21112005

Mycotoxin Prevention in Cereal Crops by Enhanced Host Plant Resistance

UMB; Åsmund Bjørnstad, Helge Skinnes, et alBIOFORSK: Sonja Klemsdal, Erik Lysøe et al.

UMN: Jim Anderson, Howard Rines, Ruth Dill-Macky, Brian Steffenson

University of Minnesota Small Grains Scab Researchers

Funding:National Science FoundationUSDA-NRI Plant Genome Program U.S. Wheat and Barley Scab Initiative, via USDA- ARSMinnesota Small Grains Initiative (Minnesota Agricultural Experiment Station)

Department of Plant Pathology Ruth Dill-Macky, Small grainsBrian Steffenson, cereals & wild relativesYanhong Dong, Mycotoxin AnalysesCorby Kistler, USDA-ARS Yue Jin, USDA-ARS Char Hollingsworth, Crookston

U.S. Wheat & BarleyScab Initiative

Department of Agronomy & Plant Genetics Jim Anderson, Wheat BreedingKevin Smith, Barley BreedingGary Muehlbauer, Wheat & Barley Mol. GeneticsDave Garvin, USDA-ARS Wheat GeneticsHoward Rines, USDA-ARS, Oat GeneticsDeon Stuthman, Oat Breeding

Researcher Fusarium Head Blight Research AreasAnderson Wheat Breeding, DNA Markers, Germplasm Screening, QTL Cloning

Dill-Macky Epidemiology, Disease Forecasting, Host Resistance Screening

Dong Mycotoxin Analyses

Garvin Genetic Resistance Characterization

Hollingsworth Chemical control

Kistler Fungal Gene Expression, F. graminearum population heterogeneity

Muehlbauer Barley & Wheat Molecular Genetics

Rines Oat Genetics, DNA markers

Smith Barley breeding, DNA markers

Steffenson Hordeum germplasm screening and mapping

Stuthman Oat Breeding

Total Annual Funding ~ $1.2 million

Hard Red Winter Hard Red Spring White Soft Red Winter Durum

USA wheat production -

shown by class 21 million ha, 64 million Mt

Hard Red Winter Hard Red Spring White Soft Red Winter Durum

USA wheat production areas -

shown by class

1993-1996Yield losses>2.3 billion

USD

Hard Red Winter Hard Red Spring White Soft Red Winter Durum

USA wheat production areas -

shown by class

1996 -

DONConversion of

all white wheat to

feed

Hard Red Winter Hard Red Spring White Soft Red Winter Durum

USA wheat production areas -

shown by class

Chronic DON contamination

sig. yield losses 1996-2003

Disease SeverityDisease Severity

Grain SoundnessGrain Soundness

Fusarium Head Blight (Scab) EvaluationFusarium Head Blight (Scab) Evaluation

DNA Marker Screening

PCR prepPCR prepGel LoadingGel Loading

GrindingGrindingTissue HarvestTissue Harvest

14.214.2LODLOD

gwm274Dgwm274DBARC73BARC73BARC101CBARC101Cfba91fba91gwm533bgwm533bBARC102BARC102BARC87BARC87gwm493gwm493gwm533agwm533aSUN2SUN2BARC75BARC75bcd907zbcd907zgwm389gwm389bcd907rbcd907r

0.00.0

cMcM00

5050

100100

3.03.0

DNA Marker MappingDNA Marker MappingAssociation of DNA Association of DNA markers (and linked markers (and linked genes) with scab genes) with scab resistanceresistance

NoNoAssociatioAssociatio

nn

Marker Assisted Evaluation of FHB Resistance : good diagnostic markers

for the important QTL Fhb1

No.

o

f

Xgwm389 alleles

One diagnostic allele!

