Species-specific PCR primers for the detection of poorly distinguishable Xanthomonas euvesicatoria

Crop Protection 127 (2020) 104978

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Short communication

Species-specific PCR primers for the detection of poorly distinguishable Xanthomonas euvesicatoria � a, Miroslav Bara �nek a, Filip Gazdík a, Lucia Ragasova � a, Jakub Pe�cenka a, *, M� aria Kocanova a a b a � �a �zova � , Jana Cechov � , Pavel Beran , Ale�s Eichmeier Eli�ska Pen a a b

Mendeleum – Institute of Genetics, Mendel University in Brno, Valticka 337, 69144, Lednice, Czech Republic Department of Plant Production, University of South Bohemia, 37005, Ceske Budejovice, Czech Republic

A R T I C L E I N F O

A B S T R A C T

Keywords: Xanthomonas euvesicatoria Bacterial spot BSX Detection Primer design ZnDoF/ZnDoR

New species-specific PCR primers for detection of bacterium Xanthomonas euvesicatoria, the causal agent of bacterial spot of tomato and pepper, were designed in this work. Developed PCR primer pair ZnDoF/ZnDoR partially targets the coding sequence of Zn-dependent oxidoreductase gene. The primer pair was tested on various bacterial strains including bacteria from the bacterial spot-causing xanthomonads (BSX) group. Designed primers showed to be species-specific for the samples containing DNA of X. euvesicatoria. Specificity of the designed primers was also compared with the primer pairs already published. Subsequently, sensitivity of ZnDoF/ZnDoR primers was determined by ten-fold dilution of standardized sample. Detection capability of developed primers was also investigated on artificially infected pepper plants and seeds collected from bacterial spot-like peppers. Primers proved to be species-specific for in planta and in seed detection as well as to distinguish X. euvesicatoria from other bacterial strains used in this assay.

1. Introducion Bacterial spot is an important disease of pepper and tomato world­ wide. The disease is caused by four Xanthomonas species referred to as bacterial spot-causing xanthomonads (BSX). Causal agent of this disease was first discovered in South Africa and named as Bacterium vesicatorium (Doidge, 1921). Later studies reported the existence of another bacterial spot strains which were divided by Jones et al. (2004) to four groups: Xanthomonas euvesicatoria (Group A), X. vesicatoria (Group B), X. perfo­ rans (Group C) and X. gardneri (Group D). Geographical distribution of these groups can vary and changes in populations of BSX strains have been reported as well (Potnis et al., 2011). Moreover, a pathogenicity of individual BSX groups is diverse. Group C is pathogenic only on tomato and groups A, B and D are pathogenic on tomato and pepper (Beran and Mr� az, 2013). Although, X. perforans was also isolated from the pepper plant (Potnis et al., 2015). According to Barak et al. (2016) strains of X. euvesicatoria and X. perforans should be grouped to one species, X. euvesicatoria. Because bacterial spot is economically important disease (Jones et al., 2004; Moretti et al., 2009) reducing yield and crop quality (Abbasi et al., 2003; Paret et al., 2012), rapid and cheap methods for detection of

BSX pathogens are needed. There are number of detection techniques based on bacteria cultivation on semi-selective media (McGuire et al., 1986), physiological, chemical and serological methods (Bouzar et al., 1994) or metabolic profiling (Stoyanova et al., 2014). However, PCR methods are still the most reliable detection tools because of their simplicity and specificity and lower time consumption. Development of PCR detection systems for BSX strains has been reported using rhs family gene (Obradovic et al., 2004; Park et al., 2009), or housekeeping genes as well as genes dnaK, fyuA, gyrB, rpoD (Almeida et al., 2010; Young et al., 2008). According to Potnis et al. (2011) BSX groups A and B are distributed worldwide and PCR detection systems for the group B (X. vesicatoria) have been published (Araújo et al., 2012; Beran and Mr� az, 2013; Park et al., 2009; Koenraadt et al., 2009; Park et al., 2009, 2009) as well as for the group A (X. euvesicatoria) (Koenraadt et al., 2009; Moretti et al., 2009; Obradovic et al., 2004; Park al., 2009). Moreover, based on reclassification of BSX groups (Barak et al., 2016) it can be expected that X. euvesicatoria strains are probably the most widespread according to Burlakoti et al. (2018), Giovanardi et al. (2018) and Roach et al. (2018). On the base of these newly raised questions a new species-specific PCR primer pair for sensitive and reliable detection of X. euvesicatoria was

* Corresponding author. E-mail address: [email protected] (J. Pe�cenka). https://doi.org/10.1016/j.cropro.2019.104978 Received 28 March 2019; Received in revised form 1 October 2019; Accepted 3 October 2019 Available online 6 October 2019 0261-2194/© 2019 Elsevier Ltd. All rights reserved.

