Pine Hosts Research Articles

Two decades of epidemiological surveillance of the pine wood nematode in France reveal its absence despite suitable conditions for its establishment

Key messageThis study takes stock of the first 20 years (2000–2019) of monitoring the pine wood nematode (PWN) in metropolitan France. While PWN was never found in the wild during this period, it was reported in some wood-based commodities entering or circulating on French territory. This stresses the importance of remaining extremely vigilant, as the conditions found in France, especially weather conditions, could be particularly suitable for the pest’s establishment.ContextThe pine wood nematode (PWN) Bursaphelenchus xylophilus, responsible for pine wilt disease (PWD), is one of the most important forest tree pests worldwide. It is thus the focus of many monitoring programmes. In the European Union, for example, it is categorised as a priority quarantine pest, so each member state is obliged to monitor the PWN on its territory.AimsThe first objective of this paper was to describe PWN monitoring in metropolitan France, namely how it is organised and whether it has led to the nematode’s detection. Secondly, we wished to investigate what the levels of PWD expression for host pines infected by B. xylophilus would be in France. Thirdly, we wanted to find out whether other Bursaphelenchus species had been found on French territory during these two decades of PWN monitoring.MethodsWe analysed data from samples collected in the framework of the monitoring programme between 2000 and 2019 to track the PWN in its host pines, its insect vector (Monochamus spp.) and in wood-based commodities imported into or circulating in metropolitan France. We also generated risk maps of PWD expression based on an evapo-transpiration model using climate data for the period 2000–2019.ResultsThis monitoring, which was regularly reinforced from 2000 to 2019, consisted of sampling and analysing around 18,000 wood samples and 66,000 insects over this period. Although the PWN was not detected in pine stands or within its insect vector, some wood-based commodities were found to be contaminated. Risk maps of PWD expression show that in the most recent years (2015–2019), the weather conditions in a large fraction of metropolitan France were suited to PWD expression, mostly with a delay (i.e., latency) between infection and observable wilt symptoms. PWN monitoring has also revealed the presence of other Bursaphelenchus species, most of which were discovered for the first time in metropolitan France and are described herein.ConclusionWhile metropolitan France is still free of the PWN, this study emphasises the need to remain cautious as the French territory appears particularly suitable for this pest’s establishment. Furthermore, our research has led us to propose some ideas on how to improve PWN monitoring.

Field ready: Development of a rapid LAMP‐based colorimetric assay for the causal agent of white pine blister rust, Cronartium ribicola

AbstractThe invasive fungal pathogen Cronartium ribicola causes white pine blister rust which is considered one of the most destructive diseases of five‐needle (white) pines in North America. The disease has a life cycle that requires two hosts: white pines and Ribes spp., although other non‐Ribes species, including Castilleja and Pedicularis, have been demonstrated as alternate hosts as well. Detection of this disease can be difficult because of the ephemeral nature of sporulation on pine hosts with ambiguity in other symptoms, and the alternate hosts for C. ribicola can also be an alternate host for other pine rust species. We used the previously published C. ribicola genome and species‐specific real‐time PCR assay to develop a field‐ready loop‐mediated isothermal amplification (LAMP) specific colorimetric assay for this pathogen. Specificity results across regionally identified pine rust pathogens showed the assay is highly specific to C. ribicola and can detect as little as 40 pg of pathogen DNA. We also developed a simple DNA extraction method that works with several tissue types (bark/phloem, aeciospores, and urediniospores/telia) to prepare the DNA samples for the LAMP assay. The DNA extraction and LAMP assay take ~70 min to complete and require a relatively small investment in equipment. This tool enables quick and efficient detection of white pine blister rust.

Seasonal Phenology and Climate Associated Feeding Activity of Introduced Marchalina hellenica in Southeast Australia.

