Coprinopsis asiaticiphlyctidospora sp. nov., an agaric ammonia fungus from Amami and Okinawa,southern Japan

Coprinopsis asiaticiphlyctidospora (Basidiomycota, Psathyrellaceae), is a new Coprinoid ammonia fungus from Amami and Okinawa, Japan. In macro- and micro-morphology, this species is similar to C. phlyctidospora and C. austrophlyctidospora, but differs in having smaller basidiospores and spiny basidiospore ornamentation. Phylogenetic analysis with the nuclear ribosomal internal transcribed spacer (ITS) data also supports this new taxon.     

<Emphasis Type="Italic">Coprinopsis austrophlyctidospora</Emphasis> sp. nov., an agaric ammonia fungus from Southern Hemisphere plantations and natural forests

A new ammonia fungus, Coprinopsis austrophlyctidospora, is described from Nothofagus and Pinus forests in New Zealand and from Eucalyptus forest in Australia. In ecology and macro-morphology, this species is similar to the Northern Hemisphere species C. phlyctidospora, but the new species differs in morphological characters of the basidiospore, i.e., in having a plage, more minute surface warts, and the smaller size of the basidiospore.     

First successful isolation of Entoloma clypeatum species complex from basidiospores

The Entoloma clypeatum species complex, known as “Harushimeji” in Japan, associates with Rosaceae and Ulmaceae plant species. In this study, we successfully isolated cultures of this fungal group via basidiospore isolation from tentative four Harushimeji species using modified Norkrans's C (MNC) medium and MNC medium containing n-butyric acid. Colony formations were observed on 22 of 25 basidioma samples; however, most exhibited slow and unsteady growth. The isolated mycelia contained dikaryotic hyphae and were identified through molecular phylogenetic analyses of the internal transcribed spacer region of fungal ribosomal RNA. Six isolates showing steady growth were deposited in the fungal culture collection of the Fungus/Mushroom Resource and Research Center, Faculty of Agriculture, Tottori University, Japan. This result indicates that basidiospore isolation is a useful method for obtaining Harushimeji strains.     

Spore formation and karyological characterization of basidiosporogenesis,meiosis, post-meiotic and nuclear migration mitosis in Coprinus comatus

In Coprinus comatus, we sampled and observed different maturity stages of basidium to find the possible causes of binucleate basidiospores by microscopy using the HCI-Giemsa and DAPI staining methods. In basidium, following karyogamy, a single nucleolus was observed. We found evidence suggesting post-meiotic mitosis following nuclear migration in the spores. Post-meiotic mitosis occurred in the basidiospores, resulting in four binucleate basidiospores. This indicates that the basidiospore is a monokaryon, and that C. comatus shows the same pattern of basidiospore formation as Coprinopsis cinerea. This type of nuclear behavior was defined as pattern D, one of six distinct patterns (pattern A–F) of nuclear behavior during basidiosporogenesis have been described for basidiomycetes by Campos and Costa in 2010.     

Sistotrema subconfluens sp. nov. (Cantharellales,Basidiomycota) from Changbaishan Nature Reserve,northeastern China

Sistotrema, typified by S. confluens, is characterized mainly by its urniform basidia mostly with 6–8 sterigmata as well as by a monomitic hyphal system, oil rich hyphae and smooth basidiospores. The species of this genus have various hymenophore configurations and basidiospore shapes. During a field trip in 2011, two specimens with urniform basidia were collected from Changbaishan Nature Reserve, northeastern China. After careful morphological and molecular studies, they are described and illustrated here as a new species, S. subconfluens. The new species shares a terrestrial habit, stipitate basidiocarps and poroid hymenophores with S. confluens, besides urniform basidia. These characters make the two species different from all other species of Sistotrema with resupinate basidiocarps on wood. Sistotrema subconfluens differs from the type by having larger basidiocarps, shorter basidiospores and consistent poroid hymenophores. In phylogeny inferred from nuclear large subunit rDNA, the two species were sister taxa but clearly separated. The difference of internal transcribed spacer sequences between the two species was 3.6%.     

