Temporal Transcriptome Profiling of Pinus densiflora Infected with Pine Wood Nematode Reveals Genetically Programmed Changes upon Pine Wilt Disease.

Pine, an evergreen conifer, is widely distributed worldwide. It is economically, scientifically, and ecologically important. However, pine wilt disease (PWD) induced by the pine wood nematode (PWN) adversely affects pine trees. Many studies have been conducted on the PWN and its beetle vectors to prevent the spread of PWD. However, studies providing a comprehensive understanding of the pine tree transcriptome in response to PWN infection are lacking. Here, we performed temporal profiling of the pine tree transcriptome using PWD-infected red pine trees, Pinus densiflora, inoculated with the PWN by RNA sequencing. Our analysis revealed that defense-responsive genes involved in cell wall modification, jasmonic acid signaling, and phenylpropanoid-related processes were significantly enriched 2 weeks after PWD infection. Furthermore, some WRKY-type and MYB-type transcription factors were upregulated 2 weeks after PWD infection, suggesting that these transcription factors might be responsible for the genome-wide reprogramming of defense-responsive genes in the early PWD stage. Our comprehensive transcriptome analysis will assist in developing PWD-resistant pine trees and identifying genes to diagnose PWD at the early stage of infection, during which large-scale phenotypic changes are absent in PWD-infected pine trees.

Nocardioides pini sp. nov. and Nocardioides pinisoli sp. nov., two novel actinomycetes isolated from Pinus densiflora.

Two novel Gram-positive bacteria designated as strains STR2T and STR3T were isolated from the rhizosphere of a Pinus densiflora sample collected from Goyang-si, Republic of Korea. Strains STR2T and STR3T were aerobic, rod shaped, non-sporulated, catalase negative, oxidase negative and non-motile bacteria. They grew at 15-37 °C (optimum, 25-30 °C), at pH 6.0-11.0 (optimum, pH 7.0) and in the presence of 0-2% NaCl (optimum, 0 %, w/v). The chemotaxonomic and morphological characteristics of the novel strains were consistent with those of the members of Nocardioides. The phylogenetic analysis of the 16S rRNA gene sequences revealed that STR2T was closely related to N. cavernae YIM A1136T (99.3 %) and N. flavus Y4T (99.1 %), and STR3T was closely related to N. exalbidus DSM 22017T (99.0 %), N. baculatus G10T (98.8 %) and N. hwasunensis HFW-21T (98.7 %). The average nucleotide identity, average amino acid identity and digital DNA-DNA hybridization values of STR2T and STR3T with the most closely related strains that have publicly available whole genomes were 83.1-89.8 %, 80.9-89.6% and 26.2-39.1 %, respectively. The cell-wall peptidoglycan of strain STR2T and STR3T contained ll-diaminopimelic acid as the diagnostic amino acid. The major fatty acids in STR2T and STR3T were iso-C16 : 0 and C17 : 1 ω8c, and the predominant quinone was MK-8(H4). Their polar lipid profile consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol and other polar lipids. The draft genome sequences showed that the genomic DNA G+C content of STR2T and STR3T were both 72.2 mol%. Physiological and biochemical tests and 16S rRNA sequence analysis clearly revealed that STR2T and STR3T could represent novel Nocardioides species. Their proposed names were as follows: Nocardioides pini sp. nov. for strain STR2T (=KACC 22784T=TBRC 16336T) and Nocardioides pinisoli sp. nov. for strain STR3T (= KACC 22785T=TBRC 16337T).

Anti-Amnesia-like Effect of Pinus densiflora Extract by Improving Apoptosis and Neuroinflammation on Trimethyltin-Induced ICR Mice.

