Annals of Sri Lanka Department of Agriculture. 2007.9:143-148.
DNA FINGERPRINTING OF Ralstonia solanacearum E.F. SMITH, THE CAUSATIVE ORGANISM OF BACTERIAL WILT IN SOLANACEOUS CROPS P.K. SAMARAJEEWA and H.M. LIYANAGE Plant Genetic Resources Center, Gannoruwa, Peradeniya, Sri Lanka
ABSTRACT Bacterial wilt caused by Ralstonia solanacearum is reported to be one of the most destructive diseases in Solanaceous crops. The pathogen is known to be soil borne, complex and heterogeneous group of bacterial species with a wide host range. It is composed of quite distinct strains and biovars with high genetic variability and hence identification of the pathogen causes difficulties. In the case of potato, Erwinia infection causes symptoms similar to the bacterial wilt caused by Ralstonia and is difficult to distinguish. In the present study, 20 isolates from three Solanaceous crops, tomato, brinjal and potato were used and the pathogen identified on molecular basis. Primers specific to Ralstonia solanacearum were used as a specific and sensitive PCR (Polymerase Chain Reaction) based detection method that uses primers targeting the gene coding the flagella subunit, fliC. With this primer system, a specific 400bp PCR product was obtained from all the isolates of Ralstonia showing the potential on an ideal marker to identify the pathogen. Erwinia isolate extracted from infected potato was used as a negative control, which did not give any amplification product with this specific primer pair. KEYWORDS: Bacterial wilt, DNA fingerprinting, Solanaceous crops.
INTRODUCTION Ralstonia solanacearum bacterium is known to be a complex and heterogeneous group of species (Palleroni et al., 1973). It has a wide host range affecting many Solanaceous crops (He et al., 1983). In Sri Lanka, it has been reported to infect potato, tomato, brinjal, chilli and several other nonSolanaceous crops and plants (Kelaniyangoda, 1995). According to available literature, it is obvious that the pathogen is composed of a number of distinct strains and biovars (Senevirathna, 1969; Bandara, 1983; Kelaniyangoda, 1995). Previous studies carried out locally have indicated that there are several biovars of the pathogen affecting several Solanaceous crops cultivated in different agro ecological regions of the country, and mostly biovar type 3 was found to be associated with the bacterial wilt disease in tomato (Kelaniyangoda, 1995). The location effect has also been observed with respect to disease incidence which attribute to different strains of the pathogen or climate and soil characteristics in each location (Hayward, 1991). Such diversity of the pathogen and the location specific nature of resistance pose many problems, particularly in producing resistant cultivars (Joseph et al., 1975).
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Reports indicate that the varieties developed for resistance to bacterial wilt have shown to be susceptible in certain regions of Sri Lanka. Earlier work carried out by analyzing the bacterial DNA using Randomly Amplified Polymorphic DNA (RAPD) revealed the existence of a vast genetic variation of the pathogen collected from different locations (Gunathilake et al., 2004). This might be due to the exchange of megaplasmid among strains. However, despite their great variation, the genome is still expected to posses many similar regions of identity. Therefore, several approaches mainly based on the amplification of ribosomal gene sequences (i.e. 16s/16s-23s intergenic spacer region of the ribosomal DNA [rDNA]) (Seal et al., 1993.) have been attempted to identify the bacterial pathogen. However, due to the high degree of conservation of the ribosomal genes within the genes Ralstonia, 16s rDNA sequences similarities between species is expected to be as high as 98% (Seal et al., 1993). In view of this, approaches that use a primer targeting the gene encoding the flagella subunit fliC has been established in order to find a common band, possesed in all the isolates of R. solanacearum as an effective way to identify the pathogen. The main objective of the present study was to identify R. solanacearum in different Solanaceous hosts with a PCR based array using a flagella specific primer pair for subsequent use in disease diagnosis, quarantine inspections and seed certification. MATERIALS AND METHODS Collection of isolates Plant samples of tomato, potato and brinjal, with typical symptoms of bacterial wilt, were collected from farmer fields in several locations. The infected plant stems were thoroughly washed separately, first with tap water and then with sterilized distilled water. The basal piece of the stem (5cm) was removed and placed in a test tube containing 20ml sterilized distilled water. The bacterial ooze from the stem tissues was allowed to stream into sterilized distilled water. This milky bacterial suspension was then plated on culture plate containing SPA (Sucrose Peptone Agar) medium (Gunathilake et al., 2004). pH was adjusted to 7.2 with 40% NaCl prior to autoclaving at 121째C for 20 minutes. After 24 hours, the cultures were sub cultured to obtain single colonies. Extraction of bacterial DNA Overnight grown 20 cultures of R. solanacearum and one culture of Erwinia (received from HORDI) in liquid broth were used for DNA extraction. Genomic DNA of those strains was obtained by SDS (Sodium
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Dodicyl Sulfate) and proteinase K cell lysis, selective precipitation of cell debris and polysaccharides with CTAB and isopropanol precipitation of DNA according to the protocol of Wilson (1997), with slight modifications. The extracted DNA was dissolved in 50µl of Tris-EDTA buffer (TE) and diluted to obtain a final concentration of 20ng/µl using the spectrophotometer reading at the 260nm and 280nm wavelengths. Polymerase chain reaction (PCR) amplification For PCR amplification of R. solanacearum fliC gene fragment, Rsol_fliC primer system (forward and reverse) giving an amplicon size of 400bp was used. The reaction mixture contained Ex Taq buffer 2.5µl, 0.2mM each dNTPs, 3.75mM MgCl2, 100nM concentration of each primer (forward and reverse) and 2U of ampli Taq in a total volume of 25µl. PCR cycle consisted of initial denaturation step of 5 minutes at 94 °C and 30 cycles with denaturation, 30 seconds at 94°C, primer annealing, 1 minute at 56°C and extension, 1 minute at 72°C followed by a final extension of 8 minutes at 72°C and soaking at 4°C. The amplified products were analyzed by gel electrophoresis in 1.4% weight/volume agarose gels in 0.5 x TBE buffer at 5V/cm for 180 minutes. The gels were stained with ethidium bromide 0.5µg/ml for 15 minutes. Observations were made under the BIO-RAD gel documentation system with Quantity one software package. RESULTS AND DISCUSSION There was a high yield of DNA from each isolate. Therefore each sample was diluted to obtain 20ng/ml concentrations for better amplification. This was subsequently used for PCR amplification to obtain Ralstonia specific band. Figure 1 shows the gel profile obtained for the specific primer pair. According to the gel profile, DNA of all the isolates (from tomato, potato and brinjal) (Fig. 1, Lane 1-10) gave amplification at 400bp size fragment with primer Rsol_fliC, revealing the primer as a potential marker to detect R. Solanacearum. Furthermore, this primer did not give any amplification with respect to Erwinia isolate, extracted from potato (Fig. 1, Lane 11). Thus the results suggest that this primer can be used to identify Ralstonia from Erwinia, which is mostly found co-infected with Ralstonia in potato.
