Browsing by Author "Castlebury, L.A."

Now showing 1 - 2 of 2

Molecular phylogeny of Bipolaris and Curvularia species associated with browntop millet.
(International Research Symposium on Pure and Applied Sciences, 2017 Faculty of Science, University of Kelaniya, Sri Lanka., 2017) Manamgoda, D. S.; Bauchan, G. R.; Berry, A.; Walker, S.; Castlebury, L.A.
Browntop millet (Urochloa ramosa, Poaceae) is a warm season grass commonly used as a cover crop in pasture management systems. This species is inexpensive compared to other forage crops and its ability to easily reseed and to remain viable in the soil for years makes browntop millet an excellent regenerating food plot for wildlife. The genera Bipolaris and Curvularia (Pleosporaceae) are closely related and comprise many pathogenic species associated with poaceous hosts. Of these, only B. setariae and C. hawaiiensis have previously been reported from U. ramosa. The taxa belonging in these sister genera are often difficult to identify based on morphology alone, especially when co-occurring on a single host. In May 2015, infected seeds of U. ramosa grown in Tifton, Georgia, USA were observed and collected. Conidia and conidiophores were observed using compound and dissecting light microscopy. Single spore isolation techniques were used to obtain pure cultures, which were also observed scanning electron microscopy. Genomic DNA was extracted from isolates of Bipolaris and Curvularia using the DNeasy Plant Mini Kit (Qiagen). The ITS, GPDH, TEF and RPB gene regions were amplified and the resulting amplicons were sequenced using the BigDye Terminator v. 3.1 on an Applied Biosystems 3130xl Genetic Analyzer. Phylogenetic analyses were carried out using maximum parsimony and maximum likelihood methods. Five Curvularia and nine Bipolaris cultures were isolated, and a total of 42 new DNA sequences were generated in this study. GPDH gene region resolved most species in both genera. Maximum parsimony analysis of combined ITS, GPDH and TEF loci for Bipolaris included 58 isolates and 81 isolates for Curvularia. Based on morphological and phylogenetic data, the isolates found on the seeds of U. ramosa are identified as Bipolaris yamadai, Curvularia geniculata and a previously undescribed species of Curvularia. To our knowledge, these are the first records of these fungal species in association with U. ramosa. Surface ornamentation and texture of Curvularia conidia were clearly observed under SEM than under light microscope. Although other conidial cells are smooth, basal cell of the conidium can be either smooth or verrucose and this was used as a morphological character to delimit some members of the “geniculata” group. Curvularia geniculata, C. urochloae and B. yamadai had conidia with smooth basal cell under SEM. Therefore SEM imaging will be useful to observe micromorphological characters that are difficult to distinguish using conventional light microscopy. The discovery of these potentially pathogenic fungi on an economically important host provides new insights for disease surveillance to plant pathologists, quarantine officials and others with interests in plant disease management.
Molecular systematics and comparative draft genome analysis of strawberry leaf blight fungus.
(International Research Symposium on Pure and Applied Sciences, 2017 Faculty of Science, University of Kelaniya, Sri Lanka., 2017) Udayanga, D.; Castlebury, L.A.
The leaf blight fungus, formerly known as “Phomopsis” obscurans originally described from the United States, is a severe pathogen of strawberry. Morphological characters are often inadequate for identification of this species. Although this fungus is currently classified as a Phomopsis sp. (=Diaporthe), preliminary analyses of this study indicated it is not congeneric with Diaporthe. Phylogenetic analyses with representative species in the order Diaporthales were performed using five nuclear loci (28S, 18S, internal transcribed spacer regions with 5.8Sof rDNA, translation elongation factor and DNA directed RNA polymerase II) to infer its evolutionary relationships. Genomic libraries of leaf blight fungal DNA were prepared for next generation whole genome sequencing. DNA and RNA were extracted and paired-end libraries were generated with Nextera library preparation kits (Illumina) and sequenced on Illumina Miseq instrument. Whole genome and transcriptome sequences obtained were initially analyzed with CLC Genomic Workbench and comparative analyses were performed using methods similar to the US-DOE Joint Genome Institute annotation pipeline. Results of phylogenetic analyses determined that “P.” obscurans represents a unique evolutionary lineage and possibly an undescribed genus within a new family. The whole genome assembly consisted of 5638 contigs (≥500 bp) with an estimated genome size of 48 Mbp. The N50 contig length is 13853 bp and the G+C content is 52%. The output resulting from MAKER-p, predicted a total of 12431 genes and was modeled with Cryphonectria parasitica EP155 genome. The estimated mitochondrial genome size is 98658 bp which is comparatively larger than the average sized mitogenomes of fungi. Analysis with the CAZy database (dbCAN) identified in total 778 putative carbohydrate active enzyme encoding genes, including 341 glycoside hydrolases (GHs), 112 glycosyl transferases, 25 polysaccharide lyases, 138 carbohydrate esterases, 51 carbohydrate-binding modules, and 111 auxiliary activities. Therefore, “P.” obscurans is equipped with all necessary enzymes to breach plant cell wall, penetrate, successfully infect and cause severe plant disease. Among GHs, majority (i.e. 20 from each) belong to GH3, GH5, GH16, and GH28 families that possess xylanase, cellulase, chitinase, polygalacturanases which are also potentially involved in maceration and soft-rot of plant tissues including fruits.