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Fluorescence in situ hybridization for phytoplasma and endophytic bacteria localization in plant tissues
Journal of Microbiological Methods 87 (2011) 220–223
Contents lists available at ScienceDirect
Journal of Microbiological Methods j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j m i c m e t h
Fluorescence in situ hybridization for phytoplasma and endophytic bacteria localization in plant tissues Daniela Bulgari a, Paola Casati a, Franco Faoro a, b,⁎ a b
Dipartimento di Produzione Vegetale, Università degli Studi di Milano, Via Celoria 2, 20133 Milano, Italy CNR, Istituto di Virologia Vegetale, Strada delle Cacce 73, 10135 Torino, Italy
a r t i c l e
i n f o
Article history: Received 5 July 2011 Received in revised form 31 July 2011 Accepted 1 August 2011 Available online 7 August 2011
a b s t r a c t In the present study, we developed a rapid and efficient fluorescence in situ hybridization assay (FISH) in nonembedded tissues of the model plant Catharanthus roseus for co-localizing phytoplasmas and endophytic bacteria, opening new perspectives for studying the interaction between these microorganisms. © 2011 Elsevier B.V. All rights reserved.
Keywords: Video-confocal microscope DNA probe Vibroslice Periwinkle
Phytoplasmas are obligate parasitic bacteria that cause economically relevant yield losses in different low- and high-value annual and perennial crops worldwide (Bertaccini and Duduk, 2009; Lee et al., 2000). Despite their relevance, research aimed at understanding phytoplasma–host interactions and at finding effective control strategies has been slow due to the inability of isolating these pathogens on culture media. A possible utilization of beneficial bacteria as candidates for developing biocontrol strategies of phytoplasmas has recently been proposed (Bulgari et al., 2009; Gamalero et al., 2010). These microorganisms may reduce disease severity directly by antibiosis, competition for nutrients and niches or indirectly by activating systemic resistance (Lugtenberg and Kamilova, 2009). In the case of phytoplasmas, it should be firstly necessary to investigate if endophytes may directly interact with phytoplasmas because of their possible co-localization in the same plant tissues. Fluorescence in situ hybridization (FISH) technique, performed by means of specific cDNA fluorescent probes, has been utilized to localize different bacterial species in plant tissues (Watt et al., 2006; Iverson and Maier, 2009; Nabti et al., 2010). However, only one attempt has been reported for localizing phytoplasmas (Italian Clover Phyllody, taxonomic group 16SrI) in host plants (Webb et al., 1999). In the present study, we set up a FISH assay able to specifically co-localize phytoplasmas of taxonomic group 16SrV and endophytic bacteria in the tissues of the model plant Catharanthus roseus L. (G. Don). Periwinkles (C. roseus L.) were inoculated by grafting with phytoplasma reference strains: ‘Ca. Phytoplasma asteris’ strains SAY (South American aster yellows, subgroup 16SrI-B) and CPh (Clover phyllody, ⁎ Corresponding author at: via Celoria, 2, 20133 Milano, Italy. Tel.: + 39 0250316786; fax: + 39 0250316781. E-mail address: [email protected] (F. Faoro). 0167-7012/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.mimet.2011.08.001
subgroup 16SrI-C), ‘Ca. Phytoplasma mali’ strain AT (Apple proliferation, subgroup 16SrX-A), stolbur phytoplasma strain STOL (subgroup 16SrXIIA), alder yellows phytoplasma strain ALY (subgroup 16SrV-C), ‘Ca. Phytoplasma ulmi’ strain Ulw (Elm witches broom, subgroup 16SrV-A) and Flavescence dorée phytoplasma strain FD-D (FDp) (subgroup 16SrVD, kindly provided by E. Boudon-Padieu, INRA Dijon, France). Shoots of periwinkle plants, inoculated by grafting with the above strains, were fixed in 0.25% glutaraldehyde and 4% paraformaldehyde in Piperazine-1,4-Bis-2- Ethanesulfonic acid (PIPES) and cut in 50– 70 μm slides with a vibroslice (World