cursus cost zurich 2015 - diagnosispp1 jjanse sept … · 19-9-2015 1 diagnosis in...

19-9-2015

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DIAGNOSIS IN PHYTOBACTERIOLOGY 1

Dr. Jaap D. Janse

Department Laboratory Methods and Diagnostics

Dutch General Inspection Service (NAK) Emmeloord, The Netherlands

[email protected]

COST FA1104 Training School

Molecular diagnostics of bacterial diseases, Zürich, Switzerland, 2015-09-21-25 – Jaap D. Janse

• Thomas J. Burrill (1839-1916) - fireblight USA

• Jan Hendrik Wakker (1859-1927) - yellow disease of hyacinth, Netherlands

• Erwin Smith, USA founder of phytobacteriology

• Stapp, Germany

• Hellmers, Denmark

• Dowson, UK

DIAGNOSIS

HISTORY



ERWIN SMITH (1854-1938), FOUNDER OF PHYTOBACTERIOLOGY

HISTORY

DIAGNOSIS

Yellow disease

of hyacinth

Xanthomonas

hyacinthi



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DIAGNOSIS

Plant pathogenicgenera

Acidovorax

AgrobacteriumArthrobacterBurkholderia

ClavibacterCurtobacteriumErwinia

HerbaspirillumSerratiaLiberibacterPantoea

PseudomonasRathayibacterRhizobacter

RhodococcusPectobacteriumSphingomonas

StreptomycesXanthomonasXylophilusXylella



STEPS IN THE DIAGNOSIS OF BACTERIAL DISEASES OF PLANTS

DIAGNOSIS

A ASSESSMENT OF SYMPTOMS

B ISOLATION OF PATHOGENIC BACTERIA

C PURE CULTURE OF ISOLATED BACTERIA

D IDENTIFICATION OF PURE CULTURE

E PATHOGENICITY TEST

F REISOLATION FROM INOCULATED PLANTS

G REIDENTIFICATION OF PURE CULTURE

H DIAGNOSIS REPORT



A. SYMPTOMS OF BACTERIAL DISEASES OF PLANTS

DIAGNOSIS

LEAF SPOTS

EXCRESCENCES AND GALLS

TUMOURS

WILTING (VASCULAR DISEASES)

NECROSIS AND CANKERS

ROTTING

BACTERIA EMBEDDED IN SLIME



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B. ISOLATION

DIAGNOSIS



B. ISOLATION

DIAGNOSIS

RESULT



B. ISOLATION

DIAGNOSIS

ISOLATION MEDIA

NON-SELECTIVE - water and non-defined nutrients (e.g.

peptones, beef extract, yeast extract) or defined inorganic salts and organic nutrients,

suitable for growth of many bacterial species.

SEMI - (S)ELECTIVE - substances to enhance production of pigments and/or substances used by certain

bacteria and/or inhibitors (e.g. antibiotics) for

non-desired bacteria.

SELECTIVE - nutrients and inhibitors, which by their quantity and quality, allow only one bacterial

species to grow in/on the medium (in an ideal

situation).



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CLAVIBACTER MICHIGANENSIS SUBSP. MICHIGANENSIS AND

PANTOEA AGGLOMERANS ON YEAST-PEPTONE-GLUCOSE AGAR

B. ISOLATION

DIAGNOSIS

NON-SELECTIVE



PSEUDOMONAS CICHORII ON KING’S MEDIUM B AND P. SYRINGAE

PV. PHASEOLICOLA ON 5% SUCROSE AGAR: LEVAN FORMATION

B. ISOLATION

DIAGNOSIS

SEMI-(S)ELECTIVE



STREPTOMYCES SCABIEI ON STREPTOMYCES ISOLATION AGAR

B. ISOLATION

DIAGNOSIS

SEMI-(S)ELECTIVE

Low nutrients andBrown pigment fromtyrosine



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C. PURE CULTURE

DIAGNOSIS



C. PURE CULTURE

DIAGNOSIS

RESULT



D. DETECTION AND IDENTIFICATION

DIAGNOSIS

DETECTION is tracing of plant pathogenicbacteria in or on plant material, especially

when they occur subclinically (latent), withoutcausing symptoms.

It should be clearly distinguished from

IDENTIFICATION, which is characterisationand naming of bacteria.



