Introgression of type IV trichomes and zingiberene into tomato from S. habrochaites,

LA2329: Current statusJohn Snyder, Mohammad Dawood and Ammar Al-Bayati

University of Kentucky

Lexington, KY

snyder@uky.edu

“Figure 1. TYLCV distribution pattern among B. tabaci B and Q biotypes in China. The survey was

carried out in the field season of 2009. The white and red dots represent B. tabaci B and the Q biotype, respectively. The black portion of the dots denotes TYLCV-infected B. tabaci population and its respective infection rate. Inset: Outline map of China showing all the provinces. Surveyed provinces are highlighted in a blue shade.doi:10.1371/journal.pone.0034817.g001”

Pan H, Chu D, Yan W, Su Q, Liu B, et al. (2012) Rapid Spread of Tomato Yellow Leaf Curl Virus in China Is Aided Differentially by Two InvasiveWhiteflies. PLoS ONE 7(4): e34817

Project Objectives

• Transfer the production of high levels of zingiberene and high type IV trichome density from the wild tomato Solanum habrochaites to the cultivated tomato using conventional breeding methods

• Recover good reproductive fitness

• Evaluate resistance to spidermites and other arthropod species in the breeding population

Luckwill, 1943

Trichome diversity in Lycopersicon

Luckwill, 1943

Type IV is present on S. habrochaites

(L. hirsutum) and absent on S.

lycopersicum (L. esculentum)

Type V is present on S.

lycopersicum (L. esculentum)

and absent on S. habrochaites

(L. hirsutum).

The morphology of the type VI gland

differs between the two species.

S. lycopersicum

S. habrochaites

0

200

400

600

800

1000

1200

1400

1600

To

tal N

um

ber

of

Ap

hid

s

Accesssion Number

ES

HS

S

MR

HR

R

Aphid Resistance of S. habrochaites

accessions Beijing, 2002

In previous tests, LA2329 had

intermediate densities of type IV

and type VI trichomes.

LA2329 was resistant to

B. argentifollii

Primary methods

• Crossing scheme is a modified backcross

• 7-epizingiberene measured by gas chromatography (GC)

• Trichome density assessed visually at 50X on abaxial surfaces

min0 2 4 6 8 10

counts

0

20000

40000

60000

80000

100000

120000

FID1 A, (6-23-16\B116.D)

0.3

42

0.4

55

0.4

83

0.5

16

2.6

34

3.1

71

4.1

39

4.8

62

4.9

99

5.2

46

5.3

84

5.7

60

5.8

30

5.9

99

6.5

95

6.8

26

6.9

15

7.2

34

7.3

31

7.5

32

7.6

18

7.7

34

7.8

64

7.9

46

8.1

68

8.2

75

8.4

54

8.6

26

8.7

31

8.8

97

8.9

51

9.0

81

9.8

04

10.5

00

10.9

48

11.2

25

11.4

69

To measure zingiberene, 10 to 20 cm² of leaf tissue is collected and placed in a scintillation vial. Two ml of hexane + internalstandard are added, mixed and then an aliquot is removed for analysisby GC. Leaf area is determined from scanned images of leaf tissue. Zingiberene is expressed as GC area units/ cm².

Internal Std.

Scan of leaflet tissue

~10 % self –fruit set, 10 %

High type IV density

Currently at BC5

Results

LA2329 BC4F2Tomato

IS

IS

ISMT MTZGB

ZGB

• Presence of zingiberene is dominant. High concentration is recessive

• Plants with very low or 0 concentrations of monoterpenes appear to be homozygous for zingiberene synthase

• Transgressive segregation for monoterpenes

Results for BC4F2

• For the BC4F2, the segregation for presence:absence of zingiberenewas 277:115 (2.4:1), X²=3.93

• Concentration of zingiberene ranged from 0 to 1.52 X 10E8 AU/cm² of leaflet tissue

• Mean was 1.0 X 10E7

• Most but not all set fruit in the field

0

10

20

30

40

50

60

70

5.00E+06

1.50E+07

2.50E+07

3.50E+07

4.50E+07

5.50E+07

6.50E+07

7.50E+07

8.50E+07

9.50E+07

1.05E+08

1.15E+08

1.25E+08

1.35E+08

No

. of

See

d/F

ruit

Zin

gib

ere

ne

co

nce

ntr

atio

n (

FID

are

a u

nit

/cm

²)

