Biopesticides, including those containing various Trichoderma species, may provide improved control of fungal diseases of woody perennials including grapevines. Thus, Trichoderma isolates were obtained from grapevines in California, U.S.A., with eight strains demonstrated to provide biological control against fungal pathogens. These were determined via phylogenetic analyses to be one of six different species: Trichoderma asperellum, Trichoderma capillare, Trichoderma harzianum, Trichoderma saturnisporopsis, and two novel Trichoderma species (Trichoderma sp. DL1-3 and Trichoderma sp. PAR10). However, little is known about strain genetic diversity. Therefore, efforts were undertaken to characterize and compare obtained mitochondrial genomes with those previously published from related Trichoderma species. For this effort, the mitochondrial genomes of T. asperellum strain TLI, T. capillare strains KC2-2 and SLO1-1, and T. saturnisporopsis strain RSI were obtained and annotated, and compared to the other Californian strains and 23 other Trichoderma species genomes. All analyzed species had the same major protein coding genes, rRNA regions, and tRNA coding sequences. Further, specific introns were observed to be associated with distinct clades within the Trichoderma genus, and these could be used to develop markers to quickly assign an unknown Trichoderma into a specific clade. Knowledge of Trichoderma mitochondrial genomes should improve understanding about this fungal species.

The circular mitochondrial genome of Kolla paulula, an insect vector of Xylella fastidiosa causing grape Pierce's disease (PD) in Taiwan, is 14,575 bp. The genome encodes genes for 13 proteins, 2 ribosomal RNAs (rRNAs), and 22 transfer RNAs (tRNAs).

The two-spotted spider mite, Tetranychus urticae, poses significant agricultural challenges due to its rapid population growth and high capacity for developing chemical resistance. This study evaluated the acaricidal activity of bacterial strain TAM1, isolated from naturally deceased mites in Taiwan. In bioassays, TAM1 caused over 90% adult mite mortality within 48 h. Infected mites showed symptoms of body darkening and deformation of the ventral abdominal crest lines. Enzymatic analysis confirmed significant chitinase and gelatinase activities. Whole genome sequence of TAM1 was acquired with a 5,066,903 bp circular chromosome (CP120954) and a 164,574 bp circular plasmid (CP120955). Refined functional profiling identified a sophisticated enzymatic arsenal including core chitin-active families (GH18, GH20, AA10) and 157 proteases, with a high prevalence of metallopeptidases that correlate with the detected gelatinase activities. Secretome analysis predicted 42 extracytoplasmic proteases primarily utilizing the Sec-dependent pathway, while the presence of multiple CBM50 modules suggests a potential for targeted substrate anchoring. These genomic insights provide a plausible molecular basis for the observed enzymatic potential and the localized ultrastructural disruption of the T. urticae cuticle. The alignment between phenotypic observations, microscopic evidence, enzymatic activities, and genomic data suggests that TAM1 utilizes synergistic, multi-target mechanisms to exert its acaricidal effects. Based on analyses of whole-genome sequence and 16S rRNA gene sequence, TAM1 was tentatively designated as a strain of Kosakonia sacchari. The bacterial strain reported here represents a promising microbial agent for integrated pest management (IPM) programs.

The complete mitochondrial genomes of Trichoderma sp. strains DL1-3 (29,197 bp), KC1-1 (27,631 bp), and PAR10 (29,122 bp), isolated from grapevine leaves, were obtained. All strains contained 25 or 26 tRNA genes, two rRNA genes, and 15 core mitochondrial protein coding genes. Phylogenetic trees made with housekeeping region and mitochondrial sequences placed these strains in the T. harzianum clade, with DL1-3 and PAR10 as potential novel species, and KC1-1 identified as a strain of T. harzianum. This suggests that complete mitochondria genome comparisons provide valuable information in resolving questions of species identification, including putatively identifying those that are novel.

Xylella fastidiosa (Xf) is an endophytic bacterial pathogen that causes bacterial leaf scorch in pecan (Carya illinoinensis). DNA was isolated from a symptomatic pecan tree (cultivar Byrd) in Albany, Georgia. High-throughput sequencing (Illumina PE150) was performed. Assembly was performed and referenced to available GenBank sequences with high read-mapping rates. The genome of strain Oak 35784 (ASM2145990v1, CP090511.1) had the highest percentage of mapped reads and was selected as the reference for a guided assembly. The final assembly of the GaT2 metagenomic-assembled genome (MAG) (ASM4286244v1, GCA_042862445.1) was 2,735,877 bp in 89 contigs, with an N50 of 848,678 bp, a GC content of 51.88%, a BUSCO score of 100% (domain bacteria_odb10) and 83.5% (lineage "Xanthomonadales"), and an average nucleotide identity of 99.33% to 11 Xf subsp. multiplex genome assemblies. The GaT2 genome sequence is the first for Xf from pecan and will be a valuable resource for future research.Copyright (c) 2025 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

A putative entomopathogenic fungus, Allocanariomyces sp. GWSS32 was isolated from glassy-winged sharpshooter, an insect vector of the Pierce's disease bacterium Xylella fastidiosa. This genome resource describes the 33,494 bp mitochondrial genome of this fungus, which should allow improved overall characterization of entomopathogenic fungi for Pierce's disease management.

