Comparative Secretome and Functional Analyses Reveal Glycoside Hydrolase Family 30 and Cysteine Peptidase as Virulence Determinants in the Pinewood Nematode Bursaphelenchus xylophilus.

Pine wilt disease, caused by the pinewood nematode, Bursaphelenchus xylophilus, is one of the world's most serious tree diseases. Although the B. xylophilus whole-genome sequence and comprehensive secretome profile have been determined over the past decade, it remains unclear what molecules are critical in pine wilt disease and govern B. xylophilus virulence in host pine trees. Here, a comparative secretome analysis among four isolates of B. xylophilus with distinct virulence levels was performed to identify virulence determinants. The four candidate virulence determinants of B. xylophilus highly secreted in virulent isolates included lipase (Bx-lip1), glycoside hydrolase family 30 (Bx-GH30), and two C1A family cysteine peptidases (Bx-CAT1 and Bx-CAT2). To validate the quantitative differences in the four potential virulence determinants among virulence groups at the protein level, we used real-time reverse-transcription polymerase chain reaction analysis to investigate these determinants at the transcript level at three time points: pre-inoculation, 3 days after inoculation (dai), and 7 dai into pine seedlings. The transcript levels of Bx-CAT1, Bx-CAT2, and Bx-GH30 were significantly higher in virulent isolates than in avirulent isolates at pre-inoculation and 3 dai. A subsequent leaf-disk assay based on transient overexpression in Nicotiana benthamiana revealed that the GH30 candidate virulent factor caused cell death in the plant. Furthermore, we demonstrated that Bx-CAT2 was involved in nutrient uptake for fungal feeding via soaking-mediated RNA interference. These findings indicate that the secreted proteins Bx-GH30 and Bx-CAT2 contribute to B. xylophilus virulence in host pine trees and may be involved in pine wilt disease.

Secretome Analysis of the Pine Wood Nematode Bursaphelenchus xylophilus Reveals the Tangled Roots of Parasitism and Its Potential for Molecular Mimicry.

Since it was first introduced into Asia from North America in the early 20(th) century, the pine wood nematode Bursaphelenchus xylophilus has caused the devastating forest disease called pine wilt. The emerging pathogen spread to parts of Europe and has since been found as the causal agent of pine wilt disease in Portugal and Spain. In 2011, the entire genome sequence of B. xylophilus was determined, and it allowed us to perform a more detailed analysis of B. xylophilus parasitism. Here, we identified 1,515 proteins secreted by B. xylophilus using a highly sensitive proteomics method combined with the available genomic sequence. The catalogue of secreted proteins contained proteins involved in nutrient uptake, migration, and evasion from host defenses. A comparative functional analysis of the secretome profiles among parasitic nematodes revealed a marked expansion of secreted peptidases and peptidase inhibitors in B. xylophilus via gene duplication and horizontal gene transfer from fungi and bacteria. Furthermore, we showed that B. xylophilus secreted the potential host mimicry proteins that closely resemble the host pine's proteins. These proteins could have been acquired by host-parasite co-evolution and might mimic the host defense systems in susceptible pine trees during infection. This study contributes to an understanding of their unique parasitism and its tangled roots, and provides new perspectives on the evolution of plant parasitism among nematodes.

Pine wood nematode, Bursaphelenchus xylophilus.

After devastating vast areas of pine forests in Asian countries, the pine wilt disease spread into European forests in 1999 and is causing worldwide concern. This disease involves very complicated interactions between a pathogenic nematode, its vector beetle, host pine species, and fungi in dead hosts. Pathogenicity of the pine wood nematode is determined not only by its physical and chemical traits but also by its behavioral traits. Most life history traits of the pine wood nematode, such as its phoretic relationship with vector beetles, seem to be more effective in virulent than in avirulent isolates or species. As the pathogenicity determinants, secreted enzymes, and surface coat proteins are very important, they have therefore been studied intensively. The mechanism of quick death of a large pine tree as a result of infection by a tiny nematode could be ascribed to the dysfunction of the water-conducting system caused by the death of parenchyma cells, which must have originally evolved as an inherent resistant system.

Making headway in understanding pine wilt disease: what do we perceive in the postgenomic era?

