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1.
The pinewood nematode (PWN), Bursaphelenchus xylophilus, is the causal agent of pine wilt disease (PWD), which is a major problem in East Asia and West Europe. Quick identification of PWN is needed to prevent the dispersal of PWD to healthy forests. Various detection methods of PWN have been developed using anatomical characters and molecular markers. These methods are not suitable for rapid diagnosis because it is difficult to distinguish B. xylophilus from the non‐pathogenic species Bursaphelenchus mucronatus based on morphological characters without expertise in nematode taxonomy and most PCR or isothermal amplification detection methods require time‐consuming processes. In this study, we developed an on‐site PWN detection method using a recombinase polymerase amplification (RPA) assay with a novel extraction buffer (DAP buffer). This new PWN detection method is able to extract genomic DNA from PWN in pinewood by simple buffer consisting of sodium hydrate, polyethylene glycol 200 and dimethyl sulfoxide in 10 min without using the experimental devices and able to distinguish between B. xylophilus and other Bursaphelenchus spp. by amplifying the species‐specific 5S rDNA fragment of B. xylophilus in 10 min. Taken together, our protocol can obtain the result for the detection of PWN in pine tree samples within 30 min. This result suggests that RPA/DAP assay is much faster, easier and cheaper than the conventional methods for detecting PWN.  相似文献   

2.
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3.
Extensive ponderosa pine (Pinus ponderosa Dougl. ex Laws.) mortality associated with a widespread severe drought and increased bark beetle (Coleoptera: Curculionidae, Scolytinae) populations occurred in Arizona from 2001 to 2004. A complex of Ips beetles including: the Arizona fivespined ips, Ips lecontei Swaine, the pine engraver beetle, Ips pini (Say), Ips calligraphus (Germar), Ips latidens (LeConte), Ips knausi Swaine and Ips integer (Eichhoff) were the primary bark beetle species associated with ponderosa pine mortality. In this study we examine stand conditions and physiographic factors associated with bark beetle-caused tree mortality in ponderosa pine forests across five National Forests in Arizona. A total of 633 fixed-radius plots were established across five National Forests in Arizona: Apache-Sitgreaves, Coconino, Kaibab, Prescott, and Tonto. Prior to the bark beetle outbreak, plots with mortality had higher tree and stocking compared with plots without pine mortality. Logistic regression modeling found that probability of ponderosa pine mortality caused by bark beetles was positively correlated with tree density and inversely related with elevation and tree diameter. Given the large geographical extent of this study resulting logistic models to estimate the likelihood of bark beetle attack should have wide applicability across similar ponderosa pine forests across the Southwest. This is particularly true of a model driven by tree density and elevation constructed by combining all forests. Tree mortality resulted in significant reductions in basal area, tree density, stand density index, and mean tree diameter for ponderosa pine and for all species combined in these forests. Most of the observed pine mortality was in the 10–35 cm diameter class, which comprise much of the increase in tree density over the past century as a result of fire suppression and grazing practices. Ecological implications of tree mortality are discussed.  相似文献   

4.
Pine wilt disease (PWD), caused by the pine wood nematode (PWN) Bursaphelenchus xylophilus, leads to serious losses to pine forestry around the world. Pinus massoniana, which is vulnerable to be attacked by the PWN, is the dominant species used in pine forestry in China. The objective of this study is to develop a direct PCR‐based method for detecting B. xylophilus in the wood of P. massoniana without a separate nematode extraction step. A simple procedure was first developed for isolating B. xylophilus DNA in 5 mg pine wood tissue samples harbouring PWN for detection by PCR amplification. A B. xylophilus‐specific amplicon of 403 bp (DQ855275) was generated by PCR from the infested wood tissue. The entire procedure can be completed within 5 h with one pair of primers. This assay can serve as a rapid, cheap and environmentally friendly method to detect B. xylophilus in samples of P. massoniana.  相似文献   

