update on bayer crdf hlb project - florida citrus...

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Update on Bayer CRDF HLB Project

Florida Citrus Industry Annual Conference,Bonita Springs

June 14, 2018 / Denise Manker

== =Plant

Defense Modulator

Abandoned groves / backyard citrus

NurseryEradi-cation

Plant Health & Defense

Systemic & contact insecti-cides

Anti-bacterial solutions

NutritionManage-

ment

IrrigationManage-

ment

Monito-ring /Traps

Tree Inspec-

tion

Partne-ring

Parasitoid BeneficialsRoot

Health Manage-

ment

Regional Manage-

ment / CHMas

Orchards

Pathogen

HostPlant DefenseModulators

Antibacterial Microbes

Vector

Nutrition Manage-

ment

Induce resistance and strengthen

plant health

Avoid / Stop multiplication

of the bacterium

HLB Screening Cascade

The Project Will Pursue Three Key Elements to Find a Solution for HLB

Allows systematic search for solutions

against HLB

1 2 3

What is unique about this approach?


Allows systematic search for solutions

against HLB

1Focus on developing a validated screening cascade

• Purpose is to have a systematic approach to identify and characterize hits with confirmed in planta HLB activity

• Develop in house high through-put in vitro Liberibactercrescens assay along with other bacterial targets

• Collaborate with academic lab to optimize Liberibactersolanacearum in planta assay greenhouse for screening leads

• Fund development of a validated in planta HLB citrus greenhouse assay to select leads for field testing in 2019

Dose-response bacterial cell tests including

Liberibacter crescens

HitSelection

Planta test other bacterial diseases and citrus

usabiltiy

Citrus field trials

Lead Selection

D

HLB Specific Screening Cascade: from Cell Test to Field

Chemical judgement: novelty, chemical variability, PhysChem Biological judgement: Disease potential, activity, mode of action

Selected strains (AM)

Plant Defense Modulators (PDM)

internal external

Tox package for fieldIntellectual Property

De-selection of unwanted strains

Greenhouse test on citrus

Liberibacter solanacearumon tomato GH test

HLB specific screening cascade will allow systematic approach for testing, selecting and optimizing strains and compounds for efficacy against HLB.

What is unique about this approach?Develop pool of candidates that target both Pathogen and Host and would have relatively shorter regulatory path to market

• Host Approach: Screen synthetic compound libraries for their ability to control Liberibacter indirectly via induction of plant defense pathways

Advantages:• Indirect activity – not a pesticide/bactericide, low risk of

resistance• Bayer has a screen in place for discovering Plant

Defense Modulators (PDM) for other bacterial diseases so can rapidly identify chemical classes

Plant DefenseModulators

Induce resistance and strengthen

plant health

2

Induced systemic response• Broad spectrum• Long lasting• Low risk of resistance

• Xanthomonas cabbage assay has been optimized

• Pseudomonas tomato assays developed

• Citrus established in greenhouse for phytotoxicity tests

• In planta citrus PDM assay under development

• One chemistry class has been identified which can protect plants from bacterial disease via plant defense mechanism

A library of compounds in this class is being synthesized for further testing/optimization activity

Progress on Plant Defense Modulators

Pseudomonas on tomato, Bayer

Plant tests for other bacterial diseases and

citrus compatibility

Citrus plantlets, Bayer

• Proof of concept of multiple marker tracking• Low Throughput Screen to be routine in Q3-2018

Citrus Molecular Diagnostic – HLB disease free test

Plant Defense test in Citrus plantlets HLB disease free test

Bayer Partnership with CRDF

What would that look like?

Plant Defense Product

Foliar spray

Likely PreventativeMay need multiple applicationsFoliar spray best application method

What is unique about this approach?

