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Journal of Pathology (2012), 94 (1, Supplement), S1.105-S1.113 Edizioni ETS Pisa, 2012 S1.105

DETECTION AND IDENTIFICATION METHODS AND NEW TESTS AS DEVELOPED AND USED IN THE FRAMEWORK OF COST873 FOR PATHOGENIC TO STONE FRUITS AND NUTS

Brenneria nigrifluens and Brenneria rubrifaciens

E.G. Biosca1 and M.M. López2

1 Departamento de Microbiología y Ecología, Facultad de Biológicas, Universitat de Valencia, Dr. Moliner 50, 46100 Burjassot, Spain 2 Instituto Valenciano de Investigaciones Agrarias (IVIA), Ctra. de Moncada-Naquera, km 4.5, 46113 Moncada, Valencia, Spain

SUMMARY INTRODUCTION

Brenneria nigrifluens and B. rubrifaciens are the Brenneria nigrifluens and Brenneria rubrifaciens (syn- causal agents of shallow bark canker and deep bark onym nigrifluens and Erwinia rubrifaciens) canker, respectively, of walnut trees. Both diseases and (Hauben et al., 1998) are the causal agents of shallow their etiological agents have been described in Europe bark canker (SBC) and deep bark canker (DBC), re- where they can cause serious economic losses. Since spectively, of walnut trees (Juglans regia and other spp.). there are no effective measures to control bark cankers These two types of cankers show similarities in external caused by these bacterial pathogens, rapid, specific and symptoms and in the production of black exudates. sensitive detection methods are required. However, Both diseases and their causal agents were first de- there are only few selective media and molecular detec- scribed in California (USA) (Wilson et al., 1957, 1967) tion protocols for these bacterial , which hinders but, until the 1990’s were reported rarely and only from and delays their detection and identification. The aim of the USA. this contribution is to select and describe in detail the In Europe, B. nigrifluens was initially identified in methods utilised and/or developed in several laborato- Spain in walnut orchards affected by shallow bark ries in the framework of COST 873. The PCR protocol cankers with exudates (López et al., 1994). Subsequent- for B. nigrifluens was appropriate for both diagnosis ly, the bacterium was also detected in Italy in young and identification, but showed sensitivity problems, nursery (Saccardi et al., 1998) and adult trees probably due to the high content of phenolic com- (Saccardi et al., 1998; Morone et al., 1998; Scortichini, pounds of bark tissues, requiring the optimisation of 1999, 2008; Loreti et al., 2008) and in France (Ménard DNA extraction protocols. For analyses of symptomless et al., 2004), but its records became more frequent in walnuts, it will be also necessary to increase the sensitiv- recent years (Scortichini, 2008). On the contrary, B. ity of the assay, or develop new PCR protocols. The in- rubrifaciens has only been detected once in Europe in a formation is very limited for B. rubrifaciens diagnosis or walnut orchard in Badajoz (Spain), in trees imported detection from naturally infected samples, because the from California (González et al., 2002). bacterium still has a very restricted distribution. On the There is little information on the current incidence of contrary, the number of techniques available for B. ni- these two bacterial pathogens in the different European grifluens and B. rubrifaciens identification is quite large, countries, but symptoms similar to those earlier ob- including physiological, biochemical, pathogenicity and served and caused by B. nigrifluens, have been observed PCR tests, as well as fatty acids or MALDI-TOF analy- in several Spanish regions (Biosca et al., 2002, 2008). ses, which have shown an appropriate specificity. These pathogens can cause serious economic losses be- cause of the weakening of trees and consequent reduc- tion in the number of nuts and in timber production, Key words: walnut, Juglans, shallow bark canker, apart from the importance of walnut trees losses for the deep bark canker, isolation, API, FAME, MALDI-TOF landscape. In general, there is scarce information on the MS, PCR, DNA extraction, pathogenicity. biology and epidemiology of both bacteria, although ap- parently they usually affect plants stressed by abiotic factors, such as water stress (Teviotdale and Sibbett, 1982). As there are no effective control measures to cure or avoid bark cankers caused by these pathogens, rapid, specific and sensitive detection methods are re- quired. Unfortunately, there are only few selective me- Corresponding author: M.M. López dia and molecular detection protocols for Brenneria Fax: +34963424001 E-mail: [email protected] species which hinders and delays their detection and 017_COST(Lopez)_S105_ COL 15-06-2012 12:29 Pagina 106

