Carlos Andrés Zarate

Carlos A Zárate-Chaves
email: carlos.zarate-chaves@ird.fr

Daniela Osorio-Rodríguez

Rubén E Mora
email: remoram@unal.edu.co

Álvaro L Pérez-Quintero

Alexis Dereeper
email: alexis.dereeper@ird.fr

Silvia Restrepo
email: srestrep@uniandes.edu.co

Camilo E López
email: celopezc@unal.edu.co

Boris Szurek
email: boris.szurek@ird.fr

Adriana Bernal
email: abernal@uniandes.edu.co

C A ; Osorio-Rodríguez

D ; Mora

R E ; Pérez-Quintero

Á L ; Dereeper

Phim

Carolina Flores

Lindsay Triplett

Véronique Maillot-Lebon

Yassine Moufid

Moussa Kanté

Claude Bragard

Valérie Verdier

Lionel Gagnevin

Isabelle Robène
email: isabelle.robene@cirad.fr

TAL Effector Repertoires of Strains of Xanthomonas phaseoli pv. manihotis in Commercial Cassava Crops Reveal High Diversity at the Country Scale

Keywords: cassava bacterial blight, SWEET, host target genes, susceptibility, transcriptomics, EBE prediction Cassava Diagnostic PCR Xanthomonas phaseoli pv. manihotis Xanthomonas cassavae

New insights into the diversity and function of Transcription Activator-Like effectors of Xanthomonas phaseoli pv. manihotis, the causal agent of Cassava Bacterial Blight

Introduction

Transcription activator-like effectors (TALEs) play a significant role for pathogenesis and virulence in several xanthomonads. Disruption or knocking out of some TALE encoding genes significantly affects the virulence of the pathogen or even abolishes pathogenicity [1][2][3][4][5][6][7][8][9][10][11][12]. This family of effectors shares a particular structure evolved to selectively bind plant host promoters and recruit the RNA polymerase complex in order to initiate transcription of downstream genes [13,14]. TALE proteins include a signal for translocation through the type III secretion system (T3SS) [15], nuclear localization signals (NLS) [16] and an acidic transcriptional activation domain (AAD) to promote transcription in the host cell [17]. The central region of the effector contains modular tandem repeats mainly composed of 33-35 amino acids [18,19] whose sequence varies primarily at residues 12th and 13th (so-called "repeat variable diresidue", RVD). RVDs interact with DNA bases with a nucleotide recognition preference [20][21][22][23][24], ruling the DNA sequence recognition. Sequences targeted by TALEs are usually located within or near the promoter of target genes, and are termed Effector Binding Elements (EBEs) [1]. After secretion, TALEs interact with host transcription factors like TFIIAγ subunits, to induce polymerase II-dependent transcription [25].

Many host genes targeted by TALEs have been described for the rice-Xanthomonas oryzae pathovars oryzae (Xoo) and oryzicola (Xoc), but data are also available for wheat [26], citrus, cotton, pepper, tomato, and cassava-interacting Xanthomonas (reviewed by [4]). TALE targets whose activation during infection is promoting host disease are defined as susceptibility (S) genes, many of which are nutrient transporters. For example, TALEmediated activation of SWEET sugar transporters in rice, cotton and cassava leads to promotion of virulence potentially through nutrient hijacking [10,27,28], while activation of sulfate transporter OsSULTR3;6 in rice may alter redox status or osmotic equilibrium to interfere with defense signaling and induce water-soaking [3]. In wheat, induction by TAL8 of Xanthomonas translucens pv. undulosa of a gene involved in abscisic acid regulation alters water management in favor of the pathogen [26]. Other S genes include plant transcription factors, which control more complex host cellular processes through multiple indirect targets [2,6,9,10,12]. In pepper, AvrsBs3-dependent activation of upa20, a basic helix-loop-helix (bHLH) family member, induces hypertrophy [12], while AvrHah1dependent activation of bHLH3 and bHLH6 upregulates cell wall-degrading enzymes [10]. In citrus, PthA4-mediated (and its functional equivalents) activation of the transcription factor CsLOB1, results in pustule development, which may be dependent on activation of DNA-interacting secondary targets [2,29].

Xanthomonas phaseoli pv. manihotis (Xpm), previously known as Xanthomonas axonopodis pv. manihotis [30], is a Gram-negative vascular pathogen responsible for Cassava Bacterial Blight (CBB). CBB has been reported in all the continents where cassava is grown [31]. As other xanthomonads, Xpm uses a suite of effectors to manipulate physiological processes in host plant cells. Xpm has up to 24 effectors that are translocated to the host cytoplasm through the T3SS (termed type III effectors), including members of the TALE family [32]. Population diversity studies using TALE1 Xam (a.k.a pthB) as a probe showed that all isolates from Latin-American and African Xpm collections carry TALEs [33][34][35][START_REF] Verdier | Genetic and pathogenic variation of Xanthomonas axonopodis pv. manihotis in Venezuela[END_REF][START_REF] Gonzalez | Characterization of pathogenic and nonpathogenic strains of Xanthomonas axonopodis pv. manihotis by PCR-based DNA fingerprinting techniques[END_REF]. Illuminabased genomic sequencing of more than 60 Xpm strains later confirmed that all strains contain at least one TALE [32,[START_REF] Arrieta-Ortiz | Genomic survey of pathogenicity determinants and VNTR markers in the cassava bacterial pathogen Xanthomonas axonopodis pv. manihotis strain CIO151[END_REF]. Full Xpm TALE sequence is available for only eight effectors [8,[START_REF] Cohn | Comparison of gene activation by two TAL effectors from Xanthomonas axonopodis pv. manihotis reveals candidate host susceptibility genes in cassava[END_REF][START_REF] Castiblanco | TALE1 from Xanthomonas axonopodis pv. manihotis acts as a transcriptional activator in plant cells and is important for pathogenicity in cassava plants[END_REF], and only two complete Xpm TALomes (whole set of TALEs present in a strain) have been reported so far [8,[START_REF] Cohn | Comparison of gene activation by two TAL effectors from Xanthomonas axonopodis pv. manihotis reveals candidate host susceptibility genes in cassava[END_REF].

Information about Xpm TALEs and their target genes in cassava has been growing in the last years. The first TALE characterized in Xpm is TALE1 Xam (contains 14 repeats), which has an important role in pathogenicity [START_REF] Castiblanco | TALE1 from Xanthomonas axonopodis pv. manihotis acts as a transcriptional activator in plant cells and is important for pathogenicity in cassava plants[END_REF], but whose targets remain unknown [8,[START_REF] Cohn | Comparison of gene activation by two TAL effectors from Xanthomonas axonopodis pv. manihotis reveals candidate host susceptibility genes in cassava[END_REF][START_REF] Castiblanco | TALE1 from Xanthomonas axonopodis pv. manihotis acts as a transcriptional activator in plant cells and is important for pathogenicity in cassava plants[END_REF][START_REF] Munoz-Bodnar | RNAseq analysis of cassava reveals similar plant responses upon infection with pathogenic and non-pathogenic strains of Xanthomonas axonopodis pv. manihotis[END_REF]. Cohn and coworkers [8] deciphered the contribution of each of the five TALEs of strain Xam668, showing that mutant strains for TAL14 Xam668 and TAL20 Xam668 significantly affected pathogen fitness characterized by reduction of bacterial growth and/or symptom formation. Importantly they also demonstrated that TAL20 Xam668 induces the S gene MeSWEET10a, which codes for a clade-III sugar transporter from the SWEET family.

Studying cassava pathogens is highly relevant since this crop is one of the most important starchy root crops for food security in America, Africa and Asia [START_REF] Aristizábal | Guía Técnica para Producción y Análisis de Almidón de Yuca[END_REF]. The annual worldwide production of cassava is estimated to 270 million tons and serves as a staple food for more than 800 million people, mainly in tropical countries. This and its tolerance to drought, make it an important food security crop [START_REF] Fao | Save and Grow: Cassava, a Guide to Sustainable Production Intensification[END_REF][START_REF] Parmar | Crops that feed the world: Production and improvement of cassava for food, feed, and industrial uses[END_REF]. CBB is the most devastating bacterial disease of cassava, causing losses that range from 12% to 100% [31] depending on environmental conditions [START_REF] Lozano | Cassava Bacterial Blight: A Manageable Disease[END_REF][START_REF] Wydra | Occurrence of cassava diseases in relation to environmental, agronomic and plant characteristics[END_REF], with incidences ranging between 25% and 100% [START_REF] Wydra | Occurrence of cassava diseases in relation to environmental, agronomic and plant characteristics[END_REF][START_REF] Abaca | Cassava Pests and Diseases' Prevalence and Performance as Revealed by Adaptive Trial Sites in North Western Agro-Ecological Zone of Uganda[END_REF]. CBB symptoms include angular leaf spotting, blight, wilt, gum exudation, vascular necrosis, stem canker and dieback (reviewed by [START_REF] López | Cassava Bacterial Blight: Using Genomics for the Elucidation and Management of an Old Problem[END_REF]). In Colombia, CBB was first described in 1971 [35]. More recently, Trujillo and coworkers described the genetic structure of Xpm populations in the most prominent cassava production regions, showing high susceptibility of the most common cultivars grown and a complex pathogen population structure in some of these regions [START_REF] Trujillo | Population typing of the causal agent of cassava bacterial blight in the Eastern Plains of Colombia using two types of molecular markers[END_REF][START_REF] Trujillo | A complex population structure of the cassava pathogen Xanthomonas axonopodis pv. manihotis in recent years in the Caribbean Region of Colombia[END_REF].

Science-based crop resistance improvement and disease tackling require a compilation of knowledge to understand the cellular and molecular bases of plant-pathogen interactions. As Xanthomonas pathogenicity notably relies on TALEs, knowledge on diversity and function of these effectors, in conjunction with resistance (R) proteins, are a cornerstone for resistance breeding in the affected crops [START_REF] Blanvillain-Baufumé | Targeted promoter editing for rice resistance to Xanthomonas oryzae pv. oryzae reveals differential activities for SWEET14-inducing TAL effectors[END_REF][START_REF] Oliva | Broad-spectrum resistance to bacterial blight in rice using genome editing[END_REF][START_REF] Hutin | Kombat: The story of defense against TAL effectors through loss-of-susceptibility[END_REF][START_REF] Shantharaj | An engineered promoter driving expression of a microbial avirulence gene confers recognition of TAL effectors and reduces growth of diverse Xanthomonas strains in citrus[END_REF]. Among the mechanisms of resistance associated with TALEs [START_REF] Zhang | TAL effectors and the executor R genes[END_REF], recessive resistance is mediated by loss-of-susceptibility (LoS) alleles, where alternative alleles of an S gene that does not possess the EBE on the promoter sequence prevent the recognition and upregulation of the S gene (reviewed by [START_REF] Hutin | Kombat: The story of defense against TAL effectors through loss-of-susceptibility[END_REF]). As previously stated, only one S gene has been described for cassava (MeSWEET10a) as a direct target of TALE20 Xam668 [8]. No LoS alleles, executor genes or TALE-recognizing nucleotide-binding leucine-rich repeat (NLR) receptors in cassava have been reported so far. In this work we aimed to characterize the TALE diversity of one of the best characterized Xpm populations in the world. We sequenced the repeat regions of 46 TALEs from 18 Xpm strains, including the complete TALomes from seven strains. We also aimed to determine functional diversity of the potential TALE-targeted genes and to propose candidates that could contribute to a better understanding of this pathosystem. The TALE-affinity analyses, host target prediction, and transcriptomics on cassava plants inoculated with the pathogen resulted in potential novel targets that might be relevant for pathogenesis. This study expands our understanding of the complex interactions between Xpm and cassava by picturing the TALE diversity in the context of this pathosystem.

Materials and Methods

Bacterial Strains, Plant Material, Culture Conditions and Media

All Xanthomonas phaseoli pv. manihotis strains were previously collected in diversity studies of this pathogen from our group (Laboratorio de Micología y Fitopatología de la Universidad de los Andes, Universidad de los Andes, Colombia). All the selected strains were pathogenic to the highly CBB-susceptible variety 60444. Isolates from two geographically distant cassava productive regions in Colombia were selected through maximization of differences of neutral genetic diversity. Additionally, two strains, one per region, isolated at least one decade before the collection dates of most isolates, were selected as reference strains. Bacteria were streaked on YPG (yeast extract 5 gL -1 , peptone 5 gL -1 , glucose 5 gL -1 , agar-agar 15 gL -1 ) solid media [35] and incubated for 48 h at 28 • C or grown in Phi broth (yeast extract 1 gL -1 , peptone 10 gL -1 , casaminoacids 1 gL -1 ) [35] at 28 • C, under constant shaking at 220 rpm for 24 h. Cassava cuttings from the cultivar 60444 were planted on individual peat pots and grown under greenhouse conditions (27 • C ± 5 • C; photoperiod 12:12, relative humidity >60%). Bacterial inoculations were performed on 3-month old plants. In vitro-grown plants from the same cultivar were grown as described elsewhere [START_REF] Mora | Using in vitro plants to study the cassava response to Xanthomonas phaseoli pv. manihotis infection[END_REF].

