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Development and Application of Cloned DNA Probes for Clavibacter xyli subsp. xyli , the Causal Agent of Sugarcane Ratoon Stunting
Author(s) -
Chung C. H.,
Lin C. P.,
Chen C. T.
Publication year - 1994
Publication title -
journal of phytopathology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.53
H-Index - 60
eISSN - 1439-0434
pISSN - 0931-1785
DOI - 10.1111/j.1439-0434.1994.tb01473.x
Subject(s) - biology , recombinant dna , plasmid , dna , hybridization probe , genomic dna , microbiology and biotechnology , molecular cloning , molecular probe , digoxigenin , escherichia coli , dna–dna hybridization , bacteria , pathogen , genetics , gene , in situ hybridization , complementary dna , gene expression
Eco RI restriction fragments of genomic DNA from Clavibacter xyli subsp. xyli (CXX) were ligated with plasmid pUC18 and cloned in Escherichia coli JM109. The cloned DNA inserts from recombinant plasmids were Eco RI‐excised and labeled with non‐radioactive digoxigenin and used as probes. Ten specific DNA probes, RSD3, 15, 30, 31, 32, 35, 37, 41, 71, and 73 were selected for disease detection and pathogen differentiation. In the specificity tests, all of the 10 CXX DNA, probes differentiated Clavibacter xyli from other bacteria specifically. Seven out of the 10 CXX probes crossreacted with C. x. subsp. cynodontis (CXC) very weakly under moderate stringency wash conditions of hybridization. In the sensitivity tests, all of the 10 DNA probes detected the homologous DNA of CXX from 0.19 to 0.75 ng. To detect various cell numbers of CXX, the DNA probes detected 10 4 to 10 5 cells effectively. In Southern hybridizations, distinctly different band patterns were shown when the probes hybridized with DNA from CXX and CXC. Among these probes, RSD3, 15, 30, 31, 35, 37, and 71, efficiently detected CXX present in the sap collected from symptomless sugarcane.