017_COST(Lopez)_S105_ COL 15-06-2012 12:29 Pagina 105 Journal of Plant 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 BACTERIA 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 Erwinia 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 species, 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 plants (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).