Develop pool of candidates that target both Pathogen and Host and would have relatively shorter regulatory path to market

• Pathogen Approach: Screen microorganisms that can directly inhibit Liberibacter through production of anti-bacterial compounds

Advantages:• Active microbes can be registered through EPA’s BPPD in

an expedited process• Bayer has a collection of >150,000 microbial strains and

sophisticated in-silico screening capabilities to identify strains that are likely to be anti-bacterial

Antibacterial Microbes

Avoid / Stop multiplication of the bacterium

3

Global Biological R&D Center in West Sacramento, CA• $80 Mio facility on 10 acres

• ~170,000 sq. ft including pilot plant

• Biologics staff of 105 people

• Additional 5 acres for greenhouse

Using this method, 100 strains selected to validate assays and first 1000 strains selected for screening

In-silico screening of Bayer’s microbial library

?

AntiBac

GENOME

A TG CSequencing

FunctionalGenomics

MicrobialGenome Portal

The genetics of isolates are used to select strains predicted to have anti-bacterial biological chemistry


Progress on Antibacterial Microbes

In vitro Assays – XANTAV, PSDMTM, ERWIAM, and CORBMI with L. Crescens – under development

In Planta Assays – XANTAV and PSDMTM (RALSSO in development)

Serenade Treated Untreated

Xanthomonas Assay

• MIC assays in place for Xanthomonas, Pseudomonas, Agrobacterium

• Working on method transfer for L. crescens in vitro assay

• Agar diffusion plate assays developed for Xanthomonas, Pseudomonas, Erwinia and Clavibacter

• Plant bioassays in place for Xanthomonas and Pseudomonas

• Plant assay for Ralstonia under development

Enriched active fraction has been prepared for first plant testing in tomato and citrus tests

Advanced microbial lead chemically characterized

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Several microbial strains from advanced projects were previously identified to be active in vitro on Liberibacter crescens

AQ713 –antibacterial activity identified, concentrated samples prepared for in planta tests

AQ2808 -- initial chemical characterization is underway with good progress being made

Remaining leads awaiting further chemical analysis


What would that look like?

Anti-bacterial Microbial Product

Trunk Injection?

• How to get the microbe to the infection?• May require trunk injection• Should have direct activity on pathogen

so possibly work on infected trees?

Collaborations with University of California Davis and University of Florida

External collaborations for greenhouse screening

UC Davis, Bryce Falk, Sandra Thuy Vu. Liberibacter solanacearum assay on tomato.Psyllid inoculated. qPCR analysis. No containment required. First samples ofadvanced microbial lead are in test.

Univ. of Florida, Southwest Florida Researchand Education Center, Ozgur Batuman has developed an HLB in planta greenhouse assay on citrus. Psyllid inoculated. qPCR analysis.First samples for testing to be shipped in June.


Strain Improvement

Field trials

Field trials

PRIO

RO

TIZA

TIO

N

Project Milestones

Setup Routine Screening

Set Coll

Set Coll Method Development

potentially substituted by GH citrusRoutine Screening

Routine ScreeningField trials HLB

GH Citrus HLB

Plant Tests (not HLB)

Cell Test

PDM Screen Existing

AMIdentification of active strains

Optimization

Set Coll

Routine Screening

Set-Up + Test Phase

Follow-up advanced strains with known bactericidal activity

Setup

MilestonesMS 1 MS 2 MS 3 MS 4 MS 5

SETUP OPTIMIZATION

FIELDFIELD set-up

Joint SC Meeting STAR

T:

May

1

2017 2018 2019 2020 2021PROJECT STAGE I PROJECT STAGE II


Progress towards Milestones

Milestone 1: Q4 2017 (~ 6 months): COMPLETED Setup of staff completed according to Budget Selection / prioritization