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Crop Protection 127 (2020) 104978

Table 1 PCR assay specificity of tested oligonucleotide primers.

Table 1 (continued ) bacterium

strain

XCVF/ XCVR

Xeu2.4/ Xeu2.5

BsXeF/ Bs-XeR

ZnDoF/ ZnDoR

natural strain natural strain NCPPB 2979

–

þ

–

–

–

–

–

–

–

–

þ

–

natural strain

–

þ

þ

–

bacterium

strain

XCVF/ XCVR

Xeu2.4/ Xeu2.5

BsXeF/ Bs-XeR

ZnDoF/ ZnDoR

Xanthomonas vesicatoria Xanthomonas vesicatoria Xanthomonas vesicatoria Xanthomonas vesicatoria Xanthomonas vesicatoria Xanthomonas vesicatoria Xanthomonas vesicatoria Xanthomonas vesicatoria Xanthomonas vesicatoria Xanthomonas vesicatoria Xanthomonas vesicatoria Xanthomonas perforans Xanthomonas gardneri

NCPPB 422 NCPPB 1421 NCPPB 2044 NCPPB 2786 Xv1

–

þ

–

–

–

–

–

–

Pectobacterium carotovorum subsp. carotovorum Pantoea sp.

þ

þ

þ

–

Klebsiella sp.

–

þ

–

–

–

þ

–

–

Xv2

–

þ

–

–

Xv3

–

þ

–

–

Xv4

–

þ

–

–

Xv5

–

þ

–

–

Xv6

–

þ

–

–

Xv7

–

þ

–

–

NCPPB 4321 NCPPB 881 NCPPB 941 NCPPB 2574 NCPPB 2594 NCPPB 2968 Xav1

–

þ

–

–

–

–

–

–

þ

þ

–

þ

þ

þ

þ

þ

þ

þ

þ

þ

þ

þ

þ

þ

þ

þ

þ

þ

Xav2

þ

þ

þ

þ

Xav3

þ

þ

þ

þ

Xav4

þ

þ

þ

þ

Xav5

þ

þ

þ

þ

Xav6

þ

þ

þ

þ

Xav7

þ

þ

þ

þ

WHRI 1279a

–

–

–

–

WHRI 3811

–

–

–

–

WHRI 3971a

–

–

–

–

2.2. Primer design

natural strain

–

–

–

–

NCPPB 2683 NCPPB 281 natural strain NCPPB 2445 natural strain NCPPB 312

–

þ

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

þ

þ

–

–

–

–

–

The primer pair was designed based on sequences available in Gen­ Bank/NCBI. The genome of Xanthomonas euvesicatoria strain 85-10 (GenBank/NCBI Acc. No. AM039952.1.1) was used as a reference. Based on in-silico analysis a coding sequence of Zn-dependent oxidore­ ductase was chosen as unique for X. euvesicatoria strains. This gene belongs to zinc-dependent alcohol dehydrogenase domain (GenBank/NCBI Acc. No. WP_011347394.1) which is included in the zinc medium-chain de­ €rnvall hydrogenases/reductases (MDR) family of the MDR superfamily (Jo et al., 2010). The genes of the zinc MDR family are significantly abundant and widespread in all kingdoms of life (Woese, 1998). High abundance and multiplicity of these genes refer to low conservation (Persson et al., 2008) and predict these genes as good targets for specific PCR detection. Primer-BLAST (Ye et al., 2012) and Primer3Plus (Untergasser et al., 2007) tools were used to design ZnDoF (50 -GGTGACAAACCGTCAGGAATAG-30 )

Xanthomonas euvesicatoria Xanthomonas euvesicatoria Xanthomonas euvesicatoria Xanthomonas euvesicatoria Xanthomonas euvesicatoria Xanthomonas euvesicatoria Xanthomonas euvesicatoria Xanthomonas euvesicatoria Xanthomonas euvesicatoria Xanthomonas euvesicatoria Xanthomonas euvesicatoria Xanthomonas campestris pv. campestris Xanthomonas campestris pv. campestris Xanthomonas campestris pv. campestris Xanthomonas campestris pv. campestris Pseudomonas syringae pv. tomato Pseudomonas syringae pv. syringae Pseudomonas sp. Pseudomonas corrugata Pectobacterium sp.