This study was conducted to investigate the anti-amnestic property of Korean red pine bark extract (KRPBE) on TMT-induced cognitive decline in ICR mice. As a result of looking at behavioral function, the consumption of KRPBE improved the spatial work ability, short-term learning, and memory ability by Y-maze, passive avoidance, and Morris water maze tests. KRPBE suppressed antioxidant system damage by assessing the SOD activity, reduced GSH content, and MDA levels in brain tissue. In addition, it had a protective effect on cholinergic and synaptic systems by regulating ACh levels, AChE activity, and protein expression levels of ChAT, AChE, SYP, and PSD-95. Also, the KRPBE ameliorated TMT-induced mitochondrial damage by regulating the ROS content, MMP, and ATP levels. Treatment with KRPBE suppressed Aß accumulation and phosphorylation of tau and reduced the expression level of BAX/BCl-2 ratio and caspase 3, improving oxidative stress-induced apoptosis. Moreover, treatment with KRPBE improved cognitive dysfunction by regulating the neuro-inflammatory protein expression levels of p-JNK, p-Akt, p-IκB-α, COX-2, and IL-1ß. Based on these results, the extract of Korean red pine bark, which is discarded as a byproduct of forestry, might be used as an eco-friendly material for functional foods or pharmaceuticals by having an anti-amnesia effect on cognitive impairment.

Comparison of Antibacterial Activities of Korean Pine (Pinus densiflora) Needle Steam Distillation Extract on Escherichia coli and Staphylococcus aureus Focusing on Membrane Fluidity and Genes Involved in Membrane Lipids and Stress.

The antibacterial activity and mechanism of Pinus densiflora extracts against Escherichia coli and Staphylococcus aureus were investigated. The growth inhibition tests of paper diffusion and optical density exhibited that the extracts have potent antibacterial potentials against foodborne pathogens. The measurement of membrane fluidity by fluorescence polarization has indicated that one of the antibacterial mechanisms involves the disruption of membrane integrity resulting in an increase in the membrane fluidity in both of E. coli and S. aureus. The alteration of fatty acid composition was accompanied by the disturbance of membranes thus shifting the proportion of saturated verses unsaturated fatty acids or trans fatty acids from 1.27:1 to 1.35:1 in E. coli and 1.47:1 to 2.31:1 in S. aureus, most likely to compensate for the increased membrane fluidity by means of a higher proportion of saturated fatty acids which is known to render rigidity in membranes. Realtime q-PCR (polymerase chain reaction) analysis of fatty acid synthetic genes and bacterial stress genes revealed that there was minimal influence of P. densiflora extracts on fatty acid genes except for fab I and the stress rpos in E. coli, and relatively greater impact on fatty acid genes and the stress sigB in S. aureus.

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.

Exploring fine-scale assembly of ectomycorrhizal fungal communities through phylogenetic and spatial distribution analyses.

Ectomycorrhizal fungi (EMF) form symbiotic relationship with the roots of host plants. EMF communities are composed of highly diverse species; however, how they are assembled has been a long-standing question. In this study, we investigated from a phylogenetic perspective how EMF communities assemble on Pinus densiflora seedlings at different spatial scales (i.e., seedling scale and root tip scale). P. densiflora seedlings were collected from different habitats (i.e., disturbed areas and mature forests), and their EMF communities were investigated by morphotype sequencing and next-generation sequencing (NGS). To infer assembly mechanisms, phylogenetic relatedness within the community (i.e., phylogenetic structure) was estimated and spatial distribution of EMF root tips was analyzed. The EMF communities on pine seedlings were largely different between the two habitats. Phylogenetically restricted lineages (Amphinema, /suillus-rhizopogon) were abundant in the disturbed areas, whereas species from diverse lineages were abundant in the mature forests (Russula, Sebacina, /tomentella-thelephora, etc.). In the disturbed areas, phylogenetically similar EMF species were aggregated at the seedling scale, suggesting that disturbance acts as a powerful abiotic filter. However, phylogenetically similar species were spatially segregated from each other at the root tip scale, indicating limiting similarity. In the mature forest seedlings, no distinct phylogenetic signals were detected at both seedling and root tip scale. Collectively, our results suggest that limiting similarity may be an important assembly mechanism at the root tip scale and that assembly mechanisms can vary across habitats and spatial scales.

Spore germination and ectomycorrhizae formation of Tricholoma matsutake on pine root systems with previously established ectomycorrhizae from a dikaryotic mycelial isolate of T. matsutake.