A Microviridae phage, CLasMV1, was recently identified in 'Candidatus Liberibacter asiaticus' (CLas), the bacterium associated with citrus Huanglongbing (HLB). The CLasMV1 strain GDHZ11D (CP045566) exhibited a circular genome of 8,869 bp containing eight open reading frames (ORFs). Phages of CLas play crucial roles in regulating bacterial population diversity that have potential use for HLB control. However, knowledge of CLasMV1 population diversity is currently limited. In this study, 1,520 CLas samples were collected from 10 citrus-growing provinces in China. PCR screening revealed that 1,042 samples (69%) harbored CLasMV1. Complete genome sequences of 43 CLasMV1 strains were obtained through Illumina HiSeq sequencing with genome sizes ranging from 8,696 to 8,881 bp. Variations were mostly related to single-nucleotide polymorphisms and sequence insertions/deletions. Notably, two hypervariable regions (HVRs) were identified. Two new ORFs were detected, ORF-A (144 bp) in HVR-1 and ORF-B (342 bp) in HVR-2. Whole-genome phylogenetic analysis using 43 strains from this study, 9 previously reported Chinese strains, and 2 Florida strains (CP040636 and MZ382797) delineated five genomic groups. Chinese strains dominated Groups 1 to 4, and Group 5 contained the Florida strains. Further analyses of HVR-1 across all 1,042 CLasMV1 samples detected a 173-bp sequence with suspected mobile activity. Analysis of a PacBio long-read assembly published previously detected a concatemer of the CLasMV1 sequence, suggesting that the phage used the rolling circle mechanism for its replication. These findings significantly enhance our understanding of CLasMV1 genomic diversity and will facilitate future research on CLasMV1 classification and HLB biology.

American black elderberry, Sambucus canadensis L., is a woody deciduous shrub rich in antioxidants belonging to the Adoxaceae and is native to North America (Lee and Finn, 2007; Osman et al. 2023). S. canadensis L. is considered a homotypic synonym of Sambucus nigra subsp. canadensis (L.) Bolli), the European elderberry (Bolli, 1994). In Oklahoma, elderberries are usually harvested from wild growing plants, but recent efforts have seen an upsurge in acreage in elderberry production (Carroll, 2017). In 2024, mild to severe leaf scorch symptoms were observed on all the elderberry plants (about 5 plants within 1-3 meters of each other) in the Oklahoma State University Native Plant Corridor collections in Stillwater campus. Leaf scorch symptoms were characterized by tan to brown, irregularly shaped necrotic lesions along the leaf margins which eventually spread to the entire leaflet resulting in curling of the leaflet and defoliation . Symptomatic leaves were collected from all the five elderberry plants for pathogen identification. Samples were processed within three days. The petiole and midrib of ten elderberry leaves showing symptoms were freeze-dried for 24 hours. Thereafter, samples were ground to powder in a Genogrinder for 3 minutes. DNA was extracted from ground petiole and midrib using NucleoSpin Plant II kit (Machereey-Nagel, Duren, Germany) following the manufacturer's protocol. The ten DNA samples were tested for the presence/ absence of Xylella fastidiosa (Xf) using Q-PCR, with positive control (DNA of Xf subsp. multiplex (Xfm) strain M12 and Xf subsp. fastidiosa (Xff) strain M23 and negative control (water). SYBR-green real-time PCR with primer sets Teme150fc/Teme454rg (more specific to Xff) and Dixon454fa/Dixon1261rg (more specific to Xfm) (Chen et al., 2005), yielding Ct values ranging from 23.71-30.55 and 14.63-28.6 for both primer sets respectively, as contrasting to no Ct in control, suggesting Xfm infection (lower Ct values). For a whole genome level evaluation, next generation sequencing was carried out on one sample, STW-2 (Ct = 14.63 for Dixon454fa/Dixon1261rg), using the Illumina NovaSeqX Plus platform as described previously (Bock et al. 2025). A total of 180 M paired reads (150 bp each) was generated with a mapping rate of 11.09% to Xfm M12 (NC_010513.1) and 10.72% to Xff M23 (NC_010577.1) using Bowtie2 (Langmead and Salzberg, 2012), suggesting the Xfm identity. A BLAST search using the top 5 and bottom 5 reads in the mapped read data against the NCBI (National Center for Biotechnology Information) core-nr database showed that all the top hits were Xfm (query coverage = 100%, Percentage Identity = 100%). The assembled draft genome of STW-2 (GenBank accession number JBNVQL000000000) has an average nucleotide index (ANI) of 99.7 to M12 and 98.0 to M23. All three analyses (PCR, whole genome read-mapping, and NCBI BLAST search) confirmed the association of Xfm to STW-2. Xff was previously isolated from an elderberry plant growing in a grape vineyard in Florida and was reported to cause mild symptoms on grapevines (Hopkins, 2005). This is the first report of Xfm associated with bacterial leaf scorch on elderberry. This finding is therefore important in understanding epidemiology and management of Xfm in the increasingly important elderberry production landscape and potential spread to other horticultural plants in Oklahoma.

