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19-9-2015
1
DIAGNOSIS IN PHYTOBACTERIOLOGY 1
Dr. Jaap D. Janse
Department Laboratory Methods and Diagnostics
Dutch General Inspection Service (NAK) Emmeloord, The Netherlands
jjanse@nak.nl
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
COST FA1104 Training School
Molecular diagnostics of bacterial diseases, Zürich, Switzerland, 2015-09-21-25 – Jaap D. Janse
ERWIN SMITH (1854-1938), FOUNDER OF PHYTOBACTERIOLOGY
HISTORY
DIAGNOSIS
Yellow disease
of hyacinth
Xanthomonas
hyacinthi
COST FA1104 Training School
Molecular diagnostics of bacterial diseases, Zürich, Switzerland, 2015-09-21-25 – Jaap D. Janse
19-9-2015
2
DIAGNOSIS
Plant pathogenicgenera
Acidovorax
AgrobacteriumArthrobacterBurkholderia
ClavibacterCurtobacteriumErwinia
HerbaspirillumSerratiaLiberibacterPantoea
PseudomonasRathayibacterRhizobacter
RhodococcusPectobacteriumSphingomonas
StreptomycesXanthomonasXylophilusXylella
COST FA1104 Training School
Molecular diagnostics of bacterial diseases, Zürich, Switzerland, 2015-09-21-25 – Jaap D. Janse
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
COST FA1104 Training School
Molecular diagnostics of bacterial diseases, Zürich, Switzerland, 2015-09-21-25 – Jaap D. Janse
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
COST FA1104 Training School
Molecular diagnostics of bacterial diseases, Zürich, Switzerland, 2015-09-21-25 – Jaap D. Janse
19-9-2015
3
B. ISOLATION
DIAGNOSIS
COST FA1104 Training School
Molecular diagnostics of bacterial diseases, Zürich, Switzerland, 2015-09-21-25 – Jaap D. Janse
B. ISOLATION
DIAGNOSIS
RESULT
COST FA1104 Training School
Molecular diagnostics of bacterial diseases, Zürich, Switzerland, 2015-09-21-25 – Jaap D. Janse
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).
COST FA1104 Training School
Molecular diagnostics of bacterial diseases, Zürich, Switzerland, 2015-09-21-25 – Jaap D. Janse
19-9-2015
4
CLAVIBACTER MICHIGANENSIS SUBSP. MICHIGANENSIS AND
PANTOEA AGGLOMERANS ON YEAST-PEPTONE-GLUCOSE AGAR
B. ISOLATION
DIAGNOSIS
NON-SELECTIVE
COST FA1104 Training School
Molecular diagnostics of bacterial diseases, Zürich, Switzerland, 2015-09-21-25 – Jaap D. Janse
PSEUDOMONAS CICHORII ON KING’S MEDIUM B AND P. SYRINGAE
PV. PHASEOLICOLA ON 5% SUCROSE AGAR: LEVAN FORMATION
B. ISOLATION
DIAGNOSIS
SEMI-(S)ELECTIVE
COST FA1104 Training School
Molecular diagnostics of bacterial diseases, Zürich, Switzerland, 2015-09-21-25 – Jaap D. Janse
STREPTOMYCES SCABIEI ON STREPTOMYCES ISOLATION AGAR
B. ISOLATION
DIAGNOSIS
SEMI-(S)ELECTIVE
Low nutrients andBrown pigment fromtyrosine
COST FA1104 Training School
Molecular diagnostics of bacterial diseases, Zürich, Switzerland, 2015-09-21-25 – Jaap D. Janse
19-9-2015
5
C. PURE CULTURE
DIAGNOSIS
COST FA1104 Training School
Molecular diagnostics of bacterial diseases, Zürich, Switzerland, 2015-09-21-25 – Jaap D. Janse
C. PURE CULTURE
DIAGNOSIS
RESULT
COST FA1104 Training School
Molecular diagnostics of bacterial diseases, Zürich, Switzerland, 2015-09-21-25 – Jaap D. Janse
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.
