Abstract
Erwinia psidii causes bacterial blight of guava (Psidium guajava), one of the most important diseases of this crop in Brazil. Control measures are not effective, and dissemination often occurs through contaminated but asymptomatic propagating plant material. Considering the need for a reliable and sensitive method for detecting the pathogen in asymptomatic plant material, E. psidii-specific PCR primers were designed from a 355-bp fragment of the recombinase A gene (recA) amplified from E. psidii type strain. Primer pair Ep2L/2R only amplified DNA from E. psidii and its detection limit was 10−5 ng/μL of purified DNA and 10 CFU (colony forming units) of bacterial cell suspension/mL. Three methods, conventional PCR, IC-PCR, and BIO-PCR were evaluated with the selected primers for their potential to detect E. psidii on guava leaves. BIO-PCR and conventional PCR were more sensitive and less time-consuming than IC-PCR. The detection limits on extracts of macerated guava leaves spiked with bacterial suspensions at different concentrations were 10 and 103 CFU/mL for BIO-PCR and conventional PCR, respectively. The PCR method here described could be useful for developing a protocol for early detection of this pathogen in asymptomatic guava plants.
Similar content being viewed by others
References
Altschul SF, Gish W, Miller W, Meyers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410
Arriel DAA, Fonseca NR, Guimarães LMS, Hermenegildo PS, Mafia RG, Borges Júnior N, De Souza HP, Alfenas AC (2013) Wilt and die-back of Eucalyptus spp. caused by Erwinia psidii in Brazil. For Pathol. doi:10.1111/efp.12087
Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K (1995) Short protocols in molecular biology, 3rd edn. John Wiley and Sons Inc., Hoboken
Barbé S, Bertolini E, Roselló M, Llop P, López MM (2014) Conventional and Real-Time PCRs for Detection of Erwinia piriflorinigrans allow its distinction from the fire blight pathogen, Erwinia amylovora. Appl Environ Microbiol 80:2390–2398
Bereswill S, Pahl A, Bellemann P, Zeller W, Geider K (1992) Sensitive and species-specific detection of Erwinia amylovora by PCR-analysis. Appl Environ Microbiol 58:3522–3526
Coelho MVS, Mendes AP, Marques ASA (2002) Seca dos ponteiros da goiabeira causada por Erwinia psidii: levantamento e caracterização. Brasília, DF. Embrapa Recursos Genéticos e Biotecnologia. Comunicado Técnico 59
Coutinho TA, Brady CL, van der Vaart M, Venter SN, Telechea N, Rolfo M, Perez C, Wingfield MJ (2011) A new shoot and stem disease of Eucalyptus species caused by Erwinia psidii. Australas Plant Pathol 40:55–60
Doyle JJ, Doyle JL (1987) A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem Bull 19:11–15
Dreo T, Pirc M, Ravnikar M (2012) Real-time PCR, a method fit for detection and quantification of Erwinia amylovora. Trees 26:165–178
Eisen JA (1995) The recA protein as a model molecule for systematic studies of bacteria: comparison of trees of recA and 16S rDNAs from the same species. J Mol Evol 41:1105–1123
El Tassa SOM, Duarte V (2006) Identificação de Pectobacterium carotovorum subsp. brasiliensis através de PCR-RFLP do gene recA. Fitopatol Bras 31:23–28
Fatmi M, Damsteegt VD, Schaad NW (2005) A combined agar-absortion and BIO-PCR assay for rapid, sensitive detection of Xylella fastidiosa in grape and citrus. Plant Pathol 54:1–7
Gehring I, Geider K (2012) Identification of Erwinia species isolated from apples and pears by differential PCR. J Microbiol Methods 89:57–62
Gonzaga Neto L, Soares JM, Teixeira AHC, Moura MSB (2001) Goiaba: produção—aspectos técnicos. Embrapa Semi-árido, Brasília, DF. Embrapa Informação Tecnológica. Frutas do Brasil 17
Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 41:95–98
Hannou N, Llop P, Faure D, López MM, Moumni M (2013) Characterization of Erwinia amylovora strains from Middle Atlas Mountains in Morocco by PCR based on tandem repeat sequences. Eur J Plant Pathol 136:665–674
Janse JD (2012) Bacterial diseases that may or do emerge, with (possible) economic damage for Europe and the Mediterranean basin: notes on epidemiology, risks, prevention and management on first occurrence. J Plant Pathol 94(4, Supplement):S4.5
Junqueira NTV, Andrade LRM, Pereira M, Lima MM, Chaves RC (2001) Doenças da goiabeira no cerrado. Planaltina, Embrapa. pp.25. Circular Técnica 15
Kado CE, Heskett MG (1970) Selective media for isolation of Agrobacterium, Corynebacterium, Erwinia, Pseudomonas and Xanthomonas. Phytopathology 60:969–976
Khoodoo MHR, Sahin F, Fakim YJ (2005) Sensitive detection of Xanthomonas axonopodis pv. dieffenbachiae on Anthurium andreanum by immune capture-PCR (IC-PCR) using primers designed from sequence characterized amplified regions (SCARS) of the blight pathogen. Eur J Plant Pathol 112:379–390
Lin C-H, Hsu S-T, Tzeng K-C, Wang J-F (2009) Detection of race 1 strains of Ralstonia solanacearum in field samples in Taiwan using a BIO-PCR method. Eur J Plant Pathol 124:75–85
Marinho JS, Oliveira MGA, Guedes RNC, Pallini A, Oliveira CA (2008) Inibidores de proteases de hospedeiros nativos e exóticos e sua ação em intestinos de lagartas de Thyrinteina leucoceraea. Rev Árvore 32:1125–1132
Marques ASA, Coelho MVS, Ferreira MASV, Damasceno JPS, Mendes AP, Vieira TM (2007) Seca dos ponteiros da goiabeira causada por Erwinia psidii: níveis de incidência e aspectos epidemiológicos. Rev Bras Frutic 29:488–493
Oliveira JR, Ventura JA, Silva IT, Costa H (2000) Ocorrência da bacteriose da goiabeira, causada por Erwinia psidii, no estado do Espírito Santo. Fitopatol Bras 25:328
Parkinson N, Stead D, Bew J, Heeney J, Lahkim LT, Elphistone J (2009) Dickeya species relatedness and clade structure determined by comparison recA sequences. Int J Syst Evol Microbiol 59:2388–2393
Powney R, Beer SV, Plummer K, Luck J, Rodoni B (2011) The specificity of PCR-based protocols for detection of Erwinia amylovora. Australas Plant Pathol 40:87–97
Pritchard L, Humphris S, Saddler GS, Parkinson NM, Bertrand V, Elphinstone JG, Toth IK (2013) Detection of phytopathogens of the genus Dickeya using a PCR primer prediction pipeline for draft bacterial genome sequences. Plant Pathol 62:587–596
Rezende AMFA, Tomita CK, Uesugi CH (2008) Fungicidas cúpricos, cloretos de benzalcônio e composto bioativo líquido (Bokashi): fitotoxicidade e controle da seca dos ponteiros causada por Erwinia psidii em goiabeiras. Trop Plant Pathol 33:288–294
Rico A, Fuhrer ME, Ortiz-Barredo A, Murillo J (2008) Polymerase chain reaction fingerprinting of Erwinia amylovora has a limited phylogenetic value but allows the design of highly specific molecular markers. Phytopathology 98:260–269
Rodrigues Neto J, Robbs CF, Yamashiro TA (1987) A bacterial disease of guava (Psidium guajava) caused by Erwinia psidii sp. nov. Fitopatol Bras 12:345–350
Romeiro RS, Moraes RMA, Oliveira JR, Couto FAA, Resende ST (1993) Uma enfermidade da goiabeira de etiologia bacteriana no estado de Minas Gerais, Brazil. Fitopatol Bras 18:283
Rozen S, Skaletsky HJ (2000) Primer 3 on the WWW for general user and for biologist programmers. In: Krawetz S, Misener S (eds) Bioinformatics methods and protocols: methods in molecular biology. Humana Press, Totowa, pp 365–386