Isolation of a homothallic mutant in <Emphasis Type="Italic">Lentinula edodes</Emphasis>

To obtain a homothallic mutant in Lentinula edodes, basidiospores derived from the common Bmut dikaryon (A1B1mut × A2B1mut) were treated with UV irradiation. Of a total of approximately 5000 monosporous cultures recovered, a single basidiospore isolate was found to produce the hyphae bearing clamp connections without mating. This mutant strain could form fruit bodies, and all its single basidiospore isolates developed into colonies with clamp connections. Such homothallic behaviors were transmitted from the mutant strain to the next generation. During the germination and following hyphal elongation in a single basidiospore of mutant strain, clamp connections were clearly detected in multicellular hyphae, which contained two nuclei in each cell. Their clamp connections were morphologically variable, viz., pseudo, abnormal, and true clamps. Amplified fragment length polymorphism (AFLP) profiles among the basidiospore isolates of mutant strain were identical, indicating that the mutant strain produced isogenic basidiospore progeny. Contribution no. 385 from the Tottori Mycological Institute     

The Genetic Structure of Phellinus noxius and Dissemination Pattern of Brown Root Rot Disease in Taiwan

Since the 1990s, brown root rot caused by Phellinus noxius (Corner) Cunningham has become a major tree disease in Taiwan. This fungal pathogen can infect more than 200 hardwood and softwood tree species, causing gradual to fast decline of the trees. For effective control, we must determine how the pathogen is disseminated and how the new infection center of brown root rot is established. We performed Illumina sequencing and de novo assembly of a single basidiospore isolate Daxi42 and obtained a draft genome of ~40 Mb. By comparing the 12,217 simple sequence repeat (SSR) regions in Daxi42 with the low-coverage Illumina sequencing data for four additional P. noxius isolates, we identified 154 SSR regions with potential polymorphisms. A set of 13 polymorphic SSR markers were then developed and used to analyze 329 P. noxius isolates collected from 73 tree species from urban/agricultural areas in 14 cities/counties all around Taiwan from 1989 to 2012. The results revealed a high proportion (~98%) of distinct multilocus genotypes (MLGs) and that none of the 329 isolates were genome-wide homozygous, which supports a possible predominant outcrossing reproductive mode in P. noxius. The diverse MLGs exist as discrete patches, so brown root rot was most likely caused by multiple clones rather than a single predominant strain. The isolates collected from diseased trees near each other tend to have similar genotype(s), which indicates that P. noxius may spread to adjacent trees via root-to-root contact. Analyses based on Bayesian clustering, F _ST statistics, analysis of molecular variance, and isolation by distance all suggest a low degree of population differentiation and little to no barrier to gene flow throughout the P. noxius population in Taiwan. We discuss the involvement of basidiospore dispersal in disease dissemination.     

Inheritance of Strain Instability (Sectoring) in the Commercial Button Mushroom, Agaricus bisporus

The button mushroom, Agaricus bisporus, is a commercially important cultivated filamentous fungus. During the last decade, the button mushroom industry has depended mainly on two strains (or derivatives of these two strains). Using one of these highly successful strains (strain U1) we examined the phenomenon of strain instability, specifically, the production of irreversible sectors. Three “stromatal” and three “fluffy” sectors were compared with a healthy type U1 strain and with a wild-collected isolate. Compost colonization and fruit body morphology were examined. The main objective of this study, however, was to examine the meiotic stability of the sectored phenotype. Single basidiospores were isolated and subjected to a grain bioassay in which the ability to produce sectors was measured. Our results were as follows: (i) basidiospore cultures obtained from a wild-collected isolate showed no tendency to produce sectors; (ii) approximately 5% of the basidiospore cultures obtained from healthy type U1 strains produced irreversible sectors in the grain bioassay; (iii) the five primary sectors examined produced basidiospore cultures, half of which produced normal-looking growth in the grain bioassay and half of which produced some degree of sectoring; and (iv) the one sectored isolate that represented the F2 generation gave ratios similar to the 1:1 ratio observed for the F1 cultures.     

