Bacterial endophytes of flowers, fruits and seeds of grapevine:

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1 Bacterial endophytes of flowers, fruits and seeds of grapevine: identification, localization, differences with other plant parts and sources of colonization Dr. Stéphane Compant Bioresources Unit, Health & Environment Department, AIT Austrian Institute of Technology GmbH, 3430 Tulln, Austria

2 Bacterial endophytes? Where? Bryophytae, Pteridophytae, Gymnospermae Angiospermae Contain various bacterial taxa Endophytic bacteria having PGPB/ biocontrol activities

3 Bacterial endophytes? Where? Angiospermae Stem and leaves Various bacterial taxa analyzed, communities described by cultivable /metagenomic analysis Roots AEM 2005a SBB 2010 Endophytes analyzed in different organs such as roots, stems and leaves of many plants

4 Bacterial endophytes? Where? Colonization of plants by endophytes SBB 2010

5 Bacterial endophytes from plant vegetative organs Endophytes in roots, stem and leaves Some niches of colonization, diversities, relation to plant parts as well as some functions studied Adv. Agronomy in press Endophytes and vegetative plant parts

6 Endophytic bacteria from flowers, fruits and seeds? But for plant reproductive organs, is there any endophytes? diversities, relation to plant parts as well as some functions???? less studies than for vegetative organs but NOW more and more studies Endophytes and plant reproductive organs (flowers, fruits and seeds inside fruits)?

7 Endophytic bacteria from flowers, fruits and seeds? If there is some endophytes: from where they derive? Sources and niches??? rhizosphere, caulosphere, laimosphere, anthosphere, or carposphere??? Endophytes and plant reproductive organs (flowers, fruits and seeds inside fruits)?

8 Endophytic bacteria from flowers, fruits and seeds? Endophytic bacteria and plant reproductive organs of grapevine? Relation to other plant parts? Niches? Sources???? How is the diversity, what are the niches of colonization, functions and relation of these microbes to other plant parts?

9 Endophytes from flowers, fruits and seeds Grapevine plants were used Illmitz, Burgenland, Helmut Gangl wines, cv. Zweigelt

10 Cultivable communities from flowers, fruits and seeds and other plant parts CFU 16S ARDRA HaeIII and Rsa I used after PCR of 16S Microbial Ecol 2011 Endophytic bacteria in flowers, fruits and seeds: low populations and diversities but presence

11 ARDRA profils of isolates from vegetative and reproductive organs F: flowers or at flowering B: berries or berry harvest S: stalk E: endorhiza RH: Rhizosphere Pu: Pulp of berries Se: seeds Microbial Ecol 2011 Different ARDRA groups

12 ARDRA profils of isolates from vegetative and reproductive organs Microbial Ecol 2011 Different ARDRA groups, some are present in vegetative and reproductive organs

13 IGS and 16S rdna sequences of plant reproductive organs isolates Microbial Ecol 2011 Endophytic bacteria in flowers, fruits and seeds: some bacterial strains detected

14 Endophytic bacteria from flowers, pulp and seeds Are naturally colonized by endophytes (without injuries) Low population, low diversity but exist (with mainly Firmicutes) Some strains are present in vegetative and reproductive organs. Some others are only present in plant reproductive organs

15 Visualization by Fluorescence in situ hybridization of endophytes inside plant reproductive organs Fluorescence in situ hybridization Probes names EUB338 EUB338II EUB338III NONEUB LGC REX72 GAM42a Pae997 HGC69A Accession numbers pb pb pb pb pb pb pb pb pb Target Most bacteria Plantomycetes Verrucomicrobia Control probe complementary to EUB 338 Firmicutes (low G+C Gram-positive bacteria) Bacillus spp. Gammaproteobacteria Pseudomonas spp.' Actinobacteria (high G+C Gram-positive bacteria) References Amann et al., 1990 Daims et al., 1999 Daims et al., 1999 Wallner et al., 1993 Küsel et al., 1999 Felske et al., 1998 Manz et al., 1992 Amann et al., 1996 Roller et al., 1994 Endophytic bacteria in flowers, fruits and seeds: proof and niches?

16 Niches of colonization of bacterial endophytes All bacteria Specific taxa Microbial Ecol 2011 Different niches of colonization and presence of some main taxa inside flowers

17 Niches of colonization of bacterial endophytes All bacteria Specific taxa Microbial Ecol 2011 Different niches of colonization and presence of Firmicutes, in particular Bacillus spp. inside pulp and seeds

18 Sources of colonization of bacterial endophytes from plant reproductive organs? Anthosphere (carposphere? ) origins Sources of endophytes of plant reproductive organs? Stomata (ovary and pedicel) Microbial Ecol 2011 Source(s) of colonization? Anthosphere

19 Niches of colonization of bacterial endophytes Bacillus spp. Anthosphere (carposphere? ) and soil origins Stomata (ovary and pedicel) All bacteria Specific taxa Source(s) of colonization, in particular of Bacillus spp.?

20 Conclusions

21 Conclusions Are naturally colonized by endophytes (without injuries) Low population, low diversity but exist (with mainly Firmicutes) Proof and origin(s) demonstrated: Anthosphere and Rhizosphere Putative function(s)? and possibility of use?

22 ACKNOWLEDGEMENTS Dr. Angela Sessitsch Anton Grahsl Dr. Birgit Mitter Dr. Juan G. Colli Helmut Gangl AIT Austrian Intitute of Technology GmbH (Austria), Centro de Investigación Científica de Yucatán (Mexico), Bundesamt für Weinbau (Austria)

23 ACKNOWLEDGEMENTS Grazie