S1.106 B. nigrifluens and B. rubrifaciens detection and identification Journal of Plant Pathology (2012), 94 (1, Supplement), S1.105-S1.113

identification. For example, there is only one publica- Isolation from symptomless samples. Although B. tion on a PCR protocol for the molecular detection of nigrifluens and B. rubrifaciens probably have an epi- B. nigrifluens (Loreti et al., 2008) and two for B. rubrifa- phytic phase in walnut trees as it was demonstrated for ciens (McClean et al., 2008; Thapa et al., 2010). In this other Brenneria species in their respective host plants context, the main goal of this contribution is to select (Biosca et al., 2011), there are no available protocols and describe in detail the methods utilised and/or de- evaluated in COST 873 and specifically designed for veloped in several laboratories in the framework of their detection in symptomless organs of walnut trees. COST Action 873 and to mention with references other promising protocols from recent publications. Culture media. The general medium KB has been re- ported as appropriate for the isolation of both B. nigri- fluens (López et al., 1994; Menard et al., 2004) and B. HOST RANGE rubrifaciens (González et al., 2002) from exudates and cankers samples, and has been used in COST 873. The The only natural host of B. nigrifluens is walnut production of the pink rubrifacine pigment in KB and (Juglans regia) (Wilson et al., 1957). It can also affect J. YPGA media is very characteristic for B. rubrifaciens hindsii and J. sieboldiana as revealed by pathogenicity (Fig. 1B). The KB medium also allows differentiation of assays (Loreti et al., 2008). fluorescent Pseudomonas spp. from these Brenneria Natural hosts of B. rubrifaciens are J. regia, J. hindsii species, after examination under UV light. and J. nigra (Kado, 2010). Inoculation assays on 54 wal- KB medium (King et al., 1954). Per 1 liter of distilled nut cultivars from these Juglans species showed that water: 20 g proteose peptone Nº 3; 10 ml glycerol; 1.5 g

more than 70% of them were susceptible to B. rubrifa- K2HPO4; 1.5 g MgSO4 .7H2O; 15 g agar. Adjust pH to ciens, cv. Hartley being the most susceptible under field 7.0-7.2, and sterilize by autoclaving at 120ºC for 20 min. conditions (Kado, 2010). No reports are available on Other general media for isolation from symptomatic the isolation of these Brenneria species from other hosts. samples are YPGA (yeast extract peptone glucose agar) used in Spain and NSA [nutrient sucrose (5%) agar] or YDCA (yeast extract dextrose calcium carbonate agar) DETECTION AND IDENTIFICATION used by Loreti et al. (2008) and McClean et al. (2008) for B. nigrifluens or B. rubrifaciens, respectively. These au- This protocol describes isolation procedures from thors used also selective media, EMB (eosin methylene cankers and exudates and molecular detection methods. blue) agar (Difco, USA) for B. nigrifluens (Loreti et al., Isolation should be followed by bacterial identification 2008) and YDCA amended with novobiocin (30 mg ml- using appropriate techniques (Janse, 2010). 1) and bacitracin (10 mg ml-1) (YDCA+) for B. rubrifa- ciens (McClean et al., 2008). The efficiency of these last Isolation from symptomatic samples of SBC or DBC. media has not been evaluated in COST 873. Collect necrotic outer and/or inner bark tissue samples from cankers, and exudates. Select from cankers small ar- Detection by molecular methods. The same PCR eas at the border of necrotic and healthy tissues, excise protocols described for identification of B. nigrifluens aseptically and comminute in 5 ml of sterile PBS (8 g or B. rubrifaciens (see below) can be applied for their