The advent of next generation sequencing has revolutionized research approaches to biology by making entire genome sequences available and marking a new age in biology that has the potential to open innovative research avenues in various fields. Genome sequencing is now being applied in the fields of forest ecology and forest pathology, which previously had limited access to molecular techniques. One of the most advanced areas of progress is the study of "pine wilt disease", which is caused by the parasitic nematode, Bursaphelenchus xylophilus. The entire genome sequence of B. xylophilus was determined in 2011, and since then, proteomic studies have been conducted to understand the molecular basis of the parasitism and pathogenicity of B. xylophilus. These postgenomic studies have provided numerous molecular insights and greatly changed our understanding of the pathogenesis of pine wilt disease. Here, we review the recent advances in genomic and proteomic approaches that address some of the longstanding questions behind the pathogenesis of pine wilt disease and have identified future questions and directions in this regard.

Detecting nonculturable bacteria in the active mycorrhizal zone of the pine mushroom Tricholoma matsutake.

The fungus Tricholoma matsutake forms an ectomycorrhizal relationship with pine trees. Its sporocarps often develop in a circle, which is commonly known as a fairy ring. The fungus produces a solid, compact, white aggregate of mycelia and mycorrhizae beneath the fairy ring, which in Japanese is called a 'shiro'. In the present study, we used soil dilution plating and molecular techniques to analyze the bacterial communities within, beneath, and outside the T. matsutake fairy ring. Soil dilution plating confirmed previous reports that bacteria and actinomycetes are seldom present in the soil of the active mycorrhizal zone of the T. matsutake shiro. In addition, the results showed that the absence of bacteria was strongly correlated with the presence of T. matsutake mycorrhizae. The results demonstrate that bacteria, especially aerobic and heterotrophic forms, and actinomycetes, are strongly inhibited by T. matsutake. Indeed, neither bacteria nor actinomycetes were detected in 11.3% of 213 soil samples from the entire shiro area by culture-dependent methods. However, molecular techniques demonstrated that some bacteria, such as individual genera of Sphingomonas and Acidobacterium, were present in the active mycorrhizal zone, even though they were not detected in soil assays using the dilution plating technique.

Fungus symbionts colonizing the galleries of the ambrosia beetle Platypus quercivorus.

Isolations were made to determine the fungal symbionts colonizing Platypus quercivorus beetle galleries of dead or dying Quercus laurifolia, Castanopsis cuspidata, Quercus serrata, Quercus crispula, and Quercus robur. For these studies, logs from oak wilt-killed trees were collected from Kyoto Prefecture, Japan. Fungi were isolated from the: (1) entrances of beetle galleries, (2) vertical galleries, (3) lateral galleries, and (4) the larval cradle of P. quercivorus in each host tree. Among the fungus colonies which appeared on YM agar plates, 1,219 were isolated as the representative isolates for fungus species inhabiting in the galleries based on their cultural characteristics. The validity of the visual classification of the fungus colonies was checked and if necessary properly corrected using microsatellite-primed PCR fingerprints. The nucleotide sequence of the D1/D2 region of the large subunit nuclear rRNA gene detected 38 fungus species (104 strains) of which three species, i.e., Candida sp. 3, Candida kashinagacola (both yeasts), and the filamentous fungus Raffaelea quercivora were isolated from all the tree species. The two yeasts were most prevalent in the interior of galleries, regardless of host tree species, suggesting their close association with the beetle. A culture-independent method, terminal restriction fragment length polymorphism (T-RFLP) analysis was also used to characterize the fungus flora of beetle galleries. T-RFLP patterns showed that yeast species belonging to the genus Ambrosiozyma frequently occurred on the gallery walls along with the two Candida species. Ours is the first report showing the specific fungi inhabiting the galleries of a platypodid ambrosia beetle.

Expression profile of jasmonic acid-induced genes and the induced resistance against the root-knot nematode (Meloidogyne incognita) in tomato plants (Solanum lycopersicum) after foliar treatment with methyl jasmonate.

