The Bursaphelenchus xylophilus Effector BxNMP1 Targets PtTLP-L2 to Mediate PtGLU Promoting Parasitism and Virulence in Pinus thunbergii.

Pinus is an important economic tree species, but pine wilt disease (PWD) seriously threatens the survival of pine trees. PWD caused by Bursaphelenchus xylophilus is a major quarantine disease worldwide that causes significant economic losses. However, more information about its molecular pathogenesis is needed, resulting in a lack of effective prevention and treatment measures. In recent years, effectors have become a hot topic in exploring the molecular pathogenic mechanism of pathogens. Here, we identified a specific effector, BxNMP1, from B. xylophilus. In situ hybridization experiments revealed that BxNMP1 was specifically expressed in dorsal gland cells and intestinal cells, and RT-qPCR experiments revealed that BxNMP1 was upregulated in the early stage of infection. The sequence of BxNMP1 was different in the avirulent strain, and when BxNMP1-silenced B. xylophilus was inoculated into P. thunbergii seedlings, the disease severity significantly decreased. We demonstrated that BxNMP1 interacted with the thaumatin-like protein PtTLP-L2 in P. thunbergii. Additionally, we found that the ß-1,3-glucanase PtGLU interacted with PtTLP-L2. Therefore, we hypothesized that BxNMP1 might indirectly interact with PtGLU through PtTLP-L2 as an intermediate mediator. Both targets can respond to infection, and PtTLP-L2 can enhance the resistance of pine trees. Moreover, we detected increased salicylic acid contents in P. thunbergii seedlings inoculated with B. xylophilus when BxNMP1 was silenced or when the PtTLP-L2 recombinant protein was added. In summary, we identified a key virulence effector of PWNs, BxNMP1. It positively regulates the pathogenicity of B. xylophilus and interacts directly with PtTLP-L2 and indirectly with PtGLU. It also inhibits the expression of two targets and the host salicylic acid pathway. This study provides theoretical guidance and a practical basis for controlling PWD and breeding for disease resistance.

Physiological Measurements and Transcriptomics Reveal the Fitness Costs of Monochamus saltuarius to Bursaphelenchus xylophilus.

The pine wood nematode (PWN) uses several Monochamus species as vehicles, through a temporary hitchhiking process known as phoresy, enabling it to access new host plant resources. Monochamus saltuarius acts as a new and major vector of the PWN in Northeastern China, showing lower PWN carrying capacity and a shorter transmission cycle compared to established vectors. The apparently altered symbiotic relationship offers an interesting area for researching the costs and adaptions involved in nematode-beetle, a specialized phoresy. We analyzed the response and fitness costs of M. saltuarius through physiological measurements and transcriptomics. The PWN exerted adverse repercussions on the growth and development of M. saltuarius. The PWN accelerated larval development into pupae, while beetle adults carrying the PWN exhibited an elevated abnormality rate and mortality, and reduced starvation resistance. During the pupal stage, the expression of growth-related genes, including ecdysone-inducible genes (E74EA), cuticle proteins, and chitin genes (CHTs), markedly increased. Meanwhile, the induced immune response, mainly by the IMD and Toll signaling pathways, could be a contributing factor to adult abnormality and mortality. Adult gonads and trachea exhibited enrichment in pathways related to fatty acid elongation, biosynthesis, and metabolism. FASN, ELOVL, and SCD possibly contributed to resistance against PWN. Our research indicated that phoretic interactions between vector beetles and PWN vary throughout the vector's lifespan, particularly before and after entry into the trachea. This study highlighted the fitness costs of immunity and metabolism on the vector beetle, indicating the adaptation mechanisms and evolutionary trade-offs to PWN.

Research Progress on the Early Monitoring of Pine Wilt Disease Using Hyperspectral Techniques.

