Competitive control of CsNCED1-1 by CsLOB1 and CsbZIP40 triggers susceptibility to citrus canker.

Pustule formation is pivotal for the development of the Xanthomonas citri subsp. citri (Xcc)-induced citrus canker disease (CCD). Although our previous study demonstrated that the exogenous application of abscisic acid (ABA) facilitated pustule formation induced by Xcc, the precise mechanism remains elusive. The 9-cis-epoxycarotenoid dioxygenase (NCED) is a crucial enzyme in ABA biosynthesis. This study explored the role of citrus CsNCED1-1 in CCD resistance through overexpression and RNA interference of CsNCED1-1 in Wanjincheng orange (Citrus sinensis). Our findings indicated that CsNCED1-1 negatively modulated CCD resistance by fostering ABA accumulation, concomitant with an increase in jasmonic acid (JA) and a decrease in salicylic acid (SA). Plants overexpressing CsNCED1-1 displayed shortened leaves with smaller and denser stomata along with irregular and increased palisade cells. CsLOB1 is a known susceptibility gene for CCD, and CsbZIP40 positively influences resistance to this disease. We further confirmed that CsLOB1 promoted and CsbZIP40 suppressed the transcription of CsNCED1-1 by directly binding to the CsNCED1-1 promoter. Notably, CsbZIP40 and CsLOB1 showed a competitive relationship in the regulation of CsNCED1-1 expression, with CsbZIP40 exhibiting greater competitiveness. Overall, our findings highlight that CsNCED1-1 promotes susceptibility to citrus canker by disrupting JA- and SA-mediated defense mechanisms and triggering the proliferation and remodeling of palisade cells, thereby facilitating pathogen colonization and pustule formation. This study offers novel insights into the regulatory mechanisms underlying citrus canker resistance and the role of CsNCED1-1 in citrus.

Comparative mineral and biochemical characterization of Citrus reticulata fruits and leaves to citrus canker pathogens, Xanthomonas axonopodis.

Pakistan's economy greatly benefits from citrus production since these fruits are sold and consumed all over the world. Although citrus fruits are easy to cultivate, they are susceptible to diseases caused by bacteria, viruses, and fungi. These challenges, as well as difficulties in obtaining the proper nutrients, might negatively impact fruit yields and quality. Citrus canker is another complicated problem caused by the germ Xanthomonas axonopodis. This germ affects many types of citrus fruits all over the world. This study looked closely at how citrus canker affects the leaves and the quality of the fruit in places like Sargodha, Bhalwal, Kotmomin, and Silanwali, which are big areas for growing citrus in the Sargodha district. What we found was that plants without the disease had more chlorophyll in their leaves compared to the sick plants. Also, the healthy plants had better amounts of important minerals like calcium, magnesium, potassium, and phosphorus in their fruits. But the fruits with the disease had too much sodium, and the iron levels were a bit different. The fruits with the disease also didn't have as much of something that protects them called antioxidants, which made them more likely to get sick. This study helps us understand how citrus canker affects plants and fruit, so we can think of ways to deal with it.

Core-Genome Multilocus Sequence Typing for Epidemiological and Evolutionary Analyses of Phytopathogenic Xanthomonas citri.

