Dark Brown Colonies Research Articles

First report of melting out on creeping bentgrass caused by Bipolaris sorokiniana in China.

Creeping bentgrass (Agrostis stolonifera L.), is one of the major cool-season turfgrass species, which is widely planted in putting greens at golf courses in China (Zhou et al. 2022). In June 2022, an unknown disease with reddish-brown spots (2-5 cm in diameter) were observed on 'A4' creeping bentgrass putting greens at Longxi golf course in Bejing. As the disease progressed, the spots coalesced and formed irregular patches (15-30 cm in diameter). When looking closely, the leaves were wilting, yellowing and melting out from the foliar tips to the crowns. The disease incidence was estimated up to 10-20 % of each putting green and a total of five putting greens had similar symptoms as described above. Three to five symptomatic samples were collected from each green. Diseased leaves were cut into pieces, surface sterilized in 0.6% sodium hypochlorite (NaClO) for 1 min, washed three times with sterilized water, air dried and placed on potato dextrose agar (PDA) containing 50 mg L-1 streptomycin sulfate and tetracycline. Plates were incubated in the dark at 25 °C for 3 days, and fungal isolates with similar morphology (irregular cultures with dark-brown back and light-brown to white surface) was consistently recovered. Pure cultures were obtained by repeat hyphal-tip transfer. The fungus did not grow well on PDA medium, radial growth was estimated 1.5 mm/d, and dark-brown colony was surrounded by light-white margin. However, it grew fast on creeping bentgrass leaf extract (CBLE) medium, CBLE medium was produced by adding 0.75 g potato powder, 5 g agar and 20 mL creeping bentgrass leaf juice (with 1 g fresh creeping bentgrass leaf) into 250 mL sterile water. The colony was sparse and light-white, and radial growth was roughly 9 mm/d on CBLE medium. Conidia were spindle-shaped, olive to brown in color, pointy or obtuse at both ends, 4 to 8 septa, and a size range of 9.85 to 20.20 × 26.26 to 45.64 μm (14.85 μm × 40.62 μm average, n = 30). Genomic DNA of two representative isolates (HH2 and HH3) was extracted, the nuclear ribosomal internal transcribed spacer (ITS) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) region were amplified with primers ITS1/ITS4 (White et al. 1990) and gpd1/gpd2 (Berbee et al. 1999), respectively. The ITS (OQ363182 and OQ363183) and GAPDH (OQ378336 and OQ378337) sequences were deposited in GenBank. BLAST analyses revealed that the sequences shared 100% and 99% similarity with the published ITS (CP102792) and GAPDH (CP102794) of B. sorokiniana strain LK93, respectively. To complete Koch's postulates, 3 plastic pots (15 cm height × 10 cm top diameter × 5 cm bottom diameter, three replicates for the isolate HH2) were seeded with creeping bentgrass and inoculated with a spore suspension (1×105 conidia/mL) after two months of plant growths. Healthy creeping bentgrass inoculated with distilled water served as controls. All pots were covered with plastic bags, placed in a growth chamber with a 12-h day/night cycle at 30/25°C and 90% relative humidity. Disease symptoms (yellowing and melting out leaf) were noted after 7 days. B. sorokiniana was recovered from the diseased leaves and identified morphologically and molecularly as described above. To our knowledge, this is the first report of melting out on creeping bentgrass caused by B. sorokiniana in China. The report here will provide a scientific basis for developing management strategies on this disease in the future. Additional study is needed to investigate the prevalence of the disease on putting greens from golf courses in larger regions of China.

Three new species of the sea fan genus Muricea (Cnidaria, Octocorallia, Plexauridae) from the northwest region of Mexico.

Twenty-one nominal species of Muricea have been reported in the Eastern Pacific with nine of them reported in the Mexican Pacific. We describe three new species of Muricea: Muriceaambaraesp. nov. and Muriceacacaosp. nov., from rocky reefs on the central and the northern Gulf of California and the Pacific coast of Baja California Sur, and Muriceamolinaisp. nov., from the Pacific coast of Baja California Sur. Muriceaambaraesp. nov. and M.cacaosp. nov. are taxonomically allied to the nominal species Muriceafruticosa Verrill, 1869 due to the morphological similarity of colony growth patterns and the phylogenetic closeness based on the mitochondrial MutS gene (mtMutS); but differ mainly in the calyx form and composition of sclerites. The main morphological differences between the new Muricea species are in their sclerite forms and color; M.ambaraesp. nov. has orange-colored colonies, thin leaf spindles and tuberculated blunt spindles, while M.cacaosp. nov. has dark brown colored colonies, strong spinous spindles and an absence of tuberculated blunt spindles. Muriceamolinaisp. nov. is phylogenetically close and morphologically similar to Muriceasquarrosa Verrill, 1869 in the growth form of the colony and tubular calyces; but has dark brown colored colonies and has calyces from the base to the branch tips. With these three new species, the total number of Muricea species reported in the Mexican northwest region increases to twelve and a total of 24 nominal species in the Eastern Pacific.