Pine wilt disease (PWD) caused by pine wood nematode (PWN, Bursaphelenchus xylophilus) originated in North America and has since spread to Asia and Europe. PWN is currently a quarantine object in 52 countries. In recent years, pine wilt disease has caused considerable economic losses to the pine forest production industry in China, as it is difficult to control. Thus, one of the key strategies for controlling pine wilt disease is to identify epidemic points as early as possible. The use of hyperspectral cameras mounted on drones is expected to enable PWD monitoring over large areas of forest, and hyperspectral images can reflect different stages of PWD. The trend of applying hyperspectral techniques to the monitoring of pine wilt disease is analyzed, and the corresponding strategies to address the existing technical problems are proposed, such as data collection of early warning stages, needs of using unmanned aerial vehicles (UAVs), and establishment of models after preprocessing.

A Reference Genome of Bursaphelenchus mucronatus Provides New Resources for Revealing Its Displacement by Pinewood Nematode.

The Bursaphelenchus mucronatus, which was highly similar with Bursaphelenchus xylophilus in terms of morphological characteristics and biological properties-but had weaker pathogenicity to forests-was a native species often displaced by B. xylophilus when occupying the same niche. Since the draft genome of the invasive B. xylophilus has been published, the absence of a reference genome of B. mucronatus still prevents us from understanding the molecular evidences behind competitive displacement. In this study, we employed Single Molecule, Real-Time (SMRT) sequencing and a Hi-C scaffolding approach to yield a near chromosome-level assembly of B. mucronatus, including six pseudo-chromosomes. The assembly size is 73 Mb, with scaffold N50 of 11.50 Mb and contig N50 of 1.48 Mb. Comparative genomics results showed high similarity between B. xylophilus and B. mucronatus. However, the losing of orphan genes and species-specific orthologous genes in B. mucronatus may indicate weaker adaptability to the environment. The gene family contractions of GPCRs (G Protein-Coupled Receptors) and cellulases in B. mucronatus may jointly contribute to its displacement by B. xylophilus. Overall, we introduced a valuable genomic resource for molecular and evolutionary studies of B. mucronatus, especially for studying the competitive displacement by the pinewood nematode, which could help us control the pathogenicity of pine wilt diseases.

Fine mapping of root lesion nematode (Pratylenchus thornei) resistance loci on chromosomes 6D and 2B of wheat.

KEY MESSAGE: Resistance QTL to root lesion nematode (Pratylenchus thornei) in wheat (Triticum aestivum), QRlnt.sk-6D and QRlnt.sk-2B, were mapped to intervals of 3.5 cM/1.77 Mbp on chromosome 6D and 1.4 cM/2.19 Mbp on chromosome 2B, respectively. Candidate resistance genes were identified in the QTL regions and molecular markers developed for marker-assisted breeding. Two previously known resistance QTL for root lesion nematode (Pratylenchus thornei) in bread wheat (Triticum aestivum), QRlnt.sk-6D and QRlnt.sk-2B, were fine-mapped using a Sokoll (moderately resistant) by Krichauff (susceptible) doubled haploid (DH) population and six newly developed recombinant inbred line populations. Bulked segregation analysis with the 90K wheat SNP array identified linked SNPs which were subsequently converted to KASP assays for mapping in the DH and RIL populations. On chromosome 6D, 60 KASP and five SSR markers spanned a total genetic distance of 23.7 cM. QRlnt.sk-6D was delimited to a 3.5 cM interval, representing 1.77 Mbp in the bread wheat cv. Chinese Spring reference genome sequence and 2.29 Mbp in the Aegilops tauschii genome sequence. These intervals contained 42 and 43 gene models in the respective annotated genome sequences. On chromosome 2B, 41 KASP and 5 SSR markers produced a map spanning 19.9 cM. QRlnt.sk-2B was delimited to 1.4 cM, corresponding 3.14 Mbp in the durum wheat cv. Svevo reference sequence and 2.19 Mbp in Chinese Spring. The interval in Chinese Spring contained 56 high-confidence gene models. Intervals for both QTL contained genes with similarity to those previously reported to be involved in disease resistance, namely genes for phenylpropanoid biosynthetic pathway-related enzymes, NBS-LRR proteins and protein kinases. The potential roles of these candidate genes in P. thornei resistance are discussed. The KASP markers reported in this study could potentially be used for marker-assisted breeding of P. thornei-resistant wheat cultivars.

Metabolomics of soybean green stem and foliar retention (GSFR) disease using mass spectrometry and molecular networking.

