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.
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.
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.
Hawaii 7998 (foliage resistant to bacterial spot) was crossed with ‘Walter’ (susceptible) and F1, backcross, and F2 generations were derived. These genotypes were grown in the field at Bradenton, Fla. in the summers of 1984 and 1985 and inoculated with Xanthomonas campestris pv. vesicatoria, the incitant of bacterial spot. Disease severity for respective genotypes was similar both years, although somewhat greater in 1985. Disease severity in the F1 was intermediate to the parents, but slightly skewed toward resistance both years. The percentage of F2 plants with resistance comparable to Hawaii 7988 was 9.6% in 1984 and 4.6% in 1985. There was no evidence of cytoplasmic inheritance from three sets of reciprocal crosses tested in 1985. The data fit an additive-dominance genetic model, but dominance variance was negative both years, which indicates a small or negligible dominance effect. The negative dominance variance resulted in biased estimates of additive variance, narrow-sense heritability, and the number of effective factors. Nevertheless, narrow-sense heritability was moderate to high. When incorporating this resistance into new genetic backgrounds, we suggest that a modified backcrossing scheme with rigorous disease screening be used to obtain plants from homozygous resistant BCF3 lines before crossing.
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.
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.
Two hundred eight-four Lycopersicon spp. genotypes reported to have some resistance to bacterial pathogens of tomato (L. esculentum Mill.) were inoculated in the field with Xanthomonas campestris pv. vesicatoria (XCV), the incitant of bacterial spot, and rated for disease severity in summer 1982 and/or summer 1983. One line tested in 1983, Hawaii 7998, had no definite XCV lesions and later was determined to be resistant to XCV in the laboratory. Genotypes with the highest levels of resistance during 2 years of testing were: Ohio 4013-3, Ohio 4014-4, Heinz 1568-F3, [(Subarctic Delite × MH1) × H603] F5, L556, ‘Campbell-28’, PI 127813, Heinz 603-F11, PI 224573, ‘Monense’, ‘Heinz 2990’, and PI 324708. Genotypes with highest levels of resistance in one year of testing were PI 379032 and ‘Burgess Crack Proof. In 1982, PI 270248- ‘Sugar’ had a high level of resistance to XCV on fruit, but foliage was susceptible.
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.
Tomato (Lycopersicon esculentum Mill.) accession PI 270248 (‘Sugar’) had high levels of resistance to bacterial spot [incited by Xanthomonas campestris pv. vesicatoria (Doidge) Dye] on fruit, but foliage was susceptible. Hawaii 7998 (H7998) was highly resistant to foliar infection, but was intermediate in resistance to fruit infection. Fruit spot on hybrids between ‘Sugar’ and H7998 was usually intermediate to the parents. Occasionally, disease incidence of hybrids was not statistically different from one or both parents, but tended to resemble ‘Sugar’ more closely than H7998. There were no significant differences between reciprocal hybrids, indicating a lack of cytoplasmic inheritance. Under low disease incidence, hybrids between ‘Sugar’ and ‘Walter’ (susceptible to bacterial spot on fruit and foliage) had fruit spot incidence similar to ‘Sugar’ and significantly less than ‘Walter’. Thus, there was a high level of dominance for resistance to bacterial spot on fruit.
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).