Novel insights into the genomic basis of citrus canker based on the genome sequences of two strains of Xanthomonas fuscans subsp. aurantifolii

Abstract Background Citrus canker is a disease that has severe economic impact on the citrus industry worldwide. There are three types of canker, called A, B, and C. The three types have different phenotypes and affect different citrus species. The causative agent for type A is Xanthomonas citri subsp. citri, whose genome sequence was made available in 2002. Xanthomonas fuscans subsp. aurantifolii strain B causes canker B and Xanthomonas fuscans subsp. aurantifolii strain C causes canker C. Results We have sequenced the genomes of strains B and C to draft status. We have compared their genomic content to X. citri subsp. citri and to other Xanthomonas genomes, with special emphasis on type III secreted effector repertoires. In addition to pthA, already known to be present in all three citrus canker strains, two additional effector genes, xopE3 and xopAI, are also present in all three strains and are both located on the same putative genomic island. These two effector genes, along with one other effector-like gene in the same region, are thus good candidates for being pathogenicity factors on citrus. Numerous gene content differences also exist between the three cankers strains, which can be correlated with their different virulence and host range. Particular attention was placed on the analysis of genes involved in biofilm formation and quorum sensing, type IV secretion, flagellum synthesis and motility, lipopolysacharide synthesis, and on the gene xacPNP, which codes for a natriuretic protein. Conclusion We have uncovered numerous commonalities and differences in gene content between the genomes of the pathogenic agents causing citrus canker A, B, and C and other Xanthomonas genomes. Molecular genetics can now be employed to determine the role of these genes in plant-microbe interactions. The gained knowledge will be instrumental for improving citrus canker control.

In vitro organogenesis of Citrus volkameriana and Citrus aurantium

In vitro organogenesis of Citrus volkameriana and C. aurantium was studied considering three explant types: epicotyl segment, internodal segment, and hypocotyl segment with attached cotyledon fragment. The explants were cultured in medium according to Grosser and Gmitter (EME) supplemented with 0, 0.5, 1.0, 1.5, and 2.0 mg dm(- 3) 6-benzyl-aminopurine (BAP), incubated firstly in darkness for 4 weeks, and then transferred to 16-h photoperiod for 2 weeks. Comparing epicotyl and internodal segments, a higher percentage of responsive explants and a higher number of shoots per explant were obtained with epicotyl segments, regardless of the BAP concentration. For C. volkameriana the highest percentage of responsive epicotyl segments (42 %) was obtained in EME with 1.0 mg dm(-3) BAP, while for C. aurantium (59 %) in EME with 0.5 mg dm(-3) BAP. The organogenesis efficiency was the best with the use of the hypocotyl segment with attached cotyledon fragment (77 % for C. volkameriana and to 75 % for C. aurantium). With this explant the morphogenesis occurred only in the hypocotyl region. The in vitro organogenesis was characterized by histological analyses showing that the morphogenic process started in the cambium region near the explant cut end.

Evaluation of citrus somatic hybrids for tolerance to Phytophthora nicotianae and citrus tristeza virus

Somatic hybridization is a biotechnology tool that can be used in citrus breeding programs to produce somatic hybrids with the complete genetic combination of both parents. The goal of this work was to test the reaction of citrus somatic hybrids that may be useful as rootstocks to trunk and root infections caused by Phytophthora nicotianae van Breda de Haan (P parasitica Dastur) and to citrus tristeza virus (CTV). The somatic hybrids evaluated were `Caipira` sweet orange (Citrus sinensis L. Osbeck) + `Rangpur` lime (C. limonia Osbeck), `Caipira` sweet orange + `Cleopatra` mandarin (C. reshni hort. ex Tanaka), `Caipira` sweet orange + `Volkamer` lemon (C. volkameriana V Ten. & Pasq.), `Caipira` sweet orange + rough lemon (C. jambhiri Lush.), `Cleopatra` mandarin + `Volkamer` lemon, `Cleopatra` mandarin + sour orange (C. aurantium L.), `Rangpur` lime + `Sunki` mandarin (C. sunki (Hayata) hort. ex Tanaka), `Ruby Blood` sweet orange (C. sinensis L. Osbeck) + `Volkamer` lemon, `Rohde Red` sweet orange (C. sinensis L. Osbeck) + `Volkamer` lemon, and `Valencia` sweet orange + Fortunella obovata hort. ex Tanaka. For P. nicotianae trunk and root infection assays, plants of the somatic hybrids, obtained from 9-month semi-hardwood cuttings, were evaluated and compared with diploid citrus rootstock cultivars after mycelia inoculation in the trunk or spore infestation in the substrate, respectively. `Cleopatra` mandarin + sour orange, `Rangpur` lime + `Sunki` mandarin, `Cleopatra` mandarin + `Volkamer` lemon, `Ruby Blood` sweet orange + `Volkamer` lemon, `Rohde Red` sweet orange + `Volkamer` lemon, and `Caipira` sweet orange + `Volkamer` lemon had less trunk rot occurrence, whereas the somatic hybrids `Cleopatra` mandarin + `Volkamer` lemon, `Cleopatra` mandarin + sour orange, `Caipira` sweet orange + `Volkamer` lemon, and `Caipira` sweet orange + `Rangpur` lime were tolerant to root rot. For CTV assays, plants of the somatic hybrids along with tolerant and intolerant rootstocks were budded with a mild strain CTV-infected or healthy `Valencia` sweet orange budwood. Differences in average scion shoot length indicated that the hybrids `Cleopatra` mandarin + sour orange and `Valencia` sweet orange + Fortunella obovata were intolerant to CTV (c) 2007 Elsevier B.V. All rights reserved.