Xanthomonas citri subsp. citri is the cause of bacterial citrus canker, responsible for major economic losses to the citrus industry. X. citri subspecies and pathovars are responsible for diseases in soybean, common bean, mango, pomegranate, and cashew. X. citri disease has been tracked using several typing methods, but recent studies using genomic sequencing have been key to understanding the evolutionary relationships within the species, including fundamental differences among X. citri subsp. citri pathotypes. Here, we describe a core-genome multilocus sequence typing (cgMLST) scheme for X. citri based on 250 genomes comprising multiple examples of X. citri subsp. citri pathotypes A, A*, and Aw; X. citri subsp. malvacearum; X. citri pv. aurantifolii, pv. fuscans, pv. glycines, pv. mangiferaeindicae, pv. viticola, and pv. vignicola; and single isolates of X. citri pv. dieffenbachiae and pv. punicae. This data set included genomic sequencing of 100 novel X. citri subsp. citri isolates. cgMLST, based on 1,618 core genes across 250 genomes, is implemented at PubMLST (https://pubmlst.org/organisms/xanthomonas-citri/). GrapeTree minimum-spanning tree and Interactive Tree of Life (iTOL) neighbor-joining phylogenies generated from the cgMLST data resolved almost identical groupings of isolates to a core-genome single nucleotide polymorphism (SNP)-based neighbor-joining phylogeny. These resolved identical groupings of X. citri subsp. citri pathotypes and X. citri subspecies and pathovars. X. citri cgMLST should prove to be an increasingly valuable resource for the study of this key species of plant-pathogenic bacteria. Users can submit genomic data and associated metadata for comparison with previously characterized isolates at PubMLST to allow the rapid characterization of the local, national, and global epidemiology of these pathogens and examine evolutionary relationships. IMPORTANCE Xanthomonas citri is a plant pathogen that causes major economic losses to the citrus industry and sweet orange production in particular. Several subspecies and pathogens are recognized, with host ranges including soybean, common bean, mango, pomegranate, and cashew, among others. Recent genomic studies have shown that host-adapted X. citri subspecies and pathovars and X. citri subsp. citri pathotypes form distinct clades. In this study, we describe a core-genome multilocus sequence typing (cgMLST) scheme for this species that can rapidly and robustly discriminate among these ecologically distinct, host-adapted clades. We have established this scheme and associated databases containing genomic sequences and metadata at PubMLST, which users can interrogate with their own genome sequences to determine X. citri subspecies, pathovars, and pathotypes. X. citri cgMLST should prove to be an invaluable tool for the study of the epidemiology and evolution of this major plant pathogen.

A novel BLUF photoreceptor modulates the Xanthomonas citri subsp. citri-host plant interaction.

Plant-pathogen interaction is influenced by multiple environmental factors, including temperature and light. Recent works have shown that light modulates not only the defense response of plants but also the pathogens virulence. Xanthomonas citri subsp. citri (Xcc) is the bacterium responsible for citrus canker, an important plant disease worldwide. The Xcc genome presents four genes encoding putative photoreceptors: one bacteriophytochrome and three blue light photoreceptors, one LOV and two BLUFs (bluf1: XAC2120 and bluf2: XAC3278). The presence of two BLUFs proteins is an outstanding feature of Xcc. In this work we show that the bluf2 gene is functional. The mutant strain, XccΔbluf2, was constructed demonstrating that BLUF2 regulates swimming-type motility, adhesion to leaves, exopolysaccharide production and biofilm formation, features involved in the Xcc virulence processes. An important aspect during the plant-pathogen interaction is the oxidative response of the host and the consequent reaction of the pathogen. We observed that ROS detoxification is regulated by Xcc bluf2 gene. The phenotypes of disease in orange plants produced by WT and XccΔbluf2 strains were evaluated, observing different phenotypes. Altogether, these results show that BLUF2 negatively regulates virulence during citrus canker. This work constitutes the first report on BLUF-like receptors in plant pathogenic bacteria.

The Expansin Gene CsLIEXP1 Is a Direct Target of CsLOB1 in Citrus.

Transcription activator-like effectors are key virulence factors of Xanthomonas. They are secreted into host plant cells and mimic transcription factors inducing the expression of host susceptibility (S) genes. In citrus, CsLOB1 is a direct target of PthA4, the primary effector associated with citrus canker symptoms. CsLOB1 is a transcription factor, and its expression is required for canker symptoms induced by Xanthomonas citri subsp. citri. Several genes are up-regulated by PthA4; however, only CsLOB1 was described as an S gene induced by PthA4. Here, we investigated whether other up-regulated genes could be direct targets of PthA4 or CsLOB1. Seven up-regulated genes by PthA4 were investigated; however, an expansin-coding gene was more induced than CsLOB1. In Nicotiana benthamiana transient expression experiments, we demonstrate that the expansin-coding gene, referred here to as CsLOB1-INDUCED EXPANSIN 1 (CsLIEXP1), is not a direct target of PthA4, but CsLOB1. Interestingly, CsLIEXP1 was induced by CsLOB1 even without the predicted CsLOB1 binding site, which suggested that CsLOB1 has other unknown binding sites. We also investigated the minimum promoter regulated by CsLOB1, and this region and LOB1 domain were conserved among citrus species and relatives, which suggests that the interaction PthA4-CsLOB1-CsLIEXP1 is conserved in citrus species and relatives. This is the first study that experimentally demonstrated a CsLOB1 downstream target and lays the foundation to identify other new targets. In addition, we demonstrated that the CsLIEXP1 is a putative S gene indirectly induced by PthA4, which may serve as the target for genome editing to generate citrus canker-resistant varieties.

