Within-wood Spatial Dispersion of the Pinewood Nematode,Bursaphelenchus xylophilus

Pinewood nematode, Bursaphelenchus xylophilus (Steiner &Buhrer) Nickle, spatial dispersion was determined in Scots pine, Pinus sylvestris L., bolts infested with the pine sawyer beetle, Monochamus carolinensis (Olivier) and in bolts without M. carolinensis. According to Taylor''s power law and Green''s index of dispersion, nematode dispersion was aggregated in both sets of bolts. The degree of aggregation did not differ significantly between beetle-infested and noninfested bolts, suggesting that the presence of M. carolinensis does not affect nematode dispersion within a bolt. Nematode population densities differed radially in bolts not infested with pine sawyers, but in a nonregular pattern. Moisture content of the bolts was correlated with population density of B. xylophilus, suggesting that nematode aggregates occur in areas of high moisture content.     

Pathogenesis in Pine Wilt Caused by Pinewood Nematode, Bursaphelenchus xylophilus

The progression of events in the development of pine wilt disease following the invasion by Bursaphelenchus xylophilus is reviewed from early migration through pine tissues until symptom development on foliage. Disease resistance in pines, especially the hypersensitive reaction that is successful in controlling many potential pests and pathogens, is explored. Pathologies resulting from the activities of pinewood nematode include cortical trails and cavities; formation of cambial gaps and traumatic resin cysts; browning and death of cortex, phloem, cambium, and ray tissues; granulation and shrinkage of cell cytoplasm in rays; and destruction of resin canal epithelial and ray parenchyma cells. Death of parenchyma, production of toxins, and leakage of oleoresins and other material into tracheids are typical of the hypersensitive reaction occurring in pines following migration of small numbers of pinewood nematodes. The hypothesis presented is that a spreading hypersensitive reaction results in some of the observed pathologies and symptoms and eventually causes pine death. The growth-differentiation balance hypothesis is used to help explain predisposition, oleoresin production and toxicity, susceptibility and resistance, and the effects of variation in climate on host pines as related to pinewilt disease.     

Incubation Temperature and Time Effects on Life Stages of Bursaphelenchus xylophilus in Wood Chips

Wood chips of Pinus strobus inoculated with Bursaphelenchus xylophilus were incubated at 3, 12, 30, or 40 C during intervals of 47, 82, and 130 days to determine the effects of incubation temperature and time on total number of nematodes and occurrence of each life stage. Nematodes did not survive at 40 C; the greatest number of nematodes was maintained at 3 C. The number and percentage of juveniles in the propagative cycle were greatest at 3 C after 47 days, but the percentage was greatest at 30 C after 130 days. More third-stage dispersal larvae, with percentages as high as 85%, were extracted at 3 and 12 C than at 30 C by the end of the study. Dauer larvae were extracted from the chips, but percentages never exceeded 5%. The percentage of adults was greater at 30 C than at 3 and 12 C after 82 and 130 days. When a 1-week heat treatment at 30 C was applied to samples at 3 and 12 C,. numbers and percentages of adults increased. Percentages of dauer larvae increased very slightly when the heat treatment was applied after 47 days, but numbers and percentages of juveniles and dispersals were affected erratically.     

Population Development and Influence of Bursaphelenchus xylophilus on Gliocladium virens

Gliocladium virens was isolated from slash pine trees symptomatic and asymptomatic for pine wilt disease with frequencies of 24% and 10%, respectively. Populations of Bursaphelenchus xylophilus, the nematode incitant of this disease, reproduced on this fungus and inhibited its growth. Growth inhibition of the fungus was characterized by an absence of sporulation and by the formation of chains of dark, thick-walled, chlamydospore-like cells. Population increase during a 12-day period following infestation of cultures of the fungus with 10,000 nematodes averaged 3-fold at 16 C, 9-fold at 20 C, and 24-fold at 24 C. In greenhouse studies, nematode recovery from slash pine seedlings coinoculated with both organisms was significantly greater than that obtained from seedlings inoculated with the nematode alone.     