Availability of soil water under tillage systems, mulch management and citrus rootstocks

The increased availability of soil water is important for the management of non-irrigated orange orchards. The objective of this study was to evaluate the availability of soil water in a Haplorthox (Rhodic Ferralsol) under different tillage systems used for orchard plantation, mulch management and rootstocks in a "Pêra" orange orchard in northwest Paraná, Brazil. An experiment in a split-split-plot design was established in 2002, in an area cultivated with Brachiaria brizantha grass in which three tillage systems (no tillage, conventional tillage and strip-tillage) were used for orchard plantation. This grass was mowed twice a year between the rows, representing two mulch managements in the split plots (no mulching and mulching in the plant rows). The split-split-plots were represented by two rootstocks ("Rangpur" lime and "Cleopatra" mandarin). The soil water content in the plant rows was evaluated in the 0-20 cm layer in 2007 and at 0-20 and 20-40 cm in 2008-2009. The effect of soil tillage systems prior to implantation of orange orchards on soil water availability was less pronounced than mulching and the rootstocks. The soil water availability was lower when "Pêra" orange trees were grafted on "Cleopatra" mandarin than on "Rangpur" lime rootstocks. Mulching had a positive influence on soil water availability in the sandy surface layer (0-20 cm) and sandy clay loam subsurface (20-40 cm) of the soil in the spring. The production of B. brizantha between the rows and residue disposal in the plant rows as mulch increased water availability to the "Pêra" orange trees.

Availability of soil water under tillage systems, mulch management and citrus rootstocks

The increased availability of soil water is important for the management of non-irrigated orange orchards. The objective of this study was to evaluate the availability of soil water in a Haplorthox (Rhodic Ferralsol) under different tillage systems used for orchard plantation, mulch management and rootstocks in a "Pera" orange orchard in northwest Parana, Brazil. An experiment in a split-split-plot design was established in 2002, in an area cultivated with Brachiaria brizantha grass in which three tillage systems (no tillage, conventional tillage and strip-tillage) were used for orchard plantation. This grass was mowed twice a year between the rows, representing two mulch managements in the split plots (no mulching and mulching in the plant rows). The split-split-plots were represented by two rootstocks ("Rangpur" lime and "Cleopatra" mandarin). The soil water content in the plant rows was evaluated in the 0-20 cm layer in 2007 and at 0-20 and 20-40 cm in 2008-2009. The effect of soil tillage systems prior to implantation of orange orchards on soil water availability was less pronounced than mulching and the rootstocks. The soil water availability was lower when "Pera" orange trees were grafted on "Cleopatra" mandarin than on "Rangpur" lime rootstocks. Mulching had a positive influence on soil water availability in the sandy surface layer (020 cm) and sandy clay loam subsurface (20-40 cm) of the soil in the spring. The production of B. brizantha between the rows and residue disposal in the plant rows as mulch increased water availability to the "Pera" orange trees.

