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.     

Factors affecting ring rot development in root-inoculated potato plants originating from stem cuttings

Some of the factors that affected ring rot development in potato plants grown from stem cuttings and root-inoculated withCoryncbacterium sepedonicum were investigated. Isolates ofC. sepedonicum cultured and stored for a, year on agar media were as virulent as those that had been cultured for almost 5 months. Isolates cultured for periods over 2 years gradually lost their virulence; however, decline in virulence was not as rapid as had been previously reported. One isolate became more virulent by inoculating and recovering it from a susceptible potato plant. Ring rot symptoms developed more rapidly in the cultivar Red Pontiac than in the cultivar Netted Gem; however, disease severity eventually reached the same level in both cultivars. The more mature the plants were when inoculated, the greater was the rate of ring rot development. Duration of exposure of wounded roots to inoculum of the pathogen did not affect disease development. No ring rot bacteria were recovered from plants originating from apical cuttings of ring-rot-infected potato plants that exhibited partial wilting. By this technique, plants infected withC. sepedonicum were freed from this pathogen quickly and effectively.     

Multiplication ofPseudomonas solanacearum in resistant potato plants and the establishment of latent infections

When 1-mo-old plants of a wilt-resistant clone ofSolanum phureja (1386.15) were stem-inoculated with three strains ofPseudomonas solanacearum (K60, S123, and S206), the bacteria multiplied rapidly at the point of inoculation and then moved in the vascular system to other parts of the stem. Resistant plants showed a remarkable ability to support relatively high populations of the bacterium in the absence of disease symptoms. Although multiplication in this resistant clone was substantially less than in susceptible Russet Burbank potato plants, large numbers of bacteria (up to 624 × 10⁴ cells of K60 per 5-cm stem segment) reached the base of the stem of plants maintained at high temperature (28°C) for 20 days after stem inoculation. From the base of the stem, the bacteria moved rapidly into the roots and tubers. Strains ofP. solanacearum differed in their ability to cause latent tuber infection in different resistant potato clones. When 11S.phureja ×S. tuberosum hybrids were stem-inoculated, maintained at 28°C for 3 wk and then grown to maturity at 20°C., most of the clones yielded tubers infected by one or more strains. The race 1 strain (K60) was the most infectious; 53.8% of all tubers harvested from all plants inoculated with this isolate carried latent infections. Because one clone (BR 53.1) never yielded infected tubers, there appear to be genetic factors which may be useful in breeding programs aimed at eliminating latent tuber infection.     

Effect of temperature and latent viruses on atypical ring rot symptoms of Russet Burbank potatoes

The ring rot bacterium,Corynebacterium sepedonicum (Spieck. and Kotth.) Skapt. and Burkh. [Clavibacter michiganense subsp.sepedonicum (Spieck et Kotth.) Davis et al.], and latent potato viruses (potato virus S and potato virus X) were investigated for their effect on atypical (ATYP) ring rot symptom development on Russet Burbank potato plants at different temperatures. Plants grown at 21 C from stem cuttings root-inoculated withC. sepedonicum developed typical wilting and chlorotic symptoms of ring rot that were equally severe on virus-free (VF) and virus-infected (VI) plants. All VF and VI plants grown at 15 C from inoculated stem cuttings exhibited ATYP symptoms that included extreme stunting, resetting, and chlorotic symptoms of ring rot. More severe ATYP symptoms developed on VI than on VF plants. Up to 5 wk after inoculation withC. sepedonicum, ATYP symptom severity ratings of both VF and VI plants increased and declined thereafter. The ATYP severity ratings were highly correlated with fresh weight of plants with high severity ratings being associated with low fresh weights. These results emphasize the need to determine the role of temperature and viral pathogens on ring rot symptomology in existing and newly developed potato cultivars and thereby enable better field detection of bacterial ring rot.     

