Bacterial canker of mango (or bacterial black spot), caused by Xanthomonas citri pv. mangiferaeindicae, is an economically important disease in tropical and subtropical producing areas (1). X. citri ...pv. mangiferaeindicae can cause severe infection in a wide range of mango cultivars and induces raised, angular, black leaf lesions, sometimes with a chlorotic halo. Several months after infection, leaf lesions dry and turn light brown or ash gray. Severe leaf infection may result in abscission. Fruit symptoms appear as small water-soaked spots on the lenticels. These spots later become star shaped, erumpent, and exude an infectious gum. Often, a "tear stain" infection pattern is observed on the fruit. Severe fruit infections will cause premature fruit drop. Twig cankers are potential sources of inoculum and weaken resistance of branches to wind damage. Leaf lesions with suspected bacterial canker were collected in January 2010 from mango trees cv. Keitt in several blocks at the Integrated Tamale Fruit Company, Ghana. Non-pigmented Xanthomonas-like bacterial colonies were isolated on Kasugamycin-Cephalexin semiselective agar medium (3). On the basis of IS1595-Ligation Mediated-PCR data, 16 strains from Ghana produced identical fingerprints and were identified as X. citri pv. mangiferaeindicae (4). The haplotype corresponding to the Ghanaian strains had not been previously reported. On the basis of multidimensional scaling (4), this haplotype clustered together with a group of strains from multiple origins and the analysis was not informative as an aid for tracing back the outbreak. Five Ghanaian strains (LH2-3, LH2-6, LH2-8, LH2-11, and LH2-15) were compared by multilocus sequence analysis to the type strain of X. citri and the pathotype strain of several X. citri pathovars, including pvs. anacardii and mangiferaeindicae. This assay targeted the atpD, dnaK, efp, and gyrB genes as described previously (2). Nucleotide sequences were 100% identical to those of the pathotype strain of X. citri pv. mangiferaeindicae whatever the gene assayed, but differed from any other assayed X. citri pathovar. Mango cv. Maison Rouge leaves from the youngest vegetative flush were infiltrated (10 inoculation sites per leaf, three replicate plants) using inoculum of each of the same five Ghanaian strains made from suspensions in Tris buffer containing ~1 × 10
CFU/ml. Negative control treatments consisted of leaves infiltrated with sterile Tris buffer. Typical symptoms of bacterial canker were observed for all assayed strains a week after inoculation. No lesions were recorded from the negative control. One month after inoculation, mean X. citri pv. mangiferaeindicae population sizes ranging from 4 × 10
to 1 × 10
CFU/lesion were recovered from leaf lesions, typical of a compatible interaction (1). High disease prevalence was observed in Ghana, indicating the suitability of environmental conditions in this region for the development of mango bacterial canker. The budwood for these blocks was imported from Burkina Faso in 2002 and symptoms were observed in these blocks shortly after establishment. To our knowledge, this is the first report of mango bacterial canker in Western Africa. References: (1) N. Ah-You et al. Phytopathology 97:1568, 2007. (2) L. Bui Thi Ngoc et al. Int. J. Syst. Evol. Microbiol. 60:515, 2010. (3) O. Pruvost et al. J. Appl. Microbiol. 99:803, 2005. (4) O. Pruvost et al. Phytopathology. Online publication. DOI:10.1094/PHYTO-11-10-0304, 2011.
1 CIRAD, UMR Peuplements Végétaux et Bioagresseurs en Milieu Tropical CIRAD-Université de la Réunion, Pôle de Protection des Plantes, 7 chemin de l'Irat, 97410 Saint Pierre, La Réunion, France
2 ...Unité Biodiversité des Bactéries Pathogènes Emergentes, Institut Pasteur, 25–28 rue du Dr Roux, 75724 Paris Cedex 15, France
3 Ecologie Microbienne, UMR CNRS 5557/USC INRA 1193, Université Claude Bernard-Lyon 1, Villeurbanne, France
Correspondence O. Pruvost olivier.pruvost{at}cirad.fr
We have used amplified fragment length polymorphism (AFLP), multilocus sequence analysis (MLSA) and DNA–DNA hybridization for genotypic classification of Xanthomonas pathovars associated with the plant family Anacardiaceae. AFLP and MLSA results showed congruent phylogenetic relationships of the pathovar mangiferaeindicae (responsible for mango bacterial canker) with strains of Xanthomonas axonopodis subgroup 9.5. This subgroup includes X. axonopodis pv. citri (synonym Xanthomonas citri ). Similarly, the pathovar anacardii , which causes cashew bacterial spot in Brazil, was included in X. axonopodis subgroup 9.6 (synonym Xanthomonas fuscans ). Based on the thermal stability of DNA reassociation, consistent with the AFLP and MLSA data, the two pathovars share a level of similarity consistent with their being members of the same species. The recent proposal to elevate X. axonopodis pv. citri to species level as X. citri is supported by our data. Therefore, the causal agents of mango bacterial canker and cashew bacterial spot should be classified as pathovars of X. citri , namely X. citri pv. mangiferaeindicae (pathotype strain CFBP 1716) and X. citri pv. anacardii (pathotype strain CFBP 2913), respectively. Xanthomonas fuscans should be considered to be a later heterotypic synonym of Xanthomonas citri .
