determining the impacts of acylsugar characteristics on...
TRANSCRIPT
Determining the Impacts of Acylsugar Characteristics on
Whitefly Response
Brian Leckie, Darlene DeJong, Martha Mutschler Dept. of Plant Breeding and Genetics
Cornell UniversityOctober 12, 2011
Acylsugars and Insect Resistance
• Acylsugars have broad spectrum insect resistance activity
• Acylsugars produced by a number of wild Solanaceous species
• Breeding programs transferring ability to produce acylsugars to crops for insect control S. pennellii LA716
Acylsugar chemotypes• Glucose or sucrose
• Substitutions vary• 3 or 4 fatty acids • Straight chain fatty acids
C8, C10, or C12• Odd or even carbon
number branched chain fatty acids
• Fatty acids 4 or 5 to 11 or 12 carbons long
Variation of Acylsugars among S. pennellii Accessions
Distribution of S. pennellii Acylsugar Chemotypes
Shapiro, Steffens, Mutschler, 1994 Biochem System & Ecol
• Northern accessions• blend of acylglucose and
acylsucrose• levels half of the high
producing accessions• ~ 55% i-C5)
• Southern accessions • highest levels of acylsugars• chiefly acylglucoses• ~ 40% i-C4
Why the high levels of diversity?
Could Fatty Acid Profile Impact Acylsugar Efficacy
Effect on Spruce Budworm Oviposition(Grant et al. 2000, Environ. Entomol. )
Effect on green peach aphid settling behavior(Greenway et al. 1978, Entomol Exp Appl)
Tests of free fatty acids suggest possible differences in insect perception/response
Previous Studies Testing Sugar Ester Chemotypes
• Initial studies evaluated synthesized sugar esters as contact insecticides (Chortyk et al. 1996)• Straight chains • Sugar moieties• Identified sucrose esters with 7-8 carbon fatty acids as most
effective
www.naturalforcesllc.com
• Glucose octanoate and sucrose octanote show high residual activity on Mustard aphid (Koul et al. 1996)
• A synthetic acylsugar, SucraShield, available as a contact biopesticide
• A mixture of mono-, di- and tri-substituted sucrose octanoate
Benchmark Acylsugar Line: CU071026• Tomato breeding program
transferred acylsugar production to tomato lines
• Current benchmark line is BC6derived line CU071026
• Contains ca. 10% of the S .pennellii genome
• Accumulates 15% of total acylsugar level of S. pennellii
• Produces acylsucrose exclusively• ~ 40% iso-C5 and 40% n-C12• Controls silverleaf whitefly in past
field trials in Florida
Project Objectives
• Identify QTL for modifying acylsugars• Level• Chemotype
• Determine level of acylsugars needed for effective control in breeding lines
• Evaluate differences in acylsugar chemotypes and their impacts on target insects
• Produce breeding lines with different levels and/or types of acylsugars for in planta tests of their impacts on insects
Generation of Backcross Population
12cM
84cM
7cM
18cM
9cM
I
12cM
84cM
7cM
18cM
9cM
2 3 4 5 6
7 8 9 10 11 12
Acylsugar parental line: CU 071026
Generation of Backcross Population
12cM
84cM
7cM
18cM
9cM
I 2 3 4 5 6
7 8 9 10 11 12
F1: CU 071026 X S. pennellii LA716
Generation of Backcross Population
12cM
84cM
7cM
18cM
9cM
I
12cM
84cM
7cM
18cM
9cM
2 3 4 5 6
7 8 9 10 11 12
BC1F1: CU 071026 X (CU 071026 X S. pennellii LA716)
BC1F1 Mapping/QTL Population
• BC1F1 population consisting of 154 plants + controls
• Plants phenotyped for acylsugar level, sugar moiety, and fatty acid profiles
• 95 PCR based markers placed in segregating regions• Chromosomes 3 and 10 each split into 2 linkage groups by the
CU071026 introgressions on those chromosomes
• Map generated with Map Manager QTX
• QTL Network 2.1 used for• Composite interval mapping• Two-way epistatic interactions
• Three-way epistatic interactions analyzed by ANOVA
QTL for Total Acylsugar
Trait Chromosome of QTL (position)
AdditiveEffecta
Support Interval F-valueb %
Explained
Total Acylsugar
4 (19.7) 0.36 1.0 - 32.9 14.4 13.9
5 (76.3) -0.15 68.4 - 84.3 14.0 17.2
6 (50.2) 0.18 44.7 - 61.2 17.5 13.4
10.1 (10.3) 0.16 0.0 - 10.3 11.1 10.7
11 (69.6) 0.11 52.5 - 80.9 8.6 6.0
a Positive values indicate that the S. pennellii allele has positive effects on the value of trait.b 0.01 alpha level F-value thresholds for total acylsugar was 7.7.
