Cotton Blue Research Articles

The Editor takes a closer look at some of this month's articles

Clinical & Experimental AllergyVolume 44, Issue 3 p. 301-301 Editor's ChoiceFree Access The Editor takes a closer look at some of this month's articles First published: 22 February 2014 https://doi.org/10.1111/cea.12285AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat In vivo confocal microscopy: a window to the allergic eye Vernal keratoconjunctivitis is a severe form of allergic eye disease which can cause lasting damage if not adequately controlled. The mechanism is presumed to involve adaptive type 2 immune responses to allergens, and therefore, the role of dendritic cells is of particular interest. In a novel study, Liu et al. (pp. 362-370), have used a technique called in vivo confocal microscopy to identify and enumerate dendritic cells in the conjunctiva, limbus and peripheral cornea before and after treatment with immunosuppressive eye drops. They observed increased numbers before treatment compared with healthy subjects and a return towards normal within 3 months of treatment. This technique offers a novel biomarker to study the effectiveness and mechanism of action of new treatments for allergic eye disease. Chien Jien-Wen In vivo confocal microscopy image of dentritic cells. (See figure 4 of M. Liu et al., pp. 362–370). Allergic bronchopulmonary mycoses (ABPM): more than just Aspergillus fumigatus There is increasing interest in the role of fungal allergy in asthma, particularly in its more severe manifestations (1). Most attention has been focused on A. fumigatus as the causative organism although in a recent study 27 species of thermotolerant fungi were cultured from the sputum of people with asthma (2). Many people with severe asthma present with features suggestive of ABPM, but there is no evidence of sensitization to fungi and sputum culture is negative. However, there are relatively few commercially available fungal allergens compared with the thousands of potentially allergenic fungi which we can use to test for fungal allergy. In addition many fungi, particularly basidiomycetes, are difficult to culture in vitro. One such basidiomycete that has been associated with ABPM, particularly in the far east, is Schizophyllum commune. Toyotome and colleagues (pp. 450-457) have advanced the field by characterizing the first allergen from this fungus, Sch c 1 which is a homologue of glucoamylase. This reagent can now be used to help investigate in detail the role of S. commune in asthma. Takahito Toyotome Aspergillus fumigatus (conidial head) stained with cotton blue. [Photo courtesy of Takahito Toyotome]. Occupational asthma: potential dangers of disinfectants in health care workers There is an increased incidence of asthma in healthcare workers. Healthcare workers are exposed to a number of known allergens and irritants such as latex, aldehydes and cleaning products including chlorine-based cleaners (bleach) and quaternary ammonium compounds (QACs). Exposure to the latter has been associated with anaphylaxis to general anaesthetics. Gonzales and colleagues (pp. 393-406) undertook a survey of healthcare workers to determine whether QACs could be responsible for asthma in healthcare workers. They found a 7.5-fold increase in the incidence of asthma and a 3.2-fold increase in the incidence of rhinitis in healthcare workers exposed to QACs with the highest risk associated with dilution of disinfectants. No link was found to other potential agents, and the risk was not associated with increased IgE to QACs. This important study emphasizes the importance of identifying healthcare workers at risk of occupational asthma and rhinitis due to QACs and the development of processes to minimize exposure. Maria Gonzalez A disinfectant containing QAC's. (Photo credit: © Tokumeigakarinoaoshima; Wikimedia Commons). References 1Agarwal R, Chakrabarti A, Shah A et al. Allergic bronchopulmonary aspergillosis: review of literature and proposal of new diagnostic and classification criteria. Clin Exp Allergy 2013; 43: 850– 73. 2Agbetile J, Fairs A, Desai D et al. Isolation of filamentous fungi from sputum in asthma is associated with reduced post-bronchodilator FEV1. Clin Exp Allergy 2012; 42: 782– 91. Caption to cover illustration: Vernal conjunctivitis (Photo credit: Wikimedia Commons). This logo highlights the Editor's Choice articles on the cover and the first page of each of the articles. Volume44, Issue3March 2014Pages 301-301 ReferencesRelatedInformation

