DETECTION OF XYLELLA FASTIDIOSA INTERLABORATORY VALIDATION OF MOLECULAR AND SEROLOGICAL METHODS DIAGNOSTIC PROTOCOLS.

DETECTION OF XYLELLA FASTIDIOSA INTERLABORATORY VALIDATION OF MOLECULAR AND SEROLOGICAL METHODS DIAGNOSTIC PROTOCOLS 19-26 Maggio 2015 Data 18/5/2015 Page 1

CONTENT 1. DETECTION OF XYLELLA FASTIDIOSA 2. OBJECTIVE 3. INTERLABORATORY VALIDATION 3.1 Laboratories involved 3.2 Methods and procedures to be evaluated and compared 3.3 Plant Samples 3.4 Tissue Preparation 4. LIST OF THE SAMPLES PROVIDED 5. FORMAT FOR RESULT REPORTING ANNEX I: Enzyme-Linked ImmunoSorbent Assay (ELISA) ANNEX II: Direct tissue blot immunoassay for detection of Xylella fastidiosa in olive trees (KIT AGRITEST) ANNEX III: Direct tissue blot immunoassay for detection of Xylella fastidiosa in olive trees (Enbiotech XylPrint Kit) ANNEX IV: CTAB-based total nucleic acid extraction ANNEX V: Protocol for conventional PCR ANNEX VI -VII: Protocols for quantitative real-time PCR ANNEX VIII-IX: Protocols for LAMP PCR REFERENCES Data 18/5/2015 Page 2

1. DETECTION OF XYLELLA FASTIDIOSA Xylella fastidiosa is a regulated plant pathogen in many parts of the world. Traditional detection of X. fastidiosa relies on symptom observations, isolation and culturing on selective media although approaches relying on serological and molecular assays have been recently developed and proved to be more suitable for screening large number of samples. Inspections of crops suspected to be infected by X. fastidiosa are fundamental to point out early symptoms of infection. Disease symptoms. Development of symptoms induced by X. fastidiosa vary according to the susceptible hosts, with leaf scorching (LS) being the most common syndrome associate to the bacterial infections in almond, coffee, elm, oak, oleander pear, and sycamore. Indeed, strains of this bacterium are the causal agent of Pierce's disease (PD) of grapes, citrus variegated chlorosis (CVC), phony peach disease, plum leaf scald. Leaf scorch symptoms appear in late summer to early fall and can be distinguished from scorch-like symptoms caused by other factors (drought, salt injury, wilt diseases, etc.) by the presence of a yellow halo between the area of marginal leaf necrosis and green leaf tissue. Infected, symptomatic trees will drop leaves prematurely, and can develop dieback and irreversible decline. PD in grapevine, consists in a sudden drying of large parts of the green leaves, evolving in brown and necrotic and the surrounding tissues become yellow to red. The necrosis is often present at the leaf margins. Scorched (burnt-like) leaves usually drop from the distal and not from the usual basal end of the petiole, leaving bare petioles attached to canes, often well after normal leaf fall. PD can be confused with other disorders such as salt toxicity, boron, copper or phosphorus deficiency. Unlike the majority of the X. fastidiosa diseases, CVC symptoms do not include scorched leaves, but typical irregular chlorosis in mature leaves recognized by interveinal yellowing on the upper side of the leaf and corresponding brownish gumlike material over the side. Indeed, there are several hosts that may carry the pathogen with, but more often without showing symptoms, such as grasses, sedges and trees. In recent years (2009-2011), X. fastidiosa was detected in olive trees in California exhibiting leaf scorch and branch dieback (Krugner et al., 2014). Indeed, in 2013 the bacterium has been consistently associated with a novel olive disease reported in southern Italy (Nigro et al., 2013; Saponari et al., 2013). The disease denoted olive quick decline syndrome is characterized by the presence of leaf scorch and scattered desiccation of twigs and small branches, which as time passes, become increasingly severe and extend to the rest of the crown, which acquires a burned look and desiccates in a short while. Besides olives, in the same outbreak area, cherry and almond trees were also found infected by the same strain of X. fastidiosa, identified as X. fastidiosa subsp. pauca strain CoDiRO. Symptoms in both hosts appeared only during summer and consisted of typical leaf scorch alterations. In addition, several ornamental species proved to be susceptible hosts of the CoDiRO strain, with infections causing typical leaf scorching (e.g. in oleander, myrtle-leaf milkwort, myrtle, rosemary) and/or dieback (e.g. Westringia fructicosa, Spartium junceum and Acacia saligna). Data 18/5/2015 Page 3