NaCl; 0.2 g KH2PO4; 1.15 g Na2HPO4; 0.2 g KCl; pH detection in symptomatic plant material. However, the 7.2 per 1 liter of distilled water) or sterile saline solution practical efficiency of these protocols for naturally in- (SS) (0.9% NaCl in distilled water, pH 7.0) in a sterile fected walnut samples requires further evaluation be- Petri dish. Wait for 10-15 min before directly plating 50 cause it is necessary to eliminate inhibitors, present in µl (and also its dilutions) on King’s B medium (KB) (King the extracts from walnut tissues, by a DNA extraction et al., 1954) plus cycloheximide (250 mg/ml), or on an- step before amplification. other general medium (see below). The exudates can be The DNA extraction protocol by Llop et al. (1999) processed separately, diluted in 1 to 5 ml of sterile SS or was utilised in the framework of COST 873 according PBS. The remaining comminuted and exudate samples to the following: take 500 µl of comminuted sample pre- are held on ice prior to transfer into sterile Eppendorf pared as mentioned above and centrifuge at 10.000 g for tubes, which are stored at -20ºC in glycerol 20% (v/v) for 10 min at room temperature. Resuspend the pellet in subsequent PCR analysis. This procedure was also suc- 500 µl of extraction buffer (200 mM Tris HCl, pH 7.5; cessfully applied for isolation of other Brenneria species 250 mM NaCl; 25 mM EDTA; 0.5% SDS; 2% PVP, (Biosca et al., 2003, 2006). After incubation for 48 to 72 h sterilized by filtration). Vortex and shake for 1 h at at 26-28°C, Brenneria-like colonies (Fig. 1A and B) can room temperature. Centrifuge at 5.000 g for 5 min. be selected and purified for identification by physiologi- Transfer 450 µl of the supernatant to a clean tube and cal, biochemical, pathogenicity and PCR tests as de- add 450 µl of cold isopropanol. Mix gently and leave for scribed below. 1 h at room temperature. Centrifuge the mixture at 017_COST(Lopez)_S105_ COL 15-06-2012 12:29 Pagina 107

Journal of Plant Pathology (2012), 94 (1, Supplement), S1.105-S1.113 Biosca and López S1.107

Fig. 1. Colonies of B. nigrifluens NCPPB 564T (A) and B. rubrifaciens NCPPB 2020T (B) on KB plates after 48 h of incubation at 26-28ºC, which are light cream, circular, smooth and raised with entire margins. B. rubrifaciens produces the pink soluble pigment rubrifacine when grown on KB medium (Photos by E.G. Biosca).

