泌抗玉米赤霉烯酮的单克隆抗体杂交瘤细胞株。间接酶联免疫吸附试验测定细胞上清抗体效价为1:2084(4H8)、1:256(6H9、4H3、2H5、2C8)、1:16(3F10);腹水抗体效价为10~9(4H3、4H8)、10~8(2H5)、10~7(6H9)、10~5(3H10)。竞争间接酶联免疫吸附试验测定六株单克隆抗体对玉米赤霉烯酮的敏感度为0.3—0.8ng/ml。六株抗体与玉米亦霉烯醇的交叉反应率为1.3—9.0％。六株单克隆抗体均属IgG类。细胞体外传代培养和冻存复苏后分泌抗体稳定。纯化抗体在37℃保存12天稳定,-30℃保存90天抗体滴度不变。用该抗体建立竞争间接酶联免疫吸附试验检测掺合玉米赤霉烯酮的玉米、小麦、饲料,平均回收率分别为105％、90％、103％,平均批间变异系数为5.8％、2.8％、6.8％,批内变异系数为3.8％、12.7％、15.7％。样品中玉米赤霉烯酮掺合量与竞争间接酶联免疫吸附试验检出量有良好相关性(r≥0.9996)。     

Traits of Masson Pine Affecting Attack of Pine Wood Nematode

Masson pine characteristics were analyzed in five sample plots in Zhejiang Province, China. Bursaphelenchus xylophilus （Steiner et Buhrer） Nickle （pine wood nematode, PWN） carried by Monochamus alternatus predominately attacked Masson pines in the lower diameter classes. Among the 10 tree characteristics examined, mean crown width, percentage of bole with crown, 5-year cumulative diameter growth, and resin amount showed significant variation between successfully attacked and unattacked trees. The attacked trees had a lower percentage of the bole covered with tree crown, lower crown width, lower radial growth in the last 5 years, and produced less induced resinosis than unattacked trees. Results allowed for effective ranking of the pine forest based on individual tree resistance to PWN. This index of resistance should be considered throughout the development of an ＂Evaluation Criterion and Indicator System＂. The preceding ranking can be used to evaluate the resistance and resiliency of the pine forest ecosystem to PWN＇s invasion, which is similar to Pest Risk Analysis （PRA）.     

Biological control of the pinewood nematode <Emphasis Type="Italic">Bursaphelenchus xylophilus</Emphasis> by application of the endoparasitic fungus <Emphasis Type="Italic">Esteya vermicola</Emphasis>

Esteya vermicola (Ophiostomataceae) is the first reported endoparasitic fungus of the pinewood nematode (PWN), Bursaphelenchus xylophilus (Nematoda: Aphelenchoidoidea). It has high in vitro infectivity. In this study, the nematocidal effect of E. vermicola in logs was investigated and evaluated. Two months after inoculation of pine wilt-killed Pinus densiflora logs with E. vermicola conidia suspensions of 3 × 10⁸ and 3 × 10⁶ ml⁻¹, the density of nematodes decreased by approximately 79% and 47%, respectively. When the fungus was sprayed on to four-year-old pine seedlings one month before PWN inoculation, the survival index of seedlings reached 0.67 compared with only 0.067 for control seedlings without fungal spraying. These results suggest that conidia spraying of E. vermicola can, to some extent, protect pine trees from wilt disease. Moreover, infected nematodes and hyphae of E. vermicola were observed in the treated wood sections.     

一种改进的分泌抗半抗原抗体杂交瘤细胞株筛选方法简

目的：研究解决半抗原分子单克隆抗体制备技术路径中遇到的在阳性杂交瘤细胞株筛选时无法排除载体蛋白问交叉反应影响的问题,以半抗原去甲肾上腺素（norepinephrine,NE）为例。方法：在NE完全抗原免疫小鼠实施细胞融合后,分别包被牛血清白蛋白（BSA）、卵清白蛋白（OVA）、BSA-NE、OVA-NE等4种不同抗原的酶标板平行检测细胞培养上清液;挑选BSA、OVA检测结果为阴性,BSA-NE、OVA-NE检测结果为阳性的孔内细胞进行克隆化筛选单克隆细胞。结果：本筛选方法可一次性从8板96孔板中筛选到13个符合要求的阳性孔,经3次克隆化后获得6株特异性强的杂交瘤细胞株。结论：本方法避免了载体蛋白间交叉反应对筛选的影响,改进了传统的单一指标筛选方法,筛选效率更高。     