Aggressiveness Assays and Bacterial Growth Curves

Bacterial aggressiveness was quantified as the leaf lesion area formed by Xpm upon inoculation of 3-month-old cassava plants. Bacteria were cultured in liquid media and washed with 10 mM MgCl 2 sterile solution. Cell density was adjusted to an OD 600 of 0.2 (c.a. 2 × 10 8 cfu/mL). Adjusted bacterial suspensions were inoculated by placing a 10-µL drop of inoculum over a 2-mm (Ø) hole made with a cork borer through the leaf tissue. Plants were kept in a greenhouse under the same conditions described above. Lesions were individually photographed at 15 days post-inoculation (dpi) in a stereoscope and areas were measured using Image-J software (version 1.48) [START_REF] Schneider | NIH Image to ImageJ: 25 Years of image analysis[END_REF]. Each treatment was inoculated once in three individual plants, and the aggressiveness assay was replicated three times.

Bacterial growth in planta was assessed as described elsewhere [START_REF] Medina | The role of type III effectors from Xanthomonas axonopodis pv. manihotis in virulence and suppression of plant immunity[END_REF] except that inoculations were performed on the variety 60444 and 0, 5, and 10 dpi were evaluated. Each treatment was inoculated once in three different plants and the bacterial growth assessment was replicated two times.

Genomic DNA Extraction, Restriction Fragment Length Polymorphism (RFLP), and Southern Blot Analysis

A single colony of each Xpm strain was grown on Phi broth overnight. DNA was extracted from 2-mL bacterial pellets using the Genelute TM Bacterial Genomic DNA kit (Sigma-Aldrich, St. Louis, MO, USA) according to manufacturer's instructions. Genomic DNA concentration was determined by gel quantification and spectrophotometry on a NanoDrop TM 1000 (Thermo Fisher Scientific, Waltham, MA, USA). Ten micrograms of each genomic DNA were digested with 120 units of BamHI-HF (NEB, Ipswich, MA, USA) sequentially added (3 times 40 units) over a 24-h incubation period. BamHI-digested DNA was precipitated using sodium acetate and ethanol [START_REF] Sambrook | Molecular Cloning: A Laboratory Manual[END_REF] and then resuspended in TE solution. Electrophoresis of digested DNA was performed on a 0.8% agarose gel on 0.5× TBE (Thermo Fisher Scientific, Waltham, MA, USA); migration was performed at a constant power of 5 Watts for 20 h, in a cold room and with periodic buffer renewal. DNA blotting was performed according to Roche's instruction manual for Digoxigenin application to filter hybridization [START_REF] Eisel | DIG Application Manual for Filter Hybridization[END_REF]. The probe was synthesized from a conserved portion of the TALE1 Xam (from nucleotide 2909 to 3409) DNA contained in pF3, a pBluescript derivative with a 5.4-kb fragment from CFBP1851 plasmid p44 containing the full TALE1 Xam gene [35]. The probe was labeled with digoxigenin by means of the Random Primed DNA Labeling kit (Roche Diagnostics GmbH, Mannheim, Germany). Luminescent detection was achieved using chloro-5-substituted adamantyl-1, 2-dioxetane phosphate (CSPD) according to manufacturer's instructions. Chemiluminescent images were acquired on the ChemiDoc XRS + System (BioRad, Hercules, CA, USA) with the Chemi Hi Resolution protocol and signal accumulation mode.

Isolation and Sequencing of TALEs

The isolation of TALE genes was achieved by two different approaches. The first one consisted of PCR amplification of the central repeat region using GoTaq polymerase (Promega, Madison, WI, USA) and subsequent cloning into pGEM ® -T easy (Promega, Madison, WI, USA). Amplification was performed using 200 nM of primers 427 Fw (5 ′ -CGGTGGAGGCAGTGCATG-3 ′ ) and 428 Rv (5 ′ -ATCAGGGCGAGATAACTGGGC-3 ′ ); 1× GoTaq Green buffer, 1.5 mM MgCl2; 100 µM dNTPs, 20 mM betaine, 0.025 GoTaq polymerase units/µL and a total amount of 40 ng of template DNA in a final volume of 20 µL. PCR conditions were as follows: Initial denaturation at 94 C for 3 min and 30 s. Cloning into pGEM ® -T easy was performed according to manufacturer's instructions. Ligation products were electroporated into E. coli DH5α cells. Screen of clones harboring inserts was performed using blue/white screening on LB agar using 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) and a PCR-based confirmation (as described above).

The second approach was based on direct cloning of TALE genes into pBlueScript. RFLP data was used to calculate the position of each TALE in the electrophoresed BamHIdigested DNA. For each strain, 70 µg of DNA were digested. Electrophoresis was carried out in a 0.8% agarose gel on 1× TAE; migration was performed at a constant power of 5 Watts for 5 h at room temperature. Gels were stained using a 3× GelRed (Biotium, Fremont, CA, USA) pool. Gels were quickly visualized in a ChemiDoc XRS+ System (BioRad, Hercules, CA, USA), regions where the expected TALE bands were excised and recovered using the Zymoclean™ Gel DNA Recovery Kit (ZymoResearch, Irvine, CA, USA) according to manufacturer's instructions. pBlueScript was digested with BamHI-HF (NEB, Ipswich, MA, USA) and dephosphorylated with Antarctic Phosphatase (NEB, Ipswich, MA, USA) according to manufacturer's instructions. Eluted DNA was ligated with the prepared vector with T4 DNA ligase (Thermo Fisher Scientific, Waltham, MA, USA) at 22 • C overnight. Transformation of ligations, screening and confirmation were performed as described earlier.

Sequencing of isolated TALE genes was carried out by standard Sanger chemistry (Macrogen Inc., Seoul, Korea) and the assembly of reported fragments was performed using the Geneious software (version R11). In the case of TALE genes obtained by PCR, they were only considered accurate if they were found in at least two rounds of independent amplifications and cloning, or if they were also cloned directly from digested DNA.

TALE Characterization and Clustering

Assembled TALE sequences were treated and classified in variants using an in-house script on R (version 3.6.1). TALEs were clustered by predicted DNA binding specificity using FuncTAL software (version 1.1) [START_REF] Pérez-Quintero | A suite of tools to classify and compare TAL effectors functionally and phylogenetically[END_REF]; default parameters were used. TALEs were also clustered by nucleotide repeat composition using DisTAL software (version 1.1) [START_REF] Pérez-Quintero | A suite of tools to classify and compare TAL effectors functionally and phylogenetically[END_REF]; default parameters were used.

In-Vitro Plant Inoculation and Cassava RNA Extraction

Xpm strains were cultured in liquid media and prepared, as described earlier. Inocula were adjusted to an OD 600 of 0.02 (ca. 2 × 10 7 cfu/mL) in a 10-mM MgCl 2 solution. In-vitro propagated plants were inoculated with bacterial suspensions or mock solution (10-mM MgCl 2 ) with a swab on axial and abaxial surfaces of punctured leaves (nine needle punctures per leaf); each treatment was inoculated on three leaves per plant and on three different plants. Tissue surrounding inoculated punctures was collected at 50 hpi using a 3-mm diameter cork borer. Total RNA was extracted with the Invitrap Spin plant RNA minikit (STRATEC, Birkenfeld, Germany), using the RP buffer per manufacturer's instructions. Total RNAs were treated with RNase-free DNase I (Thermo Fisher Scientific, Waltham, MA, USA) and quality was controlled with the RNA 6000 Nano Kit (Agilent, Santa Clara, CA, USA).

RNA Sequencing and Transcriptome Analysis

Barcoded, paired-end (150-bp inserts) libraries were constructed with the TruSeq RNA Library Prep Kit (Illumina, Vancouver, British Columbia, Canada), per manufacturer's instructions. RNA libraries were pooled and sequenced on four flow cells of the NextSeq500 System. RNAseq analyses were performed using the Kallisto pseudo-mapper [START_REF] Bray | Near-optimal probabilistic RNA-seq quantification[END_REF] and EdgeR [START_REF] Robinson | A Bioconductor package for differential expression analysis of digital gene expression data[END_REF] for differential expression analysis by comparing against the mock-inoculated treatment on R (version 3.6.1). GO-term enrichment analyses were performed using the topGO package [START_REF] Rahnenfuhrer | Enrichment Analysis for Gene Ontology[END_REF].

TALE Target Prediction and Candidate Analysis

TALE targets were predicted using four different software: TALVEZ [START_REF] Grau | Computational Predictions Provide Insights into the Biology of TAL Effector Target Sites[END_REF], TAL Effector Nucleotide Targeter 2.0 (TALENT 2.0) [START_REF] Erkes | PrediTALE: A novel model learned from quantitative data allows for new perspectives on TALE targeting[END_REF], TALgetterLong [START_REF] Wang | Effectors Drive Transcription Bidirectionally in Plants[END_REF] and PrediTALE [START_REF] Kuon | Haplotype-resolved genomes of geminivirus-resistant and geminivirus-susceptible African cassava cultivars[END_REF]. The Manihot esculenta promoterome (1-kb sequences preceding annotated translational start sites) was extracted from Phytozome's cassava genome version 6.1 [START_REF] Lin | A pthA homolog from a variant of Xanthomonas axonopodis pv. citri enhances virulence without inducing canker symptom[END_REF], by means of the Biomart tool, and it was used as input for target prediction. All the algorithms were run using the default parameters. Output data were merged and compared on R (version 3.6.1) using an in-house script.

Semi-Quantitative and Quantitative RT-PCR

Xpm strains were cultured in liquid media and prepared as described earlier. Bacterial inoculum was adjusted to an OD 600 of 0.5 (c.a. 5 × 10 8 ufc/mL) in a 10-mM MgCl 2 solution. Bacterial suspensions or the mock solution (10-mM MgCl 2 ) were infiltrated into leaves of 3-month old adult plants grown from stakes, by means of a needleless syringe; each treatment was inoculated on one leaflet per plant and on three different individuals. Infiltrated tissue was collected at 50 hpi in sterile tubes containing RNA-free glass beads, samples were frozen with liquid nitrogen and ground by vortexing. Total RNA was extracted as described earlier. cDNA synthesis was performed with the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Walthman, MA, USA), per manufacturer's instructions. Semi-quantitative RT-PCRs were performed with a 20-µL reaction mix per tube containing 1× of 5× GoTaq Green Buffer, 1.5 mM of MgCl 2 , 100 µM of a dNTP mix, 0.2 µM of each primer, 5 ng/µL of cDNA, and 0.025 U/µL of GoTaq polymerase (Promega, Madison, WI, USA). Amplification was programmed as follows: one step at 95 • C for 3 min, followed by a variable number of cycles (20 to 28, as needed) of 95 • C for 30 s, 60 • C for 40 s, and 72 • C for 30 s. Amplicons were resolved in 1% agarose gels on 0.5× TBE. RT-qPCRs were performed on a 7500 Fast & 7500 Real-Time PCR System. Each well contained 10 µL of the following reaction mix: 1× of 2× SsoFastTM EvaGreen Supermix with Low ROX (BioRad, Hercules, CA USA), 300 nM of each primer, and 20 ng/µL of cDNA. PCR cycling was as follows: one step at 95 • C for 30 s, followed by 40 cycles of 95

Statistical Analysis and Packages

All the statistical analyses were performed in R (version 3.6.1). Aggressiveness data was analyzed using an in-house script through a linear mixed model-packages lme4 [START_REF] Sambrook | Molecular Cloning: A Laboratory Manual[END_REF] and multcomp [START_REF] Eisel | DIG Application Manual for Filter Hybridization[END_REF]-for log-transformed data, where strain was used as predictor variable with fixed effects, and replicates in time were included as the random effect. All the statistical comparisons were performed at a 0.95 significance level. Correlation analyses were performed by using ggpubr [START_REF] Kassambara | ggplot2" Based Publication Ready Plots[END_REF]. RT-qPCR data were analyzed using pcr package [START_REF] Ahmed | An R package for quality assessment, analysis and testing of qPCR data[END_REF], where a two-tailed t-test (alpha 0.05) was applied to the normalized expression values (normalized by using tubulin gene as reference) of each target gene in the inoculated treatment versus the mock-inoculated treatment. Boxplots, barplots, dotplots, and heatmaps were created with ggplot2 [START_REF] Wickham | ggplot2: Elegant Graphics for Data Analysis[END_REF], gplots [START_REF] Warnes | gplots: Various R Programming Tools for Plotting Data[END_REF] package. EBEs were depicted in the promoter contexts by using the gggenes package [START_REF] Wilkins | Draw Gene Arrow Maps in "ggplot2[END_REF] or Geneious software (version R11). Distance-based trees were modified on Geneious software (version R11).