Clavibacter michiganensis subsp. michiganensis Bacillus sp.

Bacterial strains used for the testing of ZnDoF/ZnDoR primers specificity. Re­ sults were compared with primers XCVF/XCVR Park et al. (2009), Xeu2.4/Xeu2.5 Moretti et al. (2009) and Bs-XeF/Bs-XeR Koenraadt et al. (2009).

developed in this work. 2. Materials and methods 2.1. Bacterial strains, growth conditions and plant material Thirty-eight bacterial strains (Table 1) were used for the evaluation of tested primers specificity. Reference and type bacterial strains of X. euvesicatoria, X. vesicatoria, X. gardneri and X. perforans were obtained from National Collection of Plant Pathogenic Bacteria (NCPPB, UK). Three strains of Pseudomonas ssp. (NCPPB 281, 2445, 2683), one strain of Pectobacterium sp (NCPPB 312) and one strain of Clavibacter sp. (NCPPB 2979) were selected as non-BSX pathogens of tomato and pepper. Three strains of X. campestris pv. campestris were obtained from University of Warwick (WHRI, UK). BSX strains marked as Xav (Xan­ thomonas axonopodis pv. vesicatoria) and Xv (Xanthomonas vesicatoria) were collected as natural strains and are deposited at University of South � � Bohemia in Cesk e Bud� ejovice (Czech Republic). Other bacteria were obtained as a strains isolated from nature as part of microbiome of commonly grown field crops. These natural strains were firstly used in a study of Eichmeier et al. (2017). BSX strains were cultivated at 28 � C for two days on Phyto Xcv Agar Base (peptone 10 g l 1, potassium bromide 10 g l 1, boric acid 0.1 g l 1, calcium chloride anhydrous 0.25 g l 1, agar 15 g l 1) enriched with NNB supplement (nystatin 35 mg l 1, neomycin 40 mg l 1, bacitracin 100 mg l 1) (HiMedia, India), remaining bacterial strains were cultured on LB agar (Sigma Aldrich, USA) at the same conditions. To avoid false positives by amplification of the targets derived from the host genome the DNA isolated from pepper cultivars of ‘Sandra’, ‘Demetra’, ‘Patricie’, ‘Citrina’, ‘Priscila’, ‘Andrea’ and ‘Koral’ (MoravoSeed CZ) was also tested.

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10 min as a final extension step. To confirm the uniformity of results, all reactions were prepared in three replicates and using two types of thermocyclers (MJ Mini, Biorad, USA and TProfessional Gradient, Bio­ metra, USA). PCR products were separated using 1.2% agarose gel (Serva, Germany) and sequenced according to Eichmeier et al. (2010). Obtained sequences were aligned using CLC Main Workbench 6.5 (Qiagen, Germany) and evaluated by BLAST (Version 2.2.31þ). The sequences were deposited in GenBank/NCBI under accession numbers MK628532-MK628542. In addition, specificity of ZnDoF/ZnDoR primers was compared with primer pairs (Table 2) frequently used for specific detection of X. euvesicatoria developed by Koenraadt et al. (2009), Moretti et al. (2009) and Park et al. (2009) (Table 1).

Table 2 List of the oligonucleotide primer sets. primer name

primer sequence

target sequence

product length

reference

annealing temperature

XCVF

AGA AGC AGT CCT TGA AGG CA AAT GAC CTC GCC AGT TGA GT CTG GGA AAC TCA TTC GCA GT GTG GCG CTC TTA TTT CCT CAT GAA GAA CTC GGC GTA TCG GTC GGA CAT AGT GGA CAC ATA C GGT GAC AAA CCG TCA GGA ATA G CGC ACT GGC ACG TTA TCA

rhs family genes

517 bp

Park et al. (2009)

58 � C

hypothetical protein XCV3137

208 bp

Moretti et al. (2009)

64 � C

non-coding sequence

173 bp

Koenraadt et al. (2009)

