Search

ABSTRACT The quarantine nematode Bursaphelenchus xylophilus is a causative agent of the pine wilt disease. In Europe, the nematode is vectored by Monochamus galloprovincialis which is also a natural vector of the native, nonpathogenic B. mucronatus. Based on the established regulations, long‐term detection surveys of pine wood and vector insects for the presence of B. xylophilus are obligatory in all EU member countries. The main objective of the presented research was to use the real‐time PCR technique for the fast and precise detection, and identification of Bursaphelenchus nematodes associated with M. galloprovincialis ssp. pistor occurring in Poland. The research conducted on beetles collected in forest stands in Poland revealed exclusive presence of the native, nonpathogenic nematode B. mucronatus. At the beginning of the beetle flight season (June–July), the use of the real‐time PCR allowed detection of B. mucronatus, in the case of its physical presence in the body of the dissected M. galloprovincialis. However, at the end of the flight season (September–October), the DNA of B. mucronatus was also found in some beetles with no physical presence of nematodes in tracheas or under elytra. The univoltine biology of the beetle, gradual decline in dispersal juveniles to zero in/on the beetle body, and positive results of real‐time PCR reaction could indicate that in some of the beetles earlier loaded with nematodes, at the end of the season, only their remains were left after the live nematodes' departure during the vector's maturation feeding and/or egg laying. The obtained results confirm extremely high sensitivity of the real‐time PCR method used in detection and identification of both nematode species which may simultaneously occur in M. galloprovincialis beetles. It allows for precise detection of the nematodes based on their remains left in/on the vector insects. [ABSTRACT FROM AUTHOR]

Bursaphelenchus mucronatus Mamiya & Enda, 1979 is a wood nematode widely distributed over Palearctic coniferous forests. It has two subspecies, that is, B. mucronatus mucronatus and B. mucronatus kolymensis, which present molecularly different East Asian and European genotypes, respectively. The European subspecies is found mainly in Europe and Siberia, while the East Asian subspecies occupies mostly Eastern regions of Asia. However, local isolates of both subspecies have been occasionally reported from various localities in Europe and Asia. Our field isolation and molecular (DNA ITS‐RFLP) identification of 21 recently collected populations of B. mucronatus revealed only 4 isolates representing clearly East Asian or European genotypes, while the remaining 17 isolates showed intermediate genotypes with electrophoretic band characters of both the above types. Further individual crossbreeding, in vitro, of nematodes representing European, East Asian, and intermediate genotypes provided experimental evidence for the process of intraspecific hybridization between both subspecies, spontaneously taking place in the forest. Such a widening of the B. mucronatus genetic variation may have its effect on a range of reported in the literature direct interactions between this native, nonpathogenic nematode species and the genetically similar, causative agent of the pine wilt disease B. xylophilus which can colonize the same host trees, use the same insect vectors, compete, and crossbreed inter‐specifically. [ABSTRACT FROM AUTHOR]

The pinewood nematode (PWN), Bursaphelenchus xylophilus, is the causal agent of pine wilt disease (PWD). This nematode is considered to be an indigenous to North America and was introduced to Japan in the late 19th century. Subsequently, it has spread throughout Japan and in many other countries, China, Taiwan, and South Korea. In 1999, B. xylophilus was discovered in Portugal, and in 2008 in Spain. So far the studies have revealed that the pathogenicity of B. xylophilus varies between different isolates. The conducted study compared the pathogenicity of five isolates of B. xylophilus, originating from different parts of Japan, to 3-year-old Pinus sylvestris, and their ability to reproduce in the seedlings. The results revealed diverse virulence of B. xylophilus resulting in plant mortality. Three isolates S10, Ka4, and T4 caused 100% mortality of plants within three months while at the same time, the other two isolates, C14-5 and OKD-1 did not cause any disease symptoms on plants. After seven months, some dieback occurred on two seedlings, but similar symptoms were also found on the control plant. Moreover, a significant positive correlation was found between nematode virulence and the number of nematodes reproducing on pine seedlings.