COST FA1104 Training School
Molecular diagnostics of bacterial diseases, Zürich, Switzerland, 2015-09-21-25 – Jaap D. Janse
19-9-2015
6
• 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
COST FA1104 Training School
Molecular diagnostics of bacterial diseases, Zürich, Switzerland, 2015-09-21-25 – Jaap D. Janse
BIOCHEMICAL TESTS: OFFERING BACTERIA CARBON/NITROGEN SOURCE AND CHECK FOR ENZYMES OF BACTERIUM BY Ph CHANGE IN MEDIUM
CLASSICAL IDENTIFICATION
DIAGNOSIS
COST FA1104 Training School
Molecular diagnostics of bacterial diseases, Zürich, Switzerland, 2015-09-21-25 – Jaap D. Janse
BIOCHEMICAL TESTS:
MINIATURISED FORMAT, API
SYSTEM, 48H TEST, MAINLY
FOR HUMAN BACTERIA
CLASSICAL IDENTIFICATION
DIAGNOSIS
COST FA1104 Training School
Molecular diagnostics of bacterial diseases, Zürich, Switzerland, 2015-09-21-25 – Jaap D. Janse
19-9-2015
7
HYDROLYSIS OF PECTIN (CAVITY FORMATION) BY PECTOBACTERIUM CAROTOVORUM AND HYDROLYSIS OF FAT BY XANTHOMONAS CAMPESTRIS
CLASSICAL IDENTIFICATION
DIAGNOSIS
COST FA1104 Training School
Molecular diagnostics of bacterial diseases, Zürich, Switzerland, 2015-09-21-25 – Jaap D. Janse
PIGMENT FORMATION ON ELECTIVE KING’S MEDIUM B
CLASSICAL IDENTIFICATION
DIAGNOSIS
COST FA1104 Training School
Molecular diagnostics of bacterial diseases, Zürich, Switzerland, 2015-09-21-25 – Jaap D. Janse
ANTIBIOTIC SENSITIVITY TEST AND TEST FOR PRODUCTION OF TOXIN BY PSEUDOMONAS SYRINGAE STRAINS USING GEOTHRICHUM FUNGUS
CLASSICAL IDENTIFICATION
DIAGNOSIS
COST FA1104 Training School
Molecular diagnostics of bacterial diseases, Zürich, Switzerland, 2015-09-21-25 – Jaap D. Janse
19-9-2015
8
PRINCIPLE OF SEROLOGY AND CROSS-REACTIVIY OF POLYCLONAL ANTISERA
CLASSICAL IDENTIFICATION
DIAGNOSIS
COST FA1104 Training School
Molecular diagnostics of bacterial diseases, Zürich, Switzerland, 2015-09-21-25 – Jaap D. Janse
CLASSICAL IDENTIFICATION
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
COST FA1104 Training School
Molecular diagnostics of bacterial diseases, Zürich, Switzerland, 2015-09-21-25 – Jaap D. Janse
SEROLOGICAL TECHNIQUES USED IN PLANT BACTERIOLOGY
CLASSICAL IDENTIFICATIONDIAGNOSIS
CONJUGATE + SPECIFIC SERUM + BACTERIUM
COST FA1104 Training School
Molecular diagnostics of bacterial diseases, Zürich, Switzerland, 2015-09-21-25 – Jaap D. Janse
19-9-2015
9
PRINCIPLE OF IMMUNO-FLUORESCENCE MICROSCOPY
CLASSICAL IDENTIFICATIONDIAGNOSIS
COST FA1104 Training School
Molecular diagnostics of bacterial diseases, Zürich, Switzerland, 2015-09-21-25 – Jaap D. Janse
PRINCIPLE OF IMMUNO-FLUORESCENCE MICROSCOPY
CLASSICAL IDENTIFICATIONDIAGNOSIS
RESULT
COST FA1104 Training School
Molecular diagnostics of bacterial diseases, Zürich, Switzerland, 2015-09-21-25 – Jaap D. Janse
PRINCIPLE OF IMMUNO-FLUORESCENCE MICROSCOPY
CLASSICAL IDENTIFICATIONDIAGNOSIS
Differences between antigenic types, includingflagella can be made visible by IF
COST FA1104 Training School
Molecular diagnostics of bacterial diseases, Zürich, Switzerland, 2015-09-21-25 – Jaap D. Janse
19-9-2015
10
CLASSICAL IDENTIFICATION
DIAGNOSIS
IF uses microscopeslide andIF microscope andfluorochrome
Enzme-linked ImmunoSorbent Assay (ELISA)uses elisa plate, elisareader and enzyme
COST FA1104 Training School
Molecular diagnostics of bacterial diseases, Zürich, Switzerland, 2015-09-21-25 – Jaap D. Janse
PRINCIPLE OF DNA DOT/SLOT-HYBRIDISATION
MOLECULAR DETECTION
DIAGNOSIS
COST FA1104 Training School
Molecular diagnostics of bacterial diseases, Zürich, Switzerland, 2015-09-21-25 – Jaap D. Janse
PRINCIPLE OF DNA DOT/SLOT-HYBRIDISATION
MOLECULAR DETECTION
DIAGNOSIS
RESULT: BLACKDOTS OF DNAON AN X-RAYFILM