A Qualitative and Quantitative Study Monitoring Airborne Fungal Flora in the Kidney Transplant Unit

BackgroundSolid organ transplantation patients are at high risk for opportunistic air-borne fungal infections due to using the potent immunosuppressive agents.ObjectivesThe current study aimed to qualitatively and quantitatively evaluate the fungal flora present in the air of Kidney transplant unit of Baqiyatallah hospital.Materials and MethodsIn this prospective study, air samples from patient room, baths site, ICU and isolated room, corridor site and outside the ward were obtained by settled plate technique using plates containing Sabouraud's dextrose agar medium. In the current study, 36 agar plates containing Sabouraud dextrose agar medium were used. The plates were exposed for 20 min at height of 100-150 cm above the ground in units of hospital. Immediately after collection, samples were incubated at 27 ± 2ºC for four weeks. The slide culture method and Lacto-phenol cotton blue were used for definitive identification and staining fungal cultures, respectively.ResultsThe mean of colony forming units (CFUs) on indoor and outdoor plates was 6.6 ± 1.3 and 6 ± 1.9 / plate respectively. Statistical analysis showed that the observed difference is not significant. Also, the results showed that the mean of CFUs in the air of patient's rooms (6.8 ± 1.7), halls (4.5 ± 1.7), bathrooms (6.8 ± 1.5), and ICU rooms (3.2 ± 1.8) were not significantly different. The mean of different fungal genera isolated from indoor and outdoor plates were 1.9 ± 0.2 and 4 ± 0.5 genera/plate respectively, that indicates significant difference between indoor and outdoor air quality (P < 0.001).ConclusionsLack of difference between quantity of outdoor and indoor air fungi indicates inefficiency of air control measures, and indoor lower genus diversity compared to outdoor air shows that there may be conditions that facilitate fungal growth in the environment of kidney transplantation unit.

Subcutaneous Phaeohyphomycosis Caused by Exophiala salmonis

We report a case of subcutaneous infection in a 55-yr-old Korean diabetic patient who presented with a cystic mass of the ankle. Black fungal colonies were observed after culturing on blood and Sabouraud dextrose agar. On microscopic observation, septated ellipsoidal or cylindrical conidia accumulating on an annellide were visualized after staining with lactophenol cotton blue. The organism was identified as Exophiala salmonis by sequencing of the ribosomal DNA internal transcribed spacer region. Phaeohyphomycosis is a heterogeneous group of mycotic infections caused by dematiaceous fungi and is commonly associated with immunocompromised patients. The most common clinical manifestations of subcutaneous lesions are abscesses or cystic masses. To the best of our knowledge, this is the first reported case in Korea of subcutaneous phaeohyphomycosis caused by E. salmonis that was confirmed by molecular analysis and identification of morphological characteristics. This case suggests that E. salmonis infections are no longer restricted to fish.

A case of otomycosis caused by Lichtheimia corymbifera (syn. Absidia corymbifera, Mycocladus corymbifer) in a healthy immunocompetent individual

A case of otomycosis caused by Lichtheimia corymbifera from an immunocompetent patient with no known predisposing factor is reported. A 55-year-old, otherwise healthy male was presented to us with history of left-sided earache and yellowish-white ear discharge since 10 days. Ear discharge and bits of necrotic tissue were collected from ear through otoscope and processed. Direct wet mount by potassium hydroxide (10% KOH) was performed which showed broad, aseptate and branched hyphae suggestive of zygomycosis. On culture after 48 hours, cottony, wooly and fluffy growth was observed. Culture was subjected to Lactophenol Cotton Blue (LCB) mount which confirmed the presence of L. corymbifera. The patient responded well to suction clearance and debridement followed by drug therapy with amphotericin B.