Invasive insects pose an increasing risk to global agriculture, environmental stability, and public health. Giant pine scale (GPS), Marchalina hellenica Gennadius (Hemiptera: Marchalinidae), is a phloem feeding scale insect endemic to the Eastern Mediterranean Basin, where it primarily feeds on Pinus halepensis and other Pinaceae. In 2014, GPS was detected in the southeast of Melbourne, Victoria, Australia, infesting the novel host Pinus radiata. An eradication program was unsuccessful, and with this insect now established within the state, containment and management efforts are underway to stop its spread; however, there remains a need to understand the insect's phenology and behaviour in Australia to better inform control efforts. We documented the annual life cycle and seasonal fluctuations in activity of GPS in Australia over a 32 month period at two contrasting field sites. Onset and duration of life stages were comparable to seasons in Mediterranean conspecifics, although the results imply the timing of GPS life stage progression is broadening or accelerating. GPS density was higher in Australia compared to Mediterranean reports, possibly due to the absence of key natural predators, such as the silver fly, Neoleucopis kartliana Tanasijtshuk (Diptera, Chamaemyiidae). Insect density and honeydew production in the Australian GPS population studied varied among locations and between generations. Although insect activity was well explained by climate, conditions recorded inside infested bark fissures often provided the weakest explanation of GPS activity. Our findings suggest that GPS activity is strongly influenced by climate, and this may in part be related to changes in host quality. An improved understanding of how our changing climate is influencing the phenology of phloem feeding insects such as GPS will help with predictions as to where these insects are likely to flourish and assist with management programs for pest species.

A LAMP Assay for Rapid Detection of the Pitch Canker Pathogen Fusarium circinatum.

The pine pitch canker pathogen Fusarium circinatum is endemic in the southeastern United States and Central America and represents an invasive threat globally. This ecologically adaptable fungus readily infects all parts of its pine hosts, leading to widespread mortality of nursery seedlings and decline in the health and productivity of forest stands. Because trees infected by F. circinatum can remain asymptomatic for long periods of time, accurate and rapid tools are needed for real-time diagnostics and surveillance at ports, in nurseries, and in plantations. To meet this need and to limit the spread and impact of the pathogen, we developed a molecular test using loop-mediated isothermal amplification (LAMP), a technology that allows for the rapid detection of pathogen DNA on portable, field-capable devices. LAMP primers were designed and validated to amplify a gene region unique to F. circinatum. Using a globally representative collection of F. circinatum isolates and other closely related species, we have demonstrated that the assay can be used to identify F. circinatum across its genetic diversity and that it is sensitive to as few as 10 cells from purified DNA extracts. The assay can also be used with a simple, pipette-free DNA extraction method and is compatible with testing symptomatic pine tissues in the field. This assay has the potential to facilitate diagnostic and surveillance efforts both in the laboratory and in the field and, thus, to reduce the spread and impact of pitch canker worldwide.

Discovery of entomopathogenic fungi across geographical regions in southern China on pine sawyer beetle Monochamus alternatus and implication for multi-pathogen vectoring potential of this beetle.

Entomopathogen-based biocontrol is crucial for blocking the transmission of vector-borne diseases; however, few cross-latitudinal investigations of entomopathogens have been reported for vectors transmitting woody plant diseases in forest ecosystems. The pine sawyer beetle Monochamus alternatus is an important wood borer and a major vector transmitting pine wilt disease, facilitating invasion of the pinewood nematode Bursaphelenchus xylophilus (PWN) in China. Due to the limited geographical breadth of sampling regions, species diversity of fungal associates (especially entomopathogenic fungi) on M. alternatus adults and their potential ecological functions have been markedly underestimated. In this study, through traditional fungal isolation with morphological and molecular identification, 640 fungal strains (affiliated with 15 genera and 39 species) were isolated from 81 beetle cadavers covered by mycelia or those symptomatically alive across five regional populations of this pest in southern China. Multivariate analyses revealed significant differences in the fungal community composition among geographical populations of M. alternatus, presenting regionalized characteristics, whereas no significant differences were found in fungal composition between beetle genders or among body positions. Four region-representative fungi, namely, Lecanicillium attenuatum (Zhejiang), Aspergillus austwickii (Sichuan), Scopulariopsis alboflavescens (Fujian), and A. ruber (Guangxi), as well as the three fungal species Beauveria bassiana, Penicillium citrinum, and Trichoderma dorotheae, showed significantly stronger entomopathogenic activities than other fungi. Additionally, insect-parasitic entomopathogenic fungi (A. austwickii, B. bassiana, L. attenuatum, and S. alboflavescens) exhibited less to no obvious phytopathogenic activities on the host pine Pinus massoniana, whereas P. citrinum, Purpureocillium lilacinum, and certain species of Fusarium spp.-isolated from M. alternatus body surfaces-exhibited remarkably higher phytopathogenicity. Our results provide a broader view of the entomopathogenic fungal community on the vector beetle M. alternatus, some of which are reported for the first time on Monochamus spp. in China. Moreover, this beetle might be more highly-risk in pine forests than previously considered, as a potential multi-pathogen vector of both PWN and phytopathogenic fungi.

