The Role of Seed Endophytes on Plant
Microbiome Composition
5th Dutch Seed Symposium
4 October 2016, Leo van Overbeek
Introduction
Is there is a microbial balance in seeds
and in plants (plant microbiome)?
What is that balance?
What is it telling us?
What can we do with it?
2
Developments in Biology
Microbiome
Linnaeus
classification
Darwin
Mendel
Watson & Crick
evolution
genetics
DNA
3
The Microbiome; contemporary events
Informatics/ Mathematical modelling
Stability
(micro) Biology
‘Chaos’
Resilience
‘Noise’
(unexplained) variation
Pivoting points
‘Archetypes’
(Motives, laws,
higher level order)
Vast datasets
Complex systems
Multidisciplinary approaches
4
Topics
 What is a microbiome?
● What is the
Microbiome of seed
(plant)?
 How to analyse microbial
(seed) communities?
 How to manipulate the
seed microbiome?
 In summary
5
What is a Microbiome?
6
Definition...?
Metagenome
..the micro-organisms in a particular environment..
.. the combined genetic material of the microorganisms in a particular environment..
.. collective genome of all organisms in a particular environment..
.. collective genome of indigenous microbes..
‘A characteristic microbial community occupying a reasonably
well defined habitat which has distinct physico-chemical properties’
John Wipps, 1998
7
Next Generation Sequencing
Classification of ‘life’
8
Tip of the Iceberg
Dominant (most abundant)
taxons
Detection Limit
Taxons lower in Abundance
Relevance for the system?
9
The Human Microbiome
10
Microbiome dynamics
11
Human Microbiome ‘State of Science’
Characterization
Yes, to a certain extent
Interpretation
Sometimes; start to
understand
Manipulation (controlled)
12
No!
What is the Microbiome of a Seed?
Change in microbial seed communities over generations?
13
Vertical and Horizontal transmission of
micro-organisms in seeds
seed
seed
seed
vertical transmission
Seed (core) microbiome
Etc.
0
1st
2nd
External (horizontal) acquisition of new species
(accessory microbiome)
14
Generation
Definitions
Permanent in seeds over many generations
Obligate endophytes?
Required for plant growth & development
Plant (endo) symbionts?
Single habitat (small genome size)?
Core
accessory
15
Not always present
Acquired (Environment)
Required for the plant?
Context dependence?
Horizontally transmitted?
Multiple habitats (large genome size)
Endophyte: Bacterium of fungus living at least a
part of their life cycle inside plants without causing
(visible) harm to the plant.
Thus NOT a phytopathogen!
Rhizobium & Mycorrhiza often excluded from this
definition
What is the life cycle of a Plant?
Plant growth
Reproduction
Gamete fusion
Seedling emergence
Embryo formation
Propagation
Embryo growth
Seed filling
Survival/ hibernation
Energy Production
Seed ripening/ drying
Polymer hydration
Imbibition
16
Microbial Communities Associated with Seed
Systemic spread
Internalization
Plant colonization
Crossing Maternal tissue/
Embryo barrier
Vertically transmitted
Plant Microbiome
Incorporation in seed
Via seed/ other propagation
Material
Competition
Part of the Core
Microbiome?
Drought tolerant/
Silenced/ inactive/
DORMANT!
Growth/ Reproduction
Activation/ Resuscitation
17
Plant associated communities
Plant Beneficials: ‘the good’
Plant Pathogens: ‘the bad’
Human Pathogens ‘the ugly’
18
Serratia marcescens endophyte strain A2
Frankliniella occidentalis
bioassay 0165/direct contact females WFT
100
% mortality
80
control
60
A2 total culture
40
A2 supernatant
20
0
1
2
3
4
5
6
7
days after treatment
Tetranychus urticae
bioassay 0201 / A2 - direct spray adult spider mites
100
% mortality
80
control
60
40
20
0
1
2
3
4
5
6
days after treatment
19
7
Phylogenetic tree E. coli ‘plant’ isolates
Based on MLST, 6 loci: adh,fumC,gyrB,icd,mdh,recA
20
Three hypotheses for occurrence of
‘human pathogens’ in plants
I
II
III
Common ancestor
(diversification)
Invasion from elsewhere
21
Integral part of the
Holobiome
How to analyse Microbial (seed)
communities
22
How to analyse Microbial Communities in
Seeds?