Linoleic acid from the endophytic fungus Diaporthe sp. HT-79 inhibits the growth of Xanthomonas citri subsp. citri by destructing the cell membrane and producing reactive oxygen species (ROS).

Citrus canker disease caused by Xanthomonas citri subsp. citri (Xac) severely influences the quality and quantity of citrus fruits. The current management of this disease mainly relies on the application of copper-associated chemicals, which poses a threat to human health and the environment. The present study isolated an endophytic fungus HT-79 from the healthy navel orange tree, whose crude fermentation product significantly inhibited the growth of Xac. The strain HT-79 was identified as a species of the Diaporthe genus. The petroleum ether extract (PEE) of the crude fermentation product of HT-79 exhibited remarkable activity against Xac with a MIC (minimum inhibitory concentration) value of 0.0625 mg/mL, significantly better than the positive control CuSO4 (MIC = 0.125 mg/mL). Bioassay-guided isolation of PEE resulted in the discovery of one highly potent anti-Xac subfraction, namely fraction 5 (MIC = 0.0156 mg/mL). Gas chromatography-mass spectrometry (GC-MS) analysis revealed that fraction 5 mainly consisted of palmitic acid (18.17%), ethyl palmitate (15.66%), linoleic acid (6.80%), oleic acid (18.32%), ethyl linoleate (21.58%), ethyl oleate (15.87%), and ethyl stearate (3.60%). Among these seven compounds, linoleic acid (MIC = 0.0078 mg/mL) was found to be the most potent against Xac, followed by oleic acid (MIC = 0.0156 mg/mL), while all others were less pronounced than CuSO4. Linoleic acid highly inhibited the growth of Xac via the destruction of the cell membrane and overproduction of reactive oxygen species (ROS). A preliminary in vivo experiment revealed that linoleic acid was effective in the control of citrus canker disease.

Unleashing the Potential of EIL Transcription Factors in Enhancing Sweet Orange Resistance to Bacterial Pathologies: Genome-Wide Identification and Expression Profiling.

The ETHYLENE INSENSITIVE3-LIKE (EIL) family is one of the most important transcription factor (TF) families in plants and is involved in diverse plant physiological and biochemical processes. In this study, ten EIL transcription factors (CsEILs) in sweet orange were systematically characterized via whole-genome analysis. The CsEIL genes were unevenly distributed across the four sweet orange chromosomes. Putative cis-acting regulatory elements (CREs) associated with CsEIL were found to be involved in plant development, as well as responses to biotic and abiotic stress. Notably, quantitative reverse transcription polymerase chain reaction (qRT-PCR) revealed that CsEIL genes were widely expressed in different organs of sweet orange and responded to both high and low temperature, NaCl treatment, and to ethylene-dependent induction of transcription, while eight additionally responded to Xanthomonas citri pv. Citri (Xcc) infection, which causes citrus canker. Among these, CsEIL2, CsEIL5 and CsEIL10 showed pronounced upregulation. Moreover, nine genes exhibited differential expression in response to Candidatus Liberibacter asiaticus (CLas) infection, which causes Citrus Huanglongbing (HLB). The genome-wide characterization and expression profile analysis of CsEIL genes provide insights into the potential functions of the CsEIL family in disease resistance.

Xylose Isomerase Depletion Enhances Virulence of Xanthomonas citri subsp. citri in Citrus aurantifolia.

