Development Of Pine Wilt Disease Research Articles

Research progress on the resistance mechanism of host pine to pine wilt disease

AbstractPine wilt disease, caused by Bursaphelenchus xylophilus, is a major quarantine forest disease that has resulted in massive economic losses as well as ecological disaster. Therefore, it is imperative to study the defence response mechanism of host pine trees to infection by B. xylophilus to understand further the internal causes of pine tree death and to find control strategies. For many years, systematic research has been carried out on the pathophysiological response of pine trees to pine wilt disease. However, due to the complexity of the occurrence and development of pine wilt disease, the specific response mechanisms of different tree species have remained unclear. This paper attempts to provide an overview of the resistance mechanism of host pine trees against B. xylophilus infection through the perspectives of histopathological characteristics, physiological and biochemical responses, and molecular response mechanisms, which provide a theoretical reference for further investigating the disease resistance mechanism of pine trees to pine wilt disease and lay the foundation for the prevention and quarantine of pine wilt disease.

New discovery on the nematode activity of aureothin and alloaureothin isolated from endophytic bacteria Streptomyces sp. AE170020

Endophytic bacteria, a rich source of bioactive secondary metabolites, are ideal candidates for environmentally benign agents. In this study, an endophytic strain, Streptomyces sp. AE170020, was isolated and selected for the purification of nematicidal substances based on its high nematicidal activity. Two highly active components, aureothin and alloaureothin, were identified, and their chemical structures were determined using spectroscopic analysis. Both compounds suppressed the growth, reproduction, and behavior of Bursaphelenchus xylophilus. In in vivo experiments, the extracts of strain Streptomyces sp. AE170020 effectively suppressed the development of pine wilt disease in 4-year-old plants of Pinus densiflora. The potency of secondary metabolites isolated from endophytic strains suggests applications in controlling Bursaphelenchus xylophilus and opens an avenue for further research on exploring bioactive substances against the pine wood nematode.

Screening, isolation and evaluation of a nematicidal compound from actinomycetes against the pine wood nematode, Bursaphelenchus xylophilus.

Bursaphelenchus xylophilus is a migratory endoparasitic nematode known to cause severe environmental damage and economic losses in pine forest ecosystems. This present study investigated the nematicidal metabolites of actinomycetes in vitro and evaluated the disease control efficacy of the active compound and metabolites under greenhouse and field conditions. Five thousand types of actinobacteria from Korean forest soil samples were screened to identify novel nematicidal agents against the pine wood nematode. Streptomyces sp. AN091965 showed the strongest nematicidal activity. One active compound, spectinabilin, was obtained by nematicidal asssy-directed fractionation, and it showed significant nematicidal activity against B. xylophilus, with an LC50 value of 0.84 µg mL-1 . Spectinabilin effectively suppressed the development of pine wilt disease in 5-year-old Pinus densiflora trees, even at 0.9mg per tree under greenhouse conditions. Moreover, the acetone extract of the active strain's mycelia efficiently suppressed the development of pine wilt disease under field conditions. To the best of our knowledge, this the first report to describe the nematicidal activity of spectinabilin against B. xylophilus. The cell extracts described herein merit further field studies as potential nematicides against the pine wood nematode. © 2018 Society of Chemical Industry.

Bacterial Diversity and Community Structure in the Pine Wood Nematode Bursaphelenchus xylophilus and B. mucronatus with Different Virulence by High-Throughput Sequencing of the 16S rDNA.

Bursaphelenchus xylophilus is the pathogen of pine wilt disease. Bursaphelenchus mucronatus is similar to B. xylophilus in morphology. Both species share a common niche, but they are quite different in pathogenicity. Presently, the role of bacteria in pine wilt disease development has been widely speculated. The diversity of bacteria associated with B. xylophilus and B. mucronatus with different virulence remains unclear. In this study, virulence of four B. xylophilus and four B. mucronatus strains were evaluated by inoculating Pinus thunbergii. High-throughput sequencing targeted 16S rDNA of different virulence nematode strains was carried out. The associated bacterial community structures of the eight strains were analyzed. The results showed that 634,051 high-quality sequences were obtained from the eight nematode strains. The number of OTUs of bacteria associated with B. mucronatus was generally greater than those of B. xylophilus. The richness of the community of bacteria associated with high virulent B. xylophilus ZL1 and AmA3 was higher than moderately virulent B. xylophilus AA3, HE2, and all B. mucronatus strains. While the diversity of bacteria associated with B. mucronatus was higher than B. xylophilus. Stenotrophomonas, Pseudomonadaceae_Unclassified or Rhizobiaceae_Unclassified were predominant in the nematode strains with different virulence. Oxalobacteraceae and Achromobacter were found more abundant in the low virulent B. xylophilus and non-virulent B. mucronatus strains.