Cultivation
DNA/ RNA based technologies
PCR amplicon fingerprinting
Metagenomics
Next Generation Sequencing/ Microbiome
23
Microbial Community Analysis
Microbial
community
Cultivation-dependent approaches
Cultivation-independent approaches
Direct cell visualization
Serology
DNA/ RNA extraction
Fatty acids/ Proteins/
enzymes etc.
Living cell extraction:
Colony growth
Enrichment
Always alive
Directed,
Subsequent experimentation (interaction with plants),
Relatively undirected (unbiased) approach
Near to full community approached
Holistic (‘effects of environmental changes’)
Genomics
Metagenomics
Strongly biased approach
Minor fraction of the community accessed (< 1%)
Dead or alive???
No subsequent experimentation possible
Studies are often descriptive
24
Metagenomic Analysis of Endophytes
PCR amplification step
No amplification required
25
Hardoim et al.,
2016
Chloroplast (cell organelle) separation from
Plant/Seed DNA
Plant/ Seed DNA
Optimization DNA
extraction technology
Chloroplast DNA
Microbial community
DNA
PCR ampification with specific primers
(e.g for bacteria PCR primer #799)
Chelius & Triplett, 2001. Microbiol. Ecol.
41: 252 – 263.
26
Next Generation Sequencing
Next Generation Sequencing Platforms
Life Sciences (Roche) 454 (2004) ,
Illumina Miseq/ Hiseq,
Thermo Fisher Ion torrent
Pacific Biosciences (Roche) PacBio
Fragment sizes
150 bp (454)
> 20 kb (PacBio)
27
accuracy
0.1 – 1%
Principle of Metagenomics
Metagenome
Extraction and sequencing
DNA fragment reads
28
Genomics versus metagenomics
Metagenome
Genome
scaffold
Genome assembly
Metagenome assembly
Closely resembling species, but also
DNA acquired via horizontal gene transfer
Events!
29
How to Manipulate the Seed Microbiome?
30
Seed treatments
31
Back ground fungal community in Pea seed
Without chlorine
Seed treatment
Without chlorine
With chlorine
With chlorine
A
55
62
B
57
61
32
%
84.8
93.2
Without disinfection
repetition
Ascochyta sp
Stemphylium sp.
Fusarium sp.
Epicoccum sp.
Alternaria sp.
Botrytis cin.
Penicillium sp.
Cladosporium sp.
Aspergillus sp.
Mucor
A
4
1
3
10
26
2
21
5
3
1
B
3
1
3
1
19
0
16
4
1
2
Out of 132 tested seeds
%
5.3
1.5
4.5
8.3
34.1
1.5
28.0
6.8
3.0
2.3
95.5% infection!
33
After disinfection with chlorine
repetition
Ascochyta sp
Stemphylium sp.
Fusarium sp.
Epicoccum sp.
Alternaria sp.
Botrytis cin.
Penicillium sp.
Cladosporium sp.
Aspergillus sp.
Mucor sp.
A
0
0
1
0
5
0
1
1
0
0
B
1
0
1
0
5
0
1
1
0
0
Out of 132 tested seeds
%
0.8
0.0
1.5
0.0
7.6
0.0
1.5
1.5
0.0
0.0
12.9% infection!
34
Advantages of a Microbe on/ in seed
First contact with seed radicle
Avoidance of microbial competition for free spaces; first come, first served
Does it make sense:
To be inside the seeds,
or to be attached to
the seed coat?
To be an endophyte,
or not?
To be incorporated
in a biofilm?
