Change in medium components and colony morphology due to mycelial growth of ectomycorrhizal fungusTricholoma bakamatsutake

Mycelia ofTricholoma bakamatsutake isolate No. 4 grew at temperatures ranging from 10 to 30°C, and the optimum was around 25°C. In well-buffered media of initial pH 5.0 and 6.0, No. 4 mycelia secreted gluconic acid and lowered medium pH. Mycelial growth then accelerated slightly; and with the exhaustion of glucose, growth and secretion of gluconic acid stopped. In 10 different media of initial pH 4.0–7.0, No. 4 mycelia showed higher gluconic acid secretion with higher initial pH. No. 4 mycelial grew best in pH 5.0 media, in which gluconic acid secretion was low. Mycelia of 29 isolates including No. 4 grew better in the media in which less glucose, total carbon and total nitrogen remained, and almost all isolates secreted gluconic acid. Most of the 29 isolates showed irregular colony shapes with rough mycelial fronts, brown pigmentation and aerial hypha on colony surfaces, and brown pigmentation of media under colonies. Dissimilarities were calculated with coded morphological characters on colonies, and similarity between isolates was found not to correlate with proximity of origin. Chlamydospores were observed on every colony of the 29 isolates. Chlamydospores were present on colonies of No. 4, reaching to 2 mm from the mycelial front, where brown pigmentation had not yet developed, and the numbers of chlamydospores incresed with mycelial aging.     

Polyethylene Glycol-induced Uptake of Bacteria into Yeast Protoplasts

Cells of the nitrogen-fixing bacterium Azotobacter vinelandii and the unicellular cyanobacterium Anacystis nidulans were introduced into protoplasts of Saccharomyces cerevisiae by the polyethylene glycol (PEG) method. Factors influencing the uptake frequency were examined, and experimental conditions were established for maximizing the uptake frequency. Under optimal conditions, each protoplast took-up a few bacterial cells. Electron-microscopic studies showed the localization of integrated bacterial cells in membrane-bound vesicles of the cytoplasm or large vacuoles. The protoplasts at the intermediate stages of uptake revealed two major mechanisms of uptake: (a) “endocytosis” by a single protoplast and (b) “cell fusion” between two or more protoplasts. Some bacterial cells disintegrated during the subsequent incubation period through a heterophagy-like process.     

The (oxalato)aluminate complex as an antimicrobial substance protecting the “shiro” of Tricholoma matsutake from soil micro-organisms

Tricholoma matsutake, a basidiomycete, forms ectomycorrhizas with Pinus densiflora as the host tree. Its fruiting body, “matsutake” in Japanese, is an edible and highly prized mushroom, and it grows in a circle called a fairy ring. Beneath the fairy ring of T. matsutake, a whitish mycelium-soil aggregated zone, called “shiro” in Japanese, develops. The front of the shiro, an active mycorrhizal zone, functions to gather nutrients from the soil and roots to nourish the fairy ring. Bacteria and sporulating fungi decrease from the shiro front, whereas they increase inside and outside the shiro front. Ohara demonstrated that the shiro front exhibited antimicrobial activity, but the antimicrobial substance has remained unidentified for 50 years. We have identified the antimicrobial substance as the (oxalato)aluminate complex, known as a reaction product of oxalic acid and aluminum phosphate to release soluble phosphorus. The complex protects the shiro from micro-organisms, and contributes to its development.     

大百合小孢子发生和雄配子体发育研究

用常规石蜡切片技术和压片法对大百合小孢子发生和雄配子体发育进行观察。结果表明：花药4室,花药壁由表皮、药室内壁、中层和腺质绒毡层组成,花药壁发育方式为单子叶型,药室内壁部分细胞发育后期发生纤维状加厚。小孢子母细胞减数分裂过程的胞质分裂为连续型,四分体多数为左右对称型,偶有四面体型。成熟花粉为2细胞型,具1个萌发沟。经TTC法检验,成熟花粉生活力为86.3%。从小孢子的发生及雄配子体发育的整个过程看,未见异常现象,能形成大量正常的成熟花粉。     

Phylogenetic species delimitation in ectomycorrhizal fungi and implications for barcoding: the case of the Tricholoma scalpturatum complex (Basidiomycota)