"Candidatus Liberibacter asiaticus" (CLas) is associated with citrus huanglongbing, a severe disease with global importance that affects citrus production in Brazil. This study reports the first complete genome of a Brazilian strain of CLas. The genomic structure comparison of strain 9PA with those of 13 complete CLas genomes revealed 9,091 mismatches and 992 gaps/insertions, highlighting eight locally colinear blocks, among which six are in the prophage region. Phylogenetic analysis categorized 13 CLas genomes into two clusters with 9PA clustered with strains from China and the United States. Whole-genomic comparison identified diverse hypervariable genomic regions (HGRs). Three HGRs in the chromosomal region and three in the prophage region were selected and investigated by polymerase chain reaction. HGRs assessed from 68 samples, from medium- to high-huanglongbing incidence areas in Sao Paulo state, were grouped into haplotypes A to P. Haplotype A, which includes strain 9PA, is the second most prevalent, representing 19.1% of the samples. Haplotype B, the most common, accounts for 42.6%. Together with haplotype C, these make up 72% of the evaluated samples. The 9PA strain has prophage P-9PA-1, both integrated and circularized, and P-9PA-3, only found in a circularized form. Prophages show high identity with SC1 (83%) and P-JXGC-3 (98%). Co-occurrence of both type 1 and 3 prophages was observed in field samples. The approach employed provides insights into the Brazilian CLas population, providing markers for population studies and highlighting the prevalence of type 1 and 3 prophages in the population. IMPORTANCE CLas is a destructive pathogen responsible for causing the severe citrus disease known as huanglongbing. Our study presents the first fully sequenced Brazilian strain of CLas, designated as 9PA, and includes an analysis of two prophages occurring in this strain. The main objective of our research was to compare the genome features of this Brazilian strain with other fully sequenced genomes and to identify its hypervariable genetic regions. These regions were subsequently used to assess genomic variability within both the chromosomal and prophage regions in Brazilian isolates of CLas. Our findings offer valuable insights into the diversified adaptation of CLas.

Xylella fastidiosa ssp. fastidiosa (Xff) is the causal agent of Pierce's disease of grapevine, a management-intensive and potentially deadly disease. However, different stains and other subspecies, such as Xylella fastidiosa ssp. multiplex (Xfm), exist in the same regions and vary in capacity to cause disease. All strains differ in the fatty acids that comprise cell membranes, as these allow adaptations to specific host microenvironments. Therefore, studies were initiated to observe the fatty acid profiles of different Californian Xf isolates via fatty acid methyl ester (FAME) analysis. Observations revealed that the four Xff strains had similar FAME profiles that were distinct from those of the three Xfm strains, even in isolates that originated from the same host plant species. These data show consistent differences between Xff and Xfm strains and demonstrate the potential that FAME profiling has for Xylella subspecies identification of novel isolates. The author(s) have dedicated the work to the public domain under the Creative Commons CC0 "No Rights Reserved" license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law, 2024.

Prophages/phages are important components of the genome of 'Candidatus Liberibacter asiaticus' (CLas), an unculturable alphaproteobacterium associated with citrus huanglongbing (HLB) disease. Phage variations have significant contributions to CLas strain diversity research, which provide critical information for HLB management. In this study, prophage variations among selected CLas strains from southern Texas were studied. The CLas strains were collected from three different CLas inhabitant environments: citrus leaf, citrus root, and Asian citrus psyllid (ACP), the vector of CLas. Regardless of the different habitats and time span, more than 80% of CLas strains consistently had both Type 1 and Type 2 prophages, the same prophage type profile as in CLas strains from Florida but different to those reported in California and China. Further studies were performed on prophage type diversity. Analyses on Type 1-specific PCR amplicon sequences (encoding an endolysin protein) revealed the presence of two groups: Type 1-A, clustered around prophage SC1 originating from Florida, and Type 1-B, clustered with prophage P-SGCA5-1 originating in California. Type 1-B strains were mostly from ACP of nearby citrus orchards. On the other hand, analyses on Type 2-specific PCR amplicon sequences (encoding a putative hypothetical protein) showed a single group clustering around prophage SC2 originated from Florida, although a different Type 2 prophage has been reported in California. The presence of two distinct Type 1 prophage groups suggested the possibility of two different CLas introductions in southern Texas. The results from this study provide an initial baseline of information on genomic and population diversity of CLas in Texas.

Cottony ash psyllid (CAP, Psyllopsis discrepans) is an important, invasive insect pest of ash trees in North America, where it has established populations and is the host of a newly identified strain of ‘ Candidatus Liberibacter solanacearum’. However, not much is known about the diversity of its introduced population. In this study, a CAP mitochondrial genome (mitogenome) sequence was obtained from a collection in Saskatoon, Saskatchewan, Canada. The CAP mitogenome is a circular DNA of 18,824 bp, encoding 13 protein-coding genes, 21 transfer RNA genes, and two ribosomal RNA genes. BLAST search using the CAP mitogenome as a query against the GenBank sequence database showed the mitogenome of Euphyllura phillyreae (15,202 bp) was the most similar (query coverage = 77%; percentage identity = 78.90%). The CAP mitogenome is significantly different from other known psyllid mitogenomes with the presence of a 4,357-bp control region. The mitogenome sequence will further the genomic understanding of CAP. [Formula: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 “No Rights Reserved” license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law, 2024.

