Asiatic Citrus Canker Research Articles

Genetic diversity of Xanthomonas citri subsp. citri in citrus orchards in northwest Paraná state, Brazil

Xanthomonas citri subsp. citri , which causes Asiatic citrus canker (ACC), is an important pathogen of citrus in Brazil and elsewhere. The genetic diversity of X. citri subsp. citri pathotype ‘A’ has not been studied in Brazil at a local scale (up to 300 km). Forty isolates were sampled from lesions of ACC on citrus in three orchards in Parana state, Brazil. Twelve minisatellite markers were used to characterize the genetic diversity of the isolates. An Unweighted Paired Group Method of Arithmetic Averages tree was used for identifying unique multilocus haplotypes but there was no association between haplotypes and source locations. An analysis of molecular variance among populations showed that 98% of the variance was accounted for within the populations, and only 2% was accounted for among populations. Differences among populations was not significant (Φ=0.018, P =0.2). The relatively high, yet uniform, genetic diversity among isolates and low degree of spatial differentiation between populations of X. citri subsp. citri suggests that the populations in Parana state have a common origin and strong historical epidemiological links.

Complete Genome Sequences of Six Copper-Resistant Xanthomonas citri pv.citri Strains Causing Asiatic Citrus Canker, Obtained Using Long-Read Technology.

ABSTRACTThe gammaproteobacterium Xanthomonas citri pv. citri causes Asiatic citrus canker. Pathotype A strains have a broad host range, which includes most commercial citrus species, and they cause important economic losses worldwide. Control often relies on frequent copper sprays. We present here the complete genomes of six X. citri pv. citri copper-resistant strains.

Xanthomonas citri pv. citri Pathotypes: LPS Structure and Function as Microbe-Associated Molecular Patterns.

Xanthomonas citri pv. citri is the pathogen responsible for Asiatic citrus canker, one of the most serious citrus diseases worldwide. The lipopolysaccharide (LPS) molecule has been demonstrated to be involved in X. citri pv. citri virulence. Despite enormous progress in investigations of the molecular mechanisms for bacterial pathogenicity, determination of the detailed LPS structure-activity relationship is limited, as the current knowledge is mainly based on structural determination of one X. citri pv. citri strain. As X. citri pv. citri strains are distinguished into three main pathogenicity groups, we characterized the full structure of the LPS from two pathotypes that differ in their host-range specificity. This revealed an intriguing difference in LPS O-chain structure. We also tested the LPSs and isolated lipid A moieties for their ability to act as microbe-associated molecular patterns in Arabidopsis thaliana. Both LPS/lipid As induced ROS accumulation, but no difference was observed between the two pathotypes.

First Report of Copper-resistant Xanthomonas citri pv. citri Pathotype A Causing Asiatic Citrus Canker in Réunion, France