Citrus canker, caused by the bacterium Xanthomonas citri (Xcc), is one of the most devastating diseases for the citrus industry. Xylose is a constituent of the cell wall of plants, and the ability of Xcc to use this carbohydrate may play a role in virulence. Xcc has two genes codifying for xylose isomerase (XI), a bifunctional enzyme that interconverts D-xylose into D-xylulose and D-glucose into D-fructose. The aim of this work was to investigate the functional role of the two putative XI ORFs, XAC1776 (xylA1) and XAC4225 (xylA2), in Xcc pathogenicity. XI-coding genes of Xcc were deleted, and the single mutants (XccΔxylA1 or XccΔxylA2) or the double mutant (XccΔxylA1ΔxylA2) remained viable. The deletion of one or both XI genes (xylA1 and/or xylA2) increased the aggressiveness of the mutants, causing disease symptoms. RT-qPCR analysis of wild strain and xylA deletion mutants grown in vivo and in vitro revealed that the highest expression level of hrpX and xylR was observed in vivo for the double mutant. The results indicate that XI depletion increases the expression of the hrp regulatory genes in Xcc. We concluded that the intracellular accumulation of xylose enhances Xcc virulence.

CsLOB1 regulates susceptibility to citrus canker through promoting cell proliferation in citrus.

Citrus sinensis lateral organ boundary 1 (CsLOB1) was previously identified as a critical disease susceptibility gene for citrus bacterial canker, which is caused by Xanthomonas citri subsp. citri (Xcc). However, the molecular mechanisms of CsLOB1 in citrus response to Xcc are still elusive. Here, we constructed transgenic plants overexpressing and RNAi-silencing of CsLOB1 using the canker-disease susceptible 'wanjincheng' orange (C. sinensis Osbeck) as explants. CsLOB1-overexpressing plants exhibited dwarf phenotypes with smaller and thicker leaf, increased branches and adventitious buds clustered on stems. These phenotypes were followed by a process of pustule- and canker-like development that exhibited enhanced cell proliferation. Pectin depolymerization and expansin accumulation were enhanced by CsLOB1 overexpression, while cellulose and hemicellulose synthesis were increased by CsLOB1 silence. Whilst overexpression of CsLOB1 increased susceptibility, RNAi-silencing of CsLOB1 enhanced resistance to canker disease without impairing pathogen entry. Transcriptome analysis revealed that CsLOB1 positively regulated cell wall degradation and modification processes, cytokinin metabolism, and cell division. Additionally, 565 CsLOB1-targeted genes were identified in chromatin immunoprecipitation-sequencing (ChIP-seq) experiments. Motif discovery analysis revealed that the most highly overrepresented binding sites had a conserved 6-bp 'GCGGCG' consensus DNA motif. RNA-seq and ChIP-seq data suggested that CsLOB1 directly activates the expression of four genes involved in cell wall remodeling, and three genes that participate in cytokinin and brassinosteroid hormone pathways. Our findings indicate that CsLOB1 promotes cell proliferation by mechanisms depending on cell wall remodeling and phytohormone signaling, which may be critical to citrus canker development and bacterial growth in citrus.

Biallelic Editing of the LOB1 Promoter via CRISPR/Cas9 Creates Canker-Resistant 'Duncan' Grapefruit.

Citrus canker caused by Xanthomonas citri subsp. citri is one of the most devastating citrus diseases worldwide. Generating disease-resistant citrus varieties is considered one of the most efficient and environmentally friendly measures for controlling canker. X. citri subsp. citri causes canker symptoms by inducing the expression of canker susceptibility gene LOB1 via PthA4, a transcription activator-like (TAL) effector, by binding to the effector binding element (EBE) in the promoter region. In previous studies, canker-resistant plants were generated by mutating the coding region or the EBE of LOB1. However, homozygous or biallelic canker-resistant plants have not been generated for commercial citrus varieties, such as grapefruit (Citrus paradisi), which usually contain two alleles of LOB1 and thus, have two types of LOB1 promoter sequences: TI LOBP and TII LOBP. Two different sgRNAs were used to target both EBE types. Both 35S promoter and Yao promoter were used to drive the expression of SpCas9p to modify EBEPthA4-LOBP in grapefruit. Using 'Duncan' grapefruit epicotyls as explants, 19 genome-edited grapefruit plants were generated with one biallelic mutant line (#DunYao7). X. citri subsp. citri caused canker symptoms on wild-type and nonbiallelic mutant plants but not on #DunYao7. XccPthA4 mutant containing the designer TAL effector dLOB1.5, which recognizes a conserved sequence in both wild-type and #DunYao7, caused canker symptoms on both wild-type and #DunYao7. No off-target mutations were detected in #DunYao7. This study represents the first time that CRISPR-mediated genome editing has been successfully used to generate disease-resistant plants for 'Duncan' grapefruit, paving the way for using disease-resistant varieties to control canker.

