The Inherited Microbiome of Oak

The Fruit Microbiome: A New Frontier The inherited microbiome of oak

2

Ahmed Abdelfattah 1 Postdoctoral Fellow Department of Ecology, Environment and Plant Sciences 3 Stockholm University Plants, with no exception thus far, are associated with an abundant collection of microbes. 1876 Pasteur

The tissues of healthy plants are sterile (Pasteur 1876).

Louis Pasteur 1882 -1895 1887 Galippe

• Isolated bacteria from various plants parts.

• He suggested that those microbes originate from the soil.

Marie Louis Victor Galippe (1848-1922). A pioneer on microbes colonizing plants and their soil origin. 1888 Hellriegel and Wilfarth

Discovered the symbiotic association between Nitrogen fixing bacteria and legumes.

Hermann Hellriegel Hermann Wilfarth (1831-1895) (1853-1904)

The first indication of microbial endophytism.

Hellriegel and Wilfarth “divided-root experiment” with pea plants. 1904 Hiltner

• Coined the term rhizosphere to describe the area in close proximity to the plant roots which interactions between bacteria and legumes roots occurs the most

• He also suggested that the quality of plant products may be dependent on the composition of the root microflora Lorenz Hiltner (1862- 1923), Soil Bacteriologist and Professor of Agronomy at the Technical College of Munich, coined the term "rhizosphere" and emphasized the critical role of microbial activities in this root zone in relation to the nutrition and general health of plants 1956 Jakoba Ruinen DNA sequencing timeline and milestones

Carl Richard Woese 1928-2012

Kary Mullis 1944-2019

Timeline showing advances in microbial communities studies from Leeuwenhoek to NGS

(Ottman et al., 2012; Yarza et al., 2014). Frederick Sanger 1918-2013 The plant microbiome • Growth promoters e.g. (Cytokinin and Gibberellins)

• Carbon, nitrogen, and phosphorus fixation

• Disease tolerance through induced Growth + resistance or by producing inhibitory compounds

• Tolerance to abiotic stresses Water availability • Drought: Increasing water availability Induce resistance • Cold: heat production • Detoxification Production of pathogen • UV tolerance (Pseudomonas biofilm) inhibitory compounds Hots genotype Hots genotype is one of the main determinants of the microbial community composition.

Species 2 Species 2

Species 1 Species 1 e.g. PCA Correlation between plant phylogeny and microbial community composition. Is soil the source of all plant microbes?

Microbiome Selection Could Spur Next-Generation Plant Breeding Strategies (Gopal and Gupta 2016) Can seed help the continuity of the plant microbes? Studies on seed microbial community Plant References Herbaceous and woody species Mundt and Hinkle 1976 Soybean Kremer 1987 Broadleaf weed species Kremer 1987 Maize Dunleavy 1989 Bean Pleban et al. 1995 Cauliflower Pleban et al. 1995 Wild mustard Pleban et al. 1995 Rice Mukhopadhyay et al. 1996 Rapeseed Granér et al. 2003 Wheat Coombs and Franco 2003 Sugar beet Dent et al. 2004 Norway spruce Cankar et al. 2005 Coffee Vega et al. 2005 Garden pea Smerda et al. 2005 Strawberry Kukkurainen et al. 2005 Grasses Ikeda et al. 2006 Eucalyptus Ferreira et al. 2008 Tobacco Mastretta et al. 2009 Cactus Puente et al. 2009 Ash Donnarumma et al. 2010 Grape vine Compant et al. 2011 Pumpkin Fürnkranz et al. 2012 Peanut Sobolev et al. 2013 Thale cress Truyens et al. 2013 Tomato Xu et al. 2014 Hologenome theory of evolution (Ilana Zilber-Rosenberg & Eugene Rosenberg 2008) The holobiont (host with its endocellular and extracellular microbiome) can function as a distinct biological entity. Hologenome

(1) All animals and plants establish symbiotic relationships with microorganisms. 1- This principle was based on the obligate or extreme symbionts (Mitochondria, (2) Symbiotic microorganisms are Chloroplast, Wolbachia, Mycorrhiza) transmitted between generations. 2- Until now, there is no actual evidence for inheritance in plants. (3) The association between host and symbionts affects the fitness of the holobiont within its environment. (4) Changing either the host genome or the microbiome results in variations in the holobionts . Main findings • Most seeds contain one species of fungus • Pollen endophytes can be transmitted to ripe seeds and leaves

Limitations 1- The work was based on PDA-isolation 2- Roots were not analyzed Parent

Main findings 1- Substantial contributions of both or either Hybrid parent in the shaping of the bacterial seed endophytes of the offspring. Limitations 1- Used Terminal-Restriction Fragment Length Polymorphism (T-RFLP) 2- They used seeds from seed bank and didn’t track their transmission to offspring (seedlings) Main findings A significant proportion of the mother microbial community is transmitted to the daughters

Ground-ivy

Limitations 1- A clonal plant was used as plant model 2- The experiment was carried out in a greenhouse To prove microbial inheritance (in theory) we need:

• Mature Seed, endophytes, preferably embryonic • Eliminate the risk of contamination from the surrounding environment • Avoid seeds that had contact with soil • Harsh surface sterilization • Grow plants in sterile soil (substrate) • Grow plants in sterile air • Avoid above- and below-ground cross talk Surface-sterilized acorns 8% NaClO 30 min.

40 Acorns 40 Acorns Planted in microcosms

Pericarps Embryos

Amplicon sequencing ITS rDNA Illumina MiSeq V3 Main findings Most abundant fungal phyla Rare phyla

100% 0.0035%

90% 0.0030% 80%

70% 0.0025%

60% 0.0020% 50% 0.0015%

40% Relative Relative abundance% 30% Relative abundance% 0.0010%

20% 0.0005% 10%

0% 0.0000% Embryo Pericarp Root Leaf Embryo Pericarp Root Leaf Ascomycota Basidiomycota unidentified Glomeromycota Mucoromycota • Embryos were dominated by • Glomus, an arbuscular mycorrhiza Basidiomycetes fungi (AMF) in embryos and roots • While they were absent in pericarps Main findings

Roots

• Leave resembled the fungal community of embryo in both structure and composition Leaves

Embryo • Roots and pericarps seemed to have a distinct community. Pericarps

Embryo Leaves Roots Pericarps

PCoA plot of Beta diversity (Bray Curtis index) Main findings

• Most abundant taxa were shaped between all samples • No bottleneck effect is clear • Unique taxa were in embryos and pericarps • Unique taxa in leaves and roots? • Pericarp contributed equally to roots and phyllosphere community • Embryo had very little contribution to the roots • Explains why embryos and leave had a similar community

27%

32%

6%

33% Remarks

• Seed microbiome represents the source and initial microbiota to the plant root and phyllosphere • Studying seed microbiome can provide an explanation for the genotype effect and the continuity of the plant microbiome • The presence/absence and identity of the primary symbiont in each seed affects the survival of the host in the vulnerable stages of seed dispersal, germination, emergence and young growth (Primary Symbiont Hypothesis) • Maternal transmission is universal in kingdom animalia but it has to be yet proved in plant • While our understanding of the seed microbiota has lagged far behind that of the rhizosphere and phyllosphere, many advances are now being made Remarks Apple-Biome The origin, distribution, and dissemination of the apple microbiome https://applebiome.com/ Thank you for you attention!

Questions?

Contact me at [email protected]

Follow me at @AhmedAb60567245 https://applebiome.com/