Post on 02-May-2020
Samir Droby
Dept. Postharvest ScienceARO, The Volcani Center
Utilizing the Fruit Microbiome for Biocontrol of Postharvest Diseases
Michael Wisniewski
Appalachian Fruit Research Station, USDA-ARS
Fruit microbiome workshop , 2019
Major Market Drivers for Postharvest Biocontrol Products
❖ Food marketers/consumer demand – Low chemical residues or even chemical free
❖ Global biocontrol market is steadily growing
❖ Very strict legislation on pesticide residues in food
❖ Sustainability - more production with less input and less impact
Biological Control of Postharvest Pathogens
❖ Postharvest environment and disease etiology – conducive to microbial antagonists
❖ Controlled conditions
❖ Defined target
High prospects of success:
Washed Commodities Decay More Rapidly Than Unwashed Commodities
Washings From Citrus Surface
DilutedConcentrated
Isolating Antagonists from Fruit Surfacesthe Silver bullet approach
Single antagonist
Wilson, Wisniewski, Droby, Chalutz. 1993, Scienta Hort. 53: 1831-1889.
Candida sake CPA-1Liquid formulation
Pantovital
Pantoea agglomerans
Freeze-dried formulation
InovaCure
SHEMER
Developed jointly by ARO (Droby et al) and AgroGreen
Postharvest Biocontrol Products
Post-harvest diseases of apples, pears, citrus fruits and bananas.
Aureobasidium pullulans
Metschnikowia fructicola
Candida sake
Candida saitoana
Botector®
Product development was successful
Full commercial potential has not been realized yet
Pre and postharvest applications
Commercial Product for Managing Pre and Post-harvest Pathogens
Acquisition
EPA approval in the US (2018) ; EFSA approval 2019
AgroGreen
Expected product launching 2020 – trade name NOLI
Registration in Europe:
First phaseFrance, Italy, Belgium, Netherlands.
Second phaseGermany, Poland and others
Registration for:Soft fruit, stone fruit and grapes
NOLI
Developed jointly by ARO (Droby et al) and AgroGreen
Major Shortcoming of Postharvest Biocontrol Agents
Inconsistency in performance
Enhancement and broadening biocontrol activity
Extensive research activity
Additives Physical means
Combinations New formulations MOA
The one fruit-one microbe model
• Competition for limiting nutrients (sugars, Iron)• Competition for space (colonization)• Biofilm formation• Volatile and diffusible antimicrobials• Mycoparasitism (CW hydrolases)• ROS tolerance• ROS production• Induced resistance
Moving from Simplicity to Complexity
Tri-trophic interactions Quatro-trophic/multi-trophicinteractions
MOAs
Pre & Postharvest treatments
IS there a possibility that the application of a single microorganism can modify microbiota assembly on fruit surfaces – creating “healthy microbiome”?
Spadaro and Droby, 2015, Trends in Food Science and Technology
Postharvest Application of M. fructicola Modulate Microbial Diversity of Pink Lady Apples
Bacteria Fungi
Mf Untreated Cont.
Water Cont. Mf Untreated Cont.
Water Cont.
Mf Water cont. Untreated cont.
Beta Diversity Analysis of Microbial Composition of Apples (cv. Pink Lady) Treated with M. fructicola and Untreated Fruit
Bacteria Fungi
• Competition for limiting nutrients (sugars, Iron)• Competition for space (colonization)• Biofilm formation• Volatile and diffusible antimicrobials• Mycoparasitism (CW hydrolases)• ROS tolerance• ROS production• Induced resistance
Moving from Simplicity to Complexity
Tri-trophic interactions Quatro-trophic/multi-trophicinteractions
MOAs
Pre & Postharvest treatments
Spadaro and Droby, 2015, Trends in Food Science and Technology
Modulation of fruit surface microbiome?
What are the shifts taking place?
What are the interactions between the different microbial components?
The use of Probiotic Microbial Consortia for Biocontrol of Postharvest Pathogens
Target:Fruits (Both Tree Fruits and Soft Fruits)
Sources:Kefir
(Fermented Foods Containing a Consortia of Probiotic Organisms)
Near harvest application (3-5 days before harvest)
Scanning Electron Micrographs of kefir Grains
Mei, Jun; Guo, Qizhen; Wu, Yan; Li, Yunfei (2014): PLOS ONE, https://doi.org/10.1371/journal.pone.0111648.g002.