Precision Instruments, Germany) without the paraffin-embedding step. The elimination of this step did not influence the probe hybridization and tissue structure preservation and greatly speeded up specimen preparation. Fresh tissue sections were washed in cold PIPES and then permeated as described by Webb et al. (1999). R16(V)F1 probe 200 ng/ml, usually employed in PCR reaction as 16SrV group specific primer (Lee et al., 1998), was applied to the sections to localize phytoplasmas belonging to 16SrV group in the plant tissues (Lherminier et al., 1999). The probe was labeled with FAM (Primm, Milan Italy) or Marina Blue (MB) (Invitrogen, Milan Italy) at 5′ terminus. We tested these dyes emitting respectively at 518 nm and 459 nm to find out the appropriate wavelengths that interfere at least with tissue auto-fluorescence. Sections permeated through proteinase K treatment were incubated in hybridization buffer (40 mM PIPES, 0.1% PVP 10 K, 0.1% Ficoll 4000, 140 mM NaCl, pH 7.8) containing R16(V)F1 probe for four hours at 50 °C in a humid chamber. Sections were then rapidly washed in 2× SSC (1× SSC=150 mM NaCl and 15 mM tri-sodium citrate, pH 7.0) and in 1× SSC for 30 min, each at 42 °C, followed by washing in 0.2× SSC at room temperature for 30 min. Finally, sections were washed in 1× PBS, mounted in glycerol 70% and visualized by video-confocal microscope (Nikon, Italy) using the corresponding
D. Bulgari et al. / Journal of Microbiological Methods 87 (2011) 220–223
channels. As 4′,6-diamidino-2-phenylindole (DAPI), usually used for dsDNA detection, is also able to stain phytoplasmas in plant tissues (Seemuller, 1976), some serial sections were also treated with DAPI to confirm the co-localization of this staining with the probe. As FISH allows co-localization of diverse nucleic acid sequences by the use of different labeled probes, we incubated serial sections also with the bacterial universal probe pB-00542 (5′-GACGGGCGGTGTGTACA-3′, probeBase website http://www.microbial-ecology.de/probebasesearch.asp, Liu et al., 2001) 200 ng/ml for localizing endophytic bacteria. The universal
221
probe was labeled with a fluorophore emitting in the far-red (i.e. Cy5) and with Carboxy-X-Rhodamine (ROX) emitting at 602 nm (Primm). Bacterial universal probe was added together with R16(V)F1 to the hybridization buffer. Following hybridization and washing the detection of targeted bacteria and phytoplasmas were performed with at the video-confocal microscope. The antisense R16(V)F1 and pB-00542 probes were employed as negative controls to verify non specific binding; as a further control, we visualized the samples without probes to detect false positive signals and auto-fluorescence. To assess the reproducibility of obtained
A
B
C
D
E
F
G
H
Fig. 1. Fluorescence in situ hybridization (FISH) of periwinkle shoots using 16SrV phytoplasma specific probes visualized by a video-confocal microscope; in all images xylem is autofluorescent at each tested wavelength while red signal, when present, is due to chlorophyll emission (bars: in A, B, G, H = 100 μm; in C = 50 μm; in D = 70 μm; in E = 500 μm; in F = 200 μm). Green signal of hybridized FAM-labeled probe (arrows) is visible in periwinkle inoculated with: (A) ‘Ca. Phytoplasma ulmi’ strain Ulw, elm witches broom (subgroup 16SrV-A), (B) alder yellows phytoplasma strain ALY (subgroup 16SrV-C) and (C) Flavescence dorée phytoplasma strain FD-D (FDp, subgroup 16SrV-D). (D) Blue signal of hybridized Marina blue-labeled probe (arrows) is present in periwinkle shoots inoculated with Flavescence dorée phytoplasma strain FD-D (subgroup 16SrV-D). (E) No hybridization signal is visible in healthy periwinkle shoots and (F) in periwinkle inoculated with stolbur phytoplasma strain STOL (subgroup 16SrXII-A). (G, H) Comparison between FISH (G) and DAPI (H) staining in the same section of periwinkle infected with FDp: phytoplasma signal overlaps in the same phloem cells (arrows), while DAPI also stains nuclei (arrowhead).