Molecular Phylogeny of Geographical Isolates of Bursaphelenchus xylophilus: Implications on the Origin and Spread of this Species in China and Worldwide

The genetic diversity and phylogeny of 26 isolates of Bursaphelenchus xylophilus from China, Japan, Portugal and North America were investigated based on the D2/3 domain of 28S rDNA, nuclear ribosomal Internal Transcribed Spacer (ITS) sequences, and random amplified polymorphic DNA (RAPD) analysis. The genetic diversity analysis showed that the D2/3 domain of 28S rDNA of isolates of B. xylophilus from China, Portugal, Japan and the US were identical and differed at one to three nucleotides compared to those from Canada. ITS sequences of isolates from China and Portugal were the same; they differed at one or two nucleotides compared to those of Japanese isolates and at four and 23 nucleotides compared to those from the US and Canada, respectively. The phylogenetic analysis indicated that Chinese isolates share a common ancestor with one of the two Japanese clades and that the Canadian isolates form a sister group of the clade comprised of isolates from China, Portugal, Japan, and the US. The relationship between Japanese isolates and those from China was closer than with the American isolates. The Canadian isolates were the basal group of B. xylophilus. This suggests that B. xylophilus originated in North America and that the B. xylophilus that occurs in China could have been first introduced from Japan. Further analysis based on RAPD analysis revealed that the relationship among isolates from Guangdong, Zhejiang, Shandong, Anhui provinces and Nanjing was the closest, which suggests that pine wilt disease in these Chinese locales was probably dispersed from Nanjing, where this disease first occurred in China.     

Pathotypes of the Pinewood Nematode Bursaphelenchus xylophilus

An isolate of Bursaphelenchus xylophilus from Pinus sylvestris in Missouri infected and reproduced in 2-3-year-old seedlings of P. sylvestris and to some extent in seedlings of P. nigra. Wilting, however, occurred only in P. sylvestris. B. xylophilus isolated from P. strobus in Vermont infected and reproduced only in P. strobus seedlings. P. taeda seedlings were resistant to both of these isolates. Phytotoxin production was seen only in susceptible seedling species-nematode combinations. Significant water loss occurred only in those seedlings that were wilted because of infection by a compatible nematode isolate. Our results suggest that these isolates are pathotypes of B. xylophilus.     

Genetic Variation in Indian Populations of Scirpophaga incertulas as Revealed by RAPD-PCR Analysis

Scirpophaga incertulas, commonly referred to as yellow stem borer, is a predominant pest of rice causing serious losses in its yield. Genetic variation among populations of Scirpophaga incertulas collected from 28 hotspot locations in India was examined using the randomly amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR). In all, 32 primers were used and 354 amplification products were observed. No RAPD-PCR bands diagnostic to the pest population from any specific region were identified. Cluster analysis using UPGMA showed that, with the exception of the pest population from Pattambi, all the populations cluster as one group with GD values in the range of 6–22%, suggesting that gene flow between populations is independent of geographic distance and appears to be unrestricted. The relatively high GD value of 48% exhibited by the pest population from Pattambi was the only exception.     

Ribosomal DNA haplotype distribution of Bursaphelenchus xylophilus in Kyushu and Okinawa islands, Japan

Ribosomal DNA region sequences (partial 18S, 28S and complete ITS1, 5.8S, and ITS2) of the pinewood nematode (Bursaphelenchus xylophilus) were obtained from DNA extracted directly from wood pieces collected from wilted pine trees throughout the Kyushu and Okinawa islands, Japan. Either a 2569bp or 2573bp sequence was obtained from 88 of 143 samples. Together with the 45 rDNA sequences of pinewood nematode isolates previously reported, there were eight single nucleotide polymorphisms and two indels of two bases. Based on these mutations, nine haplotypes were estimated. The haplotype frequencies differed among regions in Kyushu island (northwest, northeast and center, southeast, and southwest), and the distribution was consistent with the invasion and spreading routes of the pinewood nematode previously estimated from past records of pine wilt and wood importation. There was no significant difference in haplotype frequencies among the collection sites on Okinawa island.     

