The highly invasive field bindweed (Convolvulus arvensis), native to Europe and Asia, competes with crops, reduces yields, and acts as a reservoir for plant pathogens, increasing the potential for disease spread in agricultural systems (Sosnoskie et al. 2020; Wenninger et al. 2019). In July 2023, bindweed plants exhibiting little leaf, leaf discoloration, thickening, and overall stunted growth were observed in a 0.3 ha green cherry orchard in Kharja, Bani Kenanah, Irbid Governorate, Jordan (32°37'37.883"N, 35°52'49.185"E) (Fig. 1). Twenty out of 35 plants displayed symptoms, resulting in a 57% incidence rate. Since the symptoms resembled those associated with phytoplasma infections and bindweed's known role as a phytoplasma host (Jovic et al. 2021), DNA was extracted from six symptomatic plants and two healthy controls using the Qiagen DNeasy Plant Mini Kit, and PCR assay was conducted with the primer pair P1A/16S-SR to amplify near full-length phytoplasma 16S rDNA (about 1.5 kb) as described by Lee et al. (2004). All healthy controls tested negative, while all symptomatic plants tested positive. Amplicons were cloned into the pCRII-TOPO vector, sequenced, and a representative sequence was deposited in GenBank (Accession number PQ497569). Using iPhyClassifier (Zhao et al. 2009), the strain was identified as a 'Candidatus Phytoplasma omanense' (GenBank accession: EF666051, 16SrXXIX-A)-related strain, with 98.95% sequence identity, belonging to the 16SrXXIX group. The similarity coefficients were 0.93 and 0.97 compared to reference strains 16SrXXIX-A (EF666051) and 16SrXXIX-B (OL873126, Abu Alloush et al. 2023a), respectively. The phylogenetic analysis indicated that the newly detected field bindweed little leaf phytoplasma strain (designated FBLL1) is genetically distinct from these known subgroups and represents a novel subgroup, 16SrXXIX-C (Fig. 2). Further characterization of the FBLL1 strains was performed by PCR amplification of the rp (1.9 kb) and secY (1.3 kb) gene segments using specially designed primers: FBRP-F2/FBRP-R4 (5'-TTGGCCGCCTTCAAATCCTA-3'/5'-TCGGAGGAGAAGTTTTGGCT-3') for rp, and FBSecYF1/FBSecR1 (5'-CTTCTTTTGGTGATATCCCA-3'/5'-TGGCGGAAGTATTGAGATAAGAA-3') for secY. The resulting rp and secY gene sequences were deposited in GenBank (Accession numbers PQ505136 and PQ505137). BLASTN analysis revealed that the rp gene's top hit was 'Ca. P. pruni' (CP119306) with 75.60% identity, while the secY gene's top hit was 'Ca. P. phoenicium' (MN076652), with 73.47% identity. Compared to the 16S rRNA gene, the lower percent identity is due to the limited availability of 16SrXXIX rp and secY gene sequences for comparison. Field bindweed hosts several phytoplasmas, such as 'Ca. P. solani' (16SrXII-A), which is linked to Bois noir (BN) in grapevines, and 'Ca. P. convolvuli' (16SrXII-H), associated with bindweed yellows (Jovic et al. 2021; Abu Alloush et al. 2023b). 'Ca. P. omanense' has also been documented in bindweed in Lebanon (Fossaic et al. 2019) and Iran (Esmailzadeh Hosseini et al. 2016). As a confirmed reservoir of the BN phytoplasma, bindweed supports the pathogen's persistence and vector-mediated transmission to grapevines (Jovic et al. 2021). This report marks the first identification of 'Ca. P. omanense'-related strains (16SrXXIX-C, novel subgroup) in bindweed in Jordan. These findings suggest that bindweed may serve as a potential reservoir for a broader range of phytoplasmas than previously recognized, with a wider geographical distribution. Its invasive growth near crops, such as grapevines, raises concerns about the potential phytoplasma spillover. This highlights the need for improved monitoring and management, which includes targeted herbicide application, mechanical removal, and biological control to protect agricultural biosecurity.