Xanthomonas citri pv. citri causes Asiatic citrus canker (ACC), which induces erumpent, callus-like lesions on all aerial organs, and consequently defoliation, premature fruit drop, and twig dieback under high inoculum pressure. Present in most tropical and subtropical citrus producing regions, the disease can lead to important decreases in yield and limitations to export markets due to the quarantine status of the pathogen in some countries ( Graham et al. 2004 ). Strains assigned to genetic lineage 1 within pathotype A, which has the widest host range comprising most citrus cultivars and citrus relatives, were shown to be primarily involved in the worldwide expansion of the bacterium over the 20th century ( Pruvost et al. 2014 ). ACC has been considered endemic in Reunion Island since the 1970s, but was likely present earlier and was not subjected to any eradication attempt. Most citrus growers on the island frequently apply copper-based pesticides as part of integrated pest management programs. A recurring lack of ACC control in several citrus groves was investigated in 2014 using our lab collection of authenticated X. citri pv. citri strains. A total of 501 strains from active epidemics as well as 123 historical strains isolated from 1978 onward were tested for copper resistance using PCR assays targeting copL ( Behlau et al. 2013 ). A total of 115 strains produced the expected amplicon whereas others (including all historical strains) and the negative control (H 2 O) did not. Phenotypic tests were carried out on low-complexing mineral salts casitone-yeast extract-glycerol-agar (CYE) plates ( Zevenhuizen et al. 1979 ) flooded with bacterial suspensions ( ≈ 5 × 10 6 cfu ml –1 ) in sterile tris buffer (pH 7.2), allowed to dry for 15 min., inoculated with a sterile CuSO 4 , 5H 2 O solution (10 mg ml –1 ) with an easySpiral plater used to create a gradient of log-increasing copper concentrations toward the plate center and incubated for 2 days at 28°C. Using this assay, copL + strains grew over a radius (measured from the plate periphery where the concentration of copper are lowest) >23 mm whereas copL – strains growth radius were constantly lower. Copper-resistant strains (CRS) had minimum inhibitory concentrations for copper sulfate ranging from 128 to 256 μ l ml –1 ( Pruvost et al. 1998 ). A subset of 20 CRS and 40 copper-susceptible strains (CSS) (including historical strains) were all assigned to genetic lineage 1 by minisatellite typing (performed as reported by Pruvost et al. 2014 ) and split into two multilocus haplotypes shared also with CSS. CRS were widespread in most citrus production areas of the island where they coexisted with CSS. The oldest CRS dated from 2010. Because no extreme hurricane hit the island between 2010 and 2014, the fast spread of CRS over the island was very likely related to their presence in a major citrus nursery, as confirmed by our data. In Reunion, the European Conformitas Agraria Communitatis (CAC) standards (directive 92/34/EEC) certify the disease-free status of citrus nursery plants (assessed by visual inspections for ACC). CAC is presently recommended but not compulsory. This report suggests that a strict implementation of this procedure would have helped limiting the spread of CRS in Reunion. (Resume d'auteur)

Management of Asiatic Citrus Canker Under Field Conditions in Saudi Arabia Using Bacteriophages and Acibenzolar-S-Methyl.

Applications of formulated bacteriophages with skim milk and sucrose or nonformulated bacteriophages combined with acibenzolar-S-methyl (ASM) were compared with copper bactericides applications for suppressing Asiatic citrus canker (ACC) caused by Xanthomonas citri subsp. citri (Xcc) on leaves under greenhouse and field conditions in Saudi Arabia. Bacteriophages were applied one day prior to inoculation of Mexican lime (Citrus aurantifolia) plants with Xcc, then twice a week until the end of the trials. Copper hydroxide was applied once prior to inoculation and then every seven days afterward, whereas ASM was applied one week prior to inoculation and then every 21 days afterward. Under greenhouse conditions, the incidence of ACC on leaves was reduced significantly from 75.2 to 12.8% or 18.3% for plants treated with copper hydroxide or bacteriophages in combination with ASM, respectively. Applications of formulated phages in combination with ASM as soil drench under field conditions significantly decreased disease incidence by 14.8% (Trial 1) and 16.8% (Trial 2) compared with untreated control plants. Overall, the Xcc-inoculated plants treated with bacteriophages + ASM combination showed significant ACC reduction under greenhouse and field conditions. The bacteriophages + ASM combination tested in these trials can be an effective tool in the integrated management programs of Asiatic citrus canker disease.

Adaptation of genetically monomorphic bacteria: evolution of copper resistance through multiple horizontal gene transfers of complex and versatile mobile genetic elements

Copper-based antimicrobial compounds are widely used to control plant bacterial pathogens. Pathogens have adapted in response to this selective pressure. Xanthomonas citri pv. citri, a major citrus pathogen causing Asiatic citrus canker, was first reported to carry plasmid-encoded copper resistance in Argentina. This phenotype was conferred by the copLAB gene system. The emergence of resistant strains has since been reported in Réunion and Martinique. Using microsatellite-based genotyping and copLAB PCR, we demonstrated that the genetic structure of the copper-resistant strains from these three regions was made up of two distant clusters and varied for the detection of copLAB amplicons. In order to investigate this pattern more closely, we sequenced six copper-resistant X.citri pv.citri strains from Argentina, Martinique and Réunion, together with reference copper-resistant Xanthomonas and Stenotrophomonas strains using long-read sequencing technology. Genes involved in copper resistance were found to be strain dependent with the novel identification in X.citri pv.citri of copABCD and a cus heavy metal efflux resistance-nodulation-division system. The genes providing the adaptive trait were part of a mobile genetic element similar to Tn3-like transposons and included in a conjugative plasmid. This indicates the system's great versatility. The mining of all available bacterial genomes suggested that, within the bacterial community, the spread of copper resistance associated with mobile elements and their plasmid environments was primarily restricted to the Xanthomonadaceae family.

