Unidentified Isolates Research Articles

Fungi from decayed wood as ectomycorrhizal symbionts of western hemlock

Four wood-inhabiting fungi, Byssoporiaterrestris, Cenococcumgeophilum, Pilodermabicolor, and an unidentified isolate, formed mycorrhizae with western hemlock (Tsugaheterophylla (Raf.) Sarg.) in pure culture. Field observations indicate that Cantharellusinfundibuliformis, Chroogomphustomentosus, Galerina sp., and Russula sp. are also mycorrhizal with hemlock growing in rotten wood.

New records of Phytophthora on trees in Britain.

Phytophthora megasperma var. megasperma and unidentified isolates of Pbytophthora rescmbling P. cambivora were obtained from dead and dying roots of Horsc chestnut trccs with sparse foliage, small chlorotic leaves and branch dieback. P. citricola was isolatcd from soil around dead roots of similarly affected trccs and from around dcad feeder roots of a tree suffering from the common but unexplained Leaf scorch (marginal leaf necrosis) of Horse chestnut. Both P. cactorum and P. citricola were isolated from oozing acrial stem lesions on Horse chestnut trees. Inoculations with these species reproduced the damage.

DNA Homologies Among Heterothallic Species ofNeurospora

Highly purified DNA from three reference strains representing authentic heterothallic species: Neurospora crassa, Neurospora intermedia and Neurospora sitophila, and from several unidentified Neurospora isolates was characterized by spectrophotometry, and by DNA:DNA reassociation using hydroxyapatite chromatography. These known heterothallic species and isolates were closely related on the basis of morphology, physiology and DNA characteristics such as base composition, thermal denaturation profiles and occurrence of repeated DNA sequences. They were, however, distinguishable by DNA:DNA homology studies. It was concluded that N. crassa is closer to N. intermedia than to N. sitophila. Neurospora sp. Gianjor, a 4-spored pseudohomothallic species, was significantly different from N. crassa and N. intermedia, but had greater homology with N. sitophila. All of the Neurospora isolates which crossed readily with N. crassa, and produced 95% black ascospores, were indistinguishable from that species by DNA homology studies. It was possible to assign some of the unidentified isolates to species based on DNA homology studies.

DNA Homologies among Heterothallic Species of Neurospora

Highly purified DNA from three reference strains representing authentic heterothallic species: Neurospora crassa, Neurospora intermedia and Neurospora sitophila, and from several unidentified Neurospora isolates was characterized by spectrophotometry, and by DNA:DNA reassociation using hydroxyapatite chromatography. These known heterothallic species and isolates were closely related on the basis of morphology, physiology and DNA characteristics such as base composition, thermal denaturation profiles and occurrence of repeated DNA sequences. They were, however, distinguishable by DNA:DNA homology studies. It was concluded that N. crassa is closer to N. intermedia than to N. sitophila. Neurospora sp. Gianjor, a 4-spored pseudohomothallic species, was significantly different from N. crassa and N. intermedia, but had greater homology with N. sitophila. All of the Neurospora isolates which crossed readily with N. crassa, and produced 95% black ascospores, were indistinguishable from that species by DNA homology studies. It was possible to assign some of the unidentified isolates to species based on DNA homology studies.

Histopathology of Pythium infected strawberry roots

Host–parasite interactions between eight Pythium species, previously isolated from necrotic strawberry root tissue, and Fragaria × ananassa Duch. runner plant main roots were studied. When excised roots were placed in water on petri dish cultures, P. rostratum and P. hypogenum infection was limited to the root tip epidermis. Pythium irregulare, P. perniciosum, P. dissotocum, P. sylvaticum, P. ultimum, and unidentified isolate 1E penetrated the epidermis several centimeters along the zone of maturation. Penetration occurred most frequently through root hairs by hyphae; resulting infections usually stopped in the epidermis or adjacent cortex, and here hyphae formed sporangium-like structures, oogonia, and oospores. Extensive cortical infection by hyphae was primarily limited to the zone of elongation. Extent of epidermal and cortical infection in roots grown in soil with P. irregulare, P. dissotocum, and P. sylvaticum was similar to petri dish test results. Stelar infection by P. sylvaticum and P. dissotocum occurred mainly in phloem cells, but did not advance far into the region of maturation. Numerous oogonia formed in infected steles and usually in adjacent cortical cells. Infected roots ranged from a white to grey, water-soaked color. Extensively infected root tips typically were unhealthy, succulent, and thin.