Dichorhavirus is a recently accepted plant virus genus within the family Rhabdoviridae. Species assigned to the genus consist of bi-segmented, negative sense, single-stranded RNA viruses and are transmitted by Brevipalpus spp. Currently, there are five recognized species and two unclassified members in the genus Dichorhavirus. Four out of seven-orchid fleck virus (OFV), citrus leprosis virus N, citrus chlorotic spot virus, and citrus bright spot virus-can infect citrus and produce leprosis disease-like symptoms. The E-probe Diagnostic for Nucleic Acid Analysis (EDNA) was developed to reduce computational effort and then integrated within Microbe-Finder (MiFi®) online platform to design and evaluate e-probes in raw High Throughput Sequencing (HTS) data. During this study, Dichorhavirus genomes were downloaded from public databases and e-probes were designed using the MiProbe incorporated into the MiFi® platform. Three different sizes of e-probes, 40, 60, and 80 nucleotides, were developed and selected based on whole genome comparisons with near-neighbor genomes. For curation, each e-probe was searched in the NCBI nucleotide sequence database using BLASTn. All the e-probes that had hits with non-target species with ≥90% identities were removed. The sensitivity and specificity of Dichorhavirus genus, species, strain, and variant-specific e-probes were validated in vivo using HTS meta-transcriptomic libraries generated from Dichorhavirus-suspected citrus, orchid, and ornamentals. Through downstream analysis of HTS data, EDNA not only detected the known hosts of OFV but also discovered an unknown host leopard plant (Farfugium japonicum), and the possible existence of a new ornamental strain of OFV in nature.

A Ralstonia-infecting jumbo phage was isolated from soil in Maryland, USA. The complete genome sequence was determined to be 225,638 bp and has a G+C content of 51.8%.

The oomycete Phytophthora palmivora infects the fruit of cacao trees (Theobroma cacao) causing black pod rot and reducing yields. Cacao genotypes vary in their resistance levels to P. palmivora, yet our understanding of how cacao fruit respond to the pathogen at the molecular level during disease establishment is limited. To address this issue, disease development and RNA-Seq studies were conducted on pods of seven cacao genotypes (ICS1, WFT, Gu133, Spa9, CCN51, Sca6 and Pound7) to better understand their reactions to the post-penetration stage of P. palmivora infection. The pod tissue-P. palmivora pathogen assay resulted in the genotypes being classified as susceptible (ICS1, WFT, Gu133 and Spa9) or resistant (CCN51, Sca6 and Pound7). The number of differentially expressed genes (DEGs) ranged from 1625 to 6957 depending on genotype. A custom gene correlation approach identified 34 correlation groups. De novo motif analysis was conducted on upstream promoter sequences of differentially expressed genes, identifying 76 novel motifs, 31 of which were over-represented in the upstream sequences of correlation groups and associated with gene ontology terms related to oxidative stress response, defense against fungal pathogens, general metabolism and cell function. Genes in one correlation group (Group 6) were strongly induced in all genotypes and enriched in genes annotated with defense-responsive terms. Expression pattern profiling revealed that genes in Group 6 were induced to higher levels in the resistant genotypes. An additional analysis allowed the identification of 17 candidate cis-regulatory modules likely to be involved in cacao defense against P. palmivora. This study is a comprehensive exploration of the cacao pod transcriptional response to P. palmivora spread after infection. We identified cacao genes, promoter motifs, and promoter motif combinations associated with post-penetration resistance to P. palmivora in cacao pods and provide this information as a resource to support future and ongoing efforts to breed P. palmivora-resistant cacao.

IntroductionAntimicrobial peptides (AMPs) are promising alternatives to traditional antibiotics for combating plant pathogenic bacteria in agriculture and the environment. However, identifying potent AMPs through laborious experimental assays is resource-intensive and time-consuming. To address these limitations, this study presents a bioinformatics approach utilizing machine learning models for predicting and selecting AMPs active against plant pathogenic bacteria.MethodsN-gram representations of peptide sequences with 3-letter and 9-letter reduced amino acid alphabets were used to capture the sequence patterns and motifs that contribute to the antimicrobial activity of AMPs. A 5-fold cross-validation technique was used to train the machine learning models and to evaluate their predictive accuracy and robustness.ResultsThe models were applied to predict putative AMPs encoded by intergenic regions and small open reading frames (ORFs) of the citrus genome. Approximately 7% of the 10,000-peptide dataset from the intergenic region and 7% of the 685,924-peptide dataset from the whole genome were predicted as probable AMPs. The prediction accuracy of the reported models range from 0.72 to 0.91. A subset of the predicted AMPs was selected for experimental test against Spiroplasma citri, the causative agent of citrus stubborn disease. The experimental results confirm the antimicrobial activity of the selected AMPs against the target bacterium, demonstrating the predictive capability of the machine learning models.DiscussionHydrophobic amino acid residues and positively charged amino acid residues are among the key features in predicting AMPs by the Random Forest Algorithm. Aggregation propensity appears to be correlated with the effectiveness of the AMPs. The described models would contribute to the development of effective AMP-based strategies for plant disease management in agricultural and environmental settings. To facilitate broader accessibility, our model is publicly available on the AGRAMP (Agricultural Ngrams Antimicrobial Peptides) server.