Cage Study to Test Acylsugar Level vs. Control
12 Feet
48 F
eet
• 6 acylsugar lines differ in acylsugar level and presence of chr 6 and 10 QTL
• Lines and controls greenhouse grown, and transplanted at 5 weeks
• Two cages used to replicate experiment
• Bemisia argentifollii infested plants placed in cages after 4 weeks
• Plants sampled weekly for 3 weeks
• Insects counted on 4 leaves per/plant/week
• Acylsugar assay performed
• Trichomes counted
Chromosome 6/10 Sibling Lines For Cage Experiment
Markers*4.
0140
5.00
805.
0190
5.02
655.
0350
5.04
405.
0510
5.06
20
6.06
256.
0645
6.06
906.
0730
6.07
406.
0748
6.08
006.
0805
6.08
076.
0830
6.09
256.
0980
6.10
10
8.05
508.
0670
10.0
250
10.0
329*
*10
.035
010
.041
5
chr.
9
Seed Source
SSR
43
TG62
3TG
441
2g01
410
SSR
115
3g55
120
4g24
830
Pto
4g24
690
1G12
060
5G21
160
1g65
700
5g22
620
5G22
820
TG27
9SP 1g
2990
01g
1864
01G
1687
01G
6952
3TG
221
SSR
38
CT1
48
2g33
990
pXag
tSS
R 2
48U
2417
00
SW5
NC123S 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 +
CU071026 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 -
101313 3 3 1 1 3 3 3 3 1 1 1 1 1 1 1 1 1 1 1 1 1 3 3 1 1 1 1 +101307 3 3 1 1 3 3 3 3 3 3 3 3 1 1 1 1 1 1 1 1 1 3 3 1 1 1 1 +101302 3 3 1 1 3 3 3 3 3 3 3 3 3 3 3 3 3 3 1 1 1 3 3 1 1 1 1 +
101308 3 3 3 3 3 3 3 3 1 1 1 1 1 1 1 1 1 1 1 1 1 3 3 3 3 3 3 +101301 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 1 1 1 1 1 3 3 3 3 3 3 +101310 3 3 3 3 3 3 3 3 1 1 1 1 3 3 3 1 3 3 3 3 3 3 3 3 3 3 3 +
Chromosome: 4 5 6 8 10 9
1 = Homozygous S. lycopersicum alleles
3 = Homozygous S. pennellii alleles
* = Locations based on EXPEN 2000 map** = Estimated location
Lines Description
Regions Introgressed
Leaflet Average
Chr 6 Chr 5+10Total
AcylsugarTrichomeDensity
Mean Whiteflies
101305 Tomato - - 1.9 E 0.0 C 82.2 A
101313 SW5 AS line - - 6.0 D 77.4 B 11.4 B
101307 SW5 AS line - - 6.4 D 73.7 B 11.1 B
CU071026 Benchmark - - 7.5 D 98.0 A 9.6 CD
101308 SW5 AS line - + 10.9 C 78.6 B 2.4 CD
101302 SW5 AS line + - 11.1 C 105.5 A 1.5 DE
101301 SW5 AS line + + 14.5 B 102.8 A 0.3 E
101310 SW5 AS line + + 17.9 A 100.4 A 0.5 E
Means followed by different letters differ significantly (p < 0.05)
2011 Insect Cage Study Results
2011 Insect Cage Study Results
Lines Description
Regions Introgressed Leaflet Average
Chr 6
Chr 5+10 Eggs 1st
instar2/3
instar4th
InstarPupal Cases
Clustered eggs (%)
101305 Tomato - - 45.5 A 26.5 A 6.9 A 2.8 A 0.6 A 2
101313 SW5 AS line - - 8.3 B 2.9 B 0.2 B 0.0 B 0.0 B 54
101307 SW5 AS line - - 6.8 BC 4.0 BC 0.3 B 0.0 B 0.0 B 43
CU071026 Benchmark - - 5.4 CD 3.9 CD 0.3 B 0.0 B 0.0 B 57
101308 SW5 AS line - + 1.8 DE 0.6 CDE 0.0 B 0.0 B 0.0 B 41
101302 SW5 AS line + - 1.1 DE 0.4 DE 0.0 B 0.0 B 0.0 B 68
101301 SW5 AS line + + 0.3 E 0.1 E 0.1 B 0.0 B 0.0 B 60
101310 SW5 AS line + + 0.5 E 0.1 E 0.0 B 0.0 B 0.0 B 49
Means followed by different letters differ significantly (p < 0.05)
QTL Modifying Sugar Moiety of Acylsugars
Chromosome MeanAcylglucosea
Percent Acylglucose 3 4 11
LP LP LP 6.5 A 36.3
LP LL LP 0.5 B 5.3
LP LP LL 0.3 C 0
LL LP LL 0.0 C 0
LL LL LP 0.0 C 0
LP LL LL 0.0 C 0
LL LP LP 0.0 C 0
LL LL LL 0.0 C 0a µmol/gram dry weight
Total Acylsugar (µmol/g)
0 10 20 30 40 50 60 70
Acy
lglu
cose
(µm
ol/g
)0
2
4
6
8
10
