Pseudomonas syringae causing bacterial canker on cherries and sweet cherries in Poland

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1 Pseudomonas syringae causing bacterial canker on cherries and sweet cherries in Poland Monika Kałużna and Joanna Puławska Research Institute of Horticulture, Pomology Division, Pomologiczna 18 str., Skierniewice, Poland

2 It occurs in all fruit trees growing areas in the world, causing the most damage in orchards and nurseries of stone fruit trees caused by Pseudomonas syringae polyphagous this pathogen can decrease trees resistance to frost cankers developing on the branches and main trunk may lead to death of whole trees losses caused by the disease on susceptible varieties in young orchards can reach even 75% in 2007 and 2011 the disease was observed in the high intensity in Poland

3 The aim of our study: to characterize the population of bacteria causing bacterial canker on stone fruits in Poland to design primers enabling specific detection of bacteria belonging to 2 races of P.s. pv. morsprunorum originating from different symptomatic tissue of stone fruits in Poland.

4 Symptoms

5 Bacterial cankerin Poland Pseudomonas syringae pv. syringae Pseudomonas syringae pv. morsprunorum race 1 and 2 NO Pseudomonas syringae pv. avii Pseudomonas syringae pv. persicae

6 Isolates Of 767 samples collected (316 from cherry, 179 from sweet cherry, 145 fromplum,107frompeach,14fromapricot,3fromnectarineand3frommirabelle) 150 isolates of Pseudomonas syringae were obtained: 5 from peach,20fromsweetcherry,47fromplumand78fromcherry were obtained 112 isolates were classified to pv. syringae 32 isolates to pathovar morsprunorum race 1 6isolatestomorsprunorumrace2

7 Stepsof identification and differentiation Aspects of disease etiology I. Phenotypic characteristics (morphology, biochemistry, physiology)-lopat tests, GATTa tests, L-lactate utilization II. Pathogenicity and virulence determination on sweet cherry fruitlets

8 Stepsof identification and differentiation III. Syringomycin production growth inhibition of Rhodotorula pilimanae MUCL IV. Study on synergism between bacteria and frost Pss without exposition to frost isolates Psmi Pss exposed to frost

9 Stepsof identification and differentiation V. Molecular study, genetic diversity of bacteria a. DNA isolationby modifiedmethodof Aljanabiii Martinez (1997) b. Detectionof the genes encodingtoxincoronatine, syringomycine and siderophore yersiniabactin c. Geneticdiversity: rep-pcr(box, ERIC, REP and IS50), PCR MP, MLST (gyrb, gapa, gltaand rpod)

10 Detection of the genes encoding toxin coronatine, syringomycine andsiderophoreyersiniabactin M of Psmrace 1 possess cflgene encoding for coronatine M Product 650 bp 5 strains of Psm race 2 possess irp gene encoding for yersiniabactin. M Product 943 bp Product 752bp Over 40 strains of Pss possess syrb gene encoding for syringomycine

11 Genetic diversity: repetitive PCR, PCR MP P.s. pv. syringae Psmrasa 2 P.s. pv..? Psmrasa 1 Amplification of a limited number of DNA fragments, whose number and size compared after electrophoresis gives information about the genetic differences between the investigated strains. Rep-PCRs and PCR MP methods confirmed the homogeneity of races within pathovar morsprunorum and the diversity within the isolates belonging to pathovar syringae

12 Multilocus sequence typing (MLST) 4 housekeeping genes: gyrb, gapa, glta and rpod rpoD 77rpoD 417rpoD 3800rpoD rpoD 240rpoD 236rpoD 234rpoD 233rpoD 24 68rpoD 264rpoD 437rpoD 222rpoD 242rpoD rpoD rpoD 256rpoD rpoD rpoD 239rpoD rpoD rpoD rpoD rpoD 657rpoD 1247rpoD rpoD 663rpoD 258rpoD rpoD 106rpoD 110rpoD rpoD 109rpoD 2905rpoD 103rpoD 141rpoD rpoD 959rpoD 91rpoD 122rpoD 211rpoD rpoD 970arpoD 1021rpoD 215rpoD 25B rpod 217rpoD 250rpoD Psmrace 1 283rpoD 1061rpoD 2222rpoD Psmrace 2 Pseudomonas fluorescens LMG5831 Pss Pss homogenic group 0.02

13 Detection-primer designing two primer pairs designed for the detection of strains of race 1 and race 2 ofpsm they were specific to the group for which were designated the designed primers constitute the first system for specific detection of Psmrace 1 and 2. PCR amplification of the 793-bp fragment specific for the strains of Pseudomonas pv. morsprunorum race 1 PCR amplification of the 398-bp fragment specific for the strains of Pseudomonas pv. morsprunorum race 2

14 Control Prevention treatments: Afterfruit harvest cut and removed from the orchard infested stems, branches with reserve: cm and even whole trees if neccesary Protect the wound after cutting by using Funaben03 PA or white emulsion supplemented with 1% of copper Use the slips from healthy trees, nursery stock must be absolutely free of bacterial cancer

15 Chemical control: Use of coppercompoundsin spring and autumn The range of chemicals mainly based on copper compounds-occurring of bacteria resistance The copper compounds are good bactericides however work only on the surface and do not treat/cure infected plants They can cause side effect such as flowers or buds russeting of the fruits

16 Conclusions Bacterial canker in Poland are maily caused by Pssand Psmrace 1 Among Pss and Psm race 1 some mutants with lack of syringomycin and coronatine respectively were found The pathogenicity test on immature sweet cherry fruits cv. Napoleon divided the tested strains into two groups: one causing black brown necroses and second water soaked superficial lesions Results of rep-pcr and PCR MP confirmed the homogeneity of races within pathovar morsprunorum and the diversity within the isolates belonging to pathovar syringae However, among isolates of pathovar syringae originating from sour cherry, the homogenous group of isolates showing atypical pathogenic abilities (similar to those of pathovar morsprunorum) was found. This may suggest that they belong to an intermediate form between pathovars syringae and morsprunorum or to a new patovar

17 Thank you very much for your attention