5.
Dothistroma needle blight (DNB) is among the most serious foliar diseases affecting Pinus spp. globally. Infected needles were collected from potential host species in four locations in western Ukraine and in four locations in eastern Georgia during spring–summer 2015 to update the knowledge on pathogen distribution in these countries. Dothistroma spp. were detected using isolation, sequencing and species‐specific priming (SSPP) PCR. Two new hosts for Dothistroma spp. were recorded in western Ukraine: D. septosporum on Pinus nigra var. australica and D. pini on P. nigra var. mollet. D. septosporum was found on 15‐year‐old P. strobus in western Ukraine. New hosts for D. septosporum were recorded in Georgia on 5‐ to 10‐year‐old naturally regenerated P. sylvestris var. hamata and on 40‐ to 50‐year‐old P. ponderosa trees. D. pini was found for the first time in Georgia on 30‐ to 40‐year‐old P. nigra trees. The work confirmed the presence of both D. septosporum and D. pini in western Ukraine and Georgia, and demonstrated new hosts for both Dothistroma species.  相似文献   

6.
Studies on felled stems of the black pine showed the water content, the process of putrefaction by fungi (Ceratostomella, Fomes) and the attacks by beetles developing in the same direction. The most important species of beetles were:Ips sexdentatus Boern.,Pityogenes quadridens Hrtg. (Scolytidae),Arbopalus rusticus L.,Acanthocinus aedilis L. (Cerambycidae). The infection byCeratostomella always started from the holes of entrance of bark beetles.  相似文献   

7.
Pine plantations in Argentinian Patagonia cover ca. 95,000 ha in Chubut, Río Negro and Neuquén provinces. Exotic bark beetles (Orthotomicus laricis, Hylastes ater and Hylurgus ligniperda) commonly occur in freshly cut logs, stumps and slash. These beetles are vectors of “ophiostomatoid” fungi which include primary tree pathogens as well as important agents of blue stain. The aim of this study was to identify these beetle‐associated fungi. Sawing mills and pine plantations were surveyed three consecutive years. Fungal isolates from stained logs, processed wood and insect galleries were identified based on morphological and DNA sequence comparisons of ITS and β‐tubulin gene regions. Two Grosmannia, one Graphilbum and three Ophiostoma species were identified. Ophiostoma piliferum and O. peregrinum sp. nov. were the most frequently isolated taxa. O. peregrinum occurred in all provinces, colonizing different conifer species and, interestingly, also the native broadleaved species Nothofagus dombeyi. Pine plantation forestry in southern South America includes Argentina, Brazil, Chile, Paraguay and Uruguay. Emerging data from Argentina, Chile and Uruguay revealed some coincidences between these countries, but also several differences, probably, as a result of multiple introduction events.  相似文献   

8.
The pine wood nematode (PWN), Bursaphelenchus xylophilus, causes huge economic losses in pine forests. The plant‐parasitic nematodes have a complex life cycle that includes the secretion of effector proteins through a stylet into the host cell to promote parasitism. In this study, SignalP 4.1 and TMHMM 2.0 were used in preliminary screens for candidate effectors and were expressed in Nicotiana benthamiana through the PVX virus expression vector. The yeast signal sequence trap system was used to further study the function of the signal peptide of an effector, BxSapB2. In situ hybridization was conducted to investigate the localization of BxSapB2, followed by RNA interference technology (RNAi) to assess the functions of BxSapB2. The results demonstrate that BxSapB2 is a secreted protein that induces cell death in N. benthamiana and is highly expressed in esophageal gland cells and amphids of B. xylophilus. BxSapB2 was determined to be related to the pathogenicity of B. xylophilus. The results of this work indicate that BxSapB2 plays an important role in the interactions between B. xylophilus and the hosts.  相似文献   

