Abstract
Obtaining complete phytoplasma genomes is difficult due to the lack of a culture system for these bacteria. To improve genome assembly, a non-ionic, low- and iso-osmotic iodixanol (Optiprep™) density gradient centrifugation method was developed to enrich for phytoplasma cells and deplete plant host tissues prior to deoxyribonucleic acid (DNA) extraction and high-throughput sequencing (HTS). After density gradient enrichment, potato infected with a ‘ Candidatus Phytoplasma australasia’-related strain showed a ∼14-fold increase in phytoplasma HTS reads, with a ∼1.7-fold decrease in host genomic reads compared to the DNA extracted from the same sample without density gradient centrifugation enrichment. Additionally, phytoplasma genome assemblies from libraries equalized to 5 million reads were, on average, ∼15,000 bp larger and more contiguous (N50 ∼14,800 bp larger) than assemblies from the DNA extracted from the infected potato without enrichment. The method was repeated on capsicum infected with Sweet Potato Little Leaf phytoplasma (‘ Ca. Phytoplasma australasia’-related strain) with a lower phytoplasma titer than the potato. In capsicum, ∼threefold more phytoplasma reads and ∼twofold less host genomic reads were obtained, with the genome assembly size and N50 values from libraries equalized to 3.4 million reads ∼137,000 and ∼4,000 bp larger, respectively, compared to the DNA extracted from infected capsicum without enrichment. Phytoplasmas from potato and capsicum were both enriched at a density of 1.049–1.058 g/ml. Finally, we present two highly contiguous ‘ Ca. Phytoplasma australasia’ phytoplasma reference genomes sequenced from naturally infected Solanaceae hosts in Australia. Obtaining high-quality phytoplasma genomes from naturally infected hosts will improve insights into phytoplasma taxonomy, which will improve their detection and disease management.
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Claims 10
Claim · model-written summary
Enrichment increased phytoplasma HTS reads from infected potato by about 14-fold.
- Taxon
- potato
Solanum tuberosume0 - Interactor
- ‘ Candidatus Phytoplasma australasia’-related straine0pathogen
- Outcome
- phytoplasma HTS readse0
- Direction
- increasee0
- Value
- ∼14-folde0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“potato infected with a ‘ Candidatus Phytoplasma australasia’-related strain showed a ∼14-fold increase in phytoplasma HTS reads”
Claim · model-written summary
Enrichment decreased potato host genomic reads by about 1.7-fold.
- Taxon
- potatoSolanum tuberosume1
- Outcome
- host genomic readse0
- Direction
- decreasee0
- Value
- ∼1.7-folde0
- Comparator
- DNA extracted from the same sample without density gradient centrifugation enrichmente0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“a ∼1.7-fold decrease in host genomic reads compared to the DNA extracted from the same sample without density gradient centrifugation enrichment.”
e1“potato infected with a ‘ Candidatus Phytoplasma australasia’-related strain”
Claim · model-written summary
Enrichment increased potato phytoplasma assembly size by about 15,000 bp.
- Taxon
- potatoSolanum tuberosume0
- Outcome
- phytoplasma genome assembliese0
- Direction
- largere0
- Value
- ∼15,000 bpe0
- Comparator
- assemblies from the DNA extracted from the infected potato without enrichmente0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“phytoplasma genome assemblies from libraries equalized to 5 million reads were, on average, ∼15,000 bp larger and more contiguous (N50 ∼14,800 bp larger) than assemblies from the DNA extracted from the infected potato without enrichment.”
Claim · model-written summary
Enrichment increased potato phytoplasma assembly N50 by about 14,800 bp.
- Taxon
- potatoSolanum tuberosume0
- Outcome
- N50e0
- Direction
- largere0
- Value
- ∼14,800 bpe0
- Comparator
- assemblies from the DNA extracted from the infected potato without enrichmente0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“phytoplasma genome assemblies from libraries equalized to 5 million reads were, on average, ∼15,000 bp larger and more contiguous (N50 ∼14,800 bp larger) than assemblies from the DNA extracted from the infected potato without enrichment.”
Claim · model-written summary
Enrichment produced about threefold more phytoplasma reads in capsicum.
CapsicumOrganisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“In capsicum, ∼threefold more phytoplasma reads and ∼twofold less host genomic reads were obtained”
Claim · model-written summary
Enrichment produced about twofold fewer host genomic reads in capsicum.
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“In capsicum, ∼threefold more phytoplasma reads and ∼twofold less host genomic reads were obtained”
Claim · model-written summary
Enrichment increased capsicum phytoplasma genome assembly size by about 137,000 bp.
- Outcome
- genome assembly sizee0
- Direction
- largere0
- Value
- ∼137,000e0
- Comparator
- DNA extracted from infected capsicum without enrichmente0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“genome assembly size and N50 values from libraries equalized to 3.4 million reads ∼137,000 and ∼4,000 bp larger, respectively, compared to the DNA extracted from infected capsicum without enrichment.”
Claim · model-written summary
Enrichment increased capsicum phytoplasma assembly N50 by about 4,000 bp.
- Outcome
- N50 valuese0
- Direction
- largere0
- Value
- ∼4,000 bpe0
- Comparator
- DNA extracted from infected capsicum without enrichmente0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“genome assembly size and N50 values from libraries equalized to 3.4 million reads ∼137,000 and ∼4,000 bp larger, respectively, compared to the DNA extracted from infected capsicum without enrichment.”
Claim · model-written summary
Phytoplasmas from potato and capsicum were enriched at a density of 1.049–1.058 g/ml.
- Taxon
- potatoSolanum tuberosume0
- Outcome
- Phytoplasmase0
- Value
- 1.049–1.058 g/mle0
- Qualifier
- capsicume0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“Phytoplasmas from potato and capsicum were both enriched at a density of 1.049–1.058 g/ml.”
Claim · model-written summary
The authors provide two contiguous phytoplasma reference genomes from infected Solanaceae hosts.
Verbatim
e0“two highly contiguous ‘ Ca. Phytoplasma australasia’ phytoplasma reference genomes sequenced from naturally infected Solanaceae hosts in Australia.”