Designer TALEs enable discovery of cell death-inducer genes.

Transcription activator-like effectors (TALEs) in plant-pathogenic Xanthomonas bacteria activate expression of plant genes and support infection or cause a resistance response. PthA4AT is a TALE with a particularly short DNA-binding domain harboring only 7.5 repeats which triggers cell death in Nicotiana benthamiana; however, the genetic basis for this remains unknown. To identify possible target genes of PthA4AT that mediate cell death in N. benthamiana, we exploited the modularity of TALEs to stepwise enhance their specificity and reduce potential target sites. Substitutions of individual repeats suggested that PthA4AT-dependent cell death is sequence specific. Stepwise addition of repeats to the C-terminal or N-terminal end of the repeat region narrowed the sequence requirements in promoters of target genes. Transcriptome profiling and in silico target prediction allowed the isolation of two cell death inducer genes, which encode a patatin-like protein and a bifunctional monodehydroascorbate reductase/carbonic anhydrase protein. These two proteins are not linked to known TALE-dependent resistance genes. Our results show that the aberrant expression of different endogenous plant genes can cause a cell death reaction, which supports the hypothesis that TALE-dependent executor resistance genes can originate from various plant processes. Our strategy further demonstrates the use of TALEs to scan genomes for genes triggering cell death and other relevant phenotypes.

A serralysin-like protein of Candidatus Liberibacter asiaticus modulates components of the bacterial extracellular matrix.

Huanglongbing (HLB), the current major threat for Citrus species, is caused by intracellular alphaproteobacteria of the genus Candidatus Liberibacter (CaL), with CaL asiaticus (CLas) being the most prevalent species. This bacterium inhabits phloem cells and is transmitted by the psyllid Diaphorina citri. A gene encoding a putative serralysin-like metalloprotease (CLIBASIA_01345) was identified in the CLas genome. The expression levels of this gene were found to be higher in citrus leaves than in psyllids, suggesting a function for this protease in adaptation to the plant environment. Here, we study the putative role of CLas-serralysin (Las1345) as virulence factor. We first assayed whether Las1345 could be secreted by two different surrogate bacteria, Rhizobium leguminosarum bv. viciae A34 (A34) and Serratia marcescens. The protein was detected only in the cellular fraction of A34 and S. marcescens expressing Las1345, and increased protease activity of those bacteria by 2.55 and 4.25-fold, respectively. In contrast, Las1345 expressed in Nicotiana benthamiana leaves did not show protease activity nor alterations in the cell membrane, suggesting that Las1345 do not function as a protease in the plant cell. Las1345 expression negatively regulated cell motility, exopolysaccharide production, and biofilm formation in Xanthomonas campestris pv. campestris (Xcc). This bacterial phenotype was correlated with reduced growth and survival on leaf surfaces as well as reduced disease symptoms in N. benthamiana and Arabidopsis. These results support a model where Las1345 could modify extracellular components to adapt bacterial shape and appendages to the phloem environment, thus contributing to virulence.

Different Responses in Mandarin Cultivars Uncover a Role of Cuticular Waxes in the Resistance to Citrus Canker.

'Okitsu' is a mandarin cultivar showing substantial resistance to X. citri subsp. citri (X. citri). We have previously shown that this cultivar has significantly lower canker incidence and severity than 'Clemenules', particularly during early stages of leaf development in the field. This differential response is only seen when the leaves are inoculated by spraying, suggesting that leaf surface contributes to resistance. In this work, we have studied structural and chemical properties of leaf surface barriers of both cultivars. Ultrastructural analysis showed a thicker cuticle covering epidermal surface and guard cells in young 'Okitsu' leaves than in 'Clemenules'. This thicker cuticle was associated with a smaller stomatal aperture and reduced cuticle permeability. These findings correlated with an accumulation of cuticular wax components, including primary alcohols, alkanes, and fatty acids. None of these differences were observed in mature leaves, where both cultivars are equally resistant to the bacterium. Remarkably, mechanical alteration of cuticular thickness of young 'Okitsu' leaves allows canker development. Furthermore, cuticular waxes extracted from young 'Okitsu' leaves have higher antibacterial activity against X. citri than 'Clemenules'. Taken together, these data suggest that a faster development of epicuticular waxes in 'Okitsu' leaves play a central role in its resistance to X. citri.

