Year-round dynamics of arbuscular mycorrhizal fungi communities in the roots and surrounding soils of Cryptomeria japonica.

Arbuscular mycorrhizal fungi (AMF) live simultaneously inside and outside of host plant roots for a functional mycorrhizal symbiosis. Still, the year-round dynamics and relationships between soil properties and AMF communities of trees in forest ecosystems remain unclear. We collected paired root and soil samples of the same Cryptomeria japonica trees at two forest sites (five trees at each site) every 2 months over a year. Total DNA was extracted from roots and soil separately and soil physicochemical properties were measured. With Illumina's next-generation amplicon sequencing targeting the small subunit of fungal ribosomal DNA, we clarified seasonal dynamics of soil properties and AMF communities. Soil pH and total phosphorus showed significant seasonality while total carbon, nitrogen, and C/N did not. Only pH was a good predictor of the composition and dynamics of the AMF community. The total AMF community (roots + soil) showed significant seasonality because of variation from May to September. Root and soil AMF communities were steady year-round, however, with similar species richness but contained significantly different AMF assemblages in any sampling month. Despite the weak seasonality in the communities, the top two dominant OTUs showed significant but different shifts between roots and soils across seasons with strong antagonistic relationships. In conclusion, few dominant AMF taxa are dynamically shifting between the roots and soils of C. japonica to respond to seasonal and phenological variations in their microhabitats. AMF inhabiting forest ecosystems may have high environmental plasticity to sustain a functional symbiosis regardless of seasonal variations that occur in the soil.

Chionobium takahashii, gen. et sp. nov., associated with snow blight of conifers in Japan.

Gremmenia abietis (Dearn.) Crous (syn: Phacidium abietis) was originally described in North America to accommodate the species associated with snow blight of Abies and Pseudotsuga spp. In Japan, this species was first observed on the dead needles on Abies sachalinensis and Picea jezoensis var. jezoensis in 1969. However, the identity of Japanese species was unclear due to the lack of molecular data and the absence of anamorph description. In this study, we collected fresh specimens from various conifer species (A. sachalinensis, A. veitchii, Pic. jezoensis var. jezoensis, Pic. jezoensis var. hondoensis, Pinus koraiensis, and Pin. pumila) in Japan and revised the taxonomy based on morphological and phylogenetic analyses. Phylogenetic analyses based on nuc rDNA internal transcribed spacer ITS1-5.8S-ITS2 (ITS), nuc 28S rDNA (28S), and RNA polymerase II second largest subunit (RPB2) regions indicated that the species belongs to Phacidiaceae. Conidiomata formed in vitro produced pyriform, hyaline conidia without mucoid appendage, which distinguished the species from phylogenetically related genera. Consequently, we established Chionobium takahashii to accommodate the snow blight fungus in Japan. Further phylogenetic analyses also indicated that C. takahashii includes several distinct clades corresponding to the host genera (Abies, Picea, Pinus). Morphological differences among those clades were unclear, suggesting that C. takahashii may contain host-specific cryptic species.

Physiological and Transcriptome Responses of Pinus massoniana Seedlings Inoculated by Various Ecotypes of the Ectomycorrhizal Fungus Cenococcum geophilum during the Early Stage of Drought Stress.

The impact of drought stress on plant growth in arid regions is a critical concern, necessitating the exploration of strategies to enhance plant drought resistance, particularly during the early stages of drought stress. This study focuses on the ectomycorrhizal fungus Cenococcum geophilum, renowned for its extensive genetic diversity and broad host compatibility, making it a crucial ally for host plants facing external stresses. We utilized Pinus massoniana seedlings inoculated with different ecotypic strains of C. geophilum under drought stress. The results showed that the inoculation of most strains of C. geophilum enhanced the drought resistance of P. massoniana seedlings under the early stages of drought stress, by influencing the water content, photosynthesis, accumulation of osmotic adjustment substances, and antioxidant enzyme activities in both shoots and roots of seedlings. Transcriptome analysis showed that mycorrhizal seedlings mainly regulated energy metabolism and reduction-oxidation reaction to resist early drought stress. Notably, the level of drought resistance observed in mycorrhizal seedlings was irrespective of the level of drought tolerance of C. geophilum strains. This study contributes essential data for understanding the drought response mechanisms of mycorrhizal P. massoniana seedlings inoculated by distinct C. geophilum ecotypes and guidance on selecting candidate species of ectomycorrhizal fungi for mycorrhizal afforestation in drought areas.

