Non-sporulating Fungus Research Articles

The performance and costs of XTT, resazurin, MTS and luciferin as viability dyes in in vitro susceptibility testing of Madurella mycetomatis.

in vitro susceptibility testing for the non-sporulating fungus Madurella mycetomatis is performed with a hyphal suspension as starting inoculum and a viability dye for endpoint reading. Here we compared the performance of four different viability dyes for their use in in vitro susceptibility testing of M. mycetomatis. To compare the reproducibility and the agreement between the viability dyes 2,3-bis-(2-methoxy-4-nitro-5-sulfphenyl)-2H-tetrazolium-5-carboxanilide salt (XTT), resazurin, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt (MTS) and luciferin, the in vitro susceptibilities of 14 genetically diverse M. mycetomatis isolates were determined for itraconazole and amphotericin B. The reproducibility, agreement, price and ease of use were compared. Each of the four dyes gave highly reproducible results with >85.7% reproducibility. Percentage agreement ranged between 78.9% and 92.9%. Resazurin was the most economical to use (0.0009 €/minimal inhibitory concentration [MIC]) and could be followed in real time. Luciferin omitted the need to transfer the supernatant to a new 96-well plate, but cost 6.07 €/MIC. All four viability dyes were suitable to determine the in vitro susceptibility of M. mycetomatis against itraconazole and amphotericin B. Based on the high reproducibility, high percentage agreement, price and possibility to monitor in real time, resazurin was the most suited for routine in vitro susceptibility testing in the diagnostic laboratory in mycetoma-endemic countries. Because luminescence could be measured directly without the need to transfer the supernatant to a new 96-well plate, luciferin is suitable for drug-screening campaigns. To determine the in vitro susceptibility testing in the non-sporulating fungus Madurella mycetomatis, a viability dye is needed for endpoint reading. In this study we tested the viability dyes XTT, resazurin, MTS and luciferin for their use in in vitro susceptibility testing. It appeared that they all could be used but there were differences in time to result and costs associated with them.

Development of a microplate absorbance assay for assessing fungicide sensitivity of filamentous fungi and comparison to an amended agar assay

Assessment of the sensitivity of non-sporulating fungi to fungicides through amended-media assays is labor intensive. As an alternative, we developed an absorbance assay using 96-well microplates to assess the sensitivity of Clarireedia jacksonii, a non-sporulating fungus, to the fungicide propiconazole based on the change in absorbance corresponding to fungal growth. This microplate assay can allow for the assessment of multiple isolates of C. jacksonii at different concentrations of a fungicide with many technical replications in a single plate. Three methods for inoculating microplate wells were compared. The “microplug” method was the simplest to perform, requiring only a micropipette with 1 ml tips. EC50 values from this microplate assay were compared to those of a traditional amended agar assay using 30 isolates of C. jacksonii with varying sensitivity to propiconazole. The non-transformed relationship between the two assays was low but weakly significant (R2 = 0.137, p = 0.037). However, correlation of log10 transformed EC50 values from both assays revealed a stronger and highly significant relationship (R2 = 0.56, p < 0.001). Additionally, the microplate assay appears to be more sensitive in detecting resistance (EC50 > 0.1 μg/ml), and revealed five of the assessed isolates to be resistant to propiconazole that were not found as such with the amended agar assay. These results imply that EC50 results from the microplate assay were not exactly equivalent to the amended agar assay for estimating EC50 values, but it may be useful in assigning or confirming general sensitivity classifications.

Gloeostereum cimri, a novel shelf fungus isolated from a human pulmonary cyst

ABSTRACT Filamentous basidiomycetes are uncommon agents of human diseases, despite their ubiquitous presence in the environment. We present a case of symptomatic pulmonary infection in a 38-year-old male with cough and fever; a thin-walled cyst in the posterior left upper pulmonary lobe was revealed by radiography. A non-sporulating fungus was isolated from sputum and biopsy material from the cyst. ITS and LSU sequences placed the fungus phylogenetically in Agaricales, family Cyphellaceae, and identified it as a member of shelf fungi in Gloeostereum, but without identity to any known species. The new species is described as Gloeostereum cimri. The clinical strain showed high MIC to voriconazole (>8 µg/ml) but had low MIC to amphotericin B (0.5 µg/ml).