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L1 1
2 3 4
5 6 7
8
9 10 L2 11 10000bp
1000 bp 400 bp 300 bp 200 bp 100 bp
400 bp 300 bp 200 bp 100 bp 50 bp
Specific amplification at 400bp
Figure 1. Gel profile showing 400bp specific band produced by the isolates extracted from tomato, potato and brinjal with the specific primer pair. Lane 1-7 isolates extracted from different tomato cultivars. Lane 8 and 10 isolates extracted from potato. Lane 9 isolate extracted from brinjal. Lane 11, Erwinia isolate extracted from potato. L1, 100 bp DNA low ladder. L2 wide range DNA ladder.
According to the observations with random primers, it is quite evident that the genetic variability of the pathogen is very high. Moreover; we found that it was quite difficult to obtain a common band for Ralstonia using the RAPD technique (Gunathilake et al., 2004). Furthermore a virulence specific primer was tested and that resulted in a ladder of bands (unpublished data). Therefore, there was a need to have a specific primer pair to fingerprint. Hence the primers that had been designed for the sequence of flagella sub unit fli C was found successful (Schonfeld et al., 2003). This was based on fliC gene sequence of Ralstonia solanacearum strain K60 (available at Gene Bank under accession number AF283285 the Ral_fliC primer system (Forward [5’ CCTCAGCCTCAATASCAACATC 3’] and reverse [5’CATGTTCGACG TTTCMGAWGC 3’]), giving an amplicon size of 724bp. To synthesize a more specific primer, 724bp product had been used and the designed Rsol_fliC primer, (Forward [5’GAACGCCAACGGTGCGAACT3’] and reverse [5’ GGCGGCCTTCAGGGAGGTC 3’] yielded an amplicon size of 400bp. This primer was designed in order to contain more than 60% GC for proper separation. Due to their structure, which is conserved in the terminal region that flanks a variable central region, flagella genes are regarded as good candidates for PCR based detection of pathogen (Winstanley and Morgen, 1997). The selected 400bp fragment is located within the central region of the fliC gene, where the greatest sequence variability between strains can be expected because this region codes for a non-functional domain of the protein
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(flagellin). Furthermore, the gene is not a part of the pathogenicity island, nor is found to be transmitted by horizontal gene transfer (Winstanley and Morgen, 1997). Hence it is a good and a powerful phylogenic marker. Another useful aspect of this approach is the possibility of detecting avirulant forms of the pathogen. Although flagellum dependant motility was shown to play an important role in virulence during the early stages of disease manifestation, i.e., invasion and dissemination, (TransKersten et al., 2001) the genes needed for flagellum constitution are not directly involved in virulence, i.e., they are not part of the virulence regulatory network (Allen et al., 1997 and Clough et al., 1997). Therefore, 400bp Rsol_fliC fragment is detectable in all the strains irrespective of their virulence. PCR based disease diagnosis in plants is more efficient compared to traditional methods where time consuming steps such as culturing, re-inoculation etc. are involved. This method can be used to detect infections in other important crops such as potato especially during disease diagnosis, quarantine inspections and seed certification. Moreover this identification system would enable to avoid difficulties during differentiating the pathogen such as Erwinia that causes similar pathogenic conditions in potato. CONCLUSIONS The present study obtained the specific 400bp band for all the Ralstonia solanacearum isolates from all the Solanaceous plants tested, revealing it as a potential marker for efficient identification of the pathogen. ACKNOWLEDGEMENTS Authors wish to acknowledge the funds received from SLUSDA under project number 04. Acknowledgement is also due to staff of HORDI, Gannoruwa, for providing Erwinia cultures. REFERENCES Allen, C., J. Gay and L. Simon-Buela. 1997. A regulatory locus, phesr controls polygalacturonase production and other functions in Ralstonia solanacearum. Plant microbe interaction 10:1054-1064. Bandara, J.M.R.S. 1983. Biotype distribution of vascular wilt pathogen Pseudomonas solanacearum in Sri Lanka. Journal of National Science Council of Sri Lanka 11: 65-76.
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