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A Multifaceted Systems Approach Identifies Polymicrobial Cause of AOD Stem Necrosis

A Multifaceted Systems Approach Identifies Polymicrobial Cause of AOD Stem Necrosis

Acute Oak Decline presentation

Forest Research

October 03, 2017
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  1. A Multifaceted Systems Approach Identifies Polymicrobial
    Cause of AOD Stem Necrosis
    S. Denman
    J. Doonan, M. Broberg, S. Plummer, A. Griffiths, K. Scarlett, M. Kaczmarek, J. Foster, J.E. McDonald
    Programme 2 Annual Report-back Meeting January 2017, Roslin

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  2. 11/09/2017
    2
    AOD – distinctive condition on
    native oak

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  3. 11/09/2017
    3
    Internal symptoms

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  4. Isolation and culture studies
    • Revealed novel bacterial species
    • Led to description of 2 new genera and 13 new species
    • Shift in the microbiome
    of diseased vs healthy trees
    • Enterobacteriaceae dominated
    diseased vs Pseudomonadaceae
    in healthy trees
    • 3 spp - high occurrence
    • G. quercinecans (Gram –ve, Enterobacteriaceae) - closely related to genus
    Serratia
    • B. goodwinii (Gram –ve, Enterobacteriaceae) - most closely related to B.
    rubrifaciens, – pathogen of walnut
    • Rahnella victoriana
    • Almost exclusively in symptomatic trees

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  5. Many organisms are not tractable to culture
    Confirmation of detection and abundance of Bg, R. vic, Gq in field sampled oak
    metagenomes vs extremely low presence or absence in healthy metagenomes
    Symptomatic trees (AOD lesions)
    Healthy
    trees
    Martin
    Broberg

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  6. 11/09/2017
    6
    Impact of stem lesions – carbohydrate and water allocation

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  7. Decline Diseases - concept
    11/09/2017
    7
    Decline is the interaction of interchangeable, specifically ordered abiotic and biotic
    factors to produce a general deterioration, often ending in death of trees (Manion
    and Lachance, 1991)
    A Decline is a progressive deterioration, often ending in death, for which single
    exclusive causes have not been isolated (Sinclair, 1967)
    A Decline results from an interacting set of factors (Manion, 1981)
    Concept of Declines is still evolving (Manion, 1991)
    Decline: Arises from factors that interact sequentially or simultaneously and have a cumulative effect
    over time. Certain factors may have key roles. Predisposition/altered host condition probably essential.

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  8. Test for necrotic capability of organisms associated with the AOD lesions
    Koch’s Postulates
    Model: 1 pathogen = 1 disease
    1. Isolation of the putative pathogen (the micro-organism occurs in every case
    of the disease)
    2.The putative pathogen is not found in healthy organisms
    3.Once in pure culture, the putative pathogen is reintroduced into a healthy
    host and causes disease anew
    4.The micro-organism is re-isolated from the experimentally infected host
    Problems with the above model in polymicrobial, secondary disease
    pathosystems (Decline – diseases)
    1. More than a single putative pathogen is isolated, microbiome effect not
    taken into account
    2. The putative pathogen(s) may or may not be found in healthy hosts (at very
    low levels)
    3. Re-introducing pathogen into a healthy host to create disease anew –
    Premise of Decline diseases is that the host is predisposed to infection
    Cause of stem necrosis

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  9. Microbiome Effects - Questions to address in disease development investigations - Gilbert et al. 2016:
    1. Taxonomic composition of the microbiome
    • Have all participants been detected?
    • Can they be cultured?
    2. Selection of species for testing
    • Consistently occurring micro-organisms on lesion margin (KP1&2)
    • Micro-organisms that occur in a benign state - transform behaviour under certain circumstances?
    3. Testing
    • Relative abundance of different species
    • Sequence of presence and function of micro-organisms
    • Collective gene effect – could the same functional genes be supplied by more than one organism ?
    • Specific lineages or metabolic pathways
    4. Host metabolome – the chemicals produced by the host
    • What does predisposition look like?
    • Effects of functional bacterial genes on host – host response?
    • Host response – what effect on microbial communities?

