Shin-ichiro Ito

Seasonal and environmental changes of mycorrhizal associations and heterotrophy levels in mixotrophic Pyrola japonica (Ericaceae) growing under different light environments

PREMISE OF THE STUDY Mixotrophy is a strategy whereby plants acquire carbon both through photosynthesis and heterotrophic exploitation of mycorrhizal fungi. In Euro-American Pyroleae species studied hitherto, heterotrophy levels vary according to species, sites of study, and possibly light conditions. We investigated mycorrhizal association and mixotrophy in the Asiatic forest species Pyrola japonica, and their plasticity under different light conditions. METHODS Pyrola japonica was sampled bimonthly in sunny and shaded conditions from a deciduous broadleaf forest. We microscopically assessed the rate of fungal colonization and sequenced the ITS to identify the mycorrhizal fungi. We measured (13)C and (15)N isotopic abundances in P. japonica as compared with neighboring autotrophic and mycoheterotrophic plants, to evaluate P. japonicas heterotrophy level. KEY RESULTS Pyrola japonica formed arbutoid mycorrhizas devoid of fungal mantles, with intracellular hyphal coils and a Hartig net. It tended to be more colonized by mycorrhizal fungi in spring and summer. Most associated fungi belonged to ectomycorrhizal taxa, and 84% of identified fungi were Russula spp. Rate of mycorrhizal colonization and Russula frequency tended to be higher in shaded conditions. Both δ(13)C and δ(15)N values of P. japonica were significantly higher in autotrophic plants, showing that about half of the carbon on average was received from mycorrhizal fungi. Both isotopic values negatively correlated with light availability, suggesting higher heterotrophy levels in shaded conditions. CONCLUSIONS The mixotrophic P. japonica undergoes changes in mycorrhizal symbionts and carbon nutrition according to light availability. Our results suggest that during Pyroleae evolution, a tendency to increased heterotrophy emerged in the Pyrola/Orthilia clade.

Temporal variations in endophytic fungal assemblages of Ginkgo biloba L.

We isolated endophytic fungi from living healthy leaves, petioles, and current-year twigs of Ginkgo biloba L. from April to November 2004 with the objective of identifying the dominant endophytic fungal taxa, and monitoring their occurrence and frequency. A total of 9 fungal taxa were identified to the genus level. Diversity measures inferred from the Shannon–Wiener, Morisita–Horn, and Sørensen indices indicated that leaves and petioles harbored more diverse endophytic fungal assemblages than twigs, and that fungal taxa involved in twigs shared less with those in leaves and petioles. Among the organs, the occurrence pattern of overall endophytic fungi differed significantly, and two taxa, Phomopsis sp. and Phyllosticta sp., were the most frequently isolated and thus regarded as the dominant endophytic fungi. Phomopsis sp. was isolated frequently from twigs (84.8%) but rather few from leaves (16.1%) and petioles (24.3%). Phyllosticta sp. was isolated frequently from leaves (72.9%) and petioles (65.7%) but was never isolated from twigs. Temporal changes in relative frequency of total endophytic fungi tended to differ among sampling dates for all three organs. The occurrence of Phyllosticta sp. in both leaves and petioles was first detected in August and peaked in October. Phomopsis sp. was detected in twigs throughout the growing season. These results suggest that the distribution of the two dominant endophytic fungi was organ-specific and differed within seasons.

Effects of sodium chloride on growth of ectomycorrhizal fungal isolates in culture

We studied the tolerance of ectomycorrhizal (ECM) fungi to sodium chloride (NaCl) to find the best fungus to aid growth of Pinus thunbergii. Four ECM fungi, Cenococcum geophilum, Pisolithus tinctorius, Rhizopogon rubescens, and Suillus luteus, were grown in liquid MMN media with five different concentrations of NaCl for 30 days, and their mycelial weights were determined. Mycelial weights of P. tinctorius and R. rubescens were not significantly different between 0 mM and 200 mM, whereas those of C. geophilum and S. luteus decreased with increasing NaCl concentration, indicating that the former two species were more tolerant to higher NaCl concentrations than the latter species. We further studied the intraspecific differences in NaCl tolerance of nine P. tinctorius isolates. They were grown on MMN agar media with six different concentrations of NaCl for 21 days, and their radial growth was measured. In total, the hyphal growth at 25 mM NaCl was significantly higher than those at the other NaCl concentrations, and EC50 values were confirmed at between 50 mM and 200 mM. Among the isolates, Pt03 and Pt21 showed measurable growth at 200 mM; the growth of Pt03 was not significantly different between 0 mM and 200 mM. The results indicate that there are intraspecific variations in NaCl tolerance of Pisolithus species.