EVALUATION OF CULTURE MEDIA FOR THE GROWTH OF ALTERNARIA BRASSICICOLA

Alternaria leaf spot is one of the devastating diseases of cabbage caused by Alternaria brassicola. The experiment was conducted to understand the most effective culture media for the growth and multiplication of disease pathogens. Eight different culture media and four replications of each were employed under aseptic conditions for 12 days. Potato Dextrose Agar showed the highest radial growth (88.90mm) with a brownish to greenish colony color followed by Malt Extract Agar (87.45mm) with a whitish irregular margin of the dark brown colony. Sabouraud Dextrose Agar and Corn Meal Agar showed regular margins in their colony whereas Oat Meal Agar and Potato sucrose Agar showed irregular ones. Carrot Agar showed the lowest radial growth (53.12mm) with dark brown colony color at the center and dull reddish brown towards the margin which was followed by Nutrient Agar (59.62mm). This study will be a guide for further research on the physiology, taxonomy, and management of disease pathogens.

Potential of Local Isolates of Entomopathogenic Fungi Metarhizium sp. and Beauveria sp. on Spodoptera frugiperda J.E. Smith (Lepidoptera: Noctuidae) Larvae

Spodoptera frugiperda is an important pest which is an invasive insect that attacks maize plants. One alternative control that can be used is entomopathogenic fungi. The aim of this study was to analyze the potency of local isolates and flour formulations of the entomopathogenic fungi Metarhizium sp. and Beauveria sp. against S. frugiperda larvae in maize. This study used randomized block design (RBD) which consisted of five treatments (control, local isolates of the entomopathogenic fungi Metarhizium sp. and Beauveria sp., and flour formulations of the entomopathogenic fungi Metarhizium sp. and Beauveria sp. that comes from a pesticide shop) which was held from September 2021 until January 2022 in screen house and Laboratory of Plant Pest and Disease, Faculty of Agriculture, University of Sumatera Utara at an altutide of ±25 mdpl. Each treatment used a spores density of 108 spores/ml/ FAW’s larvae with five replications. The results showed that the highest larvae mortality (96,00%) was found on local isolate of Beauveria sp. The symptoms of infection with Beauveria sp. were the body of the larvae of S. frugiperda overgrown with white fungal colonies and infection with Metarhizium sp. were the body of the larvae of S. frugiperda overgrown with green or dark brown fungal colonies. The shortest lethal time (LT50) (4,75 days) was found on local isolates of Beauveria sp and the longest one (5,65 days) was found on flour formulations of Metarhizium sp. The highest attack intensity (56,40%) was found on flour formulations of Metarhizium sp. while the lowest one (28,40%) was found on local isolate of Beauveria sp.

Preparation and Analysis of Faba Bean pods Agar with identification of C. neoformans

Background: Recently researchers used agar that prepared from natural products and can be used in biological labs to grow bacteria and fungi such as C. neoformans. Objective: This study aimed to prepare a cultural media agar based on extract from pods of fava bean and use it to cultured and identify C. neoformans and compare it with negative control (C. albicans). Materials and Methods:C. neoformans and C. albicans isolates were obtained from laboratory of Biology Department, College of Science, University of Mustansyria, and then the pods were grinded, dissolved in sterile distilled water, boiled for 10 min. The extract was then filtered and sterilized in autoclave, then cooled and poured into sterile Petri dishes. C. albicans and C. neoformans were incubated for 48 hours. Also, and after treating the extract with methanol, and the HPLC was taken to investigate its components. Results: C. neoformans showed dark brown colonies, while C. albicans showed white colonies, and the HPLC showed that the fava bean pods content acids such as Ascorbic, Gallic, Caffeic, and Methyl gallat. Conclusions: Fava bean pods agar used to identify C. neoformans based on producing phenoloxidase enzyme which oxidizes phenolic compounds in the medium.

