Development of an indirect competitive enzyme-linked immunosorbent assay applied to the Botrytis cinerea quantification in tissues of postharvest fruits

BMC Microbiology - Tập 11 Số 1 - 2011
Martín A. Fernández‐Baldo1, Jorge Fernández1, Sirley V. Pereira1, Germán A. Messina1, Eloy Salinas1, Julio Raba1, María Isabel Sanz Ferramola1
1INQUISAL, Departamento de Química, Universidad Nacional de San Luis, CONICET, Chacabuco 917, D5700BWS, San Luis, Argentina

Tóm tắt

AbstractBackgroundBotrytis cinereais a phytopathogenic fungus responsible for the disease known as gray mold, which causes substantial losses of fruits at postharvest. This fungus is present often as latent infection and an apparently healthy fruit can deteriorate suddenly due to the development of this infection. For this reason, rapid and sensitive methods are necessary for its detection and quantification. This article describes the development of an indirect competitive enzyme-linked immunosorbent assay (ELISA) for quantification ofB. cinereain apple (Red Delicious), table grape (pink Moscatel), and pear (William's) tissues.ResultsThe method was based in the competition for the binding site of monoclonal antibodies betweenB. cinereaantigens present in fruit tissues andB. cinereapurified antigens immobilized by a crosslinking agent onto the surface of the microtiter plates. The method was validated considering parameters such as selectivity, linearity, precision, accuracy and sensibility. The calculated detection limit was 0.97 μg mL-1B. cinereaantigens. The immobilized antigen was perfectly stable for at least 4 months assuring the reproducibility of the assay. The fungus was detected and quantified in any of the fruits tested when the rot was not visible yet. Results were compared with a DNA quantification method and these studies showed good correlation.ConclusionsThe developed method allowed detects the presence ofB. cinereain asymptomatic fruits and provides the advantages of low cost, easy operation, and short analysis time determination for its possible application in the phytosanitary programs of the fruit industry worldwide.

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Tài liệu tham khảo

Dewey F, Hill M, DeScenzo R: Quantification of Botrytis and laccase in wine grapes. Am J Enol Vitic. 2008, 59: 47-54.

Dewey F, Meyer U: Rapid quantitative tube immunoassays for on-site detection of Botrytis, Aspergillus and Penicillium antigens in grape juice. Anal Chim Acta. 2004, 513: 11-19. 10.1016/j.aca.2003.11.088.

Muñoz C, Gómez Talquenca S, Volpe M: Tetra primer ARMS-PCR for identification of SNP in β-tubulin of Botrytis cinerea, responsible of resistance to benzimidazole. J Microbiol Meth. 2009, 78: 245-246. 10.1016/j.mimet.2009.06.007.

Mosbach A, Leroch M, Mendgen KW, Hahn M: Lack of evidence for a role of hydrophobins in conferring surface hydrophobicity to conidia and hyphae of Botrytis cinerea. BMC Microbiology. 2011, 11: 10-21. 10.1186/1471-2180-11-10.

De Kock S, Holz G: Blossom-end rot of pears: systemic infection of flowers and immature fruit by Botrytis cinerea. J Phytopathol. 1992, 135: 317-327. 10.1111/j.1439-0434.1992.tb04317.x.

Jarvis W: Latent infections in the pre- and postharvest environment. Hort Science. 1994, 29: 749-751.

Lavy-Mair G, Barkai-Golan R, Kopeliovitch E: Initiation at the stage of postharvest Botrytis stem-end rot in normal and non-ripening fruits. Ann Appl Biol. 1988, 112: 393-396. 10.1111/j.1744-7348.1988.tb02077.x.

McNicol R, Williamson B: Systemic infection of black currant flowers by Botritis cinerea and its possible involvement in premature abscission of fruits. Ann Appl Biol. 1989, 114: 243-254. 10.1111/j.1744-7348.1989.tb02101.x.

Morales-Valle H, Silva L, Paterson R, Oliveira J, Venâncio A, Lima N: Microextraction and Gas Chromatography/Mass Spectrometry for improved analysis of geosmin and other fungal "off" volatiles in grape juice. J Microbiol Meth. 2010, 83: 48-52. 10.1016/j.mimet.2010.07.013.

Thompson J, Latorre B: Characterization of Botrytis cinerea from table grapes in Chile using RAPD-PCR. Plant Dis. 1999, 83: 1090-1094. 10.1094/PDIS.1999.83.12.1090.

Eckert J, Ogawa J: The chemical control of postharvest diseases: subtropical and tropical fruits. Annu Rev Phytopathol. 1988, 23: 421-454.

Spotts R, Cervantes L: Population, pathogenicity, and benomyl resistance of Botrytis spp., Penicillium spp., and Mucor piriformis in packinghouses. Plant Dis. 1986, 70: 106-108. 10.1094/PD-70-106.

Ragsdale N: The impact of the food quality protection act on the future of plant disease management. Annu Rev Phytopathol. 2000, 38: 577-596. 10.1146/annurev.phyto.38.1.577.

Sansone G, Calvente V, Rezza I, Benuzzi D, Sanz M: Biological control of Botrytis cinerea strains resistant to Iprodione. Postharvest Biology and Technology. 2005, 35: 229-339. 10.1016/j.postharvbio.2004.10.005.

Dewey F, Yohalem D: Detection, quantification and immunolocalisation of Botrytis species. Botrytis: Biology, Pathology and Control. Edited by: Elad Y, et al. 2007, London, Chapter 11: 181-194.