In vitro ectomycorrhizal synthesis of Tricholoma matsutake with host plants has been widely conducted to elucidate fungal symbiotic properties for future cultivation practices. Here, we report on the importance of basidiospore inocula for this fungus to provide ectomycorrhizal seedlings in vitro. Ectomycorrhizal pine seedlings synthesized in vitro with cultured mycelium of T. matsutake (isolate #45 or #84) in a 250-mL culture vessel (soil volume) were transplanted to a large 1-L culture vessel. Fresh basidiospores of this fungus were aseptically inoculated on the ectomycorrhizal root system. The ectomycorrhizal seedlings in the 1-L vessel were grown for 9 months, and some plants were further grown for 6 more months under non-aseptic conditions in 4.1-L jars. The ectomycorrhizal seedlings previously inoculated with isolate #84 in the 1-L vessel showed significant ectomycorrhizal biomass (mycorrhizal root length) after spore inoculation. The ectomycorrhizal seedlings in the 4.1-L vessel showed large shiro structures (> 10 cm in diameter). PCR amplification of intergenic spacer 1 of the rRNA gene and long terminal repeat retroelement of T. matsutake in ectomycorrhizal root tips in both the 1-L vessels and 4.1-L jars revealed the presence of amplicons of the previously inoculated culture isolate of T. matsutake and the new genet(s) that established via germination of the inoculated basidiospores. This is the first report that inoculated basidiospores of T. matsutake germinated and colonized the host root to generate ectomycorrhizae in vitro.

Mycorrhizal synthesis, morpho-anatomical characterization of mycorrhizae, and evaluation of mycorrhiza-forming ability of Hydnum albidum-like species using monokaryotic and dikaryotic cultures.

Despite the economic and ecological importance of Hydnum species, in vitro synthesis of ectomycorrhizae of this genus has not been reported due to difficulties in establishing pure cultures. We inoculated pure cultures of 12 monokaryotic and 3 dikaryotic mycelial strains of an undescribed Hydnum albidum-like species on roots of axenic Pinus densiflora seedlings to synthesize ectomycorrhizae and to evaluate their mycorrhiza-forming ability. Six months after inoculation, both monokaryotic and dikaryotic strains formed ectomycorrhizae with Hartig net hyphae at the root cortex. Monokaryotic and dikaryotic strains exhibited similar morpho-anatomical characteristics of ectomycorrhizae, with the exception for clamped septa of emanating and outer mantle hyphae in the latter. Between monokaryotic and descendant dikaryotic strains, there were no significant differences in number of mycorrhizae in pine seedlings, whereas monokaryotic strains showed a greater total number of root tips and lower colonization rates than the descendant dikaryotic strains. These results indicate that both monokaryotic and dikaryotic mycelia of the H. albidum-like species can form mycorrhizae under axenic condition, and that can be applied toward the cultivation of hedgehog mushrooms.

Anti-Gastritis and Anti-Lung Injury Effects of Pine Tree Ethanol Extract Targeting Both NF-κB and AP-1 Pathways.

An ethanol extract (Pd-EE) of Pinus densiflora Siebold and Zucc was derived from the branches of pine trees. According to the Donguibogam, pine resin has the effects of lowering the fever, reducing pain, and killing worms. The purpose of this study is to investigate whether Pd-EE has anti-inflammatory effects. During in vitro trials, NO production, as well as changes in the mRNA levels of inflammation-related genes and the phosphorylation levels of related proteins, were confirmed in RAW264.7 cells activated with lipopolysaccharide depending on the presence or absence of Pd-EE treatment. The activities of transcription factors were checked in HEK293T cells transfected with adapter molecules in the inflammatory pathway. The anti-inflammatory efficacy of Pd-EE was also estimated in vivo with acute gastritis and acute lung injury models. LC-MS analysis was conducted to identify the components of Pd-EE. This extract reduced the production of NO and the mRNA expression levels of iNOS, COX-2, and IL-6 in RAW264.7 cells. In addition, protein expression levels of p50 and p65 and phosphorylation levels of FRA1 were decreased. In the luciferase assay, the activities of NF-κB and AP-1 were lowered. In acute gastritis and acute lung injury models, Pd-EE suppressed inflammation, resulting in alleviated damage.

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.