Novelties in the Corticiales: Vuilleminia nilsii sp. nov. and Dendrominia gen. nov. (Basidiomycota)

Vuilleminia nilsii sp. nov. is described based on collections made in Argelès-sur-Mer communal forest, France. It is characterized by a resupinate, smooth, whitish, decorticating basidiocarp, cylindrical cystidia, heavily crystallized matrix, and guttulate, allantoid spores. It can be distinguished from V. coryli by basidiospore size, substrate preferences, abundance of dendrohyphidia, and crystal in the context. Phylogenetic analyses place Vuilleminia ericae, Dendrothele dryina (= V. dryina) and Dendrothele maculata in a distinct, well-defined clade. The new genus Dendrominia is proposed for this clade. The new combinations Dendrominia dryina, D. ericae, and D. maculata are proposed. Dendrothele corticola is regarded as a synonym of D. maculata. A lectotype is selected for V. macrospora. An updated key to the genus Vuilleminia and a key to Dendrominia species are provided.     

Changes caused by air-drying and alcohol dehydration of spread insect sperm and fungal spore chromosome fibres

The morphology and width of chromatin fibres from Schizophyllum commune basidiospore and Drosophila melanogaster sperm were studied using a modified whole-mount technique. As revealed by electron micrographs, the native chromatin fibre organization and width, about 130 Å in sperm and 30 Å in spore, were preserved if samples were stained immediately after spreading and drying the material without alcohol dehydration. Following air-drying and alcohol dehydration, visible changes of fibre morphology took place: air-drying before staining produced unspecific side-by-side aggregates of fibres, and a coarse fibrillar network was formed as a consequence of alcohol dehydration.     

Utilization of Ammonia Nitrogen by Intestinal Bacteria Isolated from Pigs

In a medium containing ammonia, proteose peptone, and cysteine as nitrogen sources, 17 of 24 Bacteroidaceae strains, 3 of Selenomonas strains, 1 of 7 curved rods, 3 of 7 Spirochaetaceae strains, 8 of 20 Eubacterium strains, 8 of 13 Peptococcaceae strains, 3 of 4 Clostridium strains, 19 of 20 Enterobacteriaceae strains, and 1 of 8 Streptococcus strains utilized ammonia nitrogen preferentially to proteose peptone nitrogen. To determine the ability of intestinal microbes to synthesize amino acids from ammonia, ammonia utilization by Bacteroides ruminicola strain 9 was studied in defined media containing ammonia and other nitrogen sources. In another medium containing ammonia, proteose peptone, and cysteine as nitrogen sources, ammonia was preferentially utilized even when the proteose peptone nitrogen content was eight times greater than that of ammonia nitrogen. In a medium containing ammonia, an amino acid, and cysteine, the lowest uptake of ammonia nitrogen was observed when the medium contained aspartic acid, glutamic acid, threonine, or alanine; but ammonia was utilized more effectively than any of the amino acids. Incorporation of ¹⁵N from [¹⁵N]ammonia into bacterial amino acids was studied. ¹⁵N was incorporated into every amino acid of B. ruminicola strain 9, and the highest uptake was observed in aspartic acid and alanine.     

Cytological studies of <Emphasis Type="Italic">Agaricus brasiliensis</Emphasis>

Agaricus brasiliensis is a medicinal mushroom native to Brazil. It was first identified as Agaricus blazei and its scientific name continues to be debated. We examined the cytology of different Brazilian commercial strains of A. brasiliensis and the nuclear behavior of strain CS1 during basidiospore development using fluorescent microscopy. All strains have multinucleate hyphae and no significant differences in nuclei numbers were observed between them. Basidia from A. brasiliensis strain CS1 are typically tetrasporics and produce binucleate basidiospores, demonstrating that a postmeiotic mitosis occurs during basidiospore development. This result suggests that A. brasiliensis is primarily a heterothallic species.     

