Assembly and Role of Microbes on Above-Ground Parts of Plants. Steven Lindow

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1 Assembly and Role of Microbes on Above-Ground Parts of Plants Steven Lindow University of California, Berkeley Department of Plant and Microbial Biology

2 Vein Epiphytic Bacteria: cells/cm 2 Glandular trichome Fungal hypha Bacterial aggregate 20 um From: Brandl & Lindow, Appl. Environ. Microbiol. 69: (2003)

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4 Culturable foliar bacteria reflect total community assessed by pyrosequencing of community DNA The taxa found by culture-independent methods on romaine lettuce are often culturable Plant-associated bacteria are more culturable than those in soil and water etc Romaine Soil Water Cultureindependent Cultured bacteria

5 The study of epiphytes has been driven by studies of plant disease- Plant pathogens exist as epiphytes on healthy plants before inciting disease 100% 0 Bacterial Populations (Log cells/leaf)

6 Bacterial Ice Nucleation

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8 Bacteria (10 7 cells/g) Carbon availability limits bacterial numbers on plants Composition of Bean Leaf Exudates ug/leaf Sucrose 5.3 Glucose 1.9 Fructose 1.6 Galactose 0.2 Amino Acids 0.7

9 Methylotrophs are very abundant on leaves: methanol a major carbon compound released from plants C. Marx - Harvard

10 Bacterial exploitation of fungal mutualism to obtain limiting nutrients Sugars Sugars N-Formyl Loline ` Neotyphodium

11 Ultraviolet radiation an obvious feature of plant surfaces- Selects for pigmented bacteria and those proficient in DNA repair and dessication tolerance

12 Who is not there? Most bacteria do not survive on leaves Population sizes of epiphytic bacteria and non-plantassociated bacteria on plants after moist and subsequent dry incubation conditions Strain Log cells/g fr.wt. Moist Moist/dry P. syringae 22R 6.9 a 5.0 a P. syringae 821R 6.9 a 5.1 a P. syringae 714R 6.6ab 5.2 a Aeromonas hydrophila 6.6 ab 3.6 b E. coli 6.5 ab 3.5 b Salmonella typhimurium 6.4 ab 3.6 b Rhizobium meliloti 3.0 d 2.1 c Non-epiphytes died upon drying From: O Brien & Lindow, Phytopathology 79: (1989)

13 Proteome of phyllosphere of rice is very distinct from that of rhizosphere but similar in communities on different plant species at a given site Soil bacteria are not major source of leaf colonists Clover Knief et al. ISME J. 6:1378 (2012) Delmotte et al. PNAS 106:16428 (2009)

14 About 60% of genes of Pseudomonas syringae were differentially expressed in planta compared to minimal media - and half of these were altered specifically in epiphytic vs endophytic sites

15 Majority of bacteria live in large aggregates Objective on [1] leaves [2] [3] [4]

16 IAA production by epiphytic bacteria : a story of resource conversion and plant modification

17 Most bacteria on plants produce IAA

18 Epiphytic IAA producers can alter auxin-mediated responses in plants Fruit Russet Flower and fruit abscission

19 cwinv Diffusion/ Leakage Diffusion/ Leakage Working Model: Bacterial IAA in the phyllosphere Glucose Fructose Pa299R Sucrose Leaf Cuticle Glucose Fructose Glucose Fructose Sucrose Sucrose Mesophyll cell SUT Apoplast Phloem cell 1. Microbial Key features IAA of promotes the model: plant invertase activity 2. Invertase Auxin-dependent cleaves apoplastic changes sucrose, in epiphytic yielding hexose sugars that diffuse to leaf surface and apoplast Increased fructose availability 3. Preferential Decreased use of glucose sucrose and availability fructose at the low concentrations Resource found conversion leaves makes and adaptive apoplast sense

20 Biosurfactants prominent on leaves Syringafactin

21 Syringafactin contributes to nutrient acquisition: alters cuticular permeablility Effects on Plant Fitness? Measure movement of 3 H 2 O Diffusion across waxy cuticle Apply surfactant Syringafactin added Isolated cuticle 3 H 2 O Method: Schreiber et. al., 2005

22 Many epiphytes can consume waxes deplete cuticular waxes accelerate plant senescence?

23 Considerable evidence for role of epiphytes in defense against pathogens

24 Many epiphytes produce acyl homoserine lactone signal molecules stimulators 12% produce 3-oxo hexanoyl HSL Alter behavior of neighboring AHL-perceiving bacteria AHL deficient P. syringae indicator strain Response of plant to AHLs: Activation of innate defense?

