Antigenic anomaly in a naturally occurring nonfluidal strain ofCorynebacterium sepedonicum

A nonfluidal strain (SSNF50) ofCorynebacterium sepedonicum (=Clavibacter michiganense subsp.sepedonicum) was isolated from potato (Solanum tuberosum) stems from Idaho. A precipitin band common to other strains was not detected in Ouchterlony double diffusion (ODD) tests of SSNF50. SSNF50 was indistinguishable from fluidal strain SS20 ofC. sepedonicum with respect to latent period in eggplant or potato, reaction in latex agglutination and fluorescent antibody stain, and level of population in infected potato stems. Latent period of infection in potato from nonfluidal strain SSNF201, isolated in the laboratory from a culture of fluidal strain SS20, was longer than that of SS20 or SSNF50 despite similar pathogen population levels. A precipitin band common to ODD tests of SS20 and SSNF50 was not detected in tests of SSNF201. SSNF50 is the first nonfluidal strain ofC. sepedonicum to be isolated from potatoes.     

A simple and efficient method to assess susceptibility of potato to stem rot byErwinia carotovora subspecies

Limited development of blackleg resistant potato cultivars has been ascribed to the lack of an effective and reliable test for large scale blackleg resistance screening. A method using leaf petioles was developed for assessing susceptibility of potato stems to blackleg pathogens (Erwinia carotovora subspeciesatroseptica andE. c. subspeciescarotovora). Fifty grams of sterile autoclaved silica sand in Magenta jars (GA-7 vessel, Magenta Corp., Chicago, Illinois) was drenched with bacterial suspension, (17 ml, 2.6 × 10 to 2.6 × 10⁷ cfu/ml) and freshly cut petioles from 5 to 6 week-old plants were inserted to about 5 mm into the sand. The Magenta jars with lids closed were kept on a laboratory bench (20 ±2 C, 16 hours light). The arrangement provided near ideal post-inoculation incubation environment of high humidity for pathogen multiplication and rot development in the petioles. Length of rot was recorded 72 hours after inoculation. Linear regression of rot development on inoculum concentration had R² ≥ 0.80. The petioles inoculated and incubated by this method produced measurable rot lesions with a bacterial concentration as low as 2.6 × 10⁴ cfu/ml and differentiated between the resistant cultivar Russet Burbank and the susceptible cultivar Sangre at all the inoculum concentrations used. Furthermore, susceptibility ranking of five cultivars obtained by this method agreed in general with their reported field reactions. The method is simple and sensitive, and could be used for large scale screening for stem rot resistance in wildSolanum species and early generation breeding populations.     

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.     

Detection of potato viruses X and S in tissue culture plantlets

The latex agglutination test (LAT) and enzyme-linked immunosorbent assay (ELISA) were evaluated in separate studies for their ability to detect potato virus X (PVX) and potato virus S (PVS) in tissue culture plantlets. Healthy and infected clonal lines of several potato cultivars were used. LAT was unsatisfactory because only low levels of agglutination were obtained with infected samples, and because variable and inconsistent results were obtained with both healthy and infected clones. ELISA, however, consistently gave high spectrophotometric readings and intense visual reactions for infected but not for healthy clones. The results indicate that ELISA can be used to detect PVX and PVS in tissue culture plantlets, and in programs where tissue culture is employed, early detection and elimination of infected plantlets is possible.     

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.     

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.     

Inoculum potential of verticillium—infested potato cultivars

Viable propagules ofVerticillium albo-atrum andV. dahliae were recovered from potato stems before and after burial in the field during 1968–1971. Of 14 cultivars tested: Red Pontiac and Superior ranked highest in numbers of propagules ofV. albo-atrum. Russet Burbank, Kennebec, and Irish Cobbler ranked highest withV. dahliae, respectively. Survival ofV. albo-atrum in infected stems was evident (5 ? 34 × 10³ ppg) even after burial in field soil for 17 months. In general, survival ofV. albo-atrum (47–69%) was higher thanV. dahliae (23 – 61%) following field burial of infected stems for 7 months.     

Detection of potato viruses S and X in dormant potato tubers by the latex agglutination test

A simple procedure for detection of potato viruses S and X from dormant tubers by a latex agglutination test was developed. Eyes were pricked with a toothpick after having been covered with 50μ1 of buffer. Approximately 20μ1 of buffer then were drawn into a capillary pipette containing 10μ1 of antibody-sensitized latex and a latex agglutination test was performed. Both viruses were detected in all infected tubers tested. For potato virus S, the mean agglutination value was shown to vary significantly among cultivars. Tests on one eye were sufficient for detection with some cultivars, but tests on three eyes were necessary for detection with other cultivars. Eye location on tubers did not appear to influence test results, but better results were obtained when eyes were tested than when other tuber locations were tested.     

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.     

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.     

A detached-leaf method to evaluate late blight resistance in potato and tomato

We developed a detached leaf method for evaluating potato (Solanum tuberosum L.) and tomato (Lycopersicon esculentum Mill.) germplasm for reactions toPhytophthora infestans, the causal organism of late blight. Primary leaves from the third to the sixth node of potato plants, and the fourth to sixth node of tomato plants were excised at the stem. Their petioles were inserted into 14 mm × 100 mm floral aqua tubes containing 9 ml of sterilized distilled water. The leaves in the aqua tubes were placed with abaxial sides down on galvanized metal hardware cloth (12.5 × 12.5 mm mesh). A 12.5 mm sensi-disc containing 50μl of 2 × 10⁴ zoospores was placed in the centers of the terminal leaflet and the second leaflet pair of the potato leaf. A single disc was placed on the center of the tomato leaf. The supporting hardware cloth was placed 2.5 cm above distilled water (2.5 cm deep) in 31 cm × 17 cm × 8 cm clear plastic boxes with tight fitting lids. Leaves on intact tomato and potato plants were inoculated in a similar manner and placed in a mist chamber. Lesion growth was determined 4, 5, and 6 days following inoculation. There were no significant differences in reactions to isolates ofP. infestans on detached and intact leaves of potato cultivars Green Mountain and Kennebec and the tomato cultivar Bonnie Best.     

Crop rotation and N fertilization effects on growth,yield, and disease incidence in potato

Crop rotation can be an effective mechanism for reducing disease incidence and contributing nitrogen (N) to succeeding crops. Interactions of plant pathogen suppression and soil nutrient availability may also exist, adding to the cropping system complexity. This study examined the impact of crop rotation, N fertilization, and their interaction on growth, yield, andRhizoctonia solani incidence in potato (Solanum tuberosum L. Norwis). Potato was grown continuously and in two-year rotations with annual alfalfa (Medicago sativa L. Nitro), hairy vetch (Vicia villosa Roth), white lupin (Lupinus albus L. Ultra), and oat (Avena sativa Astro). Fertilizer was banded at potato planting with 0, 45, 90, 135, 180, or 225 kg N ha^?1 as (NH₄)₂SO₄. Approximately 58% of continuous potato possessed stem lesions caused byR. solani, but only 12 to 22% of potato stems from other rotations possessed lesions. Tuber dry weight was affected by crop rotation in 1989, a dry year, but not in 1990. Apparent N fertilizer replacement values for hairy vetch, Nitro alfalfa, white lupin, and oat were 65, 43, 26, and 11 kg N ha^?1, respectively. All crop rotations studied appeared to enhance potato production by reducing stem infection byR. solani. Vetch and alfalfa provide additional benefits through their N contributions.     

Studies on the sensitivity of a latex agglutination test for the serological detection of potato virus S and potato virus X in Wisconsin

American Journal of Potato Research - A latex agglutination test using capillary pipettes as reaction vessels was a sensitive serodiagnostic procedure for detecting potato virus S (PVS) and potato...     

