@article{cai_nunziata_srivastava_wilson_chambers_rivera_nakhla_costanzo_2022, title={Draft Genome Sequence Resource of AldY-WA1, a Phytoplasma Strain Associated with Alder Yellows of Alnus rubra in Washington, USA}, ISSN={["1943-7692"]}, DOI={10.1094/PDIS-10-21-2350-A}, abstractNote={HomePlant DiseaseVol. 106, No. 7Draft Genome Sequence Resource of AldY-WA1, a Phytoplasma Strain Associated with Alder Yellows of Alnus rubra in Washington, U.S.A. PreviousNext RESOURCE ANNOUNCEMENT OPENOpen Access licenseDraft Genome Sequence Resource of AldY-WA1, a Phytoplasma Strain Associated with Alder Yellows of Alnus rubra in Washington, U.S.A.Weili Cai, Schyler O. Nunziata, Subodh K. Srivastava, Telissa Wilson, Nathaniel Chambers, Yazmín Rivera, Mark Nakhla, and Stefano CostanzoWeili CaiUnited States Department of Agriculture Animal and Plant Health Inspection Service, Plant Protection and Quarantine, Science and Technology, Plant Pathogen Confirmatory Diagnostics Laboratory, Laurel, MDDepartment of Entomology and Plant Pathology, North Carolina State University, Raleigh, NCSearch for more papers by this author, Schyler O. NunziataUnited States Department of Agriculture Animal and Plant Health Inspection Service, Plant Protection and Quarantine, Science and Technology, Plant Pathogen Confirmatory Diagnostics Laboratory, Laurel, MDSearch for more papers by this author, Subodh K. Srivastavahttps://orcid.org/0000-0003-0845-0199United States Department of Agriculture Animal and Plant Health Inspection Service, Plant Protection and Quarantine, Science and Technology, Plant Pathogen Confirmatory Diagnostics Laboratory, Laurel, MDDepartment of Entomology and Plant Pathology, North Carolina State University, Raleigh, NCSearch for more papers by this author, Telissa Wilsonhttps://orcid.org/0000-0003-2683-4081Washington State Department of Agriculture, Plant Pathology and Molecular Diagnostic Lab, Olympia, WASearch for more papers by this author, Nathaniel ChambersWashington State Department of Agriculture, Plant Pathology and Molecular Diagnostic Lab, Olympia, WASearch for more papers by this author, Yazmín RiveraUnited States Department of Agriculture Animal and Plant Health Inspection Service, Plant Protection and Quarantine, Science and Technology, Plant Pathogen Confirmatory Diagnostics Laboratory, Laurel, MDSearch for more papers by this author, Mark NakhlaUnited States Department of Agriculture Animal and Plant Health Inspection Service, Plant Protection and Quarantine, Science and Technology, Plant Pathogen Confirmatory Diagnostics Laboratory, Laurel, MDSearch for more papers by this author, and Stefano Costanzo†Corresponding author: S. Costanzo; E-mail Address: stefano.costanzo@usda.govhttps://orcid.org/0000-0003-0330-3827United States Department of Agriculture Animal and Plant Health Inspection Service, Plant Protection and Quarantine, Science and Technology, Plant Pathogen Confirmatory Diagnostics Laboratory, Laurel, MDSearch for more papers by this author AffiliationsAuthors and Affiliations Weili Cai1 2 Schyler O. Nunziata1 Subodh K. Srivastava1 2 Telissa Wilson3 Nathaniel Chambers3 Yazmín Rivera1 Mark Nakhla1 Stefano Costanzo1 † 1United States Department of Agriculture Animal and Plant Health Inspection Service, Plant Protection and Quarantine, Science and Technology, Plant Pathogen Confirmatory Diagnostics Laboratory, Laurel, MD 2Department of Entomology and Plant Pathology, North Carolina State University, Raleigh, NC 3Washington State Department of Agriculture, Plant Pathology and Molecular Diagnostic Lab, Olympia, WA Published Online:26 May 2022https://doi.org/10.1094/PDIS-10-21-2350-AAboutSectionsPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat Phytoplasmas are wall-less bacteria in the class Mollicutes that cannot be cultured in vitro and cause systemic plant