16S-23S ITS rdna PCR-RFLP APPROACH AS A TOOL FOR IDENTIFICATION AND DIFFERENTIATION OF BACTERIAL SPOT-CAUSING XANTHOMONAS

Transcription

1 Journal of Plant Pathology (2016), 98 (3), Edizioni ETS Pisa, Short Communication 16S-23S ITS rdna PCR-RFLP APPROACH AS A TOOL FOR IDENTIFICATION AND DIFFERENTIATION OF BACTERIAL SPOT-CAUSING XANTHOMONAS Y. Kizheva 1 *, T. Vancheva 1 *, M. Stoyanova 2, N. Bogatzevska 2, P. Moncheva 1 and P. Hristova 1 1 Faculty of Biology, Sofia University St. Kliment Ohridski, Sofia, Bulgaria 2 Institute of Soil Science, Agrotechnologies and Plant Protection Nikola Poushkarov, Sofia, Bulgaria * These authors contributed equally to this work. SUMMARY Bacterial spot of pepper and tomato plants is one of the most important constraints limiting crops yield in Bulgaria and Macedonia. Therefore, early identification of the pathogen is necessary for the control and prevention of the disease. In order to explore the strength of 16S-23S ITS rdna PCR-RFLP as an approach for identification and differentiation of the causative agents of bacterial spot 262 Bulgarian and Macedonian strains pathogenic of pepper and tomato plants were used. The strains were previously identified as Xanthomonas euvesicatoria (132 strains), Xanthomonas vesicatoria (115 strains), and Xanthomonas gardneri (15 strains). Each restriction analysis resulted in two profiles that grouped the used strains. The restriction with AluI endonuclease differentiated X. vesicatoria isolates from the other three species. The enzyme HpaII separated the X. euvesicatoria strains. A combination of three restriction endonucleases (AluI, MboI, HpaII) successfully differentiated the four species described as causative agents of bacterial spot. Keywords: bacterial disease, pepper and tomato plants, Xanthomonas species, interspacer region restriction. The genus Xanthomonas was first proposed by Dowson in It comprises Gram-negative, yellow-pigmented bacteria that cause plant-diseases worldwide. These phytopathogenic bacteria infect many economically important plants such as cotton, beans, citrus etc (Simoes et al., 2007). One of the most spread disease caused by the xanthomonads is bacterial spot of tomato and pepper plants. The group of bacterial spot-causing xanthomonads (BSX) have been put under reclassification many times since 1994 Corresponding author: T. Vancheva Fax: tacavancheva@gmail.com (Stall et al., 1994; Vauterin et al., 1995; Jones et al., 2004) and at present it is considered that these pathogens are grouped in four validly described species, i.e., Xanthomonas vesicatoria, Xanthomonas euvesicatoria, Xanthomonas perforans and Xanthomonas gardneri. Xanthomonas axonopodis pv. vesicatoria is no longer a valid name (Bull et al., 2010). Recent phylogenetic analyses based on DNA sequence similarity between single and multiple gene loci (Young et al., 2008; Parkinson et al., 2009; Hamza et al., 2010) support three distinct species, showing X. euvesicatoria and X. perforans to be highly related to (if not synonymous with) each other and also with the recently described Xanthomonas alfalfa. BSX are quarantine organisms in the European Union (EPPO A2 list) and their accurate detection and identification in infected plants and asymptomatic seeds are very important for effective control. Over the years, different criteria such as: semi-selective media (Mc- Guire et al., 1986), serovar and fatty acid profiles (Bouzar et al., 1994), capability to hydrolyze the starch and pit the pectate (Stall et al., 1994; Bouzar et al., 1996), phenotypic diversity including carbon substrate utilization patterns for identification and differentiation of these pathogens have been explored. However, these phenotypic, biochemical and morphological characteristics are not sufficient