Pinewood Nematode Species Complex: Interbreeding Potential and Chromosome Number

Interbreeding potential, chromosome number, and host range were compared among several isolates and species of Bursaphelenchus from diverse geographic areas. Some isolates from North America, Japan, and France had a wide-ranging interbreeding potential, whereas others were restricted in their potential to hybridize with other isolates. Although interbreeding occurred in the laboratory between some "M" and "R" forms of B. xylophilus, interbreeding of B. xylophilus and B. mucronatus was rare. The hybrids had the pathogenicity of the parent with the broader host range. This fact suggests that virulence may be inherited as a dominant character or that increased virulence may have resulted from differences in hybrid vigor. The haploid chromosome number of the different isolates separated the isolates into three groups and distinguished B. xylophilus from B. mucronatus. The findings suggest that the pinewood nematode species complex consists of sibling species that have evolved by reproductive isolation, that the French isolate is a new species, and that B. xylophilus and B. mucronatus have evolved from a common ancestor.     

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.     

Inhibitory Effect of Bursaphelenchus mucronatus (Nematoda: Aphelenchoididae) on B. xylophilus Boarding Adult Monochamus alternatus (Coleoptera: Cerambycidae)

Inhibitory effects of Bursaphelenchus mucronatus on the number of B. xylophilus carried by an adult Monochamus alternatus were investigated using artificial pupal chambers. When pupal chambers were infested with either B. xylophilus or B. mucronatus, the load of B. xylophilus onto the beetle was greater (P < 0.001) than that of B. mucronatus. However, within the pupal chamber there was no difference in the abundance of the third-stage dispersal juveniles, which would molt to the fourth-stage dispersal juveniles to board beetles. The nematode load on beetles that emerged from pupal chambers infested with both Bursaphelenchus species was smaller (P = 0.015) than that of beetles with B. xylophilus alone but greater (P < 0.001) than that of beetles with B. mucronatus alone, suggesting an inhibitory effect of B. mucronatus. As a result of this study, the rate of inhibition of B. mucronatus on molting of third-stage dispersal juveniles of B. xylophilus to fourth-stage dispersal juveniles was 0.65, which resulted in great inhibition on boarding beetles at a rate of 0.7.     

Change in Water Status of Pinus thunbergii Parl. Inoculated with Species of Bursaphelenchus

Maximum and minimum xylem pressure potentials of needles were measured to evaluate water status of Pinus thunbergii Parl. after inoculation with the virulent or avirulent populations of Bursaphelenchus xylophilus or B. mucronatus. In virulent B. xylophilus-inoculated pines, the water status changed abruptly and needle chlorosis occurred by day 29 after inoculation. Similar changes were not seen in B. mucronatus-inoculated and uninoculated control pines. Oleoresin flow ceased in virulent B. xylophilus-inoculated pines. Avirulent B. xylophilus-inoculated pines responded very little to nematode invasion by a slight decrease in oleoresin flow. Oleoresin flow did not vary in B. mucronatus-inoculated and uninoculated control pines. A decrease in soil water potential below field capacity seemed to accelerate the development of pine wilt disease.     

Temperature-mediated Behavioral Relationships in Bursaphelenchus xylophilus, B. mucronatus,and Their Hybrids

The influence of temperature on reproduction and movement was examined for seven geographic isolates of Bursaphelenchus xylophilus, three of B. mucronatus, and two of their interspecific hybrids. All nematode isolates tended to be more active and fecund the higher the temperature, with the isolates of B. xylophilus reaching a reproductive peak at higher temperatures than isolates of B. mucronatus. Most isolates of B. xylophilus and B. mucronatus did not produce significantly more progeny at higher male-to-female ratios. The interspecific hybrids appear to possess temperature-related characteristics of either B. xylophilus or both of the parents.     

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.     

Comparison of complete mitochondrial genomes of pine wilt nematode Bursaphelenchus xylophilus and Bursaphelenchus mucronatus (Nematoda: Aphelenchoidea) and development of a molecular tool for species identification

We determined the complete mitochondrial genome sequences for Bursaphelenchus mucronatus, one species of pinewood nematode. The genome is a circular-DNA molecule of 14,583 bp (195 bp smaller than its congener Bursaphelenchus xylophilus) and contains 12 protein-coding genes (lacking atp8), 22 tRNA genes, and 2 rRNA genes encoded in the same direction, consistent with most other nematodes. Based on sequence comparison of mtDNA genomes, we developed a PCR-based molecular assay to differentiate B. xylophilus (highly pathogenic) and B. mucronatus (relatively less virulent) using species-specific primers. The molecular identification system employs multiplex-PCR and is very effective and reliable for discriminating these Bursaphelenchus species, which are economically important, but difficult to distinguish based on morphology. The comparison of the mitochondrial genomes and molecular identification system of the two species of Bursaphelenchus spp. should provide a rich source of genetic information to support the effective control and management (quarantine) of the pine wilt disease caused by pinewood nematodes.     