The use of cotton blue stain to improve the efficiency of picking and identifying chironomid head capsules

Cotton blue was added to sediment samples at least 2 h before chironomid head capsules were picked under a binocular microscope and mounted on slides for identification. The use of stain greatly increased the visibility of chironomid head capsules during picking and enhanced the contrast of various parts of the head capsules (pores, ventromental plates, striations on ventromental plates), which could aid identification. In the seven samples studied, there was no significant difference between the percentages of taxa found in stained and unstained samples. The number of taxa were also similar in stained and unstained samples. This method allowed samples to be picked faster.

Investigation of the Electrocoagulation Treatment of Cotton Blue Dye Solution using Aluminium Electrodes

AbstractThis study was performed to investigate the variables that influence the removal efficiency of an acid dye, i. e., cotton blue (CB) (chemical name: aniline blue WS) dye, from aqueous solution by an electrocoagulation (EC) technique. Batch EC studies were performed using aluminum electrodes to evaluate the influences of various experimental parameters, i. e., initial pH (pH0): 3–11, electrolysis time (t): 0–30 min, initial concentration (C0): 100–1000 mg/L, electrode gap (g): 0.5–1.3 cm, number of electrodes (N): 4–10, and applied voltage (Vap): 7–11 V, on the removal of CB dye. The optimum values of pH0, Vap and t for CB removal were found to be 6.0, 11 V and 15 min, respectively. The removal efficiency increased with decreasing values of C0 and g. For a CB solution having C0 = 100 mg/L, 97% removal efficiency was obtained at the optimized conditions. It was found that the EC sludge can be dried and thermally degraded. The bottom ash obtained after its combustion can be blended with cementitious mixtures. This approach for EC sludge disposal ensures energy recovery along with safe disposal of the EC sludge.

Biodegradation of triphenylmethane dye cotton blue by Penicillium ochrochloron MTCC 517

Triphenylmethane dyes belong to the most important group of synthetic colorants and are used extensively in the textile industries for dying cotton, wool, silk, nylon, etc. They are generally considered as the xenobiotic compounds, which are very recalcitrant to biodegradation. Penicillium ochrochloron decolorizes cotton blue (50 mg l −1) within 2.5 h under static condition at pH 6.5 and temperature 25 °C. TLC, FTIR and HPLC analysis confirms biodegradation of cotton blue. FTIR spectroscopy and GC–MS analysis indicated sulphonamide and triphenylmethane as the final products of cotton blue degradation. The pH, temperature and maturity of biomass affected the rate of decolorization. Presence of lignin peroxidase, tyrosinase and aminopyrine N-demethylase activities in the cell homogenate as well as increase in the extracellular activity of lignin peroxidase suggests the role of these enzymes in the decolorization process. The phytotoxicity and microbial toxicity studies of extracted metabolites suggest the less toxic nature of them.

Phytophthora cinnamomi

<i>Phytophthora cinnamomi</i>

Evaluation of Interspecific Crossing Experiments in Facultatively Apomictic Blackberries (Rubus subgen. Rubus) Using DNA Fingerprinting

A series of interspecific crossing experiments in Rubus was evaluated with DNA fingerprinting to assess the proportion of apomictic vs sexual seed set. A total of 71 seedlings and their putative parents were thus analyzed by hybridization of HaeIII-digested DNA samples to a minisatellite DNA probe derived from the M13 phage. Of the offspring derived from non-hybridogenous seed parents, only about 10% appear to emanate from sexual seed set. On the contrary, offspring derived from pollination of hybridogenous plants were in 6 cases out of 10 sexually derived. Pollen stainability with cotton blue was also investigated and could, though to a more limited extent, be used as an indicator of reproductive mode. Taxonomic treatment of Rubus and other apomictic plant groups would clearly benefit from more data on reproduction and dispersal, which may be obtained by DNA fingerprinting.Hilde Nybom, Balsgard-Department of Horticultural Plant Breeding, Swedish University of Agricultural Sciences, Fjalkestadsvagen 123-1, S-291 94 Kristianstad, Sweden

First Report of Powdery Mildew of Greenhouse Pepper Caused by Leveillula taurica in British Columbia, Canada.