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• Isolation on media from infected tissue

• Inoculation of extract in host plant (use hostas selective medium)

• Serological techniques such as immuno-fluorescence, ELISA

• Plating or serological techniques on extractsof plant material with possible latent infectioninvolving:

extraction of bacteria by soaking or macerationconcentration of bacteria by centrifugation or (immuno-) trapping

DIAGNOSIS

CLASSICAL DETECTION



BIOCHEMICAL TESTS: OFFERING BACTERIA CARBON/NITROGEN SOURCE AND CHECK FOR ENZYMES OF BACTERIUM BY Ph CHANGE IN MEDIUM

CLASSICAL IDENTIFICATION

DIAGNOSIS



BIOCHEMICAL TESTS:

MINIATURISED FORMAT, API

SYSTEM, 48H TEST, MAINLY

FOR HUMAN BACTERIA


DIAGNOSIS



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HYDROLYSIS OF PECTIN (CAVITY FORMATION) BY PECTOBACTERIUM CAROTOVORUM AND HYDROLYSIS OF FAT BY XANTHOMONAS CAMPESTRIS


DIAGNOSIS



PIGMENT FORMATION ON ELECTIVE KING’S MEDIUM B


DIAGNOSIS



ANTIBIOTIC SENSITIVITY TEST AND TEST FOR PRODUCTION OF TOXIN BY PSEUDOMONAS SYRINGAE STRAINS USING GEOTHRICHUM FUNGUS


DIAGNOSIS



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PRINCIPLE OF SEROLOGY AND CROSS-REACTIVIY OF POLYCLONAL ANTISERA


DIAGNOSIS




DIAGNOSIS

Table 11

Effect of antiserum dilution on the occurrence of

cross-reactions with an antiserum against

Clavibacter michiganensis subsp. sepedonicus

BACTERIUM ANTISERUM DILUTION

10 20 40 80 16

0

32

0

64

0

12

80

2560 512

0

Target C. m.

sepedonicus

+ + + + + + + + +

(titre)

±

Cross reactive

bacterium 1

+ + + + - - - - - -

Cross reactive

bacterium 2

+ + + + + + ± - - -

DANGER ZONE DILUTIONS

PREFERRED



SEROLOGICAL TECHNIQUES USED IN PLANT BACTERIOLOGY

CLASSICAL IDENTIFICATIONDIAGNOSIS

CONJUGATE + SPECIFIC SERUM + BACTERIUM



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PRINCIPLE OF IMMUNO-FLUORESCENCE MICROSCOPY






RESULT





Differences between antigenic types, includingflagella can be made visible by IF



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DIAGNOSIS

IF uses microscopeslide andIF microscope andfluorochrome

Enzme-linked ImmunoSorbent Assay (ELISA)uses elisa plate, elisareader and enzyme



PRINCIPLE OF DNA DOT/SLOT-HYBRIDISATION

MOLECULAR DETECTION

DIAGNOSIS



PRINCIPLE OF DNA DOT/SLOT-HYBRIDISATION

MOLECULAR DETECTION

DIAGNOSIS

RESULT: BLACKDOTS OF DNAON AN X-RAYFILM



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MOLECULAR DETECTIONDIAGNOSIS

FLUORECENT

IN-SITU

HYBRIDISATION

(FISH)



MOLECULAR DETECTION

DIAGNOSIS

RESULT: ALL BACTERIA REACT WITH EU-PROBE (GREEN), TARGET BACTERIUM REACTS WITH SPECIFIC PROBE (RED)

FISH



PRINCIPLE OF POLYMERASE CHAIN REACTION (PCR)

MOLECULAR DETECTION

DIAGNOSIS



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PRINCIPLE OF POLYMERASE CHAIN REACTION (PCR)

MOLECULAR DETECTION

DIAGNOSIS

RESULT OF PCR WITH PRIMERS FOR DNA OF RALSTONIA

SOLANACERUM – PRODUCT OF 288 BP

ALWAYS INCLUDE CONTROLS !



