Genes homologous to members of the MRP gene family in Caenorhabditis elegans are important in drug resistance.To further explore the molecular mechanism of drug resistance in pine wood nematode(Bursaphelenchus xylophi...Genes homologous to members of the MRP gene family in Caenorhabditis elegans are important in drug resistance.To further explore the molecular mechanism of drug resistance in pine wood nematode(Bursaphelenchus xylophilus),we used bioinformatics approaches to analyze genomic data for B.xylophilus and identified Bx-MRP genes.We predicted the structure and function of the genes and encoded proteins.Using bioinformatics programs to predict and analyze various properties of the predicted proteins,including hydrophobicity,transmembrane regions,phosphorylation sites,and topologically isomeric structures,of these Bx-MRP genes,we determined that they function in transmembrane transport.From the results of RT-qPCR,the Bx-MRP family members confer significant differential resistance to different drug treatments.After treatment with different concentrations of emamectin benzoate,avermectin and matrine,the expression of each gene increased with increasing drug concentrations,indicating that the family members play a positive role in the regulation of multidrug resistance.展开更多
Bursaphelenchus xylophilus,causal agent of pine wilt disease,causes extensive damage worldwide.Strate-gies are needed to inhibit population growth or block the spread of the invasive nematode to control pine wilt dis-...Bursaphelenchus xylophilus,causal agent of pine wilt disease,causes extensive damage worldwide.Strate-gies are needed to inhibit population growth or block the spread of the invasive nematode to control pine wilt dis-ease.The gene daf-8 plays crucial roles in larval develop-ment and dauer formation in Caenorhabditis elegans,but little is known about its orthologue in B.xylophilus.In the present molecular characterization and functional analysis of daf-8 in B.xylophilus(Bx-daf-8),RT-qPCR showed that the expression of Bx-daf-8 gradually increased during the embryonic stage,peaked in the second-stage juvenile(J2),then dramatically dropped in the J3,and remained at that low level for the rest of its life.Bx-daf-8-transgenic C.elegans was employed to mimic the spatiotemporal expression of Bx-daf-8,which was expressed close to the pharynx during early embryogenesis and weakly throughout the whole body during late embryogenesis.It was observed in head neurons and tail ganglions throughout all postembryonic stages.B.xylophilus embryos were severely abnormal,and hatching rate decreased sharply after Bx-daf-8 knockdown.daf-16-1 and da-f16-2,downstream genes in the IIS pathway,also dropped sharply after Bx-daf-8 knockdown.We propose that TGFβmay crosstalk with the IIS pathway upstream of Bx-daf-16 and that daf-8 may act as a master regulator of daf-16 in B.xylophilus.However,knockdown of Bx-daf-8 did not lead to constitutive developmental arrest at the dauer larval stage,indicating that dauer entry in B.xylophilus might be controlled by several genes and is more complicated than in C.elegans.Bx-daf-8 alone did not control the dauer entry in B.xylophilus,but it was indispensable for embryogen-esis,providing a potential target for suppressing population growth of B.xylophilus.展开更多
Pine wilt disease(PWD)is a devastating disease affecting the growth of Pinus massoniana,often leading to withering and death.To reveal the changes involved during disease progression,we investigated the mRNA expressio...Pine wilt disease(PWD)is a devastating disease affecting the growth of Pinus massoniana,often leading to withering and death.To reveal the changes involved during disease progression,we investigated the mRNA expression profile of P.massoniana infested by Bursaphelenchus xylophilus.The infestation resulted in the downregulation of genes involved in interactions with pathogenic pathways such as disease resistance gene,CC-NBS-LRR resistancelike protein,and the gene encoding a putative nematode resistance protein.Increased infestation pressure(number of nematodes inoculated)caused a continuous decline in the gene expression of stem samples.An infestation of P.massoniana also resulted in a pathway enrichment of genes involved in phenylpropanoid metabolism and flavonoid biosynthesis,which in turn reduced the levels of total phenols and total flavonoids.A downregulation of auxin responsive family protein was observed in infested samples,which resulted in a suppression of plant growth.Thus,upon B.xylophilus infestation,a downregulation of genes associated with the recognition of pathogens,PWD resistance,and growth regulation was observed in P.massoniana,together with a decrease in the levels of phytoalexinlike secondary substances,all of which resulted in withering and ultimately death of P.massoniana.展开更多
