European Journal of Plant Pathology

, Volume 137, Issue 1, pp 5–9

Specific primers for Xanthomonas vesicatoria, a tomato bacterial spot causal agent

Authors

  • Edivânio R. Araújo
    • Departamento de Fitopatologia, Instituto de BiologiaUniversidade de Brasília
    • Laboratório de Fitopatologia, Embrapa Hortaliças
  • Marisa A. S. V. Ferreira
    • Departamento de Fitopatologia, Instituto de BiologiaUniversidade de Brasília
    • Laboratório de Fitopatologia, Embrapa Hortaliças
Short Communications

DOI: 10.1007/s10658-013-0225-4

Cite this article as:
Araújo, E.R., Ferreira, M.A.S.V. & Quezado-Duval, A.M. Eur J Plant Pathol (2013) 137: 5. doi:10.1007/s10658-013-0225-4

Abstract

Xanthomonas vesicatoria is a member of the species complex associated with tomato bacterial spot. New and specific primers for X. vesicatoria were developed and validated. The primers were highly specific and detection was positive using purified bacterial DNA, bacterial suspensions and foliar lesions. These primers represent an additional tool for detection and identification of one of the species involved in this important disease complex.

Keywords

Solanum lycopersicumXanthomonas vesicatoriaMolecular detection

Bacterial spot has a worldwide occurrence and causes serious economic losses (Jones et al. 1991). Xanthomonas vesicatoria is one of the species that compose the complex causing bacterial spot of tomato. Before the proposal of reclassification by Jones et al. (2004), the causal agents of the disease were classified as two distinct groups A and B, namely Xanthomonas axonopodis pv. vesicatoria and X. vesicatoria (Vauterin et al. 1995). Thus, the diversity within these two groups was still not well understood. Currently, four species are recognized as causal agents of tomato bacterial spot: X. vesicatoria, X. euvesicatoria, X. perforans, and X. gardneri (Jones et al. 2004). The most recent reports concerning X. vesicatoria were in the Russian Federation (Ignatov et al. 2009), the Indian Ocean region (Hamza et al. 2010), Tanzania (Mbega et al. 2012), and Brazil (Pereira et al. 2011; Costa et al. 2012). Xanthomonas campestris pv. raphani (Vicente et al. 2006), and X. arboricola (Mbega et al. 2012) may also be associated with lesions on tomato leaves, but biological and epidemiological aspects of these agents need to be better elucidated.

Specific primers are often used as molecular tools for the detection of various plant pathogens (Robène-Soustrade et al. 2010; López et al. 2012), including the tomato bacterial spot species complex (Koenraadt et al. 2009). However the genetic diversity of microorganisms can be generated and/or maintained by punctual deletions or insertions in the genome or by horizontal gene transfer (Arber 2000). Thus, to avoid false negatives in the identification process it is advisable to use more than one specific primer, whenever available. To overcome this constraint, primers designed to amplify conservative genome areas are being applied, such as the gyrB gene, which is rarely transmitted horizontally and is distributed ubiquitously among bacterial species (Kasai et al. 1998). This study aimed to develop specific primers from the partial DNA sequence of gyrB gene for Xanthomonas vesicatoria, and to validate them using a collection of X. vesicatoria from Brazil in assays with purified DNA, bacterial suspensions and symptomatic tomato leaves.

Fifteen strains of X. vesicatoria from three Brazilian regions (Midwest, Southeast and South) were used for the primers validation. The following strains were used as references: X. vesicatoria, IBSBF 2364 (Instituto Biológico de Campinas, São Paulo = XV1111, from New Zealand); X. euvesicatoria, IBSBF 2363 (Instituto Biológico de Campinas, São Paulo = Xvp197, from the USA), and EH 1996–212; X. perforans, IBSBF 2370 (Instituto Biológico de Campinas, São Paulo = ATCC BAA-983, from the USA), and EH 2009–111; X. gardneri, IBSBF 2373 (Instituto Biológico de Campinas, São Paulo = XCGA2, from Yugoslavia), and EH 2010–64. These strains are maintained in the work collection of the Laboratory of Plant Pathology of the Brazilian Vegetable Research Center (Embrapa Hortaliças), Brasília, DF, Brazil. Brazilian isolates were identified by: i) cultural characteristics in NA – nutrient agar medium (Schaad et al. 2001), ii) comparison of genomic profiles generated by BOX-PCR (Versalovic et al. 1991) with reference strain IBSBF 2364, and iii) pathogenicity tests on tomato (var. Yuba). Additionally, in order to attest the specificity of the new primers, 32 bacterial and fungal strains pathogenic to tomato or other hosts, from different collections (Embrapa Hortaliças – EH; Instituto Agronômico do Paraná – IAPAR, Instituto Biológico de Campinas – IBSBF and Universidade de Brasília – UnB), and 10 bacterial isolates representative of epiphytic and/or endophytic populations on tomato leaves were used (Table 1). Finally, the primers were evaluated with the DNA of tomato plants (vars. Floradade and Ponderosa).
Table 1