222
D. Bulgari et al. / Journal of Microbiological Methods 87 (2011) 220–223
results, FISH experiments, described above, were replicated five times on different periwinkle plants at 4 and 8 weeks after grafting. The R16(V)F1 probe specificity was tested in periwinkle plants infected with genetically different phytoplasmas. The fluorescent signals of both hybridized FAM-labeled and MB-labeled R16(V)F1 probes were visualized only in periwinkle plants infected with phytoplasma strains belonging to group 16SrV such as Ulw, ALY and FDp (Fig. 1A–D and Fig. 1 in Supplementary material). As expected, fluorescent signal was detected in the phloem tissues of diseased plants. Instead, no signals were visible in healthy periwinkle (Fig. 1E) and in periwinkles infected by (i) ‘Ca. Phytoplasma asteris’ strain SAY and (ii) CPh (subgroup 16SrI-C), (iii) ‘Ca. Phytoplasma mali’ strain AT (subgroup 16SrX-A), and (iv) stolbur phytoplasma strain STOL (subgroup 16SrXII-A) (e.g. Fig. 1F). Both FAM and MB dyes used for labeling oligonucleotide probes were suitable for in situ hybridization assay but they differed in stability and brightness. In particular, MB hybridization signal was fainter and more difficult to detect due the brightness of xylem auto-fluorescence (Fig. 1D). Thus, FAM was preferred in the subsequent co-localization experiment. To confirm the correct localization of FAM signal we stained some serial section also with DAPI. The double DAPI and FAM signals co-localized in the same phloem cells of 16SrV phytoplasma infected periwinkles. However, only DAPI signal was present in periwinkle infected with other phytoplasma strains not hybridized by FAM-labeled probe (Fig. 1G, H, and Fig. 1 in Supplementary material). In contrast with previous experiments using in situ hybridization with a biotin-labeled probe (ISH), that were unsuccessful in localizing phytoplasmas in periwinkle (Lherminier et al., 1999), this study demonstrated that the R16(V)F1 probe employed in FISH assays was able, not only to detect phytoplasmas, but also to recognize specifically phytoplasmas belonging to 16SrV group. However, our final goal was to set up a co-localization technique for phytoplasmas and endophytic bacteria in order to study their possible interaction in the infected plants. DAPI staining is not suitable for this since it stains
both phytoplasmas and other bacterial DNA (Kapuscinski, 1995; Oliveira et al., 2009). Moreover, bacteria colonize and reside mainly in the vascular system, so there is possible overlap with phytoplasma signals. To detect endophytic bacteria with FISH techniques we used a bacterial universal probe (pB-00542), labeled with different fluorochromes. Among them, Cy5 was shown to be an excellent reporter molecule for in situ hybridization analysis in plant leaf tissues confirming previously reported data (Bonfiglioli et al., 1996; Webb et al., 1999). The fluorescent signals of hybridized Cy5-labeled probe were detected in healthy (not shown) and FDp infected periwinkles (Fig. 2A), mostly in the xylem but also in the phloem tissues as scattered spots, suggesting a random distribution of these bacteria, without large accumulation in specific cells. In particular, in the infected periwinkles no intense labeling of sieve tubes and adjacent parenchyma cells was observed (Fig. 2A), as with phytoplasmal probe, indicating that pB0052 does not label phytoplasmal DNA. No signal was observed when the hybridization was carried out at the same time with both sense and antisense probe (Fig. 2B). Less clear cut results were obtained when the universal probe was labeled with ROX as the signal was almost indistinguishable from tissue auto-fluorescence, thus this fluorophore was not used in