Evaluation of a mass inoculation method of potatoes withPseudomonas solanacearum

Wounding of potato stems by the application of clean sand at a 45° angle at 550 kPa pressure followed by a spray-mist ofPseudomonas solanacearum proved to be the most reliable method of rapidly inoculating a large quantity of potato plants to incite bacterial wilt in the field. The technique was 100% efficient (320/320) and it was estimated that 10,000 plants/hr could be inoculated by using this method. Because of its hygroscopic nature, sand coated withP. solanacearum created too many mechanical problems to be of practical value for field use even though the amount of wilt after 21 days was not significantly different from that of the best treatment. In addition, viability of inoculum as a bacterial suspension in water was greater than as coated sand.     

Behavioral responses of adult potato psyllid, Bactericera cockerelli (Hemiptera: Triozidae), to potato germplasm and transmission of Candidatus Liberibacter psyllaurous

The potato psyllid, Bactericera cockerelli (Sulc) (Hemiptera: Triozidae), is a major pest of potatoes that can cause yield loss by direct feeding on crop plants and by transmitting a bacterial pathogen, Candidatus Liberibacter psyllaurous (a.k.a. Candidatus Liberibacter solanacearum) associated with zebra chip disease of the crop. In recent years, there have been no studies regarding resistance of potato to the potato psyllid or the bacterial pathogen that the psyllid transmits. Thus, the objectives of this study were to determine the effects of potato germplasm on adult potato psyllid behavior and transmission of Ca. L. psyllaurous. A total of twenty-two potato (Solanum tuberosum L.) breeding clones and varieties were examined. Plant genotype significantly affected the occurrence and duration of psyllid probing, the duration of psyllid cleaning, resting and the amount of time psyllids spent off the potato leaflet as well as transmission of Ca. L. psyllaurous. For the potato genotypes in which there were significant decreases in transmission compared to controls, there was often an unclear relationship between the occurrences and duration of behaviors and subsequent bacterial transmission. We discuss the implications of our results for an integrated pest management program for the potato psyllid and Ca. L. psyllaurous control on potatoes.     

An <Emphasis Type="Italic">In Vitro</Emphasis> Assay Method for Resistance to Bacterial Wilt (<Emphasis Type="Italic">Ralstonia solanacearum</Emphasis>) in Potato

To develop an in vitro assay method for bacterial wilt resistance in potato, resistant and susceptible standard genotypes were grown in vitro, and different conditions of inoculation with Ralstonia solanacearum phylotype I/biovar 4 were examined. The optimal condition was the inoculation of 6–8 leaf stage plants with a bacterial concentration of 10² CFU ml^?1 and an incubation temperature of 28 °C. Evaluation of stem wilting was more reliable than that of leaf wilting. Using this method, nine genotypes with different resistance levels in the field were evaluated. Lower disease indices were obtained for genotypes with high resistance levels in the field, suggesting that this assay is useful for evaluating bacterial wilt resistance in a controlled environment.     

Infection of aerial parts of potato plants byColletotrichum coccodes and its effects on premature vine death and yield

