DIAGNOSIS OF BACTERIAL DISEASES WITH SPECIAL ATTENTION TO PSEUDOMONAS SPP.

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1 DIAGNOSIS Dr. J.D. Janse DIAGNOSIS OF BACTERIAL DISEASES WITH SPECIAL ATTENTION TO PSEUDOMONAS SPP. 1. HISTORY 2. STEPS IN DIAGNOSIS 3. ISOLATION AND ISOLATION METHODS 4. DETECTION METHODS NON-MOLECULAR 5. PURE CULTURE 6. IDENTIFICATION METHODS NON-MOLECULAR 7. DISCRIMINATION OF PSEUDOMONAS FORMS FOUND ON STONE FRUITS AND SOME OTHER HOSTS 1

2 HISTORY Thomas J. Burrill ( ) 1916) - fireblight USA Jan Hendrik Wakker ( ) 1927) - yellow disease of hyacinth, Netherlands Erwin Smith, USA founder of phytobacteriology Stapp,, Germany Hellmers,, Denmark Dowson, UK HISTORY Yellow disease of hyacinth Xanthomonas hyacinthi ERWIN SMITH ( ), FOUNDER OF PHYTOBACTERIOLOGY 2

3 Plant pathogenic genera Acidovorax Agrobacterium Arthrobacter Burkholderia Clavibacter Curtobacterium Erwinia Herbaspirillum Serratia Liberobacter Pantoea Pseudomonas Rathayibacter Rhizobacter Rhodococcus Sphingomonas Streptomyces Xanthomonas Xylophilus Xylella STEPS IN THE DIAGNOSIS OF BACTERIAL DISEASES OF PLANTS A ASSESSMENT OF SYMPTOMS B ISOLATION OF PATHOGENIC BACTERIA C PURE CULTURE OF ISOLATED BACTERIA D IDENTIFICATION OF PURE CULTURE E F PATHOGENICITY TEST REISOLATION FROM INOCULATED PLANTS G REIDENTIFICATION OF PURE CULTURE H DIAGNOSIS REPORT 3

4 A. SYMPTOMS OF BACTERIAL DISEASES OF PLANTS LEAF SPOTS EXCRESCENCES AND GALLS TUMOURS WILTING (VASCULAR DISEASES) NECROSIS AND CANKERS ROTTING BACTERIA EMBEDDED IN SLIME B. ISOLATION 4

5 B. ISOLATION RESULT B. ISOLATION ISOLATION MEDIA NON-SELECTIVE - Contain usually water and non-defined nutrients (e.g. peptones, beef extract, yeast extract) or defined inorganic salts and organic nutrients, making them suitable for growth of many bacterial species. SEMI - (S)ELECTIVE - Contain substances to enhance production of pigments and/or substances which can only be used by certain bacteria and/or inhibitors (e.g. antibiotics) for non-desired bacteria. SELECTIVE - Contain nutrients and inhibitors, which by their quantity and quality, allow only one bacterial species to form colonies in/on the medium (in an ideal situation). 5

6 B. ISOLATION NON-SELECTIVE CLAVIBACTER MICHIGANENSIS SUBSP. MICHIGANENSIS AND ERWINIA HERBICOLA ON YEAST-PEPTONE-GLUCOSE AGAR B. ISOLATION SEMI-(S)ELECTIVE PSEUDOMONAS CICHORII ON KING S MEDIUM B AND P. SYRINGAE PV. PHASEOLICOLA ON 5% SUCROSE AGAR: LEVAN FORMATION 6

7 B. ISOLATION SEMI-(S)ELECTIVE STREPTOMYCES SCABIEI ON STREPTOMYCES ISOLATION AGAR C. PURE CULTURE 7

8 C. PURE CULTURE RESULT D. DETECTION AND IDENTIFICATION DETECTION is tracing of plant pathogenic bacteria in or on plant material, especially when they occur subclinically (latent), without causing symptoms. It should be clearly distinguished from IDENTIFICATION, which is characterisation and naming of bacteria. 8

9 CLASSICAL DETECTION Isolation on media from infected tissue Inoculation of extract in host plant (use host as selective medium) Serological techniques such as immuno- fluorescence, agglutination, ELISA disease of hyacinth, Netherlands directly on extracts of infected material CLASSICAL DETECTION Plating or serological techniques on extracts of plant material with possible latent infection involving: extraction of bacteria by soaking or maceration concentration of bacteria by centrifugation 9

10 BIOCHEMICAL TESTS: OFFERING BACTERIA CARBON/NITROGEN SOURCE AND CHECK FOR ENZYMES OF BACTERIUM BY Ph CHANGE IN MEDIUM BIOCHEMICAL TESTS: MINIATURISED FORMAT, API SYSTEM, 48H TEST, MAINLY FOR HUMAN BACTERIA 10

11 HYDROLYSIS OF PECTIN (CAVITY FORMATION) BY PECTOBACTERIUM CAROTOVORUM AND HYDROLYSIS OF FAT BY XANTHOMONAS CAMPESTRIS PIGMENT FORMATION ON ELECTIVE KING S MEDIUM B 11

12 ANTIBIOTIC SENSITIVITY TEST AND TEST FOR PRODUCTION OF TOXIN BY PSEUDOMONAS SYRINGAE STRAINS USING GEOTHRICHUM CANDIDUM FUNGUS PRINCIPLE OF SEROLOGY AND CROSS-REACTIVIY OF POLYCLONAL ANTISERA 12

13 Table 11 BACTERIUM Target C. m. sepedonicus Cross reactive bacterium 1 Cross reactive bacterium 2 Effect of antiserum dilution on the occurrence of cross-reactions with an antiserum against Clavibacter michiganensis subsp. sepedonicus ANTISERUM DILUTION ± (titre) ± DANGER ZONE DILUTIONS PREFERRED 13