@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{srivastava_knight_nakhla_abad_2022, title={Genome Resources for the Ex-Type of Phytophthora citricola, and Well-Authenticated Isolates of P. hibernalis, P. nicotianae, and P. syringae}, volume={112}, ISSN={["1943-7684"]}, DOI={10.1094/PHYTO-04-21-0167-A}, abstractNote={Phytophthora is one of the most important genera of plant pathogens, with many members causing high economic losses worldwide. To build robust molecular identification systems, it is very important to have information from well-authenticated specimens and, in preference, the ex-type specimens. The reference genomes of well-authenticated specimens form a critical foundation for genetics, biological research, and diagnostic applications. In this study, we describe four draft Phytophthora genome resources for the ex-type of Phytophthora citricola BL34 (P0716 WPC) (118 contigs for 50 Mb), and well-authenticated specimens of P. syringae BL57G (P10330 WPC) (591 contigs for 75 Mb), P. hibernalis BL41G (P3822 WPC) (404 contigs for 84 Mb), and P. nicotianae BL162 (P6303 WPC) (3,984 contigs for 108 Mb) generated with MinION long-read high-throughput sequencing technology (Oxford Nanopore Technologies). Using the quality reads, we assembled high-coverage genomes of P. citricola with 291× coverage and 16,662 annotated genes; P. nicotianae with 205× coverage and 29,271 annotated genes; P. syringae with 76× coverage and 23,331 annotated genes, and P. hibernalis with 42× coverage and 21,762 annotated genes. With the availability of genome sequences and their annotations, we predict that these draft genomes will be accommodating for various basic and applied research, including diagnostics to protect global agriculture.}, number={4}, journal={PHYTOPATHOLOGY}, author={Srivastava, Subodh K. and Knight, Leandra M. and Nakhla, Mark and Abad, Z. Gloria}, year={2022}, month={Apr}, pages={953–955} }
 @article{abad_burgess_redford_bienapfl_srivastava_mathew_jennings_2022, title={IDphy: An International Online Resource for Molecular and Morphological Identification of Phytophthora}, ISSN={["1943-7692"]}, DOI={10.1094/PDIS-02-22-0448-FE}, abstractNote={ Phytophthora, with 203 species, is a genus of high importance in agriculture worldwide. Here, we present the online resource “IDphy”, developed to facilitate the correct identification of species of Phytophthora using the type specimens from the original descriptions wherever possible. IDphy emphasizes species of high economic impact and regulatory concern for the United States. IDphy presents an interactive Lucid key and a tabular key for 161 culturable species described as of May 2018, including 141 ex-types and 20 well-authenticated specimens. IDphy contains standard operating procedures for morphological and molecular characterization, as well as a glossary, image gallery, and numerous links. Each of the 161 factsheets includes access to nomenclature and morphological and molecular features, including sequences of the internal transcribed spacer ribosomal DNA, cytochrome C oxidase subunit I (barcoding genes), YPT1, β-tubulin, elongation factor 1a, L10, heat shock protein 90, and other genes. IDphy contains an innovative in silico BLAST and phylogenetic sequence analysis using NCBI. The IDphy mobile app, released in August 2021 (free for Android or iOS), allows users to take the Lucid key into the laboratory. IDphy is the first online identification tool based on the ex-types implemented for plant pathogens. In this article, we also include information for 21 new species and one hybrid described after the publication of IDphy, the status of the specimens of the types and ex-types at international herbaria and culture collections, and the status of genomes at the GenBank (currently 153 genome assemblies which correspond to 42 described species, including 16 ex-types). The effectiveness of the IDphy online resource and the content of this article could inspire other researchers to develop additional identification tools for other important groups of plant pathogens. }, journal={PLANT DISEASE}, author={Abad, Z. Gloria and Burgess, Treena I. and Redford, Amanda J. and Bienapfl, John C. and Srivastava, Subodh and Mathew, Reny and Jennings, Krysta}, year={2022}, month={Jul} }
