Protocol

Author

Kale Prashant B.

Overview

Plant pathogenic bacteria cause diseases and economic losses to crop plants. Precise identification of bacterial pathogens is necessary for correct disease diagnosis. It is time consuming by conventional techniques but with molecular techniques it is becoming less time consuming and increasingly reliable. PCR based diagnosis requires genomic DNA isolation and it requires about two hours. To minimize the time required for PCR analysis one can use following protocol.

Background

• Isolation of plant pathogenic bacteria.
• Incubation of pure culture.
• Preparation of template for PCR i.e. Boiling of single pure colony
• Use of template in PCR.

Materials/Equipment

• Diseased plant tissue
• Nutrient Agar or Broth media i.e. NA or NB
• PCR thermal cycler
• Centrifuge
• Doubled distilled water
• PCR reaction Mixture

Procedure

1. Isolation of plant pathogenic bacteria.
   • Infected tissue is surface sterilized and used for bacterial pathogen isolation on NA medium.
   • Suspected colonies are subjected for purification.
   • Purified bacterial pathogen is incubated and single colony is used for next experiment.
2. Preparation of template Boiling of single pure colony:
3. Log phase single colonies of pure bacterial plant pathogen are taken in the PCR tube aseptically.
4. Double distilled water, 25 μl is added to the each tube and vortexed for a minute for washing purpose.
5. Centrifugation at 8-10000 rpm for a minute sediments bacterial colony.
6. Supernatant is removed aseptically with fine tip and freshly 25 μl, double distilled water is added to the tube.
7. The tubes are vortexed for a minute to mix pellet properly in DDW
8. Then put tubes properly in thermal cycler for 1 cycle of 99 ⁰C for 10 minute and hold at 4⁰C for 5 minute.
9. Heat treated bacteria further centrifuged at 8-10000 rpm for a minute to remove cell debris
10. Take supernatant and can be diluted according to need i.e. dilution depends on the size of colony take during first step centrifugation steps and handeling
11. The supernatant can be used as a template in PCR

Template from the method is useful for the conventional PCR, Box, Multiplex and Real Time PCR. The method hence helps in reducing the diagnosis time.

Notes and troubleshooting tips

Instead of Double distilled water one can use 0.85% NaCl solution during washing the colony and TE buffer i.e. EDTA 1.0 mM Tris-HCl 10 mM, pH 8.0 during boiling see. Araujo et. al. 2004.

Literature references

Araujo WL, de Angellis DA and Azevedo JL (2004) Direct RAPD Evaluation of Bacteria without Conventional DNA Extraction. Braz. arch. biol. technol. vol.47 (3)

Ghasemie E, Kazempou MN, Padasht F (2008) Isolation and Identification of Xathomonas Oryzae pv. oryzae the Causal Agent of Bacterial Blight of Rice in Iran J. Plant protection research 48(1)