Xylella taiwanensis (Xt) is a nutritionally fastidious bacterial pathogen causing pear leaf scorch disease (PLSD) in Taiwan. The disease causes early defoliation, loss of tree vigor, and reduction in fruit yield and quality. No cure for PLSD is available. The only option for growers to control the disease is to use pathogen-free propagation material, which requires early and accurate detection of Xt. Currently, only one simplex PCR method is available for the diagnosis of PLSD. We developed five Xt-specific TaqMan quantitative PCR (TaqMan qPCR) systems (primers-probe sets) for the detection of Xt. The PCR systems target three conserved genomic loci commonly used in bacterial pathogen detection: the 16S rRNA gene (rrs), the 16S-23S rRNA intergenic transcribed sequence (16S-23S rRNA ITS), and the DNA gyrase gene (gyrB). BLAST analysis using the GenBank nr sequence database, including whole genome sequences of 88 Xanthomonas campestris pv. campestris (Xcc) strains, 147 X. fastidiosa (Xf) strains, and 32 Xt strains, showed that all primer and probe sequences were specific only to Xt. Single nucleotide polymorphisms (SNPs) provided the primer/probe specificity to Xt. The PCR systems were evaluated by using DNA samples from pure cultures of two Xt strains, one Xf strain, one Xcc strain, and 140 plant samples collected from 23 pear orchards in four counties in Taiwan. The two-copy rrs and 16S-23S rRNA ITS-based PCR systems (Xt803-F/R, Xt731-F/R, and Xt16S-F/R) showed higher detection sensitivity than the two single-copy gyrB-based systems (XtgB1-F/R and XtgB2-F/R). A metagenomic analysis of a representative PLSD leaf sample detected the presence of non-Xt proteobacteria and fungal pathogens that should be taken into consideration in PLSD, as they might interfere with diagnosis.

Objectives “ Candidatus Liberibacter asiaticus” (CLas) is an un-culturable α-proteobacterium that caused citrus Huanglongbing (HLB), a destructive disease threatening citrus production worldwide. In China, the presence of HLB was first reported in Chaoshan region of Guangdong province, China around a century ago. Thus, whole genome information of CLas strains from Chaoshan area become the most important resource to understand the population diversity and evaluation of CLas in China. Data description CLas strain GDCZ was originally from Chaozhou city (Chaoshan area) and sequenced using PacBio Sequel long-read sequencing and Illumina short-read sequencing. The genome of strain GDCZ comprised of 1,230,507 bp with an average G + C content of 36.4%, along with a circular CLasMV1 phage: CLasMV1_GDCZ (8,869 bp). The CLas strain GDCZ contained a Type 2 prophage (37,452 bp) and encoded a total of 1,057 open reading frames and 53 RNA genes. The whole genome sequence of CLas strain GDCZ from the historical HLB endemic region in China will serve as a useful resource for further analyses of CLas evolution and HLB epidemiology in China and world.

Background Trichoderma is a diverse genus of fungi that includes several species that possess biotechnological and agricultural applications, including the biocontrol of pathogenic fungi and nematodes. The mitochondrial genome of a putative strain of Trichoderma harzianum called PAR3 was analyzed after isolation from the roots of Scarlet Royal grapevine scion grafted to Freedom rootstock, located in a grapevine vineyard in Parlier, CA, USA. Here, we report the sequencing, comparative assembly, and annotation of the nuclear genome of PAR3 and confirm its identification as a strain of T . harzianum . We subsequently compared the genes found in T. harzianum PAR3 to other known T. harzianum strains. Assembly of Illumina and/or Oxford Nanopore reads by the popular long-read assemblers, Flye and Canu, and the hybrid assemblers, SPAdes and MaSuRCA, was performed and the quality of the resulting assemblies were compared to ascertain which assembler generated the highest quality draft genome assembly. Results MaSuRCA produced the most complete and high-fidelity assembly yielding a nuclear genome of 40.7 Mb comprised of 112 scaffolds. Subsequent annotation of this assembly produced 12,074 gene models and 210 tRNAs. This included 221 genes that did not have equivalent genes in other T. harzainum strains. Phylogenetic analysis of ITS, rpb2 , and tef1a sequences from PAR3 and established Trichoderma spp. showed that all three sequences from PAR3 possessed more than 99% identity to those of Trichoderma harzianum , confirming that PAR3 is an isolate of Trichoderma harzianum . We also found that comparison of gene models between T. harzianum PAR3 and other T. harzianum strains resulted in the identification of significant differences in gene type and number, with 221 unique genes identified in the PAR3 strain. Conclusions This study gives insight into the efficacy of several popular assembly platforms for assembly of fungal nuclear genomes, and found that the hybrid assembler, MaSuRCA, was the most effective program for genome assembly. The annotated draft nuclear genome and the identification of genes not found in other T. harzainum strains could be used to investigate the potential applications of T . harzianum PAR3 for biocontrol of grapevine fungal canker pathogens and as source of anti-microbial compounds.

