Sy Nguyen, Mohammed Ziaul Hoque and Peer Schenk
Abstract
Ralstonia solanacearum (Smith) is an important pathogen worldwide. A new pair of primers, UDP-F and UDP-R, was designed based on the 159 bp target fragment of Chen et al. (2010) which is originated from the sequence of the upstream region of the UDP-3-O-acyl-GlcNAc deacetylase gene conserved only in R. solanacearum. The result indicated that this pair of primers could be used to detect R. solanacearum directly from the bacterial suspensions by using conventional polymerase chain reaction (PCR) technique. However, these primers had so far only been tested with close relatives (Pseudomonas syzygii and the blood disease bacterium (BDB)) of R. solanacearum, so further experimentation should be carried out to optimize the PCR technique and check the specificity and sensitivity of primers and the protocol. Furthermore, the application of these primers in real time PCR should be investigated.
Introduction
Ralstonia solanacearum (Smith) is a soil-borne pathogen which causes bacterial wilt of numerous important crop plants in the world (Anonymous 2004). R. solanacearum is defined as a species complex because the relatedness between isolates of this species is less than 70% in DNA-DNA homology (Fegan & Prior 2003). Previously, R. solanacearum strains were classified into five races based on the host range and six biovars on the differences of biochemical features (Fegan & Prior 2003). By analyzing restriction fragment length polymorphism (RFLP), R. solanacearum can be separated into two divisions: divisions 1 including biovars 3, 4 and 5 which were isolated from Asia; division 2 with biovars 1, 2 and N2 which were detected in the Americas (Fegan & Prior 2003).
According to Anonymous (2004), accurately identifying bacterium R. solanacearum includes observations of wilt symptoms on leaves and stems; isolation on SMSA and Kelman’s tetrazolium medium; identification based on the biochemical characteristics, such as the utilization of maltose, lactose, cellobiose; and pathogenicity tests on susceptible host plants. This conventional diagnostic procedure is time consuming.
The development of biotechnology in recent decades, especially polymerase chain reaction (PCR) techniques, has allowed the detection, but also further classified this bacterium into races, biovars and subgroups (Horita, Yano & Tsuchiya 2004; Ito et al. 1998; Pastrik, Elphinstone & Pukall 2002; Pastrik & Maiss 2000; Van der Wolf et al. 2004; Villa et al. 2003; Weller et al. 2000). A pair of primers 759 and 760 was designed by Opina et al. (1997) which demonstrated to be specific for R. solanacearum and two of its close relatives (Pseudomonas syzygii and the blood disease bacterium (BDB)). The PCR protocol for and specificity of these primers were checked through studies of Ito et al. (1998) and Villa et al. (2003). However, it is impossible to distinguish R. solanacearum, P. syzygii and BDB by using PCR with this pair of primers alone (Villa et al. 2003). Chen et al. (2010) developed a pair of primers for a real time PCR assay to quantify and detect R. solanacearum in soil and plant tissue samples. These primers were designed based on the sequence of the upstream region of the UDP-3-O-acyl-GlcNAc deacetylase gene which is conserved only in R. solanacearum, but they have not been tested with P. syzygii and BDB.
To assess the ability of using PCR technique alone to detect bacterium R. solanacearum, a new pair of primers (UDP-F/UDP-R) was designed based on the target fragment of Chen et al. (2010) to be expected to amplify a 96bp fragment. This experiment will investigate if primer UDP-F/UDP-R is specific to detect R. solanacearum from infected plant material and bacterial suspensions by using PCR method and in comparing it to primer 759/760.
Materials and methods
One diseased tomato plant (M14), four bacterial suspensions (M10, M13, M15, M16) and two isolates of R. solanacearum (egg plant 5446 and tomato 5818) were provided by DEEDI, Queensland. The bacterial suspensions were prepared from the diseased plant following the protocol of Fegan (2005).
With sample M14, three or four small pieces of vascular tissue from the diseased plant were cut and put in 1 ml sterile distilled water in 1.5 ml Effendorf tubes for 10 – 15 min until oozing can be seen out of the tissue. Then the ooze was directly used as template to run PCR.
To detect the presence of bacterium R. solanacearum in the plant and provided bacterial suspensions, PCR technique was used with two pairs of primers, 759 (5’-GTCGCCGTCAACTCACTTTCC-3’) and 760 (5’-GTCGCCGTCAGCAATGCGGGAATCG-3’); and UDP-F (5’-TGCCTGCCTCCAAAACGAC-3’) and UDP-R (5’-GATGACGCAACGGCATGG-3’). The reaction for primers 759 and 760 was set up and optimized by Opina et al. (1997) including 1X PCR buffer with MgCl2, 200µM of dNTPs, 0.5 pmol/ µl of each primer, 0.01UI/µl of Taq DNA polymerase. 2 µl of each bacterial suspension were added as template. PCR conditions were as follows: preincubation at 94oC for 3 min, annealing at 53oC for 1 min and extension at 72oC for 1.5 min, followed by 30 cycles of 940C for 15s, 60oC for 15s 72oC for 15s, and completion by an elongation step at 72oC for 5 min(Opina et al. 1997). The reaction for primers UDP-F and UDP-R is almost the same as above, except the concentration of Taq DNA polymerase is 1 UI/reaction for the 25 µl reaction. PCR conditions for primers UDP-F and UDP-R included preincubation at 95oC for 5 min, followed by 35 cycles of 950C for 1 min, annealing at 60oC for 1 min and extension at 72oC for 1min, and completion by an elongation step at 72oC for 10 min. The PCR technique was carried out three times for each set of primers.