Copyright of Progress in Plant Protection is the property of Institute of Plant Protection and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

The process of inter‐specific hybridization between the invasive, highly pathogenic pinewood nematode, Bursaphelenchus xylophilus and the Eurasian‐native, relatively harmless B. mucronatus has been examined with the aid of the B. xylophilus Roller (Bxy‐rol(tom3) mutation (Tomalak & Filipiak, 2019) as a morphological marker, in the breeding protocol. Validity of the morphologically based selection of the inter‐specific hybrids has been confirmed by examination of individual nematodes with the multiplex PCR technique with specific primers (Filipiak et al., 2017). The research showed that hybridization between both species was possible in F1 and to some extent in subsequent offspring generations. In the general body plan, the hybrids resembled the wild‐type parental species but differed in various morphological/anatomical details. The changes ranged from seemingly indistinct to heavily distorted or occasional missing of particular structures. The most affected were spicules, female tails, the development of gonads and the position of the excretory pore. The hybridization led to various distortions of mating, gametogenesis, embryogenesis and juvenile viability, and development in F1, F2 and subsequent generation offspring, which severely affected the nematode reproductive potential. Potential consequences of the spontaneous inter‐specific hybridization between both species are discussed. [ABSTRACT FROM AUTHOR]

Bursaphelenchus xylophilus is an emerging pathogenic nematode that is responsible for a devastating epidemic of pine wilt disease worldwide, causing severe ecological damage and economic losses to forestry. Two forms of this nematode have been reported, i.e., with strong and weak virulence, commonly referred as virulent and avirulent strains. However, the pathogenicity-related genes of B. xylophilus are not sufficiently characterized. In this study, to find pathogenesis related genes we re-sequenced and compared genomes of two virulent and two avirulent populations. We identified genes affected by genomic variation, and functional annotation of those genes indicated that some of them might play potential roles in pathogenesis. The performed analysis showed that both avirulent populations differed from the virulent ones by 1576 genes with high impact variants. Demonstration of genetic differences between virulent and avirulent strains will provide effective methods to distinguish these two nematode virulence forms at the molecular level. The reported results provide basic information that can facilitate development of a better diagnosis for B. xylophilus isolates/strains which present different levels of virulence and better understanding of the molecular mechanism involved in the development of the PWD. [ABSTRACT FROM AUTHOR]

Summary: The first spontaneous morphological/anatomical and behavioural mutation Bxy-rol (tom3) has been identified and characterised in the pine wood nematode, Bursaphelenchus xylophilus. The mutation is recessive to its wild-type counterpart. The mutant phenotype designated as Roller (Rol) is distinctive for the helical twist of the whole body around its longitudinal axis. The twist of the nematode body wall cuticle is followed by a similar twist of hypodermis, muscles and other organs and cuticular structures. These morphological and anatomical changes are responsible for the altered pattern of the nematode movement. Besides these changes, the Bxy-rol (tom3) mutation does not visibly affect any other essential character or function of B. xylophilus. Both wild-type and Rol nematodes present similar morphometrics, life cycle length, and reproductive potential. The Rol mutant easily cross-bred with individuals from any other tested strains/isolates of B. xylophilus and produced viable intra-specific hybrid offspring in vitro , on B. cinerea /potato dextrose agar plates. The mutants also seem to be well adapted to the nematode's natural environment in pine wood. They dispersed inside pine seedlings, successfully mated, produced stable and fully viable offspring in this environment, and were as pathogenic to the host tree as the wild-type B. xylophilus. Potential of the characterised mutation as a marker for direct comparative study on strain/species reproductive compatibility, competition for space, food and vector insect, and for intra- and inter-specific hybridisation of B. xylophilus and B. mucronatus , is discussed. [ABSTRACT FROM AUTHOR]

Differentiation between Bursaphelenchus xylophilus and other related, non-pathogenic species can be ambiguous when based exclusively on morphological characters. The morphology of B. mucronatus and B. fraudulentus most closely resembles that of B. xylophilus. Moreover, all of these nematodes are found in both Asia and Europe and can colonise various species of pine. Therefore, for phytosanitary purposes it is necessary to identify the three species precisely and rapidly. We report the results of a multiplex PCR that utilises four primers to identify and discriminate the three Bursaphelenchus species simultaneously. The multiplex PCR yielded DNA fragments of 767, 305 and 132 bp, for B. xylophilus, B. mucronatus and B. fraudulentus, respectively. This primer combination has produced reliable results in multiplex PCR assays with a number of different populations of the listed species, and no cross-reactions were observed with other Bursaphelenchus species. The described approach is simple, reliable and cheaper than other molecular methods presently used for simultaneous identification of the above three species within the xylophilus group. [ABSTRACT FROM AUTHOR]

The real-time PCR-HRM analysis was developed for the detection and discrimination of the quarantine nematode Bursaphelenchus xylophilus and Bursaphelenchus mucronatus. A set of primers was designed to target the ITS region of rDNA. The results have demonstrated that this analysis is a valuable tool for differentiation of these both species. [ABSTRACT FROM AUTHOR]

Bursaphelenchus trypophloei sp. n. is described from weakened and dead twigs of living or recently fallen aspen trees, Populus tremula, in Poland. All propagative stages of the nematode were present in larval galleries of a bark beetle, Trypophloeus asperatus, and its dauer juveniles could be found in the haemocoel of older larvae, pupae and adult beetles. Characteristic morphology of the male spicules with distinct cucullus, extended anterior vulval lip, lateral fields with four incisures, and number and arrangement of male caudal papillae indicate that B. trypophloei sp. n. is closely related to the xylophilus group. This relation has been further confirmed by DNA sequencing and phylogenetic analysis of the 28S and ITS-1 rDNA region. Bursaphelenchus trypophloei sp. n. can be separated from other species in the xylophilus group by the morphology of spicules which have a shorter capitulum and unique rostrum which is pointed somewhat anteriorly, relatively thick vulval flap, which is straight, parallel to the body long axis or bent towards the body wall at its distal end, and other morphological and morphometric characters, i.e., shape of female tail, position of excretory pore at or posterior to nerve ring, spicule length of 25.7 (23.1-28.0) μm (as measured along arc) and shape, moderate length, i.e., L = 702 (603-946) μm in females and L = 679 (543-828) μm in males, and slender body (a = 39.2 (33.0-45.2) and 40.9 (32.0-46.4) in female and male, respectively). The taxonomic separation of the new species is also confirmed by the unique molecular profile of the ITS region (ITS-RFLP). Full reproductive incompatibility of B. trypophloei sp. n. with B. xylophilus, B. mucronatus, B. fraudulentus, B. populi and B. doui has been demonstrated in in vitro diallelic cross-breeding. The newly described nematode revealed limited ability to develop and reproduce on Botrytis cinerea cultures although it produced large populations on laboratory cultures of Cytospora chrysosperma - a fungus naturally associated with galleries of the nematode vector, T. asperatus. [ABSTRACT FROM AUTHOR]

The article examines the sensitivity of the species-specific polymerase chain reaction primers for Bursaphelenchus fraudulentus. Results of the DNA sequencing showed that the amplified fragment of Bursaphelenchus fraudulentus DNA has complete regions of ITS-1, 5.8S and ITS-2, along with parts of the 18S and 28S regions. Researchers found that Bursaphelenchus fraudulentus-specific primer was not redycd when using a combination of Bursaphelenchus fraudulentus, Bursaphelenchus xylophilus and Bursaphelenchus mucronatus DNA samples.

Copyright of Progress in Plant Protection is the property of Institute of Plant Protection and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)