Genome Sequence Resource of Xanthomonas citri pv. citri from Formalin-Fixed Citrus Leaves Specimen Showing Canker Lesions Collected in 1982.

Xanthomonas citri pv. citri is the causative agent of citrus canker, one of the most devastating diseases threatening global citrus production. Here, we present the genome sequence of X. citri pv. citri strain GD82 from a formalin-fixed citrus leaf specimen showing canker lesions collected in 1982 in Guangdong Province, China. The GD82 genome consisted of 5,197,217 bp with G+C content of 64.8%, along with four circular plasmids: pXAC33 (32,377 bp), pXAC64 (63,972 bp), pXAC47 (47,810 bp), and pGD82.1 (219,560 bp). This is the oldest X. citri pv. citri genome from historical citrus canker specimens in China, which will enrich the current X. citri pv. citri genome database and facilitate genomic evolution research of X. citri pv. citri.

Evaluation of calcium hydroxide, calcium hypochlorite, peracetic acid, and potassium bicarbonate as citrus fruit sanitizers.

Xanthomonas citri (X. citri) is a quarentenary plant pathogen and the causal agent of the citrus canker. X. citri forms biofilms and remains fixed on the surface of plant tissues, especially on leaves and fruits. Considering this, all the citrus fruits have to be sanitized before they can be commercialized. NaOCl is the main sanitizer used to decontaminate fruits in the world. Due to its toxicity, treatment with NaOCl is no longer accepted by some Europe Union countries. Therefore, the aim of this work was to evaluate potassium bicarbonate (KHCO3), calcium hydroxide (Ca(OH)2), calcium hypochlorite (Ca(OCl)2) and peracetic acid (CH3CO3H) as alternatives to NaOCl for the sanitization of citrus fruit. By monitoring cell respiration and bacterial growth, we determined that peracetic acid and calcium hypochlorite exhibit bactericidal action against X. citri. Time-response growth curves and membrane integrity analyses showed that peracetic acid and calcium hypochlorite target the bacterial cytoplasmatic membrane, which is probably responsible for cell death in the first minutes of contact. The simulation of the sanitization process of citrus fruit in packinghouses showed that only peracetic acid exhibited a performance comparable to NaOCl. Among the tested compounds, peracetic acid constitutes an efficient and safer alternative to NaOCl.

Time-calibrated genomic evolution of a monomorphic bacterium during its establishment as an endemic crop pathogen.

Horizontal gene transfer is of major evolutionary importance as it allows for the redistribution of phenotypically important genes among lineages. Such genes with essential functions include those involved in resistance to antimicrobial compounds and virulence factors in pathogenic bacteria. Understanding gene turnover at microevolutionary scales is critical to assess the pace of this evolutionary process. Here, we characterized and quantified gene turnover for the epidemic lineage of a bacterial plant pathogen of major agricultural importance worldwide. Relying on a dense geographic sampling spanning 39 years of evolution, we estimated both the dynamics of single nucleotide polymorphism accumulation and gene content turnover. We identified extensive gene content variation among lineages even at the smallest phylogenetic and geographic scales. Gene turnover rate exceeded nucleotide substitution rate by three orders of magnitude. Accessory genes were found preferentially located on plasmids, but we identified a highly plastic chromosomal region hosting ecologically important genes such as transcription activator-like effectors. Whereas most changes in the gene content are probably transient, the rapid spread of a mobile element conferring resistance to copper compounds widely used for the management of plant bacterial pathogens illustrates how some accessory genes can become ubiquitous within a population over short timeframes.