Isolation. Isolation by pathogen cultivation in vitro is the most definitive and direct for pathogen detection and identification because whole bacterial cells and their biochemical and physiological properties are observed. However, cultivation of X. fastidiosa in the laboratory is time consuming, ranging from 3 to 20 days, and labor intensive, particularly when a large number of samples are involved. Serology. Serological methods target the unique properties of bacterial cell surface; tests include ELISA (Sherald and Lei, 1991), dot immunobinding assay (DIBA), western blotting and immunofluorescence. Among them, enzyme-linked immunosorbent assay (ELISA) is commonly used and has a high throughput capacity because of the simplicity in sample preparation and the use of the 96-well plate format. In addition, Tissue Blot Immunoassay (DTBIA) can represent a useful diagnostic tool for field based detection (Djelouah et al., 2014). Molecular tests. Molecular techniques include PCR and PCR derivatives including RFLP and RAPD analysis, as well are real-time PCR and loop-mediated isothermal amplification (LAMP). Extraction of X. fastidiosa DNA from culture and host species for PCR and related molecular analyses has been achieved from tissue by both standard commercial column kits and by basic CTAB or, in the case of cultures, Tris-EDTA-Sarkoysl techniques. The available whole genome sequences of X. fastidiosa strains make it feasible to design PCR primers at various levels of specificity. Several specific PCR primer sets are currently available for X. fastidiosa detection including the most thoroughly tested RST31/33 primer set, derived from the RNA polymerase genomic locus, those derived from16s rrna gene and gyrase genes (gyrb), and those genomic-specific targeting the conserved hypothetical HL protein. The use of LAMP assay for X. fastidiosa has been reported by Harper et al. (2010) and D Onghia et al. (2014), proved to be highly specific and with a level of sensitivity acceptable for first-instance screening (greater than conventional PCR but lower than real-time PCR). Indeed, the LAMP assay also is rapid, being able to detect X. fastidiosa extracted from infected tissue in approximately one hour. Data 18/5/2015 Page 4

2. OBJECTIVE The current ring test aims to evaluate the suitability of novel diagnostic tools for the identification of the CoDiRO strain in olives and other susceptible hosts. Specifically, new serological reagents (antisera against the CoDiRO strain is now commercially available) will be tested and novel approaches will be compared with currently adopted protocols: - ELISA will be performed with 2 commercial kits; - DTBIA assays will be evaluated for the identification of X. fastidiosa in olives; - LAMP PCR will be assessed for the identification of X. fastidiosa in olives and oleander plants. The protocols herein reported refer to procedures described in a peer reviewed journals, either included in the EPPO Diagnostic protocols for regulated pests (PM 7/24(1)) or referring to the most recently developed techniques. It is important to remark the general requirements for accredited diagnostic laboratories: - they shall use only validated procedures for confirming that the examination procedures are suitable for intended use; - the validation shall be as extensive as are necessary to meet the needs in the given application or field of application; - procedures need to be periodically revalidated in view if changing conditions and technical advances (in the light of scientific progress). The present inter-laboratory validation reviewed current procedures for detection of Xylella fastidiosa strain CoDiRO in plant tissues. Molecular and serological tools are evaluated in interlaboratory (collaborative) studies in order to obtain a reliable estimation of the performance characteristics of each method. This work follows two earlier inter-laboratory validations completed in November 2013 and September 2014 (Loconsole et al., 2014a, 2014b). The previous validations included ELISA, conventional and quantitative real-time PCR as means for bacterial detection in infected olive tissues and other susceptible host plants (oleander, almond, cherry and some ornamentals). Data 18/5/2015 Page 5