subsequent co-localization experiments. Co-localization assays, carried out with FAM labeled FDp and Cy5 labeled pB0052, highlighted cells infected with phytoplasmas or with other bacteria on the same section (Fig. 2C, D). Phytoplasmal probe was detected only in the phloem cells, mainly sieve tubes and adjacent companion and parenchyma cells, usually in large patches in individual cells, while universal bacterial probe produced scattered spots both in the xylem and phloem. No co-localization of the two probes was found in the same cell. Data obtained in the present study underline that FISH can be employed for the specific identification of phytoplasma taxonomic groups, subgroups and/or strains, besides for the localization of other bacteria. FISH could be therefore applied for studying phytoplasma colonization patterns, phytoplasma population dynamics in response to the environment, and phytoplasma–endophytes
A
B
C
D
Fig. 2. Fluorescence in situ hybridization (FISH) of phytoplasmas and/or endophytic bacteria in periwinkle shoots visualized by a video-confocal microscope (all bars = 50 μm). (A) FDp infected periwinkle hybridized with Cy5-labeled bacterial universal probe: scattered red spots are present in the xylem and in the adjacent phloem tissues indicating the presence of randomly distributed bacteria. (B) Labeling negative control hybridized with both sense plus antisense probe: no signal is observed (C) Flavescence dorée phytoplasma strain FD-D (FDp, subgroup 16SrV-D) infected periwinkle: co-localization of phytoplasmas with FAM-labeled probe (arrow) and bacteria with Cy5 probe (red spots); green signal is detected in the phloem tissue while red signal, specific for bacteria, is identified in xylem, phloem and parenchyma tissues. (D) A serial section as in C in which the confocal image is overlapped to bright field image to show the tissue localization of both probes; arrows indicate phytoplasma hybridization signal in the phloem.
D. Bulgari et al. / Journal of Microbiological Methods 87 (2011) 220–223
interactions, as previously reported for plant growth promoting rhizobacteria (PGPR) colonization patterns and antagonism mediated by endophytic bacteria (Oliveira et al., 2009; Iverson and Maier, 2009; Nabti et al., 2010). Supplementary materials related to this article can be found online at doi:10.1016/j.mimet.2011.08.001. Acknowledgments We gratefully thank Dr. Fabio Quaglino for critical reading of the manuscript and for his useful comments. This work was supported by University of Milan funding for research (PUR 2008). References Bertaccini, A., Duduk, B., 2009. Phytoplasma and phytoplasma diseases: a review of recent research. Phytopathol. Mediterr. 48, 355–378. Bonfiglioli, R.G., Webb, D.R., Symons, R.H., 1996. Tissue and intra-cellular distribution of coconut cadang cadang viroid and citrus exocortis viroid determined by in situ hybridization and confocal laser scanning and transmission electron microscopy. Plant J. 9, 457–465. Bulgari, D., Casati, P., Brusetti, L., Quaglino, F., Brasca, M., Daffonchio, D., Bianco, P.A., 2009. Endophytic bacterial diversity in grapevine (Vitis vinifera L.) leaves described by 16S rRNA gene sequence analysis and length heterogeneity-PCR. J. Microbiol. 47, 393–401. Gamalero, E., D'Amelio, R., Musso, C., Cantamessa, S., Pivato, B., D'Agostino, G., Duan, J., Bosco, D., Marzachì, C., Berta, G., 2010. Effects of Pseudomonas putida S1Pf1Rif against Chrysanthemum yellows phytoplasma nfection. Phytopathology 100, 805–813. Iverson, S.L., Maier, R.M., 2009. Effects of compost on colonization of roots of plants grown in metalliferous mine tailings, as examined by fluorescence in situ hybridization. Appl. Environ. Microbiol. 75, 842–847.