We report here the draft genome sequence of Xylella fastidiosa strain ATCC 35874. The strain was originally isolated from infected red oak in Washington, DC, and obtained from the American Type Culture Collection. The ATCC 35874 genome contains 2,543,332 bp and has a G + C content of 51.72%.

Polyamines (PA) cellular levels are maintained through a balance between synthesis and catabolism, achieved by two classes of enzymes polyamine oxidases (PAOs) and copper amine oxidases (CuAO). Here we investigated the occurrence, molecular evolution and role(s) of PAOs and CuAO gene families in aquatic duckweed and their comparison with other aquatic plants -sea eelgrass, bladderwort, and Lotus. We identified eight bona fide PAO genes (SpPAO1–SpPAO8) and one SpCuAO1 in the greater duckweed genome from three genome assemblies. Interestingly, duckweed PAO genes increased their number through a tandem duplication event, while contrary to this CuAO genes were significantly lost to a single gene SpCuAO1. Phylogenetic analysis revealed that tandemly duplicated SpPAO2-7 share close similarity to well-known terminal catabolism (TC) pathway PAO genes while SpPAO1 and SpPAO8 seem to segregate along with back conversion (BC) participating known PAO genes, suggesting that all tandem duplicated PAOs are involved in TC pathway which is contrary to known trend in land plants where CuAOs are mainly involved in TC pathway. Comparative transcript abundance studies indicated that all eight PAOs and one CuAO gene respond to multiple stresses and principal component analysis identifies SpPAO4 as a highly active gene in response to multiple stresses. Results showed that oxidation of higher polyamines (SPD/SPM) through the TC pathway is diversified in duckweeds. Taken together this study reveals unique insights into the genomic losses and gains of polyamine metabolism possibly involved in achieving the structural and physiological adaptations required for aquatic lifestyle of duckweeds.

Objectives: We present the protocol for a new data pooling initiative to investigate metabolomic markers of animal-based protein foods (ABPF) and omnivorous dietary patterns using metabolomics and machine learning. This initiative will strengthen scientific understanding of how consuming ABPF and omnivorous dietary patterns influence metabolomic and cardiometabolic responses of human research participants. Additionally, this protocol calls for a formalized collaboration in the form of a research consortium, called Metabol(om)ic Response to Omnivorous Dietary Patterns Using Randomized Controlled Feeding Trials (MET-OMNIV) to curate and expand this dataset. Methods: Data from 4 randomized controlled feeding trials (RCfT) will be pooled that met the following criteria: 1) foods and beverages provided, 2) crossover design with 4-6 week feeding phases, 3) primary objective to investigate cardiometabolic effects of consuming ABPF in the context of various dietary patterns, mostly weight-maintenance, and 4) detailed descriptions of foods and dietary patterns, including specifics to protein source and degree and type of processing. Data related to participant characteristics, menus, and cardiometabolic measures at baselines and post-feeding will be harmonized. Results: Data from 4 randomized controlled feeding trials (RCfT) will be pooled that met the following criteria: 1) foods and beverages provided, 2) crossover design with 4-6 week feeding phases, 3) primary objective to investigate cardiometabolic effects of consuming ABPF in the context of various dietary patterns, mostly weight-maintenance, and 4) detailed descriptions of foods and dietary patterns, including specifics to protein source and degree and type of processing. Data related to participant characteristics, menus, and cardiometabolic measures at baselines and post-feeding will be harmonized. Conclusions: This pooled dataset will provide an excellent resource for those who wish to study plasma metabolomic markers of food consumption, especially the consumption of ABPF. Funding Sources: Beef Checkoff Agricultural Research Service SCINet and AI Center of Excellence ORISE Fellowship Program Agriculture Research Service, USDA.

