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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)

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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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

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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.