Genomic Differences among Pathotypes of Bursaphelenchus xylophilus

Total genomic DNA from Bursaphelenchus xylophilus pathotypes MPSy-1 and VPSt-1 and from B. mucronatus was digested with restriction endonucleases. DNA fragments were electrophoretically separated, Southern blotted to nitrocellulose, and hybridized to genomic DNA from one of the isolates. The resulting hybridization patterns indicate genomic differences in repetitive DNA sequences among these populations. Greatest differences were seen between B. xylophilus and B. mucronatus, but genomic differences were also apparent between B. xylophilus pathotypes MPSy-1 and VPSt-1 and between a population from P. nigra in New Jersey and a population of a mucronate form from Abies balsamea in Quebec, Canada.     

Effect of Monochamus carolinensis on the Life History of the Pinewood Nematode,Bursaphelenchus xylophilus

The development of Bursaphelenchus xylophilus in pine wood infested with and free of Monochamus carolinensis was investigated. Formation of third-stage dispersal juveniles occurred in the presence and absence of pine sawyer beetles. The proportion of third-stage dispersal juveniles in the total nematode population was negatively correlated with moisture content of the wood. Formation of nematode dauer juveniles was dependent on the presence of the pine sawyer beetle. Dauer juveniles were present in 3 of 315 wood samples taken from non-beetle-infested Scots pine bolts and 81 of 311 samples taken from beetle-infested bolts. Nematode densities were greater in wood samples taken adjacent to insect larvae, pupae, and teneral adults compared with samples taken from areas void of insect activity. Nematodes recovered from beetle larvae, pupae, and teneral adults were mostly fourth-stage dauer juveniles, although some third-stage dispersal juveniles were also recovered. Dauer juvenile density was highest on teneral adult beetles.     

Survival and Infectivity of Bursaphelenchus xylophilus in Wood Chip-Soil Mixtures

To determine the effect of soil environment on the life stages and total numbers of Bursaphelenchus xylophilus, nematode-infested wood chips alone and mixed with soil were incubated at 12 and 20 C. Nematodes were extracted at 2-week intervals for 12 weeks. Numbers of nematodes and percentage of third-stage dispersal larvae were greater at 12 C and in chips without soil. Percentage of juveniles of the propagative cycle was greater at 20 C and in chips with soil. Although B. xylophilus survived in chips with soil for 12 weeks, nematode numbers and life stage percentages changed little over time. To determine if B. xylophilus was capable of infecting wounded roots, infested and uninfested chips were mixed with soil in pots with white and Scots pine seedlings. Trees were maintained at 20 and 30 C and harvested at mortality or after 12 weeks. Only seedlings treated with infested chips contained nematodes. In field experiments, planted seedlings were mulched with infested chips to determine if nematodes would invade basal stem wounds. Among these trees, Scots pine was more susceptible than white or red pines to infection and mortality.     

Development of Bursaphelenchus xylophilus Populations in Wood Chips with Different Moisture Contents

Bags of Pinus strobus wood chips with moisture contents of 38, 92, 164, and 217% (oven dry weight) were inoculated with Bursaphelenchus xylophilus and incubated at 30 C in order to determine the effect of wood moisture on nematode population development. Nematodes were extracted after 2, 4, 8, and 12 weeks. Population levels were greatest in wood chips with a moisture content of 38% and decreased successively with each higher moisture content. In chips with the three lower moisture contents, populations peaked at 2 weeks, but at 217% moisture, they peaked at 8 weeks. By 12 weeks, nematode populations had declined in wood chips with 92 and 164% moisture contents. The fungi most frequently isolated from the wood chips were Alternaria, Fusarium, Gliocladium, Graphium, Penicillium, Trichoderma, and Mucorales.     

