michael csukai

BioDundee

Sustainably growing more with less: fungal control solutions and technology gaps

Classification: PUBLIC

2

Issues!Gaps!

Outline

● Global food security challenges

● Syngenta

● Fungicide Active Ingredient Pipeline

- MoA diagnosis

- Biokinetics

- Resistance assessment

● Knowledge gap


3

Increasing demand for food

● World population growth- 7.0 billion today- 9.3 billion by 2050

• Large error bars!

● Increasing wealth driving increased demand for animal products- Significant effect on primary crop demand- 80% of agricultural land is used for animal production

● Bottom line: food demand in 2050 will be 70% higher than in 2006- Ignoring impacts of climate change, biofuels and the additional

production required to eliminate hunger


4

Pressures on existing global farmland

● Pressures on farmland from urbanisation

● Reduction in soil fertility due to poor agronomic practices


● Pressures to satisfy demand for non-food crops

- Food, feed, fibre, fuel, feedstocks, fine chemicals

- 40% of the US corn crop in 2011 was used to produce biofuels

● It would be an ecological disaster to convert wild lands to farmland on a significant scale

● Bottom line: Large increases in global farmed area are unlikely

- Indeed we may struggle to retain the amount of farmland we currently have!

5

● Evolution of resistance in existing pests- Weed resistance to glyphosate

● Evolution of new pests- Soybean rust, Ug-99

● Hopelessly unscientific pesticide legislation!- Hazard-based cut-offs- Preferential treatment of natural

products

● Bottom line: Agricultural pests are evolving to become more virulent- The political situation is making things

worse

Pressures from pests


6

Pressures on farming inputs

● Fertiliser- Nitrogen prices are linked to the price of oil- Phosphorus supplies are concentrated in

North Africa (> 75%)● Water

- 70% of the world’s fresh water is used by agriculture

- Already 25% of the world’s rivers do not reliably reach the sea!

● Economic inputs & infrastructure- Credit and insurance are increasingly difficult

to obtain

● Bottom line: Current practices are unsustainable


7

Uncertainties around climate change

● Crop yields- Probably already being impacted

● Global farmed area- Shifts in cropping regions- Sea level rise

● Extreme weather events● Economic, social and political stability

● Bottom line: There are significant uncertainties about the degree of impact of climate change on agriculture, and the timescale of impact- But it could well be “profoundly negative” and “soon”!

Science 9 January 2009: vol. 323 no. 5911 240-244


8

How to produce more food? New and better technologies

- Understanding and mitigating against abiotic stresses• Drought, heat stress, cold stress, nutrient deficiency

- New and better crop protection technologies• More effective, resistance management, new MOA


Actual yield

Pests

Stresses

Current Future?

Cro

p yi

eld

● Three strategies

- Increase intrinsic yield potential• Convert annual to

perennial crops• C3 to C4 photosynthesis

• Understanding and de-bottlenecking yield

9

It is not necessarily all about yield....

● Technologies to reduce agricultural inputs and impacts

- “Precision” technologies

- Water & nutrient use efficiency • Crops producing their own nitrogen

- Technologies to reduce GHG emissions• Increase soil carbon

● Technologies to produce “better” food

- More nutritious, better tasting, more “appealing”


10

Outline


● Syngenta


- MoA diagnosis

- Biokinetics


● Knowledge gap


11

Helping the world grow more from less

World

Our ambition:To bring greater food security in an environmentally sustainable way to an increasingly populous world by creating a worldwide step-change in farm productivity.

8Mlarge-scale

farms>100 Ha

450Msmallholder

farms~1.0 Ha

Classification: INTERNAL USE ONLY

12

Market overview 2010


Global market: ~ $75 bn

Source: Syngenta Analysis, Philips McDougall

25%

16%

8%

51%

Chart Title Conventional Seeds(~ $17 bn)

Crop Protection* (~ $40 bn)

GM Seeds(~ $12 bn)

Non-Crop Chemicals (~ $6 bn)

*Includes Seed Treatment

13

Competitors in 2010


Syngen

ta

Monsa

nto

Bayer

DuPont

BASFDow

0

2000

4000

6000

8000

10000

12000

14000

CP Seeds/Traits L&G

$ m

ill.

sa

les

Sources: Companies Annual/Quarterly reports, Phillips McDougall, Agreworld, Syngenta Analysis

14

Syngenta sales 2010: $ 11.6 bn


Crop Protection Seeds

$ 8.8 bn (77%)

$ 2.8 bn(23%)

Selective Herbicides (26%)

Fungicides (30%)

Insecticides (17%)

Non-selective Herbicides (11%)

Crop Protection Seeds

Seed Care (9%)

Professional Products (5%)

Other (2%)

