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Pseudomonas syringae on cherries

disease symptoms, identification, detection, differentiaton

Monika Kałużna 

Research Institute of Horticulture, Pomology Division, Pomologiczna 18 str., Skierniewice, Poland 

COST FA1104 Training Course On Molecular Diagnostics Of Bacterial Diseases, Switzerland 21‐25.09. 2015

Pseudomonas syringae pv. syringae

Pseudomonas syringae pv. morsprunorum race 1 and 2

Pseudomonas syringae pv. avii

Pseudomonas cerasi sp. nov.

Pseudomonas syringae on cherries

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P.syringae

Pseudomonas syringae is polyphagous bacterium causing diseases of over 180 plant species 

Causal agent of bacterial canker

60 pathovars within P. syringae species

Genomospecies determined by DNA:DNA hybridization(Gardan et al., 1999): gs 1 – P. s. pv. syringae (Pss) gs 2 – P. s. pv. morsprunorum race 1 (Psm race1), gs 3 – P. s. pv. morsprunorum race 2 (Psm race 2), P. s.

pv. avii (Psa)

It occurs in all regions of stone fruits cultivation in the worldcausing greatest damages in young orchards and nurseries ofsweet and sour cherry

Bacterial canker

Bacteria causing bacterial canker as epiphyte

Synergism of bacteria with frost thispathogen can decrease trees resistance tofrost Pss without 

exposition to frost 

Pss – exposed to frost

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Symptoms

Attacks all above‐ground parts of plants

(Fot. Monika Kałużna) (Fot. Monika Kałużna)

(Fot. Monika Kałużna) (Fot. Piotr Sobiczewski)

Symptoms

Cankers developing on branches and trunk can lead to death of the trees

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Cherry leaf spot – (Blumeriella jaapii) 

(fot. Monika Michalecka)(fot. Monika Michalecka)(fot. Monika Michalecka)

Bacterial canker (Pseudomonas syringae)

(Fot. Monika Kałużna)

????

??

????

Blumeriella jaapii

Prunus necrotic ringspot virus (PNRSV)(Fot. Mirosława Cieślińska)

PNRSV sourcherry

PNRSV sweetcherry

(Fot. Mirosława Cieślińska)(Fot. Mirosława Cieślińska)

Bacterial canker (Pseudomonas syringae)

(Fot. Monika Kałużna)

PNRSV

????

PNRSV sweetcherry

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Coryneum Blight (Shothole)(Wilsonomyces carpophilus)

https://utahpests.usu.edu/ipm/htm/fruits/fruit‐insect‐disease&pub‐cherry‐pests

Bacterial canker (Pseudomonas syringae)

Shothole(Clasterosporium carpophilum)

http://agronomija.rs/2014/rupicavost‐lista‐stigmina‐carpophila/

(Fot. Monika Kałużna)

Brown rot on peach and sour cherry Monilinia spp. 

Bacterial canker (Pseudomonas syringae)

Prunus necrotic ringspot virus on Shirofugen

(Fot. Mirosława Cieślińska)

(Fot. Monika Kałużna and Piotr Sobiczewski)

(Fot. Anna Poniatowska) 

(Fot. Anna Poniatowska) 

Physiological gummosis

(Fot. Monika Kałużna) 

????

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Bitter rot (Colletotrichum acutatum complex)

(Fot. Monika Michalecka)

(Fot. Monika Michalecka)

Bacterial canker (Pseudomonas syringae)

(Fot. Piotr Sobiczewski)True

True????

????

Isolation

• King B medium

• Nutrient Agar 5% sucrose

NA with 5% sucrose

King B

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Classification based on:

– hypersensitive reaction (HR) on tobacco plants

1. Gram determination with 3% KOH or staining

2. LOPAT tests

– levan production from sucrose (L)

– presence of oxidase (O) 

– ability to cause rot on potato tubers (P‐ pectolytic activity)

– presence of arginine dihydrolase (A)

Phenotypic tests – results interpretation

LOPAT group Levan formation

Oxidase reaction

Pectolyticcapability 

Arginine dihydrolase

Tobacco hypersensitivity

Species

Ia + ‐ ‐ ‐ + P. syringaeIb ‐ ‐ ‐ ‐ + P. syringae pv. 

savanastoi; P. delphini

II ‐/+ ‐ + ‐ + P. viridiflava

III ‐ + ‐ ‐ + P. cichorii

IVa + + + + ‐ P. marginalis 

IVb ‐ + + + ‐ P. fluorescens

Va ‐ + ‐ + ‐ P. tolaasii; saprophytic pseudomonads

Vb + + ‐ + ‐ P. fluorescens; saprophytic pseudomonads

Determination of green fluorescent Pseudomonas species by the LOPAT scheme(Lelliot et al., 1966)

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3. GATTa tests

– gelatine hydrolysis (G)

– aesculin hydrolysis (A‐ activity of the β‐glucosidase)

4. test of L‐lactate utilization

Classification and diversity based on:

– tyrosinase activity (T)

– utilization of tartrate (Ta)

5. Syringomycin production – growth inhibition of

Rhodotorula pilimanaeMUCL 30397

Classification and diversity based on..

