Asiatic Citrus Canker Research Articles

Amplified fragment length polymorphism and multilocus sequence analysis-based genotypic relatedness among pathogenic variants of Xanthomonas citri pv. citri and Xanthomonas campestris pv. bilvae

Three pathogenic variants (i.e. pathotypes) have been described within Xanthomonas citri pv. citri, the causal agent of Asiatic citrus canker. Pathotype A strains naturally infect a wide range of Citrus species and members of some related genera. In contrast, pathotypes A* and A(w) have narrow host ranges within the genus Citrus and have been isolated from Mexican lime (Citrus aurantifolia L.) and from Mexican lime and alemow (Citrus macrophylla L.), respectively. We used amplified fragment length polymorphism (AFLP) and multilocus sequence analysis (MLSA) based on four partial housekeeping gene sequences (atpD, dnaK, efp and gyrB ) for the genotypic classification of Xanthomonas citri pv. citri and the poorly characterized citrus pathogen Xanthomonas campestris pv. bilvae. A Mantel test showed that genetic distances derived from AFLP and MLSA were highly correlated. X. campestris pv. bilvae showed a close relatedness to the type strain of X. citri, indicating that this pathovar should be reclassified as X. citri pv. bilvae. All pathotype A* and A(w) strains were most closely related to X. citri pv. citri strains with a wide host range (pathotype A), confirming previous DNA-DNA hybridization data. Pathotype A(w) should be considered a junior synonym of pathotype A* on the basis of pathogenicity tests, AFLP, MLSA and PCR using pathovar-specific primers. Evolutionary genome divergences computed from AFLP data suggested that pathotype A* (including A(w) strains) is a group of strains that shows a wider genetic diversity than pathotype A.

Pathotype Identification of Xanthomonas citri pv. citri Strains Causing Citrus Canker in Vietnam.

Asiatic citrus canker caused by Xanthomonas citri pv. citri is a bacterial disease of major economic importance in tropical and subtropical citrus-producing areas. It probably originated in Asia (2). X. citri pv. citri induces erumpent, callus-like lesions with a water-soaked margin on aerial organs of the plants. Severe attacks cause premature fruit drop and twig dieback. This pathogen has consequently been subjected to international quarantine regulation and eradication efforts. Two pathogenic variants of X. citri pv. citri can be separated by their host range. X. citri pv. citri pathotype A strains cause severe infection worldwide in a wide range of citrus species; grapefruit (Citrus paradisi) is particularly susceptible. More recently, another group of strains from different areas of West Asia has been designated as X. citri pv. citri pathotype A* (4). These A* strains are genetically related to X. citri pv. citri, but their host range is primarily restricted to Mexican lime (C. aurantifolia) and they do not infect grapefruit. Strains similar in host range were later reported in Florida, Thailand, and Cambodia (2). In this study, we investigated the distribution of X. citri pv. citri pathotypes in Southeast Asia. A large survey on citrus was conducted in 14 provinces in the north (Ha Noi, Hung Yen, Nghe Han, Ha Ting, and Phu Tho) and south (Can Tho, Long An, Dong Nai, Tien Giang, Vinh Long, Ben Tre, Dong Thap, Vung Tau, and Lam Dong) of Vietnam. We collected 557 X. citri pv. citri isolates, after cultivation on KC semiselective medium (3), from citrus species, including 60 strains from Mexican lime in eight provinces. Ligation mediated (IS-LM)-PCR analysis using primers targeting three insertion sequences (1) was done on all Vietnamese strains and on additional reference strains of X. citri pv. citri-A, -A*, and X. citri pv. aurantifolii. IS-LM-PCR indicated that all Vietnamese isolates were pathotype A and did not include any with a restricted host range (X. citri pv. citri-A* and X. citri pv. aurantifolii). Amplified fragment length polymorphism (AFLP) analysis was carried out on a subset of 84 X. citri pv. citri strains, including 22 strains from Mexican lime from seven provinces. AFLP was carried out using SacI/MspI and four primer pairs (unlabeled MspI +1 [A, C, T or G] primers and 5'-labeled - SacI + C primer for the selective amplification step) (2) and the data confirmed that all Vietnamese X. citri pv. citri strains were genetically related to pathotype A strains. Mexican lime and Duncan grapefruit or pineapple sweet orange leaves were inoculated with 25 strains from lime (representative of the genetic diversity) using a detached leaf assay (3) and they produced typical canker lesions on both host species. In spite of the presence of pathotype A* strains in neighboring countries (2), no strains genetically or pathogenically related to this pathotype were identified in this collection. A survey of commercial Mexican lime orchards, especially in Vietnamese provinces bordering Cambodia, should be undertaken to detect and eradicate A* strains because these are known to strongly impact lime production in other parts of Asia (e.g., Thailand).