In 2003, powdery mildew of greenhouse pepper (Capsicum annuum L.) was reported simultaneously in two commercial greenhouses at two separate locations near Langley in southern British Columbia. Trace amounts of mildew on the foliage of cv. Zamboni in one operation, and 10 to 80% of the foliage of cv. Triple Four was affected in an organic operation, with losses in fruit yield of 2 to 4 kg/m2, were noted. The greenhouse pepper industry in British Columbia in 2001 comprised 76 ha with sales of $41.3 million (Canadian). Affected areas appeared as a white, powdery coating on the abaxial leaf surface of older foliage while diffuse chlorotic spots were present at corresponding locations on the adaxial surface. In some cases, pale yellow spots appeared on the adaxial portion of the affected foliage during later stages of disease development (2). Hyaline, single-celled pyriform and cylindrical conidia were present in 40:60 proportion, respectively. Both conidia had a network of crests and granules sometimes with tiny thorn-like projections on the surface. These projections were evident also on the lower half of conidiophores. Pyriform conidia ranged in length from 53.8 to 79.5 μm (mean = 68.0, SE = 0.8), and in width from 12.9 to 28.0 μm (mean = 20.1, SE = 0.6, n = 50) with a mean length to width ratio of 3.5. Cylindrical conidia had a length from 48.2 to 84.0 μm (mean = 66.0, SE = 0.9), and in width from 13.4 to 25.2 μm (mean = 18.6, SE = 0.4, n = 50) with a mean length to width ratio of 3.6. Short conidial chains borne on conidiophores consisted of a basal pyriform conidium followed by cylindrical conidium. No cleistothecia were observed. Stomatal penetration and extensive endophytic mycelial growth in the mesophyll layer were evident in foliar tissue cleared with glacial acetic acid/EtOH (1:2) and stained with lactophenol cotton blue. To confirm pathogenicity, a suspension of 2 × 104 conidia/ml from infected pepper leaves was applied onto the abaxial and adaxial leaf surfaces of 60-, 42-, and 28-day-old greenhouse pepper cv. Cubico, tomato cv. Trust, and cucumber cv. Corona, respectively. Plants were maintained in a growth chamber at 25/21°C day/night temperature and 80% relative humidity. Control plants in the same chamber were treated identically but not inoculated. After 21 days, inoculated pepper plants developed foliar powdery mildew symptoms on the abaxial surface, including sporulation similar to that of naturally infected plants. Occurrence of the fungus on the abaxial surface of pepper foliage, presence of endophytic mycelium, and the morphological characteristics of the imperfect state confirm the identity of the fungus (2). To our knowledge, this is the first report of Leveillula taurica (Lév.) G. Arnaud on pepper in British Columbia. The disease was first reported in Ontario, Canada in 1999 (1).

First Report of Powdery Mildew of Greenhouse Pepper Caused by Leveillula taurica in Canada.