Keratophilic fungi and the prevalence of dermatomycoses in Orissa, India

A survey of rural and urban student populations was undertaken to determine the prevalence of dermatomycoses in the district of Puri (Orissa). Tinea corporis was found to be the predominant disease in rural area whereas t. cruris was dominant in the urban area. T. capitis was absent in both areas.Except for M. gypseum no other dermatophyte was isolated from soil. T. rubrum (87·5%) was the most commonly isolated fungus. Among the geophilic fungi isolated from soil, species of Chrysosporium were found to be the most common.Arachniotus flavoluteus, Petalosporus anodosus, Pseudoarachniotus citrinus, P. hyalinosporus and one unidentified Gymnoascacean were often associated with superficial mycosis. Asexual spores were observed in the isolates of P. nodulosus, P. citrinus and in the unidentified Gymnoascaous isolate.

Enterobacteriaceae Isolated from Cowbirds (Molothrus ater) and Other Species of Wild Birds in Michigan

SUMMARY The prevalence of Enterobacteriaceae was studied in 60 wild birds (45 cowbirds, Molothrus ater, and 15 other specimens belonging to 11 other species). The birds were collected in central Michigan between 1966 and 1969. Seventy-eight different bacterial isolates were recovered (Ewing classification): 28 belonged to the tribe Escherichieae, 22 to Klebsielleae, 20 to Salmonelleae (tribe), and eight to Proteeae. Found in addition were three isolates of Pseudomonas fluorescens, one of Alcaligenes fecalis, and seven unidentified isolates of gram-negative bacteria. The findings emphasized that wild birds, particularly healthy Brown-headed cowbirds, may constitute a potential source and reservoir of pathogenic enteric bacteria and may play a role in their transmission to man and susceptible animals.

Biochemical Activities of Nocardia

It seems almost inevitable that the microbiologist searching for new reactions and products will become addicted to a specific genus. This chapter is an attempt to bring attention to a group of microorganisms that are believed to have considerably more potential in microbial transformations than has previously been recognized. The chapter describes the biochemical activities of the gebus nocardia because of their ability to carry out hydroxylations and cleave aromatic rings, reactions that in many instances involve the mechanism of direct addition of molecular oxygen to a substrate. In this regard, they seem to have an advantage over the somewhat more destructive pseudomonads in the accumulation of products. On the other hand, the isolation and characterization of oxygenases and mixed function oxidases, which are responsible for molecular oxygen incorporation, have been almost exclusively confined to the pseudomonads. Seventeen identified and many unidentified soil isolates of the genus Nocardia have been examined for their potential in transformations of hydrocarbons, nonhydrocarbon aromatics, herbicides, and steroids. Of the cultures surveyed, most appear to prefer paraffinic over aromatic hydrocarbons as a growth substrate.

Enumeration of Byssochlamys and Other Heat-Resistant Molds

Methods for the detection of low numbers of heat-resistant molds on fruits were studied by using cultures of Byssochlamys and a number of unidentified mold isolates. Ascospore dormancy had a marked effect on viable recoveries, and the medium in which ascospores were heated influenced activation rates. Best results were obtained when fruit homogenates were heated for 60 min at 70 C in Concord grape juice, followed by culturing on acidified Potato Dextrose Agar.

Enumeration of Byssochlamys and Other Heat-Resistant Molds 1

Methods for the detection of low numbers of heat-resistant molds on fruits were studied by using cultures of Byssochlamys and a number of unidentified mold isolates. Ascospore dormancy had a marked effect on viable recoveries, and the medium in which ascospores were heated influenced activation rates. Best results were obtained when fruit homogenates were heated for 60 min at 70 C in Concord grape juice, followed by culturing on acidified Potato Dextrose Agar.