COST FA1104 Training School
Molecular diagnostics of bacterial diseases, Zürich, Switzerland, 2015-09-21-25 – Jaap D. Janse
19-9-2015
11
MOLECULAR DETECTIONDIAGNOSIS
FLUORECENT
IN-SITU
HYBRIDISATION
(FISH)
COST FA1104 Training School
Molecular diagnostics of bacterial diseases, Zürich, Switzerland, 2015-09-21-25 – Jaap D. Janse
MOLECULAR DETECTION
DIAGNOSIS
RESULT: ALL BACTERIA REACT WITH EU-PROBE (GREEN), TARGET BACTERIUM REACTS WITH SPECIFIC PROBE (RED)
FISH
COST FA1104 Training School
Molecular diagnostics of bacterial diseases, Zürich, Switzerland, 2015-09-21-25 – Jaap D. Janse
PRINCIPLE OF POLYMERASE CHAIN REACTION (PCR)
MOLECULAR DETECTION
DIAGNOSIS
COST FA1104 Training School
Molecular diagnostics of bacterial diseases, Zürich, Switzerland, 2015-09-21-25 – Jaap D. Janse
19-9-2015
12
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 !
COST FA1104 Training School
Molecular diagnostics of bacterial diseases, Zürich, Switzerland, 2015-09-21-25 – Jaap D. Janse
DIAGNOSIS
PRINCIPLE OFTAQMANREAL-TIME PCR
COST FA1104 Training School
Molecular diagnostics of bacterial diseases, Zürich, Switzerland, 2015-09-21-25 – Jaap D. Janse
MOLECULAR DETECTION
PRINCIPLE OF TAQMAN REAL-TIME PCR
MOLECULAR DETECTION
DIAGNOSIS
RESULT OF TAQMAN PCR WITH DILUTION SERIES OF RALSTONIASOLANACERUM
BASELINE
normalised reporterfluorescence
COST FA1104 Training School
Molecular diagnostics of bacterial diseases, Zürich, Switzerland, 2015-09-21-25 – Jaap D. Janse
19-9-2015
13
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
COST FA1104 Training School
Molecular diagnostics of bacterial diseases, Zürich, Switzerland, 2015-09-21-25 – Jaap D. Janse
• 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
COST FA1104 Training School
Molecular diagnostics of bacterial diseases, Zürich, Switzerland, 2015-09-21-25 – Jaap D. Janse
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
(EARLY)MOLECULAR DETECTION and IDENTIFICATION
DIAGNOSIS
COST FA1104 Training School
Molecular diagnostics of bacterial diseases, Zürich, Switzerland, 2015-09-21-25 – Jaap D. Janse
19-9-2015
14
PRINCIPLE OF ELECTROPHORESIS OF PROTEINS
MOLECULAR IDENTIFICATION
DIAGNOSIS
PURECULTURE
EXTRACTIONOFPROTEINS
COST FA1104 Training School
Molecular diagnostics of bacterial diseases, Zürich, Switzerland, 2015-09-21-25 – Jaap D. Janse
MOLECULAR IDENTIFICATION
DIAGNOSIS
POLYACRYLAMIDE GEL-ELECTROPHORESIS (PAGE):OBTAIN PROFILE
COST FA1104 Training School
Molecular diagnostics of bacterial diseases, Zürich, Switzerland, 2015-09-21-25 – Jaap D. Janse
PRINCIPLE OF ELECTROPHORESIS OF PROTEINS
MOLECULAR IDENTIFICATION
DIAGNOSIS
PROFILE SCANNING, IDENTIFICATION AND CLUSTERINGWITH COMPUTER SOFTWARE
COST FA1104 Training School
Molecular diagnostics of bacterial diseases, Zürich, Switzerland, 2015-09-21-25 – Jaap D. Janse
19-9-2015
15
PRINCIPLE OF FATTY ACID ANALYSIS
MOLECULAR IDENTIFICATION
DIAGNOSIS
COST FA1104 Training School
Molecular diagnostics of bacterial diseases, Zürich, Switzerland, 2015-09-21-25 – Jaap D. Janse
PRINCIPLE OF FATTY ACID ANALYSIS
MOLECULAR IDENTIFICATION
DIAGNOSIS
pv. fraxinipv. oleae
pv. nerii
COST FA1104 Training School
Molecular diagnostics of bacterial diseases, Zürich, Switzerland, 2015-09-21-25 – Jaap D. Janse
Repetative extragenic palindromic (rep) PCR fingerprintingCOST FA1104 Training School
Molecular diagnostics of bacterial diseases, Zürich, Switzerland, 2015-09-21-25 – Jaap D. Janse
MOLECULAR IDENTIFICATION
DIAGNOSIS
19-9-2015
16
MOLECULAR IDENTIFICATION
DIAGNOSIS
extragenic palindromic (rep) PCR fingerprinting
COST FA1104 Training School
