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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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