12
14
16
Acylglucose vs. Total Acylsugar
Means followed by different letters differ significantly (p < 0.05)
LL = locus with homozygous S. lycopersicum alleles LP = heterozygous locus
QTL Increasing Extended Branched Fatty Acids
Chromosome of QTL Average Fatty Acids (%) %
Extended Chain2 5 i-C4 ai-C5 i-C5 n-C10 i-C11 n-C12
LL LL 1.5 AB 11.6 A 45.5 A 5.2 A 0.1 C 36.1 B 41.4 BLL LP 0.6 C 6.0 B 39.6 B 6.5 A 0.1 C 47.1 A 53.7 ALP LL 1.7 A 12.8 A 41.5 AB 4.9 A 15.4 B 23.8 C 44.1 BLP LP 1.1 BC 7.9 B 38.5 B 0.7 B 45.4 A 6.4 D 52.5 A
LL = locus with homozygous S. lycopersicum alleles; LP = heterozygous locus
Acylsugar Isolation and Purification from S. pennellii Accessions
• Seed of S. pennellii accessions obtained from Tomato Genetics Resource Center
• Multiple plants of each S. pennellii accessions grown in greenhouse
• Plants tested for:• moiety with acylsugar assay• fatty acid profile with GS/MS
• Tissue washed and purified with dry column vacuum chromatography
Chromatogram Plots
5 10 15 20 minutes
0.0
0.5
1.0
1.5
MCounts
0.0
0.5
1.0
1.5
MCounts
0.0
0.5
1.0
1.5
MCounts
35WAX.SMS Ions: 102.0:103.0+116.0:117.0+130.0:131.0+144.0:145.0+158.0:159.0+172.0:173.0+186.0:187.0+200.0:201.0+214.0:215.0 Filtered40:350
36wax.sms Ions: 102.0:103.0+116.0:117.0+130.0:131.0+144.0:145.0+158.0:159.0+172.0:173.0+186.0:187.0+200.0:201.0+214.0:215.0 Filtered40:350
37wax.sms Ions: 102.0:103.0+116.0:117.0+130.0:131.0+144.0:145.0+158.0:159.0+172.0:173.0+186.0:187.0+200.0:201.0+214.0:215.0 Filtered40:350
i4:0
ai5:0
i5:0
n10:0n12:0
i11:0
ai5:0
i5:0
n10:0n12:0
i11:0
i10:0
Acylsugars from S. pennellii Accessions Tested on Whitefly
• Acylsugar suspensions applied to tomato leaves with airbrush
• Mating pairs of SLW whitefly evaluated in clip cages
• Survival • Oviposition• Development
• Tests on Thrips being performed at NCSU
• Field cage evaluations planned for summer 2012 and 2013
Impact of acylsugar chemotypes on efficacy
CU 071026 & S. pennellii accessions
Acce
ssio
n
sub-
regi
on
% G
luco
se % Fatty Acids
i-C4 ai-C5 i-C5 i-C8 i-C10 n-C10 i-C11 n-C12
CU071026 - 0.0 0.8 17.9 41.6 0.0 0.0 0.9 0.1 37
LA1376 N 49.4 3.9 3.3 84.8 0.0 0.1 4.0 0.8 3.0LA2560 N 58.0 3.0 8.5 62.2 0.0 0.2 1.4 1.5 23.2
LA1732 S 95.6 88.7 6.9 1.3 3.2 0.0 0.0 0.0 0.0LA716 S 89.0 65.7 8.7 2.4 0.0 17.5 3.9 0.3 1.4
Current Work For Changing Fatty Acid Profile
• Progeny from selected BC1F1 plants will be trialed to:• Confirm fatty acid QTL on Chromosomes 2 and 5• Confirm fatty acid QTL on Chromosomes 3, 4, and 11
• Adding QTL to current acylsugar line using IL lines as donor of additional introgressions
• Populations being constructed to add QTL on chromosomes 3, 4, and 11
• Populations being constructed to add QTL on chromosomes 2 and 5
• As these lines are produced they will be tested for impact of alteration on infestation by whitefly and thrips and virus transmission
Acylsugar Cooperators and ContributorsMartha A. Mutschler
Mutschler Lab:Darlene DeJongStephen SouthwickRichard Gaisser
Andre Kessler (Chemical Ecology) Cornell UniversityResearch Associate Rayko Halitschke
Larry Robertson (Cage) USDA ARS
George Kennedy (Thrips) NCSU
This work supported in part with funds from – Hatch funds, the Vegetable Breeding Institute, Gates Foundation.