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.     

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.     

Multi-year evaluation ofClavibacter michiganensis subsp.sepedonicus disease symptoms in cultivated potato genotypes

The expression of foliar and tuber bacterial ring rot (BRR) symptoms in 108 cultivated potato genotypes inoculated withClavibacter michiganensis subsp.sepedonicum was examined in field experiments during 1986 to 1996. Considerable variation in the incidence of foliar BRR symptoms was observed in all plant cultivars, ranging from no detectable symptoms to complete necrosis of the leaves. Similar variability was observed in the incidence of tuber BRR symptoms, and severity of tuber symptoms ranged from no detected symptoms to complete breakdown of the vascular ring that extended throughout the tuber. A decline in the incidence and severity of tuber, but not foliar symptoms, was observed in the later years of the study (P<0.05). Foliar and tuber BRR symptoms generally were positively correlated (P<0.01) prior to 1993 but not in subsequent years (P>0.05) when tuber symptoms declined. Strong correlations were observed between the incidence and severity of foliar BRR symptoms (r=0.41, P<0.01) and between the incidence and severity of tuber BRR symptoms (r=0.96, P<0.001). During the 11 year period, no BRR symptoms were observed in 2,620 uninoculated control plants.     

The effect of ethylene and abscisic acid on symptom expression of bacterial ring rot in eggplant and potato

Studies were performed to determine the effect that plant hormones involved with plant senescence (i.e., ethylene and abscisic acid) and photoperiod have on disease development and symptom expression of bacterial ring rot (BRR) caused byCorynebacterium sepedonicum (Spieck & Kotth.) Skapt & Burkh. Potato plants were grown either under a long (14 hr.) or short (10 hr.) photoperiod, while eggplants were grown only under a short (10 hr.) photoperiod. Disease severity ratings of BRR were found to be significantly higher (P=0.05) on potato plants grown under a short photoperiod compared to a long photoperiod. Plant heights of BRR infected plants were found to be significantly greater under the long photoperiod. Endogenous levels of ethylene were found to be significantly (P=0.05) greater in inoculated potato plants grown under a long photoperiod than inoculated plants grown under a short photoperiod. Results suggest that the plant hormones ethylene and abscisic acid do not significantly affect the disease development and symptom expression of BRR.     

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 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.     

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.     

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.     

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.     

Evaluation of tuber-bearingSolanum species for symptomology,as diagnostic hosts,and sources of immunityto potato virus Y necrotic strain (PVYN)

A total of 258 Plant Introductions (PI) belonging to 69Solarium species were evaluated in the greenhouse for their reaction to the tobacco veinal necrosis strain of potato virus Y (PVY N). One hundred and thirty-one (50.7%) of the PI accessions produced mosaic symptoms ranging from mild to severe. Local lesion and veinal necrosis symptoms were observed in 19 PI accessions (7.3%) and a variety of other symptoms were observed in another 11 PI accessions (4.2%). Only 97 PI accessions (37.5%) were symptomless carriers of PVY^N. PI accession 473505 ofS. sparsipilum and PI accession 498021 ofS. brachycarpum developed local lesions and veinal necrosis with PVY^N, but necrotic spots and mosaic with PVY^o. Common mechanically-transmitted potato viruses A, S, M, and X did not interfere with PVY symptom development inS. sparsipilum andS. brachycarpum. Thus, PI 473505 and PI 498021 can be used as indicator plants for specific identification of PVY^N. PI accession 472819 ofS. chacoense developed local lesions with systemic spread in PVY^o, but without systemic spread in PVY^N. Thus, this can be used as a differential host plant for PVY strains. Two PI accessions ofS. stoloniferum, PI 160372 and 161171 were immune to PVY^N.     

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.     

