Genomic and Secretomic Analyses of the Newly Isolated Fungus Perenniporia fraxinea SS3 Identified CAZymes Potentially Related to a Serious Pathogenesis of Hardwood Trees.

Perenniporia fraxinea can colonize living trees and cause severe damage to standing hardwoods by secreting a number of carbohydrate-activate enzymes (CAZymes), unlike other well-studied Polyporales. However, significant knowledge gaps exist in understanding the detailed mechanisms for this hardwood-pathogenic fungus. To address this issue, five monokaryotic P. fraxinea strains, SS1 to SS5, were isolated from the tree species Robinia pseudoacacia, and high polysaccharide-degrading activities and the fastest growth were found for P. fraxinea SS3 among the isolates. The whole genome of P. fraxinea SS3 was sequenced, and its unique CAZyme potential for tree pathogenicity was determined in comparison to the genomes of other nonpathogenic Polyporales. These CAZyme features are well conserved in a distantly related tree pathogen, Heterobasidion annosum. Furthermore, the carbon source-dependent CAZyme secretions of P. fraxinea SS3 and a nonpathogenic and strong white-rot Polyporales member, Phanerochaete chrysosporium RP78, were compared by activity measurements and proteomic analyses. As seen in the genome comparisons, P. fraxinea SS3 exhibited higher pectin-degrading activities and higher laccase activities than P. chrysosporium RP78, which were attributed to the secretion of abundant glycoside hydrolase family 28 (GH28) pectinases and auxiliary activity family 1_1 (AA1_1) laccases, respectively. These enzymes are possibly related to fungal invasion into the tree lumens and the detoxification of tree defense substances. Additionally, P. fraxinea SS3 showed secondary cell wall degradation capabilities at the same level as that of P. chrysosporium RP78. Overall, this study suggested mechanisms for how this fungus can attack the cell walls of living trees as a serious pathogen and differs from other nonpathogenic white-rot fungi. IMPORTANCE Many studies have been done to understand the mechanisms underlying the degradation of plant cell walls of dead trees by wood decay fungi. However, little is known about how some of these fungi weaken living trees as pathogens. P. fraxinea belongs to the Polyporales, a group of strong wood decayers, and is known to aggressively attack and fell standing hardwood trees all over the world. Here, we report CAZymes potentially related to plant cell wall degradation and pathogenesis factors in a newly isolated fungus, P. fraxinea SS3, by genome sequencing in conjunction with comparative genomic and secretomic analyses. The present study provides insights into the mechanisms of the degradation of standing hardwood trees by the tree pathogen, which will contribute to the prevention of this serious tree disease.

Nitrous Oxide Emission in Response to pH from Degrading Palsa Mire Peat Due to Permafrost Thawing.

N2O, a greenhouse gas, is increasingly emitted from degrading permafrost mounds of palsa mires because of the global warming effects on microbial activity. In the present study, we hypothesized that N2O emission could be affected by a change in pH conditions because the collapse of acidic palsa mounds (pH 3.4-4.6) may result in contact with minerogenic ground water (pH 4.8-6.3), thereby increasing the pH. We compared the effects of pH change on N2O emission from cultures inoculated with peat suspensions. Peat samples were collected on a transect from a still intact high part to the collapsing edge of a degrading palsa mound in northwestern Finland, assuming the microbial communities could be different. We adjusted the pH of peat suspensions prepared from a collapsing palsa mound and compared the N2O emission in a pH gradient from 4.5 to 8.5. The collapsing edge had the highest N2O emission from the peat suspensions among all points on the transect under natural acidic conditions (pH 4.5). The N2O emission was reduced with a moderate rise in pH (pH 5.0-6.0) by approximately 85% compared with natural acidic level (pH 4.5). The bacterial communities in acidic cultures differed considerably from those in alkaline cultures. When pH was adjusted to alkaline conditions, N2O-emitting bacteria different from those present in acidic conditions appeared to emit N2O. The bacterial communities could be characterized by changing pH conditions after thawing and collapse of permafrost have contrasting impacts on N2O production that calls for further attention in future studies.

An Unknown Non-denitrifier Bacterium Isolated from Soil Actively Reduces Nitrous Oxide under High pH Conditions.

A nitrous oxide (N2O)-consuming bacterium isolated from farmland soil actively consumed N2O under high pH conditions. An acetylene inhibition assay did not show the denitrification of N2 to N2O by this bacterium. When N2O was injected as the only nitrogen source, this bacterium did not assimilate N2O. A polymerase chain reaction demonstrated that this bacterium did not have the typical nosZ gene. This bacterium belonged to Chitinophagaceae, but did not belong to known families that include bacteria with the atypical nosZ. This is the first study to show that a non-denitrifier actively reduces N2O, even under high pH conditions.

Ligninolytic Activity at 0 °C of Fungi on Oak Leaves Under Snow Cover in a Mixed Forest in Japan.