Genetic improvements of solanaceous crops for quality and stress responsive traits are needed because of the central role vegetables and fruits have in providing nutrients to human diets. Copper amine oxidase (CuAO) encoding genes involved in metabolism of primary/di-amine nitrogenous compounds, play a role in balancing internal nitrogen (N) pools especially when external N supply fluctuates during growth, development and environmental stresses. In the present study, we investigated the occurrence, molecular evolution and possible role(s) of these unknown genes in tomato crops. Multiple genome-wide bioinformatics approaches led to the identification of eight bona fide CuAO genes (SlCuAO1-SlCuAO8) in the tomato genome with gene numbers like those in Arabidopsis and rice indicating their conserved functional relevance with a tandemly duplicated SlCuAO6-SlCuAO7 pair at chr.9. A conserved intron-exon size and phase distribution for SlCuAO2, 3, 4 pairs are similar to a recently identified single duckweed SpCuAO1 orthologue gene indicating its evolutionary conservation. Synteny analysis showed their closest association to Arabidopsis and but not with rice. Transcriptome data indicated that gene expression for about six genes (SlCuAO1, 2, 3, 4, 6, 7) is root specific, fruit specific for SlCuAO5 and flower specific for SlCuAO8 thus indicating amine oxidation is variable across tissues with a prominance in the root tissue. The majority of CuAO genes are negatively regulated by methyl jasmonate. Positive regulation, however, involves CuAO3/8. Transcript analysis of the ethylene-deficient transgenic lines indicated that ethylene is required for activation of SlCuAO4. CuAO4 and CuAO5 exhibited most significant tissues-independent gene expression responses across various nitrogen regimes. Drought, heat and N stress identified CuAO5 as an overlapping highly expressed gene that corroborates with putrescine accumulation for free and conjugated forms with an opposite abundance of bound forms. Taken together our study highlights new insights into the roles of copper amine oxidation genes and identifies CuAO5 as a multiple stress induced gene that can be used in genetic improvement programs for combining heat, drought and nitrogen use efficiency related traits.

Phytoplasmas are small, intracellular bacteria that infect a vast range of plant species, causing significant economic losses and impacting agriculture and farmers’ livelihoods. Early and rapid diagnosis of phytoplasma infections is crucial for preventing the spread of these diseases, particularly through early symptom recognition in the field by farmers and growers. A symptom database for phytoplasma infections can assist in recognizing the symptoms and enhance early detection and management. In this study, nearly 35,000 phytoplasma sequence entries were retrieved from the NCBI nucleotide database using the keyword “phytoplasma” and information on phytoplasma disease-associated plant hosts and symptoms was gathered. A total of 945 plant species were identified to be associated with phytoplasma infections. Subsequently, links to symptomatic images of these known susceptible plant species were manually curated, and the Phytoplasma Disease Symptom Database (iPhyDSDB) was established and implemented on a web-based interface using the MySQL Server and PHP programming language. One of the key features of iPhyDSDB is the curated collection of links to symptomatic images representing various phytoplasma-infected plant species, allowing users to easily access the original source of the collected images and detailed disease information. Furthermore, images and descriptive definitions of typical symptoms induced by phytoplasmas were included in iPhyDSDB. The newly developed database and web interface, equipped with advanced search functionality, will help farmers, growers, researchers, and educators to efficiently query the database based on specific categories such as plant host and symptom type. This resource will aid the users in comparing, identifying, and diagnosing phytoplasma-related diseases, enhancing the understanding and management of these infections.

Through the recent advances of modern high-throughput sequencing technologies, the "one microbe, one disease" dogma is being gradually replaced with the principle of the "pathobiome". Pathobiome is a comprehensive biotic environment that not only includes a diverse community of all disease-causing organisms within the plant but also defines their mutual interactions and resultant effect on plant health. To date, the concept of pathobiome as a major component in plant health and sustainable production of alfalfa (Medicago sativa L.), the most extensively cultivated forage legume in the world, is non-existent. Here, we approached this subject by characterizing the biodiversity of the alfalfa pathobiome using high-throughput sequencing technology. Our metagenomic study revealed a remarkable abundance of different pathogenic communities associated with alfalfa in the natural ecosystem. Profiling the alfalfa pathobiome is a starting point to assess known and identify new and emerging stress challenges in the context of plant disease management. In addition, it allows us to address the complexity of microbial interactions within the plant host and their impact on the development and evolution of pathogenesis.

