Marianela Rodriguez-Carres

Phylogeny and phenotypic characterization of pathogenic Cryptococcus species and closely related saprobic taxa in the Tremellales.

ABSTRACT The basidiomycetous yeasts Cryptococcus neoformans and Cryptococcus gattii are closely related sibling species that cause respiratory and neurological disease in humans and animals. Within these two recognized species, phylogenetic analysis reveals at least six cryptic species defined as molecular types (VNI/II/B, VNIV, VGI, VGII, VGIII, and VGIV) that comprise the pathogenic Cryptococcus species complex. These pathogenic species are clustered in the Filobasidiella clade within the order Tremellales. Previous studies have shown that the Filobasidiella clade also includes several saprobic fungi isolated from insect frass, but information evaluating the relatedness of the saprobes and pathogens within this cluster is limited. Here, the phylogeny encompassing a subset of species in the Tremellales lineage that clusters closely with the pathogenic Cryptococcus species complex was resolved by employing a multilocus sequencing approach for phylogenetic analysis. Six highly conserved genomic loci from 15 related basidiomycete species were sequenced, and the alignments from the concatenated gene sequences were evaluated with different tree-building criteria. Furthermore, these 15 species were subjected to virulence and phenotype assays to evaluate their pathogenic potential. These studies revealed that Cryptococcus amylolentus and Tsuchiyaea wingfieldii, two nonpathogenic sibling species, are the taxa most closely related to the pathogens C. neoformans and C. gattii and together with Filobasidiella depauperata form a Cryptococcus sensu stricto group. Five other saprobic yeast species form the Kwoniella clade, which appears to be a part of a more distantly related sensu lato group. This study establishes a foundation for future comparative genomic approaches that will provide insight into the structure, function, and evolution of the mating type locus, the transitions in modes of sexual reproduction, and the emergence of human pathogenic species from related or ancestral saprobic species.

An ABC Transporter and a Cytochrome P450 of Nectria haematococca MPVI Are Virulence Factors on Pea and Are the Major Tolerance Mechanisms to the Phytoalexin Pisatin

The fungal plant pathogen Nectria haematococca MPVI produces a cytochrome P450 that is responsible for detoxifying the phytoalexin pisatin, produced as a defense mechanism by its host, garden pea. In this study, we demonstrate that this fungus also produces a specific ATP-binding cassette (ABC) transporter, NhABC1, that enhances its tolerance to pisatin. In addition, although both mechanisms individually contribute to the tolerance of pisatin and act as host-specific virulence factors, mutations in both genes render the fungus even more sensitive to pisatin and essentially nonpathogenic on pea. NhABC1 is rapidly induced after treatment with pisatin in vitro and during infection of pea plants. Furthermore, NhABC1 was able to confer tolerance to the phytoalexin rishitin, produced by potato. NhABC1 appears to be orthologous to GpABC1 of the potato pathogen Gibberella pulicaris and, along with MoABC1 from Magnaporthe oryzae, resides in a phylogenetically related clade enriched with ABC transorters involved in virulence. We propose that NhABC1 and the cytochrome P450 may function in a sequential manner in which the energy expense from pisatin efflux by NhABC1 releases the repression of the cytochrome P450, ultimately allowing pisatin tolerance by two mechanisms. These results demonstrate that a successful pathogen has evolved multiple mechanisms to overcome these plant antimicrobial compounds.

Mobile elements and mitochondrial genome expansion in the soil fungus and potato pathogen Rhizoctonia solani AG-3

The soil fungus Rhizoctonia solani is an economically important pathogen of agricultural and forestry crops. Here, we present the complete sequence and analysis of the mitochondrial genome of R. solani, field isolate Rhs1AP. The genome (235 849 bp) is the largest mitochondrial genome of a filamentous fungus sequenced to date and exhibits a rich accumulation of introns, novel repeat sequences, homing endonuclease genes, and hypothetical genes. Stable secondary structures exhibited by repeat sequences suggest that they comprise functional, possibly catalytic RNA elements. RNA-Seq expression profiling confirmed that the majority of homing endonuclease genes and hypothetical genes are transcriptionally active. Comparative analysis suggests that the mitochondrial genome of R. solani is an example of a dynamic history of expansion in filamentous fungi.

The Supernumerary Chromosome of Nectria haematococca That Carries Pea-Pathogenicity-Related Genes Also Carries a Trait for Pea Rhizosphere Competitiveness

ABSTRACT Fungi are found in a wide range of environments, and the ecological and host diversity of the fungus Nectria haematococca has been shown to be due in part to unique genes on different supernumerary chromosomes. These chromosomes have been called “conditionally dispensable” (CD) since they are not needed for axenic growth but are important for expanding the host range of individual isolates. From a biological perspective, the CD chromosomes can be compared to bacterial plasmids that carry unique genes that can define the habits of these microorganisms. The current study establishes that the N. haematococca PDA1-CD chromosome, which contains the genes for pea pathogenicity (PEP cluster) on pea roots, also carries a gene(s) for the utilization of homoserine, a compound found in large amounts in pea root exudates. Competition studies demonstrate that an isolate that lacks the PEP cluster but carries a portion of the CD chromosome which includes the homoserine utilization (HUT) gene(s) is more competitive in the pea rhizosphere than an isolate without the CD chromosome.

