Armillaria jezoensis,a new symbiont ofGaleola septentrionalis (Orchidaceae) in Hokkaido

NineArmillaria isolates obtained from the roots ofGaleola septentrionalis in Hokkaido were identified asA. jezoensis by means of mating tests. Cultures of these isolates were similar in colony morphology, mycelial growth and rhizomorph formation on each of malt extract-dextrose agar (MDA), potato-dextrose agar (PDA), andG. septentrionalis root extractdextrose agar (GDA) media, showing better mycelial growth and rhizomorph formation on GDA medium.     

The mycology of the freeliving baboon (Papio sp.)

Summary Three field trips were conducted to study the mycoflora of the baboon in its native habitat, East Africa. The flora of the throat, nose, ear, rectum, vagina, and skin of over 300 male and female baboons was studied. A total of 1102 yeast isolates was obtained. None of the molds isolated were of known significant pathogenicity, while yeast isolates were found to belong to the following major genera:Candida, Cryptococcus, Rhodotorula, Saccharomyces, Torulopsis, andTrichosporon. Candida albicans was found to be the yeast most frequently isolated in both 1964 and 1966, while species ofRhodotorula andCryptococcus were more frequently found in 1968. It is our belief that various ecological and geographical conditions have an effect on the mycoflora of the animals, specifically from the quantitative point of view.     

Vegetative compatibility groups of Japanese isolates ofVerticillium dahliae

Japanese isolates ofVerticillium dahliae were examined for vegetative compatibility relationships using nitrate-nonutilizing mutants. Four levels of vegetative compatibility were differentiated according to the degree of compatibility between the tester mutants ofnit1 and NitM. Wild-type growth with a complementation line greater than 5 mm wide was defined as “strong reaction (++)”, i.e., compatible. Ten out of 15 isolates showed compatibility and were separated into three groups, provisionally designated as VCGJ1, VCGJ2, and VCGJ3, depending upon their reactions. This method was used to estimate, genetic diversity within a local population ofV. dahliae. Another 12 isolates from Gunma Pref. were paired with tester isolates of the three vegetative compatibility groups proposed. Eight Gunma isolates were assigned to VCGJ1 or VCGJ2. Two isolates were incompatible with all testers. The remaining 2 isolates were self-incompatible. Thus, 18 out of 27 Japanese isolates ofV. dahliae were assigned to VCGs: 8 to VCGJ1, 7 to VCGJ2, and 3 to VCGJ3. VCGJ1 was compatible with both VCGJ2 and VCGJ3, but VCGJ2 and VCGJ3 showed a weak reaction with each other. Japanese isolates ofV. dahliae were thus demonstrated to form a VC group comprising three subgroups.     

Ploidy differences in Cryptococcus albidus

Presumed haploid and diploid cultures ofCryptococcus albidus were analysed for their DNA content per cell. A ratio of approximately 1:2 was obtained by relating the DNA content per cell of the two phases to ploidy. As the diplophase formed neither longitudinally septated cells nor ballistoconidia, the earlier suggestions thatCryptococcus is closely related toTremella seems less likely. On the assumption that the metabasidia ofCryptococcus are gastromycetoid, a closer relationship between this genus and the tulasnelloid fungi appears more probable.Microbiology Research Group, South African Council for Scientific and Industrial Research, Pretoria, South Africa.     

Assessment of vegetative compatibility of race-2 tomato wilt isolates ofVerticillium dahliae in Japan