Results

Aggressiveness of Selected Xpm Strains Is Homogeneous on Cassava Cultivar 60444

Xpm Colombian strains were selected based on three criteria: year of collection, the edaphoclimatic zone (ECZ) of CBB-endemic cassava-growing lands [35], and previous AFLP-and MLVA-based diversity information [START_REF] Trujillo | Population typing of the causal agent of cassava bacterial blight in the Eastern Plains of Colombia using two types of molecular markers[END_REF][START_REF] Trujillo | A complex population structure of the cassava pathogen Xanthomonas axonopodis pv. manihotis in recent years in the Caribbean Region of Colombia[END_REF]. From the two geographically distant ECZs, which harbor genetically differentiated Xpm populations [START_REF] Trujillo | A complex population structure of the cassava pathogen Xanthomonas axonopodis pv. manihotis in recent years in the Caribbean Region of Colombia[END_REF], a total of 18 strains representative of the Colombian Xpm diversity were selected (Table 2 and Supplementary Figure S1). It is also worth noting that strains UA318 and UA681 were previously used in mapping studies for QTL of resistance in cassava against CBB [START_REF] Soto-Sedano | Major Novel QTL for Resistance to Cassava Bacterial Blight Identified through a Multi-Environmental Analysis[END_REF]. Also, a subset of them was sequenced by Illumina (strains CIO151, CFBP1851, UA226, and UA306) [32,[START_REF] Arrieta-Ortiz | Genomic survey of pathogenicity determinants and VNTR markers in the cassava bacterial pathogen Xanthomonas axonopodis pv. manihotis strain CIO151[END_REF], and/or pathotyped on a set of cassava varieties (strains UA226, UA306, UA318, UA531 and UA681) [START_REF] Trujillo | A complex population structure of the cassava pathogen Xanthomonas axonopodis pv. manihotis in recent years in the Caribbean Region of Colombia[END_REF].

Five TALE Clusters with Similar Predicted DNA-Binding Affinities

To investigate potential DNA-binding affinities and redundant functions for TALE variants found among these isolates, we used the program FuncTAL. FuncTAL clusters TALEs based on correlations between potential target DNA sequences predicted from each RVD string [START_REF] Pérez-Quintero | A suite of tools to classify and compare TAL effectors functionally and phylogenetically[END_REF]. Figure 3 shows the dendrogram that resulted from the analysis of the 18 TALE variants reported in Table 3. The color code used to represent RVD types in the analyzed TALEs highlights interesting general features: (i) the first repeat invariably contains NI as RVD, and most of the effectors initiate the repeat sequence with a NI-NG-NI-NN repeat block; (ii) the last half-repeat is consistently NG, and the repeat region of and they collectively contain eight different TALE variants, allowing to cover four out of the five binding affinity clusters (see Figure 3), as well as the "orphan" variants TALE13A and TALE14E. Moreover, strain UA1061 lacks a TALE20 variant but it is as aggressive as others that contain TALE20, therefore, we aimed at investigating alternative explanations for this aggressiveness that could provide insight into the pathogenesis of Xpm. To this end, a mock solution (10-mM MgCl 2 ) or Xpm strains were inoculated into two-month old cassava plants propagated in-vitro, and tissue was harvested at 50 hpi. As illustrated in Figure 4A, expression profiles are remarkably similar and some of the most up-(Log 2 FoldChange ≥ 2 and p-value < 0.001) and down-regulated (Log 2 FoldChange ≤ -2 and p-value < 0.001) genes are shared by both treatments (upregulated genes Manes.06G123400, Manes.17G063800, Manes.07G120000; downregulated genes Manes.17G042600, Manes.05G151100, Manes.18G029800). However, treatment with UA681 significantly affected the transcription level of 588 genes, while UA1061 significantly affected that of 1021 genes. When analyzed as a whole, most of the differentially expressed genes (DEGs) shared by both treatments show the same transcriptomic profile (271 out of 273 genes, Figure 4B). The top three upregulated DEGs common to both treatments are Manes.06G123400, Manes.17G063800 and Manes.07G120000 which respectively encode the S gene MeSWEET10a, an oxidoreductase 2OG-Fe II oxygenase family protein, and a wallassociated receptor kinase galacturonan-binding. The top three common downregulated DEGs encode a tyrosinase (Manes.17G042600), a cytochrome P450 71B21-related protein (Manes.05G151100) and a protein that belongs to the mitochondrial calcium uniporter family (Manes.18G029800). The Gene Ontology (GO) term enrichment analysis (Fisher's test, α = 0.01) performed on shared DEGs (Figure 4C) shows that, as defined by cell compartment, most of the differential transcriptional activity accounts for proteins that localize to the cell membrane or the apoplast. Moreover, the molecular function and biological process classifications point towards a response that involves a significant number of genes altering cell redox status, glucan metabolism, and nitrogen transport.

Potential TALE Targets Are Involved in the Manipulation of Host Cell Redox Status and Nutrient Transport

Correlation of inoculated host gene expression profiles with EBE prediction is a powerful approach to find genes targeted by TALEs. The promoterome (defined as the 1-kb regions that flank annotated translational start sites) of cassava inbred line AM560-2 [START_REF] Bredeson | Sequencing wild and cultivated cassava and related species reveals extensive interspecific hybridization and genetic diversity[END_REF] was used as input to predict the EBEs of each of the 18 TALE variants discovered in this study. We used four different on-line available software: TALVEZ [START_REF] Pérez-Quintero | An Improved Method for TAL Effectors DNA-Binding Sites Prediction Reveals Functional Convergence in TAL Repertoires of Xanthomonas oryzae Strains[END_REF], TAL Effector Nucleotide Targeter 2.0 (TALENT) [START_REF] Doyle | TAL Effector-Nucleotide Targeter (TALE-NT) 2.0: Tools for TAL effector design and target prediction[END_REF], TALgetter [START_REF] Grau | Computational Predictions Provide Insights into the Biology of TAL Effector Target Sites[END_REF], and PrediTALE [START_REF] Erkes | PrediTALE: A novel model learned from quantitative data allows for new perspectives on TALE targeting[END_REF]. The output of each program was restricted to the first 400 results and the rank of each predicted EBE was used as a homogenized score to enable comparisons. We cross-referenced the DUGs with the set of predictions for the TALomes of each inoculated strain. Results for UA1061 show that from the 320 DUGs, only 36 were predicted to have at least one EBE on the promoter region, while for UA681 the ratio was 69 out of 396.

The study of common DEGs could shed light on potential hubs used by the pathogen to manipulate the host. When predictions for the two TALomes and transcriptomics from plants inoculated with both strains were compared, we detected six candidate genes (Figure 5A, first six genes from the top to the bottom of the figure) with high-quality EBE predictions whose transcription could be upregulated by TALEs from clusters 1, 2, and 4 (see threshold in Figure 5A). Table 4 details the annotation and the prediction quality obtained for the proposed candidates. In the analysis of individual TALomes (Supplementary Figure S4), a clear signature prediction/expression for probable candidates was observed for 19 additional genes in the case of UA681, and 7 additional genes for UA1061 (see Table 4). Distance to the translation start site and orientation of EBEs are two factors known to affect transcriptional activation rate driven by TALEs [3,[START_REF] Wang | Effectors Drive Transcription Bidirectionally in Plants[END_REF]. Cernadas and coworkers [3] found that EBEs in real targets ranked consistently on the first 200 predictions, and their distance to annotated transcriptional start site (TSS) generally ranged between 152 bp upstream and 63 bp downstream; being this latter parameter the most predictive a Gene identifiers and annotations were extracted from Phytozome's cassava genome version 6.1. b T stands for TALE. The bracketed alphanumeric code corresponds to the cluster number derived from FuncTAL analysis (Section 3.3 and Figure 3). NC, non-clustered; C1 to C4, cluster 1 through 4. c The quality of EBE prediction for a given candidate was calculated as the sum of the four determined ranks (one per software). Totals were then categorized as E (Excellent, combined rank = 1 to 400), VG (Very Good, combined rank = 401 to 800), G (Good, combined rank = 801 to 1200), and F (Fair, combined rank = 1201 to 1600). d These EBE predictions were not found in the corresponding 60444 promoter sequence. e EBE distance to TSS/TLS between 152 bp and 63 bp. a Gene identifiers and annotations were extracted from Phytozome's cassava genome version 6.1. b T stands for TALE. The bracketed alphanumeric code corresponds to the cluster number derived from FuncTAL analysis (Section 3.3 and Figure 3). NC, non-clustered; C1 to C4, cluster 1 through 4. c The quality of EBE prediction for a given candidate was calculated as the sum of the four determined ranks (one per software). Totals were then categorized as E (Excellent, combined rank = 1 to 400), VG (Very Good, combined rank = 401 to 800), G (Good, combined rank = 801 to 1200), and F (Fair, combined rank = 1201 to 1600). d These EBE predictions were not found in the corresponding 60444 promoter sequence. e EBE distance to TSS/TLS between 152 bp and 63 bp.

Discussion

This study started with a selection of Xpm strains based on neutral genetic diversity at a country scale that allowed us to depict the TALE variation among strains isolated from different cassava productive regions over different years in Colombia. Southern blot analysis showed that Xpm strains carry TALomes with a bimodal size distribution, and sequence analysis showed thirteen novel TALE variants. Sequence clustering by predicted DNA-binding affinity allowed us to condense this variability into five clusters and four unrelated TALE variants. A complementary transcriptomic approach allowed us to move from diversity to function and to screen host genes that might act as CBB susceptibility determinants. Transcriptomics showed that redox processes and nutrient transport are upregulated during infection, and some of the involved genes are potentially targeted by TALEs. Importantly, our results also suggest that TALEs different from TALE20 might activate the transcription of MeSWEET10a and potentially confer aggressiveness to those strains lacking TALE20 in Xpm populations. This study sets new bases for assessing unidentified TALE targets in the Xpm-cassava interaction, which could be important factors that define the fate of the infection.

TALEs play major roles in the pathogenesis of several xanthomonads (reviewed by [4]), mainly by conferring bacteria the ability to create a favorable niche in the plant vascular system. In some cases, Xanthomonas spp. pathogenicity is drastically altered by the activity of a given TALE [START_REF] Lin | A pthA homolog from a variant of Xanthomonas axonopodis pv. citri enhances virulence without inducing canker symptom[END_REF][START_REF] Ye | tale-Based Genetic Diversity of Chinese Isolates of the Citrus Canker Pathogen Xanthomonas citri subsp. citri[END_REF]. Bart and coworkers [32] found a positive trend between the number of TALEs per TALome vs. aggressiveness and virulence assessed in 18 strains of Xpm. In our study, we profiled the aggressiveness of 18 Xpm strains and found that only CFBP1851 shows reduced capacity to cause lesions. Interestingly, this strain does not carry TALE20 or any TALE20 variant that could activate the S gene MeSWEET10a [8], which could explain reduced aggressiveness. Strains UA1061 and UA1069 also lack this TALE variant, but their aggressiveness is average. However, UA1061 possess two variants of TALE22, one of them with an RVD sequence similar to that of the TALE20B variant plus a duplication of the first two repeats (Figures 2 and3). This new TALE22 variant (TALE22D) could explain the fact that strain UA1061, and potentially UA1069, do not have a decrease in aggressiveness even if they lack TALE20 variants. The DisTAL analysis (Supplementary Figure S3) indicates that TALE22D shares ancestry with TALE20B, and, based on the transcriptomic profile of UA1069-inoculated plants and the EBE predictions, this TALE seems to be able to induce MeSWEET10a. However, despite the reduced ability of CFBP1851 to cause symptoms, it still induces water-soaked lesions, indicating that there might be mechanisms that do not rely on MeSWEET10a transcriptional activation to promote water soaking, yet less efficiently.

