Mycotoxin Prevention in Cereal Crops by Enhanced Host Plant … · 2007-10-16 · 2111 2005...
Transcript of Mycotoxin Prevention in Cereal Crops by Enhanced Host Plant … · 2007-10-16 · 2111 2005...
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