In 1999, powdery mildew on cvs. Oberon and Triple 4 of greenhouse pepper (Capsicum annum L.) and cv. FireFlame of chili pepper was reported simultaneously in two commercial greenhouses at two separate locations, Leamington and Vineland, geographically separated by 290 km, in southern Ontario. Losses of 10 to 15% each in the 2 and 3.1 ha greenhouse pepper operations were noted. The greenhouse pepper industry in Canada consists of 89.4 ha with sales of $43.6 million (Canadian). Lesions appeared as a white, powdery coating on the abaxial leaf surface only, generally on the lower foliage of pepper plants, while diffuse chlorotic spots were present at corresponding locations on the adaxial surface. In chili pepper, this chlorosis was restricted to interveinal tissue, causing the leaves to have a somewhat netted appearance. In some cases, pale yellow spots appeared on the adaxial portion of the affected foliage during later stages of disease development. No cleistothecia were observed. Pyriform and cylindrical, hyaline, single-celled conidia were present in equal numbers. Both conidia had a network of crests and granules containing tiny, thornlike projections on the surface. These projections were also evident on the lower half of conidiophores. Pyriform conidia ranged in length from 61.6 to 84.0 μm (mean = 70.8, SE = 0.7) and in width from 14.0 to 25.8 μm (mean = 21.1, SE = 0.4, n = 50), with a mean length to width ratio of 3.4. Cylindrical conidia ranged in length from 54.9 to 80.1 μm (mean = 66.3, SE = 0.9) and in width from 15.7 to 24.1 μm (mean = 18.6, SE = 0.3, n = 50), with a mean length to width ratio of 3.1. Short conidial chains borne on conidiophores consisted of a pyriform conidium first followed by cylindrical conidium. Stomatal penetration and extensive endophytic mycelial growth in the mesophyll layer were evident in foliar tissue cleared with glacial acetic acid:EtOH (1:2) and stained with lactophenol cotton blue. To confirm pathogenicity, conidia from infected pepper leaves were dusted onto the water-misted abaxial leaf surface of 41-day-old greenhouse pepper cvs. Cubico and Edison, and onto the similarly treated adaxial leaf surface of 58-day-old field pepper cv. Renegade. Plants were enclosed in plastic bags for 24 h on a bench. Control plants were treated identically but not inoculated. Inoculated plants developed foliar powdery mildew symptoms, including sporulation similar to that of naturally infected plants. Occurrence of the fungus on the abaxial surface of pepper foliage, presence of endophytic mycelium, and the morphological characteristics of the imperfect state confirm the identity of the fungus. This is the first report of Leveillula taurica (Lév.) G. Arnaud on pepper in Canada. Reference: J. Palti. Bot. Rev. 54:423, 1988.