Effect of inoculum dose and preparation,strain variation,and plant growth conditions on the eggplant assay for bacterial ring rot

Quantitative differences among strains ofCorynebacterium sepedonicum (Spieck. and Kotth.) Skapt. and Burkh. (=Clavibacter michiganense subsp.sepedonicum) were distinguished in eggplant (Solanum melongena L. ‘Black Beauty’) bioassays with a simple leaf axil inoculation technique. Latent period of infection (time from inoculation to first appearance of symptoms) varied inversely with inoculum dose. Latent period of infection varied significantly among strains ofC. sepedonicum. Symptoms developed more rapidly in plants grown under controlled conditions in a growth chamber than those grown in a greenhouse. Plants treated with inoculum prepared from broth cultures exhibited symptoms more rapidly than those treated with inoculum grown on agar.     

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.     

Insect transmission of the bacterial ring rot pathogen

Three species of potato-infesting insects were evaluated under laboratory and field conditions for their capability to transmitCorynebacterium sepedonicum (Spieck. & Kotth.) Skapt. & Burkh., the bacterial ring rot (BRR) pathogen of potatoes. Laboratory and field studies confirmed the vector capability of the Colorado potato beetle (CPB),Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae) and the green peach aphid (GPA),Myzus persicae (Sulzer) (Homoptera: Aphididae). Both adults and larvae of the CPB proved to be vectors of the pathogen. Only adult GPA were evaluated. Bacterial transmission was most likely mechanical: analyses of the CPB mouthparts stained by the indirect immunofluorescence antibody staining (IFAS) procedure confirmed the presence of the bacterium after the beetles were exposed to infected plant tissue. Eggs, haemolymph, feces, regurgitations, and macerates of the digestive tract failed to show the presence of the bacterium. The aster leafhopper,Macrosteles fascifrons (St∮al) (Homoptera: Cicadellidae) failed to acquire and transmitCorynebacterium sepedonicum.     

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.     

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     

Freeing Russet Burbank potato plants from ring rot by stem cutting and tuber propagation

Apical stem cuttings removed from potato plants (cv. Russet Burbank) infected withCorynebacterium sepedonicum and expressing mild symptoms of bacterial ring rot were demonstrated to be free from the pathogen. This material remained free of ring rot through two additional stem cutting generations and when grown in the field for four successive tuber generations.     

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.     

对SMV不同抗性的大豆品种体内过氧化物酶活性变化的研究

采用ＳＭＶ－ａ析系为毒源接种３人不同抗性类型的大豆品种，在接种后８，２４，４８，７小时及在３，６，９，１２，１５，１８ １１次连续取样与相应ＣＫ一并测定过氧化物酶的活性后发现：未经接种的抗感品种体内ＰＯ酶活性差异较大，病品种中ＰＯ酶活性水平高于抗性品种２倍以上。     

Evaluation of common bean lines for their reaction to tomato yellow leaf curl virus-Ir2

Tomato yellow leaf curl virus (TYLCV) can cause an economically relevant disease in the major tomato growing regions, and no resistance has been identified in commercial and wild tomato cultivars in Iran. In the present study, 34 common bean lines were screened for their reaction to TYLCV-Ir2 under greenhouse conditions. The experiment was conducted in a randomized complete block design with three replications. Bean plants were inoculated at the first trifoliate stage with viruliferous whiteflies (Bemisia tabaci biotype B) and were then sprayed with an insecticide and maintained in whitefly-proof greenhouse. The inoculated plants were monitored for the development of symptoms and the presence of viral DNA by polymerase chain reaction (PCR) four weeks after inoculation. Results revealed that out of the 34 lines, five were resistant. These lines exhibited either no symptoms or very mild symptoms and no viral DNA was detected in them by PCR. Two lines, which showed no typical disease symptoms but contained viral DNA, were identified as tolerant to TYLCV-Ir2. The vector feeding preference for common bean lines was assessed. Results indicated a significant difference in adult whitefly numbers among bean lines but there was no relationship between the number of whiteflies and disease symptom severity. The resistance to TYLCY-Ir2 expressed in common bean lines may be useful as source of resistance for the development of resistant commercial common bean cultivars.