Crop Sci. 43: 760 - 766 (2003)

Sum

ai 3 190

180170160150140130

120

110

100

190180170160150140130

120

110

100

Dissecting a Genetically Complex Dissecting a Genetically Complex TraitTrait

0

10

20

30

40

50

60

70

1 2 3 4 5 6 7 8 9 10

% F

HB

Sev

erity

Homozygous Recombinants

ResistantResistant

SusceptibleSusceptible

Qfhs.ndsu-3B

S

Narrow down the QTL regionClosing in on Cloning a Closing in on Cloning a Scab Resistance GeneScab Resistance Gene

Xgwm533Xgwm533

Xgwm493Xgwm493

MarkerMarker

6.2 cM6.2 cM

Xgwm533Xgwm533

Xgwm493Xgwm493

MarkerMarker

0.3 cM0.3 cM

Fall 2003 Fall 2004

Qfh

s.nd

suQ

fhs.

ndsu

-- 3B

S3B

S

0.08 cM0.08 cM

Fall 2005 Spring 2007

22--7 candidate7 candidategenesgenes

Xgwm533 Xgwm493

0.06 cM 0.02 cM

M2

BAC1

3B-3

34M

1

BAC33

3B-3

55

3B-3

00

Fhb1

Genes Identified in the Fhb1 Region

20 Kb

1 2 4 5 6 7 8 91011123

NO

RW

EGIAN

UN

IVERSITY OF LIFE SCIEN

CES

www.umb.no

The current challenge of mycotoxins in Norwegian cereals

Order of concern: Oats> spring wheat> winter wheat >barley

Priority disease in wheat (after powdery mildew)

Priority disease in oats: Often infected by F. langsethii whichproduces the tenfold more toxic T-2 and HT-2 toxins

Oats: In 2004 up to 25000 µg/kg of DON with severe germination problems in certified seed (cv. ’Bessin’ )In 2005 it was difficult to obtain acceptable quality oats for horse feed infected by T2- and HT-2 producing FusariumlangsethiiIn 2006 F. graminearum severely reduced germination ability in cv. ’Gere’

NO

RW

EGIAN

UN

IVERSITY OF LIFE SCIEN

CES

www.umb.no

The current project status (1) UMB:

UMB: Norwegian Research Council award, 2007-10: Mycotoxinprevention through resistant wheat and oats. 9 786000 NOK.

Includes a post doc from 2008-10, to be advertised autumn 2007.

A Ph.D. studentship (2008-2010(11)) from the Department of Plant and Environmental Sciences, UMB also to be advertised Autumn 2007

Sabbatical, 2008-09, Åsmund Bjørnstad

UMB Health Programme : a small grant for mycotoxin assessment in Ethiopia, toxin analyses in UMN.

Norwegian Crop Research Institute, Norwegian Research Council award, 2006-09: Molecular biology of the Fusarium pathogenicity, Dr. Corby Kistler UMN

9-month stay of post doc Erik Lysøe in UMN from Sept. 1st 2007.

21112005

Post doc work at Cereal Disease Laboratory

Supervisor: Dr. H. Corby Kistler

Post doc: Dr. Erik Lysøe, Norway

Working on genes related to pathogenicity and mycotoxin production in the

plant pathogen fungi F. graminearum

NO

RW

EGIAN

UN

IVERSITY OF LIFE SCIEN

CES

www.umb.no

Regulation of trichothecene mycotoxins

Microarray studies of a mutant in F. graminearum that produce

< 1% of the trichothecenes as the wild type

NO

RW

EGIAN

UN

IVERSITY OF LIFE SCIEN

CES

www.umb.no

The current project in UMB: (1) Marker assisted selection of Fusarium resistance in Norwegian wheat : Fhb1

• Introduce the Fhb1 gene from Sumai-3

• Poor agronomy!

• Employ marker assisted selection in Norway to Graminor

• Use the good diagnostic markers (linkage disequilibrium) for the 3BS and 5AS QTL

• A recombinant line from Hungary: ‘5’ (Sgv/Nobeokabozu/Mm/Sumai3 )

Contains the NB marker alleles in the Fhb1 locus

Is the NB gene the same as in ’Sumai-3’?