9.
Four treatments (control, burn-only, thin-only, and thin-and-burn) were evaluated for their effects on bark beetle-caused mortality in both the short-term (one to four years) and the long-term (seven years) in mixed-conifer forests in western Montana, USA. In addition to assessing bark beetle responses to these treatments, we also measured natural enemy landing rates and resin flow of ponderosa pine (Pinus ponderosa) the season fire treatments were implemented. All bark beetles were present at low population levels (non-outbreak) for the duration of the study. Post-treatment mortality of trees due to bark beetles was lowest in the thin-only and control units and highest in the units receiving burns. Three tree-killing bark beetle species responded positively to fire treatments: Douglas-fir beetle (Dendroctonus pseudotsugae), pine engraver (Ips pini), and western pine beetle (Dendroctonus brevicomis). Red turpentine beetle (Dendroctonus valens) responded positively to fire treatments, but never caused mortality. Three fire damage variables tested (height of crown scorch, percent circumference of the tree bole scorched, or degree of ground char) were significant factors in predicting beetle attack on trees. Douglas-fir beetle and pine engraver responded rapidly to increased availability of resources (fire-damaged trees); however, successful attacks dropped rapidly once these resources were depleted. Movement to green trees by pine engraver was not observed in plots receiving fire treatments, or in thinned plots where slash supported substantial reproduction by this beetle. The fourth tree-killing beetle present at the site, the mountain pine beetle, did not exhibit responses to any treatment. Natural enemies generally arrived at trees the same time as host bark beetles. However, the landing rates of only one, Medetera spp., was affected by treatment. This predator responded positively to thinning treatments. This insect was present in very high numbers indicating a regulatory effect on beetles, at least in the short-term, in thinned stands. Resin flow decreased from June to August. However, resin flow was significantly higher in trees in August than in June in fire treatments. Increased flow in burned trees later in the season did not affect beetle attack success. Overall, responses by beetles to treatments were short-term and limited to fire-damaged trees. Expansions into green trees did not occur. This lack of spread was likely due to a combination of high tree vigor in residual stands and low background populations of bark beetles.  相似文献   

10.
Modified White's solution (1 g HgCl2/l H2O) is widely used to surface disinfest bark beetles of their phoretic fungi. We investigated the effectiveness of this solution at disinfesting adult Ips pini from its associated ophiostomatoid fungi. A treatment for 1, 4 or 8 min does not completely rid beetles of phoretic fungi, but does substantially reduce the amount of fungi they carry externally. Sterilizing with modified White's solution caused limited mortality (<16%).  相似文献   

11.
Beetles (Scolytinae) form intimate associations with a taxonomically and functionally diverse suite of nematodes that are phytopathogens, fungal feeders, and entomoparasites. Despite their ubiquity, the ecological significance of nematodes in the lifecycles of economically important bark and ambrosia beetle species (Curculionidae: Scolytinae) and associated plant diseases remains largely unexplored. Thousand cankers disease (TCD) is caused by the walnut twig beetle (WTB, Pityophthorus juglandis Blackman) and the fungus Geosmithia morbida (Kolařík, Freeland, Utley & Tisserat; Ascoymycota: Hypocreales) and causes foliar senescence, progressive crown dieback, and mortality in black walnut (Juglans nigra L.) throughout western North America. In this study, nematodes recovered from P. juglandis and J. nigra in Idaho (ID) and Washington (WA) were identified morphologically and by constructing multilocus phylogenies to infer taxonomic relationships to taxa for which molecular data were available. We conducted assays to determine the extent to which nematodes feed and reproduce on G. morbida and other fungi commonly found in galleries of P. juglandis. Inoculation experiments were conducted to determine the effect of nematodes on the area of subdermal necrotic lesions (cankers) caused by G. morbida in branches of mature J. nigra and stems of seedlings. The phoretic nematode Bursaphlenhus juglandis (Ryss, Parker, Alvarez-Ortega, Nadeler & Subbotin) was frequently found under elytra of WTB in all locations, and a free-living nematode (Panagrolaimus sp.) was also widespread and found in the bark of mature trees. Both B. juglandis and Panagrolaimus sp. reduced the size of cankers caused by G. morbida in seedlings and branches of mature trees, respectively. However, these species may play opposite roles as disease synergists and antagonists based on the observation that exudates and/or microbiota associated with Panagrolaimus sp., but not B. juglandis destroyed G. morbida colonies in culture. Furthermore, B. juglandis contributed to foliar symptoms in seedlings inoculated with G. morbida. An entomoparasitic nematode (Aphelenchoididae), most closely resembling an Ektaphelenchus sp., was also found in the haemocoel of WTB. Infection rates were positively related to beetle population sizes as inferred from emergence rates. Ditylenchus sp. was also found in incubated walnut wood in WA and Rhabtidolaimus sp. was phoretic on P. juglandis and found in incubated walnut wood in WA and ID. The community of nematodes in J. nigra in WA and ID differed substantially from what has been observed associated with J. nigra in its native range.  相似文献   