As seen in Southern blot analysis, Xpm TALomes are characterized by a limited and bimodal range of variant sizes. This bimodal size distribution could be explained by a recent acquisition of TALEs from two different ancestors or the fixation of two useful alleles that then diverged. Our data favors the second hypothesis since the repeat-based phylogeny (Supplementary Figure S3) shows two clades that group TALEs by size, both emerging from the TALE13 variants. This is probably why there are shared repeat block patterns between the two TALE size groups (Figure 3). Ferreira and collaborators [START_REF] Ferreira | A TALE of Transposition: Tn3-Like Transposons Play a Major Role in the Spread of Pathogenicity Determinants of Xanthomonas citri and Other Xanthomonads[END_REF] created a TALE-based phylogeny with 122 coding sequences from Xanthomonas spp. TALEs. Xanthomonas phaseoli pv. phaseoli and Xanthomonas citri pv. fuscans, two pathovars closely related to Xpm, show unimodal and continuous TALE size distribution that ranges from 18 to 23 repeats [START_REF] Ruh | Xanthomonas adaptation to common bean is associated with horizontal transfers of genes encoding TAL effectors[END_REF]. N-, C-terminal and TALE flanking region analysis suggests that these TALE variants come from a unique common ancestor [START_REF] Ruh | Xanthomonas adaptation to common bean is associated with horizontal transfers of genes encoding TAL effectors[END_REF]. X. campestris (Xc) exhibits a more complex TALE size distribution which however seems to be even from 11 to 22 repeats. Yet, analysis of N-, C-terminal and repeats indicate that Xc TALEs emerged from two different ancestors [START_REF] Denancé | Two ancestral genes shaped the Xanthomonas campestris TAL effector gene repertoire[END_REF], where smaller TALEs (variants with 12, 14 and 15 repeats) have structurally different repeats from larger TALEs with 21 and 22 repeats. Since N-and C-terminal sequences are not available in our study and repeat structure is highly homogeneous, we could not perform such analyses. TALE sequence diversity was assessed by potential DNA-binding affinity using Func-TAL [START_REF] Pérez-Quintero | A suite of tools to classify and compare TAL effectors functionally and phylogenetically[END_REF], resulting in five DNA affinity groups and four orphan (non-clustered) effectors. Among them, there are several examples of deletion/duplication, like the case of TALE22D from cluster 1 (discussed earlier), TALE14B in the cluster 3 and TALE13A when compared to TALE14E (see Figure 3). Repeat recombination patterns are also observed; for example, TALE14A variant shows the most common starting RVD block NI-NG-NI-NN, but the remaining block of ten RVDs can be found in TALE22A and B variants. Repeat swaps (non-synonymous point mutations affecting the RVD) seem to be a key variability driver for TALE diversity in Xpm, since all the clusters show at least two variants with one to four swaps (see Figure 3). These evolutionary mechanisms for TALEs have been well documented [START_REF] Erkes | Evolution of Transcription Activator-Like Effectors in Xanthomonas oryzae[END_REF][START_REF] Schandry | TALE-Like Effectors Are an Ancestral Feature of the Ralstonia solanacearum Species Complex and Converge in DNA Targeting Specificity[END_REF], and several examples of deletion/duplication, repeat swap and recombination can be found among Xanthomonas spp. TALEs [6,[START_REF] Ruh | Xanthomonas adaptation to common bean is associated with horizontal transfers of genes encoding TAL effectors[END_REF][START_REF] Denancé | Two ancestral genes shaped the Xanthomonas campestris TAL effector gene repertoire[END_REF].

To elucidate the effects of this functional diversity, we selected two strains whose TALomes covered most of the TALE diversity. Strains UA681 and UA1061 were inoculated in 60,444 varieties to obtain a transcriptomic snapshot of the infection. Briefly, plant response to infection with either of the strains shows a marked alteration of oxidoreductase activity, DNA-transcription factor activity, interaction with metals such as iron and copper, and carbon catabolism. Muñoz-Bodnar and collaborators compared the transcriptomic profiles of cassava plants from the variety MCOL2215 inoculated with two Xpm strains with contrasting virulence [START_REF] Munoz-Bodnar | RNAseq analysis of cassava reveals similar plant responses upon infection with pathogenic and non-pathogenic strains of Xanthomonas axonopodis pv. manihotis[END_REF]. In that study, Xpm transcriptomic profiles did not significantly differ and showed a transcriptional alteration of genes related to catabolic processes, cellular glucan metabolism, transcription regulation and response to oxidative stress, among other categories. A transcriptomic study in Xanthomonas oryzae pv. oryzicola (Xoc) showed altered expression of genes involved in catalytic and oxidoreductase activities, which suggest an active reactive oxygen species detoxification and cellular redox status control [3]. Taken together, this indicates that alteration of the redox environment and catabolic processes are common to the compatible interactions between Xanthomonas spp. and their hosts, highlighting their relevance in pathogenesis.

The analysis of the DUGs coupled to EBE predictions resulted in several candidates that could be important for Xpm pathogenesis. Some of these candidate genes were previously validated as actual targets of Xpm TALEs (see Table 4), which supports our findings. The sugar transporter encoding MeSWEET10a is a DUG highly overexpressed in both treatments (UA681 and UA1061), and it has been shown to play a key role in symptom formation and bacterial growth in planta [8]. Our data provide indirect evidence that the new TALE20-derived TALE22D variant is functional and capable of targeting this important S gene. In this regard, we explored if the addition of these two repeats at the beginning would negatively affect the predicted DNA-binding affinity on the already known EBE (EBE for TAL20: TATAAACGCTTCTCGCCCATC). Results show that the two nucleotides located right before the EBE for TAL20 are the same first two nucleotides of the EBE (EBE for TAL22D: TATATAAACGCTTCTCGCCCATC), allowing then a perfect match for the new variant. We foresee that these changes might affect TALE-driven induction dynamics, but it is difficult to establish if these will result beneficial for the molecular interaction. Repeats located near to the N-terminal region contribute more to the specific DNA recognition and are more important to the overall affinity of the TALE-DNA interaction [START_REF] Meckler | Quantitative analysis of TALE-DNA interactions suggests polarity effects[END_REF][START_REF] Wan | Molecular Dynamics Simulations of DNA-Free and DNA-Bound TAL Effectors[END_REF], but, paradoxically, increasing the number of repeats results in lower specificity due to more off-target activation [START_REF] Guilinger | Broad specificity profiling of TALENs results in engineered nucleases with improved DNA-cleavage specificity[END_REF]. What implications this change could have in the intensity of binding induction of the gene or in the adaptation of the pathogen to mutations in the promoter needs to be determined. A comparison of MeSWEET10a induction in plants treated with strains expressing these two TALE variants would indicate if this mutation has a significant impact on transcriptional activity of the S gene, and the search for this novel variant among populations would shed light on evolutionary dynamics of major TALEs.

The ammonium transporter encoding gene Manes.08G141800 is one of the most interesting candidates targeted by this new TALE variant. Grewal and coworkers demonstrated that rice response after a few hours of being inoculated with Xanthomonas oryzae pv. oryzae, a vascular pathogen responsible for the bacterial leaf blight in rice, encompasses the transcriptional alteration of several transporters, including ammonium transporters. Activity of these transporters change ion fluxes and affect the membrane potential, disturbing uptake of other channels and altering activation of defense response. In this regard, they point out that ammonium transporter activity affects K + uptake by cells, which would lead to modified cation balances [START_REF] Grewal | oryzae triggers immediate transcriptomic modulations in rice[END_REF]. It is possible that these ammonium transporters have an analogous effect to that hypothesized for the sulfate transporter coding gene OsSULTR3;6 induced by Tal2g in the Xoc-rice pathosystem [3]. The expression of these genes might allow the bacteria to alter antioxidant capacity through ion leaking, which would result in interference with redox signaling and defense or in the promotion of water soaking through alteration of osmotic equilibrium.

Functional redundancy by convergence has been demonstrated for TALEs in other pathosystems. In those cases, important susceptibility genes are targeted by two or more TALEs that have potentially evolved from different ancestors and converge on EBEs present in the same promoter region [1,7,[START_REF] Hutin | Kombat: The story of defense against TAL effectors through loss-of-susceptibility[END_REF][START_REF] Yang | Diverse Members of the AvrBs3/PthA Family of Type III Effectors Are Major Virulence Determinants in Bacterial Blight Disease of Rice[END_REF][START_REF] Streubel | Five phylogenetically close rice SWEET genes confer TAL effector-mediated susceptibility to Xanthomonas oryzae pv. oryzae[END_REF][START_REF] Zhou | Gene targeting by the TAL effector PthXo2 reveals cryptic resistance gene for bacterial blight of rice[END_REF]. Our data suggested that candidate genes Manes.04G033900 and Manes.13G045100 are targeted by TALE22 variants (which was already validated by Cohn and coworkers [8]) and also by the TALE21A variant found in CFBP1851, through overlapped EBEs. Our RT-PCR results indicate that Manes.13G045100 is actually upregulated in planta after inoculation with CFBP1851 strain (Figure 7B), showing that this is a potential case of functional convergence and a possible relevant target for the pathogen. However, even if at a first glance TALE21A variant has important structural differences when compared to TALE22 variants, DisTAL analysis indicates that variant TALE21A is very close to TALE22 variants, which would reflect the history of close evolving effectors that maintain affinities. Manes.13G045100 encodes for a CLAVATA3/ESR (CLE) peptide, a protein family of ligands involved in definition of cell fate with a key role in cellular division control in the shoot apical meristem [START_REF] Betsuyaku | The Function of the CLE Peptides in Plant Development and Plant-Microbe Interactions[END_REF][START_REF] Strabala | Gain-of-function phenotypes of many CLAVATA3/ESR genes, including four new family members, correlate with tandem variations in the conserved CLAVATA3/ESR domain[END_REF]. This ligand family has been shown to be important in symbiotic processes through the regulation of nodule formation, and in pathogenic interactions where phytopathogens hijack the CLE-mediated developmental signaling pathway to improve fitness and colonization rate (reviewed by [START_REF] Yamaguchi | CLE peptides and their signaling pathways in plant development[END_REF]). Nematodes Globodera rostochiensis [START_REF] Guo | Mechanisms of molecular mimicry of plant CLE peptide ligands by the parasitic nematode Globodera rostochiensis[END_REF] and Heterodera glycines [START_REF] Wang | A parasitism gene from a plant-parasitic nematode with function similar to CLAVATA3/ESR (CLE) of Arabidopsis thaliana[END_REF] secrete effectors that mimic CLE functions which allow them to reprogram cell growth at the point of infection. To our knowledge, there are no reports of phytopathogenic bacteria using this pathway as a virulence factor, but there are other examples of cell fate-related transcriptional factors, UPA20 and UPA7 [12,[START_REF] Marois | The Xanthomonas Type III Effector Protein AvrBs3 Modulates Plant Gene Expression and Induces Cell Hypertrophy in the Susceptible Host[END_REF], which are also transcriptionally activated by TALEs (AvrBs3). Taking all this into consideration, validation and further studies on this TALE target could shed light on new host susceptibility pathways.

Conclusions

This work describes for the first time the TALE variability on Xpm populations. TALome sequences allowed us to make some evolutionary and functional inferences on TALE biology for the Xpm-cassava pathosystem. Functional clustering of TALEs unveiled traces of recombination, repeat swaps and repeat duplication/deletion. Transcriptomics indicate that new TALE20-derived TALE22D variant preserves the ability to activate the S gene MeSWEET10a. RNAseq with two Xpm strains showed that common up-and down-regulated genes have virtually the same behavior in terms of expression magnitude and regulation, and differentially expressed genes fall consistently in oxidoreductase and catabolic activity categories. Our results pinpoint new potential TALE targets that could have a role in pathogenesis. Further studies are needed to validate the TALE-dependent activation of these targets through the use of mutant strains inactivated in these TALEs and characterize their role during infection using artificial TALEs.

