ارزيابي آزمون LAMP با خصوصيت رنگ‌سنجي جهت رديابي چشمي Ralstonia solanacearum در محموله‌هاي سيب‌زميني ايستگاههاي قرنطينه‌اي ايران

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

نژاد 3، بیووار 2 از كمپلكس گونه‏ای Ralstonia solanacearum خسارات اقتصادی قابلتوجهی را به سیبزمینی در سراسر دنیا وارد می‏سازد. بنابراین دستیابی به روش ردیابی حساس و اختصاصی بهمنظور حذف مواد گیاهی آلوده در مراكز تحقیقات و ایستگاههای بازرسی قرنطینه گیاهی ضروری می‏باشد. در مطالعه حاضر پس از جداسازی كلنی‏های شبیه R. solanacearum از غده‏های آلوده سیبزمینی در محیط تترازولیوم كلراید، تاییدمولكولی گونه با استفاده از آغازگرهای اختصاصی انجام گرفت. براساس آزمون استفاده از منابعكربنی و واكنشزنجیره‏ایپلیمراز اختصاصی نیز، جدایه‏های بدستآمده متعلق به بیووار 2 و فیلوتایپ II می‏باشند. آزمون بیماریزایی بر روی گیاهچه‏های سیبزمینی و گوجهفرنگی برای جدایه‏ها انجام و بیماریزایی جدایهها بررسی گردید. ارزیابی غده‏های آلوده و یا مشكوك به آلودگی با اعمال PCR سنتی و واكنش LAMP بر روی ژن fliC بااستفاده از DNA‏ی استخراجشده و عصاره آلوده سیبزمینی انجامگرفت. الگوی نردبانی محصولات LAMP و ردیابی چشمی رسوب پیروفسفات و یا تغییررنگ ایجادشده در نمونه‏های آلوده به واسط كاربرد كالسئین، دلالتبر ردیابی موفق R. solanacearum توسط واكنش LAMP دارد. اگرچه حساسیت آزمون LAMP (CFU/ml 104) مساوی و یا كمتر از PCR سنتی است، اما دقت واكنش مزبور جهت تایید قابل اطمینان وجود R. solanacearum در غده‏های سیبزمینی كافی می‏باشد. درمجموع، آزمون LAMP به همراه مرحله آشكارسازی كارایی چون استفاده از كالسئین، اطلاعات اولیه مناسبی را جهت غربالگری غده‏های آلوده قبل از انبارداری و در حین حملونقل استانی مهیا می‏سازد.

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.