Mechanisms of inhibition of fungi in agar by streptomycetes.

SUMMARY: Of 20 unidentified Streptomyces isolates tested, all inhibited Mucor ramannianus and 18 inhibited Glomerella cingulata in agar. Agar discs from inhibition zones, or paper discs placed beneath inhibition zones, of nine of the Streptomyces isolates caused new inhibition zones when transferred to fresh, seeded agar plates. Transferable inhibition zones were not produced by the other 11 isolates. Inhibitory substances were produced in liquid cultures by eight of the nine isolates which produced them in agar media, whereas no antibiotics were detected in liquid cultures of the other 11 isolates. Glomerella cingulata conidia, which do not require exogenous nutrients for germination, germinated in liquid cultures of non-antibiotic-producing streptomycetes, but failed to germinate in cultures of antibiotic-producing streptomycetes. Inhibition zones produced by non-antibiotic streptomycetes decreased in size with increasing concentration of nutrients, whereas those of antibiotic streptomycetes were unchanged. Glucose and glutamic acid levels in agar rapidly decreased adjacent to streptomycete colonies. Agar, leached of nutrients by sterilized distilled water running slowly through a groove cut in the agar, developed clear inhibition zones. Therefore, inhibition of fungi by streptomycetes in agar, in some cases, appears to be due to nutrient deprivation.

Nuclei of the Genus Emericellopsis

Nuclei of twelve Einericellopsis entities were successfully demonstrated by staining with HCI-Giemsa following treatment in 0.002 M 8-hydroxyquinoline and fixation in Carnoy's with chloroform. The hyphal cells of all known species of Emericellopsis were demonstrated to be uninucleate, with the exception of Emericellopsis synnematicola where binucleate cells were also observed. Conidia and ascospores were found to be uninucleate, thus confirming primary homothallism, as often suggested by the culturing of single spores. In some species, a high frequency of anastomosis and migrating nuclei reflected a means of establishing heterokaryons. It was observed that interphase nuclei, typically elliptical, were extremely elongate near hyphal tips. The presence of chromatin bodies (2-4 per haploid nucleus) and division figures suggested a mitotic mode of division in the somatic hyphae of Emericellopsis. The ascomycetous genus Emericellopsis was established in 1940, (van Beyma Thoe Kingma), when two species were described. Seven additional species have now been described and a number of unidentified isolates of Emericellopsis have been reported (Benson, 1966). Since the original description of the genus relatively little has been done to elucidate the nuclear situation. The nuclear staining procedures employed on Emericellopsis by Elander (1960) and Fantini (1962) served as adjuncts to their major research efforts and were limited to a small number of species. In both cases nuclear detail was lacking and ascospore nuclei were not observed. In this study numerous fixing and staining techniques were employed with hyphae, conidia, and ascospores of Emericellopsis species in an attempt to give a more complete picture of the nuclear situation and to provide evidence for the type of homothallism present in this

Gossypol detoxication by fungi.

An unidentified Diplodia isolate representative of several fungi tested and grown for 13 days on gossypol-containing cottonseed reduced the amount of free gossypol by 90 percent and detoxified the cottonseed for weanling rats and chickens. Residual free gossypol was also less toxic than equivalent amounts of normal free gossypol.

Comparative sensitivity of various tissue cultures for isolation of coxsackievirus B3.