Detection and Identification of Bursaphelenchus Species with DNA Fingerprinting and Polymerase Chain Reaction

We have evaluated the potential of DNA-based methods to identify and differentiate Bursaphelenchus spp. and isolates. The isolation of a DNA probe, designated X14, and development of a DNA fingerprinting method for the identification and differentiation of Bursaphelenchus species and strains is described. Polymerase chain reaction (PCR) amplification of DNA isolated from Bursaphelenchus species using two primers derived from the sequence of the cloned repetitive DNA fragment X14 resulted in multiple band profiles. A 4-kb fragment thus amplified from B. xylophilus DNA was not amplified from B. mucronatus or B. fraudulentus DNA. In addition to this fragment, several other fragments are amplified from the three species. The banding patterns obtained allowed species identification and may have value in determining taxonomic affinities.     

Relationship of Bursaphelenchus xylophilus Population Density to Mortality of Pinus sylvestris

Seven-month-old Scots pine seedlings were inoculated with water or culture filtrate (controls), with 10,000, or 20,000 (experiment 1), and with 2,500 (experiment 2) Bursaphelenchus xylophilus B.C. isolate nematodes and maintained under defined experimental conditions. Controls did not develop pine wilt disease over a 2-month period. In experiment 1, less than 50% of the inoculum was recovered from the nematode-inoculated seedlings in the first 48 hours, after which the nematode population of both treatments increased exponentially resulting in pine death and approximately equal populations at 216 hours after inoculation. In the second experiment, plant mortality, which was always preceded by 2-3 days of chlorosis and associated stem vascular necrosis, first occurred 14 days after inoculation. The nematode population increased until about day 40 after inoculation and declined thereafter. Nematodes extracted from the roots 2 weeks after inoculation accounted for ca.15% of the total number of nematodes per pine. The study indicates that the rate of nematode reproduction is a factor in pine wilt disease. However, the lack of a linear correlation between the number of nematodes and the timing of pine mortality suggests that the timing of pine death may also depend on the location of nematode damage to the host tissue.     

The Loss of Genetic Diversity in Sichuan Taimen as Revealed by DNA Fingerprinting

Species endangerment often derives from the “endangerment” of genetic diversity, thus loss of genetic diversity is an important cause of species extinction. Since historical specimens were unavailable, previous studies mainly described the genetic diversity status in the current population rather than the loss of genetic variation over time. In this study, we collected samples during1998–1999 and obtained historical specimens from 1957 to 1958. Based on the two sets of fish, we determined the changes in genetic diversity of Sichuan taimen using DNA fingerprinting. The differences in genetic parameters between the present samples and historical taimens revealed their loss of genetic variation. As a result, the existing populations have lower viability, and proper management has to be implemented to preserve genetic diversity.     

Effect of Bursaphelenchus xylophilus on the Assimilation and Translocation of 14 C in Pinus sylvestris

The effect of wound, wound + water, wound + Bursaphelenchus xylophilus culture filtrate, or wound + lethal B. xylophilus doses on the assimilation and translocation of ¹⁴C by 8-month-old Pinus sylvestris seedlings was tested. In two separate experiments, pine seedlings were exposed to 28.35 μCi of ¹⁴CO₂ for 20 minutes below or above (to the pine shoot leader) the point of nematode inoculation. After 2 and 4 hours of dark adaptation, 80% ethanol soluble ¹⁴C tissue extracts were determined by liquid scintillation counting. Nematode infection significantly (P = 0.05) decreased ¹⁴C assimilation. Treatments translocated less than 6% of the total amount of the fixed ¹⁴C and translocation generally decreased with increasing size of nematode inoculum. However, infected pines translocated a greater proportion of the amount of ¹⁴C fixed per gram of exposed nematode-plant tissue than did the control pines. The lower levels of photoassimilate entering the plant system probably resulted in a reduced metabolic capacity in B. xylophilus-infected pine seedlings. The effect on photosynthesis could be one of the key factors leading to death of pines through starvation, and it is possible that it was preceded by an effect on related physiological processes such as water uptake.     