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.

First Report of Xanthomonas citri pv. citri Pathotype A Causing Asiatic Citrus Canker in Martinique, France

HomePlant DiseaseVol. 100, No. 9First Report of Xanthomonas citri pv. citri Pathotype A Causing Asiatic Citrus Canker in Martinique, France PreviousNext DISEASE NOTES OPENOpen Access licenseFirst Report of Xanthomonas citri pv. citri Pathotype A Causing Asiatic Citrus Canker in Martinique, FranceD. Richard, C. Boyer, S. Javegny, K. Boyer, P. Grygiel, O. Pruvost, A. L. Rioualec, V. Rakotobe, J. Iotti, R. Picard, C. Vernière, C. Audusseau, C. François, V. Olivier, A. Moreau, and A. ChabirandD. RichardSearch for more papers by this author, C. BoyerSearch for more papers by this author, S. JavegnySearch for more papers by this author, K. BoyerSearch for more papers by this author, P. GrygielSearch for more papers by this author, O. PruvostSearch for more papers by this author, A. L. RioualecSearch for more papers by this author, V. RakotobeSearch for more papers by this author, J. IottiSearch for more papers by this author, R. PicardSearch for more papers by this author, C. VernièreSearch for more papers by this author, C. AudusseauSearch for more papers by this author, C. FrançoisSearch for more papers by this author, V. OlivierSearch for more papers by this author, A. MoreauSearch for more papers by this author, and A. ChabirandSearch for more papers by this authorAffiliationsAuthors and Affiliations D. Richard C. Boyer S. Javegny K. Boyer P. Grygiel O. Pruvost , CIRAD UMR PVBMT, F-97410 Saint-Pierre, La Réunion, France A. L. Rioualec V. Rakotobe J. Iotti , DAAF Service de l’Alimentation, F-97264 Fort de France, Martinique R. Picard , FREDON, F-97224 Ducos, Martinique C. Vernière , CIRAD UMR BGPI, F-34398 Montpellier Cedex 5, France C. Audusseau C. François V. Olivier , ANSES Laboratoire de la Santé des Végétaux, F-49044 Angers, France A. Moreau A. Chabirand , ANSES Laboratoire de Santé des Végétaux, F-97410 Saint-Pierre, La Réunion, France. Published Online:17 Jun 2016https://doi.org/10.1094/PDIS-02-16-0170-PDNAboutSections ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat Asiatic canker, caused by Xanthomonas citri pv. citri, is a major threat to worldwide citriculture. Three pathotypes differing in host range and hypersensitive reactions toward citrus species have been defined. Whereas pathotypes Aw and A* have a restricted host range, X. citri pv. citri pathotype A infects a broader range including most commercial citrus species and hybrids and can cause important economic losses in tropical and subtropical areas. Pathotype A strains, especially those assigned to lineage 1, were implicated in the major geographical expansion of X. citri pv. citri during the 20th century from their native area, Asia (Pruvost et al. 2014). X. citri pv. citri is listed as a quarantine pathogen in the European Union (EU) – Directive 2000/29/EC annex II A1. Martinique (France) is an outermost region of the EU in the eastern Caribbean Sea. Canker lesions were first observed at Morne Rouge, Martinique in June 2014 on grapefruit (Citrus paradisi), mandarin (C. reticulata), Tahiti lime (C. latifolia), and Valencia and Washington Navel oranges (C. sinensis). Official diagnostics, including bacterial isolations on YPGA or KC semiselective medium (Pruvost et al. 2005), PCR-based identification with 4/7 primers (Hartung et al. 1993), and pathogenicity tests, were performed following the EPPO standard PM7/44 (www.eppo.int) and identified isolates as X. citri pv. citri. Three strains isolated in Martinique in 2014 from grapefruit or Tahiti lime were further characterized (LL074-4, LL077-2, and LL079). Multilocus sequence analysis (MLSA) targeting six housekeeping genes (atpD, dnaK, efp, gltA, gyrB, and lepA) (Almeida et al. 2010; Bui Thi Ngoc et al. 2010) identified Martinique strains as X. citri pv. citri with 100% sequence identity to the type strain LMG 9322. Using MLVA-31 targeting 31 minisatellites, Martinique strains were assigned to lineage 1 composed of pathotype A strains (Pruvost et al. 2014). All strains were inoculated by a detached leaf assay onto Mexican lime SRA 140 (C. aurantifolia), sweet orange Washington Navel SRA 102, and grapefruit Henderson SRA 336 (Bui Thi Ngoc et al. 2010). All inoculated leaves produced typical erumpent, callus-like tissue at wound sites. Xanthomonas-like colonies were reisolated from lesions that had developed. Boiled suspensions were assayed by PCR with 4/7 primers and produced the expected amplicon, fulfilling Koch’s postulates. No lesions developed on the negative control consisting of sterile 0.01M tris buffer pH 7.2. This is the first report of X. citri pv. citri in Martinique and to our knowledge in the Caribbean region. Surprisingly, all strains collected to date in Martinique grew on YPGA supplemented with 300 mg liter−1 copper sulfate even when no extensive copper spray programs have been used, suggesting that copper-resistant strains may have been introduced. Disease is contained by tree removal and burning and the situation is presently under apparent control although positive trees were sporadically detected in 2015 in backyards or small orchards at Le Lorrain and Saint Pierre. An extensive surveillance program is currently implemented in Martinique for quarantine pathogens of citrus.

