Post on 17-Nov-2018
transcript
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
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
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.
– 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.
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%
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