The aim of the present study was to develop a very fast and simple genomic DNA isolation method for Ralstonia solanacearum which infest potato tubers. One hundred potato tubers were collected and ten composite samples were prepared having 10 tubers each. Four different DNA isolation methods were used for bacterial genomic DNA isolation present in tubers. PCR with R. solanacearum specific primers and pathogenicity tests were performed. Out of four methods two gave PCR amplifiable DNA. The simplest method was boiling the cell lysate for 5 min, vortexing for 2 min then extraction with phenol chloroform method. This method provides significant amount of DNA which is free from contaminants thus rendering the DNA amicable to PCR amplification. The developed method would be useful for quick and sensitive detection of this pathogen in seed potatoes and would be beneficial to stop the further spread of pathogen.
The potato is the most popular non-cereal food crop of the world and ranks fourth in importance after rice, wheat and maize. Potato is the most widely consumed vegetable in India. India is the second largest producer of potato contributing to 10% - 11% of the world potato production after China, and contributes a share of 22% of the world production [
The ability of the bacterium to infect a large number of hosts makes its control rather difficult. To date, no effective control methods exist for bacterial wilt disease.
Resistant varieties, field sanitation, crop rotation and use of bactericides have met, if at all, with only limited successes [
Use of healthy seeds is the most effective way to avoid introduction and dissemination of this pathogen in disease free areas. In order to have healthy seeds very sensitive and accurate detection methods are required. For quicker detection of the pathogen, immunodiagnostics [
Despite the high sensitivity and speed of PCR method, detection of R. solanacearum in potato tubers using PCR assay is not certain [
In the present study we are reporting a very fast and effective bacterial genomic DNA isolation method for PCR detection of R. solanacearum in potato tubers.
For standardization of DNA isolation method, one hundred tubers were collected from wilt infested fields of Central Potato Research Institute, Shimla, India during one cropping season. Tubers were randomly selected from heaps at harvest, at the beginning of the storage period, between 4 and 12 weeks after harvest.
The tubers were washed under running water and airdried. One hundred tubers were randomly divided into 10 composite samples of 10 tubers each. The skin was removed at the heel end of the tuber with a clean and disinfected vegetable knife so that the vascular tissues first became visible. A small conical core (3 - 5 mm diameter) of vascular tissue at the heel end was carefully cut out. The amount of nonvascular tissue was kept to a minimum. A composite sample of heel ends of 10 tubers was crushed together with the help of pestle and mortar and collected in a sterile Falcon tube containing 5 mL of double distilled water making a homogenate then samples were allowed to stand for 30 min.
The supernatant was gently decanted in a centrifuge tube and centrifuged at 7000 ×g for 15 min at 10˚C. The pellet was resuspended in 1-mL sterile water and a cell homogenate was prepared.
Following four different methods were used for bacterial chromosomal DNA isolation from cell homogenate.
a) The cell homogenate was boiled for 5 min and directly used for PCR amplification.
b) The cell homogenate was boiled for 5 min then vortexed for 2 min and DNA was extracted with phenol: chloroform:isoamylalcohol (25:24:1) and precipitated by adding 0.1 volume of ammonium acetate and 2 volumes of chilled ethanol [
c) The cell homogenate was treated with Murmur’s method [
In this method, to 1-mL of cell homogenate 100 µL of extraction medium (0.15 M NaCl; 0.1 M Na2 EDTA) and 100 µL of lysozyme (10 mg·mL–1) was added, incubated at 37˚C for 30 min shaking occasionally. After incubation the lysis was completed by adding 200 µL of 25% SDS solution. The mixture was heated at 60˚C in a water bath then cooled to room temperature. To this mixture sufficient amount of 5 M sodium per chlorate was added to a final concentration of 1 M. An equal volume of chloroform:iso amyl alcohol (24:1) was added to the lysed preparation suspended in 1 M sodium per chlorate and shaked slowly (30 - 60 oscillations·min–1) for 30 min at room temperature. The resulting emulsion was separated by centrifuging for 5 min at 10,000 rpm at room temperature. After centrifugation top clear aqueous phase was pipetted out and placed in a fresh eppendorf tube. DNA was precipitated out by adding 2 volumes of 95% ethanol to the clear aqueous phase. This crude DNA preparation was dissolved in 900 µL of dilute (10–1 fold) saline citrate (0.15 M sodium chloride, 0.015 M tri sodium citrate) and an even solution was prepared. To this even solution 100 µL of 3 M sodium acetate and 1 mM EDTA pH 7.0 solution was added and a uniform suspendsion was prepared. The prepared suspension was transferred to another tube containing 540 µL of isopropanol and DNA was precipitated out. The DNA was washed first with 70% ethanol and then with 95% ethanol. Removed alcohol from the precipitated DNA by blotting with a clean piece of filter paper and then finally dissolved the DNA in 100 µL of ultra pure sterile water.