Supplementary Materials:

The following are available online at https://www.mdpi.com/2076-260 7/9/2/315/s1, Figure S1: Two edaphoclimatic zones (ECZs) where cassava is grown in Colombia and strain origins. The blue region corresponds to the Caribbean coast ECZ delimited as two departments: Córdoba and Sucre. The yellow region corresponds to the Eastern plains ECZ delimited as two departments: Casanare and Meta. Black dots show municipalities where strains were collected. The list of strains from each ECZ includes the collection date in parentheses. Figure S2: Southern blots for the characterized strains. The upper panel shows lanes for strains from the Caribbean coast and strains isolated from the department Meta (Eastern plains). The lower panel shows lanes for the strains from the department Casanare (Eastern plains). The ID of each strain is marked above each lane. MWM = molecular weight marker; TALE1 Xam = pF3 vector containing TALE1 Xam . This vector was also digested with BamHI and loaded into each gel as a positive control and size indicator. The molecular weight marker is presented as seen in the stained gel to estimate sizes. Figure S3: Repeat-based phylogeny created by DisTAL. Unrooted cladogram showing the phylogenetic relationship among nucleotide variants according to nucleotide composition of repeats after masking positions coding for RVDs. Labels indicate the variant classification according to the RVD string (T stands for TALE) and the strain where the gene was isolated. Colors are related to the clusters defined in Figure 3: blue = Cluster 1, magenta = Cluster 2, red = Cluster 3, cyan = Cluster 4, yellow = Cluster 5; orphans are all in black. Values on branches correspond to distances obtained from pair-wise alignments for repeat-based coded TALEs calculated by DisTAL. Figure S4: TALE-targeted candidate prediction for A) UA681 and B) UA1061 DUGs. These heatmaps show the best optioned candidates and their EBEprediction quality from each software used. Top labels indicate the TALE variant that is predicted to bind each gene and the DNA-binding affinity cluster where it belongs. Rank indicates the position of the prediction (which is a reflect of the score) among the set of the top 400 predictions. Genes over the dashed horizontal line are considered as the best optioned candidates (see text). Figure S5: Selective GO-term enrichment analysis showing the cellular component (CC), molecular function (MF) and biological process (BP) categories that are significantly enriched with DEGs carrying EBEs on their promoters in UA681-or UA1061-treated plants. Discontinuous lines show the p-value thresholds corresponding to 0.05 (blue) and 0.01 (dark gray). Table S1. EBE position comparison between the promoter context of genes reported for varieties AM560-2 and 60444 genomes. Table S2. Potential cases of functional convergence among DUGs with predicted EBEs. Candidates are sorted from the top to the bottom according to the EBE prediction quality. Table S3. ID and short description of GO Terms displayed on Figure 4 Data Availability Statement: Publicly available datasets were analyzed in this study. TALE nucleotide sequences can be found at GenBank under accession numbers MW413757-MW413802. RNA-Seq data can be found in the SRA database under the BioProject No. PRJNA688032, BioSample No. SAMN17167922 (https://www.ncbi.nlm.nih.gov/sra/PRJNA688032).

Acknowledgments:

The Xanthomonas strains were collected and investigated under the contract for access to genetic resources from the Colombian Environmental Ministry Number 261 or 2019 with Universidad de los Andes. We acknowledge the contribution of undergraduate students from Universidad de los Andes Luisa Jaimes Niño and María Camila Buitrago during their internships. We also thank Adolfo Amezquita for statistical advice.

Conflicts of Interest:

The authors declare no conflict of interest.

Introduction

Cassava (Manihot esculenta, Crantz) is a staple crop for one billion people in the tropics. It is equally important as a source of feed and industrial applications, and is also a source of energy [1]. Cassava Bacterial Blight (CBB) is the predominant and most economically significant bacterial disease of cassava [2]. Its causal agent, formerly known as Xanthomonas axonopodis pv. manihotis, was recently reassessed taxonomically [3], and the accepted nomenclature is now Xanthomonas phaseoli pv. manihotis (Xpm). Xpm is a bacterial vascular pathogen that penetrates the host through hydathodes, stomata and/or wounds, causing angular leaf spots, leaf wilting, stem gum exudates, vascular necrosis of the stem, and shoot dieback [4]. Transmission of CBB is mainly driven by contaminated farming tools and rain splashing, while infected stakes allow long-distance dissemination. Other factors influencing Xpm epidemiology include its epiphytic behavior, survival on plant debris and reservoir plants [5,6].

Cassava Bacterial Necrosis (CBN), which is caused by Xanthomonas cassavae (Xc) has so far only been reported in Africa [7,8]. This contrasts with CBB, which was first reported in Latin America in the early 1900s and is now present throughout the tropics [9]. Xc provokes foliar symptoms somehow similar to those caused by Xpm, except that no vascular colonization has been observed for Xc. Despite many common biochemical characteristics, Xpm and Xc differ in the presence of xanthomonadin, a yellow pigment typical of xanthomonads, which is conserved in Xc. In contrast, the cluster of genes responsible for xanthomonadin biosynthesis is mutated in Xpm resulting in a white phenotype [10].

The major control strategies against CBB include the use of healthy propagative plant material, removal of infected plant material, regular surveillance, monitoring of regional outbreaks, determining highly affected areas, and predicting zones under immediate threat. These strategies rely on the ability to efficiently detect and identify the pathogen in plant material. The diversity of the pathogen has to be taken https://doi.org/10.1016/j.pmpp.2018.07.005 Received 28 November 2017; Received in revised form 21 July 2018; Accepted 23 July 2018 into account for the development of such tools. Several studies based on biochemical, serological, pathological and molecular approaches clearly distinguished Xc from Xpm strains and showed intra-species variability [11][12][13][14]. Nevertheless, for Xpm, genetic variability was shown to be pronounced in strains from South America, but was limited in strains from other regions such as in Africa [11,14]. Yet other studies suggest that genetic variability of Xpm in this continent might have been underestimated [4]. Recently, a full-genome SNP-based phylogeny of 65 geographically and temporally diverse Xpm genomes showed a strong clustering by geographic origin with only a few outliers [15].

Systematic diagnostic studies began in the 1990s, following different approaches from the use of selective growth medium to molecular characterization. The development of a semi-selective agar medium greatly improved the recovery of Xpm from plant material and soil [16]. Nevertheless, culture-based methods are not specific enough and require complementary identification methods. A monoclonal antibody was also used for an ELISA assay to detect Xpm from plant material, but some false negative results, as well as cross-reactions with non-target strains, were obtained [17]. Different molecular tests were also developed for the detection of Xpm: a dot-blot assay [18], a PCR assay [19] and a nested-PCR assay [20], all targeting a gene encoding a major Xpm pathogenicity factor. No molecular test has been developed until now for the detection of Xc.

Next generation sequencing technologies have produced a wealth of Xanthomonas spp. genomes, including draft sequences of Xpm strains [10,15] and of a single strain of Xc [21]. Based on these resources we searched in silico for appropriate targets and tested them against a collection of Xpm and Xc strains. This allowed us to develop a novel duplex-PCR diagnostic tool that successfully detects and distinguishes Xpm and Xc from symptomatic cassava plant material.

Materials and methods

Bacterial strains, culture conditions and DNA extraction

The bacterial strains used in this study include 50 strains of Xpm, 23 strains of Xc and 32 strains belonging to other genera, species or pathovars, including saprophytic strains isolated from cassava (Table 1). Strains were stored at -80 °C in 10% glycerol for long-term storage, and routinely grown on YPGA medium (5 g/l yeast extract, 5 g/l peptone, 5 g/l glucose, 15 g/l agar) at 28 °C for 48-72 h. Bacterial cultures were also grown in liquid medium Φ (1 g/l yeast extract, 1 g/l acidhydrolyzed casein) in 5 ml tubes, prior to DNA extraction or preparation of bacterial suspensions. For this, a single colony was picked on YPGA medium with a sterile toothpick and dropped into Φ liquid medium. The tubes were incubated at 28 °C for 48 h in Ecotron shaker (INFORS HT) at a speed of 160 RPM.

Genomic DNA was extracted from 1 ml liquid culture using the Wizard genomic DNA purification kit (Promega) according to supplier's instructions. DNA concentration was measured with a NanoDrop ND-1000 spectrophometer and adjusted to 1 ng/μl. To prepare boiled bacterial suspensions, 1 ml of liquid culture was adjusted spectrophotometrically to approximately 10 8 CFU/ml (O.D 600 = 0.2) using a WPA biowave CO8000 Cell Density Meter, heated at 95 °C for 10 min in a water bath (F25, Jubalo) and chilled on ice.

Pathogenicity tests

The pathogenicity of Xpm (CIX1088, CIAT1120, CIAT1135, CIO151, CIAT1111, CI4, CIAT1206, ORST2, K419b, CFBP 2603) and Xc strains (UPB008, UPB029, UPB033, UPB043, UPB046, UPB047, UPB054, UPB059, UPB146) was evaluated under greenhouse conditions at 28 °C and a relative humidity of 80% on 6-week-old cassava plants of the variety SG107-15. Strains grown for 48 h in Φ medium were used to prepare bacterial suspensions with sterile pure water and adjusted spectrophotometrically to O. D 600 = 0.2. About 500 μl of bacterial suspensions were infiltrated into the abaxial side of the leaf by means of a needleless syringe. For each strain, three infiltrations were performed in each lobe of the leaf. This experiment was repeated three times. The observations of symptoms were performed up to 7 days after inoculation.

Design and screening of primers

To develop diagnostic primers that differentiate between Xpm and Xc, unique sequences were identified using in-house primer pipeline named Uniqprimer version 0.5.0 (L. Triplett and J.E. Leach, in preparation) that was scripted to complement the speed and utility of the nucmer sequence aligner in the MUMmer 3.0 package [22]. The pipeline runs a series of nucmer alignments (default conditions except for the options -minmatch 300, -maxgap 1) to align whole or draft genomic sequence of a diagnostic target with genomes of non-target strains and additional target strains, and parses the output to identify coordinates of regions aligning to target genomes but not to non-target genomes. Sequences are retrieved into a multifasta file that is the template for Primer3 primer design [23]. Primer3 outputs are then aligned to the target and non-target genomes using the Primersearch program from the EMBOSS package [24] to ensure amplification specificity (cutoffs = 0% mismatch with target sequence, 20% mismatch with non-target sequence). The source code, instructions, and further validation of this pipeline will be made publicly available through two publications in preparation.

The Xpm primers were designed from the complete draft sequence of the template genome UG23, and aligned against the genome sequences of X. cassavae strain CFBP4642, Xanthomonas oryzae PXO99A, Xanthomonas vasicola NCPPB206, Xanthomonas citri pv. citri Aw12879, Xanthomonas campestris pv. campestris ATCC33913, Xanthomonas phaseoli pv. fuscans 4834-R and Xanthomonas axonopodis pv. citrumelo F1 (NCBI accession numbers ATMC00000000.1, NC_010717, AKBM00000000, CP003778.1, NC_003902, FO681494.1, and CP002914, respectively). The draft genome of Xc strain CFBP4642 was used as a template for the selection of Xc primers. The specified nontarget genomes were those of Xpm strain UG23 (NCBI accession AKEV00000000), and reference strains of X. oryzae pv. oryzicola, X. campestris pv. campestris and X. vasicola named above. Six primer pairs for Xpm and eight for Xc were selected (Table 2).

In silico screening

Blastn searches performed on the NCBI nucleotide collection (nr/nt) database with the primers and target sequences showed no significant sequence similarities with non-Xanthomonas bacteria (data not shown). The primers and target sequences were then used in BLASTn queries against the 901 genome sequences of different strains belonging to the Xanthomonadales order that were available in the NCBI database (16/ 08/17), including 66 Xpm and one Xc genome sequences (Microbial genome Blast, Databases = All genomes/Draft genomes, Organism: Xanthomonadales (taxid:135614), Program selection: blastn). Some types of primer-template mismatches are known to have no significant effect on PCR efficiency. We relied on previous studies [25,26] to predict PCR amplifications based on different criteria, mainly the nature, the number and the location of the mismatches. We only took into account template/primer duplexes including up to two mismatches internal or located at primer 5′ terminal base. No mismatch located in the primer 3′ terminal base was tolerated and for the 3 bases before 3′ terminal base, we did not consider the mismatches known to have a significant effect on PCR yield (A:G, G:A, and C:C and A:A).

Experimental screening

The primer pairs were tested using boiled culture suspensions of a subset of 10 Xpm (CIX1088, CIAT1120, CIAT1135, CIO151, CIAT1111, CI4, CIAT1206, ORST2, K419b, CFBP 2603) and 9 Xc (UPB008, UPB029, UPB033, UPB043, UPB046, UPB047, UPB054, UPB059, UPB146) strains (Table 1). The selected strains were temporally and especially geographically diverse in order to cover as much as possible the genetic diversity of the bacteria, as shown by Bart et al. for Xpm [15].

We also tested a set of eight non-target strains including strains that were positive in the in silico evaluation (LMG 876, CFBP2053, LMG695, LMG9055, LMG8014, CFBP2868, CFBP2903, CFBP2905) (Tables 1 and4).

PCR assays

Simplex PCR reactions were performed in a final volume of 10 μl containing 0.5 μM of each forward and reverse primers, 2 mM dNTPs, 2 mM MgCl 2 , 1,25 U GoTaq DNA polymerase (Promega), buffer 1X and For the duplex-PCR, preliminary results showed the Xpm target to be preferentially amplified. Optimization experiments were therefore conducted in order to homogeneously amplify both targets. For this, various concentrations of Xpm1 F/R and Xc1 F/R primers were tested: 0.5 μM Xpm1/0.5 μM Xc1, 0.25 μM Xpm1/0.5 μM Xc1 and 0.125 μM Xpm1/0.5 μM Xc1 in the same final mix as for the simplex PCR. The duplex-PCR assay was tested on gDNA (1 ng/μl) or boiled suspensions of Xpm strain CIO151 and Xc strain CIX739 mixed at ratios 1:1 and 1:2. This experiment was repeated thrice. PCR amplifications were carried out in a thermal cycler (Applied Biosystems 2720). After 2 min of denaturation at 95 °C, 35 cycles of 45 s of denaturation at 95 °C, 45 s of annealing at 61 °C and 1.5 min of extension at 72 °C were performed, with a final extension at 72 °C for 5 min. Aliquots containing the PCR amplification products were subjected to electrophoresis in 1.5% agarose gels at 50 V for 1 h and visualized by ultraviolet light after staining with ethidium bromide.