diseases in more than 1,000 plant species, including many economically important crops (Jurga and Zwolińska 2020; Rao et al. 2018). Phytoplasmas are vectored by different sap-sucking insects, primarily leafhoppers, planthoppers, and psyllids (Weintraub and Beanland 2006).Typically, phytoplasma-infected plants show symptoms of phyllody, stunting, yellowing, witches’-broom, leaf roll, and generalized decline, although some may remain symptomless (Kumari et al. 2019). The inability to culture Phytoplasmas has limited the generation of genome sequences and, currently, only 42 are listed in NCBI’s Genome database.The the elm yellows group (16SrV group) represents the third largest phytoplasma cluster after the aster yellows and X-disease phytoplasma groups. The phytoplasmas of group 16SrV are associated with serious diseases such as elm yellows, grapevine yellows, Rubus stunt, cherry lethal yellows, peach yellows, jujube witches’-broom, and alder yellows. In August 2018, a phytoplasma strain in the 16SrV group was identified from a red alder (Alnus rubra) displaying leaf yellowing symptoms. Primer pair P1/16S-SR followed by P1A/16S-SR were used in a seminested PCR to amplify the phytoplasma 16S ribosomal DNA region (Deng and Hiruki 1991; Lee et al. 2004) and the resulting sequence was deposited in GenBank (accession MZ557341). Results from the NCBI BLASTn search of the 16S ribosomal RNA (rRNA) gene sequence MZ557341 showed 99.9% identities with the reference strain of ‘Candidatus Phytoplasma vitis’ (FD70, AF176319) (Davis and Dally 2001), followed by 99.7% identities with the reference strain of ‘Ca. P. ulmi’ (EY1, AY197655) (Lee et al. 2004) and ‘Ca. P. rubi’ (99.5%, RuS, AY197648) (Lee et al. 2004). Additional phytoplasma-positive samples were collected from red alder trees at public sites in many Washington State counties (T. M. Wilson, N. I. Chambers, W. Cai, S. K. Brown, S. Dickerson, J. S. Falacy, and S. Costanzo, unpublished data).Here, we report the first draft genome sequence of the ‘Ca. Phytoplasma’ strain alder yellows-Washington 1 (AldY-WA1), which provides a resource for comparative genomics of phytoplasmas in the elm yellows group.Genomic DNA was extracted from symptomatic leaf tissue using the DNeasy PowerPlant Pro kit (Qiagen, Valencia, CA). The phytoplasma titer level present in the host tissue was estimated using a 23S rRNA gene-based real-time quantitative PCR with primers and probe for the “universal” assay (JH-F 1, JH-F, JH-R, and JH-P uni) as described by Hodgetts et al. (2009). A genomic DNA sample producing the lowest threshold cycle (CT) value (CT14), indicating a higher pathogen titer, was selected for DNA library preparation. Both Illumina short-read and Nanopore long-read sequencing technologies were used to generate the draft genome. The Illumina paired-end sequencing libraries were prepared using the Illumina Nextera DNA Flex library prep kit. Illumina (2 × 300-bp paired-end) sequencing was performed on the Illumina MiSeq platform (Illumina, Inc., San Diego, CA). The Nanopore single-end library was prepared using Nanopore ligation sequencing kit SQK-LSK109. Nanopore sequencing was performed on the MIN106 flow cell R9.4 using the MinION portable device (Oxford Nanopore Technologies PLC, Oxford, U.K.). Basecalling for Nanopore sequencing was done in real-time using MinKNOW (version 19.06.7).In total, 4.91 × 107 reads with a mean read length of 269 bp were generated from MiSeq sequencing and 1,673,188 reads with a mean read length of 2,615.9 bp were obtained from Nanopore sequencing.Illumina reads were trimmed using Trimmomatic v0.39 (Bolger et al. 2014). Nanopore reads were trimmed using Porechop v0.2.1 (Wick et al. 2017). The trimmed reads from both technologies were mapped to the ‘Ca. P. ziziphi’ genome (NZ_CP025121) (Wang et al. 2018) using