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Dickeya Solani’ Proceedings Crop Protection in Northern Britain 2012 DEVELOPMENT OF A REAL-TIME PCR ASSAY FOR THE DETECTION OF ‘DICKEYA SOLANI’ R M Kelly1, 3, G Cahill1, J G Elphinstone2, W J Mitchell3, V Mulholland1, N M Parkinson2, L Pritchard4, I K Toth4 and G S Saddler1 1Science and Advice for Scottish Agriculture (SASA), Edinburgh, EH12 9FJ 2The Food and Environment Research Agency (Fera), Sand Hutton, York, YO41 1L0Z 3School of Life Sciences, Heriot-Watt University, Edinburgh, EH14 4AS 4The James Hutton Institute (JHI), Dundee, DD2 5DA E-mail: [email protected] Summary: ‘Dickeya solani’ is a recently emerged bacterial pathogen of potato that has had a great impact on the potato industry of Israel and many European countries and poses a significant threat to GB production. ‘D. solani’ is highly aggressive and results in symptoms and disease similar to that of blackleg. Current diagnostic methods are time-consuming and expensive. Work is being carried out to reduce the time taken to make a diagnosis and to develop a single, specific test for ‘D. solani’. A real-time PCR assay, based on the fusA gene has been developed and evaluated against a panel of 110 representative strains from the genera Dickeya and Pectobacterium, alongside two other real-time PCR assays, developed independently and jointly by JHI and Fera. All assays performed equally well and were shown to positively identify ‘D. solani’ with only a small number of false positives and only one false negative. Further evaluation of these assays is underway to ensure they are suitable for routine use. INTRODUCTION Members of the genus Dickeya (formerly Erwinia chrysanthemi) cause diseases on numerous crop and ornamental plants World-wide. The first disease reports of Dickeya spp. in European potatoes were probably attributable to D. dianthicola; originally detected in the Netherlands in the 1970s then spreading to other countries. Since 2004-5 a new, highly aggressive pathogen, when compared against D. dianthicola and the more established European blackleg pathogens, Pectobacterium atrosepticum and P. carotovorum subsp. carotovorum, has been spreading across Europe and to Israel via trade in seed potatoes and is causing increasing economic losses (Toth et al., 2011). Once established, ‘D. solani’ can rapidly displace these other established pathogens to become the principal cause of wilting and blackleg symptoms in potato. Although disease symptoms caused by ‘D. solani’ are often indistinguishable from typical blackleg it is evident that ‘D. solani’ can produce symptoms at lower inoculum levels and is considerably more aggressive than D. dianthicola and Pectobacterium spp.. ‘D. solani’ can cause disease over a wide temperature range and also has a higher optimal temperature for disease development, which may explain, in part, the domination of this pathogen in certain European countries (van der Wolf et al., 2007). The aggressiveness of ‘D. solani’ is thought to increase with increasing ambient temperature and it also benefits from increased rainfall as the flooding of fields allows for the bacteria to spread between plants; spread also occurs vascularly from 201 mother to daughter tubers (Czajkowski et al., 2010). In 2007 ‘D. solani’ was responsible for a loss of 20% of potato stocks during certification in the Netherlands either as a consequence of downgrading or rejection at a cost of €25million (Toth et al., 2011). Scotland and Northern Ireland are currently the only countries in Europe to enforce a ‘nil tolerance’ for Dickeya spp., in an attempt to keep their potato production free from this pathogen. Irrespective whether other countries adopt a similar approach or rely on existing control measures enshrined in their seed certification schemes, reliable diagnosis of the pathogen is the first step towards effective control. Because symptoms alone are unreliable as a means of determining the presence of ‘D. solani’ some form of diagnostic test is therefore essential. The polymerase chain reaction (PCR) is increasingly used for the specific detection and identification of bacterial plant pathogens. Currently diagnostic protocols for Dickeya spp., based at least in part on PCR, are expensive, time-consuming and it can take up to two weeks to perform a reliable diagnosis. The most widely used procedure at present for identifying Dickeya spp. involves initial enrichment and/or selective isolation steps prior to PCR using the ADE primers (ADE1/ADE2) from the pectate lyase (pel) gene (Nassar et al., 1996) to detect all known members of the genus. Species identification to ‘D. solani’ can only be achieved by sequencing either the recA (Parkinson et al., 2009) or dnaX genes (Sławiak et al., 2009), then comparing against database entries or by sequencing alongside known reference strains. The aim of this study, therefore, is to develop a diagnostic protocol