for accurate classification of the species. Different parts of xanthomonads s genome have also been analyzed: 16S rdna (Hauben et al., 1997), rpfb and atpd genes (Simoes et al., 2007), hrp genes (Leite et al., 1994). Some of these gene loci such as 16S are found to be less discriminative because of their highly conservative structure. Another part of the genome of these species, internal transcribed spacer (ITS), is considered to be 10 times more variable than 16S (Leblond-Bourget et al., 1996). Most bacteria are thought to have structure of ribosomal operon as follows: 5-16S-23S- 5S-3 with two ITSs inside (Kabadjova et al., 2002). The ITS located between 16S and 23S is considered to be under less evolutionary pressure and thus this region is proper for developing DNA-based techniques for identification of bacteria at species level. The structure of this region among xanthomonads was observed and a common structure of ITS was documented. Its length varies in size from 492 to 578 bp with two trnas (trna ala and trna Ile )

2 646 Differentiation tool for bacterial spot xanthomonads Journal of Plant Pathology (2016), 98 (3), Fig. 1. PCR-RFLP analysis of 16S-23S rdna of representative Xanthomonas strains with AluI. М: DNA ladder, 100 bp; lanes 7, 15: X. gardneri representative strains forming profile I; lanes 14, 19: X. euvesicatoria representative strains forming profile I; lanes 1-6, 8-13, 16-18: X. vesicatoria representative strains forming profile II; lane 20: X. vesicatoria NBIMCC 2427 (type strain). included. The phylogenetic tree based on ITS sequences grouped xanthomonads into six major clusters indicating high level of diversity among the studied species (Goncalves and Rosato, 2002). In order to investigate the variability of the 16S-23S ITS region among the bacterial spot pathogens 262 bacterial strains isolated in the period , 161 of which isolated from pepper plants with symptoms of bacterial spot originating from Bulgaria and Macedonia, 74 from tomato plants and 27 from weeds of tomato plantations from Bulgaria were included in this study. All analyzed strains were previously identified as follows: 115 strains as X. vesicatoria, 132 as X. euvesicatoria, and 15 strains as X. gardneri (Kizheva et al., 2013; Vancheva et al., 2015). No strains belonging to the species X. perforans were identified. The type cultures X. vesicatoria NBIMCC 2427 (=DSM-22252), X. euvesicatoria NBIMCC 8731 (=DSM-19128), X. perforans NBIMCC 8729 (=DSM-18975) and X. gardneri NBIMCC 8730 (=DSM-19127) were used as controls. Genomic DNA was extracted from bacterial suspensions with OD 600 = 1 by GeneMATRIX Tissue and Bacterial DNA Purification Kit (EURx Ltd., Gdansk, Poland). Control of yield and purity of the obtained DNA was performed by measuring with a spectrophotometer Nanodrop 2000 (Thermo Scientific) at 230 nm, 260 nm, 280 nm, and 320 nm. The amplification of 16S-23S rdna was performed by primer pair 16S-p2 (5 -CTTGTACA- CACCGCCCGTC-3 ) and 23S-p7 (5 -GGTACTTAGAT- GTTTCAGTTC-3 ) (Kabadjova et al., 2002). PCR was performed in a thermal cycler Techne TC-312. Fig. 2. PCR-RFLP analysis of 16S-23S rdna of representative Xanthomonas strains with AluI. М: DNA ladder, 100 bp; lanes 1, 2, 5, 8-11, 13, 16, 18-20: X. euvesicatoria representative strains forming profile I; lanes 3, 4, 6, 7, 12, 14, 15, 17: X. vesicatoria representative strains forming profile II. Amplification was carried out in a total volume of 50 μl containing (final concentrations) 5 μl of 10 PCR buffer (STS); 1.5 mm MgCl 2 ; 0.75 μl of each dntp 10mM; 0.4 U Taq DNA polymerase (STS); 10 pmol of each primer; 1 μl of template DNA, under the following reaction conditions: a denaturation step at 95 C for 5 min, followed by 30 cycles at 94 C for 45 s, 58 C for 45 s, and 72 C for 45 s, and a final step at 72 C for 7 min. The PCR products were separated electrophoretically in 1.5% agarose gel in TBE buffer (Tris-Boric acid-edta; 30 min at 100 V), stained with ethidium bromide and photographed under UV light. The DNA 100 bp marker (Thermo Scientific) was used. The amplified product had length of about 900 bp and included part of 16S rdna (150 bp), ITS (about 550 bp) and part of 23S rdna (207 bp). For differentiation of the strains nine endonucleases were used: AluI, MboI, HpaII, Csp6I, XbaI, TaqI, Tru1I, TasI, and Bsp143I (Thermo Scientific). The restriction endonuclease digestion was performed in a total volume of 25 μl containing 10 μl enzyme mix (containing, final concentrations: 10 U enzyme and 1 Buffer Tango in nuclease-free water) and 15 μl PCR product for 3 h at 37 C (65 C for TasI) according to the recommendation of the manufacturer. The products of the restrictions were separated electrophoretically at 100 V for 40 min in 2.5% agarose gel in TBE buffer, stained with ethidium bromide and photographed under UV light. Restriction with Csp6I, TaqI, XbaI, Tru1I, and Bsp143I generated profiles which did not distinguish the four species. The analysis of the 16S-23S interspacer region with endonuclease

3 Journal of Plant Pathology (2016), 98 (3), Kizheva et al. 647 Fig. 3. PCR-RFLP analysis of 16S-23S rdna of representative Xanthomonas strains with MboI. М: DNA ladder, 100 bp; lanes 14, 19: X. euvesicatoria representative strains forming profile I; lanes 1-6, 8-13, 16-18: X. vesicatoria representative strains forming profile II; lanes 7, 15: X. gardneri representative strains forming profile II; lane 20: X. vesicatoria NBIMCC 2427 (type strain). AluI divided the strains into two groups, according to the obtained restriction profiles. The all 147 pepper and tomato isolates (from Bulgaria and Macedonia) identified as X. euvesicatoria and X. gardneri, the type cultures X. perforans NBIMCC 8729, X. euvesicatoria NBIMCC 8731 and X. gardneri NBIMCC 8730 generated the first RFLP profile (consisting of four fragments with approximate length of 400, 200, 100 bp and a fragment with a length of less than 100 bp, respectively). The remaining 115 isolates, identified as X. vesicatoria (isolated from pepper plants from Bulgaria and Macedonia and Bulgarian tomato and weed strains) as well the type culture of X. vesicatoria NBIMCC 8731 formed the second RFLP profile, which was closely related to the first one, but the largest fragment length was about 300 bp (Fig. 1; Fig. 2). The restriction with MboI also separated the strains in two RFLP groups. The first group united the type cultures of X. euvesicatoria NBIMCC 8731 and X. perforans NBIMCC 8729 and also 132 of our strains, all identified as X. euvesicatoria. This profile had four fragments (about 400, 170, 150 and 126 bp, respectively). The second profile grouped 115 of our strains identified as X. vesicatoria and 15 as X. gardneri. This profile shared the length of the three smaller fragments with the first one. The length of the largest fragment was about 300 bp (Fig. 3). The RFLP analysis of the 16S-23S interspacer region by HpaII differentiated the strains also in two groups. The first group had a profile with two fragments (about approximate length of 700 and 100 bp) and was characteristic for the type culture X. Fig. 4. PCR-RFLP analysis of 16S-23S rdna of representative Xanthomonas strains with HpaII. М: DNA ladder, 100 bp; lane 1: X. perforans NBIMCC 8729 (type strain); lane 2: X. euvesicatoria NBIMCC 8731 (type strain); lane 5: X. gardneri NBIMCC 8730 (type strain); lane 8: X. vesicatoria NBIMCC 2427 (type strain); lanes 3, 4: X. euvesicatoria representative strains forming profile I; lanes 6, 7: X. gardneri representative strains forming profile II; lanes 9, 10: X. vesicatoria representative strains forming profile II. euvesicatoria NBIMCC 8731 and all 132 isolates, identified as X. euvesicatoria (one tomato and 131 pepper isolates). These strains were also grouped together by restriction with MboI and AluI. The second HpaII profile consisted of three fragments (500, 200 and a fragment with a length of less than 100 bp) and was generated from the type cultures X. perforans NBIMCC 8729, X. vesicatoria NBIMCC 2427, X. gardneri NBIMCC 8730, and from the remaining 130 isolates, identified as X. vesicatoria and X. gardneri (Fig. 4; Fig. 5). The enzyme AluI differentiated the species X. vesicatoria from the other three species, but did not distinguish between X. perforans, X. euvesicatoria and X. gardneri. Restriction with HpaII differentiated X. euvesicatoria from the remaining three species. X. vesicatoria and X. gardneri can be distinguished using the enzymes AluI and MboI, and X. euvesicatoria and X. perforans by MboI and HpaII. The combination of the three restriction endonucleases AluI, MboI and HpaII successfully differentiated the four species, causal agents of bacterial spot of tomato and pepper and can be used as an alternative method for identification of these pathogens. The analysis of the 16S-23S interspacer region with the used endonucleases

4 648 Differentiation tool for bacterial spot xanthomonads Journal of Plant Pathology (2016), 98 (3), to their species. The results of our investigations confirm the potential of 16S-23S rdna ITS region to differentiate Xanthomonas strains on species level when applied for the causative agents of bacterial spot of tomato and pepper. Three of the used endonucleases, AluI, MboI and HpaII, are suitable for identification of X. vesicatoria, X. euvesicatoria, X. gardneri and X. perforans. AKNOWLEDGEMENTS This study was supported by project DFNI B02/4 by the National Science Fund of Bulgaria. REFERENCES Fig. 5. PCR-RFLP analysis of 16S-23S rdna of representative Xanthomonas strains with HpaII. М: DNA ladder, 100 bp; lane 1: X. perforans NBIMCC 8729 (type strain); lane 2: X. vesicatoria NBIMCC 2427 (type strain); lane 3: X. gardneri NBIMCC 8730 (type strain); lane 4: X. euvesicatoria NBIMCC 8731 (type strain); lanes 5-11: X. euvesicatoria representative strains forming profile I. was not discriminative on intraspecies level, because all strains of each species formed the same RFLP pattern. The high level of similarity of the 16S genes among the xanthomonads was the reason to search other phylogenetic molecular markers for differentiation of these species (Goncalves and Rosato, 2002). ITS which are located between 16S and 23S genes are considered much more variable than 16S genes (Leblond-Bourget et al., 1996). Goncalves and Rosato (2002) used several restriction enzymes for ITS digestion and discovered variable similarity (between 30-87%) among the Xanthomonas species. After sequencing of the ITS regions the authors established variations in the length of the product ( nucleotides) and similarity between 63-99%. The topology of the phylogenetic tree of 16S-23S rdna ITS was similar to the one of 16S rdna reported by Hauben et al. (1997), but revealed more clusters due to the higher diversity in the ITS sequences. Another comparison of ITS sequences held, by Quezado-Duval et al. (2004), grouped several Xanthomonas strains and the relevant type cultures according Bouzar H., Jones J.B., Minsavage G.V., Stall R.E., Scott J., Proteins unique to phenotypically distinct groups of Xanthomonas campestris pv. vesicatoria revealed by silver staining. Phytopathology 84: Bouzar H., Jones J.B., Somodi G.C., Stall R.E., Daouzli N., Lambe R.C., Felix-Gastelum R., Trinidad-Correa R., Diversity of Xanthomonas campestris pv. vesicatoria in tomato and pepper fields of Mexico. Canadian Journal of Plant Pathology 18: Bull C.T., De Boer S.H., Denny T.P., Firrao G., Fisher-le Saux M., Saddler G.S., Scortichini M., Stead D.E., Takikawa Y., Comprehensive list of names of Plant pathogenic Bacteria Journal of Plant Pathology 92: Dowson W.J., On the systematic position and generic names of the gram negative bacterial plant pathogens. Zentralblatt für Bakteriologie, Parasitenkunde, Infektionskrankenheit und Hygiene Abt. II 100: Goncalves E.R., Rosato Y.B., Phylogenetic analysis of Xanthomonas species based upon 16S-23S rdna intergenic spacer sequences. International Journal of Systematic and Evolutionary Microbiology 52: Hamza A.A., Robene-Soustrade I., Jouen E., Gagnevin L., Lefeuvre P., Genetic and pathological diversity among Xanthomonas strains responsible for bacterial spot on tomato and pepper in the Southwest Indian Ocean Region. Plant Disease 94: 993. Hauben L., Vauterin L., Swings J., Moore E.R.B., Comparison of 16S ribosomal DNA sequences of all Xanthomonas species. International Journal of Systematic Bacteriology 47: Jones J.B., Lacy G.H., Bouzar H., Stall R.E., Schaad N.W., Reclassification of the Xanthomonas associated with bacterial spot disease of tomato and pepper. Systematic and Applied Microbiology 27: Kabadjova P., Dousset X., Le Cam V., Prevost H., Differentiation of closely related Carnobacterium food isolates based on 16S-23S ribosomal DNA intergenic spacer region polymorphism. Applied and Environmental Microbiology 68: Kizheva Y., Vancheva T., Hristova P., Stoyanova M., Stojanovska M., Moncheva P, Bogatzevska N., Identification of

5 Journal of Plant Pathology (2016), 98 (3), Kizheva et al. 649 Xanthomonas strains from tomato and pepper and their sensitivity to antibiotics and copper. Bulgarian Journal of Agricultural Science 19: Leblond-Bourget N., Philippe H., Mangin I., Decaris B., S rdna and 16S to 23S internal transcribed spacer sequence analyses reveal inter- and intraspecific Bifidobacterium phylogeny. International Journal of Systematic Bacteriology 46: Leite Jr. R.P., Minsavage G.V., Bonas U., Stall R.E., Detection and identification of phytopathogenic Xanthomonas strains by amplification of DNA sequences related to the hrp genes of Xanthomonas campestris pv. vesicatoria. Applied and Environmental Microbiology 60: McGuire R.G., Jones J.B., Sasser M., Tween media for semi-selective isolation of Xanthomonas campestris pv. vesicatoria from soil and plant material. Plant Disease 70: Parkinson N., Cowie C., Heeney J., Stead D., Phylogenetic structure of Xanthomonas determined by comparison of gyrb sequences. International Journal of Systematic and Evolutionary Microbiology 59: Quezado-Duval A.M., Leite Jr. R.P., Truffi D., Camargo L.E.A., Outbreaks of bacterial spot caused by Xanthomonas gardneri on processing tomato in central-west Brazil. Plant Disease 88: Simoes T.H.N., Goncalves E.R., Rosato Y.B., Mehta A., Differentiation of Xanthomonas species by PCR-RFLP of rpfb and atpd genes. FEMS Microbiology Letters 271: Stall R.E., Beaulieu C., Egel D., Hodge N.C., Leite R.P., Minsavage G.V., Bouzar H., Jones J.B., Alvarez A.M., Benedict A.A., Two genetically diverse groups of strains are included in Xanthomonas campestris pv. vesicatoria. International Journal of Systematic Bacteriology 44: Vancheva T., Phytopathogenic xanthomonads of pepper plants (Capsicum annuum). Ph.D. Thesis. Sofia University St. Kliment Ohridski, Sofia, Bulgaria. Vauterin L., Hoste B., Kersters K., Swings J., Reclassification of Xanthomonas. International Journal of Systematic Bacteriology 45: Young J.M., Park D.-C., Shearman H.M., Fargier E., A multilocus sequence analysis of the genus Xanthomonas. Systematic and Applied Microbiology 31: Received May 1, 2016 Accepted July 25, 2016

6