DNA samples used in this study and Xv-gyrB-F/Xv-gyrB-R-based PCR assay specificity

DNA Samples

Host

PCR

Xanthomonas vesicatoria

  

  EH 1995–95; EH 1995–99; EH 1995–101;

  

  EH 1994–102; EH 1995–103; EH 1995–104;

  

  EH 1995–105; EH 1995–107; EH 1995–108;

Tomato

+

  EH 2009–42; EH 2010–08; EH 2010–24;

  

  EH 2010–25; EH 2010–41; EH 2010–59; IBSBF 2364a

  

X. euvesicatoria

Tomato

  EH 1996–212; IBSBF 2363 a

  

X. perforans

Tomato

  EH 2009–111; IBSBF 2370 a

  

X. gardneri

Tomato

  EH 2010–64; IBSBF 2373 a

  

X. raphani, IAPAR 11300; IBSBF 1590

Brassica sp.

X. campestris pv. campestris, UnB 828

Brassica sp.

X. axonopodis pv. manihotis, UnB 1159

Cassava

X. campestris pv. vitians, UnB 110; UnB 830

Lettuce

X. citri pv. anacardii, IBSBF 2579

Cashew

X. campestris pv. armoraciae, IBSBF 1102

Iberis sp.

X. campestris pv. viticola, UnB 1318

Grape

X. citri pv. mangiferaeindicae, IBSBF 2586

Mango

Pseudomonas marginalis, IBSBF 1240

Tomato

P. cichorii, IBSBF 402; IBSBF 1748

Tomato

P. syringae pv. syringae, IBSBF 451; IBSBF 375; IBSBF 281

Tomato/Lilac

P. syringae pv. tomato, IBSBF 836; IBSBF 432; EH 75

Tomato

Pseudomonas viridiflava, IBSBF 1464

Tomato

Clavibacter michiganensis subsp. michiganensis, UnB 1151

Tomato

Ralstonia solanacearum, UnB 1273

Tomato

Acidovorax avenae subsp. citrulli, 646-2

Melon

Erwinia chrysanthemi, UnB 336

Chard

Erwinia psidii, IBSBF 1347; IBSBF 453

Guava

Pectobacterium carotovorum subsp. carotovorum, IBSBF 776

Tomato

Alternaria solani, IBSBF 1940

Tomato

Corynespora cassiicola, IBSBF 1828

Tomato

Stemphylium sp., EH 502

Tomato

Unknown epiphytic and/or endophytic bacteria (10)

Tomato

DNA tomato

+, amplification; – no amplification

aXanthomonas species reference strains associated with tomato bacterial spot, IBSBF 2363 originated from the USA, IBSBF 2364 from New Zealand, IBSBF 2370 from the USA and IBSBF 2373 from Yugoslavia

DNA extraction was performed according to Wilson (1999) and/or Mahuku (2004). DNA samples were diluted up to the approximately concentration of 50 ng/μl and kept at – 20 °C until used. For amplification reactions with bacterial suspensions, we recovered the colonies grown on NA for 48 h at 28 °C. Bacterial suspensions were prepared in sterile distilled water, and its concentration was adjusted in spectrophotometer (OD600 = 0.3) to approximately 5 × 108 CFU/ml (Jones et al. 2000). We used both purified DNA and bacterial suspensions (2 μl) for PCR reaction.

