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MOLECULAR TYPING OF DUTCH ISOLATES OF XANTHOMONAS ARBORICOLA pv. PRUNI ISOLATED FROM ORNAMENTAL CHERRY LAUREL M. Bergsma-Vlami1, W. Martin2, H. Koenraadt3, H. Teunissen3, J.F. Pothier4, B. Duffy4 and J. van Doorn2 1Plant
Protection Service, P.O. Box. 9102, 6700 HC Wageningen, The Netherlands University and Research Centre, P.O. Box. 85, 2160 AB Lisse, The Netherlands 3Naktuinbouw, P.O. Box. 40, 2370 AA Roelofarendsveen, The Netherlands 4Agroscope Changins-Wädenswil ACW,Plant Protection Division, Schloss 1, CH-8820 Wädenswil, Switzerland 2Wageningen
SUMMARY
Xanthomonas arboricola pv. pruni (Xap) has been found in several cherry laurel (Prunus laurocerasus) nurseries in the Netherlands, causing leaf spot. As no information is available yet about the epidemiology of this quarantine bacterium in cherry laurel, molecular typing of Xap isolates can considerably improve our understanding of pathogen spread between various cherry laurel production systems in different regions of the Netherlands and pathogen relatedness among different disease outbreaks. In this study, the genotypic diversity within a population of 25 Xap isolates isolated from different cherry laurel cultivars grown in different locations in the Netherlands between 2008-2010, was assessed using Multiple-locus variable-number analysis (MLVA). The identity of these Xap isolates was initially determined based on the EPPO standard PM 7/64. Confirmation of the identity of these Xap isolates was additionally achieved with diverse methodologies, including gyrase subunit B (gyrB) sequence typing, BOXand ERIC-PCR, AFLP, and Xap-specific PCR’s: one based on the previously described Pagani primers (2004) (conventional-PCR and its TaqMan-PCR variant) and one based on the recently described Pothier primers (2011c). Based on the results of the MLVA analysis, the Dutch population of Xap isolates could be divided into two groups; however no correlation with the geographical origin or any other character of these isolates could be established. Additionally, based on colony morphology, a panel of 5 look-a-likes were isolated from symptomatic leaves of P. laurocerasus which reacted in the Xap-specific PCR described by Pagani (2004) but that did not react in the Xap-specific PCR described by Pothier et al. (2011c). Further characterisation of these look-a-like isolates with AFLP, BOXand ERIC-PCR, and gyrB sequencing showed that the Xap-specific PCR described by Pagani does not discriminate between Xap and the look-a-like isolates. Similarly to Pagani PCR, the performance of a pathogenicity test Corresponding author: M. Bergsma-Vlami Fax: +31.317.421701 E-mail:
[email protected]