PthA4AT , a 7.5-repeats transcription activator-like (TAL) effector from Xanthomonas citri ssp. citri, triggers citrus canker resistance.

Transcription activator-like effectors (TALEs) are important effectors of Xanthomonas spp. that manipulate the transcriptome of the host plant, conferring susceptibility or resistance to bacterial infection. Xanthomonas citri ssp. citri variant AT (X. citri AT ) triggers a host-specific hypersensitive response (HR) that suppresses citrus canker development. However, the bacterial effector that elicits this process is unknown. In this study, we show that a 7.5-repeat TALE is responsible for triggering the HR. PthA4AT was identified within the pthA repertoire of X. citri AT followed by assay of the effects on different hosts. The mode of action of PthA4AT was characterized using protein-binding microarrays and testing the effects of deletion of the nuclear localization signals and activation domain on plant responses. PthA4AT is able to bind DNA and activate transcription in an effector binding element-dependent manner. Moreover, HR requires PthA4AT nuclear localization, suggesting the activation of executor resistance (R) genes in host and non-host plants. This is the first case where a TALE of unusually short length performs a biological function by means of its repeat domain, indicating that the action of these effectors to reprogramme the host transcriptome following nuclear localization is not limited to 'classical' TALEs.

Plant responses underlying nonhost resistance of Citrus limon against Xanthomonas campestris pv. campestris.

Citrus is an economically important fruit crop that is severely afflicted by citrus canker, a disease caused by Xanthomonas citri ssp. citri (X. citri); thus, new sustainable strategies to manage this disease are needed. Although all Citrus spp. are susceptible to this pathogen, they are resistant to other Xanthomonas species, exhibiting non-host resistance (NHR), for example, to the brassica pathogen X. campestris pv. campestris (Xcc) and a gene-for-gene host defence response (HDR) to the canker-causing X. fuscans ssp. aurantifolii (Xfa) strain C. Here, we examine the plant factors associated with the NHR of C. limon to Xcc. We show that Xcc induced asymptomatic type I NHR, allowing the bacterium to survive in a stationary phase in the non-host tissue. In C. limon, this NHR shared some similarities with HDR; both defence responses interfered with biofilm formation, and were associated with callose deposition, induction of the salicylic acid (SA) signalling pathway and the repression of abscisic acid (ABA) signalling. However, greater stomatal closure was seen during NHR than during HDR, together with different patterns of accumulation of reactive oxygen species and phenolic compounds and the expression of secondary metabolites. Overall, these differences, independent of Xcc type III effector proteins, could contribute to the higher protection elicited against canker development. We propose that Xcc may have the potential to steadily activate inducible defence responses. An understanding of these plant responses (and their triggers) may allow the development of a sustained and sustainable resistance to citrus canker.

The histone-like protein HupB influences biofilm formation and virulence in Xanthomonas citri ssp. citri through the regulation of flagellar biosynthesis.

Citrus canker is an important disease of citrus, whose causal agent is the bacterium Xanthomonas citri ssp. citri (Xcc). In previous studies, we found a group of Xcc mutants, generated by the insertion of the Tn5 transposon, which showed impaired ability to attach to an abiotic substrate. One of these mutants carries the Tn5 insertion in hupB, a gene encoding a bacterial histone-like protein, homologue to the ß-subunit of the Heat-Unstable (HU) nucleoid protein of Escherichia coli. These types of protein are necessary to maintain the bacterial nucleoid organization and the global regulation of gene expression. Here, we characterized the influence of the mutation in hupB regarding Xcc biofilm formation and virulence. The mutant strain hupB was incapable of swimming in soft agar, whereas its complemented strain partially recovered this phenotype. Electron microscope imaging revealed that impaired motility of hupB was a consequence of the absence of the flagellum. Comparison of the expression of flagellar genes between the wild-type strain and hupB showed that the mutant exhibited decreased expression of fliC (encoding flagellin). The hupB mutant also displayed reduced virulence compared with the wild-type strain when they were used to infect Citrus lemon plants using different infection methods. Our results therefore show that the histone-like protein HupB plays an essential role in the pathogenesis of Xcc through the regulation of biofilm formation and biosynthesis of the flagellum.