Pyramiding the antimicrobial PR1aCB and AATCB genes in 'Tarocco' blood orange (Citrus sinensis Osbeck) to enhance citrus canker resistance.

Citrus canker, caused by Xanthomonas citri subsp. citri (Xcc), is a major bacterial disease responsible for substantial economic losses in citrus-producing areas. To breed transgenic citrus plants with enhanced resistance to citrus canker, two antimicrobial peptide genes, PR1aCB and AATCB, were incorporated into 'Tarocco' blood orange (Citrus sinensis Osbeck) plants via co-transformation and sequential re-transformation. The presence of PR1aCB and AATCB in double transgenic plants was confirmed by PCR. The expression of PR1aCB and AATCB in double transformants was demonstrated by quantitative real-time PCR. An in vivo disease resistance assay involving the injection of Xcc revealed that the double transformants were more resistant to citrus canker than the single gene transformants and wild-type plants. An analysis of the bacterial population indicated that the enhanced citrus canker resistance of the double transformants was due to inhibited Xcc growth. These results proved that the pyramiding of multiple genes is a more effective strategy for increasing the disease resistance of transgenic citrus plants than single gene transformations.

Endophytic bacterial community diversity in two citrus cultivars with different citrus canker disease resistance.

The selective infection of Xanthomonas citri pv. citri (Xcc) to citrus cultivars is universally known, but the relationship between endophytic bacteria and the resistance of host variety to canker disease remains unclear. In this study, endophytic bacterial populations of two citrus cultivars-the resistant satsuma mandarin and the susceptible Newhall navel orange-were analyzed through high-throughput sequencing. The results showed that endophytic bacterial community of satsuma mandarin was more abundant than that of Newhall navel orange. In addition, bacterial abundance was the highest in the spring samples, followed by that in summer and winter samples, in both the varieties. In all samples, the predominant phyla were Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes; the major genera were Bacillus and Stenotrophomonas, and the main species was Bacillus subtilis. According to the analysis of the predominant bacteria in the two citrus cultivars, B. subtilis with potential antagonistic characteristics against Xcc existed universally in all samples. However, the susceptible Newhall navel oranges were abundant in Bacillus subtilis and had a relatively large number of canker-causing cooperative bacteria such as Stenotrophomonas. The results suggested that endophytic bacterial community of the two citrus cultivars had some differences based on the season or plant tissue, and these differences were mainly in the quantity of bacteria, affecting citrus canker disease occurrence. In conclusion, the differences in endophytic bacteria on citrus cultivars might be related to host resistance or susceptibility to citrus canker disease.

Molecular Analysis of 14-3-3 Genes in Citrus sinensis and Their Responses to Different Stresses.

14-3-3 proteins (14-3-3s) are among the most important phosphorylated molecules playing crucial roles in regulating plant development and defense responses to environmental constraints. No report thus far has documented the gene family of 14-3-3s in Citrus sinensis and their roles in response to stresses. In this study, nine 14-3-3 genes, designated as CitGF14s (CitGF14a through CitGF14i) were identified from the latest C. sinensis genome. Phylogenetic analysis classified them into ε-like and non-ε groups, which were supported by gene structure analysis. The nine CitGF14s were located on five chromosomes, and none had duplication. Publicly available RNA-Seq raw data and microarray databases were mined for 14-3-3 expression profiles in different organs of citrus and in response to biotic and abiotic stresses. RT-qPCR was used for further examining spatial expression patterns of CitGF14s in citrus and their temporal expressions in one-year-old C. sinensis "Xuegan" plants after being exposed to different biotic and abiotic stresses. The nine CitGF14s were expressed in eight different organs with some isoforms displayed tissue-specific expression patterns. Six of the CitGF14s positively responded to citrus canker infection (Xanthomonas axonopodis pv. citri). The CitGF14s showed expressional divergence after phytohormone application and abiotic stress treatments, suggesting that 14-3-3 proteins are ubiquitous regulators in C. sinensis. Using the yeast two-hybrid assay, CitGF14a, b, c, d, g, and h were found to interact with CitGF14i proteins to form a heterodimer, while CitGF14i interacted with itself to form a homodimer. Further analysis of CitGF14s co-expression and potential interactors established a 14-3-3s protein interaction network. The established network identified 14-3-3 genes and several candidate clients which may play an important role in developmental regulation and stress responses in this important fruit crop. This is the first study of 14-3-3s in citrus, and the established network may help further investigation of the roles of 14-3-3s in response to abiotic and biotic constraints.

