Draft Genome Sequence of a Washington Isolate of “Candidatus Phytoplasma pruni”

ABSTRACT

Illumina sequencing of a Prunus avium tree with X-disease symptoms was performed to obtain a draft genome of “Candidatus Phytoplasma pruni.” The genome consists of 14 contigs covering 588,767 bp. This is the first metagenome to be sequenced from the current X-disease epidemic in stone fruit in the Pacific Northwest.

ANNOUNCEMENT

The causative agent of X-disease in Prunus avium (cherry) and Prunus persica (peach/nectarine), which has reached epidemic levels in stone fruit in the Pacific Northwest, is “Candidatus Phytoplasma pruni” (1, 2). In peaches and nectarines, infection by “Ca. Phytoplasma pruni” causes premature yellowing of leaves and spots of necrosis, producing a shot-hole-like appearance. The pathogen also causes limb dieback and, within approximately 5 years of infection, tree death (1). In cherries, the fruit is affected; at harvest, the fruit is small and misshapen, with poor color and taste and often a reduction in sugar content (1, 2). Like other phytoplasmas, “Ca. Phytoplasma pruni” is a cell wall-less bacterium that currently cannot be cultured independently of its host (3). Developing improved genomic resources for this pathogen is crucial to understanding and managing this disease. To that end, an isolate from the current epidemic was sequenced.

Cuttings from a sweet cherry tree expressing classic X-disease symptoms were collected from a commercial orchard in Zillah, Washington. DNA was extracted from woody stem tissue because this is the tissue in which phytoplasma titers are highest (4). Briefly, 100 mg of tissue was frozen at −80°C for at least 1 h prior to homogenization to a fine powder by bead-beating in a TissueLyser (Qiagen, Germantown, MD) at 20 beats/s for 30 s. DNA was then extracted with a DNeasy Plant minikit (Qiagen) according to the manufacturer’s instructions. Phytoplasma presence was confirmed by quantitative PCR (qPCR) as described (4), and the DNA concentration was determined using a Qubit fluorometer (Thermo Fisher Scientific, Hampton, NH). DNA was submitted to Genewiz (South Plainfield, NJ) for library preparation using a NEBNext Ultra library preparation kit (New England Biolabs, Ipswich, MA) and 250-bp paired-end sequencing with a single HiSeq flow cell on a HiSeq 2500 system (Illumina Inc., San Diego, CA, USA).

Raw reads were processed with CLC Genomics Workbench v21.0.5 (Qiagen, Hilden, Germany) using default settings unless stated otherwise. Sequencing yielded 309,073,036 raw reads, which were reduced to 309,072,714 reads after adaptor removal and quality assessment; this was performed using the Trim Reads pipeline with the default quality limit of 0.05. De novo assembly was performed on the trimmed reads using the CLC Genomics Workbench de novo assembly tool. Minimum contig length was set to 200 bp, word size to 26, and bubble size to 246. The assembly consisted of 343,722 contigs, with an N₅₀ value of 2,935 bp. BLASTn analysis with a word size of 50 was used to identify 371 phytoplasma-like sequences, which were then imported into Geneious v2022.0.2 (Biomatters, Inc., Auckland, New Zealand) to find open reading frames (ORFs). ORF identification was performed with a minimum size set to 300 bp and the genetic code set to standard. Genes were confirmed and where possible assigned a function based on homology to known phytoplasma genes using BLASTp.

Of the 371 contigs, 14 were large enough to contain ORFs; the remainder were small fragments that might have been the result of repetitive regions that could not be resolved by Illumina sequencing. These 14 contigs were composed of a total of 588,767 bp, with a GC content of 27.1%. The average sequencing depth was 18,538×, with 45,212,112 reads mapped to the contigs. Through the Geneious ORF prediction tool and manual curation, 469 genes were annotated. These genes were assigned functions based on homology to genes in other phytoplasma genomes.

The estimated size of the “Ca. Phytoplasma pruni” genome is 677 kb (5). This draft genome represents approximately 87% of the full genome. It is similar in size to the draft genome published previously (6); however, there are fewer contigs in the genome of this metagenome. The genome sequenced previously (6) was from a strain that was originally from an infected peach tree in Connecticut (7) and was maintained in periwinkle, making it temporally and geographically separate from the Washington sample. The genome presented here was sequenced directly from an infected cherry in Zillah, Washington, and is representative of the current epidemic in the Pacific Northwest. Additional work, including comparative genomic analysis among geographically diverse isolates, may explain why the current epidemic is so devastating.

Data availability.

Raw reads have been submitted to the SRA under BioProject accession number PRJNA857992. The assembled and annotated genome has been deposited in GenBank with the accession number JANIEM000000000.