Verticillium dahliae race-2 can invade the resistant cultivars of tomato possessing theVe gene. This new race was recently found in several regions in Japan, and 10 isolates ofV. dahliae race-2 from these regions were used in our study. Pathogenicity tests identified these isolates as the tomato pathotype (B). We examined the vegetative compatibility of 8 of these 10 Japanese isolates ofV. dahliae race-2 to estimate their genetic relatedness with the testers of Japanese vegetative compatibility group previously proposed (VCGJ) usingnit mutants. Compatiblenit1 and NitM mutants were obtained from allV. dahliae race-2 isolates. Selected representativenit1 and NitM mutants of eachV. dahliae race-2 isolates were paired with VCGJ testers. All isolates ofV. dahliae race-2 showed a strong reaction with VCGJ2, i.e., tomato pathotype. All isolates ofV. dahliae race-2 except for isolate To22 reacted weakly to VCGJ1 and J3. Japanese isolates ofV. dahliae race-2 were assigned as VCGJ2 and were hence vegetatively closely related with those ofV. dahliae race-1. The origin of Japanese isolates ofV. dahliae race-2 was discussed.     

Application of a colorimetric DNA-DNA hybridization method for identification ofAeromonas genomic species,isolated from diarrheal stools

The colorimetric DNA-DNA hybridization method for the identification of 18 strains ofAeromonas spp. isolated from human stools was used. Bacterial isolates were also examined by phenotypic characteristics. On the basis of biochemical tests 13 strains were included in phenogroupA. caviœ and 5 strains inA. sobria. Identification to the species level was obtained by colorimetric hybridization method. DNA-DNA similarity values showed that isolates ofA. caviœ group belong to hybridization group (HG) 4 whereas isolates ofA. sobria belong to HG 8/10. DNA relatedness results obtained by the colorimetric method showed good agreement with values detected by the spectrophotometric method. The background in the colorimetric method is lower than in the spectrophotometric one. Results of this study indicate the usefulness of the colorimetric DNA-DNA hybridization in microplates method for the identification ofAeromonas genomic species, isolated from human diarrheal stools.     

Direct plate isolation method for cryptococcus neoformans from the soil

Summary An in vitro technique was developed for the isolation ofCryptococcus sp. from soil. Cycloheximide was sused to suppress the growth of many of the saprophytic fungi, includingCryptococcus sp. cells. These cells are then selectively recovered by neutralizing the effect of the cycloheximide, using a hydrolysis product of protein (usually peptone) to adsorb the antibiotic off the walls, allowing the cells to grow.Presented at the National Meeting of the American Society for Microbiology, May 1–5, 1966, in Los Angeles, California.     

The origin of some species of the genus Candida in non-pseudomycelium-producing anascosporogenous yeasts

Summary One hundred thirty-one cultures of anascosporogenous yeasts isolated from the human body late in 1947 were all typical members of the genusCryptococcus when examined in 1947 and early in 1948. No mycelium was produced in repeated tests in corn meal agar scratch plates or in beef peptone gelatin stabs. By 1950, 17 of the 131 cultures had definitely become morphologically identical with members of the genusCandida, showing typical pseudomycelial growth with blastospores sprouting from the mycelium on the above mentioned media. It is concluded that some species ofCandida can be morphological dissociates ofCryptococcus. The status and the varied phylogeny of the genusCandida is discussed.

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Sumario Todos los 131 cultivos de una colección de levaduras imperfectas recientemente aisladas de la piel de seres humanos eran miembras típicas del géneroCryptococcus en 1947. No producían ningún micelio, aunque lo buscabamos repeditas veces en gelosa de harina de maíz y en gelatina de carne y peptona. Ahora en 1950, 17 de los 131 cultivos se han convertido en formas identicas morfológicamente con miembras del generoCandida. En estas medias las 17 levaduras ya poseen un seudomicelio conspicuo de que muchas blastoesporas brotan. Se deduce que unas especies deCandida pueden ser formas deCryptococcus producidas por disociación microbiana. Se trata del estado y de la filogenia multiple del géneroCandida.

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El origen de unas especies del géneroCandida en levaduras imperfectas que no formaban seudomicelio

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This investigation was supported in part by funds provided for biological and medical research by the State of Washington Initiative Measure No. 171.     

