Klaus Naumann

Untersuchungen zur aussagegenauigkeit und sicherheit des nachweises von Corynebacterium sepedonicum (Spieck. et Kotth.) Skapt. et Burkh. mit Hilfe mikroskopisch-zytologischer und serologischer teste

Summary Some several methods for the identification of Corynebacterium sepedonicum (Spieck. et Kotth.) Skapt. et Burkh., the causal agent of the potato ring rot, were tested for their usefulness. It could be confirmed that under our conditions the analyzing of symptoms alone does not allow an errorless diagnosis. The considerably sensitive microscopical diagnosis by the Gram staining (minimum concentration 4 × 105 cells/ml) does also not give clear results, because a differentiation of C. sepedonicum from saprophytic coryneform bacteria naturally occurring in potato tubers is not possible on this way. Further investigation with three serological methods for the identification of C. sepedonicum demonstrated that — the microagglutination test using slide technique is not specific enough, for with the applicable antisera the causal agent of the black leg disease, Erwinia carotovora subsp. atroseptica, and some identified and not identified coryneform bacteria gave cross reactions, too, besides this technique was not useful for the direct identification of the causal agent in plant pressure juice, — in contrary, the double diffusion test of Ouchterlony and the radial immuno-diffusion assay both basing on the precipitation technique were considerably better suitable for the C. sepedonicum diagnosis. By using good antisera these methods did not reveal cross reactions with saprophytic coryneforms and Gram negative soft-rotting bacteria. Their sensitivity, however, was low (≥ 109 cells/ml) and remarkable differences between C. sepedonicum isolates we tested could be observed. The double diffusion assay was also suitable for the direct identification of C. sepedonicum in plant material infected. The practical applicability of the results we obtained is discussed.

Diagnose und Vorkommen bakterieller Naßfäuleerreger in Kartoffellagerbeständen

Summary From 1971 to 1976 soft-rotting potato tubers out of 6 districts of the GDR were studied upon the occurrence of bacterial soft rot pathogens. For this purpose bacterial strains were isolated from diseased tissue after enrichment on potato slices and identified (1971–1974) or bacteria isolated out of soft-rotted tissue were directly differentiated on various selective media without pre-enrichment (1975–1976). Tuber samples from 39 different sites were analyzed and more than 2,400 pathogenic bacterial strains were isolated thereout. By both methods in the most years 30–40% of the isolates obtained could be identified as Erwinia carotovora subsp. atroseptica, the causal agent of the black-leg disease of potato. Further E. carotovora subsp. carotovora (3–33%), pectolytic Pseudomonas (2–29%) and Bacillus spp. (9–82%) frequently were found. In contrast, Clostridium strains could be isolated continuously but in a lower frequency (2%). Bacteria from other genera (Enterobacter, Klebsiella, Alcaligenes and others) occurred only very seldom. The results we obtained show, that out of the 26 possible combinations and 5 single variants of soft rot pathogens, only 18 in the samples studied were realized. In all combinations frequently found, the strains of both subspecies of Erwinia carotovora participated as a component. In the extreme dry year 1976 a considerable alteration of the normal pathogen population was to observe. Under these conditions Bacillus strains occupied 80 per cent of the pectolytic isolates. The importance of the results obtained for successful control of tuber soft rot is discussed, concerning the fact that all soft rot pathogens found can infect the tubers only pathing wounds or lenticells.

Comparison of different serological methods for the detection of the fire blight pathogen, Erwinia amylovora (burrill) winslow et al.

Summary 1. Three serological methods (slide agglutination test for colonies isolated by a semiselective medium (Hahn agar), immunofluorescence technique (IFT) and enzyme-linked immunosorbent assay (DAS-ELISA) were compared for detecting Erwinia amylovora, the causal agent for fireblight, in fluids resp. on twigs contaminated and stored up for some days and by different temperatures. 2. By all techniques the causal agent could be surely detected in concentrations of 105 cells/ml; but by isolation on the semiselective agar medium (Hahn agar), still 102 cells/ml of E. amylovora were to find out easily. In many cases isolates of E. herbicola, a frequent saprophytic inhabitant of plant surfaces, showed cross reactions with antisera against E. amylovora, if agglutination test was used, but such reactions could not be completely eliminated by employing the IFT, too. However, in ELISA cross reactions with E. herbicola strains were not observed. 3. In all experiments the results of IFT and DAS-ELISA essentially agreed. 4. After few days of storing by different temperatures a decrease of the cell concentration in suspensions of E. amylovora could be demonstrated by the isolation method and by IFT. By short-time storing of suspensions (24 h) preservation by 22°C (laboratory) or 4 to 6°C (refrigerator) induced smaller losses of cell concentration than freezing (−21°C). By storing longer than 24 hours in samples preserved in a refrigerator (4 to 6°C) the slightest reduction of pathogen density could be stated. 5. In contrast to, on twigs contaminated cell populations of the causal agent persisted without diminishing.