Visualization of root extracellular traps in an ectomycorrhizal woody plant (Pinus densiflora) and their interactions with root-associated bacteria.

MAIN CONCLUSION: Extracellular traps in the primary root of Pinus densiflora contribute to root-associated bacterial colonization. Trapped rhizobacteria induce the production of reactive oxygen species in root-associated, cap-derived cells. Ectomycorrhizal (ECM) woody plants, such as members of Pinaceae and Fagaceae, can acquire resistance to biotic and abiotic stresses through the formation of mycorrhiza with ECM fungi. However, germinated tree seedlings do not have mycorrhizae and it takes several weeks for ectomycorrhizae to form on their root tips. Therefore, to confer protection during the early growth stage, bare primary roots require defense mechanisms other than mycorrhization. Here, we attempted to visualize root extracellular traps (RETs), an innate root defense mechanism, in the primary root of Pinus densiflora and investigate the interactions with root-associated bacteria isolated from ECM and fine non-mycorrhizal roots. Histological and histochemical imaging and colony-forming unit assays demonstrated that RETs in P. densiflora, mainly consisting of root-associated, cap-derived cells (AC-DCs) and large amounts of root mucilage, promote bacterial colonization in the rhizosphere, despite also having bactericidal activity via extracellular DNA. Four rhizobacterial strains retarded the mycelial growth of a pathogenic strain belonging to the Fusarium oxysporum species complex in dual culture assay. They also induced the production of reactive oxygen species (ROS) from host tree AC-DCs without being excluded from the rhizosphere of P. densiflora. Applying three Paraburkholderia strains, especially PM O-EM8 and PF T-NM22, showed significant differences in the ROS levels from the control group. These results reveal the indirect contributions of rhizobacteria to host root defense and suggest that root-associated bacteria could be a component of RETs as a first line of defense against root pathogens in the early growth stage of ECM woody plants.

Analysis of the distribution pattern of the ectomycorrhizal fungus Cenococcum geophilum under climate change using the optimized MaxEnt model.

Cenococcum geophilum (C. geophilum) is a widely distributed ectomycorrhizal fungus that plays a crucial role in forest ecosystems worldwide. However, the specific ecological factors influencing its global distribution and how climate change will affect its range are still relatively unknown. In this study, we used the MaxEnt model optimized with the kuenm package to simulate changes in the distribution pattern of C. geophilum from the Last Glacial Maximum to the future based on 164 global distribution records and 17 environmental variables and investigated the key environmental factors influencing its distribution. We employed the optimal parameter combination of RM = 4 and FC = QPH, resulting in a highly accurate predictive model. Our study clearly shows that the mean temperature of the coldest quarter and annual precipitation are the key environmental factors influencing the suitable habitats of C. geophilum. Currently, appropriate habitats of C. geophilum are mainly distributed in eastern Asia, west-central Europe, the western seaboard and eastern regions of North America, and southeastern Australia, covering a total area of approximately 36,578,300 km2 globally. During the Last Glacial Maximum and the mid-Holocene, C. geophilum had a much smaller distribution area, being mainly concentrated in the Qinling-Huaihe Line region of China and eastern Peninsular Malaysia. As global warming continues, the future suitable habitat for C. geophilum is projected to shift northward, leading to an expected expansion of the suitable area from 9.21% to 21.02%. This study provides a theoretical foundation for global conservation efforts and biogeographic understanding of C. geophilum, offering new insights into its distribution patterns and evolutionary trends.

Identification of microRNAs involved in ectomycorrhizal formation in Populus tomentosa.

The majority of woody plants are able to form ectomycorrhizal (ECM) symbioses with fungi. During symbiotic development, plants undergo a complex re-programming process involving a series of physiological and morphological changes. MicroRNAs (miRNAs) are important components of the regulatory network underlying symbiotic development. To elucidate the mechanisms of miRNAs and miRNA-mediated mRNA cleavage during symbiotic development, we conducted high-throughput sequencing of small RNAs and degradome tags from roots of Populus tomentosa inoculated with Cenococcum geophilum. This process led to the annotation of 51 differentially expressed miRNAs between non-mycorrhizal and mycorrhizal roots of P. tomentosa, including 13 novel miRNAs. Increased or decreased accumulation of several novel and conserved miRNAs in ECM roots, including miR162, miR164, miR319, miR396, miR397, miR398, novel-miR44 and novel-miR47, suggests essential roles for these miRNAs in ECM formation. The degradome analysis identified root transcripts as miRNA-mediated mRNA cleavage targets, which was confirmed using real-time quantitative PCR. Several of the identified miRNAs and corresponding targets are involved in arbuscular mycorrhizal symbioses. In summary, increased or decreased accumulation of specific miRNAs and miRNA-mediated cleavage of symbiosis-related genes indicate that miRNAs play important roles in the regulatory network underlying symbiotic development.

Paired Root-Soil Samples and Metabarcoding Reveal Taxon-Based Colonization Strategies in Arbuscular Mycorrhizal Fungi Communities in Japanese Cedar and Cypress Stands.

Arbuscular mycorrhizal fungi (AMF) in the roots and soil surrounding their hosts are typically independently investigated and little is known of the relationships between the communities of the two compartments. We simultaneously collected root and surrounding soil samples from Cryptomeria japonica (Cj) and Chamaecyparis obtusa (Co) at three environmentally different sites. Based on molecular and morphological analyses, we characterized their associated AMF communities. Cj was more densely colonized than Co and that root colonization intensity was significantly correlated with soil AMF diversity. The communities comprised 15 AMF genera dominated by Glomus and Paraglomus and 1443 operational taxonomic units (OTUs) of which 1067 and 1170 were in roots and soil, respectively. AMF communities were significantly different among sites, and the root AMF communities were significantly different from those of soil at each site. The root and soil AMF communities responded differently to soil pH. At the genus level, Glomus and Acaulospora were abundant in roots while Paraglomus and Redeckera were abundant in soil. Our findings suggest that AMF colonizing roots are protected from environmental stresses in soil. However, the root-soil-abundant taxa have adapted to both environments and represent a model AMF symbiont. This evidence of strategic exploitation of the rhizosphere by AMF supports prior hypotheses and provides insights into community ecology.

Nitrogen fixation and nifH gene diversity in cyanobacteria living on feather mosses in a subalpine forest of Mt. Fuji.

In the boreal forests, feather mosses such as Hylocomium splendens and Pleurozium schreberi are colonized by cyanobacteria, which provide large amounts of nitrogen to forest ecosystems through nitrogen fixation. Although these feather mosses are also ubiquitous in subalpine forests of East Asia, little is known regarding their associated cyanobacteria and their ability to fix nitrogen. In this study, we investigated (1) whether cyanobacteria co-exist and fix nitrogen in the two species of feather mosses that cover the ground surface in a subalpine forest of Mt. Fuji, (2) whether cyanobacteria belonging to a common cluster with boreal forests are found in feather mosses in Mt. Fuji, and (3) whether moss-associated nitrogen fixation rates differed among moss growing substrates, canopy openness, and moss nitrogen concentrations in the same forest area. Our results showed that cyanobacteria colonized feather mosses in the subalpine forests of Mt. Fuji and acetylene reduction rates as an index of nitrogen fixation tended to be higher in H. splendens than in P. schreberi. Based on analysis of the nifH gene, 43 bacterial operational taxonomic units (OTUs) were identified, 28 of which represented cyanobacteria. Among the five clusters of cyanobacteria classified based on their nifH gene and identified in northern Europe, four (Nostoc cluster I, Nostoc cluster II, Stigonema cluster, and nifH2 cluster) were also found at Mt. Fuji. The acetylene reduction rate differed depending on the moss growing substrate and the total nitrogen concentration of moss shoots, and a strong negative correlation was observed with the total nitrogen concentration.

Estimation of the most suitable nitrogen concentration for sporocarp formation in Laccaria japonica colonizing Pinus densiflora seedlings through in vitro mycelial culture.

Many ectomycorrhizal (ECM) fungi produce commercially valuable edible sporocarps. However, the effects of nitrogen (N) application on ECM fungal sporocarp formation remain poorly understood. In this study, we investigated the effect of application of various N concentrations (0, 5, 25, 50, 100, and 200 mg/L) on the growth of Laccaria japonica mycelia in vitro for 1 month. The results showed that L. japonica mycelial biomass was highest in the 50 mg/L treatment and was significantly inhibited at N concentrations higher than 200 mg/L. Next, we investigated the effects of N application on mycorrhizal colonization and sporocarp formation in L. japonica colonizing Pinus densiflora seedlings in pots. The seedlings were watered with nutrient solutions containing 0, 5, 25, 50, or 100 mg N/L. The biomass, photosynthetic rate, and mycorrhizal colonization rates of the seedlings were measured at 45 days (first appearance of primordia), 65 days (sporocarp appearance on the substrate surface), and 4 months after seedlings were transplanted. The numbers of primordia and sporocarps were recorded during the experimental period. Total carbon (C) and N content were determined in seedlings at 4 months after transplantation, and in L. japonica sporocarps. Both mycelial growth and sporocarp production reached their maximum at an N application concentration of 50 mg/L, suggesting that the most suitable N concentration for ECM fungal sporocarp formation can easily be estimated in vitro during mycelial growth. This finding may help determine the most suitable N conditions for increasing edible ECM fungus sporocarp production in natural forests.

Physiological and transcriptional responses of the ectomycorrhizal fungus Cenococcum geophilum to salt stress.

Ectomycorrhizal (ECM) fungi improve the host plant's tolerance to abiotic and biotic stresses. Cenococcum geophilum (Cg) is among the most common ECM fungi worldwide and often grows in saline environments. However, the physiological and molecular mechanisms of salt tolerance in this fungus are largely unknown. In the present study, 12 isolates collected from different ecogeographic regions were used to investigate the mechanism of salt tolerance of Cg. The isolates were classified into four groups (salt-sensitive, moderately salt-tolerant, salt-tolerant, and halophilic) based on their in vitro mycelial growth under 0, 50, 125, 250, and 500 mM NaCl concentrations. Hence, the Na, Ca, P, and K concentrations of mycelia and the pH of the culture solution were determined. Compared with salt-tolerant isolates, treatment with 250 mM NaCl significantly increased the sodium concentration and decreased the potassium concentration of salt-sensitive isolates. RNA-sequencing and qRT-PCR analysis were conducted to identify differentially expressed genes (DEGs) involved in transmembrane transport and oxidoreductase activity pathways. The hydrogen peroxide concentration and activities of peroxidase and superoxide dismutase in mycelia were determined, and the accumulation and scavenging of reactive oxygen species in the salt-sensitive isolates were more active than those in the salt-tolerant isolates. The results supply functional validations to RNA-seq and qRT-PCR analysis. This study provides novel insights into the salt-stress response of Cg isolates and provides a foundation for elucidation of the salt-tolerance mechanism of ECM fungi.