The epidemiological significance of post-packinghouse survival of Xanthomonas citri subsp. citri for dissemination of Asiatic citrus canker via infected fruit

The risk of introduction of Xanthomonas citri subsp. citri ( Xcc) to new, unaffected citrus producing areas is a major concern for those citrus industries attempting to remain free of citrus canker. Citrus fruit, as a potential pathway for Xcc to enter and become established in these areas, are assumed to be a risk. However, there is little information relative to the potential of harvested fruit to act as an inoculum source. A multi-national research team was established to investigate the potential of bacterial survival in infected citrus fruit lesions and as surface contaminants on symptom-free fruit, and to examine the potential of infected fruit as a viable inoculum source. Experiments were conducted in various locations in Florida and Argentina. Bacterial recovery and culture plating were problematic due to the presence of non-pathogenic bacteria with cultural characteristics that were difficult to distinguish from Xcc. Therefore, in all experiments, although culturing on semi-selective agar media was used as an indication of overall bacterial populations, bioassays were conducted via needleless injection and infiltration of suspect bacterial suspensions into susceptible cv. Duncan grapefruit leaves. Inoculation sites were subsequently assessed for symptoms of citrus canker and lesions were individually enumerated to confirm the presence of Xcc. In commercial packing lines in Florida and northwest Argentina, prewashing the fruit to remove dirt and debris reduced surface bacterial populations. As anticipated, recovery of Xcc from fruit surfaces increased when active citrus canker lesions were present but total bacterial recovery decreased after processing, and bioassays demonstrated that the quantity of viable Xcc declined as fruit remained in cold storage, or as they aged on the trees. Bioassays demonstrated that the highest incidence of Xcc from fruit after the packing line antimicrobial treatment occurred with symptomatic fruit (2.5–50.6 lesions leaf −1), and zero to very low levels with fruit from apparently healthy trees (0–1.74 lesions leaf −1). Furthermore, the proportion of injection–infiltration bioassay sites that developed lesions consistently decreased with time after processing in each of the three packinghouse studies, also showing that as fruit senesce and lesions age the ability of fruit to generate or sustain Xcc bacteria was increasingly compromised. The packing line process reduced canker lesion activity by as much as 50% compared to unprocessed fruit. Xcc survived in wounds on mature fruit attached to the tree, but Xcc populations declined in wounds of processed or non-processed harvested fruit. Discarded canker-infected fruit in cull piles was ineffective as a source of inoculum for dispersal. Transmission from cull piles of packing line-processed fruit to surrounding trap plants, even less than 1 m away, did not occur under natural conditions. However, with severely infected piles of culled fruit subject to extreme simulated wind (25 m s −1) and rain conditions, only a single lesion, associated with leaf injury, developed on a trap plant immediately downwind of the cull pile, suggesting an exceedingly low risk of spread. Taken as a group, this series of experiments demonstrate that harvested and packinghouse-disinfested citrus fruit are extremely unlikely to be a pathway for Xcc to reach and infect susceptible citrus and become established in canker-free areas.

First Report of Xanthomonas citri pv. citri-A* Causing Citrus Canker on Lime in Ethiopia.

Asiatic citrus canker caused by Xanthomonas citri pv. citri hinders national citrus markets in tropical and subtropical areas and international trade. The bacterium induces erumpent, callus-like lesions causing defoliation, premature fruit drop, and twig dieback. Because of the damage caused by infection and reduced marketability of fruit, several countries have undergone eradication. Strains with different host ranges have been described. Pathotype A strains are the most widespread and produce canker in a wide range of citrus species. Pathotype A* strains with a host range restricted to Mexican lime (Citrus aurantifolia), Tahiti lime (C. latifolia), and alemow (C. macrophylla), but not infecting the susceptible species grapefruit (C. paradisi), were described in different areas of Asia (4). Reemergence of X. citri pv. citri pathotype A was recently described in Africa as affecting citrus production in Mali and Somalia. Canker-like infected citrus trees with symptoms on leaves, fruits, and stems were first observed in 2004 in Ethiopia in the Rift Valley Region. After a survey conducted in 2008, the disease was recorded in different areas of the Rift Valley located in the lowlands (altitude <1300 m, daily mean temperatures 24 to 29°C) and confirmed to only affect Mexican lime orchards with disease incidence as much as 80%. Ten canker-like infected leaves were collected during this survey from eight different orchards distributed along the infected area. Isolations were performed using KC semiselective medium (3), and Xanthomonas-like isolates were further characterized. PCR was used to check the identity of these isolates by using X. citri pv. citri strain CFBP 2525 from New Zealand as the positive control and distilled water as the template for the negative control. The DNA fragment typical of X. citri pv. citri was obtained from all the bacterial isolates using the diagnostic primer pair 4/7 (2). Amplified fragment length polymorphism (AFLP) analysis of the 80 Ethiopian isolates and additional reference isolates from X. citri pv. citri-A, -A*, and pv. aurantifolii using SacI/MspI and four primer pairs (unlabeled MspI + 1 [A, C, T, or G] primers and 5'-labeled-SacI + C primer for the selective amplification step) (1) grouped all the Ethiopian isolates in a cluster that was comprised of only X. citri pv. citri pathotype A* strains. On the basis of the AFLP, Ethiopian isolates were only distantly related to X. citri pv. aurantifolii. When inoculated to Mexican lime and Duncan grapefruit by a detached leaf assay (4), all of the Ethiopian strains produced canker on lime only. This confirms the larger geographical distribution of pathotype A*, and to our knowledge, is the first report of its presence on the African continent. This could allow studying the epidemiology of pathotype A* strains in a unique situation where they do not compete with pathotype A strains. The molecular characterization of Ethiopian strains suggests that this introduction event is not related to the recent introduction of citrus canker in neighboring Somalia where X. citri pv. citri pathotype A was identified. Ethiopia will have to prevent the introduction of this wide host range pathotype to avoid further negative impacts on citrus production.