Cryopreservation of the Pinewood Nematode,Bursaphelenchus spp.

Populations of three isolates of Bursaphelenchus xylophilus, the pinewood nematode, and one of B. mucronatus were treated with three cryoprotectants at -70 C for 24 hours followed by deep freezing at -180 C in liquid nitrogen for different periods of time. A solution of 15% glycerol, 35% buffer S, and 50% M9, or 1% aqueous solution of dimethylsulfoxide (DMSO), or a mixture of 60% M9 and 40% S buffer were used as cryoprotectants. A significantly larger number of juveniles than adults survived deep freezing. Significantly more nematodes were motile after cryopreservation in the 15% glycerol-S-M9 soludon than in the M9-S buffer solution or the DMSO aqueous solution. When cryopreserved nematodes that had been treated with glycerol solution were plated onto Botrytis cinerea, they reproduced rapidly over several generations. Cryopreserved nematodes were as pathogenic as untreated nematodes to Scots pines.     

Cytotype diversity and genome size variation in eastern Asian polyploid Cardamine (Brassicaceae) species

Background and Aims

Intraspecific ploidy-level variation is an important aspect of a species'' genetic make-up, which may lend insight into its evolutionary history and future potential. The present study explores this phenomenon in a group of eastern Asian Cardamine species.

Methods

Plant material was sampled from 59 localities in Japan and Korea, which were used in karyological (chromosome counting) and flow cytometric analyses. The absolute nuclear DNA content (in pg) was measured using propidium iodide and the relative nuclear DNA content (in arbitrary units) was measured using 4,6-diamidino-2-phenylindole fluorochrome.

Key Results

Substantial cytotype diversity was found, with strikingly different distribution patterns between the species. Two cytotypes were found in C. torrentis sensu lato (4x and 8x, in C. valida and C. torrentis sensu stricto, respectively), which displays a north–south geographical pattern in Japan. Hypotheses regarding their origin and colonization history in the Japanese archipelago are discussed. In Korean C. amaraeiformis, only tetraploids were found, and these populations may in fact belong to C. valida. C. yezoensis was found to harbour as many as six cytotypes in Japan, ranging from hexa- to dodecaploids. Ploidy levels do not show any obvious geographical pattern; populations with mixed ploidy levels, containing two to four cytotypes, are frequently observed throughout the range. C. schinziana, an endemic of Hokkaido, has hexa- and octoploid populations. Previous chromosome records are also revised, showing that they are largely based on misidentified material or misinterpreted names.

Conclusions

Sampling of multiple populations and utilization of the efficient flow cytometric approach allowed the detection of large-scale variation in ploidy levels and genome size variation attributable to aneuploidy. These data will be essential in further phylogenetic and evolutionary studies.     

New Insights into the Phylogeny and Worldwide Dispersion of Two Closely Related Nematode Species,Bursaphelenchus xylophilus and Bursaphelenchus mucronatus

The pinewood nematode, Bursaphelenchus xylophilus, is one of the greatest threats to coniferous forests worldwide, causing severe ecological damage and economic loss. The biology of B. xylophilus is similar to that of its closest relative, B. mucronatus, as both species share food resources and insect vectors, and have very similar morphological characteristics, although little pathogenicity to conifers has been associated with B. mucronatus. Using both nuclear and mitochondrial DNA markers, we show that B. xylophilus and B. mucronatus form distinct phylogenetic groups with contrasting phylogeographic patterns. B. xylophilus presents lower levels of intraspecific diversity than B. mucronatus, as expected for a species that evolved relatively recently through geographical or reproductive isolation. Genetic diversity was particularly low in recently colonised areas, such as in southwestern Europe. By contrast, B. mucronatus displays high levels of genetic diversity and two well-differentiated clades in both mitochondrial and nuclear DNA phylogenies. The lack of correlation between genetic and geographic distances in B. mucronatus suggests intense gene flow among distant regions, a phenomenon that may have remained unnoticed due to the reduced pathogenicity of the species. Overall, our findings suggest that B. xylophilus and B. mucronatus have different demographic histories despite their morphological resemblance and ecological overlap. These results suggest that Bursaphelenchus species are a valuable model for understanding the dispersion of invasive species and the risks posed to native biodiversity and ecosystems.     