Relationship between pine wilt disease development in asymptomatic carrier trees of Bursaphelenchus xylophilus (Nematoda: Apelenchoididae) and their use by Monochamus alternatus (Coleoptera: Cerambycidae)

To clarify the effect of asymptomatic carrier trees on the epidemic spread of pine wilt disease, we inoculated Bursaphelenchus xylophilus (Steiner and Buhrer) Nickle into ~10-year-old Pinus thunbergii Parl. trees to make asymptomatic carrier trees of the nematodes, and then we examined the relationship between pine wilt disease development in these trees and their use by Monochamus alternatus Hope. Mature adults of M. alternatus were attracted by and oviposited on trees that died in summer and fall, regardless of whether they died in the year of nematode infection or were asymptomatic during the infection year and died the next year. These results suggest that trees that die in summer and fall should be disposed of to help control M. alternatus and limit epidemic spread of the disease. In addition, we suggest the possibility that M. alternatus larvae may not grow and develop in trees that die after the infection year or that M. alternatus adults that emerge from such trees do not carry many B. xylophilus.

Bacterial role in pine wilt disease development - review and future perspectives.

Mutualistic and beneficial relationships between nematodes and bacteria are highly present in nature, mostly occurring because of nutritional dependence and pathogen protection, and intrinsically related with the environment, the ecological conditions and the nematode life stages. Thirty-four years have passed since the first hypothesis suggesting a bacterial role in pine wilt disease (PWD), associated with the pinewood nematode (PWN), Bursaphelenchus xylophilus. In 1980, researchers reported that bacteria associated with the PWN could produce toxins that lead to PWD development in pine seedlings. It was also suggested a double vector system for PWD, where bacteria were vectored by the PWN and the PWN vectored by an insect from the Monochamus genus. Presently, the specific involvement of bacteria in such complex disease is still controversial, even though the increased number of studies focused on the potential bacteria role has increased considerably. This review is an up-to-date comprehensive perspective and brings new insights on the role of PWN-associated bacteria in PWD.

Suppressive effects of Bursaphelenchus mucronatus on pine wilt disease development and mortality of B. xylophilus-inoculated pine seedlings

Bursaphelenchus xylophilus causes pine wilt disease in Pinus trees whereas B. mucronatus has no or little virulence to the trees. Interspecific crossing experiments conducted so far suggest reproductive interference between the two nematode species. Theory predicts that one of the two competing species populations quickly displaces the other through reproductive interference in a frequency-dependent manner. Thus, it is anticipated that B. mucronatus suppresses the virulence of B. xylophilus against pine trees when B. mucronatus heavily outnumber B. xylophilus. To determine the suppressive effects of B. mucronatus, the two nematode species were inoculated simultaneously on 30 3-year-old Pinus thunbergii seedlings at three combinations of different numbers, and B. xylophilus alone was inoculated on 30 other seedlings at the corresponding numbers in early August. Seedlings were observed at intervals of 4 or 6 weeks and two stem sections were sampled from each seedling to determine the density and species composition of nematode populations after death or in December. Inoculation of B. mucronatus significantly retarded the speed of foliage discolouration from 0.170 ± 0.024 week−1 to 0.061 ± 0.017 week−1 and significantly prolonged the survival time of seedlings. Bursaphelenchus mucronatus significantly reduced the nematode density from 1799.7 ± 305.0 to 521.0 ± 148.4 (g dried seedling stem)−1. Analysis of rDNA genotype showed 1846 B xylophilus, no B. mucronatus and one hybrid.