First Report of Xanthomonas citri pv. citri-A* Causing Citrus Canker on Lime in Cambodia.

Asiatic citrus canker caused by Xanthomonas citri pv. citri (X. citri pv. citri-A) is detrimental to citrus production in tropical and subtropical areas. The bacterium can cause severe infection on many citrus species, initially causing water-soaked leaf lesions that become erumpent and necrotic, often with a chlorotic halo. Severe infection causes premature fruit drop and twig dieback. X. citri pv. citri-A has consequently been subject to eradication and international quarantine regulations. In the 1990s, strains with a host range restricted to Mexican lime (Citrus aurantifolia), but not infecting grapefruit (C. paradisi), were described in different areas of Southwest Asia (4). This variant was designated X. citri pv. citri-A* because of its phenotypic and genetic similarities with X. citri pv. citri. Lime leaves with canker lesions were collected in 2007 from a citrus nursery in Kandal Province, Cambodia and isolations were performed with KC semiselective medium (3). Four Xanthomonas-like strains were further characterized by PCR alongside positive control strain CFBP 2525 from New Zealand. The expected DNA fragment was obtained using primer pair 4/7 (2) from the bacterial strains but not when distilled water was used as a template. Amplified fragment length polymorphism (AFLP) analysis of the four X. citri pv. citri strains from Cambodia and reference strains X. citri pv. citri-A (CFBP 2525, CFBP 2900, LMG 9322), -A* (CFBP 2911, JF90-2, JK2-10, JK143-1, JM47-2), and X. citri pv. aurantifolii (CFBP 2866, CFBP 2868, CFBP 2901) using SacI/MspI and four primer pairs (1) separated the Cambodian strains into two distinct haplotypes (i.e., AFLP fingerprint patterns). One haplotype was closely related (evolutionary genome divergences [EGD] ≤0.006 [1]) to X. citri pv. citri-A strains with a wide host range and the other was most genetically related to a strain of X. citri pv. citri-A* from Thailand (EGD of 0.003). On the basis of AFLP, the Cambodian isolates were not related to X. citri pv. aurantifolii (EGD values of >0.060). When inoculated to Mexican lime and Duncan grapefruit using a detached leaf assay in which inoculum droplets containing ∼1 × 106 CFU were deposited on wounds (4), the strains genetically related to X. citri pv. citri-A produced typical canker lesions on both citrus species a week after inoculation, whereas the Cambodian strains related to X. citri pv. citri-A* by AFLP analysis only produced canker lesions on lime. Our finding extended the geographical distribution of pathotype A*. Identification of both pathotypes from a few samples collected in a nursery suggests a potential for large-scale distribution of these strains within the citrus orchards in Cambodia, where the most important citrus crop is sweet orange, suggesting that the occurrence of X. citri pv. citri-A* is of moderate economic significance, in contrast with X. citri pv. citri-A strains with a wide host range. Diseased citrus nursery plants are a major source of primary inoculum in developing countries. Sanitation of citrus nurseries against citrus canker in Cambodia is a prerequisite for improved management of the disease.

Modelling the progress of Asiatic citrus canker on Tahiti lime in relation to temperature and leaf wetness

The combined effect of temperature (15°C, 20°C, 25°C, 30°C, 35°C, 40°C and 42°C) and leaf wetness duration (0, 4, 8 12, 16, 20 and 24 h) on infection and development of Asiatic citrus canker (Xanthomonas citri subsp. citri) on Tahiti lime plant was examined in growth chambers. No disease developed at 42°C and zero hours of leaf wetness. Periods of leaf wetness as short as 4 h were sufficient for citrus canker infection. However, a longer leaf duration wetness (24 h) did not result in much increase in the incidence of citrus canker, but led to twice the number of lesions and four times the disease severity. Temperature was the greatest factor influencing disease development. At optimum temperatures (25–35°C), there was 100% disease incidence. Maximum disease development was observed at 30–35°C, with up to a 12-fold increase in lesion density, a 10-fold increase in lesion size and a 60-fold increase in disease severity.

