The yeast <Emphasis Type="Italic">Candida railenensis</Emphasis> in the fruits of English oak (<Emphasis Type="Italic">Quercus robur</Emphasis> L.)

The cotyledons of whole intact acorns were shown to contain yeasts; their number increased sharply before acorn germination. The yeasts in the cotyledons are mainly represented by one species, Candida railenensis, with the number in the germinating cotyledons reaching 10⁷ CFU/g. After germination or exocarp destruction, the cotyledons were colonized by the usual epiphytic and litter yeasts Cryptococcus albidus, Rhodotorula glutinis, and Cystofilobasidium capitatum.     

Seasonal Dynamics in a Yeast Population on Leaves of the Common Wood Sorrel Oxalis acetosella L.

Analysis of an epiphytic yeast population on the leaves of the evergreen common wood sorrel Oxalis acetosella L. throughout a year showed that the density and the species composition of this population underwent regular seasonal changes. There were almost no yeasts on the young spring leaves. However, the yeast population on the mature leaves tended to increase in the autumn, reaching a maximum after the formation of continuous snow cover. Then the yeast population on the leaves tended to decrease, reaching a minimum in the spring. The species diversity of the yeasts was maximum in the autumn. The population of the epiphytic yeast species Cystofilobasidium capitatum, Rhodotorula fujisanensis, Leucosporium scottii, and Cryptococcus flavus peaked in the autumn. On the other hand, the population of the widespread epiphytic species Cryptococcus laurentii on the wood sorrel leaves peaked in January. The relative abundance of the red-pigmented phytobionts Rhodotorula glutinis and Sporobolomyces roseus virtually did not change throughout the year. The relative abundance of the euribiotic species Cryptococcus albidus showed irregular monthly variations. The data obtained show that the epiphytic microbial population of various plants can be comprehensively studied only by analyzing this population throughout the vegetative period of the plants.     

Cytoskeletal structures,ultrastructural characteristics and the capsule of the basidiomycetous yeast <Emphasis Type="Italic">Cryptococcus laurentii</Emphasis>

The cytoskeleton, capsule and cell ultrastructure were studied during the cell cycle of Cryptococcus laurentii. In an encapsulated strain, cytoplasmic microtubules and a mitotic spindle were detected. Mitosis was preceded by migration of the nucleus into the bud. F-actin failed to be visualised by rhodamine-phalloidin (RhPh) in encapsulated cells and therefore an acapsular strain was used. The following actin structures were found: actin dots, actin cables and cytokinetic ring. Ultrastructural studies showed the presence of a nucleus in the bud before mitosis. A collar-shaped structure was seen at the base of bud emergence. A lamellar cell wall and a rough outer surface of the cells were detected. Cytoskeletal structures found in C. laurentii are similar to those in Cryptococcus neoformans, which is a serious human pathogen.     

Intra- and Interspecies Virus Transfer in Aspergilli via Protoplast Fusion

Intra- and interspecies transfer of dsRNA viruses between blackAspergilliandAspergillus nidulansstrains has been investigated using protoplast fusion. We found interspecies transfer of virus in all combinations of blackAspergillusandA. nidulansstrains and vice versa. Using the same conditions, intraspecies virus transfer among heterokaryon incompatible strains was also tested. Whereas such transfer was always found amongA. nidulansstrains, transfer among blackAspergilliwas frequently unsuccessful. The lack of virus transfer between blackAspergillusisolates was further investigated by using a mitochondrial oligomycin resistance marker as a positive control for cytoplasmic exchange. These experiments showed independent transfer of the oligomycin resistance and dsRNA viruses during protoplast fusion of heterokaryon incompatible blackAspergilli. The inefficient transfer of dsRNA viruses between blackAspergilliis not caused by absolute resistance to viruses but may be related to heterokaryon incompatibility reactions that operate intraspecifically. Consequences for the dynamics of mycoviruses in populations of blackAspergilliare discussed.     

Selection of Optimal Host Strain for Molecular Pathogenesis Studies on <Emphasis Type="Italic">Cryptococcus gattii</Emphasis>

Encapsulated yeast, Cryptococcus gattii (Cg) is a primary and emerging fungal pathogen in North America. It has a predilection for invading the central nervous system of both healthy and immunocompromised humans and animals. Recently, we initiated molecular pathogenesis studies in Cg strain NIH444 (ATCC 32609). In this report, we compared the biology and pathogenic potential of NIH444 to those of WM276, an Australian environmental isolate that is being used for the whole genome-sequencing project. Our data indicated that NIH444 is comparatively more virulent in a mouse model of cryptococcosis than is WM 276. We found robust mating of NIH444, and no mating of WM276, when tested against Cg MATa strain, NIH198. WM276 but not NIH444 was defective in filamentation and sporulation (haploid fruiting). Interestingly, NIH444 has a VGII/AFLP6 genotype similar to that of the genotype of the recent outbreak strains from Vancouver Island, British Columbia, Canada. Additionally, comparisons of nucleotide sequences of various genes also showed differences between NIH444 and WM276. Based on these observations, we conclude that NIH444 should remain the strain of choice for understanding Cg pathogenesis, especially on the North American continent.     

Cryptococcus haglerorum,sp. nov., an anamorphic basidiomycetous yeast isolated from nests of the leaf-cutting ant Atta sexdens

A yeast strain (CBS 8902) was isolated from the nest of a leaf-cutting ant and was shown to be related to Cryptococcus humicola. Sequencing of the D1/D2 region of the 26S ribosomal DNA and physiological characterization revealed a separate taxonomic position. A novel species named Cryptococcus haglerorum is proposed to accommodate strain CBS 8902 that assimilates n-hexadecane and several benzene compounds. Physiological characteristics distinguishing the novel species from some other members of the C. humicola complex are presented. The phylogenetic relationship of these strains to species of the genus Trichosporon Behrend is discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Cryptococcus silvicola nov. sp. from nature reserves of Russia and Portugal

Nitrate-positive strains of a filobasidiaceous anamorphic yeast related to Cryptococcus cylindricus were isolated from forest litter in a Russian nature reserve and from a lichen in Portuguese one. Mycocinotyping and rDNA sequence analysis revealed that the strains represent a novel species, for which the name Cryptococcus silvicola (type strain VKM Y-2939=CBS 10099) is proposed. An erratum to this article can be found at     

The killer phenomenon in Ustilago: Electron microscopy of the dsRNA encapsidated in individual virus particles

From earlier studies with the Ustilago maydis virus and other dsRNA viruses it is known that discrete dsRNA segments typical of each virus are obtained by extraction. A variation exists with respect to the encapsidation of these segments among different viruses. The encapsidation of the genome in individual particles of the Ustilago virus was examined by electron microscopy after disruption of virus particles. The study included the P6 wild-type and 2 mutants containing only part of the genome. The results indicate that most virus particles of the wild-type and the mutants contain up to 12–14×10⁶ daltons of dsRNA. Since the largest extracted molecule is 3.2×10⁶D these findings suggest that an individual particle may contain more than one segment of dsRNA. Free linear molecules that exceed in size the extracted segments were also found following the disruption of each of the 3 virus types examined. Thus, the viral genome seen segmented after extraction is organized as a concatamer in the capsid and each virus particle can contain an entire viral genome consisting of each type of the segments seen after extraction, a repeat of a single segment or a random assortment. In each case the information may be organized as a concatamer.     

dsRNA-induced gene silencing in Moniliophthora perniciosa, the causal agent of witches’ broom disease of cacao

The genome sequence of the hemibiotrophic fungus Moniliophthora perniciosa revealed genes possibly participating in the RNAi machinery. Therefore, studies were performed in order to investigate the efficiency of gene silencing by dsRNA. We showed that the reporter gfp gene stably introduced into the fungus genome can be silenced by transfection of in vitro synthesized gfpdsRNA. In addition, successful dsRNA-induced silencing of endogenous genes coding for hydrophobins and a peroxiredoxin were also achieved. All genes showed a silencing efficiency ranging from 18% to 98% when compared to controls even 28 d after dsRNA treatment, suggesting systemic silencing. Reduction of GFP fluorescence, peroxidase activity levels and survival responses to H₂O₂ were consistent with the reduction of GFP and peroxidase mRNA levels, respectively. dsRNA transformation of M. perniciosa is shown here to efficiently promote genetic knockdown and can thus be used to assess gene function in this pathogen.     