Fairway patch - a serious emerging disease of couch (syn. bermudagrass) [Cynodon dactylon] and kikuyu (Pennisetum clandestinum) turf in Australia caused by Phialocephala bamuru P.T.W. Wong &amp; C. Dong sp. nov.

Since 2005, a serious emerging disease called fairway patch has occurred on the fairways, tees and green surrounds of a number of golf courses in New South Wales, Queensland, Victoria and Western Australia. It occurs most commonly on couch or bermudagrass (Cynodon dactylon) but has also been found on kikuyu (Pennisetum clandestinum) fairways at one golf course in Sydney. The disease begins as small patches (5–10 cm diam.) of yellow to tan-coloured grass. The patches enlarge into tan-coloured rings up to 1 m in diameter, which often coalesce to form unsightly brown networks of dead and dying grass. The patches are present all the year round but are most pronounced from late spring to late autumn. Fungal isolation from diseased roots and pathogenicity tests have proven that a slow-growing (ca. 2 mm/day on PDA at 25 °C), dark, septate, non-sporulating fungus is the cause of fairway patch. Phylogenetic analysis of the pathogen’s rDNA ITS and partial 28S sequences has shown that it is a new taxon and is described as Phialocephala bamuru P.T.W. Wong & C. Dong sp. nov. It belongs to a heterogeneous clade that includes Phialocephala, Acephala, Vibrissea, Phaeomollisia and Mollisia spp., but, as it is phylogenetically closest to Phialocephala spp. (94–95 % ITS affinities), it has been described as a Phialocephala species although it has not been shown to produce spores in culture. Research is continuing to study the biology and ecology of this ectotrophic root-infecting fungal pathogen and develop practical strategies to manage the disease.

Mechanism of induced systemic resistance against anthracnose disease in cucumber by plant growth-promoting fungi

Plant growth-promoting fungi (PGPF) such as Phoma sp. (isolates GS8-1, GS8-2 and GS8-3) and non-sporulating fungus (isolate GU21-2) were tested for their ability to induce systemic resistance against Colletotrichum orbiculare in cucumber. These isolates, used as colonized barley kernels to natural soils, induced systemic resistance in the greenhouse as well as in the field. Different elicitors from PGPF such as cell walls retaining or lacking protein and lipids, cell walls lipid fraction, and three fractions of culture filtrate (CF) with different molecular weight ranges were tested for their ability to elicit the defense response of cucumber plants under controlled conditions. Cell wall fraction lacking protein and lipids and the cell wall lipid fraction from root colonizing isolates GS8-1, GS8-2, and GS8-3 protected plants against C. orbiculare. While, only cell wall lipid fraction and CF fractions of different molecular weight ranges of the isolate GU21-2 protected plants against C. orbiculare infection. Methanol-soluble substances from CF fractions with molecular weight greater than 12,000 and less than 8000 consistently protected plants against the pathogen. Germination of C. orbiculare spores decreased significantly at 72 h of incubation on leaves of PGPF-protected plants. Isolate GU21-2 was the most effective in inhibiting spore germination. On the other hand, isolates GS8-1 and GU21-2 induced lignifications in the hypocotyls of seven-day-old cucumber seedlings after challenge inoculation with C. orbiculare. Three-week-old cucumber plants treated with isolate GU21-2 and challenged with C. orbiculare showed increased activities of exo- and endo-forms of glucanase and chitinase, as well as peroxidase and polyphenol oxidase in the second true leaves. Induction treatment with isolate GS8-1 also increased the activities of these enzymes with the exception of exo-glucanase. This study shows that the inoculation of PGPF or its CF resulted in additive effect on the suppression of anthracnose disease in cucumber.