Ligation-mediated PCR, a fast and reliable technique for insertion sequence-based typing ofXanthomonas citripv.citri

Asiatic citrus canker, caused by Xanthomonas citri pv. citri, is a major disease threatening citrus crops throughout the world. The most common methods for strain differentiation of this pathogen are repetitive element sequence-based PCR (rep-PCR) and pulsed field gel electrophoresis (PFGE), using rare-cutting restriction enzyme analysis. We developed a ligation-mediated PCR targeting three insertion sequences (IS-LM-PCR) present as several copies in the genome of the fully sequenced strain 306 of X. citri pv. citri. This technique amplifies DNA fragments between an insertion sequence element and an MspI restriction site. The analysis of strains can be conducted within 24 h, starting from very small amounts of bacterial DNA, which makes IS-LM-PCR much less labor-intensive than PFGE. We used IS-LM-PCR to analyze a collection of 66 strains of X. citri pv. citri from around the world. The overall reproducibility of IS-LM-PCR reached 98% in this data set and its discriminatory power was markedly superior than rep-PCR. We suggest that IS-LM-PCR could be used for the global surveillance of non-epidemiologically related strains of X. citri pv. citri.

Control of Citrus Canker and Citrus Bacterial Spot with Bacteriophages.

Bacteriophages, alone or in combination with copper bactericides, were evaluated for managing Asiatic citrus canker and citrus bacterial spot incited by Xanthomonas axonopodis pathovars citri and citrumelo, respectively. In a set of five greenhouse experiments, phage treatment provided consistent control of citrus canker, causing an average of 59% reduction in disease severity. However, treatment with phage was ineffective if applied with skim milk, a protective formulation, which increases phage residual activity. In nursery settings, phage treatment also reduced disease but was less effective than copper-mancozeb, a chemical bactericide. The integration of phage and copper-mancozeb resulted in equal or less control than copper-mancozeb application alone. Phage treatments were evaluated in a commercial citrus nursery for reducing citrus bacterial spot caused by natural inoculum. Phage treatment provided significant disease reduction on moderately sensitive Valencia oranges in two trials (48 and 35%); however, on the highly susceptible grapefruit host it was ineffective. In an experimental citrus nursery, phage treatment provided significant control of citrus bacterial spot caused by a phage-sensitive strain, but was equally or less effective than copper-mancozeb. The combination of phage and copper-mancozeb did not increase control compared with copper-mancozeb alone.

New insights into the resistance of Nagami kumquat to canker disease

Asiatic citrus canker, caused by the bacterial species Xanthomonas axonopodis pv. citri ( Xac A), has had a detrimental impact on citrus production in various regions of the world. It has been suggested, based on reported field observations and other anecdotal evidence, that some citrus relatives, e.g. kumquats, have higher levels of tolerance to Xac A compared to commercial varieties. However, this has not been confirmed with rigorous scientific evidence. In this study, the growth rate of Xac A in challenged kumquat leaves was significantly hindered after 4 days of infection compared to that in susceptible grapefruit leaves. Cell death was observed in kumquat leaves however, typical symptoms of a hypersensitive response (HR) were not apparent until 3–4 days post-inoculation. Abscission of the leaves occured 5–7 days after infection, depending on the age of the leaf. The HR was accompanied by differential gene expression: two suppression subtractive cDNA libraries were constructed using Nagami kumquat mRNA to identify genes that were differentially expressed due to infection. A number of cDNA clones were randomly selected for sequencing. Homologues to components such as transcription factors, receptors and defense genes known to be involved in plant–pathogen interactions in general and plant cell death were identified.

Spatial Pattern Analysis of Citrus Canker-Infected Plantings in São Paulo, Brazil, and Augmentation of Infection Elicited by the Asian Leafminer