Zum Nachweis des Erregers der Bakteriellen Blatt- und Fruchtfleckenkrankheit, Pseudomonas syringae pv. tomato (Okabe) young, dye & wilkie, am tomatensaatgut

Summary A technique for the detection of Pseudomonas syringae pv. tomato , the causal agent of bacteria speck disease, on tomato seeds is described. The method is based on the enrichment of the pathogen by adding of the seeds under test to nutrient broth for 1–3 days at 28 °C. The seeds can be used single or in lots (5, 10, 50 or 100 ones). For identification a small amount of the broth is to transmit to agar plates, especially to Kings Agar B, or to inoculate in young tomato seedlings. On agar plates suspectable colonies (small, convex, hyaline, fluorescent) must be exactly identified by a specific antiserum (agglutination test). In bioassay the causal agent produces typical symptoms on the leaves. In three years lasting tests including 6,000 tomato trade seeds only in the sample of 1980 Ps. syringae pv. tomato could be detected (until to the May of the following year). The addition of selective substances (penicillin and actidione) to the enrichment broth considerably improved the detection and identifying of the typical colonies of Ps. syringae pv. tomato on agar plates. However, the method of enriching in normal nutrient broth and transmitting on the selective medium recommended by Bashan et al. (1981) is also very effective.

Modellversuche zur Isolierung bakterieller Naßfäuleerreger mittels pektinhaltiger nährböden

Summary 1. Under aerobic conditions bacterial soft rots are caused in the most cases by pectolytic Erwinia, Pseudomonas , and Bacillus spp. 2. Using 7 soft rotting strains several selective media were tested for their ability to sure a quick and exact isolation and differentiation of these pathogens. By bile salt-lactose-medium all Erwinia spp. under test could be isolated. For isolation of pectinolytic Pseudomonas strains the D4 medium of Kado and Heskett was suitable as the best one. 3. By the use of substrats containing pectic substances the results of isolation could be essentially improved: So, a sure differentiation of soft rotting bacteria and saprophytic organisms already upon the substrate and in addition, the isolation of pectinolytic Bacillus strains also became possible. The pectinolytic activity of the test strains on pectin-double layer-media was dependent upon the composition of the basal medium. 4. On the base of the results we obtained an isolation scheme is proposed, that allowed to indicate the pectolytic active bacilli (after heating to 80°C for 10 min) on thioglycollate medium covered by a pectin layer, the Erwinia spp. on Stewart -Medium covered by pectin layer and the pectolytic pseudomonads on FPA-medium described by Sands, Hankin and Zucker (containing citrus pectin) to the equal time. 5. The trials also demonstrated, that for the preparing of double layer-media instead of sodium polypectate mostly being recommended in literature lower estered pectic compounds can be used successfully.

Vergleichende untersuchungen an bohnensaatgut auf befall mit dem erreger der fettfleckenkrankheit, Pseudomonas syringae pv. phaseolicola(Burkholder) Young et al., mit hilfe verschiedener erfassungsmethoden: I. Nachweis durch isolierung mittels bakteriennährböden und serologische methoden

Summary 1. The halo blight of beans is one of the most important seedborne diseases of vegetables. Thus, by ascertainment of the number of bean seeds being attacked or contaminated by the causal agent of this bacteriosis, Pseudomonas syringae pv. phaseolicola, and elimination of lots containing such infested seeds, the transmission can be interrupted. 2. The mostly used methods for testing bean seeds described in literature are compiled. 3. By comparing experiments following results could be obtained: — Precultivation of seed samples in tap water containing penicillin G (17,000 IE/l), novobiocin (45 ppm) and cycloheximide (45 ppm) did not enough inhibit the saprophytic organisms frequently occurring in bean seeds; among them Pseudomonas putida and Erwinia herbicola could be identified. — However, submerging and shaking of bean seeds in sterile tap water (6 h, 22 °C) allows a sure detection of pathogen cells. — Out of 13 media under test, nutrient agar containing saccharose (5%) (NASA), NASA + crystal violet (2 ppm) described by Ercolani and Crosse (1966), Kings agar B and a modification of this one (Kings agar BF) were most suitable for isolating and identifying colonies of Ps. syringae pv. phaseolicola. On this way still — The pathogen could also be very well discovered by immuno fluorescence technique. By this method the seed rinsing water must contain nearly 105 cells/ml for a exact detection. If collecting samples containing 100 seeds were used, results completely agreeing with the agar test could be obtained. — Enzyme-linked immunosorbent assay (ELISA) was also successfully used for identifying the causal agent in rinsing water. By this technique a minimal concentration of 105 cells/ml is necessary for detection. However, testing of collecting samples containing 100 seeds brought the equal results like agar tests.