13.000 g for 10 min. Discard the supernatant and dry Storage of isolates and reference strains. Pure cul- the pellet on air. Resuspend in 50 µl of sterile distilled tures from selected isolates and reference strains should water. Store at 4°C, or at - 20ºC for long term. be cryopreserved at -80oC in glycerol (25% v/v) after 48 This simple isopropanol extraction method proved h of growth at 26-28ºC on KB plates. more efficient in preliminary experiments than the ultra Type strains should be used as reference strains in all clean plant DNA kit isolation (Mo Bio, USA), designed the assays: for DNA extraction from plant material. DNA extracts NCPPB 564T Brenneria nigrifluens (Juglans regia; were diluted 1/10 and 1/100 before use for amplifica- California, USA, 1957). tion to decrease potential PCR inhibitors. NCPPB 2020T Brenneria rubrifaciens (Juglans regia; From our experience, walnut bark samples make diffi- California, USA, 1966). cult PCR detection, probably because of the presence of These type strains are also available from collections high concentrations of inhibitors (phenolic compounds other than that of the National Collection of Plant Path- among others). Further work is necessary to optimize ogenic Bacteria indicated above. DNA extraction to improve Brenneria spp. diagnosis from bark samples. The PCR protocol from Loreti et al. Identification by biochemical tests. The characteris- (2008) for B. nigrifluens detection, gave inconsistent re- tics of B. nigrifluens and B. rubrifaciens, according to sults from bark samples of some cultivars even when the Wilson et al. (1957, 1967), Hauben et al. (1998), Schaad above described DNA extraction step was applied. et al. (2001) and Biosca et al. (2003), are reported in The conventional PCR protocol of McClean et al. Table 1, whereas Table 2 shows the results obtained (2008) for B. rubrifaciens was asssayed only in USA with with tests of the miniaturized strips API 20E, utilised as DNA from sap samples from a walnut orchard exhibit- recommended by the manufacturer (BioMérieux, ing DBC symptoms, spiked greenhouse soil and infil- France), but incubated 48 h at 26-28ºC, or 24 h at 25ºC trated walnut leaf tissue (although B. rubrifaciens has (Biosca et al., 2003). not yet been found in natural conditions in soil and leaves). A masterpure total DNA extraction kit (Epicen- Identification by serological techniques. Commercial tre, USA) was used, with some modifications (McClean antisera or monoclonal antibodies specific for B. nigri- et al., 2008). DNA from spiked soil samples was extract- fluens and/or B. rubrifaciens are not available to the best ed as described by Wechter et al. (2003). Sensitivity of of our knowledge. Several antisera obtained at IVIA PCR in walnut leaves was around 182 CFU per mg leaf have been evaluated for specificity in indirect immuno- disc with primers sets BR, GSP1 and GSP2. A B. rubri- fluorescence and ELISA; these antisera against both faciens DNA concentration of 1 ng was detected in wal- bacteria, obtained using whole cells and O antigens nut sap using a BR primer set and real-time PCR. How- were not species-specific, showing cross-reactions with ever, detection of B. rubrifaciens from naturally infected other Brenneria species. bark samples was not reported by these authors. 017_COST(Lopez)_S105_ COL 15-06-2012 12:29 Pagina 108

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Identification by automated biochemical techniques. identification of B. nigrifluens and B. rubrifaciens, this FAME (fatty acid methyl esters profiles). Qualitative and method has been applied to the type strains of both quantitative cellular fatty acid analyses can be per- species. Bacterial cells from KB medium at 26ºC for 48 formed as described by Sasser (1990). FAMEs were ex- h were harvested to obtain the biomass for protein tracted using hexane/methyl-tert butyl ether (MTBE) analysis directly from whole cells. The analysis was per- and analyzed with the Microbial Identification System formed following the protocol recommended by Bruker (MIS, Hewlett-Packard, USA) and the corresponding Daltonics (http://www.bdal.de) by ethanol/formic acid software (MIDI, USA) as previously described (Biosca extraction and MALDI-TOF MS using a Reflex IV et al., 2003) for Brenneria spp. The fatty acid composi- mass spectrometer (Bruker Daltonics, Germany) tion of type strains of these species is shown in Table 3. equipped with a N2 laser. Spectra were acquired in pos- MALDI-TOF MS (Matrix-Assisted Laser Desorption/ itive linear ion mode, as the sum of 240 shots per target, Ionization Time-of-Flight Mass Spectrometry) protein and between 12-16 targets by strain. The mass range mass fingerprints. MALDI-TOF MS is a well established used for analysis was 2.000-20.000 Da, as described in method for peptide and protein mass profiles (mainly Maier et al. (2006). Results obtained with the type ribosomal proteins), which is increasingly being used in strains of these Brenneria species are in Fig. 2, which bacterial identification because of its rapidity and dis- shows that the type strains of B. nigrifluens and B. rubri- criminatory power. To evaluate its accuracy for rapid faciens have most peaks in common, but also some

Fig. 2. MALDI-TOF MS protein mass fingerprints from type strains of B. nigrifluens (A) and B. rubrifaciens (B) that show charac- teristic whole-cell profile of normalized peaks from M/Z (Da) 1.000-12.000. Normalized peak intensity of ions is shown on the y axis and the masses (Da) of the ions are shown on the x axis. The m/z values represent mass-to-charge ratios. 017_COST(Lopez)_S105_ COL 15-06-2012 12:29 Pagina 109

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Table 1. General physiological and biochemical characteristics of type strains of Brenneria nigrifluens and Brenneria rubrifaciens.