ABSTRACT Eradication of Asiatic citrus canker (ACC) has become increasingly difficult over the last decade, following the introduction of the Asian leafminer into Brazil and Florida, which has led to changes in the eradication protocols. The present study, undertaken in Brazil, was aimed at characterizing the spatial patterns of ACC in commercial citrus plantings to gain better understanding of the dynamics of the disease subsequent to introduction of the leafminer. The spatial patterns of ACC were mapped in 326 commercial citrus plantings and statistically assessed at various spatial dimensions. The presence of "within-group" aggregation in each plot was examined via beta-binomial analysis for groups of trees parsed into three-by-three-tree quadrats. The relative intensity of aggregation was expressed as a binomial index of dispersion (D) and heterogeneity among plots expressed as the intracluster correlation coefficient, rho. The population of data sets was found to fall into three D categories, D < 1.3, 1.3 </= D = 3.5, and D > 3.5. These categories then were related to other spatial characteristics. The binary form of Taylor's power law was used to assess the overdispersion of disease across plots and was highly significant. When the overall population of plots was parsed into D categories, the Taylor's R (2) improved in all cases. Although these methods assessed aggregation well, they do not give information on the number of foci or aggregations within each plot. Therefore, the number of foci per 1,000 trees was quantified and found to relate directly to the D categories. The lowest D category could be explained by a linear relationship of number of foci versus disease incidence, whereas the higher two categories were most easily explained by a generalized beta function for the same relationship. Spatial autocorrelation then was used to examine the spatial relationships "among groups" composed of three-by-three-tree quadrats and determine common distances between these groups of ACC-infected trees. Aggregation was found in >84% of cases at this spatial level and there was a direct relationship between increasing D category and increasing core cluster size, and aggregation at the among-group spatial hierarchy was generally stronger for the within-row than for the across-row orientation. Clusters of disease were estimated to average between 18 and 33 tree spaces apart, and the presence of multiple foci of infection was commonplace. The effectiveness of the eradication protocol of removing all "exposed" trees within 30 m surrounding each "ACC-infected tree" was examined, and the distance of subsequent infected trees beyond this 30-m zone from the original focal infected tree was measured for each plot. A frequency distribution was compiled over all plots to describe the distance that would have been needed to circumscribe all of these outliers as a theoretical alternative protocol to the 30-m eradication protocol. The frequency distribution was well described by a monomolecular model (R(2) = 0.98) and used to determine that 90, 95, and 99% of all newly infected trees occurred within 296, 396, and 623 m of prior-infected trees in commercial citrus plantings, respectively. These distances are very similar to previously reported distances determined for ACC in residential settings in Florida.

First Report in Thailand of Xanthomonas axonopodis pv. citri-A* Causing Citrus Canker on Lime.

Asiatic citrus canker, caused by Xanthomonas axonopodis pv. citri (Xac-A), is a bacterial disease of economic importance in tropical and subtropical citrus-producing areas. Xac-A can cause severe infection in a wide range of citrus species and induces erumpent, callus-like lesions with a water-soaked margin. Severe attacks cause premature fruit drop and twig dieback. It has consequently been submitted to eradication efforts and international regulations. Recently, a group of strains with a host range restricted to Mexican lime (Citrus aurantifolia), but not infecting grapefruit (C. paradisi) known to be very susceptible to Xac-A, was described in different areas of southwest Asia, including Saudi Arabia, Oman, Iran, and India (3). Phenotypic and genetic similarities with Xac-A designated it as a variant called X. axonopodis pv. citri-A*. A variant with a similar restricted host range, designated X. axonopodis pv. citri-Aw, was also detected in Florida and likely originated from India (2). Amplified fragment length polymorphism (AFLP) analysis of 26 isolates from Thailand and additional reference isolates from Xac-A, -A*, -Aw, and X. axonopodis pv. aurantifolii (2,3) using SacI/MspI and four primer pairs (unlabeled MspI + 1 [A, C, T, or G] primers and 5'-labeled SacI + C primer for the selective amplification step), separated the Thai isolates into two distinct groups. A group composed of 20 isolates from different citrus species, including five isolates from lime, was closely related to strains with a large host range and previously identified as Xac-A. A second group, solely composed of six isolates from lime, was genetically related to Xac-A*. All Thai isolates were collected before 1991. Isolates genetically closed to Xac-A* originated from the central and northern provinces, whereas isolates related to Xac-A originated from all sampled provinces. On the basis of AFLP, no Thai isolate was related to X. axonopodis pv. aurantifolii. A specific X. axonopodis pv. citri nested-PCR assay (1) produced the expected fragments for all Thai isolates. Mexican lime leaves inoculated with Thai isolates of Xac-A and Xac-A* using a detached leaf assay (3) showed typical canker symptoms 1 week after inoculation. When inoculated to grapefruit or sweet orange, the Thai isolates genetically related to Xac-A* by AFLP analysis did not induce any canker symptoms, while isolates related to Xac-A produced canker symptoms on these two citrus species. In Thailand, Xac-A* induced severe symptoms on lime trees, including extensive defoliation and numerous twig cankers that often developed as diebacks. The Xac-A* variant appears epidemiologically important on lime, consistent with previous reports from southwest Asia. The detection of Xac-A* in Thailand makes it necessary to evaluate its geographic distribution in southeast Asia.

