Hyerim Han

Comparative analysis of complete mitochondrial genome sequences confirms independent origins of plant-parasitic nematodes

BackgroundThe nematode infraorder Tylenchomorpha (Class Chromadorea) includes plant parasites that are of agricultural and economic importance, as well as insect-associates and fungal feeding species. Among tylenchomorph plant parasites, members of the superfamily Tylenchoidea, such as root-knot nematodes, have great impact on agriculture. Of the five superfamilies within Tylenchomorpha, one (Aphelenchoidea) includes mainly fungal-feeding species, but also some damaging plant pathogens, including certain Bursaphelenchus spp. The evolutionary relationships of tylenchoid and aphelenchoid nematodes have been disputed based on classical morphological features and molecular data. For example, similarities in the structure of the stomatostylet suggested a common evolutionary origin. In contrast, phylogenetic hypotheses based on nuclear SSU ribosomal DNA sequences have revealed paraphyly of Aphelenchoidea, with, for example, fungal-feeding Aphelenchus spp. within Tylenchomorpha, but Bursaphelenchus and Aphelenchoides spp. more closely related to infraorder Panagrolaimomorpha. We investigated phylogenetic relationships of plant-parasitic tylenchoid and aphelenchoid species in the context of other chromadorean nematodes based on comparative analysis of complete mitochondrial genome data, including two newly sequenced genomes from Bursaphelenchus xylophilus (Aphelenchoidea) and Pratylenchus vulnus (Tylenchoidea).ResultsThe complete mitochondrial genomes of B. xylophilus and P. vulnus are 14,778 bp and 21,656 bp, respectively, and identical to all other chromadorean nematode mtDNAs in that they contain 36 genes (lacking atp8) encoded in the same direction. Their mitochondrial protein-coding genes are biased toward use of amino acids encoded by T-rich codons, resulting in high A+T richness. Phylogenetic analyses of both nucleotide and amino acid sequence datasets using maximum likelihood and Bayesian methods did not support B. xylophilus as most closely related to Tylenchomorpha (Tylenchoidea). Instead, B. xylophilus, was nested within a strongly supported clade consisting of species from infraorders Rhabditomorpha, Panagrolaimomorpha, Diplogasteromorpha, and Ascaridomorpha. The clade containing sampled Tylenchoidea (P. vulnus, H. glycines, and R. similis) was sister to all analyzed chromadoreans. Comparison of gene arrangement data was also consistent with the phylogenetic relationships as inferred from sequence data. Alternative tree topologies depicting a monophyletic grouping of B. xylophilus (Aphelenchoidea) plus Tylenchoidea, Tylenchoidea plus Diplogasteromorpha (Pristionchus pacificus), or B. xylophilus plus Diplogasteromorpha were significantly worse interpretations of the mtDNA data.ConclusionsPhylogenetic trees inferred from nucleotide and amino acid sequences of mtDNA coding genes are in agreement that B. xylophilus (the single representative of Aphelenchoidea) is not closely related to Tylenchoidea, indicating that these two groups of plant parasites do not share an exclusive most recent common ancestor, and that certain morphological similarities between these stylet-bearing nematodes must result from convergent evolution. In addition, the exceptionally large mtDNA genome size of P. vulnus, which is the largest among chromadorean nematode mtDNAs sequenced to date, results from lengthy repeated segments in non-coding regions.

Microsatellite variation in the pinewood nematode, Bursaphelenchus xylophilus (Steiner and Buhrer) Nickle in South Korea

The pinewood nematode Bursaphelenchus xylophilus is the causative agent of pine wilt disease, which has caused heavy economic losses to the South Korean forest industry. In this study, we investigated the genetic variation among South Korean pinewood nematodes using newly developed microsatellite loci. In order to ensure sufficient templates for the amplification of multiple loci required for individual identification, we employed an amplifying step of restricted fragments during the microsatellite development procedure. We found atypical genetic patterns in this non-native pest species: high allelic diversity and population structure. The large number of alleles may be the result of continuous and/or large-scaled introduction, which apparently went unnoticed before the first official report of pine wilt disease in Korea in 1998, or may come from gene pools of closely related species through genetic introgression after hybridization. Ecological properties of this species, such as a vector-mediated life cycle, may have contributed to its population structure, which may be enhanced by governmental efforts to prevent dispersal of this disease. As a geographic population structure was not observed, geographic patterns of genetic variation appear to be more affected by anthropogenic mediation than by natural dispersion through vector insects. And genotypes of Korean populations were compared to genotypes found in neighboring countries such as China and Japan.