64 � C

zinc-binding dehydrogenase

100 bp

This paper

55 � C

XCVR

Xeu2.4

Xeu2.5 Bs-XeF

Bs-XeR

ZnDoF

ZnDoR

2.4. Sensitivity of primers, X. euvesicatoria detection in seeds and in planta Sensitivity of the designed primers was evaluated by PCR with tenfold dilution of DNA sample adjusted to 50 ng μl 1 according to Mor­ etti et al. (2009). For X. euvesicatoria detection in the seeds the plants of pepper cv. ‘Citrina’ with visible bacterial spot symptoms were used as a source. Obtained seeds were analysed in four doses (4 � 2500 seeds) according to ISTA (2019) using stomacher method. Occurrence of X. euvesicatoria in samples was firstly evaluated by PCR with BSX-XeF/BSX-XeR primer pair (Koenraadt et al., 2009). Then, the same samples were analysed by PCR with newly developed ZnDoF/ZnDoR primers. For in planta detection the plants of pepper cv. ‘Andrea’ with four true leaves were artificially infected by X. euvesicatoria (NCPPB 2968). Fresh bacterial inoculum grown for 24 h in Mueller-Hinton broth (beef infusion 3 g l 1, casein acid hydrolysate 17.5 g l 1, starch 1.5 g l 1) (HiMedia, India) was adjusted by sterile saline to equivalent of 1 � 108 CFU ml 1 and pressed into the leaf parenchyma using syringe. Symptomatic tissue was processed according to EPPO protocol (2013) and PCR with developed primers ZnDoF/ZnDoR was used for detection of X. euvesicatoria.

and ZnDoR (50 -CGCACTGGCACGTTATCA-30 ) primers, IDT OligoAnalyzer (https://eu.idtdna.com) and CLC Workbench 6.5 (Qiagen, Germany) were used for the tuning of primers parameters and in silico evaluation. 2.3. DNA isolation and quantification, PCR, sequencing

3. Results and discussion

Bacteria were harvested from the plates and total DNA was extracted with NucleoSpin Tissue (Macherey Nagel, Germany) by following manufacturer’s protocol. Concentration of DNA was measured using SPECTROstar Nano (BMG LABTECH, Germany). DNA from pepper cul­ tivars was extracted also with NucleoSpin Tissue. DNA samples were adjusted to concentration of 50 ng μl 1 and stored at 20 � C. Designed primers were tested for the species-specificity with DNA of all the mentioned bacterial strains and seven pepper varieties. All the reactions were carried out with GoTaq® G2 Flexi DNA Polymerase kit (Promega, USA), content of the PCR mixture was 10.5 μl of water (HPLC purity), 4 μl Colourless flexi buffer, 1.2 μl MgCl2 (25 mM), 0.2 μl dNTPs (10 mM), 1 μl of each primer (10 μM), 0.7 μl Green flexi buffer, 0.2 μl of GoTaq® polymerase and 2 μl of DNA template per each sample. PCR conditions consisted of 94 � C for 5 min for initial denaturation and 25 cycles of 95 � C for 40 s, 55 � C for 30 s, 72 � C for 40 s followed by 72 � C for

Specificity of ZnDoF/ZnDoR primer pair was evaluated on all the bacterial strains listed in Table 1. PCR conditions as the most significant factor for sensitive and specific amplification were thoroughly tuned and finally set to 25 cycles of 95 � C for 40 s, 55 � C for 30 s and 72 � C for 40 s with the initial denaturation at 94 � C for 5 min and 72 � C for 10 min as final extension step. As expected, PCR with newly designed primers amplified 100 bp DNA fragment of all NCPPB X. euvesicatoria strains and none of the other bacterial nor pepper DNA (Figs. 1 and 2). The amplicons of interest were also obtained for all natural X. euvesicatoria strains which corresponds to their previous characterization Xantho­ monas axonopodis pv. vesicatoria (Xav), presently X. euvesicatoria (Table 1). Despite small length of the PCR product, it was well distin­ guishable from primer dimers on the agarose gel. In confrontation with other testing methods published by Park et al. (2009), Koenraadt et al. (2009), Moretti et al. (2009) and Park et al. (2009) (Table 1) it reveals,

Fig. 1. BSX strains used to evaluate X. euvesicatoria DNA specificity of developed ZnDoF/ZnDoR primers. X. euvesicatoria NCPPB strains (lanes 2–5), X. euvesicatoria strains Xav1 to Xav7 (lanes 6–12). X vesicatoria NCPPB strains (lanes 13–16), X. perforans (lane 17), X. gardneri (lane 18), X. vesicatoria strains Xv1 to Xv7 (lanes 19–25), negative control (lane 26) 100bp DNA Ladder (lanes 1 and 27). 3