with a pure culture of the isolate was not always discriminative between Xap and the look-a-like isolates, unraveling a complexity in Xanthomonas pathogenicity. Therefore, in routine screening based on the EPPO standard PM 7/64, complementary techniques such as BOX- ERIC-PCR, gyrB sequencing, Xap-specific PCR described by Pothier (2011c), MLVA and AFLP should be used to obtain a reliable diagnosis of Xap and avoid false positive results. Key words: Xanthomonas arboricola pv. pruni, VNTR, MLVA, AFLP, BOX- and ERIC-PCR, gyrB sequence analysis, pathogen-specific PCR, genotypic diversity. Bacterial spot due to Xanthomonas arboricola pv. pruni (Xap) is a serious disease of stone fruits, mainly in peach and plum. The disease was described for the first time on plum in the United States by Smith (1903), and was introduced into Europe, first in Italy (Petri, 1934; Zaccardelli et al., 1998; Stefani, 2010). In most countries, including the entire EU and EPPO region, Xap is regulated as a quarantine pathogen (Anonymous, 2000, 2006; EPPO/CABI, 1997). In the Netherlands Xap has until now only been detected in the ornamental species P. lauroceracus (cherry laurel) (Tjou-Tam-Sin et al., 2012). The number of Xap-positive samples diagnosed by the Bacteriology laboratory of the Dutch PPS has gradually increased from 63 in 2009 to 80 in 2010, infections found in different cultivars and locations (unpublished data, Plant Protection Service, The Netherlands). The aim of this work was to obtain a better understanding of Xap epidemiology by using molecular-based techniques to characterise the Dutch population of Xap isolates. With this knowledge, effective measures can possibly be implemented to eradicate this contagious bacterial disease in P. lauroceracus. Bacterial strains were isolated from symptomatic leaves of cherry laurel (Fig. 1) by plating three 10-fold dilutions on Wilbrink (Sands et al., 1986) or mXCP1 media. The mXCP1 medium (Xanthomonas campestris pv. phaseoli medium) was adapted from McGuire et al. (1986) to allow better recovery of seed transmittable xanthomonads of bean (H. Koenraadt, unpublished information). Modified mXCP1 medium was prepared by
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dissolving 10.0 g l-1 KBr, 0.25 g l-1 CaCl2, 10.0 g l-1 soluble potato starch, 10.0 g l-1 peptone, with 15.0 g l-1 of agar and adding after autoclaving 0.15 ml crystal violet, 35 mg l-1 nystatin, 10 mg l-1 cephalexin, 3 mg l-1 fluorouracil, 0.112 mg l-1 tobramycin and 10 ml l-1 Tween 80. In order to discriminate between other bacterial diseases induced in P. laurocerasus, by Pseudomonas syringae pv. morsprunorum and P. s. pv. syringae (Kaluzna et al., 2010), a TaqMan PCR assay was developed, based on the primers and sequence described by Pagani (2004). TaqMan PCR was developed with forward primer Y17CoF3 (5’-TCA AGG TGG CAG AAT GAG TG-3’), reverse primer Y17CoR (5’-GAC GTG GTG ATG ATG ATC TGC-3’) and a FAM-TAMRA probe (5’-FAM-CTC GCA GAT CCC GGT TAT T-3’). PCR was carried out in a total volume of 25 µl containing 12.5 µl Brilliant II QPCR Master Mix 2X (Stratagene Europe, The Netherlands), 1 µl DNA, 0.75 µl of
Fig. 1. Typical necrotic symptoms with water soaked rims, caused by Xanthomonas arboricola pv. pruni in Prunus laurocerasus cv. Rotundifolia.
Table 1. Bacterial strains of Xanthomonas arboricola pv. pruni and other Xanthomonas spp. used in this study.
The Q in the Qxx numbers denotes the quarantine status of the isolates in the collection of PPO-Lisse. The K in the Kxxxx numbers denotes that the isolates in the collection of the Plant Protection Service were kept in vials at -80oC.