3. INTERLABORATORY VALIDATION 3.1 Laboratories involved Validation of the molecular and serological assays is carried out by the Laboratories listed below, under the supervision of the reference laboratory CNR-UNIBA. CNR-UNIBA: Istituto per la Protezione Sostenibile delle Piante CNR, UOS Bari e, Università degli Studi Aldo Moro, Bari (Italy) (UNIBA); CRSFA: Centro di Ricerca, Sperimentazione e Formazione in Agricoltura Basile Caramia, Locorotondo (BA); IAMB: Istituto Agronomico Mediterraneo, Valenzano (BA). Dipartimento di Scienze Agroambientali, Chimica e Difesa Vegetale - Università degli Studi di Foggia. 3.2 Methods and procedures to be evaluated and compared The following procedures will be tested on a panel of blind samples (see next paragraph): SEROLOGICAL ASSAYS Technique ELISA (KIT AGRITEST) ELISA (KIT LOEWE) DTBIA (KIT AGRITEST) DTBIA (KIT ENBIOTECH) Protocol ANNEX I ANNEX I ANNEX II ANNEX III Samples/Tissues TABLE 1 TABLE 1 TABLE 2 TABLE 2 MOLECULAR ASSAYS Technique PCR Real-time PCR (Harper et al., 2010) Template Real-time PCR KIT QUALIPLANTE LAMP - KIT ENBIOTECH LAMP - KIT QUALIPLANTE ANNEX VIII Plant extracts Plant extracts (Annex IV) (Annex IV) Protocol ANNEX V ANNEX ANNEX VII ANNEX VIII ANNEX IX VI TABLE 3 TABLE3 TABLE3 TABLE4 TABLE3 Data 18/5/2015 Page 6

3.3 Plant Samples Samples must be received by the diagnostic labs in good conditions, with the bags properly sealed, clearly labeled and accompanied by the proper documents. During the registration of the samples, the bag (especially those harboring weed samples) must be inspected for the presence of any winged insects and bags contaminated with bugs should be annotated, kept separately from the other sample bags and managed carefully. All sample bags must be stored at 4 C for at least 3h prior processing the plant tissues. Bags annotated as containing bugs should be opened in a dark room under a light trap. 3.4 Tissue Preparation Leaf peduncles and midribs excised from mature leaves are the most suitable tissues for X. fastidiosa detection in perennial crops. For annual herbaceous plants stem and leaf peduncles and veins from basal leaves should be used. For each sample, at least 0,5-0,8 gr of tissue are recovered from 5-10 leaves (according to the leaf size and consistency) and used for DNA extraction or ELISA sap preparation. Samples should be inspected for symptoms and if present symptomatic leaves (showing leaf scorching and necrosis), selected and processed, removing the necrotic and dead tissue. Alternatively, i.e. for DTBIA and/or LAMP (Annexes II, III, VIII) young cuttings are used to prepare the printed membranes or to recover small pieces to be subjected directly to DNA extraction for LAMP assays. Data 18/5/2015 Page 7

4. LIST OF THE SAMPLES PROVIDED The panel of blind samples includes plant tissues from naturally infected olives and oleander plants. In addition, the panel of sample includes extracts (PBS-extracts lyophilized for ELISA, and total nucleic acids for PCR and qpcr tests) of grapes, oak and citrus artificially spiked with inactivated bacterial suspension. Specifically, the inactivated suspension (adjusted to final concentration of 10 7 CFU/ml) was added to the PBS-extracts obtained from healthy grapes, oak and citrus; and to the CTABextracts obtained from healthy grapes, oak and citrus for conventional and real-time PCR. Data 18/5/2015 Page 8

Table 1: SAMPLES TO BE TESTED IN ELISA ID SAMPLES Host species Material provided Notes X1 Olive Cuttings Use 8-10 petioles X2 Olive Cuttings Use 8-10 petioles X3 Olive Cuttings Use 8-10 petioles X4 Olive Cuttings Use 8-10 petioles X5 Olive Cuttings Use 8-10 petioles X6 Olive Cuttings Use 8-10 petioles X7 Olive Cuttings Use 8-10 petioles X8 Olive Cuttings Use 8-10 petioles X9 Olive Cuttings Use 8-10 petioles X10 Olive Cuttings Use 8-10 petioles X11 Olive Cuttings Use 8-10 petioles X12 Olive Cuttings Use 8-10 petioles MND1 Almond Lyophilized prep Add 4ml of distilled water MND2 Almond Lyophilized prep Add 4ml of distilled water MND3 Almond Lyophilized prep Add 4ml of distilled water MND4 Almond Lyophilized prep Add 4ml of distilled water MND5 Almond Leaves Use 8-10 petioles LEC1 Oak Lyophilized prep Add 6ml of distilled water LEC2 Oak Lyophilized prep Add 6ml of distilled water LEC3 Oak Lyophilized prep Add 6ml of distilled water LEC4 Oak Lyophilized prep Add 6ml of distilled water LEC5 Oak Leaves Use 8-10 petioles VIT1 Grape Lyophilized prep Add 6ml of distilled water VIT2 Grape Lyophilized prep Add 6ml of distilled water VIT3 Grape Lyophilized prep Add 6ml of distilled water VIT4 Grape Lyophilized prep Add 6ml of distilled water VIT5 Grape Leaves Use 5-6 petioles AGR1 Citrus Lyophilized prep Add 6ml of distilled water AGR2 Citrus Lyophilized prep Add 6ml of distilled water AGR3 Citrus Lyophilized prep Add 6ml of distilled water AGR4 Citrus Lyophilized prep Add 6ml of distilled water AGR5 Citrus Leaves Use 8-10 petioles X33 Oleander Leaves Use 5-6 petioles X34 Oleander Leaves Use 5-6 petioles X35 Oleander Leaves Use 5-6 petioles X36 Oleander Leaves Use 5-6 petioles Data 18/5/2015 Page 9

