PCR detection of Guignardia bidwellii, causal agent of grape black rot

Magnolia Moreno-Velázquez, Lervin Hernández-Ramos, Ana Karen Preuss-Angeles, Liliana Elizabeth Ronces-Frutos, Israel Morales-González, Nayeli Carrillo-Ortiz, Antonio Cárcamo-Rodríguez

Abstract


Guignardia bidwellii (anamorph Phyllosticta ampelicida) is the etiological agent of grape black rot, a disease of economic importance in Europe and a quarantine pest for Mexico. The identification of the anamorphic state by morphological characteristics is complicated, due to the similarity among species of the same genus, for example with the cosmopolitan endophyte G. endophyllicola (P. capitalensis), therefore it is necessary to have quick and specific diagnostic tools. For this purpose, a molecular marker based on single nucleotide polymorphisms (SNP) in the ITS region of the rDNA was developed, where the Bidwell and Ampel2 primers were designed. The PCR amplification generates a fragment of 173 bp, specific to G. bidwellii f. euvitis. The validation of the method showed that there is no cross amplification with closely related phytopathogenic fungi or with the genome of grapevine. The technique was sensitive to detect up to 30 pg ?L-1 of DNA from monosporic cultures and in mixture of plant tissue. This diagnostic procedure is presented as a fast and specific option for the monitoring, and detection of G. bidwellii f. euvitis in support of prevention, management, quarantine and eradication strategies of the disease.

Keywords


Phyllosticta ampelicida; molecular diagnosis; ITS; Vitis spp

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References


Altschul FS, Gish W, Miller W, Myers WE and Lipman JD. 1990. Basic local alignment search tool. Journal of Molecular Biology 215: 403-410. https://doi.org/10.1016/ S0022-2836(05)80360-2

Ayyadevara S, Thaden JJ and Shmookler RJ. 2000. Discrimination of primer 3?-nucleotide mismatch by Taq DNA Polymerase during Polymerase Chain Reaction. Analytical Biochemistry 284: 11-18. https://doi.org/10.1006/ abio.2000.4635

Bonants JM Peter, Carroll CG, Weerdt M, Brouwershaven RI and Baayen PR. 2003. Development and validation of a fast PCR-based detection method for pathogenic isolates of the citrus black spot fungus, Guignardia citricarpa. European Journal of Plant Pathology 109: 503–513. https:// doi.org/10.1023/A:1024219629669

Cenis LJ. 1992. Rapid extraction of fungal DNA for PCR amplification. Nucleic Acids Research 20 (9): 2380. https:// www.ncbi.nlm.nih.gov/pmc/articles/PMC312363/pdf/ nar00083-0171.pdf

Crous PW, Verkleij GJM, Groenewald JZ, J Houbraken. 2005. Laboratory Manual Series No. 1: Fungal Biodiversity. CBS Fungal Biodiversity Centre. 425 pp. http://www.westerdijkinstitute.nl/News/Category/Laboratory%20Manual%20Series

Doyle JJ and Doyle JL. 1987. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochemical Bulletin 19: 11-15. https://webpages.uncc.edu/~jweller2/pages/BINF8350f2011/BINF8350_Readings/Doyle_plantDNAextractCTAB_1987.pdf

Everett RK and George Rees J. 2006. Species-specific PCR primers for Guignardia citricarpa and Guignardia mangiferae. New Zealand Plant Protection 59: 141-145. http:// citeseerx.ist.psu.edu/viewdoc/download?doi=10.1.1.902.2 94&rep=rep1&type=pdf

Hall TA. 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/ NT. Nucleic Acids Symposium Series 41: 95–98. http:// brownlab.mbio.ncsu.edu/jwb/papers/1999hall1.pdf

Humber AR. 1997. Fungi: Preservation of cultures. Pp. 269279. In: Lawrence A. Lacey (Ed.). Manual of techniques in insect pathology. Academic Press. 409p. https://doi. org/10.1016/B978-012432555-5/50015-4

Hyun JW, Peres NA, Yi SY, Timmer LW, Kim KS, Kwon HM and Lim HC. 2007. Development of PCR assays for the identification of species and pathotypes of Elsinoë causing scab on citrus. Plant Disease 91: 865-870. https://doi. org/10.1094/PDIS-91-7-0865

Luttrell SE. 1946. Black rot of muscadine grapes. Phytopathology 36: 905–924. https://naldc.nal.usda.gov/download/ IND43894446/PDF

Manchester KL. 1995. Value of A260/A280 ratios for measurement of purity of nucleic acids. BioTechniques 19 (2): 208-210. https://jglobal.jst.go.jp/en/detail?JGLOBAL_ ID=200902136544040775&rel=0

Matveeva VO, Shabalina AS, Nemtsov AV, Tsodikov DA, Gesteland RF and Atkins FJ. 2003. Thermodynamic calculations and statistical correlations for oligo-probes design. Nucleic Acids Research 31 (14): 4211-4217. https://www. ncbi.nlm.nih.gov/pmc/articles/PMC167637/

Motohashi Keiichi, Shigeki Inaba, Kozue Anzai, Susumu Takamatsu and Chiharu Nakashima. 2009. Phylogenetic analyses of Japanese species of Phyllosticta sensu stricto. Mycoscience 50: 291–302. https://doi.org/10.1007/ S10267-009-0487-Z

Okane I, Lumyong S, Nakagiri A and Ito T. 2003. Extensive host range of an endophytic fungus, Guignardia endophyllicola (anamorph: Phyllosticta capitalensis). Mycoscience 44 (5): 353–363. https://doi.org/10.1007/s10267-0030128-x