Isolation of Cryptococcus laurentii from Canada Goose guano in rural upstate New York

Cryptococcus neoformans and Cryptococcus gattii are etiologic agents of cryptococcal pneumonia and meningitis, potentially lethal syndromes associated with AIDS. A related species, Cryptococcus laurentii, has recently been implicated in several cases of human disease. Guano from Canada Goose (Branta canadensis), an organism that lives closely beside man and inhabits recreational space in rural and suburban areas, might be a significant environmental reservoir of Cryptococcus organisms in non-urban areas. Cryptococcal organisms were isolated from Canada Goose guano from a site in rural northern New York, with identification based upon colony and microscopic morphology, ability to metabolize l-Dopa to melanin, and positive reaction with a commercial anti-cryptococcal capsular polysaccharide latex bead agglutination test. DNA sequences from five positive isolates were identical to each other, and identical to the ITS1-5.8S-ITS2 sequences of C. laurentii strain CBS7140 (Accession AY315665) across a 511 bp sequence. All five isolates of C. laurentii possess three of the known virulence factors common to cryptococcal organisms that cause human disease: capsule, ability to grow at 37 °C, and laccase activity.     

Phylogenetic relationships ofCryptococcus neoformans and some related basidiomycetous yeasts determined from partial large subunit rRNA sequences

The genusCryptococcus was found to be heterogeneous on the basis of partial rRNA sequences. The human-pathogenic speciesC. neoformans, comprising 4 serotypes and havingFilobasidiella neoformans andF. bacillispora as teleomorphs, was found at a relatively large distance fromFilobasidium. Serotypes B and C had identical sequences, while in A and D they were different, with D closer to B and C than to A.Filobasidiella depauperata, which lacks a yeast-like anamorph, clustered withF. neoformans.The genusFilobasidium was clearly separated fromFilobasidiella and clustered withC. albidus, C. kuetzingii, C. gastricus, C. lupi, C. vishniaciae, C. bhutanensis, C. aerius, C. terreus andC. ater. The latter may represent the anamorph ofFilobasidium elegans. The organe to red species ofCryptococcus, as well asC. aquaticus andC. yarrowii, were found completely unrelated with these taxa,C. macerans being affiliated toCystofilobasidium capitatum.The genusTrichosporon was found relatively homogeneous; it includesC. humicola, C. curvatus and the filamentous speciesHyalodendron lignicola. Cryptococcus flavus andC. dimennae probably belong to the Tremellales, though distances between these species are large. The positions ofC. laurentii andC. luteolus remains to be determined.     

Double-stranded RNA replicons associated with chloroplasts of a green alga,Bryopsis cinicola

Double-stranded RNAs (dsRNAs) associated with chloroplasts and mitochondria have been found in the coenocytic green alga Bryopsis cinicola. In this study we report molecular properties of the four chloroplast-associated dsRNAs (BDRC1 to BDRC4) The longest dsRNA molecule (BDRC1) was sequenced entirely (1959 bp) and a single large ORF of 1722 bp was found within it. Database searches revealed similarities between the deduced amino acid sequence of this ORF and RNA-dependent RNA polymerase (RdRp) sequences from several RNA viruses. The most similar sequence in the database was the RdRp of beet cryptic virus 3. Phylogenetic analysis revealed that the RdRp-like sequence of BDRC1 can be placed in the Partitiviridae clade. To detect autonomous replication of these dsRNAs, RdRp assays were carried out with actinomycin D, which is an inhibitor of DNA-dependent RNA synthesis. Incorporation of [-³²P]UTP was detected specifically in the chloroplast and mitochondrial dsRNAs, indicating that both the chloroplast dsRNAs (BDRCs) and the mitochondrial dsRNA (BDRM) of B. cinicola are RNA replicons. The green alga B. cinicola harbors different dsRNA replicons in its chloroplasts and mitochondria.     

