Real-time PCR detection of quarantine plant pathogenic bacteria in potato tubers and olive plants Simultaneous detection of Ralstonia solanacearum race 3 and Clavibacter michiganensis subsp. sepedonicus in potato tubers by a multiplex real-time PCR assay

This chapter focuses on molecular detection of quarantine plant pathogenic bacteria associated with potato and olive plants. A real-time PCR for detection of two bacteria of potato in Europe: Ralstonia solanacearum race 3 and Clavibacter michiganensis subsp. sepedonicus , is described. This method allows the simultaneous detection of both species in a single PCR reaction with an internal control from potato. Described protocol is sensitive and specific and can be used in large scale screening tests. Quarantine pest Xylella fastidiosa is recently detected in Europe for the first time. Considering the importance of early detection, procedure for real-time PCR detection of Xylella fastidiosa in olive plant tissue is described.

) and ring-rot ( Figure 2) of potato, respectively. These diseases represent a serious threat to potato (Solanum tuberosum) production in temperate climates. Both bacteria are listed as A2 pests in the EPPO region and as zero-tolerance quarantine organisms in the European Union. These bacteria remain latent for a long time in asymptomatic potato tubers which are one of the main factors for the disease's dissemination. The existing phytosanitary regulations rely on the availability of pathogen-free seed tubers.

Materials, Methods and Notes
Since the protocols involve detection of quarantine organisms and include the use of viable cultures of Rs andCms, it is necessary to perform the procedures under suitable quarantined conditions with adequate waste disposal facilities and under the conditions of appropriate licenses as issued by the official plant quarantine authorities.

Sample preparation -potato tubers
Note: -The standard sample size is 200 tubers per test. Larger numbers of tubers in the sample will lead to inhibition or difficult interpretation of the results. However, the procedure can be conveniently applied for samples with less than 200 tubers where fewer tubers are available. -Detection methods described below are based on testing of samples of 200 tubers. -Optional pre-treatment in advance to sample preparation: wash the tubers.
Use appropriate disinfectants (chlorine compounds when PCR-test is to be used in order to remove eventual pathogen DNA) and detergents between each sample. Air-dry the tubers. -This washing procedure is particularly useful (but not required) for samples with excess soil and if a PCR-test or direct isolation procedure is to be performed.
2.1.1. Remove with a clean and disinfected scalpel or vegetable knife the skin at the heel end of each tuber so that the vascular tissue becomes visible. Carefully cut out a small core of vascular tissue at the heel end and keep the amount of non-vascular tissue to a minimum.  Note: If during removal of the heel end core suspect symptoms of ring rot are observed, the tuber should be visually inspected after cutting near the heel end. Any cut tuber with suspected symptoms should be suberised at room temperature for two days and stored under quarantine (at 4 to 10°C) until all tests have been completed. 2.1.2. Collect the heel end cores in unused disposable containers which can be closed and/or sealed (in case containers are reused they should be thoroughly cleaned and disinfected using chlorine compounds). Preferably, the heel end cores should be processed immediately. If this is not possible, store them in the container, without addition of buffer, refrigerated for not longer than 72 hours or for not longer than 24 hours at room temperature. Drying and suberisation of cores and growth of saprophytes during storage may hinder detection of the brown rot and ring rot bacterium. 2.1.3. Process the heel end cores by one of the following procedures: either, (a) cover the cores with sufficient volume (approximately 40 ml) of extraction buffer (see recipe below) and agitate on a rotary shaker (50 to 100 rpm) for four hours below 24°C or for 16 to 24 hours refrigerated; or (b) homogenize the cores with sufficient volume (approximately 40 ml) of extraction buffer, either in a blender (e.g. Waring or Ultra Thurax) or by crushing in a sealed disposable maceration bag (e.g. Stomacher or Bioreba strong gauge polythene, 150 mm × 250 mm; radiation sterilized) using a rubber mallet or suitable grinding apparatus (e.g. Homex, Bioreba). Note: -The risk of cross-contamination of samples is high when samples are homogenized using a blender. Take precautions to avoid aerosol generation or spillage during the extraction process. Ensure that freshly sterilized blender blades and vessels are used for each sample. If the PCR test is to be used, avoid carry-over of DNA on containers or grinding apparatus. Crushing in disposable bags and use of disposable tubes is recommended where PCR is to be used. -Recipe for extraction buffer (50 mM phosphate buffer): Na 2 HPO 4 (anhydrous), 4.26 g; KH 2 PO 4 , 2.72 g; distilled water, 1 L. Dissolve ingredients, adjust pH to 7.0 and sterilize by autoclaving at 121°C for 15 min.
2.1.4. Decant the supernatant. If excessively cloudy, clarify either by slow speed centrifugation (at not more than 180 g for 10 minutes at a temperature between 4 to 10°C) or by vacuum filtration (40 to 100 μm), washing the filter with additional (10 ml) extraction buffer.
2.1.5. Concentrate the bacterial fraction by centrifugation at 7 000 g for 15 minutes (or 10 000 g for 10 minutes) at a temperature between 4 to 10°C and discard the supernatant without disturbing the pellet. 2.1.6. Resuspend the pellet in 1,5 ml pellet buffer (see recipe below). Use 500 μl to test for Rs, 500 μl for Cms, and 500 μl for reference purposes. Add sterile glycerol to final concentration of 10 to 25 % (v/v) to the 500 μl of the reference aliquot and to the remaining test aliquot, vortex and store at -16 to -24°C (weeks) or at -68 to -86°C (months). Preserve the test aliquots at 4 to 10°C during testing. Repeated freezing and thawing is not advisable. If transport of the extract is required, ensure delivery in a cool box within 24 to 48h.
Note: Note: -It is also recommended to prepare one decimal dilution of sample DNA extract (1:10 in sterile distilled water) for PCR analysis. -Other DNA extraction methods, e.g. Qiagen DNeasy Plant Kit, could be applied providing that they are proven to be equally as effective in purifying DNA from control samples containing 10 3 to 10 4 pathogen cells per ml.