X37 Oleander Leaves Use 5-6 petioles X38 Oleander Leaves Use 5-6 petioles PC Olive Cuttings Use 8-10 petioles HC (Olive) Olive Cuttings Use 8-10 petioles TABLE 2: SAMPLES TO BE TESTED BY DTBIA ID SAMPLES Material Kit Agritest Kit BIONAT X1 Olive Cuttings X2 Olive Cuttings X3 Olive Cuttings X4 Olive Cuttings X5 Olive Cuttings X6 Olive Cuttings X7 Olive Cuttings X8 Olive Cuttings X9 Olive Cuttings X10 Olive Cuttings X11 Olive Cuttings X12 Olive Cuttings 2spots/cutting, use tongs to squeeze the cuttings 2spots/cutting Data 18/5/2015 Page 10

TABLE 3: SAMPLES TO BE TESTED BY PCR, qpcr and LAMP (kit Qualiplante) ID SAMPLES Material Notes X1 Olive Cuttings Use 8-10 petioles X2 Olive Cuttings Use 8-10 petioles X3 Olive Cuttings Use 8-10 petioles X4 Olive Cuttings Use 8-10 petioles X5 Olive Cuttings Use 8-10 petioles X6 Olive Cuttings Use 8-10 petioles X7 Olive Cuttings Use 8-10 petioles X8 Olive Cuttings Use 8-10 petioles X9 Olive Cuttings Use 8-10 petioles X10 Olive Cuttings Use 8-10 petioles X11 Olive Cuttings Use 8-10 petioles X12 Olive Cuttings Use 8-10 petioles MND2 Almond CTAB-extract Use directly the total nucleic acid MND3 Almond CTAB-extract Use directly the total nucleic acid MND4 Almond Leaves Use 8-10 petioles LEC3 Oak CTAB-extract Use directly the total nucleic acid LEC4 Oak CTAB-extract Use directly the total nucleic acid LEC5 Oak Leaves Use 8-10 petioles VIT1 Grape CTAB-extract Use directly the total nucleic acid provided VIT2 Grape CTAB-extract Use directly the total nucleic acid provided VIT5 Grape Leaves Use 5-6 petioles AGR1 Citrus CTAB-extract Use directly the total nucleic acid AGR2 Citrus CTAB-extract Use directly the total nucleic acid AGR5 Citrus Leaves Use 8-10 petioles X33 Oleander Leaves Use 5-6 petioles X34 Oleander Leaves Use 5-6 petioles X35 Oleander Leaves Use 5-6 petioles X36 Oleander Leaves Use 5-6 petioles X37 Oleander Leaves Use 5-6 petioles X38 Oleander Leaves Use 5-6 petioles PC Olive Cuttings Use 8-10 petioles HC (Olive) Olive Cuttings Use 8-10 petioles Data 18/5/2015 Page 11

TABLE 4: SAMPLES TO BE TESTED BY LAMP (KIT ENBIOTECH) ID SAMPLES Material Notes X1 Olive Cuttings X2 Olive Cuttings X3 Olive Cuttings X4 Olive Cuttings X5 Olive Cuttings X6 Olive Cuttings X7 Olive Cuttings X8 Olive Cuttings X9 Olive Cuttings X10 Olive Cuttings See annex VIII X11 Olive Cuttings X12 Olive Cuttings X33 Oleander Leaves X34 Oleander Leaves X35 Oleander Leaves X36 Oleander Leaves X37 Oleander Leaves X38 Oleander Leaves PC Olive Cuttings HC (Olive) Olive Cuttings Data 18/5/2015 Page 12