Effect of total light energy on symptoms and growth of ring rot-infected Red Pontiac potato plants

The effect of total light energy levels on development of bacterial ring rot symptoms caused byCorynebacterium sepedonicum (Spieck. and Kotth.) Skapt. and Burkh., and growth (plant height) of cv. Red Pontiac was investigated by growing inoculated and uninoculated rooted stem cuttings under different pre-inoculation and post-inoculation total light energy levels. Infected plants grown under a 45 mW cm^?2 hr pre-inoculation total light energy level had a higher (P< 0.01) mean ring rot severity rating than plants grown under 103 mW cm^?2 hr. Disease severity increased as the postinoculation total light energy decreased. Plants inoculated withC. sepedonicum showed little growth under a post-inoculation total light energy of 45 mW cm^?2 hr, however, under 103 mW cm^?2 hr; significantly greater growth occurred. Uninoculated plants grew more rapidly than infected plants under all pre-and post-inoculation total light energy level combinations, except those grown at pre-inoculation levels of 45 mW cm^?2 hr.     

Comparison of inoculation methods on ring rot development in potatoes

Symptoms of bacterial ring rot (Corynebacterium sepedonicum) developed earlier and were more severe in root-inoculated potato plants grown from stem cuttings treated with indole-3-butyric acid (IBA) than in those grown from stem cuttings not so treated or from tuber seed pieces. Disease expression was more rapid and severe in root-inoculated plants than in plants from inoculated seed pieces. The use of IBA-treated stem cuttings provided uniform plant material for ring-rot studies.     

Phytophthora erythroseptica in Peru: Its identification and pathogenesis

EightPhytophthora isolates cultured from diseased potato plants and tubers in Peru were identified asPhytophthora erythroseptica Pethyb. var.erythroseptica. This pathogen was not previously reported from South America. The isolates were pathogenic to potato plants but they differed in virulence. Injuring roots prior to inoculation enhanced infection and disease development but all isolates infected noninjured roots. Continuous high soil moisture during incubation favored disease development. Under these conditions, disease developed at soil temperatures from 10 C to 30 C, but most rapidly at 25 C and 30 C. Plants grown 60 days before inoculation developed symptoms earlier and more rapidly than plants grown 15-, 30-, and 45 days. Four concentrations of mycelial-suspension inocula did not alter disease development, but zoospore inoculum induced symptoms and plant death more rapidly than mycelial suspensions at 25 C.     

Reevaluation ofSolanum species accessions showing resistance to bacterial ring rot

One hundred and fifty-six plant accessions, including susceptible checks, from the IR-1 collection were evaluated for resistance/immunity to bacterial ring rot (BRR). Each accession selected had been previously rated as highly resistant to BRR based on symptom expression but, in our tests, 57 of them yielded no plants which failed to produce symptoms. However, the remaining 99, represented by a total of 2589 inoculated plants, yielded 1000 plants which failed to produce BRR symptoms after eight weeks. Immunofluorescent antibody stain testing showed that 679 of the symptomless plants supported detectable numbers ofCorynebacterium sepedonicum cells. Comparison of root-inoculated and tuber-inoculated accessions showed that root-inoculations yielded significantly fewer symptomless and bacterial cell-free plants. No significant differences were found in BRR reactions of fiveSolanum spp. when inoculated with two different strains ofC. sepedonicum. Appreciable numbers of symptomless plants were obtained from inoculated progeny of crosses with resistant cultivars but none of those retested proved to be immune.     

Resistance toPseudomonas solanacearum in potato: Infectivity titrations in relation to multiplication and spread of the pathogen

When several wilt-resistant potato clones were tested against a highly virulent strain from Mexico (No. 276) by standard stem inoculation methods, only one clone ofSolanum phureja was resistant. When different inoculum concentrations were introduced quantitatively for infectivity titrations, however, different levels of resistance were clearly defined among clones previously rated as susceptible. Linear regression was used to estimate the ED₅₀ values (dosage required to wilt 50% of the population) for each of seven clones. These values ranged from 3 and 100 colony-forming units (CFU) for Katahdin and Russet Burbank, respectively, to 2.1 x 10⁶ CFU for S.phureja clone 1386.15. The distribution of bacteria in stems of Russet Burbank plants inoculated with the compatible strains 276 and K-60 and the incompatible strains B1 and S-210 was very different. In all cases, incompatible bacteria were not detected 10 cm above the inoculation point in the stem, even by 12 days after inoculation, whereas compatible strains multiplied rapidly at this site by 6 days. At the inoculation site and at sites immediately adjacent to it, however, incompatible bacteria attained populations that only differed by one order of magnitude from those of compatible bacteria. Similar results were obtained when a resistant clone (MS 118.24) was inoculated with the virulent strain K-60. Thus, resistance was characterized both by reduced acropetal spread and by tolerance to large numbers of the bacterium.     