Nematicidal Activity of Cassia and Cinnamon Oil Compounds and Related Compounds toward Bursaphelenchus xylophilus (Nematoda: Parasitaphelenchidae)

The nematicidal activity of two cassia, Cinnamomum cassia, oils (Especial and true), four cinnamon, Cinnamomum zey-lanicum, oils (technical, #500, bark and green leaf), and their compounds (e.g., trans-cinnamaldehyde and trans-cinnamic acid) toward adult Bursaphelenchus xylophilus was examined by a direct contact bioassay. Results were compared with those of 34 related compounds. As judged by 24-hour LC₅₀ values, two cassia oils (0.084–0.085 mg/ml) and four cinnamon oils (0.064–0.113 mg/ml) were toxic toward adult B. xylophilus. Of 45 test compounds, trans-cinnamaldehyde (0.061 mg/ml) was the most active nematicide, followed by ethyl cinnamate, α-methyl-trans-cinnamaldehyde, methyl cinnamate and allyl cinnamate (0.114–0.195 mg/ml). Potent nematicidal activity was also observed with 4-methoxycinnamonitrile, trans-4-methoxycinnamaldehyde, trans-2-methoxy-cinnamaldehyde, ethyl α-cyanocinnamate, cinnamonitrile and cinnamyl bromide (0.224–0.502 mg/ml). Structure-activity relationships indicate that structural characteristics, such as types of functional groups, saturation and carbon skeleton, appear to play a role in determining the toxicities to adult B. xylophilus. Cassia and cinnamon oils and test compounds described merit further study as potential nematicides or leads for the control of pine wilt disease caused by B. xylophilus.     

Nemtaode-Vector Relationships in the Pine Wilt Disease System

Pinewood nematode, Bursaphelenchus xylophilus, is the causal agent of pine wilt disease in North America and Japan. Dispersal stage dauer larvae are transported to new host trees on the body surface and within the tracheal system of several beetle species. Worldwide, 21 species of Cerambycidae, 1 genus of Buprestidae, and 2 species of Curculionidae are known to carry pinewood nematode dauer larvae upon emerging from nematode-infested trees. Five species of cerambycids in the genus Monochamus are known to transmit dauer larvae to new host trees, four North American species and one Japanese species. Primary transmission to healthy trees occurs through beetle feeding wounds on young branches. Secondary transmission to stressed trees or recently cut logs occurs through Monochamus oviposition sites.     

Effect of Monochamus carolinensis on Bursaphelenchus xylophilus Dispersal Stage Formation

Bursaphelenchus xylophilus and its insect vector, Monochamus carolinensis, both develop within rapidly degrading xylem tissue of dying or recently cut trees of Pinus spp. The influence of Monochamus development on B. xylophilus dispersal stage formation was investigated. Nearly all nematodes extracted from wood surrounding beetle galleries were third-stage dispersal juveniles (J3). Formation of fourth-stage dispersal juveniles (J4) occurred almost exclusively in the presence of M. carolinensis late pupae and callow adults. This pattern was observed with live insects in naturally formed galleries, diet-reared insects in artificial galleries, and pulverized insects in artificial galleries. The molt from J3 to J4 appeared to be related to adult eclosion in M. carolinensis. We hypothesize that a genus-specific substance(s) associated with Monochamus adult eclosion ensures the Monochamus-B. xylophilus association.     

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.     

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.     

Random Amplified Polymorphic DNA Analysis of Heterodera cruciferae and H. schachtii populations

Heterodera schachtii and H. cruciferae are sympatric in California and frequently occur in the same field upon the same host. We have investigated the use of polymerase chain reaction (PCR) amplification of nematode DNA sequences to differentiate H. schachtii and H. cruciferae and to assess genetic variability within each species. Single, random oligodeoxyribonucleotide primers were used to generate PCR-amplified fragments, termed RAPD (random amplified polymorphic DNA) markers, from genomic DNA of each species. Each of 19 different random primers yielded from 2 to 12 fragments whose size ranged from 200 to 1,500 bp. Reproducible differences in fragment patterns allowed differentiation of the two species with each primer. Similarities and differences among six different geographic populations of H. schachtii were detected. The potential application of RAPD analysis to relationships among nematode populations was assessed through cluster analysis of these six different populations, with 78 scorable markers from 10 different random primers. DNA from single cysts was successfully amplified, and genetic variability was revealed within geographic populations. The use of RAPD markers to assess genetic variability is a simple, reproducible technique that does not require radioisotopes. This powerful new technique can be used as a diagnostic tool and should have broad application in nematology.     