Evaluation of Resistance to Asiatic Citrus Canker among Selections of Pêra Sweet Orange (Citrus sinensis).

Asiatic citrus canker (ACC, caused by the bacterium Xanthomonas citri subsp. citri) is a destructive disease of citrus in Brazil and in several other citrus-producing countries. ACC management is problematic, and bactericides such as copper can be reasonably efficacious but do not completely control the disease. Furthermore, injury by citrus leaf miner (CLM) can exacerbate severity of ACC. Host resistance is the most desirable solution for management of ACC; however, evaluations of germplasm indicate that resistance is limited in many popular species and cultivars that are grown commercially. Limited evaluations have been made of sweet orange (Citrus sinensis) selections. We evaluated resistance of 25 Pêra sweet orange selections to X. citri subsp. citri by wound inoculation and measuring lesion diameter under greenhouse conditions (wound inoculation indicates mesophyll resistance which will be valuable in areas where CLM exists). ACC severity was assessed on the same 25 selections at three locations in the field in Brazil, relying on natural inoculum and conditions to cause disease. In the greenhouse experiments, the selections EEL, Bianchi/CC, Ipiguá, Olimpia, IAC 2000/1, and Ovale Siracusa consistently had the smallest diameter lesions, indicating greatest resistance, although differences in lesion diameter were small. Results from the field experiments were less conclusive, although EEL and Ovale Siracusa were consistently numerically least affected by ACC. These results indicate selections of sweet orange that might be preferable to consider in canker-prone areas in Brazil and elsewhere.