Japewiella gen. nov., a New Lichen Genus and a New Species from Mexico

The genus Japewiella Printzen, gen. nov. is separated from Japewia Tynsberg and placed in the family Lecanoraceae. Japewiella pacifica Printzen, sp. nov. is described as new. The species is closely related to the southwestern European J. carrollii (Coppins & P. James) Printzen, comb. nov. and the East Asian J. djagensis (Zahlbr.) Printzen, comb. nov. Differences between Japewia and Japewiella are discussed. Like the closely related Megalaria Hafellner and Tasmidella Kantvilas, Hafellern, & Elix, Japewiella seems to be a genus of humid temperate and subtropical regions, whereas the two known species of Japewia show a boreal-(sub)arctic-alpine distribution. The lichen genus Lecidea Ach. is one of the most heterogeneous of Zahlbruckner's artificial system and a large number of genera have been separated from it, especially during the last 30 years. Most systematic studies on Lecidea have been concerned with saxicolous species groups, but a number of recent studies deal with the non-saxicolous species groups as well. One of these groups was treated by Tensberg (1990). In his paper, he assigned J. tornoensis (Nyl.) Tensberg and J. carrollii (Coppins & P. James) Tensberg, which were formerly kept under Lecidea, and a new species, J. subaurifera Muhr & Tensberg to the newly described genus Japewia. The three species of this new genus were separated from the rest of Lecidea s. lat. predominantly on the basis of their thick-walled ascospores. In the original description, Tensberg (1990) also emphasized the following characters: asci with a conspicuous axial body, branched and anastomosing paraphyses and excipular hyphae, and the presence of a reddish brown epihymenium formed by pigmented caps surrounding the paraphysis tips. Although a young and still small genus, Japewia is already riddled with systematic and nomenclatural problems. The systematic position of Lecidea tornoensis Nyl. had been subject to discussion for years. Generic affinities with Mycoblastus (Anderson & Carmer 1974) or Miltidea (James in Poelt & Vtzda 1977) were proposed. When describing L. carrollii Coppins & P. James, they (Coppins & James 1979) discussed similarities between this species and L. tornoensis. Hafellner (1984) proposed to unite these two species in a new genus; T~nsberg (1990) finally did this by describing Japewia. Recently, doubts have been expressed whether J. carrollii was really congeneric with J. tornoensis (Kalb & Hafellner 1992). For nomenclatural reasons, Printzen (1997) proposed to conserve Lecidea carrollii against L. tavaresiana H. Magn. Earlier, L. djagensis Zahlbr. from China proved to be related to J. carrollii (Printzen 1995). This and the finding of another undescribed species of this group from Mexico showed that J. tornoensis and J. subaurifera on one hand and J. carrollii and allied species on the other hand differ in distribution. While the first two are boreal-(sub)arctic, more or less circumpolar species, the latter group occurs in humid temperate and subtropical regions. As the generic position of the new species had to be clarified, a closer investigation of Japewia and similar species was necessary and the results are presented below. MATERIAL AND METHODS The study is based on material deposited in ASU, BG, BM, GZU, LISU, M, MIN, SANT, UPS, and the private herbaria of A. Aptroot (Baarn, The Netherlands), J. Etayo (Pamplona, Spain), J. Hafellner (Graz, Austria), K. Kalb (Neumarkt, Germany), and C. Printzen (Koln, Germany). Species were hand-sectioned and mounted in water, Lugol's solution or lactophenol cotton blue. Spore measurements were made on sections mounted in water; measurements of ascomatal structures follow Printzen (1995). Spore measurements are given as (smallest single measurement-) smallest mean-largest mean (-largest single measurement). Spot test reactions were carried out on hand sections of thalli and apothecia under the microscope. Lichen substances were identified by HPTLC (Arup et al. 1993) and HPLC (Feige et al. 1993). Specimens of Japewia tornoinsis examined.-SWEDEN. TORNE LAPPMARK. Abisko, Tal von Ballinviggi, 1991, Printzen 2226 (hb. Printzen); Abisko, Opferplatz Paddustievva SE von Abisko, 1991, Printzen 2314 (hb. Printzen). LULE LAPPMARK. Jokkmokk, Aktse, ramulos Abietis, 1871, Hellbom (M). Specimens of Japewia aff tornoinsis examined.CANADA. BRITISH COLUMBIA. Vancouver Island, Victoria Watershed, 1994, Aptroot 35092, 35121 (hb. Aptroot). U.S.A. CALIFORNIA. Del Norte Co., Patrick Creek Campground, US Hwy 199, 1936, Harris 3570 (MIN). WASHINGTON. Cowlitz Co., 8 km SW of summit of Mount St. Helens, E of Goat Mt., N of Goat Marsh Lake, 1994, Tynsberg 20060a (BG). 0007-2745/99/714-719$0.75/0 This content downloaded from 157.55.39.255 on Mon, 23 May 2016 06:04:39 UTC All use subject to http://about.jstor.org/terms 1999] PRINTZEN: JAPEWIELLA 715

Infection and morphogenesis of Pythium marinum in species of Porphyra and other red algae

A strain of Pythium marinum (Peronosporales: Pythiaceae) from Puget Sound, Washington, was isolated from lesions of Porphyra nereocystis. The fungus grew on a modified Vishniac medium, from temperatures of 4 to 25 °C, although growth was slow at the lowest temperature. Sexual and asexual reproduction also occurred within this temperature range. Mycelium diluted in seawater initiated zoospore release within 16 h and continued to release zoospores for over 200 h at temperatures from 4 to 20 °C. Zoospore encystment on several species of marine red, brown, and green algae was readily monitored following staining with lactophenol – cotton blue. Pythium marinum zoospore encystment occurred on rhodophyceaen species, including Porphyra (gametophytes), Gigartina exasperata (tetrasporophyte), Mastocarpus papillatus (gametophyte), Prionitis lanceolata (nonfertile), and Iridaea heterocarpa (gametophyte and tetrasporophyte), but not on Nereocystis leutkeana or Ulva lactuca. Over 50% of zoospores held in half-strength seawater at 4 and 20 °C encysted within 24 h, whereas those held at 12 °C reached 50% encystment only after 32 h. For 4-mm diameter discs of Porphyra nereocystis and Porphyra perforata (formerly Porphyra sanjuanensis) blades, there was only a transient relationship between cell damage and number of encysted zoospores. Zoospores did not attach to the conchocelis phase of two species of Porphyra. Sequential extraction of carbohydrates from the blades of Porphyra perforata implicated separate chemical signals for zoospore encystment and appressorium formation prior to the initiation of blade invasion. Addition of diverse monosaccharides and polysaccharides to zoospore suspensions suggested that these chemical signals are specific, with the attachment–encystment signal chemically related to polysaccharides consisting of sulfated or nonsulfated galactose and 3,6-anhydrogalactose found in commercial agars and carrageenans. There was no consistent relationship between zoospore encystment and the amount of 3,6-anhydrogalactose present in the blade phase of several species of red algae. Key words: Pythium, Porphyra, zoospore, encystment, sulfated galactans, 3,6-anhydrogalactose.