d) The cell homogenate was treated with ProteinaseK-SDS lysis miniprep method for genomic DNA extraction [
For genomic DNA isolation 1.5 mL of cell homogenate was centrifuged for 2 min in a micro centrifuge. Resuspended the pellet in 567 μL TE buffer by repeated pipetting then added 30 μL of 10% SDS and 3 μL of 20 mg·mL–1 proteinase K to give a final concentration of 100 μg·mL–1 proteinase K in 0.5% SDS. Mixed thoroughly and incubated for 1 hr at 37˚C. To this mixture added 100 μL of 5 M NaCl, mixed thoroughly then added 80 μL of CTAB/NaCl solution. Mixture was incubated for 10 min at 65˚C. An approximately equal volume (0.7 to 0.8 mL) of Phenol:Chloroform:Isoamyl alcohol (25:24:1) was added and mixed thoroughly, and spun for 4 to 5 min in a micro centrifuge. The aqueous, viscous supematant was removed to a fresh microcentrifuge tube, leaving the interface behind. An equal volume of chloroform:isoamyl alcohol (24:1) was added to extract thoroughly, and spun in a microcentrifuge for 5 min. The aqueous phase was transferred to a fresh tube. DNA was extracted with 0.6 vol. isopropanol. The precipitate was washed with 70% ethanol. The supernatant was removed and pellet was briefly dried. The pellet was resuspended in 100 μL TE buffer. The precipitated DNA pellet was washed with 70% ethanol and resuspended in 100 μL sterile ultra pure water.
Quantity and quality of DNA preparations were checked by standard spectrophotometry and gel electrophoresis and all the samples were diluted to 10 ng DNA per microlitre.
Ralstonia solanacearum infested potato tubers were used as positive control and potato tubers that were clean to Ralstonia solanacearum were included as negative control.
Total 10 DNA isolate mixtures were used in amplification. PCR was performed in a reaction volume of 25 μL containing 1X Taq polymerase buffer with 1.5 mmol·L–1 of MgCl2 (PE Applied Biosystem, Foster City, CA, USA), 100 μL·mol·L–1 of each dNTP (PE Applied Biosystem), 0.5 pmol of primer, 10 ng of genomic DNA and 1.0 U of Taq DNA polymerase (AmpliTaq; PE Applied Biosystem).
PCR amplifications were performed in a thermal cycler (GENAMP PCR SYSTEM 9700; Applied Biosystems). Primers specific to R. solanacearum 16S rDNA (Y2-OLI 1) [
Reproducibility of PCR reactions was checked by performing duplicate reactions for each template DNA isolated. DNA templates used in the study were able to give reproducible PCR amplification results in duplicated experiment. Amplified products were separated by electrophoresis in 1.0% agarose gels with 0.5 μg·mL–1 of ethidium bromide at 4 V·cm–1 constant voltages for 2 h. The gels were scanned in FluorS MultiImager (Bio-Rad Laboratories, Hercules, CA, USA).
All the Positive results of PCR were confirmed by pathogenicity test. To check the pathogenicity around 100 R. solanacearum isolates were isolated from the same infected potato (Solanum tuberosum L.) tubers which were used for bacterial genomic DNA isolation, by standard procedure on casamino acid peptone glucose (CPG) agar medium [
Biovar of each isolate was determined by standard procedure [
Race identification of the isolates was done based on the procedure described by He et al. [
Pathogenicity test was performed by inoculating the tomato plants as described by Winstead and Kelman [
Appearance of purple color within 10 - 60 s in oxidase test; violet color in urease test; formation of a fine threadlike slime in KOH test indicated that all the isolates belonged to R. solanacearum. All 100 isolates belonged to race 1 and biovar III.
The wilting was recorded in seedlings up to 2 - 3 weeks after inoculation.
The specific primers Y2 and OLI 1 amplified a specific DNA fragment of size approximately 288 bp with bacterial genomic DNA which was extracted through method b and method d. DNA isolated through protocol a and c was not PCR amplifiable.
Ralstonia solanacearum causes extensive losses to crops in subtropical and tropical areas thus the rapid identification of the pathogen is most important for disease management. To our information, there was no fast bacterial genomic DNA isolation method from potato tubers that can be used in PCR assay detecting directly the pathogen R. solanacearum from potato tubers without culture; although many related methods were reported [10,21]. In the present study four different protocols were screened, and found that PCR amplifiable DNA samples were obtained with the method (b) and (d) but the fastest method was boiling the cell homogenate, vortexing and then purification with phenol chloroform (b). This method takes only 30 min to isolate PCR amplifiable bacterial genomic DNA from potato tubers. One DNA sample was also amplified in method a, lane 16; however the amplification results were not reproducible and the reliability of each DNA extraction method was defined in terms of consistency of the results in the three replicates.
Theoretically, the PCR technique is able to detect as few as single copy of target DNA. In practice, however, sensitivity of PCR reaction depends upon recovery of target DNA sequence and efficiency of the PCR reaction. Taq DNA polymerase enzyme is sensitive to inhibition by compounds present in biological samples [
tuber homogenate by crude cell lysis without purification step but the results were inconsistent. Pastrik and Maiss (2000) [
Co-solvents have been reported to be useful for some PCR reactions [
The authors are grateful to Director AIB, Amity University, Noida UP for providing necessary facilities to undertake this study.