Evaluation of Colorimetric LAMP Assay for Visual Detection of Ralstoniasolanacearum in Potato Shipments at Quarantine Stops in Ira

Document Type : Research Article

Authors

1 Department of Plant Protection, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran

2 Assistant Professor, Seed and Plant certification and registration institute. Agricultural Research, Education and Extension organization (AREEO), Tehran, Iran

3 Ferdowsi

Abstract

Introduction: Race 3/ biovar 2 of this pathogen causes bacterial blight of solanaceous plants especially potato in both tropical and temperate regions and results in great economic losses worldwide. Infection is prevented via quarantine or incineration of infected plant materials. However, the use of healthy seed tubers is the most effective way to avoid dissemination of this harmful plant pathogenic bacterium to pathogen-free areas. Amplification of functional genes such as endoglucanase and hrpB and fliChas been used as an alternative to study R. solanacearumspecies complex. In order to facilitate detection of R. solanacearumin imported seed tubers and identify high-risk fields and stores where inoculums population is low, loop-mediated isothermal amplification (LAMP) reaction as a potentially fast and cost-effective method was used. The attention of the present study wason evaluation of latent infection in potato tubers with R. solanacearum bacterium targeting the fliC gene by colorimetric LAMP assay. The LAMP protocol was compared with the conventional PCR which routinely used at most quarantine stops.
Materials and Methods: In this study, bacterial strains were isolated on tetrazolium chloride (TZC) agar medium. Pathogenicity assay was carried out on tomato and potato seedlings under greenhouse conditions. Total DNA of bacterial strains was prepared using Chen and Kao (1993) protocol. In some cases, the boiled filtrated potato extract was used directly in molecular experiments. Identification of R. solanacearum strains at species and phylotype levels and biovar determination were done based on literature. The PCR products were analyzed on 1.2 % agarose gels in TBE buffer and visualized with UV light. To detect R. solanacearumin symptomatic and symptomless tissues, conventional PCR and LAMP assay according to fliC gene were performed and compared with each other. In order to check amplified LAMP products in visual assessment, the existence of magnesium pyrophosphate precipitate in tested tubes was analyzed. Furthermore, change in colourdue to the reaction was evaluated bynaked eye and UV treatmentafter adding the calcein. Finally, the LAMP products were examined by electrophoresis through 2% agarose gel after staining with green viewer. To determine limit of the LAMP assay, seven dilution series (2×107 to 2×10 CFU/ml) were prepared and 2 μl of each dilution was used for LAMP.
Results and Discussion: Bacterial colonies showed mucous and opaque appearance with red centre and whitish periphery on TZC agar medium were selected for further study.In plant bioassay two weeks after bacterial inoculation, different levels of wilting were observed on tomato and potato seedlings.The expected 281 and 372 bp PCR-amplified fragments was observed in all strains supporting species and phylotype identification, respectively. Moreover, utilization of carbon sources indicated that the strains were related to biovar 2. Furthermore, all strains from potato were screened using Ral-fliC and Rsol-fliC primers. A 400 bp PCR product specific to R. solanacearum was obtained from all strains. Sequencing three purified PCR products confirmed the right amplification of fliC gene specific to R. solanacearum.
The amplified products were detected by visual observation which the white turbidity of the reaction mixture by magnesium pyrophosphate was seen after 55 min. An alternative indicator to visually check the positive reactionwas calceinwhich was based onobservation ofyellow (green) in colour at the absence (presence) of UV light in infected samples and clear colour in negative control. Detection limits in pure cultures and infected potato extract were also determined. In conventional fliC-PCR, the detection limit rangedapproximately from 10 3 to 10 4cfu ml−1in both infected potato extract and pure cultures. Moreover, the lowest amount of consistently tested positive through LAMP assay was 10 4cfu ml−1 for both cases.
Although the sensitivity of the fliC LAMP assay wasequal or lower than that of the conventional PCR, the accuracy of fliC LAMP seems to be sufficient toreliably confirmthe presence of R. solanacearum in potato samples. In addition, LAMP protocol assay is time-consuming procedure, does not require expensive equipments, provides visually detection of positive reactions and can apply to survey possible infection in host plants.
Conclusion: Consequently, LAMP assay with ashort nucleic acid extraction step like as boiling treatment and efficient visualization processes such as calcein provide suitable preliminary data for screening of pathogen–free tubers prior to storage and during transportation.