Resistance of Auto- and Allotetraploid Triticeae Species and Accessions to Meloidogyne chitwoodi based on Genome Composition

The Columbia root-knot nematode Meloidogyne chitwoodi parasitizes several plant species, including grasses that have been developed for semiarid environments, and substantially reduces the productivity of cereals and the longevity of perennial grasses growing under semiarid conditions throughout the intermountain region. Thirty-two auto- and allotetraploid (2n = 28) taxa in the perennial Triticeae were evaluated as possible sources of resistance to M. chitwoodi. Low levels of root galling were observed on roots of all accessions; root-gall indices ranged from 0 (no galls) to 1.95 in the grasses compared to 4.67 for the susceptible ''Ranger'' alfalfa check on a scale of 1 to 6. Even though the gall ratings were low, significant (P < 0.01) differences among accessions of the same species, among species, and among genera with different genomes were observed. Within the reproductive indices, which ranged from 0.01 to 1.20 in the grasses compared to 65.38 for the alfalfa check, there was no difference among genera with different genomes and accessions within the same species and genome; however, there was a significant (P < 0.05) difference among species with the same genomes. This variation can be traced to Thinopyrum nodosum (Jaaska-19), which was the only accession with a reproductive factor greater than 1.00. Based on the data, all auto- and allotetraploids are considered resistant to M. chitwoodi.     

Carbohydrate Concentration in Pine as Affected by Inoculation with Bursaphelenchus xylophilus

Pines responded to inoculation with Bursaphelenchus xylophilus by changes in reducing and nonreducing carbohydrate concentrations dependent on the pine species and the pathotype of B. xylophilus with which the trees were inoculated. Carbohydrate concentrations, in compatible pine-nematode pathotype combinations, decreased initially after inoculation and then increased slightly before decreasing to approximately 10% of the control levels as the seedlings wilted. In compatible nematode pathotype-pine species combinations, carbohydrate concentrations decreased and then increased as the nematode population densities declined.     

Current Research on the Major Nematode Problems in Japan

Among important nematode species occurring in Japan, current research achievements with the following four nematodes are reviewed: 1) Soybean cyst nematode (SCN), Heterodera glycines - breeding for resistance, race determination, association with Cephalosporium gregatum in azuki bean disease, and isolation of hatching stimulant. 2) Potato-cyst nematode (PCN), Globodera rostochiensis - pathotype determination (Ro 1), breeding for resistance, and control recommendations. 3) Pinewood nematode (PWN), Bursaphelenchus xylophilus - primary pathogen in pine wilt disease, life cycle exhibiting a typical symbiosis with Japanese pine sawyer, Monochamus alternatus, and project for control. 4) Rice root nematodes (RRN), Hirschmanniella imamuri and H. oryzae - distribution of species, population levels in roots, and role of these nematodes in rice culture.     

Specific and Functional Diversity of Endophytic Bacteria from Pine Wood Nematode Bursaphelenchus Xylophilus with Different Virulence

Pine wilt disease (PWD) caused by the pine wood nematode (PWN), Bursaphelenchus xylophilus, is one of the most devastating diseases of Pinus spp. The PWN was therefore listed as one of the most dangerous forest pests in China meriting quarantine. Virulence of the PWN is closely linked with the spread of PWD. However, main factors responsible for the virulence of PWNs are still unclear. Recently epiphytic bacteria carried by PWNs have drawn much attention. But little is known about the relationship between endophytic bacteria and virulence of B. xylophilus. In this research, virulence of ten strains of B. xylophilus from different geographical areas in six provinces of China and four pine species were tested with 2-year-old seedlings of Pinus thunbergii. Endophytic bacteria were isolated from PWNs with different virulence to investigate the relationship between the bacteria and PWN virulence. Meanwhile, the carbon metabolism of endophytic bacteria from highly and low virulent B. xylophilus was analyzed using Biolog plates (ECO). The results indicated that ten strains of PWNs showed a wide range of virulence. Simultaneously, endophytic bacteria were isolated from 90% of the B. xylophilus strains. The dominant endophytic bacteria in the nematodes were identified as species of Stenotrophomonas, Achromobacter, Ewingella, Leifsonia, Rhizobium, and Pseudomonas using molecular and biochemical methods. Moreover, S. maltophilia, and A. xylosoxidans subsp. xylosoxidans were the predominant strains. Most of the strains (80%) from P. massoniana contained either S. maltophilia, A. xylosoxidans, or both species. There was a difference between the abilities of the endophytic bacteria to utilize carbon sources. Endophytic bacteria from highly virulent B. xylophilus had a relatively high utilization rate of carbohydrate and carboxylic acids, while bacteria from low virulent B. xylophilus made better use of amino acids. In conclusion, endophytic bacteria widely exist in B. xylophilus from different pines and areas; and B. xylophilus strains with different virulence possessed various endophytic bacteria and diverse carbon metabolism which suggested that the endophytic bacteria species and carbon metabolism might be related with the B. xylophilus virulence.