Evaluation of ‘Hamlin’ sweet orange + ‘Montenegrina’ mandarin somatic hybrid for tolerance to Xanthomonas axonopodis pv. citri and Xylella fastidiosa

Asiatic citrus canker (ACC), caused by Xanthomonas axonopodis Starr & Garces pv. citri (Hasse) Vauterin et al., and citrus variegated chlorosis (CVC), caused by Xylella fastidiosa Wells et al., are considered the main diseases affecting sweet orange scion varieties in Brazil. Among commercial varieties, mandarins and tangerines are recognized as tolerant to these pathogens. We report herein the production of ‘Hamlin’ sweet orange ( Citrus sinensis L. Osbeck) + ‘Montenegrina’ mandarin ( Citrus deliciosa Ten.) allotetraploid somatic hybrid plants by protoplast fusion with improved disease tolerance that could be used as a donor of resistance genes in interploid hybridisation. Somatic hybridisation was confirmed by leaf morphology, flow cytometry and RAPD analyses. The somatic hybrid was propagated by grafting and cultivated in a screenhouse for tolerance assays. For X. axonopodis pv. citri assays, buds were collected from both ‘Hamlin’ sweet orange and the somatic hybrid and grafted onto ‘Cleopatra’ mandarin ( Citrus reshni hort. ex Tanaka). As a negative control, buds from ‘Mexerica Tardia’ mandarin ( C. deliciosa) were collected and grafted onto ‘Cleopatra’ mandarin. Two-month old plants with at least one young vegetative flush were individually spray-inoculated with a 10 6 CFU mL −1 X. axonopodis pv. citri suspension and incubated in a growth chamber, at 27 °C, under 16-h photoperiod. The somatic hybrid showed a statistically significant reduction in susceptibility to ACC 30 days after inoculation. Compared to ‘Hamlin’ sweet orange, disease severity was reduced by 70%, with similar tolerance to that of the mandarin negative control. For X. fastidiosa assays, buds were collected from the somatic hybrid and its parental plants and grafted onto ‘Rangpur’ lime ( Citrus limonia Osbeck). The developed plants were needle-inoculated with a X. fastidiosa suspension (8.7 × 10 10 CFU mL −1) into the new growth flush stem. Bacterial population was quantified both at 4 (at the inoculation point) and 8 months (50 cm above the inoculation point) after inoculation. The first evaluation detected X. fastidiosa in 63% of ‘Hamlin’ sweet orange and ‘Hamlin’ + ‘Montenegrina’ mandarin samples. In the second evaluation, X. fastidiosa was detected in 47.4% of ‘Hamlin’ sweet orange and 10.5% of ‘Hamlin’ + ‘Montenegrina’ somatic hybrid samples, suggesting that bacterial movement was restricted in the somatic hybrid. X. fastidiosa was not detected in both evaluations in samples collected from leaves of ‘Montenegrina’ mandarin. These results indicate that the ‘Hamlin’ sweet orange + ‘Montenegrina’ mandarin somatic hybrid has potential for improved disease tolerance that should enhance its value regarding future use in citrus breeding programs.

Post-hurricane Analysis of Citrus Canker II: Predictive Model Estimation of Disease Spread and Area Potentially Impacted by Various Eradication Protocols Following Catastrophic Weather Events

The impact of 2005 Hurricane Wilma on the dissemination of Xanthomonas axonopodis pv. citri (Xac), the cause of Asiatic citrus canker (ACC), and subsequent disease development was examined and predictions for the areas into which Xac was likely to have spread from known sources of infection were developed. In addition, the effect of the current 579-m (1900-ft) ACC eradication protocol, resulting in removal of all “exposed trees” with a 579-m radius of a known Xac-infected tree, was calculated via GIS analysis and expressed as the predicted “impacted area.” The GIS calculations were based on the extension of the previous published wind-rain index vector (WRIV) model. The model extension consisted of the incorporation of an estimate of distance of spread due to various combinations of wind and rain from data collected during the 2004 hurricane season. An inverse power law dissemination function was used to describe regional dispersal from a point focus of Xac infection. Alternative eradication protocol (distances) to the 579-m protocol were evaluated in association with the GIS analyses and used to examine the effect of eradication distance on predicted “impacted area.” The results of these analyses were used by state and federal regulatory agencies and commercial citrus producer groups to evaluate the feasibility of continued ACC eradication. Accepted for publication 23 January 2007. Published 5 April 2007.

Effect of citrus leaf-miner damage, mechanical damage and inoculum concentration on severity of symptoms of Asiatic citrus canker in Tahiti lime

Citriculture in São Paulo State, Brazil, is threatened by Asiatic citrus canker ( Xanthomonas axonopodis pv. citri). The introduction of the Asian citrus leaf miner ( Phyllocnistis citrella [CLM]) has resulted in an increase in the number of disease foci and has changed the spatial pattern of citrus canker symptomatic trees from strong aggregation to intermediate aggregation and random patterns. We evaluated the effect of inoculum concentration (10 1, 10 2, 10 4 and 10 6 colony forming units (cfu) ml −1) on infection of leaves of Tahiti lime ( Citrus latifolia) with three distinct treatments: (1) intact leaves, (2) mechanically wounded leaves, and (3) leaves exhibiting injuries from CLM at the egg stage, first instar, third instar, and pupal stages. The minimum inoculum concentration to cause symptom development in intact leaves was 10 4 cfu ml −1; in mechanically wounded leaves and in leaves with CLM injury at the egg stage and first instar stage, 10 2 cfu ml −1; in leaves with CLM injuries at the third instar and pupa stage, 10 1 cfu ml −1. The injuries from the third instar and pupa stages resulted in greater disease severity than other treatments at all inoculum concentrations (five times higher than in the intact leaf). Disease severity in leaves with mechanical wounding and with CLM injuries caused at the egg and first instar stages did not differ from intact leaves.

