Citrus Canker Disease Research Articles

Identification of an Extracellular Endoglucanase That Is Required for Full Virulence in Xanthomonas citri subsp. citri

Xanthomonas citri subsp. citri causes citrus canker disease, which is characterized by the formation of water-soaked lesions, white or yellow spongy pustules and brown corky canker. In this work, we report the contribution of extracellular endoglucanase to canker development during infection. The ectopic expression of nine putative cellulases in Escherichia coli indicated that two endoglucanases, BglC3 and EngXCA, show carboxymethyl cellulase activity. Both bglC3 and engXCA genes were transcribed in X. citri subsp. citri, however, only BglC3 protein was detected outside the cell in western blot analysis. The deletion of bglC3 gene resulted in complete loss of extracellular carboxymethyl cellulase activity and delayed the onset of canker symptoms in both infiltration- and wound-inoculation assays. When growing in plant tissue, the cell density of bglC3 mutant was lower than that of the wild type. Our data demonstrated that BglC3 is an extracellular endoglucanase required for the full virulence of X. citri subsp. citri.

Reduced Susceptibility to Xanthomonas citri in Transgenic Citrus Expressing the FLS2 Receptor From Nicotiana benthamiana.

Overexpression of plant pattern-recognition receptors by genetic engineering provides a novel approach to enhance plant immunity and broad-spectrum disease resistance. Citrus canker disease associated with Xanthomonas citri is one of the most important diseases damaging citrus production worldwide. In this study, we cloned the FLS2 gene from Nicotiana benthamiana cDNA and inserted it into the binary vector pBinPlus/ARS to transform Hamlin sweet orange and Carrizo citrange. Transgene presence was confirmed by polymerase chain reaction (PCR) and gene expression of NbFLS2 was compared by reverse transcription quantitative PCR. Reactive oxygen species (ROS) production in response to flg22Xcc was detected in transgenic Hamlin but not in nontransformed controls. Low or no ROS production was detected from nontransformed Hamlin seedlings challenged with flg22Xcc. Transgenic plants highly expressing NbFLS2 were selected and were evaluated for resistance to canker incited by X. citri 3213. Our results showed that the integration and expression of the NbFLS2 gene in citrus can increase canker resistance and defense-associated gene expression when challenged with X. citri. These results suggest that canker-susceptible Citrus genotypes lack strong basal defense induced by X. citri flagellin and the resistance of these genotypes can be enhanced by transgenic expression of the flagellin receptor from a resistant species.

The Role of Image Enhancement in Citrus Canker Disease Detection

Digital image processing is employed in numerous areas of biology to identify and analyse problems. This approach aims to use image processing techniques for citrus canker disease detection through leaf inspection. Citrus canker is a severe bacterium-based citrus plant disease. The symptoms of citrus canker disease typically occur in the leaves, branches, fruits and thorns. The leaf images show the health status of the plant and facilitate the observation and detection of the disease level at an early stage. The leaf image analysis is an essential step for the detection of numerous plant diseases. The proposed approach consists of two stages to improve the clarity and quality of leaf images. The primary stage uses Recursively Separated Weighted Histogram Equalization (RSWHE), which improves the contrast level. The second stage removes the unwanted noise using a Median filter. This proposed approach uses these methods to improve the clarity of the images and implements these methods in lemon citrus canker disease detection.

Integration of Pseudomonas fluorescens and salicylic acid improves citrus canker disease management caused by Xanthomonas citri subsp citri-A*

The individual and combined effects of Pseudomonas fluorescens (Pf) and salicylic acid (SA) were investigated for control of citrus bacterial canker (CBC). Both treated plants with copper hydroxide and untreated ones were used as controls. Mexican lime (Citrus aurantifolia) seedlings were treated with SA at 10 mM, Pf and distilled water. Plants were initially inoculated with Xanthomonas citri subsp citri 72 h post treatments. Results indicated that the Pf and SA treatment controlled CBC more effectively compared to separately applying Pf or SA. The application of Pf in combination with SA significantly reduced lesion number per leaf (72%) and disease severity (84%). Significant changes in the activities of peroxidase and catalase were found. In conclusion, the integration of Pf with SA complements each other and can be applied to manage citrus canker disease in conjunction with other control programmes.