ABSTRACTAttempted virus isolation from fecal specimens using four different cell cultures in parallel: primary human embryonic kidney (PHEK) cells, human embryonic lung diploid (DHEL) cells, human cancer HEp‐2 cell line and African green monkey kidney VERO cell line. Of 266 rectal swabs obtained from children, ages 0 to 11 from a small town in northern Honshu during August 1966, 74 (27.8%) yielded viral isolates. These included 58 coxsackievirus B3, 7 coxsackievirus B4, 3 echovirus 6, 3 adenoviruses, and 3 unidentified isolates. Of special interest is that practically no clinical disease was associated with this evident outbreak of coxsackievirus B3 infection. Our results provide evidence that HEp‐2 and VERO cells are most sensitive, PHEK cells are less sensitive, DHEL cells are the least sensitive for isolation of coxsackievirus B3, isolation rates were 19.2, 12.8, and 7.5%, respectively. Sensitivity differences were revealed by time of appearance and rapidity of progress of the cytopathic effect, and the infectious titer attained in the passages of the isolates.

Serum Fraction Requirements of Mycoplasma gallisepticum

Yoder and Hofstad (2) found that certain Mycoplasma serotypes survived eight passages in serum-free broth whereas an unclassified isolate persisted in serum-free media. Mycoplasma gallisepticum, however, was found to require serum for growth. Their report recalls an experience with an unsatisfactory serum fraction lot in this laboratory that justifies report since it represents a potential hazard in research and diagnostic procedure. One particular lot of PPLO serum fraction (Difco) obtained from an intermediary dealer failed to support growth of stock cultures of M. gallisepticum. Meanwhile, 3 unidentified Mycoplasma isolates grew readily in broth medium containing the offending lot. These three isolates did not produce sinusitis, other signs of disease, or M. gallisepticum agglutinins in turkeys or chickens when inoculated by appropriate respiratory routes. Further, unlike M. gallisepticum, they did not agglutinate chicken red blood cells.

Influence of exchangeable ions on germinability of bacterial spores.

Rode, L. J. (The University of Texas, Austin), and J. W. Foster. Influence of exchangeable ions on germinability of bacterial spores. J. Bacteriol. 91:1582-1588. 1966.-Native spores of Bacillus megaterium Texas, and H-spores produced by titration of native spores to pH 4 with mineral acid, did not germinate in a solution of alanine and inosine unless a strong electrolyte was present. Ca-spores prepared from either H-spores or native spores did germinate efficiently in the same solution without a strong electrolyte. Of several other bivalent cations tested, only strontium and barium could substitute for calcium in conditioning spores for subsequent germination in the absence of an electrolyte. Variable responses were obtained with different metal ion forms of 62 unidentified soil isolates and several stock species of Bacillus. Although the pattern of response was not uniform in all organisms, ions played a crucial role in the germinability of the great majority of strains tested.

Parasitism of the Chytrid Dictyomorpha Dioica

SUMMARYThe chytrid, Dictyomorpha dioica, was found capable of infecting 8 identified species of Achlya, 2 unidentified isolates of Achlya, and Thraustotheca clavata. All isolates tested of Saproleg...

Fungistasis as a factor in the rhizosphere phenomenon.

IN the course of work in progress on soil inhibitory factors, it has been observed that the spores of certain fungi which are unable to germinate when introduced into fresh fertile soil on glass slides coated with 0.5 per cent peptone in 2.0 per cent agar, owing to the presence in the soil of a fungistatic factor, may germinate readily when seedling roots grow in their immediate vicinity, that is, within a distance of some 2 mm. This observation has been made with conidia of Gliocladium roseum and Paecilomyces marquandii on glass slides buried beneath germinating peas so that the seedling roots grew into contact with the slides; and also with Fusarium solani and two unidentified isolates of Fusarium in similar experiments using seedlings of pea, wheat and lettuce. Fusarium solani and one of the unidentified Fusaria show peculiar behaviour; in the presence of soil their conidia germinate normally, but usually produce terminal chlamydospores on short germ tubes and cease further development. Park1 has reported similar formation of chlamydospores by Fusarium roseum in response to an inhibitory factor produced by Bacillus macerans. In the presence of seedling roots the chlamydospores germinate and growth is resumed (Fig. 1). According to the abstract of a thesis by Burke2 on root rot of Phaseolus vulgaris, Fusarium solani f. phaseoli may exhibit the same type of behaviour in naturally infested root-rot soils. It has further been observed with Gliocladium roseum and with the Fusaria studied that the germ tubes show a strong tropic growth towards the roots, although those of Paecilomyces marquandii do not appear to show such directed growth.