Bacterial ring rot disease development in resistant and susceptible cultivars

In two greenhouse tests, we compared cell populations ofCorynebacterium sepedonicum in inoculated cultivars which had failed to show symptoms in field inoculation tests against cultivars that produce well-defined symptoms. Both population studies showed that cell numbers as high as 1 × 10⁹ colony-forming units per gm of fresh weight (cfu gm^-1) were reached in some cultivars prior to symptom development. In one of these tests, conducted when day length averaged 14 1/2 hrs, some cultivars failed to show symptoms 11 wks after inoculation but were found to have cell populations as high as 3.2 × 10⁸ cfu gm^-1 7 wks after inoculation. In another study, root and seed piece inoculations were compared in susceptible and resistant cultivars and only minor differences in symptom expression due to mode of entry of the pathogen were found.     

An agglutination test for the serodiagnosis ofCorynebacterium sepedonicum

Antisera specific forCorynebacterium sepedonicum were produced in three rabbits and in a goat. Bacterial cell agglutination titers of 640, 1280, and 1280 were recorded for the rabbit sera and 320 for the goat antiserum. All antisera reacted equally well with twelve isolates ofC. sepedonicum obtained from Canada, California, Montana, Oregon, Washington, and Wisconsin. No cross-reactivity was observed when the anti-C. sepedonicum sera were tested against five otherCorynebacterium spp.and seven other species of plant pathogenic bacteria. Using the antisera collected from rabbits, an effective and reliable agglutination test was developed for the detection ofC. sepedonicum in potato stem and tuber tissue. Extracts from tubers affected by other maladies incudingErwinia spp. (soft rot),Fusarium spp. (dry rot),Phytopthora erythroseptica (waterrot),and Pythium debaryanum (leak) or tubers otherwise free fromC. sepedonicum infection did not produce an agglutination reaction when tested with anti-C. sepedonicum serum.     

Effects of moldboard plowing,chisel plowing and rotation crops on the Rhizoctonia disease of white potato

Two tillage practices, chisel plowing (30 cm) and deep moldboard plowing (22 cm), and five rotation crops (oats, lupine, buckwheat, broccoli and peas) were studied for their effects on the soil population ofRhizoctonia solani AG-3 and on Rhizoctonia disease on potato. All rotation crops were harvested except buckwheat, which was treated as a green manure crop. Chisel plowing significantly reduced (p = 0.05) the incidence and severity of stem lesions on potato caused byR. solani AG-3. In 1990, oats after moldboard plowing significantly increased disease when compared to other crops and broccoli after chisel plowing decreased disease severity. Soil populations ofR. solani AG-3 were significantly lower with chisel plowing. No interactions between tillage and rotation crops were observed. Rhizoctonia solani Kühn is a soil inhabiting plant pathogen found worldwide that affects many plant species including white potato (Solanum tuberosum L.).R. solani attacks potato at one or more stages in development resulting in distinct disease symptoms (25) often termed the Rhizoctonia disease complex of potato. In Maine, only strains AG-3 and AG-5 ofR. solani (4, 5) have been identified as attacking potato and causing four distinct types of symptoms: 1) black scurf (sclerotia) on tubers, 2) stem cankers, 3) aerial tubers and top rosetting, and 4) killing of sprouts. Crop rotation has been reported to reduce the incidence and severity ofR. solani on potato, but no single rotation method controls completely or to a high degree of reliability (11, 23, 24, 26). Deep moldboard plowing has been shown to reduce diseases caused byR. solani andSclerotium rolfsii Sacc. in crops other than potatoes (2, 7, 15, 19, 20). However, Gudmestadet al. (6) reported that deep moldboard plowing increased the severity ofR. solani on stems and stolons of potatoes. The reduction of diseases caused byR. solani andS. rolfsii by moldboard plowing is attributed to low inoculum densities in the upper soil layer by the burial of sclerotia to depths where germination and infection were prevented (14, 15, 19, 20). However, disking to a depth of 5–7 cm did not affect disease as the inoculum remained in the root zone (14, 15). Gurkin (7) states that the rationale for deep moldboard plowing is to promote decay of organic matter, remove organic matter from the infection court and to bury the sclerotia below the infection court. Cultural control techniques are largely preventive and are designed to reduce the quantity or the activity of inoculum by means of crop rotation, tillage practices, green manure crops, etc. (22). This study was conducted to determine individual effects and possible interactions of deep moldboard plowing versus chisel plowing in various rotation crops on the presence ofR. solani AG-3 in the soil and on the incidence of Rhizoctonia disease complex of potato.     