Peres NA, Harakava R, Carroll CG, Adaskaveg EJ and Timmer WL. 2007. Comparison of molecular procedures for detection and identification of Guignardia citricarpa and G. mangiferae. Plant Disease 91 (5): 525-531. https://doi. org/10.1094/PDIS-91-5-0525

Rinaldi AP, Paffetti D, Comparini C, Broggini ALG, Gessler C and Mugnai L. 2017. Genetic variability of Phyllosticta ampelicida, the agent of black rot disease of grapevine. Phytopathology 107: 1406-1416. https://doi.org/10.1094/ PHYTO-11-16-0404-R

Rychlik W. 1995. Selection of Primers for Polymerase Chain Reaction. Molecular Biotechnology 3 (2): 129-134. https:// doi.org/10.1007/BF02789108

Sambrook J and Russell DW. 2001. Molecular Cloning: A Laboratory Manual. 3ª ed. Ed Cold Spring Harbor Laboratory Press. Nueva York. USA. 2344.

Schoch LC, Seifert AK, Huhndorf S, Robert V, Spouge LJ, Levesque A and Chen W. 2012. Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi. Proceedings of the National Academy of Sciences of the United State of America 109 (16): 6241–6246. https://doi.org/10.1073/pnas.1117018109

SENASICA. 2019. Pudrición negra de la vid (Guignardia bidwellii–Phyllosticta ampelicida). Servicio Nacional de Sanidad, Inocuidad y Calidad Agroalimentaria. Dirección General de Sanidad Vegetal. Programa de Vigilancia Epidemiológica Fitosanitaria. México, D.F. Ficha Técnica No 63. 15p. Consultado en línea el 22/07/2019 a través del portal: https://prod.senasica.gob.mx/SIRVEF/ContenidoPublico/Fichas%20tecnicas/Pudrici%C3%B3n%20 Negra%20de%20la%20Vid%20(Guignardia%20bidwellii).pdf

SIAP. 2018. Servicio de Información Agroalimentaria y Pesquera. Anuario estadístico de la producción agrícola: producción de vid. Consultado online en Abril de 2019 a través de https://nube.siap.gob.mx/cierreagricola/

Stadhouders R, Paz DS, Anber J, Voermans J, Mes MHT and Schutten M. 2010. The effect of primer-template mismatches on the detection and quantification of nucleic acids using the 5? nuclease assay. Journal of Molecular Diagnostics 12 (1): 109-117. https://doi.org/10.2353/jmoldx.2010.090035

Ullrich IC, Kleespies GR, Enders M and Koch E. 2009. Biology of the black rot pathogen, Guignardia bidwellii, its development in susceptible leaves of grapevine Vitis vinifera. Journal für Kulturflanzen 61 (3): 82–90.

Untergasser A, Cutcutache I, Koressaar T, Ye J, Faircloth BC, Remm M and Rozen SG. 2012. Primer3 - new capabilities and interfaces. Nucleic Acids Research 40(15): e115. https://doi.org/10.1093/nar/gks596

Van der Aa H. 1973. Studies in Phyllosticta. Studies in Mycology 5: 1–110

White JT, Bruns T, Lee S and Taylor J. 1990. Amplification and direct sequencing of fungal ribosomal RNA Genes for phylogenetics. PCR Protocols: a guide to methods and applications. 315-322. https://nature.berkeley.edu/brunslab/papers/white1990.pdf

Wicht B, Petrini O, Jermini M, Gessler C and Lodovico BGA. 2012. Molecular, proteomic and morphological characterization of the ascomycete Guignardia bidwellii, agent of grape black rot: a polyphasic approach to fungal identification. Mycologia 104(5): 1036–1045. https://www.ncbi. nlm.nih.gov/pubmed/22492405

Wicht B, Jermini M, Gessler C and Lodovico BGA. 2014. Microsatellite markers for population studies of the ascomycete Phyllosticta ampelicida, the pathogen causing grape black rot. Phytopathologia Mediterranea 53(3): 470?479. http://www.jstor.org/stable/43871799

Wikee S, Lombard L, Nakashima C, Motohashi K, Chukeatirote E, Cheewangkoon R, McKenzie EH, Hyde KD and Crous PW. 2013a. A phylogenetic re-evaluation of Phyllosticta (Botryosphaeriales). Studies in Mycology 76: 1–29. https://dx.doi.org/10.3114%2Fsim0019

Wikee S, Lombard L, Crous WP, Nakashima C, Motohashi K, Chukeatirote E, Alias AS, McKenzie HCE and Hyde DK. 2013b. Phyllosticta capitalensis, a widespread endophyte of plants. Fungal Diversity 60: 91–105. https://doi. org/10.1007/s13225-013-0235-8

Ye Jian, Coulouris G, Zaretskaya I, Cutcutache I, Rozen S and Madden LT. 2012. Primer-BLAST: A tool to design targetspecific primers for polymerase chain reaction. BMC Bioinformatics 13: 134. https://doi.org/10.1186/1471-210513-134

Zhang Ke, Ning Zhang and Lei Cai. 2013. Typification and phylogenetic study of Phyllosticta ampelicida and P. vaccinii. Mycologia 105(4): 1030–1042. https://doi. org/10.3852/12-392

Zuker M. 2003. mFold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Research 31: 3406–3415. https://www.ncbi.nlm.nih.gov/pmc/articles/ PMC169194/




DOI: http://dx.doi.org/10.18781/R.MEX.FIT.1905-5

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