Bacterial blight in field pea

Transcription

1 Bacterial blight in field pea Efficient phenotyping method developed in controlled environment for pre-breeding and breeding Dr Pragya Kant- Horsham, Vic Bacterial blight epidemic in Rupanyup, Vic, 2012

2 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

3 Causal pathogen: Pseudomonas syringae Two pathovars: Ps. pv. syringae and Ps. pv. pisi (1-8 Races) 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) 3

4 Pathogen occurrence Major pathogen pv. syringae (Richardson and Hollaway 2011) Data from historic isolate collection at Horsham Before 2000: mainly pv. pisi (Race 3,6) 2000 now: mainly pv. syringae pv. syringae resistance is quantitative pv. Pisi Race 6?(No resistance genes have been identified in cultivars tested) Emphasis 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 4

5 Bacterial blight in field trials 2012 cv. Kaspa cv. PBA Percy cv. PBA Oura cv. Kaspa 5

6 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 Field screening nursery Yield loss due to bacterial blight damage 2011 % Yield loss Both pisi race 6 syringae Mean yield loss Variety rating PBA Pearl R PBA PERCY R OZP MR OZP MR PBA OURA MR OZP MR STURT MS KASPA MS PBA WHARTON MS MORGAN MS OZP MS PBA COOGEE MS PARAFIELD MS PBA GUNYAH MS YARRUM S MAKI S Peter Kennedy and Kurt Lindbeck 6

7 Advantages: A new glasshouse screening method for Bacterial blight 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

8 Methodology of Bacterial blight screen Bacteria was grown in Nutrient Yeast Broth. The P. syringae pv. syringae conc. 5.2 x 10 7 cfu/ml. P. syringae pv. pisi conc. approximately 1.4 x 10 8 cfu/ml. Pre-inoculated plants were kept under high light to ensure the opening of stomata. The surfactant Pulse Penetrant was added to the suspensions at 0.055% (v/v). Bacterial suspension was sprayed on seedling in glasshouse with overhead misting and disease was assessed 7-9 days after inoculation. 8

9 Disease rating scale used to assess severity of symptoms caused by P. syringae pv. syringae and pv. pisi Ratings a 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 a Ratings R: resistant; MR: moderately resistant; MS: moderately susceptible; S: susceptible; VS: Very susceptible. 9

10 Mean disease Score Effect of misting and surfactant on disease development 9 c b a a 0 No surfactant Tween (0.1%) with misting Pulse (0.05%) without misting Pulse (0.05%) with misting Treatments Susceptible variety Kaspa 10

11 P. syringae pv. Pisi A: Without misting B: with misting A B P. syringae pv. syrinage A: Without misting B: with misting 1 C D 11

12 Symptoms in the glasshouse based protocol are similar as observed in field Symptoms in field Symptoms in glasshouse 12

13 Glasshouse ranking Glasshouse ranking Glasshouse and field correlation Field ranking Field ranking 13

14 Phenotyping of diverse germplasm identified new resistant genotypes pv. pisi Race 6 Genotype Mean disease scores ATC PBA Percy 2.75 ATC ATC ATC ATC ATC ATC ATC ATC PBA Oura 4.17 ATC ATC ATC ATC ATC ATC ATC Kaspa 6.42 ATC pv. syrinage Genotype Mean disease score ATC PBA Percy 3.33 ATC ATC ATC PBA Oura 4.75 ATC ATC ATC ATC ATC ATC ATC ATC ATC ATC ATC Kaspa 7.92 ATC ATC

15 Whole genome sequencing of Bacterial blight isolates VPRI Location Year Host Plant Comments Identity Plum (Prunus domestica ) Psy Impatients sp Psy NSW 2003 Pisum sativum (Field Pea) Stem/leaf diseased plant P. viridiflava NSW 1987 Ranunculus sp. (Buttercup?) From spots on stem/leaf P. viridiflava NSW 1986 Lactuca sativa (Lettuce) Race 7 P. cichorii Race 3 pisi isolates NSW 1981 Citrus sinensis (Orange) Leaf Psy NSW 1978 Phaseolus vulgaris (French Bean) From spot on leaf Psy QLD <1985? Non-pathogenic; lemon -'ve Psy SA 1984 Pisum sativum (Field Pea) Stem/leaf Psy NSW 2003 Pisum sativum (Field Pea) Stem/leaf diseased plant Psy NSW 2003 Pisum sativum (Field Pea) Stem/leaf diseased plant Psy USA 1944 Pisum sativum (Field Pea) Ppi R Vic Pisum sativum (Field Pea) Pea seed Ppi R USA 1975 Pisum sativum (Field Pea) Ppi R NSW 1992 Pisum sativum (Field Pea) Ppi R USA 1975 Pisum sativum (Field Pea) Ppi R TAS 1976 Pisum sativum (Field Pea) Ppi R NSW 2003 Pisum sativum (Field Pea) Stem/leaf diseased plant Ppi R Vic Pisum sativum (Field Pea) Ppi R NSW 2003 Pisum sativum (Field Pea) Crop failure Ppi R Crop seed sample 1993 Pisum sativum (Field Pea) Ppi R VIC 1993 Pisum sativum (Field Pea) Ppi R6 pisi isolates P. viridiflava P. cichorii UniRef 90 syringae isolates 15 Jason Shiller, P. Kant, M. Rodda,A. Stephens- Agribio

16 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 Pulse Molecular Maker Project- Agribio

17 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

18 Results and implications A new glasshouse based high-throughput phenotyping protocol has been established. Manuscript submitted to Aust. Plant Pathology New landrace genotypes were identified with higher resistance to both pathovars Phenotyping protocol has been applied to field pea breeding program for routine screening Utilization for identification and validation of molecular markers for bacterial blight Needs improvements to understand the pathovars syringae and Pisi Race 6 resistance

19 VIC-Govt. Matthew Rodda Grant Hollaway Jason Shiller Peter Kennedy Acknowledgements NSW DPI Kurt Lindbeck Eric Armstrong Gerard O connor Garry Rosewarne Tony Slater Amber Stephens Noel Cogan Australian Grains Gene bank 19