The following points highlight the top five techniques for detection of bacterial pathogens. The techniques are: 1. Visual Separation of Infected Seed 2. Phage Method 3. Serological Techniques 4. Growing on Test 5. Indicator Test.
Technique # 1. Visual Separation of Infected Seed:
Parker and Dean (1968) found that bean seeds infected with Pseudomonas phaseollcola did not show external symptoms unless there was a very massive concentration. Exposure of most such white or light colored seed coat bean seed lots to UV emission in subdued light or darkness revealed varying percentage of seed with were incited to fluorescence.
Seeds showing the bright bluish white fluorescence most frequently produced halo blight infected plants. The seeds which cannot be recognized as infected by routine examination under ordinary light conditions, when scanned under the near UV light show tiny diffuse fluorescent patches.
Later Jansing and Rudolph (1990) developed a sensitive and quick test to determine the seed infection by Pseudomonas phaseolicola:
1. Seeds are soaked in Physiological saline solution for 20 hours at 4-6°C.
2. Saline solution, concentrated by centrifugation is streaked on a semisolid selective medium which supports the growth of all strains of the pathogen.
3. The pathovars are distinguished by determining phaseotoxin production by Escherichia coli bioassay.
4. The pathogen may be detected even when 1 out of 50,000 seeds are infected by the bacterial pathogens.
Technique # 2. Phage Method:
The technique is based on the multiplication and increase in the number of specific bacteriophage particles in the presence of the susceptible bacterium. Surface sterilized seeds are aseptically blended in nutrient broth and the macerated sample is incubated for 24 hours to permit the multiplication of phage sensitive cells of the suspected bacterial pathogen.
Aliquots of this sample suitably diluted are plated out with a definite number of phage particles and the number of plaques counted. The plating is repeated at the end of 6-12 hours of incubation and an increase in the number of plaques to a significant extent indicates the presence of the susceptible bacterial pathogen.
Katznelson and Sutton (1951) first detected and identified seed borne X. compestris pv. phaseoli. or Ps. Phaseolicola.
Technique # 3. Serological Techniques:
Guthrie (1965) developed serological technique for detection of halo blight in bean seed samples. Young, virulent cultures were suspended in formalized saline and injected into test animals.
The anti Ps. phaseolicola serum was produced with bi-weekly interveinous or sub-cutaneous injection of rabbits and sheep according to a schedule lasting eight weeks. Animals were sacrificed from the clotter blood by low speed centrifugation.
Straw coloured serum was placed in a plastic container and frozen. Infected seed was detected serologically by surface sterilization and testing the supernatant with the anti serum. The anti Ps. phaseolicola serum gave positive results only with Ps. phaseolicola and not with any other plant pathogenic and non-pathogenic bacterial cultures.
Technique # 4. Growing on Test:
Srinivasan (1973) developed a standardized method for detection of X. compestris pv. compestris in seed sample of cauliflower.
Procedure:
1. Seeds are soaked for 3-4 hours in a 20 ppm solution of Aurefungin (antifungal antibiotic), which is very effective against wide range of fungi including seed borne fungi.
2. Treated seeds are placed on plain agar plates (Bacto agar 1.5%) and plates are incubated at 20°C.
3. First observation is recorded after 8 days of incubation. The normal healthy seedlings grow rapidly, while diseased seeds show delayed germination and poor growth.
4. Emerging hypocotyls and Cotyledons appear yellowish and pulpy with the seedling collapsing on the agar surface.
5. The infected parts show prolific oozing of the bacteria as yellowish mass.
6. In infected seedlings V-shaped marginal lesions develop on emerging hypocotyls which show the bacterial ooze on microscopic examination.
7. Percentage of infected seedling is recorded by scanning the plates under the stereo binocular microscope.
Technique # 5. Indicator Test:
Main objective of this method is to produce symptoms on healthy seedlings or mature plants (Indicator plants) by using inoculum of infected or contaminated seeds. The inoculum from seeds slightly by X. compestris pv. phaseoli when inoculated, to indicator plants by hypodermic injection gave more sensitive results than serological techniques.
Procedure:
1. Seeds are pre-treated in 2.6% sodium hypo chloride for 15 minutes and rinsed with sterile water.
2. Sample is incubated for 18-24 hours at room temperature after adding sterile water.
3. From the remainder of the liquid, injections are made into the Primary leaf node of 10 day old bean seedlings.
4. Appearance of large lesions followed by systemic necrosis is the positive reaction.