PDM chemistry starting points AMs to be screened first

Milestone 2: Aug 2018 (~ 15 months) Screening Cascade Cell test: workflow established -- Validation in process Lsol tomato test: cooperation / workflow established--

operational GH citrus test: identification of collaborator, contract setup

and method development initiated – Ahead of schedule PDM + AM Screening results of first examples in initial tests

obtained -- UNDERWAY Activity of advanced strains in Lsol system known – FIRST

LEADS BEING TESTED


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Greenhouse* and field trials

evaluation of solutions against HLB

Host

Induce resistance & strengthen plant health

Pathogen

Avoid/Stop multiplication of the bacterium

=

Antibacterial Microbes (AM)

Strain selection by genomic dereplicationDose response against

bacterial diseases

=Plant Defense

Modulators (PDM)

Optimization of chemistry against bacterial diseases

on planta testWith citrus compatibility

Highlights first thirteen months:

Plant Defense• Process for selecting PDM hits and optimizing chemistry in place• 1 Lead compound in Hit Exploration ph 0.1 – ready for testing in

tomato and citrus plant assays• Seven additional PDM chemical families identified for further work

Anti-bacterial Microbes• In vitro assays, including Liberibacter crescens, established,

429 of first 500 strains screened• Total of 1000 strains selected for screening using genomic

information• Activity in one advanced strain identified

Screening cascade• Research agreement with UCD* in place, assay and application

methods being validated, advanced microbe lead extract in test• Research agreement with UF** in place, trees infected, assay

being validated, advanced microbe extract lead ready for shipping

*UF : University of Florida, UC Davis

CRDF COLLABORATIONCross-Technology & Cross-Sites Project to bring our newly established bacterial diseases platform to the next level

Bayer CRDF agreement -- 13 month summary

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• On track to produce leads for 2019 field trials vs HLB

• Screening cascade expected to be ready for routine testing in Q3

• Good progress on collaborator in planta assays vs Liberibacter (CA, FL)

• Promising PDM chemical class to be tested in greenhouse Liberibacter assays in July

• Validation and optimization of primary in vitro screen in process in Sacramento

• Activity identified in advanced microbial lead with concentrated material in test in tomato Lsol assay

• 1000 strains selected through genomic analysis for in vitro screening

Silver bullet is unlikely for a solution to HLB

/// IHC 2018 /// August 201820

The complexity and speed of spreading the disease shows clearly that multiple tools will be needed to manage HLB

We will need all available tools working together to address this challenge

Below are examples of approaches being explored by other parties

Psyllid control innovations

Breeding resistant citrus trees

Using genome editing and transgenic trees

Endophytes

ThankYou!

Bayer and Monsanto will create a global leader in sustainable agriculture

22

InnovationEngine

SocialResponsibility

Commitment to Sustainability


Focus on Sustainable Horticulture through Innovation

Why citrus matters to us…

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Focus• With 25%, horticulture accounts for the largest share of our business. • Leader of the global citrus crop protection segment.• Citrus is our No. 1 horticulture crop in the US and Brazil.• Leading company in crop protection research and technology.

Innovation• Our company mission is the foundation of what we strive to achieve within

agriculture. • We help to secure the supply of high-quality, affordable food.• We invest about 10 percent of our annual sales in R&D, and a significant part into

horticulture.

Commitment for sustainable horticulture:• The current Citrus Greening management practices are not sustainable.• To secure future citrus fruit supply, the way of cultivation needs to change. • Sustainable agriculture is the only way to overcome the challenge.


How will divestment affect Bayer’s CRDF project?

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Effect on CRDF Agreement

Divestment announcement

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The divestment of Morrisville Traits research affected CRDF agreement by:

-- Loss of one FTE, transferred to BASF

-- Loss of site/access to High Throughput Screening (HTS)

Activities to mitigate these effects:

Replacement FTE hired, internal post-doc transferred to project allowing for quick start

Transfer and validation of HTS currently underwayTaking opportunity to modify process to enhance discovery of active microbial metabolites

RESULT: Approximately two months behind in screening AM’s in vitro

Long term gains: More efficient process – no shipping of strains and extracts between CA and NCMedia optimized for metabolite production


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