Development and comparative validation of genomic-driven PCR-based assays to detect Xanthomonas citri pv. citri in citrus plants.

BACKGROUND: Asiatic Citrus Canker, caused by Xanthomonas citri pv. citri, severely impacts citrus production worldwide and hampers international trade. Considerable regulatory procedures have been implemented to prevent the introduction and establishment of X. citri pv. citri into areas where it is not present. The effectiveness of this surveillance largely relies on the availability of specific and sensitive detection protocols. Although several PCR- or real-time PCR-based methods are available, most of them showed analytical specificity issues. Therefore, we developed new conventional and real-time quantitative PCR assays, which target a region identified by comparative genomic analyses, and compared them to existing protocols. RESULTS: Our assays target the X. citri pv. citri XAC1051 gene that encodes for a putative transmembrane protein. The real-time PCR assay includes an internal plant control (5.8S rDNA) for validating the assay in the absence of target amplification. A receiver-operating characteristic approach was used in order to determine a reliable cycle cut-off for providing accurate qualitative results. Repeatability, reproducibility and transferability between real-time devices were demonstrated for this duplex qPCR assay (XAC1051-2qPCR). When challenged with an extensive collection of target and non-target strains, both assays displayed a high analytical sensitivity and specificity performance: LOD95% = 754 CFU ml- 1 (15 cells per reaction), 100% inclusivity, 97.2% exclusivity for XAC1051-2qPCR; LOD95% = 5234 CFU ml- 1 (105 cells per reaction), 100% exclusivity and inclusivity for the conventional PCR. Both assays can detect the target from naturally infected citrus fruit. Interestingly, XAC1051-2qPCR detected X. citri pv. citri from herbarium citrus samples. The new PCR-based assays displayed enhanced analytical sensitivity and specificity when compared with previously published PCR and real-time qPCR assays. CONCLUSIONS: We developed new valuable detection assays useful for routine diagnostics and surveillance of X. citri pv. citri in citrus material. Their reliability was evidenced through numerous trials on a wide range of bacterial strains and plant samples. Successful detection of the pathogen was achieved from both artificially and naturally infected plants, as well as from citrus herbarium samples, suggesting that these assays will have positive impact both for future applied and academic research on this bacterium.

Photodynamic inactivation of the phytopathogenic bacterium Xanthomonas citri subsp. citri.

The present work intended to evaluate the applicability of photodynamic inactivation (PDI) of Xanthomonas citri subsp. citri with toluidine blue O (TBO), a commercial photosensitizer, as a strategy to control citrus canker. Assays were conducted with cell suspensions and biofilms, constructed either on polypropylene microtubes (in vitro assays) or on the surface of orange leaves (ex vivo assays), in the presence of TBO and under irradiation with artificial white light or natural sunlight. PDI assays using TBO alone caused a maximum 5·8 log10 reduction of X. citri viable cells in suspensions, and a much smaller inactivation (1·5 log10) in biofilms. However, concomitant use of KI potentiated the TBO photosensitization. Biofilms were inactivated down to the detection limit (>6 log10 reduction) with 5·0 µmol l-1 TBO + 10 mmol l-1 KI (in vitro) or 5·0 µmol l-1 TBO + 100 mmol l-1 KI (ex vivo) after artificial white light irradiation. Under natural sunlight, a reduction down to the detection limit of the Miles-Misra method was achieved with 50 µmol l-1 TBO and 100 mmol l-1 KI. PDI has potential to be applied in the control of citrus canker in field conditions although further studies are needed to show that there are no risks to plant physiology or fruit quality. SIGNIFICANCE AND IMPACT OF THE STUDY: Xanthomonas citri subsp. citri is a major cause of disease in citrus orchards. Because of the low efficacy and high environmental toxicity of copper-based treatments, there is growing interest on more sustainable phytosanitary approaches. Photodynamic inactivation (PDI) is being successfully used to control infectious agents and literature reports indicate that it is effective against some fungi and bacteria attacking fruit crops. The results of the present work open the perspective of using a low-cost photosensitizer and sunlight, as energy source, to control of the causative agent of citrus canker.