Corn & Soybean (58%)

Vegetables (10%)

Diverse Field Crops (23%)

Flowers (9%)

15

Syngenta R&D

Over 5000 Syngenta employees

work in Research and Development

We spent around $1 billion in

2010 on R&D


16

Major R&D sites located on three continents

Other sites

● Marker-assisted and seed breeding capabilities

● Global field station network

Global R&D capabilities


GREENSBOROFormulationEnvironmental Science

SBIBiotechnology

R&D

JEALOTT’S HILLChemical DiscoveryWeed ControlFormulationBioscienceEnvironmental Science

STEINFungicides, Insecticides &Professional Products

GOAChemistry

BEIJINGBiotechnologyR&D

17 Classification: PUBLIC

18

Lawn & Garden*

Flowers

Growing Media**

Chemical Controls**

Turf & Ornamentals**

Syngenta was focused three businesses


Crop Protection

Selective herbicides

Non-selective herbicides

Fungicides

Insecticides

Seed care

Seeds

Corn & Soybean

Diverse Field Crops

Vegetables

* Newly established in 2008 - Financial reporting for product lines in 2008 under Crop Protection and Seeds respectively** Reported as Professional Products under Crop Protection

19

New Syngenta strategy is crop focused


RiceSpecialty

Divers Field CropsCerealsSugar caneSoybeanVegetablesCorn

20

Integrated solutions addressing growers’ needs comprehensively

Weedcontrol

Insectcontrol

Diseasecontrol

Nematodecontrol

Yield potential

Nitrogen efficiency

Drought Quality traits

Labor shortage

Post harvest

Breeding

Native traits

GM traits

Seed care

Crop protection

Nutrients, water

Machinery

Biological solutions

Chemical solutions

Growers’ needs

Technology


Services

Innovating across technologies to transform the way crops are grown

21

Outline


● Syngenta


- MoA diagnosis

- Biokinetics


● Knowledge gap



Why look for novel fungicides

● Likely to remain the mechanism to deliver fungal control of disease complexes in many crops

● Looking for novel Modes of Action (MoA)- resistance management- cleaner toxicological & environmental profile- broader antifungal spectrum

● New products sell better than old products


Screening up to 300,000 compounds a year


Discovery pipeline

Activity?

Activity Profile

Hits leads

Performance Profile

Research Development

Tox Profile

launch

Financial profile

candidates

Registration requirements


Discovery pipeline

Activity?

Activity Profile

Hits leads

Research Development launch

Financial profile

candidates

Registration requirements

Performance ProfileTox IssuesRegulatory issues

26

Outline


● Syngenta


- MoA diagnosis

- Biokinetics


● Knowledge gap



MoA impact on AI pipeline

● MoA determination:

- Intrinsic potency measurement

- Structure based design strategies

- Valuable for Tox and resistance risk assessment


Early projects

Collect and review information on “physiological effects”“symptomology”Haploinsufficiency / OERSpectrumPhys Chem

KnownMoA

UnknownMoAMoA1

Tools will generallybe available.Use external collaborationfor speed, in-house capabilityfor SAR support

Mode of action diagnosis


Target site elucidation

M

M

M

More than 25 assays

Identify activity at known target sites (+/-)

Identify hits with interesting activity or symptomology


Early projects


KnownMoA

UnknownMoAMoA1



Structure Activity Relationships (SAR)plant pathogen vs mammalian intrinsic potency


Haploinsufficiency and over-expression induced resistance both exploit gene dosage methods

Haploinsufficiency and over-expression induced resistance


Basis of the assays

OverexpressingLess sensitive to 25ppm

Haploinsufficient More sensitive to 25ppm

IC50 25ppmNormal cell Target protein

http://images.google.co.uk/imgres?imgurl=www.rhino3d.co.kr/gallery/cartoons/pacman.jpg&imgrefurl=http://www.rhino3d.co.kr/gallery/cartoons/cartoons.htm&h=375&w=500&prev=/images?q=Pacman&svnum=10&hl=en&lr=&ie=UTF-8&oe=UTF-8











Chemical Target identified

Haploinsufficiency OEIR

Tunicamycin

F-2003-035

Cerulenin

Rapamycin

Oxytriazine ND

Flutriafol

Fenpropimorph

Soraphen

Eupolauridine

Haploinsufficiency and OEIR are complementary


How do we utilise Haploinsufficiency and OER data

• Data and literature review

• Express proteins and determine if small molecule interact

• Mutate genes and determine if resistant clone can be produced

Typically a small gene group is identified. Which is target?

Could a data integration approach improve this process?Would like to have the ability to do OER in appropriate plant pathogens


Early projects


KnownMoA

UnknownMoAMoA1


Testable, quality MoA hypothesis?