6. colour of growth on Nutrient Brothwith 5% sucrose

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Test Pss Psm race 1 Psm race 2 Psa Psp

Fluorescence on King’s B medium + ‐ ‐/+ ‐ ‐

Fluorescence on CSGM + + + nd +

Gelatin hydrolysis (G) + ‐ + + ‐

Aesculin hydrolysis (A) + ‐ ‐ / + ‐ ‐

Tyrosinase activity (T) ‐ + ‐ /±/+ ‐ ‐

Utilization of tartaric acid L (+) (Ta)  ‐ + ‐ ‐??? ‐

Utilization of lactic acid + ‐ ‐/+ nd nd

Nutrient sucrose broth growth Yellow White Yellow/White intermediate colour

Anthranilate ‐ + nd ‐

Syringomycin production  +/‐ ‐ ‐ nd nd

Ice nucleation activity + ‐ ‐ ‐ +

Discrimination of the P. syringae pathovars syringae, morsprunorum race 1 and morsprunorumrace 2 by the GATTa tests and other tests (Crosse and Garrett, 1966; Schaad, 2001; Vicente andRoberts, 2007; Gilbert et al., 2009)

Phenotypic tests – results interpretation

Advantages:

Allow for determination of species and division into pathovarsand races

Disadvantages:

Require the implementation of a number of laborious and time‐consuming test

Not always clear results

Phenotypic test

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Detection of the genes encoding:‐ toxin coronatine (cfl), syringomycine(syrB and syrD)

‐ siderophore yersiniabactin (irp1)

Classification based on genetic analyses:

coronatineM  1  2   3   4  5   6  7  8  

Product 650 bp 

Disadvantages:

Detection of the genes encoding toxins production: need the estimation of product on gels

Detection of the genes encoding toxins production: mutants with lack of genes responsible for toxin production NOT GOOD FOR IDENTIFICATION!

Advantages:

If present you have luck

M   1 2 3   4 5 6 7   8   9

Product 752 bp

syringomycine

PCR fingerprints:‐ Repetitive PCRs ‐ ERIC, BOX REP and IS50

‐ Melting Profile PCR

Multilocus Sequence Typing (MLST):

‐ mainly gapA, gltA, gyrB, rpoB and rpoDcommonly used

Classification and genetic diversity based on:

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Advantages:

PCR fingerprints and MLST:  Allow for identification and determination of affiliation to taxon

Accurate and repeatable

MLST: Allow for comparisonwith sequences from database

Disadvantages:

PCR fingerprints and MLST: need the estimation of product on gels

PCR fingerprints: require  the reference strain for pattern comparison

in case of heterogenic Pss not good for classification; rep‐PCR the problem with reproducibility between laboratories occur

Classification and genetic analyses

Looking for fast and reliable methods

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MLST and                 MALDI TOF MS

New pathogenic group of polish

isolates from sour cherry

Psm1

Psm2

P.cerasi

Pss

Pss

Pss

Pss

Pss

MALDI TOF MS – ROTATION (FLIP)

Psm1

Psm2

Pss(A)Psm1

Psm2

Pss(A)

Psm1

Psm2

Pss(A)

Pss

Pss

Pss

Pss

Pss

Pss

Pss

Pss

Pss

Pss

Pss

Pss

Pss

Pss

Pss

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Detection‐primer designing

PCR amplification of the 793‐bpfragment specific for the strains of P.s.pv.morsprunorum race 1

PCR amplification of the 410‐bpfragment specific for the strains ofP.s. pv.morsprunorum race 2

4 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 first system for specific detection of Psm race 1 and 2

Sensitivity‐limit of detection

(LOD) 100 –101 in case of 

bacterial suspension, 

101 ‐ 102 in plant material and 

pg when using genomic DNA

Real time PCR

Sensitivity – limit of

detection (LOD)

100 in case of

bacterial suspension

and in plant

material and 10‐100

fg when using

genomic DNA

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Conclusions

Diagnosis based on symptoms of disease is not simple, especially in lastyears because of climate conditions changing.

There are a few of methods available for characterization anddifferentiation of causal agent of bacterial canker, but majority of themneed the time for evaluation and sometimes the results are difficult forinterpretation.

The developing of new fast reliable methods allowing for identification(Especially in case of heterogenic taxons) within a shorter andreasonable period of time are important as they allow to applyappropriate programs to prevent and control the disease.

This work was supported by National Science Centre grantNo UMO‐2013/08/M/NZ9/00138.

I want to thank Mrs. Halina Kijańska for excellent technical help andall colleagues from Department of Plant Protection for kind supply ofthe photo of the diseases

ACKNOWLEDGEMENTS

The part of work presented here was carried out within the framework of COST FA1104 “Sustainable production of high‐quality cherries for the European market”

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Thank you very much for your attention