5.1 ELISA TEST KIT LOEWE Date SAMPLES X1 X2 X3 X4 X5 X6 X7 X8 X9 X10 X11 X12 MND1 MND2 MND3 MND4 MND5 LEC1 LEC2 LEC3 LEC4 LEC5 VIT1 VIT2 VIT3 VIT4 VIT5 AGR1 AGR2 AGR3 AGR4 AGR5 X33 X34 OD405 60 min OD405 120 min 5. FORMAT FOR RESULT REPORTING OD405 180 min STATUS Positive/negative* Data 18/5/2015 Page 13

X35 X36 X37 X38 HC PC *Positive sample= the OD absorbance value is at least four times higher than the OD value of the NC negative control and >0.4 OD. 5.2 ELISA TEST KIT AGRITEST Date SAMPLES OD405 60 min OD405 120 min OD405 180 min STATUS Positive/negative* X1 X2 X3 X4 X5 X6 X7 X8 X9 X10 X11 X12 MND1 MND2 MND3 MND4 MND5 LEC1 LEC2 LEC3 LEC4 LEC5 VIT1 VIT2 VIT3 VIT4 VIT5 AGR1 Data 18/5/2015 Page 14

AGR2 AGR3 AGR4 AGR5 X33 X34 X35 X36 X37 X38 HC PC *Positive sample= the OD absorbance value is at least three times higher than the OD value of the NC negative control. Data 18/5/2015 Page 15

5.3 DTBIA Date ID SAMPLES KIT AGRITEST Positive*/negative KIT ENBIOTECH Positive*/negative X1 X2 X3 X4 X5 X6 X7 X8 X9 X10 X11 X12 * If positive, indicate the number of positive spots for each sample. Data 18/5/2015 Page 16

5.4 PCR TESTS Date X1 X2 X3 X4 X5 X6 X7 X8 X9 X10 X11 X12 MND2 MND3 MND4 LEC3 LEC4 LEC5 VIT1 VIT2 VIT5 AGR1 AGR2 AGR5 X33 X34 X35 X36 X37 X38 PC SAMPLES RST31/33 (723bp) Positive/ negative qpcr Harper et al. (2010) Cq values - Positive/negative qpcr (Kit Qualiplante) Cq values - Positive/negative LAMP (Kit Qualiplante) Cq values - Positive/negative Data 18/5/2015 Page 17

HC (Olive) NTC (non template control) 5.5 LAMP TEST (KIT ENBIOTECH) Date X1 X2 X3 X4 X5 X6 X7 X8 X9 X10 X11 X12 X33 X34 X35 X36 X37 X38 PC HC (Olive) SAMPLES LAMP (Kit Enbiotech) Cq values - Positive/negative Data 18/5/2015 Page 18

ANNEX I Enzyme-Linked ImmunoSorbent Assay (ELISA) FOR EACH KIT SEE THE TECHNICAL SHEET FOR ADDITIONAL INFORMATION The procedure hereafter described refers to the ELISA test performed with two commercially available kits, from Agritest and Loewe, to be used for X.f. detection in infected tissues from olive, oleander, almond, cherry, etc. The following steps must be followed: 1. Coat the plate Dilute the IgG (anti -Xf.-IgG) 1:200 (Loewe)/1:500 (Agritest) in coating buffer, and load 100 or 200 µl to each well of the microtiter plate. Cover the plate tightly and place it in a humid box. Incubate the plate at 37 C for 4 h. 2. Washing step Remove the sap from the wells and wash 4 times the plates using the washing buffer, remove any liquid by blotting the plate on paper towels. 3. Plant sap preparation and Antigen incubation Homogenize the samples in extraction buffer 1:10 (w/v): weigh at least 0,5 g of leaf petioles and basal portion of the leaves, cut in small pieces using a razor blade (while processing the samples, sterilize the blade between samples). Transfer the plant tissue into the extraction bags and add 5 ml of extraction buffer; crush with a hammer and grind by a semi-automated homogenizer (i.e. Homex). Transfer 1 ml of sap into a microcentrifuge tube that store at 4 C until use, allowing plant debris precipitation. Load 100 or 200 µl of plant extract to each well of the microtiter plate. Cover the plate and incubate at 4 C overnight in a humid box. 4. Washing step Repeat step 2. 5. Add the detection antibody Dilute enzyme-conjugated antibodies (anti-xf.-apconjugate) 1:200 (Loewe)/1:500 (Agritest) in conjugate buffer. Add 100 or 200 µl to each well of the microtiter plate. Cover the plate and incubate at 37 C for 4h in a humid box. 6. Washing step Repeat step 2. 7. Add Substrate Dissolve the p-nitrophenylphosphate (0.6-1 mg/ml) in substrate buffer and add 100 or 200 µl per well. Incubate at room temperature (18-25 C) till the yellow color reaction start to develop and read the plate at 60-120-180 min (if necessary, prolong the reaction over-night) using a plate Data 18/5/2015 Page 19