RATIONALE: The nematode Aphelenchoides besseyi is the causal agent of green stem and foliar retention, a soybean disease recently described in Brazil. This condition can reduce soybean yield by up to 100%. However, little is known about chemical interactions between the plant and pathogen. Therefore, this work aimed to investigate metabolites from healthy soybean roots and from soybean roots that were inoculated with A. besseyi. METHODS: A. besseyi were multiplied in vitro with Fusarium sp. colonies in Petri dishes for 25 days, and were axenically inoculated into hydroponics healthy soybean plants. The metabolites were extracted from the roots of healthy and A. besseyi-infected plants 16 days post-inoculation. These extracts were analyzed using an untargeted metabolomic method with an ultra-high-performance liquid chromatography/electrospray ionization /tandem mass spectrometry (UHPLC/ESI-MS/MS) and molecular networking approach. RESULTS: Roots from infected plants showed morphological alterations such as shrinkage, darkening, and arching. Similarly, they also showed an increased presence of flavonoids, compared with healthy roots. Compounds such as neobavaisoflavone, glycitin, genistin, and genistein were putatively identified and had greater intensity in inoculated roots. These compounds are linked to the defensive mechanisms in plants against nematodes. Moreover, coumaric acid, also exclusively putatively identified in inoculated roots, shows activity related to inhibition of root growth. CONCLUSIONS: Liquid chromatography, mass spectrometry, and molecular networking approaches proved to be a powerful tool for the metabolomic study of GSFR. This study showed metabolomics differences of protective substances in the roots, evidencing a quick response of the plant to the attack of A. besseyi.

Thaumatin-Like Protein-1 Gene (Bx-tlp-1) Is Associated with the Pathogenicity of Bursaphelenchus xylophilus.

The pine wood nematode Bursaphelenchus xylophilus is a destructive species affecting pine trees worldwide; however, the underlying mechanism leading to pathogenesis remains unclear. In this study, a B. xylophilus gene encoding thaumatin-like protein-1 (Bx-tlp-1) was silenced by RNA interference to clarify the relationship between the Bx-tlp-1 gene and pathogenicity. The in vitro knockdown of Bx-tlp-1 with double-stranded RNA (dsRNA) decreased B. xylophilus reproduction and pathogenicity. Treatments with dsRNA targeting Bx-tlp-1 decreased expression by 90%, with the silencing effect maintained even in the F3 offspring. Pine trees inoculated with B. xylophilus treated with Bx-tlp-1 dsRNA decreased the symptom of wilting, and the disease severity index was 56.7 at 30 days after inoculation. Additionally, analyses of the cavitation of intact pine stem samples by X-ray microtomography revealed that the xylem cavitation area of pine trees inoculated with B. xylophilus treated with Bx-tlp-1 dsRNA was 0.46 mm2 at 30 days after inoculation. Results from this study indicated that the silencing of Bx-tlp-1 has effects on B. xylophilus fitness. The data presented here provide the foundation for future analyses of Bx-tlp-1 functions related to B. xylophilus pathogenicity.

Silencing of cyp-33C9 Gene Affects the Reproduction and Pathogenicity of the Pine Wood Nematode, Bursaphelenchus xylophilus.

Cytochrome P450 genes are very important for plant-parasitic nematodes to reproduce and to metabolize xenobiotic compounds generated by their host plants. The pine wood nematode (PWN), Bursaphelenchus xylophilus, causes very high annual economic losses by killing large numbers of pine trees across Asia and into Europe. In this study, we used RNA interference (RNAi) to analyze the function of the cyp-33C9 gene of PWN. Our results showed that expression of the cyp-33C9 gene was suppressed successfully after soaking nematodes for 24 h in cyp-33C9 double-stranded RNA (dsRNA). The silencing of the cyp-33C9 gene significantly decreased the feeding, reproduction, oviposition and egg hatch of B. xylophilus. Meanwhile, the migration speed of B. xylophilus in Pinus thunbergii was reduced in the early stages when the cyp-33C9 gene was silenced in the nematodes. Moreover, knockdown of the cyp-33C9 gene in B. xylophilus caused a decrease in pathogenicity to pine trees. These results suggest that the cyp-33C9 gene plays an important role in the reproduction and pathogenicity of B. xylophilus. This discovery identified several functions of the cyp-33C9 gene in B. xylophilus and provided useful information for understanding the molecular mechanism behind pine wilt disease caused by PWN.