The non-climacteric octoploid strawberry (Fragaria x ananassa Duchesne ex Rozier) was used as a model to study its regulation during fruit ripening. High performance liquid chromatography electrospray tandem-mass spec-trometry (HPLC-ESI-MS/MS) was employed to profile 28 different endogenous phytohormones in strawberry. These include auxins, cytokinins (CKs), abscisic acid (ABA), ethylene precursor 1-aminocyclopropane-1-carbox-ylic acid (ACC), jasmonates, and phenolic compounds salicylic acid (SA), benzoic acid (BzA) and phenylacetic acid (PAA) together with their various metabolic forms that have remained largely unexplored thus far. ABA, ACC and CK N6-(A2-isopentenyl)adenine (iP) were found to be associated with ripening while ABA catabolites 9-hydroxy-ABA and phaseic acid mimicked the pattern of climacteric decline at the turning phase of strawberry ripening. The content of other CK forms except iP decreased as fruit ripened, as also that of auxins indole-3-acetic acid (IAA) and oxo-IAA, and of jasmonates. Data presented here also suggest that both the transition and pro -gression of strawberry fruit ripening are associated with N6-(A2-isopentenyl)adenosine-5 '-monophosphate (iPRMP)-* N6-(A2-isopentenyl)adenosine (iPR)-* iP as the preferred CK metabolic pathway. In contrast, the ethylene precursor ACC was present at higher levels, with its abundance increasing from the onset of ripening to the red ripe stage. Further investigation of ripening-specific ACC accumulation revealed the presence of a large ACC synthase (ACS) encoding gene family in octoploid strawberry that was previously unknown. Seventeen ACS genes were found differentially expressed in fruit tissues, while six of them showed induced expression during strawberry fruit ripening. These data suggest a possible role(s) of ACC, ABA, and iP in strawberry fruit ripening. These data add new dimension to the existing knowledge of the interplay of different endogenous phytohor-mones in octoploid strawberry, paving the way for further investigation of their individual role(s) in fruit ripening.

Thread blight disease (TBD) is a serious emerging threat to global cacao production, causing leaf blight symptoms and necrosis in the canopy of infected trees. Though previously thought to be caused primarily by the fungus Marasmiellus scandens, recent work has revealed that a variety of pathogenic Marasmiaceae are responsible for TBD, with Marasmius tenuissimus identified as the dominant causal agent of TBD in Ghanaian cacao plantations. M. tenuissimus has also been reported in remote organic cacao plantations in the Amazonas Department of Peru, infecting 90 to 100% of the trees surveyed. Here, we have assembled and annotated the nuclear genome of M. tenuissimus isolate GH-37. This resource is a first attempt at a high-quality draft genome utilizing both second- and third-generation sequencing for M. tenuissimus and extends the current understanding of the evolution, ecology, and virulence of this pathogen. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Thirteen draft genome assemblies are presented for four Colletotrichum gloeosporioides complex species, namely, Colletotrichum aeschynomenes , Colletotrichum asianum , Colletotrichum fructicola , and Colletotrichum siamense , which were isolated from tropical tree hosts as endophytes.

Pseudohyphozyma bogoriensis is gaining attention as a microbial source of high-value sophorolipids. We report here on its genomic sequence, which will improve our understanding of its metabolic pathways and allow the development of genome manipulation systems. PacBio sequencing was performed, yielding a 26-Mbp genome with 57% GC content and encoding 7,847 predicted proteins.

Phytoplasmas are small phloem-restricted and insect-transmissible bacteria that infect many plant species, including important crops and ornamental plants, causing severe economic losses. Our previous studies screened phytoplasmas in hundreds of leafhoppers collected from natural habitats worldwide and identified multiple genetically different phytoplasmas in seven leafhopper species (potential insect vectors). As an initial step toward determining the impact of these phytoplasmas on the ecosystem, ribulose 1,5-biphosphate carboxylase large subunit (rbcL), a commonly used plant DNA barcoding marker, was employed to identify the plant species that the phytoplasma-harboring leafhoppers feed on. The DNA of 17 individual leafhoppers was PCR amplified using universal rbcL primers. PCR products were cloned, and five clones per amplicon were randomly chosen for Sanger sequencing. Moreover, Illumina high-throughput sequencing on selected PCR products was conducted and confirmed no missing targets in Sanger sequencing. The nucleotide BLAST results revealed 14 plant species, including six well-known plant hosts of phytoplasmas such as tomato, alfalfa, and maize. The remaining species have not been documented as phytoplasma hosts, expanding our knowledge of potential plant hosts. Notably, the DNA of tomato and maize (apparently cultivated in well-managed croplands) was detected in some phytoplasma-harboring leafhopper species sampled in non-crop lands, suggesting the spillover/spillback risk of phytoplasma strains between crop and non-crop areas. Furthermore, our results indicate that barcoding (or metabarcoding) is a valuable tool to study the three-way interactions among phytoplasmas, plant hosts, and vectors. The findings contribute to a better understanding of phytoplasma host range, host shift, and disease epidemiology.

