Microbiota and neurologic diseases: potential effects of probiotics
From plant microbiota to plant probiotics - hevs.ch · From plant microbiota to plant probiotics...
Transcript of From plant microbiota to plant probiotics - hevs.ch · From plant microbiota to plant probiotics...
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.