A pathogenic strain of Xanthomonas campestris pv. vesicatoria, causal agent of bacterial spot of tomato (Lycopersicon esculentum Mill.), was genetically engineered to bioluminesce. In planta growth of the bioluminescent strain was similar to that of its parental strain. Movement and growth of the bioluminescent strain in susceptible tomato seedlings after wound inoculation was followed over time with a liquid-N-cooled, charge-coupled device camera. Highly significant differences in bioluminescent bacterial growth were observed in the four tomato cultivars used. Systemic bacterial movement was most pronounced in `Sunny', which showed population development not only at the inoculation sites but also on several sites in the leaves and at the leaf margins. Bacterial bioluminescence levels in `Campbell 28' remained significantly lower than those observed in `Walter' and `Sunny'. The technique offers unique possibilities for studying host-pathogen interactions and bacterial pathogenesis.
Fenny Dane and Marten H. Dane
Jeffrey A. Anderson, Niels O. Maness, and Robert E. Stall
Bell pepper (Capsicum anuum L.) leaves inoculated with Race 1 of Xanthomonas campestris pv. vesicatoria (XCV) produced more ethylene and methanol than water-infiltrated controls in studies with leaves attached or detached during inoculation and dissipation of water-soaking. `Early Calwonder 20R'. a pepper genotype resistant to Race 1 of XCV, evolved more ethylene and methanol than `Early Calwonder 10R' (susceptible) following syringe inoculation of detached leaves with ≈ 7 × 107 cells/ml. A light intensity of ≈ 500 μmol· m-2·s-1 during dissipation of water-soaking of attached leaves triggered more ethylene and methanol than covering inoculated leaves with aluminum foil. Volatile hydrocarbon production from leaves infiltrated with distilled water was not significantly affected by light intensity during dissipation of water-soaking. The lipid peroxidation products, ethane and pentane, were not detected by headspace sampling following bacterial inoculation.
J.W. Scott, J.B. Jones, G.C. Somodi, and R.E. Stall
Tomato (Lycopersicon esculentum Mill.) accessions were tested for hypersensitivity and rated for resistance following field inoculation with tomato race 3 (T3) of the bacterial spot pathogen Xanthomonas campestris pv. vesicatoria (Doidge) Dye (Xcv) in 1992 and 1993. Hawaii 7981, PI 126932, PI 128216, and selections of the latter two expressed hypersensitivity. Hawaii 7981, only tested in the field in 1993, was nearly symptomless and developed significantly less disease than any other accession. PI 128216 had a level of disease similar to susceptible `Solar Set' when tested in 1993. However, a selection from it (PI 126218-S) was significantly more resistant than `Solar Set' in both years. Although PI 126932 had a level of disease similar to `Solar Set' in both years, a selection from it (PI 126932-1-2) was significantly more resistant than `Solar Set' in 1993. Other accessions without hypersensitive responses but more resistant than `Solar Set' for two seasons were PI 114490, PI 126428, PI 340905-S, and PI 155372. Hawaii 7975 was significantly more resistant than `Solar Set' in the one season it was tested.
Suparna R. Mundodi, Jeffrey A. Anderson, Niels O. Maness, Michael W. Smith, Bjorn Martin, Marlee L. Pierce, and Andrew J. Mort
The hypersensitive response in resistant plants exposed to incompatible pathogens involves structural changes in the plant cell wall and plasma membrane. Cell wall changes may include pectin deesterification resulting in release of methanol. The time course of methanol production was characterized from `Early Calwonder 20R' pepper (Capsicum annuum L.) leaves infiltrated with the incompatible pathogen, Xanthomonas campestris pv. vesicatoria (Doidge) Dye race 1 (XCV). In the first time course experiment, leaves were infiltrated with either 108 colony-forming units/mL of XCV or water control. Leaf panels (1 × 5 cm) were excised after dissipation of water soaking, then incubated in vials at 24 °C. Headspace gas was analyzed at 6-hour intervals up to 24 hours. The rate of methanol production from resistant pepper leaves infiltrated with XCV was greatest during the first 12 hours after excision. In another experiment, leaf panels were harvested at 6-hour intervals up to 24 hours after inoculation and incubated for 12 hours at 24 °C to determine the relationship between the interval from inoculation to leaf excision and methanol production. The highest rate of methanol production was obtained when the interval between bacterial infiltration and leaf excision was 18 hours. The relationship between methanol release and changes in the degree of methylesterification (DOM) of cell wall pectin was determined in near isogenic lines of `Early Calwonder' pepper plants resistant (20R) and susceptible (10R) to XCV race 1. Cell walls were prepared from resistant and susceptible pepper leaves infiltrated with XCV or water. XCV-treated resistant leaves had 18% DOM and 9.7 nmol·g-1·h-1 of headspace methanol, and the susceptible leaves had 48% DOM with 0.2 nmol·g-1·h-1 methanol. Susceptible and resistant control leaves infiltrated with water had 55% and 54% DOM, respectively, with no detectable methanol production. Increased methanol production in resistant pepper leaves inoculated with XCV coincided with an increase in cell wall pH. Intercellular washing fluid of resistant pepper leaves had a significantly higher pH (6.9) compared to susceptible leaves (pH 5.1) and control leaves infiltrated with water (pH 5.1). Both 10R and 20R pepper leaves infiltrated with buffer at increasing pH's of 5.1, 6.9 or 8.7 had increased methanol production. Since deesterified pectin is more susceptible to degradation, demethylation may facilitate formation of pectic oligomers with defensive signalling activity.