Modification of the PthA4 effector binding elements in Type I CsLOB1 promoter using Cas9/sgRNA to produce transgenic Duncan grapefruit alleviating XccΔpthA4:dCsLOB1.3 infection.

Citrus canker caused by Xanthomonas citri subspecies citri (Xcc) is a severe disease for most commercial citrus cultivars and responsible for significant economic losses worldwide. Generating canker-resistant citrus varieties will provide an efficient and sustainable solution to control citrus canker. Here, we report our progress in generating canker-resistant grapefruit by modifying the PthA4 effector binding elements (EBEs) in the CsLOB1 Promoter (EBEPthA4 -CsLOBP) of the CsLOB1 (Citrus sinensis Lateral Organ Boundaries) gene. CsLOB1 is a susceptibility gene for citrus canker and is induced by the pathogenicity factor PthA4, which binds to the EBEPthA4 -CsLOBP to induce CsLOB1 gene expression. There are two alleles, Type I and Type II, of CsLOB1 in Duncan grapefruit. Here, a binary vector was designed to disrupt the PthA4 EBEs in Type I CsLOB1 Promoter (TI CsLOBP) via epicotyl transformation of Duncan grapefruit. Four transgenic Duncan plants with targeted modification of EBEPthA4 -T1 CsLOBP were successfully created. As for Type I CsLOB1 promoter, the mutation rate was 15.63% (#D13), 14.29% (#D17), 54.54% (#D18) and 81.25% (#D22). In the presence of wild-type Xcc, transgenic Duncan grapefruit developed canker symptoms similarly as wild type. An artificially designed dTALE dCsLOB1.3, which specifically recognizes Type I CsLOBP, but not the mutated Type I CsLOBP or Type II CsLOBP, was developed to infect Duncan transformants. Consequently, #D18 had weakened canker symptoms and #D22 had no visible canker symptoms in the presence of XccΔpthA4:dCsLOB1.3. Our data suggest that activation of a single allele of susceptibility gene CsLOB1 by PthA4 is sufficient to induce citrus canker disease, and mutation in the promoters of both alleles of CsLOB1 is probably required to generate citrus canker-resistant plants. This work lays the groundwork to generate canker-resistant citrus varieties via Cas9/sgRNA in the future.

Bacteria from the citrus phylloplane can disrupt cell–cell signalling in Xanthomonas citri and reduce citrus canker disease severity

Xanthomonas citri subsp. citri (Xcc) is the causal agent of citrus canker, a disease that affects almost all types of citrus crops. Production of particular Xcc pathogenicity factors is controlled by a gene cluster rpf, which encodes elements of a cell–cell communication system called quorum sensing (QS), mediated by molecules of the diffusible signal factor (DSF) family. Interference with cell–cell signalling, also termed quorum quenching, either by signal degradation or over‐production, has been suggested as a strategy to control bacterial disease. In this study, three bacterial strains were isolated from citrus leaves that displayed the ability to disrupt QS signalling in Xcc. Pathogenicity assays in sweet orange (Citrus sinensis) showed that bacteria of the genera Pseudomonas and Bacillus also have a strong ability to reduce the severity of citrus canker disease. These effects were associated with alteration in bacterial attachment and biofilm formation, factors that are known to contribute to Xcc virulence. These quorum‐quenching bacteria may represent a highly valuable tool in the process of biological control and offer an alternative to the traditional copper treatment currently used to treat citrus canker disease.