HomePlant DiseaseVol. 107, No. 3Draft Genome Sequence Resource of CBPPT1, a 'Candidatus Phytoplasma trifolii'-Related Strain Associated with Potato Purple Top Disease in the Columbia Basin, U.S.A. PreviousNext RESOURCE ANNOUNCEMENT OPENOpen Access licenseDraft Genome Sequence Resource of CBPPT1, a 'Candidatus Phytoplasma trifolii'-Related Strain Associated with Potato Purple Top Disease in the Columbia Basin, U.S.A.Wei Wei, Jonathan Shao, Kristi D. Bottner-Parker, and Yan ZhaoWei Weihttps://orcid.org/0000-0003-3561-913XMolecular Plant Pathology Laboratory, USDA-ARS, Beltsville, MD 20705, Jonathan ShaoStatistics Group, Northeast Area Bioinformatics, USDA-ARS, Beltsville, MD 20705, Kristi D. Bottner-ParkerMolecular Plant Pathology Laboratory, USDA-ARS, Beltsville, MD 20705, and Yan Zhao†Corresponding author: Y. Zhao; E-mail Address: [email protected]https://orcid.org/0000-0002-0032-7535Molecular Plant Pathology Laboratory, USDA-ARS, Beltsville, MD 20705AffiliationsAuthors and Affiliations Wei Wei1 Jonathan Shao2 Kristi D. Bottner-Parker1 Yan Zhao1 † 1Molecular Plant Pathology Laboratory, USDA-ARS, Beltsville, MD 20705 2Statistics Group, Northeast Area Bioinformatics, USDA-ARS, Beltsville, MD 20705 Published Online:31 Dec 2022https://doi.org/10.1094/PDIS-08-22-1788-AAboutSectionsPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat Phytoplasmas are a large group of phloem-inhabiting, insect-transmitted plant pathogenic bacteria lacking a cell wall. They are etiological agents of diseases affecting more than a thousand plant species (Gasparich et al. 2020). Having descended from an ancestral gram-positive, low G + C walled bacterium, phytoplasmas underwent rapid evolution through substantial genome size reduction and recurrent horizontal gene acquisition (Wei et al. 2008; Zhao et al. 2014). Due to loss of many essential genes encoding diverse biosynthetic pathways (Kube et al. 2012; Oshima et al. 2004), phytoplasmas possess limited metabolic capacities and have a strong host dependency. Despite numerous attempts, pure phytoplasma culture has yet to be established in cell-free media. Although extant phytoplasmas have the same habitat specificity and life mode (Zhao et al. 2015) and comprise a monophyletic clade in 16S rRNA gene sequence-based phylogeny, the breadth of their genetic diversity is remarkable (Zhao et al. 2010). As unculturable bacteria, phytoplasmas are currently accommodated in a provisional genus 'Candidatus Phytoplasma' (The IRPCM Phytoplasma/Spiroplasma Working Team-Phytoplasma Taxonomy Group 2004). To date, 48 'Candidatus Phytoplasma' species have been formally described (Wei and Zhao 2022). Phytoplasmas are also classified into groups and subgroups based on collective actual enzymatic (Lee et al. 1993, 1998) or computer-simulated (Wei et al. 2007) restriction fragment length polymorphism (RFLP) profiles of their 16S rRNA genes. Thirty-seven such groups have been established in the classification scheme (Jones et al. 2021; Wei and Zhao 2022), with each group containing at least one 'Candidatus Phytoplasma' species. Potato purple top (PPT) is a worldwide-occurring potato disease complex attributed to infection by phytoplasmas. Common symptoms of the disease include purple discoloration of terminal shoots and formation of aerial tubers. Affected plants often wilt and die prematurely, causing substantial tuber yield and quality losses (Munyaneza et al. 2005). At least five mutually distinct 'Candidatus Phytoplasma' species, including 'Ca. P. asteris', 'Ca. P. aurantifolia', 'Ca. P. pruni', 'Ca. P. trifolii', and 'Ca. P. americanum', have been linked to PPT disease incidences that have occurred in various geographic areas (Lee et al. 2004, 2006; Santos-Cervantes et al. 2010). The Columbia Basin potato purple top (CBPPT) phytoplasma is the etiological agent responsible for the PPT epidemics that repeatedly occurred in the Pacific Northwest region of the U.S. since 2002 (Munyaneza et al. 2005). The CBPPT phytoplasma is a 'Ca. P. trifolii'-related strain and is classified in the clover proliferation phytoplasma (CP) group, subgroup A (16SrVI-A). The phytoplasma has a broad plant host range and is capable of infecting potato, tomato, broccoli, carrot, radish, and many other vegetable crops (Lee et al. 2004, 2006; Santos-Cervantes et al. 2010). Since the CBPPT phytoplasma induces virescence symptoms in several hosts and is transmitted from