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.     

Effect ofUlocladium consortiale,a nonpathogen,on the severity of tuber blight caused byAlternar1a solani

Inoculation withAlternaria solani andUlocladium consortiale caused significantly more severe infection of tubers than inoculations ofA. solani alone.U. consortiale, when inoculated alone, caused no significant tuber infection. Infection resulting from a plug-type inoculation was significantly greater than that from a flap-type inoculation.     

Latent infection of potato tubers byPseudomonas solanacearum

Strains ofPseudomonas solanacearum differed in their ability to infect tubers of different resistant potato clones grown in infested soil. When eight resistant clones (Solanum phureja orS.phureja ×S. tuberosum hybrids) were grown at 24–28°C in soil infested with a race 1 or a race 3 strain of the bacterium, relatively few plants had wilt symptoms at harvest, but 26.7% and 9.2% of the tubers harvested from plants infected with the race 1 and race 3 strains, respectively, carried latent infections. Some infected clones never yielded diseased tubers, however. The development of symptoms above ground was not correlated with the incidence of tuber infection in any particular clone. No tuber infection occurred in tolerant or resistant clones grown in infested soil at cool (12–22°C) temperatures. Tubers were inoculated directly in an attempt to evaluate the ability of bacteria to multiply in these tissues at different temperatures. Highly virulent strains ofP. solanacearum survived in susceptible tubers in higher numbers and for longer periods than in resistant ones. Low temperature (4°C) had a deleterious effect on survival of the bacterium in tubers, but did not completely eliminate the pathogen even after 40 days.     

Soil survival and thiabendazole sensitivity ofHelminthosporium solani isolates from Alberta,Canada

Summary Survival of the pathogen causing silver scurf of potato (Helminthosporium solani) in Alberta soils was studied by evaluatingH. solani infection in the progeny ofH. solani-free nuclear seed potato tubers planted in fields where potatoes had never been grown or were grown 1, 2, 3, or 4 years previously. Daughter tubers from all the fields developed silver scurf lesions, andH. solani was isolated from infected tubers. This is the first report of survival ofH. solani in Alberta soils. Soil-borne inoculum appears to have a role in the epidemiology of the disease and in the introduction of the pathogen into silver scurf-free potato seed stock. Of 31 plant species tested, only potato was found to be a host ofH. solani. Most of theH. solani isolates from north central Alberta were more sensitive to thiabendazole than those from southern Alberta, where thiabendazole is much more commonly used.     

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.     

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     

Evaluation of an avirulent strain ofPseudomonas solanacearum for biological control of bacterial wilt of potato

An avirulent strain ofPseudomonas solanacearum, B82, was tested for its ability to protect the potato cultivar, Ontario, from bacterial wilt caused by virulent strains of this bacterium. Strain B82 was not antagonistic to 124 virulent strains ofP. solanacearum and was not bacteriocinogenic. When potato seedpieces were soaked for 5 hr in suspensions of B82 (10⁸ to 10⁹ cfu/ml), reduction in disease severity (up to 50%) was noted in some experiments. The treated plants, grown in the greenhouse or growth room for 4–5 wk, were challenged by stem inoculation, soil drenching, or root-to-root infection with virulent strains (276 or Br5) of the bacterium. Protection was not obtained consistently, however, and much of the variability could be attributed to differences in ambient temperature and light conditions in the greenhouse between experiments. In general, high ambient temperatures and/or reduced sunlight resulted in no protection. Protection appeared to depend on the ability of strain B82 to multiply in the vascular system in the crown region and to colonize the rhizosphere of treated plants. Soil temperatures above 24 C affected the ability of strain B82 to survive in the rhizosphere. The close dependence of protection on environmental conditions suggests that this method of control may not be practical under field conditions.     

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.