Investigating the Modes of Action of the Antimicrobial Chalcones BC1 and T9A.

Xanthomonas citri subsp. citri (X. citri) is an important phytopathogen and causes Asiatic Citrus Canker (ACC). To control ACC, copper sprays are commonly used. As copper is an environmentally damaging heavy metal, new antimicrobials are needed to combat citrus canker. Here, we explored the antimicrobial activity of chalcones, specifically the methoxychalcone BC1 and the hydroxychalcone T9A, against X. citri and the model organism Bacillus subtilis. BC1 and T9A prevented growth of X. citri and B. subtilis in concentrations varying from 20 µg/mL to 40 µg/mL. BC1 and T9A decreased incorporation of radiolabeled precursors of DNA, RNA, protein, and peptidoglycan in X. citri and B. subtilis. Both compounds mildly affected respiratory activity in X. citri, but T9A strongly decreased respiratory activity in B. subtilis. In line with that finding, intracellular ATP decreased strongly in B. subtilis upon T9A treatment, whereas BC1 increased intracellular ATP. In X. citri, both compounds resulted in a decrease in intracellular ATP. Cell division seems not to be affected in X. citri, and, although in B. subtilis the formation of FtsZ-rings is affected, a FtsZ GTPase activity assay suggests that this is an indirect effect. The chalcones studied here represent a sustainable alternative to copper for the control of ACC, and further studies are ongoing to elucidate their precise modes of action.

Canine Olfactory Detection of a Non-Systemic Phytobacterial Citrus Pathogen of International Quarantine Significance.

For millennia humans have benefitted from application of the acute canine sense of smell to hunt, track and find targets of importance. In this report, canines were evaluated for their ability to detect the severe exotic phytobacterial arboreal pathogen Xanthomonas citri pv. citri (Xcc), which is the causal agent of Asiatic citrus canker (Acc). Since Xcc causes only local lesions, infections are non-systemic, limiting the use of serological and molecular diagnostic tools for field-level detection. This necessitates reliance on human visual surveys for Acc symptoms, which is highly inefficient at low disease incidence, and thus for early detection. In simulated orchards the overall combined performance metrics for a pair of canines were 0.9856, 0.9974, 0.9257 and 0.9970, for sensitivity, specificity, precision, and accuracy, respectively, with 1-2 s/tree detection time. Detection of trace Xcc infections on commercial packinghouse fruit resulted in 0.7313, 0.9947, 0.8750, and 0.9821 for the same performance metrics across a range of cartons with 0-10% Xcc-infected fruit despite the noisy, hot and potentially distracting environment. In orchards, the sensitivity of canines increased with lesion incidence, whereas the specificity and overall accuracy was >0.99 across all incidence levels; i.e., false positive rates were uniformly low. Canines also alerted to a range of 1-12-week-old infections with equal accuracy. When trained to either Xcc-infected trees or Xcc axenic cultures, canines inherently detected the homologous and heterologous targets, suggesting they can detect Xcc directly rather than only volatiles produced by the host following infection. Canines were able to detect the Xcc scent signature at very low concentrations (10,000× less than 1 bacterial cell per sample), which implies that the scent signature is composed of bacterial cell volatile organic compound constituents or exudates that occur at concentrations many fold that of the bacterial cells. The results imply that canines can be trained as viable early detectors of Xcc and deployed across citrus orchards, packinghouses, and nurseries.