 @article{srivastava_zeller_sobieraj_nakhla_2021, title={Genome Resources of Four Distinct Pathogenic Races Within Fusarium oxysporum f. sp. vasinfectum that Cause Vascular Wilt Disease of Cotton}, volume={111}, ISSN={["1943-7684"]}, DOI={10.1094/PHYTO-07-20-0298-A}, abstractNote={ Whole genome sequence (WGS) based identifications are being increasingly used by regulatory and public health agencies to facilitate the detection, investigation, and control of pathogens and pests. Fusarium oxysporum f. sp. vasinfectum is a significant vascular wilt pathogen of cultivated cotton and consists of several pathogenic races that are not each other’s closest phylogenetic relatives. We have developed WGS assemblies for isolates of F. oxysporum f. sp. vasinfectum race 1 (FOV1), race 4 (FOV4), race 5 (FOV5), and race 8 (FOV8) using a combination of Nanopore (MinION) and Illumina sequencing technology (Mi-Seq). This resulted in assembled contigs with more than 100× coverage for each of the F. oxysporum f. sp. vasinfectum races and estimated genome sizes of FOV1 52 Mb, FOV4 68 Mb, FOV5 68 Mb, and FOV8 55 Mb. The AUGUSTUS gene prediction program predicted 16,263 genes in FOV1, 20,259 genes in FOV4, 20,375 genes in FOV5 and 16,615 genes in FOV8. We were able to identify 525 genes unique to FOV1, 570 unique to FOV4, 1,242 unique to FOV5, and 383 unique to FOV8. We expect that these findings will help in comparative genomics and in the identification of unique genes as candidate targets for diagnostic marker and methods development to permit rapid differentiation of F. oxysporum f. sp. vasinfectum subgroups. }, number={3}, journal={PHYTOPATHOLOGY}, author={Srivastava, Subodh K. and Zeller, Kurt A. and Sobieraj, James H. and Nakhla, Mark K.}, year={2021}, month={Mar}, pages={593–596} }
 @article{srivastava_abad_knight_zeller_mavrodieva_nakhla_2020, title={Draft Genome Resource for the Ex-types of Phytophthora ramorum, P. kernoviae, and P. melonis, Species of Regulatory Concern, Using Ultra-Long Read MinION Nanopore Sequencing}, volume={33}, ISSN={["1943-7706"]}, DOI={10.1094/MPMI-12-19-0342-A}, abstractNote={ Phytophthora ramorum, P. kernoviae, and P. melonis are each species of current regulatory concern in the United States, the United Kingdom, and other areas of the world. Ex-type material are cultures and duplicates of the type that was used to describe each species and that are deposited in additional culture collections. Using these type specimens as references is essential to designing correct molecular identification and diagnostic systems. Here, we report a whole genome sequence for the Ex-type material of P. ramorum, P. kernoviae, and P. melonis generated using high-throughput sequencing via the MinION third generation platform from Oxford Nanopore Technology. We assembled the quality filtered reads into contigs for each species. We assembled the continuous contigs of P. ramorum, P. kernoviae, and P. melonis (1,322, 545, and 2,091 contigs, respectively). The ab initio prediction of genes from these species reveals that there are 16,838, 12,793, and 34,580 genes in P. ramorum, P. kernoviae, and P. melonis, respectively. Of the 34,580 P. melonis genes, 10,164 genes were conserved among all three of these Phytophthora species which may include pathogenicity genes. We compared the ex-type of P. ramorum EU1 lineage assembly with another selected isolate of EU1 available at the National Center for Biotechnology Information and found 251,859 single nucleotide polymorphisms (SNPs) genome-wide; the comparison with the EU2 lineage genome isolate revealed 441,859 SNPs genome-wide. This genome resource of the ex-types of P. ramorum, and P. kernoviae is a significant contribution as these species are among the most important pathogens of regulatory concern in different regions of the world. }, number={6}, journal={MOLECULAR PLANT-MICROBE INTERACTIONS}, author={Srivastava, Subodh K. and Abad, Z. Gloria and Knight, Leandra M. and Zeller, Kurt and Mavrodieva, Vessela and Nakhla, Mark}, year={2020}, month={Jun}, pages={794–797} }
 @article{rivera_zeller_srivastava_sutherland_galvez_nakhla_poniatowska_schnabel_sundin_abad_2018, title={Draft Genome Resources for the Phytopathogenic Fungi Monilinia fructicola, M. fructigena, M. polystroma, and M. laxa, the Causal Agents of Brown Rot}, volume={108}, ISSN={["1943-7684"]}, DOI={10.1094/PHYTO-12-17-0418-A}, abstractNote={ Fungi in the genus Monilinia cause brown rot disease of stone and pome fruits. Here, we report the draft genome assemblies of four important phytopathogenic species: M. fructicola, M. fructigena, M. polystroma, and M. laxa. The draft genome assemblies were 39 Mb (M. fructigena), 42 Mb (M. laxa), 43 Mb (M. fructicola), and 45 Mb (M. polystroma) with as few as 550 contigs (M. laxa). These are the first draft genome resources publicly available for M. laxa, M. fructigena, and M. polystroma. }, number={10}, journal={PHYTOPATHOLOGY}, author={Rivera, Yazmin and Zeller, Kurt and Srivastava, Subodh and Sutherland, Jeremy and Galvez, Marco and Nakhla, Mark and Poniatowska, Anna and Schnabel, Guido and Sundin, George and Abad, Z. Gloria}, year={2018}, month={Oct}, pages={1141–1142} }