Molecular diagnostics of bacterial diseases, Zürich, Switzerland, 2015-09-21-25 – Jaap D. Janse
MOLECULAR IDENTIFICATION
DIAGNOSIS
extragenic palindromic (rep) PCR fingerprinting
COST FA1104 Training School
Molecular diagnostics of bacterial diseases, Zürich, Switzerland, 2015-09-21-25 – Jaap D. Janse
AFLPfingerprinting
R = restrictionsite
A,B,C = variablelength DNAfragments
MOLECULAR IDENTIFICATION
DIAGNOSIS
COST FA1104 Training School
Molecular diagnostics of bacterial diseases, Zürich, Switzerland, 2015-09-21-25 – Jaap D. Janse
19-9-2015
17
MOLECULAR IDENTIFICATION
DIAGNOSIS
AFLP fingerprinting: result with Xanthomonas strains
COST FA1104 Training School
Molecular diagnostics of bacterial diseases, Zürich, Switzerland, 2015-09-21-25 – Jaap D. Janse
• 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
COST FA1104 Training School
Molecular diagnostics of bacterial diseases, Zürich, Switzerland, 2015-09-21-25 – Jaap D. Janse
MOLECULAR IDENTIFICATION
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
COST FA1104 Training School
Molecular diagnostics of bacterial diseases, Zürich, Switzerland, 2015-09-21-25 – Jaap D. Janse
MOLECULAR IDENTIFICATION
DIAGNOSIS
19-9-2015
18
• 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
(EARLY)MOLECULAR DETECTION and IDENTIFICATION
DIAGNOSIS
COST FA1104 Training School
Molecular diagnostics of bacterial diseases, Zürich, Switzerland, 2015-09-21-25 – Jaap D. Janse
• 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
COST FA1104 Training School
Molecular diagnostics of bacterial diseases, Zürich, Switzerland, 2015-09-21-25 – Jaap D. Janse
• 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
COST FA1104 Training School
Molecular diagnostics of bacterial diseases, Zürich, Switzerland, 2015-09-21-25 – Jaap D. Janse
19-9-2015
19
MOLECULAR IDENTIFICATION
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
COST FA1104 Training School
Molecular diagnostics of bacterial diseases, Zürich, Switzerland, 2015-09-21-25 – Jaap D. Janse
MOLECULAR IDENTIFICATION
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
COST FA1104 Training School
Molecular diagnostics of bacterial diseases, Zürich, Switzerland, 2015-09-21-25 – Jaap D. Janse
MOLECULAR IDENTIFICATION
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
COST FA1104 Training School
Molecular diagnostics of bacterial diseases, Zürich, Switzerland, 2015-09-21-25 – Jaap D. Janse
19-9-2015
20
MOLECULAR IDENTIFICATION
DIAGNOSIS
EXPERIMENTAL ERROR
60°C
SPECIFIC
55°C
NON-SPECIFIC
40°C
NON-SPECIFIC
SPECIFIC PROBEEU-PROBE
COST FA1104 Training School
Molecular diagnostics of bacterial diseases, Zürich, Switzerland, 2015-09-21-25 – Jaap D. Janse
•
• 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
MOLECULAR IDENTIFICATION
COST FA1104 Training School
Molecular diagnostics of bacterial diseases, Zürich, Switzerland, 2015-09-21-25 – Jaap D. Janse
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
COST FA1104 Training School
Molecular diagnostics of bacterial diseases, Zürich, Switzerland, 2015-09-21-25 – Jaap D. Janse
19-9-2015
21
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 !
COST FA1104 Training School
Molecular diagnostics of bacterial diseases, Zürich, Switzerland, 2015-09-21-25 – Jaap D. Janse
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
COST FA1104 Training School
Molecular diagnostics of bacterial diseases, Zürich, Switzerland, 2015-09-21-25 – Jaap D. Janse
• 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
COST FA1104 Training School
Molecular diagnostics of bacterial diseases, Zürich, Switzerland, 2015-09-21-25 – Jaap D. Janse
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