Isolation, storage and management of plant pathogenic bacterial strains. 4 March 2008 COST 873, York

Transcription

1 Isolation, storage and management of plant pathogenic bacterial strains

2 Collect of samples This first step is very important because all other steps depend on this step. Moreover, the person who performs this step is often not the person who isolates the bacterium. different kind of samples can be colected Always collect disease and healthy parts of plant as samples for isolation

3 Types of sample Collect in the field Photo S. Süle Photo S. Süle

4 Type of sample Collect in the field Photo S. Süle Photo S. Süle

5 Type of sample Collect in the field

6 Material for isolation Photo Horky

7 Isolation Photo S. Süle Photo S. Süle

8 Isolation Crushing and /or washing in buffer Photo S. Süle

9 Isolation

10 Isolation (6/6) Photo Horky Photo Horky Photo Horky

11 Plating on agar medium 1 2 3

12 Cloning of bacterial strains Photo Horky

13 Cloning of bacterial strains

14 UMR Pathologie Vˇgˇ tale - 42, rue Georges Morel BP 57, Beaucouzˇ c edex, France Expˇditeur : Date de prˇl vement : Dˇpartement : Commune : Nom du propriˇtaire : Plante attaquˇe : Esp ce : Variˇtˇ : å ge : Nature du sol : Profond, superficiel, sableux, caillouteux, rocheux, bourbeux, calcaire, silicieux, argileux Altitude : Exposition : Pente : v nements anormaux, climatiques, biologiques des derni res annˇes : Sˇc heresse, gelˇe, incendie, attaques de maladies ou ravageurs : Conditions de vˇgˇ tation de l Ōannˇe : Nature des cultures prˇcˇde ntes (en cas dõassolement ou de reconversion) : Traitements phytosanitaires appliquˇs : Dates : Maladie Localisation sur la plante : racine, tige, branche, feuilles, fleurs, fruits : tat de la plante attaquˇe ou malade : sur pied, coupˇe, entreposˇe, mise en Ļuvre : Description dˇtaillˇe des dˇg ts au moment du prˇl vement : Date dõapparition des dˇg ts : Importance des dˇg ts : Intensitˇ : - dans lõexploitation : - dans la rˇgion : tendue : - dans lõexploitation : - dans la rˇgion : La rˇgion a-tõelle connu des attaques semblables : oui, non ; si oui, quand? : Observations diverses : Des ˇchantillons ont-ils ˇtˇ envoyˇs un autre laboratoire? oui, non ; si oui,lequel? Fig. 2. Fiche de renseignements

15 Diagnostic, first steps Cytology Gram coloration Classical KOH Motility

16 Biochemical features Glucose metabolism O F I A Yellow w Yellow Bluegreen Blue Yellow Bluegreen Bluegreen Bluegreen O : oxydation, F :Fermentation, I : no utilisation, A : Alkalinisation

17 Biochemical features Nitrate reductase Cytochrome oxydase Florescent pigment

18 Hypersensitivity on non-host plants

19 Complementary biochemical features Carbone hydrate utilisation Beta-glucosidase Aesculine hydolase

20 Rapide identification of bacterial genus Gram + - Utilisation du glucose (Hugh et Leifson) Oxydatif ou inactif Oxydatif ou inactif - + Mobilitˇ Flagelles Fluorescence Rˇduc tion des nitrates Oxydase HR sur Tabac - + ou Genres Clavibacter Rhodococcus exemples d'esp ces ou de Groupes C. michiganensis R. fascians Particularitˇs Couleur va riable suivant subsp. (subsp. nebraskensis : orange) Colonies d aspect sec, orange vif Rhatayibacter R. rathayi Colonies jaunes Curtobacterium (inactif) ou ou - Agrobacterium (inactif) ou - Xanthomonas C. flaccumfaciens A. tumefaciens A. vitis Etc 19 espèces + 66 pathovars , 2 de Le liott et al. 1 (oxydatif) + + ou Pseudomonas 3 P. cichorii Couleur va riable suivant pv. (pv. oortii: jaune) Colonies crème muqueuses Colonies jaunes très muqueuses Colonies blanc-crème 4, 5 (phytopathogènes + ou ou - et saprophytes) + ou Colonies blanches (alcalinisant) + ou - + Acidovorax A. valerianellae généralement ovales Colonies jaunes, croissance (inactif) - - Xylophilus X. ampelinus - + ou - très lente (inactif) + + Ralstonia R. solanacearum Colonies crème (inactif) + ou - + ou - Fermentatif ou ou - Burkholderia B. caryophylli Colonies crème Erwinia Pectobacterium E. amylovora Colonies blanches muqueuses E. rhapontici Colonies roses P. carotovorum Colonies dentelées blanccrème Brenneria B. rubrifaciens Colonies roses Pantoea P. agglomerans Colonies jaunes (saprophytes) Enterobacter E. pyrinus Colonies blanches

21 Storage of bacterial cultures

22 Storage of bacterial cultures Under water layer On agar sland under oil layer Freezed Deep freezed lyophilized

23 Storage of bacterial cultures Under water layer Store at +4 C Until one year Easy to use

24 Storage of bacterial cultures On agar sland under oil layer Store at room temperature 2-3 years Easy to use

25 Storage of bacterial cultures Freezed Store at -20 C Store at -80 C Bacterial suspension in nutrient broth (2/3) and sterile glycerol (1/3) Possibility to use glass beds several years Better reliability at -80 C than -20 C

26 Storage of bacterial cultures Deep-freezed Store at -196 C in liquid nitrogen Bacterial suspension in nutrient broth (2/3) and sterile glycerol (1/3) Possibility to use glass beds several years Better reliability at than at -20 C or -80 C because no risk of electric failure cryovials Scelled capillary

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28 Storage of bacterial cultures Lyophilized - in vials In scelled glass tube Strain#

29 What is Lyophilization? Lyophilization is a technique of dehydration by refrigeration and vacuum. A balance between the pressure and temperature helps in drying aqueous preparation while respecting the integrity of living cells (e.g.: bacteria), as well as complex and delicate structures (e.g.: enzymes, proteins).

30 Advantages of lyophilization: Lyophilization helps in: *Preserving delicate substances in aqueous solution (mainly of biological nature) *Obtaining a chemical stability (e.g.: biological reagents) *Resolving the problems of stocking, marketing and use of product

31 Lyophilization

32 Retreiving bacterial culture from lyophilized bacterial suspension QuickTime et un décompresseur mpeg4 sont requis pour visionner cette image.

33 management E.g. CFBP An homemade software developed under filemaker Pro c to manage strains information, stocks and distribution A Web site for customers

34 Management

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