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each primer (300 nm), 0.25 µl probe and 9.75 µl nuclease-free water using an initial denaturation step of 10 min at 95°C, followed by 40 cycles of 15 sec at 95°C and 1 min at 60°C after 2 min at 50°C. The sensitivity of this TaqMan assay was within the range of 103-104 CFU ml-1 leaf extract, as determined by spiking leaf extract samples with a range of Xap isolates at different concentrations (data not shown). A second Xap-specific PCR was applied based on the primers and sequence described by Pothier et al. (2011c). BOX- and ERIC-PCR were performed according to a protocol slightly adapted from Versalovic et al. (1991), whereas AFLP analysis was carried out as described by Zaccardelli et al. (1999). Additionally, gyrB sequence typing was performed by using the single locus sequence typing protocol described by Parkinson et al. (2009). Genotypic diversity was assessed using MLVA on a total of 25 Xap isolates (Table 1) recovered from different Dutch locations and different P. laurocerasus cultivars between 2008 and 2010. Bacterial isolates were cultured for 48 h in Nutrient broth (Oxoid, UK). One ml of cell suspension was centrifuged at 12,000 rpm for 10 min and the resulting pellet resuspended in 50 µl of lysis buffer (Agowa, Germany). DNA isolations were then carried out using magnetic particle isolation (King Fisher system, Thermo Scientific, USA) in combination with an AGOWA magnetic plant kit (AGOWA/LCG, Germany). DNA concentrations were estimated by reading absorbance at OD 260 nm; samples were then diluted with nuclease-free water to give similar target concentrations in the range of 1-4 µg ml-1. To confirm the identity of isolates used in this analysis, gyrB sequence typing and BOX- and ERIC-PCR were performed beforehand. Sequence analysis of the gyrB gene (Parkinson et al., 2009) of the Xap isolates revealed in all cases a 99.6% homology with the Xap reference strain ICMP51, when fragments between 727 and 730 bp were used (accession No. EU498953) (Young et al., 2008). Patterns acquired by BOX- and ERIC-PCR among the Xap isolates showed a very high degree of homology (Fig. 3A and 3B). Additionally, all 25 Xap isolates gave a positive PCR reaction with the primers described by Pagani (2004) and by Pothier et al. (2011a, 2011c). Based on the whole genome sequence of the Xap reference strain CFBP 5530 (Pothier et al., 2011b), 51 possible variable number tandem repeat (VNTR) loci were identified at Agroscope, Switzerland (Pothier et al., in preparation). Primers were designed on the flanking regions of target loci and tested with a limited number of Xap, X. arboricola pv. juglandis and X. arboricola pv. corylina strains. Twenty eight of these VNTR were tested with a larger group of X. arboricola strains (Pothier et al., in preparation). Based on this work, 12 loci were selected for the MLVA analysis of the 25 Xap isolates found on P. laurocerasus in the Netherlands. Information regarding motif sequence, repeat number in Xap reference strain CFBP
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5530 and primer sequences were provided by Agroscope (Pothier et al., in preparation). Primers used in MLVA analysis were synthesised by Biolegio BV (The Netherlands). All reactions were carried out in a total reaction volume of 25 µl containing 0.4 µM forward and reverse primers, 12.5 µl Master Mix 2X [50 units/ml Taq DNA polymerase, 400 µM dNTPs and 3 µM MgCl2 (Promega, USA)] 2.5 µl template DNA and 8 µl nuclease-free water. Amplification programs varied depending on Tm of primers: initial denaturation at 94°C for 5 min followed by 35 cycles of denaturation at 94°C for 30 sec, annealing at 60°C, 64°C or 66°C for 30 sec and extension at 72°C for 45 sec. Final extension was 10 min at 72°C. Products were analysed on a 2% agarose gel with 0.2 µg ml-1 ethidium bromide and visualized with a UV imager. From the 12 selected loci, 10 primer combinations
Fig. 2. Dendrogram of 25 Xanthomonas arboricola pv. pruni isolates used in MLVA analysis. It was constructed with UPGMA (POPTREE2), 14ST’s were found. The letters A-D correspond to different geographical parts of The Netherlands, from which the isolates originated. Bar represents the estimated relative distance between isolates.
