The diagnosis and copper sensitivity of Pseudomonas syringae pv. syringae isolates from sweet cherry in Turkey Hatice Ozaktan

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1 The diagnosis and copper sensitivity of Pseudomonas syringae pv. syringae isolates from sweet cherry in Turkey Hatice Ozaktan Damla Ertimurtaş Kazım Eğerci University of Ege, Faculty of Agriculture, Department of Plant protection, 35100, Bornova, İzmir TURKEY

2 Cherry production in Turkey

3 cv. Salihli Turkish cherry growers have rapidly upgraded their orchards and horticultural skills.

4 SOME IMPORTANT DISEASES of CHERRY TREES IN TURKEY Causal agent Monilinia fructigena, Monilinia laxa disease BROWN ROT AND MONILINIA Armillaria mellea Taphirina deformans Agrobacterium tumefaciens Pseudomonas syringae pv. syringae P. syringae pv. morsprunorum PDV, ilarivirus nepovirus, CLRV ARMILLARIA ROOT AND CROWN ROTS LEAF CURL CROWN GALL BACTERIAL CANKER PRUNE DWARF VıRUS CHERRY LEAF ROLL VIRUS

5 Symptoms of bacterial canker Pseudomonas syringae Stone and pome fruits stone fruits Pseudomonas syringae pv. syringae (Pss) Pseudomonas syringae pv. morsprunorum (Psm) Psm race 1, Psm race 2

6 BLOSSOM BLAST SYMPTOMS Disease symptoms include blossom blast and spur dieback, leaf and fruit lesions, cankers with associated gummosis of woody tissue, loss of scaffold limbs, and overall decreased fruit yields.

7 Bacterial canker fruit lesions (A) and leaf spot symptoms (B) caused by Pseudomonas syringae pv. syringae on sweet cherry. B

8 Dieback of the young twigs

9 Bacterial canker and Gummosis on sweet cherry trees

10 AIM OF THIS STUDY The purpose of this study was to diagnose of Pseudomonas syingae pathovars which were isolated from the bacterial canker symptoms on cherry trees by some physiological & biochemical, pathogenicity and molecular tests, and to evaluate the potential of copper resistance by screening a number of P. syringae pv. syringae strains collected in Izmir, Turkey. The screening technique was based on the growth of strains on agar medium.

11 Steps of identification and differentiation of bacterial canker agents of sweet cherry PSS / PSM

12 Phenotypic characteristics (physiological and biochemical tests) and molecular tests LOPAT TESTS (Levan production, Oxidase, Pectolytic activity on potato slices, Arginine dehydrolase, HR on tobacco leaves) GATTa TESTS (Gelatine hydrolysis, Aesculine hydrolysis, Tyrosine activity, Tarataric Acid usage) Utilization of carbon sources (mannitol, sorbitol, inositol, erythritol, lactic acid), Pathogenicity and virulence determination on sweet cherry fruitlets INA (Ice nucleation activity) Syringomycin production growth inhibition of Rhodotorula pilimanae strain MUCL Molecular study

13 Phenotypic characters- LOPAT Test results + - Levan production from sucrose (L) Presence of oxidase (O) Pectolytic activity on potato tubers (P) Presence of arginin dihydrolase (A) Hypersensitive (HR) reaction on tobacco leaves LOPAT: (PSS / PSM) 40 isolates belonged to Pseudomonas syringae species

14 GATTa Test results Gelatine hydrolysis (G) Aesculine hydrolysis Tyrosine activity Using of tartaric acid According to GATTa test results, all of the tested P. syringae isolates were recorded as P. syringae pv syringae GATTa: PSS (+ -/+ - -/+)

15 Utilization of some carbohydrates K (-) Mannitol Sorbitol K (-) P11/2 P 32/2-b P40/3 Erythritol P95/2 P29/2 Da4 P32/2-b Badem Sorbitol Da2 Da3(1) P39/3 psm P40/3 Da1 Da3(1) Da3(2) P11/2 P32/2-b P29/2 K (-) Badem Badem Yalova P40/3 P39/2 Da3(1) şeftali çiçek İnositol Lactic acid P11/2 K (-) K (-)

16 Pathogenicity tests on sweet cherry fruitlets cv. Salihli Each cherry fruitlet was inoculated by bacterial suspension (10 9 cfu per ml, 10µl) after pricking with sterile needle Control (+) Pss (Poland) and Psm (Poland) Control (-) sterile distilled water The incubation lasted 4 days at 24 0 C, RH over 90%.