J.W. Scott, J.B. Jones, G. Cameron Somodi, and R.E. Stall
Resistant Hawaii 7981 (P1) was crossed with susceptible Fla. 7060 (P2), and F1, BCP1, BCP2, and F2 generations were obtained. Hypersensitive reactions (Hr) were measured 24 and 48 hours after inoculation in growth chambers at 24 and 30C. At 30C, there was no Hr. At 24C and 24 hours, 100% of Hawaii 7981 plants, 54.2% of BCP1 plants, and 21.7% of F2 plants had Hr. At 24C and 48 hours, 100% of Hawaii 7981, the F1, and BCP1 plants; 50% of BCP2 plants; and 73.3% of F2 plants had Hr. Other plants were inoculated and rated for race T3 in the field. Disease for each generation was significantly different (P < 0.05) and their order from most to least resistant was P1, BCP1, F1, F2, BCP2, and P2. The F1s were distributed between the parents with slight overlaps. BC plants had bimodal peaks similar to the F1 and their respective parents. The F2 had three peaks corresponding to P1, F1, and P2. The data suggest Hr and field resistance are controlled by the same incompletely dominant gene.
G. Cameron Somodi, J.B. Jones, J.W. Scott, and J.P. Jones
A `spray-inoculation seedling screening procedure was developed for detecting resistance to Xanthomonas campestris pv. vesicatoria (Doidge) Dye, causal agent of bacterial spot of tomato (Lycopersicon esculentum Mill.). Two-week-old transplants were preconditioned under 95% humidity for 16 hours before spray inoculation and then rated for bacterial spot 2 weeks later. Resistant plants could also be distinguished from susceptible genotypes using a modified bacterial speck [Pseudomonas syringae pv. tomato (Okabe) Young, Dye, and Wilkie] screening procedure (cotyledon-dip technique). When results of both screening methods were compared to field ratings from three previous seasons, significant correlations were more frequently observed for the spray-inoculation method. In Summer 1991, individual plants were evaluated by the spray-inoculation technique and then were placed in the field to determine susceptibility under field conditions. Correlations (r = 0.28 to 0.34) between spray-inoculation seedling screening ratings and field ratings, although low, were significant (P ≤ 0.0001). More than 90% of susceptible plants could be eliminated, saving labor, space, and time.
J.E. Flaherty, G.C. Somodi, J.B. Jones, B.K. Harbaugh, and L.E. Jackson
A mixture of host-range mutant (h-mutant) bacteriophages specific for tomato race 1 (T1) and race 3 (T3) of the bacterial spot pathogen, Xanthomonas campestris pv. vesicatoria (Doidge) Dye was evaluated for biological control of bacterial spot on `Sunbeam' tomato (Lycopersicon esculentum Mill.) transplants and field-grown plants for two seasons (Fall 1997 and Fall 1998). Foliar applications of bacteriophages were compared with similar applications of water (control) and of copper/mancozeb bactericides, the commonly used chemical control strategy for tomato seedling and field production. In 1997, the incidence of bacterial spot on greenhouse-grown seedlings was reduced from 40.5% (control) to 5.5% or 0.9% for bactericide- or bacteriophage-treated plants, respectively. In 1998, the incidence of bacterial spot was 17.4% on control plants vs. 5.5% and 2.7% for bactericide- and bacteriophage-treated plants, respectively, although these differences were not statistically significant at P ≤ 0.05. Applications of bacteriophages to field-grown tomatoes decreased disease severity as measured by the area under the disease progress curve (AUDPC) by 17.5% (1997) and 16.8% (1998) compared with untreated control plants. Preharvest plant vigor ratings, taken twice during each field season, were higher in the bacteriophage-treated plants than in either bactericide-treated plants or nontreated controls except for the early vigor rating in 1998. Use of bacteriophages increased total weight of extra-large fruit 14.9% (1997) and 24.2% (1998) relative to that of nontreated control plants, and 37.8% (1997) and 23.9% (1998) relative to that of plants treated with the chemical bactericides. Chemical names used: manganese, zinc, carboxyethylene bis dithiocarbamate (mancozeb).
Brent Rowell, R. Terry Jones, William Nesmith, and John C. Snyder
Bacterial spot epidemics, caused by Xanthomonas campestris pv. vesicatoria (Doidge) Dye, continue to plague bell pepper (Capsicum annuum L.) growers in a number of southern and midwestern states. A 3-year study designed to compare cultivars and breeding lines under induced bacterial spot epidemic and bacterial spot-free conditions began soon after the first release of cultivars having the Bs2 gene for resistance to races 1 to 3 of the pathogen. Bacterial spot epidemics were created by transplanting `Merlin' plants (inoculated with races 1 to 3) into plots of each test cultivar at an isolated location in eastern Kentucky. Plots of the same trial entries at a second location were kept free of bacterial spot for 2 of the 3 years of trials; however, a moderate natural epidemic occurred at this location in 1996. Bacterial spot resistance had the greatest impact on yields and returns per acre in the inoculated trials. Cultivars with only Bs1 or a combination of Bs1 and Bs3 were highly susceptible in the inoculated trials. There were statistically significant and economically important differences in resistance among cultivars and breeding lines having the Bs2 gene; some were nearly as susceptible as susceptible checks. Although many Bs2-gene cultivars showed satisfactory levels of resistance, only a few were highly resistant, horticulturally acceptable, and comparable in yields to the best susceptible hybrids in a bacterial spot-free environment.
C.S. Kousik, D.C. Sanders, and D.F. Ritchie
The impact of a single hail storm injury in combination with bacterial spot caused by Xanthomonas campestris pv. vesicatoria was assessed on three commercial pepper (Capsicum annuum) cultivars—King Arthur, Jupiter, and Rebell. In addition, the effectiveness of copper plus maneb sprays on hail-damaged plants to suppress bacterial spot was evaluated. A hail storm of ≈5-min duration severely damaged and defoliated the pepper plants. Severe bacterial spot was observed 10 days later on all plants. Disease ratings taken 2 weeks after the hail storm were significantly greater than ratings before the storm. Unsprayed plots of all three cultivars had the greatest disease and the least yield. Plots sprayed weekly (7-day schedule) had a significantly greater yield and less disease compared to unsprayed and biweekly sprayed (14-day schedule) plots for all three cultivars. The combination of hail damage and bacterial spot resulted in a 6-fold reduction in yield in the absence of copper plus maneb sprays and a 2-fold reduction with weekly sprays when compared to the previous season with no hail injury, but similar levels of bacterial spot disease. Disease ratings were less and yields were greater for `King Arthur', than for `Jupiter' and `Rebell'. A judicious copper plus maneb spray program can suppress bacterial spot and help recovery of a young pepper crop when hail damage occurs.
J.W. Scott, S.A. Miller, R.E. Stall, J.B. Jones, G.C. Somodi, V. Barbosa, D.L. Francis, and F. Sahin
Thirty-two tomato (Lycopersicon esculentum Mill.) or L. pimpinellifolium (L.) Mill. accessions were inoculated with race T2 of Xanthomonas campestris pv. vesicatoria (Xcv) in a field experiment at Wooster, Ohio, in 1995. Plants from accessions which segregated for race T2 resistance in greenhouse tests were selected and these are designated by hyphenated extensions below. The eight most resistant accessions from 1995 and PI 262173 were retested in 1996. Lycopersicon esculentum accession PI 114490-1-1 had virtually no Xcv symptoms either year. Lycopersicon pimpinellifolium accessions LA 442-1-Bk and PI 128216-T2 expressed a high level of resistance in 1995, but only partial resistance in 1996. Accessions with partial resistance for both seasons were PI 79532-S1, PI 155372-S1, PI 126428, PI 271385, PI 195002, PI 262173, Hawaii 7998, and Hawaii 7983. PI 79532-S1 is a L. pimpinellifolium accession and the remaining seven are L. esculentum. Twenty accessions tested in 1995 for T2 plus 10 other accessions were also tested for race T1 resistance in Presidente Prudente, Sao Paulo, Brazil, in 1993. Hawaii 7983, PI 155372-S1, PI 114490, PI 114490-S1, and PI 262173 had greater resistance to T1 than the susceptible control, `Solar Set'. Comparisons with earlier experiments, in which accessions were inoculated with race T1 or T3, indicated that the most consistent source of resistance to all three races was PI 114490 or selections derived from it.