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  10. 11/09/2017
    10
    Sequence whole genomes of isolated key putative pathogens
    Transcriptomes of field infected material and inoculated
    material
    Proteomes of field infected material and inoculated material

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  11. Genomics
    Hypothesis: Gq and Bg are necrotic pathogens of oak
    Aim: Identify genes or gene families causing pathogenesis and/or necrosis
    Pathogenicity genes:
    • Plant Cell-Wall Degrading Enzymes (PCWDEs)
    • Secretion systems
    • Oxidative stress
    • Iron acquisition
    • Quorum sensing
    • Effectors
    Virulence factors:
    Many eg:
    Iron acquisition enzyme
    James
    Doonan

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  12. Detection of Bg, Gq & Rv in oak metagenomes and metatranscriptomes

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  13. Gallic acid Tannic acid
    Carboxymethyl cell.
    Cellobiose
    Starch
    Nutrient agar
    Avicel
    Xylan
    Lignin
    Substrate Brenneria
    goodwinii
    Gibbsiella
    quercinecan
    s
    Starch ✔ ✔
    Cellobiose ✔ ✔
    CMC ✔ ✔
    Avicel ✗ ✗
    Xylan ✗ (1/27)
    Lignin ✗ ✗
    Gallic acid ✔ ✔
    Tannic acid ✗ ✔
    Phenotypic tests: Plant Cell-Wall Degrading Enzymes

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  14. NB
    Phloem
    NB
    Sapwood
    NB only
    Gq only x3
    Bg only x3
    Gq & Bg x3
    Transcriptome analysis of Bg and Gq grown on oak tissue
    1. How do Bg and Gq respond to growth on oak tissue?
    2. Do Bg and Gq interact?
    • Growth on nutrient broth + sapwood or phloem
    • Analysis of gene expression

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  15. Axenic Gq
    Below Co-culture Bg+Gq
    Growth on oak tissue and in co-culture with Bg alters gene expression of
    Gq
    Sapwood Phloem
    2h
    6h
    Sapwood Phloem
    Axenic Bg
    2h
    6h

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  16. Field sampled oak microbiome analysis:
    functional composition (metagenomics)
    • 67-95% of predicted genes in AOD lesions
    were bacterial
    • 0.6-6% in non-symptomatic samples were
    bacterial
    • 627 genes common to all symptomatic
    samples (not in non-symptomatic trees)
    • Genes were associated with bacterial
    virulence and plant defence

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  17. Metatranscriptome analysis

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  18. Causality: Koch’s Postulates is the standard
    Overall aims were to test the necrogenic capability of the bacterial species singly and in combination,
    with or without the addition of Agrilus biguttatus
    Hypotheses
    1. Enterobacterial species consistently isolated from AOD symptomatic oak can cause necrosis of oak
    stem tissue
    2. Combinations of bacterial species cause more severe tissue necrosis (reflected in larger lesions),
    than individual species only,
    3. The interaction between Agrilus larvae (derived from eggs) and bacteria, leads to the development
    of AOD symptoms.
    Ojectives
    1. To determine necrogenic ability of bacterial species singly and in combination,
    2. To determine necrogenic ability of Agrilus eggs on their own and in combination with bacteria,
    3. To reproduce on logs, the symptoms and signs that characterise AOD.