Four New Species of Discomycetes (Pezizales) from West Greenland

A stay in 1970-73 at the Arctic Station in Qegerttarssuaq (Godhavn), West Greenland made it possible for the Danish ecologist P. Milan Petersen to make more than 100 collections of operculate Discomycetes along the west coast of Greenland. The main purpose of this presentation is to describe four new species found in this material, viz. Lamprospora leptodictya, Scutellinia megalosphaera, Trichophaea arctica, and Urnula groenlandica. All descriptions are based on dried material. Before microscopic examination the material was placed overnight in tapwater. The revived material was cut on a freezing microtome, in general in 15 Asm thick sections, then stained in cotton blue. Some edaphic factors for Urnula groenlandica, viz. pH, conductivity, and loss of ignition were investigated in a preliminary way. A full survey of Discomycetes from 29 collecting sites distributed all over Greenland will be given elsewhere (Dissing, 1980). The material constitutes in total 30 genera and about 70 species.

Polychromatic staining with toluidine blue O for studying the host-parasite relationships in wheat leaves of Erysiphe graminis f. sp. tritici

For studying host-parasite relationships of Erysiphe graminis f. sp. tritici in wheat leaves, staining with toluidine blue O proved superior to that obtained with the commonly used stain cotton blue. Toluidineblue O gives polychromatic staining and all the fungal structures (such as conidia, germ tubes, haustoria and hyphae) as well as cells and halo responses of the host are stained consistently and are clearly identifiable. This stain may also prove useful in studying other host-parasite systems.

Some Experiences with the Use of Cresyl Blue in the Determination of Polypores

Summary. Thick walled hyphae of polypores react in cresyl blue with a wide variety of colours, not only according to the species involved, but also according to the provenance of the dye used. In most cases, the resulting colours are constant for a particular species although there are slight individual variations. In some of the genera investigated all or nearly all species reacted similarly, whilst in others rather wide differences in the manner of reaction occurred. These differences, taken with other characters, may indicate that taxonomical relationships inside a particular genus (e.g. Amyloporia, Piptoporus) are not as close as at present considered. Metachromatic reaction with cresyl blue which was first applied in agarics (Kiihner, 1934; Singer, 1962), has been used only rarely in polypores. Teston (1953) cites the hyphae of some species of Leptoporus as being metachromatic. This reaction is emphasized as a particular character of the hyphae in Leptotrimitus semipileatus (Pouzar, 1966a), of the cystidia in Climacocystis borealis (Kotlaba & Pouzar, 1958) and of hyphae and cystidia in two species of the genus Auriporia (David et al., 1974). The possibility has existed that reactions with cresyl blue might be of considerable help in the study of polypores, like those with Melzer's reagent or cotton blue. However, one must keep in mind that the techniques of chemical colour reactions have been developed to supplement the small number of natural morphological characters and, if used in taxonomy, must be evaluated with caution and only in connexion with other characters. Although many species have not yet been tested, some interesting results have been obtained which justify this preliminary account.

A method for staining infection hyphae in pine leaves.