Evaluation of specificity and sensitivity of the different PCR assays

To determine the specificity, we evaluated both the criteria of inclusivity, i.e. the ability of the different primers to detect all strains of the target organism, and of exclusivity, i.e. no amplicon acquisition of the expected size from non-target strains.

The specificity of the simplex PCRs directed by the primer pairs Xpm1 F/R and Xc1 F/R and of the duplex-PCR (combining the two sets of primers) was evaluated on the collection of Xpm, Xc and non-target strains (Table 1), using 1 μl of genomic DNA (1 ng/μl) and/or boiled liquid cultures as templates. The primer pairs were also tested on an historical DNA collection including 39 Xpm and Xc strains (20 ng/μl) collected in the 1970s [27] (Supplemental Table S1).

The sensitivity of the duplex-PCR was assayed for both bacterial pure cultures and plant extracts. Xpm strain CIO151 and Xc strain CIX739 were grown overnight in Φ liquid medium, were suspended at O. D 600 = 0.2 and serially diluted in Tris buffer pH 7.2. Negative control received only Tris buffer. The different suspensions were boiled as described and tested with the duplex-PCR assay. Before boiling, bacterial cells concentration was checked by plating 100 μl of the 10 4 ,10 5 and 10 6 -fold dilutions on YPGA (10 replicates per dilution). For determination of the sensitivity in a plant matrix, the dilution series were added to plant homogenates. Cassava leaves were ground using mortar and pestle in Tris buffer pH 7.2, 2% polyvinylpyrrolidone (PVP) (20 ml buffer per 0.2 g of leaf material), and total DNA was extracted from 2 ml samples using the DNeasy Plant Mini Kit (Qiagen), according to the supplier's instructions and was tested using the duplex-PCR assay. This experiment was performed three time independently (including the preparation of the initial bacterial suspensions).

Detection of Xpm and Xc from plant samples

To determine the ability of the primers to detect the presence of Xpm and Xc in infected plant tissues, cassava leaves from the variety SG107-15 were artificially inoculated with Xpm strain CIO151 or Xc strain CIX739 as described above. Leaves were collected 7 dpi and angular lesions were cut from the leaves and macerated for 2 min in 0.5 ml of sterile distilled water [19]. Ten-fold dilution series were performed in distilled water down to 10 8 -fold, and 1 μl of each dilution was used as a template in simplex and duplex-PCR assays. Total DNA was also extracted from CBB and CBN symptoms obtained after inoculation of different Xpm (CIO151 and CIX1088) and Xc (UPB059, UPB146, UPB029, UPB043, UPB046) strains using the DNeasy Plant Mini Kit (Qiagen) as described above, and 1 μl of DNA extract was used as a template in duplex-PCR assays.

The presence of Xpm and Xc was also tested from diseased leaf samples collected in the 1970s and 80s in different countries, all maintained at the Earth and Life Institute (Université catholique de Louvain, Belgium), at room temperature (20-25 °C), under dry conditions (Table 3). At the time of sample collection, bacterial colonies had been isolated from these samples and characterized by morphological, physiological, biochemical features and pathogenicity tests. Some isolates were also characterized by protein gel electrophoregrams and DNA:DNA hybridizations [28]. Typical symptomatic lesions were sampled and serially diluted down to 10 5 -fold as described above, or total DNA was extracted from lesions using the DNeasy Plant Mini Kit (Qiagen). Duplex-PCR was performed using 1 μl of each dilution or 1 μl DNA extract.

Results

Search for unique primers differentiating Xpm and Xc

Comparative genomic analysis of target and non-target genomes using the Uniqprimer pipeline 0.5.0 successfully generated candidate primer pairs which are a priori specific to each of the two pathogens (Table 2). The screening of these primer pairs was performed based on both in silico and experimental tests. The pathogenicity of the subset of Xpm and Xc strains (n = 19) used for PCR screening was previously confirmed by inoculation assays (Fig. S1).

In silico analyses showed 100% of inclusivity for all Xpm primer pairs that were selected except for Xpm6. Indeed, while primer pairs Xpm1 to 5 matched with the 66 Xpm genome sequences available at NCBI (Table 2), only two genomes were putatively detected by primer pair Xpm6. When tested experimentally, pairs of primers Xpm1 to 5 all resulted in amplification of products of the expected sizes for all 10 Xpm target strains that originate from diverse geographic areas. In contrast, only one Xpm strain among 10 was tested positive using the pair Xpm6 (Table 4).

In silico analyses showed that Xpm1 primers displayed the best exclusivity value, with only one strain of X. fuscans pv. aurantifolii being potentially detected among 901 Xanthomonadales genomes. This was not the case for other primer pairs which resulted in amplification from other xanthomonads, including strains of the phylogenetically-close pathovars syngonii, dieffenbachiae and phaseoli of the species X. phaseoli (Table 2).

When tested experimentally on a set of non-target strains, Xpm1 primers showed the expected exclusivity with only strains of X. fuscans pv. aurantifolii being amplified (n = 2). As predicted by in silico analyses, the other primer pairs generated amplicons of the expected sizes from non-target strains DNA, and particularly with X. phaseoli pathovars for Xpm2 and Xpm3 primers. Importantly, Xpm pairs of primers led to no amplification when tested on Xc strains (Table 4).

In silico analyses were less informative for Xc primers, because only one Xc genome sequence was available at the time of this study and formed the basis of all Xc primers. Nevertheless, these results showed high exclusivity value for most of Xc primers with only a few non-target strains potentially detected (for Xc3 and Xc4 primer pairs) among the 901 Xanthomonadales genomes (Table 2).

Most primer pairs generated an amplicon of the expected size when tested on the nine Xc target strains of our collection (Table 4). Exceptions are pairs of primers Xc3 and Xc8 for which no amplification was obtained in 1 and 7 samples, respectively. The experimental results obtained on the set of non-target strains reflected partially in silico predictions, with high specificity of the different primer pairs, but yielded an unexpected nonspecific amplification (600 bp amplicon) for X. phaseoli pathovars and Xpm strain CFBP2603 when using primer pair Xc3. Importantly, no amplification of the expected size was observed when Xpm strains were used as templates using any of the tested Xc pairs of primers.

In conclusion, the Xpm1 and Xc1 primer pairs were chosen as candidates for the diagnosis of Xpm and Xc. Also, the difference in amplicon sizes obtained for Xpm1 and Xc1 (203 bp and 314 bp, respectively) was compatible with the development of a duplex-PCR Fig. 1. Optimization of the duplex-PCR assay. The duplex-PCR was performed using various concentrations of Xpm1 F/R and Xc1 F/R primers. A, B and C: agarose gel electrophoresis of PCR products. A, Xpm1 0.5 μM, Xc1 0.5 μM. B, Xpm1 0.25 μM, Xc1 0.5 μM. C, Xpm1 0.125 μM, Xc1 0.5 μM. Lane 1, water. Lanes 2 and 3, gDNA (1 ng/μl) of Xanthomonas cassavae (Xc) strain CIX739 and Xanthomonas phaseoli pv. manihotis (Xpm) strain CIO151, respectively. Lanes 4 and 5, mix of gDNA (1 ng/μl) of CIX739 and CIO151 at ratios of 1:1 and 2:1, respectively. Lanes 6 and 7, boiled suspensions (OD = 0.2) of CIX739 and CIO151, respectively. Lanes 8 and 9, mix of boiled suspensions (OD = 0.2) of CIX739 and CIO151 at ratios of 1:1 and 2:1, respectively. M: Size marker 100 bp (Promega). product from any of the non-target strains with the exception of two X. fuscans pv. aurantifolii B strains, leading to a high exclusivity value of 96.4% (Table 5).

Sensitivity of the duplex-PCR assay

The concentrations of the CIO151 and CIX739 bacterial dilution series used to determine the limit of detection were estimated by spread plate method. The suspensions adjusted to OD 600 = 0.2 corresponded to ≈10 8 CFU/ml (1.02 ± 0.26 × 10 8 CFU/ml) and ≈10 7 CFU/ml (1.79 ± 0.65 × 10 7 CFU/ml) for CIO151 and CIX739, respectively.

The duplex-PCR assay tested on pure bacterial suspensions could reliably detect Xpm strain CIO151 to a concentration of 10 5 CFU/ml (3/ 3 positive replicates) which corresponds to a quantity of 100 bacterial cells per reaction. At 10 4 CFU/ml, weak amplification was obtained for only one among the three replicates. For Xc strain CIX739, the sensitivity of the assay was slightly lower with only two replicates among three tested positive for 10 5 CFU/ml and a weak amplification in only one among the three replicates at 10 4 CFU/ml (Fig. 3A).

When tested with total DNA extracted from a plant matrix spiked with different concentrations of the bacteria, the reliable threshold of detection was lowered to 10 3 CFU/ml for Xpm (3/3 positives replicates) which corresponds here to 20 bacterial cells per reaction after the step of DNA extraction (Fig. 3B). We did not take into account the signals obtained for the lower bacterial concentrations because they were very weak and non-reproducible. For Xc, the reliable threshold of detection was 10 4 CFU/ml (3/3 positive replicates).

Detection of Xpm and Xc in plant tissues

To address the question whether the duplex-PCR diagnostic tool was efficient in detecting Xpm and Xc within cassava plant tissues, we artificially inoculated Xpm strain CIO151 and Xc strain CIX739 into leaves of cassava plants grown under greenhouse conditions. Our results demonstrated consistent detection of Xpm. Nevertheless, detection was only possible with macerated lesions that were diluted prior to the PCR assay, for dilutions ranging from 10 2 to 10 5 -fold dilutions (Fig. 4A). As expected, no amplification was demonstrated when using Xc1 primers on the same set of samples (Fig. 4B). Xpm detection from total DNA extracted from CBB lesions was also demonstrated (Fig. 5).

Conversely, no amplification occurred with macerated lesions from plants inoculated with Xc strain regardless of the dilution factor. However, detection of Xc could be achieved when the duplex-PCR was performed with total DNA extracted from CBN lesions (Fig. 5).

When performing the duplex-PCR on cassava leaves collected in the 1970s -80s, Xpm was detected in several samples showing characteristic CBB symptoms and previously declared as infected by Xpm as is the case of samples 1-4 and 8-14 (Table 3). As with tissues inoculated artificially, dilution of the tissue macerates was necessary prior to performing the duplex-PCR assay (dilutions from 10 2 to 10 5 -fold), with optimal results at 10 3 -fold dilution for the majority of samples. No positive signals could be obtained using this dilution method with CBN symptoms (samples 5-7). However, like for artificially contaminated lesions, Xc detection could be achieved after a step of DNA extraction (Table 3). A. Ten-fold diluted boiled suspensions of Xpm strain CIO151 (top) and Xc strain CIX739 (bottom) were used as templates for duplex-PCR. M: Size marker 100 bp (Promega). Lane 1, water; lanes 2-8: bacterial suspensions ranging from 10 2 CFU/ml to 10 8 CFU/ml for Xpm strain CIO151 and 10 1 CFU/ml to 10 7 CFU/ml for Xc strain CIX739; lane 9, CIO151 and lane 10, CIX739 gDNA used as positive controls. B. Total DNA extracted from cassava leaf homogenates spiked with ten-fold diluted suspensions of Xpm strain CIO151 (top) and Xc strain CIX739 (bottom) were used as templates for duplex-PCR. M: Size marker 100 bp (Promega). Lane 1, water; lane 2, Plant SG-107-5 gDNA, lanes 3-9, bacterial suspensions in plant matrix ranging from 10 1 CFU/ml to 10 7 CFU/ml for Xpm strain CIO151, and 10 0 CFU/ml to 10 6 CFU/ml for Xc strain CIX739; lane 10, CIO151 gDNA and lane 10, CIX739 gDNA used as positive controls.

Discussion

This paper describes the development of a duplex-PCR able to differentially detect X. phaseoli pv. manihotis and X. cassavae, both pathogenic on cassava. CBB is a major threat in the main cassava producing areas in the tropics, while until now, CBN has only been reported in Africa [7]. Nevertheless, the foliar symptoms caused by these two pathogens are very similar, and it is essential to be able to distinguish them in order to deploy adapted control strategies.