Morphological and Genomic Characterization of Filobasidiella depauperata: A Homothallic Sibling Species of the Pathogenic Cryptococcus Species Complex

The fungal species Cryptococcus neoformans and Cryptococcus gattii cause respiratory and neurological disease in animals and humans following inhalation of basidiospores or desiccated yeast cells from the environment. Sexual reproduction in C. neoformans and C. gattii is controlled by a bipolar system in which a single mating type locus (MAT) specifies compatibility. These two species are dimorphic, growing as yeast in the asexual stage, and producing hyphae, basidia, and basidiospores during the sexual stage. In contrast, Filobasidiella depauperata, one of the closest related species, grows exclusively as hyphae and it is found in association with decaying insects. Examination of two available strains of F. depauperata showed that the life cycle of this fungal species shares features associated with the unisexual or same-sex mating cycle in C. neoformans. Therefore, F. depauperata may represent a homothallic and possibly an obligately sexual fungal species. RAPD genotyping of 39 randomly isolated progeny from isolate CBS7855 revealed a new genotype pattern in one of the isolated basidiospores progeny, therefore suggesting that the homothallic cycle in F. depauperata could lead to the emergence of new genotypes. Phylogenetic analyses of genes linked to MAT in C. neoformans indicated that two of these genes in F. depauperata, MYO2 and STE20, appear to form a monophyletic clade with the MAT a alleles of C. neoformans and C. gattii, and thus these genes may have been recruited to the MAT locus before F. depauperata diverged. Furthermore, the ancestral MAT a locus may have undergone accelerated evolution prior to the divergence of the pathogenic Cryptococcus species since several of the genes linked to the MAT a locus appear to have a higher number of changes and substitutions than their MATα counterparts. Synteny analyses between C. neoformans and F. depauperata showed that genomic regions on other chromosomes displayed conserved gene order. In contrast, the genes linked to the MAT locus of C. neoformans showed a higher number of chromosomal translocations in the genome of F. depauperata. We therefore propose that chromosomal rearrangements appear to be a major force driving speciation and sexual divergence in these closely related pathogenic and saprobic species.

Vascular Streak Dieback of cacao in Southeast Asia and Melanesia: in planta detection of the pathogen and a new taxonomy

Vascular Streak Dieback (VSD) disease of cacao (Theobroma cacao) in Southeast Asia and Melanesia is caused by a basidiomycete (Ceratobasidiales) fungus Oncobasidium theobromae (syn. =Thanatephorus theobromae). The most characteristic symptoms of the disease are green-spotted leaf chlorosis or, commonly since about 2004, necrotic blotches, followed by senescence of leaves beginning on the second or third flush behind the shoot apex, and blackening of infected xylem in the vascular traces at the leaf scars resulting from the abscission of infected leaves. Eventually the shoot apex is killed and infected branches die. In susceptible cacao the fungus may grow through the xylem down into the main stem and kill a mature cacao tree. Infections in the stem of young plants prior to the formation of the first 3-4 lateral branches usually kill the plant. Basidiospores released from corticioid basidiomata developed on leaf scars or along cracks in the main vein of infected leaves infect young leaves. The pathogen commonly infects cacao but there are rare reports from avocado. As both crops are introduced to the region, the pathogen is suspected to occur asymptomatically in native vegetation. The pathogen is readily isolated but cultures cannot be maintained. In this study, DNA was extracted from pure cultures of O. theobromae obtained from infected cacao plants sampled from Indonesia. The internal transcribed spacer region (ITS), consisting of ITS1, 5.8S ribosomal RNA and ITS2, and a portion of nuclear large subunit (LSU) were sequenced. Phylogenetic analysis of ITS sequences placed O. theobromae sister to Ceratobasidium anastomosis groups AG-A, AG-Bo, and AG-K with high posterior probability. Therefore the new combination Ceratobasidium theobromae is proposed. A PCR-based protocol was developed to detect and identify C. theobromae in plant tissue of cacao enabling early detection of the pathogen in plants. A second species of Ceratobasidium, Ceratobasidium ramicola, identified through ITS sequence analysis, was isolated from VSD-affected cacao plants in Java, and is widespread in diseased cacao collected from Indonesia.