Isolation, molecular identification, and phylogenetic evaluation of Cryptococcus neoformans isolated from pigeon lofts, Psittaciformes, and Passeriformes in Ahvaz, Iran

Cryptococcus neoformans, the main pathogen in immunocompromised patients, is a ubiquitous free-living fungus that can be isolated from avian excreta, soils, and plant material. This study was carried out to determine the infection rate of pigeon lofts, Passeriformes, and Psittaciformes in Ahvaz, the capital of Khuzestan province in Iran and to determine varieties of Cryptococcus neoformans (C. neoformans). The 80 samples were collected from pigeon lofts. Also, 163 feces of captive birds (Passeriformes and Psittaciformes) which kept in Ahvaz pet shops, and the 70 cloacal swabs of pet birds (Passeriformes and psittaciformes) referring to the department of avian medicine (the faculty of veterinary medicine of Shahid Chamran University of Ahvaz) were analyzed. The samples were directly inoculated on niger seed agar (NSA) and also enriched in brain heart infusion broth and then inoculated on NSA. Dark brown colonies suspected to C. neoformans subcultured on saborouds dextrose agar and pure cultures subjected to molecular (polymerase chain reaction (PCR)) diagnosis. For detection of C. neoformans, primer sets that targeting the CNLAC1 gene were selected and nested PCR was conducted. For identification of C. neoformans varieties, a primer set targeting the STR1 gene was selected. For more accurate confirmation, the purified PCR products of some isolates were also sequenced, and based on the gene sequences, all of the isolates belonged to C. neoformans variety grubii (var. grubii)(serotype A). Totally 16 out of 80 pigeon samples (20%) were contaminated with C. neoformans. The results in pigeons disclosed a 98.64% identity when compared with other strains of C. neoformans (CN1525, T4, and T1) which were previously deposited in GenBank from Italy and Thailand. Also, 21 out of 233 samples from Psittaciformes (9.01%) were contaminated with C. neoformans. The results in Psittaciformes disclosed a 99.7% identity when compared with other strains of C. neoformans (TIMM1313, IFM5882, CN1525, etc.) which were previously deposited in GenBank from Japan and Italy, etc. In the present study, the samples belonging to the passerine order were free of C. neoformans infection. According to the results, C. neoformans is prevalent in pigeon flocks and pet birds including Psittaciformes in the Ahvaz area, and should be considered by pigeon and captive bird breeders, veterinarians, and public health organizations in Ahvaz. The cryptococcus species isolated from captive birds and pigeons could be potential pathogens in humans.

First report of Rhizoctonia solani Kühn AG 2-2 LP in Zoyzia japonica Steud in Brazil

ABSTRACT The use of cultivated grasses in Brazil has grown by 40% between 2010 and 2015, and the species Zoysia japonica Steud, especially the cultivar ‘Esmeralda’, corresponds to 81% of the grass market in the country. The most important disease affecting zoysia grass, known as large patch, is caused by Rhizoctonia solani and occurs in the Brazilian lawns particularly during winter months. The aim of this study was to contribute to the identification and characterization of the anastomosis group of R. solani isolates from lesions typical of large patch collected from ‘Esmeralda’ grass at gardens and golf courses in the states of São Paulo and Bahia, Brazil. All 12 obtained isolates presented dark-brown colonies with aerial mycelial growth, multinucleated hyphae and absence of concentric zonation or sclerotia, and showed their greatest mycelial growth rate at 25°C. In pathogenicity experiments, except three out of R. solani isolates, reduced the growth of zoysia grass. Based on the analysis of sequences of the rDNA-ITS region, the isolates clustered with reference isolates of the anastomosis group AG 2-2 LP. Phylogenetic inference showed that the Brazilian isolates are grouped into two clades that shared the same common ancestral with 96% bootstrap. One of the clades includes only Brazilian isolates while the other one also includes American and Japanese R. solani isolates AG 2-2 LP. This is the first report and characterization of R. solani AG 2-2 LP in zoysiagrass in Brazil.

First Report of Soft Rot Caused by Aspergillus niger sensu lato on Mother-in-law's Tongue in China.