Pinus densiflora bark extract ameliorates 2,4-dinitrochlorobenzene-induced atopic dermatitis in NC/Nga mice by regulating Th1/Th2 balance and skin barrier function.

Korean red pine (Pinus densiflora) bark has been traditionally used in Korea and other parts of East Asia to relieve inflammatory diseases. Although many studies using P. densiflora bark have been reported, its effect on atopic dermatitis (AD) has not been elucidated. Thus, we investigated whether the P. densiflora bark extract (PBE) has potential to attenuate AD symptoms and elucidated the molecular mechanism. Oral administration of PBE to mice with 2,4-dinitrochlorobenzene (DNCB)-induced AD lessened dermatitis scores and scratching behavior and significantly reduced measures of epidermal thickness, infiltration of mast cells and eosinophils, levels of immunoglobulin E (IgE), and IgG1 /IgG2a ratio in serum. PBE not only inhibited IL-4, IL-5, and IL-13 but also increased IFN-γ in splenic production. Furthermore, PBE significantly suppressed mRNA expression of thymic stromal lymphopoietin and further downregulated the mRNA expression of Th2 and Th17 cytokines such as IL-4, IL-13, IL-17, IL-31, and TNF-α. In addition, the protein expressions of filaggrin, involucrin, and loricrin in lesional skin were recovered by PBE. These results suggest that PBE attenuates DNCB-induced AD via regulating Th1/Th2 balance and skin barrier function.

Two new triterpenoids from the roots of Pinus densiflora.

Chemical investigation of the roots of Pinus densiflora led to the isolation of two new triterpenoids, (24S)-3ß-methoxy-24,25-epoxy-lanost-9(11)-ene (1) and 29-acetoxy-3α-methoxyserrat-14-en-21α-ol (2), together with three known serratene-type triterpenoids (3-5) and four known diterpenoids (6-9). Their structures were determined by spectroscopic analyses.

Growth and photosynthetic responses of ectomycorrhizal pine seedlings exposed to elevated Cu in soils.

It is still controversial whether ectomycorrhizal (ECM) mycelia filter out toxic metals in nutrient absorption of host trees. In this study, pine (Pinus densiflora) seedlings colonized by Cu-sensitive and Cu-tolerant ECM species were exposed to a wide spectrum of soil Cu concentrations to investigate functions of ECM fungi under Cu stress. The photosynthetic rates of intact needles were monitored in situ periodically. The biomass and elements of plants were also measured after harvest. The ameliorating effect of ECM infection on host plants exposed to toxic stress was metal concentration specific. Under lower-level Cu stress, ECM fungi increased seedling performance, while ECM seedlings accumulated more Cu than nonmycorrhizal (NM) seedlings. Under higher-level Cu stress, photosynthesis decreased well before visible symptoms of Cu toxicity appeared. The reduced photosynthesis and biomass in ECM seedlings compared to NM seedlings under higher Cu conditions were also accompanied by lower phosphorus in needles. There was no marked difference between the two fungal species. Our results indicate that the two ECM fungi studied in our system may not have an ability to selectively eliminate Cu in nutrient absorption and may not act as effective barriers that decrease toxic metal uptake into host plants.

De Novo Transcriptome Sequencing Analysis of cDNA Library and Large-Scale Unigene Assembly in Japanese Red Pine (Pinus densiflora).

Japanese red pine (Pinus densiflora) is extensively cultivated in Japan, Korea, China, and Russia and is harvested for timber, pulpwood, garden, and paper markets. However, genetic information and molecular markers were very scarce for this species. In this study, over 51 million sequencing clean reads from P. densiflora mRNA were produced using Illumina paired-end sequencing technology. It yielded 83,913 unigenes with a mean length of 751 bp, of which 54,530 (64.98%) unigenes showed similarity to sequences in the NCBI database. Among which the best matches in the NCBI Nr database were Picea sitchensis (41.60%), Amborella trichopoda (9.83%), and Pinus taeda (4.15%). A total of 1953 putative microsatellites were identified in 1784 unigenes using MISA (MicroSAtellite) software, of which the tri-nucleotide repeats were most abundant (50.18%) and 629 EST-SSR (expressed sequence tag- simple sequence repeats) primer pairs were successfully designed. Among 20 EST-SSR primer pairs randomly chosen, 17 markers yielded amplification products of the expected size in P. densiflora. Our results will provide a valuable resource for gene-function analysis, germplasm identification, molecular marker-assisted breeding and resistance-related gene(s) mapping for pine for P. densiflora.