Symbiotic microbes aid host adaptation by metabolizing a deterrent host pine carbohydrate d -pinitol in a beetle-fungus invasive complex

The red turpentine beetle (RTB) is one of the most destructive invasive pests in China and solely consumes pine phloem containing high amounts of d-pinitol. Previous studies reported that d-pinitol exhibits deterrent effects on insects. However, it remains unknown how insects overcome d-pinitol during their host plant adaptation. We found that d-pinitol had an antagonistic effect on RTB, which mainly relied on gallery microbes to degrade d-pinitol to enhance host adaptation with mutualistic Leptographium procerum and two symbiotic bacteria, Erwinia and Serratia, responsible for this degradation. Genomic, transcriptomic, and functional investigations revealed that all three microbes can metabolize d-pinitol via different branches of the inositol pathway. Our results collectively highlight the contributions of symbiotic microbes in RTB's adaptation to living on pine, thereby facilitating outbreaks of RTB in China. These findings further enrich our knowledge of symbiotic invasions and contribute to the further understanding of plant-insect interactions.

Body size as a magic trait in two plant-feeding insect species.

When gene flow accompanies speciation, recombination can decouple divergently selected loci and loci conferring reproductive isolation. This barrier to sympatric divergence disappears when assortative mating and disruptive selection involve the same "magic" trait. Although magic traits could be widespread, the relative importance of different types of magic traits to speciation remains unclear. Because body size frequently contributes to host adaptation and assortative mating in plant-feeding insects, we evaluated several magic trait predictions for this trait in a pair of sympatric Neodiprion sawfly species adapted to different pine hosts. A large morphological dataset revealed that sawfly adults from populations and species that use thicker-needled pines are consistently larger than those that use thinner-needled pines. Fitness data from recombinant backcross females revealed that egg size is under divergent selection between the preferred pines. Lastly, mating assays revealed strong size-assortative mating within and between species in three different crosses, with the strongest prezygotic isolation between populations that have the greatest interspecific size differences. Together, our data support body size as a magic trait in pine sawflies and possibly many other plant-feeding insects. Our work also demonstrates how intraspecific variation in morphology and ecology can cause geographic variation in the strength of prezygotic isolation.

Host and abiotic constraints on the distribution of the pine fungal pathogen Sphaeropsis sapinea (= Diplodia sapinea)

Plant fungal pathogens are an increasing emerging threat as climate change progresses. Sphaeropsis sapinea (syn. Diplodia sapinea), the causal fungal agent of Diplodia tip blight, is a major pathogen of pines of forestry and ornamental relevance in Europe and worldwide. Here, we combined molecular-based field surveys in a common-garden setting and across an elevation gradient with historical records, cultivation-based growth experiments and microscopy to report on host and abiotic constraints on the distribution of S. sapinea. Using the arboretum at the Botanical Garden Berlin, Germany, to control for environmental variability, S. sapinea was detected on all seven host Pinus species we studied. However, P. sylvestris is the only species in which the fungus was detected in symptomless needles at the arboretum, and the most frequently recorded host for over a century, suggesting that it is the main, and perhaps, potential original host. In addition, sampling symptomatic needles in four out of the seven same species across a gradient from 200 to 2,100 m of elevation in the French Alps, S. sapinea was not detected at elevation higher than 800 m. Abiotic constraints were also supported by reduced growth of isolates of S. sapinea at low temperature under controlled conditions, but a 35°C prior stress exposure increased the subsequent growth of S. sapinea within its optimal temperature range (20-30°C). Altogether, our study thus not only suggests that S. sapinea is more likely to cause tip blight in P. sylvestris compared to the other species we studied, but also that in the current context of global climate change with predicted temperature increases, the fungus could infect a wider range of pine hosts and locations worldwide.