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Kapuscinski, J., 1995. DAPI: a DNA-specific fluorescent probe. Biotech. Histochem. 70, 220–233. Lee, I.-M., Gundersen-Rindal, D.E., Davis, R., Bartoszyk, I.M., 1998. Revised classification scheme of Phytoplasmas based on RFLP analyses of 16S rRNA and ribosomal protein gene sequences. Int. J. Syst. Bacteriol. 48, 1153–1169. Lee, I.-M., Davis, R.E., Gundersen-Rindal, D.E., 2000. Phytoplasma: phytopathogenic mollicutes. Annu. Rev. Microbiol. 54, 221–255. Lherminier, J., Bonfiglioli, R.G., Daire, X., Symons, R.H., Boudon-Padieu, E., 1999. Oligodeoxynucleotides as probes for in situ hybridization with transmission electron microscopy to specifically localize phytoplasma in plant cells. Mol. Cell. Probes 93, 41–47. Liu, W.T., Mirzabekov, A.D., Stahl, D.A., Stahl, D.A., 2001. Optimization of an oligonucleotide microchip for microbial identification studies: a non-equilibrium dissociation approach. Environ. Microbiol. 3, 619–629. Lugtenberg, B., Kamilova, F., 2009. Plant-growth-promoting rhizobacteria. Annu. Rev. Microbiol. 63, 541–556. Nabti, E., Sahnoune, M., Ghoul, M., Fischer, D., Hofmann, A., Rothballer, M., Schmid, M., Hartmann, A., 2010. Restoration of growth of durum wheat (Triticum durum var. waha) under saline conditions due to inoculation with the rhizosphere bacterium Azospirillum brasilense NH and extracts of the marine alga Ulva lactuca. J. Plant Growth Regul. 29, 6–22. Oliveira, A.L.M., Stoffels, M., Schmid, M., Reis, V.M., Baldani, J.I., Hartmann, A., 2009. Colonization of sugarcane plantlets by mixed inoculations with diazotrophic bacteria. Eur. J. Soil Biol. 45, 106–113. Seemuller, E., 1976. Investigations to demonstrate mycoplasmalike organisms in diseased plants by fluorescence microscopy. Acta Horticulturae 67, 109–112. Watt, M., Hugenholtz, P., White, R., Vinall, K., 2006. Numbers and locations of native bacteria on field-grown wheat roots quantified by fluorescence in situ hybridization (FISH). Environ. Microbiol. 8, 871–884. Webb, D.R., Bonfiglioli, R.G., Carraro, L., Osler, R., Symons, R.H., 1999. Oligonucleotides as hybridization probes to localize phytoplasmas in host plants and insect vectors. Phytopathology 89, 894–901.
Contents lists available at ScienceDirect
Journal of Microbiological Methods j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j m i c m e t h
Fluorescence in situ hybridization for phytoplasma and endophytic bacteria localization in plant tissues Daniela Bulgari a, Paola Casati a, Franco Faoro a, b,⁎ a b
Dipartimento di Produzione Vegetale, Università degli Studi di Milano, Via Celoria 2, 20133 Milano, Italy CNR, Istituto di Virologia Vegetale, Strada delle Cacce 73, 10135 Torino, Italy
a r t i c l e
i n f o
Article history: Received 5 July 2011 Received in revised form 31 July 2011 Accepted 1 August 2011 Available online 7 August 2011
a b s t r a c t In the present study, we developed a rapid and efficient fluorescence in situ hybridization assay (FISH) in nonembedded tissues of the model plant Catharanthus roseus for co-localizing phytoplasmas and endophytic bacteria, opening new perspectives for studying the interaction between these microorganisms. © 2011 Elsevier B.V. All rights reserved.