Reaction of detached leaves from different varieties of sweet orange to inoculation with Xanthomonas citri subsp. citri

ABSTRACT Citrus genotypes show a broad range of resistance to infection with Xanthomonas citri subsp citri (Xcc), the causal agent of Asiatic citrus canker (ACC). The aim of this study was to evaluate a detached leaf inoculation method to compare nine different citrus genotypes that exhibit varied resistance levels to ACC, and to evaluate infection, colonization and symptom development of the varieties by using Scanning Electron Microscopy (SEM). Inoculation of Xcc in the leaves was carried out by producing injuries with needles. Lesion diameters were measured at 7, 10 and 13 days after inoculation (DAI). The variety Pera IAC was most resistant to the pathogen and had lesions with smaller diameters, while the variety Washington was most susceptible and had the largest lesions, indicating low resistance to Xcc. SEM images were taken from the lesion development, and sections were obtained from the disease lesions in the tested varieties. The most resistant variety, Pera IAC, had smaller stomatal openings and produced greater quantity of foliar exudates, compared with the variety Washington. We hypothesize that the exudates may contain Xcc toxic compounds and thus may be a defense mechanism against infection by Xcc, but further research is required to establish any antibiotic effects of the exudates against citrus canker.

A comprehensive analysis of the Asiatic citrus canker eradication programme in São Paulo state, Brazil, from 1999 to 2009

Asiatic citrus canker (ACC), caused by Xanthomonas citri subsp. citri, has been controlled by exclusion and eradication measures in São Paulo state (SP), Brazil, since it was first detected in 1957. Several protocols of eradication have been used since then, but the protocol applied from 1999 to 2009 was the most successful and well documented of these. During that time, if the incidence of trees with symptoms in a citrus block was ≤0.5%, the infected trees and the symptomless trees within a 30 m radius were removed. However, if the incidence was >0.5%, the entire block was eliminated. Subsequently, inspections were carried out to monitor the affected blocks during quarantine. Although this protocol did not eliminate the disease from SP, it suppressed the incidence of affected blocks to very low annual levels (<0.20%) for a decade. Of over 5000 foci detected, 65.1% were eliminated by removing the block, either when disease was first detected (47.8%) or at a subsequent detection (17.3%), and 28.4% were eradicated by removing trees within a 30 m radius. The latter method was not an effective stand‐alone measure for ACC eradication and the 0.5% threshold was the key factor in suppression of disease statewide. Currently, a re‐emergence of ACC is occurring in SP. The information presented here will aid the establishment of protocols for the eradication of ACC in other citrus‐growing areas with low incidence of the disease or at risk of pathogen introduction.

A comparison of resistance to Asiatic citrus canker among different genotypes ofCitrusspp. in a long-term canker-resistance field screening experiment in Brazil

A comparison of resistance to Asiatic citrus canker among different genotypes of<i>Citrus</i>spp. in a long-term canker-resistance field screening experiment in Brazil

Bridgehead invasion of a monomorphic plant pathogenic bacterium: Xanthomonas citri pv. citri, an emerging citrus pathogen in Mali and Burkina Faso.

Molecular epidemiology studies further our understanding of migrations of phytopathogenic bacteria, the major determining factor in their emergence. Asiatic citrus canker, caused by Xanthomonas citri pv. citri, was recently reported in Mali and Burkina Faso, a region remote from other contaminated areas. To identify the origin and pathways of these emergences, we used two sets of markers, minisatellites and microsatellites, for investigating different evolutionary scales. Minisatellite typing suggested the introduction of two groups of strains in Mali (DAPC 1 and DAPC 2), consistent with microsatellite typing. DAPC 2 was restricted to Bamako district, whereas DAPC 1 strains were found much more invasive. The latter strains formed a major clonal complex based on microsatellite data with the primary and secondary founders detected in commercial citrus nurseries and orchards. This suggests that human activities played a major role in the spread of DAPC 1 strains via the movement of contaminated propagative material, further supported by the frequent lack of differentiation between populations from geographically distant nurseries and orchards. Approximate Bayesian Computation analyses supported the hypothesis that strains from Burkina Faso resulted from a bridgehead invasion from Mali. Multi-locus variable number of tandem repeat analysis and Approximate Bayesian Computation are useful for understanding invasion routes and pathways of monomorphic bacterial pathogens.

Comparison of Resistance to Asiatic Citrus Canker Among Different Genotypes of Citrus in a Long-Term Canker-Resistance Field Screening Experiment in Brazil.