Diplodia sapinea infection reprograms foliar traits of its pine (Pinus sylvestris L.) host to death.

Infection with the necrotrophic fungus Diplodia sapinea (Fr.) Fuckel is among the economically and ecologically most devastating diseases of conifers in the northern hemisphere and is accelerated by global climate change. This study aims to characterize the changes mediated by D. sapinea infection on its pine host (Pinus sylvestris L.) that lead to the death of its needles. For this purpose, we performed an indoor infection experiment and inoculated shoot tips of pine seedlings with virulent D. sapinea. The consequences for foliar traits, including the phytohormone profile, were characterized at both the metabolite and transcriptome level. Our results showed that D. sapinea infection strongly affected foliar levels of most phytohormones and impaired a multitude of other metabolic and structural foliar traits, such as reactive oxygen species scavenging. Transcriptome analysis revealed that these changes are partially mediated via modified gene expression by fungal exposure. Diplodia sapinea appears to overcome the defense reactions of its pine host by reprogramming gene expression and post-transcriptional controls that determine essential foliar metabolic traits such as the phytohormone profile, cell wall composition and antioxidative system.

Studies on the Requirement of Transthyretin Protein (BxTTR-52) for the Suppression of Host Innate Immunity in Bursaphelenchus xylophilus

The pinewood nematode, Bursaphelenchus xylophilus, has been determined as one of the world's top ten plant-parasitic nematodes. It causes pine wilt, a progressive disease that affects the economy and ecologically sustainable development in East Asia. B. xylophilus secretes pathogenic proteins into host plant tissues to promote infection. However, little is known about the interaction between B. xylophilus and pines. Previous studies reported transthyretin proteins in some species and their strong correlation with immune evasion, which has also been poorly studied in B. xylophilus. In this study, we cloned and functionally validated the B. xylophilus pathogenic protein BxTTR-52, containing a transthyretin domain. An in situ hybridization assay demonstrated that BxTTR-52 was expressed mainly in the esophageal glands of B. xylophilus. Confocal microscopy revealed that BxTTR-52-RFP localized to the nucleus, cytoplasm, and plasma membrane. BxTTR-52 recombinant proteins produced by Escherichia coli could be suppressed by hydrogen peroxide and antioxidant enzymes in pines. Moreover, silencing BxTTR-52 significantly attenuated the morbidity of Pinus thunbergii infected with B. xylophilus. It also suppressed the expression of pathogenesis-related genes in P. thunbergii. These results suggest that BxTTR-52 suppresses the plant immune response in the host pines and might contribute to the pathogenicity of B. xylophilus in the early infection stages.

Identification and Characterization of a QTL for Growth of Fusarium circinatum on Pine-Based Medium.

Fusarium circinatum is an economically important pathogen of pine and resides in the Fusarium fujikuroi species complex. Here we investigated the molecular processes underlying growth in F. circinatum by exploring the association between growth and the nutritional environment provided by the pine host. For this purpose, we subjected a mapping population consisting of F. circinatum X F. temperatum hybrid progeny to an analysis of growth rate on a pine-tissue derived medium. These data, together with the available genetic linkage map for F. circinatum, were then used to identify Quantitative Trait Loci (QTLs) associated with growth. The single significant QTL identified was then characterized using the available genome sequences for the hybrid progeny's parental isolates. This revealed that the QTL localized to two non-homologous regions in the F. circinatum and F. temperatum genomes. For one of these, the F. circinatum parent contained a two-gene deletion relative to the F. temperatum parent. For the other region, the two parental isolates encoded different protein products. Analysis of repeats, G+C content, and repeat-induced point (RIP) mutations further suggested a retrotransposon origin for the two-gene deletion in F. circinatum. Nevertheless, subsequent genome and PCR-based analyses showed that both regions were similarly polymorphic within a collection of diverse F. circinatum. However, we observed no clear correlation between the respective polymorphism patterns and growth rate in culture. These findings support the notion that growth is a complex multilocus trait and raise the possibility that the identified QTL contains multiple small-effect QTLs, of which some might be dependent on the genetic backgrounds. This study improved our current knowledge of the genetic determinants of vegetative growth in F. circinatum and provided an important foundation for determining the genes and processes underpinning its ability to colonize its host environment.