Diplogasteroides asiaticus n. sp. is Associated with Monochamus alternatus in Japan.

Diplogasteroides asiaticus n. sp. is described and illustrated, and its molecular profile and phylogenetic status within the family Diplogastridae are inferred. Morphologically, the new species is characterized by its stomatal structure, a tube-like stoma with three small, rod-like dorsal teeth and two subventral ridges; a spicule clearly ventrally bent at 1/3 from the anterior end; a gubernaculum with a rounded anterior end and sharply pointed distal end in lateral view; nine pairs of genital papillae with an arrangement of ; a short tail spike in males; and a well-developed receptaculum seminis, i.e., the antiparallel blind sacs of the uteri beyond the vulva region and elongated conical tail in females. This new species is morphologically similar to D. haslacheri, but it can be distinguished by the morphology of the somewhat shorter tail in females. D. asiaticus n. sp. shares high sequence conservation with D. andrassyi as there is only one base pair difference in the nearly full-length 18S rDNA and seven base pair differences in the D2-D3 expansion segments of the 28S rDNA. Despite this sequence conservation, the species status of D. asiaticus n. sp. was confirmed using the biological species concept, as D. asiaticus n. sp. and D. andrassyi failed to generate viable F2 progeny in hybridization tests.

Red Pine Bark Extract Alleviates Akt/GSK-3ß Signaling Disruption in the Hippocampus of Streptozotocin-Induced Diabetic Sprague-Dawley Rats.

This study investigates whether red pine (Pinus densiflora Sieb. et Zucc.) bark extract (PBE) can alleviate diabetes and abnormal apoptosis signaling pathways in the hippocampus of streptozotocin (STZ)-induced diabetic Sprague-Dawley (SD) rats. Two dosages of PBE (15 and 30 mg/kg of body weight/day) were administered orally to STZ-induced diabetic SD rats for 20 days. Blood glucose level and body weight were measured once per week. After 20 days of oral administration of PBE, the rat hippocampus was collected, and the production of Akt, p-Akt, GSK-3ß, p-GSK-3ß, tau, p-tau, Bax, and Bcl-2 proteins were determined by western blot analysis. A decrease in blood glucose level and recovery of body weight were observed in PBE-treated diabetic rats. In the Akt/GSK-3ß/tau signaling pathway, PBE inhibited diabetes-induced Akt inactivation, GSK-3ß inactivation, and tau hyperphosphorylation. The protein production ratio of Bax/Bcl-2 was restored to the control group level. These results suggest that PBE, rich in phenolic compounds, can be used as a functional food ingredient to ameliorate neuronal apoptosis in diabetes mellitus.

The anti-amoebic activity of Pinus densiflora leaf extract against the brain-eating amoeba Naegleria fowleri.

Naegleria fowleri invades the brain and causes a fatal primary amoebic meningoencephalitis (PAM). Despite its high mortality rate of approximately 97%, an effective therapeutic drug for PAM has not been developed. Approaches with miltefosine, amphotericin B, and other antimicrobials have been clinically attempted to treat PAM, but their therapeutic efficacy remains unclear. The development of an effective and safe therapeutic drug for PAM is urgently needed. In this study, we investigated the anti-amoebic activity of Pinus densiflora leaf extract (PLE) against N. fowleri. PLE induced significant morphological changes in N. fowleri trophozoites, resulting in the death of the amoeba. The IC50 of PLE on N. fowleri was 62.3±0.95 µg/ml. Alternatively, PLE did not significantly affect the viability of the rat glial cell line C6. Transcriptome analysis revealed differentially expressed genes (DEGs) between PLE-treated and non-treated amoebae. A total of 5,846 DEGs were identified, of which 2,189 were upregulated, and 3,657 were downregulated in the PLE-treated amoebae. The DEGs were categorized into biological process (1,742 genes), cellular component (1,237 genes), and molecular function (846 genes) based on the gene ontology analysis, indicating that PLE may have dramatically altered the biological and cellular functions of the amoeba and contributed to their death. These results suggest that PLE has anti-N. fowleri activity and may be considered as a potential candidate for the development of therapeutic drugs for PAM. It may also be used as a supplement compound to enhance the therapeutic efficacy of drugs currently used to treat PAM.