The risk of pine wilt disease in Poland

The pine wood nematode (PWN) Bursaphelenchus xylophilus (Steiner et Buhrer) Nickle (Aphelenchida, Parasitaphelenchidae), causing the pine wilt disease (PWD), is a quarantine species of Europe that was found in Portugal in 1999. High ambient temperature (above 20° C) is considered to be one of the main factors enhancing the development of PWD in pine stands. In the early 90s of the last century, Poland was considered a country of low risk of PWN establishment. The aim of our study was to verify whether this status has changed due to the effects of climate change. We analysed changes in monthly mean ambient temperatures in June, July and August from 75 weather stations in various regions of Poland from 2005– 2010. Additional analyses of changes in monthly mean temperatures in July and August in the period 1991– 2010 were performed for the weather station where the highest temperatures were recorded. Multi-year (2005– 2010) average of mean temperatures in June did not exceed 20° C at any of the meteorological stations. However, in July it was higher than the threshold temperature at 26 meteorological stations located in the central part of Poland. The highest multi-year average temperatures were recorded at the station in Kolo (52° 12’ N, 18° 38’ E). A detailed analysis of July and August temperature changes at this station during the period 1991– 2010 revealed increasing trends that were described by polynomial functions. The most substantial increase has been observed since 2001. The mean temperature in July and August over the last decade (2001– 2010) has increased by 2.4 and 1.5° C respectively when compared to the previous decade. It is our conclusion that, although the PWN has not yet been found in Poland, the country should no longer be regarded as an area of low risk due to the effects of summer temperature increase. Other factors favourable for the pine wilt disease development and spread were discussed.

Bacteria associated with the pinewood nematode Bursaphelenchus xylophilus collected in Portugal

In this study, we report on the bacterial community associated with the pinewood nematode Bursaphelenchus xylophilus from symptomatic pine wilted trees, as well as from long-term preserved B. xylophilus laboratory collection specimens, emphasizing the close bacteria-nematode associations that may contribute to pine wilt disease development.

Applying a spread model to identify the entry points from which the pine wood nematode, the vector of pine wilt disease, would spread most rapidly across Europe

Pine wilt disease, which can rapidly kill pines, is caused by the pine wood nematode, Bursaphelenchus xylophilus. It is expanding its range in many countries in Asia and measures are being taken at the EU level to prevent its spread from Portugal. Due to the threat to European forests, it is important to prevent additional introductions and target surveillance to the points of entry that pose the greatest risk. In this study, we present a model to identify the European ports from which the nematode can spread most rapidly across Europe. This model describes: (1) the potential spread of the pine wood nematode based on short-distance spread (the active flight of the vector beetles) and long-distance spread (primarily due to human-mediated transportation), and (2) the development of pine wilt disease based on climate suitability and the potential spread of the nematode. Separate introductions at 200 European ports were simulated under various climate change scenarios. We found that the pine wood nematode could invade 19–60% of the study area (30°00 N–72°00 N, 25°00 W–40°00 E) by 2030, with the highest spread from ports located in Eastern and Northern Europe. Based on climate change scenarios, the disease could affect 8–34% of the study area by 2030, with the highest spread from ports located in South-Eastern Europe. This study illustrates how a spread model can be used to determine the critical points of entry for invasive species, so that surveillance can be targeted more accurately and control measures prioritised.

Pyochelin isolated from Burkholderia arboris KRICT1 carried by pine wood nematodes exhibits phytotoxicity in pine callus

Abstract Pine wilt disease is a very complex disease known to be caused by the pine wood nematode, Bursaphelenchus xylophilus, and its accompanying bacteria. The phytotoxin-producing bacteria have been reported to be involved in the development of pine wilt disease. In this study, we attempted to characterise phytotoxins produced by Burkholderia arboris KRICT1, which was carried by the pine wood nematode. It produced phytotoxic compounds in the pine seedling assay, and the ethyl acetate (EtOAc) layer of the fermentation broth of the strain displayed phytotoxic activity on the pine callus of Pinus densiflora. One active compound was isolated from the EtOAc layer by repeated Sephadex LH-20 column chromatography and preparative TLC. Using mainly mass spectroscopy and nuclear magnetic resonance (NMR) spectroscopy, the chemical structure was determined to be that of pyochelin (a mixture of two interconvertible diastereoisomers, pyochelin I and II). Pyochelin exposure reduced the viability of pine callus. Compared with phenylacetic acid, a phytotoxin produced by Bacillus spp. that was transmitted by B. xylophilus in Japan, pyochelin showed much stronger phytotoxicity. The results suggested that pyochelin might play a role in the wilting process of pine wilt disease.

Guidance on sampling to detect pine wood nematode Bursaphelenchus xylophilus in trees, wood and insects

The pine wood nematode Bursaphelenchus xylophilus is the causal agent of pine wilt disease. It is a quarantine pest for most countries in the world. Surveys for the occurrence of the nematode may be necessary in the framework of internationally agreed phytosanitary standards, in order to delimit infested areas or to demonstrate the absence of B. xylophilus and thus to identify pest free areas. Import inspections of wood and wood products also need sound monitoring systems. It is obvious that the quality of survey/monitoring, as well as the results of diagnosis, are strongly dependent on the sampling procedure. Although, in the literature, numerous results of surveys and monitoring are documented, sampling procedures vary and there is no scientific or statistically based sampling system published for B. xylophilus. The current paper relates background information concerning the biology of the nematode, its vector beetles and the development of pine wilt disease and the influence of these factors on sampling procedures, and attempts to define a more systematic methodology.