Keywords: Video-confocal microscope DNA probe Vibroslice Periwinkle
Phytoplasmas are obligate parasitic bacteria that cause economically relevant yield losses in different low- and high-value annual and perennial crops worldwide (Bertaccini and Duduk, 2009; Lee et al., 2000). Despite their relevance, research aimed at understanding phytoplasma–host interactions and at finding effective control strategies has been slow due to the inability of isolating these pathogens on culture media. A possible utilization of beneficial bacteria as candidates for developing biocontrol strategies of phytoplasmas has recently been proposed (Bulgari et al., 2009; Gamalero et al., 2010). These microorganisms may reduce disease severity directly by antibiosis, competition for nutrients and niches or indirectly by activating systemic resistance (Lugtenberg and Kamilova, 2009). In the case of phytoplasmas, it should be firstly necessary to investigate if endophytes may directly interact with phytoplasmas because of their possible co-localization in the same plant tissues. Fluorescence in situ hybridization (FISH) technique, performed by means of specific cDNA fluorescent probes, has been utilized to localize different bacterial species in plant tissues (Watt et al., 2006; Iverson and Maier, 2009; Nabti et al., 2010). However, only one attempt has been reported for localizing phytoplasmas (Italian Clover Phyllody, taxonomic group 16SrI) in host plants (Webb et al., 1999). In the present study, we set up a FISH assay able to specifically co-localize phytoplasmas of taxonomic group 16SrV and endophytic bacteria in the tissues of the model plant Catharanthus roseus L. (G. Don). Periwinkles (C. roseus L.) were inoculated by grafting with phytoplasma reference strains: ‘Ca. Phytoplasma asteris’ strains SAY (South American aster yellows, subgroup 16SrI-B) and CPh (Clover phyllody, ⁎ Corresponding author at: via Celoria, 2, 20133 Milano, Italy. Tel.: + 39 0250316786; fax: + 39 0250316781. E-mail address: [email protected] (F. Faoro). 0167-7012/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.mimet.2011.08.001
subgroup 16SrI-C), ‘Ca. Phytoplasma mali’ strain AT (Apple proliferation, subgroup 16SrX-A), stolbur phytoplasma strain STOL (subgroup 16SrXIIA), alder yellows phytoplasma strain ALY (subgroup 16SrV-C), ‘Ca. Phytoplasma ulmi’ strain Ulw (Elm witches broom, subgroup 16SrV-A) and Flavescence dorée phytoplasma strain FD-D (FDp) (subgroup 16SrVD, kindly provided by E. Boudon-Padieu, INRA Dijon, France). Shoots of periwinkle plants, inoculated by grafting with the above strains, were fixed in 0.25% glutaraldehyde and 4% paraformaldehyde in Piperazine-1,4-Bis-2- Ethanesulfonic acid (PIPES) and cut in 50– 70 μm slides with a vibroslice (World Precision Instruments, Germany) without the paraffin-embedding step. The elimination of this step did not influence the probe hybridization and tissue structure preservation and greatly speeded up specimen preparation. Fresh tissue sections were washed in cold PIPES and then permeated as described by Webb et al. (1999). R16(V)F1 probe 200 ng/ml, usually employed in PCR reaction as 16SrV group specific primer (Lee et al., 1998), was applied to the sections to localize phytoplasmas belonging to 16SrV group in the plant tissues (Lherminier et al., 1999). The probe was labeled with FAM (Primm, Milan Italy) or Marina Blue (MB) (Invitrogen, Milan Italy) at 5′ terminus. We tested these dyes emitting respectively at 518 nm and 459 nm to find out the appropriate wavelengths that interfere at least with tissue auto-fluorescence. Sections permeated through proteinase K treatment were incubated in hybridization buffer (40 mM PIPES, 0.1% PVP 10 K, 0.1% Ficoll 4000, 140 mM NaCl, pH 7.8) containing R16(V)F1 probe for four hours at 50 °C in a humid chamber. Sections were then rapidly washed in 2× SSC (1× SSC=150 mM NaCl and 15 mM tri-sodium citrate, pH 7.0) and in 1× SSC for 30 min, each at 42 °C, followed by washing in 0.2× SSC at room temperature for 30 min. Finally, sections were washed in 1× PBS, mounted in glycerol 70% and visualized by video-confocal microscope (Nikon, Italy) using the corresponding