reader at λ =405 nm. The enzymatic reactions can be stopped by adding 25 µl 3 M NaOH (Sodium Hydroxide) to each well. BUFFERS REQUIRED for ELISA PBS ( ph 7,4) NaCl 8 g KH2 PO4 anhydrous 0,2 g Na2HPO4 anhydrous 1,15 g KCl 0,2 NaN3 (optional) 0,2 g Bring final volume to 1 L with distilled water Washing buffer (PBST) PBS 1 L Tween-20 0,5 ml Store at room temperature Coating buffer (1 L; ph 9,6) Na2CO3 anhydrous 1,59 g NaHCO3 2,93 g NaN3 (optional) 0,2 g Store at 4 C Extraction buffer/conjugate buffer (1L; ph 7,4) PBST 1 L Polyvinylpyrrolidone (PVP-25) 20 g Bovin serum albumin (BSA) 2g Store at 4 C Substrate buffer (1 L; ph 9,8) Diethanolamine 97 ml MgCl2 x 6H2O 0,2 g NaN3 (optional) 0,2 g Adjust ph with HCl and bring to final volume of 1 L with distilled water Store at 4 C Data 18/5/2015 Page 20

ANNEX II (KIT AGRITEST SEE THE TECHNICAL SHEET FOR ADDITIONAL INFORMATION) Direct tissue blot immunoassay for detection of Xylella fastidiosa in olive trees 1. Preparation of the imprints Use the membrane of nitrocellulose (0.45µm) with the premarked grid for positioning the individual sample. Select four mature twigs from each sample; make a fresh clean cut with a shears across each twig, then after positioning the tongs just above the end of the twig, squeeze and press gently the surface on the membrane. Prepare 2 spots for each twig, a total of eight spots should be done for each sample. Sterilize the shears between samples using a 10% bleach solution. Dry the membrane for at least 30 min at room temperature. 2. Blocking Prepare the blocking solution 1% non-fat milk in PBS. Place the membrane in a box of the appropriate size and cover with the blocking solution. Incubate 2 hours at room temperature (alternatively overnight at 4 C) 3. Washing Discard the blocking solution Wash the membrane with an appropriate volume of washing buffer, for 3 minutes at room temperature, on a shaker (optional) Repeat the washing step three times 4. Incubation Dilute the Xf-specific antibodies 1:250 in conjugate buffer and incubate the membrane with an appropriate volume (covering the membrane) for 2 hours at room temperature 5. Washing Repeat step 3. 6. Substrate development Dissolve 1 tablet of BCIP-NBT (Sigma Fast) in 10ml of distilled water. Cover the membrane with this solution and incubate at room temperature until the appearance of purple-violet colour in the positive samples (about 5 to 10 min.) Stop the reaction by washing the membrane with distilled water 7. Results recording Dry the membrane and observe the imprints using a low power magnification (x10-x20) The presence of purple-violet precipitates in the imprints indicates the presence of the pathogen. Data 18/5/2015 Page 21

PREPARATION OF THE MEMBRANE DTBIA RESULTS INTERPRETATION: Example of membrane after development Purple coloration indicates positive reactions The absence of purple coloration in the spot(s) indicates negative reactions Data 18/5/2015 Page 22

BUFFERS REQUIRED for DTBIA PBS (ph 7,4) NaCl 8 g KH2 PO4 anhydrous 0,2 g Na2HPO4 anhydrous 1,15 g KCl 0,2 NaN3 (optional) 0,2 g Bring final volume to 1 L with distilled water Washing buffer (PBST) PBS 1 L Tween-20 0,5 ml Store at room temperature Conjugate buffer ( 1 L; ph 7,4) PBST 1 L PVP-25 20 g BSA 2 g Store at 4 C Data 18/5/2015 Page 23

ANNEX III (Enbiotech XylPrint Kit) Direct tissue blot immunoassay for detection of Xylella fastidiosa in olive trees Data 18/5/2015 Page 24