"Mother-in-law's tongue" (MLT) [Dracaena trifasciata (Prain) Mabb. (syn. Sansevieria trifasciata Prain.)], also known as "Saint George's sword", "snake plant", "tiger's tail orchid", etc., is an evergreen perennial ornamental plant grown worldwide. In September 2016, severe soft rot occurred on the leaves of MLT in a flower market in Nanyang city (32º56´N, 112º32´E), Henan province, China with 25% disease incidence (n=100). Water-soaked spots initially appeared on the leaf margin, enlarged rapidly, and became soft rot under excessively watered conditions. A blight zone was visualized at the margin of a developing lesion in backlit conditions. Severely affected leaves folded down from the lesions. Lesion expansion stopped under dry conditions. Grey or dark brown mycelia were frequently seen on the lesions. Tissue pieces (4×4 mm2) at the margin of lesions were cut out, treated with 75% ethanol for 10 s, followed by 70 s in 0.1% HgCl2, rinsed eight times with sterile water, and plated on potato dextrose agar (PDA) medium. Pure Aspergillus cultures were obtained from the surface-disinfected lesions after 4 days of incubation at 26°C. Two single-spore-derived isolates (An-1 and An-2) were randomly selected and used for morphological and molecular identifications as well as pathogenicity tests. The isolates formed round dark brown colonies with a large number of conidia after 5 days of incubation on PDA at 28°C. Conidia were subsphaeroidal or oblate, unicellular, dark brown, 2.9-4.2(3.5) × 1.9-3.4(2.7) μm in size (n=100), developed from a two-series of strigmata born on a conidial head, with ridge or stab-shaped prominences. For pathogenicity tests, the two isolates were separately grown on oatmeal agar and incubated at 30°C for 6 days. Mycelial plugs (5 mm diam.) were inoculated on the scalpel incision X-shaped wounds of surface-disinfected leaves of MLT. The inoculated leaves were kept on a two-layer of wet napkin in a steel basin covered with a plastic film. Soft rot symptoms developed from the wounds 6 days after incubation, similar to those observed on naturally affected leaves. The An-1- and An-2-inoculated unwounded leaves remained symptomless during the pathogenicity tests. Fungal cultures with the same phenotypes as the inocula were consistently reisolated from the lesions of the leaves inoculated by each of the two isolates, verifying the isolates as the causal agent of the disease based on Koch's postulates. Both β-tubulin gene and rDNA-ITS (internal transcribed spacer) sequences of the two isolates were separately amplified and sequenced. Sequences were submitted to GenBank with accession numbers MN259522 and MN259523 for the β-tubulin gene sequences, and accession numbers MN227322 and MN227324 for the rDNA-ITS sequences of An-1 and An-2, respectively. Both An-1 and An-2 were clustered with members of Aspergillus niger van Tieghem in the phylogenetic tree of rDNA-ITS, clearly separated from other Aspergillus spp. In the phylogenetic tree of β-tublin gene, both An-1 and An-2 formed a subclade inside a large clade consisting of members of A. niger in strict sense. Based on the molecular and morphological results, both An-1 and An-2 clearly separated from other Aspergillus spp. and can be considered as A. niger sensu lato. Foliar diseases of MLT are known to be caused by a few fungal species such as Chaetomella spp. (Li et al. 2014) and Colletotrichum sansevieriae (Nakamura et al. 2006). This is the first report of A. niger sensu lato causing soft rot on MLT in China.

The utility of banana peel extract agar in the presumptive identification of Cryptococcus neoformans

We prepared a newer growth medium, banana peel extract agar (BPEA) containing the extracts of chopped banana peels for the selective cultivation of Cryptococcus neoformans. Over the medium, the growth resulted in the development of light to the dark brown coloured colonies indicating the chromogenic potential of the BPEA. The organism grown over BPEA was subsequently confirmed as C. neoformans by phenotypic as well as by molecular method. This medium, being cost-effective, may be used in resource-poor settings of the developing or underdeveloped countries for selective isolation of C. neoformans.

Septonema lohmanii G. Delgado & O. Koukol, sp. nov., a New Species in Mytilinidiales (Dothideomycetes) and the Phylogenetic Position of S. fasciculare (Corda) S. Hughes