We investigated what gene(s) in the plant roots have the positive role against repressing root-knot nematode (RKN) infection. We investigated the interaction between RKN infection and gene expression in the plant roots induced by methyl jasmonate (MeJA). We focused on the induced resistance response and the duration after foliar treatment with MeJA of 0.1, 0.5, 1.0, and 5.0mM at 1, 24, 48, and 72h prior to the inoculation of RKN. As a result, the foliar treatment with MeJA at 0.5mM or higher concentrations significantly reduced the infection of RKN in plants and the effect lasted for about 1 week. The repressing effect on RKN population declined to the lowest level in two weeks after MeJA treatment. The expression of proteinase inhibitors (PIs) and multicystatin (MC) were induced while the repressing effect on RKN was valid and a negative correlation was found between the expression of PIs or MC and RKN infection. In addition, when tomato plants no longer expressing MC and PIs were treated again with MeJA, the repressing effect revived. These phenomena appeared to be regardless of the existence of Mi-genes or isolate of RKN. Our results indicate that the expression level of MC and PIs may be effective as marker genes for estimating the induced resistance response against RKN infection.

Comparison of the surface coat proteins of the pine wood nematode appeared during host pine infection and in vitro culture by a proteomic approach.

Pine wilt disease, caused by the pine wood nematode (PWN), Bursaphelenchus xylophilus, has become of worldwide quarantine concern in recent years. Here, we disclosed the surface coat (SC) proteins of the PWN which are thought to be one of the key components in pine wilt development. This is the first report that focused on the SC proteins and thoroughly identified those proteins of a plant-parasitic nematode using the proteomic approach. In this study, SC protein profiles were compared for PWNs grown on the fungus Botrytis cinerea and in host pine seedlings. The results demonstrated that the gross amount of PWN SC proteins drastically increased during infection of the host pine. Thirty-seven protein bands showed significant quantity differences between fungus-grown and host-origin PWNs, and were used for identification by matrix-assisted laser desorption ionization time of flight mass spectrometry analysis. These included several proteins that are presumed to be involved in the host immune response; for example, regulators of reactive oxygen species (ROS) and a ROS scavenger. These results might suggest that the PWN SC proteins are crucial in modulating or evading host immune response. Our data provide a new insight into the mechanism of pine wilt disease and the biological role of the SC proteins of plant-parasitic nematodes.

Biocontrol of Meloidogyne incognita on tomato using antagonistic fungi, plant-growth-promoting rhizobacteria and cattle manure.

BACKGROUND: Biocontrol achieved by a single biocontrol agent is generally inconsistent under field conditions. The aim of the present study was to increase the competitiveness and efficacy of biocontrol agents by using them together with cattle manure. RESULTS: The effects of antagonistic fungi [Aspergillus niger v. Teigh., Paecilomyces lilacinus (Thom) Samson and Penicillium chrysogenum Thom] and plant-growth-promoting rhizobacteria (PGPR) [Azotobacter chroococcum Beijer., Bacillus subtilis (Ehrenberg) Cohn and Pseudomonas putida (Trev.) Mig.] were assessed with cattle manure on the growth of tomato and on the reproduction of Meloidogyne incognita (Kof. & White) Chitwood. Application of antagonistic fungi and PGPR alone and in combination with cattle manure resulted in a significant increase in the growth of nematode-inoculated plants. The highest increase (79%) in the growth of nematode-inoculated plants was observed when P. putida was used with cattle manure, followed by use of P. lilacinus plus cattle manure. Paecilomyces lilacinus resulted in a high reduction in galling and nematode multiplication, followed by P. putida, B. subtilis, A. niger, A. chroococcum and P. chrysogenum. The combined use of P. lilacinus with cattle manure resulted in a maximum reduction in galling and nematode multiplication. CONCLUSION: Application of P. lilacinus or P. putida with cattle manure was useful to achieve greater biocontrol of M. incognita on tomato.

Distribution of ectomycorrhizal and pathogenic fungi in soil along a vegetational change from Japanese black pine (Pinus thunbergii) to black locust (Robinia pseudoacacia).