Detection and Characterization of a New Strain of Citrus Canker Bacteria from Key/Mexican Lime and Alemow in South Florida.

In the Wellington and Lake Worth areas of Palm Beach County, FL, citrus canker appeared on Key/Mexican lime (Citrus aurantiifolia) and alemow (C. macrophylla) trees over a period of about 6 to 7 years before detection, but nearby canker-susceptible citrus, such as grapefruit (C. × paradisi) and sweet orange (C. sinensis), were unaffected. Colonies of the causal bacterium, isolated from leaf, stem, and fruit lesions, appeared similar to the Asiatic group of strains of Xanthomonas axonopodis pv. citri (Xac-A) on the nutrient agar plate, but the growth on lima bean agar slants was less mucoid. The bacterium produced erumpent, pustule-like lesions of typical Asiatic citrus canker syndrome after inoculation into Key/Mexican lime, but brownish, flat, and necrotic lesions on the leaves of Duncan grapefruit, Madame Vinous sweet orange, sour orange (C. aurantium), citron (C. medica), Orlando tangelo (C. reticulata × C. × paradisi), and trifoliate orange (Poncirus trifoliata). The bacterium did not react with the Xac-A specific monoclonal antibody A1 using enzyme-linked immunosorbent assay (ELISA) and could not be detected by polymerase chain reaction (PCR)-based assays using primers selected for Xac-A. DNA reassociation analysis confirmed that the pathogen, designated as Xac-AW, was more closely related to Xac-A and Xac-A* strains than X. axonopodis pv. aurantifolii or the citrus bacterial spot pathogen (X. axonopodis pv. citrumelo). The strain can be easily differentiated from Xac-A and Xac-A* using ELISA, PCR-based tests, fatty acid analysis, pulsed-field gel electrophoresis of genomic DNA, and host specificity.

Screening Triploid Hybrids of 'Lakeland' Limequat for Resistance to Citrus Canker.

Resistance of citrus genotypes to Xanthomonas axonopodis pv. citri, the cause of Asiatic citrus canker (ACC), was evaluated by injection infiltration of 103 and 104 CFU/ml through stomates on the abaxial surface of immature leaves. Citrus genotypes for screening comprised two autotetraploids and nine triploid hybrids of 'Lakeland' limequat (Citrus aurantifolia × Fortunella japonica) and their progenitors ('Lakeland' limequat, the autotetraploids 'Femminello' lemon (Citrus limon) and 'Giant Key' lime (C. aurantifolia), and the somatic hybrids 'Key' [also known as 'Mexican'] lime + 'Valencia' orange and 'Hamlin' orange + 'Femminello' lemon). 'Meiwa' kumquat (Fortunella crassifolia) and 'Pineapple' sweet orange (C. sinensis) were used as known resistant and susceptible standards, respectively. Lesion number per inoculation site and bacterial population per lesion were recorded 15 to 19 days after inoculation. The assay was performed four times during a spring-summer-fall period under greenhouse conditions. Canker lesions were consistently produced by stomatal inoculation with 104 but not 103 CFU/ml. Susceptible and resistant genotypes were separated based on lesion number per inoculation site and bacterial population per lesion. Spearman's rank correlation analysis for lesion numbers on 15 genotypes common to all four assays showed significant correlations among the genotype rankings. Genotype rankings were also significantly correlated between the two bacterial population assays. Lesion number per inoculation site is sufficient for assessment of resistance of citrus genotypes to ACC without the necessity of conducting bacterial population assays. 'Lakeland' limequat is a promising seed parent for breeding acid citrus fruit that is resistant to ACC.

Protecting Agriculture: The Legal Basis of Regulatory Action in Florida.

The Division of Plant Industry of the Department of Agriculture and Consumer Services of Florida is empowered to protect the state's agriculture from both introduction and intrastate movement of pests. This responsibility is shared by similar regulatory agencies of the federal government and other states. The legal basis of such regulatory action, police power and eminent domain, is reviewed. The regulatory processes by the State of Florida of two important citrus pests, the burrowing nematode and Asiatic citrus canker, are presented. It was concluded that Florida's regulatory power to take property is of great importance to the agricultural industry and to Florida's economy. This power must be exercised within the constitutional limitation that "no person shall be deprived of life, liberty, or property without due processes of law." However, the imminence of an emergency may justify state action without a prior hearing or just compensation of valueless items.