Eriksson R, Jobs M, Ekstrand C, Ullberg M, Herrmann B, Landegren U, Nilsson M, Blomberg J: Multiplex and quantifiable detection of nucleic acid from pathogenic fungi using padlock probes, generic real time PCR and specific suspension array readout. J Microbiol Meth. 2009, 78: 195-202. 10.1016/j.mimet.2009.05.016.

Gao X, Jackson K, Lambert S, Hartman G, Niblack T: Detection and quantification of Fusarium solani in soybean roots with real-time quantitative polymerase chain reaction. Plant Dis. 2004, 88: 1372-1380. 10.1094/PDIS.2004.88.12.1372.

Leisova L, Minarikova V, Kucera L, Ovesna J: Quantification of Pyrenophora teres in infected barley leaves using real-time PCR. J Microbiol Meth. 2006, 67: 446-455. 10.1016/j.mimet.2006.04.018.

Lopez M, Bertolini E, Olmos A, Caruso P, Gorris M, Llop P, Penyalver R, Cambra M: Innovative tools for detection of plant pathogenic viruses and bacteria. Int Microbiol. 2003, 6: 233-243. 10.1007/s10123-003-0143-y.

McCartney H, Foster S, Fraaije B, Ward E: Molecular diagnostics for fungal plant pathogens. Pest Manag Sci. 2003, 59: 129-142. 10.1002/ps.575.

Savazzini F, Oliveira Longa C, Pertot I, Gessler C: Real-time PCR for detection and quantification of the biocontrol agent Trichoderma atroviride strain SC1 in soil. J Microbiol Meth. 2008, 73: 185-194. 10.1016/j.mimet.2008.02.004.

Schaad N, Frederick R: Real-time PCR and its application for rapid plant disease diagnostics. Can J Plant Pathol. 2002, 24: 250-258. 10.1080/07060660209507006.

Ward E, Foster S, Fraaije B, McCartney H: Plant pathogen diagnostics: immunological and nucleic acid-based approaches. Ann Appli Biol. 2004, 145: 1-16. 10.1111/j.1744-7348.2004.tb00354.x.

Xie Z, Thompson A, Kashleva H, Dongari-Bagtzoglou A: A quantitative real-time RT-PCR assay for mature C. albicans biofilms. BMC Microbiology. 2011, 11: 93-100. 10.1186/1471-2180-11-93.

Serrano R, Gusmão L, Amorim A, Araujo R: Rapid identification of Aspergillus fumigatus within the section Fumigati. BMC Microbiology. 2011, 11: 82-88. 10.1186/1471-2180-11-82.

He F, Soejoedono RD, Murtini S, Goutama M, Kwang J: Complementary monoclonal antibody-based dot ELISA for universal detection of H5 avian influenza virus. BMC Microbiology. 2010, 10: 330-338. 10.1186/1471-2180-10-330.

Rigano LA, Marano MR, Castagnaro AP, Do Amaral AM, Vojnov AA: Rapid and sensitive detection of Citrus Bacterial Canker by loop-mediated isothermal amplification combined with simple visual evaluation methods. BMC Microbiology. 2010, 10: 176-183. 10.1186/1471-2180-10-176.

Dewey F, Ebeler S, Adams D, Noble A, Meyer U: Quantification of Botrytis in grape juice determined by a monoclonal antibody-based immunoassay. Am J Enol Vitic. 2000, 51: 276-282.

Meyer U, Spotts R, Dewey F: Detection and quantification of Botrytis cinerea by ELISA in pear stems during cold storage. Plant Dis. 2000, 84: 1099-1103. 10.1094/PDIS.2000.84.10.1099.

Obanor F, Walter M, Waipara N, Cernusko R: Rapid method for the detection and quantification of Botrytis cinerea in plant tissues. New Zealand Plant Protection. 2002, 55: 150-153.

Obanor F, Williamson K, Mundy D, Wood P, Walter M: Optimisation of PTA-ELISA detection and quantification of Botrytis cinerea infections in grapes. New Zealand Plant Protection. 2004, 57: 130-137.

Ricker R, Marois J, Dlott R, Morrison J: Immunodetection and quantification of Botrytis cinerea on harvested wine grapes. Phytopathology. 1991, 81: 404-411. 10.1094/Phyto-81-404.

González C, Noda J, Espino J, Brito N: Drill-assisted genomic DNA extraction from Botrytis cinerea. Biotechnol Lett. 2008, 30: 1989-1992. 10.1007/s10529-008-9790-6.

Muñoz C, Gómez Talquenca S, Oriolani E, Arias F: Identificación rápida de distintas razas de Botrytis cinerea en cultivos de vid. Enologia. 2008, 6: 5-7.

Giraud T, Dominique F, Levis C, Leroux P, Brygoo Y: RFLP Markers show genetic recombination in Botrytinia Fuckeliana (Botrytis cinerea) and transposable element reveal two sympatric species. Mol Biol Evol. 1997, 11: 1177-1185.

Giraud T, Fortini D, Levis C, Lamarque C, Leroux P, Lo Buglio K, Brygoo Y: Two sibling species of the Botrytis cinerea complex, transposa and vacuma, are found in sympatry on numerous host plants. Phytopathology. 1999, 89: 967-973. 10.1094/PHYTO.1999.89.10.967.

Fernández-Baldo M, Messina GA, Sanz MI, Raba J: Microfluidic immunosensor with micro magnetic beads coupled to Carbon-based Screen-Printed Electrodes (SPCEs) for determination of Botrytis cinerea in tissue of fruits. J Agric Food Chem. 2010, 58: 11201-11206. 10.1021/jf1025604.

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Tập 11 Số 1

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