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.

PCR-RFLP Identification of Three Major Meloidogyne Species in Korea

Abstract Partial mitochondrial DNA from single female or second stage juvenile (J2) of root-knot nematodes was amplified by PCR (polymerase chain reaction), and the further analysis by RFLP (restriction fragment length polymorphism) provided discriminatory profiles useful for three major Meloidogyne species, M. arenaria, M. incognita , and M. hapla , in Korea. The nematode DNA was extracted individually in nuclease-free water. The sizes of PCR product (1.7 kb and 500 bp) and restriction patterns obtained from single female nematodes were consistent with the results from single J2 within the same species. M. hapla was easily differentiated from the two other root-knot nematode species by the size of the PCR products. A fragment of 500 bp was generated from M. hapla , while M. arenaria , and M incognita produced an 1.7 kb fragment in PCR amplification. M. arenaria could be distinguished from M. incognita by analysis of restriction enzyme digestion with Hinf I or Alu I. Hinf I had no digestion site in mitochondrial DNA of M. arenaria; however, it generated 1.3 kb and 400 bp fragments in M. incognita. Alu I digestion resulted in 1 kb, 460 bp, and 250 bp fragments in M. arenaria , but showed different digestion patterns in M. incognita by generating 800 bp, 460 bp, 250 bp, and 150 bp fragments.

Comparison in disease development and gas exchange rate of Pinus densiflora seedlings artificially inoculated with Bursaphelenchus xylophilus and B. mucronatus

Four‐year‐old seedlings of Pinus densiflora were inoculated with a suspension of B. xylophilus, or B. mucronatus adjusted to 3,000 nematodes per 50 μL sterilized distilled water in a greenhouse on July 21, 2008 to evaluate initial symptom development and the changes of gas exchange rate. B. xylophilus and B. mucronatus were distinguished by four restriction enzymes except Rsa I, and the result of ITS‐RFLP of B. xylophilus used in this experiment was completely matched to that of pine wood nematode reported previously. Needle dehydration and subsequent yellowing were observed in all seedlings inoculated with B. xylophilus isolate while the appearance of the seedlings was normal in all seedlings inoculated with B. mucronatus and control. Needle dehydration was observed in most seedlings inoculated with B. xylophilus between 2 and 3 weeks after inoculation. In seedlings inoculated with B. xylophilus, continuous decrease in photosynthetic rate was observed after 6 days of inoculation. Photosynthetic rate decreased more markedly after 12 days of inoculation when external symptoms appeared in most seedlings, and ceased almost completely 19 days after inoculation. Photosynthetic decline in seedlings inoculated with B. mucronatus was only observed at 19 days after inoculation but recovered above control level after 25 days of inoculation. We found that disease development and the changes of gas exchange rate in the seedlings of Pinus densiflora inoculated with B. xylophilus were not observed on those inoculated with B. mucronatus.

Amplified fragment length polymorphism analysis and genetic variation of the pinewood nematode Bursaphelenchus xylophilus in South Korea

Abstract The pinewood nematode Bursaphelenchus xylophilus causes pine wilt disease and is a serious economic concern for the forest industry of South Korea. To achieve effective control with limited resources, it is necessary to clarify the transmission routes and mechanisms of dispersal of this organism. Highly polymorphic and easy-to-use molecular markers can be used for investigating this aspect. In this study, we evaluated the usefulness of amplified fragment length polymorphisms (AFLPs) for investigating the genetic variations of B. xylophilus and related individuals from China, Japan, and South Korea. The AFLP patterns obtained in our study were similar to the microsatellite patterns reported in a previous study; our AFLP patterns indicated high genetic variability and cryptic genetic structure, but did not indicate any peculiar geographic structure. Moreover, the genetic distances between individuals suggested that the Korean population was affected to a greater extent by the Chinese population than the Japanese population. Further, the gene flow among the related species appeared to be limited; however, there may be also the possibility of genetic introgression among species. These results confirm the usefulness of AFLPs for understanding the epidemiology of pine wilt disease, thereby contributing to the effective control of this disease.

