A new species of <Emphasis Type="Italic">Ophiodothella</Emphasis> on <Emphasis Type="Italic">Casearia</Emphasis> from Venezuela

 Ophiodothella caseariae sp. nov. from leaves of Casearia tremula in Venezuela is described and illustrated. Received: February 19, 2002 / Accepted: April 30, 2002     

Isolation of the <Emphasis Type="Italic">B</Emphasis> incompatibility factor mutants in <Emphasis Type="Italic">Pleurotus ostreatus</Emphasis>

 B incompatibility factor mutants (Bmut) in Pleurotus ostreatus were recovered from common-B mating heterokaryons resulted from matings between wild-type monokaryons with different A but the same B factors (A1B2 and A2B2) after NTG mutagenesis. The mutant monokaryons such as A1B2mut and A2B2mut were observed to have regularly uninucleated hyphal cells and to be compatible with each other. Matings between A1B2mut and A2B2mut monokaryons produced stable heterokaryons (A1B2mut + A2B2mut) that had binucleated hyphal cells with true clamp connections and formed normal fruit-bodies. Mating tests using basidiospore progeny from each of these heterokaryons revealed the bipolar mating pattern. Genetic analysis suggested that the mutation of B factor in P. ostreatus might occur in the B incompatibility factor genes. Received: August 3, 2001 / Accepted: January 18, 2002     

<Emphasis Type="Italic">Erysiphe patagoniaca</Emphasis>: a new species of <Emphasis Type="Italic">Erysiphe </Emphasis>sect. <Emphasis Type="Italic">Uncinula </Emphasis>from Patagonia,Argentina

 A new species of Erysiphe sect. Uncinula is described and illustrated from Patagonia, Argentina. Erysiphe patagoniaca sp. nov., found on leaves of Nothofagus × antarctica, is similar to E. nothofagi and E. kenjiana, but differs in its appendages being twisted throughout their length and the number of appendages, asci, and ascospores. The two endemic species of Erysiphe sect. Uncinula, E. magellanica and E. nothofagi, coexisted on the same leaves together with Erysiphe patagoniaca. Received: September 19, 2002 / Accepted: November 28, 2002 Acknowledgments The authors are grateful to Ms. Seiko Niinomi for providing the micrographs of ascomata of Erysiphe spp. on Nothofagus. Correspondence to:S. Takamatsu     

Highly efficient <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation of celery (<Emphasis Type="Italic">Apium graveolens</Emphasis> L.) through somatic embryogenesis

A protocol was developed for rapid and efficient production of transgenic celery plants via somatic embryo regeneration from Agrobacterium tumefaciens- inoculated leaf sections, cotyledons and hypocotyls. These explants were excised from in vitro seedlings of the cvs. XP166 and XP85 and inoculated with A. tumefaciens strain EHA105 containing the binary vector pBISN1. PBISN1 has the neomycin phosphotransferase gene (nptII) and an intron interrupted β-glucuronidase (GUS) reporter gene (gusA). Co-cultivation was carried out for 4 d in the dark on callus induction medium (CIM): Gamborg B5 + 2.79 μM kinetin + 2.26 μM 2,4-dichlorophenoxyacetic acid (2,4-D) supplemented with 100 μM acetosyringone. Embryogenic calluses resistant to kanamycin (Km) were then recovered on CIM + 25 mg l⁻¹ Km + 250 mg l⁻¹ timentin after 12 weeks. Subsequently, a large number of Km-resistant and GUS-positive transformants, tens to hundreds per explant were regenerated via somatic embryogenesis on Gamborg B5 + 4.92 μM 6 (γ,γ-dimethylallylamino)-purine (2iP) + 1.93 μM α-naphthaleneacetic acid (NAA) + 25 mg l⁻¹ Km + 250 mg l⁻¹ timentin after 8 weeks. Using this protocol, the transformation frequency was 5.0% and 5.0% for leaf sections, 17.8% and 18.3% for cotyledons, and 15.9% and 16.7% for hypocotyl explants of cvs. XP85 and XP166, respectively. Stable integration of the model transgenes with 1–3 copy numbers was confirmed in all ten randomly selected transgenic events by Southern blot analysis of gusA. Progeny analysis by histochemical GUS assay showed stable Mendelian inheritance of the transgenes. Thus, A. tumefaciens-mediated transformation of cotyledons or hypocotyls provides an effective and reproducible protocol for large-scale production of transgenic celery plants.     