Keywords


Boudazin G., Le Roux A.C., Josi K., Labarre P., and Jouan B. 1999. Design of division specific primers of Ralstonia solanacearum and application to the identification of European isolates. European Journal of Plant Pathology. 105:373–380.
2-Chen W.P., and Kuo T.T. 1993. A simple and rapid method for the preparation of gram-negative bacterial genomic DNA. Nucleic Acids Research. 21: 2260.
3-Fegan M., and Prior P. 2005. How complex is the Ralstonia solanacearum species complex? In: Allen C, Prior P, Hayward AC, eds. Bacterial Wilt: The Disease and the Ralstonia solanacearum Species Complex. St. Paul, MN, U.S.A.: American Phytopathological Society Press, 449-461.
4-Fu S., Qu G,, Guo S,, Ma L,, Zhang N. 2011. Applications of loop-mediated isothermal DNA amplification. Applied Biochemistry and Biotechnology, 163: 845–850.
5-Hayward A.C., 1991. Biology and epidemiology of bacterial wilt caused by Pseudomonas solanacearum. Annual Review of Phytopathology. 29: 65–87.
6-Hayward A.C. 1994. Systematics and phylogeny of Pseudomonas solanacearum and related bacteria. In: Hayward AC, Hartman GL, eds. Bacterial Wilt: the Disease and its Causative Agent, Pseudomonas solanacearum. Wallingford, UK: CAB International, 123–35.
7-Kaneko H., Kawana T., Fukushima E., and Suzutani T. 2007. Tolerance of loop-mediated isothermal amplification to a culture medium and biological substances. Journal of Biochemistry and Biophysical Methods, 70: 499-501.
8-Kubota R., Vine B.G., Alvarez A.M., and Jenkins D.M. 2008. Detection of Ralstonia solanacearum by loop-mediated isothermal amplification. Phytopathology, 98:1045–1051.
9-Lenaric R,, Morisset D,, Pirc M,, Llop P,, Ravnikar M., and Dreo T. 2014. Loop-mediated isothermal amplification of specific endoglucanase gene sequence for detection of the bacterial wilt pathogen Ralstonia solanacearum. PLoS ONE, 9: e96027
10-Machado J., Grimont F., and Grimont P.A.D. 2000. Identification of Escherichia coli flagellar types by restriction of the amplified fliC gene. Research Microbiology, 151: 535–546.
11-Moradi A., Nasiri J., Abdollahi H., Almasi A. 2012. Development and evaluation of a loop-mediated isothermal amplification assay for detection of Erwinia amylovora base on chromosomal DNA. European Journal of plant pathology, 133: 609. Doi: 10.1007/s10658-012-9939-y
12-Morgan J.A.W., Bellingham N.F., Winstanley C., Ousley M.A., Hart C.A., and Saunders J.R., 1999. Comparison of flagellin genes from clinical and environmental Pseudomonas aeruginosa isolates. Applied and Environmental Microbiology, 65:1175–1179.
13-Notomi T., Okayama H., Masubuchi H., Yonekawa T., Watanabe K., Amino N., and Hase T. 2000. Loop-mediated isothermal amplification of DNA. Nucleic Acids Research, 28: E63.
14-Nouri S., Bahar M., and Fegan M. 2008. Diversity of Ralstonia solanacearum causing potato wilt in Iran and the first record of phylotype ΙΙ/biovar 2T strain outside South America. Plant Pathology, 58: 243– 249.
15-Opina N., Tavner F., Holloway G., Wang J.F., Li T.H., Maghirang R., Fegan, M., Hayward A.C., Krishnapillai V., Hong W.F., Holloway B.W., and Timmis J.N. 1997. A novel method for development of species and strain-specific DNA probes and PCR primers for identifying Burkholderia solanacearum (formerly Pseudomonas solanacearum). Asia-Pacific Journal of Molecular Biology and Biotechnology. 5:19-30.
16-Pastrik K.H., and Maiss E. 2000. Detection of Ralstonia solanacearum in potato tubers by polymerase chain reaction. Journal of Phytopathology, 148: 619–626.
17-Pastrik K.H., Elphinstone J.G., and Pukall R. 2002. Sequence analysis and detection of Ralstonia solanacearum by multiplex PCR amplification of 16S-23S ribosomal intergenic spacer region with internal positive control. European Journal of Plant Pathology, 108:831-842.
18-Poussier S., Prior P., Luisetti J., Hayward C., and Fegan M. 2000. Partial sequencing of the hrpB and endoglucanase genes confirms and expands the known diversity within the Ralstonia solanacearum species complex. Systematic and Applied Microbiology, 23:479-486.
19-Schaad N.W., Jones J.B., and Chun W. 2001. Laboratory Guide for Identification of Plant Pathogenic bacteria. 3rd ed. American Phytopathological Society St. Paul, MN, USA, 378 pp.
20-Schonfeld J., Gelsomino A., van Overbeek L.S., Gorissen A., Smalla K., and van Elsas J.D. 2003. Effects of compost addition and simulated solarisation on the fate of Ralstonia solanacearum biovar 2 and indigenous bacteria in soil. FEMS Microbiology and Ecology. 43:63–74.
21-Seal S.E., Jackson L.A., Young J.P., and Daniels M.J. 1993. Differentiation of Pseudomonas solanacearum, Pseudomonas syzygii, Pseudomonas pickettii, and the blood disease bacterium by partial 16S rRNA sequencing: construction of oligonucleotide primers for sensitive detection by polymerase chain reaction. Journal of General Microbiology, 139: 1587–1594.
22-Tasteyre A., Karjalainen T., Avesani V., Delmee M., Collignon A., Bour-lioux P., and Barc M.C. 2000. Phenotypic and genotypic diversity of the flagellin gene (fliC) among Clostridium difficile isolates from different sero-groups. Journal of Clinical Microbiology, 38: 3179-3186.
23- Tomita N., Mori Y., Kanda H., Notomi T. 2008. Loop-mediated isothermal amplification (LAMP( of gene sequences and simple visual detection of products. Nature Protocols, 3, 877–882.
24-Ward L., Harper S., Clover G. 2010. Development of a LAMP assay for Xylella fastidiosa. MAF Biosecurity New Zealand Technical Paper No: 2010/14.
25-Wicker E., Grassart L., Coranson-Beaudu R., Mian D., Guilbaud C., Fegan M. 2007. Ralstonia solanacearum Strains from Martinique (French WestIndies) Exhibiting a New Pathogenic Potential. Applied and Environmental Microbiology. 71: 6790–6801.
26-Yasuhara-Bell J., Marrero G., De Silva A., Alvarz A.M. 2016. Specific detection of Pectobacterium carotovorum by loop-mediated isothermal amplification. Molecular Plant Pathology, DOI: 10.1111/mpp.12378.