Can be answered when the gene is cloned in UMN

NO

RW

EGIAN

UN

IVERSITY OF LIFE SCIEN

CES

www.umb.no

(2) Genetic mapping of Fusarium resistance in wheat in UMB

The cross between Arina x NK93604:

• NK93604 (Norwegian spring wheat, moderate resistance)

Arina (Swiss winter wheat, moderate resistance)

Published in Genome and Crop Science 2006/07

Moderate resistance is also available in adapted gene pool and may be recombinedMarkers and QTL need to be verified for consistency

NO

RW

EGIAN

UN

IVERSITY OF LIFE SCIEN

CES

www.umb.no

Linkage map for AxNK93604 (group1) Black = SSRs, blue = AFLPs, green = DArTs

P36/M60-1590.0wPt-57072.9wPt-39833.3wPt-80726.3wPt-94026.4wPt-94236.6P36/M49-2589.9Xbarc26323.3wPt-215024.0P34/M49-9826.3P37/M59-17530.2Xwmc33637.9P34/M59-12939.5Xgwm26444.4P34/M49-16248.9wPt-390454.6P44/M60-26756.1Xbarc119**66.2Xgwm66673.2Xgwm164**73.3Xcfd59**74.2Xbarc28**75.8Xwmc611*76.3wPt-9757*91.1Xwmc30492.2Xwmc31293.6Xcfa212995.2P46/M62-94107.0XDuPw38120.3P35/M48-141124.1P34/M59-277129.6Xwmc59135.2wPt-5577140.0Xbarc213142.7Xbarc17145.8

1AP46/M50-328**0.0Xwmc495.3wPt-89308.7wPt-41079.0wPt-205210.1wPt-2230**11.2Xbarc128*13.2wPt-375318.4wPt-388924.4wPt-097424.6P44/M48-28727.3wPt-783329.0(Xbarc119)**41.3wPt-556248.1P46/M59-40549.0Xwmc61955.8wPt-567856.6P37/M47-17858.4(XDuPw532)65.1Xwmc61166.7P34/M59-19268.3Xgwm1168.7Xbarc13769.1Xbarc18769.7(Xbarc120)70.9wPt-137472.2P37/M61-17376.5XDuPw214.277.4P34/M61-45681.5wPt-816882.6wPt-345183.5Xbarc18183.7Xgwm27487.3XDuPw214.187.5Xbarc18891.7Xwmc76692.1wPt-140392.3wPt-697592.7P33/M59-47494.6P43/M62-40096.4wPt-3475107.3wPt-0944107.4Xwmc44119.5Xwmc367136.9wPt-6142wPt-4721138.7Xwmc728139.6P33/M60-102140.7

1B

P43/M48-85**0.0Xbarc14917.8Xwmc14723.6Xwmc43233.5Xbarc152**46.4wPt-3738**48.7Xgwm84866.0wPt-041371.5wPt-703871.6wPt-938071.9Xgwm19172.5Xgwm33773.4wPt-550374.2Xcfd59.275.3Xwmc59075.8(Xgwm603)76.9Xbarc162.2**78.8Xbarc162.186.5wPt-885490.0Xwmc3699.2Xcfd48.2107.1(Xgwm55)114.7Xgwm820118.9P45/M60-325123.7P36/M59-205**135.2Xgwm642**135.8P45/M60-118**137.3

1D

NO

RW

EGIAN

UN

IVERSITY OF LIFE SCIEN

CES

www.umb.no

Same (pleiotropic) QTL affecting both FHB (symptoms) and DON: 1A

1A

0

1

2

3

4

5

6

7

Xw

mc3

12X

cfa2

129

Xw

mc5

9w

Pt-5

577

Xba

rc21

3

LOD

FHB DON

NO

RW

EGIAN

UN

IVERSITY OF LIFE SCIEN

CES

www.umb.no

Phenotypic variation in anther extrusion

Line 20055: susceptible

Line 20054:resistant

Parent Arina: moderate

Parent NK:moderate

NO

RW

EGIAN

UN

IVERSITY OF LIFE SCIEN

CES

www.umb.no

The Arina x NK cross: Anther extrusion is available from both parents and can be combined

0

5

10

15

20

25

1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0

Anther extrusion rating

Perc

enta

ge o

f DH

line

s

Arina

NK93604(c)

NO

RW

EGIAN

UN

IVERSITY OF LIFE SCIEN

CES

www.umb.no

How is anther extrusion related to FHB and DON?

Old observations since 1921, but disregarded

Pierce (1976): Anthers are the best substrate for F. graminearum

Hypothesis: Extrusion removes the favourite source ofnutrition for the pathogen

If anthers are trapped, other mechanisms of resistance areneeded.