Bursaphelenchus xylophilus is the pathogen that causes pine wilt disease, which has greatly damaged forests and ecosystems in countries of East Asia and Europe.Bursaphelenchus mucronatus is closely related to B. xylop...Bursaphelenchus xylophilus is the pathogen that causes pine wilt disease, which has greatly damaged forests and ecosystems in countries of East Asia and Europe.Bursaphelenchus mucronatus is closely related to B. xylophilus in morphology and host plant specificity. A longrunning debate has existed regarding whether these two species can successfully produce hybrid offspring. In the present study, we performed in the laboratory, hybridization of two B. xylophilus nematode isolates from China and Japan and three B. mucronatus isolates from China, Japan and France. Nematode isolates of B. xylophilus were successfully crossed with B. mucronatus isolates, and the rate of hybridization was relatively high; however, some hybrid offspring died. Successful hybridization occurred between B. xylophilus and B. mucronatus isolates from China, and 22 generations of hybrids were produced. All F1 hybrids could be backcrossed with their parents and produce offspring. Variation in mucro length among the hybrid offspring and their parents was observed. The hybrid offspring and their parents were inoculated into 3-months-old black pine(Pinus tunbergii) seedlings. Weaker pathogenicity of hybrid offspring was observed compared with that of their parents, and significantly fewer offspring nematodes than parents were reisolated from pines. Therefore, the offspring of B. xylophilus and B. mucronatus may exist in the forest and could influence disease epidemics.展开更多
Pine wilt disease caused by pine wood nema-todes is a deadly disease of the genus Pinus requiring strong quarantine measures.Since its discovery,it has been widely distributed throughout the world.China is one of the ...Pine wilt disease caused by pine wood nema-todes is a deadly disease of the genus Pinus requiring strong quarantine measures.Since its discovery,it has been widely distributed throughout the world.China is one of the coun-tries with a severe rate of infections due to its abundant pine resources.In this study,nematode-trapping fungi were collected from pine trees in Ninghai City,Zhejiang Prov-ince,which is the key area of pine wilt control in Febru-ary,May,September,October and November.The results showed that nematode-trapping fungi of pine are abundant,especially the number and species detected in each month and are quite different;species of fungi in July,Septem-ber and November were more numerous and had higher separation rates.The dominant species in November was Arthrobotrys oligospora followed by Arthrobotrys cladodes;July and September it was A.cladodes.In each part of the tree,A.cladodes was the most widely distributed with a high separation rate.Therefore,it is the dominant fungal species in the area.Arboreal Bursaphelenchus xylophilus-trapping fungi are dependent on pine wood nematodes,and none were isolated from healthy pine trees.More arboreal B.xylophilus-trapping fungi were found under the bark than in the xylem.There is a close relationship between arboreal B.xylophilus-trapping fungi and pine wood nematodes.How to make use of these resources to reduce the damage of pine wood nematode fusarium is the focus of future research.展开更多
Pine wood nematode (PWN), Bursaphelenchus xylophilus (Steiner et Buhrer) Nickle is an important invasive alien species in forests of China and has become one of the most destructive forest diseases. In order to im...Pine wood nematode (PWN), Bursaphelenchus xylophilus (Steiner et Buhrer) Nickle is an important invasive alien species in forests of China and has become one of the most destructive forest diseases. In order to improve the resistance and resilience of pine forest ecosystems against B. xylophilus invasion and make the pine forest ecosystem more timely responsive to PWN invasion, we made some recommendations based on five years of intensive observations. We advocate a set of management measures with the theme “Prevention is priority, but integrated with curative techniques and ecological resilience” on the pine forest ecosystem invaded by B. xylophilus; details of accurate measures are proposed. The aim is to discover the underlying problems of present pine forest ecosys-tems and to take, correspondingly, administrative measures and strategies, which will encourage the pine forest ecosystem to develop in a benign way.展开更多