Mycorrhizal fungi associated with Monotropastrum humile (Ericaceae) in central Japan.

We explored the diversity of mycorrhizal fungi associated with Monotropastrum humile in the central part of Japans main island. We collected 103 M. humile individuals from 12 sites with various forest types. We analyzed the DNA sequences of the internal transcribed spacer region from fungal and plant nuclear ribosomal DNAs to assess the genetic diversity of the fungi associated with M. humile roots and to position the plant with respect to known Monotropoideae groups, respectively. The plants formed a monophyletic clade with other members of M. humile but were separated from M. humile var. glaberrimum and other monotropes (97% bootstrap support). Of the 50 fungal phylotypes, 49 had best matches with the Russulales, and the other had highest similarity with the Thelephoraceae. Our phylogenetic analysis suggests that M. humile roots have a highly specialized association with fungal partners in the Russulaceae. Moreover, a few fungal phylotypes from the M. humile roots had positions neighboring those from Monotropa uniflora roots. These results indicated that the genetic diversity of mycorrhizal fungi of M. humile was highly specific to the Russulaceae, but with high diversity within that family, and that the fungi associated with M. humile differ from those associated with M. uniflora.

Spatial distribution and genetic structure of Cenococcum geophilum in coastal pine forests in Japan

The asexual ectomycorrhizal fungus Cenococcum geophilum has a wide geographic range in diverse forest ecosystems. Although its genetic diversity has been documented at a stand or regional scale, knowledge of spatial genetic structure is limited. We studied the genetic diversity and spatial structure of C. geophilum in eight Japanese coastal pine forests with a maximum geographic range of 1364 km. A total of 225 samples were subjected to phylogenetic analysis based on the glyceraldehyde 3-phosphate dehydrogenase gene (GAPDH) followed by microsatellite analysis with five loci. The phylogenetic analysis based on GAPDH resolved three groups with most isolates falling into one dominant lineage. Microsatellite analyses generated 104 multilocus genotypes in the overall populations. We detected significant genetic variation within populations and genetic clusters indicating that high genetic diversity may be maintained by possible recombination processes at a stand scale. Although no spatial autocorrelation was detected at a stand scale, the relationship between genetic and geographic distances among the populations was significant, suggesting a pattern of isolation by distance. These results indicate that cryptic recombination events at a local scale and unknown migration events at both stand and regional scales influence spatial distribution and genetic structure of C. geophilum in coastal pine forests of Japan.

Manganese hyperaccumulation from non-contaminated soil in Chengiopanax sciadophylloides Franch. et Sav. and its correlation with calcium accumulation

Abstract The objective of this study was to explore the ability of Chengiopanax sciadophylloides Franch. et Sav. to hyperaccumulate manganese (Mn). We were particularly interested in (1) Mn uptake ability relative to soil-Mn availability, (2) the potential use of this species in phytomining, and (3) potential key physiological factors associated with Mn hyperaccumulation ability. Among plants sampled from 17 locations in their native Japan provenance, the maximum foliar Mn concentration was 23,000 mg kg−1 dry weight (dw; mean: 11,000 mg kg−1). Soils from all sites examined contained low to normal Mn concentrations. There were no significant relationships detected between several soil factors and foliar Mn concentrations, although we found a strong positive correlation between foliar Mn and calcium (Ca) concentrations. Half of the foliar Mn was readily extractable with water, and almost all Mn was extractable with hydrochloric acid (HCl). Furthermore, a highly purified Mn compound was precipitated from leaf ash solutions when pH was adjusted to 8–10. We demonstrated that C. sciadophylloides has an extraordinary Mn-accumulating ability in non-contaminated soil and that Ca accumulation is correlated with Mn hyperaccumulation. We also showed that collecting Mn from C. sciadophylloides is easy and straightforward. We discuss the potential for using the plant in Mn phytomining.