plant to plant by beet leafhopper (Circulifer tenellus, Munyaneza et al. 2010), it has a trivial name, beet leafhopper-transmitted virescence agent or BLTVA.The CBPPT phytoplasma and its alternative host, tomato, have been used as a model system to study phytoplasma–host interactions. Studies revealed that (i) a single infection of the CBPPT phytoplasma was able to induce multiple mutually distinct symptoms in tomato sequentially and (ii) the symptom type on a given branch varied depending on the developmental stage of the apex when it became affected by the phytoplasma (Wei et al. 2013, 2019). Such findings led to a hypothesis that phytoplasma infection can derail the genetically preprogrammed fate of a developing meristem, thereby changing the growth pattern and morphology of the host (Wei et al. 2013). Conceivably, phytoplasma pathogenesis is a multifaceted process that involves complex interactions between the pathogen and its plant host. Previous studies in our laboratory suggested that, in CBPPT phytoplasma-infected tomato plants, the homeostasis of auxin, cytokinin, and gibberellin was disrupted, as the biosynthesis, signaling, and distribution of these phytohormones were altered. Studies also revealed that CBPPT phytoplasma infection in tomato significantly changed the expression profiles of numerous genes including defense-related genes, key meristem switching genes, bioactive gibberellin synthesis genes, marker genes involved in premature leaf senescence, and axillary bud release (Ding et al. 2013; Wei et al. 2013; Wei and Zhao 2022). However, it remains unknown what pathogenicity factor(s) the CBPPT phytoplasma possesses and how these factors trigger the above- observed host responses. As the first step toward answering these questions, the genome of a representative strain of the CBPPT phytoplasma, CBPPT1, was sequenced.The phytoplasma strain CBPPT1 was originally identified in diseased potato plants growing in Washington State (Lee et al. 2004) and was transmitted to Madagascar periwinkle (Catharanthus roseus) via dodder (Cuscuta campestris), a parasitic vine. Once established in periwinkle, the strain was maintained in a greenhouse via serial shoot graft transmission. Over the years, the phytoplasma has consistently induced the same set of characteristic symptoms in the experimental host, indicating its pathogenicity remains essentially unchanged. Infected periwinkle shoots exhibiting phyllody symptom were used for genomic DNA extraction using a modified DNA extraction protocol as described by Lee et al. (1991). The DNA was sent to BGI-Americas for library construction and sequencing on the Illumina HiSeq 4000 platform. A total of 468,647,184 clean reads, with an average length of 150 bp (Q20 = 96.19%), were obtained. Genome assembly and analysis were performed at the USDA-ARS' in-house bioinformatics facility. The phytoplasma reads were separated from the plant host reads by mapping the reads to available phytoplasma genomes using the bowtie2 program (v2.4.5, Langmead and Salzberg 2012). All completed and draft phytoplasma genomes in the National Center for Biotechnology Information (NCBI) BioProject database (https://www.ncbi.nlm.nih.gov/bioproject/) were used in the mapping. The assembly was performed using the Velvet assembler (v1.2.10, Zerbino and Birney 2008) and the SPAdes assembler (v3.14.0, Prjibelski et al. 2020). The phytoplasma contigs were further verified based on their nucleotide sequence identity scores with previously sequenced phytoplasma genomes using the BLASTN program.The assembled draft genome of CBPPT1 consists of 71 contigs totaling 512,902 bp with an average coverage of 1,434 and an N50 value of 78,175. Approximately 90% of the assembled sequences are in the 10 largest contigs. The G + C content of the CBPPT1 draft genome is 22.62%. One RNA operon and 26 tRNA genes were identified using software Barrnap (v0.8) and tRNAscan (v1.4), respectively (Fichant and Burks 1991; Seemann 2013). The rRNA gene sequence of CBPPT1 shares 99.61% sequence identity with that of CP (AY390261), the reference strain of 'Ca. P. trifolii' and the representative strain of group 16SrVI, confirming CBPPT1 is a 'Ca. P. trifolii'-related strain. The CBPPT1 is the first 'Ca. P. trifolii'-related strain and the first 16SrVI phytoplasma whose genome is sequenced.The completeness of the CBPPT1 draft genome assembly was assessed by examining