OPM<sub>05</sub>-M<sub>2100</sub>,the specific RAPD fragment of Bursaphelenchus xylophilus,was collected from agarose gels and purified.Then,the purified fragment was inserted into the pGEM<...OPM<sub>05</sub>-M<sub>2100</sub>,the specific RAPD fragment of Bursaphelenchus xylophilus,was collected from agarose gels and purified.Then,the purified fragment was inserted into the pGEM<sup>R</sup> -T Vector that was transformed into Escherichia.coli and cloned and sequenced.Based on the sequence of RAPD marker,the sequences characterized amplified region(SCAR) primers were designed by the aid of the software Oligo5.0.The forward primer is M<sub>05</sub>F<sub>2</sub> (5’-CGGGT CATGG CTGGA GGTAT CGT-3’),and the backward primer is M<sub>05</sub>R<sub>1</sub>(5’-TGGCT CAATG GCAAA TCCTT CGTA-3’.The specific fragment (OPM<sub>05</sub>-M<sub>2100</sub>) was successfully converted to SCAR marker(SCAR-M<sub>05</sub>-X<sub>600</sub>) by using M<sub>05</sub> F<sub>2</sub>/R<sub>1</sub>,which was the specific markers of B.xylophilus.Then, the DNA of 92 isolates of Bursaphelenchus,B. mucronatus,B.hofmanni,Aphelenchoides macronucleatus and Seinura sp.which were isolated from dead pines,were marked,and the DNA of a single nematode extracted with a simple method was detected using this set of specific primers.The results indicated that the PCR product of all 81 isolates of B.xylophilus was a clear and bright fragment about 600 bp with M<sub>05</sub> F<sub>2</sub>/R<sub>1</sub>.But eight isolates of B. mucronatus,one B.hofmanni,one A.macronucleatus and one Seinura sp.had no any fragments.Assay M<sub>05</sub> F<sub>2</sub>/R<sub>1</sub> also successfully detected single pinewood nematode.Therefore,the specific pairwises would be used for constructing identification kits of B. xylophilus,implementing the aim of quick detection, and achieving the purpose of identify juvenile successfully.展开更多
This article summarizes the results of the research papers presented at the International Symposium on pine wilt disease (IUFRO Working Party Meeting 4.04.03) held in July 2009, at Nanjing, China. The general topics...This article summarizes the results of the research papers presented at the International Symposium on pine wilt disease (IUFRO Working Party Meeting 4.04.03) held in July 2009, at Nanjing, China. The general topics covered were on pine wilt disease (PWD), its causal organism, the pinewood nematode (PWN) Bursaphelenchus xylophilus, plus other PWN-associated microorganisms that play a significant role in PWD such as bacteria (e.g. Pseudomonasfluorescens). Most of the papers that are reviewed are based on work on PWD-PWN in East Asia and Russia. Specific topics covered include: 1) the fundamental conceptions of PWD development, 2) pathogenicity, 3) host-parasite relationships including the histopathology of diseased conifers and the role of toxins from bacteria-nematode ecto-symbionts, 4) PWN life cycle and transmission, 5) B. xylophilus dissemination models, 6) associations (with other nematodes), 7) diagnostics, 8) quarantine and control of the PWN and 9) biocontrol of the PWN.展开更多
Pine wilt disease (PWD), which is caused by pine wood nematodes (PWN), is one of the most serious forest diseases worldwide. To clarify the mechanism of resistance to PWD, we compared metabolites from resistant and su...Pine wilt disease (PWD), which is caused by pine wood nematodes (PWN), is one of the most serious forest diseases worldwide. To clarify the mechanism of resistance to PWD, we compared metabolites from resistant and susceptible Japanese black pine (Pinus thunbergii) families after inoculation with PWN. After 2 weeks to 1 month post inoculation, the number of PWN dramatically increased in susceptible plants, but not in resistant plants. At this PWN-proliferation phase, ethyl acetate soluble fractions extracted from PWN-inoculated plants were analyzed by gas chromatogramphy-mass spectrometry (GC-MS). Although most compounds were qualitatively and quantitatively similar between resistant and susceptible plants, resistant plants accumulated 2.0-fold more linoleic acid (LA) than susceptible plants. On the other hand, benzoic acid (BA) was barely detected in resistant plants, but it accumulated in susceptible plants as the number of PWN increased. Susceptible plants contained greater levels of the nematicidal compounds pinosylvin and pinosylvin monomethyl ether, compared with resistant plants. These results suggested that LA is involved in the resistance reaction against PWN-proliferation, and that BA could be a good biomarker for PWD.展开更多
Pine wilt disease was first discovered in Dongtang town,Liaoning Province,China,in 2017.Monochamus saltuarius Gebler is a new vector of pinewood nematode and the only known vector in Liaoning Province.The biology of t...Pine wilt disease was first discovered in Dongtang town,Liaoning Province,China,in 2017.Monochamus saltuarius Gebler is a new vector of pinewood nematode and the only known vector in Liaoning Province.The biology of this pest has not been reported thus far;therefore,it is necessary to study its life history.During 2018 and 2019,we collected 138 and 491 adult M.saltuarius beetles,respectively,to analyze their eclosion from larva to adult stage.In mid-March,overwintering larvae began to feed(on xylem)and seek nutrition in preparation for pupation and eclosion.The adults began to appear in mid-April,and the population reached its peak in late May.The life span of the adults was 28-76 days.After approximately 1 week of supplemental nutrition(feeding on twigs),adults began to mate and lay eggs.The egg stage of M.saltuarius lasted 4-8 days.The larvae in Dongtang town have 4 instars and overwinter in tunnels as 3rd-4th instars.The 1st-instar stage lasted 3-9 days,the 2nd-instar stage lasted 11-23 days,the 3rd-instar stage lasted 30-130 days,and the 4th-instar stage lasted 44-180 days.The pupal stage lasted 7-12 days,and the life span of the adults was 28-76 days.In this study we systematically monitored the life history of M.saltuarius for the first time.Our objective was to lay a foundation for improving control of this pinewood nematode vector.展开更多
