SIMPLE, RAPID AND ACCURATE PCR-BASED DETECTION ... - SIPaV

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Journal of Plant Pathology (2012), 94 (3), 663-667

Edizioni ETS Pisa, 2012

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SHORT COMMUNICATION SIMPLE, RAPID AND ACCURATE PCR-BASED DETECTION OF PANTOEA ANANATIS IN MAIZE, SORGHUM AND DIGITARIA sp. J.E.F. Figueiredo1 and L.D. Paccola-Meirelles2 1 Molecular

Biochemistry Laboratory, Embrapa Milho e Sorgo, Rod MG 424, Km 65, Cx. Postal 151, CEP 35701-970, Sete Lagoas, MG, Brazil 2 Departamento de Biologia Geral, Universidade Estadual de Londrina, Rod PR 445 Km 380, Cx. Postal 6001, CEP 86051-980, Londrina, PR, Brazil SUMMARY

Detection of Pantoea ananatis in the early growing season is important for disease prediction and management. We developed a simple molecular marker based on PCR for conclusive diagnosis of P. ananatis in maize, sorghum and Digitaria sp. A pair of primers was used for amplifying only one of the seven internal transcribed spacer (ITS) regions of P. ananatis 16S-23S rRNA genes. Sixty-one strains of P. ananatis from diverse ecogeographical origins; total DNA of pool of maize white spot (MWS) lesions and MWS-like lesions from sorghum and Digitaria sp.; reference strains of P. agglomerans, P. ananatis, P. stewartii, P. allii, P. vagans, P. anthophila, P. eucalypti, P. deleyi, P. rodasii, P. rwandensis and P. wallisii were used for testing the specificity of the primers. A single amplicon per sample, 361 bp or 389 bp in size, was obtained from P. ananatis from different sources and P. allii isolated from Allium cepa and the identity of all amplicons was confirmed by DNA sequencing. The present results provide a rapid and reliable tool for the accurate identification of P. ananatis isolates and for direct PCR-based diagnosis of P. ananatis associated with maize, sorghum and Digitaria sp. Key words: Maize white spot disease, plant pathogen detection, molecular diagnosis, PCR.

Pantoea ananatis (Pa) the causal agent of maize white spot (MWS) disease (Paccola-Meirelles et al., 2001), is becoming a serious problem for maize producers in many countries (Alippi and López, 2010; Krawczyk et al., 2010; Pérez-y-Terrón et al., 2009; Pomini et al., 2007). Pa is also associated with severe economic losses in a broad range of agricultural crops as well as forest tree species worldwide, including honeydew melon (Wells et al., 1987), cantaloupe (Bruton et al., 1991),

Corresponding author: J.E.F. Figueiredo Fax: +55.31.30271188 E-mail: [email protected]

onion (Gitaitis and Gay, 1997; Gitaitis et al., 2002), sundangrass (Azad et al., 2000), eucalyptus (Coutinho et al., 2002), rice (Cortesi and Pizzatti, 2007; Cother et al., 2004), netted melon (Kido et al., 2008) and sorghum (Cota et al., 2010). Pa can be found as an epiphyte, endophyte or pathogen during different life-cycle stages of host plants. Thus, latent Pa infections in the host leaves usually serve as inoculum for epidemic outbreaks (Coutinho and Venter, 2009), the same as Pa living as a saprophyte in plant debris. Detection of initial inoculum in the early growing season is important for disease prediction and management (Huang et al., 2011). Traditionally, the tentative identification of Pantoea species has been based on different phenotypic characters and biochemical tests. These methods, however, are time consuming and laborious and require specialized taxonomic knowledge, so that the correct identification of bacterial isolates at the species level and the resolution of the taxonomic framework of the genus are difficult (Brady, 2005). Therefore, a rapid and sensitive diagnostic test for Pantoea species is desirable. Species-specific primers targeting the rRNA genes of Pa recognize also Pantoea stewartii subsp stewartii, a species closely associated with maize plants (Gitaitis et al., 2002; Walcott et al., 2002). We now describe a simple, rapid, sensitive and accurate PCR-based test for conclusive Pa identification in maize, sorghum and Digitaria sp. which can be performed by any laboratory technician and which does not require taxonomic expertise. Bacterial isolates from sorghum, pools of MWS lesions from maize and of MWS-like lesions from sorghum and Digitaria sp. were collected from growing plants in the experimental fields of Embrapa Maize and Sorghum at Sete Lagoas (Minas Gerais, Brazil). Pa isolates of epiphytic or saprophytic origin (maize culture residues), or obtained from MWS lesions of field-grown maize plants and from MWS-like lesions from sorghum and Digitaria sp. were deposited in the microorganism collection of the fungal laboratory of the State University of Londrina (Paraná, Brazil) and identified by morphological, biochemical and rDNA sequencing methods. FTA cards (Whatman, USA) with preserved DNA