Integrating Multi-Scale Remote-Sensing Data to Monitor Severe Forest Infestation in Response to Pine Wilt Disease

Pine wilt disease (PWD) is one of the most destructive forest diseases that has led to rapid wilting and mortality in susceptible host pine trees. Spatially explicit detection of pine wood nematode (PWN)-induced infestation is important for forest management, policy making, and practices. Previous studies have mapped forest disturbances in response to various forest diseases and/or insects over large areas using remote-sensing techniques, but these efforts were often constrained by the limited availability of ground truth information needed for the calibration and validation of moderate-resolution satellite algorithms in the process of linking plot-scale measurements to satellite data. In this study, we proposed a two-level up-sampling strategy by integrating unmanned aerial vehicle (UAV) surveys and high-resolution Radarsat-2 satellite imagery for expanding the number of training samples at the 30-m resampled Sentinel-1 resolution. Random forest algorithms were separately used in the prediction of the Radarsat-2 and Sentinel-1 infestation map induced by PWN. After data acquisition in Muping District during August and September 2021, we first verified the ability of a deep-learning-based object detection algorithm (i.e., YOLOv5 model) in the detection of infested trees from coregistered UAV-based RGB images (Average Precision (AP) of larger than 70% and R2 of 0.94). A random forest algorithm trained using the up-sampling UAV infestation map reference and corresponding Radarsat-2 pixel values was then used to produce the Radarsat-2 infestation map, resulting in an overall accuracy of 72.57%. Another random forest algorithm trained using the Radarsat-2 infestation pixels with moderate and high severity (i.e., an infestation severity of larger than 0.25, where the value was empirically set based on a trade-off between classification accuracy and infection detectability) and corresponding Sentinel-1 pixel values was subsequently used to predict the Sentinel-1 infestation map, resulting in an overall accuracy of 87.63%, where the validation data are Radarsat-2 references rather than UAV references. The Sentinel-1 map was also validated by independent UAV surveys, with an overall accuracy of 76.30% and a Kappa coefficient of 0.45. We found that the expanded training samples by the integration of UAV and Radarsat-2 strengthened the medium-resolution Sentinel-1-based prediction model of PWD. This study demonstrates that the proposed method enables effective PWN infestation mapping over multiple scales.

Feeding Preferences and Responses of Monochamus saltuarius to Volatile Components of Host Pine Trees.

Simple SummaryBursaphelenchus xylophilus causes pine wilt disease, which is one of the most devastating forest diseases in the world. However, B. xylophilus cannot spread naturally and must be carried by vector insects from infested trees to healthy ones. Monochamus saltuarius is a B. xylophilus vector that has caused massive pine tree mortality in Japan, South Korea, and China’s middle temperate zone. In China, there have been only a few reports of this species. The feeding preference of M. saltuarius on different host pine trees was determined, and the volatile components of the host pine trees were analyzed. Furthermore, the responses of M. saltuarius to different host volatiles were determined, and the correlation between M. saltuarius preference and host pine tree volatiles was investigated. This study will serve as a reference for the development and application of M. saltuarius attractants, which will aid in the control of M. saltuarius and pine wilt disease.Monochamus saltuarius is a vector of Bursaphelenchus xylophilus and has caused massive mortality in pine trees in Japan, South Korea, and the middle temperate zone of China. In this study, the feeding preferences of M. saltuarius to four common host pine species in the middle temperate zone of China were investigated in a laboratory. The volatiles of the four host pine species were sampled and analyzed by gas chromatography and mass spectrometry. The responses of M. saltuarius to host pine tree volatiles were measured using a four-arm olfactometer. The results showed that: (1) the preference of M. saltuarius for Pine tabulaeformis was significantly higher than the other ones. (2) The composition and content of the volatiles of the four tested host pine twigs were different, and the relative content of α-pinene was the highest. (3) M. saltuarius was highly sensitive to α-pinene, β-pinene, limonene, and 3-carene, and the attracting effect was enhanced by the mixture of volatile components. This study provides a reference for the development and application of attractants for M. saltuarius. Our results would be helpful in controlling M. saltuarius and pine wilt disease.

