Transcriptomic changes in Echinochloa colona in response to treatment with the herbicide imazamox.

MAIN CONCLUSION: Presented here is the first Echinochloa colona leaf transcriptome. Analysis of gene expression before and after herbicide treatment reveals that E. colona mounts a stress response upon exposure to herbicide. Herbicides are the most frequently used means of controlling weeds. For many herbicides, the target site is known; however, it is considerably less clear how plant gene expression changes in response to herbicide exposure. In this study, changes in gene expression in response to herbicide exposure in imazamox-sensitive (S) and- resistant (R) junglerice (Echinochloa colona L.) biotypes was examined. As no reference genome is available for this weed, a reference leaf transcriptome was generated. Messenger RNA was isolated from imazamox-treated- and untreated R and S plants and the resulting cDNA libraries were sequenced on an Illumina HiSeq2000. The transcriptome was assembled, annotated, and differential gene expression analysis was performed to identify transcripts that were upregulated or downregulated in response to herbicide exposure for both biotypes. Differentially expressed transcripts included transcription factors, protein-modifying enzymes, and enzymes involved in metabolism and signaling. A literature search revealed that members of the families represented in this analysis were known to be involved in abiotic stress response in other plants, suggesting that imazamox exposure induced a stress response. A time course study examining a subset of transcripts showed that expression peaked within 4-12 h and then returned to untreated levels within 48 h of exposure. Testing of plants from two additional biotypes showed a similar change in gene expression 4 h after herbicide exposure compared to the resistant and sensitive biotypes. This study shows that within 48 h junglerice mounts a stress response to imazamox exposure.

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.

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.

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.

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.

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.

Seasonal Prevalence of Species of Binucleate Rhizoctonia Fungi in Growing Medium, Leaf Litter, and Stems of Container-Grown Azalea.

Rhizoctonia web blight is an annual problem on container-grown azalea (Rhododendron spp.) in the southern and eastern United States but little is documented about the distribution or persistence of Rhizoctonia spp. in container-grown azalea. Sixty web-blight-damaged azalea plants ('Gumpo White') were collected in August 2005 and 2006 and arranged in a completely randomized design on an outdoor irrigation pad. A nylon mesh bag containing 30 necrotic leaves collected from web-blight-damaged 'Gumpo White' azalea plants were placed on the surface of the medium under the plant canopy in each container to simulate leaf litter. Ten plants were destructively sampled into eight zones by dividing stems into three zones (lengths of 0 to 2, 4 to 6, and 9 to 15 cm above the medium surface), bagged leaves into one leaf litter zone, and the medium into four zones (three horizontal layers: 1 to 3, 3 to 7, and 7 to 10 cm below the medium surface, with the middle layer further divided by removing the central 7.5-cm-diameter core) in December, February, and May. Only the three stem zones were sampled from 10 plants in early and late June and late July. Of 8,940 total isolations, 3,655 fungi with morphological characteristics of a Rhizoctonia sp. were recovered. Percent recovery differed from the eight zones (P < 0.0001) but did not differ between years (P = 0.3950) and sampling times (P = 0.1896). Frequency of recovery of Rhizoctonia spp. was highest from the lower stem and the leaf litter, and decreased with distance from the leaf litter. Recovery from stems over the six sample times was analyzed separately. Percent recovery differed between stem zones (P < 0.0001), sample times (P = 0.0478), and experiment years (P < 0.0001). In both years, mean recovery of Rhizoctonia spp. was higher from the lower stem and decreased with distance to the upper stem layer. From a subsample of 145 isolates, 95.1% were identified as binucleate Rhizoctonia (BNR) anastomosis groups (AGs)-A, -G, -K, -R, -S, and -U (-P), and 2.8 and 2.1% were Rhizoctonia solani AG-2 and an uncultured Laetisaria sp., respectively. Based on frequency analysis, recovery of BNR AGs differed by plant zone (P < 0.0001) but not over sample times (P = 0.4831). The six AGs of BNR are the predominant Rhizoctonia fungi occupying the habitat niches in container-grown azalea, with little change in population frequency and composition from fall to summer; thus, BNR pathogenic and nonpathogenic to azalea have established a mixed Rhizoctonia community on container-grown azalea.

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 MATa 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 MATa locus may have undergone accelerated evolution prior to the divergence of the pathogenic Cryptococcus species since several of the genes linked to the MATa locus appear to have a higher number of changes and substitutions than their MATalpha 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.

The genome of Nectria haematococca: contribution of supernumerary chromosomes to gene expansion.

The ascomycetous fungus Nectria haematococca, (asexual name Fusarium solani), is a member of a group of >50 species known as the "Fusarium solani species complex". Members of this complex have diverse biological properties including the ability to cause disease on >100 genera of plants and opportunistic infections in humans. The current research analyzed the most extensively studied member of this complex, N. haematococca mating population VI (MPVI). Several genes controlling the ability of individual isolates of this species to colonize specific habitats are located on supernumerary chromosomes. Optical mapping revealed that the sequenced isolate has 17 chromosomes ranging from 530 kb to 6.52 Mb and that the physical size of the genome, 54.43 Mb, and the number of predicted genes, 15,707, are among the largest reported for ascomycetes. Two classes of genes have contributed to gene expansion: specific genes that are not found in other fungi including its closest sequenced relative, Fusarium graminearum; and genes that commonly occur as single copies in other fungi but are present as multiple copies in N. haematococca MPVI. Some of these additional genes appear to have resulted from gene duplication events, while others may have been acquired through horizontal gene transfer. The supernumerary nature of three chromosomes, 14, 15, and 17, was confirmed by their absence in pulsed field gel electrophoresis experiments of some isolates and by demonstrating that these isolates lacked chromosome-specific sequences found on the ends of these chromosomes. These supernumerary chromosomes contain more repeat sequences, are enriched in unique and duplicated genes, and have a lower G+C content in comparison to the other chromosomes. Although the origin(s) of the extra genes and the supernumerary chromosomes is not known, the gene expansion and its large genome size are consistent with this species' diverse range of habitats. Furthermore, the presence of unique genes on supernumerary chromosomes might account for individual isolates having different environmental niches.

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

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.