Movement of Xanthomonas campestris pv. vitians in the stems of lettuce and seed contamination
Article first published online: 9 AUG 2002
Volume 51, Issue 4, pages 506–512, August 2002
How to Cite
Barak, J. D., Koike, S. T. and Gilbertson, R. L. (2002), Movement of Xanthomonas campestris pv. vitians in the stems of lettuce and seed contamination. Plant Pathology, 51: 506–512. doi: 10.1046/j.1365-3059.2002.00730.x
- Issue published online: 9 AUG 2002
- Article first published online: 9 AUG 2002
- Accepted 6 February 2002
- bacterial leaf spot of lettuce;
- Lactuca sativa;
- seed contamination;
- vascular movement;
- Xanthomonas campestris pv. vitians
Xanthomonas campestris pv. vitians, the causal agent of bacterial leaf spot of lettuce (BLS), can be seedborne, but the mechanism by which the bacteria contaminates and/or infects lettuce seed is not known. In this study, the capacity of X. campestris pv. vitians to enter and translocate within the vascular system of lettuce plants was examined. The stems of 8- to 11-week-old lettuce plants were stab-inoculated, and movement of X. campestris pv. vitians was monitored at various intervals. At 4, 8, 12 and 16 h post-inoculation (hpi), X. campestris pv. vitians was recovered from 2 to 10 cm above (depending on stem length) and 2 cm below the inoculation site. Xanthomonas campestris pv. vitians was also recovered from surface-disinfested stem sections of spray-inoculated plants. Together, these results are consistent with X. campestris pv. vitians invading and moving systemically within the vascular system of lettuce plants. To investigate the mechanism of seed contamination, lettuce plants at the vegetative stage of growth were spray-inoculated with X. campestris pv. vitians and allowed to develop BLS. Seed collected from these plants had a 2% incidence of X. campestris pv. vitians external colonization, but no bacteria were recovered from within the seed.
Beginning in the early 1990s, bacterial leaf spot of lettuce (BLS), caused by Xanthomonas campestris pv. vitians, has been occurring with increasing frequency in commercial fields throughout coastal (San Benito, Santa Cruz, Monterey, San Luis Obispo, Santa Barbara and Ventura) and inland (Fresno and Imperial) counties of California, USA. This suggests that BLS has become established in California, although relatively little is known of the inoculum sources involved in these outbreaks. Xanthomonas campestris pv. vitians can be seedborne, and may survive in association with seed for extended periods (Umesh et al., 1996; Sahin & Miller, 1997). However, X. campestris pv. vitians was not recovered from representative lettuce seed lots used to establish commercial fields in California (1992–96), including lots used to establish fields that later developed BLS (Umesh et al., 1996). One explanation for this was that lettuce seed was internally infected with X. campestris pv. vitians and that this inoculum was not detected with seed wash assays, but was able to colonize germinated seedlings and cause outbreaks of BLS.
In the present study we investigated the capacity of X. campestris pv. vitians to enter and translocate in the vascular system of lettuce plants. In addition, we examined whether lettuce seed, produced from symptomatic plants that had been spray-inoculated with X. campestris pv. vitians at the vegetative stage of growth, were contaminated externally and/or infected internally.
Materials and methods
Preparation of inoculum
Xanthomonas campestris pv. vitians (strain Sal) has been described previously (Barak et al., 2001). Bacterial cells were recovered from storage by streaking on plates of 523 medium (Kado & Heskett, 1970). All bacteria were cultured aerobically in Luria–Bertani (LB) broth (Fisher Scientific, Pittsburgh, PA, USA). Flasks were placed on a rotary shaker and incubated at 28°C for 30 h. Cell suspensions were adjusted to an optical density of 0·4 at 600 nm (c. 108 CFU mL−1) with water.