The primers were designed with Primer-BLAST Primer designing tool (http://www.ncbi.nlm.nih.gov), to amplify a 104-bp fragment from the partial sequence of the gyrase beta subunit (gyrB) gene of strain LMG 920 of X. vesicatoria, available at GenBank (access number EU015399.1). By using BLAST bases similarity (http://www.ncbi.nlm.nih.gov), 100 % query cover was obtained only for X. vesicatoria strains. The oligonucleotide sequences were: Xv-gyrB-F (5′-ATACGCGTTGGGCGAGCCT-3′) and Xv-gyrB-R (5′-CATCGCTGAAGATGGCCACG GCT-3′). PCR was performed in a thermocycler My CyclerTM (BIO RAD) with the following program: 95 °C for 4 min followed by 30 cycles of 95 °C for 30 s, 68 °C for 30 s, 72 °C for 30 s, and a final extension at 72 °C for 3 min. The reactions consisted of: 1.62 mM of MgCl2; 0.2 mM of each dNTPs; 1 μM of each primer; 1.26 U of Taq DNA polymerase; approximately 50 ng/μl of DNA; and Milli-Q® water to a final volume of 12 μl.

PCR products were analyzed by agarose gel (1.5 %) electrophoresis in 0.5X TBE buffer conducted at 5 V/cm for 2 h and scanned using the imaging system L-PIX ST (Loccus Biotecnologia, São Paulo, Brazil).

Bacterial suspensions (≈5 × 108 CFU/ml) were prepared in magnesium sulphate solution (10 mM) and homogeneously sprayed over leaf surfaces (two plants with strain EH 2010–25) up to the run-off point. Plants were then incubated for 48 h in moist chambers consisting of plastic bags previously moistened with tap water. After symptom development, a circular section with a diameter of 10 mm contained one individual leaf spot was macerated in 200 μl of distilled sterilized water 10 days after the inoculation. For PCR reaction, 2 μl of the undiluted leaf crude extract and its dilutions of 1:10 and 1:100 were used. The visualization of the fragments was as previously described. For all assays, each reaction was performed at least twice.

The primers were specific, amplifying the purified DNA and bacterial suspensions of all strains of X. vesicatoria used (Fig. 1) but not DNA from the other species associated with tomato bacterial spot (Table 1). Similarly, there was no DNA amplification from other plant pathogens tested, or from tomato leaves.
https://static-content.springer.com/image/art%3A10.1007%2Fs10658-013-0225-4/MediaObjects/10658_2013_225_Fig1_HTML.gif
Fig. 1

Specificity of Xv-gyrB-F/Xv-gyrB-R-based PCR assay for the detection of Xanthomonas vesicatoria in A: purified DNA (≈ 50 μl/ml); B: Bacterial suspension (≈ 5 × 108 CFU/ml); C: Symptomatic leaves of tomato. PC: Positive control (IBSBF 2364); CE: Crude extract of symptomatic leaves of tomato; 1:10: 10-fold dilution of the crude extract of symptomatic leaves; 1:100: 100-fold dilution of the crude extract of symptomatic leaves; NI: Uninoculated leaves; NC: Negative control; M: molecular marker (100 bp DNA Ladder, Invitrogen)

The development of specific primers for Xanthomonas species associated with tomato bacterial spot has been reported in some studies. All four species can be identified and distinguished by the primers designed by Koenraadt et al. (2009). Xanthomonas perforans can be identified indirectly with primers (RST 88/89) developed by Astua-Monge et al. (2000) to amplify avirulence gene avrXv3. In the same way, X. euvesicatoria can be identified indirectly with primers (RST 27/28) designed by Bouzar et al. (1994) to amplify the gene avrRxv and also with specific primers (Xeu2.4/Xeu2.5) developed by Moretti et al. (2009). For Xanthomonas vesicatoria, besides the pair specific primers of Koenraadt et al. (2009), new specific primers were designed by Beran and Mráz (2013). These primers were developed from partial DNA sequences of atpD, and were validated with purified bacterial DNA. Primers Xv-gyrB-F/Xv-gyrB-R, here reported, are an additional tool for confirming X. vesicatoria identification, that can be also used for direct detection of the pathogen on its host tissue were, speeding up the process of diagnosis (Fig. 1).

The crude extracts samples inhibited PCR, but from a 1:10 dilution it was possible to detect the pathogen through a positive PCR signal. Inhibition of PCR when using crude extracts as templates is often observed because plant polyphenols can form complexes with nucleic acids (Koonjul et al. 1999). The studies of Moretti et al. (2009) for X. euvesicatoria, and of Araújo et al. (2012), for the four tomato bacterial spot Xanthomonas have also shown the applicability of specific primers directly in symptomatic plants.

The primers developed in this study need to be further evaluated for their sensitivity and reproducibility for routine use. However, their specificity for X. vesicatoria and a detection limit that allows the amplification of the pathogens DNA in a diluted crude extract from a lesion, reveal their potential to be also used in diagnostic assays with infected seeds and seedlings.

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© KNPV 2013