During independent surveys of microfungi associated with Pinus spp. in the United States and the Czech Republic, a distinct fungus matching the generic concept of Septonema Corda was collected. It is characterized by distinctly ornamented conidiophores, branches, conidia and hyphae, ranging from verruculose to strongly verrucose with prominent rounded warts, yellowish brown to brown or reddish brown in color and forming densely floccose, dark brown or dark reddish brown colonies on pine wood and bark. Conidia are cylindrical or subcylindrical and produced in short, simple or branched acropetal chains. Multigene phylogenetic analyses including nuclear ribosomal (LSU) and protein coding gene (EF1-α) sequence data suggest that both collections are conspecific and belong to the order Mytilinidiales (Dothideomycetes, Ascomycota) where they group distant from other mytilinidiaceous fungi with known septonema-like anamorphs. To provide a proper name based on phylogenetic placement and to possibly circumscribe Septonema sensu stricto, a non-sporulating, putative strain belonging to S. secedens Corda, the generic type, was included in the analyses. DNA sequence data placed this strain within the family Venturiaceae (Venturiales, Dothideomycetes) but morphological examination of the corresponding herbarium specimen revealed that it belongs instead to S. fasciculare (Corda) S. Hughes. Because of the polyphyletic nature of the genus and the unknown phylogenetic position of its type species, our fungus is accommodated in Septonema as a new species named S. lohmanii G. Delgado & Koukol, sp. nov.

A crustoid graptolite lithoimmured inside a Middle Ordovician nautiloid conch from northern Estonia

A light grey nautiloid conch has a dark brown colony attached to its internal surface. This colonial fossil resembles hederellids and bryozoans, but is in fact a crustoid graptolite ( Hormograptus ? sp.). The colony has been lithoimmured inside this nautiloid conch by early cementation. Crustoid graptolites were a part of the encrusting communities in the Middle Ordovician of Baltica, but their abundance among encrusters of biogenic substrates reached a peak in the middle Sandbian. The cryptic mode of life appeared very early in the evolution of the crustoids. The discovery of this crustoid graptolite in a nautiloid conch indicates that the Baltic Middle Ordovician cryptic communities were taxonomically more diverse than was known previously. The nautiloid conch studied is sparsely encrusted with an encrustation density that is similar to those of other Middle Ordovician cryptic surfaces described from Estonia.

Anew Medium for Isolation and Diagnosis of Cryptococcus neoformans

Melanin produce by Cryptococcus neoformans yeastwas used to characterize this yeast.In present study,we used red onion extract in prepared a new medium.The isolates of yeast were cultivation. After 48 hours results show,the isolates of C.neoformans were appear in dark brown colonies while the colonies on SDA not produce any pigment.Also analysis resultsof red onion extract by usedUV-Spectometerand FT-IR appear this extract content phenolic compounds which causes to produce brown pigment.thus, this medium Red Onion Agar (RO A) useful for rapid identification of yeast C.neoformans

FIRST REPORT OF CURVULARIA LUNATA CAUSING LEAF SPOTS ON LOQUAT FROM PAKISTAN

The loquat (Eriobotrya japonica Lindl.) is achieving significant value in Pakistan. During the year 2014-2015, reddish brown circular spots were observed on leaves; these spots increased in size and coalesced to form oblong lesions. Samples were cut into small pieces (3 mm2), surface sterilized with sodium hypochlorite and transferred on Malt Extract Agar (MEA). These samples were incubated at 24±2°C and dark brown colonies with large stroma were observed. Septate condiophores were unbranched or simple and geniculate with dark brown scar. Brown conidia were straight to pyriform with 3-4 cells (large and curved center cell), ranging in size from 6.9 to 20.99 μm and produced apically in a sympodial manner. Morphological characterization was similar to Curvularia lunata (Ellis, 1971). PCR was used for amplification of rDNA region (White et al., 1990) and sequences of PAK34 and PAK35 was submitted to GenBank under accession Nos. KT280007 and KT280008. Both isolates were exhibiting 99% genetic homology with previously reported C. lunata isolates (KM246260, KM246262). Spore suspension (1 × 106 spore/ml) was sprayed on healthy loquat seedlings, kept in glasshouse at 30°C and plants developed lesions similar to those observed on naturally infected plants. Isolation was performed from inoculated loquat plants and morphological characterizations were similar to naturally isolated pathogens. Curvularia leaf spot diseases caused by C. lunata have been previously found on small evergreen tree. To our knowledge, this is the first report worldwide of C. lunata causing leaf spots on loquat.

