Abstract
The 4′,6-diamidino-2-phenylindole (DAPI) stain technique is a simple method that was developed for confirming the presence of phytoplasmas in hand-cut or freezing microtome sections of infected tissues. DAPI binds AT-rich DNA preferentially, so that phytoplasmas, localized among phloem cells, can be visualized in a fluorescence microscope. The procedure is quick, easy to use, inexpensive, and can be used as a preliminary or quantitative method to detect or quantify phytoplasma-like bodies in infected plants.
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References
Hogenhout SA et al (2008) Phytoplasmas: bacteria that manipulate plants and insects. Mol Plant Pathol 9:403–423
Lee I-M, Davis RE, Gundersen-Rindal DE (2000) Phytoplasma: phylogenetic mollicutes. Annu Rev Microbiol 54:221–255
Weintraub PG, Beanland L (2006) Insect vectors of phytoplasmas. Annu Rev Entomol 51:91–111
Lepka P et al (1999) Effect of phytoplasmal infection on concentration and translocation of carbohydrates and amino acids in periwinkle and tobacco. Physiol Mol Plant Pathol 55:59–68
Bertamini M, Grando MS, Nedunchezhian N (2003) Effects of phytoplasma infection on pigments, chlorophyll-protein complex and photosynthetic activities in field grown apple leaves. Biol Plant 47:237–242
Ambrožič-Dolinšek J et al (2008) Phytoplasma infection may affect morphology, regeneration and pyrethrin content in pyrethrum shoot culture. Sci Hortic 116:213–218
Bertaccini A, Duduk B (2009) Phytoplasma and phytoplasma diseases: a review of recent research. Phytopathol Mediterr 48:355–378
Deeley J, Stevens WA, Fox RTV (1979) Use of Dienes’ stain to detect plant diseases induced by mycoplasmalike organisms. Phytopathology 69:1169–1171
Hogenhout SA, Šeruga MM (2010) Phytoplasma genomics, from sequencing to comparative and functional genomics—what have we learnt? In: Weintraub PG, Jones P (eds) Phytoplasmas: genomes, plant hosts and vectors. CABI Publishing, Oxfordshire, pp 19–36
Eriksson S et al (1993) Binding of 4′,6-diamidino-2-phenylindole (DAPI) to AT regions of DNA: evidence for an allosteric conformational change. Biochemistry 32:2987–2998
Jansen K, Nordén B, Kubista M (1993) Sequence dependence of 4′6-diamidino-2-phenylindole (DAPI)–DNA Interactions. J Am Chem Soc 115:10527–10530
Dale JI (1988) Rapid compression technique for detecting mycoplasmalike organisms in leaf midrib sieve tubes by fluorescence microscopy. Phytopathology 78:118–120
Sinclair WA et al (1992) Detection of ash yellows mycoplasmalike organisms in different tree organs and in chemically preserved specimens by a DNA probe vs. DAPI. Plant Dis 76:154–158
Thomas S, Balasundaran M (1998) In situ detection of phytoplasma in spike-disease-affected sandal using DAPI stain. Curr Sci 74:989–993
Ghosh DK et al (1999) Association of a phytoplasma with witches’ broom, a new disease of acid lime (Citrus aurantifolia). Curr Sci 77:174–177
Bricker JS, Stutz JC (2005) Host range and distribution of the phytoplasma causing Arizona ash decline. J Arboric 3:257–262
Fletcher S, Melcher U, Wayadande A (2006) The phytopathogenic spiroplasmas. In: Falkow S, Dworkin M, Rosenberg E et al (eds) The prokaryotes: a handbook on the biology of bacteria: Firmicutes, Cyanobacteria, vol 4, 3rd edn. Springer, New York, pp 905–947
Arismendi AS et al (2010) Presence of a phytoplasma associated with witches’-broom disease in Ugni molinae Turcz. and Gaultheria phillyreifolia (Pers.) Sleumer determined by DAPI, PCR, and DNA sequencing. Chil J Agric Res 70:26–33
Jung H et al (2003) Detection and treatment of mycoplasma contamination in cultured cells. Chang Gung Med J 26:250–258
Mabuchi T, Nishikawa S (1991) Selective staining with two fluorochromes of DNA fragments on gels depending on their AT content. Nucl Acids Res 18:7461–7462
Hamada S, Fujita S (1983) DAPI staining improved for quantitative cytofluorometry. Histochem Cell Biol 79:219–226
Liu J-Y et al (2004) A new chromosome fluorescence banding technique combining DAPI staining with image analysis in plants. Chromosome 113:16–21
Romero J (2001) XI Curso Internacional Teórico-Práctico de Detección e Identificación de Virus, Viroides y Fitoplasmas. Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA). Madrid, p 2–9
Lichtman JW, Conchelo JA (2005) Fluorescence microscopy. Nat Methods 2:910–919
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Andrade, N.M., Arismendi, N.L. (2013). DAPI Staining and Fluorescence Microscopy Techniques for Phytoplasmas. In: Dickinson, M., Hodgetts, J. (eds) Phytoplasma. Methods in Molecular Biology, vol 938. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-089-2_10
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DOI: https://doi.org/10.1007/978-1-62703-089-2_10
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