Collectotrichum coccodes, the pathogen causing black dot disease of potato, was investigated to determine its potential for causing symptoms on the aerial parts of potato plant, premature vine death, and reduction in yield under greenhouse and field conditions. Spray inoculations with conidial suspensions (10⁶ colony forming units/ml) ofC. coccodes caused sunken, dark, necrotic lesions on stems, leaves and petioles, vein and leaf necrosis, dieback and premature vine death under growth chamber and greenhouse conditions. Field inoculations in 1986 and 1987 provided evidence for an increased incidence of premature vine death in seven potato genotypes (Russet Burbank, Norgold Russet, Superior, Butte, NDA 8694-3, A68113-4 and A66107-51). Late season application of metribuzin at 0.84 kg/ha significantly increased the incidence of stems with black dot symptoms in cv. Russet Burbank at one location. Field inoculations in 1987 at two locations resulted in yield reduction in the late-maturing cv. Russet Burbank (6.3 and 6.5 t/ha) and clone A68113-4 (5.0 t/ha), but not in the early maturing cv. Norgold Russet. Similar results were obtained in greenhouse studies following soil inoculations withC. coccodes: yield reduction was observed in cv. Russet Burbank but not in cvs. Norgold Russet, Superior and Katahdin. Colonization of stem ends of tubers byC. coccodes from spray inoculated field plots was higher than in tubers from uninoculated plots at one of the two locations. These results point to a much broader potential ofC. coccodes as a potato pathogen, than hitherto documented. Colletotrichum coccodes (Wallr.) Hughes (syn.C. atramentarium (Berk. & Br. Taub.) was described in 1825 (21) and has a wide host range (5). Potato and tomato are the more economically important hosts. This fungus has been variously referred to as an unspecialized, minor pathogen or secondary invader (18), weak pathogen (22), specialized parasite (5), or superficial colonizer and widespread epiphyte (19). The symptoms produced by this pathogen on potato have been described as black dot (10, 14) on stems, stolons, tubers and roots, sometimes associated with leaf scorch or wilt (24), rubbery tuber wilt (1), and tuber skin necrosis (17). The fungus is commonly associated with senescent, decaying root and stem tissue, especially at the end of the season. In Idaho, instances of premature death of potato without typical symptoms of Verticillium wilt, but accompanied by a high incidence of stems with black dot symptoms, have been observed in potato crops grown on fields fumigated with metam sodium. Additional observations by growers have also implicated the possible interaction of the herbicide metribuzin with the incidence of black dot. Although it was observed by some workers to accelerate leaf senescence (12, 22), Schmiedeknecht (21) concluded thatC. coccodes was unable to infect the green leaves of potato plant. Even thoughVerticillium dahliae is generally considered the major factor in potato early dying (20), several workers (7, 11, 13, 19, 22) have investigated the possible role ofC. coccodes in causing premature death of potato (8). However, these studies examinedC. coccodes only as a soilborne, root and tuber pathogen of potato; its effects on shoot portions (aerial parts) of the plant were not considered. In addition to causing anthracnose of fruits of tomato, pepper and squash (18), recent reports indicate thatC. coccodes causes foliage blights on eastern black nightshade (2) and velvetleaf (25). The objectives of this study were: (1) to examine the potential ofC. coccodes to cause symptoms on aerial parts of potato plant, (2) to investigate the effect of spray inoculation in the field withC. coccodes on incidence of premature vine death, tuber colonization and yield of potato, and (3) to document the effect of soil inoculation withC. coccodes alone or in combination withV. dahliae on potato varieties under greenhouse conditions.     

Potato leafroll virus purification and antiserum preparation for enzyme-linked immunosorbent assays

Potato leafroll virus (PLRV) was purified from potato foliage and stems with an average yield of 0.14 mg of PLRV/kg of potato. Modifications of an existing purification procedure are reported. Five low dosage (38-118 μg of PLRV) intravenous injections were used to produce a PLRV antiserum for use in enzyme-linked immunosorbent assays (ELISA) from tubers. PLRV was readily detected in ELISA testing of potato tubers and leaves and inPhysalis floridana Rybd. Non-specific reactions were low with all tissues. In parallel tests, a Canadian antiserum produced higher nonspecific reactions with tuber and leaf tissue. The results indicated that the use of low dosage-intravenous injections might be necessary methodology for producing PLRV antiserum for use in ELISA diagnostic tests with tuber tissue where high non-specific reactions have been reported.     

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 limited     

Prevalence of Verticillium wilt in potatoes in the Red River Valley area of Minnesota

Verticilium spp. were present in over 60% of potato fields in Clay, Marshall, Norman, and Polk Counties in the Red River Valley in Minnesota. ThreeVerticillium spp. were isolated from 27% of the potato stems collected in commercial fields during 1968–1972.Verticillium alboatrum andV. dahliae were recovered from infected stems in approximately equal amounts (51% to 47%), butV. nigrescens was recovered from less than 2% of all host-infected plants. Of 12 cultivars sampled, Superior, Kennebec, Red Pontiac, Sebago, Irish Cobbler, and Norland were most frequently infected.