specific for ‘D. solani’ which must be cost-effective, fast and accurate and which can be used to test direct from potato plants, tubers and environmental samples. A previously designed multi-locus sequence typing (MLST) system (Kowalewska et al., 2010), developed to characterise D. dianthicola strains but which also included a number of ’D. solani’ strains was utilized to design the real-time PCR assay. MATERIALS AND METHODS Design of fusA primers The fusA gene was selected as a target for further study after visual inspection of phylogenetic trees, generated previously (Kowalewska et al., 2010), showed good separation between ‘D. solani’ isolates and known reference strains from the genus Dickeya and Pectobacterium. Using the ClustalW method of the MegAlign program in Lasergene v7.0.0 (DNASTAR Inc.) the fusA sequences of 62 Dickeya and Pectobacterium strains and 20 ‘D. solani’ isolates were aligned. From these data a TaqMan® ‘D. solani’ specific probe was designed, in addition to flanking primer sequences. The assay was evaluated using the protocol described below. In addition, this assay was compared against two other real-time assays developed by JHI and Fera, jointly and independently of this study, by exploiting draft genome assemblies from 16 Dickeya isolates and reference strains (Pritchard et al., unpublished data). The accuracy, effectiveness and efficiency of the three assays were analysed and the results are described here. Real-time PCR The fusA real-time PCR assay, based on the probes and primers described (Table 1), was evaluated against two previously designed assays (SOL-C & SOL-D; Pritchard et al., unpublished data), using a collection of 110 Dickeya and Pectobacterium strains. These strains 202 had been stored on freezer beads at -80oC. Strains were streaked onto crystal-violet pectate medium (CVPM) plates and incubated for 48 hours at 36oC. Cultures were then re-streaked onto Nutrient Agar and incubated overnight at 36 oC. A loopful of colonies was suspended in 500µl of sterile water (Sigma) and boiled at 100 oC for 5 minutes. The sample was then ready to be used in the PCR reaction or stored at -20 oC until needed. Table 1. Primers and probes used in fusA real-time assay. Assay Forward primer Reverse primer Probe fusA GGTGTCGTTGACCTG ATAGGTGAAGGTCAC TGAAAGCC GTGAAA ACCCTCATC ATCAACTG GAATGATT C Master mix (24 µl; Table 2) was added to each well of a 96-well plate (MicroAmp optical well plate with barcode) with an electronic pipette (Autorep) in a flow hood for each of the three tested. Boiled cells (1µl) were added to each well (1 µl of sterile H2O (Sigma) for negative controls). The plate was sealed with PCR optical film. Table 2. PCR reaction mix. 1x Reaction Taqman master mix 12.5 µl Forward primer (5 pmol) 1.5 µl Reverse primer (5 pmol) 1.5 µl Probe (5 pmol) 0.5 µl Template 1 µl Sterile H2O (Sigma) 8 µl TOTAL REACTION 25µl AMOUNT Table 3. Real-time PCR cycle. Temperature Time Number of cycles 95oC 10 mins x 1 cycle 95oC 15 s x 40 cycles 60oC* 1 mins The cycle was run on Applied Biosystems 7900HT real time PCR machine in standard mode, detecting FAM/TAMRA and using ROX as the passive reference. Data was taken at the extension (*) step only. 203 RESULTS Initial screening and comparison of the three real-time assays was carried out against 110 samples of known Dickeya and Pectobacterium spp., the results of which are shown in Table 4. Real-time, or quantitative, PCR is typically used to measure the amount of DNA expressed in the reaction, based on the critical threshold (Ct) value. The Ct value is the cycle at which the fluorescence becomes detectable and is inversely proportional to the logarithm of the initial number of target cells; however, in this case the Ct value is used qualitatively to indicate either positive or negative identification of ‘D. solani.’ Ct values less than or equal to 25 signified the sample to be positive as ‘D. solani’ and Ct values greater than 25 or “undetermined” were denoted as being negative and therefore, not ‘D. solani.’ In this study 97% of the strains received as ‘D. solani’ were correctly identified using all three assays and only 5-7.5% of non- ‘D.solani’ strains were incorrectly identified (false positives). Both the fusA and SOL-C assays gave identical results, with SOL_D performing marginally better through a smaller number of false positives. Table 4. Results from initial screening of three real-time assays against Dickeya and Pectobacterium spp, with Ct values <25 recorded as positive and Ct>25 or “undetermined” denoted as negative. fusA SOL-C SOL-D Species Tested Positive Negative Positive Negative Positive Neg ativ e D. solani 29 1 29 1 29 1 D. dianthicola 2 11 2 11 1 12 D. zeae 2 14 2 14 1 15 D. dadantii 0 5 0 5 0 5 Other Dickeya and Pectobacterium spp. 2 44 2 44 2 44 Total 35 75 35 75 33 77 All three real-time assays produce similar results.
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