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from bacterial cultures of the following reference species: Pantoea agglomerans (strains: PNG 06-3, PNG 09-1 and PNG 97-2), P. ananatis (strains: PNA 08-2, PNA 97-5 and PNA 99-13), P. stewartii (strains: ES 021 and ES 02-2) and P. allii (strains: Bsf 24, HH 24, BD 380 and BD 390T= LMG 24248T) were kindly provided by Prof. R.D. Gitaitis (University of Georgia, USA) Genomic DNAs of P. vagans (strain: 105T= LMG 24199T), P. allii (strain: BD 390T= LMG 24248T), P. anthophila (strain: 689T= LMG 2558T), P. eucalypti, (strain: 76T= LMG 24197T), P. deleyi (strain: BD767T= LMG 24280T), P. rodasii (strain: 518T= LMG 26293T), P. rwandensis (strain: 571T= LMG 26275T) and P. wallisii (strain: 682T= LMG 26277T) were kindly provided by Prof. T.A. Coutinho (University of Pretoria, South Africa). Genomic DNA was extracted from Brazilian bacterial isolates according to Sambrook et al. (1989), and total DNA from 0.5 g of a pool of surface-sterilized lesions was extracted by the hexadecyltrimethylammonium bromide (CTAB) method (Doyle and Doyle, 1990). Samples in FTA cards were prepared according to the manufacturer’s instructions (Whatman, USA) for direct use in PCR reactions. The species-specific forward primer ANAF: 5’-CGTGAAACTACCCGTGTCTGTTGC-3’, designed by sequence alignments of the ITS region of 16S-23S rRNA genes of Pantoea spp. from GenBank, anneals to one of the seven copies of rDNA of Pa in a region containing a genomic rearrangement. The reverse universal primer EC5: 5’-TGCCAGGGCATCCACCGTGTACGCT-3’ (Gürtler and Stanish, 1996) was modified by addition of eight nucleotides at the 3’ end (in bold). The speciesspecificity of the primers was checked against all DNA sequences deposited in GenBank by the Primer-BLAST program (http://www.ncbi.nlm.nih.gov/tools/primerblast/). PCR reactions and incubation were done according to Gitaitis et al. (2002) except for the use of 20 ng of genomic DNA as a template, 60°C annealing temperature and 0.5 unit of Taq DNA polymerase (Phoneutria, Brazil). Loading dye plus 1 µl of Gel Red (Biotium, USA) were added to PCR products and loaded on the 1% (w/v) agarose gel with Tris-Acetate-EDTA (TAE) running buffer at 60 V for 90 min. Amplicons excised from agarose gels were purified with the DNA gel extraction kit (Fermentas, USA) and sequenced four times each (forward and reverse) with the Big Dye Terminator Cycle Sequencing within the ABI Prism 3100 automatic sequencer according to the manufacturer’s instructions (Applied Biosystems, USA). Contig sequences were generated with CAP3 Program (Huang and Madan, 1999) and similarity was evaluated by the BlastN program (Altschul et al., 1997) run against all available DNA sequences deposited in GenBank (http:// www.ncbi.nlm.nih.gov). All seventy-two nucleotide se-