Pine wilt disease,caused by Bursaphelenchus xylophilus and Bursaphelenchus mucnatus,is a serious quarantined disease.Arboreal nematode-trapping fungi of P inus spp.are effective predators on nematodes and have strong ...Pine wilt disease,caused by Bursaphelenchus xylophilus and Bursaphelenchus mucnatus,is a serious quarantined disease.Arboreal nematode-trapping fungi of P inus spp.are effective predators on nematodes and have strong host adaptability.The development of these fungal resources may be an effective way to control pine wood nematodes.We collected 515 samples of pine wilt disease from the areas of Ninghai City(Zhejiang province),Shuangbai County(Yunnan province),and Daxing'anling(Heilongjiang province),China.Through isolation,culture and identification,6 species of nematode-trapping fungi(A rthrobotrys cladodesr,A.oligospora,A.musiformis,A.dendroides,Dactylellina ellipsospora,Monacrosporium thaumasium)were identified for predation against B.xylophilus,and 9 species(Arthrobotrys dactyloides,A.cladodes r A.oligospora A.dendroides,Dactylellina ellipsospora,Dactylella asthenopaga,D.leptospora,Arthrobotrys superba,Monacrosporium drechseri)were identified for predation against B.mucnatus.This study provides information in the classification of arboreal predator nematodes and provides an important basis for future biological control of pine wood nematodes.展开更多
To accelerate the breeding and selection of Pinus densiflora Siebold and Zucc. resistance to pine wilt disease, a micropropagation system was established and nematode resistance evaluated in vitro. Cotyledon-hypocotyl...To accelerate the breeding and selection of Pinus densiflora Siebold and Zucc. resistance to pine wilt disease, a micropropagation system was established and nematode resistance evaluated in vitro. Cotyledon-hypocotyl explants from 28-day-old seedlings were first cultured on Gresshoff and Doy medium supplemented with 4.0 mg L^(-1) 6-benzyladenine and 0.2 mg L^(-1) a-naphthaleneacetic acid(NAA) to stimulate the formation of buds. Induced buds were subsequently subcultured on Gupta and Durzan medium supplemented with 0.1%(w/v)activated charcoal for elongation. Stem sections derived from shoots were used as explants for the further multiplication. Roots were formed from shoots transferred to woody plant medium containing 0.2 mg L^(-1) NAA for4 weeks. The nematode resistance test showed that symptoms in micropropagated shoots after infection with pine wood nematode(PWN) were similar to those in plants infected in the field. The wilting rate varied from 20 to100% among different clones 18 days after inoculation.The most susceptible clone was Clone 6-4 with a 100%wilting rate, while Clone 8-4 showed a relatively high resistance with a 20% wilting rate. The number of nematodes recovered from Clone 8-4 shoots was significantly lower(P = 0.05) than from Clones 5-10 and 16-4. This work contributes to the breeding of PWN resistance in P.densiflora.展开更多
Pine wilt is a serious and fatal disease in pine forests. It is caused by the pine wood nematode, Bursaphelenchus xylophilus, transmitted by the Japanese saw beetle Monochamus alternatus. Control of this disease depen...Pine wilt is a serious and fatal disease in pine forests. It is caused by the pine wood nematode, Bursaphelenchus xylophilus, transmitted by the Japanese saw beetle Monochamus alternatus. Control of this disease depends on reducing its vector population. The public is demanding environmentally friendly control methods and biological control has become increasingly seen as an impor-tant, safe and effective approach. Through our investigations both in the field and by breeding in our laboratory, a new predator of M. alternatus, Tetrigus lewisi (Coleoptera: Elateridae), was found for the first time in China. Given our continuous observations, we are assured that the predatory behavior of the elater can be separated into four parts, i.e., foraging and sensing, killing, eating and excre-tion. They always took several hours to eat the preys that have been killed momentarily. We finally found that most of the elaters preferably attacked in the stomach and abdomen of the larva of M. alternatus.展开更多
基金supported by the National Key Research and Development Program of China(2017YFD0600101).