FIRST REPORT OF LEAF SPOT CAUSED BY BARTALINIA ROBILLARDOIDES ON PARTHENOCISSUS QUINQUEFOLIA IN CHINA

In late August 2013, leaf spot symptoms were first noticed on Virginia creeper [Parthenocissus quinquefolia (L.) Planch], an ornamental cum medicinal plant of vitaceae family, in the Forest Park, Luoyang City of Henan Province, China. Symptoms appeared as small circular brown dots on the leaves, gradually expanding into circular or irregularly-shaped yellow-brown to dark brown spots with raised margins and black dots in the center of the lesions. A fungus was carefully isolated from the leaf spot and cultured on potato dextrose agar, which produced dark brown colonies. Pycnidia were brown, ovoid, 13.3 μm in diameter. Conidiophores were cylindric and colorless, 8.1-10.l × 3.8-4.0 μm. Conidia (n = 50) were 18.1-20.8 × 2.5-4.2 μm, 2 to 4 septate, with apical cell hyaline, other cells pale brown. One eccentric pedicel, hyaline, 8-10.6 × 1.2 μm. Apical appendages 1 to 4, 13.3-16.0 × 1.2 μm, unbranched. Based on the isolate characteristics (Sutton, 1980; Barnett and Hunter, 1998), the fungus was subsequently confirmed as Bartalinia robillardoides by DNA sequence analysis of internal transcribed spacer, which was 100% identical to other known B. robillardoides isolates (GenBank Accession No. KF656706.1). To confirm pathogenicity of the fungus, ten surface disinfected healthy leaves of Virginia creeper were inoculated with a fresh conidial suspension (40 μl, 5 × 106 conidia/ml) of B. robillardoides at 26°C. Leaf necrosis symptoms appeared on the inoculated leaves within 7 days. The same fungus was successfully reisolated from the lesions, confirming Koch’s postulates, whereas, control plants showed no symptoms. To our knowledge, this is the first report of B. robillardoides infecting P. quinquefolia in China.

Molecular typing of environmental Cryptococcus neoformans/C. gattii species complex isolates from Manaus, Amazonas, Brazil.

Cryptococcus neoformans and Cryptococcus gattii are the main causative agents of cryptococcosis, a systemic fungal disease that affects internal organs and skin, and which is acquired by inhalation of spores or encapsulated yeasts. It is currently known that the C. neoformans/C. gattii species complex has a worldwide distribution, however, some molecular types seem to prevail in certain regions. Few environmental studies of Cryptococcus have been conducted in the Brazilian Amazon. This is the first ecological study of the pathogenic fungi C. neoformans/C. gattii species complex in the urban area of Manaus, Amazonas, Brazil. A total of 506 samples from pigeon droppings (n = 191), captive bird droppings (n = 60) and tree hollows (n = 255) were collected from June 2012 to January 2014 at schools and public buildings, squares, pet shops, households, the zoo and the bus station. Samples were plated on niger seed agar (NSA) medium supplemented with chloramphenicol and incubated at 25°C for 5 days. Dark-brown colonies were isolated and tested for thermotolerance at 37°C, cycloheximide resistance and growth on canavanine-glycine-bromothymol blue agar. Molecular typing was done by PCR-RFLP. Susceptibility to the antifungal drugs amphotericin B, fluconazole, itraconazole and ketoconazole was tested using Etest(®) strips. In total, 13 positive samples were obtained: one tree hollow (C. gattiiVGII), nine pigeon droppings (C. neoformansVNI) and three captive bird droppings (C. neoformansVNI). The environmental cryptococcal isolates found in this study were of the same molecular types as those responsible for infections in Manaus.

Environmental isolation of Cryptococcus gattii VGII from indoor dust from typical wooden houses in the deep Amazonas of the Rio Negro basin.

Cryptococcosis is a human fungal infection of significant mortality and morbidity, especially in the meningoencephalitis form. Cryptococcosis is distributed worldwide and its agents, C. neoformans and C. gattii, present eight major molecular types—VNI-VNIV and VGI-VGIV respectively. The primary cryptococcosis caused by molecular type VGII (serotype B, MAT alpha) prevails in immunocompetent patients in the North and Northeast of Brazil, revealing an endemic regional pattern to this molecular type. Since 1999, C. gattii VGII has been involved in an ongoing outbreak in Canada, and is expanding to the Northwest of the United States, two temperate regions. Exposure to propagules dispersed in the environment, related to various organic substrates, mainly decomposing wood in and around dwellings, initiates the infection process. The present study investigated the presence of the agents of cryptococcosis in dust from dwellings in the upper Rio Negro, municipality of Santa Isabel do Rio Negro in Amazonas state. Indoor dust was collected from 51 houses, diluted and plated on bird seed agar. Dark brown colonies were identified phenotypically, and genotypically by URA5 restriction fragment length polymorphism analysis and multilocus sequence typing (MLST). The mating type was identified using pheromone-specific primers. Three of the 51 houses were positive for C. gattii molecular type VGII, MATα and MATa, showing a high prevalence of this agent. MLST studies identified eight subtypes, VGIIb (ST7), VGIIa (ST20), (ST5) and 5 new subtypes unique to the region. For the first time in the state of Amazonas, C. gattii VGII MATα and MATa were isolated from the environment and correlates with endemic cryptococcosis in this state. This is the first description of MLST subtypes on environmental isolates in the Brazilian Amazon, indicating domiciliary dust as a potential source for human infection with different subtypes of C. gattii VGII MATα and MATa.