Testa B. nigrifluens B. rubrifaciens NCPPB 564T NCPPB 2020T Gram stain or KOH 3% test - - Oxidase - - Catalase + + O/F glucose metabolism +/+ +/+ Nitrate reduction - - Arginine dihydrolase - - Urease - - Indole - - Esculin hydrolysis + + Pectate degradation - - β-galactosidase + - Acid production from: L-arabinose + + Raffinose + - Xylose + - Growth at 36ºC+- Growth at 39ºC-- a Results after 48 h of incubation at 26-28ºC.

unique ones in the range from 3.000 to 11.000 Da, that USA)] 0.5 µl, forward primer F1 (10 µM) 0.5 µl, reverse distinguish B. nigrifluens from B. rubrifaciens, and both primer C3 (10 µM) 0.5 µl, Taq DNA polymerase from of them from the type strains of B. alni and B. salicis Biotools (5 U µl-1) 0.4 µl, DNA template (heated bacter- (data not shown). More strains of these species should ial culture, SS or PBS) 5.0 µl, in a total volume of 50 µl. be tested, however, to substantiate these findings. DNA polymerase (Code No. R001 A) from Takara (Spain) was used for plant material (this polymerase

Identification by molecular techniques does not require addition of MgCl2 since it is provided A. Conventional PCR and real-time PCR protocols. in Takara PCR 10X buffer). Amplification conditions Modified protocol from Loreti et al. (2008) for B. nigri- according to Loreti et al. (2008) were: an initial denatu- fluens. Loreti et al. (2008) selected the F1-C3 primers in ration cycle at 96ºC for 3 min; 30 cycles of 1 min at a 1 kb DNA sequence from an amplicon of B. nigrifluens 94ºC, 1 min at 57ºC, 45 sec at 72ºC, and a final exten- genomic DNA obtained using the minisatellite M13 sion at 72ºC for 5 min. Appropriate positive and nega- primer. The expected product of the F1-C3 primers is a tive controls should be included. 250 bp DNA band. The specificity of the method for PCR products were analyzed by electrophoresis in detection of B. nigrifluens, was assessed and confirmed 1% agarose gels (w/v) in 1X TAE buffer (National Di- for 6 Spanish strains as well as for 8 strains of closely relat- agnostics, USA) by loading 10 µl of each amplicon plus ed Brenneria and non-Brenneria species (Agrobacterium, 2 µl of loading buffer. A molecular weight marker [100 Pantoea, Escherichia and Pseudomonas). Sensitivity was bp DNA ladder (Invitrogen, USA)] was included. Gel evaluated using pure cultures of the B. nigrifluens type can be run at 90 V for about 1 h, then stained with strain and two Spanish strains (ranging from 1 to 108 ethidium bromide for 15 min and visualized with UV CFU ml-1). Amplifiable DNA from pure cultures of B. light. A DNA fragment of the predicted size (about 250 nigrifluens was obtained by heat-treatment of bacterial bp) indicates the presence of B. nigrifluens DNA in the suspensions at 95°C for 5 min and subsequent cooling sample, provided that the negative controls are negative. on ice. The sensitivity observed with pure cultures was Protocol from McClean et al. (2008) for B. rubrifaciens. 104 CFU ml-1. Primers sequences are reported in Table This protocol has not been evaluated in the framework of 4. Mastermix (per 50 µl reaction) for conventional PCR COST 873 but the sequences of the primers are listed in for B. nigrifluens, used in our laboratories (according to Table 4. BR primers were designed from the 16S rRNA Loreti et al., 2008) but optimized for Thermocycler gene and GSP1 and GSP2 primers from sequences of a 2720 (Applied Biosystems, USA), was: ultrapure sterile gene involved in rubrifacine production. According to water 36.3 µl, PCR 10X buffer (Biotools, Spain) 5.0 µl, McClean et al. (2008), the same reactions and cycling

MgCl2 (50 mM) 1.8 µl, dNTP mix [10 mM, (Invitrogen, conditions were used for all three primers pairs. Ampli- 017_COST(Lopez)_S105_ COL 15-06-2012 12:29 Pagina 110

S1.110 B. nigrifluens and B. rubrifaciens detection and identification Journal of Plant Pathology (2012), 94 (1, Supplement), S1.105-S1.113

Table 2. Physiological and biochemical characteristics of type strains of Brenneria nigrifluens and Brenneria rubrifa- ciens by using the API 20E system.