Molecular biological characterization of bursaphelenchus xylophilus and bursaphelenchus mucronatus in Korea

Pine wood nematode (PWN), Bursaphelenchus xylophilus, is a causal organism to induce pine wilt disease (PWD) in many varieties of pine trees. PWD was first introduced to Korea in 1988 but, the damage has been dramatically increased since 2000. Recently PWD is newly reported in Korean pine tree (Pinus koraien‐sis) and is considered one of the most important forest diseases in Korea. Fifteen isolates of B. xylophilus, 2 isolates of B. mucronatus, and 6 unidentified Bursaphelenchus spp. were collected from different geographical locations and hosts in Korea. All the isolates were cultured in fungal medium and nematodes were harvested for further experiment. First, all the isolates were characterized by both ITS and D2D3 rDNA sequence analysis. Template DNA was prepared by DNA extraction from single female nematode. ITS and D2D3 region were amplified by PCR and followed by cloning and sequence. As a result, all the sequences of ITS and D2D3 from B. xyophilus isolates were identical and there is no intraspecific variation. However, 2 genotypes of B. mucronatus were found, which one from P. thunbergii was East Asian type and the other one from P. koraiensis was European type. ITS‐RFLP phenotype was also proposed to discriminate different species and genotypes and 5 enzymes, Hinf I, Alu I, Msp I, Hae III, and Rsa I. were selectively treated.

Development of Monoclonal Antibodies Specific to Galectin of Pine Wood Nematode, Bursaphelenchus xylophilus (Steiner and Buhrer) Nickle and Their Utilization for Detection of Pine Wood Nematodes

Currently, there is no available tool that rapidly diagnoses pine wood nematode (PWN)-infected pine trees in the field. In this study, we synthesized and purified PWN Galectin, which might be an antigen specific to PWN, using the Baculovirus expression system. We used PWN Galectin as an antigen for generating 1,464 fusion hybridoma cell lines secreting monoclonal antibodies (Mabs). Among them, we selected 62 fusion hybridoma cell lines showing high reactivity to PWN Galectin. We further selected 12 fusion hybridoma cell lines showing high reactivity to the standard PWN-infected pine tree phosphate buffered saline (PBS) extract. Additionally, two fusion hybridoma cell lines showing no or extremely low reactivity were used as controls. The selected fusion hybridoma cell lines were subjected to limiting dilutions for selecting and establishing Mab-secreting cell lines showing higher reactivity to the standard PWN-infected pine tree extract than to the standard normal pine tree PBS extract. Moreover, the selected fusion hybridoma cell lines were further selected based on their higher reactivity to PWN protein extracts than to three non-pathogenic nematode protein extracts. The Mab-secreting cell lines established in this study could be used to develop rapid diagnostic tools that can be used in the field or in laboratories for detecting PWN-infected pine trees or PWN.

Dispersal capacity of Monochamus saltuarius on flight mills

Pine wilt disease (PWD) is caused by pine wood nematode (PWN), Bursaphelenchus xylophilus (Steiner & Buhrer) Nickle, transmitted by Monochamus saltuarius (Gebler) (Coleoptera: Cerambycidae) to healthy Korean white pine, Pinus koraiensis Siebold & Zucc. (Pinaceae). It is a serious threat to forest ecosystems worldwide. To better understand and predict the spread of PWD in Korean white pine forests, we investigated flight distance, time, and velocity of M. saltuarius adults using computer‐linked flight mills under laboratory conditions. In addition, we investigated the impact of biological characteristics on flight distance of this vector insect. Flight distance and time were individually measured during flight mill experiments (30 min per session) and repeatedly measured twice a week. Our results revealed large variation in flight distance within the population. Half of the beetles tested in this study flew more than 1 km and no difference was found between males and females when only fliers were considered. The average flight velocity of females was higher than that of males, but intermittent flights were observed more frequently in males compared to females. Both unmated male and female M. saltuarius adults can fly more than 5 km during their entire lifespan based on these flight mill experiments. Consequently, guidelines for managing dead pines in infected areas should consider a large buffer zone around PWN‐invaded areas so that uninfected areas are beyond the dispersal capacity of M. saltuarius.