Summer School on Advanced BiotechnologySion 3-6 September 2017

From plant microbiota to plant probiotics

Anna Maria PugliaLaboratory of Molecular Microbiologyand Biotechnology

STEBICEF

Microorganisms are the most abundantorganisms on the whole planet

Eukaryotes do not live alone

Plant microbiota influences:

biomass accumulation

metabolite production

drought tolerance

flowering time

resistance to pathogens

Shade et al.Current Opinion in Microbiology 2017,37.15-22

Healthy plants are associated with their microorganisms by metabolic co-operation and exchange of signals,hormons and nutrients.Diseases are characterized by a microbial dysbioses

Bacterial phylaActinobacteria

Bacteroidetes and Firmicutes

ProteobacteriaBulgarelli et al. (2013). Annu. Rev. Plant Biol. 64, 807–838

PLANT MICROBIOTA

Metagenomicanalysis

(pyrosequencing)

Culture dependent method

Strain isolation and

16S rDNA PCR

Identification of seed

microbiota

Surface-sterilized

seeds

Culture independent

method

Fluorescence in situ hybridization (FISH)

DNA sequencing& Bioinformatic

analysis

Identification and characterization of the seed microbiota

The seed microbiota of Anadenanthera colubrina

Alibrandi et al. Plant and Soil 2017

Anadenantheracolubrina

Methylobacteria spp.

Staphylococcus spp.

Culture-dependent method

Methylobacterium Phylogenetic Analysis

G2_6

Masterthesis

II

Methylobacteria spp.

Staphylococcus spp.

Staphylococcus Phylogenetic AnalysisMasterthesis

II

Methylobacteria spp.

Staphylococcus spp.

Next Generation Sequence Technology analysis (NGS) of metagenomic DNAby pyrosequencing of 16S rDNA

Phylogenetic analysis of A. colubrina seed microbiota from bacterial V3–V5 16S rRNA gene sequencing

0

10

20

30

40

50

60

70

80

90

Relative Abundance

Seed G Seed B

Next Generation Sequence Technology analysis (NGS) of metagenomic DNAby pyrosequencing of 16S rDNA

Fluorescence in situ hybridization–confocal laser scanning microscopy (FISH-CLSM) analysis

pa = parenchyma of the seed coat hg = hourglass-cellsb = bacteria

Fluorescence in situ hybridization –confocal laser scanning microscopy (FISH-CLSM) analysis

vt = vascular tissues of the tracheid barb = bacteria

Stress Test

PGP Test

(Plant GrowthPromoting)

• Saline Stress (NaCl)• Water Stress (PEG)

• Organic and inorganic P solubilization• Nitrogen Fixation• ACC-Deaminase production• IAA production• Siderophore production

Staphylococcus spp

Methylobacterium spp

Microbiological

Assay• Agar-Diffusion Test

Microbiological Assay

tester strain : Kokuria rizophila

Methylobacterium spp. Staphylococcus epidermidis

tester strain : Escherichia coli

Stress Test

Isolate Nearest relativeANaCl (%) PEG (%)

2,5 5 7,5 15

G2_2 Methylobacterium variabile - - - -

G2_6 Methylobacterium extorquens - - - +

G2_7 Methylobacterium hispanicum - - - -

B6_7 Methylobacterium rhodesianum - - - -

G2_1G

2_3G

2_5

G2_9

Staphylococcus epidermidis + + + +

B6_5

G_10Staphylococcus aureus + + + +

B6_1

G2_4Staphylococcus pasteuri + + + +

A Closest relative species 16S rDNA sequence databaseNaCl = sodium chloride; PEG=polietilenglicol

PGP Test

Isolate Nearest relative CaP* AlP FeP PHY NF ACC IAA SID

G2_2 Methylobacterium variabile+ - + + + + - -

G2_6 Methylobacterium extorquens- - + + + + - -

G2_7 Methylobacterium hispanicum+ - + + + + - -

B6_7 Methylobacterium rhodesianum+ - + + + + - -

G2_1;G2_3G

2_5;G2_9Staphylococcus epidermidis

+ - + - - - + +

B6_5;G_10 Staphylococcus aureus + - + - - - + +

B6_1;G2_4 Staphylococcus pasteuri + - + - - - + +

*CaP= Calcium phosphate mobilization; AlP= Aluminium phosphate mobilization; FeP= Iron phosphate mobilization;PHY= Phytate mobilization; NF= Nitrogen Fixation; ACC= 1-aminocyclopropane-1-carboxylate -Deaminase production; IAA= Indole-Acetic-Acid production; SID= Siderophore production

All Staphylococcus epidermidis tested produce antimicrobial substances

All Staphylococcus strains tested are resistant to salt and water stresses, solubilize CaP and FeP and produce auxin and siderophores

Methylobacterium extorquens is resistant to water stress and solubilizesFeP, while others Methylobacteria solubilize CaP, FeP and Phytate

All Methylobacterium strains tested produce ACC-Deaminase and fix nitrogen

The seeds of Anadenantheracolubrina host a complex microbialcommunity.

The results of stress-and PGP-tests indicate that this community. might play a role in promoting/protectingthe plant, thussuggesting that the plant actively selectsthe beneficial bacteriafor the nextgeneration via seedtransmission.