12.
Pitch canker is a destructive disease of pine caused by the fungus Fusarium circinatum. This taxon is listed as a quarantine fungus for several regional plant protection organizations throughout the world. Whereas long‐distance spread of the disease is made possible through the trade of infected pine seeds, local spread is caused by aerial dispersion or insect transportation of the fungal conidia. Developing a reliable and efficient tool to detect of F. circinatum in insects would be very useful to monitor the local spread of the pathogen. This tool would also provide the means to assess the range of insect species that could serve as potential vector of the fungus. A DNA extraction protocol was optimized and combined with a real‐time PCR test to detect F. circinatum on pine beetles. Using artificially contaminated Ips sexdentatus, it was shown that the test was able to detect down to 10 F. circinatum conidia per individual, and 20 conidia per batch of 10 insects, which is below the lowest inoculum load occurring in nature. With this technique, several batches of up to 10 insects may be analysed simultaneously, with a timescale for analysis reduced to <5 h and without the need for expertise in Fusarium taxonomy. This tool may be useful to monitor potential spread of the pathogen across regions. Using this method, to date, despite F. circinatum foci occurred in Northern Spanish regions across the border in France, the pathogen was not found on I. sexdentatus.  相似文献   

13.
Pine wilt disease (PWD), caused by the pinewood nematode (PWN), Bursaphelenchus xylophilus (Steiner and Buhrer) Nickle 1970 , is a serious threat to susceptible pine forests of the world. The PWN is primarily vectored by Monochamus species (Coleoptera: Cerambycidae). The first occurrence of PWD was reported from Japan in the early 1900s. Following this report, Japanese scientists documented the community of bark‐ and phloem‐inhabiting insects associated with the nematodes in dying trees to determine possible vectors of the nematode. Monochamus alternatus was reported to be the most effective vector in Japan. The primary vector in North America is Monochamus carolinensis, and in Europe, it is Monochamus galloprovincialis. Further studies have been expanded through the nematode‐invaded countries of Korea, Taiwan, China and Portugal. There is an interspecific association between the PWN and its insect vectors, and it is an obligatory component of the disease cycle. It is crucial to understand this relationship as well as the population ecology of the beetle to aid in monitoring and control of this worldwide threat to pine forests. Studies to date indicate a remarkable similarity among beetle species around the globe for a variety of life‐history traits, including lifespan, adult emergence numbers, flight capability, nematode transmission rates and attraction to pine volatiles. Wherever pines are found, there is a beetle species capable of transmitting the nematode. Although flight performance and range is generally poor for this group of beetle vectors, the cryptic nature of the species and the lack of interest in the beetles by countries in the absence of the nematode have led to the disease establishing a foothold in a variety of countries such as Portugal. In this paper, studies conducted in different countries on Monochamus vector species of the PWN are compared and discussed.  相似文献   

14.
The pinewood nematode (PWN), Bursaphelenchus xylophilus, is a serious quarantine pest first detected in Portugal and Europe in 1999. It is the causal agent of pine wilt disease (PWD). A resistance breeding programme has been initiated to contribute to control the evolution of the disease. Five hundred and four adult maritime pine, Pinus pinaster, trees were phenotypically selected as candidate trees for this programme from an area affected by PWD. To identify tolerance to the nematode, the selected trees were monitored monthly. Over the course of 1 year, 57 candidate trees died and were tested for the presence/absence of the PWN. As accuracy of detection is of major importance, an ITS‐PCR‐based method applied directly to wood from adult maritime pine trees was tested and compared with a standard morphological identification method. The results showed that the use of PCR to detect the pathogen provided more rapid and accurate results in comparison with the standard morphological identification. Thus, this method is suitable to be used in the survey of the breeding population for resistance/tolerance to PWD.  相似文献   