In vitro efficacy of different botanicals, bioagents, chemicals against Xanthomonas axonopodis pv.citri by turbidiometrical method

Citrus canker disease of acid lime caused by Xanthomonas axonopodis pv. citri is an important disease in many parts of MH region .The bacterium infects the twigs, petioles, fruit stalks and fruits. Action of botanicals, bioagents, chemicals against Xac was studied by turbidiometrically at 24hrs. intervals upto 96hrs by spectrophotometer at 620nm. At 96 hours of incubation least bacterial growth(0.232OD) was exhibited in copperoxychloride + streptomycin sulphate (0.2 % + 200ppm) followed with copper-oxychloride + streptomycin sulphate (0.2% + 100ppm) 0.266 OD statistically superior over all treatments. Similar findings are observed after 24h growth in copper oxychloride + streptomycin sulphate (0.2 % + 200ppm) followed with copper-oxychloride + streptomycin sulphate (0.2 % + 100ppm) (0.303, 0.306, respectively. In botanicals and bioagents neem seed kernel extract (5%) was effective in reducing the growth of bacteria with 0.446 OD at 96 h followed by Pseudomonas flourescence 1x10 8 cell and Bacillus subtilis 1x10 8 cell with 0.506 and 0.486 OD, respectively.

Efficient production of marker-free transgenic ‘Tarocco’ blood orange (Citrus sinensis Osbeck) with enhanced resistance to citrus canker using a Cre/loxP site-recombination system

Marker-free transgenic plants alleviate concerns regarding the biosafety of genetically modified organisms and promote their commercialization. In this study, a transformation vector pLI35SAAT, harboring a Cre/loxP-mediated recombination system combined with the isopentenyl transferase (ipt) selectable marker gene and an anti-bacterial peptide gene AATCB, was used to produce marker-free transgenic ‘Tarocco’ blood orange (Citrus sinensis Osbeck) with enhanced resistance to citrus canker. Using ipt positive selection, a transformation efficiency of 21.4 % was achieved. When the DNA between two loxP sites was excised, phenotypically normal shoots gradually appeared from 74.8 % of the transgenic ipt shoots. Their marker-free transgenic nature was confirmed using PCR and sequencing analyses. In vitro evaluations of citrus canker disease resistance revealed that marker-free transgenic plants exhibited an enhanced resistance to Xanthomonas axonopodis pv. citri. The marker-free transgenic plants appeared phenotypically normal under greenhouse conditions. Thus, marker-free transgenic citrus plants with targeted traits can be efficiently produced using a Cre/loxP-mediated recombination system combined with ipt positive selection.

Production of sweet orange somaclones tolerant to citrus canker disease by in vitro mutagenesis with EMS

Xanthomonas citri subsp. citri (Xcc) is the causal agent of citrus canker, a quarantine disease worldwide. This disease is hard to be completely controlled by chemical sprays, and the selection of citrus genotypes resistant to the pathogen becomes one of the most important ways to control the disease. The limited genotypes that have been found up to now tolerant to Xcc are not commercially cultivated. Sweet orange [Citrus sinensis (L.) Osbeck] is one of the most important citrus species widely cultivated in world and all the orange cultivars are susceptible to canker disease. Therefore, this study aims at the establishment of an efficient protocol for in vitro ethyl methane sulphonate (EMS) mutagenesis and the selection of ‘Bingtang’ sweet orange somaclones tolerant to citrus canker disease. Mutation was introduced by treating the cell suspension of embryogenic callus with 1.5 % of EMS for 1 h (the lethal concentration). A crude extract produced from the pathogen solution was used as the selection agent. Sweet orange leaves inoculated by Xcc-crude extract showed the symptoms similar to those inoculated by Xcc bacterial inoculum. The young shoots of susceptive genotypes cultured in 10 % of Xcc-crude extract solution become brown and died in 1 week, while the resistant citron C-05 grew normally under the same conditions. After two steps (cell suspension and plants) of selection by Xcc-crude extract solution, the survival plants were tested by in vitro and in vivo inoculation with Xcc. One somaclone, named DG-2, was identified to be resistant to the canker disease. The results suggested that the established protocol for somaclones variation by treating sweet orange callus with EMS and in vitro selection of the somaclones tolerant to canker disease by the pathogen crude extract was effective. The gained sweet orange somaclone tolerant to X. citri subsp. citri will go into further cultivar selection procedures.