Genotype

Zingiberene concentration (solid bars, left axis) and seed set (hatched bars, right axis) for 27 selected BC4F2 interspecific genotypes. Of ~550 BC4F2 interspecific hybrids evaluated for type IV trichome density and zingibereneconcentration, the 27 depicted in this graph were those having type IV density and zingiberene concentration approximately equal to or greater than those on the wild resistant donor parent S. habrochaites LA2329. Seeds were obtained from 16 hybrids. Thus frequency of individuals in the BC4F2 having type IV trichome density and zingibereneconcentration similar to those of wild parent, and had the ability to produce seeds was nearly 3%.

O32 fruit - 2017~3.4 x 107 zingiberene/cm2

High IV density~25 seed per fruit

High zingiberene, no IV’s

LINE N Zingiberene (AU/cm²)

H21 29 2.2E+07 A

G85 33 1.7E+07 B

G82 35 1.5E+07 B

H20 34 1.1E+07 C

Zingiberene differences among four BC3F4 families

• Reproductive Fitness

Dwarfing can be extreme

Exerted Stigma!!!BC4F3January 2018

More stigma problems!

Reproductive fitness

• Seeds

• Seeds per fruit

• No. of flowers

• Flowers per truss

• Pollen

• Stigma problems?

• Seed germination

• Pollen stainability is significantly correlated with fruit set (r=0.82).

• Seed germination is a problem.

Some valuable phenotypes?

• Small Stature (~20 inches tall

• Erect, 1 major branch (no staking?)

• Very concentrated set (~50 fruit in two weeks)

• Other phenotypes with value• Very high brix (2x normal)

• High flavor

Columnar Plants

Thanks to:

Belinda Labadie

Mohammad Dawood

Ammar Al-Bayati

Dr. Barbara Liedl

Trichomes on wild and

cultivated eggplant

snyder@uky.edu

A NEW SESQUITERPENE ALCOHOL FROM WILD TOMATO

REPELLED THE TWO-SPOTTED SPIDER MITE, TETRANYCHUS

URTICAE

Mohammad H Dawood: PhD Student

Advise by Dr. John Snyder

Horticulture Department

April 6th 2018

INTRODUCTION• Tomato Solanum lycopersicum is one of the most

economically important world-wide grown vegetable.

• However, tomato is a host for numerous pests that

reduce productivity.

• Tomato breeders have focused more on increasing fruit

quantity and quality and less on enhancing crop

resistance to herbivores.

Ric Bessin,

UKY

• Because of many pathogens and pests, tomato cultivation

in most cases considered a high risk crop.

• Because it needs intensive use of insecticides and

pesticides.

ashmkn.com

Levels of cultivar insect resistance are not enough to

decrease the amount of insecticides applied.

Alternative approach:

decrease the chemical sprays, and

allows for a smaller environmental impact.

Why wild type?

S. habrochaites

Do other compounds present in trichome secretions have

repulsive activities against the two spotted-spider mite

Tetranychus urticae, similar to that of zingiberene?

OBJECTIVES

Ric Bessin,

UKY

MATERIAL AND METHODS

Plant materials

• LA2329 accession Solanum habrochaites

2016-2017

• Florida Materials 2015

Spot test on TLC plate,

UV illumination

Column Chromatography

Silica gel,

MTBE,

Hexane

GC/MS

(GC)

SPIDER MITES (TETRANYCHUS URTICAE

KOCH) AND BIOASSAY

Spider mites

nurtured on bean

Phaseolus vulgaris

L.

Hexane

zingiberene

RESULTS AND DISCUSSION

Florida materials

LA2329 F accessionLA2329 A accession

After Separation

CONCLUSION

• LA2329 (A) which has ZGB-OH had activity against

spider mite behavior more than LA2329 F that included

ZGB only.

• After separation of two major components, the ZGB-OH

affected spider mite behavior more than ZGB.