quences generated with primers ANAF and EC5-modified were deposited in GenBank with the accession Nos JN622029 to JN622037 and JQ288776 to JQ288839. The adonitol fermentation test was made with nutrient broth base medium (peptone 10 g/l, beef extract 1 g/l, sodium chloride 5 g/l, adonitol 10 g/l, pH 7.4). All bacterial isolates from maize, sorghum and Digitaria sp. used in this study and Klebsiella pneumoniae (positive control) were cultured at 30°C for 24-48 h. Virtual analysis with primer-blast program (http:// www.ncbi.nlm.nih.gov/tools/primer-blast) to estimate PCR products with ANAF and the modified EC5 primers revealed two possible amplicons for the same ITS region of the Pa genome (360 bp for strain LMG20103 (De Maayer et al., 2010) or 388 bp for strain AJ13355 (accession No. AP012032.1). In silico, two amplicons (3487 bp and 4643 bp) were also expected to be amplified with DNA of the non-plant-associated bacterium Clostridium difficile, a commensal bacterium of the human intestine (Harvey et al., 2011). As predicted by Primer-BLAST analysis, 72 PCR products amplified with ANAF and the modified EC5 primers showed a single DNA fragment per sample with approximately the expected size (361 or 389 bp) for Pa isolates from maize, sorghum and Digitaria sp. and four Pa reference strains (Table 1). A single amplicon per sample with 361 bp or 389 bp was also obtained from MWS lesions from maize, MWS-like lesions from sorghum and Digitaria sp. and four strains of P. allii (Table 1). P. allii (formerly Pa) is a new species of Pantoea isolated from onion (Brady et al., 2011). In the last decade, members of the genus Pantoea isolated from onion (Allium cepa) with center rot disease in the USA and onion seeds in South Africa were routinely identified as Pa based on phenotypic data (Gitaitis and Gay, 1997; Walcott et al., 2002) and sequence analysis of the 16S rRNA gene (Goszczynska et al., 2006). Recently, MLSA (multilocus sequence analysis) and AFLP (amplified fragment length polymorphism) analyses have shown that the US/South Africa group of Pantoea isolated from onion consisits of two different although closely related species: i.e. Pa proper and a new species named by P. allii (Brady et al., 2011). So far, P. allii has been reported only from the USA and South Africa, (Brady et al., 2011). The ability of P. allii to use adonitol is a useful phenotypic features to distinguish it from Pa (Brady et al., 2011). Due to insufficient information about the host range and the geographic distribution of this new species and considering the great genetic similarity with Pa, the adonitol fermentation test was applied to all bacterial isolates from maize, sorghum and Digitaria sp. used in this study and previously identified as Pa by biochemical tests and sequencing of 16S and ITS region of 16S-23S rRNA genes. The adonitol test was positive (yellow color) for Klebsiella pneumoniae used as positive control


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Table 1. Size of PCR amplicons obtained with primers ANAF and EC5 modified for detection of P. allii and P. ananatis in different sources and ecogeographical regions. Strains and isolates

Bacterial species Host/Source

PNA 97-1

P. ananatis

PNA 99-13

P. ananatis

PNA 200-5

P. ananatis

PNA 08-2

P. ananatis

LMG 24248T (BD390T)

P. allii (formerly P. ananatis)

Allium cepa ----Allium cepa bulbs Allium cepa leaf Allium cepa bulbs Allium cepa Britex seed

Country

Amplicon size (bp)

US

389

US

389

US

389

US

389

ZA

361

Allium cepa seeds from US 389 bolting plants Allium cepa HH 24 P. allii seeds from bolting US 361 plants Allium cepa BD 380 P. allii ZA 361 Britex seed Zea mays PR234 to PR237, PR240 to PR246 P. ananatis BR 389 Epiphytic PR238, PR239 Zea mays BR 361 P. ananatis Epiphytic Zea mays BR 389 PR258 to PR260, PR262, PR264 P. ananatis Crop debris Zea mays PR257, PR261 P. ananatis BR 361 Crop debris MS281, MS282, MS284, MG286, PR248, PR249, PR251 Zea mays P. ananatis BR 389 to PR254, PR 271, PR272, PR276, PR279, PR280 MWS lesions PR247, PR250, PR255, PR256, PR270, PR273 to PR275, Zea mays P. ananatis BR 361 PR277, PR278, PR283 MWS lesions Zea mays BR 389 -----------------------------------------------pool of MWS lesions Sorghum bicolor PA10121, MG287, GO290 P. ananatis BR 389 MWS-like lesions Sorghum bicolor GO288, GO289, MG285, MG292 to MG297 P. ananatis BR 361 MWS-like lesions Sorghum bicolor BR 389 -----------------------------------------------pool of MWS-like lesions Digitaria sp. PR265, PR266, MG302 P. ananatis BR 389 MWS-like lesions Digitaria sp. -----------------------------------------------pool of MWS-like BR 389 lesions US= United States of America; ZA= South Africa; BR= Brazil. GO, MG, MS, and PR are the Brazilian States of Goiás, Minas Gerais, Mato Grosso do Sul and Paraná, respectively. PA= strain from Minas Gerais State. Pool of MWS and MWS-like lesions were collected in maize and sorghum fields in the Minas Gerais State. Bsf 24

