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G.W. Hesseltine. 5407 N. Isabell, Peoria, IL 61614 New food ,fermentations: ragi and related products. In the Orient there are several different classes of food fermentations. One class including ragi, bubod, murcha, Chinese yeast, and look-pang are based upon solid dry inoculum used to start a variety 03 beverages and food fermentations. They are mixed cultures containing bacteria, yeast and fungi. A study of 114 strains of Mucor from 45 of these starters showed that they belong to Mucor circinelloides (82) and g. indicus (32) with 6 not identified. Identification was based, in part, upon sexual reactions with known tester strains. None of the 56 Mucor strains tested utilized starch. fifty-three isolates from the amylase starters from 7 regions showed that picros~orus (B. chinensis) and B. orvzae were the species present, and all of them caused hydrolysis of starch. A third group of RhiZoDuS strains (7) were intermediate between B. orvzae and - R. micros~orus. Fourteen strains of RhiZoDuS tested for growth under anaerobic conditions grew bud did not sporulate. The third mold present was Amvlomvces rouxii, found in 7 countries; 70 strains were isolated: and these produced amylases. When various combinations of microorganisms, isolated from amylase starters, were tested as mixed-culture fermentations, the only combinations that produced a good lao-chao product were the combinations of Saccharomvces fibuliaera with Mucor circinelloides, M. indicus, Rhizo~us micros~orus and Amvlomvces rouxii. is more closely related to the Polyporaceae than to the Tricholocataceae. Morphological characters (dimitic hyphae and hyphal pegs) are consistent with this hypothesis. The results also suggest that neither Lentinus or the Polyporaceae is monophyletic. T.*, H. C. HOCH, and B. TERHUNE. '~ept. of Biology, Rhodes College, Memphis, TN 38112, and Dept. of Plant Pathology, N. Y. State Agric. Exp. Sta., Cornen Univ., Geneva NY 14456. lmmunolocalization of Coiled Cytoplasmic Filaments in Uromyces appendiculatus uredospore germlings. Uredospore germlings of Uromyces appendiculatus were homo- genized with glass beads, and a microparticulate fraction was produced by differential ultracentrifugation. The material was further fractionated on a column of Sephacryl S-1000, and a peak containing mainly flexuous coiled filaments was obtained. Their diameter was about 13 nm, and their typical maximum length was 500 - 540 nm. These appear similar to the "WFR" class of "virus-like particle" described by McDonald and Heath (1978. Can. J. Bot. 56:963-975). The major polypeptide of the filament fraction has a molecular weight of about 36 kD, as measured by SDS-PAGE, with weaker bands between 29 and 34 kD. Monoclonal antibodies were produced that were shown by immunoblotting to recognize an antigen that co-migrates with a 34 kD protein of the filament fraction. With indirect immuno- fluorescence microscopy, the antibody labels fusiform structures distributed throughout the cytoplasm. With EM immuno- gold labeling, the antibody binds to bundles of cytoplasmic filaments most closely resembling structures previously identified as "filamentous distalsomes" (Hoch and Staples. 1983. Mycologia 75:795-824). David S. Hibbett and Rytas Vilgalys. Department of Botany, Duke University, ~ukham, NC 27706. - Taxonomic relationships of Lentinus to the W. E. HIhTZ, J. C. ROYER, M. HUBBES and P. A. HORGEN. Polyporaceae: evidence from restriction Center for Plant Biotechnology, University of Toronto. analysis of enzymatically amplified Transformation-mediated insemonal mutagenesis of the dimorphic DNA. pathogen Ophoswma ulmi. Evolutionary relationships of Jentinus Fr. to the Polyporaceae were elucidated using restriction analysis of ribosomal DNA (rDNA). Five species in Mntinus sensu Pegler, three species in the Polyporaceae and two species in the Tricholomataceae were examined. RDNA phenotypes were determined by restriction analysis of enzymatically amplified rDNA (PCR Fingerprinting). This method generates Restriction Fragment Length Polymorphism (RFLP) data without Southern hybridization or autoradiography. With five different four- base restriction enzymes, one hundred unique restriction fragments were resolved. Among the 16 individuals examined, there were twelve unique rDNA phenotypes. A similarity matrix based on presence or absence of comigrating restriction fragments was analyzed by clustering (UPGMA) and distance algorithms (Fitch-Margoliash analysis). Results of all analyses were highly consistent and strongly suggest that Lentinus Rotoplasts of Ophiostoma ulmi, the causal agent of Dutch Elm disease, were aansfmed to hygromycin resistance using a DNA vector (vPS57) containing a promoter for i~openicillin N synthetase from ~enicillibn chryso@& fused to a bac'terial gene f& hygromycin phosphotransferase. The transformation efficiency was 5 X 103 transformants per ug DNA per 10' protoplasts. The transforming DNA was stably integrated (mitotically) in single or tandem copies at unique or several &spersed sites within the transformant genornes. There appeared to be no targetting of specific integration sites within the genome for the transforming DNA. This provided a useful approach for generating mutations by the insemonal disruption of functional genes. The crippled genes were conveniently tagged by the transforming DNA and could be recovered from genomic libraries. We have generated a developmental mutant (U9) in which the dimorphic phenotype has been altered. Unlike the untransformed parental strain the U9 mutant fails to switch from yeast-like growth to mycelial pwth when transferred to selective medium. The transforming DNA integrated at a single locus and we hypothesize that the transforming DNA interupted one of the genes or regulatory regions essential to phenotype transition. We are currently analysing the site of inteption.