<Emphasis Type="Italic">Exobasidium dubium</Emphasis> and <Emphasis Type="Italic">E. miyabei</Emphasis> sp. nov. causing Exobasidium leaf blisters on <Emphasis Type="Italic">Rhododendron </Emphasis>spp. in Japan

 Two Exobasidium species causing Exobasidium leaf blister on Rhododendron spp. are described. An Exobasidium leaf blister on Rhododendron yedoense var. yedoense f. yedoense has been recognized in Hokkaido Prefecture, Japan, since the first report was issued in 1950. The causal fungus is identified with Exobasidium dubium from the morphology of its hymenial structure and mode of germination of the basidiospores. Another Exobasidium leaf blister on Rhododendron dauricum has been observed in Hokkaido Prefecture, Japan. In comparison with morphology based on hymenial structure and mode of germination of the basidiospores of the 100 validly described taxa, this fungus differs from those known taxa in the size of basidia and basidiospores, the numbers of sterigmata and septa of basidiospores, and the mode of germination of basidiospores. Thus, a new species, Exobasidium miyabei, is established and illustrated. Received: February 13, 2002 / Accepted: September 25, 2002  Present address: National Institute of Agrobiological Sciences, Tsukuba 305-8602, Japan Acknowledgments We profoundly appreciate the cooperation of Dr. V. Melnik in providing Russian papers and Dr. L. Vasilyeva for translating them into English. We thank Prof. H. Takahashi for loaning the materials in the Herbarium of the Hokkaido University Museum and Dr. W. Abe, Graduate School of Science, University of Hokkaido, for his kind help with the sampling of R. dauricum in Teshikaga, Hokkaido Prefecture. This study was supported in part by a Grant-in-Aid for Scientific Research (B) (No. 13460019), Japan Society for the Promotion of Science (JSPS). Contribution No. 171, Laboratory of Plant Parasitic Mycology, Institute of Agriculture and Forestry, University of Tsukuba. Correspondence to:M. Kakishima     

Chlamydospore formation of <Emphasis Type="Italic">Entoloma clypeatum</Emphasis> f. <Emphasis Type="Italic">hybridum </Emphasis>on mycorrhizas and rhizomorphs associated with <Emphasis Type="Italic">Rosa multiflora</Emphasis>

 Chlamydospores of Entoloma clypeatum f. hybridum were described on the mycorrhizas and rhizomorphs associated with Rosa multiflora. Their developmental pattern seems to be the Nyctalis type. This is the first report on chlamydospore formation on the mycorrhizae in entolomatoid fungi. Received: January 17, 2002 / Accepted: November 5, 2002 Acknowledgments K.H. is grateful to Emeritus Professor N. Sagara in Kyoto University, in whose laboratory part of this study was undertaken. Thanks are due to Mr. D. Sakuma for allowing the specimens to be kept in Osaka Museum of Natural History. Correspondence to:H. Kobayashi     

New species of <Emphasis Type="Italic">Clitocybe </Emphasis>and <Emphasis Type="Italic">Crepidotus </Emphasis>(Agaricales) from eastern Honshu,Japan

 Three new species of Agaricales from eastern Honshu, Japan, are described and illustrated. (1) Clitocybe minutella sp. nov. (section Vernae), having white, very small, clitocyboid basidiomata with white, strigose mycelial tomentum at the base of the stipe and trichodermial elements in the pileipellis, occurs on leaf litter in deciduous oak forests. (2) Crepidotus longicomatus sp.nov. (section Echinospori), forms very small (up to 3.5 mm in diameter), reniform basidiomata densely covered overall with white to pale yellow, erect, thick-walled, long hairs. It has melleous, echinulate basidiospores, and was found on fallen dead branches in lowland oak forests. (3) Crepidotus virgineus sp. nov. (section Crepidotus), forming pure white, reniform basidiomata, has smooth, ellipsoid basidiospores, subfusiform-pedicellate to irregularly cylindrical cheilocystidia, and abundant clamp connections. It occurs on fallen dead branches in lowland oak forests. Received: August 22, 2002 / Accepted: January 8, 2003 Acknowledgments I am grateful to Dr. Yousuke Degawa (KPM) for allowing the specimens cited to be kept in the Kanagawa Prefectural Museum of Natural History. Correspondence to:H. Takahashi     