Identification of a novel effector BxSapB3 that enhances the virulence of pine wood nematode Bursaphelenchus xylophilus.

Pine wilt disease, caused by the pine wood nematode Bursaphelenchus xylophilus, leads to severe damage to pine forests in China. In our previous study, effectors secreted by this pathogen were shown to play roles in the different infection stages of pine wilt disease, and a series of candidate effectors were predicted by transcriptome sequencing. This study identified and characterized a novel effector, BxSapB3, which was among these candidate effectors. Agrobacterium-mediated transient expression was used to identify BxSapB3. BxSapB3 was secreted by B. xylophilus and found to be capable of inducing cell death in Nicotiana benthamiana. Quantitative real-time PCR (qRT-PCR) analysis revealed that BxSapB3 was upregulated in a highly virulent strain of B. xylophilus and expressed at lower levels in a weakly virulent strain at the early stages of infection. When BxSapB3 was silenced in B. xylophilus, the process of infection was delayed. These results indicate that BxSapB3 acts as an effector and contributes to virulence at the early stages of B. xylophilus infection.

Bacterial Communities and Virulence Associated with Pine Wood Nematode Bursaphelenchus xylophilus from Different Pinus spp.

Bursaphelenchus xylophilus, the causal agent of pine wilt disease, is a destructive threat to pine forests. The role of bacteria associated with B. xylophilus in pine wilt disease has attracted widespread attention. This study investigated variation in bacterial communities and the virulence of surface-sterilized B. xylophilus from different Pinus spp. The predominant culturable bacteria of nematodes from different pines were Stenotrophomonas and Pseudomonas. Biolog EcoPlate analysis showed that metabolic diversity of bacteria in B. xylophilus from P. massoniana was the highest, followed by P. thunbergii and P. densiflora. High-throughput sequencing analysis indicated that bacterial diversity and community structure in nematodes from the different pine species varied, and the dominant bacteria were Stenotrophomonas and Elizabethkingia. The virulence determination of B. xylophilus showed that the nematodes from P. massoniana had the greatest virulence, followed by the nematodes from P. thunbergii and P. densiflora. After the nematodes were inoculated onto P. thunbergii, the relative abundance of the predominant bacteria changed greatly, and some new bacterial species emerged. Meanwhile, the virulence of all the nematode isolates increased after passage through P. thunbergii. These inferred that some bacteria associated with B. xylophilus isolated from different pine species might be helpful to adjust the PWN's parasitic adaptability.

Enhancement of oxidative stress contributes to increased pathogenicity of the invasive pine wood nematode.

Reactive oxygen species (ROS) play important roles in defence response of host plants versus pathogens. While generation and detoxification of ROS is well understood, how varied ability of different isolates of pathogens to overcome host ROS, or ROS contribution to a particular isolate's pathogenicity, remains largely unexplored. Here, we report that transcriptional regulation of the ROS pathway, in combination with the insulin pathway, increases the pathogenicity of invasive species Bursaphelenchus xylophilus. The results showed a positive correlation between fecundity and pathogenicity of different nematode isolates. The virulent isolates from introduced populations in Japan, China and Europe had significantly higher fecundity than native avirulent isolates from the USA. Increased expression of Mn-SOD and reduced expression of catalase/ GPX-5 and H2O2 accumulation during invasion are associated with virulent strains. Additional H2O2 could improve fecundity of Bu. xylophilus. Furthermore, depletion of Mn-SOD decreased fecundity and virulence of Bu. xylophilus, while the insulin pathway is significantly affected. Thus, we propose that destructive pathogenicity of Bu. xylophilus to pines is partly owing to upregulated fecundity modulated by the insulin pathway in association with the ROS pathway and further enhanced by H2O2 oxidative stress. These findings provide a better understanding of pathogenic mechanisms in plant-pathogen interactions and adaptive evolution of invasive species. This article is part of the theme issue 'Biotic signalling sheds light on smart pest management'.