Cold-adapted yeasts as producers of cold-active polygalacturonases

Eight cold-adapted, polygalacturonase-producing yeasts belonging to four species were isolated from frozen environmental samples in Iceland. They were identified as Cystofilobasidium lari-marini, Cystofilobasidium capitatum, Cryptococcus macerans and Cryptococcus aquaticus species by sequence analysis of rDNA regions. Growth behavior of the isolates was investigated. All strains could grow at 2 degrees C. Addition of glucose to pectin-containing culture medium had a repressive effect on enzyme production except for C. aquaticus, which showed increased polygalacturonase activity. Optimal temperature for enzyme production for the Cystofilobasidium strains was 14 degrees C, while that for the Cryptococcus strains was lower. Among the isolates, C. lari-marini S3B produced highest levels of enzyme activity at pH 3.2. Preliminary characterization of the polygalacturonases in the culture supernatant showed the enzyme from Cystofilobasidium strains to be optimally active at 40 degrees C and pH 5, and that from the Cryptococcus strains at 50 degrees C and pH 4. The polygalacturonase from C. macerans started to lose activity after 1 h of incubation at 40 degrees C, while that from the other strains had already lost activity at 30 degrees C. All the strains except C. aquaticus produced isoenzymes of polyglacturonase. In addition to polygalacturonase, the Cystofilobasidium strains produced pectin lyase, C. aquaticus pectin esterase, and C. macerans pectin lyase, pectate lyase and pectin esterase.     

<Emphasis Type="Italic">Cryptococcus randhawai</Emphasis> sp. nov<Emphasis Type="Italic">.,</Emphasis> a novel anamorphic basidiomycetous yeast isolated from tree trunk hollow of <Emphasis Type="Italic">Ficus religiosa</Emphasis> (peepal tree) from New Delhi,India

A novel anamorphic Cryptococcus species is described, which was isolated in New Delhi (India) from decaying wood of a tree trunk hollow of Ficus religiosa. On the basis of sequence analysis of the D1/D2 domains of the 26S rRNA gene and the internally transcribed spacer (ITS)-1 and ITS-2 region sequences, the isolate belonged to the Cryptococcus albidus cluster (Filobasidiales, Tremellomycetes) and was closely related to Cryptococcus saitoi, Cryptococcus cerealis and Cryptococcus friedmannii with 98% sequence identity. Phenotypically, the species differed from C. saitoi with respect to growth temperature (up to 37^oC), presence of a thin capsule, ability to grow in the absence of vitamins, and inability to assimilate citrate and ethylamine. With respect to C. friedmannii, it differed in growth temperature, ability to assimilate lactose, raffinose, l-rhamnose, myo-inositol, and inability to utilize citrate. Furthermore, our isolate also differed from C. cerealis in growth temperature, presence of capsule and inability to assimilate l-sorbose. In view of the above phenotypic differences and unique rDNA sequences, we consider that our isolate represents a new species of Cryptococcus, and therefore, a new species, Cryptococcus randhawai is proposed for this taxon. The type strain J11/2002 has been deposited in the culture collection of the Centraalbureau voor Schimmelcultures (CBS10160) and CABI Biosciences (IMI 393306).     

Identification of Pns12 as the second silencing suppressor of <Emphasis Type="Italic">Rice gall dwarf virus</Emphasis>

RNA silencing is a conserved mechanism found ubiquitously in eukaryotic organisms. It has been used to regulate gene expression and development. In addition, RNA silencing serves as an important mechanism in plants’ defense against invasive nucleic acids, such as viruses, transposons, and transgenes. As a counter-defense, most plants, and some animal viruses, encode RNA silencing suppressors to interfere at one or several points of the silencing pathway. In this study, we showed that Pns12 of RGDV (Rice gall dwarf virus) exhibits silencing suppressor activity on the reporter green fluorescent protein in transgenic Nicotiana benthamiana line 16c. Pns12 of RGDV suppressed local silencing induced by sense RNA but had no effect on that induced by dsRNA. Expression of Pns12 also enhanced Potato virus X pathogenicity in N. benthamiana. Collectively, these results suggested that RGDV Pns12 functions as a virus suppressor of RNA silencing, which might target an upstream step of dsRNA formation in the RNA silencing pathway. Furthermore, we showed that Pns12 is localized mainly in the nucleus of N. benthamiana leaf cells.     

