Scientific Report Short Term Scientific Mission (STSM)

Transcription

1 Scientific Report Short Term Scientific Mission (STSM) COST Action F1103 Dr. Matthias Döring Host institution: AIT GmbH in Tulln an der Donau (Austria) Period: 15/07/2015 to 31/08/2015 Reference code: COST-STSM-ECOST-STSM-FA STSM Topic: Molecular-histological analyses of Rhabdocline pseudotsugae and bacterial endosymbionts in callus cultures and seeds of Douglas fir (i) Abstract The focus of this STSM was the molecular-microscopical detection of the latent phytopathogen Rhabdocline pseudotsugae and bacteria inside seeds and callus cultures of Douglas Fir. R. pseudotsugae is an ascomycetous phytopathogen of needles and it could be detected also as latent and symptomless inside seeds and in in vitro cultures of this plant species via PCR. During this STSM project plant material were fixated and labelled with microbial DNA-probes for FISH. Sections from seed tissues were made for histology and also detection of fungal DNA. The DNA-labeled sections from seeds and single cells from callus were analyzed via confocal laser scanning microscopy. (ii) Purpose of the STSM This STSM should confirm the PCR results for R. pseudotsugae inside seeds of Douglas Fir published by Morgenstern et al. (2013, 2014) and provide the proof of bacteria inside seeds of this tree species. The main goal of this STSM was to learn techniques for detection of endophytic microbes in plant tissues and to get an overview in this field. The supervisor of this STSM Dr. Stephane Compant from AIT in Tulln has many experiences in this modern detection technique. (iii) Description of the work carried out during the STSM Plant material: Seeds of Douglas Fir with different origins 8880 (West- and South Germany) and 8884 (Southwestern Germany) Different Douglas Fir clones (FB1, K12, E11-32) as callus material established from embryonal tissues 1

2 Fluorescence microscopy: Staining of bacterial DNA in plant tissue by SYTO9 staining according to manufacturer's instructions. Fluorescence in situ hybridization (FISH)-analyse: Probes for Rhabdocline were constructed via Stellaris Probe Designer Software and MathFish programme based on sequence data of NCBI-database in Internet. Following FISH-probes for bacteria were ALSO used: Probe names Accession numbers Targets References EUB338 EUB338II EUB338III NONEUB ALF1B BET42a GAM42a LGC HGC69a pb pb pb pb pb pb pb pb pb Most bacteria Planctomycetes Verrumicrobia Control probe Alphaproteobacteria Some Deltaproteobacteria Some Spirochetes Betaproteobacteria Gammaproteobacteria Firmicutes Actinobacteria Amann et al., 1990 Daims et al., 1999 Daims et al., 1999 Wallner et al., 1993 Manz et al., 1992 Manz et al., 1992 Manz et al., 1992 Küsel et al., 1999 Roller et al., 1994 Tab. 1: Probes used for fluorescence in situ hybridization of bacteria Fixation, lysozyme treatment, dehydration with different Ethanol concentrations, hybridization with probes (Tab. 1), posthybridization, washing and air drying of seed halves of Douglas Fir were carried out according Glassner et al. (2015). The samples were observed with a confocal microscope (Olympus Fluoview FV1000 with multiline laser FV5-Lamar-2 HeNe(G)laser FV10-LAHEG230-2), Imari software and reconstruct with Image J. (iv) Description of the main results obtained Microscopy of callus material: The DNA of endophytic bacteria could be detected especially in dead callus cells of all 3 Douglas Fir clones. Young and intact callus cells were colonized by no bacteria (Fig. 1a), very few single bacteria (Fig. 1b) or completely (Fig. 1c) Unique fungal structures could be not detected in callus cultures (data not shown). 2

3 Fig. 1. Callus cells with no (a), few (b) and many bacteria (arrows) (c), stained with Syto9 Microscopy of seeds: Bacteria, stained their DNA via Syto 9, were detected on and in seed coat tissues (Fig. 2a), in cotyledons (Fig. 2b) and hypocotyl-root axis (Fig. 2c). Seed coat Cotyledon Embryo hypocotyl-root axis A A B C Fig. 2: CSLM/SYTO9 of Douglas fir clone 8884 showing bacteria (arrows) in different tissues. The DOPE-FISH analyses of bacteria and Rhabdocline pseudotsugae in Douglas-Fir seeds had following results: All active living bacteria were detected on the outer surface and inside of seed coats of Douglas Fir (Fig. 3a). Many bacteria colonized the cotyledons (Fig. 3b) and partly the embryo hypocotyl-root axis (Fig. 3c). The hybridization of seed material with NONEUB-ATTO488 resulted in no specific detection of bacteria (Fig. 3d-f). Representatives of Firmicutes were detected intercellular and on the outer surface of seed coat (Fig. 3g). Few bacteria of it colonized the cotyledons (Fig. 3h) and hypocotyl-root axis (Fig. 3i). Actinobacteria were also detected especially in seed coat (Fig. 3j) and in few parts of cotyledons (Fig. 3k) and hypocotyl-root axis (Fig. 3l). Alphaproteobacteria colonized intracellular the seed coat (Fig. 4a) and were detected intracellular in cotyledons (Fig. 4b) and hypocotyl-root axis (Fig. 4c). Few Betaproteobacteria (Fig. 4d-f) and Gammproteobacteria (Fig. 4g-l) colonized the seed coat, cotyledons and hypocotyl-root axis. 3

