New targets for plant breeding and the challenge of sustainable intensificationsustainable intensification

Royal Swedish Academy of Agriculture and Forestry, Stockholm, 29-30/01/13

J A Pickett Rothamsted ResearchJ.A. Pickett, Rothamsted Research

New directions for agriculture in the 21st Century

There is a pressing need for the ‘sustainable intensification’ of global agriculture in which yields are increased without adverse environmental impact and without the cultivation of more land

A second green revolution which is knowledge intensive rather than input intensive?rather than input intensive?

Royal Society (2009) Policy Document 11/9

SustainabilitySustainability

Now Future

No loss to pests, diseases and weeds because carbon footprint from land

Delivery, via the seed/planting material, of yield traits nutrition and cropfootprint from land

preparation, fertilizers, seed and pesticides, and their delivery is already committed

traits, nutrition and crop protection, plus move from annual to perennial crops (new issues of pest control indelivery, is already committed (new issues of pest control in rhizosphere)

Influence of different crop plant species on organic : conventional yield ratios

Seufert et al. (2012), Nature, 485: 229

Reconciling Food Production and Biodiversity Conservation: Land Sharing d L d S i C dand Land Sparing Compared

Abstract

The question of how to meet rising food demand at the least cost to biodiversityrequires the evaluation of two contrasting alternatives: land sharing, whichintegrates both objectives on the same land; and land sparing in which high yieldintegrates both objectives on the same land; and land sparing, in which high-yieldfarming is combined with protecting natural habitats from conversion toagriculture. To test these alternatives, we compared crop yields and densities ofbird and tree species across gradients of agricultural intensity in southwest Ghanabird and tree species across gradients of agricultural intensity in southwest Ghanaand northern India. More species were negatively affected by agriculture thanbenefited from it, particularly among species with small global ranges. For bothtaxa in both countries land sparing is a more promising strategy for minimizingtaxa in both countries, land sparing is a more promising strategy for minimizingnegative impacts of food production, at both current and anticipated future levelsof production.

Phalan et al. (2011), Science 333: 1289

Production of Behaviour-Controlling Chemicals by Crop Plants

Abstract

The possibilities of breeding crop plants that produce insect pheromonesThe possibilities of breeding crop plants that produce insect pheromonesand other behaviour-controlling chemicals and of obtaining antifeedantsfrom natural sources for protection against invertebrate pests arediscussed The possible role of genetic manipulation in these approaches todiscussed. The possible role of genetic manipulation in these approaches tocrop protection is also considered.

Pickett (1985), Phil. Trans. Royal Soc. B 310: 235

Zeyaur Khan Charles yMidega

Push-Pull or Stimulo-Deterrent Diversionary Strategy (Vuta Sukuma)

Main CropMain Crop

Trap Crop

Attract moths

Attract naturalenemies

Moths are pushed away

Intercrops – Melinis, Desmodiump ,Cook et al. (2007), Ann. Rev. Ent. 52: 375;

Hassanali et al. (2008), Phil. Trans. Royal Soc. Lond. B, 363: 611; Tamiru et al. (2011), Ecol. Lett. 14: 1083

Commercial decision support

Hooper et al. (2007), Tet. Lett. 48: 5991

Current highly effective pesticides are small lipophilic molecules (SLMs) derived from natural product leads and, for some, are natural products

Insecticide Target Natural product lead

pyrethroid sodium channel/activators pyrethrin Ipyrethroid sodium channel/activators pyrethrin I

indoxacarb/ sodium channel/blockers xmetaflumizone

organophosphate/ acetylcholinesterase/inhibitors xcarbamate physostigmine

neonicotinoid nAChR nicotine/epibatidine

spinosad nAChR spinosyns

cyclodiene/ chloride channel/gaba xfiproles

abamectin chloride channel/glutamate avermectins

diamide calcium release channel (muscle) (ryanodine)

tetronic acid acetyl CoA carboxylase/inhibitor x

Tanacetum cinariifolium

pyrethrin I

HYDROXAMIC ACIDS IN WHEAT (benzoxazinones)