Abbreviations: AFLP, amplified fragment length polymorphism; CBS, cashew bacterial spot; DDH, DNA–DNA hybridization; EGD, evolutionary genome divergence; MBC, mango bacterial canker; ML, maximum-likelihood; MLSA, multilocus sequence analysis; NJ, neighbour-joining; nsps, nucleotide substitutions per site; rep-PCR, repetitive extragenic palindromic PCR
The GenBank/EMBL/DDBJ accession numbers for the partial 16S rRNA gene sequences of X. citri pv. mangiferaeindicae CFBP 1716, X. citri pv. anacardii CFBP 2913 and X. axonopodis pv. spondiae CFBP 2547 are respectively EF989732 , EF989733 and EF989734 . Those of the partial sequences used in the MLSA study are EU015124 –EU015156, EU015158 –EU015215 and EU333904 –EU333906 ( atpD ), EU015216 –EU015248, EU015250 –EU015307 and EU333907 –EU333909 ( dnaK ) and EU015308 –EU015340, EU015342 –EU015399 and EU333910 –EU333912 ( gyrB ).
Details of strains and primers and ML trees derived from partial atpD , dnaK and gyrB sequences are available as supplementary material with the online version of this paper.
We screened the genome of Xanthomonas citri pv. citri strain 306 for tandem repeats. A multiplex polymerase chain reaction protocol was used to assess the genetic diversity of 239 strains of X. citri ...pv. citri from Asia. The total number of alleles per locus ranged from three to 20. Using pooled data sets, 223 different haplotypes were identified. Successful amplifications were obtained at most loci for seven other X. citri pathovars. This typing scheme is expected to be useful at different spatial scales for population studies of pathovars of X. citri, several of which cause plant diseases of economic importance.
•Producers’ practices affected the nutritional composition of cowpea-based doughnuts.•Lipid content is lower in doughnuts from whole than from dehulled-soaked cowpea.•Oil absorption during frying ...induced passive reduction of other nutrient contents.•60 % of folate were lost during Ata production, of which around 20 % were degraded.•Ata-doco production reduced alpha-galacto-oligosaccharide contents compared to Ata.
Doughnuts made from cowpea, a highly nutritious pulse, are frequently consumed in West Africa. As processing may affect their nutritional composition, cowpea processing into two doughnut types (ata and ata-doco) was characterized, and samples collected from 12 producers in Cotonou, Benin. Proximate composition, folate, mineral, phytate, and alpha-galacto-oligosaccharide contents were determined in the raw material, intermediate products, and doughnuts. Mass balance was assessed during ata production to monitor folate and alpha-galacto-oligosaccharides distribution, and to determine what steps most influenced their concentration. Ata was prepared with dehulled-soaked seeds, and ata-doco with whole or partially dehulled, non-soaked and dry-milled seeds. After both types of doughnuts production, lipid content increased by 11–33 times compared with raw seeds, due to oil absorption during deep-frying. Milling led to an increase of iron content by 50–57 % (ata) and 21–75 % (ata-doco production). Alpha-galacto-oligosaccharide contents decreased by 22–57 % after whipping during ata-doco, but not during ata production. The mass balance assessment showed significant reductions of folate (-50 %) and alpha-galacto-oligosaccharides (-33 %) after dehulled seed washing and soaking during ata production. This study showed that the impact of traditional processing on the nutritional value of cowpea-based doughnuts is strong, but highly variable depending on the doughnut type and producers’ practices.
Leafy vegetable sauces from Burkina Faso were assessed as a potential vehicle for food fortification. First, iron and zinc bioaccessibility were measured by dialysability method in amaranth and Jew's ...mallow sauces and in traditional whole dishes consisting of maize paste plus leafy vegetable sauces. Iron dialysability and solubility were higher in amaranth than in Jew's mallow sauce, pointing to a marked effect of the matrix. Iron dialysability was hardly affected by the maize paste contrary to zinc dialysability, which was reduced. Second, iron and zinc bioaccessibility was assessed in the same sauces fortified with NaFeEDTA or iron sulfate. Added iron,
i.e.
iron supplied by fortification, represented 60% of total iron at the low fortification level and 80% at high level. In amaranth sauces with the high level of fortification using NaFeEDTA and iron sulfate, fractional dialysable iron reached respectively 66% and 26% compared to only 8.1% in the unfortified sauce. Similarly, in Jew's mallow sauces, fractional dialysable iron was 57% and 5% respectively with NaFeEDTA and iron sulfate and less than 1% in the unfortified sauce. Concomitantly, fractional dialysable zinc increased by respectively 20% and 40% in amaranth and Jew's mallow sauces fortified with NaFeEDTA whereas it remained unchanged with iron sulfate. Iron fortification could be an efficient way to greatly increase the available iron content of green leafy vegetable sauces and for this purpose NaFeEDTA is more effective than iron sulfate whatever the food matrix.
Leafy vegetable sauces from Burkina Faso were assessed as a potential vehicle for food fortification.
Xanthomonas citri pv. mangiferaeindicae is the causal agent of bacterial canker of mango (Mangifera indica, Anacardiaceae), a disease of international importance. Since the original description of ...the bacterium in the 1940s, the status of cashew (Anacardium occidentale, Anacardiaceae) as a host species has been unclear. Here, we report the first outbreak of a cashew bacterial disease in Burkina Faso (Western Africa) where X. citri pv. mangiferaeindicae recently emerged on mango. A comprehensive molecular characterization, based on multilocus sequence analysis, supplemented with pathogenicity assays of isolates obtained during the outbreak, indicated that the causal agent on cashew in Burkina Faso is X. citri pv. mangiferaeindicae and not X. citri pv. anacardii, which was previously reported as the causal agent of a cashew bacterial leaf spot in Brazil. Pathogenicity data supported by population biology in Burkina Faso suggest a lack of host specialization. Therefore, the inoculum from each crop is potentially harmful to both host species. Symptoms induced on cashew leaves and fruit by X. citri pv. mangiferaeindicae and nonpigmented strains of X. citri pv. anacardii are similar, although the causative bacteria are genetically different. Thus, xanthomonads pathogenic on cashew may represent a new example of pathological convergence in this bacterial genus.
Multilocus variable-number tandem-repeat analysis (MLVA) is efficient for routine typing and for investigating the genetic structures of natural microbial populations. Two distinct pathovars of ...Xanthomonas oryzae can cause significant crop losses in tropical and temperate rice-growing countries. Bacterial leaf streak is caused by X. oryzae pv. oryzicola, and bacterial leaf blight is caused by X. oryzae pv. oryzae. For the latter, two genetic lineages have been described in the literature. We developed a universal MLVA typing tool both for the identification of the three X. oryzae genetic lineages and for epidemiological analyses. Sixteen candidate variable-number tandem-repeat (VNTR) loci were selected according to their presence and polymorphism in 10 draft or complete genome sequences of the three X. oryzae lineages and by VNTR sequencing of a subset of loci of interest in 20 strains per lineage. The MLVA-16 scheme was then applied to 338 strains of X. oryzae representing different pathovars and geographical locations. Linkage disequilibrium between MLVA loci was calculated by index association on different scales, and the 16 loci showed linear Mantel correlation with MLSA data on 56 X. oryzae strains, suggesting that they provide a good phylogenetic signal. Furthermore, analyses of sets of strains for different lineages indicated the possibility of using the scheme for deeper epidemiological investigation on small spatial scales.
West Asia has been recognized as a major centre for the diversification of Xanthomonas citri pv. citri, a citrus quarantine pathogen of considerable economic importance. However, little genotyping ...data is available mainly due to the paucity of microbial resources in this region. Using a comprehensive strain collection, several genotyping techniques and a pathogenicity assay, the status of strains causing Asiatic citrus canker in Iran, an internationally significant citrus‐producing country, was clarified. All strains were genetically related to X. citri pv. citri pathotype A* (i.e. strains with a host range restricted to Mexican lime and related species) but not to pathotype A (i.e. strains with a wide host range among rutaceous species). The findings were based on discriminant analysis of the principal components of MLVA‐31 data and were further confirmed by pathogenicity data. Two genetically, geographically and pathologically separate groups of strains in Iran were identified. One of the groups had never been previously reported anywhere in the world. A very strong genetic structure was found (RST = 0·938), consistent with their geographical isolation. Strains from these two groups also differed in terms of their type III effector repertoire. The atypical host range of one of these groups could explain why some Iranian strains had previously been mistakenly identified as pathotype A. This study suggests the absence of invasive pathotype A strains in Iran (known as DAPC 1), which account for most of the economically important outbreaks internationally.
