S. H. De Boer

Bacterial ring rot testing with the indirect fluorescent antibody staining procedure

The indirect fluorescent antibody staining (IFAS) procedure was successfully used in place of the Gram stain procedure to confirm diagnosis of bacterial ring rot. Antisera produced against untreatedCorynebacterium sepedonicum cells, glutaraldehyde-fixed cells, heat-treated glutaraldehydefixed cells, and a cell extract had useful IFAS titers and similar specificity. Optimum antiserum dilution was critical for a reliable test; at high antiserum dilution optimum antigen excess caused poor staining, while at low antiserum dilution non-specific reactions occurred. Although the IFAS procedure was useful as a confirmatory test, its realiability as a diagnostic test is seriously questioned.

Evaluation of immunofluorescence with monoclonal antibodies for detecting latent bacterial ring rot infections

Corynebacterium sepedonicum was detected in symptomless potato stems and tubers with immunofluorescence using monoclonal antibodies specific for the bacterial ring rot pathogen. The concentration of bacterial cells in potato tissue preparations ranged from >500 cells/microscope field to 1 cell per preparation. Symptomless tubers containing ring rot bacteria planted in field plots yielded plants with ring rot symptoms, plants with latent ring rot infections, or plants with no detectable levels of ring rot bacteria. Tubers with the greatest number of bacteria were most likely to develop plants expressing ring rot symptoms, but even some seed tubers with a low number of bacteria developed into plants with symptoms. Some seed tubers with high levels of ring rot bacteria produced plants with only low numbers ofC. sepedonicum.ResumenSe detectóCorynebacterium sepedonicum en tallos y tubérculos de papa que no mostraban sÍntomas de la enfermedad, mediante inmunofluorescencia que utilizaba anticuerpos monoclonales específicos para el patógeno de la pudrición anular. La concentración de células bacterianas en las preparaciones de tejidos de papa varió de >500 células/campo del microscopio a 1 célula por preparación. Los tubérculos sin sÍntomas, portando la bacteria de la pudrición anular, y sembrados en parcelas en el campo, desarrollaron plantas con síntomas de pudrición anular, plantas con infecciones latentes, o plantas sin niveles détectables de la bacteria. Los tubérculos con el mayor número de bacterias fueron los más propensos a desarrollar plantas mostrando sÍntomas de pudrición anular, pero aún así, algunos tubérculos-semillas con bajo número de bacterias produjeron plantas con síntomas. Algunos tubérculos-semillas con altos niveles del patógeno produjeron plantas con solo ún bajo contenido deC. sepedonicum.

Evaluation of procedures for detection of pectolyticErwinia spp. on potato tubers

The effectiveness of a tuber incubation method for detection ofErwinia carotovora var.atroseptica andE. carotovora var.carotovora in potato tubers was compared with a lenticel sampling procedure. In the first method, tubers were injured by puncturing lenticels with sterile toothpicks, then wrapped in moist paper towels and polyvinylidene film, and placed in closed chambers flushed with N2. In later experiments, wrapping tubers in two layers of polyvinylidene film and incubation in air was found to be as effective as the single layer of polyvinylidene and incubation in chambers flushed with N2. Isolations were made on a selective crystal violet pectate (CVP) medium from homogenized samples of tissue removed from soft rot lesions developing around injured lenticels. In the second method, 10 lenticels/tuber were aseptically removed with a scalpel and homogenized in distilled water; the suspension was plated on CVP. The first method was less tedious and slightly more effective than the lenticel sampling method. In a preliminary survey, these methods were used to detectErwinia infestations in small samples of certified seed potato tubers from Maine, Minnesota, Montana, New York, North Dakota, and Wisconsin. PectolyticErwinia spp. were detected in at least one sample from each state except Montana. The percentage of tubers withErwinia infestations varied from 0–100% among samples. Characterization ofErwinia isolates showed that bothE. carotovora var.carotovora andE. carotovora var.atroseptica were present. PectolyticErwinia spp. on symptomless potato seed tubers may serve as inoculum sources for blackleg and soft rot diseases.ResúmenSe comparó la eficacia de un método de incubación de tubérculos con otro de muestreo de lenticelas para detectarErwinia carotovora var.atroseptica y E. carotovora var.carotovora. Para el primer método, los tubérculos fueron heridos punzando las lenticelas con mondadientes esterilizados, luego fueron envueltos en toallas de papel húmedas y en una lámina de polivinilideno (Pv.) y colocados en cámaras cerradas inundadas con N2. En los experimentos posteriores, se determinó que era tan efectivo envol ver los tubérculos en dos capas de Pv. con incubatión en aire como la capa simple de Pv. con incubación en cámaras inundadas con N2. Se hicieron aislamientos en un medio selectivo de cristal violeta pectato (CVP) de muestras homogenizadas de tejido extirpado de lesiones de podredumbre blanda en desarrollo alrededor de lenticelas heridas. En el segundo método, se extirparon asepticamente 10 lenticelas por tubérculo con un bisturí, y se homogenizaron en agua destilada; la suspensión fue sembrada en CVP. El primer método fue menos tedioso y ligeramente más efectivo que el método de muestreo de lenticelas. En una encuesta preliminar, estos métodos fueron usados para detectar infestaciones deErwinia en pequeñas muestras de semilla certificada procedente de los Estados de Maine, Minnesota, Montana, Nueva York, Nor-Dakota y Wisconsin. Se determinóErwinia spp. en por lo menos una muestra de cada Estado, exceptuando a Montana. El porcentaje de tubérculos conErwinia varió de 0–100% entre muestras. La caracterización de los aislamientos deErwinia demostraron la presencia de ambosE. carotovora var.carotovora yE. carotovora var.atroseptica. Las especies pectolíticas deErwinia en tubérculos de semilla de papa sin s’intomas pueden actuar como fuentes de inóculo para las enfermedades piema negra y podredumbre blanda.

Nonpathogenic bacteria associated with potato stems cross-react withCorynebacterium sepedonicum antisera in immunofluorescence

Forty-three and 16% of stem smears from ostensibly healthy potato plants tested in 1980 and 1981, respectively, by immunofluorescence usingCorynebacterium sepedonicum antisera, had fluorescing bacterial cells. Eight different bacteria that cross-reacted withC. sepedonicum antisera in immunofluorescence were isolated from stems. Four of these bacteria were Gram negative, three were Gram positive, and one was Gram variable. All bacteria differed fromC. sepedonicum in morphological and biochemical characteristics except the Gram variable bacteria which were morphologically similar toC. sepedonicum at some growth stages. None of the cross-reacting bacteria was pathogenic on eggplant (Solanum melongena L. cv. Black Beauty). Three of the bacteria also formed precipitin bands in double diffusion withC. sepedonicum antiserum. Adsorption of antiserum with any one of the cross-reacting bacteria did not prevent immunofluorescence staining of all the isolated strains. Due to the cross-reactions, reliability of immunofluorescence for detection of latent bacterial ring rot infection was limitedResumenCuarenta y tres y 16% de los frotis de tallos provenientes de plantas sanas de papas probados en 1980 y 1981, respectivamente, por medio de immunofluorescencia usando antisuero deCorynebacterium sepedonicum presentaron células bacterianas fluorescentes. Ocho diferentes bacterias que reaccionaron con antisuero deC. sepedonicum en immunofluorescencia fueron aisladas de los tallos. Cuatro de estas bacterias fueron Gram positivo, tres fueron Gram negativo y una fue Gram variable. Todas estas bacterias fueron diferentes deC. sepedonicum en sus características morfológicas y bioquímicas, excepto la Gram variable cuyas características morfológicas fueron similares a las deC. sepedonicum el algunos de sus estados de desarrollo. Ninguna de estas bacterias fue patogénica en berenjena (Solanum melongena L. cv. Black Beauty). Tres de estas bacterias también formaron bandas de precipitación en doble difusión con antisuero deC. sepedonicum. La absorción de antisuero por cualquiera de estas bacterias no previno el teñido por immunofluorescencia de todos los strains aislados. Debido a estas reacciones cruzadas, la confiabilidad de la immunofluorescencia para detectar infección latente de pudrición anular fue limitada

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.CompendioSe sembraron en parcelas en el campo varios cultivares de papa utilizando segmentos de tubérculos-semillas inoculados por infiltración al vacío conCorynebacterium sepedonicum. Se registraron los síntomas foliares durante la temporada de cultivo y los síntomas en los tubérculos al momento de la cosecha. Los cultivares fueron clasificados de acuerdo a la frecuencia con la cual los tubérculos progenie expresaban síntomas externos de la purdrición anular bacteriana. Aquellos que rindieron 50–100% de tubérculos con síntomas fueron colocados en la clase de expresión de síntomas (CES) 1; los que produjeron 25–49%, 10–24%, y 9% de tubérculos con síntomas fueron colocados en CES 2, 3 y 4, respectivamente. La incidencia de infecciones latentes de pudrición anular en tallos y tubérculos fue similar entre las diferentes clases de CES de los cultivares. Las densidades de las poblaciones bacterianas, cuantificadas por inmunofluorescencia, fueron tan altas como 1010 células/g en tallos de cultivares de todas las clases CES. Las poblaciones medias en los tubérculos variaron de 106 a 107 células/g y no difirieron significativamente entre los cultivares.

Survival ofErwinia carotovora in Wisconsin soils

Erwinia carotovora var.atroseptica (van Hall) Dye andE. carotovora var.carotovora (Jones) Dye were detected in agricultural soils in Wisconsin using baiting and enrichment techniques. These soft rot bacteria could not be detected in soils of potato fields prior to planting of crops in the spring period using standard soil dilution plating techniques on a crystal violet pectate medium, however. A procedure involving incubation of samples in pectate enrichment broth followed by preparation of smears on slides and treatment with a fluorescent antibody stain specific forE. carotovora var.atroseptica was the most sensitive of the methods tested for detecting the blackleg pathogen.Erwinia carotovora was isolated more frequently during the spring from fields in which potatoes had been grown the previous year than from fields in which other crops had been grown. It was also isolated from potato tubers and stems that had overwintered in the field. The presence ofE. carotovora could not be detected in root zone samples of weed plants using the dilution plating method.ResumenEn suelos agrícolas del estado de Wisconsin, EE. UU. de NA, se detectaron ambosErwinia carotovora var.atroseptica (van Hall) Dye yE. carotovora var.carotovora (Jones) Dye por medio de técnicas de cebo y enriquecimiento. Sin embargo, estas bacterias que causan pudrición blanda, no pudieron ser detectadas en suelos de campos de papa antes de la siembra de cultivos en la época de primavera por medio del método reconocido de serie de dilución con siembra en placas de medio pectato cristal violeta. Un procedimiento que involucra la incubación de muestras en un medio de enriquecimiento con pectato seguido de preparación de un frotis sobre portaobjetos y tratamiento con una tinción fluorescente específica paraE. carotovora var.atroseptica, fue el mas sensible de los métodos probados para la detección del patógeno de la pierna negra de la papa.E. carotovora fue aislado con mas frecuencia durante la primavera, de campos en los cuales se cultivó papa el año anterior que de campos en los cuales hubieron otros cultivos. Tambien fue aislado de tubérculos y tallos de papa que habían invernado en el campo. No se pudo detectar la presencia deE. carotovora en muestras de la zona radicular de malas hierbas mediante la técnica de dilución en placas.

Current status and future prospects of bacterial ring rot testing

The bacterial ring rot disease of potato (Corynebacterium sepedonicum) can be detected in seed potato lots by serological tests with greater accuracy than by visual field inspections. Indexing tests can make use of highly specific immunofiuorescence probes, enzyme-linked immunosorbent assays, and perhaps other procedures. Sensitivity and specificity of these procedures need to be determined in order to estimate the confidence with which ring rot can be detected in a seed lot.In addition to the laboratory tests, the sampling strategy determines the efficacy and sensitivity of the indexing procedure. Samples might consist of stems, leaf petioles, or tubers in single or composite units. Disease incidence and number of plants sampled also determine the confidence level with which ring rot can be detected in a potato seed lot. Although research has been conducted on many aspects of ring rot testing procedures, further work needs to be done. Nevertheless, it is anticipated that indexing potato seed lots for the presence of ring rot will be a significant factor in control and eradication of the disease.

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

SummaryAn 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.

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

SummaryThe 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.

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

SummaryPotato 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.