Sustained interestin Chemistry?

Test using reverse geneticsS. cerevisiaeM. graminicola P. InfestansEtc.

yes

yesReverse Genetics



Early projects


KnownMoA

UnknownMoAMoA1


Generate resistant strain S. cerevisiae M. graminicolaP. infestans,M. grisea

Identify mutant gene

no

successful Forward Genetics



Test using reverse geneticsS. cerevisiaeM. graminicola P. InfestansEtc

yes

yesReverse Genetics


Collaboration with researchers at the

James Hutton Institute & Dundee University

instrumental to Phytophthora infestans work


Early projects


KnownMoA

UnknownMoAMoA1


Generate resistant strain S. cerevisiae M. graminicolaP. infestans,M. grisea

no



Test using reverse geneticsS. cerevisiaeM. graminicola P. InfestansEtc

yes

yesReverse Genetics


Develop affinity assay

Embark on protein purificationdirected by affinity assay or affinity chromatographysupported by proteomics

successful

Generate SAR and correlate to biology

Affinity Based

unsuccessful Forward Genetics


Plant pathogen platform for MoA Discovery

Efficient transformation method

Good homologous recombination efficiency

– for reverse genetics construct generation

Inducible/titratible promoter

– for protein expression, overexpression induced resistance screens and lethal KO determination

Good predicted protein database

– for peptide mass fingerprinting in affinity purification studies

Ability to map resistant mutants

– direct identification of target protein by forward genetics


M. graminicola, the most important cereal disease in northern European countries

● ~17% of cultivated land is planted with wheat (worldwide)

● Estimated annual yield loss between 30 and 50% during an outbreak

- It represents loss of >9 millions tons worldwide

● Ascomycete (Dothedomycete, like : Alternaria, Pyrenophora, Stagnospora)

● Pathogen is semi-biotrophic with a stealth growth

● Infection and spread via ascospores (airborne) and conidiospores (water splash)

- Germ tube hyphae penetrate the leaves via stomata

- First phase: biotrophic almost no growth, no symptoms for ~1 to 2 weeks

- Second phase: necrotrophic, mediate PCD in plants, fast growth

Treated

Photo: GHJ Kema, Wageningen Univerisity and Research Centre, Plant Research International B.V., Wageningen, The Netherlands.

Non treated

No 1 pathogen in No1 fungicide market!


M. graminicola plant pathogen platform

Efficient transformation method

Good homologous recombination efficiency

Inducible/titratible promoter

– inducible promoter still needed

Good predicted protein database

Ability to map resistant mutants

41 Internal use only

Published – methods and tools to stimulate external research

42

Outline


● Syngenta

● Syngenta seeds and R genes


- MoA diagnosis

- Biokinetics


● Knowledge gap


43

What they do …

spray droplet characteristics and behaviour (microscopy)

leaf surface redistribution

foliar spray retention, uptake, rainfastness, uv photo-stability

vapour movement

xylem and phloem systemicity and movement to new growth

metabolism in model systems and target organisms

(maize cell culture, SEPTTR cell culture – looking to expand these to wheat and oomycete)

biokinetics in the lab, glasshouse, field, and on the farm

The study of all processes which occur following initial contact between a pesticide and a target crop / fungus and delivery of the toxophore to the target site

Biokinetics and Microscopy


44

What they do …

spray droplet characteristics and behaviour (microscopy)

leaf surface redistribution

foliar spray retention, uptake, rainfastness, uv photo-stability

vapour movement

xylem and phloem systemicity and movement to new growth

metabolism in model systems and target organisms

(maize cell culture, SEPTTR cell culture – looking to expand these to wheat and oomycete)

biokinetics in the lab, glasshouse, field, and on the farm

The study of all processes which occur following initial contact between a pesticide and a target crop / fungus and delivery of the toxophore to the target site

Biokinetics and Microscopy



Biokinetic issues

● Gaps

- Ability to follow movement of compounds within cell!

- Range of efflux reporter strains in a plant pathogen

- Range of metabolism reporter strains in a plant pathogen

- Understanding metabolic capacity of plant pathogens

46

Outline


● Syngenta


- MoA diagnosis

- Biokinetics


● Knowledge gap



Resistance assessment

● Frequently asked to address if a new compound have a high or low resistance risk?

● UV mutagenesis and selection – but limited numbers are covered compared to the field.

Gap:Tools for better quality resistance assessment


Guide selection of chemical inputs

Historically driven by diverse inputs (Combi-chem approaches)

Can we be smarter at identifying biologically relevant chemical space?