bowtie2 v2.3.5.1 (Langmead & Salzberg 2012) for Illumina reads (14.20% mapped) and minimap2 v2.18 (Li 2018) for Nanopore reads (2.35% mapped). Reads mapping to the reference genome were extracted using samtools v1.9 (Li et al. 2009) and used to create a hybrid genome assembly using the SPAdes assembler v 3.15.2 (Bankevich et al. 2012), generating 50 contigs, ranging from 204 to 84,212 bp (N50 = 27,562 bp). Resulting contigs were aligned to the A. glutinosa genome (GCA_003254965.1) using blastn, and no contigs were identified as originating from the host. The contigs consisted of a total length of 457,625 bp, with a G+C content of 22.36%. The completeness of the genome is estimated at 89.4% by BUSCO version 4.0.6 (Seppey et. al., 2019), with the mollicutes database consisting of 151 BUSCOs. Annotation was performed using Prokka v1.14.5 (Seemann 2014). The genome included 376 protein-coding sequences, 34 transfer RNAs, and two complete rrn operons (16S, 23S, and 5S rRNA). The coverage depth obtained from Illumina and Nanopore sequencing was 383× and 137×, respectively.Data AvailabilityThe AldY-WA1 draft genome sequence has been deposited under BioProject and BioSample SAMN19836676. This Whole Genome Shotgun project has been deposited at DNA Data Bank of Japan/European Nucleotide Archive/GenBank under the accession JAHRHX000000000. The version described in this article is version JAHRHX010000000. Data has also been deposited in the Sequence Read Archive (SRA) database, under SRA accessions SRR16501719 and SRR16501720.The author(s) declare no conflict of interest.Literature CitedBankevich, A., Nurk, S., Antipov, D., Gurevich, A., Dvorkin, M., Kulikov, A. S., Lesin, V., Nikolenko, S., Pham, S., Prjibelski, A., Pyshkin, A., Sirotkin, A., Vyahhi, N., Tesler, G., Alekseyev, M. A., and Pevzner, P. A. 2012. 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Genomics 3:e000132. https://doi.org/10.1099/mgen.0.000132 Crossref, ISI, Google ScholarThe findings and conclusions in this publication are those of the authors and should not be construed to represent any official United States Department of Agriculture or U.S. Government determination or policy.Funding: This research was supported by the United States Department of Agriculture Animal and Plant Health Inspection Service.The author(s) declare no conflict of interest.DetailsFiguresLiterature CitedRelated Vol. 106, No. 7 July 2022SubscribeISSN:0191-2917e-ISSN:1943-7692 Download Metrics Article History Issue Date: 8 Jul 2022Published: 26 May 2022Accepted: 15 Mar 2022 Pages: 1971-1973 Information© 2022 The American Phytopathological SocietyFundingUnited States Department of Agriculture Animal and Plant Health Inspection ServiceKeywords16SrValderelm yellows groupgenomephytoplasmasThe author(s) declare no conflict of interest.PDF download}, journal={PLANT DISEASE}, author={Cai, Weili and Nunziata, Schyler O. and Srivastava, Subodh K. and Wilson, Telissa and Chambers, Nathaniel and Rivera, Yazmin and Nakhla, Mark and Costanzo, Stefano}, year={2022}, month={May} }
 @article{salgado-salazar_romberg_blomquist_nunziata_cai_rivera_2022, title={Lifestyle, mating type and mitochondrial genome features of the plant pathogen Calonectria hawksworthii (Hypocreales, Nectriaceae) as revealed by genome analyses}, ISSN={["1715-2992"]}, DOI={10.1080/07060661.2022.2065534}, abstractNote={Abstract In 2019, fungal necrotic spots were observed on the cotyledons of grafted avocado (Persea americana) seedlings from a nursery in Ventura County, California. Morphology and comparison of DNA sequences from eight loci identified the isolate as Calonectria hawksworthii, a species in the Nectriaceae not yet recorded in the United States. Calonectria hawksworthii is a necrotrophic fungal pathogen in the C. cylindrospora species complex. Most species