To investigate the ability of X. campestris pv. vitians to translocate in the vascular system, lower stems of 8–11-week-old lettuce plants (stem elongation stage) were stabbed with a sterile dissecting needle, 0·5 cm above the soil line, to a depth of 5 mm. X. campestris pv. vitians cell suspension (20 µL) was introduced into the wound with a Pipetman (P20). Negative control plants were stabbed in a similar fashion, and 20 µL sterile LB was introduced into the wound. A total of 20 plants were inoculated (16 with X. campestris pv. vitians and four with LB). Four hours postinoculation (hpi) and at 4 h intervals up to 16 hpi, four plants inoculated with X. campestris pv. vitians and one negative control plant were sampled, and stems were assayed for bacteria. To detect populations of X. campestris pv. vitians inside stem tissue, leaves were removed and stems were cut into 2 cm sections with a sterile razor blade. Sections were surface-disinfested in 5% Physan 20 (active ingredient, 10%n-alkyl dimethyl benzyl ammonium chloride and 10%n-alkyl dimethyl erthylbenzyl ammonium chloride; Maril, Tustin, CA, USA) for 10 min, and rinsed with sterile distilled water for 2 min. Each section was placed in a Universal extraction bag (Bioreba Ag, Reinach, Switzerland) and crushed with a Lenze Homex 6 homogenizer (Bioreba Ag). Sterile water (1 µL) was added to each bag, mixed with the crushed sample by hand, and 100 µL of the resulting suspension was plated (two plates per section) on XCS medium (Williford & Schaad, 1984) adjusted to pH 6·6 with 5 N NaOH. This experiment was repeated three times.
To determine the approximate location of X. campestris pv. vitians within lettuce stems, the cut surface of each stem piece from each of the stab-inoculation experiments was gently pressed onto the surface of an XCS plate for 10 s. The outlines of the stem sections were traced on the undersurface of the Petri plate with a marker. Plates were incubated at 28°C for 3 days, and the appearance and position, relative to the outline of the stem, of xanthomonad-like colonies was recorded. The identity of representative colonies as X. campestris pv. vitians was determined by polymerase chain reaction (PCR) analysis with the B162 primer pair (described below).
To investigate the ability of X. campestris pv. vitians to enter the vascular system in the absence of wounding, 8-week-old lettuce plants were spray-inoculated with a cell suspension of X. campestris pv. vitians (108 CFU mL−1) in water with a plastic spray-bottle until run-off. Negative control plants were sprayed with sterile water. Plants were kept in a greenhouse that was maintained at c. 28–30°C. A total of 12 plants were inoculated (nine with X. campestris pv. vitians and three with water). At mid-heading, stem elongation, and flower formation growth stages (about every 3 weeks postinoculation), three plants inoculated with X. campestris pv. vitians and one negative control plant were sampled, and the surface and inside of stems were assayed for bacteria.
To determine whether X. campestris pv. vitians had colonized the stem surface of lettuce plants following spray-inoculation, stem sections were assayed with a wash-sonication dilution-plating method. Plants were sampled, leaves were removed, and stems were cut into 2 cm sections with a sterile razor blade. Three stem sections, chosen randomly along the length of the stem, were weighed and placed in a 100 mL flask containing 75 mL chilled 0·85% NaCl buffer (hereafter referred to as NaCl buffer), and flasks were sonicated for 20 min in an ultrasonic bath (Fisher Scientific, Pittsburgh, PA, USA). The tissue/buffer suspension was filtered through two layers of sterile cheesecloth into a 250 mL polypropylene centrifuge bottle (Nalgene; Fisher Scientific), and bottles were centrifuged for 10 min at 10 000 g. The resulting pellet was suspended in 10 mL NaCl buffer, serial dilutions (10–10−4) were prepared, and 100 µL aliquots (two plates per dilution) were plated onto XCS medium. The remaining stem sections were pooled and surface-disinfested. Half of these stem sections were weighed and washed (described above) to monitor surface disinfestation, and the other half of the stem sections were weighed and assayed for internal populations of X. campestris pv. vitians as previously described. This experiment was repeated three times.
To confirm the identity of xanthomonad-like colonies recovered from lettuce plants, PCR with an X. campestris pv. vitians-specific primer pair was used. The X. campestris pv. vitians primer pair B162 directs the amplification of a c. 700 bp DNA fragment from all X. campestris pv. vitians strains that have been tested (including strain Sal; Barak et al., 2001). The template for the PCR was a boiled cell extract prepared from a bacterial cell suspension made by adding a loopful of bacteria from an individual colony into 1 mL sterile water in a 1·5 mL Eppendorf tube. The tubes were boiled for 10 min, and 2 µL of this cell extract was used in the PCR. The PCR was carried out as previously described (Barak et al., 2001) in a Thermal Cycler 480 (Perkin-Elmer Cetus, Emeryville, CA, USA). The amplification profile was 35 cycles of 94°C for 1 min, 55°C for 2 min, and 72°C for 3 min. Amplified DNA fragments were analysed by gel electrophoresis in 1·5% agarose in 0·5 × Tris–borate EDTA buffer. Gels were stained with ethidium bromide, and DNA was visualized with a gel-imaging system (Alpha Innotech Corp., San Leandro, CA, USA).