D. Bulgari et al. / Journal of Microbiological Methods 87 (2011) 220–223
channels. As 4′,6-diamidino-2-phenylindole (DAPI), usually used for dsDNA detection, is also able to stain phytoplasmas in plant tissues (Seemuller, 1976), some serial sections were also treated with DAPI to confirm the co-localization of this staining with the probe. As FISH allows co-localization of diverse nucleic acid sequences by the use of different labeled probes, we incubated serial sections also with the bacterial universal probe pB-00542 (5′-GACGGGCGGTGTGTACA-3′, probeBase website http://www.microbial-ecology.de/probebasesearch.asp, Liu et al., 2001) 200 ng/ml for localizing endophytic bacteria. The universal
221
probe was labeled with a fluorophore emitting in the far-red (i.e. Cy5) and with Carboxy-X-Rhodamine (ROX) emitting at 602 nm (Primm). Bacterial universal probe was added together with R16(V)F1 to the hybridization buffer. Following hybridization and washing the detection of targeted bacteria and phytoplasmas were performed with at the video-confocal microscope. The antisense R16(V)F1 and pB-00542 probes were employed as negative controls to verify non specific binding; as a further control, we visualized the samples without probes to detect false positive signals and auto-fluorescence. To assess the reproducibility of obtained
A
B
C
D
E
F
G
H
Fig. 1. Fluorescence in situ hybridization (FISH) of periwinkle shoots using 16SrV phytoplasma specific probes visualized by a video-confocal microscope; in all images xylem is autofluorescent at each tested wavelength while red signal, when present, is due to chlorophyll emission (bars: in A, B, G, H = 100 μm; in C = 50 μm; in D = 70 μm; in E = 500 μm; in F = 200 μm). Green signal of hybridized FAM-labeled probe (arrows) is visible in periwinkle inoculated with: (A) ‘Ca. Phytoplasma ulmi’ strain Ulw, elm witches broom (subgroup 16SrV-A), (B) alder yellows phytoplasma strain ALY (subgroup 16SrV-C) and (C) Flavescence dorée phytoplasma strain FD-D (FDp, subgroup 16SrV-D). (D) Blue signal of hybridized Marina blue-labeled probe (arrows) is present in periwinkle shoots inoculated with Flavescence dorée phytoplasma strain FD-D (subgroup 16SrV-D). (E) No hybridization signal is visible in healthy periwinkle shoots and (F) in periwinkle inoculated with stolbur phytoplasma strain STOL (subgroup 16SrXII-A). (G, H) Comparison between FISH (G) and DAPI (H) staining in the same section of periwinkle infected with FDp: phytoplasma signal overlaps in the same phloem cells (arrows), while DAPI also stains nuclei (arrowhead).
222
D. Bulgari et al. / Journal of Microbiological Methods 87 (2011) 220–223
results, FISH experiments, described above, were replicated five times on different periwinkle plants at 4 and 8 weeks after grafting. The R16(V)F1 probe specificity was tested in periwinkle plants infected with genetically different phytoplasmas. The fluorescent signals of both hybridized FAM-labeled and MB-labeled R16(V)F1 probes were visualized only in periwinkle plants infected with phytoplasma strains belonging to group 16SrV such as Ulw, ALY and FDp (Fig. 1A–D and Fig. 1 in Supplementary material). As expected, fluorescent signal was detected in the phloem tissues of diseased plants. Instead, no signals were visible in healthy periwinkle (Fig. 1E) and in periwinkles infected by (i) ‘Ca. Phytoplasma asteris’ strain SAY and (ii) CPh (subgroup 16SrI-C), (iii) ‘Ca. Phytoplasma mali’ strain AT (subgroup 16SrX-A), and (iv) stolbur phytoplasma strain STOL (subgroup 16SrXII-A) (e.g. Fig. 1F). Both FAM and MB dyes used for labeling oligonucleotide probes were suitable for in situ hybridization assay but they differed in stability and brightness. In particular, MB hybridization signal was fainter and more difficult to detect due the brightness of xylem auto-fluorescence (Fig. 1D). Thus, FAM was preferred in the subsequent co-localization experiment. To confirm the correct localization of FAM signal we stained some serial section also with DAPI. The double DAPI and FAM signals co-localized in the same phloem cells of 16SrV phytoplasma infected periwinkles. However, only DAPI signal was present in periwinkle infected with other phytoplasma