in this group are associated with Eucalyptus, but the type host for C. hawksworthii is waterlilies (Nelumbo nucifera). In this study, the C. hawksworthii genome was sequenced using Illumina technologies. The draft assembly of 64.8 Mb contained 18 703 predicted gene models, of which 70% could be assigned to a GO functional category. The mating type loci indicated this species is heterothallic. Approximately, 3.79% of the draft genome consists of transposable elements (TE), and close to 36% of the predicted proteins were homologous to those known to be involved in pathogenicity in other fungal species. The C. hawksworthii genome displays elements typical of a necrotrophic lifestyle based on the composition of predicted genes encoding for carbohydrate-active enzymes, number of predicted secretory proteins including effectors, secondary metabolite biosynthesis clusters and cytochrome oxidase P450 (CYP) genes. The annotated mitochondrial genome was found to be 35.7 kb with very few intra or intergenic introns. This report constitutes the first draft genome of a Calonectria species belonging to the C. cylindrospora species complex, as well as the first report of C. hawksworthii species occurring in the United States on avocado.}, journal={CANADIAN JOURNAL OF PLANT PATHOLOGY}, author={Salgado-Salazar, Catalina and Romberg, Megan K. and Blomquist, Cheryl and Nunziata, Schyler and Cai, Weili and Rivera, Yazmin}, year={2022}, month={May} }
 @article{cai_shao_zhao_davis_costanzo_2020, title={Draft Genome Sequence of 'Candidatus Phytoplasma pini'-Related Strain MDPP: A Resource for Comparative Genomics of Gymnosperm -Infecting Phytoplasmas}, volume={104}, ISSN={["1943-7692"]}, DOI={10.1094/PDIS-10-19-2127-A}, abstractNote={ ‘Candidatus Phytoplasma pini’-related strain MDPP, the reference strain of subgroup 16SrXXI-B, is a pathogen associated with witches’ broom disease of Pinus spp. in North America. Here, we report the first draft genome sequence of ‘Ca. Phytoplasma pini’ strain MDPP, which consists of 474,136 bases, with a G + C content of 22.22%. This information will facilitate comparative genomics of gymnosperm-infecting phytoplasmas. }, number={4}, journal={PLANT DISEASE}, author={Cai, Weili and Shao, Jonathan and Zhao, Yan and Davis, Robert E. and Costanzo, Stefano}, year={2020}, month={Apr}, pages={1009–1010} }
 @article{cai_nunziata_costanzo_kumagai_rascoe_stulberg_2020, title={Genome Resource for the Huanglongbing Causal Agent 'Candidatus Liberibacter asiaticus' Strain AHCA17 from Citrus Root Tissue in California, USA}, volume={104}, ISSN={["1943-7692"]}, DOI={10.1094/PDIS-08-19-1735-A}, abstractNote={ ‘Candidatus Liberibacter asiaticus’ is the unculturable causative agent of citrus huanglongbing disease. Here, we report the first citrus root metagenome sequence containing the draft genome of ‘Ca. L. asiaticus’ strain AHCA17, obtained from a pummelo tree in California. The assembled genome was 1.2 Mbp and resulted in 37 contigs (N50 = 158.7 kbp) containing 1,057 predicted open reading frames and 45 RNA-coding genes. This draft genome will provide a valuable resource in further study of ‘Ca. L. asiaticus’ genome diversity and pathogen epidemiology. }, number={3}, journal={PLANT DISEASE}, author={Cai, Weili and Nunziata, Schyler and Costanzo, Stefano and Kumagai, Lucita and Rascoe, John and Stulberg, Michael J.}, year={2020}, month={Mar}, pages={627–629} }
 @article{cai_nunziata_rascoe_stulberg_2019, title={SureSelect targeted enrichment, a new cost effective method for the whole genome sequencing of Candidatus Liberibacter asiaticus}, volume={9}, ISSN={["2045-2322"]}, DOI={10.1038/s41598-019-55144-4}, abstractNote={Abstract}, journal={SCIENTIFIC REPORTS}, author={Cai, Weili and Nunziata, Schyler and Rascoe, John and Stulberg, Michael J.}, year={2019}, month={Dec} }