First Report of Black Leaf Mold of Tomato Caused by Pseudocercospora fuligena in Ohio.

Diseased tomato (Solanum lycopersicum L. cvs. Geronimo, Rebelski, and Big Dena) plants were received for diagnosis from a home gardener in Wayne County, Ohio, in August 2013 and from a 0.14-ha greenhouse in Brown County, Ohio, in September 2013. Approximately 10 and 60% of leaf area was diseased in the home garden and greenhouse, respectively. One or more lesions, each with an indistinct border, were observed on the leaves. Black fungal growth was observed on both sides of the leaf in association with the lesions. Microscopic examination revealed Cercospora-like conidia (2). Three symptomatic leaves from each location were surface-sterilized with 0.5% NaClO for 1 min and cultured on V8 juice agar medium at room temperature under continuous fluorescent lighting. One isolate was selected from each of Wayne Co. (SAM33-13) and Brown Co. (SAM34-13). The fungus produced small, dark-brown colonies within 2 weeks of plating. Mycelium was olive brown and septate, producing fascicles of conidiophores. Conidia were cylindrical, 2 to 14 septate, and 25.8 to 109.7 × 6.5 μm. Genomic DNA was extracted from colonies of isolate SAM33-13 grown on V8 juice agar medium using the Wizard SV Genomic DNA Purification System (Promega, Madison, WI). The internal transcribed spacer (ITS) region of rDNA was amplified by PCR using primer pair ITS1 and ITS4 (5), and the purified amplicon was sequenced (OARDC Molecular and Cellular Imaging Center, Wooster, OH). The ITS sequence was 99% identical to those of GenBank accessions of Pseudocercospora fuligena from Korea (JX290079) and Thailand (GU214675). The sequence was deposited in GenBank (KF931141). Based on morphology (4) and sequence analysis, the fungus was identified as P. fuligena (Roldan) Deighton (basionym Cercospora fuligena). To satisfy Koch's postulates, three 4-week-old tomato plants each of the cultivars L390 (AVRDC, Taiwan) and Mountain Spring (Siegers Seed Co., Holland, MI) were sprayed with a suspension of 1 × 103 conidia/ml of isolates SAM33-13 or SAM34-13 prepared from 3-week-old cultures growing on V8 juice agar medium. Three non-inoculated control plants were sprayed with sterilized water. Plants were maintained in a growth chamber at 25 to 30°C, 80% RH, and a 12 h/12 h day/night cycle. The first symptoms appeared 3 weeks after inoculation as light yellow foliar lesions. The lesions enlarged and turned black due to fungal growth, and the infected leaves dried. Disease severity was 70 and 10% of leaf area for cvs. L390 and Mountain Spring, respectively, for each isolate. Non-inoculated control plants were symptomless, and no fungus was re-isolated from the leaves. P. fuligena was isolated from symptomatic leaves of inoculated plants as described above, and the identity was confirmed based on morphology. In the United States, C. fuligena has not been reported infecting tomato since the first report in Florida in 1974 (1). To our knowledge, this is the first report of black leaf mold of tomato caused by P. fuligena in Ohio. Resistant cultivars, crop sanitation, and fungicides are recommended to manage the disease (3).

Primary Cutaneous mycosis in an Immunocompetent Parrot Keeper Due to Cryptococcus neoformans

The prime objective of this paper is to delineate the etiologic significance of Cryptococcus neoformans in primary cutaneous mycosis of a 34- year- old male immunocompetent person, who was occupationally exposed to the excreta of a caged parrot. The biopsy obtained from the cutaneous lesion of a parrot keeper was examined in Indian ink mount, and was cultured onto the plates of Pal’s sunflower seed medium. In addition, the urine and blood of patient was also inoculated on Pal’s sunflower seed medium to rule out the systemic infection. In order to establish the source of infection, samples of parrot droppings, and wooden box were examined on Pal’s sunflower seed medium. Microscopic examination of clinical specimen in India ink preparation revealed circular, wide, thickly encapsulated yeast cells of Cryptococcus neoformans . Numerous dark brown colored colonies of C. neoformans grew from biopsied tissue on Pal’s sunflower seed medium. The microscopic morphology of the fungal isolates was done in Narayan stain. There was no growth of C. neoformans from blood, and urine of the patient on Pal’s sunflower seed medium. The pathogen was easily recovered from the parrot droppings, and wooden box on Pal’s sunflower medium. These findings conclusively established that the patient acquired primary cutaneous cryptococcosis from his immediate environment as revealed by the presence of C. neoformans in the excreta, and wooden cage of the caged parrot.