Truyens et al.2014 Environmental microbiology reports

Citrus limon

B1

B2

B3

The isolated bacterial

strains belong to the genus

Staphylococcus

Bacterial and fungal strains isolated by culture-dependent method from surface–sterilized seeds

F1

F2

F3

F4

Aspergillus

Quambalaria

Aspergillus

Efibula

The isolated fungal strains belonged to

genera:

F1

F3

F2

F4

Bacterial and fungal strains isolated by culture-dependent method from surface–sterilized seeds

Fluorescence in situ hybridization –confocal laser scanning microscopy (FISH-CLSM)analysis of C. limon seed sections

Parenchyma

Tegument

Vasculartissues

Rhodamine red–labelleduniversal

FISH–probes EUB338MIX

Cy5–labelled Firmicutes–specificprobe LGC354MIX

Merge

Fluorescence in situ hybridization –confocal laser scanning microscopy (FISH-CLSM)analysis of seed embryo region

bacterial colonization of seed embryo region

Ryan et al 2008 FEMS Microbiology Letters .

Biofertilizers

Towards next-generation agriculture

Seed microbiotaas probiotic

Schlaeppi and Bulgarelli 2015,MPMI28,212-217

Laboratory of Molecular Microbiology and Biotechnology .

University of Palermo:

Giuseppe GalloTeresa FaddettaPasquale AlibrandiPaolo CinàIvana La MendolaCarla MaraglianoMarco Grande

Carlotta De Filippo,Francesco StratiComputational Biology Research UnitFondazione Edmund Mach, Trento

Massimiliano CardinaleInstitute of Applied Microbiology,Justus-LiebigUniversity of Giessen, Germany

Mirella Ciaccio CNR, Palermo

Marta De VianaBanco de Germoplasma de Especies nativas, Instituto de Ecologia Universidad National de Salta, UNSA , Argentina

Francesco MercatiLoredana AbbateIstituto di Bioscienze e Biorisorse (IBBR) CNR, Palermo

Acknowledgments

• A Bio fertilizer (also bio-fertilizer) is a substance which contains living microorganisms which, when applied to seeds, plant surfaces, or soil, colonize the rhizosphere or the interior of the plant and promotes growth by increasing the supply or availability of primary nutrients to the host plant.[1] Bio-fertilizers add nutrients through the natural processes of nitrogen fixation, solubilizing phosphorus, and stimulating plant growth through the synthesis of growth-promoting substances. Bio-fertilizers can be expected to reduce the use of chemical fertilizers and pesticides. The microorganisms in bio-fertilizers restore the soil's natural nutrient cycle and build soil organic matter. Through the use of bio-fertilizers, healthy plants can be grown, while enhancing the sustainability and the health of the soil. Since they play several roles, a preferred scientific term for such beneficial bacteria is "plant-growth promoting rhizobacteria" (PGPR). Therefore, they are extremely advantageous in enriching soil fertility and fulfilling plant nutrient requirements by supplying the organic nutrients through microorganism and their byproducts. Hence, bio-fertilizers do not contain any chemicals which are harmful to the living soil.

• Bio-fertilizers provide "eco-friendly" organic agro-input. Bio-fertilizers such as Rhizobium, Azotobacter, Azospirilium and blue green algae (BGA) have been in use a long time. Rhizobiuminoculant is used for leguminous crops. Azotobacter can be used with crops like wheat, maize, mustard, cotton, potato and other vegetable crops. Azospirillum inoculations are recommended mainly for sorghum, millets, maize, sugarcane and wheat. Blue green algae belonging to a general cyanobacteria genus, Nostoc or Anabaena or Tolypothrix or Aulosira, fix atmospheric nitrogen and are used as inoculations for paddy crop grown both under upland and low-land conditions. Anabaena in association with water fern Azolla contributes nitrogen up to 60 kg/ha/season and also enriches soils with organic matter.[2][3]

• Other types of bacteria, so-called phosphate-solubilizing bacteria, such as Pantoea agglomerans strain P5 or Pseudomonas putida strain P13,[4] are able to solubilize the insoluble phosphate from organic and inorganic phosphate sources.[5] In fact, due to

bacterial colonization of seedembryo region.Bacterial cells (red), stained with the Cy3-labelled EUB338MIX FISH probe, weredetected in and around the plant vasculartissues (blue, autofluorescence).

Fluorescence in situ hybridization –confocal laser scanning microscopy (FISH-CLSM)analysis of C. limon seed sections

Fluorescence in situ hybridization –confocal laser scanning microscopy (FISH-CLSM)analysis of C. limon seed sections

Parenchyma

Tegument

Vasculartissues

Rhodamine red–labelleduniversal

FISH–probes EUB338MIX

Cy5–labelled Firmicutes–specificprobe LGC354MIX Merge

CLSM-images showing FISH–stained bacteria inside C. limon seed cryosections. Bacterialcells (red), stained with the Rhodamine red-labelled universal FISH-probe EUB338MIX,were detected inside seeds, at different sites. FISH signals (green) were obtained withCy5–labelled Firmicutes–specific probe LGC354MIX. Arrows indicate Firmicutes cells withstaphylococci-compatible morphology.