Inoculation of tomato plants with rhizobacteria enhances the performance of the phloem-

feeding insect Bemisia tabaci Roee Shavit1,2, Maya Ofek-Lalzar3, Saul Burdman2 and Shai Morin1

1Department of Entomology, The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot, Israel.2Department of Plant Pathology and Microbiology, The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot, Israel.3Institute of Soil, Water and Environmental Sciences, Agricultural Research Organization of Israel, Bet Dagan, Israel.

Pseudomonas fluorescens

WCS417r

Two forms of induced resistance that are systemically expressed in the plant

SAR ISR

et al,. 2009) (Pieterse

Callose deposition

Enhanced expression of JA/ET-responsive genes

Pseudomonas fluorescens

WCS417rPieterse et al., Nature Chemical Biology, 2009

Hypothesis and complexity

• As JA/ET are key regulators of both ISR and plant defenses against herbivorous insects, it has been hypothesized that ISR induced by PGPR should benefit plants in their battle against insect herbivores.

• Indeed, studies with A. thaliana plants that were pre-inoculated with the PGPR P. fluorescens WCS417r showed that the growth and development of the generalist caterpillar Spodoptera exigua was negatively affected by the PGPR treatment (Van Oosten et al. 2008).

• However, it was recently found that P. fluorescens WCS417r has a positive effect on the performance (weight gain and intrinsic rate of increase) of the generalist phloem-feeding aphid Myzus persicae on A. thaliana (Pineda et al., 2012).

Broad host range, extending over 600 plant species from over 70 plant families including many

crop species

Bemisia tabaciVirus vector Physiological

disorders Honeydew excretion

Chlorosis

SA

JA

Kempema et al., 2007. Plant physiology 143: 849-865

Zarate et al., 2007. Plant physiology 143: 866-875

A. thaliana defense responses to B. tabaci

Arabidopsis mutants

SA

JA

Higher nymphal

development

Lower nymphal

development

Tomato defense responses to B. tabaci

Puthoff et al. (J. Chem. Ecol., 2010, 36:1271–1285)

Gene Protein Pathway

Pseudomonas fluorescens

WCS417r

We hypothesized that the relationship between the players in the B. tabaci - tomato - P. fluorescens system will add a general perspective to the complexity reported for the M. persicae - A. thaliana - P. fluorescens system.

(I) Control plants, not inoculated with P. fluorescens and without B. tabaci infestation (P-/B- plants)

(II) P. fluorescens pre-inoculated plants without B. tabaci infestation (P+/B- plants)

(III) Non-inoculated plants with B. tabaci infestation (P-/B+ plants);

(IV) P. fluorescens pre-inoculated plants with B. tabaci infestation (P+/B+ plants).

Reproductive performance of B. tabaci on P. fluorescens WCS417r pre-

inoculated and non-inoculated tomato plants

0

10

20

30

40

50

60

70

P. fluorescens Control

Nu

mb

er

of

eg

gs N.S.

0

0.2

0.4

0.6

0.8

1

P. fluorescens Control

Nym

ph

su

rviv

al

*

0

0.05

0.1

0.15

0.2

0.25

0.3

P. fluorescens ControlPro

po

rtio

n o

f e

me

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g

ad

ult

s a

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r 1

7 d

ays

*

Salicylic Acid Pathway

SA

PR2PR5

Jasmonic Acid/Ethylene

Pathways

JA

PI1

ET

PI2

The expression level of SA and JA/ET responsive genes transcripts in tomato before and after B.

tabaci infestation

‘Day 0' (before B. tabaci infestation) ‘Day 1' (the effect of 24h of adults' feeding and egg laying) ‘Day 6' (the beginning of egg hatching, minimal infestation effect) ‘Day 12' (presence of B. tabaci 2nd and 3rd feeding nymphs)

Gene

'day 1' 'day 6' 'day 12'