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gave sufficient universal amplification for MLVA analysis of the Dutch isolates. Data from 6 of these loci (Xap1, Xap2, Xap4, Xap7, Xap10 and Xap12) were sufficient to discriminate among all 25 Xap isolates. Table 2 shows the allele number of tandem repeats at selected loci and the discriminatory level of each VNTR. Based on sequence data alone, 7 distinct different allele numbers were found at the locus Xap1. This locus gave the highest discriminatory level among the Dutch isolates tested. This was consistent with results obtained from the COST collection of Xap isolates of worldwide origin where Xap1 was also found to be the most discriminatory locus (Pothier et al., in preparation). From the 25 Xap isolates, 14 distinct profiles (ST’s) were found. A dendrogram was constructed (Fig. 2) using Poptree 2 software with the UPGMA (Unweighted pair group method witha mean) algorithm (Sneath and Sokal, 1973). The analysis of polymorphic data with the UPGMA algorithm showed that these 14 profiles clustered into two distinct groups (Fig. 2). Some of the isolates also shared identical profiles: K2804, K2841, K2771, K2957, K2826, Q24, Q11, Q13, Q14 (profile 1) and K2810, K2761, K2839, Q12 (profile 10). Based on
the analysis of the 25 Xap isolates, no correlation with the geographical origin or any other character of these isolates was found. The two groups may represent different introductions of Xap by importation of P. laurocerasus from other parts of Europe. To substantiate this hypothesis a larger group of isolates, obtained from other European countries is currently being analysed. A highly specific PCR system, especially for quarantine pathogens, is a prerequisite for a reliable routine detection and identification (Maes, 1993; Palacio-Bielsa et al., 2010; López et al., 2010; Doorn et al., 2010). However, in the case of Xap, a routine setting cross reaction was occasionally observed with the Pagani (2004) PCR or/and the derived TaqMan PCR based on the Pagani primers (Y17CoF3 and Y17CoR). This cross reaction occurred not only with X. arboricola pv. juglandis strain LMG 745 and X. arboricola pv. corylina strain LMG 688, but also with a panel of 5 look-a-likes isolated in 2010 from symptomatic leaves from different cultivars of P. laurocerasus grown in the Netherlands. All look-a-like isolates showed the typical yellow, shiny colony phenotype on both Wilbrink and mXCP1 media. As indicated by BOX-, ERIC-PCR, AFLP and gyrB
Fig. 3. Agarose gel with ERIC-PCR (A) and BOX-PCR (B) fingerprints from Xanthomonas arboricola pv. pruni isolates that were isolated from symptomatic leaves of P. laurocerasus. Lanes 1-6 and lanes 9-20 refer to Xap isolates and lane 24 to the 100 bp size marker, respectively. Lane 22 corresponds to the Italian Xap reference strain PD 740, clearly showing an additional band in ERICPCR (3A). Additionally, the ERIC-PCR fingerprint pattern (3A) in lane 18 was erratic. Lanes 21 and 23 were empty. Isolates in lanes 7 (strain 162) and 8 (strain 162A), representing look-a-likes, gave atypical fingerprints when compared to the fingerprints of the Xap isolates.
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sequence typing, these 5 look-a-likes proved to be false positives in the PCR test and are not considered to be Xap. BOX- and ERIC-PCR fingerprints (Fig. 3A and 3B) already showed significant differences between the Xap isolates and the look-a-likes. AFLP data confirmed this observation (Fig. 4). Pathogenicity tests were not always conclusive, unraveling a complexity in Xanthomonas pathogenicity (Young et al., 2010). Analysis of the 727 bp fragment from the gyrB gene of the 5 look-a-likes did not match with any of the published Xap gyrB sequences in the NCBI database. Isolates 77 and 158 shared 99.0% and 99.3% homology with Xanthomonas arboricola refer-