The Detection of Pine Wilt Disease: A Literature Review.

Pine wilt disease (PWD) is a global quarantine disease of forests that mainly affects Pinaceae species. The disease spreads rapidly. Once infected, pine trees have an extremely high mortality rate. This paper provides a summary of the common techniques used to detect PWD, including morphological-, molecular-, chemical- and physical-based methods. By comprehending the complex relationship among pinewood nematodes, vectors and host pine trees and employing the available approaches for nematode detection, we can improve the implementation of intervention and control measures to effectively reduce the damage caused by PWD. Although conventional techniques allow a reliable diagnosis of the symptomatic phase, the volatile compound detection and remote sensing technology facilitate a rapid diagnosis during asymptomatic stages. Moreover, the remote sensing technology is capable of monitoring PWD over large areas. Therefore, multiple perspective evaluations based on these technologies are crucial for the rapid and effective detection of PWD.

Mapping edaphic soils' conditions to identify conservation targets for pine barren and sandplain ecosystems in New York State.

Small habitat patches can be important reservoirs for biodiversity, capable of hosting unique species that are largely absent from the surrounding landscape. In cases where such patches owe their existence to the presence of particular soil types or hydrologic conditions, local‐scale edaphic variables may be more effective components for models that identify patch location than regional‐scale macroclimatic variables often used in habitat and species distribution models. We modeled the edaphic soil conditions that support pine barren, sandplain, and related ecosystems in New York State with the purpose of identifying potential locations for biodiversity conservation. We quantified soil percent sand and soil depth of 156 known high‐quality remnant pine barren and sandplain ecosystems to calculate threshold soil characteristics. We then mapped all soils in the state that were at least as sandy and deep as the threshold values we calculated. The total area of our map of suitable soil conditions was over 9500 km2, made up of forested (57%), urban (26%), agricultural (13%), and open (4%) land covers. Our analysis nearly doubled the recognized area of barren, shrubland, and grassland habitat on deep, sandy soils in New York State. Extensive forested and even agricultural cover on these soils could also be the subject of restoration to further support the biodiversity of these unique ecosystems. The presence of extensive soils in coastal and interior New York that, with the appropriate disturbance regime, have the potential to host pine barren and sandplain ecosystems offers a new perspective on these ecosystems' distribution in the past—and about how to better align conservation and restoration to preserve the future.

Research Progress on Biocontrol of Pine Wilt Disease by Microorganisms

Pine wilt disease is a malady caused by a complex interaction of various factors such as pine wood nematodes, host plants, vector insects, associated fungi and bacteria, human economic and logistics activities, and environmental factors. The use of microorganisms to biologically control pine wilt disease is a potentially environmentally friendly means for the prevention and control of the disease. In this study, we carried out a systematic review of the progress in research on the biocontrol of pine wilt disease, by focusing on the pathogenic pine wood nematode, its vector beetle, and the host pine tree species. Then, we discuss the implementation prospects and research trends associated with the biocontrol of pine wood disease. This study provides reference information for the understanding and application of various biocontrol microorganisms in the prevention and control of pine wood disease and for the establishment of an environmentally friendly prevention and control strategy.

A Bursaphelenchus xylophilus Effector, BxSCD3, Suppresses Plant Defense and Contributes to Virulence.

Bursaphelenchus xylophilus is the most economically important species of migratory plant-parasitic nematodes (PPNs) and causes severe damage to forestry in China. The successful infection of B. xylophilus relies on the secretion of a repertoire of effector proteins. The effectors, which suppress the host pine immune response, are key to the facilitation of B. xylophilus parasitism. An exhaustive list of candidate effectors of B. xylophilus was predicted, but not all have been identified and characterized. Here, an effector, named BxSCD3, has been implicated in the suppression of host immunity. BxSCD3 could suppress pathogen-associated molecular patterns (PAMPs) PsXEG1- and INF1-triggered cell death when it was secreted into the intracellular space in Nicotiana benthamiana. BxSCD3 was highly up-regulated in the early infection stages of B. xylophilus. BxSCD3 does not affect B. xylophilus reproduction, either at the mycophagous stage or the phytophagous stage, but it contributes to the virulence of B. xylophilus. Moreover, BxSCD3 significantly influenced the relative expression levels of defense-related (PR) genes PtPR-3 and PtPR-6 in Pinus thunbergii in the early infection stage. These results suggest that BxSCD3 is an important toxic factor and plays a key role in the interaction between B. xylophilus and host pine.