Incidence ofCandida species isolated from human skin and nails: A survey

Distribution ofCandida species was investigated by examining 245 samples from skin lesions and nails. The isolates were identified using standard laboratory methods including germ tube test, micromorphology of colonies on rice agar, the commercial kit, saccharide assimilation and fermentation tests. Eight species ofCandida were identified:C. albicans accounted for 56.4% of the isolates,C. parapsilosis 29.1,C. tropicalis 7.8,C. pulcherrima 2.9,C. guilliermondii 1.5,C. krusei andC. zeylanoides for 0.9% each, andC. robusta for 0.5%. The factors significantly associated with colonization were prolonged antibiotic therapy, parenteral nutrition, low birth body mass of infants, intubation, duration of stay in hospital, indwelling intravenous catheter, malignancies, diabetes, surgery, and obesity.     

Comparison ofRhizoctonia solani isolates from web blight and basal canker of cowpea and from soil

Summary Isolates ofRhizoctonia solani from web blight and stem basal canker of cowpea and those obtained from soil had similar linear growth rates on potato dextrose agar (PDA) at various temperatures but differed in other features. The web blight isolates differed from the basal canker and the soil isolates in cultural appearance on PDA and on potato marmite agar (PMA). The web blight isolates readily formed discrete sclerotia on PDA, PMA and soil but the other isolates did not. In greenhouse tests, the former were generally the most virulent in inciting foliage and stem basal necrosis and damping-off of seven crop species. Of the plants tested, the legumes were the most susceptible toR. solani.     

A natural focus ofHistoplasma capsulatum var.duboisii is a bat cave

The natural reservoir ofHistoplasma capsulatum var.duboisii, the etiological agent of histoplasmosis duboisii (African histoplasmosis) is not yet known. We report the isolation ofH. capsulatum var.duboisii from soil admixed with bat guano and from the intestinal contents of a bat in a sandstone cave in a rural area, Ogbunike in Anambra State of Nigeria. Eight of 45 samples of soil admixed with bat guano yieldedH. capsulatum var.duboisii. Of the 35 bats belonging to the speciesNycteris hispida andTadirida pumila examined, only one (N. hispida) yielded this fungus from its intestinal contents. Identification of the isolates asHistoplasma was confirmed by exoantigen tests and by mating with tester strains ofH. capsulatum. In vitro conversion to large yeast from suggestive ofH. capsulatum var.duboisii was obtained on brain heart infusion agar supplemented with sheep blood and glutamine or cysteine. Pathogenicity tests with mice for all the isolates confirmed their identity by the demonstration of large yeast forms (8–15 µm in diameter) within giant cells in the infected tissues. Investigations on the possible occurrence of human infections in the area are in progress.A poster based on this work was presented at the 11th ISHAM Congress in Montreal, Canada (22–28 June 1991), La-Hoffman Roche, Basel, Switzerland kindly financed the trip of one of us (H.C.G) for the Congress.     

Non-leguminous root nodules in Japan

Summary Among plants native to Japan, nodule formation is confirmed in 14 species and varieties of Alnus, 10 of Elacagnus, 2 of Myrica and 1 of Coriaria, in a number of instances for the first time. Plants of 20 foreign species, in 8 genera, which bear nodules in their native countries, were raised in the nursery in Tokyo; only species of Alnus, Myrica and Ceanothus formed nodules. No nodules were found on native plants ofDryas octopetala var.asiatica. In a trial extending over 12 years evidence was obtained that the growth ofPinus thunbergii was benefited by underplanting withMyrica rubra, a result attributed to nitrogen fixation in the root nodules of the latter species. In the attempted isolation of the endophytes from the nodules of Alnus and other non-legume Angiosperms, although actinomycetes peculiar to the host species were usually obtained from the nodules, none of the isolates induced nodules in re-inoculation tests. Also from Podocarpus nodules actinomycetal and bacterial strains were commonly isolated; re-inoculation tests with these are in progress.     