<Emphasis Type="Italic">Umbelopsis gibberispora</Emphasis> sp. nov. from Japanese leaf litter and a clarification of <Emphasis Type="Italic">Micromucor ramannianus </Emphasis>var. <Emphasis Type="Italic">angulisporus</Emphasis>

 Umbelopsis gibberispora is described as a new species in the genus Umbelopsis, Umbelopsidaceae, Mucorales. The species differs from others in this genus by ellipsoidal sporangiospores with unilaterally thickened walls. Phylogenetic analyses based on nuclear large subunit ribosomal DNA (nLSU rDNA) partial sequences suggest that U. gibberispora, U. swartii, and U. westeae form a clade together with the strains of Umbelopsis ramanniana. The ex-type strain of Micromucor ramannianus var. angulisporus is found to be very close to Umbelopsis vinacea, whereas other isolates identified under the former name in the sense of Linnemann fall in the U. ramanniana subclade. For these isolates, a new species, Umbelopsis angularis, is introduced. Phylogenetic relationships among Umbelopsis species are discussed related to their attributes of the sporangial wall and mature spore shapes. Received: August 27, 2002 / Accepted: March 11, 2003 Acknowledgments We thank Dr. Takashi Ohsono, Graduate School of Agriculture, Kyoto University, Japan, for providing the strain of U. gibberispora (CBS 109328). We also thank Dr. Wieland Meyer, University of Sydney, Australia for access to the phylogenetic tree based on ITS sequence data before publishing, and Dr. Richard C. Summerbell, Centraalbureau von Schimmelcultures, the Netherlands, for linguistic corrections.     

<Emphasis Type="Italic">Haliphthoros milfordensis</Emphasis> isolated from black tiger prawn larvae (<Emphasis Type="Italic">Penaeus monodon</Emphasis>) in Vietnam

 A marine fungus was isolated from the black tiger prawn Penaeus monodon at Nha Trang, Vietnam, on March 20, 2001 and named isolate NJM 0131. The fungus was identified as Haliphthoros milfordensis from the characteristics of asexual reproduction, and its physiological characteristics were investigated. Although the optimum temperature for growth of the isolate was 25°–30°C, the fungus grew at a wide range of temperatures (15°–40°C). H. milfordensis grew well in 50%–100% seawater, but poorly in PYG agar containing 1.0%–5.0% NaCl and KCl. The fungus grew at a wide range of pH (4.0–11.0) with the optimum pH value of 7.0–9.0. The isolate also showed pathogenicity to swimming crab larvae (Portunus trituberculatus) by artificial infection, but mortality was not high. This is the first report of disease in the black tiger prawn P. monodon in Vietnam caused by H. milfordensis. Received: July 22, 2002 / Accepted: January 21, 2003 Correspondence to:K. Hatai     

Genetic analysis of <Emphasis Type="Italic">Vrn-B1</Emphasis> for vernalization requirement by using linked dCAPS markers in bread wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

To identify a molecular marker closely linked to Vrn-B1, the Vrn-1 ortholog on chromosome 5B, sequence polymorphism at four orthologous RFLP loci closely linked to the Vrn-1 gene family was analyzed by using near-isogenic lines of ”Triple Dirk.” At Xwg644, a RFLP locus, three types of nucleotide sequence differing by the number of (TG) repeats, two or three times, and base changes were detected. A (TG)₃-type sequence proved to be specific to chromosome 5B by nulli-tetrasomic analysis, and substitution of single nucleotide (C/T) was detected between TD(B) carrying the former Vrn2 allele and TD(C) carrying the vrn2 allele. A mismatch primer was designed for dCAPS analysis of this single nucleotide polymorphism (SNP). Polymorphism was successfully detected between two NILs, through nested PCR by using a (TG)₃-specific primer (1st) and a dCAPS primer (2nd) followed by a NsiI digest. The analysis of a BF₂ population [(TD(B)//TD(C)] revealed the close linkage (1.7 cM) between WG644–5B and Vrn2. It was therefore concluded that the former Vrn2 locus is located on chromosome 5B and equivalent to Vrn-B1. Received: 3 May 2001 / Accepted: 19 July 2001     