Here, we report the complete genome sequences of Xylella fastidiosa subsp. fastidiosa strain AlmaReb2 and X. fastidiosa subsp. multiplex strain AlmaRebR6, causing blueberry bacterial leaf scorch disease in Georgia, USA. The X. fastidiosa subsp. fastidiosa AlmaRebR2 chromosome is 2,549,422 bp, and the X. fastidiosa subsp. multiplex AlmaReb6 chromosome is 2,530,348 bp.

Protein biomarkers were identified from bacterial cell lysates of two sequenced strains of Xylella fas-tidiosa subsp. fastidiosa (Xff), the causal agent of Pierce's Disease of grapevine, using MALDI-TOF-TOF tandem mass spectrometry (MS/MS) and top-down proteomic analysis. Proteins were identified from their intact masses as well as sequence-specific fragment ions resulting from the aspartic acid effect detected by MS/MS and post-source decay (PSD). Two Xff strains from California were studied, M23 isolated from almond in Kern County and Stag's Leap isolated from grapevine in Napa County. The two strains are pathogenically identical or highly similar. Most of the proteins identified were highly conserved, e.g., 10 kDa chaperonin, cold-shock and/or DNA-binding, ribosomal and hypothetical proteins. The amino acid sequences and post translational modifications (PTM) of these conserved proteins were identical between the two Xff strains. However, the amino acid sequence of an outer membrane/hypo-thetical protein differed significantly between the two strains and could be a useful biomarker to distinguish between these closely related strains. In silico signal peptide analysis suggest that the outer membrane/hypothetical protein are secreted lipoproteins (SPII) with a putative prosthetic group attached to the only cysteine residue (C19) before or after removal of an 18-residue N-terminal signal peptide. However, only y-type fragment ions were detected by MS/MS-PSD which precluded confir-mation of the site of sequence truncation and/or attached PTM. The annotated genomes of the two strains indicate ten to twenty proteins that are either lipoproteins or are associated with assembly, modification, or transport of lipoproteins. In consequence, a variety of prosthetic groups, e.g. diac-ylglycerol, may be attached to C19 in the mature protein.Published by Elsevier B.V.

“Candidatus Liberibacter asiaticus” (CLas) is the causal agent of citrus Huanglongbing (HLB, also called citrus greening disease), a highly destructive disease threatening citrus production worldwide. A novel Microviridae phage (named CLasMV1) has been found to infect CLas, providing a potential therapeutic strategy for CLas/HLB control. However, little is known about the CLasMV1 biology. In this study, we analyzed the population dynamics of CLasMV1 between the insect vector of CLas, the Asian citrus psyllid (ACP, Diaphorina citri Kuwayama) and the holoparasitic dodder plant (Cuscuta campestris Yunck.); both acquired CLasMV1-infected CLas from an HLB citrus. All CLas-positive dodder samples were CLasMV1-positive, whereas only 32% of CLas-positive ACP samples were identified as CLasMV1-positive. Quantitative analyses showed a similar distribution pattern of CLasMV1 phage and CLas among eight citrus cultivars by presenting at highest abundance in the fruit pith and/or the center axis of the fruit. Transcriptome analyses revealed the possible lytic activity of CLasMV1 on CLas in fruit pith as evidenced by high-level expressions of CLasMV1 genes, and CLas genes related to cell wall biogenesis and remodeling to maintain the CLas cell envelope integrity. The up-regulation of CLas genes were involved in restriction–modification system that could involve possible phage resistance for CLas during CLasMV1 infection. In addition, the regulation of CLas genes involved in cell surface components and Sec pathway by CLasMV1 phage could be beneficial for phage infection. This study expanded our knowledge of CLasMV1 phage that will benefit further CLas phage research and HLB control.

The genome of Curtobacterium sp. strain TXMA1, isolated from a grapevine in Texas showing leaf marginal necrosis symptoms, was sequenced. The TXMA1 genome has a 3,454,876-bp, circular chromosome with a GC content of 71.74%, 3,213 open reading frames (ORFs), 47 tRNAs, and 4 complete rRNA operons (5S, 16S, and 23S).