Inducible expression of Bs2 R gene from Capsicum chacoense in sweet orange (Citrus sinensis L. Osbeck) confers enhanced resistance to citrus canker disease.

Transgenic expression of the pepper Bs2 gene confers resistance to Xanthomonas campestris pv. vesicatoria (Xcv) pathogenic strains which contain the avrBs2 avirulence gene in susceptible pepper and tomato varieties. The avrBs2 gene is highly conserved among members of the Xanthomonas genus, and the avrBs2 of Xcv shares 96% homology with the avrBs2 of Xanthomonas citri subsp. citri (Xcc), the causal agent of citrus canker disease. A previous study showed that the transient expression of pepper Bs2 in lemon leaves reduced canker formation and induced plant defence mechanisms. In this work, the effect of the stable expression of Bs2 gene on citrus canker resistance was evaluated in transgenic plants of Citrus sinensis cv. Pineapple. Interestingly, Agrobacterium-mediated transformation of epicotyls was unsuccessful when a constitutive promoter (2× CaMV 35S) was used in the plasmid construction, but seven transgenic lines were obtained with a genetic construction harbouring Bs2 under the control of a pathogen-inducible promoter, from glutathione S-transferase gene from potato. A reduction of disease symptoms of up to 70% was observed in transgenic lines expressing Bs2 with respect to non-transformed control plants. This reduction was directly dependent on the Xcc avrBs2 gene since no effect was observed when a mutant strain of Xcc with a disruption in avrBs2 gene was used for inoculations. Additionally, a canker symptom reduction was correlated with levels of the Bs2 expression in transgenic plants, as assessed by real-time qPCR, and accompanied by the production of reactive oxygen species. These results indicate that the pepper Bs2 resistance gene is also functional in a family other than the Solanaceae, and could be considered for canker control.

Resistance to citrus canker induced by a variant of Xanthomonas citri ssp. citri is associated with a hypersensitive cell death response involving autophagy-associated vacuolar processes.

Xanthomonas citri ssp. citri (X. citri) is the causal agent of Asiatic citrus canker, a disease that seriously affects most commercially important Citrus species worldwide. We have identified previously a natural variant, X. citri AT , that triggers a host-specific defence response in Citrus limon. However, the mechanisms involved in this canker disease resistance are unknown. In this work, the defence response induced by X. citri AT was assessed by transcriptomic, physiological and ultrastructural analyses, and the effects on bacterial biofilm formation were monitored in parallel. We show that X. citri AT triggers a hypersensitive response associated with the interference of biofilm development and arrest of bacterial growth in C. limon. This plant response involves an extensive transcriptional reprogramming, setting in motion cell wall reinforcement, the oxidative burst and the accumulation of salicylic acid (SA) and phenolic compounds. Ultrastructural analyses revealed subcellular changes involving the activation of autophagy-associated vacuolar processes. Our findings show the activation of SA-dependent defence in response to X. citri AT and suggest a coordinated regulation between the SA and flavonoid pathways, which is associated with autophagy mechanisms that control pathogen invasion in C. limon. Furthermore, this defence response protects C. limon plants from disease on subsequent challenges by pathogenic X. citri. This knowledge will allow the rational exploitation of the plant immune system as a biotechnological approach for the management of the disease.

Xanthan Pyruvilation Is Essential for the Virulence of Xanthomonas campestris pv. campestris.