the presence of benchmarking universal single-copy orthologs using software BUSCO v5 (Manni et al. 2021). The obtained BUSCO score, 62.1%, was then compared with those of complete phytoplasma genomes. Currently, there are 12 complete phytoplasma genomes available for comparative analysis, and the BUSCO scores of these genomes range from 58.9 to 63.9%, with an average of 61.2%. It is reasonable to conclude that the CBPPT1 draft genome is near completion as its BUSCO score is above the average score of the 12 complete phytoplasma genomes.Protein coding genes (CDS) were predicted using Genemark (v3.36) and the gene annotation was performed using the BLASTP program against the nr database at the NCBI and the Blast2GO module of the OmicsBox (v2.0.36, BioBam Bioinformatics 2019). A total of 554 CDS were predicted. Among them, 442 were assigned to at least one gene ontology (GO) term, and 402 were assigned to InterPro functional categories.The genome size of different phytoplasmas varies, ranging from 530 to 1,350 kbp (Marcone et al. 1999). Apparently, the genome size of CBPPT1 is at the low end of the spectrum. Consistent with its small size, it has only one rRNA operon and possesses much less repetitive genes when compared with other phytoplasmas such as 'Ca. P. asteris'-related strain OYM (Oshima et al. 2004) and 'Ca. P. australiense'-related strain SLY (Andersen et al. 2013). Nevertheless, the CBPPT1 genome contains multiple putative pathogenicity genes (Table 1) including those encoding homologs of SAP54 and SAP05, which are known phytoplasma virulence factors (effectors) responsible for phyllody and witches'-broom symptoms, respectively (Huang et al. 2021; MacLean et al. 2014). Among other putative CBPPT1 pathogenicity genes are those encoding more than a dozen secretory proteins, most of which belong to the SVM family (Table 1). In genomes of other phytoplasmas, SVM protein genes often reside in prophage-derived pathogenicity islands (Wei et al. 2008).Table 1. Putative effectors and other secretory proteins encoded by the CBPPT1 genomeLocus tagContig no.Length (aa)AnnotationM8044_0001532152Immunodominant membrane proteinM8044_0003626256Hypothetical proteinM8044_0003876200Hypothetical protein (SAP09-like)M8044_000392690SVM family protein (SAP44-like)M8044_0004661043Hypothetical proteinM8044_00048313125Phytoplasma effector causing phyllody symptoms (SAP54-like)M8044_00048413774ATP-dependent Zn proteaseM8044_0004931568SVM family proteinM8044_00049415111SVM family proteinM8044_00050817125SVM family proteinM8044_00050917259SVM family proteinM8044_00051822158Putative secreted protein (SAP42-like)M8044_00051922188SVM family proteinM8044_00053029188SVM family protein (SAP19-like)M8044_0005353687Hypothetical protein (SAP53-like)M8044_00053639125Hypothetical protein (SAP05-like)Table 1. Putative effectors and other secretory proteins encoded by the CBPPT1 genomeView as image HTML Since CBPPT phytoplasma is capable of inducing multiple mutually distinct and developmental stage-dependent symptoms in the host, the phytoplasma may possess multiple pathogenicity factors and each could interact with host component(s) in a stage-specific manner, leading to distinct symptoms as previously postulated (Wei et al. 2019). We hope the draft CBPPT1 genome sequence can serve as a resource for the search for and eventual identification of such pathogenicity factors.Data AvailabilityThe draft genome sequence data of the 'Candidatus Phytoplasma trifolii'-related strain CBPPT1 have been uploaded to the NCBI's BioProject database under accession number PRJNA839414. The annotated sequence has been deposited in the GenBank under accession number JANHJP000000000.The author(s) declare no conflict of interest.Literature CitedAndersen, M. T., Liefting, L. W., Havukkala, I., and Beever, R. 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The American Phytopathological Society, 2022.FundingU.S. Department of Agriculture, Agricultural Research ServiceKeywordsBLTVAclover proliferation groupgenomephytoplasmapotato purple topThe author(s) declare no conflict of interest.PDF download

Here, we report the draft genome sequence of Xylella fastidiosa strain ATCC 35873, which was obtained from the American Type Culture Collection and was originally isolated from a symptomatic American elm tree grown in Washington, DC. The ATCC 35873 genome contains 2,454,216 bp and has a GC content of 51.68%.