Rasterelektronenmikroskopische untersuchungen zur naßfäuleentwicklung an verschiedenen gemüsearten

Summary 1. The first steps of maceration taking place on carrot, cucumber fruit and onion slices after inoculating Erwinia carotovora subsp. carotovora and E. carotovora subsp. atroseptica were studied by scanning electron microscope. 2. Immediately after inoculation the cells of the pathogen lay on the surface of wound, separately. On carrot slices, 2 h post infectionem (p.i.) the first signs of cell division and development of microcolonies were to observe under anaerobic and aerobic conditions. On cucumber slices the division started under anaerobic conditions 2 h p.i. and under normal O 2 pressure 4 h p.i. On onions tested, the lag-phase lasted 6 h. An intensive multiplication of the pathogen could be proved on carrot and cucumber tissue 24 (anaerobic) resp. 48 h (aerobic) p.i. and on onions 48 (anaerobic) resp. 72 h (aerobic) p.i. 3. Simultaneously with the beginning of multiplication phase a complete slime layer composed from degradated plant cell material on the wound surface was established: Carrots and — only under anaerobic conditions — cucumbers reached this state 2–4 h p.i., however onions needed 6–8 h; under normal O 2 pressure on cucumber slices this process was started 15 h p.i. 4. First signs of lysis of the plant cell walls could be demonstrated on carrot tissue 6–8 h p.i., on onion slices 48 h p.i. 5. The E. carotovora subsp. atroseptica strain inoculated comparingly, induced pathological changes on the plant organs partly earlier than the typical vegetable soft rot pathogen E. carotovora subsp. carotovora under test.

Vergleichende untersuchungen an bohnensaatgut auf befall mit dem erreger der Fettfleckenkrankheit, Pseudomonas syringae pv. phaseolicola(Burkholder) Young et al., mit hilfe verschiedener erfassungsmethoden: II. Anwendung von biologischen verfahren

Summary 1. Following a communication concerning the usage of nutrient media and 2 serological tests (enzyme-linked immunosorbent assay (ELISA) and immunofluorescence technique (IFT) in this paper results obtained by applying biological methods for testing bean seeds on incidence by Pseudomonas syringae pv. phaseolicola, the causal agent of halo blight, are presented. 2. A direct detection of bean seeds infested by a single growingup test is recommended; seedlings infected can be found by typical lesions being developed on their cotyledons. However, because of the low percentage of seeds infected this method requires the checking of a lot of seeds. 3. Cells of the pathogen can be also indirectly detected in rinsing water, in which beans were submerged and shaked single or as collected sample for 6 h (22°C), by — submerging inoculation of healthy seedlings as a laboratory test — the inoculation of primary leaves by vacuum infiltration and abrasion technique and of trifoliate leaves by vacuum infiltration and abrasion technique in glass-house tests. The critical concentration of the different methods varies between 103 and 104 cells/ml, the vacuum infiltration technique is the most sensitive one (102 cells/ml). 4. The method of submerging seedlings in test fluid is also useful for evaluating the virulence of different isolates of Ps. syringae pv. phaseolicola, if an inoculum density of 108 cells/ml is used. 5. The cultivars “Esto”, “Dilana”, “Jutta”, “Rela” and “Berggold” were well suitable for all inoculation tests, however, “Valja” did not sensitively enough react. 6. Infusion of seeds in test fluids proved a failure, because by this method in many cases putrefaction was induced. 7. The advantages and disadvantages of the different techniques for detecting bean seeds infested by halo blight organism are discussed.

Auswirkung einer Infektion mit Corynebacterium sepedonicum (Spieckermann & Kotthoff) Skaptason & Burkholder im Feldbestand bei gleichzeitigem Befall mit Erwinia carotovora subsp. atroseptica (van Hall) Dye

Summary In 3 years lasting field and glass-house trials the effects of a single and a combined inoculation of tubers with the causal agents of potato black-leg and tuber soft rot, Erwinia carotovora subsp. atroseptica , and potato ring rot, Corynebacterium sepedonicum , on the development of potato crops and the tuber production were tested. The following results could be obtained: 1. By single inoculation of seed tubers with C. sepedonicum the typical symptoms of potato ring rot (occurrence of wilted plants with yellow or rolled dry leaves and of ring rot-diseased tubers at the end of the storage period) were caused and by certain varieties the incidence of black-leg disease was increased, too, in comparison with uninoculated control variants. That is to interpret as an activation of a latent E. carotovora subsp. atroseptica -infection triggered by C. sepedonicum . 2. By a combined inoculation with both causal agents — beside the typical ring rot — a significant increase of the black-leg disease compared with the single inoculation variants was to observe. Especially, the number of plants per plot and the yield of tubers were considerably diminished.