Correlation of environmental variables on canker disease development in commercial citrus cultivars of Pakistan

Citrus canker is an extremely costly disease causing worldwide loss of millions of dollars. The onset and development of disease depends upon the favorable environmental conditions. In the present study the crucial environmental variables were correlated. The method provides the ability to examine the evolution of an epidemic in both space and time simultaneously and led to the symptom development. So, study was conducted in research area of Department of Plant Pathology 2008-09. Fifteen commercial citrus varieties were screened against canker disease to find out degree of resistance. Jaffa, pine apple, kinnow, mungal singh, tangerine, succari were found moderately resistant. Five varieties such as chinese lime, musambi, grapefruit, blood red and mayer lime were highly susceptible to canker disease. The susceptible varieties were feutral's early, sweet lime, malta and valentia late showed moderately susceptible response against the canker disease. The disease responses of these citrus varieties were correlated with environmental factors (Air temperature (maximum and minimum), relative humidity (%) and wind velocity (km/hr). Air temperature (maximum and minimum), relative humidity and wind velocity had significant correlations with citrus canker disease development. Rainfall also had significant correlations with citrus canker disease development. Disease started increasing in July and reached maximum incidence in August to October. The current understanding of pathogen and correlation of epidemiological factors help in developing comprehensive management practices to reduce fruit losses.

Antibiotic sensitivity against Xanthomonas axonopodis pv. citri by using Hi media disc

Citrus canker disease is of regular occurrence on several citrus cultivars in varying degrees of incidence depending on the climatic conditions. The bacterium, Xanthomonas causes different symptoms ranging from pustules to necrotic lesions consisting of erumpent corky tissue surrounded by water soaked tissues and yellow halo on leaves, stems and fruits. Sensitivity of different antibiotics were tested against Xanthomonas axonopodis pv. citri isolates ( Xac ). using eight different Hi-media disc viz. , HX006, HX007, HX010, HX032, HX038, HX060, HX067, HX069. Each Hi-media disc having six different antibiotics of different concentration have been tested against Xac isolates. The causal pathogen Xanthomonas axonopodis pv. citri showed sensitivity to most of the antibiotic tested. Hi-media disc HX060 having antibiotic Imepenem (10mg) and Ticarcillin (70mg) showed maximum zone of inhibition (42mm). However, no effect of antibiotics i.e. Co-Trimoxazole, Nofloxacin, Cefoxitin, Erythromycin observed against Xac.

Induced resistance in sweet orange against Xanthomonas citri subsp. citri by hexanoic acid

Citrus canker, caused by Xanthomonas citri subsp. citri, is a serious and wide-spread disease of citrus, causing losses in fruit yield and quality. There are no highly effective citrus canker disease control measures. Repeated spray applications of copper are often employed to protect fruit from bacterial infection with consequences for copper phytotoxicity and accumulation in the soil. Alternatively, innate plant defense mechanisms can be enhanced by plant treatments with specific natural and synthetic inducers for control of bacterial diseases. In this study, hexanoic acid applied as a soil drench or foliar spray on 9-month-old potted citrus trees reduced lesions on leaves by 50% compared with control plants. Disease-reducing activity lasted up to 50 days after application. Induction of resistance mediated by hexanoic acid was demonstrated by enhanced expression of Pathogenesis-related (PR) genes and callose deposition in treated and infected plants. These findings indicated that hexanoic acid applications trigger a defensive response in the plants. The application of this natural compound may have potential for management of citrus canker in conjunction with other disease control measures and may reduce the frequency or rate of copper bactericides.

The dual nature of trehalose in citrus canker disease: a virulence factor for Xanthomonas citri subsp. citri and a trigger for plant defence responses.

Xanthomonas citri subsp. citri (Xcc) is a bacterial pathogen that causes citrus canker in susceptible Citrus spp. The Xcc genome contains genes encoding enzymes from three separate pathways of trehalose biosynthesis. Expression of genes encoding trehalose-6-phosphate synthase (otsA) and trehalose phosphatase (otsB) was highly induced during canker development, suggesting that the two-step pathway of trehalose biosynthesis via trehalose-6-phosphate has a function in pathogenesis. This pathway was eliminated from the bacterium by deletion of the otsA gene. The resulting XccΔotsA mutant produced less trehalose than the wild-type strain, was less resistant to salt and oxidative stresses, and was less able to colonize plant tissues. Gene expression and proteomic analyses of infected leaves showed that infection with XccΔotsA triggered only weak defence responses in the plant compared with infection with Xcc, and had less impact on the host plant's metabolism than the wild-type strain. These results suggested that trehalose of bacterial origin, synthesized via the otsA-otsB pathway, in Xcc, plays a role in modifying the host plant's metabolism to its own advantage but is also perceived by the plant as a sign of pathogen attack. Thus, trehalose biosynthesis has both positive and negative consequences for Xcc. On the one hand, it enables this bacterial pathogen to survive in the inhospitable environment of the leaf surface before infection and exploit the host plant's resources after infection, but on the other hand, it is a tell-tale sign of the pathogen's presence that triggers the plant to defend itself against infection.