Evaluation of <Emphasis Type="Italic">Hbr</Emphasis> (MITE) markers for assessment of genetic relationships among maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.) inbred lines

Recently, a new type of molecular marker has been developed that is based on the presence or absence of the miniature inverted repeat transposable element (MITE) family Heartbreaker (Hbr) in the maize genome. These so-called Hbr markers have been shown to be stable, highly polymorphic, easily mapped, and evenly distributed throughout the maize genome. In this work, we used Hbr-derived markers for genetic characterization of a set of maize inbred lines belonging to Stiff Stalk (SS) and Non-Stiff Stalk (NSS) heterotic groups. In total, 111 markers were evaluated across 62 SS and NSS lines. Seventy six markers (68%) were shared between the two groups, and 25 of the common markers occurred at fairly low frequency (≤0.20). Only two markers (3%) were monomorphic in all samples. Although DNA sequencing indicated that 5.5% of same-sized DNA fragments were non-homologous, this result did not affect the cluster analyses (i.e., relationships obtained from the Hbr data were congruent with those derived from pedigree information). Distance matrices generated from Hbr markers were significantly correlated (p<0.001) with those obtained from pedigree (r=0.782), RFLPs (r=0.747), and SSRs (r=0.719). Overall, these results indicated that Hbr markers could be used in conjunction with other molecular markers for genotyping and relationship studies of related maize inbred lines. Received: 26 February 2001 / Accepted: 20 April 2001     

Regeneration and<Emphasis Type="Italic"> Agrobacterium-</Emphasis>mediated transformation of hop (<Emphasis Type="Italic">Humulus lupulus</Emphasis> L.)

An efficient procedure for direct organogenesis and regeneration of hop (Humulus lupulus L.) was established. For the first time Agrobacterium-mediated genetic transformation of hop (cv. "Tettnanger") was achieved. Shoot internodes from in vitro cultures were identified as the most suitable type of explant for regeneration. Using this type of explant, a shoot-inducing medium was developed that supported direct organogenesis of approximately 50% of the explants. Plantlets were successfully rooted and transferred to the greenhouse. Overall, in less than 6 months hop cultures propagated in vitro were regenerated to plants in the greenhouse. Agrobacterium-mediated genetic transformation was performed with the reporter gene GUS (-glucuronidase). The presence and function of transgenes in plants growing in the greenhouse was verified by PCR (polymerase chain reaction) and enzyme assay for GUS activity, respectively. We have obtained 21 transgenic plants from 1,440 explants initially transformed, yielding an overall transformation efficiency of 1.5%.Abbreviations BAP 6-Benzylaminopurine - GA₃ Gibberellic acid - GUS -Glucuronidase - IAA Indole-3-acetic acid - IBA Indole-3-butyric acid - NAA -Naphthaleneacetic acid - nptII Neomycin phosphotransferase II - PCR Polymerase chain reaction - TDZ 1-Phenyl-3-(1,2,3-thiadiazol-5-yl) urea (thidiazuron)Communicated by H. Lörz     

Hyaloscyphaceae in Japan (7): <Emphasis Type="Italic">Hyaloscypha albohyalina</Emphasis> var. <Emphasis Type="Italic">monodictys </Emphasis>var. nov.

 Hyaloscypha albohyalina var. monodictys, a new variety in the family Hyaloscyphaceae, Helotiales with Monodictys anamorph is described and illustrated. Received: June 26, 2002 / Accepted: July 27, 2002 Present address: Strategic Product Portfolio Department, Sankyo Co., Ltd., 3-5-1 Nihonbashi-Honcho, Chuo-ku, Tokyo 103-8426, Japan Tel. +81-3-5255-7040 (Ext. 2528); Fax +81-3-5255-7086 e-mail: hosoya@hq.sankyo.co.jp Correspondence to:T. Hosoya     

Modification of polysaccharides from callus culture of <Emphasis Type="Italic">Silene vulgaris</Emphasis> (M.) G. using carbohydrases <Emphasis Type="Italic">in vitro</Emphasis>