Real-time PCR assay (method according to Massart et al. 2014)
This multiplex real-time PCR assay allows simultaneous detection of Rs and Cms in potato tubers. For both bacteria, the primers and probes (Table 1) were selected in the rRNA gene intergenic spacer sequences. Additionally, the reliability of this molecular diagnostic test has been improved by the simultaneous amplification of an internal control, corresponding to a potato gene co-extracted from the sample. For the internal control, primers and probes (Table 1) were designed based on chloroplastic ATP synthase beta-subunit from Solanum tuberosum. The Minor Groove Binder (MGB) probes were supplied by Applied Biosystem with a 5' covalently attached reporter dye (FAM, VIC or NED), a nonfluorescent quencher and MGB moiety at the 3' end. The composition of reaction mix and thermal cycling conditions are given in Tables 2 and 3. The proper negative and positive controls are essential for eliminating falsenegative or false-positive results. In this regard, the following negative controls should be included in the real-time PCR test: -DNA extracted from sample extract that was previously tested negative for Rs and Cms. Sample extracts should be as free as possible from soil. It could therefore, in certain cases, be advisable to prepare extracts from washed potatoes. -Buffer controls used for extracting the bacterium and the DNA from the sample, -Incorporate a negative control sample containing only PCR reaction mix and add the same source of nuclease-free water as used in the PCR mix in place of sample.    In addition, the following positive controls should be also included:

MultiRaso-F CGCGGAGCATTGATGAGAT
-DNA extracted from sample extract that was previously tested negative for Rs and Cms spiked withsuspensions of Rs and Cms(several dilutions) -DNA extracted from suspension of 10 6 cells per ml of Rsand Cms in water from a virulent reference strain (e.g. NCPPB 4156 = PD 2762 = CFBP 3857 for Rs; NCPPB 2140 or NCPPB 4053 for Cms). -If possible use also DNA extracted from positive control samples in the PCR test.
To avoid potential contamination prepare positive controls in a separate environment from samples to be tested.

Acknowledgements
This work was funded by EU Commission project AREA, no. 316004. We would like to thank Dutch General Inspection Service and NAK Institute in Emmeloord, especially Miriam Kooman and Jaap Janse for arranging our visit, Robert Vreeburg and Robert Bollema for laboratory support.

References
Massart

Introduction
The olive quick decline syndrome (OQDS) is a disease that appeared suddenly a few years ago in the province of Lecce (Italy). In 2013, it has been found that the most relevant factor for this disease is a quarantine pathogen Xylella fastidiosa. This was the first confirmed record in the European Union. In addition, almond, oleander, cherry and several other perennial ornamentals have been reported as hosts (Cariddi et al. 2014, EPPO 2016. Isolation and culturing of the bacterium on media are fundamental in phytobacteriology, but considering that some Xylella subspecies are very slow-growth, molecular and serological techniques showed as more suitable methods for screening a large number of samples. The purpose of this manuscript is to describe procedure for real-time PCR detection of X. fastidiosa in plant tissue.

Materials, Methods and Notes
Since the protocols involve detection of a quarantine organisms and include the use of viable cultures of X. fastidiosa, it is necessary to perform the procedures under suitable quarantined conditions with adequate waste disposal facilities and under the conditions of appropriate licenses as issued by the official plant quarantine authorities.

Collecting samples
During the training, we collected mostly symptomatic or asymptomatic olive plant material for X. fastidiosa isolation. Typical symptoms for OQDS are the presence of leaf scorch ( Figure 1) and scattered desiccation of twigs and small branches. In the early stages of the infection, symptoms prevail on the upper part of the canopy. Later, these symptoms become increasingly severe and progress into the rest of the crown, which becomes blighted.

Sample preparation
Extraction of X. fastidiosa DNA from culture and plant tissue for molecular analyses has been achieved by both standard commercial column kits and by basic CTAB buffer. Basal leaf portion and peduncles excised from mature leaves in total weight between 0,5-0,8 g are used for DNA extraction. Selected leaves should be representative of the whole sample. Symptomatic leaves have priority.