Xanthan, the main exopolysaccharide (EPS) synthesized by Xanthomonas spp., contributes to bacterial stress tolerance and enhances attachment to plant surfaces by helping in biofilm formation. Therefore, xanthan is essential for successful colonization and growth in planta and has also been proposed to be involved in the promotion of pathogenesis by calcium ion chelation and, hence, in the suppression of the plant defense responses in which this cation acts as a signal. The aim of this work was to study the relationship between xanthan structure and its role as a virulence factor. We analyzed four Xanthomonas campestris pv. campestris mutants that synthesize structural variants of xanthan. We found that the lack of acetyl groups that decorate the internal mannose residues, ketal-pyruvate groups, and external mannose residues affects bacterial adhesion and biofilm architecture. In addition, the mutants that synthesized EPS without pyruvilation or without the external mannose residues did not develop disease symptoms in Arabidopsis thaliana. We also observed that the presence of the external mannose residues and, hence, pyruvilation is required for xanthan to suppress callose deposition as well as to interfere with stomatal defense. In conclusion, pyruvilation of xanthan seems to be essential for Xanthomonas campestris pv. campestris virulence.

Closely-related Xanthomonas citri subsp. citri isolates trigger distinct histological and transcriptional responses in Citrus limon

Citrus canker, caused by Xanthomonas citri subsp. citri (Xcc), has an important economic impact on the citrus industry. Extensive information is available about the disease but, nevertheless, the study of plant-pathogen interactions could provide new information in the understanding of citrus canker disease. A new isolate has been identified, Xcc AT, which has a high genetic similarity (> 90 %) to the virulent Xcc T strain based on genetic clustering analyses of the rep-PCR fingerprinting patterns, but it does not produce cankerous lesions in Citrus limon. In this study, we compared C. limon responses to Xcc AT and to the virulent Xcc T strain at both histological and transcriptional levels. Histologically, leaves inoculated with Xcc AT exhibited neither a typical disordering of the spongy mesophyll, nor a swelling of epidermis. A particular content (undetermined) was also found in mesophyll cells near the stomata, together with increased starch accumulation. The transcriptomic profiles were compared by cDNA-AFLP technique. A total of 121 fragments derived from transcript (TDF) were either specifically induced or repressed by the isolates, and 62 were sequenced. Analysis of global expression identified different classes of genes known to be involved in plant-pathogen interactions. This study constitutes the first approach of the specific interaction between the avirulent Xcc AT isolate and C. limon.

Closely-related Xanthomonas citri subsp. citri isolates trigger distinct histological and transcriptional responses in Citrus limon

Citrus canker, caused by Xanthomonas citri subsp. citri (Xcc), has an important economic impact on the citrus industry. Extensive information is available about the disease but, nevertheless, the study of plant-pathogen interactions could provide new information in the understanding of citrus canker disease. A new isolate has been identified, Xcc AT, which has a high genetic similarity (> 90 %) to the virulent Xcc T strain based on genetic clustering analyses of the rep-PCR fingerprinting patterns, but it does not produce cankerous lesions in Citrus limon. In this study, we compared C. limon responses to Xcc AT and to the virulent Xcc T strain at both histological and transcriptional levels. Histologically, leaves inoculated with Xcc AT exhibited neither a typical disordering of the spongy mesophyll, nor a swelling of epidermis. A particular content (undetermined) was also found in mesophyll cells near the stomata, together with increased starch accumulation. The transcriptomic profiles were compared by cDNA-AFLP technique. A total of 121 fragments derived from transcript (TDF) were either specifically induced or repressed by the isolates, and 62 were sequenced. Analysis of global expression identified different classes of genes known to be involved in plant-pathogen interactions. This study constitutes the first approach of the specific interaction between the avirulent Xcc AT isolate and C. limon.(AU)

Novel set of real-time PCR primers for simultaneous detection of Liberibacter species associated with citrus Huanglongbing