Presence of pathogenic cryptococci on trees situated in two recreational areas in South Africa

Knowledge of the environmental prevalence of members of the Cryptococcus neoformans/Cryptococcus gattii species complex is important, since cryptococcal infection is acquired from the environment. We determined whether trees located in two South African recreational areas harboured pathogenic cryptococci and compared the isolates to clinical isolates obtained from Western Cape hospitals with molecular typing techniques. The majority of isolates originating from trees in a public park in Cape Town (PPCT) were C. gattii sensu stricto, followed by C. neoformans sensu stricto genotype AFLP1/VNI. The PPCT trees might be a source of infection, since all genotype AFLP1/VNI isolates from these trees and one clinical isolate belonged to the same sequence type (ST), i.e. ST23. Recombination and basidiospore production might be occurring in PPCT trees that contained C. gattii s.s. isolates belonging to both mating types. The presence of C. gattii s.s. in PPCT trees might therefore pose a risk to human health.     

Life cycle and nuclear behavior in three rust fungi (Uredinales)

Kuehneola japonica has a microcyclic life cycle with a regular alternation of generations. Single basidiospore inoculations onto Rosa wichuraiana resulted in teliospore production, indicating its homothallic nature. Dikaryotization in a vegetative mycelium in the host seemed to occur through nuclear division that was not followed by septum formation. Karyogamy and meiosis took place through teliospore and metabasidium development; this fungus was considered to reproduce genetically homogeneous progenies. Puccinia lantanae and P. patriniae were also microcyclic in their life cycle; however, these fungi differed from K. japonica in the mode of nuclear behavior. In the former two fungi, both vegetative and reproductive cells were uninucleate. No karyogamy was observed, and nuclear division in the metabasidium development was thought to be mitotic. In P. lantanae, a basidiospore was formed on a sterigma, whereas a whiplike hypha emerged from each metabasidium cell in P. patriniae. Inoculations of Justicia procumbens with a single basidiospore of P. lantanae resulted in teliospore production. The fungus seemed to remain uninucleate, either haploid or diploid, throughout the life cycle. Thus, reproduction was considered to be apomictic. Received: August 16, 2001 / Accepted: October 1, 2001     

曝气充氧条件下污染河道氨挥发特性模拟

以污染河道为研究对象,模拟研究污染河道在曝气充氧(底泥曝气,ES组;水曝气,EW组)条件下氨挥发的特性,探讨主要影响因素及其作用过程.研究表明,污染河道水体具有一定氨挥发潜力,在实验室模拟条件下,氨挥发速率平均为2.51mg·m-2·h-1,相当于0.50 kgN· hm-2·d-1;曝气污染河道水体的氨挥发有一定的促进作用,与对照相比(EC组)氨挥发速率和累积氨挥发量存在显著差异(P＜ 0.05);不同曝气方式对氨挥发过程影响不同,氨挥发速率存在显著差异(P＜0.05);至实验结束,EW组的累积挥发量为2809.76 mg/m2,分别是ES组和EC组的1.17和2.25倍;各实验组的氨挥发累积量用一级动力学方程能很好地拟合,根据模型可以预测氨挥发量;同一温度条件下,pH值、铵氮浓度和通气频率是影响氨挥发的主要因素;曝气可以通过增加通气频率和提高水体pH值来促进氨挥发进行;在曝气作用下随着硝化过程的进行对氨挥发有一定的限制作用;曝气条件下,氨挥发作用在硝化过程启动阶段最为明显.     

Phylogenetically Diverse Denitrifying and Ammonia-Oxidizing Bacteria in Corals Alcyonium gracillimum and Tubastraea coccinea