Asiatic citrus canker (ACC) is caused by Xanthomonas citri subsp. citri. The disease results in yield loss and renders fruit unfit for the fresh market. A 6-year study in Paraná State, Brazil, was conducted to compare the susceptibility of 186 genotypes of citrus representing sweet orange (Citrus sinensis), mandarin (C. reticulata), Mediterranean mandarin (C. deliciosa), Clementine mandarin (C. clementina), Satsuma mandarin (C. unshiu), sour orange (C. aurantium), lemon (C. limon), sweet lime (C. aurantifolia), grapefruit (C. paradisi), and four hybrids (C. reticulata × Citrus sp., C. reticulata × C. paradisi, C. reticulata × C. sinensis, and C. unshiu × C. sinensis). Sweet orange (C. sinensis) was represented by the most genotypes (n = 141). The number of lesions per leaf was assessed 18 times from 2005 to 2010 (up to 4 times per year). The data were analyzed using mixed-model analysis of fixed and random effects, which showed a total of six resistance-susceptibility groupings of species and hybrids. Based on species, the most resistant genotypes, on average, included Satsuma and lemon (mean lesions per leaf = 4.32 and 4.26, respectively), and the most susceptible genotypes were grapefruit and sweet lime, with 14.84 and 10.96 lesions per leaf, respectively. Genotypes of mandarin, sour orange, Mediterranean mandarin, and sweet orange had intermediate severity (5.48 to 9.56 lesions per leaf). The hybrids also showed a range of ACC severity but all were in the more resistant groupings (5.26 to 7.35 lesions per leaf). No genotype was immune to ACC. The most resistant genotype was 'Muscia' (C. reticulata) and the most susceptible was 'Valencia Frost' (C. sinensis) (1.86 and 14.78 lesions per leaf, respectively). Approximately one-sixth of the genotypes showed a negative relationship of mean lesions per leaf with time, suggesting increasing resistance as they aged, due to a reduction in either new flush or plant size and structure. These results of the relative susceptibility of different citrus genotypes can be used in future research and to assist in varietal selection or for breeding purposes both within Brazil and other regions where ACC is an issue.

Novel insights into the Citrus sinensis nonhost response suggest photosynthesis decline, abiotic stress networks and secondary metabolism modifications.

Plants are constantly exposed to stress factors. Biotic stress is produced by living organisms such as pathogens, whereas abiotic stress by unfavourable environmental conditions. In Citrus species, one of the most important fruit crops in the world, these stresses generate serious limitations in productivity. Through biochemical and transcriptomic assays, we had previously characterised the Citrus sinensis (L.) Osbeck nonhost response to Xanthomonas campestris pv. vesicatoria (Doidge), in contrast to Asiatic citrus canker infection caused by Xanthomonas citri subsp. citri (Hasse). A hypersensitive response (HR) including changes in the expression of several transcription factors was reported. Here, a new exhaustive analysis of the Citrus sinensis transcriptomes previously obtained was performed, allowing us to detect the over-representation of photosynthesis, abiotic stress and secondary metabolism processes during the nonhost HR. The broad downregulation of photosynthesis-related genes was correlated with an altered photosynthesis physiology. The high number of heat shock proteins and genes related to abiotic stress, including aquaporins, suggests that stresses crosstalk. Additionally, the secondary metabolism exhibited lignin and carotenoid biosynthesis modifications and expression changes in the cell rescue GSTs. In conclusion, novel features of the Citrus nonhost HR, an important part of the plants' defence against disease that has yet to be fully exploited in plant breeding programs, are presented.

First Report of Xanthomonas citri pv. citri Pathotype A Causing Asiatic Citrus Canker in Grande Comore and Anjouan.