Trade patterns and policies of CDCC countries in rice, legumes, ground provisions, fresh vegetables and citrus products and identification of areas for joint action

Includes bibliography

The antimicrobial effects of Citrus limonum and Citrus aurantium essential oils on multi-species biofilms

The aim of this study was to evaluate the effects of Citrus limonum and Citrus aurantium essential oils (EOs) compared to 0.2% chlorhexidine (CHX) and 1% sodium hypochlorite (NaOCl) on multi-species biofilms formed by Candida albicans, Enterococcus faecalis and Escherichia coli. The biofilms were grown in acrylic disks immersed in broth, inoculated with microbial suspension (106 cells/mL) and incubated at 37°C / 48 h. After the biofilms were formed, they were exposed for 5 minutes to the solutions (n = 10): C. aurantium EO, C. limonum EO, 0.2% CHX, 1% NaOCl or sterile saline solution [0.9% sodium chloride (NaCl)]. Next, the discs were placed in sterile 0.9% NaCl and sonicated to disperse the biofilms. Tenfold serial dilutions were performed and the aliquots were seeded onto selective agar and incubated at 37°C / 48 h. Next, the number of colony-forming units per milliliter was counted and analyzed statistically (Tukey test, p ≤ 0.05). C. aurantium EO and NaOCl inhibited the growth of all microorganisms in multi-species biofilms. C. limonum EO promoted a 100% reduction of C. albicans and E. coli, and 49.3% of E. faecalis. CHX was less effective against C. albicans and E. coli, yielding a reduction of 68.8% and 86.7%, respectively. However, the reduction of E. faecalis using CHX (81.7%) was greater than that obtained using C. limonum EO. Both Citrus limonum and Citrus aurantium EOs are effective in controlling multi-species biofilms; the microbial reductions achieved by EOs were not only similar to those of NaOCl, but even higher than those achieved by CHX, in some cases.

The influence of citrus rootstocks on the relationship between the mite Brevipalpus phoenicis and citrus leprosis disease

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Study of packing lines for stone fruits and citrus using two instrumented spheres in some cooperatives in the region of Murcia (Spain).

Two instrumented spheres IS 100 were used to evaluate the quality of post-harvest operations. Results obtained from measurements made with both IS (8.8 cm 0 and 6.2 cm 0) show significant differences. Both IS measure the same values of the same variables for soft materials, but not for hard surfaces. Four packing lines belonging to different cooperatives of the region of Murcia (two for stone fruits and two for citrus) were tested. IS values obtained in transfers belonging to the tested lines lay well above 50 g's in most of them Much higher impact intensities are registered in citrus lines than in stone fruit packing lines. To study the incidence of a certain packing line on different products an interaction fruit-packing line test was perf01med. In all cases, more than 50% of fruits belonging to the post-handling sample showed some kind of damage. Bruises evolve after 48 hours storage at room temperature.

Effects of precooling and degreening treatments on the susceptibility of peach and citrus to handling damage.

Mechanical damage to fruits results from a combination of fruit properties and of damage inflicting effects by the handling equipment. Treatments were applied to fruits which affect mechanical damage susceptibility: precooling for stone fruits and degreening to citrus fruits. Laboratory tests (compression, impact, tumbling, abrasion) and field tests (damage in the handling lines) were applied to (3) peach, (2) apricot, (2) orange and (1) lemon varieties. Hydroor-air-cooling influence positively peach and apricot firmness and cause a significant reduction in: number of bruised fruits, and size of visible bruise, when combined with a low level of loading during handling. Degreening also affects the resistance of citrus fruits to handling, when compared to green fruits.

Optimisation of analytical methods for the characterisation of oranges, clementines and citrus hybrids cultivated in Spain on the basis of their composition in ascorbic acid, citric acid and major sugars

Three HPLC methods were optimised for the determination of citric acid, succinic acid and ascorbic acid using a photodiode array detector and fructose, glucose and sucrose using a refractive index in twenty eight citrus juices. The analysis was completed in <16 min. Two different harvests were taken into account for this study. For the season 2011, ascorbic acid content was comprised between 19.4 and 59 mg vitamin C/100 mL; meanwhile for the season 2012, the content was slightly higher for most of the samples ranging from 33.5 to 85.3 mg vitamin C/100 mL. Moreover, the citric acid content in orange juices ranged between 9.7 and 15.1 g L−1, while for clementines the content was clearly lower (i.e. from 3.5 to 8.4 g L−1). However, clementines showed the highest sucrose content with values near to 6 g/100 mL. Finally, a cluster analysis was applied to establish a classification of the citrus species.

Detection of mechanical and disease stresses in citrus plants by fluorescence spectroscopy

We have investigated the detection of mechanical and disease stresses in citrus plants (Citrus limonia [L.] Osbeck) using laser-induced fluorescence spectroscopy. Due to its economic importance we have chosen to investigate the citrus canker disease, which is caused by the Xanthomonas axonopodis pv. citri bacteria. Mechanical stress was also studied because it plays an important role in the plant's infection by such bacteria. A laser-induced fluorescence spectroscopy system, composed of a spectrometer and a 532 nm 10 mW excitation laser was used to perform fluorescence spectroscopy. The ratio of two chlorophyll fluorescence bands allows us to detect and discriminate between mechanical and disease stresses. This ability to discriminate may have an important application in the field to detect citrus canker infected trees. (c) 2008 Optical Society of America.