HomePhytopathology®Vol. 112, No. 11A Genome Resource for Xylella fastidiosa subsp. multiplex Strain P5A2 Causing Phony Peach Disease in the Southeastern United States PreviousNext Resource Announcement OPENOpen Access licenseA Genome Resource for Xylella fastidiosa subsp. multiplex Strain P5A2 Causing Phony Peach Disease in the Southeastern United StatesKendall A. Johnson, Clive H. Bock, Phillip M. Brannen, and Jianchi ChenKendall A. JohnsonDepartment of Plant Pathology, University of Georgia, 2105 Miller Plant Sciences Building, Athens, GA 30602, Clive H. BockU.S. Department of Agriculture–Agricultural Research Service–Southeastern Fruit and Tree Nut Research Laboratory, Byron, GA 31008, Phillip M. Brannen†Corresponding authors: J. Chen; E-mail Address: [email protected], and P. M. Brannen; E-mail Address: [email protected]Department of Plant Pathology, University of Georgia, 2105 Miller Plant Sciences Building, Athens, GA 30602, and Jianchi Chen†Corresponding authors: J. Chen; E-mail Address: [email protected], and P. M. Brannen; E-mail Address: [email protected]https://orcid.org/0000-0003-0662-3164U.S. Department of Agriculture–Agricultural Research Service–San Joaquín Valley Agricultural Sciences Center, Parlier, CA 93648AffiliationsAuthors and Affiliations Kendall A. Johnson1 Clive H. Bock2 Phillip M. Brannen1 † Jianchi Chen3 † 1Department of Plant Pathology, University of Georgia, 2105 Miller Plant Sciences Building, Athens, GA 30602 2U.S. Department of Agriculture–Agricultural Research Service–Southeastern Fruit and Tree Nut Research Laboratory, Byron, GA 31008 3U.S. Department of Agriculture–Agricultural Research Service–San Joaquín Valley Agricultural Sciences Center, Parlier, CA 93648 Published Online:19 Nov 2022https://doi.org/10.1094/PHYTO-03-22-0081-AAboutSectionsPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat Xylella fastidiosa is a gram-negative bacterial pathogen infecting many economically important crops including alfalfa, almond, citrus, grape, olive, peach, pecan, and plum (Hopkins 1977, 1989; Purcell and Hopkins 1996; Sicard et al. 2018). In the United States, all known X. fastidiosa strains belong to one of two subspecies, X. fastidiosa subsp. fastidiosa or X. fastidiosa subsp. multiplex (Schaad et al. 2004). X. fastidiosa subsp. fastidiosa causes Pierce's disease (PD) of grapevine, which is the limiting factor for the expansion of wine grape industry in the southeastern United States, and PD is currently a significant threat to viticulture in California. Another important X. fastidiosa disease in the southeastern United States is phony peach disease (PPD) (Hopkins 1977, 1989; Hutchins et al. 1951; Johnson et al. 2021; Neal 1920), presumably caused by a strain of X. fastidiosa subsp. multiplex based on 16S rRNA gene sequence (Chen et al. 2000). Unlike PD that has attracted extensive research efforts in the past 20 years, largely due to PD outbreaks in California, PPD has received much less research attention, although peach is also an important crop in the United States, with an estimated value of over $624 million in 2021 (USDA-NASS 2022). There are currently >140 X. fastidiosa whole genome sequences deposited in the GenBank bacterial genome database, but none are from X. fastidiosa subsp. multiplex strains associated with PPD.PPD symptoms include a flattened tree canopy, shortened internodes, abnormal growth, reduced fruit size and quality, and eventual shoot dieback (Hutchins 1933; Johnson et al. 2021; Smith 1941). These are uniquely different to those symptoms of marginal necrosis or marginal leaf scorch, typical to most known X. fastidiosa diseases (Hopkins 1977, 1989). In addition, PPD bacteria reside primarily in the roots (Cochran and Hutchins 1976; Wells et al. 1980). PPD is vectored by sharpshooter insects (Turner 1949). Epidemics of PPD were particularly severe in the 1920s and 1930s, but additional sporadic outbreaks have occurred more recently (Johnson et al. 2021). Federal PPD eradication programs instigated in 1929 were terminated in the 1970s, although peach producers continue to destroy symptomatic trees annually. Currently, there are no other control measures used.X. fastidiosa is nutritionally fastidious, i.e., difficult to culture on artificial media. Based on cross-inoculation studies, the PPD strain is presumed to be identical or related to the plum leaf scald (PLS) strain (Raju et al. 1982; Wells et al. 1981). Among the known X. fastidiosa strains, the PPD strain is particularly difficult to isolate and culture in vitro in our working experience. Genome sequencing has recently emerged as an effective and critical means to study X. fastidiosa biology and taxonomy. The X. fastidiosa citrus variegated chlorosis strain 9a5c was the first plant pathogenic bacterium to be sequenced (Simpson et al. 2000). Subsequently, genome of multiple X. fastidiosa strains were sequenced and deposited in public domains including GenBank that benefited X. fastidiosa research. For example, a pear leaf scorch bacterium from Taiwan was first described as a strain of X. fastidiosa based on phenotypic data (Leu et al. 1993). Analysis of the published draft genome sequence (Su et al. 2014) revealed significant variations of this strain and led to the establishment of a new species, X. taiwanensis (Su et al. 2016). Strain variation issues, including potential differences between PPD and PLS strains, currently remain. A genome sequence of a PLS strain of X. fastidiosa (Pr8x) from Brazil has been published (Pierry et al. 2020), but no sequence exists for a strain from PPD, which would aid in resolving the X. fastidiosa strain relations.Traditionally, bacterial genome sequencing utilizes DNA from pure culture. The availability of next generation sequencing (NGS) technology has made it possible to acquire bacterial genome sequences through a DNA sample with mixed biological species, i.e., metagenomics. For example, multiple genome sequences of 'Candidatus Liberibacter asiaticus', an unculturable bacterial pathogen of citrus Huanglongbing, have been obtained from infected insect vector and plant host DNA (Duan et al. 2009; Huang et al. 2021; Zheng et al. 2014). Following the same metagenomic pipeline (Huang et al. 2021), we obtained and report here the whole genome sequence of an X. fastidiosa subsp. multiplex strain from a PPD symptomatic peach tree.A 6-year-old peach tree (Prunus persica 'Empress') showing typical PPD symptoms (Fig. 1) was identified in an orchard (32°47′06′′N 86°34′51′′W) located in Chilton County near Clanton, Alabama, U.S.A. on 23 July 2020. A sample of root 10.2 cm long and 2 mm in diameter was collected. Prior work has indicated that root tissue consistently maintains more X. fastidiosa DNA compared with other plant organs (Chen et al. 2019). The sample preparation was conducted following the protocol of Chen et al. (2019). Briefly, DNA was extracted using a Zymo Research Quick-DNA Fungal/Bacterial Miniprep Kit following the manufacture's protocol (Zymo Research Corp., Irvine, CA). PCR was performed to detect and evaluate titer of X. fastidiosa subsp. multiplex following the procedure of Chen et al. (2019). A sample designated as P5A2 (Ct = 19.1 with primer set C03Xf-gyrB) was selected. An aliquot of DNA (50 µl) was sent to the USDA-ARS San Joaquin Valley Agricultural Sciences Center at Parlier, California where the X. fastidiosa subsp. multiplex was further confirmed with an X. fastidiosa subsp. multiplex-specific primer set Dixon454fa-Dixon1261rg (Chen et al. 2005) using SYBR green-based quantitative PCR (Ct = 21.1).Fig. 1. A peach tree with typical, advanced symptoms (flattened tree canopy) of phony peach disease (PPD). A root sample of this tree was collected for the study. A healthy peach tree and a plum tree (inter-planted in the peach orchard) can be seen in the background (image captured 23 July 2020 in Clanton, Alabama). Further information on symptoms of PPD can be found in Johnson et al. (2021).Download as PowerPointTo obtain sufficient DNA (µgs) for NGS whole genome sequencing, the P5A2 DNA was enlarged using the GenomiPhi V2 DNA Amplification Kit (GE Healthcare) according to the manufacturer's instructions. Genome sequencing was performed with an Illumina NovaSeq 6000 PE150 format (Illumina, Inc.) through a commercial source. The P5A2 DNA sample yielded a total of 58,622,084,324 bp in 388,225,724 reads (read size = 151 bp) with a mean Q score = 35 (89.3% > Q30).For P5A2 genome sequence assembling, plant host chloroplast DNA reads were first identified by reference-mapping with CLC Genomic Workbench version 12.0 (Qiagen, Inc.) using a peach chloroplast genome sequence (NC_014697, Jansen et al. 2011) as a reference. The unmapped data (55,375,610,900 bp or 366,725,900 reads) were used for reference-mapping each with the five references listed in Table 1: (i) NC_010513.1, the chromosomal sequence of X. fastidiosa subsp. multiplex strain M12 (Chen et al. 2010); (ii) NC_010577.1, the chromosomal sequence of X. fastidiosa subsp. fastidiosa strain M23 and NC_010579.1, a plasmid of strain M23 (Chen et al. 2010); and (iii) NZ_CP009826.2, the chromosomal sequence of strain Pr8x and NZ_CP009827.1, a plasmid of Pr8x (Pierry et al. 2020). The default parameters (specifically, length fraction = 0.5; similarity fraction = 0.8) were used for all reference-mapping experiments except for the rrn operon region where higher stringent parameters (length fraction = 1.0; similarity fraction = 1.0) were applied to further minimize the interference of plant host DNA in sequence assembling.Table 1. A summary of reference-mapping on an Illumina NovaSeq data set generated from a root sample of a peach tree infected with Xylella fastidiosa subsp. multiplex phony peach strain P5A2 in the southeastern United StatesX. fastidiosa strainM12M23Pr8xMapping referenceNC_010513.1NC_010577.1NC_010579.1NZ_CP009826.2NZ_CP009827.1Cellular originChromosomeChromosomePlasmid/pXFAS01ChromosomePlasmid/pXF39Reference genome size (bp)2,475,1302,535,69038,2972,666,24039,580Plant hostAlmondAlmondPlumGeographic originCalifornia, U.S.A.California, U.S.A.Sao Paulo, BrazilP5A2 mapping resultsP5A2 contigs (>500 bp)51204529714Consensus genome (bp)2,461,1242,438,1146,0472,443,52634,866Mapped bp coverage (%)99.496.115.891.788.1Largest mapped contig273,383120,1301,82080,9638,180Mapped reads32,893,37232,359,700124,30332,372,475645,055Mapped reads (%)8.978.820.038.520.17Mapped bp4,966,899,1724,886,314,70018,769,7534,888,243,72597,403,305Table 1. A summary of reference-mapping on an Illumina NovaSeq data set generated from a root sample of a peach tree infected with Xylella fastidiosa subsp. multiplex phony peach strain P5A2 in the southeastern United StatesView as image HTML Selected metrics of the reference-mapping results are presented in Table 1. Among the three references, strain M12 showed the best-mapped metrics, e.g., the smallest number of contigs with 500 bp as a cutoff threshold (51 