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ence strains ICMP4939 (accession No. GQ183106) isolated from Mahonia lomariifolia and ICMP8452 isolated from Magnolia sp. (accession No. GQ183105), respectively (Young et al., 2010). Surprisingly, isolates 159 and 162 showed 99.6 % and 99.0% homology with X. arboricola pv. celebensis reference strain ICMP 1488 (accession No. EU498984), respectively (Young et al., 2008). Additionally, isolate PD 5859 was almost identical (99.7% homology) to the recently described Xanthomonas dyei reference strain ICMP 2374 (accession No. GQ183099) isolated from Aralia sp. in New Zeeland (Young et al., 2010). BOX-, ERIC-PCR and AFLP analysis of these reference strains and the look-a-likes
Table 2. VNTR loci used in MLVA analysis of 25 Xap Dutch isolates and their profiles based on allele number of tandem repeat at selected loci. Locus
Xap1
Xap12
Xap7
Xap10
Xap4
Xap2
Motif and allele No. in Xap reference strain CFBP 5530
16 (GGGAAT C)
11 (TCCCGA T)
8 (CAAG CCACA GATCG TCCGT T)
9 (TCGGCA )
4 (TTCCCG A)
9 (CCCAAT C)
Isolate
Profile
K 2812
21
5
7
8
5
6
9
K 2810
21
4
7
8
5
6
10
K 2804
12
4
6
6
4
6
1
K 2841
12
4
6
6
4
6
1
K 2774
23
9
6
6
4
8
12
K 2771
12
4
6
6
4
6
1
K 2762
13
3
7
8
5
6
6
K 2815
20
4
8
8
5
6
8
K 2761
21
4
7
8
5
6
10
K 2562
12
5
6
6
4
7
2
K 2957
12
4
6
6
4
6
1
K 2843
12
5
6
8
4
6
3
K 2839
21
4
7
8
5
6
10
K 2826
12
4
6
6
4
6
1
K 2822
23
3
7
8
5
6
13
Q9
22
4
7
8
5
6
11
Q10
23
5
7
8
5
6
14
Q15
12
6
6
6
5
6
5
Q18
18
4
7
8
5
6
7
Q21
12
5
6
6
4
6
4
Q24
12
4
6
6
4
6
1
Q11
12
4
6
6
4
6
1
Q12
21
4
7
8
5
6
10
Q13
12
4
6
6
4
6
1
Q14
12
4
6
6
4
6
1
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working initiatives, leading to a productive cooperation among research groups in the Netherlands, France and Switzerland. The authors also thank S. Cesbron, S. Gironde and C. Manceau for their valuable help in developing methods during COST Action 873. The work presented here was financed by the Dutch Ministry of Economic Affairs, Agriculture and Innovation (LNV OS 2010183), and the Dutch FES-programme ‘Versterking infrastructuur plantgezondheid’ WP3 call “Identification and Detection”. Part of the work was also supported by the Swiss State Secretariat for Education and Research (SBF C07.0139).
Fig. 4. AFLP dendrogram acquired from 4 Xanthomonas arboricola pv. pruni isolates (40, 60, 128 and 160) and from 4 look-a-likes (77, 158, 159 and 162) giving false positive reactions in conventional/TaqMan based on Pagani PCR, primers and sequence. Isolate 40 corresponds to the reference strain PD 740. All isolates were positive with the Pagani PCR but only the four Xap strains (higher part of figure) were positive with the Pothier PCR. Dendrogram has been generated using the Jaccard similarity coefficient.
should give more information about their identities. Discrimination between Xap isolates is crucial to understand the epidemiological aspects of the disease they induce within production systems on a local and regional basis (Boudon et al., 2005). Molecular typing of Xap isolates might improve our understanding of pathogen relatedness among different disease outbreaks and pathogen distribution between various crop production systems and between regions. Our MLVA analysis of the 25 Xap Dutch isolates from P. laurocerasus indicates the high discriminatory potential of this assay, differentiating isolates into two distinct groups. For future analysis it might provide an effective and efficient molecular typing method for epidemiology studies of Xap in the Netherlands and on a regional basis (Europe), through track and trace applications, hence establishing infection routes. To substantiate this hypothesis a larger group of isolates, obtained from other European countries is currently being analysed. Additionally, the use of complementary methods for the identification of suspect isolates such as BOX- and ERIC-PCR, gyrB barcoding and Xap-specific PCR described by Pothier (2011c) are strongly recommended in order to avoid the false positive results that can be obtained with the Pagani (2004) PCR or/and the derived TaqMan PCR based on Pagani’s primers.
ACKNOWLEDGEMENTS
Authors thank the European Science Foundation research network COST Action 873 for its support of net-
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