Translocatable Plant Growth Inhibitors Produced by Penicillium Thomii and Arachniotus trisporus.

The fungus cultures used in these studies were maintained as soil stocks. Conidial suspensions from seven-day-old potato-dextrose-agar slant cultures prepared from soil stocks were used to inoculate 500-ml Erlenmeyer flasks containing 100 ml of corn steepcerelose medium (Staley's corn steep liquor, 40.0 gm; cerelose, 40.0 gm; CaC03, 3.5 gm; NaN03, 3.0 gm; K2HP04, 0.5 gm; MgS04, 0.25 gm; de-ionized H20, 1000 ml). Two drops of Dow Corning Antifoam AF were added to each flask prior to autoclaving. After inoculation the flasks were placed on a reciprocating shaker (99 to 100 cpm with 3-inch strokes) at 27 to 28? C for 7 days. At the end of this time the mycelial growth in each flask was removed by filtering through Whatman No. 1 filter paper and discarded. The culture filtrates were adjusted to pH 5.0 and one drop of ? ween 80 per 100 ml (approx.) of filtrate was added. The undiluted filtrates were frozen until needed. Filtrates from approximately 700 fungi were collected in this manner. Approximately 350 of these were unidentified soil isolates obtained by routine plating-out procedures and 350 were identified as to genus and/or species. In addition, 500 aetinomycete culture filtrates obtained from cultures routinely isolated from soil were obtained from the Eli Lilly Company of Indianapolis, Indiana. With two exceptions these filtrates showed no antibiotic activity toward a wide spectrum of microorganisms. The methods used in obtaining these culture filtrates were similar to those described above for the fungus cultures. The shake-flask medium, adjusted to pH 7.0, was as follows: Bacto-peptone, 5.0 gm; glucose, 10.0 gm; molasses (Brer Rabbit Green Label) 20 ml; FeS04 ? 7 H20, 0.01 gm; and distilled water, 1000 ml. The inoculated flasks were placed on a reciprocating shaker (114 cpm with 2-inch strokes) for 5 days at 30? C. As with the fungus cultures the growth in each flask was removed by filtration and discarded. The culture filtrates were treated as described above and frozen until needed.

Ptychogaster Cubensis, A Wood-Decaying Fungus of Southern Oaks and Waxmyrtle

Routine isolations from decay in living hardwoods occasionally reveal the presence of fungi not previously known to invade living trees. The decay fungi may be species that fruit commonly on old logs or stumps, as Stereum gausapatumn Fries in oaks and Polyporus glomeratus Peck in maple, or little known fungi such as Poria Andersonii (Ellis & Ev.) Neum. and Polyporus cormnpactus Overh. in oaks. Identification of cultures of the former group do not present such a difficult problem since it is usually possible to obtain sporophore cultures for comparison, but cultures of the latter group are much more difficult to identify. However, the identity of an isolate is sometimes suggested through comparisons of some microscopic features of the culture with described characteristics of sporophores. For instance, Overholts (6) described the context of P. comwpactus as composed largely of hyaline chlamydospores, whereas the most distinguishing characteristic of cultures of this fungus are the conspicuous masses of similar chlamydospores. The identities of cultures of Poria Aindersonii and Polyporus gloimeratus were first suspected by the presence of abundant hyphal setae which are conspicuous in sporophores of these species (1). In 1935 Hepting (2) referred to 60 unidentified isolates obtained in a decay study in Mississippi Delta hardwoods. Among these was a culture from decay in the heartwood of willow oak (Quercus phellos L.) that formed abundant brown conidia. Another culture of what appeared to be the same fungus species was isolated in 1938 from heartwood of a living water oak (Q. nligra L.) from Florida. At that time a careful search of the literature disclosed the fact that brown conidia described by Patouillard (7, p. 133)