contigs), longest consensus genome size (2,461,124 bp), highest mapped base pair coverage (99.4%), largest mapped contig (273,383 bp), and largest number of mapped reads % (8.97). Therefore, the M12 reference-mapping contigs were chosen to represent the draft whole P5A2 genome. Read-mapping to M12 was performed using Bowtie 2 (version 2.4.1, Langmead and Salzberg 2012) and yielded a similar genome size (2,463,513 bp).The final draft P5A2 genome had a total of 2,461,124 bp in 51 contigs of 500 bp or larger, an average nucleotide coverage of 2,061×, N50 of 136,797 bp, L50 of 6, and G + C content of 51.9%. Benchmarking universal single-copy orthologs (BUSCO v5.2.2) analysis based on generic domain bacteria_odb10 (Manni et al. 2021) showed the results of C:100.0% [S:100.0%, D:0.0%], F:0.0%, M:0.0%, and n:124. Average nucleotide identity (ANI), a standard index for bacterial genome comparison for species determination (Goris et al. 2007; Konstantinidis and Tiedje 2005), was calculated. ANIs of P5A2 to M12, M23, and Pr8x were 99.64, 97.64, and 96.22, respectively, further confirming the X. fastidiosa subsp. multiplex status of strain P5A2 at the whole-genome level, i.e., most similar to X. fastidiosa subsp. multiplex strain M12, and demonstrated the distance between the U.S. PPD strain (P5A2) and the Brazil plum strain (Pr8x).From plasmid sequence mappings, P5A2 showed low similarity to the M23 plasmid pXFAS01, e.g., the mapped base pair coverage was only 15.8% (Table 1), suggesting the absence of any pXFAS01-like plasmid. The mapped base pair coverage of P5A2 sequence to the Pr8x plasmid pXF39 was 88.1% (34,866 bp in 14 contigs with the largest contig being 8,180 bp, Table 1), indicating the presence of sequences fragments similar to pXF39. However, more research is needed to validate the presence of pXF39-like plasmid in the P5A2 genome. The P5A2 genome sequences were annotated using the NCBI PGAP (Prokaryotic Genome Annotation Pipeline) (Tatusova et al. 2016) with 2,135 coding DNA sequences and 59 RNA genes including two sets of identical rrn operons (16S-23S-5S) and 49 rRNA genesFor sequence quality validation, six housekeeping genes in P5A2 genome, gyrB (DNA-gyrase subunit B, EC 5.99.1.3), holC (DNA-polymerase III chi subunit, EC 2.7.7.7), rfbD (dTDP-4-dehydrorhamnose 3,5-epimerase, EC 5.1.3.13), nuoL (NADH ubiquinone oxidoreductase chain A, EC 1.6.5.3), pilG (twitching motility protein), and leuA (isopropylmalate synthase, EC 2.3.3.13), were selected for BLAST search against GenBank nr sequence database. All BLAST hits showed 100% length coverage with 99 to 100% identity to those of X. fastidiosa subsp. multiplex.The P5A2 genome is the first PPD strain genome from the southeastern United States, filling a gap of current X. fastidiosa genome database in GenBank. It should be noted that the P5A2 genome was obtained through mapping to the M12 genome sequence. It is possible that sequences not covered by the M12 genome could be missed. Another contribution of the study was the use of metagenomic approach to acquire a X. fastidiosa whole genome sequence in planta that by-passed the obstacle of in vitro culture. The selection of a root sample with high X. fastidiosa subsp. multiplex titer increased the probability of successful genome sequence acquisition. The P5A2 genome was validated with six housekeeping genes and used for ANI analyses that revealed the difference between the U.S. PPD strain and a plum strain from Brazil. The P5A2 genome sequence will provide a valuable resource for further research in X. fastidiosa biology, genome-based taxonomy, and management of PPD and other X. fastidiosa diseases.Data AvailabilityThis whole-genome shotgun project has been deposited in GenBank under the accession number JAKZEN000000000 for strain P5A2 chromosome. The versions described here are the first version.AcknowledgmentsWe thank Y. Andrade for technical assistance and Edgar Vinson (Auburn University) for assistance in sample collection.The author(s) declare no conflict of interest.Literature CitedChen, C., Bock, C. H., and Brannen, P. M. 2019. Novel primers and sampling for PCR detection of Xylella fastidiosa in peach. Phytopathology 109:307-317. https://doi.org/10.1094/PHYTO-11-18-0439-FI Link, ISI, Google ScholarChen, J., Banks, D., Jarret, R. L., Chang, C. J., and Smith, B. J. 2000. Use of 16S rDNA sequences as signature characters to identify Xylella fastidiosa. Curr. 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Mention of a trademark or proprietary product is solely for the purpose of providing specific information and does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products that may also be suitable.The author(s) declare no conflict of interest.DetailsFiguresLiterature CitedRelated Vol. 112, No. 11 November 2022SubscribeISSN:0031-949Xe-ISSN:1943-7684 DownloadCaptionPierce's disease symptoms on grapevine (Vitis vinifera) mechanically inoculated with Xylella fastidiosa subsp. fastidiosa. Photograph was taken two and a half months after inoculation (Zecharia et al.). Photo credit: Ofir Bahar Metrics Article History Issue Date: 2 Dec 2022Published: 19 Nov 2022Accepted: 6 Jun 2022 Pages: 2466-2470 InformationThis article is in the public domain and not copyrightable. It may be freely reprinted with customary crediting of the source. The American Phytopathological Society, 2022.Keywordsbacterial pathogensbioinformaticsdata sciencegenomicsThe author(s) declare no conflict of interest.PDF download