Effect of Phytotoxin from Nematode-induced Pinewilt on Bursaphelenchus xylophilus and Ceratocystis ips

A phytotoxic extract from Pinus sylvestris infected with Bursaphelenchus xylophilus inhibited growth of the blue-strain fungus Ceratocystis ips and caused temporary paralysis in vitro of B. xylophilus. Although the nematodes recovered from paralysis, final population size of B. xylophilus was suppressed by the toxin. Extracts from noninfected P. sylvestris affected neither the fungus nor the nematode.     

Pinewood Nematode,Bursaphelenchus xylophilus,Associated with Red Pine, Pinus resinosa,in Western Maryland

Red pines Pinus resinosa in Garrett and Allegany counties, Maryland, were examined during 1982-84 to determine distribution of the pinewood nematode, Bursaphelenchus xylophilus, within and among trees. Approximately 25-year-old (younger) and 47-year-old (older) trees were subdivided into the following categories: 1) trees with mostly green needles; 2) trees with mostly reddish-brown needles; 3) trees lacking needles but with bark intact; 4) trees lacking both needles and bark; and 5) trees with chlorotic, bleached-green needles. Bursaphelenchus xylophilus was found infecting 68% of younger red pines and 77% of older red pines. Nematodes were not evenly distributed in trees within any given tree decadence category or in trees of the same age. Nematodes were recovered from 20% of wood samples from trunks and primary and secondary branches in younger pines and from 15 % of older red pines. On the basis of tree decadence category, the highest incidence of infection in younger trees (31%) was in bleached-green needled trees (category 5), whereas in older trees the highest infection (25%) occurred in green needled trees (category 1). At both sites trunks were infected more often than branches.     

Genetic Heterogeneity of the Giant Tiger Shrimp (Penaeus monodon) in Thailand Revealed by RAPD and Mitochondrial DNA RFLP Analyses

Genetic diversity of the giant tiger shrimp (Penaeus monodon) collected from 5 areas, Chumphon and Trat (Gulf of Thailand), and Phangnga, Satun, and Trang (Andaman Sea), was examined by randomly amplified polymorphic DNA (RAPD) and mitochondrial DNA (16S ribosomal DNA and an intergenic COI-COII) polymorphism. A total of 53 polymorphic fragments from UBC299, UBC273, and UBC268 was consistently scored across all samples. From the respective primers 26, 32, and 30 genotypes were generated. A 260-bp RAPD fragment generated by the primer UBC268 was specifically observed in 95.8% of Trat P. monodon, suggesting that this RAPD could be used as a marker for comparing phenotypic performance of P. monodon from Trat and other geographic samples. In addition, 37 mtDNA composite haplotypes were observed from restriction analysis of the same P. monodon samples. High haplotype diversity (0.855) and nucleotide diversity (3.328%) of Thai P. monodon were observed. Population differentiation of P. monodon between the Andaman Sea and Gulf of Thailand was clearly illustrated by both techniques (P < .0001). Nevertheless, contradictory results on patterns of differentiation were observed between P. monodon within the Gulf of Thailand. Analysis of nuclear DNA polymorphism (RAPD) indicated a genetically significant difference between Chumphon and Trat (P < .0001), whereas mtDNA polymorphism did not show differentiation between these samples (P= .0497). Under the presumption of selective neutrality of these markers, biased female gene flow between Trat and Chumphon P. monodon may exist and be responsible for an anomalous differentiation pattern between these geographic samples. Received October 11, 2000; accepted March 5, 2001.