P-/B- P-/B+ P+/B- P+/B+ P-/B- P-/B+ P+/B- P+/B+ P-/B- P-/B+ P+/B- P+/B+

PI-I

1.00 2.50 1.21 1.33 1.19b 0.53ab 0.53ab 0.22a 1.00 1.07 0.66 0.88

PI-II

1.00 4.37 1.24 3.64 1.00ab 1.66b 0.12a 0.16a 1.00ab 3.61b 0.12a 4.50b

PR-2

1.00a 4.45b 1.95ab 1.93ab 1.00ab 1.17b 0.64ab 0.52a 0.7 0.84 0.44 0.52

PR-5

1.00a 5.44b 0.99a 1.61ab 1.00 0.94 0.31 0.93 1.00 0.26 0.19 0.23

The expression level of SA and JA/ET responsive genes transcripts before and after B. tabaci

infestation

Eggs II,III InstarsAdults

JA/ET JA/ET

SA

JA/ET ?

S1 S2 S3

P-/B-‘day 0’

S1 S2 S3 S1 S2 S3 S1 S2 S3 S1 S2 S3 S1 S2 S3

P+/B-‘day 0’

P-/B-‘day 12’

P-/B+ ‘day 12’

P+/B-‘day 12’

P+/B+ ‘day 12’

Pseudomonas fluorescens

WCS417r

Denaturing gradient gel electrophoresis (DGGE) profiles of the bacterial communities of the tomato

rhizosphere

Cluster analysis of tomato rhizosphere bacterial community

patterns

Group I – Non-inoculated plants before infestation (‘day 0’)

Group II – Pre-inoculated, infested (‘day 12’) and un-infested (‘day 0‘, ‘day 12’) plants

Group III – Non-inoculated, infested and un-infested plants (‘day 12’)

P-/B+ ‘day 12’ S1

P-/B+ ‘day 12’ S2

P-/B+ ‘day 12’ S3

P-/B- ‘day 12’ S2

P-/B- ‘day 12’ S3

P+/B- ‘day 12’ S2

P-/B- ‘day 12’ S1

P+/B- ‘day 12’ S1

P+/B- ‘day 12’ S3

P+/B+ ‘day 12’ S2

P+/B+ ‘day 12’ S1

P+/B+ ‘day 12’ S3

P+/B- ‘day 0’ S1

P+/B- ‘day 0’ S2

P+/B- ‘day 0’ S3

P-/B- ‘day 0’ S3P-/B- ‘day 0’ S2

P-/B- ‘day 0’ S1

74

9196

94

86

97

100

82

95

9995

0.02

Time P = 0.0004Bacteria P = 0.015Insect P = 0.38

The levels, in P. fluorescens WCS417r pre-inoculated and non-inoculated plants of soluble

carbohydrates, N (nitrogen), and C (carbon)

Nutritional Content

Plant Treatment

Soluble Carbohydrates (mg/g

leaf FW SE)

N % (DW SE)

C % (DW SE)

C/N Ratio

P. fluorescens WCS417r

2.49 ± 0.15 6.08 ± 0.05 41.11 ± 0.51 6.76 ± 0.06

Control 2.14 ± 0.12 6.27 ± 0.06 42.82 ± 0.54 6.83 ± 0.12

P-value 0.047 0.035 0.023 0.60

Why do generalist phloem feeders respond differently than other insect herbivores to pre-inoculation of plant hosts with P. fluorescens WCS417r?

Alternative explanations• Down-regulation of both ET-responsive genes and JA-responsive

genes (to a lesser extent) in P. fluorescens WCS417r pre-inoculated plants (suppressed expression of both ABA1 and MYC2 in plants infested with M. persicae or PI-I, PI-II and PR-2 in plants infested with B. tabaci).

• A possible manipulation by P. fluorescens of the plant quality (in

terms of suitability for B. tabaci) through an indirect effect on the rhizosphere bacterial community (suppression of the activity of pathogenic and non-pathogenic microorganisms by microbial antagonism).

• Root colonization by P. fluorescens WCS417r seems not to promote tomato growth or nutrient uptake directly.

Yissumit A – (2008-2009)Yissumit B - (2010-2011)

Israel Science Foundation