Phylogenetic relationships of Rhizoctonia fungi within the Cantharellales

Phylogenetic relationships of Rhizoctonia fungi within the order Cantharellales were studied using sequence data from portions of the ribosomal DNA cluster regions ITS-LSU, rpb2, tef1, and atp6 for 50 taxa, and public sequence data from the rpb2 locus for 165 taxa. Data sets were analysed individually and combined using Maximum Parsimony, Maximum Likelihood, and Bayesian Phylogenetic Inference methods. All analyses supported the monophyly of the family Ceratobasidiaceae, which comprises the genera Ceratobasidium and Thanatephorus. Multi-locus analysis revealed 10 well-supported monophyletic groups that were consistent with previous separation into anastomosis groups based on hyphal fusion criteria. This analysis coupled with analyses of a larger sample of 165 rpb2 sequences of fungi in the Cantharellales supported a sister relationship between the Botryobasidiaceae and Ceratobasidiaceae and a sister relationship of the Tulasnellaceae with the rest of the Cantharellales. The inclusion of additional sequence data did not clarify incongruences observed in previous studies of Rhizoctonia fungi in the Cantharellales based on analyses of a single or multiple genes. The diversity of ecological and morphological characters associated with these fungi requires further investigation on character evolution for re-evaluating homologous and homoplasious characters.

Characterization of a Basidiomycete fungus from stored sugar beet roots

Eighteen isolates from sugar beet roots associated with an unknown etiology were characterized based on observations of morphological characters, hyphal growth at 4–28 C, production of phenol oxidases and sequence analysis of internal transcribed spacer (ITS) and large subunit (LSU) regions of the ribosomal DNA (rDNA). The isolates did not produce asexual or sexual spores, had binucleate hyphal cells with clamp connections, grew 4–22 C with estimated optimal growth at 14.5 C and formed a dark brown pigment on potato dextrose or malt extract agar amended with 0.5% tannic acid. Color changes observed when solutions of gum guiac, guiacol and syringaldzine were applied directly to mycelium grown on these media indicated that all isolates produced phenol oxidases. Sequences of ITS and LSU regions on the rDNA gene from 15 isolates were 99.2–100% identical, and analysis of sequence data with maximum likelihood and maximum parsimony suggest that the isolates from sugar beet roots are phylogenetically related to Athelia bombacina, Granulobasidium vellereum and Cyphella digitalis. High statistical support for both loci under different criteria confirmed that Athelia bombacina was consistently the closest known relative to the sugar beet isolates. Additional taxonomic investigations are needed before species can be clarified and designated for these isolates.

First report of root rot caused by binucleate Rhizoctonia anastomosis group F on Musa spp.

A research program was initiated at the University of Georgia in 2003 to identify banana cultivars suitable for production in the coastal and southern areas of the state. During a root disease survey conducted in October 2007 on bananas (Musa spp.) grown at the University of Georgia Bamboo Farm and Coastal Gardens in Savannah, GA, root lesions and root rot were observed on banana cvs. Gold Finger, Kandarian, and Manzano. Root lesions were dark brown to black and irregular in shape, with partial or entire roots affected. Lateral roots and outer layers of cord roots (roots arising from interior layers of the corm) of infected plants were blackened and rotted. Diseased root samples were collected from three plants of each cultivar, surface sterilized with 0.6% sodium hypochlorite, and placed on tannic acid benomyl agar (TABA). Pure cultures of the fungus consistently associated with diseased tissue were obtained by subculturing hyphal tips on TABA. Mycelia of the fungus on potato dextrose agar (PDA) were light to deep brown and the hyphae tended to branch at right angles. A septum was present in each hyphal branch near the point of origin and a slight constriction at the branch was observed. The hyphae of two isolates were stained with 0.6% phenosafranin and 3% KOH and binucleate hyphal cells were observed. On the basis of these morphological features, the isolates appeared to be binucleate Rhizoctonia anamorphs (teleomorph Ceratobasidium Rogers). For molecular identification, the internal transcribed spacer (ITS) regions and the 5.8S gene from rDNA of the isolates were cloned and sequenced (GenBank Accession No. HQ168370). The ITS regions (775 bp) were 100% identical between the two isolates and 99% identical to Ceratobasidium sp. AG-F strain SIR-1 isolated from sweet potato in Japan (GenBank Accession No. AF354085). The anastomosis group of the isolates was confirmed by pairing with strain SIR-1 on PDA. On the basis of morphological and molecular characteristics and the anastomosis assay, the two isolates were identified as a Ceratobasidium sp. AG-F (1-3). Pathogenicity assays were conducted by inoculating banana plants (cv. Golden pillow, synonym = Manzano) grown in pots under greenhouse conditions (25 to 27°C). Twenty wheat seeds infested with each isolate were placed uniformly around each plant at a depth of 10 cm in the soil. The plants were incubated in the greenhouse and the roots were examined 2 months after inoculation. Brown-to-black lesions and root rot, identical to symptoms associated with field banana roots, were observed on all inoculated plants but not on the noninoculated control plants. The fungus was reisolated from affected root samples and the identity was confirmed by morphological and molecular characteristics and the anastomosis assay. To our knowledge, this is the first report of banana root rot caused by binucleate Rhizoctonia anastomosis group F. With the increased interest in producing bananas for food and ornamental purposes, the occurrence of Ceratobasidium root rot on bananas needs to be considered when designing disease management programs and searching for suitable cultivars for banana production. References: (1) L. L. Burpee et al. Mycologia 70:1281, 1978. (2) D. González et al. Mycologia 93:1138, 2001. (3) B. Sneh et al. Identification of Rhizoctonia Species. The American Phytopathological Society, St. Paul, MN. 1991.