First record of citrus canker, caused by Xanthomonas citri subsp. citri in Solomon Islands

Citrus canker disease was detected for the first time in Solomon Islands on Guadalcanal Island. The bacterium was isolated from diseased citrus plants exhibiting distinct citrus canker symptoms consisting of crater-like lesions on the rind of fruit and corky yellow-brownish lesions on leaves. Xanthomonas citri subsp. citri was identified using biochemical and molecular tests and confirmed as the causal agent by fulfilling Koch’s postulates.

Nutrient Management Options for Florida Citrus: A Review of NPK Application and Analytical Methods

Citrus production in Florida is ranked first in the United States. Success of the citrus industry in the state relies heavily on sound water and nutrient management practices. Recently, citrus production has been declining due to the escalating prevalence of the citrus greening (Liberibacter asiaticus) and canker (Xanthomonas axonopodis) diseases. One option being explored is the manipulation of nutrient management scenarios to increase and enhance tree productivity. The paper presents a review of the management, analytical and application methods of three major nutrients Nitrogen (N), Phosphorus (P), and Potassium (K) on Florida's sandy soils with low organic matter (OM) and high leaching potential due to heavy annual rains (∼1200 mm). The NPK management options for Florida citrus are compared with those of other citrus producing regions around the world. Also, the critical tissue and soil nutrient concentrations for optimal and high citrus production are discussed. The review paper should provide important nutrient management guidelines to citrus growers in Florida and other regions with similar climatic and soil conditions.

Activity of Secondary Bacterial Metabolites in the Control of Citrus Canker

This study investigated the protective effects of secondary bacterial metabolites, produced by Pseudomonas sp. (bacterium strain LN), on citrus canker disease caused by Xanthomonas axonopodis pv. citri (Xac 306). The LN bacteria strain was cultured in liquid medium and the supernatant free-cells was treated with methanol (AMF) and ethyl acetate (AEF), respectively, and then the extract was concentrated, filtrated, lyophilized and fractionated by vacuum liquid chromatography (VLC). After VLC, eight fractions were obtained. All fractions’ activity against Xac 306 by agar well diffusion assay and minimum inhibitory concentration but in different concentrationswere tested. Cytotoxicity effects were observed in all fractions in 50 μg·mL-1 concentration. The comet assay demonstrated that the fractions EAF, VLC2 and VLC3 presented no genotoxic effects at tested concentrations. In plants only VLC3 showed significant results (p

Evaluation of Saudi Fluorescent Pseudomonads Isolates as a Biocontrol Agent against Citrus Canker Disease Caused by Xanthomonas citri subsp citri A*

The goal of this study was to determine whetherbacterial antagonists could be used to control Xanthomonas citri subspcitri (Xcc), the causal agent of bacterial citrus canker disease. Atotal of 22 potentially bacterial antagonists isolated as epiphytes from thephylloplane of healthy citrus trees were screened for their in vitro biologicalcontrol capability against Xcc. These strains were identified as Pseudomonas fluorescens onthe basis of biochemical and physiological tests and 16S rDNA. Out of these 22potentially bacterial antagonists, five strains (KSA1, KSA9, KSA14, KSA17, andKSA20) showed high potential growth inhibition capabilities against Xcc.The KSA1 strain was selected for further studies to test its in vivocapability to control bacterial citrus canker pathogen. It was sprayed in asuspension of 107CFU ml-1on citrus leaves which were subsequently inoculated after 72 h with 108 CFU ml-1 suspension of Xcc strain JQ890095.According to the in vivo biocontrol tests, the putative antagonist KSA1significantly reduced the symptoms on the leaves of Mexican lime seedlingscompared with untreated inoculated plants.