The causal agent of Asiatic citrus canker, Xanthomonas citri pv. citri, is a bacterium of major economic importance in tropical and subtropical citrus-producing areas. X. citri pv. citri pathotype A can cause severe infection in a wide range of citrus species and induces erumpent, callus-like lesions with water-soaked margins evolving to corky cankers and leading to premature fruit, leaf drop, and twig dieback on susceptible cultivars. This quarantine organism can strongly impact citrus markets so it has consequently been subjected to eradication efforts and international quarantine regulations. Asiatic citrus canker occurs on most islands in the Southwest Indian Ocean region including the Mascarene and Seychelles archipelagos. In the Comoros archipelago, the disease was observed for the first time in Mohéli island in 1966 (2), but had not yet been reported in neighboring islands, Grande Comore and Anjouan. In September 2013, leaves of key lime (Citrus aurantifolia) and sweet orange (C. sinensis) showing symptoms of citrus canker were collected from Anjouan, Grande Comore, and Mohéli. Nine Xanthomonas-like strains (three from each of the three islands) were isolated using KC semi-selective medium (5) from diseased samples (LK126-3, LK127-7, LK128-2, LK131-10, LK137-1, LK141-3, LK144-5, LK145-5, LK146-2). Based on a specific PCR assay with 4/7 primers (4), all Xanthomonas-like strains were tentatively identified as X. citri pv. citri. All strains produced a 468-bp amplicon similar to X. citri pv. citri strain IAPAR 306 used as a positive control. Negative control reactions with sterile tris buffer did not produce amplicons. Multilocus sequence analysis (MLSA) targeting six housekeeping genes (atpD, dnaK, efp, gltA, gyrB, and lepA) (1,3) fully identified all strains from the Comoros as X. citri pv. citri. More specifically, eight strains were identified as sequence type ST2 composed of pathotype A strains of X. citri pv. citri (3) (including all strains from the Southwest Indian Ocean region) while one of them (LK141-3 from Mohéli) was identified as a new sequence type based on a non-synonymous single nucleotide polymorphism in gyrB (accession KJ941208). All strains were inoculated by a detached leaf assay (3) onto Mexican lime SRA 140 (C. aurantifolia), Tahiti lime SRA 58 (C. latifolia), sweet orange New Hall Navel SRA 343 (C. sinensis), grapefruit Henderson SRA 336 (C. paradisi), and Ortanique tangor SRA 110 (C. reticulata × C. sinensis). All citrus species inoculated produced typical erumpent, callus-like tissue at wound sites. Xanthomonas-like yellow colonies were re-isolated from lesions produced on Mexican lime. Boiled bacterial suspensions were assayed by PCR with 4/7 primers (4) and produced the expected amplicon, fulfilling Koch's postulates. No lesions developed on the negative control consisting of inoculations with sterile tris buffer. This is the first report of X. citri pv. citri-A causing Asiatic citrus canker in Grande Comore and Anjouan islands confirming the wide distribution of the pathogen in Southwest Indian Ocean islands. Canker-free nurseries and grove sanitation should be implemented to decrease the prevalence of Asiatic canker in the Comoros.

Lessons from a case of successful eradication of citrus canker in a citrus-producing farm in São Paulo state, Brazil.

Asiatic citrus canker (ACC), caused by the bacterium Xanthomonas citri subsp. citri, is one of the most serious diseases affecting citrus worldwide. In areas under quarantine/eradication, such as Sao Paulo state (SPS), Brazil, the impact of ACC is related to the production costs for inspections and tree removal. Several protocols for eradication were adopted in SPS since the first detection of ACC in 1957. Among these, the one carried out from 1999 to 2009 was the most effective for maintaining the disease at a very low level throughout the state. That protocol mandated periodic inspections and removal of symptomatic and asymptomatic trees based on disease incidence in the block. After 2009 the eradication protocols were less stringent and ACC incidence continued to increase. This study aimed at identifying the key factors for the successful eradication of ACC in a farm located in SPS. Data on the outbreaks from 2006 to 2010 were collected and the related control measures adopted. The farm remained under quarantine from July 2006 to March 2010. Twelve disease foci were detected from July 2006 to January 2008 and incidence of symptomatic tress ranged from 0.05% and 0.43%. The successful eradication of ACC in this farm is attributed to the relatively low number of diseased trees in all foci detected in the course of frequent and well-executed inspections. The results of this case study may be used to guide containment protocols in other citrus production areas of Brazil or countries where ACC is either absent or has a low incidence.