To date, the association of coral–bacteria and the ecological roles of bacterial symbionts in corals remain largely unknown. In particular, little is known about the community components of bacterial symbionts of corals involved in the process of denitrification and ammonia oxidation. In this study, the nitrite reductase (nirS and nirK) and ammonia monooxygenase subunit A (amoA) genes were used as functional markers. Diverse bacteria with the potential to be active as denitrifiers and ammonia-oxidizing bacteria (AOB) were found in two East China Sea corals: stony coral Alcyonium gracillimum and soft coral Tubastraea coccinea. The 16S rRNA gene library analysis demonstrated different communities of bacterial symbionts in these two corals of the same location. Nitrite reductase nirK gene was found only in T. coccinea, while both nirK and nirS genes were detected in A. gracillimum, which might be the result of the presence of different bacterial symbionts in these two corals. AOB rather than ammonia-oxidizing archaea were detected in both corals, suggesting that AOB might play an important role in the ammonia oxidation process of the corals. This study indicates that the coral bacterial symbionts with the potential for nitrite reduction and ammonia oxidation might have multiple ecological roles in the coral holobiont, which promotes our understanding of bacteria-mediated nitrogen cycling in corals. To our knowledge, this study is the first assessment of the community structure and phylogenetic diversity of denitrifying bacteria and AOB in corals based on nirK, nirS, and amoA gene library analysis.     

Genetic Differentiation and Spatial Structure of Phellinus noxius,the Causal Agent of Brown Root Rot of Woody Plants in Japan

Phellinus noxius is a pathogenic fungus that causes brown root rot disease in a variety of tree species. This fungus is distributed in tropical and sub-tropical regions of Southeast and East Asia, Oceania, Australia, Central America and Africa. In Japan, it was first discovered on Ishigaki Island in Okinawa Prefecture in 1988; since then, it has been found on several of the Ryukyu Islands. Recently, this fungus was identified from the Ogasawara (Bonin) Islands, where it has killed trees, including rare endemic tree species. For effective control or quarantine methods, it is important to clarify whether the Japanese populations of P. noxius are indigenous to the area or if they have been introduced from other areas. We developed 20 microsatellite markers from genome assembly of P. noxius and genotyped 128 isolates from 12 of the Ryukyu Islands and 3 of the Ogasawara Islands. All isolates had unique genotypes, indicating that basidiospore infection is a primary dissemination method for the formation of new disease foci. Genetic structure analyses strongly supported genetic differentiation between the Ryukyu populations and the Ogasawara populations of P. noxius. High polymorphism of microsatellite loci suggests that Japanese populations are indigenous or were introduced a very long time ago. We discuss differences in invasion patterns between the Ryukyu Islands and the Ogasawara Islands.     

Molecular Characterization of Branchial aquaporin 1aa and Effects of Seawater Acclimation,Emersion or Ammonia Exposure on Its mRNA Expression in the Gills,Gut, Kidney and Skin of the Freshwater Climbing Perch,Anabas testudineus

We obtained a full cDNA coding sequence of aquaporin 1aa (aqp1aa) from the gills of the freshwater climbing perch, Anabas testudineus, which had the highest expression in the gills and skin, suggesting an important role of Aqp1aa in these organs. Since seawater acclimation had no significant effects on the branchial and intestinal aqp1aa mRNA expression, and since the mRNA expression of aqp1aa in the gut was extremely low, it can be deduced that Aqp1aa, despite being a water channel, did not play a significant osmoregulatory role in A. testudineus. However, terrestrial exposure led to significant increases in the mRNA expression of aqp1aa in the gills and skin of A. testudineus. Since terrestrial exposure would lead to evaporative water loss, these results further support the proposition that Aqp1aa did not function predominantly for the permeation of water through the gills and skin. Rather, increased aqp1aa mRNA expression might be necessary to facilitate increased ammonia excretion during emersion, because A. testudineus is known to utilize amino acids as energy sources for locomotor activity with increased ammonia production on land. Furthermore, ammonia exposure resulted in significant decreases in mRNA expression of aqp1aa in the gills and skin of A. testudineus, presumably to reduce ammonia influx during ammonia loading. This corroborates previous reports on AQP1 being able to facilitate ammonia permeation. However, a molecular characterization of Aqp1aa from A. testudineus revealed that its intrinsic aquapore might not facilitate NH₃ transport. Hence, ammonia probably permeated the central fifth pore of the Aqp1aa tetramer as suggested previously. Taken together, our results indicate that Aqp1aa might have a greater physiological role in ammonia excretion than in osmoregulation in A. testudineus.