Esteya vermicola has shown promise as an efficient biological control agent against pine wilt disease, a devastating disease in pine forests caused by the pinewood nematode (PWN, Bursaphelenchus xylophilus). However, the in vivo interactions among E. vermicola, PWN, and pine hosts are less understood, both at the population and molecular levels. In this study, we performed a series of bioassays to investigate E. vermicola colonization patterns in pine xylem and its population responses to PWN invasion in healthy and PWN-induced wilting trees. Our results demonstrated that although E. vermicola exhibits slow growth, its conidia germinate and grew along the pine tracheid, even producing lunate conidia capable of initiating PWN infections within the xylem. Interestingly, while fungal hyphae became undetectable in pine sapling xylem after inoculation, the E. vermicola population increased immediately in response to PWN invasion. Furthermore, we observed a \"leap-frog\" dispersal pattern of fungal colonization in PWN-induced wilting pines, facilitated by the migration of fungal-infected nematodes. Moreover, we explored the molecular mechanisms underlying fungal tolerance to pine defense systems using transcriptomic analysis. Comparative transcriptomics revealed that carbohydrate metabolism and abiotic stress-induced oxidoreductive activities are involved in the fungal tolerance to the pine defense compound β-pinene. This study enhances our understanding of how E. vermicola colonizes and persists within pine xylem, its molecular responses to plant defense compounds, and its population dynamics upon PWN invasion, validating its efficacy as a biocontrol agent against pine wilt disease.

Adults of the pine sawyer Monochamus alternatus are the primary vector of Bursaphelenchus xylophilus, the causative agent of pine wilt disease. A sawyer subspecies in Taiwan (abbreviated \'T\') has two generations a year (bivoltinism) due to facultative diapause, whereas another subspecies in Japan (abbreviated \'J\') has a one- or two-year life cycle due to obligate diapause. T, with two infection periods a year, will cause more severe disease epidemics than J if it is introduced into Japan. Inter-subspecies hybridization may inhibit the expression of bivoltinism because many F1 hybrids induce diapause. To predict the effects of introducing T into Japan, the present study investigated copulation duration and late-male sperm precedence to fertilize eggs. The results indicated that a single copulation for more than 65 s supplied sufficient sperm to fertilize a lifetime production of eggs. The incidence of larval diapause was 0.15 for the offspring of T females that mated with a T male and increased to 0.292-0.333 after remating with a J male, while the incidence of larval diapause was 0.900-1.000 for hybrids from T females mated with a J male. Consequently, the estimated proportion of second-male sperm used by T females was 0.185-0.217. The effects of introducing T populations into Japan on the severity of disease epidemics were also discussed.

ShK domain-containing proteins are peptides found in different parasitic and venomous organisms. From a previous transcriptomic dataset from Bursaphelenchus xylophilus, a plant-parasitic nematode that infects forest tree species, we identified 96 transcripts potentially as ShK domain-containing proteins with unknown function in the nematode genome. This study aimed to characterize and explore the functional role of genes encoding ShK domain-containing proteins in B. xylophilus biology. We selected and functionally analyzed nine candidate genes that are putatively specific to B. xylophilus. In situ hybridization revealed expression of one B. xylophilus ShK in the pharyngeal gland cells, suggesting their delivery into host cells. Most of the transcripts are highly expressed during infection and showed a significant upregulation in response to peroxide products compared to the nematode catalase enzymes. We reported, for the first time, the potential involvement of ShK domain genes in oxidative stress, suggesting that these proteins may have an important role in protecting or modulating the reactive oxygen species (ROS) activity of the host plant during parasitism.

UNASSIGNED: The pinewood nematode (PWN) is responsible for causing pine wilt disease (PWD), which has led to the significant decline of conifer species in Eurasian forests and has become a globally invasive quarantine pest. Manipulating plant-associated microbes to control nematodes is an important strategy for sustainable pest management. However, it has proven difficult to find pine-associated bacteria that possess both nematocidal activity and the ability to colonize pine tissues.
UNASSIGNED: The stress experiments with turpentine and pine tissue extract were carried out to screen for the desired target strain that could adapt to the internal environment of pine trees. This strain was used to construct an engineered nematocidal strain. Additionally, a fluorescent strain was constructed to determine its dispersal ability in Pinus massoniana seedlings through plate separation, PCR detection, and fluorescence microscopy observations. The engineered nematocidal strain was tested in the greenhouse experiment to assess its ability to effectively protect P. massoniana seedlings from nematode infection.
UNASSIGNED: This study isolated a Bacillus toyonensis strain Bxy19 from the healthy pine stem, which showed exceptional tolerance in stress experiments. An engineered nematocidal strain Bxy19P3C6 was constructed, which expressed the Cry6Aa crystal protein and exhibited nematocidal activity. The fluorescent strain Bxy19GFP was also constructed and used to test its dispersal ability. It was observed to enter the needles of the seedlings through the stomata and colonize the vascular bundle after being sprayed on the seedlings. The strain was observed to colonize and spread in the tracheid after being injected into the stems. The strain could colonize the seedlings and persist for at least 50 days. Furthermore, the greenhouse experiments indicated that both spraying and injecting the engineered strain Bxy19P3C6 had considerable efficacy against nematode infection.
UNASSIGNED: The evidence of the colonization ability and persistence of the strain in pine advances our understanding of the control and prediction of the colonization of exogenously delivered bacteria in pines. This study provides a promising approach for manipulating plant-associated bacteria and using Bt protein to control nematodes.