THANKS

Department of Horticulture

The Role of Trichome Secretions and Densities in Spider Mite Behavior for an Interspecific Population of Tomato

Advisor Dr. John SnyderPresented by

Ammar AL-Bayati, PhD candidate

Overview

Introduction

Hypothesis

Objectives

Materials and Methods

Results

Conclusion

Introduction

There is significant necessity for assay platforms to assess and identify levelsof resistance and insect performance, as demonstrated by recent publicationsreporting this subject.

Our hypothesis: Are behavioral differences of mites associated with thepresence or absence of leaf compounds and trichome densities?

Objectives

1- Observe the differences of mite behavior among a range of hybrids compared to

the positive and negative controls.

2- Identify the interactions between genotypes and leaf surfaces with spider mites

with respect to presence and absence of type IV trichomes.

3- Determine the interaction of zingiberene content on mite performance.

Plant Materials and Methods

An interspecific populationbetween Solanum lycopersicumand a wild relative, Solanumhabrochaites LA2329, used inthis research was maintained inthe greenhouse and field insummer 2017 at the Universityof Kentucky, Lexington, Ky.

13 tomato genotypes (BC3F3 &BC3F4) were selected based onpresence or absence of trichometype IV, and ZGB.

Whole Leaf Bioassay

Parameters Measured

Leaflet position (1-5) and surface infested by mites

Mite webbing score and Feeding damage score (0-3)

Leaflet position of webbing and feeding damage (1-5)

Egg density (Egg No. per cm2 of total leaf area)

ZGB and monoterpene (MTP) content using gas chromatography (GC-FID).

Type IV trichome counting for abaxial and adaxial surfaces using a microgrid.

Data were analyzed by SAS using GLM & CORR procedures.

Results

Mean (n=3) for Trichome Type IV Density per 4.3 mm2 Microgrid

and Phytochemical concentration per cm2

Genotype Generation

Type IV

Density

Lower

Surface

Monoterpene

(AU/cm2 leaf

area)

Zingiberene

(AU/cm2 leaf

area)

LA2329 Wild Parent 45 0.0E+00 4.5E+06

LA2329OH Wild Control 65 0.0E+00 8.6E+06

PI127826 Wild Control 350 0.0E+00 1.2E+07

W126 Tomato 0 2.3E+06 0.0E+00

W129 Tomato Control 0 3.1E+05 0.0E+00

W160 Tomato Parent 0 4.8E+05 0.0E+00

X155 BC3F3 203 6.0E+06 0.0E+00

X166 BC3F3 173 0.0E+00 1.1E+07

X71 BC3F3 208 1.4E+05 5.7E+07

Z116 BC3F3 116 6.6E+06 0.0E+00

Z120 BC3F3 57 1.8E+06 0.0E+00

Z161 BC3F4 0 0.0E+00 2.6E+06

Z58 BC3F4 0 0.0E+00 4.8E+06

Correlations among parameters after Combining Variables by Surface

Results

Spider mite Webbing and Feeding Damage

W129

Z58W

160

X71

W126

Z120Z161

LA2329

LA2329O

H

Z116X155

X166

PI127826

Tomato Genotypes

0.0

0.5

1.0

1.5

2.0

2.5W

eb

bin

g S

co

re,

Day 7

Results

Spider mite Webbing and Feeding Damage

Results

Two-spotted spider mites with eggs

Tetranychus urticae Koch

https://agfax.com/2017/08/04/iowa-corn-soybeans-control-options-for-twospotted-spider-mites/

W129

W160

W126

Z58Z120

Z161

Z116

LA2329

X155

X166

X71

LA2329O

H

PI127826

Tomato Genotypes

0

2

4

6

Day 7

Egg D

en

sity

(N

o/c

m2 l

eaf

are

a),

Results

Susceptible line

Web

Resistant Line

Results

Conclusion

Based on reduced mite success on some of the genotypes, we concluded:

Resistance has been successfully transferred from the wild to the interspecific population.

This bioassay demonstrated behavioral differences of mites associated with the presence or absence of

leaf compounds and trichome densities.

New plant versions that can produce toxic or repellent chemicals on their own can defend themselves

against certain types of arthropods and insects which in turn reduce or eliminate extensive synthetic

pesticide utilization and cost.