and negative (red color) for all maize, sorghum and Digitaria sp. isolates, reinforcing their identity as Pa. In onion plants, ANAF and EC5 modified primers can be used as a first step to distinguish P. allii and Pa from other Pantoea species, whereas simple biochemical tests (Brady et al., 2011) can differentiate P. allii from Pa. Pantoea vagans is another new Pantoea species that

P. allii

causes brown stalk rot in maize (Brady et al., 2009). Although P. vagans has been isolated simultaneously with Pa from maize in South Africa (Brady et al., 2009), in Brazil P. vagans or other Pantoea species has not been isolated from MWS lesions, as demonstrated by morphological, biochemical and molecular analysis. In this study, no amplicons were obtained using primers ANAF


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and EC5-modified and DNA samples of P. vagans, even when the annealing temperature of primers was reduced to 50°C. This result confirmed our previous analysis performed using the Primer-BLAST program. Besides, an exhaustive analysis of complete genome of P. vagans, strain C9-1 (Smits et al., 2010), confirmed the lack of similarity between the nucleotide sequence of the primer ANAF and the genome of P. vagans. The present study demonstrates that ANAF and EC5-modified primers represent a powerful tool for the rapid and dependable identification of Pa isolates from maize, sorghum and Digitaria sp., thus for a reliable diagnosis. ACKNOWLEDGEMENTS

We would like to thank Prof. R.D. Gitaitis (College of Agricultural and Environmental Sciences, University of Georgia, Tifton, GA, USA) and Prof. T.A. Coutinho (Department of Microbiology and Plant Pathology, Forestry and Agricultural Biotechnology Institute, University of Pretoria, South Africa) for providing DNA samples of reference strains. We are also grateful to Prof. E. Kalapothakis from the Federal University of Minas Gerais State (Brazil) for sequencing facilities and to Dr. Á.V. Folgueras Flatschart for valuable comments and suggestions during the preparation of this article. This study was supported by Embrapa, FUNARBE and Fundação Araucária. REFERENCES Alippi A.M., López A.C., 2010. First report of leaf spot disease of maize caused by Pantoea ananatis in Argentina. Plant Disease 94: 487. Altschul S.F., Madden T.L., Shaffer A.A., Zhang J., Zhang Z., Miller W., Lipman D.J., 1997. Gapped BLAST and PSIBLAST: a new generation of protein database search programs. Nucleic Acids Research 25: 3389-3402. Azad H.R., Holmes G.J., Cooksey D.A., 2000. A new leaf blotch disease of sudangrass caused by Pantoea ananas and Pantoea stewartii. Plant Disease 84: 973-979. Brady C.L., 2005. Taxonomy and relatedeness of Pantoea strains recovered from Eucalyptus from South Africa, South America and Uganda. Ph.D. Thesis. University of Pretoria, Pretoria, Gauteng, South Africa. Brady C.L., Venter S.N., Cleenwerck I., Engelbeen K., Vancanneyt M., Swings J., Coutinho T., 2009. Pantoea vagans sp. nov., Pantoea eucalypti sp. nov., Pantoea deleyi sp. nov. and Pantoea anthophila sp. nov. International Journal of Systematic and Evolutionary Microbiology 59: 2339-2345. Brady C.L., Goszczynska T., Venter S.N., Cleenwerck I., De Vos P., Gitaitis R.D., Coutinho T., 2011. Pantoea allii sp. nov., isolated from onion plants and seed. International Journal of Systematic and Evolutionary Microbiology 61: 932-937.

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Received August 30, 2011 Accepted April 13, 2012

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