Field inoculation of Actinidia sp. with Pseudomonas syringae var. actinidiae biovar 3 (Psa)

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Ursula Weaver
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1 Field inoculation of Actinidia sp. with Pseudomonas syringae var. actinidiae biovar 3 (Psa) Stephen Hoyte S. Kabir, B. Parry and E. Karlsson

2 Glasshouse & Laboratory inoculation bioassays vs. Field inoculated kiwifruit vines Bioassay development and phenotyping for Psa resistance commenced winter of 2011 Overall Aim: provide a suite of phenotyping bioassays to support breeding for resistance AIM for this project: study phenotypic response in Glasshouse/Lab vs. Field grown vines Two inoculation methods Woody stem bioassay + Mechanical stem inoculator Using 5 genotype classes 2 were clonal = A. chinensis var. chinensis ( Hort16A ) and ( Zesy002 = Gold3) 3 were seedlings = A red A. chinensis var. chinensis (Red chinensis) (controlled cross) A. deliciosa var. deliciosa ( Hayward OP) (open pollinated) A. chinensis var. chinensis x A. arguta (arguta chinensis hybrid OP) (open pollinated) Ranking for Resistance..Tolerance Susceptibility (it s a continuum )

3 Methods: Woody Stem Bioassay Index (lab) Wound and inoculate dormant canes (two inoculation doses 10 6 & 10 9 ) After 3 weeks, cut the bark to expose lesion measure Then after 24 h score ooze development + Psa No Psa

4 Methods: Woody Stem Bioassay Index (lab) Wound and inoculate dormant canes (two inoculation doses 10 6 & 10 9 ) After 3 weeks, cut the bark to expose lesion measure Then after 24 h score ooze development lesion (mm) Index Ooze Index >20 50 WSBI = Lesion index + Ooze index (Relative scale 0 100) 1:1 weighting for lesion and ooze + Psa No Psa

5 Woody stem bioassay index 5 genotypes Number of bioassays

Methods: Mechanical stem inoculation (GH) Mechanical inoculator developed in 2013 Needles puncture wounds into plant stem Delivers Psa directly into the vascular system 5 x 10 8 CFU / ml inoculum

6 Methods: Mechanical stem inoculation (GH) Mechanical inoculator developed in 2013 Needles puncture wounds into plant stem Delivers Psa directly into the vascular system 5 x 10 8 CFU / ml inoculum Incubate plants in plastic tents inside a containment glasshouse for 3 weeks Assessed for a wide range of Psa symptoms Lesion / Ooze / Leaf necrosis / Leaf wilt / Collapse (Tip/Stem/Whole plant) (= Susceptibility) Callus and swelling at the wound and Regrowth (= Resistance) Composite score of Psa severity on a 0 12 scale

7 Resistance responses... Callus & swelling at the wound A. arguta A. chinensis var. chinensis x A. arguta

8 Resistance responses... Callus & swelling at the wound Regrowth A. arguta A. chinensis x A. arguta Hayward OP (A. chinensis var. deliciosa)

9 Susceptible responses... Systemic leaf infection

10 Susceptible responses... Systemic leaf infection Ooze

11 Susceptible responses... Stem collapse Systemic leaf infection Ooze Total collapse

12 Mechanical stem inoculation (GH) potted plants

13 Mechanical stem inoculation (GH) potted plants

14 Methods: Mechanical inoculation of field grown vines 50 plants x 5 genotypes Planted at Te Puke Research Orchard, spring 2014 Exposed to natural Psa inoculum for one season Vine survival and Psa symptoms monitored the following spring November shoots/plant inoculated (5 x 10 8 CFU / ml) with Psa using the mechanical inoculator (indeterminate non-fruiting shoots) Psa symptoms scored after 5 weeks using same assessment as GH

15 Field for 15 months - Psa symptoms natural infection

16 Field for 15 months - Psa symptoms natural infection

17 Field for 15 months - Psa symptoms natural infection

18 Field - Post inoculation: Lesion length

19 Field - Post inoculation: Stem collapse Hort16A

20 Field - Post inoculation: Callus and swelling at wound arguta chinensis hybrid OP

21 Correlations - Bioassays vs. Field Woody stem vs. Field Potted plant vs. Field

22 Summary Mechanical inoculator suitable for both glasshouse and field inoculations Psa symptoms and severity observed on five genotypes in GH and Lab bioassays strongly correlates with 15 month-old field vines exposed to natural inoculum and when inoculated Mechanical inoculation can effectively cull out the most susceptible Mechanical inoculation offers significant potential for breeders to improve the efficiency of their breeding pipeline in the search for Psa resistant cultivars This research represents just one piece in the large and challenging puzzle that needs to be solved in order to overcome Psa

23 Acknowledgements PFR Ruakura bioassay team for all the woody stem bioassays PFR Research Orchard Network team at Te Puke for maintaining the vines PFR Palmerston North and Motueka propagation teams for supplying seedlings PFR Kiwifruit Royalty Investment Programme (KRIP) for funding this research

KPCS target organisms, controls and symptom guide

KPCS target organisms, controls and symptom guide 1. Virus Virus included: I. Cherry leafroll virus (CLRV) II. Actinidia seed-borne latent virus (ASBLV) III. Monitor for other unusual symptoms n 2017,