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  19. Three experiments were carried out over 3 consecutive years as testing could only be done annually
    when beetle eggs were available. Two of the three trials were carried out in growth chambers and the
    third trial in the field, where young plantation oak (25 years old) were used instead of logs.
    Treatment
    No.
    Bacterial Treatments – Applied with and without Agrilus biguttatus eggs
    1 Brenneria goodwinii
    2 Gibbsiella quercinecans
    3 Lonsdalea quercina britanica
    4 Rahnella victoriana
    5 Rahnella variigena
    6 Water (control)
    7 Egg only
    8 Erwinia billingiae
    9 B. goodwinii + G. quercinecans
    10 B. goodwinii + G. quercinecans + L. quercina britainca
    11 B. goodwinii + G. quercinecans + R. victoriana
    12 B. goodwinii + G. quercinecans + R. variigena
    13 B. goodwinii + G. quercinecans + L. quercina britainca + R. victoriana + R. variigena

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  22. 11/09/2017
    22
    0
    10
    20
    30
    40
    50
    60
    70
    80
    90
    100
    0
    50
    100
    150
    200
    250
    300
    Percentage data points out of total
    uncontaminated inoculation points used (%)
    Mean Lesion Area mm^2
    Treatment
    Pathogenicity tests: bacteria only and bacteria plus egg
    treatments lesion area and % usable data points
    Area % data points used p=0.08 p<0.007
    • Agrilus increased lesion size and bacteria were re-isolated along gallery length – spread in tree
    • Significantly larger lesions with Bg+Gq and with Gq+egg; Bg+egg; Bg=Gq+egg and Lqb+egg
    • Co-inoculation of Bg+Gq greatly increased back isolation of Bg from 17% to 66% - synergistic effect

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  23. 11/09/2017
    23

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  24. Undiluted
    Bg FRB
    186
    Undiluted
    R.vic BRK
    18a
    Bg +
    R.vic 1:1
    ratio
    R.vic + Bg
    1:10 ratio
    Bg + Rvic
    1:10 ratio
    Hypersensitivity reaction tests

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  25. 0
    1
    2
    3
    4
    5
    6
    7
    8
    9
    10
    Bg (undiluted) Rvic (undiluted) Bg + Rvic (1:1) Bg + Rvic (1:10) Rvic + Bg (1:10)
    HR (MM)
    INOCULATION COMBINATIONS
    HR of Bg vs Rvic vs Bg + Rvic
    Series1 Series2 Series3

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  26. Microbiome Effects - Questions to address in disease development investigations - Gilbert et al. 2016:
    1. Taxonomic composition of the microbiome
    • Have all participants been detected?
    • Can they be cultured?
    2. Selection of species for testing
    • Consistently occurring micro-organisms on lesion margin (KP1&2)
    • Micro-organisms that occur in a benign state - transform behaviour under certain circumstances ?
    • Relative abundance of different species *
    • Sequence of presence and function of micro-organisms *
    • Collective gene effect – could the same functional genes be supplied by more than one organism ?
    • Specific lineages or metabolic pathways
    3. Host metabolome – the set of chemicals produced by the host
    • What does predisposition look like? *
    • Effects of functional bacterial genes on host – host response? *
    • Host response – what effect on microbial communities? *

    View Slide

  27. Conclusions
    • Bg, Gq and Rv are consistently detected as abundant members of the cultivable and
    ‘omics’-based AOD microbiome.
    • Integrated multi-omics of AOD lesions revealed the dominance of a bacterial
    community and concomitant detection of host defence responses.
    • Bg, Gq and Rv have genome-encoded virulence genes found in canonical plant
    pathogens belonging to the Enterobacteriaceae
    • Genes and transcripts of Bg, Gq and Rv are abundant in the lesion microbiome and
    experiments confirm functionality.
    • Log inoculation tests confirm the ability of Bg and Gq to produce lesions and
    demonstrate synergistic effects.
    • Microbiome analysis of AOD lesions and artificial inoculation has provided compelling
    evidence of a polymicrobial disease with by Bg, Gq and Rv as key agents of disease.
    • We have adopted a systems approach to analysis of the stem bleeds and have
    developed a template for analysis of complex disease symptoms, which is still likely to
    develop especially with regard to taking account of host predisposition.

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  28. Thank you to our funders for their
    generous support and for all the
    technical support from TSU as well as
    help from landowners and managers
    Working hard to future-proof the resilience of oak

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