G. F. Friques et al., ,2015 Chronic administration of the probiotic kefir improves the endothelial function in spontaneously hypertensive rats. Journal of Translational Medicine. 13 .10.1186s/12967-015-0759-7.
Diversity and Relative Abundance of Bacteria And Yeasts genera and Species in kefir
FG = Fresh grains
MA = Milk activated
Bacteria Yeasts FungiBacteria
Culture dependent Amplicon based
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Control of postharvest Decay of Strawberries by Near-harvest Application of Yeasts and Bacterial Consortium
70%84%68%64%63% 68%
Commercial chemicalcontrol
Biocontrol consortium
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Experiment 1 Experiment 2
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Control 2B4+B40+K198
**p<0.01
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01234567
Control 2B4+B40+K198
Bacteria
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Yeasts
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After storage
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Filamentous fungi
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The US of bacterial and yeast Consortium for the Control of Postharvest Decay of Raspberry
Near-Harvest Application
The Functional Fruit Microbiome Network
❑ Degradation of aromatic compounds and sugars
❑ Production of resistance inducing compounds/peptides
❑ Production of antimicrobial substances
❑ Production of surfactants and biofilms
The holobiont concept
Moving from the single antagonist approach to the holobiont concept
Studying Fruit Microbiomes
Sample collection
Community DNA extraction
Amplicon sequencing
genera, species??, number, abundance, composition
“Who is there”?
Metagenome sequencing
Community function
“What can they do”?
Community RNA, protein, metabolites extraction
Meta –Transcriptome sequencing
Meta-proteome sequencing
Metabolome analysis
Community function
“What are they doing?
Global Apple Fruit Microbiome Project
Silvana Vero
Neus Teixido &Rosario Torres
Davide Spadaro
Andreas Pulman
Okan Ozkaya
Achour Amiri
Michael Wisniewski Samir Droby
Shawkat AliWalid Ellouz
Awis Khan
Studying Fruit Microbiomes
Sample collection
Community DNA extraction
Amplicon sequencing
Taxa, genera, species??, number, abundance, composition
“Who is there”?
Metagenome sequencing
Community function
“What can they do”?
Community RNA, protein, metabolites extraction
Meta –Transcriptome sequencing
Metaproteomesequencing
Metabolome analysis
Community function
“What are they doing?
The Epiphytic Apple Metagenome Project
❖ Repeats are co-clustered.
❖ American apples are highly similar and different from Israeli fruits.
❖ Differences between organic vs conventional.
Beta Diversity Analysis of Epiphytic Microbial Composition of Organic and Conventional Royal Gala Apples in the US and Israel
How can we associate changes with relevant functional modifications and produce predictions ?
16S
correlation matrix
ITS
correlation matrix
Common OTUs Calyx, Stem and Peel
Both fungi and bacteria are included
CalyxPeelStem
Common OTUs CalyxBoth fungi and bacteria are included
(Syn=Cryptococcus victoriae)
(Syn=Cryptococcus magnum)
(Syn=Acremonium strictum)
Sooty mold fungi
(fungal symbionts, mycorhiza)
contains largest number of entomopathogenic fungi
Spearman correlation matrix-taxa groups
Spearman correlation
among interesting
genera
Questions that can be asked:
What is the impact of different species on the community function?
Knockout simulations allow predicting the functional significance of each taxonomic group
What is the impact of different species on the network expansion?
Network location and connectivity of removed edge determines - the effect of its removal
Designing Beneficial Microbiomes for Biocontrol
Bottom-up approach
Collection of individual microorganisms from specific host/habitat
Role of distinct species/ strains in complex interactions
Selection of strains based on phylogeny
Experimental testing Selection based on functional interaction networks
Selection of essential nodes
Function testing on fruit
Decay Healthy
Community ecology
AcknowledgementsARO, Dept. Postharvest Science
Dr. Elena Levin
Dr. Yeka Zhimo
Dr. Amit Kishore Singh
Dr. Antonio Biasi
Dr. Ajay Kumar
Ginat Raphael
Oleg Feygenberg
Isaschar Giladi
Yaara Danon
USDA-ARS, Appalachian Fruit Research Station, Kearneysville, WV
Dr. Michael Wisniewski
Dr. John Norelli
ARO, Plant Sciecnse and Newe-Ya'ar Res. Center
Dr. Shiri FrielichDr. Adi Faigenboim
Stockholm University
Dr. Ahmed Abdelfattah
Davide SpadaroEdoardo Piombe