15.
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16.
The pinewood nematode, Bursaphelenchus xylophilus, is the causal agent of pine wilt disease and is transmitted to new host trees by beetles of the genus Monochamus. The increasing interest in imported wood chips from North America for paper production and energy purposes and the corresponding phytosanitary risk of non‐vector transmission of B. xylophilus has been discussed since 1984, the year of the first interception of B. xylophilus in wood chips in the European Union. The long‐term survival of B. xylophilus in wood chips and its non‐vector spread from infested wood chips to non‐infested trees were studied. Pinus sylvestris logs were inoculated with a suspension of B. xylophilus to produce infested wood chips. During the long‐term storage test, B. xylophilus in P. sylvestris wood chips were examined. Four variants, including sealed and openly stored wood chips at both 15°C and 25°C, were studied. For the test of non‐vector spread, B. xylophilus ‐infested wood chips were placed on three‐ to four‐year‐old P. sylvestris saplings under different conditions. Bursaphelenchus xylophilus survived for more than 1 year at both temperatures in the sealed wood chips, which was significantly longer than for the openly stored variant at 25°C. Temperature, tree condition and wood chip location all influenced non‐vector spread through wood chips. Of the 480 trees that were in contact with infested wood chips and showed clear symptoms of pine wilt disease, B. xylophilus were extracted from 42 pines at 25°C and one pine at 15°C. The highest B. xylophilus infestation rates resulting in clear pine wilt disease symptoms (75%) were found in infested wood chips directly attached to stem‐wounded trees at 25°C. However, more variants exhibited B. xylophilus infestation at this temperature; trees with stem or root injuries plus direct contact with infested wood chips to the wounded part were primarily affected. Moreover, non‐vector spread was also detected in stem‐ and root‐injured pines without any direct contact with infested wood chips. Our results confirmed that B. xylophilus can survive for long periods in wood chips and can be transmitted from infested wood chips to damaged trees, but the likelihood of such PWN establishment should be low compared to spread through vectors. These findings must be considered in the pest risk analysis of B. xylophilus, and studies using outdoor trials should be carried out to complete this pest risk analysis.  相似文献   

17.
Migration of the pine wood nematode (PWN), Bursaphelenchus xylophilus, in susceptible and resistant pines was investigated at the tissue level. PWN was inoculated onto the top cross‐cut surface of 20‐cm stem cuttings of susceptible Pinus thunbergii and resistant pines (P. strobus, P. rigida and P. thunbergii of a resistant family Namikata‐(t)‐73 (half‐sib)). PWNs were mainly distributed in cortical resin canals of susceptible P. thunbergii down to 15 cm from the inoculated surface by 6 h after inoculation (HAI) and all tissues (including cortical and xylem resin canals) down to the bottom at 192 HAI. In P. strobus, P. rigida and P. thunbergii family Namikata‐(t)‐73 (half‐sib), PWN was distributed in cortical resin canals down to 5 cm by 6 HAI and down to the base at 192 HAI. However, the distribution of PWN in xylem resin canals of the resistant pines was restricted near inoculated surfaces down to 5 cm, even at 192 HAI. These results demonstrated that migration of PWN in resistant pines was slowed in cortical resin canals and restricted in xylem axial resin canals, features which may be associated with the resistance.  相似文献   