Isolation ofAcholeplasma spp. from coconut palms affected by lethal yellowing disease in Jamaica

A total of 35 isolates ofAcholeplasma spp. were recovered from phloem sap and rotting tissues of lethal yellowing-diseased coconut palms. The highest isolation rate, 33% of samples from two palms, was obtained in a conventional mycoplasma medium supplemented with Tween 80; no isolates were recovered from healthy palm tissues or from uninoculated media constituents. Metabolic and serological tests on uncloned isolates showed that about two-thirds were strains ofA. axanthum and the remainder were related toA. oculi. These results strongly suggest that acholeplasmas occur in or on plant tissues either as pathogens, epiphytes, or saprophytes.     

Evaluation of Propionibacterium acnes Isolates Using Contour-clamped Homogeneous Electric Field Gel Electrophoresis

Contour-clamped homogeneous electric field electrophoresis was performed to compare strains ofPropionibacterium acnes isolated from patients with chronic postoperative endophthalmitis. Propionibacterium acnes isolates were obtained from the vitreous humor of nine patients with chronic postoperative endophthalmitis following cataract surgery. In two of the patients, P. acnes isolates were also obtained from the aqueous humor as well as from the vitreous humor. Bacterial DNA was digested using Not I and Spe I restriction endonucleases. The DNA fragments were then subjected to contour-clamped homogeneous electric field electrophoresis and the DNA banding patterns were analysed. Eight nonidentical banding patterns were identified among the nine vitreous isolates of P. acnes. In each of the two cases from which aqueous and vitreous isolates were recovered from the same eye, the banding patterns were identical. Contour-clamped homogeneous electric field electrophoresis is a powerful method to distinguish P. acnes isolates based on DNA banding patterns and could be used in the epidemiological study of clinical processes caused by this organism.     

A Multivariate Analysis of Soil Yeasts Isolated from a Latitudinal Gradient

Yeast isolates from soil samples collected from a latitudinal gradient (>77°S to >64°N) were subjected to multivariate analysis to produce a statistical foundation for observed relationships between habitat characteristics and the distribution of yeast taxa (at various systematic levels) in soil microbial communities. Combinations of temperature, rainfall (highly correlated with net primary productivity), and electrical conductivity (EC) could explain up to ca. 44% of the distribution of the predominant yeast species, rainfall and pH could explain ca. 32% of the distribution of clades in the most common orders (Filobasidiales and Tremellales), whereas vegetation type (trees, forbs, and grass) played the same role for orders. Cryptococcus species with appropriate maximum temperatures for growth predominated in most soils. Cryptococcus species in the Albidus clade of the Filobasidiales predominated in desert soils; Cryptococcus species of other clades in the Filobasidiales and Tremellales predominated in wetter and more-vegetated soils, with Tremellalean species favored in soils of lower pH or higher EC. The predominance of Cryptococcus species in soils has been attributed to their polysaccharide capsules, particularly important when competing with bacteria in arid soils.     

Characterization of rhizobia fromLeucaena

Seventy-six rhizobia were isolated from the nodules ofLeucaena plants of various genotypes growing in a wide range of soil types and climatic regions. The isolates were fast-growing and acid-producing. In establishing a serological grouping for the isolates, the intrinsic antibiotic resistance (IAR) patterns to low concentrations of eight antibiotics was helpful for selecting the strains for immunization purposes. Eight distinct somatic serogroups ofLeucaena rhizobia were identified by using strain-specific fluorescent antibodies. The results indicated that use of serological markers is a more specific technique than IAR pattern for strain identification. Strains from some different serogroups had the same IAR patterns. The immunofluorescence cross-reactions ofLeucaena rhizobia serogroups among themselves and with other species of fast- and slow-growing rhizobia were very low. Sero-grouping is ideal for use in further ecological studies in field inoculation trials.     