The nitrogen-fixing tree black locust (Robinia pseudoacacia L.) seems to affect ectomycorrhizal (ECM) colonization and disease severity of Japanese black pine (Pinus thunbergii Parl.) seedlings. We examined the effect of black locust on the distribution of ECM and pathogenic fungi in soil. DNA was extracted from soil at depths of 0-5 and 5-10 cm, collected from the border between a Japanese black pine- and a black locust-dominated forest, and the distribution of these fungi was investigated by denaturing gradient gel electrophoresis. The effect of soil nutrition and pH on fungal distribution was also examined. Tomentella sp. 1 and Tomentella sp. 2 were not detected from some subplots in the Japanese black pine-dominated forest. Ectomycorrhizas formed by Tomentella spp. were dominant in black locust-dominated subplots and very little in the Japanese black pine-dominated forest. Therefore, the distribution may be influenced by the distribution of inoculum potential, although we could not detect significant relationships between the distribution of Tomentella spp. on pine seedlings and in soils. The other ECM fungi were detected in soils in subplots where the ECM fungi was not detected on pine seedlings, and there was no significant correlation between the distribution of the ECM fungi on pine seedlings and in soils. Therefore, inoculum potential seemed to not always influence the ECM community on roots. The distribution of Lactarius quieticolor and Tomentella sp. 2 in soil at a depth of 0-5 cm positively correlated with soil phosphate (soil P) and that of Tomentella sp. 2 also positively correlated with soil nitrogen (soil N). These results suggest the possibility that the distribution of inoculum potential of the ECM fungi was affected by soil N and soil P. Although the mortality of the pine seedlings was higher in the black locust-dominated area than in the Japanese black pine-dominated area, a pathogenic fungus of pine seedlings, Cylindrocladium pacificum, was detected in soil at depths of 0-5 and 5-10 cm from both these areas. This indicates that the disease severity of pine seedlings in this study was influenced by environmental conditions rather than the distribution of inoculum potential.

Distribution of Bacterial Species in Soil with a Vegetational Change from Japanese Black Pine (Pinus thunbergii) to Black Locust (Robinia pseudoacacia).

The effects of a nitrogen-fixing tree, black locust (Robinia pseudoacacia), on the distribution of bacterial species were examined in a Japanese black pine (Pinus thunbergii) and black locust-dominated area. DNA was extracted from the soil at depths of 0-5 and 5-10 cm, collected at the border between a Japanese black pine-dominated forest and a black locust-dominated forest, and the distribution of bacterial species was investigated by denaturing gradient gel electrophoresis (DGGE). The bacterial communities did not differ between the two forests. The distribution of some bacterial species correlated significantly with soil pH, soil carbon [C], soil nitrogen [N], and soil N/phosphate [P], but not with soil C/N or soil P. The distributional relationships between ectomycorrhizal (ECM) fungal species and bacterial species were also analyzed. A positive correlation was observed between the distribution of some ECM fungi and bacterial species. These bacteria may have some interactions with ECM fungi in the field.

Candida kashinagacola sp. nov., C. pseudovanderkliftii sp. nov. and C. vanderkliftii sp. nov., three new yeasts from ambrosia beetle-associated sources.

Three new yeast species, Candida kashinagacola (JCM 15019(T) = CBS 10903(T)), C. pseudovanderkliftii (JCM 15025(T) = CBS 10904(T)), and C. vanderkliftii (JCM 15029(T) = CBS 10905(T)) are described on the basis of comparison of nucleotide sequences of large subunit ribosomal DNA D1/D2 region (LSU rDNA D1/D2). The nearest assigned species of the three new species was Candida llanquihuensis. Candida kashinagacola and C. pseudovanderkliftii differed from C. llanquihuensis by 3.8% nucleotide substitution of the region, while C. vanderkliftii did by 4.4%. Three new species differed in a number of physiological and growth characteristics from any previously assigned species and from one another. A phylogenetic tree based on the sequences of LSU rDNA D1/D2 showed that these new species together with Candida sp. ST-246, Candida sp. JW01-7-11-1-4-y2, Candida sp. BG02-7-20-001A-2-1 and C. llanquihuensis form a clade near Ambrosiozyma species. The new species did not assimilate methanol as a sole source of carbon, which supported the monophyly of these non methanol-assimilating species which are closely related to the methylotrophic yeasts. Candida kashinagacola was frequently isolated from the beetle galleries of Platypus quercivorus in three different host trees (Quercus serrata, Q. laurifolia and Castanopsis cuspidata) located in the sourthern part of Kyoto, Japan, thus indicating that this species may be a primary ambrosia fungus of P. quercivorus. On the other hand, C. pseudovanderkliftii and C. vanderkliftii were isolated only from beetle galleries in Q. laurifolia. Candida vanderkliftii was isolated from beetle gallery of Platypus lewisi as well as those of P. quercivorus. Candida pseudovanderkliftii and C. vanderkliftii are assumed to be auxiliary ambrosia fungi of P. quercivorus.

