Collection of Bacterial Blight (BB) infected rice leaf samples

Infected rice leaves (BB) were collected from Basmati 385 fields of Punjab, Pakistan. Typical symptoms of BB (dry leaf whorls and amber colored ooze outs etc.) were verified. From each location most infected leaf was collected, cataloged, brought to center (CEMB) and preserved in specified refrigerator. A total of two hundred leaves were collected, isolates were purified which after molecular biology steps and statistical analysis restricted to twenty-one strains.

Bacterial isolates, culture media and isolation of genomic DNA

For Isolation of the pathogen Xanthomonas oryzae  pv. oryzae, the amber colored oozed outs from each leaf were composited, rinsed with sterile deionized water and grown as sets of three in Nutrient Broth (Difco). Bacterial growth was achieved by Culturing bacteria at 28°C (48 hours). The obtained opaque bacterial growth was streaked on Luria Broth agar plates and incubated at 28°C (24 hours).

From these plates; single bacterial colony was taken and each selected colony was homogenized by using 650 µl lysis buffer (Tris;100 mM-pH 8; EDTA; 100 mM-pH 8, NaCl; 250 mM, Sodium Dodecyl Sulfate 1% and Polyvinyl Pyrrolidone 1% – molecular weight 40,000). Incubation was done at 65°C (30 min). 100 µl of potassium acetate (3M- potassium and 5M- acetate, pH 4.8) was used to remove cellular proteins. DNA was precipitated by using equal amount of isopropanol and air dried. The DNA was dissolved in sterile- distilled water and quantified.

Achievement of economic viability (direct DNA extraction from infected leaves)

For achieving economic viability, DNA from bacteria were directly amplified by leaching it from infected leaves. Leaf fragments (2 cm in length) with advanced lesion tips were cut into small pieces (with flame sterilized scissors), soaked in sterile distilled water (400 µl) for 60 min. After 1 hour, the leaf fragments were disposed, and the bacterial cells were obtained. The cells were concentrated by centrifugation at 10,000 × g (2 min). Extra water was removed. Left over solution (25 µl) having suspended cells was treated with 70% ethanol (200 µl). Spun for 10 seconds. Cell debris was pellet out. After transferring the supernatant to a new tube, content was spun for 5 minutes. DNA was precipitated, dissolved in 10 µl of sterile water. 5 ml of DNA solution was used as template for PCR.

Oligonucleotide synthesis and polymerase chain reaction (PCR)

The sequence of the repetitive element IS1112 (Yun, 1991) was used for oligonucleotide synthesis (software: PC/GENE of IntelliGenetics; Mountain View; CA). Two primers (JEL1 5′-CTCAGGTCAGGTCGCC-3′) and (JEL2 5′-GCTCTACAATCGTCCGC-3′) were designed (from each end of IS1112). Opposite orientation was followed (3′ ends were directed outwards from each element).

 PCR amplification in a 25-µl volume was performed. Each of two opposite primers (0.5 µM), genomic DNA (20 ng), Each of four dNTPs (185 µM), Taq polymerase (2.5 U) and incubation buffer (Tris (100 mM) pH 8.3; KCl (500 mM); MgCl2 (1.5 mM), and gelatin (0.01% wt/vol). Dimethyl sulfoxide (10% vol/vol) and 1 M Tris pH 9.5 (7.5 µl) was added for long DNA fragments amplification (Cheng et al, 1994). For avoiding evaporation, reaction mixture was overlaid by one drop of mineral oil addition. Denaturation in PCR was done for 1 min (94°C). Subjected to 30 cycles of PCR (10 seconds denaturation; 94°C, 1 minute annealing; 62°C and 10 minutes extension; 65°C). Final extension was done for 15 minutes (65°C) in DNA thermal cycler of PerkinElmer Cetus, Foster City, CA. Each experiment was repeated twice.

Achievement of economic viability (direct PCR of bacterial exudate from infected leaves)

For achieving economic viability, PCR was done directly from DNA of bacterial exudates (leached from lesions). So, the chemicals, time and human efforts involved in isolation and cultivation of the bacteria were reduced. The total process shrank from six to two days which may help in future use of present study for taking quarantine measurements and forensics.

DNA fingerprints

Product of Polymerase Chain Reaction (10 µl) was used for generating DNA fingerprints. Three techniques were compared for best resolution of DNA fingerprints.

1.      20 ml of PCR product was loaded in the gel (0.5% agarose and 0.75% Synergel). Electrophoresis conditions were: 0.5x Tris-borate buffer; 125 V for 6 hours; staining with ethidium bromide. Prints of gel photographs (20 x 25 cm) facilitated the scoring no. of bands (regardless of intensity).20-40 fragments were detected.

2.      20 ml of PCR product was loaded in the gel (2% agarose). Electrophoresis conditions were: 0.5x Tris-borate buffer; electrophoresis on 125 V for 6 hours; staining with ethidium bromide). 20-40 fragments were detected.

3.      5 ml of PCR product was loaded in the gel (10% polyacrylamide). Electrophoresis conditions were: 0.5x Tris-borate buffer; gels were run for about 25 cm, at 45 W, stained by silver staining and photographed. More than 80 fragments were detected through PAGE.

Numerical analysis of data and dendrogram formation

Reference sample (C2) of IRRI, Philippine was used for comparison.  DNA fragments generated by PAGE were used for the banding pattern studies. Which was coded in binary form; presence of band was coded by 1 and the absence of band in comparison to C2 was coded by 0 (Yap, 1996). An index of genetic distance was calculated by using the unweighted pair-group method with arithmetic averages (UPGMA) method. 

Main objectives were achieved as given under:

1. Properly planned survey of Xoo affected rice growing areas of the Punjab, Pakistan was done, and diseased samples were collected.
2. For achieving economic viability, simplification in the experimental steps was done.

Based on bands similarity, twenty-one representative banding patterns were used for numerical analysis and comparison with IRRI strain. Dendrogram pattern was obtained and compared with IRRI strain C2 (fig.1).