The gene localization technique of Bursaphelenchus xylophilus (pinewood nematode, PWN) is used for study gene expression in PWNs. Two in situ hybridization methods, namely, whole-mount in situ hybridization and the cut-off method are used widely. To compare the effects of these two in situ hybridization methods, the present study investigated the patterns of two functional genes expression in PWNs. The Bx-vap-2 gene (GenBank accession number: OR228482), related to pathogenicity, and the fem-2 gene (GenBank accession number: OR228481), related to sex determination, were selected to map related genes in the whole-mount and amputated PWNs at different ages using these in situ hybridization methods. Based on the overall statistical comparison, we found that compared to the cut-off method, the whole-mount method exhibited higher staining rates and correct staining rates for the fem-2 gene and Bx-vap-2 gene. However, considering the correct staining aspect, the cut-off method yielded better staining effects on pinewood nematode sections than the whole-mount method, with clearer hybridization signal locations and less non-specific staining. In other words, the cut-off method demonstrated more precise gene localization. Both methods are applicable for gene localization, but considering the overall staining pattern, analysis of experimental results, and comprehensive experimental operations, we believe that the whole-mount method is more suitable for gene localization and expression analysis of development-related genes in pinewood nematodes. This is because intact pinewood nematodes are better suited for showcasing the continuous developmental process of development-related genes. On the other hand, considering the experimental time, accuracy of staining site, and the amount of non-specific staining, the cut-off method is more suitable for disease-related genes. Additionally, to achieve better performance, the cut-off method can be selectively applied to samples during the experimental process.

Many pathogens rely on their insect vectors for transmission. Such pathogens are under selection to improve vector competence for their transmission by employing various tissue or cellular responses of vectors. However, whether pathogens can actively cause hypoxia in vectors and exploit hypoxia responses to promote their vector competence is still unknown. Fast dispersal of pinewood nematode (PWN), the causal agent for the destructive pine wilt disease and subsequent infection of pine trees, is characterized by the high vector competence of pine sawyer beetles (Monochamus spp.), and a single beetle can harbor over 200,000 PWNs in its tracheal system. Here, we demonstrate that PWN loading activates hypoxia in tracheal system of the vector beetles. Both PWN loading and hypoxia enhanced tracheal elasticity and thickened the apical extracellular matrix (aECM) of the tracheal tubes while a notable upregulated expression of a resilin-like mucin protein Muc91C was observed at the aECM layer of PWN-loaded and hypoxic tracheal tubes. RNAi knockdown of Muc91C reduced tracheal elasticity and aECM thickness under hypoxia conditions and thus decreasing PWN loading. Our study suggests a crucial role of hypoxia-induced developmental responses in shaping vector tolerance to the pathogen and provides clues for potential molecular targets to control pathogen dissemination.
Various parasites, bacteria and other disease-causing pathogens are transmitted by insects. A tiny worm called the pine wood nematode, for example, is spread by pine sawyer beetles which can carry up to 280,000 worms in their trachea, the network of tubes they use to breathe. This has resulted in millions of hectares of pine forests in Asia and Europe becoming infected with the deadly disease caused by the nematodes. Pine wood nematodes, as well as other pathogens, can exploit the biological processes of the insects carrying them to make the insects transmit them more effectively. Precisely how nematodes and other disease-causing agents do this is unclear. One possibility is that they reduce the amount of oxygen being supplied to the trachea – a phenomenon known as hypoxia – which occurs naturally at specific stages in the life of an insect, and during infections. To test this theory, Tang, Zhou, Koski et al. used genetics and imaging approaches to study how pine wood nematodes affect the trachea of pine sawyer beetles. The experiments found that when the nematodes infected the beetles, their trachea did indeed develop hypoxia. This, in turn, made the beetles’ airways more elastic and made the layer of structure lining the trachea, known as the apical extracellular matrix, thicker. These changes increased the amount of pinewood nematodes the trachea could hold, allowing the beetle to spread more worms from tree to tree. Further experiments revealed that hypoxia in the trachea increased the levels of a protein called Muc91C in the apical extracellular matrix. When the levels of Muc91C were artificially decreased in the beetles, this made their airways less elastic and the apical extracellular matrix thinner. This work suggests that pine wood nematodes exploit the beetles’ normal responses to loss of oxygen supply to make the beetles more effective at transmitting the nematodes between pine trees. Other pathogens carried by insects may also use this strategy to help increase their transmission. Further studies on the Muc91C protein may provide clues for potential drug targets to control pine wood nematodes and protect pine trees from disease.