Does ectomycorrhizal fungal community structure vary along a Japanese black pine (Pinus thunbergii) to black locust (Robinia pseudoacacia) gradient?

In this study we examined the role of the nitrogen-fixing tree, Robinia pseudoacacia (black locust), in ectomycorrhizal (ECM) formation and ECM community of Pinus thunbergii (Japanese black pine) seedlings. Two 200 m(2) experimental plots were established at the border between a Japanese black pine- and a black locust-dominated area in a coastal forest. The ECM fungal community of pine seedlings was examined by PCR-RFLP and sequence analysis. We analyzed the relationship between ECM formation, ECM community, growth, and nutrient status of pine seedlings and environmental conditions using the Mantel test and structural equation model. Percentages of ECM root tips, the number of ECM fungal species and ECM diversity on pine seedlings decreased in the black locust-dominated area. Cenococcum geophilum and Russula spp. were dominant in the Japanese black pine-dominated area, whereas Tomentella spp. were dominant in the black locust-dominated area. Nitrogen (N) concentration in soils or pine seedlings strongly influenced the percentage of ECM root tips, the number of ECM fungal species and ECM fungal similarity. These results imply the long-term eutrophication caused by N-fixing trees can change ECM formation and ECM community structure.

Comparative Studies between Portuguese and Japanese Isolates of the Pinewood Nematode, Bursaphelenchus xylophilus.

Comparative studies between Portuguese (T and HF) and Japanese (S10, T4, C14-5 and OKD-1) isolates of the pinewood nematode Bursaphelenchus xylophilus have been made in order to provide information to better understand the possible origin of the Portuguese isolates, recently introduced in the European Union. The main comparative aspects investigated were pathogenicity (seedling mortality ratio), sexual compatibility, and DNA sequences of the rDNA region. Four-year-old Japanese black pine (Pinus thunbergii) seedlings were used as host plants for pathogenicity tests. The Portuguese isolates, and in particular isolate "T," propagated in higher numbers than the Japanese isolates within pine seedlings. All combinations of crossings produced viable progeny, with higher numbers obtained when crossings were made between Japanese and Portuguese isolates, a possible situation of heterosis and/or inbreeding depression. Reciprocal crossings yielded different values, which may reflect a sex effect (maternal inheritance, mtDNA). Regarding DNA sequencing, both Portuguese isolates displayed nearly identical ITS 1, ITS2, and 5.8S rDNA base sequences as the Japanese isolates. Although biologically very similar, and possibly reflecting a common origin, the Portuguese isolates may present a serious threat to Japanese black pine, due to their higher virulence.

Early embryogenesis of the pinewood nematode Bursaphelenchus xylophilus.

The early embryogenesis and cell lineage of the pinewood nematode Bursaphelenchus xylophilus was followed from a single-cell zygote to a 46-cell embryo under Nomarski optics, and elongation of the microtubules was studied by immunostaining. As a B. xylophilus oocyte matures, it passes through a passage connecting the oviduct with the quadricolumella, the distal part of the uterus, and reaches the quadricolumella where it stays for a few minutes and is fertilized. After fertilization, the germinal vesicle disappears, an eggshell is formed, and the male and female pronuclei appear. The pronuclei move toward each other and fuse at the center of the egg. Around this time, the microtubule-organizing center appears. The presumptive region of sperm entry into the oocyte becomes the future anterior portion of the embryo. This anterior-posterior axis determination is opposite to that of Caenorhabditis elegans, where the sperm entry site becomes the posterior portion of the embryo. The optimal growth temperatures of these two nematodes also differ in that temperatures of about 30 degrees C afford the fastest growth rate and highest hatching frequency in B. xylophilus. Otherwise, the lineage resembles that of C. elegans with respect to timing, positioning and the axis orientation of each cell division.