18.
The internal transcribed spacer (ITS) regions of rDNA have been routinely employed for identification and phylogenetic analysis of many nematode species. In this study, the intra‐ and interspecies ITS genetic diversity of Bursaphelenchus xylophilus and Bursaphelenchus mucronatus was evaluated. Ninety‐one isolates of the two nematode species collected from 14 Chinese provinces, Japan and Korea were used for ITS‐PCR and sequencing. An unweighted pair group cluster analysis dendrogram clustered them as two B. mucronatus and one B. xylophilus independent clades. Principal component analysis showed the phylogenetic relationship of the two nematode species more clearly; B. mucronatus isolates were separated into more than four groups, whereas B. xylophilus isolates still clustered into a group. The results of the Mantel test indicated the correlation of genetic distance matrices and geographic distance matrices was significant for both nematode species. The genetic differentiation coefficient (Gst) and gene flow (Nm) of B. mucronatus were 0.341 and 1.091, respectively, suggesting the importance of landscape heterogeneity and considerable obstacles for genetic exchange among B. mucronatus isolates in China. However, Gst and Nm of B. xylophilus were 0.188 and 2.151, respectively, very different compared to B. mucronatus. This could be owing to the short‐term introduction of B. xylophilus into China and a rapid spread through anthropogenic pathways. Our work adds to the understanding of the genetic diversity and genetic relationship of the two pine‐parasitic nematode species, and will aid in controlling them in the future.  相似文献   

19.
Bark beetles are notorious pests of natural and planted forests causing extensive damage. These insects depend on dead or weakened trees but can switch to healthy trees during an outbreak as mass-attacks allow the beetle to overwhelm tree defences. Climatic events like windstorms are known to favour bark beetle outbreaks because they create a large number of breeding sites, i.e., weakened trees and for this reason, windthrown timber is generally preventively harvested and removed. In December 1999, the southwest of France was struck by a devastating windstorm that felled more that 27 million m3of timber. This event offered the opportunity to study large-scale spatial pattern of trees attacked by the bark beetle Ips sexdentatus and its relationship with the spatial location of pine logs that were temporally stored in piles along stand edges during the post-storm process of fallen tree removal. The study was undertaken in a pure maritime pine forest of 1300 ha in 2001 and 2002. We developed a landscape approach based on a GIS and a complete inventory of attacked trees. During this study more than 70% of the investigated stands had at least one tree attacked by I. sexdentatus  . Spatial aggregation prevailed in stands with n≥15n≥15 attacked trees. Patches of attacked trees were identified using a kernel estimation procedure coupled with randomization tests. Attacked trees formed patches of 500–700 m2 on average which displayed a clumped spatial distribution. Log piles stemming from the sanitation removals were mainly distributed along the large access roads and showed an aggregated spatial pattern as well. The spatial relationship between patches of attacked trees and log pile storage areas was analyzed by means of the Ripley’s statistic that revealed a strong association at the scale of the studied forest. Our results indicated that bark beetle attacks were facilitated in the vicinity of areas where pine logs were stored. The spatial extent of this relationship was >1000 m. Similar results were obtained in 2001 and 2002 despite differences in the number and spatial distribution of attacked trees. The presence of a strong “facilitation effect” suggests that log piles should be removed quickly in order to prevent outbreaks of bark beetles.  相似文献   

20.
Interactions between the pine wood nematode (PWN), Bursaphelenchus xylophilus, and bacteria of the genus Pseudomonas were examined by cultivating axenic PWN and bacterial strains using callus of Pinus thunbergii. Ten (Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas cepacia and Pseudomonas spp.) of the 29 bacterial strains tested, significantly increased the reproduction of PWN. The rest of the bacteria (19 strains of 10 species) inhibited the reproduction of PWN completely. The growth of 18 of the 29 bacterial strains tested, including the 10 strains promoting PWN reproduction, was significantly increased by the presence of PWN. It indicated a mutualistic symbiotic relationship between PWN and the 10 bacterial strains in the genus Pseudomonas. The bacterial mutualistic symbionts are organisms, which may have co‐evolved with PWN rather than being accidentally associated. The finding provides further evidence for our hypothesis that pine wilt disease is complex, induced by both PWN and associated phytotoxin‐producing bacteria.  相似文献   

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