First Report of Rhizoctonia spp. Causing a Root Rot of the Invasive Rangeland Weed Lepidium draba in North America.

The exotic, invasive perennial rangeland weed Lepidium draba spreads rapidly and reduces native species diversity. The extensive root system of L. draba constitutes 76% of its biomass (4). Thus, searches have been done for biocontrol agents that target root tissue or that may interact with a weevil, Ceutorhynchus assimilis, that causes galls in the crown area of L. draba. An association of Rhizoctonia spp. with root tissue of plants galled by the weevil has been documented in Europe (3). The possible presence of soilborne pathogens similar to those found in the native range has been the subject of L. draba surveys in the United States. One such survey in 2008 detected a few plants with reddened and chlorotic foliage in a stand near Shepherd, MT. Such symptoms typically indicate the occurrence of soilborne diseases on L. draba in the native range of the weed (2). The site had shown a gradual increase in the range of detectable pathogens beginning with foliar pathogens in 1997. In 2010, at the Shepherd site, L. draba plants with similar (but more severe) symptoms to those seen in 2008 were noted in a different area of the stand. Excavation of the roots in both years revealed brown, sunken crown and root cankers. Pieces of root tissue were excised from the lesions and plated on acidified PDA and Ko and Hora medium. A non-sporulating fungus was isolated from three plants. Colonies of the isolates on PDA were typical of known Rhizoctonia spp. The 2010 isolates were determined to be multinucleate using DAPI and were paired with 14 tester (including subgroups) isolates of AG-1 to AG-4 on water agar. Anastomosis was observed between the multinucleate isolates and the AG-2-1 tester isolate. Sequence analysis of ITS of the rDNA of a multinucleate isolate (GenBank KJ545577) indicated 99% similarity with an accession of R. solani AG 2-1 (AB547381). The 2008 isolates were binucleate. A binucleate isolate, KJ545578, had 100% similarity with an isolate of Rhizoctonia spp. AG-A (AY927356). Pathogenicity tests consisted of planting 6-week-old seedlings of L. draba, one per pot, in ten 85-cm-diameter pots of pasteurized soil mix infested with Rhizoctonia-colonized barley grain that had been dried and milled. An inoculum level of ~8 CFU/g (1) of air-dried soil was established by most probable number calculations from fourfold dilutions of infested soil. Controls were the same number of plants in pasteurized potting mix. Results were recorded after 3 months in a greenhouse at 20-25°C. The test was repeated. Typically, R. solani caused mortality of six to eight plants, from which it was re-isolated, whereas binuclate isolates caused stunting and lower dry weight of L. draba. Control plants remained asymptomatic. This is the first report of R. solani and binucleate Rhizoctonia spp. on L. draba in North America.

Elevated expression of hydrolases, oxidase, and lyase in susceptible and resistant cucumber cultivars systemically induced with plant growth-promoting fungi against anthracnose

Susceptible and resistant cultivars of cucumber (cv. Suyo and cv. Shogoin fushinari, respectively) were evaluated for induction of systemic resistance by plant growth-promoting fungal isolates Phoma sp. GS8-1 and nonsporulating fungus GU21-2 against Colletotrichum orbiculare. Reduction in anthracnose disease was assessed at regular intervals of 0, 3, 6, and 9 days after challenge inoculation. Activities of endochitinase, β-1,3-endoglucanase, peroxidase (PO), polyphenol oxidase, and phenylalanine ammonia lyase (PAL) were assayed at the above intervals, while the activities of exochitinase and β-1,3-exogluconase were assayed at 9 days after challenge inoculation. The data showed that all the enzymes activities increased in plants treated with plant growth-promoting fungi (PGPF) compared with nontreated plants of both cultivars. The activities of endochitinase and β-1,3-endoglucanase were highly increased in PGPF-induced plants than in nontreated plants of both cultivars at different time points. Activities of PO, polyphenol oxidase, and PAL also increased between 3 and 6 days due to inoculation with PGPF and decreased thereafter. The activity of PAL at 9 days in PGPF-induced plants was at the same level as that of nontreated plants of both cultivars. Results of this investigation suggest that PGPF-induced systemic resistance is associated with not only high increases in the activities of chitinase and β-1,3-gluconases but also other plant defense-related enzymes such as PO, PPO, and PAL. Roles of other mechanisms rather than accumulation of defense-related enzymes in PGPF-mediated induced resistance in susceptible cultivars were demonstrated.

