Development of endophytic bacterial inoculants possessing plant growth

promotion traits for practical application in bio-energy plant species

Institute of Technology Carlow, Ireland

Richard Lally, Antonio Sergio Moreira, Paul Galbally, John Culhane, Nicholas Otiento, David Ryan, Kieran Germaine and

David Dowling

Presentation Overview

Introduction

Field experiment

Results

Conclusion

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

My research focus Examination of the use of microorganisms for their use as live

microbial biofertilisers

Microorganisms and agriculture

Exploitation of favourable traits for agricultural benefits

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?

In 2012 -EnviroCORE IT Carlow was awarded SFI (Science foundation Ireland) TIDA ( Technology Innovation Development Feasibility Study)

Funding provided a platform to perform a:

1. Field trial to investigate the use of live microbial biofertilisers

2. Investigation into the bacterial gene sequences of the endophytes

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

Background to our endophytes Isolated from Miscanthus giganteus

Three Pseudomonas fluorescens strains•L321, L111 and L228

Why test in the field?

Choice of crop Brassica napus – 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

The field trial

Three field trials1. First trial unsuccessful trial; uncompleted 2. Malones site examination of “Compass” variety; completed 3. Knockbeg site examination of “Castille” variety; completed

Treatments:L321L111L228F113Master mix (MM)Control

All treatments were applied to each trial by spray application after germination

Master mix contained the 3 endophytes including that of 7 other EnviroCORE strains

Trial 1: Uncompleted Used a seed coating incorporating our strains

Trial 2: Malones trial Three spray applications after germination

Trail 3: Knockbeg Trial Laboratory based coating formulation designed in IT Carlow followed by one spray application

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 Felten et al., 2010

Unsuccessful as primers were amplifying regions of similar bacteria in the field

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

Seed yield from “Compass” and “Castille”

F113 L228 L321 L111 MM Control0

500

1000

1500

2000

2500

Yiel

d Kg

/ha

Castille seed yield F113 L228 L111 L321 MM Control

2500

2600

2700

2800

2900

3000

3100

Yeild

Kg/

ha

Compass seed yield

No significant increase in seed yield

Error bars represent the standard error of the mean

“Compass” dry stem and leaf biomass

F113 L228 L111 L321 MM CONTROL0

5

10

15

20

25

Treatment Strain

Wei

ght

(g)

* *

*Significant increase over control

Conclusions

The bacterial inoculants F113 and master mix increased the stem and leaf biomass of Brassica napus variety “Compass”

Strain specific molecular markers are ineffective in the detection of Pseudomonas F113 wild type strain in the field

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

The inoculants may be increasing stem development but not contributing to the overall yield

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

AcknowledgementsThis 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

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