柑橘衰退病毒多克隆和单克隆抗体的制备及检测效果分析

通过改进提纯方法获得了柑橘衰退病毒(Citrustristezavirus,CTV)的提纯液,其产量为1mg/100g植物组织。用CTV免疫大耳白兔,获得多克隆抗体,间接ELISA效价为1∶25600。用CTV免疫小鼠,经细胞融合、ELISA筛选和克隆化培养,获得18株能稳定分泌抗CTV单克隆抗体的杂交瘤单细胞株。对其中4株单克隆腹水抗体进行分析的结果表明,这些抗体的ELISA效价为1∶51200~1∶204800,其中2G和3H的抗体类型及亚类为IgG2a,1E和4H为IgG2b。用所制备抗体对不同来源柑橘样品的CTV检测结果显示,单克隆和多克隆抗体结合使用,采用三抗体夹心ELISA(TAS-ELISA)可以获得理想的检测效果,其特异性强、灵敏度高。同时发现所分析4株单克隆抗体对不同的CTV分离物鉴别能力存在差异,但有关这些CTV分离物的特性及其血清学关系还需进一步研究。     

Intra-annual variation in habitat choice by an endemic woodpecker: Implications for forest management and conservation

The Canary Islands great spotted woodpecker Dendrocopos major canariensis is an endemic bird restricted to the Pinus canariensis forests of Tenerife and Gran Canaria. Classification tree models were applied to explore the relationship of the occurrence of this picid and habitat variables between two contrasting periods (breeding vs. non-breeding seasons) and for the entire annual cycle. During the reproductive period the availability of mature trees (DBH > 60 cm), and snags (dead trees), for nesting and roosting, characterize the breeding territory. Outside the breeding season the choice of locations was driven by a tree cover larger than 28.5% and the presence of trees taller than 8.5 m on average, a pattern explained by the availability of pine seeds in the cones of well-developed canopies, and less so by predation risk. Overall, during the annual cycle, well-developed canopy sites influenced the presence of this picidae (tree cover > 38%) and on more open sites (<38%) the presence of mature trees (DBH> 60 cm) became the second most important predictor of occurrence. We suggest that food abundance and availability could be the ultimate factor explaining the intra-annual variation observed, with the availability of snags being an important factor during nesting. In the range of this endemic, we recommend selective cuts in pine plantations, to allow the trees to set seed and improve their crops, minimizing the elimination of snags, and killing some large pine trees if the priority is to expand the distributional range of the woodpecker.     

Radioimmunoassay for the tachykinin peptide physalaemin: Detection of a physalaemin-like substance in rabbit stomach

An antiserum against the amphibian tachykinin physalaemin was specific for the NH₂-terminal region of this peptide. The cross-reactivity of antiserum PS-1 with uperolein was 2.03%, phyllomedusin, 0.36%, and kassinin, 0.013%; negligible (<0.0001%) recognition was shown for substance P as well as with numerous other polypeptide hormones. The assay readily detected 1 pg (0.79 fmol) physalaemin with 50% displacement at 6.79 ± 1.98 pg (5.29 ± 1.54 fmol). Rabbit stomach, extracted by boiling followed by homogenization in 1.0 n formic acid, contained physalaemin-like immunoreactivity. The concentration detected in the antrum was 43.0 ± 7.3 ng/g dry weight with less in the fundus, pylorus, corpus, and duodenum. Chromatography of the extracts on Bio-Gel P4 columns in 0.1 n formic acid gave a single, symmetrical peak of immunoreactivity which eluted with an apparent molecular weight of approximately 1700. These data are the first to demonstrate the presence of a peptide with physalaemin-like immunoreactivity in mammalian tissues.     

Survival of black alder (Alnus glutinosa L.), silver birch (Betula pendula Roth.) and Scots pine (Pinus sylvestris L.) seedlings in a reclaimed oil shale mining area

Early survival and growth of black alder, silver birch and Scots pine were investigated on reclaimed extremely stony and heterogeneous calcareous (pH 8) opencast oil shale mining areas (OOSMAs). Biomass allocation, production, leaf and root adaptations, and mineral nutrition in relation to tree species and soil heterogeneity were analysed. The adaptive strategies of tree species in first-year plantations on OOSMA were different. Scots pine allocated 1.5–2 times more biomass into leaves and fine roots than deciduous trees. The lower leaf/fine root biomass ratio was in proportion to the better survival (%) of seedlings, decreasing in the following order: black alder (93%) ≥ Scots pine (83%) > silver birch (64%). Deciduous trees improved mineral nutrition more by fine-root morphological adaptations than Scots pine; e.g. the mean specific root length (SRL, m g^?1) of short roots increased in the following order: Scots pine (62) < black alder (172) < silver birch (314). The effect of soil heterogeneity on growth and adaptations was minor. All studied species suffered from P and N, and deciduous species also from K deficiency. In the first year after planting, black alder was best adapted to the harsh conditions of the post-mining substrate. The approaches of this study can be used for other regions where wastelands require reclamation.     

Carbon storage in post-mining forest soil,the role of tree biomass and soil bioturbation

Carbon storage in aboveground tree biomass and soil organic matter (in depth of A layer development i.e., up to 20 cm) was studied in 22–32 year-old post-mining sites in the northwest of the Czech Republic. Four replicated sites afforested with different tree species (spruce, pine, larch, oak, lime or alder) were compared with sites left to natural regeneration which were dominated by aspen, birch and willow. No topsoil was applied at the sites; hence carbon accumulation resulted from in situ soil development on alkaline tertiary clays that were dumped on the heaps. In aboveground tree biomass, carbon storage ranged from 17.0 ± 5.9 (mean ± SEM) to 67.6 ± 5.9 t ha⁻¹ and the rate of C accumulation increased from 0.60 ± 0.09 to 2.31 ± 0.23 t ha⁻¹ year⁻¹ (natural regeneration < pine < spruce < oak < lime < alder < larch). Carbon storage in soil organic matter varied from 4.5 ± 3.7 to 38.0 ± 7.1 t ha⁻¹ and the rate of C accumulation in soil organic matter increased from 0.15 ± 0.05 to 1.28 ± 0.34 t ha⁻¹ year⁻¹ at sites in the order: natural regeneration < spruce < pine, oak < larch < alder < lime. Carbon storage in the soil was positively correlated with aboveground tree biomass. Soil carbon was equivalent to 98.1% of the carbon found in aboveground tree biomass at lime dominated sites, but only 21.8% at sites with natural regeneration. No significant correlation was found between C storage in soil and aboveground litter input. Total soil carbon storage was correlated positively and significantly with earthworm density, and occurrence of earthworm cast in topsoil, which indicated that bioturbation could play an important role in soil carbon storage. Hence, not only restoring of wood production, but also restoring of soil community is critical for C storage in soil and whole ecosystem.     

抗麻痹性贝毒素GTX2,3单克隆抗体的制备及特性分析

制备抗麻痹性贝毒GTX2,3单克隆抗体。利用醛化法将GTX2,3与载体牛血清白蛋白(BSA)偶联,制备完全抗原。免疫小鼠,取小鼠脾细胞与Sp2/0细胞融合。GTX2,3与钥孔血蓝蛋白(KLH)偶联作为检测抗原,用间接ELISA法筛选阳性克隆株。将筛选的阳性细胞株制备腹水。获得三株稳定分泌抗GTX2,3单克隆抗体的杂交瘤细胞株F4、F10、G9。间接ELISA法检测F10细胞株腹水抗体效价为1.4×10^-5。半抗原GTX2,3与载体蛋白偶联后,作为免疫原,可制备高滴度的抗GTX2,3抗血清和单克隆抗体。该抗体对于藻毒素具有高特异性和高亲和力,可用于污染海产品的麻痹性贝毒的检测。     

Effects of pine wilt disease invasion on soil properties and Masson pine forest communities in the Three Gorges reservoir region,China

Pine wilt disease (PWD) has caused significant Masson pine mortality in the Three Gorges reservoir region in central China. In this study, five uniform Masson pine stand types infected by PWD were selected and surveyed on slopes and aspects with similar environmental conditions. In sites that had been infected, soil bulk density was reduced, and the difference among the groups was statistically significant (P < 0.05) at the 0–10 cm and 10–20 cm soil layers, but not at 20–40 cm. Other soil water‐related physical properties, excluding noncapillary porosity, significantly differed among the groups in all soil layers. Additionally, the values of available phosphorus, sodium, potassium, calcium, and magnesium were higher in the invaded stands, but the total nitrogen and organic matter contents were lower. Masson pine does not become reestablished following PWD‐induced mortality but is instead replaced by broad‐leaved tree species. Among the 19 examined environmental variables, five were found to be significantly related with the ordination of plant community structure: Masson pine stumps (MPS), K⁺, capillary water holding capacity (CWHC), capillary porosity (CP), and soil water content (SWC). Among these factors, the plant community structure was principally related to MPS and K⁺. The findings of this study show that the outbreak of PWD has impacted Masson pine forest soil properties and altered forest community composition. The disease is negatively related with the presence of Masson pine and positively associated with that of broad‐leaved tree species.