25 Continuous immigration and subsequent selection and growth determine eventual communities on mature leaves

26 Bacterial numbers are usually very low on young plants: Lack of suitable colonists since young plants can support high numbers? Bean Soybean Pumpkin Tomato

27 Limitation of immigration of suitable colonists restricts bacterial population sizes on young plant tissue such as flowers Pseudomonas fluorescens inoculated flowers uninoculated flowers

28 Aerial plant surfaces are an open microbiological habitat Immigration plays an important role in structuring epiphytic communities

29 High leaf populations Differences in airborne microflora and subsequent bacterial-induced fruit russeting in a replicated pear field plot having different cover crop plant species Cover crop species Bacteria Deposited Fruit Russet (cells/petri dish/hr) (% of surface) Mixed weeds 28.0 ab 9.5 ab Annual Ryegrass 23.3 ab 13.0 a Mixed Grasses 15.0 bcd 7.9 b Perennial Clovers 18.0 cd 7.8 b Berseem Clover 12.7 cd 7.8 b Bare soil 11.0 cd 6.3 b Pea+Vetch 7.8 d 5.7 b Burr Clover 6.3 d 6.3 b

30 Dynamics of bacterial populations on some plants like citrus is driven by immigration of bacteria from nearby plants Immigration can be a quantitatively important process Total Bacteria Ice + Bacteria Bacterial populations on plants in and near citrus orchards Plant species Log cells/g Total Ice+ Henbit 8.1 a 4.8 a Annual Bluegrass 7.4 ab 3.0 bc Chickweed 7.1 b 0.7 de Malva 6.8 b 4.4 a Mustard 6.6 b 1.9 cd Navel Orange 5.3 c 0.4 de Immigration rate = 1000/100 cm 2 /day From Lindow & Andersen, AEM 62: (1996)

31 Orange trees adjacent to grass pasture Biogeography of cropping system can influence microbial ecology of leaves Crop grown in isolation few immigrant bacteria present nearby Sources of immigrant bacteria nearby

32 Bacterial populations on citrus and numbers of airborne bacteria related to proximity to vegetation with high epiphytic bacterial populations Vegetation Distance from Bacteria deposited Leaf Bacteria nearby edge of orchard per petri plate/hr (Log cells/g) (trees) NO 0 33 a 4.8 a 7 29 a 4.7 a a 4.7 a a 4.9 a a 4.7 a YES a 6.1 a 7 97 a 5.7 a b 5.8 a b 5.5 ab b 5.3 b From Lindow & Andersen, AEM 62: (1996)

33 Large influence of neighboring plant species on bacterial communities of tall fescue within a local area Selection of epiphytic communities apparently occurring from different local metacommunities While distinct communities on given plant species: Communities on fescue influenced by neighboring plants

34 PNAS 108:14288 (2011) Bacterial species assemblages on algae distinct from seawater but also differed greatly between algal samples High similarity in functional genes on different algal samples Competitive lottery model of community assembly Combination of niche- or guild-based selection and random components Species with similar trophic or other ecological properties are able to occupy the same niche within an ecosystem and the particular species that occupies a particular space is then determined by stochastic recruitment

35 How open or substitutable are surface communities on plants and are there successional changes with time? Determining drivers of community composition and function on plants Needed: Large-scale metagenomic analysis & phylogeny of inhabitants and their functional genes on many individual plants at many Locations/contexts at several different times Ascertain: Stochasticity/ ideosyncrasy of assembly vs selection Stability and degree to which community composition can be modified

36 To what extent is there a precedence effect or role of keystone species on foliar colonization by bacteria? Resident bacteria on leaves increase success of immigration of other bacteria

37 Can microbial communities on plants be changed at other times than early in plant development? Is the effect of epiphytes on plants greater or less than that of root colonists or endophytes? Are modifications of communities persistent and predictable? Are there different rules of assembly for perennial/evergreen plants and annual plants? How do we measure effects of epiphytes on plant phenotypes? Are effects of microflora on plants population-size dependent? eg. Any effects of rare taxa? To what extent are effects of microflora on plant phenologically dependent? Can we link a given taxa or their traits with a plant phenotype?