文摘Genes homologous to members of the MRP gene family in Caenorhabditis elegans are important in drug resistance.To further explore the molecular mechanism of drug resistance in pine wood nematode(Bursaphelenchus xylophilus),we used bioinformatics approaches to analyze genomic data for B.xylophilus and identified Bx-MRP genes.We predicted the structure and function of the genes and encoded proteins.Using bioinformatics programs to predict and analyze various properties of the predicted proteins,including hydrophobicity,transmembrane regions,phosphorylation sites,and topologically isomeric structures,of these Bx-MRP genes,we determined that they function in transmembrane transport.From the results of RT-qPCR,the Bx-MRP family members confer significant differential resistance to different drug treatments.After treatment with different concentrations of emamectin benzoate,avermectin and matrine,the expression of each gene increased with increasing drug concentrations,indicating that the family members play a positive role in the regulation of multidrug resistance.
基金The work was supported by the National Natural Science Foundation of China(31870633,31700565,31670652,31570638 and 31270688)National Key Research and Development Plan of China(2017YFD0600102-03).
文摘Bursaphelenchus xylophilus,causal agent of pine wilt disease,causes extensive damage worldwide.Strate-gies are needed to inhibit population growth or block the spread of the invasive nematode to control pine wilt dis-ease.The gene daf-8 plays crucial roles in larval develop-ment and dauer formation in Caenorhabditis elegans,but little is known about its orthologue in B.xylophilus.In the present molecular characterization and functional analysis of daf-8 in B.xylophilus(Bx-daf-8),RT-qPCR showed that the expression of Bx-daf-8 gradually increased during the embryonic stage,peaked in the second-stage juvenile(J2),then dramatically dropped in the J3,and remained at that low level for the rest of its life.Bx-daf-8-transgenic C.elegans was employed to mimic the spatiotemporal expression of Bx-daf-8,which was expressed close to the pharynx during early embryogenesis and weakly throughout the whole body during late embryogenesis.It was observed in head neurons and tail ganglions throughout all postembryonic stages.B.xylophilus embryos were severely abnormal,and hatching rate decreased sharply after Bx-daf-8 knockdown.daf-16-1 and da-f16-2,downstream genes in the IIS pathway,also dropped sharply after Bx-daf-8 knockdown.We propose that TGFβmay crosstalk with the IIS pathway upstream of Bx-daf-16 and that daf-8 may act as a master regulator of daf-16 in B.xylophilus.However,knockdown of Bx-daf-8 did not lead to constitutive developmental arrest at the dauer larval stage,indicating that dauer entry in B.xylophilus might be controlled by several genes and is more complicated than in C.elegans.Bx-daf-8 alone did not control the dauer entry in B.xylophilus,but it was indispensable for embryogen-esis,providing a potential target for suppressing population growth of B.xylophilus.
基金financially supported by the National Key Research and Development Program(2017YFD0600105)the National Natural Science Foundation of China(Grant No.31870641)+2 种基金the Research Foundation of Education Department of Fujian Province(No.JAT170882)Project of Financial Department of Fujian Province(Nos.K81139238 and K8911010)the Special Fund for Forestry Research in the Public Interest of China(No.201304401)
文摘Pine wilt disease(PWD)is a devastating disease affecting the growth of Pinus massoniana,often leading to withering and death.To reveal the changes involved during disease progression,we investigated the mRNA expression profile of P.massoniana infested by Bursaphelenchus xylophilus.The infestation resulted in the downregulation of genes involved in interactions with pathogenic pathways such as disease resistance gene,CC-NBS-LRR resistancelike protein,and the gene encoding a putative nematode resistance protein.Increased infestation pressure(number of nematodes inoculated)caused a continuous decline in the gene expression of stem samples.An infestation of P.massoniana also resulted in a pathway enrichment of genes involved in phenylpropanoid metabolism and flavonoid biosynthesis,which in turn reduced the levels of total phenols and total flavonoids.A downregulation of auxin responsive family protein was observed in infested samples,which resulted in a suppression of plant growth.Thus,upon B.xylophilus infestation,a downregulation of genes associated with the recognition of pathogens,PWD resistance,and growth regulation was observed in P.massoniana,together with a decrease in the levels of phytoalexinlike secondary substances,all of which resulted in withering and ultimately death of P.massoniana.
基金supported by the special research program for non-profit forestry of the State Forestry Administration(201204501)National Nature Science Foundation of China(31170600)funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions(PAPD)
文摘Bursaphelenchus xylophilus is the pathogen that causes pine wilt disease, which has greatly damaged forests and ecosystems in countries of East Asia and Europe.Bursaphelenchus mucronatus is closely related to B. xylophilus in morphology and host plant specificity. A longrunning debate has existed regarding whether these two species can successfully produce hybrid offspring. In the present study, we performed in the laboratory, hybridization of two B. xylophilus nematode isolates from China and Japan and three B. mucronatus isolates from China, Japan and France. Nematode isolates of B. xylophilus were successfully crossed with B. mucronatus isolates, and the rate of hybridization was relatively high; however, some hybrid offspring died. Successful hybridization occurred between B. xylophilus and B. mucronatus isolates from China, and 22 generations of hybrids were produced. All F1 hybrids could be backcrossed with their parents and produce offspring. Variation in mucro length among the hybrid offspring and their parents was observed. The hybrid offspring and their parents were inoculated into 3-months-old black pine(Pinus tunbergii) seedlings. Weaker pathogenicity of hybrid offspring was observed compared with that of their parents, and significantly fewer offspring nematodes than parents were reisolated from pines. Therefore, the offspring of B. xylophilus and B. mucronatus may exist in the forest and could influence disease epidemics.
基金The work was supported by the Fundamental Research Funds for the Central University(No.2572020DY11).
文摘Pine wilt disease caused by pine wood nema-todes is a deadly disease of the genus Pinus requiring strong quarantine measures.Since its discovery,it has been widely distributed throughout the world.China is one of the coun-tries with a severe rate of infections due to its abundant pine resources.In this study,nematode-trapping fungi were collected from pine trees in Ninghai City,Zhejiang Prov-ince,which is the key area of pine wilt control in Febru-ary,May,September,October and November.The results showed that nematode-trapping fungi of pine are abundant,especially the number and species detected in each month and are quite different;species of fungi in July,Septem-ber and November were more numerous and had higher separation rates.The dominant species in November was Arthrobotrys oligospora followed by Arthrobotrys cladodes;July and September it was A.cladodes.In each part of the tree,A.cladodes was the most widely distributed with a high separation rate.Therefore,it is the dominant fungal species in the area.Arboreal Bursaphelenchus xylophilus-trapping fungi are dependent on pine wood nematodes,and none were isolated from healthy pine trees.More arboreal B.xylophilus-trapping fungi were found under the bark than in the xylem.There is a close relationship between arboreal B.xylophilus-trapping fungi and pine wood nematodes.How to make use of these resources to reduce the damage of pine wood nematode fusarium is the focus of future research.
文摘Pine wood nematode (PWN), Bursaphelenchus xylophilus (Steiner et Buhrer) Nickle is an important invasive alien species in forests of China and has become one of the most destructive forest diseases. In order to improve the resistance and resilience of pine forest ecosystems against B. xylophilus invasion and make the pine forest ecosystem more timely responsive to PWN invasion, we made some recommendations based on five years of intensive observations. We advocate a set of management measures with the theme “Prevention is priority, but integrated with curative techniques and ecological resilience” on the pine forest ecosystem invaded by B. xylophilus; details of accurate measures are proposed. The aim is to discover the underlying problems of present pine forest ecosys-tems and to take, correspondingly, administrative measures and strategies, which will encourage the pine forest ecosystem to develop in a benign way.
文摘OPM<sub>05</sub>-M<sub>2100</sub>,the specific RAPD fragment of Bursaphelenchus xylophilus,was collected from agarose gels and purified.Then,the purified fragment was inserted into the pGEM<sup>R</sup> -T Vector that was transformed into Escherichia.coli and cloned and sequenced.Based on the sequence of RAPD marker,the sequences characterized amplified region(SCAR) primers were designed by the aid of the software Oligo5.0.The forward primer is M<sub>05</sub>F<sub>2</sub> (5’-CGGGT CATGG CTGGA GGTAT CGT-3’),and the backward primer is M<sub>05</sub>R<sub>1</sub>(5’-TGGCT CAATG GCAAA TCCTT CGTA-3’.The specific fragment (OPM<sub>05</sub>-M<sub>2100</sub>) was successfully converted to SCAR marker(SCAR-M<sub>05</sub>-X<sub>600</sub>) by using M<sub>05</sub> F<sub>2</sub>/R<sub>1</sub>,which was the specific markers of B.xylophilus.Then, the DNA of 92 isolates of Bursaphelenchus,B. mucronatus,B.hofmanni,Aphelenchoides macronucleatus and Seinura sp.which were isolated from dead pines,were marked,and the DNA of a single nematode extracted with a simple method was detected using this set of specific primers.The results indicated that the PCR product of all 81 isolates of B.xylophilus was a clear and bright fragment about 600 bp with M<sub>05</sub> F<sub>2</sub>/R<sub>1</sub>.But eight isolates of B. mucronatus,one B.hofmanni,one A.macronucleatus and one Seinura sp.had no any fragments.Assay M<sub>05</sub> F<sub>2</sub>/R<sub>1</sub> also successfully detected single pinewood nematode.Therefore,the specific pairwises would be used for constructing identification kits of B. xylophilus,implementing the aim of quick detection, and achieving the purpose of identify juvenile successfully.
基金supportedby a Key Program of the National Natural Science Foundation of China (Grant No. 30430580)the State Forestry Administration of China (Grant No.20070430)a review is done in frames of the project 10-04-01644-a of the Russian Foundation for Basic Research
文摘This article summarizes the results of the research papers presented at the International Symposium on pine wilt disease (IUFRO Working Party Meeting 4.04.03) held in July 2009, at Nanjing, China. The general topics covered were on pine wilt disease (PWD), its causal organism, the pinewood nematode (PWN) Bursaphelenchus xylophilus, plus other PWN-associated microorganisms that play a significant role in PWD such as bacteria (e.g. Pseudomonasfluorescens). Most of the papers that are reviewed are based on work on PWD-PWN in East Asia and Russia. Specific topics covered include: 1) the fundamental conceptions of PWD development, 2) pathogenicity, 3) host-parasite relationships including the histopathology of diseased conifers and the role of toxins from bacteria-nematode ecto-symbionts, 4) PWN life cycle and transmission, 5) B. xylophilus dissemination models, 6) associations (with other nematodes), 7) diagnostics, 8) quarantine and control of the PWN and 9) biocontrol of the PWN.
文摘Pine wilt disease (PWD), which is caused by pine wood nematodes (PWN), is one of the most serious forest diseases worldwide. To clarify the mechanism of resistance to PWD, we compared metabolites from resistant and susceptible Japanese black pine (Pinus thunbergii) families after inoculation with PWN. After 2 weeks to 1 month post inoculation, the number of PWN dramatically increased in susceptible plants, but not in resistant plants. At this PWN-proliferation phase, ethyl acetate soluble fractions extracted from PWN-inoculated plants were analyzed by gas chromatogramphy-mass spectrometry (GC-MS). Although most compounds were qualitatively and quantitatively similar between resistant and susceptible plants, resistant plants accumulated 2.0-fold more linoleic acid (LA) than susceptible plants. On the other hand, benzoic acid (BA) was barely detected in resistant plants, but it accumulated in susceptible plants as the number of PWN increased. Susceptible plants contained greater levels of the nematicidal compounds pinosylvin and pinosylvin monomethyl ether, compared with resistant plants. These results suggested that LA is involved in the resistance reaction against PWN-proliferation, and that BA could be a good biomarker for PWD.
基金This study was financially supported by the National Key R&D Program of China(2017YFD0600104)the Shenyang Science and Technology Planning Project(18-400-3-03).