API 20E test B. nigrifluens B. rubrifaciens NCPPB 564T NCPPB 2020T ONPG - - Arginine dihydrolase - - Lysine decarboxylase - - Ornithine decarboxilase - - Simmons citrate - -

Production of H2S- - Urease - - Tryptophane desaminase - - Indole - - Voges-Proskauer -a - Gelatin liquefaction - - Acid production from: Glucose + + Mannitol + + Inositol + - Sorbitol + - Rhamnose + - Sucrose + + Melibiose + - Amygdalin + - Arabinose + + a Voges-Proskauer test was negative for the type strain of B. nigrifluens but variable for Spanish strains of this species in the API 20E strip.

cons of 409, 233 and 280 bp for primers BR1/BR3, and GSP1 primers and 2.5×104 CFU ml-1 for GSP2 GSP1F/GSP1R and GSP2F/GSP2R, respectively, indi- primers, using DNA extracted from ten-fold serial dilu- cate the presence of B. rubrifaciens DNA in the sample. tions of B. rubrifaciens. Sensitivity was reported to be 2.5×102 CFU ml-1 for BR Protocol from Thapa et al. (2010) for B. rubrifaciens.

Table 3. Fatty acid composition (percentage of peak areas) of type strains of Brenneria nigrifluens and Brenneria rubrifaciens.

Fatty acid class B. nigrifluens B. rubrifaciens NCPPB 564T NCPPB 2020T Saturated fatty acids

12:0 3.24 (0.29)a 3.11 (0.61) 14:0 6.15 (0.10) 7.15 (1.68) 16:0 30.58 (1.82) 29.47 (1.01) 18:0 1.04 (0.73) 0.19 (0.28) Unsaturated fatty acids 16:1 w7c 31.04 (0.35) 29.18 (1.22) 18:1 w7c 13.97 (1.25) 12.14 (0.55) Cyclopropane fatty acids 17:0 6.63 (0.54) 1.62 (0.02) 19:0 1.29 (0.60) 0 Hydroxy fatty acids 14:0 3OH 8.99 (0.21) 9.89 (0.24) Unknown 14.503b 0.78 (0.23) 0.42 (0.59) aMean (%) and standard deviation of two extractions per each strain. bEquivalent chain length relative to the lengths of known fatty acids. 017_COST(Lopez)_S105_ COL 15-06-2012 12:29 Pagina 111