DIAGNOSIS

PRINCIPLE OFTAQMANREAL-TIME PCR



MOLECULAR DETECTION

PRINCIPLE OF TAQMAN REAL-TIME PCR

MOLECULAR DETECTION

DIAGNOSIS

RESULT OF TAQMAN PCR WITH DILUTION SERIES OF RALSTONIASOLANACERUM

BASELINE

normalised reporterfluorescence



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MOLECULAR DETECTION

RESULT OF MULTIPLEX TAQMAN PCR WITH POTATO SAMPLES CONTAINING DIFFERENT Dickeya AND Pectobacterium spp

Orange = generic probeGreen = virulent Pectobacterium carotovorumBlue = P. atrosepticumPink = Dickeya spp.

DIAGNOSIS



• On site detection using (serological) lateral flow devices

• Real-time PCR (also in multiplex), MLST

• Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS)

(EARLY)MOLECULAR DETECTION and IDENTIFICATION

DIAGNOSIS



Early detection/Identification without thermocycler• Loop-mediated isothermal amplification (LAMP) - 4-6 primers recognizing

6-8 distinct regions of target DNA. A strand-displacing DNA polymerase

initiates synthesis and 2 of the primers form loop structures to facilitate

subsequent rounds of amplification. Rapid, sensitive, magnesium

pyrophosphate produced can be seen by eye: field diagnosis.

• Strand displacement amplification (SDA) uses a strand-displacing DNA

polymerase, used in medical clinical diagnostics.

• Helicase-dependent amplification (HDA) uses ds-DNA unwinding activity

of a helicase to separate strands, and primer annealing and extension by

a strand-displacing DNA polymerase. Like PCR, only two primers. Not

much used yet.

• Nicking enzyme amplification reaction (NEAR) uses a nicking enzym

creating nicks were a strand-displacing DNA polymerase starts producing

many short nucleic acids from the target sequence. Very fast and

sensitive, detections in minutes. NEAR is already used in medical clinical

diagnostics


DIAGNOSIS



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PRINCIPLE OF ELECTROPHORESIS OF PROTEINS

MOLECULAR IDENTIFICATION

DIAGNOSIS

PURECULTURE

EXTRACTIONOFPROTEINS




DIAGNOSIS

POLYACRYLAMIDE GEL-ELECTROPHORESIS (PAGE):OBTAIN PROFILE



PRINCIPLE OF ELECTROPHORESIS OF PROTEINS


DIAGNOSIS

PROFILE SCANNING, IDENTIFICATION AND CLUSTERINGWITH COMPUTER SOFTWARE



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PRINCIPLE OF FATTY ACID ANALYSIS


DIAGNOSIS



PRINCIPLE OF FATTY ACID ANALYSIS


DIAGNOSIS

pv. fraxinipv. oleae

pv. nerii



Repetative extragenic palindromic (rep) PCR fingerprintingCOST FA1104 Training School



DIAGNOSIS

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DIAGNOSIS

extragenic palindromic (rep) PCR fingerprinting




DIAGNOSIS

extragenic palindromic (rep) PCR fingerprinting



AFLPfingerprinting

R = restrictionsite

A,B,C = variablelength DNAfragments


DIAGNOSIS



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DIAGNOSIS

AFLP fingerprinting: result with Xanthomonas strains



• Multi Locus Sequence Typing (MLST), (partial) sequencing of 16S rRNA and 16-23S rRNAintergenic spacer region and housekeeping genes such as gyrB and rpoD.

need pure culture, relatively fast , low taxonomic level, strain, comparison with database possible

MLST expensive, single locus cheap(er)

more resolution than 16S rRNA




DIAGNOSIS

• Single locus rpoD for Pseudomonas• Data by Neil Parkinson CSL/FERA

Courtesy Neil Parkinson

Pathogen Host Phylogroup

Prunus amygdali almond PG 3

P. mume Japan apricot PG 3 and 2

P. armeniaca apricot PG 3

P. cerasifera cherry plum PG 3

P. avium bird cherry PG 2

P. domestica plum PG 1

P. salicina Japan plum PG 1




DIAGNOSIS

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• Many of the recent technologies have great potential,

but their practical development in plant pathology is

still underway.

• Analyses for comparison, validation, and

standardization are strictly necessary for molecular

methods to be accepted and widely used in routine

diagnosis (Martin et al., 2000; Alvarez, 2004).