Stage-specific transcriptome of Bursaphelenchus xylophilus reveals temporal regulation of effector genes and roles of the dauer-like stages in the lifecycle.

The pine wood nematode Bursaphelenchus xylophilus is the causal agent of pine wilt disease, one of the most devastating forest diseases in East Asian and West European countries. The lifecycle of B. xylophilus includes four propagative larval stages and gonochoristic adults which are involved in the pathogenicity, and two stages of dispersal larvae involved in the spread of the disease. To elucidate the ecological roles of each developmental stage in the pathogenic life cycle, we performed a comprehensive transcriptome analysis using RNA-seq generated from all developmental stages of B. xylophilus and compared transcriptomes between stages. We found more than 9000 genes are differentially expressed in at least one stage of the life cycle including genes involved in general nematode biology such as reproduction and moulting but also effector genes likely to be involved in parasitism. The dispersal-stage transcriptome revealed its analogy to C. elegans dauer and the distinct roles of the two larval stages from each other regarding survival and transmission. This study provides important insights and resources to understand B. xylophilus parasitic biology.

Cathepsin L-like Cysteine Proteinase Genes Are Associated with the Development and Pathogenicity of Pine Wood Nematode, Bursaphelenchus xylophilus.

The pine wood nematode (PWN), Bursaphelenchus xylophilus, is the pathogen of pine wilt disease (PWD), resulting in huge losses in pine forests. However, its pathogenic mechanism remains unclear. The cathepsin L-like cysteine proteinase (CPL) genes are multifunctional genes related to the parasitic abilities of plant-parasitic nematodes, but their functions in PWN remain unclear. We cloned three cpl genes of PWN (Bx-cpls) by rapid amplification of cDNA ends (RACE) and analyzed their characteristics using bioinformatic methods. The tissue specificity of cpl gene of PWN (Bx-cpl) was studied using in situ mRNA hybridization (ISH). The functions of Bx-cpls in development and pathogenicity were investigated using real-time quantitative PCR (qPCR) and RNA interference (RNAi). The results showed that the full-length cDNAs of Bx-cpl-1, Bx-cpl-2, and Bx-cpl-3 were 1163 bp, 1305 bp, and 1302 bp, respectively. Bx-cpls could accumulate specifically in the egg, intestine, and genital system of PWN. During different developmental stages of PWN, the expression of Bx-cpls in the egg stage was highest. After infection, the expression levels of Bx-cpls increased and reached their highest at the initial stage of PWD, then declined gradually. The silencing of Bx-cpl could reduce the feeding, reproduction, and pathogenicity of PWN. These results revealed that Bx-cpls play multiple roles in the development and pathogenic processes of PWN.

Differential effects of rapamycin on Bursaphelenchus xylophilus with different virulence and differential expression of autophagy genes under stresses in nematodes.

Pine wilt disease (PWD) caused by the pine wood nematode (PWN), Bursaphelenchus xylophilus, is a devastating disease for Pinus spp. The virulence and resilience of PWN are closely linked to the spread and development of PWD. Numerous studies have shown that autophagy has important physiological and pathological functions in eukaryotes. But little is known about the relationships between autophagy and PWNs' virulence and resistance. In this study, through observation under the microscope and recording, we found the induction of autophagy by rapamycin could dramatically improve movement ability of PWNs with different virulence, and the highly virulent AMA3 isolate moved more than the low virulent YW4 isolate when autophagy was over-induced. High concentrations of rapamycin substantially improved the feeding and reproduction of AMA3 but not YW4. Conserved domains of autophagy genes BxATG3, BxATG4, and BxATG7 were first cloned from PWNs by reverse transcription-polymerase chain reaction (RT-PCR). Expression profiling of these three autophagy genes under biotic and abiotic stresses in PWNs with different virulence was determined by quantitative RT-PCR. The results revealed the expression levels of these three autophagy genes in PWNs with different virulence were increased significantly when nematodes were subject to high and low temperatures, oxidative stress, and defensive responses of pine trees. The expression levels of autophagy genes under biotic and abiotic stresses in AMA3 were higher than those in YW4, and different genes showed different performance. Our study clarified that autophagy was closely related to virulence and resistance of PWN, and the ability of a highly virulent isolate to regulate autophagy activity under stresses was stronger than that of a low virulent isolate.