Huanglongbing (HLB), a devastating citrus disease caused by the bacterium Candidatus Liberibacter spp., is now responsible for significant economic losses worldwide. Yet, no effective disease control has been found, and the non-cultivability of the bacterium has severely hampered studies on the pathogen. The 16S rDNA gene is a well-characterized sequence, essential for cell survival, and is used for bacterial identification or assignment of close relationships at the genus and species levels. Quantitative Real-Time PCR (qPCR) assays based on 16S rDNA genes are widely used in the detection of Ca. Liberibacter spp. in multiplex reactions. We have developed for the first time a set of qPCR primers based on the conserved 16S rDNA gene, which specifically and simultaneously detects in a singleplex reaction, all three bacterial species associated with HLB, and can differentiateCa.Liberibacter asiaticus or africanus from americanus by their characteristic melting curves. The assay is very sensitive, and it was possible to amplify expected DNA fragments with an efficiency of 98 % using the Syber Green system and a Ct value lower than tested methods for HLB diagnosis. The application of this fast, simple and efficient detection methodology could also be important in the detection of all species of HLB-associated Liberibacters and could contribute to early pathogen detection, a crucial step in the development of preventive strategies aimed at avoiding the dissemination of this devastating disease in HLB-free areas.(AU)

Novel set of real-time PCR primers for simultaneous detection of Liberibacter species associated with citrus Huanglongbing

Huanglongbing (HLB), a devastating citrus disease caused by the bacterium Candidatus Liberibacter spp., is now responsible for significant economic losses worldwide. Yet, no effective disease control has been found, and the non-cultivability of the bacterium has severely hampered studies on the pathogen. The 16S rDNA gene is a well-characterized sequence, essential for cell survival, and is used for bacterial identification or assignment of close relationships at the genus and species levels. Quantitative Real-Time PCR (qPCR) assays based on 16S rDNA genes are widely used in the detection of Ca. Liberibacter spp. in multiplex reactions. We have developed for the first time a set of qPCR primers based on the conserved 16S rDNA gene, which specifically and simultaneously detects in a singleplex reaction, all three bacterial species associated with HLB, and can differentiateCa.Liberibacter asiaticus or africanus from americanus by their characteristic melting curves. The assay is very sensitive, and it was possible to amplify expected DNA fragments with an efficiency of 98 % using the Syber Green system and a Ct value lower than tested methods for HLB diagnosis. The application of this fast, simple and efficient detection methodology could also be important in the detection of all species of HLB-associated Liberibacters and could contribute to early pathogen detection, a crucial step in the development of preventive strategies aimed at avoiding the dissemination of this devastating disease in HLB-free areas.

XbmR, a new transcription factor involved in the regulation of chemotaxis, biofilm formation and virulence in Xanthomonas citri subsp. citri.

Xanthomonas citri subsp. citri (Xcc) is the causal agent of citrus canker. Biofilm formation on citrus leaves plays an important role in epiphytic survival of Xcc. Biofilm formation is affected by transposon insertion in XAC3733, which encodes a transcriptional activator of the NtrC family, not linked to a gene encoding a sensor protein, thus could be considered as an 'orphan' regulator whose function is poorly understood in Xanthomonas spp. Here we show that mutation of XAC3733 (named xbmR) resulted in impaired structural development of the Xcc biofilm, loss of chemotaxis and reduced virulence in grapefruit plants. All defective phenotypes were restored to wild-type levels by the introduction of PA2567 from Pseudomonas aeruginosa, which encodes a phosphodiesterase active in the degradation of cyclic diguanosine monophosphate (c-di-GMP). A knockout of xbmR led to a substantial downregulation of fliA that encodes a σ(28) transcription factor, as well as fliC and XAC0350 which are potential member of the σ(28) regulon. XAC0350 encodes an HD-GYP domain c-di-GMP phosphodiesterase. These findings suggest that XbmR is a key regulator of flagellar-dependent motility and chemotaxis exerting its action through a regulatory pathway that involves FliA and c-di-GMP.

Identification and characterization of biofilm formation-defective mutants of Xanthomonas citri subsp. citri.

Xanthomonas citri subsp. citri (Xcc) develops a biofilm structure both in vitro and in vivo. Despite all the progress achieved by studies regarding biofilm formation, many of its mechanisms remain poorly understood. This work focuses on the identification of new genes involved in biofilm formation and how they are related to motility, virulence and chemotaxis in Xcc. A Tn5 library of approximately 6000 Xcc (strain 306) mutants was generated and screened to search for biofilm formation defective strains. We identified 23 genes not previously associated with biofilm formation. The analysis of the 23 mutants not only revealed the involvement of new genes in biofilm formation, but also reinforced the importance of exopolysaccharide production, motility and cell surface structures in this process. This collection of biofilm-defective mutants underscores the multifactorial genetic programme underlying the establishment of biofilm in Xcc.

Characterization of a variant of Xanthomonas citri subsp. citri that triggers a host-specific defense response.

Citrus is an economically important fruit crop that is severely afflicted by Asiatic citrus bacterial canker (CBC), a disease caused by the phytopathogen Xanthomonas citri subsp. citri (X. citri). To gain insight into the molecular epidemiology of CBC, 42 Xanthomonas isolates were collected from a range of Citrus spp. across 17 different orchards in Tucumán, Argentina and subjected to molecular, biochemical, and pathogenicity tests. Analysis of genome-specific X. citri markers and DNA polymorphisms based on repetitive elements-based polymerase chain reaction showed that all 42 isolates belonged to X. citri. Interestingly, pathogenicity tests showed that one isolate, which shares >90% genetic similarity to the reference strain X. citri T, has host range specificity. This new variant of X. citri subsp. citri, named X. citri A(T), which is deficient in xanthan production, induces an atypical, noncankerous chlorotic phenotype in Citrus limon and C. paradisi and weak cankerous lesions in C. aurantifolia and C. clementina leaves. In C. limon, suppression of canker development is concomitant with an oxidative burst; xanthan is not implicated in the phenotype induced by this interaction, suggesting that other bacterial factors would be involved in triggering the defense response.

HrpM is involved in glucan biosynthesis, biofilm formation and pathogenicity in Xanthomonas citri ssp. citri.

Xanthomonas citri ssp. citri (Xcc) is the causal agent of citrus canker. This bacterium develops a characteristic biofilm on both biotic and abiotic surfaces. A biofilm-deficient mutant was identified in a screening of a transposon mutagenesis library of the Xcc 306 strain constructed using the commercial Tn5 transposon EZ-Tn5 Tnp Transposome (Epicentre). Sequence analysis of a mutant obtained in the screening revealed that a single copy of the EZ-Tn5 was inserted at position 446 of hrpM, a gene encoding a putative enzyme involved in glucan synthesis. We demonstrate for the first time that the product encoded by the hrpM gene is involved in ß-1,2-glucan synthesis in Xcc. A mutation in hrpM resulted in no disease symptoms after 4 weeks of inoculation in lemon and grapefruit plants. The mutant also showed reduced ability to swim in soft agar and decreased resistance to H(2)O(2) in comparison with the wild-type strain. All defective phenotypes were restored to wild-type levels by complementation with the plasmid pBBR1-MCS containing an intact copy of the hrpM gene and its promoter. These results indicate that the hrpM gene contributes to Xcc growth and adaptation in its host plant.

Novel demonstration of RNAi in citrus reveals importance of citrus callose synthase in defence against Xanthomonas citri subsp. citri.

Citrus is an economically important fruit crop that is severely afflicted by citrus canker, a disease caused by the bacterial phytopathogen, Xanthomonas citri subsp. citri (Xcc). GenBank houses a large collection of Expressed Sequence Tags (ESTs) enriched with transcripts generated during the defence response against this pathogen; however, there are currently no strategies in citrus to assess the function of candidate genes. This has greatly limited research as defence signalling genes are often involved in multiple pathways. In this study, we demonstrate the efficacy of RNA interference (RNAi) as a functional genomics tool to assess the function of candidate genes involved in the defence response of Citrus limon against the citrus canker pathogen. Double-stranded RNA expression vectors, encoding hairpin RNAs for citrus host genes, were delivered to lemon leaves by transient infiltration with transformed Agrobacterium. As proof of principle, we have established silencing of citrus phytoene desaturase (PDS) and callose synthase (CalS1) genes. Phenotypic and molecular analyses showed that silencing vectors were functional not only in lemon plants but also in other species of the Rutaceae family. Using silencing of CalS1, we have demonstrated that plant cell wall-associated defence is the principal initial barrier against Xanthomonas infection in citrus plants. Additionally, we present here results that suggest that H2O2 accumulation, which is suppressed by xanthan from Xcc during pathogenesis, contributes to inhibition of xanthan-deficient Xcc mutant growth either in wild-type or CalS1-silenced plants. With this work, we have demonstrated that high-throughput reverse genetic analysis is feasible in citrus.