MICHAEL T. HOLMES, EDUARDO M. VADELL AND JAMES C. CAVENDER. Department of Botany, Ohio University, Athens, Ohio 45701. Ecological Aspects of Dictyostelids in Tropical Forest Soils Our study site was at Tikal, Guatemala which is located at 17.5' N. latitude. The preserve consists of approximately 355 square kilometers of Semi- Evergreen Rainforest on limestone soils with 2500 mm annual rainfall occuring mostly during the months of June through January. Twenty-five species of cellu- 'lar slime molds, including four undescribed species were Isol.ated from a relatively small area of the preserve, in and around the ancient city of Tikal. Tropical forest are of particular interest because of species richness and diversity in many taxonomic groups. Soil habitats in the tropical forest also appear to harbor equivalent species richness and diversity based on our studies of cellular slime molds. Four trips (August '89, December '88, '89 and March '90) were made in order to study seasonal variation. Three sites of different moisture conditions were chosen for similarity analysis. Ecological analyses Cere conducted using regression models and analysis of variance. Our study has focused on the explora- tion of species richness and diversity through both microdistribution and macrodistribution within the Tikal area. We have also investigated the correla- tion between cellular slime molds and the abundance of bacteria and fungi in the soil. Future regenera- tion of tropical forest may depend upon the inocula- tion of cellular slime molds along with other microorganisms into the soil. Therefore, preservation of isolated species using the lyophilization process described by Raper and Alexander (1945) was an important Dart of. th~~-studv. John S. Hopple, Jr. Department of Botany, Duke University, Durham, NC. 27706. Phylogenetic Relationships Within the Genus Co~rinus Based on Molecular and Morphological Evidence. Evolutionary relationships of select taxa within the genus m~rinus were investigated using molecular and morphological data. Co~rinus atramentarius, C. cinereus, C. cornatus, C. micaceus, C, patoui1lardii;and C. plicatilis were selected as taxa representing a wide spectrum of variation within the genus for which adequate material was available. DNA coding for the large ribosomal subunit and part of the ITS region was amplified using polymerase chain reaction (PCR). A restriction fragment map of the DNA was obtained through a combination of Southern blotting and PCR mapping. Morphological studies of the taxa were carried out through character analyses of traditionally significant taxonomic structures. Evolutionary relationships were determined using phenetic and parsimony techniques. Significance of morphological characters governing taxonomic classifications will be discussed. Preliminary evidence supports the classifications of Moser (1983) and Orton and Watling (1979) at the sectional level. Further evidence indicates that Co~rinus is not a monophyletic group, but rather is paraphyletic. W. E. Horner and S. R. Lehrer. Tulane Medical Center, 1700 Perdido St., New Orleans, LA 70112. Allergenic/antigenic cross reactivity among basidiqmycetes. Basidiospore extracts contain allergens capable of binding IgE antibodies and eliciting bronchial al- lergic responses in sensitized asthmatics. Immuno- print (IP) characterization of crude extracts of Calvatia cyathiformis (Cc) with sera from skin test and RAST positive subjects demonstrated 21 a1 lergen- ic bands. Gal c Bd9.3 (pl 9.3, 16 kD), was the most important allergen, based on the number of sera (l2/l9), reacting to it. IP inhibition showed that Cc, Coprinus quadrif idus, Psi locybe cubensis and Pleurotus ostreatus each contain a pi 9.3 allergen that cross-reacts with the other species. Spore extracts of Pisolithus tinctorius and Ganoderma meredithae did not contain a 9.3 allergen. @lc Bd9.3 was isolated and rabbit antiserum was raised against it. lmmunodiffusion analysis of this antiserum against crude Cc extract and Calf BD9.3 produced single precipitin bands that fused. IP with this serum also indicated shared epitopes for the 9.3 antigen among these four species. These results demonstrate shared antigens among four families in two orders of basidimycetes. The presence of shared antigens among basidimycetes may facilitate screening for basidiomycete allergies and aid phyletic studies. J. S. HORTON and C. A. RAPER. Department of Microbiology and Molecular Genetics, University of Vermont, Burlington, VT 05405. Pulsed-field electrophoretic analysis of $chizo~hvllum commune chromosomes. Transverse alternating field electrophoresis (TAFE) was used to separate chromosome-sized DNAs of the Basidiomycete ~chiw~hvllum commune. Six bands were observed in TAFE separations. ranging in size from 6200 to 1250 kb. Three bands appeared to be doublets, as judged by the intensity of their ethidium bromide staining, and by hybridization analysis using cloned probes. Polymorphisms in chromosome size were observed between different strains of S. Fommuns. The results of DNA hybridization experiments suggested that certain genes previously determined by genetic analysis to reside on different linkage groups may be present on the same chromosomal DNAs. The utility of pulsed-field electrophoresis and chromosome-specific probes as an aid in the isolation of genes will be discussed. P. A. HUMBER. USDA-ARS Plant, Soil & Nutrition Labora- tory, Tower Rd., Ithaca, NY 14853. Thermotolerance of Conidiobolus species. Species of Conidrobolus (Entomophthorales: Ancylistaceae) are primarily saprobes occurring in plant dewitus and soil; sweral species occur primarily as enromopathogens; a smaller number of (saprobic andlor entomopathogenic) species can cause mycoses of humans, equines, or other mammals. Vertebrate conidi- obolomvcoses are usually confined to the nasal passages and to adjacent superficial tissues, both of which are generally appreci- ably cooler than the core body temperature; these mycoses are only very rarely deep-seated or systemic.
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