Paper Information

Journal:   JOURNAL OF PLANT PROTECTION (AGRICULTURAL SCIENCE AND TECHNOLOGY)   SUMMER 2017 , Volume 31 , Number 2 #M0057; Page(s) 277 To 283.
 
Paper: 

EVALUATION OF COLORIMETRIC LAMP ASSAY FOR VISUAL DETECTION OF RALSTONIASOLANACEARUM IN POTATO SHIPMENTS AT QUARANTINE STOPS IN IRAN

 
 
Author(s):  NABAVI CHASHMI O.H., BAGHAEE RAVARI S.*, FALAHATI RASTEGAR M., MOSLEMKHANI C., JAHANBAKHSH MASHHADI V.
 
* DEPARTMENT OF PLANT PROTECTION, FACULTY OF AGRICULTURE, FERDOWSI UNIVERSITY OF MASHHAD, MASHHAD, IRAN
 
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 andhrp B and fli Chas been used as an alternative to studyR. solanacearum species complex. In order to facilitate detection of R. solanacearum in 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 withR. 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 ofR. 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 detectR. solanacearum in symptomatic and symptomless tissues, conventional PCR and LAMP assay according tofli C 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 ?l 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-fli C and Rsol- fli C primers. A 400 bp PCR product specific toR. solanacearum was obtained from all strains. Sequencing three purified PCR products confirmed the right amplification offli C 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 conventionalfli C-PCR, the detection limit rangedapproximately from 10 3to 10 4cfu ml?1in both infected potato extract and pure cultures. Moreover, the lowest amount of consistently tested positive through LAMP assay was 104cfu ml?1 for both cases.
Although the sensitivity of thefli C LAMP assay wasequal or lower than that of the conventional PCR, theaccuracy offliC 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.

 
Keyword(s): COST-EFFECTIVE METHOD, ENTRY POINTS, LATENT INFECTION, POTATO BROWN ROT, STORE
 
References: 
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