Pine wood nematode, Bursaphelenchus xylophilus, is a worldwide pest of pine trees, spreading at an alarming rate and with great ecological adaptability. In the process of causing disease, the nematode causes metabolic disorders and changes in the endophytic microbial community of the pine tree. However, the changes at the pine nidus during early nematode invasion have not been well studied, especially the differential metabolites, in Pinus massoniana, the main host of B. xylophilus in China. In this study, we analyzed the endophytic bacterial and fungal communities associated with healthy and B. xylophilus-caused wilted pine trees. The results show that 1333 bacterial OTUs and 502 fungal OTUs were annotated from P. massoniana stem samples. The abundance of bacterial communities in pine trees varies more following infection by B. xylophilus, but the abundance changes of fungal communities are less visible. There were significant differences in endophytic microbial diversity between wilted and healthy P. massoniana. In wilted pine trees, Actinobacteria and Bacteroidia were differential indicators of bacterial communities, whereas, in healthy pine trees, Rhizobiales in the Proteobacteria phylum were the major markers of bacterial communities. Meanwhile, the differential markers of fungal communities in healthy pines are Malasseziales, Tremellales, Sordariales, and Fusarium, whereas Pleosporaceae is the key marker of fungal communities in wilted pines. Our study examines the effect of changes in the endophytic microbial community on the health of pine trees that may be caused by B. xylophilus infection. In parallel, a non-targeted metabolomic study based on liquid mass spectrometry (LC-MS) technology was conducted on pine trees inoculated with pine nematodes and healthy pine trees with a view to identifying key compounds affecting early pine lesions. Ultimately, 307 distinctly different metabolites were identified. Among them, the riboflavin metabolic pathway in pine trees may play a key role in the early pathogenesis of pine wood nematode disease.

Monochamus alternatus Hope, 1842, is a major forest pest that hosts the pathogenic pinewood nematode (PWN), Bursaphelenchus xylophilus (Steiner and Buhrer, 1934) Nickle 1970. Taxonomically, M. alternatus is currently divided into two subspecies, based on morphology and geography: Monochamus alternatus alternatus Hope, 1842 in China, Taiwan, Tibet, Vietnam, and Laos and Monochamus alternatus endai Makihara, 2004 in South Korea and Japan. Despite their economic importance, the subspecies taxonomy of M. alternatus has never been tested after the first description. In this study, we aimed to reassess the subspecies taxonomy of M. alternatus using molecular and morphological data. For morphological analysis, we examined three major morphological characters (pronotal longitudinal band, granulation on humeri, and elytral proximomedial spine) from 191 individuals from China, Korea, and Taiwan. Population genetic structures were examined using 85 de novo sequences and 82 public COI sequences from China, Korea, Japan, Malaysia, Taiwan, and a few intercepted specimens from the United States. All the genetic data were aligned as three different multiple sequence alignments. Individuals from each subspecies were morphologically and genetically scattered, not clustered according to subspecies in any of the analyses. Therefore, a new synonymy is proposed: Monochamus alternatus Hope, 1842 = Monochamus alternatus endai, syn. n. This study suggests a more robust classification of M. alternatus for the first time and ultimately will pose a substantial impact on implementing quarantine or forestry policies.

Plant-parasitic nematodes (PPNs) are among the most notorious and underrated threats to food security and plant health worldwide, compromising crop yields and causing billions of dollars of losses annually. Chemical control strategies rely heavily on synthetic chemical nematicides to reduce PPN population densities, but their use is being progressively restricted due to environmental and human health concerns, so alternative control methods are urgently needed. Here, we review the potential of bacterial and fungal agents to suppress the most important PPNs, namely Aphelenchoides besseyi, Bursaphelenchus xylophilus, Ditylenchus dipsaci, Globodera spp., Heterodera spp., Meloidogyne spp., Nacobbus aberrans, Pratylenchus spp., Radopholus similis, Rotylenchulus reniformis, and Xiphinema index.

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