Insects (chewing and sucking) and

MeO O OH

DIMBOA

and sucking) and airborne pathogens

N O

OH

DIMBOA

APHIDS, artificial diets

Fungi, bacteria and nematodes

Moraes et al. (2008), Phytochem. 69: 9

Allelopathy, weeds

Intrinsic rate of population increase of the cereal aphid Rhopalosiphum padi on diploid, tetraploid and hexaploid wheat (hydroxamic acids, active against aphids and other pests and diseases, are present but only effective in ancestral species)

diploid tetraploid hexaploid

0,45

0,35

0,4

0,25

0,3

0,2

 spe

ltoides

MDR 04

9

8116

longissima

MDR0

43

8404

MDR0

50 102

MDR0

44 Ae …

MDR2

98

8150

Napier

Tasm

an

920/2

W32

3

L58

L69

920/9

920/3

T. dicoccum

Istabraq

920/10

L112

Svilena

Welford

W32

0

920/1

Turtsikum

920/4

920/11

Robigus

Solstice

MV4

Alifen

Hum

ber

L124 L61 L3

Largo

L25

PI29

4994

L122

Ae

Ae  T

Intrinsic rate of population increase of the cereal aphid Rhopalosiphum padi on diploid, tetraploid and hexaploid wheat (hydroxamic acids, active against aphids and other pests and diseases, are present but only effective in ancestral species)

diploid tetraploid hexaploid

0,45

0,35

0,4

0,25

0,3

0,2

 spe

ltoides

MDR 04

9

8116

longissima

MDR0

43

8404

MDR0

50 102

MDR0

44 Ae …

MDR2

98

8150

Napier

Tasm

an

920/2

W32

3

L58

L69

920/9

920/3

T. dicoccum

Istabraq

920/10

L112

Svilena

Welford

W32

0

920/1

Turtsikum

920/4

920/11

Robigus

Solstice

MV4

Alifen

Hum

ber

L124 L61 L3

Largo

L25

PI29

4994

L122

Ae

Ae  T

F1 hybrids between the aliens and normal wheat are treated with colchicine to double the chromosome number and produce fertile amphidiploids

F1 hybrid shedding pollen:Ae speltoides 2140008 XAe. speltoides 2140008 X Chinese Spring Euploid 94

C l hi i t t d F1 h b id l t

F1 hybrid setting seed:Ae. mutica 2130004 X Chi S i E l id 94Colchicine treated F1 hybrid plants Chinese Spring Euploid 94

Ian & Julie King, University of Nottingham (unpublished)

BX1

rye, diploid wheat

maize, hexaploid wheat

Frey et al (1997) Nomura et al (2003) Sue et al (2006)

Activation by glucosidases

Frey et al. (1997), Nomura et al. (2003), Sue et al. (2006)

Moraes et al. (2008), Phytochem. 69: 9

Ahmad et al. (2011), Plant Physiol. 157: 317

It is time now, in planning the new generation ofGMOs for delivery of pest control, to target the

l d h i b i d fnatural products that, acting by non-toxic modes ofaction, affect, in more sophisticatedways behavioural and developmental processes inways, behavioural and developmental processes inthe pest organisms. Such natural products areexemplified as insect pheromones and otherp psemiochemicals, i.e. those chemicals that affectdevelopment or behaviour of organisms generally( d ill i l d “ it hi ” f th(and will include “switching on” genes for thebiosynthesis of semiochemicals by means ofanother set of natural products that act as plantanother set of natural products that act as plantactivators).

Coupled gas chromatography-electrophysiology

EAG/SCRsample

FIDFIDsplitter

GC

air N2

a. Two hypotheses for host location:-

Plant Volatile1. Ratio-specific odour recognition:

ORN

Plant VolatilePlant Volatile

Plant VolatileORN

ORNORN

plant odour specificity is achieved by a particular ratio between constituent volatiles, distributed generally among

CNS

Plant VolatileORN

, g y gplant species

2. Species-specific odour recognition: host plant odour recognition relies upon

Plant Volatile

host plant odour recognition relies upon taxonomically characteristic volatiles not found in unrelated plant species

ORN

CNSCNS

Bruce et al. (2005), Trends in Plant Sci. 10: 269Bruce & Pickett (2011) Phytochem 72: 1605Bruce & Pickett (2011), Phytochem. 72: 1605Pickett et al. (2012), Physiol. Ent. 37: 2

Female Sitodiplosis mosellana responses to semiochemicals identified from wheat, cv. “ECO22”, in the olfactometer, ,

7

*5

6

n) *

*

2

3

4

pent

(min

0

1

2

Tim

e sp

-2

-1

0

4-comp 5-comp (natural ratio) 5-comp (unnatural ratio)-2

7ng a-pinene5ng 6-methyl-5-hepten-2-one10ng 3-carene

7ng a-pinene15ng 6-methyl-5-hepten-2-one10ng 3-careneBruce et al (2005) Trends in 10ng 3-carene