NO

RW

EGIAN

UN

IVERSITY OF LIFE SCIEN

CES

www.umb.no

If anthers are thrown out, low DON levels!

Regression of DON in 2002 on anther extrution 2005-06 for Arina x NK93604

y = 29,777e-0,2427x

R2 = 0,2823

0

10

20

30

40

50

60

70

80

0 1 2 3 4 5 6 7 8 9

AE

DO

N

Arina

NK93604

NO

RW

EGIAN

UN

IVERSITY OF LIFE SCIEN

CES

www.umb.no

Relation between anther extrusion and FHB: one identical QTL, two linked ones

7A

0

1

2

3

4

5

6

7

8

9

10

11

Xgw

m63

1P3

3/M

50-3

70X

gwm

276

XD

uPw

226

Xcf

a201

9P4

3/M

62-2

53X

wm

c525

LOD FHB AE

NO

RW

EGIAN

UN

IVERSITY OF LIFE SCIEN

CES

www.umb.no

Anther extrusion also reduces FHB in other breeding lines

Figure Breeding lines from spring wheat experiment 2006 (c.f. figure 3). Crosses with high FHB / low AE resp. low FHB / high AE.

0,001,002,003,004,00

5,006,007,008,009,00

BA

STIAN

AV

LE

BJA

RNE

ZEBR

A

Altar/Ae,squ//2*S

eri/3/Avle

Altar/Ae,squ//2*S

eri/3/Avle

Altar/Ae,squ//2*S

eri/3/Avle

Altar/Ae,squ//2*S

eri/3/Avle

Altar/Ae,squ//2*S

eri/3/Avle

Altar/Ae,squ//2*S

eri/3/Avle

Altar/Ae,squ//2*S

eri/3/Avle

Altar/Ae,squ//2*S

eri/3/Avle

Altar/Ae,squ//2*S

eri/3/Avle

Altar/Ae,squ//2*S

eri/3/Avle

Altar/Ae,squ//2*S

eri/3/Avle

T9040/WW

29323//SW

37262

T9040/WW

29323//SW

37262

T9040/WW

29323//SW

37262

T9040/WW

29323//SW

37262

T9040/WW

29323//Avle

T9040/WW

29323//Avle

T9040/WW

29323//Avle

T9040/WW

29323//Avle

T9040/WW

29323//Avle

T9040/WW

29323//Avle

0

10

20

30

40

50

60

70AE 0-9FHB %

NO

RW

EGIAN

UN

IVERSITY OF LIFE SCIEN

CES

www.umb.no

A recombinant line from China: ‘WSY’, Wangshubai/Sumai3/Yangangfanzhou (= Nobeokabozu) Strong anther extrusion!

NO

RW

EGIAN

UN

IVERSITY OF LIFE SCIEN

CES

www.umb.no

Fusarium resistance in oats derived from Avena sterilis L.

NO

RW

EGIAN

UN

IVERSITY OF LIFE SCIEN

CES

www.umb.no

Oats: an orphan crop

A cool season Northern crop, although grown world wide (> 3000 masl in Ethiopia)

Stress tolerant to poor soils, drought, waterlogging, less diseased

Positive nutritional features for humans and animals

Relatively little research

Biotechnology in its infancy, altthough transformation is possible (first achieved in UMN) and also markers (also in UMN)

NO

RW

EGIAN

UN

IVERSITY OF LIFE SCIEN

CES

www.umb.no

Infected panicles in the cv. ’Hurdal’ inoculated with F. graminearum: pink mycelium or empty spikelets with infected pedicel

NO

RW

EGIAN

UN

IVERSITY OF LIFE SCIEN

CES

www.umb.no

Infected panicles in the cv. ’Hurdal’ inoculated with F. graminearum: Degrees of seed discoloration and shrivelling

NO

RW

EGIAN

UN

IVERSITY OF LIFE SCIEN

CES

www.umb.no

Basic unanswered questions about FHB in oats

When are oats infected: at flowering or later?– Probably all the time after anthesis

How can infection be scored?– Not adequately investigated

Which pathogen species dominate?– Differes throughout the season. A succession?

– Artificial infection by F. langsethii has still proved impossible

Is there resistance available?