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ANNEX IV CTAB-based total nucleic acid extraction 1. Weigh out 0,5-0,8 g of fresh small pieces of midribs and petioles (1/4 if lyophilized) and put into Bioreba extraction bags and add 2ml of CTAB. Crush with a hammer and homogenize in Homex. The rest of the midrib pieces can be stored at -20ºC for use in the future. 2. In each bag add additional 3ml of CTAB; 3. Transfer 1ml of sap into a 2ml microcentrifuge tube. 4. Heat samples at 65 C for 30 minutes. 5. Centrifuge samples at 10,000 rpm for 5 minutes and transfer 1ml to a new 2ml microcentrifuge tube, being careful not to transfer any of the plant tissue debris. Add 1ml of Chloroform:Isoamyl Alcohol 24:1 and mix well by shaking. 6. Centrifuge sample at 13,000 rpm for 10 minutes. Transfer 750ml to a 1.5ml microcentrifuge tube and add 450ml (approximately 0.6V) of cold 2-Propanol. Mix by inverting 2 times. Incubate at 4 C or -20 C for 20 minutes. 7. Centrifuge the samples at 13.000 rpm for 20 minutes and decant the supernatant. 8. Wash pellet with 1ml of 70% ethanol. 9. Centrifuge sample at 13,000 rpm for 10 minutes and decant 70% ethanol. 10. Air dry the samples or use the vacuum. 11. Re-suspend the pellet in 100µl of TE or RNAse- and DNase-free water. 12. Extracts of total nucleic acid can be stored at 4º C for immediate use or at -20ºC for use in the future. 13. Determine the concentration at the spectrophotometer (Nanodrop 1000 or similar). Read the absorption (A) at 260nm and at 280 nm. Optimal A260/280 ratio should be close to 2 for high quality nucleic acid. 14. Adjust the concentration to 50-100ng/ l, and use 2 l (in a final volume of 25 l) to set up the conventional and real time PCR assays for X. fastidiosa detection. BUFFER REQUIRED for the extraction: CTAB BUFFER 2% CTAB (Hexadecyl trimethyl-ammonium bromide) (any vendor) Autoclaved 0.1M TrisHCl PH 8 (any vendor) Autoclaved 20mM EDTA (any vendor) Autoclaved 1.4M NaCl (any vendor) Adjust PH to 8.0 Data 18/5/2015 Page 27

ANNEX V Protocol for conventional PCR -Primers RST31 and RST33 (desalt purification), which generate a PCR product of 733 base pairs, (Minsavage et al., 1994). RST31 (forward): 5 -GCGTTAATTTTCGAAGTGATTCGATTGC-3 RST33 (reverse): 5 -CACCATTCGTATCCCGGTG-3 Two different PCR mix (A and B) can be used. Preliminary tests demonstrated that the GoTaq DNA polymerase (Promega) is the most efficient enzymes for the amplification of the targets in different plant extracts. The use of a layer of mineral oil over the PCR reactions proved to be useful to effectively prevent carryover contamination, especially when a large number of samples have to be processed routinely. REACTION MIX (OPTION A): Reagent Volume Total genomic DNA 2 µl 5X Green GoTaq Buffer (Promega) 5 µl 10 µm Forward Primer 0.5 µl 10 µm Reverse Primer 0.5 µl 10 mm dntps (any vendor) 0.4 µl GoTaq G2 DNA Polymerase (Promega, cod. M7845) 0.2 µl Molecular grade water 16.4 µl Total 25 µl REACTION MIX (OPTION B): Reagent Volume Total genomic DNA 2 µl 2X GoTaq Green Master Mix 12.5 µl (Promega, cod. M7122) 10 µm Forward Primer 0.5 µl 10 µm Reverse Primer 0.5 µl Molecular grade water 9.5 µl Total 25 µl Data 18/5/2015 Page 28

PCR conditions Primers RST31/RST33 94 C 5 min 1 cycle 94 C 30 sec 55 C 30 sec 35 cycles 72 C 45 sec 72 C 7 min 1 cycle Gel-electrophoresis Load 8-10 µl of PCR products on 1.2% Agarose gel in TAE 1X (STOCK 1lt 50X: Tris 242g, Acetic Acid 57 ml, EDTA 0,5 M-ph8 100ml) previously added of GelRed Nucleic Acis Stain (1µl/100ml of gel) (BIOTIUM, cod. 410003-0.5ml). RESULTS INTERPRETATION: Negative samples: No DNA band of the expected size is present. Positive samples: Clear DNA band of the expected size in the corresponding well. Samples which produce a faint bands should betested again in real-time PCR to confirm the first run results. Data 18/5/2015 Page 29