Invasion History of the Pinewood Nematode Bursaphelenchus xylophilus Influences the Abundance of Serratia sp. in Pupal Chambers and Tracheae of Insect-Vector Monochamus alternatus.

Pine wilt disease (PWD) has caused extensive mortality in pine forests worldwide. This disease is a result of a multi-species interaction among an invasive pinewood nematode (PWN) Bursaphelenchus xylophilus, its vector Monochamus sp. beetle, and the host pine tree (Pinus sp.). In other systems, microbes have been shown to attenuate negative impacts on invasive species after the invasion has reached a certain time point. Despite that the role of PWD associated microbes involved in the PWD system has been widely studied, it is not known whether similar antagonistic “hidden microbial players” exist in this system due to the lack of knowledge about the potential temporal changes in the composition of associated microbiota. In this study, we investigated the bacteria-to-fungi ratio and isolated culturable bacterial isolates from pupal chambers and vector beetle tracheae across five sampling sites in China differing in the duration of PWN invasion. We also tested the pathogenicity of two candidate bacteria strains against the PWN-vector beetle complex. A total of 118 bacterial species belonging to 4 phyla, 30 families, and 54 genera were classified based on 16S sequencing. The relative abundance of the genus Serratia was lower in pupal chambers and tracheae in newly PWN invaded sites (<10 years) compared to the sites that had been invaded for more than 20 years. Serratia marcescens strain AHPC29 was widely distributed across all sites and showed nematicidal activity against PWN. The insecticidal activity of this strain was dependent on the life stage of the vector beetle Monochamus alternatus: no insecticidal activity was observed against final-instar larvae, whereas S. marcescens was highly virulent against pupae. Our findings improved the understanding of the temporal variation in the microbial community associated with the PWN-vector beetle complex and the progress of PWD and can therefore facilitate the development of biological control agents against PWN and its vector beetle.

Novel Functional Analysis for Pathogenic Proteins of Bursaphelenchus xylophilus in Pine Seed Embryos Using a Virus Vector.

Pine wilt disease (PWD), which is caused by the pine wood nematode Bursaphelenchus xylophilus, is among the most serious tree diseases worldwide. PWD is thought to be initiated by sequential excessive hypersensitive responses to B. xylophilus. Previous studies have reported candidate pathogenic molecules inducing hypersensitive responses in pine trees susceptible to B. xylophilus. The functions of some of these molecules have been analyzed in model plants using transient overexpression; however, whether they can induce hypersensitive responses in natural host pines remains unclear due to the lack of a suitable functional analysis method. In this study, we established a novel functional analysis method for susceptible black pine (Pinus thunbergii) seed embryos using transient overexpression by the Apple latent spherical virus vector and investigated five secreted proteins of B. xylophilus causing cell death in tobacco to determine whether they induce hypersensitive responses in pine. We found that three of five molecules induced significantly higher expression in pathogenesis-related genes ( p < 0.05), indicating hypersensitive response in pine seed embryos compared with mock and green fluorescence protein controls. This result suggests that tobacco-based screening may detect false positives. This study is the first to analyze the function of pathogenic candidate molecules of B. xylophilus in natural host pines using exogenous gene expression, which is anticipated to be a powerful tool for investigating the PWD mechanism.

An Ips bark beetle tracks late Holocene range extension of its pinyon pine host.

The authors declare no conflict of interest. Voucher specimens in the William F. Barr Entomological Museum at The University of Idaho are available by requesting I. confusus collected by James Cane from City of Rocks National Reserve (Idaho) on 2 June and 3 June 2020. Appendix S1 Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.