Englerulaceae (Dothideomycetes)

The family Englerulaceae presently includes seven genera that produce brown to dark-brown colonies on living leaves. Ascomata are superficial and scattered on the colonies and lack ostioles. Asci are 2–8-spored, bitunicate, ovate to globose and ascospores are multi-seriate, oblong to ellipsoid, brown, and smooth-walled with a single septum. This paper brings all genera of the family together in one place with descriptions and illustrations and discusses their current taxonomic placement. The type species of Englerula, Parenglerula, Rhizotexis, Rhytidenglerula, Schiffnerula and a new species of Thrauste are redescribed and illustrated with light photomicrographs, in this paper. It is suggested that Butleria should be accommodated in Myriangiaceae. Rhizotexis is excluded from the family and moved to Dothideomycetes genera incertae sedis, as morphological characters are not similar to type genus and other genera in the family. The type specimen of Goosia is missing and is redrawn from the protologue. By illustrating and redescribing the genera we expect renewed interest for recollection and molecular study so that these dothideomycetous genera can be placed in a natural taxonomic framework.

First Report of Curvularia lunata Causing Leaf Spots on Sorghum bicolor from Pakistan.

In October 2012, reddish brown, oblong lesions with chlorotic centers were observed on the leaves of Sorghum bicolor in Punjab Province, Pakistan. Early symptoms appeared as reddish brown circular spots on the leaves. These spots increased in size and coalesced to form oblong lesions. Entire fields were severely affected by the disease. Pathogen isolations were made on malt extract agar (MEA) media. Symptomatic leaf samples were cut into 4 to 6 mm2 pieces, surface sterilized (10% bleach for 1 min, 90% ethanol for 30 sec) and rinsed in sterilized water several times, followed by air drying. These samples were plated onto 2% MEA media, supplemented with 10 mg/liter chloramphenicol, and incubated at 25°C for 6 days in the dark. A mitosporic fungus of dark brown colony, bearing large stroma, appeared on the media. Conidiophores were brown, septate, geniculate, simple or unbranched, with dark brown scar. Conidia were brown, straight to pyriform, with 3 to 4 cells, with large and curved central cells, smooth walled, ranging in size from 7.3 to 21.26 μm, and produced apically in a sympodial manner. Based on morphological characteristics, the pathogen was identified as Curvularia lunata (Wakk.) Boedijn. (1,2). Morphological identification was also confirmed by the First Fungal Culture Bank of Pakistan (FCBP), Institute of Agricultural Sciences, University of the Punjab, Lahore, Pakistan, and samples were submitted to FCBP (Accession No. 1201). The fungus was further identified by amplifying internal transcribed spacer region sequences (ITS1, rDNA, ITS2) by using ITS4 and ITS5 primers (4). The resulting 584-bp sequence was submitted to GenBank with Accession No. HG326308. This sequence showed 99% homology with C. lunata strain pingxiang (GenBank Accession No. JQ701897), causing leaf spots of lotus in China. Pathogenicity assay was conducted on 20-day-old seedlings of S. bicolor variety Indian Gold, grown from surface sterilized seeds. Fifteen replicate plants were sprayed with a spore suspension of 1 × 106 spore/ml in distilled sterilized water, prepared from 1-week-old fungal culture, grown in the dark on 2% MEA media. Five replicate plants were sprayed with distilled sterilized water as control. Plants were covered with transparent polyethylene bags to retain moisture and enhance disease development, and kept in a greenhouse at ~30°C. Bags were removed after 5 days of incubation. Inoculated plants developed lesions similar to those observed on naturally infected plants. No symptoms were observed on control plants. The pathogen was re-isolated from infected leaves, and the morphology features were again studied, matching those of the pathogen isolated from field samples. Curvularia leaf spot diseases, caused by different Curvularia species, have been previously found on many grass species worldwide (3). To our knowledge, this is the first report of C. lunata leaf spots on S. bicolor in Pakistan.