Schaad NW, Cheong SS, Tamaki S, Hatziloukas E, Panopoulos NJ (1995) A combined biological and enzymatic amplification (BIO-PCR) technique to detect Pseudomonas syringae pv. phaseolicola in bean seed extracts. Phytopathology 85:243–248
Song WY, Kim HM, Hwang CY, Schaad NW (2004) Detection of Acidovorax avenae ssp. avenae in rice seeds using BIO-PCR. J Phytopathol 152:667–676
Stoger A, Schaffer J, Ruppitsch W (2006) A rapid and sensitive method for direct detection of Erwinia amylovora in symptomatic and asymptomatic plant tissues by polymerase chain reaction. J Phytopathol 154:469–473
Taylor RK, Guilford PJ, Clark RG, Hale CN, Forster RLS (2001) Detection of Erwinia amylovora in plant material using novel polymerase chain reaction (PCR) primers. N Z J Crop Hortic Sci 29:35–43
Teixeira ACO, Ferreira MASV, Marques ASA (2008) Detecção de Erwinia psidii via enriquecimento em extrato de folhas de goiabeira e imunodifusão radial dupla. Trop Plant Pathol 33:212–218
Teixeira ACO, Marques ASA, Ferreira MASV (2009) Low genetic diversity among pathogenic strains of Erwinia psidii from Brazil. Braz J Microbiol 40:678–684
Torres P, Marques ASA, Ferreira MASV (2008) Sequenciamento e análise das sequências do gene da recombinase (rec A) em Erwinia psidii. Trop Plant Pathol 33:S95
Vanneste JL, Yu J, Boyd RJ, Cornish DA (2008) Detection of Erwinia amylovora in symptomless apple plants by BIO-PCR and BIO-DUPLEX PCR. Acta Horticult 793:517–518
Waleron M, Waleron K, Podhajska AJ, Lojkowska E (2002) Genotyping of bacteria belonging to the former Erwinia genus by PCR-RFLP analysis of a recA gene fragment. Microbiology 148:583–595
Wensing A, Zimmermann S, Geider K (2010) Identification of the corn pathogen Pantoea stewartii by spectrometry of whole-cell extracts and its detection with novel PCR primers. Appl Environ Microbiol 76:6248–6256
Wensing A, Gernold M, Geider K (2012) Detection of Erwinia species from the apple and pear flora by mass spectroscopy of whole cells and with novel PCR primers. J Appl Microbiol 112:147–158
Wetzel T, Candresse T, Macquaire G, Ravelonandro M, Dunez J (1992) A highly sensitive immune capture polymerase chain reaction method for plum pox potyvirus detection. J Virol Methods 39:27–37
Xiao W, Le Z, Fu-Shou X, Li-Han Z, Guan-Lin X (2007) Immuno-capture PCR method for detecting Acidovorax avenae subsp. citrulli from watermelon. Chin J Agric Biotechnol 4:173–179
Ye J, Coulouris G, Zaretskaya I, Cutcutache I, Rozen S, Madden T (2012) Primer-BLAST: a tool to design target-specific primers for polymerase chain reaction. BMC Bioinf 13:134
Young JM, Park DC (2007) Relationships of plant pathogenic enterobacteria based on partial atpD, carA and recA as individual and concatenated nucleotide and peptide sequences. Syst Appl Microbiol 30:343–354
Acknowledgments
C.F. Silva was supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Brazil. Conselho Nacional de Desenvolvimento Cientifico e Tecnológico (CNPq) provided scholarships to N. Pontes and P. Torres (PIBIC). We are grateful to Dr. Júlio Rodrigues Neto, IBSBF, Campinas, São Paulo, for kindly providing the strains, and José Cezar Castro, Universidade de Brasília, for technical assistance. This study was funded by Fundação de Amparo à Pesquisa do Distrito Federal (FAP-DF), grant 193.000.254/2007.
Author information
Authors and Affiliations
Corresponding author
Additional information
Section Editor: Bernardo Halfeld-Vieira
Rights and permissions
About this article
Cite this article
Silva, C.F., Torres, P., Oliveira, N.P.D. et al. PCR-based methods for detection of Erwinia psidii on guava. Trop. plant pathol. 40, 251–259 (2015). https://doi.org/10.1007/s40858-015-0020-1
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s40858-015-0020-1