Commercial risk-based survey for HLB and implications for efficacy of Citrus Health Management Areas (CHMAs)

The USDA, APHIS, PPQ has been conducting a risk-based multi-pest survey (MPS) for the past two years.&nbsp; The initial 6 statewide survey cycles were conducted on a 6 week basis, whereas the subsequent 20 cycles have been conducted on a 3-week basis.&nbsp; HLB and ACP are the two main pests addressed by the MPS but other citrus pests and diseases, i.e., Asiatic Citrus Canker (ACC), Citrus Leprosis Virus (CLiV), Citrus Black Spot (CBS) and Citrus Variegated Chlorosis (CVC) were also given lesser emphasis during the survey.

Development and Validation of Standard Area Diagrams as Assessment Aids for Estimating the Severity of Citrus Canker on Unripe Oranges.

Asiatic citrus canker (ACC) is an important disease of citrus in Brazil and elsewhere in the world. Infection with the causal pathogen, Xanthomonas citri subsp. citri, can cause severe disease on the fruit. Visual estimation of severity is the usual method used to quantify ACC on diseased fruit. The objective of this research was to construct and validate standard area diagram (SAD) sets as assessment aids for raters to improve the accuracy and reliability of visual estimates of ACC on unripe (green) fruit of sweet orange. Two SAD sets were constructed. A five-diagram SAD set had five severities depicted (0.5, 2.0, 8.0, 27.0, and 40.0%) and a six-diagram SAD set had six severities depicted (0.5, 1.0, 3.0, 9.0, 20.0, and 40.0%). Fifteen raters evaluated 40 images of cankered, unripe fruit. Both the five- and six-diagram SAD sets significantly improved the accuracy and reliability of estimates. Agreement, measured by Lin's concordance correlation coefficient, was 0.220 to 0.913 when not using SADs, 0.814 to 0.955 when using five-diagram SAD sets, and 0.863 to 0.925 when using six-diagram SAD sets. The five-diagram SAD set was significantly more accurate and reliable compared with the six-diagram set. Possible reasons for this are discussed. Based on the results, the five-diagram SAD set is preferable to use. Although the SAD set was developed for sweet orange, it doubtless has applicability to other citrus, including grapefruit. These SAD sets should be useful for research endeavors where accurate and reliable estimates of the severity of ACC are required.

A standard area diagram set to aid estimation of the severity of Asiatic citrus canker on ripe sweet orange fruit

Asiatic citrus canker (ACC) is a major disease of citrus in many tropical and subtropical citrus-growing regions. Severe disease can develop on the fruit, making it unmarketable. Research activities need accurate and reliable methods to estimate the severity of citrus canker, most often done visually by raters. The objective was to validate and compare two standard area diagram sets (SADs) as aids for raters to improve the accuracy and reliability of visual estimates of canker severity on ripe fruit of sweet orange (unripe fruit are green, and have slightly different symptoms compared to ripe fruit, warranting different SADs). A 5-diagram SAD set (0.7, 2.0, 7.0, 21.0 and 39.0 %) and a 6-diagram SAD set (0.7, 2.0, 4.0, 10.0, 21.0 and 39.0 %) were compared; the 6-diagram set had an additional diagram in the most common severity range. Fifteen raters evaluated 40 images of cankered, unripe fruit. Both the 5- and 6-diagram SADs improved the accuracy and reliability of rater estimates. Agreement, measured by Lin’s concordance correlation coefficient (ρ c ) was 0.735 to 0.906 when not using SADs, 0.931 to 0.985 when using 5-diagram SADs, and 0.879 to 0.979 when using 6-diagram SADs. Mean inter-rater reliability (R2) was 0.638 when not using SADs, 0.889 when using 5-diagram SADs, and 0.834 when using 6-diagram SADs. The 5-diagram SADs resulted in significantly more accurate and reliable estimates compared with the 6-diagram set, and is thus recommended for use. The SADs should have application for estimating ACC severity on ripe fruit of other citrus, including grapefruit.