Crystallization and preliminary X-ray diffraction analysis of the phosphate-binding protein PhoX from Xanthomonas citri.

Xanthomonas axonopodis pv. citri (X. citri) is an important bacterium that causes citrus canker disease in plants in Brazil and around the world, leading to significant economic losses. Determination of the physiology and mechanisms of pathogenesis of this bacterium is an important step in the development of strategies for its containment. Phosphate is an essential ion in all microrganisms owing its importance during the synthesis of macromolecules and in gene and protein regulation. Interestingly, X. citri has been identified to present two periplasmic binding proteins that have not been further characterized: PstS, from an ATP-binding cassette for high-affinity uptake and transport of phosphate, and PhoX, which is encoded by an operon that also contains a putative porin for the transport of phosphate. Here, the expression, purification and crystallization of the phosphate-binding protein PhoX and X-ray data collection at 3.0 Å resolution are described. Biochemical, biophysical and structural data for this protein will be helpful in the elucidation of its function in phosphate uptake and the physiology of the bacterium.

Expression of Xylella fastidiosa RpfF in citrus disrupts signaling in Xanthomonas citri subsp. citri and thereby its virulence.

Xylella fastidiosa and Xanthomonas citri subsp. citri, that cause citrus variegated chlorosis (CVC) and citrus canker diseases, respectively, utilize diffusible signal factor (DSF) for quorum sensing. DSF, produced by RpfF, are similar fatty acids in both organisms, although a different set of genes is regulated by DSF in each species. Because of this similarity, Xylella fastidiosa DSF might be recognized and affect the biology of Xanthomonas citri. Therefore, transgenic Citrus sinensis and Carrizo citrange plants overexpressing the Xylella fastidiosa rpfF were inoculated with Xanthomonas citri and changes in symptoms of citrus canker were observed. X. citri biofilms formed only at wound sites on transgenic leaves and were thicker; however, bacteria were unable to break through the tissue and form pustules elsewhere. Although abundant growth of X. citri occurred at wound sites on inoculated transgenic leaves, little growth was observed on unwounded tissue. Genes in the DFS-responsive core in X. citri were downregulated in bacteria isolated from transgenic leaves. DSF-dependent expression of engA was suppressed in cells exposed to xylem sap from transgenic plants. Thus, altered symptom development appears to be due to reduced expression of virulence genes because of the presence of antagonists of DSF signaling in X. citri in rpfF-expressing plants.

Production of Transgenic Anliucheng Sweet Orange (Citrus sinensis Osbeck) with Xa21 Gene for Potential Canker Resistance

Citrus canker, an epidemic quarantine disease caused by Xanthomonas axonopodis pv. citri, has brought a great damage in citrus production worldwide. Herein, a rice PRR (pattern recognition receptor) gene Xa21 together with GUS reporter gene and hygromycin phosphotransferase gene (HPT) was introduced into Anliucheng sweet orange (Citrus sinensis Osbeck) via Agrobacterium-mediated transformation of embryogenic callus. The transgenic calluses were screened on MT basal medium containing hygromycin (HYG) and detected by histochemical GUS staining. The transgenic plantlets were recovered through somatic embryogenesis pathway. The regenerated plantlets were accustomed to and maintained in the greenhouse. The transgene integration of recovered plantlets was identified by PCR and Southern blot hybridization. It showed that all the transgenic plantlets tested had undergone single copy integration, the expression of Xa21 in eight different transgenic lines detected by qRT-PCR can be divided into three grades, high for T5 and T6, middle for T4 and low for the rest. The tolerance to citrus canker disease of the three recovered transgenic lines T2, T4 and T6 was assessed by in vitro pin-puncture inoculation. The results showed that all the three transgenic lines conferred improved resistance to citrus canker bacterium infection and the T4 transgenic line displayed the highest resistance. The mechanism and feasibility of rice Xa21 in triggering innate immunity in citrus was briefly discussed.