Polysaccharides (pectin and intracellular and extracellular arabinogalactans) were isolated from campion callus culture cultivated on medium with varied concentrations of pectinase and beta-galactosidase. A decrease in contents of arabinose residues in pectin and arabinogalactans and of galactose residues in arabinogalactans was associated with an increase in the activities of alpha-L-arabinofuranosidase and beta-galactosidase upon addition of pectinase into the medium. Pectinase destroyed the high-molecular-weight (more than 300 kD) fraction of pectin and decreased the content of galacturonic acid residues. alpha-L-Arabinofuranosidase transformed arabinogalactan into galactan, and galactan was destroyed under the influence of galactosidase. The contents of arabinogalactan and/or galactan in the cells were decreased, and it was released into the culture medium. Pectin samples with low contents of arabinose and galactose in the side chains and galactan samples were obtained from the callus grown on the medium with beta-galactosidase. Cultivation of the plant cells on medium containing carbohydrases resulted in modification of pectin and arabinogalactan of the cell walls.     

Methods for syntheses of <Emphasis Type="Italic">N</Emphasis>-methyl-<Emphasis Type="Italic">DL</Emphasis>-aspartic acid derivatives

Summary. A novel practical method for the synthesis of N-methyl-DL-aspartic acid 1 (NMA) and new syntheses for N-methyl-aspartic acid derivatives are described. NMA 1, the natural amino acid was synthesized by Michael addition of methylamine to dimethyl fumarate 5. Fumaric or maleic acid mono-ester and -amide were regioselectively transformed into beta-substituted aspartic acid derivatives. In the cases of maleamic 11a or fumaramic esters 11b, the α-amide derivative 13 was formed, but hydrolysis of the product provided N-methyl-DL-asparagine 9 via base catalyzed ring closure to DL-α-methylamino-succinimide 4, followed by selective ring opening. Efficient methods were developed for the preparation of NMA-α-amide 13 from unprotected NMA via sulphinamide anhydride 15 and aspartic anhydride 3 intermediate products. NMA diamide 16 was prepared from NMA dimethyl ester 6 and methylamino-succinimide 4 by ammonolysis. Temperature-dependent side reactions of methylamino-succinimide 4 led to diazocinone 18, resulted from self-condensation of methylamino-succinimide via nucleophyl ring opening and the subsequent ring-transformation.     

Pleosporales in Japan (1): the genus <Emphasis Type="Italic">Lophiostoma</Emphasis>

 Seven species of the genus Lophiostoma were the subject of this study. Among these, Lophiostoma mucosum is described and illustrated as a new species. All other species, L. macrostomum, L. semiliberum, L. arundinis, L. caulium, L. caudatum, and L. winteri, are reported for the first time in Japan. A key to the species of Lophiostoma in Japan is given. Received: August 5, 2002 / Accepted: November 28, 2002 Acknowledgments We are grateful to Dr. Hideki Takahashi (curator of SAPA) for the loan of fungal material. Correspondence to:Y. Harada     

A DNA marker closely linked to the <Emphasis Type="Italic">vrs1</Emphasis> locus (row-type gene) indicates multiple origins of six-rowed cultivated barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.)

The origin of six-rowed cultivated barley was studied using a DNA marker cMWG699 closely linked to the vrs1 locus. Restriction patterns of the PCR-amplified product of the cMWG699 locus were examined in 280 cultivated (Hordeum vulgare ssp. vulgare) and 183 wild (H. vulgare ssp. spontaneum) barleys. Nucleotide sequences of the PCR products were also examined in selected accessions. Six-rowed cultivated barleys were divided into two distinct groups, types I and II. Type I six-rowed cultivated barley was distributed widely while type II six-rowed cultivated barley was found only in the Mediterranean region. The type I sequence was also found in a wild barley accession from Turkmenistan whereas the type II sequence was also found in a two-rowed cultivated barley from North Africa and a wild barley from Morocco. These results suggested that the six-rowed type I and II barleys were derived from two-rowed type I and II barleys, respectively, by independent mutations at the vrs1 locus. Received: 3 November 2000 / Accepted: 17 April 2001