vasculorum) was digested with Bgl1 instead of being sonicated, only because a sonicator was not available. The only modification was that the driver DNA ( X. The high phosphate genomic subtraction technique ( Seal et al. ![]() Digoxigenin (DIG) was used as label and anti-DIG antibodies conjugated with alkaline phosphatase and the substrate CSPD ® (disodium 3-(4-methoxyspiro-4-yl)phenyl phosphate) were added for detection by chemiluminescence. DNA labelling and detection procedures were done according to the instruction manual from Boehringer Mannheim (Mannheim, Germany). The DNA was stored in sterile distilled water at − 20 ☌.Īll other manipulations such as restriction digests, agarose gel electrophoresis, ligations, and transformation of E. The CTAB (cetyl-trimethylammonium bromide) is essential to remove all contaminating polysaccharides. After addition of phenol and chloroform, the tubes were centrifuged at 8000 g for 15–20 min. The cells were thoroughly washed with NE buffer (50 mmol l −1 EDTA, 0♱5 mmol l −1 NaCl) before lysis. DNA extractions and manipulationsĭNA was extracted according to the method of Ausubel et al. Strains from Mauritius were serotyped using polyclonal antibodies specific for either serovar.Įscherichia coli, DH5α, was cultured in LB medium at 37 ☌. Bacteria were cultured in Wilbrink broth or agar at 28 ☌ and preserved at − 80 ☌ in glycerol broth. MATERIALS and METHODS Bacterial strainsīacterial strains used are indicated in Tables 1 and 2. The probes used in this study showed specificity for the species as they did not hybridize to 10 other bacteria or to sugarcane DNA. albilineans by genomic subtraction and their characterization as potential probes for evaluating genetic polymorphism within the species. We describe here the isolation of DNA fragments of X. This method is, however, relatively cumbersome and lengthy and, in addition, it requires highly precise interpretation of complex banding patterns. Eight different groups have been defined using data from 52 banding patterns. albilineans has been done using pulsed-field gel electrophoresis of fragments restricted with the rare-cutting enzyme Spe1 ( An important study of variation within X. Have reported the identification of RAPD markers for the differentiation of X. Molecular techniques, using DNA-based methods, have proven more useful in the identification of different pathotypes for many plant pathogens. However, the use of antibodies is limited in that they can only detect the presence of a particular epitope and any polyclonal preparation is unique, thus making comparison of different studies difficult. Type I includes widely distributed strains and Type II those mostly isolated on the African continent.įound a correlation between serological and DNA groups, based on the identification of restriction fragment length polymorphism. Have defined three serotypes I, II and III. albilineans using polyclonal and monoclonal antibodies, respectively. Thus, it is imperative to have means of detecting the different forms of the pathogen during its evolution and of identifying the subpopulations. As the bacteria evolve, this interaction changes accordingly. Sugar cane varieties differ in their resistance or tolerance to the bacteria, as a result of the genetic components that define the host–pathogen relationships and thus determine whether a compatible or an incompatible interaction is established. Erwinia herbicola) shows resistance to the pathogen ( ![]() Transgenic sugar cane carrying an albicidin-resistance gene from Pantoea dispersa (syn. This has resulted in the rejection of some high yielding hybrids because of their susceptibility to the disease. The major form of control is to grow varieties that have acceptable levels of resistance to the disease. There is evidence of its increased incidence in the southern states of USA and Central America ( It has been reported in nearly all locations where sugar cane is grown and was responsible for severe epidemics in Mauritius and Florida in 1989. Leaf scald disease of sugar cane is caused by Xanthomonas albilineans, a Gram-negative bacterium.
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