Fungus-inoculated Pinus radiata leaves were fixed and then stained with periodic acid-Schiff reagent. Pieces of leaf with fungal material on the surface were removed. These pieces were stained in lactophenol cotton blue for a few minutes and then mounted in dilute lactophenol cotton blue. Microscopic examination of fungal material inside and outside the mounted leaf pieces revealed the following: condidia and germ tubes on the leaf surface were red, appressoria remained unstained, and infection hyphae within the leaf were stained blue. This differential staining method was particularly useful for distinguishing germ tubes from infection hyphae arising from appressoria.

Composition analysis of "halo" area of barley leaf epidermis induced by powdery mildew infection.

The halo area on the barley leaf epidermis induced by powdery mildew infection was examined using a cotton blue stain and an electron probe microanalyzer. The halo consisted of the faintly stained zones and the densely stained zones which alternately encircled the penetration point, when stained with cotton blue. With the scanning electron microscope, two slightly raised zones, the central and the outer ring zones, were clearly distinguished in the halo. They corresponded to the zones stained faintly with cotton blue. The composition analysis by the X-ray microanalyzer indicated that calcium accumulated around the penetration point and manganese and silicon in the central and the outer ring zones.

Zygopolaris, A New Genus of Trichomycetes Producing Zygospores with Polar Attachment

SUMMARY A new monotypic genus of the fungal order Harpellales (Genistellaceae) has been found in three localities in the United States inhabitating the guts of mayfly nymphs, Ephemerella and Baetis. Mature sporulating thalli of Zygopolaris ephemeridarum may be found protruding from the the anus of the host into the aquatic environment. Released trichospores do not appear to have basal appendages, as do most other Harpellales, but evidence of rudimentary appendage formation has been found in the generative cells. Zygospores are attached at one pole, and resemble those of Carouxella (Harpellaceae). The similarities of the two genera and two families are discussed, as well as the four types of zygospores recognized in the Harpellales. Zygopolaris, a new genus of Trichomycetes growing in the hindgut of mayfly nymphs in flowing streams, has been found in several different areas in western United States, thus indicating that it may be widespread. The genus is monotypic, like the majority of Harpellales genera (Manier and Lichtwardt, 1968; Lichtwardt, 1972). Its distinctive characteristics are attachment of zygospores at one pole, the lack of welldefined trichospore appendages, and projection of thalli from the host anus. All attempts to culture the species, Zygopolaris ephemeridarum, were unsuccessful, and all observations were made from material dissected from the living hosts. The majority of measurements and photographs were taken from distilled water mounts prior to their infiltration with lactophenol containing cotton blue.

Synonymy of Sphacelotheca ischaemicola and Syndrome on Ischaemum Species

SUMMARY Teliospore morphology, hosts and symptomatology suggest that Sphacelotheca ischaemicola is a synonym of S. tanglinensis, but possibly differing in infectivity. Two syndromes were observed affecting inflorescences of Ischaemum indicum and I. timorense, a severe and a mild one, the former being more prevalent. Hyphae in host tissues and the systemic nature of the disease are shown. A prevalent inflorescence smut of Ischaemum indicum (Houtt.) Merr. and I. timorense Kunth. occurs throughout Singapore and Malaya. The causal fungus was identified and recorded as Sphaecelotheca ischaemicola Ling sp. nov. on I. timorense (Ling, 1949a) and also on I. indicum (Thompson and Lim, 1965). Ling (1949a) reported S. ischaemicola to be an intensified form of S. tanglinensis (Tracy & Earle) Zundel, a species recorded earlier on I. ciliare Retz. (I. indicum) from the Tanglin locality of Singapore (Tracy and Earle, 1895, Ling, 1949b). Apart from these records and some references (Thompson and Johnston, 1953; Johnston, 1960), no detailed investigation has been made on this interesting smut. A study of the fungus and the disease is reported here. MATERIALS AND METHODS Diseased plants were collected from various parts of Singapore and Malaya. The infected inflorescences and teliospores were examined and hand sections (15-20,/ thick) of diseased tissues made and stained in lactophenol cotton blue. The type specimen of S. tanglinensis was loaned from New York to compare with the fungus collected during the investigation.