4 The DOPE-FISH analyse for Rhabdocline pseudotsugae with designed FISH probe 5'- TGG GAG ATC TGC CCG CTA GG -3' resulted in no specific signal for this fungal phytopathogen. EUBmix-ATTO488 HGC69A-Cy5 EUBmix-ATTO488 LGC-Cy5 NONEUB-ATTO488 EUBmix-ATTO488 Seed coat Cotyledon Embryo hypocotyl-root axis AA B C D ED F I H G 8884eubseed3.bmp J K L Fig. 3: CSLM/DOPE-FISH of Douglas fir clone 8884 with probes targeting all bacteria, no bacteria, Firmicutes and Actinobacteria. Bacteria (arrows), specific bacteria (arrow heads). 4

5 EUBmix-ATTO488 EUBmix-ATTO488 EUBmix-ATTO488 BET42A-Cy5 GAM42A-Cy5 ALF1B-Cy5 Seed coat Cotyledon A D G B E H Embryo hypocotyl-root axis C F I Fig. 4: CSLM/DOPE-FISH of Douglas fir clone 8884 with probes targeting Alphaproteobacteria, Betaproteobacteria and Gammaproteobacteria. Bacteria (arrows), specific bacteria (arrow heads). (v) Future collaboration with host institution The AIT will perform a metagenome sequencing for the bacteriome of Douglas Fir seeds in order to publish the results of it together with results of STSM and bacterial isolation experiments. Otherwise INOQ GmbH could maybe also cooperate with Dr. Stephane Compant about the detection of Rhabdocline pseudotsuage of seeds of Douglas Fir in the range of a project about this phytopathogen and therefore to continue work of this STSM. (vi) Foreseen publications/articles resulting or to result from the STSM (if applicable) The AIT and I would like to publish the results about DOPE-FISH and SYTO9 staining of bacteria inside seeds of Douglas Fir beside of results about isolation of bacteria and metagenome sequencing in the journal FEMS Microbiology Ecology. 5

6 (vii) References Amann, R.I., Binder, B.J., Olson, R.J., Chisholm, S.W., Devereux, R., & Stahl, D.A. (1990) Combination of 16S rrna targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations. Appl. Environ. Microbiol. 56: Daims, H., Brühl, A., Amann, R., Schleifer, K.H., & Wagner, M. (1999) The domain-specific probe EUB338 is insufficient for the detection of all Bacteria: development and evaluation of a more comprehensive probe set. Syst. Appl. Microbiol. 22: Glassner, H., Zchori-Fein, E., Compant, S., Sessitsch, A., Katzir, N., Portnoy, V. & Yaron, S. (2015) Characterization of endophytic bacteria from cucurbit fruits with potential benefits to agriculture in melons (Cucumis melo L.) FEMS Microb. Ecol. 91: Küsel, K., Pinkart, H.C., Drake, H.L. & Devereux, R. (1999). Acetogenic and sulfate-reducing bacteria inhabiting the rhizoplane and deep cortex cells of the sea grass Halodule wrightii. Appl. Environ. Microbiol. 65: Manz, W., Amann, R., Ludwig, W., Wagner, M., & Schleifer, K.H. (1992) Phylogenetic oligodeoxynucleotide probes for the major subclasses of proteobacteria: problems and solutions. Syst. Appl. Microbiol. 15: Morgenstern, K., Döring, M. & Krabel, D. (2013) Rhabdocline needle cast investigations on various Douglas fir tissue types. Eur. J. Plant Pathol. 137: Morgenstern, K., Döring, M. & Krabel, D. (2014) Rhabdocline needle cast most recent findings of the occurrence of Rhabdocline pseudotsugae in Douglas-fir seeds. Botany 92: Roller, C., Wagner, M., Amann, R., Ludwig, W. & Schleifer, K.H. (1994). In situ probing of Gram-positive bacteria with high DNA G + C content using 23S rrna-targeted oligonucleotides. Microbiology 140: Wallner, G., Amann, R. & Beisker, W. (1993) Optimizing fluorescent in situ hybridization with rrna-targeted oligonucleotide probes for flow cytometric identification of microorganisms. Cytometry 14: