Persistence ofCorynebacterium sepedonicum in soil and in buried potato stems

The effect of moisture content and temperature on the persistence, in soil, of cells of the ring rot bacterium,Corynebacterium sepedonicum (Spieck. and Kotth.) Skapt. and Burkh., was followed with root-inoculated, rooted potato stem cuttings. Ring rot bacteria were still viable after 284 days in a sterilized Lethbridge silt loam moistened to field capacity and incubated at 15°C. However, the cells did not survive beyond 6 days in two non-sterile silt loams at field capacity and 20°C. In non-sterile Lethbridge silt loam, persistence of the pathogen increased as soil moisture content and temperature decreased. Cells persisted for 10 and 15 days, respectively, in soil at 50% field capacity held at 15 and 10°C. Ring rot bacteria were not recovered after 88 days from a non-sterile Lethbridge silt loam moistened to the wilting point and held at a temperature fluctuating between 10 and 20°C, or from soil at field capacity and at a temperature fluctuating between ?5 and +5°C. They survived for 362 days in soil at the wilting point at mean temperatures of 0 or ?10°C, but were not viable after 278 days in soil at field capacity and a mean temperature of ?10°C. In excised, infected potato stems held in a non-sterile Lethbridge silt loam, the pathogen persisted for 325 days in soil at the wilting point and a mean temperature of 0°C. These results suggest that soil infested with ring rot bacteria should be kept well moistened during warm periods to inactivate these bacteria and to ensure that they do not contaminate healthy tubers that may be stored or grown there later.     

Observations on systemic infection of potato plants grown from stem cuttings inoculated withCorynebacterium sepedonicum

Summary Potato stem cuttings dipped in high concentrations ofCorynebacterium sepedonicum developed into plants which showed ring rot symptoms on leaves, stems and tubers after 13 weeks. Stem cuttings dipped in very low concentrations of the bacterium developed into plants which visually could not be distinguished from non-infected plants, but latent infection was detected in leaves, stems and tubers.     

Long-Term survival ofCorynebacterium sepedonicum on contaminated surfaces and in infected potato stems

The survival of the ring rot bacterium,Corynebacterium sepedonicum, on contaminated surfaces and in infected stems was investigated by root inoculating potato stem cuttings with aqueous suspensions prepared from these materials. The pathogen survived for 24 mo on contaminated surfaces of burlap, kraft paper, and polyethylene plastic held at 12% relative humidity (RH) at either 5 or 20°C. It persisted for less than 14 mo on surfaces held at 94% RH at either temperature. Infectious ring rot bacteria were also recovered from dried, infected potato stems held for 26 mo in an unheated machine shed. These results emphasize the importance of strict sanitation and disinfestation procedures in maintaining potato seed stocks free of bacterial ring rot.     

Survival ofCorynebacterium sepedonicum in potato stems and on surfaces held at freezing and above-freezing temperatures

The survival ofCorynebacterium sepedonicum, the bacterial ring rot pathogen, in infected potato stems and on burlap surfaces held at various freezing and above-freezing temperatures was investigated by root-inoculating potato stem cuttings with aqueous suspensions prepared from these materials. Infectious bacteria were recovered from dried Russet Burbank stems held for 44 mo in an unheated machine shed and from dried Warba stems held for 55 mo. Inoculum from dried stems of all cultivars held for 63 mo did not incite symptoms; however, inoculum from Russet Burbank stems incited symptomless infection in 10% of the inoculated plants. This 63-mo survival period exceeds a 26-mo survival period previously reported. Ring rot bacteria survived and remained infectious for at least 18 mo on burlap surfaces subjected to temperatures of ?40° to ?5°C, alternating temperatures of -5° to 5°C, or a constant temperature of 5°C. Inoculum from burlap stored at the lower temperatures caused the most severe symptoms. These results stress the need for keeping crop debris away from potato operations and for using proper decontamination procedures to maintain potato seed stocks free of ring rot.     

Cross-reactions betweenCorynebacterium sepedonicum andArthrobacter polychromogenes in immunofluorescence staining

Summary In a comparative serological study using indirect fluorescence antibody staining (IFAS) on three strains ofCorynebacterium sepedonicum and eleven strains of the genusArthrobacter, onlyA. polychromogenes cross-reacted withC. sepedonicum. The antiserum titre was 1∶2000 and its dilution was critical for identifying cross-reactions. At dilutions higher than 1∶500, onlyC. sepedonicum showed intense fluorescence and sharp edges of the walls; species ofArthrobacter gave reactions of uncertain interpretations. Some aspects of the immunofluorescence staining technique, the nature of common antigenic determinants, and the applicability of the immunofluorescence staining for detectingC. sepedonicum are discussed. Work supported by grant No 80.00458.06 from the Consiglio Nazionale delle Ricerche, Rome, Italy, Publication No 127.     

Detection ofCorynebacterium sepedonicum in potato cultivars with different propensities to express ring rot symptoms

Various potato cultivars were grown in field plots from seed pieces inoculated withCoryneacterium sepedanicum by vacuum infiltration. Foliage symptoms were recorded during the growing season and tuber symptoms at harvest. Cultivars were ranked according to the frequency with which the progeny tubers expressed external bacterial ring rot symptoms. Those that yielded 50–100% symptomatic tubers were placed in symptom expression class (SEC) 1; those that produced 25–49%, 10–24%, and ≤9% symptomatic tubers were placed, respectively, in SEC 2,3, and 4. The incidence of latent ring rot infections in stems and tubers were similar among cultivars in the different SEC classes. Bacterial population densities, quantified by immunofluorescence, were as high as 10¹⁰ cells/g in stems from cultivars of all SEC classes. Mean populations in tubers ranged from 10⁶ – 10⁷ cells/g and did not differ significantly among cultivars.     

Differential diagnosis ofVerticillium dahliae in potato with antisera to partially purified pathogen-produced extracellular antigens

Summary An extracellular protein-lipopolysaccaride antigen (PLP) was purified from culture fluids ofVerticillium dahliae. Antiserum produced in rabbits to the PLP detected the antigen in homogenates of tubers, stems and leaves ofV. dahliae-infected potato plants but not in extracts of healthy potato tissue or extracts of potato plants infected by other fungal pathogens. The antigen was not detectable in extracts of potato isolates ofV. tricorpus, V. nigrescens andV. nubilum or various other fungi. The antigen was shown to be different from cross-reactive antigens detected by antisera to mycelial antigens. When used as a tool for specific diagnosis ofV. dahliae infection in potato, antiserum to PLP was more reliable than that prepared against fungal body antigens. Publication of the Agricultural Research Organization No 245-E.     

Detection ofErwinia carotovora var.atroseptica in potato tubers with immunofluorescence following induction of decay

Summary The simplest method of inducing tubers to decay in order to detect contamination by the blackleg organism,Erwinia carotovora var.atroseptica, was to wound them and place them in a mist chamber for four days. Incubating the decayed tubers in air for a further two days improved detection of the blackleg organism by immunofluorescence, which was a more sensitive test than the double diffusion method.     

Comparison of three potato leafroll virus antisera and a single Beet western yellows virus antiserum for luteovirus detection in potato

Three potato leafroll virus (PLRV) antisera, representing European, British Columbian, and Californian isolates, performed similarly in detection of PLRV in ELISA tests of samples collected in three successive years at the Florida certification test plots and in tests of other samples collected in New York State. Although a range of absorbance values occurred, this was probably due to random variation in virus titers of samples rather than the occurrence of different virus strains or differential serological reactions by the antisera. Beet western yellows virus (BWYV) was detected in potato leafroll samples from nine states and provinces in North America. The BWYV-positive samples represented 40% in 1983 and 62.5% in 1984 of the total number of samples tested. These results confirm previous reports on the widespread occurrence of BWYV in potato with symptoms of leafroll.     

Enzyme-linked immunosorbent assay with single or combined antisera for viruses S and X in potato tubers and plants

Enzyme-linked immunosorbent assay (ELISA), with single or combined antisera was effective for diagnosing potato virus S (PVS), potato virus X (PVX) or both viruses in plants grown in the greenhouse or field. In dormant tubers, stolon, middle and apical end composite sampling with or without eyes and sprouted tubers produced reliable positive assays for PVX. Only tuber pieces with sprouts resulted in consistently reliable assays for PVS. Composite sampling of potato foliage was effective for detecting one PVX infected plant in a total of 100 Kennebec, Norland, or Russet Burbank plants. There were some false negative results and greater variability in composite PVS assays, but on average, one PVS infected plant can be detected in composites of 10 Kennebec, Norland, or Russet Burbank plants. Sodium diethyldithiocarbamate (0.01M NaDIECA) in phosphate buffered saline + 0.5% Tween (PBS-T) added to plant extracts enhanced specific reactions for either virus. Onceor twiceused enzyme conjugate was effective in ELISA of either virus from potato foliage.     

Waste potato dumps as sources of insects contaminated with soft rot coliform bacteria in relation to re-contamination of pathogen-free potato stocks

Summary Insects belonging to 12 genera in the Order Diptera, found at two large waste potato dumps in Scotland. were contaminated with soft rot coliform bacteria. In 1973, 5.7% and 3.2% and in 1974, 4.8% and 4.1% of the insects caught at each site yielded these organisms. The bacteria were identified mainly asErwinia carotovora var.carotovora, though some isolates wereErwinia carotovora var.atroseptica. Isolations from potato dump waste showed that at the time when fly activity was greatest the majority of soft rot coliforms in the waste wereE. carotovora var.carotovora, despite the fact that tubers originally forming the dumps were almost certainly infected mostly withE. carotovora var.atroseptica. Contaminated insects could readily transmit soft rot coliforms to damaged areas on the aerial parts of potato plants. It is suggested that potato stocks freed from these organisms by the stem cutting procedure could become re-contaminated by insects that disperse from potato dumps.

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Zusammenfassung Zw?lf Arten von Insekten der Gattung Diptera, periodisch gesammelt auf zwei grossen Kartoffelabfallhaufen in Schottland, waren mit koliformen Nassf?ulebakterien kontaminiert (Tab. 2, 3, 5 und 6). Nur zwei der Arten galten vorher als m?gliche Ubertr?ger dieser Organismen. 1973 waren 4,6% der an den beiden Orten w?hrend der Saison gesammelten Insekten mit diesen Organismen behaftet, 1974 waren es 4,5%. Nicht weniger als 9,6% der Insekten waren bei einigen F?ngen im Jahre 1973 und 14,7% im Jahre 1974 Tr?ger von Nassf?uleErwinia sp. Die Fruchtfliegen (Parascaptomyza sp. undDrosophila spp.) waren 1973 die am h?ufigsten kontaminierten Insekten (Tab. 2 und 3). Diese Gruppe von Insekten war auch 1974 meistens kontaminiert, aber am h?ufigsten wurden die Bakterien vonScatophaga spp. undDelia spp. (Tab. 5 und 6) isoliert. Sowohl 1973 als auch 1974 wurdeErwinia carotovora var.carotovora (in der FolgeE. carotovora genannt) h?ufiger von den Insekten isoliert alsErwinia carotovora var.atroseptica (in der FolgeE. atroseptica genannt) (Tab. 1 und 4), obwohl die Knollen. die ursprünglich den Abfallhaufen ausmachten, fast sicher meistens mitE. atroseptica infiziert waren. Das Verh?ltnis vonE. carotovora zuE. atroseptica, isoliert von Insekten beider Haufen, war 1973 9,5∶1 (13,5∶1 und 5,5∶1 je Abfallhaufen) und 1974 3,8∶1 (2,7∶1 und 6,8∶1 für die beiden Orte).E. atroseptica wurde 1973 nur früh in der Vegetationszeit (vor dem 30. Juli) von den Insekten isoliert (Tab. 1), 1974 nur früh (vor dem 30. Juli) oder sp?t in der Vegetationszeit (nach dem 10. Oktober) (Tab. 4).E. carotovora dagegen konnte in beiden Jahren über die ganze Vegatationszeit isoliert werden (Tab. 1 und 4). Die Wiedergewinnung vonE. atroseptica von Insekten war mit dem Vorhandensein von frisch abgelagerten faulen Knollen zur Zeit. wenn die Insekten gesammelt werden. verbunden. EinzigE. carotovora wurde von Insekten isoliert. die zu einer Zeit gesammelt wurden, wenn auf den Abfallhaufen nur?lteres verfaulendes Material vorhanden war. Isolationen von faulenden Ueberresten, die 1975 dem Abfallhaufen entnommen wurden (Tab. 7), zeigten, dass sowohlE. carotovora als auchE. atroseptica in der Regel das ganze Jahr vorhanden waren.E. atroseptica herrschte früh im Jahr (Januar bis Juni) vor und an einem Ort auch sp?t in der Zeit der Musterentnahme (Oktober bis Dezember).E. carotovora war von Juni bis Oktober der am meisten isolierte Organismus. Das Vorherrschen vonErwinia carotovora var.atroseptica war mit dem Vorhandensein von frisch weggeworfenen Kartoffelknollen früh und sp?t in der Zeit der Musterentnahme verbunden.E. carotovora schien in den faulenden Knollen l?nger zu überleben und war der am meisten isolierte Organismus aus ?lterem verwesendem Material. Die Verh?ltniswerte vonE. carotovora zuE. atroseptica. isoliert von Insekten. waren h?her alsjene, die für das verfaulende Material aus den Abfallhaufen gefunden wurden. Es ist anzunehmen, dass die Insekten selbst einen selektiven Einfluss zu Gunsten vonE. carotovora ausüben. Von einem der Abfallhaufen gesammelten Insekten übertrugen im GlashausE. carotovora sogleich auf verletzte Kartoffelstengel. Man nimmt an, dass Kartoffelbest?nde. die mit Hilfe des Stengelschnittes von koliformen Nassf?uleerregern befreit wurden, durch Insekten, die von Kartoffelabfallhaufen kommen, wieder verseucht werden k?nnen.

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Résumé 12 genres d'insectes appartenant à l'ordre des Diptères, capturés périodiquement en Ecosse. sur deux grands tas de déchets de pommes de terre se sont révélés être contaminés par des bactéries coliformes. agents de la pourriture molle (tableaux 2, 3, 5 et 6). Auparavant, seuls 2 genres avaient été cités comme vecteurs possibles de ces microorganismes. En 1973, 4.6% des insectes capturés aux 2 endroits, et en cours de saison. portaient ces parasites de la pomme de terre. En 1974. 4,5% étaient également contaminés. Pour l'ensemble de ces captures. il y a eu 9.6% des insectes en 1973. et 14.7% en 1974 qui portaientErwinia sp. Les mouches des fruits (Parascaptomyza etDrosophila spp.) ont été le plus fréquemment contaminés en 1973 (tableaux 2 et 3). Ce groupe d'insectes était également atteint en 1974. mais les bactéries on été plus fréquemment isolées à partir deScatophaga spp. etDélia spp. (tableaux 5 et 6). En 1973 et 1974.Erwinia carotovora var.carotovora (dans la suiteE. carotovora) a été plus fréquemment isolé de ces insectes qu'Erwinia carotovora var.atroseptica (dans la suiteE. atroseptica) (tableaux 1 et 4) bien que les tubercules constituant à l'origine les tas de déchets étaient certainement plus infestés parE. atroseptica. Le rapport entreE. carotovora etE. atroseptica pour les isolements sur insectes provenant des deux endroits a été 9,5/1 (respectivement 13.5/1 et 5.5/1 pour chacun des tas de déchets) en 1973 et 3,8/1 (2,7/1 et 6,8/1) en 1974.E. atroseptica n'a été isolé que t?t dans l'année (avant le 30 juillet) en 1973 (tableau 1) et 1974 (tableau 4) elle l'a été précocement (avant le 30 juillet) ou tardivement (après le 10 octobre).E. carotovora, par contre, l'a été durant toute la saison pendant les deux années (tableuaux 1 et 4) l'isolement d'E. atroseptica est lié au moment de la capture des insectes, à la présence de tubercules nouvellement pourris lors de la mise en tas. Par contreE. carotovora a été isolé à partir des insectes capturés sur les tas où il n'y avait que du matériel présentant des pourritures plus agées. En 1975, des isolements effectués sur les tas de déchets ont montré qu'E. carotovora etE. atroseptica étaient habituellement présents toute l'année (tableau 7).E. atroseptica était prédominante en début de saison (de janvier à juin). II n'y a eu qu'un seul cas où on l'a observé plus tardivement durant la période d'échantillonage (octobre à décembre).E. carotovora a été le principal organisme isolé de juin à octobre. La prédominance d'E. atroseptica était associée à la présence de tubercules nouvellement entreposés précocement et tardivement durant la période d'échantillonage.E. carotovora semblait subsister plus longuement dans les tubercules pourris et était le principal organisme isolé à partir de matériel altéré le plus agé. Les rapportsE. carotovora surE. atroseptica obtenus à partir des isolements effectués sur insectes ont été plus élévés que ceux trouvés à partir du matériel pourri en provenance des tas. Les auteurs suggèrent que les insectes euxmêmes peuvent exercer une action sélective en faveur d'E. carotovora. Les insectes collectés à partir d'un des tas peuvent transmettre facilementE. carotovora aux pommes de terre se trouvant en serre et dont les tiges sont endommagées. Les auteurs suggèrent que les lots de pommes de terre indemnes de bactéries coliformes grace au procédé de bouturage peuvent être à nouveau recontaminé à partir des tas de déchets de pommes de terre et par l'intermédiaire des insectes.

     

Detection ofClavibacter michiganensis subsp.sepedonicus in potato stems and tubers grown from seed pieces with various levels of inoculum

Summary An indirect sandwich enzyme-linked immunosorbent assay (ELISA) was used to monitor potato stems of three cultivars for the presence of the ring rot pathogen,Clavibacter michiganensis subsp.sepedonicus, during the growing season and in progeny tubers after harvest. The highest ELISA values were obtained with the highest concentration of bacteria used to inoculate seed pieces in all cultivars tested. Low ELISA values were obtained for stems and progeny tubers selected from plants grown from seed inoculated at lower bacterial concentrations. Estimates of bacterial densities in stems and progeny tubers by immunofluorescence indicated that low ELISA values were most probably caused by low bacterial numbers. It is suggested that the sensitivity of ELISA for detecting the ring rot pathogen in potato stems and progeny tubers is a function of the concentration of bacteria in individual seed pieces.     

A comparative study in five laboratories on detection ofClavibacter michiganensis subsp.sepedonicus in potato stems and tubers

Summary Potato stems and tubers grown in the field from seed tubers inoculated withClavibacter michiganensis subsp.sepedonicus, which causes bacterial ring rot, were tested by indirect, sandwich enzyme-linked immunosorbent assay (ELISA) in five laboratories. Correlation between values for each experimental treatment from the five laboratories was greater (r=0.86) than correlation between values for individual samples (r=0.71). When three or more laboratories obtained ELISA values of ≥0.200 for a sample, that sample was presumed to be positive. Conversely, when three or more laboratories obtained ELISA values <0.200, the consensus determination was regarded as negative. The percentage of stem and tuber samples that were in agreement with the consensus ELISA determination varied from 65.5 to 96.7%. Indirect immunofluorescence tests, conducted on the same samples in two laboratories, were consistent with 83.4–91.9% of the consensus ELISA determinations. Presence or absence ofC.m. sepedonicus was confirmed in some samples by an eggplant bioassay and direct isolatiion of the bacterium. The ELISA procedure was well suited for screening large numbers of samples and this study confirms it to be a promising procedure in routine indexing of seed potatoes forC.m. sepedonicus.     