• See EPPO Validation Standard


DIAGNOSIS



• Many problems due to false positive and false negatives

in detection and identification

• Leading in official EU detection schemes to obligation to

do two screening tests of different nature

• Therefore a combination of methods/tests and in official

schemes still a pathogenicity test (fulfilling Koch’s

postulates) are a condition for good identification and

more realistic phenetic/phylogenetic taxonomic trees and

diagnoses

The polyphasic approach in diagnosis, identification and taxonomy

DIAGNOSIS



• For preliminary diagnosis sometimes smell of

colonies on isolation plate is enough (Erwinia

amylovora on 5% sucrose agar: sweet scent;

Pseudomonas syringae rotting odor

• A few biochemical tests, one serological test, one

PCR using one primer pair, MLSA on one gene, too

few strains/isolates, wrong reference strain (many

type strains are not typical) can all lead to very

serious mistakes

• molecular tests have the advance of discriminating at

very low taxonomic level

The polyphasic approach in diagnosis, identification and taxonomy

DIAGNOSIS



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DIAGNOSIS

PERSPECTIVES

• Rapid, sensitive and cost effective

• Integration into certification/inspection schemes

• Commercially available, standardized test kits

• Non-culturable organisms such as Phytoplasmas can be analysed

• Genetically modified organisms can be traced in the environment more easily

• Less sensitive to mutation or variation

• Discrimination at low taxonomic level, often strain level




DIAGNOSIS

PITFALLS

• Specificity, sensitivity and reproducibility unknownor only tested to a limited extent (not validated)

• Negative influence of conditions and biochemicals (experimental error)

• Impossibility to discriminate between viable and non-viable cells and free nucleic acid in a sample

• False negatives and false positives difficult to verify, Koch’s postulates cannot be fulfilled

• In databases many errors




DIAGNOSIS

PITFALLS 2

• Changing probes/primers/enzymes/methods/chemicals may yield different (conflicting) patterns or no patterns at all

• Only small part of structural elements of an organism used (sampling error)

• Answers from automated identification systems as good as standard libraries and present-day taxonomy are

• Points of reference usually determine choice of patterns



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DIAGNOSIS

EXPERIMENTAL ERROR

60°C

SPECIFIC

55°C

NON-SPECIFIC

40°C

NON-SPECIFIC

SPECIFIC PROBEEU-PROBE



•

• In research a non-reductionistic view, realising that there is

difference between DNA/RNA of an organism and the

organism itself,– and an open field of study –gene

expression/phenotype

What do we understand about the Kingfisher when we

sequenced the genome?

DIAGNOSIS




STILL VALID, STILL GOING STRONG AND NECESSARY TO FULLFIL

KOCH’S POSTULATES

E. PATHOGENICITY TEST AND F. REISOLATION

DIAGNOSIS

Ralstonia solanacearumin tomato - fast: 4-5 days

Pseudomonas viridiflavain chicory - fast 2-4 days



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HYPERSENSITIVITY TEST ON TOBACCO: DENSE (C. 108 CELLS.ML-1) INFILTRATED BETWEEN EPIDERMI, WITHIN 24 H NECROSIS

E. PATHOGENICITY TEST and F. REISOLATION

DIAGNOSIS

FOR PSEUDOMONASSYRINGAE PVS. ANDSOME OTHER PLANTPATHOGENICBACTERIA

BUT NOT ALL !



REFERENCE COLLECTIONS

DIAGNOSIS

FOR COMPARISON, VALIDATION AND STANDARDISATION WELL PRESERVED AND DOCUMENTED REFERENCE CULTURES, AND THEREFORE CULTURE COLLECTIONS ARE INDISPENSABLE

• AGAR SLANTS IN SCREW CAP TUBES WITH

RUBBER STOPPER AT 4°C

• IN STERILE WATER AT ROOM TEMPERATURE

• FROZEN AT –20 OR -80 °C ON STERILE BEADS

• LYOPHILIZED (FREEZE DRIED) IN AMPULES AT 4°C



• Observation and assimilation remain a prerequisite

• Open eye and mind: we work for our producers

(growers), our agriculture/horticulture and

agricultural/horticultural industry – small

producers: gold mine for many countries when

educated and financial possibilities

• Cross-fertilisation of field – and lab specialists

conditional for maximum benefit in practice and

correct diagnosis in the field of plant diseases

Conclusion

DIAGNOSIS



cursus cost zurich 2015 - diagnosispp1 jjanse sept … · 19-9-2015 1 diagnosis in...

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