strains not hybridized by FAM-labeled probe (Fig. 1G, H, and Fig. 1 in Supplementary material). In contrast with previous experiments using in situ hybridization with a biotin-labeled probe (ISH), that were unsuccessful in localizing phytoplasmas in periwinkle (Lherminier et al., 1999), this study demonstrated that the R16(V)F1 probe employed in FISH assays was able, not only to detect phytoplasmas, but also to recognize specifically phytoplasmas belonging to 16SrV group. However, our final goal was to set up a co-localization technique for phytoplasmas and endophytic bacteria in order to study their possible interaction in the infected plants. DAPI staining is not suitable for this since it stains
both phytoplasmas and other bacterial DNA (Kapuscinski, 1995; Oliveira et al., 2009). Moreover, bacteria colonize and reside mainly in the vascular system, so there is possible overlap with phytoplasma signals. To detect endophytic bacteria with FISH techniques we used a bacterial universal probe (pB-00542), labeled with different fluorochromes. Among them, Cy5 was shown to be an excellent reporter molecule for in situ hybridization analysis in plant leaf tissues confirming previously reported data (Bonfiglioli et al., 1996; Webb et al., 1999). The fluorescent signals of hybridized Cy5-labeled probe were detected in healthy (not shown) and FDp infected periwinkles (Fig. 2A), mostly in the xylem but also in the phloem tissues as scattered spots, suggesting a random distribution of these bacteria, without large accumulation in specific cells. In particular, in the infected periwinkles no intense labeling of sieve tubes and adjacent parenchyma cells was observed (Fig. 2A), as with phytoplasmal probe, indicating that pB0052 does not label phytoplasmal DNA. No signal was observed when the hybridization was carried out at the same time with both sense and antisense probe (Fig. 2B). Less clear cut results were obtained when the universal probe was labeled with ROX as the signal was almost indistinguishable from tissue auto-fluorescence, thus this fluorophore was not used in subsequent co-localization experiments. Co-localization assays, carried out with FAM labeled FDp and Cy5 labeled pB0052, highlighted cells infected with phytoplasmas or with other bacteria on the same section (Fig. 2C, D). Phytoplasmal probe was detected only in the phloem cells, mainly sieve tubes and adjacent companion and parenchyma cells, usually in large patches in individual cells, while universal bacterial probe produced scattered spots both in the xylem and phloem. No co-localization of the two probes was found in the same cell. Data obtained in the present study underline that FISH can be employed for the specific identification of phytoplasma taxonomic groups, subgroups and/or strains, besides for the localization of other bacteria. FISH could be therefore applied for studying phytoplasma colonization patterns, phytoplasma population dynamics in response to the environment, and phytoplasma–endophytes
A
B
C
D
Fig. 2. Fluorescence in situ hybridization (FISH) of phytoplasmas and/or endophytic bacteria in periwinkle shoots visualized by a video-confocal microscope (all bars = 50 μm). (A) FDp infected periwinkle hybridized with Cy5-labeled bacterial universal probe: scattered red spots are present in the xylem and in the adjacent phloem tissues indicating the presence of randomly distributed bacteria. (B) Labeling negative control hybridized with both sense plus antisense probe: no signal is observed (C) Flavescence dorée phytoplasma strain FD-D (FDp, subgroup 16SrV-D) infected periwinkle: co-localization of phytoplasmas with FAM-labeled probe (arrow) and bacteria with Cy5 probe (red spots); green signal is detected in the phloem tissue while red signal, specific for bacteria, is identified in xylem, phloem and parenchyma tissues. (D) A serial section as in C in which the confocal image is overlapped to bright field image to show the tissue localization of both probes; arrows indicate phytoplasma hybridization signal in the phloem.
D. Bulgari et al. / Journal of Microbiological Methods 87 (2011) 220–223
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