Biological Control of Postharvest Diseases of Apple and Pear under Semi-commercial and Commercial Conditions Using Three Saprophytic Yeasts

The yeastsCryptococcus laurentii(strain HRA5),Cryptococcus infirmominiatus(strain YY6), andRhodotorula glutinis(strain HRB6) were tested as biocontrol agents of postharvest diseases of apple and pear in semi-commercial and commercial trials. The yeasts effectively controlled decay when applied in a drench or line spray. The yeasts were not adversely affected when treated fruits were stored in a controlled atmosphere consisting of 1% oxygen and 99% nitrogen. In a commercial trial, the most effective treatments for control of blue mold of pear were a combination ofC. laurentiiandC. infirmo-miniatus(91% control) and the commercially recommended high rate (528 μg/ml) of thiabendazole (88% control). In the commercial apple trial, the most effective treatments for blue mold wereC. infirmo-miniatuscombined with 264 μg/ml thiabendazole (91% control),C. infirmo-miniatuscombined withC. laurentii(84% control), and thiabendazole alone at 528 μg/ml (79% control). The combination ofC. laurentiiwith 264 μg/ml of thiabendazole was significantly more effective for control of blue mold on pear than thiabendazole at 528 μg/ml whenever any thiabendazole-resistant spores were present in the inoculum.     

Characterization of Mycoviruses and Analyses of Chitinase Secretion in the Biocontrol Fungus <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis>

Metarhizium anisopliae is the best-characterized entomopathogen and is used to control insect pests in sugar cane plantations in Brazil on a commercial scale. We have previously reported the infection of some M. anisopliae strains by dsRNA mycoviruses. Here we describe the purification and characterization of the viruses (MaV-A1, MaV-M5, MaV-RJ) in terms of dsRNA content, capsid proteins, electron microscopy, Western blot, and hybridization patterns. One spontaneous mutant lost some of the high molecular weight dsRNA components and showed significant alterations in colony morphology and spore production, suggesting that viral genes interfere with fungal phenotype. A comparison between dsRNA mycovirus-free and infected M. anisopliae isolates showed that virus-free isolates have increased endochitinase secretion. By comparing the following parameters: the buoyant density in CsCl of the presumed virions; the number and estimated molecular weight of the dsRNA components and the molecular mass of the capsid proteins to other mycoviruses previously described, we suggest the inclusion of MaV-A1 and MaV-M5 in the family Totiviridae and MaV-RJ in the family Partitiviridae. Received: 25 September 2001 / Accepted: 1 February 2002     

Double-Stranded RNA Mycovirus from Fusarium graminearum

Double-stranded RNA (dsRNA) viruses in some fungi are associated with hypovirulence and have been used or proposed as biological control agents. We isolated 7.5-kb dsRNAs from 13 of 286 field strains of Fusarium graminearum isolated from maize in Korea. One of these strains, DK21, was examined in more detail. This strain had pronounced morphological changes, including reduction in mycelial growth, increased pigmentation, reduced virulence towards wheat, and decreased (60-fold) production of trichothecene mycotoxins. The presence or absence of the 7.5-kb dsRNA was correlated with the changes in pathogenicity and morphology. The dsRNA could be transferred to virus-free strains by hyphal fusion, and the recipient strain acquired the virus-associated phenotype of the donor strain. The dsRNA was transmitted to approximately 50% of the conidia, and only colonies resulting from conidia carrying the mycovirus had the virus-associated phenotype. Partial nucleotide sequences of the purified dsRNA identify an RNA-dependent RNA polymerase sequence and an ATP-dependent helicase that are closely related to those of Cryphonectria hypovirus and Barley yellow mosaic virus. Collectively, these results suggest that this dsRNA isolated from F. graminearum encodes traits for hypovirulence.