1 Scientific Report – Short Term Scientific Mission (STSM) Dr. Matthias Döring COST Action F1103 Host institution: AIT GmbH in Tulln an der Donau (Austria) Period: 15/07/2015 to 31/08/2015 Reference code: COST-STSM-ECOST-STSM-FA1103-150715-060164 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
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Scientific Report – Short Term Scientific Mission (STSM)
Dr. Matthias Döring
COST Action F1103
Host institution: AIT GmbH in Tulln an der Donau (Austria)
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
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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:
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).
Probe names
EUB338 EUB338II EUB338III NONEUB
ALF1B
BET42a GAM42a
LGC HGC69a
Accession numbers
pB-00189 pB-00160 pB-00161 pB-00243 pB-00017
pB-00034 pB-00174 pB-01040 pB-00182
References
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
Targets
Most bacteria
Planctomycetes Verrumicrobia Control probe
Alphaproteobacteria Some Deltaproteobacteria
Some Spirochetes Betaproteobacteria
Gammaproteobacteria Firmicutes
Actinobacteria
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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).
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.
A
15µm
Fig. 2: CSLM/SYTO9 of Douglas fir clone 8884 showing bacteria (arrows) in different tissues.
A B C 15µm 15µm 15µm
Seed coat Cotyledon Embryo hypocotyl-root axis
Fig. 1. Callus cells with no (a), few (b) and many bacteria (arrows) (c), stained with Syto9
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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.
A B C
8884eubseed3.bmp G
EU
Bm
ix-A
TT
O4
88
E
UB
mix
-AT
TO
48
8
LGC
-Cy
5
D
EU
Bm
ix-A
TT
O4
88
HG
C6
9A
-Cy
5
NO
NE
UB
-AT
TO
48
8
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).
A C
D E F
H I
J K L
15µm 15µm 15µm
15µm 15µm 15µm
15µm
15µm 15µm 15µm
15µm 15µm
Seed coat Cotyledon Embryo hypocotyl-root axis
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(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.
EU
Bm
ix-A
TT
O4
88
ALF
1B
-Cy
5
EU
Bm
ix-A
TT
O4
88
GA
M4
2A
-Cy
5
Fig. 4: CSLM/DOPE-FISH of Douglas fir clone 8884 with probes targeting Alphaproteobacteria, Betaproteobacteria and Gammaproteobacteria. Bacteria (arrows), specific bacteria (arrow heads).
EU
Bm
ix-A
TT
O4
88
BE
T4
2A
-Cy
5
A B C
D E F
G H I 15µm 15µm 15µm
15µm 15µm 15µm
15µm 15µm 15µm
Seed coat Cotyledon Embryo hypocotyl-root axis
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(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. 56:1919–25. 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: 434-444. 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: 1-13. 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: 5117-5123. Manz, W., Amann, R., Ludwig, W., Wagner, M., & Schleifer, K.H. (1992) Phylogenetic oligo-deoxynucleotide probes for the major subclasses of proteobacteria: problems and solutions. Syst. Appl. Microbiol. 15: 593-600. Morgenstern, K., Döring, M. & Krabel, D. (2013) Rhabdocline needle cast—investigations on various Douglas fir tissue types. Eur. J. Plant Pathol. 137: 495-505.
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: 465-469.
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: 2849-2858. Wallner, G., Amann, R. & Beisker, W. (1993) Optimizing fluorescent in situ hybridization with rRNA-targeted oligonucleotide probes for flow cytometric identification of microorganisms. Cytometry 14:136–43.