Preferential freezing avoidance localised in anthers and embryo sacs in wintering Daphne kamtschatica var. jezoensis flower buds visualised by magnetic resonance imaging.

To explore diversity in cold hardiness mechanisms, high resolution magnetic resonance imaging (MRI) was used to visualise freezing behaviours in wintering Daphne kamtschatica var. jezoensis flower buds, which have naked florets and no bud scales. MRI images showed that anthers remained stably supercooled to the range from -14 to -21°C or lower while most other tissues froze by -7°C. Freezing of some anthers detected in MRI images between -14 and -21°C corresponded with numerous low temperature exotherms and also with the 'all-or-nothing' type of anther injuries. In ovules/pistils, only embryo sacs remained supercooled at -7°C or lower, but slowly dehydrated during further cooling. Cryomicroscopic observation revealed ice formation in the cavities of calyx tubes and pistils but detected no ice in embryo sacs or in anthers. The distribution of ice nucleation activity in floral tissues corroborated the tissue freezing behaviours. Filaments likely work as the ice blocking barrier that prevents ice intrusion from extracellularly frozen calyx tubes to connecting unfrozen anthers. Unique freezing behaviours were demonstrated in Daphne flower buds: preferential freezing avoidance in male and female gametophytes and their surrounding tissues (by stable supercooling in anthers and by supercooling with slow dehydration in embryo sacs) while the remaining tissues tolerate extracellular freezing.

New findings on the fungal species Tricholoma matsutake from Ukraine, and revision of its taxonomy and biogeography based on multilocus phylogenetic analyses.

Matsutake mushrooms are among the best-known edible wild mushroom taxa worldwide. The representative Tricholoma matsutake is from East Asia and the northern and central regions of Europe. Here, we report the existence of T. matsutake under fir trees in Eastern Europe (i.e., Ukraine), as confirmed by phylogenetic analysis of nine loci on the nuclear and mitochondrial genomes. All specimens from Japan, Bhutan, China, North Korea, South Korea, Sweden, Finland, and Ukraine formed a T. matsutake clade according to the phylogeny of the internal transcribed spacer region. The European population of T. matsutake was clustered based on the ß2 tubulin gene, with a moderate bootstrap value. In contrast, based on analyses of three loci, i.e., rpb2, tef1, and the ß2 tubulin gene, T. matsutake specimens sampled from Bhutan and China belonged to a clade independent of the other specimens of this species, implying a genetically isolated population. As biologically available type specimens of T. matsutake have not been designated since its description as a new species from Japan in 1925, we established an epitype of this fungus, sampled in a Pinus densiflora forest in Nagano, Japan.

Snow mold fungus Racodium therryanum is phylogenetically Herpotrichia juniperi.

Racodium therryanum is a snow mold causing snow blight and seed rot in conifers. The sexual stage of R. therryanum has not been found, however, it has been speculated that Herpotrichia juniperi, which causes brown felt blight, is the sexual stage of R. therryanum. In this study, we conducted phylogenetic analysis using 28S large subunit (LSU) rDNA, 18S small subunit (SSU) rDNA, translation elongation factor 1-alpha (tef1), and RNA polymerase II second largest subunit (rpb2). Four isolates were identified as R. therryanum based on their morphological characteristics; together with two strains of H. juniperi, they composed a clade supported by high bootstrap and Bayesian posterior probability values. Therefore, we conclude that the snow mold previously described as R. therryanum is phylogenetically H. juniperi.

The effects of co-colonising ectomycorrhizal fungi on mycorrhizal colonisation and sporocarp formation in Laccaria japonica colonising seedlings of Pinus densiflora.

Forest trees are colonised by different species of ectomycorrhizal (ECM) fungi that interact competitively or mutualistically with one another. Most ECM fungi can produce sporocarps. To date, the effects of co-colonising fungal species on sporocarp formation in ECM fungi remain unknown. In this study, we examined host plant growth, mycorrhizal colonisation, and sporocarp formation when roots of Pinus densiflora are colonised by Laccaria japonica and three other ECM fungal species (Cenococcum geophilum, Pisolithus sp., and Suillus luteus). Sporocarp numbers were recorded throughout the experimental period. The biomass, photosynthetic rate, and mycorrhizal colonisation rate of the seedlings were also measured at 45 days, 62 days, and 1 year after seedlings were transplanted. Results indicated that C. geophilum and S. luteus may negatively impact mycorrhizal colonisation and sporocarp formation in L. japonica. Sporocarp formation in L. japonica was positively correlated with conspecific mycorrhizal colonisation but negatively correlated with the biomass of seedlings of P. densiflora. The co-occurring ECM fungi largely competed with L. japonica, resulting in various effects on mycorrhizal colonisation and sporocarp formation in L. japonica. A variety of mechanisms may be involved in the competitive interactions among the different ECM fungal species, including abilities to more rapidly colonise root tips, acquire soil nutrients, or produce antibiotics. These mechanisms need to be confirmed in further studies.

Tricholoma matsutake may take more nitrogen in the organic form than other ectomycorrhizal fungi for its sporocarp development: the isotopic evidence.

Tricholoma matsutake is an ectomycorrhizal (ECM) fungus capable of in vitro saprotrophic growth, but the sources of C and N used to generate sporocarps in vivo are not well understood. We examined natural abundance isotope data to investigate this phenomenon. For this purpose, C, N and their stable isotopes (13C, 15N) content of fungal sporocarps and their potential nutrient sources (i.e., foliage, litter, fine roots, wood, and soil) were investigated from two well-studied sites in Finland and Japan. Our results show that δ13C values of T. matsutake and other fungal groups are consistent with those of most studies, but a very high δ15N value (16.8‰ ± 2.3) is observed in T. matsutake. Such isotopic pattern of fungal δ15N suggests that matsutake has a greater proteolytic potential to digest chemically complex 15N-enriched organic matter and hydrophobic hyphae. This assumption is further supported by a significant and positive correlation between δ13Ccap-stipe and δ15Ncap-stipe exclusively in T. matsutake, which suggests common C and N sources (protein) possible for isotopically enriched cap. The 13C increase of caps relative to stipe presumably reflects greater contents of 13C-enriched protein than 13C-depleted chitin. We conclude that T. matsutake is a typical ECM fungus which obtains for its sporocarp development for both C and N from a common protein source (vs. photosynthetic carbon) present in soil organic matter.

A qPCR assay that specifically quantifies Tricholoma matsutake biomass in natural soil.

Tricholoma matsutake is an ectomycorrhizal basidiomycete that produces prized, yet uncultivable, "matsutake" mushrooms along densely developed mycelia, called "shiro," in the rhizosphere of coniferous forests. Pinus densiflora is a major host of this fungus in Japan. Measuring T. matsutake biomass in soil allows us to determine the kinetics of fungal growth before and after fruiting, which is useful for analyzing the conditions of the shiro and its surrounding mycorrhizosphere, predicting fruiting timing, and managing forests to obtain better crop yields. Here, we document a novel method to quantify T. matsutake mycelia in soil by quantifying a single-copy DNA element that is uniquely conserved within T. matsutake but is absent from other fungal species, including close relatives and a wide range of ectomycorrhizal associates of P. densiflora. The targeted DNA region was amplified quantitatively in cultured mycelia that were mixed with other fungal species and soil, as well as in an in vitro co-culture system with P. densiflora seedlings. Using this method, we quantified T. matsutake mycelia not only from shiro in natural environments but also from the surrounding soil in which T. matsutake mycelia could not be observed by visual examination or distinguished by other means. It was demonstrated that the core of the shiro and its underlying area in the B horizon are predominantly composed of fungal mycelia. The fungal mass in the A or A0 horizon was much lower, although many white mycelia were observed at the A horizon. Additionally, the rhizospheric fungal biomass peaked during the fruiting season.

