Development of endophytic bacterial inoculants possessing plant growth promotion traits for practical application in b

1. Richard Lally, Antonio Sergio Moreira, Paul Galbally, John Culhane, Nicholas Otiento, David Ryan, Kieran Germaine and David Dowling Development of endophytic bacterial inoculants possessing plant growth promotion traits for practical application in bio-energy plant species Institute of Technology Carlow, Ireland

2. Presentation Overview Introduction Field experiment Results Conclusion

3. Introduction • Richard Lally • Postgraduate researcher, PhD candidate • BSc in Biotechnology with Biopharmaceuticals EnviroCORE research department • Focus on • Environmental research techniques • Environmental issues and • Environmentally effective biotechnology innovation

4. My research focus Examination of the use of microorganisms for their use as live microbial biofertilisers How do bacterial endophytes colonise and promote growth ? What are the microorganisms doing to make capable plant colonisation? Are the microbes promoting growth at a field scale? Microorganisms and agriculture Exploitation of favourable traits for agricultural benefits

5. In 2012 - EnviroCORE IT Carlow was awarded SFI (Science foundation Ireland) TIDA ( Technology Innovation Development Feasibility Study) Funding provided a platform to perform a: Field trial to investigate the use of live microbial biofertilisers 2. Investigation into the bacterial gene sequences of the endophytes

6. Background to our endophytes Isolated from Miscanthusgiganteus • Three Pseudomonas fluorescensstrains • L321, L111 and L228 Why test in the field? • Plant growth promoting traits • Siderophore production, Phosphate solubilisation, Plant hormone regulation compounds including ACC deaminase and plant hormone IAA production • Strains can colonise plant roots and rhrizoshere • Displayed numerous times in green house trials in various plant species • Have enhanced plant growth in green house trials • Shown in various experiments to enhance different aspects of plant growth

7. Choice of crop Brassicanapus – Oilseed rape • Economically important crop in Ireland • Used for bio-fuel production and fuel substitution • Used in culinary oil production • Prior experiments showed that the microorganisms colonise and significantly promote plant biomass in greenhouse trials

8. The field trial Three field trials First trial unsuccessful trial; uncompleted Malones site examination of “Compass” variety; completed Knockbeg site examination of “Castille” variety; completed All treatments were applied to each trial by spray application after germination Treatments: L321 L111 L228 F113 Master mix (MM) Control Master mix contained the 3 endophytes including that of 7 other EnviroCORE strains

9. Trial 1: Uncompleted Used a seed coating incorporating our strains Three spray applications after germination Trial 2: Malones trial Trail 3: Knockbeg Trial Laboratory based coating formulation designed in IT Carlow followed by one spray application

11. Tracking microorganisms in the field An effort was made to detect the microorganisms in the field We could not use plating techniques as we did not use genetically modified microorganisms Attempted to track F113 using molecular methods PCR amplification using sequence characterised amplification region markers developed by Von Feltenet al., 2010 Unsuccessful as primers were amplifying regions of similar bacteria in the field

12. Results The overall seed and oil yield was not impacted as a result of the biofertiliser application The “Castille” trial found there was no positive impact as a result of the treatment biofertilisers However Continuous sampling from “Compass” trial showed a significant increase in stem and leaf biomass at full growth

13. Seed yield from “Compass” and “Castille” Compass seed yield Error bars represent the standard error of the mean Castille seed yield No significant increase in seed yield

14. “Compass” dry stem and leaf biomass *Significant increase over control

15. Conclusions The bacterial inoculants F113 and master mix increased the stem and leaf biomass of Brassicanapusvariety “Compass” Strain specific molecular markers are ineffective in the detection of Pseudomonas F113 wild type strain in the field The inoculantsmay be increasing stem development but not contributing to the overall yield Three spray strategy may be more effective than a seed coating followed by a signal spray treatment A variety effect can not be ruled out as a possible colonisation effect factor

16. Future Work Effect of endophytes on stem yielding crops Stages of colonisation Data mining using bacterial annotations Molecular interaction between plants and bacteria Examination of reduced chemical treatments under biofertilisers

17. Acknowledgements • This research is funded by Science Foundation Ireland and Enterprise Ireland under the Technology Innovation Development Award (TIDA), ITC Postgraduate Scholarship Scheme and IoTI (Institute of Technology Ireland) Postgraduate Scholarship Initiative Thank you for listening