BP Singh

Detection of Bacterial Pathogens and Salvaging of Infected Gcrmplasm 153precipitate. Agglutination test can be performed in culture tubesas well as on glass slides.(b) Immunofluorescence (IF) test: Immunofluorescence is asimple and rapid test which is more commonly used in clinicaldiagnosis of the agents of human diseases. Nowadays IF testsare also used for the detection of plant pathogenic bacteria in seedsand other plant parts. P. syringae pv. phaseolico/a and X. campestrispv. phaseoli, the halo blight and bacterial blight pathogens,respectively in bean seeds can be detected by direct IF and indirectIF test. In direct IF, the slide prepared for IF test is treated withantibacterium conjugate and after mounting, the slide is examinedwith a super pressure mercury lamp and a 1-2 filter system forincident illumination with blue light for FITe excitation whichshowed clear IF positive cells. For indirect IF test, the slide is firstreacted with bacterial antiserum then with antibacterium conjugateand the mounted slides are examined for IF positive number ofbacterial cells.The IF test can also be performed on agar medium by IFcolony staining. After the appearance of the colony on agarmedium, the agar medium is treated with antibacterial conjugateantiserum and the agar can be checked with a UV microscope atlow magnification for fluorescing colonies.' The halo blightpathogen in bean and bacterial canker in tomato can be detectedby this method.(c) Immunodiffusion: The basic principle of this test is that theantigen and antiserum are placed opposite to each other at certaindistance in soft gel (0.75 to 1%) of agar. The soluble antigen andantibodies diffuse through the gel and at the place of meeting fromantigen-antibody complex, a precipitin line appears.(i) Direct double diffusion: The halo blight pathogen in beanis detected by this method. The seed extract/leachate is inoculatedin agar plates and the plates are incubated. The bacterial coloniesare observed on agar under binocular after 24-48 hr of incubation.Then wells are made in agar medium at a distance from thebacterial colonies to be tested and diluted antiserum is added tothe wells. The precipitation line may be seen after 24-48 hr.(ii) Ouchterlony double diffusion (ODD) : Clavibactermichiganensis subsp. sepedonicus which causes ring rot in potato is