Attached
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 pairedend 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 MAKERp, 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 carbohydratebinding 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