4ng acetophenone4ng 2-dodecanone

10ng 3-carene 4ng acetophenone4ng 2-dodecanone

Bruce et al. (2005), Trends in Plant Sci. 10: 269

a. Two hypotheses for host location:-

Plant Volatile1. Ratio-specific odour recognition:

ORN

Plant VolatilePlant Volatile

Plant VolatileORN

ORNORN

plant odour specificity is achieved by a particular ratio between constituent volatiles, distributed generally among

CNS

Plant VolatileORN

, g y gplant species

2. Species-specific odour recognition: host plant odour recognition relies upon

Plant Volatile

host plant odour recognition relies upon taxonomically characteristic volatiles not found in unrelated plant species

ORN

CNSCNS

Bruce et al. (2005), Trends in Plant Sci. 10: 269Bruce & Pickett (2011) Phytochem 72: 1605Bruce & Pickett (2011), Phytochem. 72: 1605Pickett et al. (2012), Physiol. Ent. 37: 2

Pests of oilseed rape

Meligethes aeneus

Psylliodes chrysocephala

Ceutorhynchus assimilis

GC-EAG of cabbage stem flea beetle, Psylliodes chrysocephala, with volatiles from oilseed rape

Biosynthesis of methionene-derived glucosinolates in Arabidopsisthaliana: methylthioalkylmalate (MAM) synthase, the condensingy y ( ) y genzyme of the chain elongation cycle

Etc

Textor et al. (2004), Planta 218: 1026

H

OPP

Pickett (1985), Phil. Trans. Royal Soc. Lond. B 310: 235

Biosynthesis of (E)-β-farnesene and (1R,4E,9S)- caryophyllene

H

b

OPP

aa

b

H

HH

H

The Eβf (alarm pheromone) synthase gene for aphid control

Mentha piperita chemotype no longer synthesising Eβf; two genes having high overall amino acid sequence identity with known Eβf synthase.

H

MpSS1, when cis-muurola-3,5-dienep ,expressed in E. coli plus FPP, gave:

,[and cis-muurola-4(14),5-diene]

MpSS2 (with 2 aas different from Eβf synthase), when expressed in E. coli plus FPP, gave nothing.

BUT with L531S (site-directed mutagenesis product restoring one of the Eβf synthase aas) it gives highly selective production of Eβfsynthase aas), it gives highly selective production of Eβf.

Prosser et al. (2006), Phytochem. 67: 1564

GC of volatiles from flowering Arabidopsis thaliana (A) wild-type and (B) transformed line: (1R,4E,9S)-caryophyllene (1), (E)-β-farnesene (2)

pA

(B) transformed line: (1R,4E,9S) caryophyllene (1), (E) β farnesene (2)

p

2000

2500

3000

A

500

1000

1500

1

min0 5 10 15 20 25 30 350

pA

3000

2

1000

1500

2000

2500

B2

min0 5 10 15 20 25 30 350

5001

Beale et al. (2006), PNAS 103: 10509

Wheat transformation

• Transformation of a plant to emit an insect pheromone was shown for the first time in Arabidopsis

• Now achieved in wheat cv ‘Cadenza’ but with a different• Now achieved in wheat, cv. Cadenza , but with a different transformation process

• Alarm pheromone-emitting GM wheat– repels aphids

tt t th i t l i– attracts their natural enemies

• The next step is to test GM wheat under field conditionsThe next step is to test GM wheat under field conditions

Analysis of GM wheat pollen in honey

Background:

• ECJ ruling 6/9/11 - honey containing pollen from unauthorised GM events could not be soldECJ ruling 6/9/11 honey containing pollen from unauthorised GM events could not be sold.• Local Hertfordshire bee-keepers concerned about GM wheat pollen.• Honey bees known to forage over several Km, but do not normally collect wheat pollen.• May occasionally collect ‘honeydew’ from aphids feeding on wheat.

We set out to answer two questions:

Does honey from local hives usually contain wheat pollen?Does honey from local hives usually contain wheat pollen?Did honey from sentinel hives intentionally placed close to GM trial contain GM wheat pollen?