One negative control (using sterile distilled water as template) and two positive control (using both DNA and suspension from bacterium R. solanacearum) are included to check the operation of reactions and the contamination. The PCR products were electrophoresed on 2% agarose gel to check the presence of PCR products (about 281-283 bp for primers 759/760 and 96 bp for primers UDP-F/UDP-R).
Result
The results showed that when running PCR with primer pairs 759/760 and UDP-F/UDP-R, targeted products (281-283 bp band and 96 bp, respectively) were obtained (Fig. 1 and Fig. 2). Consistently, two samples (M14 and M13) generated target bands in both sets of primers. It can be said that diseased tomato plant provided was caused by R. solanacearum and bacterial suspension M13 contained bacterium R. solanacearum. Sample M16 were detected by primers UDP-F/UDP-R but not by the others (Fig. 3). This sample just showed the amplification in the last time of running PCR. No amplification was obtained from sample M15.
Fig. 1. PCR amplifications of primers 759/760 with about 281-283 bp fragments
Notes: M: molecular size makers (100bp), M14: suspension extracted from diseased tomato plant, M13, M15, M16: Bacterial suspensions, N: negative control. T1: positive control with DNA of R. solanacearum (tomato 5818), T2: positive control with bacterial suspension of R. solanacearum (tomato 5818).
Fig. 2. PCR amplifications of primers UDP-F/UDP-R with 96 bp fragments
Notes: M: molecular size makers (100bp), M14: suspension extracted from diseased tomato plant, M13, M15, M16: Bacterial suspensions, N: negative control. T1: positive control with DNA of R. solanacearum (tomato 5818), T2: positive control with bacterial suspension of R. solanacearum (tomato 5818), E1: control with DNA of R. solanacearum (egg plant 5446).
Fig. 3. PCR amplifications of primers UDP-F/UDP-R with 96 bp fragments (up) and of primers 759/760 with about 281-283 bp fragments (down)
Notes: M: molecular size makers (100bp), M14: suspension extracted from diseased tomato plant, M13, M15, M16: Bacterial suspensions, N: negative control. T1: positive control with DNA of R. solanacearum (tomato 5818), T2: positive control with bacterial suspension of R. solanacearum (tomato 5818), E1: control with DNA of R. solanacearum (egg plant 5446).
However, in the first time running with primers UDP-F/UDP-R, no amplification was achieved for the positive control with bacterial suspension of R. solanacearum (T2) (Fig. 2). This could be caused by handling errors when carrying out the PCR. In addition, primers UDP-F and UDP-F amplified two unspecific bands in sample M10 (about 500bp and 600 bp) and the positive control with DNA of R. solanacearum egg plant 5446 had failed (Fig. 4).
Fig. 4. PCR amplifications of primers UDP-F/UDP-R with 96 bp fragments
Notes: M: molecular size makers (100bp), M14: suspension extracted from diseased tomato plant, M10 and M13: Bacterial suspensions, N: negative control. E1 and T1: positive controls with DNA of R. solanacearum (egg plant 5446 and tomato 5818, respectively), E2 and T2: positive controls with bacterial suspensions of R. solanacearum (egg plant 5446 and tomato 5818, respectively).
Discussion
Primers UDP-F/UDP-R were designed by Peer Schenk and Mohammed Ziaul Hoque with the purpose to apply in real time PCR. That is the reason why the size of the targeted band is quite small (96bp), yet this pair of primers worked quite well in conventional PCR technique.
Although the specificity of primers UDP-F/UDP-R was not checked with other bacterial species, the result was quite similar to the result of primers 759/760 which is demonstrated to be specific to R. solanacearum and two close relatives (P. syzygii and the BDB) (Ito et al. 1998; Opina et al. 1997; Villa et al. 2003). Fortunately, P. syzygii and the BDB are not present in Australia. Therefore, both sets of primers can be used to specifically detect bacterial wilt R. solanacearum in Australia.
Once again, the PCR protocol for primers 759/760 which is developed and optimised by Opina et al. (1997) consistently amplified the targeted band (about 281-283bp). Although there is one base pair different in the sizes of PCR products between studies of Opina et al. (1997) and Villa et al. (2003), it is impossible to distinguish them in gel. Therefore, it is not sure what the exact size of PCR product of primers 759/760 was in this experiment.
The result indicated that both sets of primers can be employed to directly detect the bacterial wilt from the ooze extraction by conventional PCR technique. The ooze should be used immediately after extracting from the diseased plant tissue. The sensitivity of reaction decreased when the extraction was put at room temperature overnight (data not shown).
The sensitivity of primers UDP-R and UDP-F has not investigated yet. Further experiments should be carried out to check the specificity and sensitivity of primers and protocol.
Acknowledgement
We thank Dr. Young Anthony for providing the diseased plant, bacterial suspensions and R. solanacearum isolates; Apollo Gomez for his useful comments for my research proposal and Dr. Jitender Singh for his consultation during this project.
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