Genetic structure and biology of Xylella fastidiosa strains causing disease in citrus and coffee in Brazil

Xylella fastidiosa is a vector-borne, plant-pathogenic bacterium that causes disease in citrus (citrus variegated chlorosis [CVC]) and coffee (coffee leaf scorch [CLS]) plants in Brazil. CVC and CLS occur sympatrically and share leafhopper vectors; thus, determining whether X. fastidiosa isolates can be dispersed from one crop to another and cause disease is of epidemiological importance. We sought to clarify the genetic and biological relationships between CVC- and CLS-causing X. fastidiosa isolates. We used cross-inoculation bioassays and microsatellite and multilocus sequence typing (MLST) approaches to determine the host range and genetic structure of 26 CVC and 20 CLS isolates collected from different regions in Brazil. Our results show that citrus and coffee X. fastidiosa isolates are biologically distinct. Cross-inoculation tests showed that isolates causing CVC and CLS in the field were able to colonize citrus and coffee plants, respectively, but not the other host, indicating biological isolation between the strains. The microsatellite analysis separated most X. fastidiosa populations tested on the basis of the host plant from which they were isolated. However, recombination among isolates was detected and a lack of congruency among phylogenetic trees was observed for the loci used in the MLST scheme. Altogether, our study indicates that CVC and CLS are caused by two biologically distinct strains of X. fastidiosa that have diverged but are genetically homogenized by frequent recombination.

Genetic linkage maps of 'Pêra' sweet orange and 'Cravo' mandarin with RAPD markers

The objective of this work was to construct linkage maps of 'Pêra' sweet orange [Citrus sinensis (L.) Osbeck] and 'Cravo' mandarin (Citrus reticulata Blanco) using RAPD markers and the pseudo-testcross strategy. The parents were chosen according to the resistance/susceptibility to citrus variegate chlorosis (CVC). The segregation of 176 markers was analyzed in 94 progeny of F1 hybrids, which were obtained from controlled crossings. The linkage map of 'Pêra' sweet orange had 117 markers defined by 12 linkage groups, which spanned 612.1 cM. Only six markers could not be linked to the linkage group and 48.7% of the markers showed segregation distortion. The linkage map of 'Cravo' mandarin had 51 markers defined by 12 linkage groups, which spanned 353.3 cM. Only two markers did not link to the groups and 15.7% showed segregation distortion. The construction of linkage maps is relevant to future mapping studies of the inheritance of CVC, citrus canker and leprosis.

The Xanthomonas citri effector protein PthA interacts with citrus proteins involved in nuclear transport, protein folding and ubiquitination associated with DNA repair

P>Xanthomonas axonopodis pv. citri utilizes the type III effector protein PthA to modulate host transcription to promote citrus canker. PthA proteins belong to the AvrBs3/PthA family and carry a domain comprising tandem repeats of 34 amino acids that mediates protein-protein and protein-DNA interactions. We show here that variants of PthAs from a single bacterial strain localize to the nucleus of plant cells and form homo- and heterodimers through the association of their repeat regions. We hypothesize that the PthA variants might also interact with distinct host targets. Here, in addition to the interaction with alpha-importin, known to mediate the nuclear import of AvrBs3, we describe new interactions of PthAs with citrus proteins involved in protein folding and K63-linked ubiquitination. PthAs 2 and 3 preferentially interact with a citrus cyclophilin (Cyp) and with TDX, a tetratricopeptide domain-containing thioredoxin. In addition, PthAs 2 and 3, but not 1 and 4, interact with the ubiquitin-conjugating enzyme complex formed by Ubc13 and ubiquitin-conjugating enzyme variant (Uev), required for K63-linked ubiquitination and DNA repair. We show that Cyp, TDX and Uev interact with each other, and that Cyp and Uev localize to the nucleus of plant cells. Furthermore, the citrus Ubc13 and Uev proteins complement the DNA repair phenotype of the yeast Delta ubc13 and Delta mms2/uev1a mutants, strongly indicating that they are also involved in K63-linked ubiquitination and DNA repair. Notably, PthA 2 affects the growth of yeast cells in the presence of a DNA damage agent, suggesting that it inhibits K63-linked ubiquitination required for DNA repair.