Spray-inoculation of lettuce plants for production of X. campestris pv. vitians-contaminated lettuce seed
To inoculate plants used for seed production, nutrient broth shake cultures of X. campestris pv. vitians were grown for 48 h. Sterile Tween 20 (60 µL per 100 mL) was added to these cultures (107 CFU mL−1, quantified by dilution plating) prior to inoculation. Lettuce plants were grown in a greenhouse kept at 22–24°C with ambient light. Approximately 40 lettuce plants (cv. Alpha) at the eight- to nine-leaf stage were spray-inoculated with this suspension with a hand-held mister until run-off. Plants were misted with water every day to enhance disease development, and extensive BLS symptoms developed on leaves and flower bracts. Seeds were collected from plants by shaking plants over buckets. Seeds were stored in brown paper sacks at 4°C until assayed. Seeds were assayed within 1 year of harvest.
Assaying lettuce seed for X. campestris pv. vitians
The lettuce seed lot was assayed for X. campestris pv. vitians by washing 15 g seed in 100 mL NaCl buffer plus 30 µL sterile Tween 20 in a 250 mL Erlenmeyer flask. The flask was shaken at 200 r.p.m. for 3 h at room temperature. The seed suspension was filtered through cheesecloth, and the supernatant was centrifuged at 10 000 g for 10 min. The resulting pellet was suspended in 10 mL NaCl buffer, serial dilutions prepared, and 100 µL aliquots (two plates per dilution) were plated onto XCS medium. Plates were incubated at 28°C for 4 days and examined for X. campestris pv. vitians-like colonies.
A seed-plating assay was also used to investigate the incidence of X. campestris pv. vitians contaminating the surface of seed and infection. To assess contamination, 1000 seeds were placed directly on the surface of plates of XCS medium (25 seeds per plate), and plates were incubated at 28°C. After 5 days the percentage seed contamination was determined by counting the number of seeds from which xanthomonad-like bacteria were observed growing in culture. The identity of bacteria as X. campestris pv. vitians was determined by PCR with the B162 primer pair. Seeds from which no bacteria or fungi grew, and/or that did not germinate, were surface disinfested with 5% Physan 20 (Maril) for 10 min, and air dried at room temperature (c. 25°C) for 2 h. Individual seeds were then placed between two paper towels, crushed with a hammer, and placed directly on the surface of plates containing XCS medium (25 crushed seeds per plate). After 5 days the percentage seed infection was determined by counting the number of crushed seeds from which xanthomonad-like bacteria grew. The identity of these bacteria as X. campestris pv. vitians was determined by PCR as described above. This seed assay was replicated three times for a total of 3000 seeds tested.
Stem sections were identified according to their maximum distance from the site of inoculation (the stem section located 0–2 cm from the inoculation site is referred to as the 2 cm section). By 4–16 hpi, xanthomonad-like bacteria were recovered from most surface-disinfested stem sections of 8-week-old plants (Table 1). However, due to the limited stem development of these plants, sections 4 cm beyond the inoculation site could not be taken. For the 9-week-old plants, xanthomonad-like bacteria were consistently recovered from surface-disinfested stem sections taken 2–6 cm above and 2 cm below the inoculation site at 4–16 hpi, but not from sections 8 cm above or 4 cm below the inoculation site (Table 1). Eleven-week-old plants had considerably longer stems than those of 8- and 9-week-old plants and, at 4–16 hpi, xanthomonad-like bacteria were recovered from surface-disinfested stem sections taken 2–10 cm above and 2 cm below the inoculation point, but not from sections 4–6 cm below the inoculation point (Table 1). Xanthomonad-like bacteria were not recovered from control plants stab-inoculated with sterile LB. For all three experiments, one representative xanthomonad-like colony recovered from each stem section was tested by PCR analysis with the B162 primer pair. All colonies tested were confirmed as X. campestris pv. vitians based on amplification of a c. 700 bp DNA fragment. Taken together, these results suggest that X. campestris pv. vitians has the capacity to move within the stems of lettuce plants, and that this movement is in the direction of the shoot apex.
|Distance from inoculation site (cm)||Hours post- inoculation (hpi)||Plant age (weeks)a|
To gain insight into the location of X. campestris pv. vitians within the stems of stab-inoculated 8-, 9- and 11-week-old lettuce plants, stem sections taken every 2 cm from the point of inoculation and at each time point (4, 8, 12, 16 hpi) were pressed onto XCS medium. Colonies of xanthomonad-like bacteria consistently grew in a circular pattern, c. 2 mm inside the circumference of the stem. The location of these colonies roughly corresponded to the location of the vascular bundles of the lettuce stem (Fig. 1). All colonies of xanthomonad-like bacteria recovered from the stem pressings that were tested were confirmed as X. campestris pv. vitians by PCR with the B162 primer pair.
