Submitted to: CDFA Pierce's Disease Control Program Research Symposium
Publication Type: Proceedings
Publication Acceptance Date: 12/14/2021
Publication Date: 12/14/2021
Citation: Chen, J., Ledbetter, C.A., O'Leary, M.L. 2021. Isolation and whole genome sequencing of Curtobacterium sp. strain TXMA1 from a grapevine showing PD-like symptoms in Texas, U.S.A. Proceedings of the 2021 CDFA Pierce's Disease Control Program Research Symposium. Sacramento, CA. p. 121.
Technical Abstract: In September 2019, samples of grapevines (cultivar Blanc du Bois) with leaf marginal necrosis symptoms resembling those of Pierce’s disease (PD) were collected in an experimental plot in Monte Alto, Texas. An initial investigation attempted to detect the presence of Xylella fastidiosa, the pathogen of PD. PCR detection of X. fastidiosa following a previously described procedure was not successful. In vitro culture experiments using PW medium found no X. fastidiosa colonies during the 30-day incubation period at 28 C. In contrast, yellow-pigmented colonies (YPCs) were observed within seven days. The YPCs were selected and triple-cloned. DNA fragments of 16S rRNA gene were amplified by PCR using an universal primer set and sequenced by Sangers’ method. BLASTn search using the 16S rRNA gene sequences against GenBank database showed sequence similarity (>96%) to multiple strains of Curtobacterium sp. The YPC strain was designated as Curtobacterium sp. strain TXMA1. DNA of strain TXMA1 was extracted and sequenced on a MinION device (Oxford Nanopore Technologies, UK). Basecalling was performed using Guppy v.5.0.11. Nanopore sequence reads were assembled using Flye v2.9. The TXMA1 genome consists of one circular contig of 3,454,876 bp with GC content of 71.74% and ~110x coverage, 3,213 open reading frames (ORFs), 47 tRNA genes, four complete 16S-23S-5S rRNA operons, and three additional noncoding RNA genes. The whole-genome sequence has been deposited in GenBank under accession number CP083910. No plasmids were identified. Average nucleotide identity (ANI) to genome assemblies of 138 Curtobacterium strains deposited in GenBank was determined using fastANI v1.1. TXMA1 has >98% ANI to two C. oceanosedimentum strains (NS263 and NS2359), and >95% ANI to C. sp. strain SGAir0471. The association of strain TXMA1 to PD-like symptoms of grapevine remains unclear. The availability of in vitro culture and whole genome sequence will facilitate future research in the bacterial characterization that would benefit grape disease management.