New Species of Madurella, Causative Agents of Black-Grain Mycetoma

A new species of nonsporulating fungus, isolated in a case of black-grain mycetoma in Sudan, is described as Madurella fahalii. The species is characterized by phenotypic and molecular criteria. Multigene phylogenies based on the ribosomal DNA (rDNA) internal transcribed spacer (ITS), the partial β-tubulin gene (BT2), and the RNA polymerase II subunit 2 gene (RPB2) indicate that M. fahalii is closely related to Madurella mycetomatis and M. pseudomycetomatis; the latter name is validated according to the rules of botanical nomenclature. Madurella ikedae was found to be synonymous with M. mycetomatis. An isolate from Indonesia was found to be different from all known species based on multilocus analysis and is described as Madurella tropicana. Madurella is nested within the order Sordariales, with Chaetomium as its nearest neighbor. Madurella fahalii has a relatively low optimum growth temperature (30°C) and is less susceptible to the azoles than other Madurella species, with voriconazole and posaconazole MICs of 1 μg/ml, a ketoconazole MIC of 2 μg/ml, and an itraconazole MIC of >16 μg/ml. Since eumycetoma is still treated only with azoles, correct species identification is important for the optimal choice of antifungal therapy.

Identification problems with sterile fungi, illustrated by a keratitis due to a non-sporulatingChaetomium-like species

A 39-year-old farm worker was injured in her right eye by a piece of wire, which resulted in a corneal ulcer unresponsive to antibiotic treatment. The clinical appearance was that of a corneal infiltrate with feathery borders resembling fungal keratitis. Corneal scrapings were collected and the patient was started on natamycin 5% eye drops, fluconazole 0.3% eye drops, and oral fluconazole. A non-sporulating fungus was isolated from the samples. Based upon macroscopic and microscopic morphologic features, it was provisionally identified as a Papulaspora species due to the fact that members of this genus generally do not form diagnostically useful conidia. However, it was found through the use of ITS sequencing that the isolate clustered within the ascomycete genus Chaetomium. The sequence did not fully match with any sequences of available ex-type strains of Chaetomium, Thielavia and Papulaspora and hence might belong to an undescribed specie. However, without diagnostic morphological features the taxon cannot be introduced as a novel member of the genus Chaetomium. Antifungal susceptibility testing was performed according to published standards. The corneal ulcer was successfully treated with six weeks of antifungal therapy.

First Report of a Root and Crown Disease Caused by Rhizoctonia solani on Centaurea stoebe in Russia.