We used a PCR-based TaqMan method to detect specific DNA sequences in honey d i d t th t land carried out three separate analyses:

1. We determined the limit of detection for the method using honey spiked with GM pollen.2. In 2011 (before the trial was planted but after local wheat had flowered), we tested2. In 2011 (before the trial was planted but after local wheat had flowered), we tested

honey from hives on the Rothamsted farm and surrounding areas for wheat pollen.3. In 2012 (after the trial and local wheat had flowered), we tested honey from sentinel

hives placed close to the GM trial for the presence of GM and non-GM wheat pollen.

Analysis of GM wheat pollen in honey

Establishing a limit of detection:Honey (collected in 2011 when no wheat was flowering) was spiked with 0 1 10 100 1000 and 10 000 GM wheat pollenspiked with 0, 1, 10, 100, 1000 and 10,000 GM wheat pollen grains (all in triplicate).

TaqMan assay applied to all samples Designed to detect 3 genes:TaqMan assay applied to all samples. Designed to detect 3 genes:1. COX (cytochrome oxidase) internal general plant control gene.2. WPAL (wheat phenylalanine ammonia-lyase), wheat-specific.3. EBF transgene.

Results:

COX gene was abundant in all samples g p(indicated by lower CT value) due to OSR and other pollen species.

Wh t PAL d t d t t dC

Tva

lue

Wheat PAL and transgene were detected in honey containing 100 pollen grains or more.

LOD lies between 10 and 100 grains.No. of pollen grains

A l i f h l d b f d d i GM t i lAnalysis of honey sampled before and during GM trial

Was GM pollen found in honey?

In summer 2012, after GM trial wheat had flowered, we collected and tested honey from six sentinel hives placed close to the trial sitesentinel hives placed close to the trial site.

No GM pollen was detected.

Does honey ever contain wheat pollen?

Honey collected from hives on the Rothamsted Farm in 2011 and from sentinel hives inHoney collected from hives on the Rothamsted Farm in 2011, and from sentinel hives in 2012, was tested for wheat pollen. None was detected in 2011 and a trace level was found in some samples from the sentinel hives in 2012.

Pollen analysis done by Fera

SLM

Pickett & Poppy (2001), Trends in Plant Sci. 6: 137 Bruce & Pickett (2007), Curr. Op. Plant Biol. 10: 387

SLM

Pickett & Poppy (2001), Trends in Plant Sci. 6: 137 Bruce & Pickett (2007), Curr. Op. Plant Biol. 10: 387

O

Bi k tt t l (2000) PNAS 97 9329Birkett et al. (2000), PNAS 97: 9329Bruce et al. (2008), PNAS 105: 4553

Characterisation of a cis-jasmone responsive promoter: t ti f t / t f i f CYP81D11construction of a promoter/reporter fusion for CYP81D11

stop

At3g28750

Start P450

At3g28750

luciferaseorGUSAt3g28750 At3g28750 GUS

= exon

Promoter

= exon

= intron Ca. 1 kb

- cis-jasmone + cis-jasmone - cis-jasmone + cis-jasmone

Matthes et al.

Matthes et al. (2010), Planta 232: 1163

(2011), Plant Sig. & Behav. 6: 1

H

Signal SLMOPP

O

g

Field trial with wheat

1.2 *1

control

cis-jasmone

P = 0.036

0.8

ds /

Tille

r

cis jasmone

0.6

n N

o. A

phid

*

0 2

0.4

Mea

n

0

0.2

28-May 8-Jun 16-Jun 24-Jun 6-Jul

Bruce et al. (2003), Pest Man. Sci. 59: 1031

Aphidius ervi foraging on cis-jasmone treated wheatp g g j

25

15

20

10

min

0

5

Significantly longer time spent on i d d l t

Treated Control

induced plants

Potential for enhancement of useful traits in crop plants by cis-jasmoneby cis jasmone

Putative biosynthesis of (E,E)- 4,8,12-trimethyl-1,3,7,11-tridecatetraene in Arabidopsis thalianatridecatetraene in Arabidopsis thaliana

CYP82G1 (Insect-induced)

CYP81D11 / CYP72A13?