Enkianthus campanulatus (Ericaceae) is commonly associated with arbuscular mycorrhizal fungi

Enkianthus is the most basal extant genus in the phylogeny of ericaceous plants. Its members harbor arbuscular mycorrhiza (AM)-like hyphal structures in their roots but, as yet, no study has surveyed the AM fungal species component. Roots from six species of Enkianthus were collected from five distantly located sites in Japan. Intracellular hyphal coils were observed in the root cortical cells of all species. Fungal DNA sequences of the small subunit ribosomal RNA gene were obtained from 73 of 75 segments of Enkianthus campanulatus roots by PCR using either AML2 or NS31/AM1primer pairs. Results indicated that all E. campanulatus trees were extensively associated with Glomus spp. A phylogenetic analysis showed that 71 root segments harbored fungi belonging to Glomus group A. Among eight delineated clades, seven did not nest with any known AM fungal species. One clade was detected in all roots at all sites at relatively high frequencies, but the rest were detected sporadically at each site. The placement of sequences from distantly located sites into a single clade without known AM fungal species suggests the common association of E. campanulatus with particular AM fungal taxa.

Extractives of Quercus crispula sapwood infected by the pathogenic fungi Raffaelea quercivora I: comparison of sapwood extractives from noninfected and infected samples.

The extracts of Quercus crispula infected by the ambrosia fungus, Raffaelea quercivora, were investigated. Phenol and tannin analyses indicated that normal sapwood (NS) contained a considerable amount of hydrolysable tannins, while infected colored sapwood (IS) contained less hydrolysable tannins and more phenols than NS. In treating pentagalloyl glucose (PGG), which is a model compound of hydrolysable tannins, with a culture medium of R. quercivora, PGG was rapidly hydrolyzed to produce gallic acid. The resulting gallic acid decreased in concentration over the subsequent cultivation period eventually disappeared. Measuring tannase and laccase activities of the culture medium of R. quercivora, tannase activity increased gradually from the beginning, while laccase activity increased rapidly at 5 days of incubation and disappeared at 8 days. An oxidative product from gallic acid treated with laccase was isolated by preparative high performance liquid chromatography, and was identified as purprogallincarboxylic acid (PGCA) by nuclear magnetic resonance spectroscopy and electron-impact mass spectrometry. PGCA was present in a 70% aqueous acetone extract of IS, and showed slight growth inhibition against R. quercivora.

The Effect of Raffaelea quercus-mongolicae Inoculations on the Formation of Non-conductive Sapwood of Quercus mongolica

Abstract In Korea, mass mortality of Quercus mongolica trees has become obvious since 2004. Raffaelea quercus-mongolicae is believed to be a causal fungus contributing the mortality. To evaluate the pathogenicity of the fungus to the trees, the fungus was multiple- and single-inoculated to the seedlings and twigs of the mature trees, respectively. In both the inoculations, the fungus was reisolated from more than 50% of inoculated twigs and seedlings. In the single inoculations, proportions of the transverse area of non-conductive sapwood at inoculation points and vertical lengths of discoloration expanded from the points were significantly different between the inoculation treatment and the control. In the multiple inoculations, no mortality was confirmed among the seedlings examined. These results showed that R. quercus-mongolicae can colonize sapwood, contribute to sapwood discoloration and disrupt sap flows around inoculation sites of Q. mongolica, although the pathogenicity of the fungus was not proven.

Arbuscular mycorrhizas on Athyrium yokoscense and A. niponicum grown at a lead-contaminated site

To investigate the occurrence of arbuscular mycorrhizas (AM) and their morphological types, four Athyrium yokoscense and five A. niponicum individuals were collected at a lead-contaminated site. Three of the A. yokoscense and four of the A. niponicum formed AM structures. However, the percentage of AM formation within both species differed significantly among individuals. Morphological types of most AM structures were the Paris-type, except for one A. yokoscense that formed both Paris-type and Arum-type-like structures in different parts of the same root section. These results demonstrate that the two Athyrium species form AM associations in lead-contaminated soil.