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This protocol has not been evaluated in the framework h culture grown on KB medium. Inoculated plants of COST 873. The authors used this protocol with sam- should be maintained for three months at 22-28°C with ples from inoculated walnut branches. It utilised light and at 70-80% RH in greenhouse or plastic tunnel. primers BrAF/BrAR (designed from sequence of as- The appearance of SBC or internal necrosis of tissues, paragine synthase gene) and 2BrIF/2rBIR (designed requires at least one month (Moretti and Buonaurio, from sequence of the autoinducer synthase gene in- 2010). However, for inoculation experiments the use of volved in rubrifacine production), previously reported older walnut plants (more than seven years old) would by McClean and Kuepfel (2009) that are shown in Table be advisable, since they are considered more susceptible 4. They yield amplicons of 537 and 671 bp, respectively. to the disease (Wilson et al., 1957). Sensitivity was reported to be 5×102 CFU ml-1 and 5×104 CFU ml-1 for primers BrAF/BrAR and 2BrIF/ B. Methods for plant material inoculation with B. 2BrIR, respectively, when using pure cultures. rubrifaciens Real-time-PCR protocol from McClean et al. (2008) Tissue culture inoculation (McClean et al., 2009). for B. rubrifaciens. This real-time PCR for B. rubrifaciens Young shoots of cvs Chandler and Hartley, growing on detection with BR1/BR3 primers using SYBR Green, DKW tissue culture medium (Driver and Kuniyuki, has not been evaluated in the framework of COST 873. 1984), can be inoculated with a sterile scalpel with B. Its sensitivity was reported to be 8 CFU ml-1 for DNA rubrifaciens cells scraped from a 3-4-day-old culture extracted from ten-fold serial dilutions of B. rubrifaciens grown on YDCA. The shoots should be incubated on cells grown in pure culture. the same medium for 10-11 weeks at 25ºC, 40% RH and 16 h of light. Hartley shoots exhibited disease B. Molecular fingerprinting. symptoms within 3 weeks, while Chandler needed 10 Several molecular techniques could be used for finger- weeks before symptoms appearance. Then, cv. Hartley printing Brenneria species from walnut but there are few should be used for a rapid virulence bioassay. This diversity studies available. Moretti et al. (2007) found method has not been evaluated in COST 873. that four B. nigrifluens isolates from two locations gener- Plant stem inoculation (González et al., 2002). Accord- ated the same fingerprint by REP-PCR as reference ing to Wilson et al. (1957) it is convenient to use walnut strains. Loreti (2007) obtained three patterns using plants older than seven years for a better expression of ERIC-PCR and two patterns using microsatellite M13 symptoms. The test was carried out on Persian walnut with a selection of B. nigrifluens strains. However, there plants by inoculating B. rubrifaciens suspensions at 108 are no reports on B. rubrifaciens diversity. For standard CFU ml-1 (48 h culture grown on KB plates) in deep rep-PCR protocols see EPPO Standard (OEPP/EPPO, wounds made in the trunk with a knife at 40 and 80 cm 2010). from the crown. Inoculated plants should be maintained for 3 months at 22-28°C with 16 h light and high RH. If external symptoms of DBC were not observed, the bark Identification by pathogenicity tests was carefully removed to observe lesions in the inner A. Methods for plant material inoculation with B. ni- part. At least, two months of incubation were required. grifluens. In all types of inoculations, a reference strain and Immature walnut fruit inoculations. The test on imma- sterile water, SS or PBS should be included as controls. ture fruits from adult walnut trees, slightly modified from Further, inoculated bacteria should be re-isolated from Moretti and Buonaurio (2010) by including a disinfection lesions to confirm the presence of colonies of either B. step, was used for assaying B. nigrifluens pathogenicity. nigrifluens or B. rubrifaciens and putative Brenneria sp. Fruits should be briefly disinfected (1 min) with 70% colonies should be identified by appropriate techniques. ethanol and repeatedly washed with sterile water prior to inoculation. Then, B. nigrifluens (108 CFU ml-1 from a 48 Hypersensitive (HR) assay. This test is negative for h culture grown on KB medium) was infiltrated into the both Brenneria species from walnut trees (López et al., mesocarp with a syringe either with needle (in an area 1994; González et al., 2002; Biosca et al., 2003). The about 1 cm in diameter) or without needle (three punc- HR inoculation can be performed on tobacco leaves tures per fruit, in areas about 0.5-0.8 cm in diameter). In- (Klement et al., 1964) with bacterial suspensions at a oculated fruits were incubated in plastic boxes for 8 days concentration of 109 CFU ml-1 in PBS. at 20°C, with a 12 h photoperiod. The test was consid- ered positive when symptoms of necrosis and exudates (starting after 2 days) appear in inoculated sites. CONCLUDING REMARKS Plant stem inoculation (Moretti et al., 2007). This test was performed on two-year-old Persian walnut plants A new integrated protocol for diagnosis and/or de- by stem inoculation (about 1 cm long wound) of 20 µl tection of B. nigrifluens and B. rubrifaciens, based on of B. nigrifluens suspensions at 108 CFU ml-1 from a 48 isolation and PCR analysis with several sets of primers 017_COST(Lopez)_S105_ COL 15-06-2012 12:29 Pagina 112

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Table 4. Primers for Brenneria nigrifluens and Brenneria rubrifaciens.