Tools based on PCR and N-PCR have been developed, but these only target Xpm, and are based on the amplification of a fragment of TALE1 Xam gene that encodes a major pathogenicity factor widely conserved in Xpm [14,19]. This gene is highly prone to horizontal transfer and recombinational events [29]. Therefore using TALE1 Xam for diagnosis purposes is not optimal because of the pitfall that some strains don't have it [30] and the risk that some non-target strains harbor it, thereby leading to false negative and positive results, respectively.

For the screening of specific primers targeting Xpm and Xc we used the genome alignment-based computational pipeline designated Uniqprimer 0.5.0 (L. Triplett and J. E. Leach, in preparation), which allows to compare multiple genomes to find unique regions. It has been successfully used for the search of specific loop-mediated isothermal amplification (LAMP) primers for the detection of X. translucens pathovars [31]. By combining rapid global alignments before primer design with stringent screening of the primers, primers specific to the target genome sequences are more efficiently produced than manual or targeted alignment techniques. However, as seen with the initial list of candidate Xc primers produced in this study, the proportion of primers that will be robust in the field may be dependent on the quantity and diversity of genomes available for primer design.

Screening of the different primer pairs was performed by exploiting genome sequences available in NCBI databases, particularly for Xpm. Our in silico predictions were globally consistent with the generated experimental data. Inclusivity results predicted in silico for the different Xpm primers were in agreement with the results obtained in our PCR assays. Xc inclusivity could not be tested in silico due to limited genomic information, so we experimentally tested the tool on a collection of 23 strains. The cross reactions of Xpm1 primers with X. fuscans pv. aurantifolii strains and of Xpm2 and Xpm3 primer pairs with the phylogenetically related strains belonging to the species X. phaseoli were also experimentally confirmed. Some in silico predictions were not experimentally confirmed (e.g. amplification of X. translucens species with primers Xpm4 and Xpm5). However, because of a lack of availability in some instances, the strains we tested in vitro were not necessarily the same isolates that those present in the NCBI databases. These two approaches were complementary and allowed us to develop a duplex-PCR assay with primer set Xpm1/Xc1 which displayed a high specificity when tested on a broad collection of target and non-target strains. Total DNA extracted from cassava leaves that were infiltrated with different Xc or Xpm strains were used as template in the duplex-PCR assay using Xc1 and Xpm1 primers. Agarose gel electrophoresis of PCR products. Lanes 1 and 12, PCR mix without DNA (negative control); lane 2, gDNA of strain CIO151 (1 ng/μl); lane 3, gDNA of Xc strain CIX739; lane 4, total DNA from healthy cassava leaf (negative control); lanes 5 to 9, total DNA extracted from leaves inoculated with Xc strains UPB059, UPB146, UPB029, UPB043, UPB046, respectively; lanes 10 and 11, total DNA from leaves inoculated with Xpm strains CIO151 and CIX1088, respectively; lane M, DNA size marker 100 bp (Promega). Indeed, the simplex or the duplex-PCR assays led to the amplification of DNA fragments of the expected sizes with all Xpm and Xc tested strains. No cross-reactions were obtained except for strains of X. fuscans pv. aurantifolii when tested with Xpm1 primers. Nevertheless, it is unlikely that these cross-reactions will interfere in the diagnosis of Xpm because most xanthomonads have a highly restricted host range [32] and the ability to induce symptoms on cassava plants seems to be specific to Xpm and Xc. The most important result is that no amplification was obtained with strains sharing the same ecological niche, such as saprophytic strains isolated from cassava leaves.

The duplex-PCR assay was able to reliably detect Xpm from pure culture down to a concentration of 10 5 CFU/ml. For Xc, the PCR sensitivity was slightly lower and the limit of detection was between 10 5 CFU/ml and 10 6 CFU/ml. The detection limit in a plant matrix was lowered to 10 3 CFU/ml for Xpm, and 10 4 CFU/ml for Xc, which corresponds to 20 and 200 CFU per reaction, respectively. This gain of sensitivity compared to pure culture is explained by the step of DNA extraction which both concentrates the DNA target and eliminates PCRinhibiting plant substances. These results are consistent with the data found in the literature for conventional PCR [33,34]. Consistently, this DNA extraction step greatly improved the diagnosis from artificially or naturally infected plants, particularly for Xc for which detection was seen only from total DNA plant extracts. Recently, Zou et al. [35] provided a review on quick and inexpensive approaches to simplify nucleic acid preparation, which could guide the further development of a simplified protocol adapted to high volume of samples, especially in low-resource settings. The level of sensitivity of the current protocol is enough for confirming the presence of the different pathogens in symptomatic lesions. For certification purposes, more sensitive molecular tools like real-time quantitative PCR assays should be next developed.

The duplex-PCR assay described here is a reliable and specific procedure that can be used for epidemiological monitoring of the cassava-pathogenic Xanthomonas strains, useful for efficient surveillance programs of these diseases, allowing for the monitoring of crop health and for deploying the control measures that are necessary to prevent severe disease epidemics.

  -NN-HD-NG-HD-NI-NG-HD-NN-NI-NI-NG 9 UA306, UA318, UA522, UA681, UA1211, UA1235, UA1245, UA1357, UA1399 TALE14A NI-NG-NI-NN-HD-HD-NG-NG-HD-NG-NG-HD-NG-NG 1 CIO151 TALE14B NI-NG-NI-NN-HD-HD-NS-NS-NS-HD-HD-NS-HD-NG 1 UA318 TALE14C NI-NG-NI-NN-NG-HD-NS-NS-NN-NG-HD-NN-NI-NG 9 UA226, UA306, UA318, UA522, UA531, UA681, UA1061, UA1183, UA1381 TALE14D NI-NG-NI-NN-NI-HD-NS-NS-NN-NG-HD-NN-NI-NG 1 CFBP1851 TALE14E NI-NS-NN-HD-NG-HD-NI-NS-NN-HD-NN-NI-NI-NG 4 UA226, UA531, UA1061, UA1069 TALE15A NI-NG-NI-NN-HD-HD-NS-NS-NS-HD-HD-NN-HD-NG-NG 1 UA1211 TALE15B NI-NG-NI-NN-HD-HD-NS-NS-NS-HD-HD-NS-HD-NG-NG 3 UA522, UA681, UA1235 TALE20A NI-NG-NI-NN-HD-HD-NN-HD-NN-NG-HD-NS-HD-NN-HD-NG-HD-NI-NG-NG 1 UA318 TALE20B NI-NG-NI-NN-NG-HD-NN-HD-NN-NG-HD-NG-HD-NN-HD-NG-HD-NN-NG--NI-NN-NG-HD-NN-HD-NN-NG-HD-NS-HD-NN-HD-NG-HD-NI-NG--NI-NN-NG-HD-NS-NS-NN-NG-HD-NG-HD-NN-HD-HD-NS-NI--NI-NN-NG-HD-NS-NS-NN-NG-HD-NS-HD-NN-HD-NN-HD-NI--HD-NG-HD-N*-NG-NG-HD-HD-NG-NG-N*-NG-HD-NG-NG-NG-HD-NG-NG 1 CFBP1851 TALE22A NI-NG-HD-NG-NG-NG-HD-HD-NG-NG-HD-NG-HD-HD-NG-NG-HD-NG-NG-HD-NG-NG 1 UA1235 TALE22B NI-NG-HD-NG-NG-NG-HD-HD-NG-NG-NG-NG-HD-HD-NG-NG-HD-NG-NG-HD-NG-NG 4 UA306, UA318, UA522, UA681 TALE22C NI-NG-HD-NG-NG-NG-HD-HD-NG-NG-NG-NN-HD-NN-NG-NG-HD-NG-NG-HD-NG--NI-NG-NI-NN-NG-HD-NN-HD-NN-NG-HD-NG-HD-NN-HD-NG-HD-NN-NG-NG 1 UA1061

  and Figure S5. The order of GO Terms matches the order displayed on the y-axis of both figures. Author Contributions: D.O.-R. performed strain selection, isolation, and sequencing of a number of TALEs; R.E.M. carried out the RNAseq experiments in in-vitro plants; Á.L.P.-Q. analyzed the RNAseq data and provided advice on TALVEZ and QueTAL suite use; A.D. processed the RNAseq data and cross-referenced this information with prediction data for further analyses. C.A.Z.-C. performed aggressiveness assessments, isolation, and sequencing of TALEs, data processing and analyses, and wrote the initial draft of the article; A.B. conceived the project, secured funding, directed experiments, and helped draft the manuscript in collaboration with C.E.L. and B.S.; S.R., C.E.L., B.S. and A.B. wrote and reviewed sections of the manuscripts. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the Colombian Ministry of Sciences (Former Colciencias), through project No. 315-2013; by Universidad de los Andes and its Basic Sciences funding through multiple research projects (seed project NUMBER assigned to CAZ and Project No. INV-2019-84-1855 assigned to AB); the Research Program on Roots, Tubers and Bananas, and the Agropolis Foundation (project #1403-073). CAZ was supported by a doctoral fellowship awarded by the Insitut de Recherche pour le Développement.

Fig. 3 .

 3 Fig. 3.Duplex-PCR assay performed on serial dilutions of Xanthomonas phaseoli pv. manihotis (Xpm) and Xanthomonas cassavae (Xc) template DNA in absence or presence of plant matrix. A. Ten-fold diluted boiled suspensions of Xpm strain CIO151 (top) and Xc strain CIX739 (bottom) were used as templates for duplex-PCR. M: Size marker 100 bp (Promega). Lane 1, water; lanes 2-8: bacterial suspensions ranging from 10 2 CFU/ml to 10 8 CFU/ml for Xpm strain CIO151 and 10 1 CFU/ml to 10 7 CFU/ml for Xc strain CIX739; lane 9, CIO151 and lane 10, CIX739 gDNA used as positive controls. B. Total DNA extracted from cassava leaf homogenates spiked with ten-fold diluted suspensions of Xpm strain CIO151 (top) and Xc strain CIX739 (bottom) were used as templates for duplex-PCR. M: Size marker 100 bp (Promega). Lane 1, water; lane 2, Plant SG-107-5 gDNA, lanes 3-9, bacterial suspensions in plant matrix ranging from 10 1 CFU/ml to 10 7 CFU/ml for Xpm strain CIO151, and 10 0 CFU/ml to 10 6 CFU/ml for Xc strain CIX739; lane 10, CIO151 gDNA and lane 10, CIX739 gDNA used as positive controls.

Fig. 4 .

 4 Fig. 4. Duplex-PCR detection of Xanthomonas phaseoli pv. manihotis (Xpm) from artificially inoculated cassava leaf tissues. Macerated lesions of cassava leaves that were infiltrated with Xpm strain CIO151 were used as template in the duplex-PCR assay using Xpm1 (A) and Xc1 (B) primers.A. Agarose gel electrophoresis of Xpm1 PCR products (negative picture). Lane 1, water; lane 2, macerate obtained from untreated leaf; lane 3, undiluted macerated lesion; lanes 4 to 11, serial dilutions of macerated lesion material ranging from 10 1 -fold to 1 × 10 8 -fold and lane 12, gDNA of Xam strain CIO151. B. Agarose gel electrophoresis of Xc1 PCR products (negative picture). Lane 1, undiluted macerated lesion material; lanes 2-8 and 10, serial dilutions of macerated lesion material ranging from 10 1 -fold to 1 × 10 8 -fold; lane 9, gDNA of Xc strain CIX196 (positive control) and lane 11, water. Lane M, DNA size marker 100 bp (Promega).

Fig. 5 .

 5 Fig. 5. Duplex-PCR detection of Xanthomonas cassavae (Xc) and Xanthomonas phaseoli pv. manihotis (Xpm) from artificially inoculated cassava leaf tissues.Total DNA extracted from cassava leaves that were infiltrated with different Xc or Xpm strains were used as template in the duplex-PCR assay using Xc1 and Xpm1 primers. Agarose gel electrophoresis of PCR products. Lanes 1 and 12, PCR mix without DNA (negative control); lane 2, gDNA of strain CIO151 (1 ng/μl); lane 3, gDNA of Xc strain CIX739; lane 4, total DNA from healthy cassava leaf (negative control); lanes 5 to 9, total DNA extracted from leaves inoculated with Xc strains UPB059, UPB146, UPB029, UPB043, UPB046, respectively; lanes 10 and 11, total DNA from leaves inoculated with Xpm strains CIO151 and CIX1088, respectively; lane M, DNA size marker 100 bp (Promega).

  • C for 3 min; then 25 cycles of 94 • C for 40 s, 60 • C for 40 s, and 72

Table 1 .

 1 • C for 5 s, and 60 • C for 30 s; data was acquired during the second step of each cycle. The melting curve was evaluated from 65 • C to 95 • C. Primers for reference and candidate genes are summarized in Table1. Primers used for RT-qPCR assays.

	Target Gene a	Type	Sequence (5 ′ →3 ′ )	Reference
	Candidate gene: Manes.06G123400 (MeSWEET10a)	Fw Rv	TCCTCACCTTGACTGCGGTG AGCACCATCTGGACAATCCCA	[8]
	Candidate gene: Manes.04G033900 (Dof domain, zinc finger)	Fw Rv	AAAGTGCCCAAGAGGTGGTG GCCTTTCACTTGAAGCTGGG	This study
	Candidate gene: Manes.13G045100 (Clavata3/ESR CLE-related protein)	Fw Rv	CCACGACGAACTTTCACCCA CGCTGGGAACTTCATGAGCT	This study
	Candidate gene: Manes.11G151300 (Serine carboxypeptidase)	Fw Rv	GCCCCAACTGTTAGATTTGTGG GGTGACCAGCTTCATACACCTT	This study
	Candidate gene: Manes.15G052000 (Beta-glucosidase)	Fw Rv	TTGAAGATATGCTCAACGACACG CGTCTGCTCCGTTCCTGATA	This study
	Reference gene: Manes.08G061700.1 (Tubulin beta-6 chain)	Fw Rv	GGAAAGATGAGCACCAAGGA ACCAGTATACCAGTGCAAGAAG	[56]
	a Gene ID for Phytozome's Cassava genome annotation version 6.1, main functional annotation is indicated in parentheses.

Table 2 .

 2 General characteristics of the Xpm strains selected in this study.

	Strain	Region of Origin (ECZ a )	Location of Origin	Year of Isolation	AFLP-Based Haplotype b	Estimated Number of TALEs c
	CFBP1851	Caribbean coast (ECZ 1)	nd	1974	3 *	2
	CIO151	Eastern plains (ECZ 2)	Meta	1995	3 *	5
	UA226	Caribbean coast (ECZ1)	Chinú	2008	5 *	3
	UA306	Caribbean coast (ECZ 1)	Palmitos	2008	7 *	4
	UA318	Caribbean coast (ECZ 1)	Ciénaga de oro	2008	3 *	4
	UA522	Caribbean coast (ECZ 1)	Chinú	2009	4 *	5
	UA531	Caribbean coast (ECZ 1)	Chinú	2009	5 *	3
	UA681	Caribbean coast (ECZ 1)	Chinú	2009	8 *	5
	UA1061	Caribbean coast (ECZ 1)	Chinú	2009	5 *	2
	UA1069	Caribbean coast (ECZ 1)	Chinú	2009	5 *	2
	UA1183	Eastern plains (ECZ 2)	Villavicencio	2011	1 **	5
	UA1211	Eastern plains (ECZ 2)	Granada	2011	2 **	5
	UA1235	Eastern plains (ECZ 2)	Villavicencio	2011	3 **	5
	UA1245	Eastern plains (ECZ 2)	Villavicencio	2011	3 **	5
	UA1357	Eastern plains (ECZ 2)	Orocué	2012	8 **	4
	UA1381	Eastern plains (ECZ 2)	Orocué	2012	6 **	4
	UA1396	Eastern plains (ECZ 2)	Orocué	2012	7 **	5
	UA1399	Eastern plains (ECZ 2)	Orocué	2012	7 **	5

a Previously described in

[35]

. b Numbers were arbitrarily assigned to clades of distance trees from data reported by

[START_REF] Trujillo | Population typing of the causal agent of cassava bacterial blight in the Eastern Plains of Colombia using two types of molecular markers[END_REF]

(*) or

[START_REF] Trujillo | A complex population structure of the cassava pathogen Xanthomonas axonopodis pv. manihotis in recent years in the Caribbean Region of Colombia[END_REF]

(**). Since analyses were performed separately, clade numbers from

[START_REF] Trujillo | Population typing of the causal agent of cassava bacterial blight in the Eastern Plains of Colombia using two types of molecular markers[END_REF][START_REF] Trujillo | A complex population structure of the cassava pathogen Xanthomonas axonopodis pv. manihotis in recent years in the Caribbean Region of Colombia[END_REF]

are not shared. c Number of TALE bands determined by RFLP. nd, no data.

Table 3 .

 3 Translated RVD strings of the TALE gene variants sequenced in this study.

Table 4 .

 4 Candidate gene targets for TALEs present in strains UA681 and UA1061 with good to excellent EBE predictions.

	Target Gene ID a	Annotations a

Table 4 .

 4 Cont.

	Target Gene ID a	Annotations a

Table 1

 1 (continued) Prefixes of strain identifiers indicate the origin ot the strain: CIAT, Xanthomonas collection of the Centro Internacional de Agricultura, Cali, Colombia; INTA, Collection from the Instituto Nacional de Tecnologia Agropecuaria, Bella Vista, Argentina; CIO, ORSTOM collection; UPB, Collection from Unité de Phytopathologie (ELI-UCL), Louvain-La-Neuve, Belgium; CFBP, Collection Française de Bactéries associées aux Plantes, Angers, France; LMG, Laboratorium voor Microbiologie Gent culture collection, Universiteit Gent, Belgium; Run, JX and LN, Bacterial strains Collection, Cirad, La Réunion, France; CIX, CPX and other strains, IRD bacterial collections, Montpellier, France.b Bold font refers to results from pathogenicity tests performed in this study on cassava variety SG107-15, normal font refers to results from unpublished data or cited references. +, pathogenic strain; -, non pathogenic strain.c Strains marked in bold were used for the screening of the different primers sets.

	Strain ID a	Other known ID	Species	Origin	Date of	Host	Pathogenicity b References
					isolation
	UPB059	LMG764	Xc	Tanzanie	1978	M. esculenta	+	[13]
	UPB146	LMG5271	Xc	Kenya	1979	M. esculenta	+	[13]
	CIX157	LMG 5264	Xc	Malawi	1951	M. esculenta	+	[20]
	CIX739	CFBP4642,	Xc	Malawi	1951	M. esculenta	+	[13]
		LMG673
	CIX196	CFBP4642	Xc	Malawi	1951	M. esculenta	+	[22]
	CPX05		Saprophytic strain	Venezuela	2015	M. esculenta	-	Flores & Szurek,
								unpublished
	LN149, LN154, LN160,		Saprophytic strains	Réunion, France	2016	M. esculenta	-	Flores & Robène,
	LN161, LN163, LN164,							unpublished
	LN168, LN170
	LMG 876		Xanthomonas translucens	USA	1933	Hordeum vulgare	NA
			pv. translucens
	CFBP2053		X. translucens pv.	Switzerland	1973	Dactylis glomerata	NA
			translucens
	LMG695		Xanthomonas phaseoli pv.	Brazil	1965	Anthurium sp.	NA
			dieffenbachiae
	CIX227		X. phaseoli pv.	NA	NA	NA	NA
			dieffenbachiae
	LMG9055		X. phaseoli pv. syngonii	USA	1984	Syngonium	NA
						podophyllum
	LMG8014		X. phaseoli pv. phaseoli	Romania	1966	Phaseolus vulgaris	NA
	CIX208		X. phaseoli	NA	NA	NA	NA
	CIX228		X. phaseoli pv. phaseoli	NA	NA	NA	NA
	LMG539		Xanthomonas axonopodis	Colombia	1949	Axonopus scoparius	NA
			pv. axonopodis
	CIX230		Xanthomonas citri pv.	NA	NA	NA	NA
			vignicola
	LMG7429		X. citri pv. malvacearum	Uganda	1965	Gossypium sp.	NA
	CFBP2525		X. citri pv. citri	New Zeland	1956	Citrus limon	NA
	CFBP2868		Xanthomonas fuscans pv.	Argentina	NA	Citrus limon	NA
			aurantifolii (type B)
	CFBP2903		X. fuscans pv. aurantifolii	Argentina	NA	Citrus limon	NA
			(type B)
	CFBP2905		X. fuscans pv. aurantifolii	Argentina	NA	Citrus limon	NA
			(type C)
	LMG901		X.axonopodis pv.	Mauritius	1960	Saccharum officinarum NA
			vasculorum
	CIX202		Xanthomonas begoniae	NA	NA	NA	NA
	CFBP2532		Xanthomonas oryzae pv.	India	1965	Oryza sativa	NA
			oryzae
	CFBP4641		Xanthomonas sacchari	Guadeloupe,	NA	Saccharum officinarum NA
				France
	LMG2804		Pectobacterium	USA	NA	Chrysanthemum ×	NA
			chrysanthemi			morifolium
	Run 215		Ralstonia solanacearum	Cameroon	NA	Gaylussacia sp.	NA
			(phylotype I)
	Run 17		Ralstonia solanacearum	Martinique,	NA	Heliconia sp.	NA
			(phylotype II)	France
	JX63B		Pseudomonas fluorescens	Réunion, France	2001	Anthurium sp.	NA
	Strains which genome sequence is available {Bart, 2012#2840;Arrieta-Ortiz, 2013#1744}.

a d Not available. e

Microorganisms 2021, 9, 315. https://doi.org/10.3390/microorganisms9020315 https://www.mdpi.com/journal/microorganisms

a +: amplification, -: no amplification, x: amplification but not the expected size for the amplicons.

Acknowledgement

This work was supported by the Agropolis Foundation grants PAIX (#1403-073) and E-SPACE (1504-004), and by the Agricultural Fund for Rural Development (EAFRD) of the European Union. CF was supported by a doctoral fellowship awarded by Fundayacucho and IRD, and CZ was supported by a doctoral fellowship awarded by IRD. We also wish to thank A. Rieux (CIRAD) and H. Maraite (UCL) for fruitful discussions and advices.

(continued on next page) a Locus tags and positions are given for the strains UG23 (X. phaseoli pv. manihotis) and CFBP 4642 (X. cassavae). b Among 901 Xanthomonadales genomes (taxid:135614). c Strain UG28 was not detected using "Microbial nBlast" on the database "draft genomes " but the target sequence was found in the raw data in the non aligned reads, using "nBlast" on the datatbase SRA ″SRR522435 ″. d NA = not applicable.

X. phaseoli pv. manihotis

X. phaseoli pv. manihotis

X. phaseoli pv. manihotis

X. phaseoli pv. manihotis

X. phaseoli pv. manihotis

X. phaseoli pv. manihotis

X. phaseoli pv. manihotis

X. phaseoli pv. manihotis

X. transluscens pv. graminis

Optimization of the duplex-PCR assay

We compared the effect of various concentrations of Xpm1 and Xc1 primer sets on the ability of the duplex-PCR to amplify uniformly both Xpm1 and Xc1 targets. The best results were obtained by reducing the concentration of Xpm1 primers in the mix PCR (Fig. 1C). Particularly, concentrations of 0.125 μM for the Xpm1 primers and 0.5 μM for the Xc1 primers generated a comparable amplification of both amplicons.

Specificity of the simplex and duplex-PCR assays

The specificity of the simplex PCR assays with primer pairs Xpm1 and Xc1, and of the duplex-PCR combining the two sets of primers was tested on the collection of 50 Xpm strains, 23 Xc strains, and 32 non-target strains, using 1 μl of genomic DNA (1 ng/μl) or boiled samples as templates. Our results evidenced 100% of inclusivity for both primer pairs, all target strains being tested positive with their respective primer pairs (Table 5 and Fig. 2). This result was consistent whether the template was purified bacterial genomic DNA or boiled cultures.

We also tested our sets of primers using 39 DNA samples from the IRD historical collection as templates (Table S1). All led to the production of an amplicon of the expected size when using the corresponding primer pair.

Exclusivity values were also very high for both primer pairs, as tested on the collection of non-target strains (n = 32), which includes other Xanthomonas species or pathovars, saprophytic strains isolated from cassava and other genera of phytopathogenic bacteria. This list also included strains of Xpm (n = 50) when testing Xc primers, and strains of Xc when testing Xpm primers (n = 23). As a result, 100% exclusivity was obtained for Xc1. The Xpm1 primers did not amplify a

Conflicts of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Author's contributions

IR and BS conceived and designed the research work. CF, CZ, LT, MK, YM and VML performed the experiments. IR, BS and CF analyzed the data. IR, CF, LG and BS wrote the manuscript. VV, CB, LG and LT critically reviewed the manuscript. All authors read and approved the final manuscript.

Appendix A. Supplementary data

Supplementary data related to this article can be found at https:// doi.org/10.1016/j.pmpp.2018.07.005.