Fine-scale distribution of ectomycorrhizal fungi colonizing Tsuga diversifolia seedlings growing on rocks in a subalpine Abies veitchii forest.

Numerous species of ectomycorrhizal (ECM) fungi coexist under the forest floor. To explore the mechanisms of coexistence, we investigated the fine-scale distribution of ECM fungal species colonizing root tips in the root system of Tsuga diversifolia seedlings in a subalpine forest. ECM root tips of three seedlings growing on the flat top surface of rocks were sampled after recording their positions in the root system. After the root tips were grouped by terminal-restriction fragment length polymorphism (T-RFLP) analysis of ITS rDNA, the fungal species representing each T-RFLP group were identified using DNA sequencing. Based on the fungal species identification, the distribution of root tips colonized by each ECM fungus was mapped. Significant clustering of root tips was estimated for each fungal species by comparing actual and randomly simulated distributions. In total, the three seedlings were colonized by 40 ECM fungal species. The composition of colonizing fungal species was quite different among the seedlings. Twelve of the 15 major ECM fungal species clustered significantly within a few centimeters. Some clusters overlapped or intermingled, while others were unique. Areas with high fungal species diversity were also identified in the root system. In this report, the mechanisms underlying generation of these ECM root tip clusters in the root system are discussed.

Spatial distribution of Raffaelea quercivora in xylem of naturally infested and inoculated oak trees.

Mass mortality of Japanese oak caused by Raffaelea quercivora due to Japanese oak wilt (JOW) has been tremendous since the late 1980s in Japan. We investigated detailed distribution of R. quercivora hyphae in a naturally infected Quercus serrata tree using fluorescein-conjugated wheat germ agglutinin and also examined spatial relationships between hyphal distribution, water conduction loss, and host reactions in xylem in inoculated Q. crispula saplings. Hyphae of R. quercivora elongated longitudinally in vessels and transversely in ray tissues in both naturally infected and inoculated Quercus trees. Hyphae were confined within a relatively small area near the inoculation site. Vessel dysfunction was also confined and overlapped with hyphal distribution. The reaction zone consisting of two types of fluorescent substance was formed surrounding the outside of the hyphal area in xylem and was always found in non-water-conductive zone. These results suggest that wilting of Quercus trees caused by JOW may not be induced by dysfunction of a small number of vessels, but by that of many vessels, and it requires that R. quercivora hyphae spread from many galleries bored by beetles during mass attacks.

Nodulation in black locust by the Gammaproteobacteria Pseudomonas sp. and the Betaproteobacteria Burkholderia sp.

Nodulation abilities of bacteria in the subclasses Gammaproteobacteria and Betaproteobacteria on black locust (Robinia pseudoacacia) were tested. Pseudomonas sp., Burkholderia sp., Klebsiella sp., and Paenibacillus sp. were isolated from surface-sterilized black locust nodules, but their nodulation ability is unknown. The aims of this study were to determine if these bacteria are symbiotic. The species and genera of the strains were determined by RFLP analysis and DNA sequencing of 16S rRNA gene. Inoculation tests and histological studies revealed that Pseudomonas sp. and Burkholderia sp. formed nodules on black locust and also developed differentiated nodule tissue. Furthermore, a phylogenetic analysis of nodA and a BLASTN analysis of the nodC, nifH, and nifHD genes revealed that these symbiotic genes of Pseudomonas sp. and Burkholderia sp. have high similarities with those of rhizobial species, indicating that the strains acquired the symbiotic genes from rhizobial species in the soil. Therefore, in an actual rhizosphere, bacterial diversity of nodulating legumes may be broader than expected in the Alpha-, Beta-, and Gammaproteobacteria subclasses. The results indicate the importance of horizontal gene transfer for establishing symbiotic interactions in the rhizosphere.