Xanthomonas campestris pv. vitians was readily recovered from the surfaces of stem sections of spray-inoculated plants (Table 2), and populations ranged from 0–105, 104−105, and 0–106 CFU g−1 stem tissue at mid-heading, stem elongation, and flower formation growth stages, respectively. No xanthomonad-like bacteria were recovered from the surface of stem sections following surface disinfestation. Xanthomonas campestris pv. vitians was readily recovered from crushed surface-disinfested stem pieces (Table 2), and populations from inside the stems ranged from 0–105, 0–106, and 0–105 CFU g−1 stem tissue at mid-heading, stem elongation and flower formation, respectively. In general, populations recovered from the surfaces of stems were greater than those recovered from inside stems. PCR analysis with the B162 primer pair was performed with one representative xanthomonad-like colony recovered from each plant sampled at each growth stage. In all cases, a c. 700 bp DNA fragment was amplified from these colonies, confirming their identity as X. campestris pv. vitians. Seed wash assays established a contamination level for seed recovered from spray-inoculated plants of c. 104 CFU g−1 seed (average of three replicates).
|Experiment||Planta||Treatment||Mid-heading||Stem elongation||Flower formation|
Direct plating of individual seeds established incidences of contamination (1·6, 2·3, 2·2%) for the three samples of 1000 seeds assayed. All the xanthomonad-like bacteria recovered from these seeds were confirmed as X. campestris pv. vitians by PCR analysis with the B162 primer pair. No xanthomonad-like bacteria grew from any of the crushed seeds.
Xanthomonas campestris pv. vitians, like many other leaf spot bacteria, can be a contaminant of seed (Umesh et al., 1996; Sahin & Miller, 1997). However, little is known of the mechanism by which X. campestris pv. vitians contaminates lettuce seed. There are two hypotheses regarding seed contamination: (i) bacteria grow epiphytically on the surface of plant reproductive organs and externally contaminate seed; or (ii) the bacteria move systemically within the plant and gain access to seed via vascular connections. This study was conducted to investigate the capacity of X. campestris pv. vitians to move within the vascular system of lettuce plants and to assess whether this might be a mechanism by which the bacteria can contaminate seed.
The results of the experiments presented here suggest that X. campestris pv. vitians can enter and translocate within the stems of lettuce plants and that the bacteria probably move in the vascular system. First, the recovery of X. campestris pv. vitians from surface-disinfested stem sections taken 10 cm from the point of stab-inoculation, 16 h after introduction of the bacteria into the stem, established that the bacteria can be transported throughout the length of the plant stem. This transport is presumably via the vascular system because it is unlikely the bacteria could have moved this distance, in such a short period of time, via epiphytic growth on the stem surface. Second, X. campestris pv. vitians was consistently recovered from surface-disinfested stem sections as long as 9 weeks after spray-inoculation and after extensive stem elongation. This is also consistent with colonization of internal stem tissues. Direct evidence that the bacteria were present in the vascular system following stab-inoculation came from stem pressing experiments in which X. campestris pv. vitians colonies consistently originated in a circular pattern that corresponded with the location of the vascular tissues. It is most likely that the bacteria move within the xylem vessels because the diameters of sieve plate pores of phloem sieve elements are too small (c. 50 nm diameter) to allow free passage of bacterial cells (c. 1 × 3 µm dimensions).
Other studies have suggested that phytopathogenic leaf spot bacteria can systemically infect plants and move within vascular tissues. Hamacher & Kurze (1996) stab- and spray-inoculated geranium (Pelargonium × hortorum) plants with X. campestris pv. pelargonii, and used immunohistochemistry to demonstrate short-distance intercellular movement (e.g. from inoculation sites on the leaf surface) and long-distance transport via xylem vessels. These results are consistent with results of our spray-inoculation experiments, in which it appeared that X. campestris pv. vitians moved intercellularly for short distances (e.g. from inoculation sites on the leaf surface) into the vascular tissue, and then longer distances inside stems.
