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

Cherry production in Turkey

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

cv. Salihli

SOME IMPORTANT DISEASES of CHERRY TREES IN TURKEY

Causal agent disease

Monilinia fructigena, Monilinia laxa

BROWN ROT AND MONILINIA

Armillaria mellea

ARMILLARIA ROOT AND CROWN

ROTS

Taphirina deformans LEAF CURL

Agrobacterium tumefaciens CROWN GALL

Pseudomonas syringae pv. syringae

P. syringae pv. morsprunorum

BACTERIAL CANKER

PDV, ilarivirus PRUNE DWARF VıRUS

nepovirus, CLRV CHERRY LEAF ROLL VIRUS

Symptoms of bacterial canker

Pseudomonas syringae

Pseudomonas syringae pv. syringae Pseudomonas syringae pv. morsprunorum

(Pss) (Psm)

Psm race 1 , Psm race 2

Stone and pome fruits

stone fruits

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.

BLOSSOM BLAST SYMPTOMS

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

B

Dieback of the young twigs

Bacterial canker and Gummosis on sweet cherry trees

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.

Steps of identification and differentiation of bacterial canker

agents of sweet cherry

PSS / PSM

– 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 30397

– Molecular study

Phenotypic characteristics (physiological and biochemical tests) and molecular tests

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

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 (+ -/+ - -/+)

Utilization of some carbohydrates K (-)

P11/2

P40/3

P29/2

Da4

P39/3 psm

K (-)

P 32/2-b

P95/2

Da2

Da3(1)

Da1

Mannitol

Sorbitol

Da3(2)

P29/2

Badem Yalova

P39/2

şeftali çiçek

K (-)

P32/2-b

Badem

P40/3

Da3(1)

P11/2

K (-)

Sorbitol

Erythritol

P32/2-b

Badem

P40/3

Da3(1)

P11/2

K (-)

Lactic acid İnositol

Pathogenicity tests on sweet cherry fruitlets cv. Salihli

Each cherry fruitlet was inoculated by bacterial suspension (109 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 240C, RH over 90%.

Pathogenicity test results

C ( - )

Deep black brown lesions were produced by Pss isolates

Water soaked superficial lesions were produced by Psm reference strain

Pss

Psm

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

Results of Syringomycin production test – growth inhibition of Rhodotorula

pilimanae by Pss

Rhodotorula pilimanae strain MUCL 30397 (Polland)

Pss Psm

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 )

Presence of syrB

1 2 3 4 5 6 7 8 9 NC M

752 bp

1 2 3 4 5 6 7 8 9 NC M

•Responsible for syringomycin production, testing by using syrB1 and syrB2 primers •The same isolates which inhibited the growth of Rhodotorula possess the syrB gene

Presence of cfl

1 2 3 4 5 6 7 8 9 Pst Pst-re NC M

650 bp

1 2 3 4 5 6 7 8 9 Pst Psm-ref NC M

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

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.

cts gene M 1 2 3 4 5 6 7

500-600 bp

gyrB gene

1 2 3 4 5 6 7

507 bp

gapA ve rpoD genes

M 1 2 3 4 5 6 7 1 2 3 4 5 6 7

gapA rpoD

476 bp 575 bp

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%

SCREENING FOR COPPER SENSITIVITY OF SELECTED PSS ISOLATES FROM SWEET

CHERRY

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

< 0.6 0.6 -1.0 1.0- 1.5 2.0 ->

Kemalpaşa (25) 7 15 3 --

Salihli (15) 6 8 1 --

TOTAL (40 strain)

13 23 4 --

Examination of a collection of P. syringae pv. syringae isolates for copper resistance showed that 68 % were resistant to cupric sulfate

27 Pss isolates

• 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

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

%6,6

X

4

cuprous oxide

%75

X

5

Three basic copper

sulfate

%3

X

6

Copper oxide Nano

powder

%7

X

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

THANK YOU FOR YOUR KIND ATTENTION

KAZIM EĞERCI DAMLA ERTIMURTAŞ