The first report of Xenorhabdus indica from Steinernema pakistanense: co-phylogenetic study suggests co-speciation between X. indica and its steinernematid nematodes.

During a survey in agricultural fields of the sub-humid region of Meerut district, India, two strains of entomopathogenic nematodes, labelled CS31 and CS32, were isolated using the Galleria baiting technique. Based on morphological and morphometric studies, and molecular data, the nematodes were identified as Steinernema pakistanense, making this finding the first report of this species from India. For the first time, we performed a molecular and biochemical characterization of the bacterial symbiont of S. pakistanense. Furthermore, a co-phylogenetic analysis of the bacteria from the monophyletic clade containing a symbiont of S. pakistanense, together with their nematode hosts, was conducted, to test the degree of nematode-bacteria co-speciation. Both isolates were also tested in a laboratory assay for pathogenicity against two major pests, Helicoverpa armigera and Spodoptera litura. The morphology of the Indian isolates corresponds mainly to the original description, with the only difference being the absence of a mucron in first-generation females and missing epiptygmata in the second generation. The sequences of bacterial recA and gyrB genes have shown that the symbiont of S. pakistanense is closely related to Xenorhabdus indica, which is associated with some other nematodes from the 'bicornutum' group. Co-phylogenetic analysis has shown a remarkable congruence between the nematode and bacterial phylogenies, suggesting that, in some lineages within the Steinernema / Xenorhabdus complex, the nematodes and bacteria have undergone co-speciation. In the virulence assay, both strains caused a 100% mortality of both tested insects after 48 h, even at the lowest doses of 25 infective juveniles per insect, suggesting that S. pakistanense could be considered for use in the biocontrol of these organisms in India.

An Effector, BxSapB1, Induces Cell Death and Contributes to Virulence in the Pine Wood Nematode Bursaphelenchus xylophilus.

The pine wood nematode (PWN) Bursaphelenchus xylophilus has caused serious damage to pine forests in China. Effectors secreted by phytonematodes play a role in host infection. We identified and characterized an effector, BxSapB1, based on the B. xylophilus transcriptome at the early stages of infection and the transient expression of proteins in Nicotiana benthamiana. BxSapB1 triggered cell death in N. benthamiana when secreted into the apoplast, and this effect was independent of N. benthamiana brassinosteroid-insensitive 1-associated kinase 1 (NbBAK1) and suppressor of BIR1-1 (NbSOBIR1). The signal peptide of BxSapB1 was proven to be functional in yeast using the yeast signal sequence trap system and BxSapB1 was strongly expressed in the subventral gland cells of B. xylophilus, as revealed by in-situ hybridization. In addition, based on local BLAST analysis, the BxSapB1 showed 100% identity to BUX.s00139.62, which was identified from the B. xylophilus secretome during Pinus thunbergii infection. BxSapB1 was upregulated in a highly virulent strain and downregulated in a weakly virulent strain of PWN at the early stages of infection. RNA interference assays showed that silencing BxSapB1 resulted in decreased expression of pathogenesis-related genes (PtPR-1b, PtPR-3, and PtPR-5) as well as delayed onset of symptoms in P. thunbergii infected by B. xylophilus. The combined data suggest that BxSapB1 can trigger cell death in N. benthamiana and that it contributes to the virulence in B. xylophilus during parasitic interaction.

Transcriptomic Profiling Reveals Differentially Expressed Genes Associated with Pine Wood Nematode Resistance in Masson Pine (Pinus massoniana Lamb.).