Parameters for specific detection ofClavibacter michiganensis subsp.sepedonicus in potato stems and tubers by multiplexed PCR-ELISA

A multiplex PCR-ELISA protocol for detection ofClavibacter michiganensis subsp.sepedonicus (Cms) was developed that is based on primers for amplification of three single-copy, unique DNA sequences, Cms50, Cms72, and Cms85. The three sequences were simultaneously amplified from the genomes of all 42 strains of Cms that were tested including variant mucoid forms, but not from strains representing five related subspecies, andRathayibacter rathayi andRhodococcus faciens. The lowest limit of detection by gel electrophoresis was estimated to be approximately 300 CFU per mL when cells were spiked into potato core fluid, but sensitivity increases approximately 10-fold using PCR-ELISA. Inclusion of a sea anemone DNA fragment engineered so it could be amplified from the Cms72 primer set provided the simultaneous signal that the system functioned properly when any sample was free of the pathogen. The addition of hydrolyzed casein to the reaction mix was demonstrated to markedly reduce or eliminate inhibition of PCR by plant cell components or contaminants. Multiplex PCR-ELISA detection of Cms was determined to be verifiable for analysis of both stems and tubers based on the amplification of multiple sites in its genome, it provides absolute specificity, and it was more sensitive than detection based on gel electrophoresis of PCR products and serological approaches.     

Physiological mechanisms associated with the development of internal necrotic disorders of potato

Physiological disorders such as Brown Center and Internal Brown Spot affect the quality of potato tubers destined for fresh and processing markets. Cultural and environmental factors giving rise to such disorders have been studied extensively. Considerably less information is available on the underpinning physiological, molecular and biochemical mechanisms. However, the cell’s calcium status clearly plays an important role in resistance responses. Whilst extreme genotypic susceptibility to these disorders exist such variation has not been exploited fully to dissect the genetics and biochemistry of the key processes. This is central to the selection of resistant germplasm or to the manipulation of existing genotypes for improved tolerance     

Changes in flavor-affecting aroma compounds during potato storage are not associated with lipoxygenase activity

When potatoes are boiled and then chill-stored they rapidly develop off-flavor. This is a result of lipoxygenase activity, and is a serious problem in food-service systems. Lipoxygenase activity may vary during winterstorage of potatoes. Raw potatoes of the variety ‘Bintje’ were therefore analyzed for lipoxygenase activity during winter storage. Furthermore, content of potential offflavors and typical potato aroma compounds were determined in raw, boiled, and in boiled/chill-stored potatoes. Four months after harvest the lipoxygenase activity started to increase, both when linoleic and linolenic acid were used as substrates. Production of typical oxidation products (aldehydes) of these fatty acids that earlier have been shown to cause off-flavor in boiled/chillstored potatoes did, however, decrease. It is therefore concluded that this was not due to changes in lipoxygenase activity     

Effect of plant density on the inflorescence production of stems and the distribution of flower production in potato

Summary A knowledge of the pattern of flowering and seed production is required for the development of large-scale field production of True Potato Seed (TPS). At the highland experimental station of the International Potato Center in Peru, data on flowering and seed production were collected from three cultivars planted at three densities. Main stems in which flowering was delayed ceased shoot growth at an earlier stage and produced fewer inflorescences. Inflorescences produced later had fewer flowers, a lower berry set and yielded less seed. Inflorescences flowering at the same time performed similarly, irrespective of their position on the plant. Increasing plant density resulted in cessation of shoot growth at an earlier stage and concentrated inflorescence and flower production at primary positions of early-flowering shoots. With cvs Renacimiento and Yungay a higher plant density increased the percentage of flowers produced in the first three weeks of the flowering period, but with cv. Atzimba the effect of plant density on the distribution of flower production was off-set by a slower stem development.