35
To be alone or mixed
with other microbes?
Role of Microbial Biofilm Formation in Plants
36
Van Overbeek & Saikkonen, 2016, Trends in Plant Science
How to get Microbes in/ on seeds?
Pericarp
Seed coat
Endosperm
Seed dressing
• Seed coating
• Encrusting
• Film coating
37
Prospects and pitfalls
Seed dressing
Endophyte application
Microbiome manipulation
In Principle applicable
Beneficial strain inside
the seed
Beneficial part of the
plant core microbiome
Microbe must be
tolerant to drought
stress.
Must be able to cross
the border between
parental and endosperm/
embryo tissue
Insufficient knowledge
Apply as integral part
of microbial consortium?
Shifts in plant microbial
communities observed
upon microbial inoculation
Is biofilm formation
on seed coat an option?
38
Examples
Seed coating with P. fluorescens
strain F113 for control of P. ultimatum
in onion.
Log Ec O104 CFU per
seed
In patented BioPolymer
Best survival of strain F113 tested was
at 4°C for 70 days (~ 107 CFU/ seed)
9
8.5
8
7.5
Log Ec O104
CFU per seed
7
2-3 Log-fold decline at 20°C!
6.5
6
0
O’Callaghan et al., 2006.
New Zealand Plant Protection Society
•
•
6
14
35
Over 6 weeks survival (100% E. coli
survival!)
Used for Field experimentation (treatment
#5)
39
E. coli O104 rhizosphere soil colonization
# positive plants (Log E. coli O104:H4 CFU/ g dry Rf soil)
Submergence 1h
5 (2.30)
3 (1.98)
2 (2.18)
0
1 (2.12)
1 (2.50)
1 (2.10)
4 (1.79)
0
Log 7.18 CFU/ seed
10 µl drop
Log 7.18 CFU/ seed
Biofilm,
48h 100% RAH
5 (1.98)
5 (2.51)
3 (2.12)
Log 7.30 CFU/ seed
Biofilm,
48h 36% RAH
5 (1.67)
2 (1.82)
5 (2.80)
1 (2.35)
5 (1.42)
0
1 (1.86)
0
4 (juvenile)
7 (flowering)
9 (pod formation)
Log 6.98 CFU/ seed
6 weeks 4°C
Log 8.49 CFU/ seed
Time (weeks)
40
13 (ripened pods
In summary
41
Overview...
 Unprecedented new opportunities for microbiome
(complex microbial communities) by Next Generation
Sequencing, in combination with other ‘omics
technologies’
● Studies with these novel technologies are still too
descriptive,
● Hard to design experimental studies using these
technologies
 Next challenge is to implement these technologies in
protocols for practical applications (industry, registration,
monitoring)
42
Plant Metagenomics
Where are we now in Science?
Stamp collecting
Descriptive
Who is there?
Functional interaction
with plants
What are they doing?
43
Plant Holobiome Concept
Other communities
Environment
(Soil)
Host Plant
Establishment,
Growth,
Metabolome/
proteome
Associated
Microbial community
Composition,
(inter-) activity
44
Challenges in seed Production?
Application of ‘seed microbiome’ knowledge
 What happens when we treat seeds?
● Disinfection
● Seed priming
● Seed pelleting/ encrusting (treatment with
fungicides)
● Produce seeds (same species/ cv.) in different
locations (soils)?
 Can we stably integrate ‘endophytes’ into seeds?
● Transfer to Next Generations of Plants
● Seed Production and Storage Survival
45
Successful application
Fungal endophyte ‘Neotyphodium coenophialum’ in Tall Fescue
Alkaloid Production
Deterring insects
Reduction in bird grazing
Airports!
Challenge is stable incorporation of the endophyte into grass seeds
All Phases in Seed Production, i.e.
• Harvesting,
• Drying, storage,
• Distribution
• Whole sale/ retail chain
46
Thanks for your
attention!
Questions?
47