Innate Immune-Enhancing Effect of Pinus densiflora Pollen Extract via NF-κB Pathway Activation.

Considering the emergence of various infectious diseases, including the coronavirus disease 2019 (COVID-19), people's attention has shifted towards immune health. Consequently, immune-enhancing functional foods have been increasingly consumed. Hence, developing new immune-enhancing functional food products is needed. Pinus densiflora pollen can be collected from the male red pine tree, which is commonly found in Korea. P. densiflora pollen extract (PDE), obtained by water extraction, contained polyphenols (216.29 ± 0.22 mg GAE/100 g) and flavonoids (35.14 ± 0.04 mg CE/100 g). PDE significantly increased the production of nitric oxide (NO) and reactive oxygen species (ROS) but, did not exhibit cytotoxicity in RAW 264.7 cells. Western blot results indicated that PDE induced the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2. PDE also significantly increased the mRNA and protein levels of cytokines and the phosphorylation of IKKα/ß and p65, as well as the activation and degradation of IκBα. Additionally, western blot analysis of cytosolic and nuclear fractions and immunofluorescence assay confirmed that the translocation of p65 to the nucleus after PDE treatment. These results confirmed that PDE increases the production of cytokines, NO, and ROS by activating NF-κB. Therefore, PDE is a promising nutraceutical candidate for immune-enhancing functional foods.

Factors associated with seedling establishment on logs of different fungal decay types-A seed-sowing experiment.

Wood decay fungi alter the abiotic and biotic properties of deadwood, which are important as nurse logs for seedling regeneration. However, the relationship between fungal decay type and seedling performance has not been evaluated experimentally. In this study, we examined the germination, growth, and survival of six arbuscular mycorrhizal (AM) and six ectomycorrhizal (ECM) tree species on three substrates (pine logs with brown and white rot and soil) by conducting seed-sowing experiments in a mixed forest dominated by Pinus densiflora and Quercus serrata. Analysis using ribosomal DNA internal transcribed spacer 1 (rDNA ITS1) sequencing revealed that the fungal community was significantly different across three substrates. The richness of operational taxonomic units (OTUs) of AM and ECM fungi was the largest on brown rot logs and soil, respectively. The substrate significantly affected the seedling performance when comparing wood decay types, but these were not consistent across the mycorrhizal status of the seedlings. Nevertheless, seedlings of some AM trees showed better growth and enhanced mycorrhizal colonization on brown rot logs than on white rot logs. The wood decay type influenced fungal communities in the logs and the performance of some seedling species partly by different mycorrhizal colonization rates. However, the effect was seedling species dependent and showed no apparent difference between AM and ECM trees.

Accurate measurement of optical properties of narrow leaves and conifer needles with a typical integrating sphere and spectroradiometer.

Accurate information on the optical properties (reflectance and transmittance spectra) of single leaves is important for an ecophysiological understanding of light use by leaves, radiative transfer models and remote sensing of terrestrial ecosystems. In general, leaf optical properties are measured with an integrating sphere and a spectroradiometer. However, this method is usually difficult to use with grass leaves and conifer needles because they are too narrow to cover the sample port of a typical integrating sphere. Although ways to measure the optical properties of narrow leaves have been suggested, they have problems. We propose a new measurement protocol and calculation algorithms. The protocol does not damage sample leaves and is valid for various types of leaves, including green and senescent. We tested our technique with leaves of Aucuba japonica, an evergreen broadleaved shrub, and compared the spectral data of whole leaves and narrow strips of the leaves. The reflectance and transmittance of the strips matched those of the whole leaves, indicating that our technique can accurately estimate the optical properties of narrow leaves. Tests of conifer needles confirmed the applicability.