Spotted knapweed (SKW), Centaurea stoebe L., is a nonindigenous species that is invasive over large areas in the United States, especially in the west. It has been estimated that infestations of SKW cause $42 million in direct and indirect economic losses annually (2), and the weed could potentially invade 13.6 million ha of rangeland in Montana alone. Extensive efforts toward the control of SKW have included the release of 12 insects for biological control, four of which attack the crowns and roots of this short-lived perennial. To focus efforts to select potential soilborne pathogens, which could be applied in combination with insects, we conducted a survey for plant pathogens in the native range of SKW associated with damage caused by any root-attacking insects. Stunted and chlorotic SKW plants, which were colonized by larvae of Cyphocleonus spp., were found in June 1994 near the Novomar'evskaya Botanical Sanctuary (45°08'49.87″N, 41°51'02.05″E) in the Caucasus Region of Russia. A nonsporulating multinucleate fungus was isolated from the lower stem, crown, and upper root tissue of one such plant. Colonies growing on potato dextrose agar and Ko and Hora media were examined microscopically and identified as Rhizoctonia solani by the occurrence of robust, thick-walled, golden hyphae with right-angled branching and constrictions at the branch points. The anastomosis grouping of the one isolate was determined to be AG 2-2 IIIB after pairing it on water agar with 11 AG tester isolates representing all subgroups of AG 1 to AG 5. The hyphal diameter at the obvious point of anastomosis was reduced and cell death of adjacent cells was observed. In 2007, pathogenicity was determined by planting 12-week-old seedlings of SKW, one per pot, into 20 15-cm-diameter pots of a steamed greenhouse soil mix composed of sphagnum peat, sand, and Bozeman silt loam (1:1:1, vol/vol), pH 6.6, infested with R. solani-colonized barley grain that had been dried and milled. An inoculum level of 8 CFU/g of air-dried soil was determined by most probable number calculations from fourfold dilutions of infested soil. Controls were planted into noninfested soil. In both greenhouse tests, the isolate caused either mortality or a 93% mean fresh weight reduction of surviving plants, relative to the controls, after 8 months. R. solani was reisolated from necrotic root and crown tissue of dead and stunted plants but not from the controls. To our knowledge, this is the first report of R. solani occurring on SKW in Europe. The characterization and pathogenicity of Fusarium spp. isolated from insect-colonized roots of SKW in Europe was reported previously (1).

Keratinophilic fungi of poultry farm and feather dumping soil in Tamil Nadu, India

Soils of 10 poultry farms from Namakkal and 12 feather dumping sites from Chennai were studied for the presence of keratinophilic fungi. A total of 34 species belonging to 19 genera and one non-sporulating fungus were recovered. Sixteen species of fungi and one non-sporulating fungi were common to both sites, eight species were specific to Namakkal and nine species were specific to Chennai. Dermatophytes and closely related fungi were represented by six species belonging to five genera. Fungal species commonly found in the soil samples included Chrysosporium keratinophilum (73%), Trichophyton mentagrophytes (68.2%), Microsporum gypseum (64%), Myceliopthora vellerea (32%), Chrysosporium state of Arthroderma tuberculatum (27.3%) and Geomyces pannorum (23%). Non-dermatophyte fungi were represented by 28 species belonging to 14 genera and one non-sporulating fungus.

Isolation and characterization of a cellobiose dehydrogenase formed by a asporogenic mycelial fungus INBI 2-26(-)

A nonsporulating fungus isolated from dioxine-containing tropical soils forms cellobiose dehydrogenase, when grown in media supplemented by a source of cellulose. The enzyme purified to homogeneity by SDS-PAGE (yield, 43%) had an M(r) of 95 kDa; its pH optimum was in the range 5.5-7.0; more than 50% activity was retained at pH 4.0-8.0 (citrate-phosphate buffer). The absorption spectrum of the enzyme in the visible range had the characteristic appearance of flavocytochrome proteins. Cellobiose dehydrogenase oxidized cellobiose and lactose (the respective K(M) values at pH 6.0 equaled 4.5 +/- 1.5 and 56 microM) in the presence of dichlorophenolindophenol (K(M) app = 15 +/- 3 microM at pH 6.0) taken as an electron acceptor. Other sugars were barely if at all oxidized by the enzyme. Neither ethyl-beta-D-cellobioside, heptobiose, nor chitotriose inhibited the enzymatic oxidation of lactose, even under the conditions of 100-fold molar excess. The enzyme was weakly inhibited by sodium azide dichlorophenolindophenol reduction and exhibited affinity to amorphous cellulose. At 55 degrees C and pH 6.0 (optimum stability), time to half-maximum inactivation equaled 99 min. The enzyme reduced by cellobiose was more stable than the nonreduced form. Conversely, the presence of an oxidizer (dichlorophenolindophenol) decreased the stability eight times at pH 6.0. In addition, the enzyme acted as a potent reducer of the single-electron acceptor cytochrome c3+ (K(M) app = 15 microM at pH 6.0).