● Target based screening still not popular concept !● Stronger focus on hypothesis driven starting point

- including relevant Pharma targets & chemistry as starting points

49

Outline


● Syngenta

● Syngenta seeds and R genes


- MoA diagnosis

- Biokinetics


● Knowledge gap


50

Major gaps in fundamental understanding of Fungal Biology


Neurospora crassa S. cerevisiae

Systematic KO projects show similar numbers of lethal genes

~ 1200~ 1200

51



• Crude calculations, BlastP, 25% identity cut-off so very permissive• Rounded numbers!


500

Overlap is surprisingly poor!!!

52





350

Compare Lethals to M. graminicola1000/1200 Neurospora lethal are in M. graminicola

850/1200 Yeast lethal are in M. graminicolaOverlap = 350

53





350

750500

150

1000/1200 Neurospora lethal are in M. graminicola850/1200 Yeast lethal are in M. graminicola

M. graminicola


A significantly better understanding

of genetics/biochemistryof plant pathogens!!!!

55

General areas of interest for Syngenta Biological Sciences

● Weed, insect and fungal resistance (including mathematical modelling)

● Metabolism in fungi, plants, nematodes and insects (including modelling and prediction)

● Predictive toxicology and selectivity between pest and non-target organisms

● Chemical biology approaches to agricultural science

● Systems and synthetic biology in agriculture

● Assay development and target identification

● Microscopy – micro-localisation of chemicals in plant, fungal and insect tissues

● In-vitro assay to whole organism translation

● Plant phenotyping

● Movement of chemicals in soil and uptake into roots

● Genetics - translation into pests from models & marker identification

● Protecting seeds from insect and fungal attack

● Plants and abiotic stress



A web portal to submit innovative ideas - opportunities and collaboration www.syngentathoughtseeders.com


Questions?

58 Classification: Confidential

Key pathogens (no particular order)

● Mycosphaerella graminicola (Zymoseptoria tritici) ● Fusarium graminearum, oxysporum & verticillioides ● Botrytis cinerea● Blumeria graminis tritici● Puccinia graminis tritici● Alternaria● Cercospora● Magnaporthe grisea● Rhizoctonia● Phytophthora infestans ● Plasmopara● Pythium● Phakopsora pachyrhizi

● No genome yet

59

Syngenta Seeds Position on R genes

Native traits approach

● Genetic resistances against fungus in plants are used for decades.

● In many crops, resistance traits have been identified and characterized. Some genes have been identified

● A range of commercial varieties exhibiting high or intermediate resistances against obligate or necrotrophic fungus.

● Frequently these resistances are linked to active immunitypathogen recognition by the plant triggers plant resistance - often seen as a hypersensitive response.


60

Resistance genes used in Tomato (Solanum lycopersicum)

Some resistance loci commonly used in commercial varieties:

● Ve Verticilium dahliae and Verticilium albo-atrum● Frl Fusarium oxysporum radici-lycopersici● I1,I2,I3 Fusarium oxysporum lycopersici● mlo Oïdium neolycopersici● Lv Leveillula taurica● St Stemphylium solani● Ph1,Ph2,Ph3 Phytophthora infestans

Not an exhaustive list


61

Issues

● Necrotrophic fungus are more complex to control by genetic means and only intermediate resistance are currently available.

● Despite the high frequency of resistance in the natural plant diversity, some groups of pathogens such as Oomycetes are more complex to control only from genetic factors

● In some pathosystems, resistance traits spread in commercial germplasm are durable whereas in some others it’s not.

● Continuous effort required to provide resistant variety to growers.


62

Perception of many!

● Pathogen diversity and evolution allow rapid breaking resistance – Making GM approaches risky and unattractive

● “Engineering would take much longer than the defeat of the R-gene in the field”.


Methods to deliver Durable Resistance

Methods to assess Durability of Resistance

Mutagenesis approaches – numbers game is a problem

65

Resistance breeding activities

The classical approach

● Search for phenotypic variability in diverse natural germplasm. ● Genetics of the most favorable phenotype is characterized via the

construction of a genetic map and BSA or QTL studies according to the genetic complexity of the resistance.

New approach ● Utilize fungus effectors to identify host receptor in natural or induced

germplasm diversity.


66 Classification: Confidential

Issues/gaps summary

● Data integration approach for yeast work?

● OER in appropriate plant pathogens

● Inducible promoter for M. graminicola work

● Ability to follow movement of compounds within cell!

● Range of efflux reporter strains in a plant pathogen

● Understanding metabolic capacity of plant pathogens

● Range of metabolism reporter strains in a plant pathogen

● Tools for better quality resistance assessment

● Tools for target prioritisation/compound follow up

michael csukai

Health & Medicine

food demand

moreappealing9 classification

unsustainable6 classification

thingsworse5 classification

hunger3 classification

better food

new moa8 classification

bn crop protection