17 Pathogenicity test results Psm C ( - ) Water soaked superficial lesions were produced by Psm reference strain Pss Deep black brown lesions were produced by Pss isolates

18 Ice Nucleation Activity (INA) of P. syringae isolates Pss (INA + ) Psm (INA - ) All tested Pss isolates showed ice nucleation activity Psm could not cause INA

19 Results of Syringomycin production test growth inhibition of Rhodotorula pilimanae by Pss Rhodotorula pilimanae strain MUCL (Polland) Pss Psm

20 Molecular study, genetic analysis of P. syringae pathovars DNA extraction of bacterial isolates Detection of the genes encoding toxin coronatine (cfl), syringomycin (syrb) by PCR Genetic diversity of bacteria by MLST Four housekeeping genes (cts, rpod, gyrb and gapa )

21 Presence of syrb NC M NC M 752 bp Responsible for syringomycin production, testing by using syrb1 and syrb2 primers The same isolates which inhibited the growth of Rhodotorula possess the syrb gene

22 Presence of cfl Pst Pst-re NC Pst Psm-ref NC M 650 bp Responsible for Coronatine production, testing by using primers cfl1 and cfl2 genes None of tested Ps isolates produced bands on 650 bp, except reference strains Psm and Pst

23 MLST (Multi Locus Sequence Typing) Results 4 housekeeping genes: cts, rpod, gapa ve gyrb Sequence analysis of cts, gapa, gyrb ve rpod genes which were performed in Molecular Biology lab of Faculty of Biological Science, Oregon State University, U.S.A. İn order to distinguish for P. syringae pathovars.

24 cts gene M bp

25 507 bp gyrb gene

26 gapa ve rpod genes M bp 575 bp gapa rpod

27 CONCLUSION Bacterial canker in Izmir, Turkey is mainly caused by Pss Pathogenicty test on sweet cherry fruitlets is very quick and reliable method for distinguishing the Pss and Psm The results of Pss isolates for syringomycine production test on agar plate and PCR were correlative each other According to the results of sequence analysis based on cts, rpod, gapa ve gyrb genes, all tested Pseudomonas syringae isolates were found similar to Pseudomonas syringae pv. syringae at the rate of 90%

28 SCREENING FOR COPPER SENSITIVITY OF SELECTED PSS ISOLATES FROM SWEET CHERRY

29 Number of Pseudomonas syringae pv. syringae strains isolated from sweet cherry trees, belonging to different categories of minimal inhibitory concentration (MIC) values for cupric sulfate (mm) MIC (mm) interval for cupric sulfate (CuSO4) Pss strains sensitive resistant < > Kemalpaşa (25) Salihli (15) TOTAL (40 strain) Pss isolates Examination of a collection of P. syringae pv. syringae isolates for copper resistance showed that 68 % were resistant to cupric sulfate

30 Bordeaux mixture, cupric hydroxide, cuprous oxide, copper salts, ammoniacal copper and tribasic copper sulfate are registered and have been used to control Pseudomonas syringae pv. syringae in Turkey. Pss isolates, resistant to copper sulphate, were also tested for the reaction to other copper based bactericides Six different formulations of copper-based bactericides were evaluated for their efficacy in reducing populations of copper-resistant strains of Pseudomonas syringae pv. syringae growing on agar medium

31 Effect of copper-based bactericides on populations of copper-resistant (Cur) Pseudomonas syringae pv. syringae strains No Name of copper-based bactericides The rate of active ingredient (%) Reaction of tested Pss isolates Resistant susceptible 1 cupric hydroxide %36 X 2 Copper oxychloride %50 X 3 Copper sulphate pentahydrate cuprous oxide 4 %6,6 X %75 X 5 Three basic copper sulfate 6 Copper oxide Nano powder %3 X %7 X

32 CONCLUSION Many strains of P. syringae pv. syringae isolated from sweet cherry trees in İzmir exhibited high levels of copper resistance in culture All Pss isolates, resistant to copper sulphate, were also found other copper based bactericides except copper oxide nano powder. These data suggest that selection of copper-resistant strains could be a major reason for control failures following management with copper bactericides. The application of copper oxide nano powder may be good solution for preventing the copper resistance of Pss strains in Izmir province

33 KAZIM EĞERCI DAMLA ERTIMURTAŞ THANK YOU FOR YOUR KIND ATTENTION