Efficient phenotyping method developed in controlled

environment for pre-breeding and breeding

Bacterial blight in field pea

Dr Pragya Kant- Horsham, Vic

Bacterial blight epidemic in Rupanyup, Vic, 2012

Overview

• Bacterial blight in Australia

– Significant limitation for Australian field pea industry

• Current screening methods

– Field nursery

– High throughput phenotyping method- New development

• Improving resistance into the future

– Introgression of land races – new resistance sources

– Marker assisted selection, Pathogen sequence analysis

2 APPS 2015

Causal pathogen: Pseudomonas syringae

• Highly virulent -pv. pisi Race 6 and pv. syrinage

• Endemic to all major field pea cropping zones

• Particular problem following frost damage

• Disease is highly seed borne, no chemical control

• Pathovar prevalence has changed with shift toward new

agronomic types (e.g Kaspa)

Two pathovars: Ps. pv. syringae and Ps. pv. pisi (1-8 Races)

3 APPS 2015

Major pathogen pv. syringae (Richardson and Hollaway 2011)

• More work needed to understand pv. syringae and pv. pisi Race 6

• Resistance need to develop in breeding material for these pathovars

• Crosses made to pyramid the partial resistances

• 2000 – now: mainly pv. syringae

Data from historic isolate collection at Horsham

• Before 2000: mainly pv. pisi (Race 3,6)

Emphasis

Pathogen occurrence

pv. syringae – resistance is quantitative

pv. Pisi Race 6 ?(No resistance genes have been identified in cultivars tested)

4 APPS 2015

Bacterial blight in field trials 2012

cv. Kaspa cv. PBA Percy cv. PBA Oura cv. Kaspa

5

Field screening nursery

• Field nursery conducted in

Wagga, NSW (> 800 lines)

• Yield loss trial conducted on

near-to-release varieties

• Infected stubble and spray

applied inoculum applied

• Inoculation co-ordinated with

frost events

• Mechanical plot damage to

encourage infection

% Yield loss

Both pisi race 6 syringae Mean yield loss Variety rating

PBA Pearl 11 8 0 6 R

PBA PERCY 9 2 16 9 R

OZP0903 10 16 16 14 MR

OZP1001 15 19 14 16 MR

PBA OURA 19 19 16 18 MR

OZP1101 13 30 14 19 MR

STURT 21 24 17 21 MS

KASPA 20 24 29 24 MS

PBA WHARTON 26 26 23 25 MS

MORGAN 28 27 26 27 MS

OZP1004 15 37 29 27 MS

PBA COOGEE 28 32 25 28 MS

PARAFIELD 31 30 24 28 MS

PBA GUNYAH 25 31 32 29 MS

YARRUM 36 51 30 39 S

MAKI 50 50 46 49 S

Yield loss due to bacterial blight damage 2011

Peter Kennedy and Kurt Lindbeck

6 APPS 2015

A new glasshouse screening method for Bacterial blight

Advantages:

• Efficient, rapid and reliable. Approx. 1

month from seed sowing to disease

scoring

• Utilizes the “Pulse Penetrant” as a

surfactant to improve infection

• Validated using single isolates, mixed

isolates and with the traditional stab

inoculation method

• Closely correlated with field

screening Screening of advanced field pea breeding material

7 APPS 2015

Methodology of Bacterial blight screen

• The P. syringae pv. syringae conc. 5.2 x 107 cfu/ml.

• P. syringae pv. pisi conc. approximately 1.4 x 108 cfu/ml.

• Pre-inoculated plants were kept under high light to ensure the

opening of stomata.

• Bacteria was grown in Nutrient Yeast Broth.

• Bacterial suspension was sprayed on seedling in glasshouse with

overhead misting and disease was assessed 7-9 days after

inoculation.

• The surfactant “Pulse Penetrant” was added to the suspensions

at 0.055% (v/v).

8 APPS 2015

Ratingsa Score Description

R 0 No signs of infection

R 1 Small dried lesions ≤5 mm in diameter on leaves

R-MR 2 Several dried lesions >5 mm in diameter on leaves

MR 3 Small dried lesions ≤5 mm in diameter on stem

MR-MS 4 Several water soaked lesions <5 mm in diameter on stem and tip damage

MS 5 Large water-soaked lesions up to one internode in length

MS-S 6 Large water-soaked lesion/s (approx. two internode in length)

S 7 Up to 60 % of plant necrotic tissue

S-VS 8 61 to 80 % of plant necrotic tissue

VS 9 81 % to 100 % of plant necrotic tissue

Disease rating scale used to assess severity of symptoms

caused by P. syringae pv. syringae and pv. pisi

aRatings R: resistant; MR: moderately resistant; MS:

moderately susceptible; S: susceptible; VS: Very susceptible.