ANNEX VI Protocol for quantitative real-time PCR Primers and probe for quantitative real-time PCR (Harper et al., 2010) XF-F(forward) 5 -CAC GGC TGG TAA CGG AAG A-3 XF-R (reverse) 5 -GGG TTG CGT GGT GAA ATC AAG-3 XF-P (probe) 5 6FAM -TCG CAT CCC GTG GCT CAG TCC-BHQ-1-3 Reagent Volume Total genomic DNA 2 µl 2X master mix for probes (compatible with the 12.5 µl instrument) 10 µm Forward Primer 0.6 µl 10 µm Reverse Primer 0.6 µl 10 µm TaqMan probe (FAM) 0.2 µl Molecular grade water 9.1 µl Total 25 µl Quantitative real-time PCR amplification conditions 50 C 2 min 1 cycle 95 C 10 min 1 cycle 94 C 10 sec 39 cycles 62 C 40 sec RESULTS INTERPRETATION: Negative samples: If a sample produces a FAM Cq =0.00 or >35.00 then it is determined to be negative for X. fastidiosa. Positive samples: If a sample produces a FAM Cq value in the range of 0.00< FAM Cq <35.00 the sample is determined to be positive for Xylella fastidiosa Samples which produce a Fam Cq value in the range of 32.01>FAM Cq >34.99 need to be tested again in real-time PCR to confirm the first run results. Data 18/5/2015 Page 30

ANNEX VII Data 18/5/2015 Page 31

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ANNEX VIII Data 18/5/2015 Page 33

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ANNEX IX Data 18/5/2015 Page 37

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REFERENCES Djelouah, K., Frasheri, D., Valentini, F., D' Onghia, A.M. & Digiaro, M. 2014. Direct tissue blot immunoassay for detection of Xylella fastidiosa in olive trees. Phytopathologia Mediterranea, 53 doi: 10.14601/Phytopathol_Mediterr-14603 D Onghia, A. M., Santoro, F., Yassen, T., Djelouah, K., Guario, A., Percoco, A., Caroppo, T., & Valentini, F. 2014. An innovative monitoring model of Xylella fastidiosa in Apulia. International Symposium on the European Outbreak of Xylella fastidiosa in Olive, Journal of Plant Pathology, 96, S4.99. Harper S.J., Ward L.I., Clover G.R.G., 2010. Development of LAMP and real-time PCR methods for the rapid detection of Xylella fastidiosa for quarantine and field applications. Phytopathology 100: 1282 1288. Loconsole, G., Potere, O., Boscia, D., Altamura, G., Djelouah, K., Elbeaino, T., Frasheri, D., Lorusso, D., Palmisano, F., Pollastro, P., Silletti, M. R., Trisciuzzi, N., Valentini, F., Savino V. & Saponari, M. (2014a). Detection of Xylella fastidiosa in olive trees by serological and molecular methods. Journal of Plant Pathology, 96, 7-14. Loconsole, G., Potere, O., Elbeaino, T., Frasheri, D., Frisullo, S., Palmisano, P., Boscia, D. & Saponari, M. (2014b). Interlaboratory validation of molecular and serological diagnosis of Xylella fastidiosa strain CoDiRO in susceptible host plants. International Symposium on the European Outbreak of Xylella fastidiosa in Olive, Journal of Plant Pathology, 96, S4.100. Minsavage G.V., Thompson C.M., Hopkins D.L., Leite R.M.V.B.C., Stall R.E., 1994. Development of a polymerase chain reaction protocol for detection of Xylella fastidiosa in plant tissue. Phytopathology 84: 446-461. Nigro, F., Boscia, D., Antelmi, I. & Ippolito, A. (2013). Fungal species associated with a severe decline of olive in southern Italy. Journal of Plant Pathology, 95, 668. Saponari M., Boscia D., Nigro F., Martelli G.P., 2013. Identification of DNA sequences related to Xylella fastidiosa in oleander, almond and olive trees exhibiting leaf scorch symptoms in Apulia (Southern Italy). Journal of Plant Pathology 95: 668. Sherald J.L., Lei J.D., 1991. Evaluation of a rapid ELISA test kit for detection of Xylella fastidiosa in landscape trees. Plant Disease 75: 200-203. Data 18/5/2015 Page 39