(CJ-induced)

Bruce et al. (2008), PNAS 105: 4553

Lee et al. (2010), PNAS 107: 21025

Matthes et al. (2010), Planta 232: 1163

Dewhirst et al. (2012), Pest Man. Sci. 68: 1419

O

Blassioli-Moraes et al. (2009), Ent. Exp. App. 131: 178

“Smart maize”: indirect defence elicited by herbivore eggs

Smart plants detect egg-laying by pests and call in natural enemies (parasitic wasps) to attack eggs and larvae before plant damage occurs

Bruce et al. (2010), Biol. Lett. 6: 314; Tamiru et al. (2011), Ecol. Lett. 14: 1083

GC profiles of systemically released headspace volatiles from a representativeheadspace volatiles from a representative maize landrace line C-2101 (Cuba), with or without Chilo partellus eggs

EAG active compounds:

(a) (E)-ocimene(a) (E) ocimene(b) (R)-linalool(c) (E)-4,8-dimethyl-1,3,7-nonatriene (DMNT)(d) methyl salicylate ( ) y y(e) decanal (f) methyleugenol(g) (E)-(1R,9S)-caryophyllene(g) ( ) ( ) y y(h) (E)-β-farnesene (i) (E,E)-4,8,12-trimethyl-1,3,7,11-tridecatetraene (TMTT)

Tamiru et al. (2011), Ecol. Lett. 14: 1083

Smart sensing to optimise farm inputs:sensitive sentinel plants detect problem, not just pests, diseases andweed competition but also depleted or excess nutrients and water, andsignal to main crop of smart plants, with natural response to signal SLMslinked to gene expression (by GM) to deal with problem (or opportunity)

Sentinel plantSentinel plantMain crop

Smart sensing to optimise farm inputs:sensitive sentinel plants detect problem, not just pests, diseases andweed competition but also depleted or excess nutrients and water, andsignal to main crop of smart plants, with natural response to signal SLMslinked to gene expression (by GM) to deal with problem (or opportunity)

Sentinel plantSentinel plantMain crop

Problem detected

Response to problem

Destruction of global soybean crop?

Phakopsora pachyrhizi, soybean rust (with C B Hoffmann(with C.B. Hoffmann-Campo and S. Lima, EMBRAPA, Brazil)

OHHO

OHThe most Striga-inhibitory chemistry from Desmodiumspp, and biosynthesis by C-glycosylation

OH Onaringenin chalcone

HOOH

FLAVANONE

spp, and biosynthesis by C glycosylation

OHO

OH O

OH

XY

OHO

OHOHHO

OH

OOH

FLAVANONE

OH O

OHOH

naringeninX

OH OHO

OH D. uncinatum CGT

CYP450X-Y = CH2CH or CH2COH

OHO

OH O

OH

2-hydroxynaringeninOHO

HOOH

HHO

OH

OHFLAVONE

OHO

OH O

OOH

HOOH

OHO

OH O

OH

R = H; apigenin

R3'Hamilton et al. (2009), Tet. Lett. 50: 565

Hooper et al. (2010), Phytochem. 71: 904R3' = H; apigeninR3' = OH; luteolinKhan et al. (2010), J. Exp. Bot. 61: 4185

Hamilton et al. (2012), Phytochem. 84: 169

Broader opportunities: delivery by the seed

• Suppression of methane production by ruminants

• Interference with N20 release from fertilized soils

CH4

CH4

Perennial crops

Perennial rice (Oryza sativa/O. longistaminata)Perennial rice (Oryza sativa/O. longistaminata)

Food Crops Research Institute, Kunming

Beijing Genetics Institute, ShengzhenBeijing Genetics Institute, Shengzhen

Perennial wheat (Thinopyrum spp)Washington State University, Pullman

Chemistry

Mik Bi k ttVisiting Scientists/

RI F llMike BirkettStephen BarasaJohn Caulfield

Keith Biology

RI Fellows

Andrea BirkeH i G l tKeith

ChamberlainTony Hooper

Patrick Mayon

Biology

Gudbjorg Inga AradottirToby Bruce

Henrique GoulartMarilene Fancelli

Salvador LimaMarlene LimpalaerPatrick Mayon

John PickettToby Bruce

Jason ChapmanJames CookSam Cook

Marlene LimpalaerCarol Moraes

Paulina PowolowskaAlessandro RiffelPlant Mol Biol Sam Cook

Sarah DewhirstHenriette ElekJanet Martin

Alessandro RiffelBenisio Silva-Filho

Jozsef Vuts

Plant Mol. Biol.

Shakoor AhmadHuw Jones Janet Martin

Lesley SmartBen Webster

Christine Woodcock

Huw JonesMichaela MatthesJohnathan Napier Retired

Technical Development

Insect Mol. Biol.

Lin Field

Margaret BlightDavid Griffiths

Wilf Powell L W dh

p

Barry PyeXiaoli He

Jing-Jiang ZhouLester Wadhams