文摘Pine wilt disease was first discovered in Dongtang town,Liaoning Province,China,in 2017.Monochamus saltuarius Gebler is a new vector of pinewood nematode and the only known vector in Liaoning Province.The biology of this pest has not been reported thus far;therefore,it is necessary to study its life history.During 2018 and 2019,we collected 138 and 491 adult M.saltuarius beetles,respectively,to analyze their eclosion from larva to adult stage.In mid-March,overwintering larvae began to feed(on xylem)and seek nutrition in preparation for pupation and eclosion.The adults began to appear in mid-April,and the population reached its peak in late May.The life span of the adults was 28-76 days.After approximately 1 week of supplemental nutrition(feeding on twigs),adults began to mate and lay eggs.The egg stage of M.saltuarius lasted 4-8 days.The larvae in Dongtang town have 4 instars and overwinter in tunnels as 3rd-4th instars.The 1st-instar stage lasted 3-9 days,the 2nd-instar stage lasted 11-23 days,the 3rd-instar stage lasted 30-130 days,and the 4th-instar stage lasted 44-180 days.The pupal stage lasted 7-12 days,and the life span of the adults was 28-76 days.In this study we systematically monitored the life history of M.saltuarius for the first time.Our objective was to lay a foundation for improving control of this pinewood nematode vector.
基金supported by the National Natural Science Foundation of China(No.30571495)。
文摘Pine wilt disease,caused by Bursaphelenchus xylophilus and Bursaphelenchus mucnatus,is a serious quarantined disease.Arboreal nematode-trapping fungi of P inus spp.are effective predators on nematodes and have strong host adaptability.The development of these fungal resources may be an effective way to control pine wood nematodes.We collected 515 samples of pine wilt disease from the areas of Ninghai City(Zhejiang province),Shuangbai County(Yunnan province),and Daxing'anling(Heilongjiang province),China.Through isolation,culture and identification,6 species of nematode-trapping fungi(A rthrobotrys cladodesr,A.oligospora,A.musiformis,A.dendroides,Dactylellina ellipsospora,Monacrosporium thaumasium)were identified for predation against B.xylophilus,and 9 species(Arthrobotrys dactyloides,A.cladodes r A.oligospora A.dendroides,Dactylellina ellipsospora,Dactylella asthenopaga,D.leptospora,Arthrobotrys superba,Monacrosporium drechseri)were identified for predation against B.mucnatus.This study provides information in the classification of arboreal predator nematodes and provides an important basis for future biological control of pine wood nematodes.
基金funded by the Special Research Program for Forestry Sectors Beneficial to Public,State Forestry Administration,China(Grant No.201204501)the Science and Technology Support Program of Jiangsu Province(BE2014405)+1 种基金the Key University Science Research Project of Jiangsu Province(15KJA220003)the Priority Academic Program Development of Jiangsu Higher Education Institutions(PAPD)
文摘To accelerate the breeding and selection of Pinus densiflora Siebold and Zucc. resistance to pine wilt disease, a micropropagation system was established and nematode resistance evaluated in vitro. Cotyledon-hypocotyl explants from 28-day-old seedlings were first cultured on Gresshoff and Doy medium supplemented with 4.0 mg L^(-1) 6-benzyladenine and 0.2 mg L^(-1) a-naphthaleneacetic acid(NAA) to stimulate the formation of buds. Induced buds were subsequently subcultured on Gupta and Durzan medium supplemented with 0.1%(w/v)activated charcoal for elongation. Stem sections derived from shoots were used as explants for the further multiplication. Roots were formed from shoots transferred to woody plant medium containing 0.2 mg L^(-1) NAA for4 weeks. The nematode resistance test showed that symptoms in micropropagated shoots after infection with pine wood nematode(PWN) were similar to those in plants infected in the field. The wilting rate varied from 20 to100% among different clones 18 days after inoculation.The most susceptible clone was Clone 6-4 with a 100%wilting rate, while Clone 8-4 showed a relatively high resistance with a 20% wilting rate. The number of nematodes recovered from Clone 8-4 shoots was significantly lower(P = 0.05) than from Clones 5-10 and 16-4. This work contributes to the breeding of PWN resistance in P.densiflora.
文摘Pine wilt is a serious and fatal disease in pine forests. It is caused by the pine wood nematode, Bursaphelenchus xylophilus, transmitted by the Japanese saw beetle Monochamus alternatus. Control of this disease depends on reducing its vector population. The public is demanding environmentally friendly control methods and biological control has become increasingly seen as an impor-tant, safe and effective approach. Through our investigations both in the field and by breeding in our laboratory, a new predator of M. alternatus, Tetrigus lewisi (Coleoptera: Elateridae), was found for the first time in China. Given our continuous observations, we are assured that the predatory behavior of the elater can be separated into four parts, i.e., foraging and sensing, killing, eating and excre-tion. They always took several hours to eat the preys that have been killed momentarily. We finally found that most of the elaters preferably attacked in the stomach and abdomen of the larva of M. alternatus.