Lack of Control of Citrus Canker by Induced Systemic Resistance Compounds.

Induced systemic resistance compounds (ISRs), acibenzolar-S-methyl (Actigard), and harpin protein (Messenger) were assayed in the greenhouse against Xanthomonas axonopodis pv. citrumelo, the cause of citrus bacterial spot (CBS), and X. axonopodis pv. citri, the cause of Asiatic citrus canker. Actigard and Messenger applied as foliar sprays 3 to 7 days before inoculation reduced numbers of lesions when either bacterium at 103 or 104 CFU/ml was injection-infiltrated into Swingle citrumelo leaves. Based on this activity, the ISRs were evaluated in southern Brazil in orchards of sweet oranges with low to moderate canker disease incidence in spray programs with and without copper oxychloride (COC) and copper hydroxide (CuOH). Actigard and Messenger were applied full season or in the first two or three sprays of a six-spray program in an attempt to reduce early canker disease on foliage and thereby reduce subsequent fruit infection and premature drop. Sprays of COC and CuOH were moderately to highly effective in reducing canker disease incidence and preventing premature fruit drop. Actigard or Messenger in combination with COC and CuOH, respectively, did not significantly reduce citrus canker incidence on foliage or fruit drop compared with Cu alone. The lack of additional control with ISRs means they cannot be recommended at this time to augment Cu programs for management of citrus canker.

Xanthomonas axonopodis pv. citri: factors affecting successful eradication of citrus canker

SUMMARY Taxonomic status: Bacteria, Proteobacteria, gamma subdivision, Xanthomodales, Xanthomonas group, axonopodis DNA homology group, X. axonopodis pv. citri (Hasse) Vauterin et al. Microbiological properties: Gram negative, slender, rod-shaped, aerobic, motile by a single polar flagellum, produces slow growing, non-mucoid colonies in culture, ecologically obligate plant parasite. Causal agent of Asiatic citrus canker on most Citrus spp. and close relatives of Citrus in the family Rutaceae. Disease symptoms: Distinctively raised, necrotic lesions on fruits, stems and leaves. Bacteria exude from lesions during wet weather and are disseminated by splash dispersal at short range, windblown rain at medium to long range and human assisted movement at all ranges. Crop loss: Severe infections cause defoliation, blemished fruit, premature fruit drop, die-back of twigs and general debilitation of the tree. Distribution: Citrus canker is not present in all subtropical to tropical regions of citriculture in the world, so considerable regulatory efforts are expended to prevent the introduction and spread of X. axonopodis pv. citri into areas in the Americas, Australia and elsewhere, with climates conducive to the disease. Limited strategies exist for suppression of citrus canker on more susceptible cultivars. Blemished fruit are unmarketable and exposed fruit are restricted in market access. The economic impact of loss of markets is much greater than that from yield and quality reductions of the crop. http://doacs.state.fl.us/canker, http://www.apsnet.org/education/lessonsplantpath/citruscanker/top.htm, http://www.apsnet.org/online/feature/citruscanker/, http://www.plantmanagementnetwork.org/pub/php/review/citruscanker/, http://www.abecitrus.com.br/fundecitrus.html, http://www.biotech.ufl.edu/PlantContainment/canker.htm, http://www.aphis.usda.gov/oa/ccanker/.

Disease Development and Symptom Expression of Xanthomonas axonopodis pv. citri in Various Citrus Plant Tissues

ABSTRACT Experimental inoculations of Xanthomonas axonopodis pv. citri in different tissues of Tahiti lime and Pineapple sweet orange were conducted monthly under natural conditions on Réunion Island. The interactions between a set of environmental and epidemic variables associated with disease expression and 184 different factor combinations were investigated to determine the parameters needed to explain Asiatic citrus canker (ACC) disease expression. Area under the disease progress curve (AUDPC), inoculation date (Id), fruit and leaf age ratings (FAR and LAR), and number of days during the first 2 weeks postinoculation for which the temperature was less than 14 degrees C (T(min)) or more than 28 degrees C (T(max)) were retained by principal component analysis and canonical correlation analysis as the most meaningful epidemic and environmental variables, respectively. AUDPC as the strongest dependent variable and combinations of the environmental variables as independent variables were used in multiple regression analyses. Tissue age rating at the time of infection was a good predictor for disease resulting from spray inoculation on fruits and leaves and also on fruits following a wound inoculation. Temperature, as expressed by T(min) or T(max), was also a significant factor in determining disease development described by AUDPC. Mature green stems were highly susceptible after wounding, similarly to leaves, but buds and leaf scars expressed the lowest susceptibility. These variations in disease expression according to the tissues will have different impacts on ACC epidemiology.