Name Sequence (5´- 3´) References Forward primer F1 CCTGCGCCATGTTGCCAGATCGCTAT Loreti et al., 2008 Reverse primer C3 ACCTGAGTAGCAGTTTCGACTATTT (B. nigrifluens) Forward primer BR1 CAGCGGGAAGTAGCTTGCTACTTTGCCGG Reverse primer BR3 TGAAAAAGTCTCTCTTAAACCTTTCC Forward primer TAGTGTTGCATTAGCCGATTTAG GSP1F Reverse primer McClean et al., 2008 GCATTTAAAGACTATGTTTCCTG GSP1R (B. rubrifaciens) Forward primer CATTACTGTTTCTCCTCGCTAATC GSP2F Reverse primer GATGTAAATTAGCCATACACGGAATG GSP2R Forward primer BrAF ATGTACGCAGTCTCTATTTGG McClean and Kuepfel, Reverse primer BrAR CCATCAGCCTGAAATAACTCA 2009; Forward primer 2BrIF CGGGATCCATGTTAGAAATATTCGATGTC Thapa et al., 2010 Reverse primer 2BrIR ATCAGCTGTCAAGCCTCTTCCTTTTTG (B. rubrifaciens)

indicated here, should be validated in different labora- ses have shown an appropriate specificity. Pathogenicity tories. Currently, both conventional and molecular as- tests are also required for ultimate diagnosis, especially says are necessary for the diagnosis and detection of when the disease and pathogen are found for the first these walnut pathogens. There is a need of comparative time and when required by the National Plant Protec- analyses of the efficiency of the available culture media tion Organizations. for isolation, but as PCR and especially real-time PCR still remain as the gold standard tests for rapid screen- ing of plant samples, new real-time PCR protocols will ACKNOWLEDGEMENTS be very useful for diagnosis laboratories. For a more ac- curate evaluation of the sensitivity of the PCR protocols The authors thank the European Science Foundation mentioned here, further comparative assays using symp- research network COST Action 873 for its support of tomatic plant material with SBC or DBC from naturally networking initiatives, leading to a productive coopera- infected hosts of different cultivars are necessary, as well tion among international research groups involved in de- as optimisation of DNA extraction protocols from tis- veloping methods and strategies for pest management of sues that are usually affected in natural conditions, such bacterial diseases of stone fruits and nuts. This work was as the bark of the walnut trunk. For analyses of symp- carried out in the framework of COST 873 “Bacterial tomless walnuts, it will be also necessary to increase the diseases of stone fruits and nuts” in collaboration be- sensitivity of the assay, or develop new PCR and/or real- tween the Instituto Valenciano de Investigaciones time protocols, LAMP assays or others. The protocol of Agrarias (IVIA) and the Universitat de Valencia. The au- Loreti et al. (2008) was appropriate for identification of thors thank R. González, S. Martín, B. Aguila and J.F. B. nigrifluens but was not sufficiently sensitive to allow Català-Senent for valuable technical assistance and E. its reliable detection from all symptomatic walnut sam- Pérez-Laorga (Generalitat Valenciana. Conselleria de In- ples tested. This is probably directly related to the high fraestructuras, Territorio y Medio Ambiente) for help in content of phenolic compounds in walnut bark tissues, field sampling. or others that act as PCR inhibitors. A duplex real-time PCR protocol for the sensitive and specific simultaneous detection of both pathogens would also be very useful. REFERENCES The information is very limited for B. rubrifaciens di- agnosis or detection from naturally infected samples, be- Biosca E.G., Català-Senent J.F., Aguila B., González R., cause the bacterium still has a very restricted distribution. Pérez-Laorga E., López M.M., 2011. Epiphytic survival Comparatively, the number of identification tech- and temporal dissemination of Brenneria quercina in a plot niques available for B. nigrifluens and B. rubrifaciens is of Quercus ilex in Spain. IV International Conference on quite large, because biochemical and molecular meth- Environmental, Industrial and Applied Microbiology, Mála- ga, Spain: 41. ods, as well as fatty acids profile or MALDI-TOF analy- 017_COST(Lopez)_S105_ COL 15-06-2012 12:29 Pagina 113

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