Passive mechanisms exist: plant height

Possibly panicle compactness, hulledness

Limited diversity in resistance in most oats

NO

RW

EGIAN

UN

IVERSITY OF LIFE SCIEN

CES

www.umb.no

Possible measures of Fusarium infection in oats.

Fusarium Head Blight (Type 1 -2)

– Difficult in oat panicles, discoloration for many reasons

Freezer blotter test (Type 1-2)

– 100 harvested seeds tested individually on filter paper. Medium heritability.

Grain shrivelling, spikelet sterility, yield effects (Type 5-6)

– Has not been investigated

– Mycotoxin determinations: Often too expensive

NO

RW

EGIAN

UN

IVERSITY OF LIFE SCIEN

CES

www.umb.no

Susceptible left (cv. ‘Hurdal’), resistant right (Z615): not easy to see differences…

NO

RW

EGIAN

UN

IVERSITY OF LIFE SCIEN

CES

www.umb.no

Quick and dirty measure seed of infection: imbibe and freeze seed, thaw, allow fungus development under ultra violet light (seeds on filter paper in plastic boxes)

NO

RW

EGIAN

UN

IVERSITY OF LIFE SCIEN

CES

www.umb.no

Seed infection in oats: left susceptible cultivar ’Belinda’, right resistant ’615-4’

NO

RW

EGIAN

UN

IVERSITY OF LIFE SCIEN

CES

www.umb.no

Freezer blotter test in oats after inoculation with Fusarium culmorum.

Commercial cvs. 20-40% infection, A. sterilis lines 5-12%

2001

05

101520253035

Lena Biri

Belind

aRoop

eB60

5XD921

-643Z59

5-7Z61

5-4Z56

2-3

2001

NO

RW

EGIAN

UN

IVERSITY OF LIFE SCIEN

CES

www.umb.no

Resistance appears highly heritable (’Hurdal x ’Z615-x’ cross) (In 2007 both in UMB and UMN)

Freezer blotter test after spray inoculation in field 2006 at Aas by Fusarium cumorum mixture.

0

5

10

15

20

25

Hurda

l/Z61

5-4Hur

dal/Z

615-4

Hurda

l/Z61

5-4Hur

dal/Z

615-4

Hurda

l/Z61

5-4Hur

dal/Z

615-4

Hurda

l/Z61

5-4Hur

dal/Z

615-4

Hurda

l/Z61

5-4Hur

dal/Z

615-4

Hurda

l/Z61

5-4Hur

dal/Z

615-4

Hurda

l/Z61

5-4Hur

dal/Z

615-4

HURDALBESSIN

B605X

% h

ighl

y +

med

ium

infe

cted

see

ds

susceptible

resistant

NO

RW

EGIAN

UN

IVERSITY OF LIFE SCIEN

CES

www.umb.no

Further poject work (1) The Ph.D. 2008-2010(11):Genetic studies of the resistance in wheat and oats

Studies of the infection process using a GM strain of Fusarium

Effect on yield (components)

The inheritance of resistance in oats

The ‘5’ x’ Avle’ cross in wheat

NO

RW

EGIAN

UN

IVERSITY OF LIFE SCIEN

CES

www.umb.no

Further project work (2) The Post.Doc.: Marker development in oats

For marker-assisted selection, markers FIRST need to be developed!

Orphan cereal- little research and less…

The DArT system is being established in oats with UMB and UMN support

Link this up with a previous map made in UMN

EST SSRs from oat (produced in UMN): 7031 ESTs gave 107 polymorphic loci and 51 now mapped in Kanota x Ogle cross

Retro-transposon based systems have been developed in Finland (’Aslak’ x ’Matilda’ cross)

NO

RW

EGIAN

UN

IVERSITY OF LIFE SCIEN

CES

www.umb.no

Questions and challenges

UMN funding for the collaboration!

Funding for guest researcher visits in UMB?

To develop more on the health aspects in oats as a food (UMN medical School already has links with University of Oslo)

Possible exchange visits by non-Norwegian students: to facilitate visa questions etc.

The Norwegian side is funded for some years (also FP7 will be invoked)

Steps towards a Nordic collaboration (NKJ) also on oatmapping