Characterization of Fusarium graminearum from Acacia and Eucalyptus using β-tubulin and histone gene sequences

During routine surveys of diseased Acacia mearnsii and Eucalyptus grandis in South Africa, isolates of an unknown, nonsporulating fungus with a red mycelium were collected. Symptoms associated with the disease included branch dieback and stem cankers on both hosts. None of the isolates of the fungus produced spores, making identification using morphological characteristics impossible. An attempt was thus made to identify the isolates using DNA sequence data for the β-tubulin and histone genes. Using this approach, the fungus was tentatively identified as Fusarium graminearum. Sequences were then compared to those for isolates of F. graminearum from cereal hosts. The relative pathogenicity of F. graminearum to A. mearnsii and an E. grandis clone was determined in pathogenicity trials. Pathogenicity tests were conducted by inoculating 18-mo-old A. mearnsii trees and 12-mo-old E. grandis clone trees under field conditions. All the isolates tested produced significant lesions on both the A. mearnsii and E. grandis clones. The isolates collected from A. mearnsii and E. grandis were further compared to other F. graminearum isolates using β-tubulin and histone gene sequence. In these comparisons the isolates collected from A. mearnsii and E. grandis consistently grouped with F. graminearum isolates. The occurrence of F. graminearum on A. mearnsii and E. grandis is intriguing, and as far as we are aware, this is the first report of the fungus associated with a disease of a woody host.

Characterization of Fusarium graminearum from Acacia and Eucalyptus Using [beta]-Tubulin and Histone Gene Sequences

During routine surveys of diseased Acacia mearnsii and Eucalyptus grandis in South Africa, isolates of an unknown, nonsporulating fungus with a red mycelium were collected. Symptoms associated with...

Fungi in Porites lutea: association with healthy and diseased corals

Healthy and diseased scleractinian corals have been reported to harbour fungi. However, the species of fungi occurring in them and their prevalence in terms of biomass have not been determined and their role in coral diseases is not clear. We have found fungi to occur regularly in healthy, partially dead, bleached and pink-line syndrome (PLS)-affected scleractinian coral, Porites lutea, in the reefs of Lakshadweep Islands in the Arabian Sea. Mostly terrestrial species of fungi were isolated in culture from these corals. Hyaline and dark, non-sporulating fungi were the most dominant forms. Fungal hyphae extended up to 3 cm within the corals. Immunofluorescence detection using polyclonal immunological probes for a dark, initially non-sporulating isolate (isolate # 98-N28) and for a hyaline, non-sporulating fungus (isolate # 98-N18) revealed high frequencies of these in PLS-affected, dead and healthy colonies of P. lutea. Total fungal biomass accounted for 0.04 to 0.05% of the weight of corals in bleached corals and was higher than in PLS-affected and healthy colonies. Scanning electron microscopy revealed the presence of fungi within the carbonate skeleton and around polyps. Fungi appear to be a regular component of healthy, partially dead and diseased coral skeleton.

Freeze-drying vegetative mycelium of Laccaria fraterna and its subsequent regeneration

Laccaria fraterna, a basidiomycetous, filamentous, non-sporulating (in vitro) fungus, was subjected to freeze-drying and tested for viability after rehydration. The optimization of the freeze-drying protocol included selection of the candidate fungus; optimizing physiological growth conditions and age; standardizing the protectant type and concentration; optimizing the pre-freezing method, freeze-drying run and extent of drying; choice of the rehydrant and the extent of rehydration. The culture retained its viability after lyophilization and when subjected to quality assurance tests gave consistent results similar to the non-lyophilized culture, indicating the stability of the product and the applicability of the developed process to freeze-dry vegetative mycelium of filamentous non-sporulating fungi.