Pine wilt disease caused by pine wood nematode (Bursaphelenchus xylophilus, PWN) is a severe forest disease of the genus Pinus. Masson pine as an important timber and oleoresin resource in South China, is the major species infected by pine wilt disease. However, the underlying mechanism of pine resistance is still unclear. Here, we performed a transcriptomics analysis to identify differentially expressed genes associated with resistance to PWN infection. By comparing the expression profiles of resistant and susceptible trees inoculated with PWN at 1, 15, or 30 days post-inoculation (dpi), 260, 371 and 152 differentially expressed genes (DEGs) in resistant trees and 756, 2179 and 398 DEGs in susceptible trees were obtained. Gene Ontology enrichment analysis of DEGs revealed that the most significant biological processes were "syncytium formation" in the resistant phenotype and "response to stress" and "terpenoid biosynthesis" in the susceptible phenotype at 1 and 15 dpi, respectively. Furthermore, some key DEGs with potential regulatory roles to PWN infection, including expansins, pinene synthases and reactive oxidation species (ROS)-related genes were evaluated in detail. Finally, we propose that the biosynthesis of oleoresin and capability of ROS scavenging are pivotal to the high resistance of PWN.

Integrative taxonomy of root-knot nematodes reveals multiple independent origins of mitotic parthenogenesis.

During sampling of several Coffea arabica plantations in Tanzania severe root galling, caused by a root-knot nematode was observed. From pure cultures, morphology and morphometrics of juveniles and females matched perfectly with Meloidogyne africana, whereas morphology of the males matched identically with those of Meloidogyne decalineata. Based on their Cox1 sequence, however, the recovered juveniles, females and males were confirmed to belong to the same species, creating a taxonomic conundrum. Adding further to this puzzle, re-examination of M. oteifae type material showed insufficient morphological evidence to maintain its status as a separate species. Consequently, M. decalineata and M. oteifae are synonymized with M. africana, which is herewith redescribed based on results of light and scanning electron microscopy, ribosomal and mitochondrial DNA sequences, isozyme electrophoresis, along with bionomic and cytogenetic features. Multi-gene phylogenetic analysis placed M. africana outside of the three major clades, together with M. coffeicola, M. ichinohei and M. camelliae. This phylogenetic position was confirmed by several morphological features, including cellular structure of the spermatheca, egg mass position, perineal pattern and head shape. Moreover, M. africana was found to be a polyphagous species, demonstrating that "early-branching" Meloidogyne spp. are not as oligophagous as had previously been assumed. Cytogenetic information indicates M. africana (2n = 21) and M. ardenensis (2n = 51-54) to be a triploid mitotic parthenogenetic species, revealing at least four independent origins of mitotic parthenogenesis within the genus Meloidogyne. Furthermore, M. mali (n = 12) was found to reproduce by amphimixis, indicating that amphimictic species with a limited number of chromosomes are widespread in the genus, potentially reflecting the ancestral state of the genus. The wide variation in chromosome numbers and associated changes in reproduction modes indicate that cytogenetic evolution played a crucial role in the speciation of root-knot nematodes and plant-parasitic nematodes in general.

Arabidopsis thaliana as a suitable model host for research on interactions between plant and foliar nematodes, parasites of plant shoot.

The rice white tip nematode (RWTN), Aphelenchoides besseyi and the chrysanthemum foliar nematode (CFN), Aphelenchoides ritzemabosi are migratory plant parasitic nematodes that infect the aboveground parts of plants. In this research, Arabidopsis thaliana was infected by RWTN and CFN under indoor aseptic cultivation, and the nematodes caused recognizable symptoms in the leaves. Furthermore, RWTN and CFN completed their life cycles and proliferated. Therefore, A. thaliana was identified as a new host of RWTN and CFN. The optimum inoculum concentration for RWTN and CFN was 100 nematodes/plantlet, and the optimum inoculum times were 21 and 24 days, respectively. For different RWTN populations, the pathogenicity and reproduction rates were different in the A. thaliana Col-0 ecotype and were positively correlated. The optimum A. thaliana ecotypes were Col-0 and WS, which were the most susceptible to RWTN and CFN, respectively. Additionally, RWTN was ectoparasitic and CFN was ecto- and endoparasitic in A. thaliana. The RWTN and CFN migrated from inoculated leaves to the entire plantlet, and the number of nematodes in different parts of A. thaliana was not correlated with distance from the inoculum point. This is a detailed study of the behavior and infection process of foliar nematodes on A. thaliana.