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Crop Protection 127 (2020) 104978

Fig. 2. Evaluation of ZnDoF/ZnDoR primers specificity on non-BSX strains. X. euvesica­ toria NCPPB strains (lanes 2–5), X. campestris pv. campestris WHRI strains (lanes 6–8), X. campestris pv. campestris natural strain (lane 9), Pseudomonas spp. strains NCPPB (lanes 10–12), Pseudomonas sp. natural strain (lane 13), Pectobacterium carotovorum subsp. car­ otovorum NCPPB strain (lane 14), Pecto­ bacterium sp. natural strain (lane 15), Clavibacter michiganensis subsp. michiganensis NCPPB strain (lane 16), natural strains of Pantoea sp., Klebsiella sp. and Bacillus sp. (lanes 17–19), pepper DNA (lanes 20–26), negative control (lane 27), 100bp DNA Ladder (lanes 1 and 28).

Fig. 3. Sensitivity of developed ZnDoF/ZnDoF primers. Ten-fold dilution of 50 ng μl 1 X. euvesicatoria DNA from 5 to 5.10 9 ng μl 1 respectively (lanes 2–11), detection limit of developed ZnDoF/ZnDoF primers 0.0005 ng μl 1 (lane 6), X. euvesicatoria DNA (50 ng μl 1) (lane 12), negative control (lane 13), 100 bp DNA ladder (lanes 1 and 14).

that newly designed primers provided most reliable results regarding correct assignment to individual bacterial species. The second best match was achieved by using XCVF/XCVR primers designed by Park et al. (2009), where only difference from newly designed primers was positive detection of NCPPB 2044 strain. Interestingly, all tested primer pairs, except newly designed ZnDoF/ZnDoR, amplified the DNA of the strain NCPPB 2044 (Table 1), although this strain is maintained at NCPPB as X. vesicatoria. Some other contradictories have been registered as well. After PCR with Xeu2.4/Xeu2.5 primers (Moretti et al., 2009) specific amplicons were present in the most of the X. vesicatoria strains, X. perforans strain as well as some of non-BSX strains. In the case of false positives, according to Moretti et al. (2009), the specificity of Xeu2.4 and Xeu2.5 primers is probably influenced by varying of Mg2þ content in the reaction. Amplification of the specific products in the case of some non-BSX strains were also registered after PCR with Bs-XeF/Bs-XeR (Koenraadt et al., 2009) primer pair (Table 1). Newly designed ZnDoF/ZnDoR primer pair was also examined for in seeds and/or in planta detection purposes. For the detection in seeds, positive results were obtained for all four doses of tested seeds. This corresponds to the results obtained by BSX-XeF and BSX-XeR primers (Koenraadt et al., 2009). For in planta detection, PCR with ZnDoF/ZnDoR primers showed positive results using the DNA extracted from the pellet sample of arti­ ficially inoculated peppers. Evaluation of sequences was carried out by BLASTn and analysis of all the 11 obtained sequences confirmed a high similarity to X. euvesicatoria excluding amplification of false positive products. Regarding the sensitivity of PCR detection by using newly designed primers, the DNA of X. euvesicatoria was detected at the equivalent of 0.0005 ng μl 1 (Fig. 3), identical to detection limit of primers Xeu2.4/Xeu2.5 by Moretti et al. (2009).

4. Conclusions In conclusion, designed primers ZnDoF and ZnDoR can be used as a tool for the rapid, cheap and simple detection of Xanthomonas euvesi­ catoria. In comparative tests of different bacterial strains, the results showed significant improvement of their reliability if compared with results obtained by using other previously published primer pairs designated for X. euvesicatoria detection. In fact, specific amplicons were present only in the samples containing DNA from verified strains of X. euvesicatoria, the other tested samples as those of BSX bacteria and of other tested bacterial strains were correctly evaluated as X. euvesicatoria negative. The newly designed ZnDoF/ZnDoR primer pair was also suc­ cessfully tested for detection of this pathogen in planta and in the seeds. Funding This research was carried out in the overall framework of the ERDF “Multidisciplinary research to increase application potential of nano­ materials in agricultural practice” (No. CZ.02.1.01/0.0/0.0/16_025/ 0007314) and project No. TJ01000274. Declaration of competing interest No conflict exists: the authors declare that they have no conflict of interest. Acknowledgements � We would like to thank Dr. Stencl for numerous valuable comments and proofreading our manuscript mainly for English editing service.

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