Role of root colonization ability of plant growth promoting fungi in the suppression of take-all and common root rot of wheat

Plant growth promoting fungal (PGPF) isolates — GS6-1, GS7-4 ( Phoma sp.) and GU23-3 (non-sporulating fungus), from zoysiagrass rhizosphere, suppressed take-all and common root rot of spring wheat caused by Gaeumannomyces graminis var. tritici (Ggt) and Cochliobolus sativus (Cs), respectively, in the greenhouse. Barley kernels colonized with pathogens (0.1%, 0.5% or 1.0% w w ) and PGPF isolates (1.0%, 1.5% or 2.0% w w ) were added to soil individually and in different combinations of pathogen and PGPF. With an increase in the rate of PGPF amendment, all three PGPF isolates suppressed take-all and common root rot ( P = 0.05), irrespective of the pathogen inoculum level. Among the PGPF isolates, GS6-1 and GS7-4 were more effective than GU23-3. When added to soil individually, PGPF isolates were recovered from 75 to 95% of root segments, and Ggt and Cs from 88–95% of root segments. Both Ggt and Cs were isolated less frequently from roots when the PGPF concentration was high (2.0%); however, the PGPF isolates were isolated from roots in high frequencies even when the pathogen inoculum level was high. Isolate GS6-1 promoted plant growth even when the pathogen inoculum rate was high, as did GS7-4, but to a lesser amount. These fungal isolates suppressed take-all and common root rot, and two of them also increased plant growth. The suppression of take-all and common root rot appeared to be due to the competitive root colonization by PGPF isolates, thus blocking sites available for infection by pathogens.

Microfungi associated with the mycorrhizal mantle and adjacent microhabitats within the rhizosphere of black spruce

Microfungi were isolated from serially washed ectomycorrhizae of Picea mariana (black spruce) collected in boreal northern Ontario. This assemblage of microfungal isolates was compared with others isolated from surface-sterilized ectomycorrhizae, adjacent suberized feeder-root surfaces, and rhizosphere soil. In addition, a comparison was carried out with microfungi isolated from serially washed assimilative rootlets of Cornus canadensis, an endomycorrhizal understory plant. The most common microfungi from spruce mycorrhizae included Mycelium radicis atrovirens alpha and an undescribed nonsporulating fungus, as well as Micromucor isabellinus, Penicillium spinulosum, and Penicillium montanense. None of the fungi isolated from ectomycorrhizae appeared to have a high degree of specificity for the mycorrhizal mantle. The assemblage of species isolated from ectomycorrhizae was relatively similar to those obtained from suberized root surfaces and from assimilative rootlets of C. canadensis.

The Genus Atropellis, and a New Genus of the Helotiales Associated with Branch Cankers of Western Hemlock

(with 15 figures) During the summer of 1960, an unusual discomycete was found associated with fusiform, cankered swellings on branches of western hemlock [Tsuga heterophylla (Raf.) Sarg.], growing in the coastal region of British Columbia. Cultural studies show it to be identical with a nonsporulating fungus previously isolated from this type of canker. We determined that this organism is Atropellis treleasei (Sacc.) Zell. & Goodd. (1930), but examination of other members of this genus shows that A. treleasei is not congeneric with either Atropellis pinicola Zell. & Goodd. (1930), the type of this genus, or with the other species assigned to Atropellis. Studies on the other species of the genus were undertaken to clarify the taxonomic position of A. treleasei and of the

Identity of a Fungus Causing Blue Stain in Balsam Fir

Blue-stained balsam fir wood [Abies balsamea (L.) Mill.] consistently yielded cultures of a nonsporulating fungus which were found to be the same as those obtained from ascospores of Amphisphaeria thujina (Peck) Sacc. collected from dead branches of this host. This blue stain appears to be identical with a previously reported but hitherto unidentified stain of conifers. The association of Amphisphaeria with stain in living trees does not appear to have been recognized before.