Measuring the Genetic Diversity of Xanthomonas axonopodis pv. manihotis Within Different Fields in Colombia

ABSTRACT Cassava bacterial blight, caused by Xanthomonas axonopodis pv. manihotis, is a widespread disease that affects cassava (Manihot esculenta). We collected 238 X. axonopodis pv. manihotis strains by intensively sampling single fields in four edaphoclimatic zones (ECZs) in Colombia. DNA polymorphism of different X. axonopodis pv. manihotis populations was assessed by restriction fragment length polymorphism (RFLP) analyses, repetitive sequence-based polymerase chain reaction (rep-PCR), and amplified fragment length polymorphism (AFLP) assays. Genetic diversity, phenetic relationships among strains, and the coefficient of genetic differentiation were determined. All strains were tested for aggressiveness on the susceptible cassava cv. MCOL 1522. Strains were also tested for virulence on cassava differentials adapted to the strains' respective ECZs. Our study showed that the Colombian X. axonopodis pv. manihotis population has a high degree of genetic diversity. The hierarchical analysis of diversity showed genotypic differentiation at all levels, among ECZs, among fields within ECZs, and among strains within fields planted to several cassava genotypes. New RFLP haplotypes were detected, leading to the characterization of a new pathotype. Dendrograms from AFLP were more robust than those from RFLP data. A close association between the strains' geographical origin and DNA polymorphism was obtained using RFLP and AFLP data. We suggest that the host played a role in causing pathogen differentiation.     

Molecular characterization of the incitant of cowpea bacterial blight and pustule, Xanthomonas campestris pv. vignicola

Strains of Xanthomonas campestris pv. vignicola (Xcv), isolated from cowpea leaves with blight or minute pustules and collected from various geographic areas, were selected on the basis of pathological and physiological features. All strains were analyzed for genotypic markers by two methods: ribotyping with EcoRI endonuclease, and RFLP analysis with a plasmid probe (pthB) containing a gene required for pathogenicity from Xanthomonas campestris pv. manihotis. Ribotyping revealed a unique pattern for all the strains that corresponded to the previously described ribotype rRNA7. Based on polymorphism detected by pthB among Xcv strains, nine haplotypes were defined. The observed genetic variation was independent of the geographic origin of the strains and of pathogenic variation. Some haplotypes were widely distributed, whereas others were localized. In some cases, we could differentiate strains isolated from blight symptoms and pustules according to haplotypic composition. However, in most cases, no significant differences were observed. Our results and the previous pathogenic and biochemical characterizations suggest that the strains isolated from leaves with blight symptoms or minute pustules belong to the same pathovar. We provide information on pathogen diversity that can be used to identify and characterize resistant germplasm.     

The use of a nerve stimulation test to confirm sacrococcygeal epidural needle placement in cats

ObjectiveTo determine if a nerve stimulation test (NST) could act as a monitoring technique to confirm sacrococcygeal epidural needle placement in cats.Study designProspective experimental trial in a clinical setting.AnimalsTwenty-four adult cats, scheduled for a therapeutic procedure where epidural anesthesia was indicated.MethodsUnder general anesthesia, an insulated needle was inserted through the S₃-Cd₁ intervertebral space guided by the application of a fixed electrical current (0.7 mA) until a motor response was obtained. The NST was considered positive when the epidural nerve stimulation produced a motor response of the muscles of the tail, whereas it was considered negative when no motor response was evoked. In the NST positive cases, 0.3 mL kg⁻¹ of 0.5% bupivacaine was administrated before needle withdrawal. Ten minutes after injection, epidural blockade was confirmed by the loss of perineal (anal), and pelvic limbs reflexes (patellar and withdrawal).ResultsThe use of a fixed electrical stimulation current of 0.7 mA resulted in correct prediction of sacrococcygeal epidural injection, corroborated by post bupivacaine loss of perineal and pelvic limb reflexes, in 95.8% of the cases.Conclusion and clinical relevanceThis study demonstrates the feasibility of using, in a clinical setting, an electrical stimulation test as an objective and in real-time method to confirm sacrococcygeal epidural needle placement in cats.     

Estimation du stock semencier dans le cadre d'un essai étudiant l'influence de systémes culturaux sur l'évolution de la flore adventice

Estimation of seed stock during a trial study of the influence of cultural systems on the development of the weed flora Twelve plots (12 × 20 m) were sampled at the En Crambade inter-institute station in 1984 before a 9-year trial (3×3 year rotations) was begun to study the influence of weed control methods, soil cultivation and crop rotation on the development of the weed flora. Three hundred cores, 25 per plot, were analysed separately. The weed species were identified and classified in accordance with their size and distribution. Sampling error as a function of the number of cores sampled, the variation and the average number of seeds per core were calculated.     

The use of electrical stimulation to guide epidural and intrathecal needle advancement at the L5‐L6 intervertebral space in dogs

ObjectiveTo determine the minimal electrical threshold (MET) necessary to elicit appropriate muscle contraction when the tip of an insulated needle is positioned epidurally or intrathecally at the L_5-6 intervertebral space (phase-I) and to determine whether the application of a fixed electrical current during its advancement could indicate needle entry into the intrathecal space (phase-II) in dogs.Study designProspective, blinded study.AnimalsThirteen (phase-I) and seventeen (phase-II) dogs, scheduled for a surgical procedure where L_5-6 intrathecal administration was indicated.MethodsUnder general anesthesia, an insulated needle was first inserted into the L_5-6 epidural space and secondly into the intrathecal space and the MET necessary to obtain a muscular contraction of the pelvic limb or tail at each site was determined (phase-I). Under similar conditions, in dogs of phase-II an insulated needle was inserted through the L_5-6 intervertebral space guided by the use of a fixed electrical current (0.8 mA) until muscular contraction of the pelvic limb or tail was obtained. Intrathecal needle placement was confirmed by either free flow of cerebrospinal fluid (CSF) or myelography.ResultsThe current required to elicit a motor response was significantly lower (p < 0.0001) when the tip of the needle was in the intrathecal space (0.48 ± 0.10 mA) than when it was located epidurally (2.56 ± 0.57). The use of a fixed electrical stimulation current of 0.8 mA resulted in correct prediction of intrathecal injection, corroborated by either free flow of CSF (n = 12) or iohexol distribution pattern (n = 5), in 100% of the cases.Conclusion and clinical relevanceNerve stimulation may be employed as a tool to distinguish epidural from intrathecal insulated needle position at the L_5-6 intervertebral space in dogs. This study demonstrates the feasibility of using an electrical stimulation test to confirm intrathecal needle position in dogs.     

Growth,blood chemistry and histology of rats fed zincsupplemented rapeseed protein concentrates

Two rapeseed protein concentrates (RPC) supplemented with zinc oxide were fed to male and female rats at two levels providing approximately half or all of the protein in a purified basal diet containing 20% protein (N×6.25). Control groups were given either a purified basal diet with casein or a laboratory chow diet. After 16 weeks, there were no significant (P<0.05) differences in body weight except the male rats fed the casein control diet had higher body weights than did other male rats. No significant differences were observed in organ weights including thyroids, hematology, bone marrow differential counts, serum glucose, urea nitrogen, total protein, albumin/globulin ratio, cholesterol, transaminases or alkaline phosphatase. The levels of serum vitamins A and E of females fed some of the RPC diets were less than in those fed the caseincontrol diet, but equal to those in females fed laboratory chow diet. Females fed either RPC or casein in the semi-purified diets, which contained zinc oxide, exhibited kidney calcification.Bureau of Nutritional Sciences, No. 112; Food Research Institute, No. 420.     

Development of a variable number of tandem repeats typing scheme for the bacterial rice pathogen Xanthomonas oryzae pv. oryzicola

Xanthomonas oryzae pv. oryzicola is an important bacterial pathogen responsible for outbreaks of bacterial leaf streak (BLS) on rice, mostly occurring in Asia and parts of Africa. To better monitor epidemics and assess population structures, efficient tools that allow the precise identification and diagnosis of pathogenic populations are needed. In this study, we explored variable numbers of tandem repeats (VNTR) as a fast, reliable, and cost-effective molecular typing tool. Screening of three X. oryzae pv. oryzicola genome sequences (Philippine strain BLS256, Chinese strain GX01, and Malian strain MAI10) predicted 28 candidate VNTR loci. Primer pairs for polymerase chain reaction (PCR) amplification of all 28 loci were designed and applied to a panel of 20 X. oryzae pv. oryzicola strains originating from Asia and Africa. Sequencing of PCR amplicons revealed 25 robust and polymorphic VNTR loci that are shared among Asian and African X. oryzae pv. oryzicola strains. A dendrogram constructed from 25 VNTR loci indicated that most Asian strains are clearly discriminated from African strains. However, in agreement with previous reports, one strain from Mali is related to Asian strains, pointing to a possible introduction of Asian strains to the African continent. The new VNTR-based tool described here is useful for studies of population structures and epidemiological monitoring of X. oryzae pv. oryzicola.     

Methods for detecting the cassava bacterial blight pathogen: A practical approach for managing the disease

Cassava bacterial blight (CBB), caused by Xanthomonas axonopodis pv. manihotis (Xam), is a particularly destructive disease in South America and Africa. The movement of infected asymptomatic stems is a major means of pathogen dispersal as well as infected seeds. The success of a cassava-seed certification program depends on the availability of reliable tests to detect the pathogen in vegetative planting materials and true seeds. We report here the different methods that permitted to detect the pathogen in cassava tissues. A polymerase chain reaction (PCR) test was developed for this pathogen. The PCR assay worked well for pathogen detection in extracts from leaf and stem lesions and the minimum number of cells that could be detected ranged from 3 × 10² to 10⁴ CFU per ml. Nested-PCR worked well for Xam detection from naturally infected seeds. This technique was specific, sensitive, and rapid for detecting Xam in cassava true seeds. The highest detection level found was 1–2 viable cells per reaction. A dot-blot assay was developed by evaluating a 898 bp DNA fragment unique to Xam strains as a diagnostic DNA probe. The probe detected Xam strains in crude extracts of leaf and stem lesions, cassava fruits and sexual seeds that were naturally infected. Overall sensitivity of the dot-blot method was about10³CFU per reaction. The dot-blot hybridization technique can be easily used for culture indexing. A monoclonal antibody (MAb) was also used for an enzyme-linked immunosorbent assay (ELISA) and tested with various infected tissues. Overall sensitivity of the method was about10³CFU per reaction. This revised version was published online in July 2006 with corrections to the Cover Date.