Despite the importance of litter decomposition under snow cover in boreal forests and tundra, very little is known regarding the characteristics and functions of litter-decomposing fungi adapted to the cold climate. We investigated the decomposition of oak leaves in a heavy snowfall forest region of Japan. The rate of litter weight loss reached 26.5% during the snow cover period for 7 months and accounted for 64.6% of the annual loss (41.1%). Although no statistically significant lignin loss was detected, decolourization portions of oak leaf litter, which was attributable to the activities of ligninolytic fungi, were observed during snow cover period. This suggests that fungi involved in litter decomposition can produce extracellular enzymes to degrade lignin that remain active at 0 °C. Fungi were isolated from oak leaves collected from the forest floor under the snow layer. One hundred and sixty-six strains were isolated and classified into 33 operational taxonomic units (OTUs) based on culture characteristics and nuclear rDNA internal transcribed spacer (ITS) region sequences. To test the ability to degrade lignin, the production of extracellular phenoloxidases by isolates was quantified at 0 °C. Ten OTUs (9 Ascomycota and 1 Basidiomycota) of fungi exhibited mycelial growth and ligninolytic activity. These results suggested that some litter-decomposing fungi that had the potential to degrade lignin at 0 °C significantly contribute to litter decomposition under snow cover.

(+)-Epogymnolactam, a novel autophagy inducer from mycelial culture of Gymnopus sp.

Mushrooms, including Ganoderma lucidum, have been used as a potential source of therapeutic compounds, and an autophagy inducer would be useful for treatment of diverse diseases in human. Reported here is a full account of screening, isolation, structural determination, and biological activity of an autophagy inducer, (+)-epogymnolactam (1) from a mycelial culture of a Gymnopus sp. strain. Its structure was elucidated by HR-ESI-MS, NMR, and its plus sign by specific rotation. It exists as a tautomeric mixture of 1a and 1b in MeOH. The major tautomer of 1 is (1R,5S)-4-butyl-4-hydroxy-3-aza-6-oxa-bicyclo[3.1.0]hexan-2-one (1a), and the minor tautomeric form is (2R,3S)-3-pentanoyloxirane-2-carboxamide (1b).

Morphological and genetic characteristics of the entomopathogenic fungus Ophiocordyceps nutans and its host insects.

We examined the morphology, genetic variation, and insect host species of the fungus Ophiocordyceps nutans. Fifty-two fruit bodies collected in Hokkaido, Japan, were grouped by host species, and 19 samples were analysed. The ranges of the lengths and breadths of all fruit body parts were similar among host groups. In the genetic analysis, the 5.8S rDNA region showed completely identical sequences, although differences of up to six nucleotides were recognized in the ITS regions. The distance values between our samples using the Kimura two-parameter model were lower than 0.007. Thus, the O. nutans examined here were concluded to form a closely related group with little detectable variability that parasitized nine hemipteran species.

Mycorrhizal associations in woody plant species at the Mt. Usu volcano, Japan.

We investigated the association between ectomycorrhizal (ECM) and arbuscular mycorrhizal (AM) fungi and pioneer woody plant species in areas devastated by the eruption of Mt. Usu, Japan, in 2000. We observed eight woody plant species at the research site, most of which were associated with ECM and/or AM fungi. In particular, dominant woody plant species Populus maximowiczii, Salix hultenii var. angustifolia and Salix sachalinensis were consistently associated with ECM fungi and erratically associated with AM fungi. We found one to six morphotypes in the roots of each ECM host and, on average, two in the roots of each seedling, indicating low ECM fungal diversity. ECM colonization ranged from 17 to 42% of root tips. Using morphotyping and molecular analyses, 15 ECM fungi were identified. ECM fungi differed greatly between hosts. However, Laccaria amethystea, Hebeloma mesophaeum, Thelephora terrestris and other Thelephoraceae had high relative colonization, constituting the majority of the ECM colonization in the roots of each plant species. These ECM fungi may be important for the establishment of pioneer woody plant species and further revegetation at Mt. Usu volcano.

Isolation of vegetable wasps and plant worms, Cordyceps nutans, from fruit-body tissue.

We isolated Cordyceps nutans from the stipe and abdominal tissues of fruit bodies using a surface sterilization method. Hyphal growth was observed in inocula from both the stipe and abdominal tissue. Some strains from discharged ascospores were obtained and colony characteristics were compared to the strains isolated from the tissues. Colonies of isolates from ascospores grew quite slowly. Isolates of 43 from the 52 examined fruit bodies formed colonies similar to those from ascospores. To confirm the success of isolation, we analyzed by PCR-RFLP of the ITS regions of rDNA samples from fruit bodies, isolates from fruit bodies, and isolates from ascospores. All the isolates obtained from stipe and abdominal tissues presented identical patterns. In this study, we report the first successful isolation of C. nutans from fruit-body tissue using a surface sterilization method.

Effects of bird ingestion on seed germination of Sorbus commixta.

To determine the effects of ingestion by birds on seed germination, we performed germination experiments in the field and laboratory with Sorbus commixta. The germination of four groups of seeds was compared: ingested seeds, seeds defecated in feces after feeding of fruits to birds; extracted seeds, seeds deliberately extracted from the fruit pulp; juiced seeds, seeds plus the juice of the pulp after seeds had been deliberately extracted from the pulp; intact seeds, seeds in untreated intact fruits. In the laboratory, intact and juiced seeds hardly germinated, but ingested and extracted seeds germinated. Thus, the pulp and its juice appeared to inhibit germination, but seeds could germinate without ingestion by birds once the seeds had been manually extracted from the pulp. In the field, intact fruits did not germinate in the first spring, because the seed was still covered with pulp. The pulp of intact seeds decomposed during the first summer, and thus, the seeds had the potential to germinate during the second spring. In fact, most intact seeds do not germinate during the second spring either, since they lose their viability during the first summer. Thus, under natural conditions, most seeds of Sorbus commixta cannot germinate without bird ingestion.