Relationship between resistance to pine wilt disease and the migration or proliferation of pine wood nematodes

To study the relationship between resistance to pine wilt disease and the migration or proliferation of pine wood nematodes (PWN) (Bursaphelenchus xylophilus), we conducted experiments using clonally-propagated Japanese black pines (Pinus thunbergii) with pre-evaluated individual resistance levels. Bark including the cortical resin canals—one of the main migration pathways of PWN—was removed by girdling, but neither the migration of PWN nor symptom development of pine wilt disease were inhibited by this treatment in non-resistant clones. Histological observations showed no significant differences in the lumen area or the number of cortical- and xylem- axial resin canals between resistant and susceptible clone groups from a half-sib family. A bioassay using methanol extracts from resistant and susceptible clones showed that extracts from both clones showed similar attractant effects to PWN, but neither had repellent effects. The resistant clones were multi-inoculated with PWN into three split points to mimic migration in the stem. The proportion of damaged plants was not significantly different from that in single-inoculated plants (control). In this experiment, the number of PWN detected from partially-damaged plants was much higher than that from non-damaged plants. An inoculation test using stem cuttings showed that the population of PWN increased in susceptible cuttings at 1–20 days after inoculation (dai), while it remained unchanged or gradually decreased in resistant cuttings. These findings suggested that the factors contributing to resistance were associated with inhibiting the proliferation of PWN, rather than inhibiting their migration.

Effects of inoculum density of pinewood nematode on the development of pine wilt disease in Japanese black pine seedlings pretreated with simulated acid rain

SummarySix‐month‐old Japanese black pine seedlings (Pinus thunbergii) were exposed to simulated acid rain (SAR) at pH 3 and 2 three times a week. After treatment for 2 months, the seedlings were inoculated with a virulent isolate (S10) of the pinewood nematode (Bursaphelenchusxylophilus), at three inoculum levels (Pi = 50, 160 or 500 nematodes per seedling). In seedlings inoculated with 500 nematodes, both population growth of nematodes and disease development were accelerated by pretreatment with SAR at pH 3 or 2. In seedlings inoculated with 50 nematodes, population growth of the nematodes was suppressed and more time was needed for seedlings to die when pretreated with pH 3 SAR. This suggests that exposure to pH 3 SAR increased not only the progress of mortality, but also simultaneously enhanced the tolerance limit of the seedlings to the pinewood nematode – the critical value of physiological burden (represented as a product of time and initial nematode population) necessary to kill a seedling. Exposure to pH 2 SAR accelerated nematode reproduction in seedlings and increased seedling mortality irrespective of the number of nematodes inoculated.

Physiology and molecular characteristics of a pine wilt nematode-trapping fungus, Monacrosporium megalosporum.

We isolated the nematode-trapping fungus Monacrosporium megalosporum from nature and examined its morphology, physiology and molecular characteristics. The nematode-trapping device of this fungus is a three-dimensional network. This fungus captures the pine wilt nematode (Bursaphelenchus xylophilus), but not a non-phytopathogenic nematode that is morphologically similar to B. xylophilus. The phylogenic relationship of the nucleotide sequence of the rDNA ITS region was close to those of M. thaumasium and Geniculifera eudermata, which also have nematode-trapping devices that are three-dimensional networks. Acidic pH inhibited both the liberation and regeneration of protoplasts. Moreover, cytoplasmic granulation of protoplasts was found below pH 6.0. Mycelial growth on agar media was also inhibited below pH 4, but not at pH 9. These results strongly suggest that the activity of this fungus is inhibited by acid rain in the field. Therefore, development of pine wilt disease might be a secondary effect of acid rain.