The Xanthomonas axonopodis pv. citri flagellum is required for mature biofilm and canker development.

Xanthomonas axonopodis pv. citri (Xac) is the causative agent of citrus canker. This bacterium develops a characteristic biofilm on both biotic and abiotic surfaces. To evaluate the participation of the single flagellum of Xac in biofilm formation, mutants in the fliC (flagellin) and the flgE (hook) genes were generated. Swimming motility, assessed on 0.25 % agar plates, was markedly reduced in fliC and flgE mutants. However, the fliC and flgE mutants exhibited a flagellar-independent surface translocation on 0.5 % agar plates. Mutation of either the rpfF or the rpfC gene, which both encode proteins involved in cell-cell signalling mediated by diffusible signal factor (DSF), led to a reduction in both flagellar-dependent and flagellar-independent surface translocation, indicating a regulatory role for DSF in both types of motility. Confocal laser scanning microscopy of biofilms produced in static culture demonstrated that the flagellum is also involved in the formation of mushroom-shaped structures and water channels, and in the dispersion of biofilms. The presence of the flagellum was required for mature biofilm development on lemon leaf surfaces. The absence of flagellin produced a slight reduction in Xac pathogenicity and this reduction was more severe when the complete flagellum structure was absent.

Rapid and sensitive detection of Citrus Bacterial Canker by loop-mediated isothermal amplification combined with simple visual evaluation methods.

BACKGROUND: Citrus Bacterial Canker (CBC) is a major, highly contagious disease of citrus plants present in many countries in Asia, Africa and America, but not in the Mediterranean area. There are three types of Citrus Bacterial Canker, named A, B, and C that have different genotypes and posses variation in host range within citrus species. The causative agent for type A CBC is Xanthomonas citri subsp. citri, while Xanthomonas fuscans subsp. aurantifolii, strain B causes type B CBC and Xanthomonas fuscans subsp. aurantifolii strain C causes CBC type C. The early and accurate identification of those bacteria is essential for the protection of the citrus industry. Detection methods based on bacterial isolation, antibodies or polymerase chain reaction (PCR) have been developed previously; however, these approaches may be time consuming, laborious and, in the case of PCR, it requires expensive laboratory equipment. Loop-mediated isothermal amplification (LAMP), which is a novel isothermal DNA amplification technique, is sensitive, specific, fast and requires no specialized laboratory equipment. RESULTS: A loop-mediated isothermal amplification assay for the diagnosis of Citrus Bacterial Canker (CBC-LAMP) was developed and evaluated. DNA samples were obtained from infected plants or cultured bacteria. A typical ladder-like pattern on gel electrophoresis was observed in all positive samples in contrast to the negative controls. In addition, amplification products were detected by visual inspection using SYBRGreen and using a lateral flow dipstick, eliminating the need for gel electrophoresis. The sensitivity and specificity of the assay were evaluated in different conditions and using several sample sources which included purified DNA, bacterium culture and infected plant tissue. The sensitivity of the CBC-LAMP was 10 fg of pure Xcc DNA, 5 CFU in culture samples and 18 CFU in samples of infected plant tissue. No cross reaction was observed with DNA of other phytopathogenic bacteria. The assay was capable of detecting CBC-causing strains from several geographical origins and pathotypes. CONCLUSIONS: The CBC-LAMP technique is a simple, fast, sensitive and specific method for the diagnosis of Citrus Bacterial Canker. This method can be useful in the phytosanitary programs of the citrus industry worldwide.