Ecology of yeasts in polluted water

The general size and composition of the extant yeast populations in 13 polluted freshwater habitats were surveyed. Subsequently the yeast populations in three of the 13 locations were quantitatively determined and compared. The three locations had (A) low pollution levels, (B) heavy industrial waste pollution, and (C) heavy domestic waste pollution.The yeast population at location A was dominated byRhodotorula andCryptococcus isolates. At station BRhodotorula andCandida were predominant.Candida isolates were in the majority at location C andRhodotorula strains were second in frequency, but were much lower in proportion of the population than at the other two habitats.These polluted waters in general had large yeast populations, ranging as high as 27,000 yeasts per 100 ml, and averaging approximately 3000 yeasts per 100 ml.The presence of human wastes was especially associated with large increases in the proportion ofCandida yeasts in the environment. The genusRhodotorula was consistently present at all locations, but the genusCryptococcus was a major component of the yeast population only in non-polluted or lightly polluted fresh water.We appreciate the assistance of the Calumet Area Surveillance Program, Federal Water Pollution Control Administration, Chicago, in the collection of water samples. This investigation was supported by U.S. Public Health Service Research Grant No. AI 04642 from the National Institute of Allergy and Infectious Diseases.     

Fungi isolated from Sclerotia ofSclerotium cepivorum and from soil and their effects upon the pathogen

Summary Data are presented on the antagonistic effects of the fungi isolated from sclerotia ofSclerotium cepivorum and from nonrhizosphere soil taken from around the roots of infected onions upon mycelial growth and sclerotial germination ofS. cepivorum. Most of the isolated fungi especiallyPenicillium species were antagonistic to mycelial growth. Sclerotial germination was slightly inhibited by diffusates of these fungal isolates. Testing the antifungal effect of someAllium extracts against the fungal isolates by the inhibition zone method showed that garlic extract has the greatest antifungal effects and onion extract is the least potent. However, spore germination tests indicated that onion extract completely inhibits the spore germination of all test fungi. The role of host-plant extracts in stimulating sclerotial germination is discussed.     

Plant growth regulatory metabolites from novel actinomycetes

Metabolites from 796 isolates of aerobic actinomycetes were screened for plant growth regulatory properties using an algal bioassay. These included 266 isolates ofStreptomyces, 28 unidentified actinomycetes, and 502 isolates of novel actinomycetes represented by 18 genera. Algal growth inhibition of 30% was observed with 60 isolates, 37 of which belonged to the genusStreptomyces. Among other inhibitors were 8 isolates ofActinomadura, 6 ofActinoplanes, 2 each of the generaThermomonospora, Streptoverticillium, andPromicromonospora, and 3 unidentified. Metabolites from 70 isolates promoted algal growth by 20%. These included 13 isolates ofMicromonospora, 11 ofStreptomyces, 6 ofNocardia, 5 ofActinomadura, and 4 each ofRhodococcus andThermomonospora. Sixteen unidentified isolates; 3 isolates ofPromicromonospora; 2 isolates each ofActinoplanes, Streptosporangium, andOerskovia; and 1 of Thermoactinomyces peptonophilus-like organism andSaccharomonospora viridis also promoted the algal growth by 20%. The plant growth inhibitory properties of 9 actinomycetes and the growth promoting properties of 6 were demonstrable during the secondary screening on higher plants using chemicals extracted from the culture broth. The metabolites fromMicromonospora, Nocardia, Rhodococcus, Streptosporangium, andOerskovia isolates were associated with plant growth promotion only; those fromStreptomyces were most frequently involved with the growth inhibition.This is Michigan Agriculture Experiment Station Journal Article No. 12191.     

Chemotaxonomic identity ofCorynebacterium matruchotii from animal and human dental plaques

Eight strains ofCorynebacterium matruchotii, isolated from the dental plaque of patas monkeys and orangutans as part of a study of the flora of animal plaque, were compared with isolates from human sources. The polypeptide patterns on sodium dodecyl sulfate-polyacrylamide gel electrophoresis were similar, and the animal isolates were not distinguished from human strains by the mean base composition of DNA, the peptidoglycan components, the cell wall carbohydrates, nor the mycolic acids. All strains appear to belong to the same species, which is distinct from other species ofCorynebacterium.