The effects of long‐term exposure to simulated acid rain on the development of pine wilt disease caused by Bursaphelenchus xylophilus

To elucidate the synergetic effects of acid rain on the development of pine wilt disease, we measured the sap flow rate in the stems and the chlorophyll content in the needles of 10‐year‐old Japanese black pine trees, Pinus thunbergii and 12‐year‐old Japanese red pine trees, Pinus densiflora, after exposure to simulated acid rain (SAR, pH 3) or tap water (TW, pH 6.3) as a control. The heat pulse method was used for the estimation of the sap flow rate. No apparent difference was found in the sap flow rate between the trees exposed to SAR and TW, but the chlorophyll content of needles at the end of the treatment was significantly higher in the trees exposed to SAR than in those exposed to TW. When the pinewood nematode, Bursaphelenchus xylophilus, the causal agent of pine wilt disease, was inoculated onto the Japanese black pines that had been exposed to SAR repeatedly for 1 year, the period to death was shortened. Japanese red pines that had been exposed to SAR for 2 years, however, did not show any development of symptoms after the nematode inoculation, suggesting that acid rain only affects pine wilt disease slightly, if at all.

Different Development of Pine Wilt Disease in Artificially Infected Pinus thunbergii Seedlings Potted together with Different Tree Species

To evaluate the effect of adjacent tree species on the susceptibility of Japanese black pine (Pinus thunbergii) to pine wilt disease, an inoculation experiment was conducted using P. thunbergii seedlings potted with seedlings of six tree species, i. e. Alnus sieboldiana, Eurya japonica, Lespedeza bicolor form acutifolia, Pinus thumbergii, Robinia pseudo-acacia and Sarothamus scoparius. About ten months after planting, they were inoculated with the pinewood nematode (Bursaphelenchus xylophilus) in early July 1992. After that, the proportion of pine seedlings with completely discolored foliage increased more quickly when the seedlings were potted with R. pseudo-acacia, S. scoparius or A. sieboldiana than when potted with P. thunbergii, L. bicolor or E. japonica. At the end of the study period, 17 weeks after inoculation, it reached 90.6%, 90.0%, 87.5%, 72.7%, 63.3%, and 59.4% when the pine seedlings were potted with R. pseudo-acacia, S. scoparius, A. sieboldiana, P. thunbergii, L. bicolor and E. japonica, respectively. This indicated that the susceptibility of P. thunbergii seedlings to pine wilt disease was influence by the species of adjacent trees.

Changes in Nematode Population and Movement in Pine Seedlings with the Development of Pine Wilt Disease : Suppressive Effect of Water Extracts from Infected Pine Seedlings on the Movement of Nematodes

Pinus thunbergii (susceptible to pine wilt) and P. taeda (resistant species) seedlings were inoculated with one of the following three Bursaphelenchus isolates; one pathogenic (B. xylophilus) and two nonpathogenic (B. xylophilus and B. mucronatus) isolates. The effect of water extracts from the nematode-inoculated seedlings on the nematodes' movement was tested in relation to the dynamic resistance induced in the host. In every water extract from seedlings of either pine species, the movement of all isolates was inhibited, however, the inhibition to pathogenic isolates diminished later on. The results indicate that some substance(s) responsible for the inhibition of nematode movement was produced in the stems of the inoculated pine seedlings. The substance was denatured as the disease progressed, and movement of the pathogenic isolate became free from inhibition, while that of two nonpathogenic isolated did not. Regardless of the nematode isolate inoculated, extracts from P. taeda were somewhat more suppressive to nematode movement than those from P. thunbergii, although the differential manner of inhibition against the three nematode isolates was similar. Nematode migration and proliferation in stems of the seedlings, decline of water content and resin exudation of the stems were confirmed only when susceptible pine seedlings were inoculated with the pathogenic isolate.

Xylem Dysfunction in Bursaphelenchus xylophilus-infected Pinus thunbergii in Relation to Xylem Cavitation and Water Status.

The occurrence of cavitation in Bursaphelenchus xylophilus-inoculated Pinus thunbergii was evaluated by analyzing acoustic emissions and water status in pine saplings. As determined from changes in the minimum xylem pressure potential, pine wilt disease development occurred in two stages. A small amount of cavitation occurred during the latter half of the first stage of disease development. As the infection progressed, cavitation increased. The occurrence of cavitation was greater after B. xylophilus inoculation when the xylem pressure potential was constant. The presence of B. xylophilus in pine increased the plant's potential for cavitation. In the second stage of disease development, a large increase in cavitations in the tracheids led to catastrophic destruction of the water conducting pathway. These findings support the idea of “runaway embolism” having a major role in mechanism of induction of pine wilt disease.

Migration speed of pine wood nematodes and activities of other microbes during the development of pine-wilt disease in Pinus thunbergii

Migration speed of pine wood nematodes and activities of other microbes during the development of pine-wilt disease in Pinus thunbergii