Population studies have revealed that the fungal ectomycorrhizal morphospecies Tricholoma scalpturatum consists of at least two genetically distinct groups that occur sympatrically in several geographical areas. This discovery prompted us to examine species boundaries and relationships between members formerly assigned to T. scalpturatum and allied taxa using phylogenetic analyses. Sequence data were obtained from three nuclear DNA regions [internal transcribed spacer (ITS), gpd and tef], from 101 carpophores collected over a large geographical range in Western Europe, and some reference sequences from public databases. The ITS was also tested for its applicability as DNA barcode for species delimitation. Four highly supported phylogenetic clades were detected. The two previously detected genetic groups of T. scalpturatum were assigned to the phylospecies Tricholoma argyraceum and T. scalpturatum. The two remaining clades were referred to as Tricholoma cingulatum and Tricholoma inocybeoides. Unexpectedly, T. cingulatum showed an accelerated rate of evolution that we attributed to narrow host specialization. This study also reveals recombinant ITS sequences in T. inocybeoides, suggesting a hybrid origin. The ITS was a useful tool for the determination of species boundaries: the mean value of intraspecific genetic distances in the entire ITS region (including 5.8S rDNA) was <0.2%, whereas interspecific divergence estimates ranged from 1.78% to 4.22%. Apart from giving insights into the evolution of the T. scalpturatum complex, this study contributes to the establishment of a library of taxonomically verified voucher specimens, an a posteriori correlation between phenotype and genotype, and DNA barcoding of ectomycorrhizal fungi.     

松口蘑深层发酵工艺的研究

对松口蘑深层发酵工艺进行系统研究。通过正交试验初步确定松口蘑适宜的培养基组成 :玉米粉 30g,葡萄糖 1 0g ,豆饼粉 1 0g ,玉米浆 1 0g ,KH₂ PO₄1g ,定容至 1L。适宜发酵条件 :最适生长温度为 2 5℃ ,摇瓶转速为 1 60r/min ,最适pH为 5 0 ,最适接种量为 1 0 % ,装液量 1 2 0mL/ 5 0 0mL摇瓶培养 1 0d ,菌体生物量达 1 2 94g/L。     

Tricholoma matsutake in a natural Pinus densiflora forest: correspondence between above- and below-ground genets, association with multiple host trees and alteration of existing ectomycorrhizal communities

Tricholoma matsutake (matsutake) is an ectomycorrhizal (ECM) fungus that produces economically important mushrooms in Japan. Here, we use microsatellite markers to identify genets of matsutake sporocarps and below-ground ECM tips, as well as associated host genotypes of Pinus densiflora. We also studied ECM fungal community structure inside, beneath and outside the matsutake fairy rings, using morphological and internal transcribed spacer (ITS) polymorphism analysis. Based on sporocarp samples, one to four genets were found within each fairy ring, and no genetic differentiation among six sites was detected. Matsutake ECM tips were only found beneath fairy rings and corresponded with the genotypes of the above-ground sporocarps. We detected nine below-ground matsutake genets, all of which colonized multiple pine trees (three to seven trees per genet). The ECM fungal community beneath fairy rings was species-poor and significantly differed from those inside and outside the fairy rings. We conclude that matsutake genets occasionally establish from basidiospores and expand on the root systems of multiple host trees. Although matsutake mycelia suppress other ECM fungi during expansion, most of them may recover after the passage of the fairy rings.     

Identification of the DNAs of the ectomycorrhizal fungusTricholoma bakamatsutake using specific oligonucleotide probes and PCR primers

Partial nucleotides of the 18S rDNAs ofTricholoma bakamatsutake were sequenced and compared with those of six ectomycorrhizal fungi and a tree. Two probes, Probes 1 and 2, and a pair of primers were designed based on the variable positions in this region. The DNAs ofT. bakamatsutake were isolated from the colonized mycelia in the soil, field-collected fruit-bodies and artifically cultured mycelia. Hybridization with Probe 1 and PCR-amplification with the primers differentiated these DNAs of this fungus from those of eight ectomycorrhizal fungi and two tree species.     

大链壶菌对白纹伊蚊及几种其它贵州蚊虫的杀灭作用的观察(英文)

采用大链壶菌在实验室内对白纹伊蚊及其它几种贵州蚊虫的幼虫进行杀灭试验。白纹伊蚊的感染率为25—89％,而致倦库蚊的感染率可达90—100％。实验结果为大链壶菌的宿主记录新增加一个属和五个种。