Citrus canker, caused by Xanthomonas citri pv. citri, is a bacterial disease of economic importance in tropical and sub-tropical citrus-producing areas (EPPO-PQR online database). X. citri pv. citri ...causes severe infection in a wide range of citrus species, and induces erumpent, callus-like lesions with water-soaked margins leading to premature fruit drop and twig dieback. It has consequently been subjected to eradication efforts and international regulations. It was first described on the African continent in South Africa at the beginning of the 20th century, from which it was eventually eradicated. Since 2006, several outbreaks caused by phylogenetically diverse strains of X. citri pv. citri have been reported from several African countries (Ethiopia, Mali, Senegal, and Somalia). In July 2011, citrus canker in Burkina Faso was suspected in the area adjacent to the Sikassso Province of Mali where X. citri pv. citri has been confirmed. In November and December 2012, leaves of clementine (Citrus clementina), lemon (C. limon), Volkamer lemon (C. volkameriana), sweet orange (C. sinensis), tangelo (C. paradisi× C. reticulata), and mandarin (C. reticulata) were collected from orchards with trees showing symptoms of citrus canker in the Comoé, Houet, and Kénédougou provinces of Burkina Faso. Isolations performed using KC semi-selective medium (4) recovered 45 Xanthomonas-like strains. All Xanthomonas-like strains were tentatively identified as X. citri pv. citri by PCR (4/7 primers) using IAPAR 306 and sterile distilled water as the positive and negative controls, respectively (3). Among these, two strains (LK4-4 and LK4-5) produced a 'fuscans'-like brown diffusible pigment, a phenotype never reported previously for X. citri pv. citri. MultiLocus Sequence Analysis targeting six housekeeping genes (atpD, dnaK, efp, gltA, gyrB, and lepA) (1,2) fully identified seven strains from Burkina Faso (LJ301-1, LJ303-1, LK1-1, LK2-6, LK4-3, LK4-4, and LK4-5) as X. citri pv. citri (and not to any other Xanthomonas pathovars pathogenic to citrus or host range-restricted pathotypes of pathovar citri), and more specifically as sequence type ST2 which is composed mostly of pathotype A strains of X. citri pv. citri (2). The same seven strains were inoculated to at least four leaves of each of grapefruit cv. Henderson, Mexican lime SRA 140 (C. aurantifolia), Tahiti lime SRA 58 (C. latifolia), and sweet orange cv. Washington Navel, using a detached leaf assay (2). All strains developed typical erumpent, callus-like tissue at wound sites on all citrus species inoculated. No lesions developed on the negative control (sterile 10 mM tris buffer). Koch's postulate was fulfilled after reisolation of Xanthomonas-like yellow colonies from symptoms on Mexican lime produced by the seven strains. Boiled bacterial suspensions were assayed by PCR with 4/7 primers (3) and produced the expected 468-bp amplicon in contrast with the PCR negative control. To our knowledge, this is the first report of X. citri pv. citri in Burkina Faso. Citrus canker-free nurseries and grove sanitation should be implemented for reducing the prevalence of Asiatic canker in Burkina Faso and a thorough survey of citrus nurseries and groves in the region should be conducted. References: (1) N. F. Almeida et al. Phytopathology 100:208, 2010. (2) L. Bui Thi Ngoc et al. Int. J. Syst. Evol. Microbiol. 60:515, 2010. (3) J. S. Hartung et al. Phytopathology 86:95, 1996. (4) O. Pruvost et al. J. Appl. Microbiol. 99:803, 2005.
Molecular fingerprinting techniques that have the potential to identify or subtype bacteria at the strain level are needed for improving diagnosis and understanding of the epidemiology of pathogens ...such as Xanthomonas citri pv. mangiferaeindicae, which causes mango bacterial canker disease. We developed a ligation-mediated polymerase chain reaction targeting the IS1595 insertion sequence as a means to differentiate pv. mangiferaeindicae from the closely related pv. anacardii (responsible for cashew bacterial spot), which has the potential to infect mango but not to cause significant disease. This technique produced weakly polymorphic fingerprints composed of approximately equal to 70 amplified fragments per strain for a worldwide collection of X. citri pv. mangiferaeindicae but produced no or very weak amplification for pv. anacardii strains. Together, 12 tandem repeat markers were able to subtype X. citri pv. mangiferaeindicae at the strain level, distinguishing 231 haplotypes from a worldwide collection of 299 strains. Multilocus variable number of tandem repeats analysis (MLVA), IS1595-ligation-mediated polymerase chain reaction, and amplified fragment length polymorphism showed differences in discriminatory power and were congruent in describing the diversity of this strain collection, suggesting low levels of recombination. The potential of the MLVA scheme for molecular epidemiology studies of X. citri pv. mangiferaeindicae is discussed.
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