Kenji Fukuda

Summary: Xylem vessels in sapwood are typically interpreted as either conductive or non-functional following embolism or occlusion, implicitly assuming that the presence of water within the lumen reflects participation in the transpiration stream. However, the relationship between lumen water content and hydraulic connectivity has rarely been examined directly at the cellular level. In this study, we combined cryo-scanning electron microscopy and fluorescence microscopy to compare lumen water status with hydraulic connectivity on the same xylem surface in current-year xylem of Carpinus tschonoskii and Aria alnifolia , using tension-driven dye uptake to identify conductive vessels. Most vessels were stained and water-filled, consistent with functional conduits, whereas unstained gas-filled vessels corresponded to embolized conduits. Notably, certain vessels retained water despite lacking dye signals, indicating hydraulic isolation from the conductive xylem network. Although these vessels represented a minor fraction of the conduit population, their occurrence suggests that the traditionally binary classification should be expanded to incorporate an additional conduit function. These vessels were observed in both species, suggesting that hydraulically disconnected vessels represent a common phenomenon across species. These findings reveal previously underrecognized heterogeneity in xylem hydraulic organisation and demonstrate that conduit status cannot always be inferred from the presence of water alone. Recognising this intermediate condition refines current interpretations of xylem function and highlights the value of integrating high-resolution imaging with hydraulic measurements.

Arbuscular mycorrhizal fungi (AMF) are potential bioinoculants to grow healthy plants in healthy soils. Although they are generalist, plant – AMF associations work along a mutualism-to-parasitism continuum where currently unresolved host – symbiont compatibility overrides the versatility of AMF functions. Here, we hypothesized that habitat, origin, and cohabitation of AMF determine compatibility and functionality in the associations. To test the hypothesis, we established two forest-inhabiting AMF isolates under Cryptomeria japonica (Cupressaceae) and inoculated them into C. japonica seedlings grown in a controlled environment. We identified the isolates as Gigaspora rosea LFB-4 and G. margarita LFB-A1 using morphological, molecular, and phylogenetic analyses. They simultaneously developed multiple germ tubes during germination, showed pre-symbiotic sporulation, erratic root colonization, and produced spores inside host root cells. When compared to controls, G. rosea and G. margarita significantly promoted the growth of C. japonica seedlings, with synergistic effects in their cohabitation. While G. rosea boosted water uptake and height growth, G. margarita improved biomass production. Together, they encouraged carbon release into the soil and delayed root growth, increasing shoot-to-root biomass ratio for faster seedling growth. We concluded that, despite erratic root colonization, forest-derived G. rosea and G. margarita worked synergistically to improve the growth of C. japonica seedlings by modulating root development. We proved that beyond taxonomic affiliation, habitat, origin, and cohabitation of AMF matter in plant – AMF compatibility for mutual benefits. Our findings imply that forests are home to potent AMF bioinoculants such as beneficial Gigaspora species and diversity improves plant – AMF associations.

During the course of pine wilt disease (PWD), xylem dysfunction is induced (in the absence of water stress) by invasion of pinewood nematodes (PWN) into resin canals, and the infected pine trees wilt. When PWNs are inoculated to Japanese black pine seedlings, massive embolisms develop at the inoculation site. The developmental process of massive embolization to form an embolized zone is not well understood. We studied the development of xylem dysfunction around the inoculation site of one-year-old stems of PWN-infected seedlings using a combination of compact MRI and cryo-SEM. We visualized pathological changes in tracheids and resin canals in the xylem, and found that embolisms occurred in PWN-inoculated seedlings at a negative hydraulic pressure of −0.5 MPa. Resin canals in healthy trees were filled with resin. In PWN-infected seedlings, abnormal embolisms and resin were observed around resin canal near the inoculation wound. Empty tracheids were observed more extensively than resin-filled tracheids. We suggest that abnormal embolisms and resin leakage occur from damages of resin canals. Resin may seep into the tracheids when cell wall of the axial and radial resin canals is crushed. These embolisms under high water potential are unavoidable under natural conditions.

The similarity between the arbuscular mycorrhizal fungi (AMF) communities of trees and neighboring understory herbs in forests remains unclear, which we aimed to clarify. We traced and collected basal roots of 20 randomly chosen Cryptomeria japonica (Cupresaceae) trees and the surrounding soil at four microsites in 1 km² of a Cr. japonica forest. One Chloranthus serratus (Chlorantaceae) herb immediately at the base of each sampled tree was excavated to collect an intact root system. We amplified a partial small subunit of fungal ribosomal DNA (18S) using Illumina MiSeq amplicon sequencing. Soil physicochemical properties were also measured. We detected 670 and 679 AMF operational taxonomic units (OTUs) in Cr. japonica and Ch. serratus, respectively, belonging to Acaulospora, Dominikia, Glomus, Microkamienskia, Rhizophagus, Septoglomus, and Sclerocystis. Seventeen OTUs were detected in the roots of both host species at average relative abundances > 1%. Among them, four dominant OTUs with an average relative abundance > 10% were concurrently detected in the roots of 17 tree–herb sets. The composition and similarity of their AMF communities were spatially varied, significantly driven by spatially varying soil pH, total C, N, C/N, and elevation, but not electroconductivity, supported by the microsite-dependent distributions of their dominant OTUs. We concluded that the similarity of AMF communities between trees and neighboring understory herbs depends on the soil physicochemical conditions that influence the distribution of their dominant AMF.

The development of xylem embolism in 1-year-old stems of Japanese black pine ( Pinus thunbergii) seedlings was monitored by compact magnetic resonance imaging (MRI) after inoculation with the pinewood nematode ( Bursaphelenchus xylophilus). In parallel, the nematode distribution and population structure in the stems were examined by isolating the nematodes using the Baermann funnel technique. The vertical length and volume of massive embolisms in each seedling were strongly correlated with the maximum relative embolized area (REA) in stem cross-sections. Embolism development and nematode reproduction were not restricted to the inoculation site, as any portion of the stem could be the initial point of a population burst. The nematode population in the stem xylem was strongly correlated with the REA and with the circumferential proportion of cambial death in cross-sections monitored by MRI. The proportion of second-stage juveniles was also correlated with the REA in the xylem. In contrast, the nematode population in bark tissue was not correlated with either the REA or cambial death. These results suggested that nematode reproduction in the cambial zone is the key step in pine wilt disease, and second-stage juveniles were suggested to induce massive embolisms in the advanced stage of the disease.

Arbuscular mycorrhizal fungi (AMF) live in a community in the roots of host plants. Still, the patterns and factors that drive their spatiality and cohabitation remain uncovered, particularly that of trees in planted forests, which we aimed to clarify in Cryptomeria japonica, a major plantation tree in Japan. We analyzed 65 paired root and soil samples of Cryptomeria japonica trees collected from 11 microsite (MS) plots at two environmentally different forest sites in central Japan and measured soil pH, total phosphorus (TP), C, N, and the carbon-to-nitrogen ratio. Root AMF communities were recovered using Illumina’s next-generation amplicon sequencing targeting the small subunit of ribosomal DNA. We detected more than 500 AMF OTUs at each site but only three belonging to Dominikia, Rhizophagus, and Sclerocystis were dominant in the roots of C. japonica, detected each at an average relative abundance higher than 20%. Two showed negatively correlated spatial distributions and different associations with soil pH. Similarly, the physicochemical properties at MSs significantly determined the AMF assemblages in the roots of C. japonica. Dominikia, Rhizophagus, and Sclerocystis coexist in the roots of C. japonica where soil physicochemical properties, particularly pH, determine their spatial dynamic, turnovers, and cohabitation patterns. These findings highlight the importance of simultaneous colonization of plants by multiple AMF.

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.

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.