9 APPS 2015

a a

b

c

0

1

2

3

4

5

6

7

8

9

No surfactant Tween (0.1%)

with misting

Pulse (0.05%)

without misting

Pulse (0.05%)

with misting

Mean

dis

ea

se S

co

re

Treatments

Effect of misting and surfactant on

disease development

Susceptible variety Kaspa

10 APPS 2015

1

A B

C D 11 APPS 2015

P. syringae pv. Pisi

A: Without misting

B: with misting

A: Without misting

B: with misting

P. syringae pv. syrinage

Symptoms in the glasshouse based protocol

are similar as observed in field

Symptoms in glasshouse Symptoms in field

12 APPS 2015

13 APPS 2015

Glasshouse and field correlation

Field ranking Field ranking

Gla

ssho

use r

an

kin

g

Gla

ssho

use r

an

kin

g

2012 2014

Phenotyping of diverse germplasm identified

new resistant genotypes

pv. pisi Race 6 pv. syrinage

Genotype Mean disease

scores

ATC2553 1.83

PBA Percy 2.75

ATC4900 3.08

ATC4983 3.25

ATC1498 3.5

ATC4391 3.5

ATC4122 3.75

ATC4984 3.75

ATC4985 3.92

ATC4622 4

PBA Oura 4.17

ATC3966 4.92

ATC1430 5

ATC4030 5.5

ATC5349 5.5

ATC3318 5.92

ATC5378 6

ATC5348 6.17

Kaspa 6.42

ATC1326 6.5

Genotype Mean disease

score

ATC1498 2.65

PBA Percy 3.33

ATC4985 3.38

ATC4622 3.5

ATC4900 4.13

PBA Oura 4.75

ATC4030 4.83

ATC4984 4.83

ATC3318 4.91

ATC1430 5.04

ATC5349 5.08

ATC2553 5.13

ATC4391 5.92

ATC4122 6.17

ATC4983 6.67

ATC3966 7

ATC5348 7.83

Kaspa 7.92

ATC5378 8

ATC1326 8.25

14

syringae isolates

Race 7

Race 3 pisi isolates

pisi isolates

P. viridiflava

P. cichorii

UniRef 90

Jason Shiller, P. Kant , M. Rodda,A. Stephens- Agribio

Whole genome sequencing of Bacterial

blight isolates

15 APPS 2015

VPRI Location Year Host Plant Comments Identity

22505 Plum (Prunus domestica ) Psy

32448 Impatients sp Psy

42146 NSW 1981 Citrus sinensis (Orange) Leaf Psy

42147 NSW 1978 Phaseolus vulgaris (French Bean) From spot on leaf Psy

42148 QLD <1985 ? Non-pathogenic; lemon -'ve Psy

42149 SA 1984 Pisum sativum (Field Pea) Stem/leaf Psy

42150 NSW 2003 Pisum sativum (Field Pea) Stem/leaf diseased plant Psy

42151 NSW 2003 Pisum sativum (Field Pea) Stem/leaf diseased plant Psy

42161 USA 1944 Pisum sativum (Field Pea) Ppi R2

42165 Vic. 1992 Pisum sativum (Field Pea) Pea seed Ppi R2

42162 USA 1975 Pisum sativum (Field Pea) Ppi R3

42166 NSW 1992 Pisum sativum (Field Pea) Ppi R3

42163 USA 1975 Pisum sativum (Field Pea) Ppi R4

42164 TAS 1976 Pisum sativum (Field Pea) Ppi R7

42167 NSW 2003 Pisum sativum (Field Pea) Stem/leaf diseased plant Ppi R6

42168 Vic. 2001 Pisum sativum (Field Pea) Ppi R6

42169 NSW 2003 Pisum sativum (Field Pea) Crop failure Ppi R6

42170 Crop seed sample 1993 Pisum sativum (Field Pea) Ppi R6

42171 VIC 1993 Pisum sativum (Field Pea) Ppi R6

42172 NSW 2003 Pisum sativum (Field Pea) Stem/leaf diseased plant P. viridiflava

42173 NSW 1987 Ranunculus sp. (Buttercup?) From spots on stem/leaf P. viridiflava

42174 NSW 1986 Lactuca sativa (Lettuce) P. cichorii

QTL Mapping of Bacterial Blight

Resistance

• 2 mapping populations – Kaspa x Parafield

• 101 RILs

• 458 markers

– Kaspa x PBA Oura

• 107 RILs

• 358 markers

• Environments – 3 glasshouse assay completed

– Future screening in both glasshouse and field

16 APPS 2015

Pulse Molecular Maker Project- Agribio

Summary

• Bacterial blight is a significant biotic stress on field pea production

in Australia

• A new high throughput screening protocol has been established

for evaluating bacterial blight in field pea breeding material

• Land race germplasm identified with improved resistance has

been delivered to breeders

• QTL analysis conducted on two mapping populations, with further

field and glasshouse validation in 2015

• Markers being validated and being applied to the breeding

program

17 APPS 2015

Results and implications

• A new glasshouse based high-throughput phenotyping protocol has been

established. Manuscript submitted to Aust. Plant Pathology

• Phenotyping protocol has been applied to field pea breeding

program for routine screening

• Utilization for identification and validation of molecular markers for bacterial

blight

• New landrace genotypes were identified with higher resistance to both

pathovars

• Needs improvements to understand the pathovars syringae and Pisi Race 6

resistance

Acknowledgements

Tony Slater

Grant Hollaway

Australian Grains Gene bank

Kurt Lindbeck

Peter Kennedy

Matthew Rodda

Garry Rosewarne

Amber Stephens

Eric Armstrong

Gerard O’connor

NSW DPI

Jason Shiller

Noel Cogan

VIC-Govt.

19