There are at least two mechanisms by which bacteria may gain access to the xylem from the leaf surface and/or stomatal chambers: via water movement during dark conditions; and/or via embolisms in the xylem. Erwinia amylovora was recovered from below the inoculation site in stab-inoculated apple seedlings, and it was suggested that the negative hydraulic pressure of the xylem following cellular disruption facilitated pathogen invasion (Bogs et al., 1998). In the present study, X. campestris pv. vitians was recovered from stem sections taken above and below the inoculation site of stab-inoculated plants (Table 1), as well as from inside stems of spray-inoculated plants (Table 2). The recovery of X. campestris pv. vitians below the inoculation site may be a result of cavitation caused by disruption of the water column during inoculation, whereas recovery of X. campestris pv. vitians within lettuce stems following spray-inoculation may be a result of the bacterial cells being drawn into the vascular system as the water column recedes from the stomatal chambers into the xylem columns.
Xanthomonas campestris pv. vitians can move within the vascular system of lettuce plants without inducing visible disease symptoms, as stab- and spray-inoculated plants did not develop BLS. This is consistent with reports for other bacterial leaf spot pathogens that can translocate via the xylem vessels. For example, McPherson & Preece (1979) found that X. campestris pv. pelargonii moved rapidly in the xylem following stab-inoculation of 4- and 15-week-old geranium plants, and that this movement occurred without development of symptoms. Fukui et al. (1998) studied the susceptibility of anthurium (Anthurium andraeanum) to systemic infection by X. campestris pv. dieffenbachiae and found that the bacteria rapidly moved to areas distal from the site of inoculation in the absence of symptom development. Kritzman & Zutra (1983) detected symptomless systemic infection of cucumber plants by Pseudomonas syringae pv. lachrymans based on recovery of bacteria from root-pressure liquid. Similarly to other bacterial leaf spot pathogens, X. campestris pv. vitians may colonize lettuce plants without inducing visible symptoms.
Plant pathogenic bacteria can contaminate seed, for example X. campestris pv. carotae and carrot seed (Kuan et al., 1985), whereas infection involves the bacterium gaining access to an internal part of the seed, for example the embryo, as reported for X. campestris pv. manihotis (Elango & Lozano, 1980); the endosperm in the case of E. stewartii (Rand & Cash, 1921); or within the seed coat as shown for X. campestris pv. malvacearum (Brinkerhoff & Hunter, 1963). In the present study, X. campestris pv. vitians was recovered only from the outside of seed, despite the fact that these seeds were derived from heavily diseased plants inoculated during the vegetative stage of growth. Thus it is concluded that, under the conditions of these experiments, X. campestris pv. vitians did not infect lettuce seed via vascular connections, but probably via external contamination via colonization of flowers and fruits.
In conclusion, the results of the experiments conducted in this study established that X. campestris pv. vitians has the capacity to enter and translocate within the vascular system of lettuce plants without inducing visible disease symptoms. Seed produced from diseased lettuce plants were contaminated at a level of c. 2%, but internally infected seeds were not detected. Although lettuce seed failed to become infected, symptomless systemic infections of lettuce stems could favour survival of X. campestris pv. vitians in association with lettuce debris, as stem tissue is likely to persist longer than leaves on or in soil (Barak et al., 2001). Symptomless systemic infections of lettuce plants could also result in external contamination of seed in the absence of disease symptoms. In such cases, field inspections of seed crops would fail to reveal this source of seed contamination. Thus it appears advisable to perform seed assays to confirm that seed lots are not contaminated with X. campestris pv. vitians, particularly if such lots are to be planted in areas where conditions are favourable for development of bacterial leaf spot disease.
- 2001. The role of crop debris and weeds in the epidemiology of bacterial leaf spot of lettuce in California. Plant Disease 85, 169–78. , , ,
- 1998. Colonization of host plants by the fire blight pathogen Erwinia amylovora marked with genes for bioluminescence and fluorescence. Phytopathology 88, 416–21. , , , ,
- 1963. Internally infected seed as a source of inoculum for the primary cycle of bacterial blight of cotton. Phytopathology 53, 1397–401. , ,
- 1980. Transmission of Xanthomonas manihotis in seed of cassava (Manihot esculenta). Plant Disease 64, 784–5. , ,
- 1998. Differential susceptibility of anthurium cultivars to bacterial blight in foliar and systemic infection phases. Plant Disease 82, 800–6. , , ,
- 1996. Licht- und elektronenmikroskopische untersuchungen zur ausbreitung von Xanthomonas campestris pv. pelargonii (Xcp) in zonalen Pelargonienhybriden. Berlin-Dahlem, Germany: Mitteilungen aus der Biologischen Bundesanstalt fuer Landund Forstwirtschaft. , ,
- 1970. Selective media for isolation of Agrobacterium, Corynebacterium, Erwinia, Pseudomonas, and Xanthomonas. Phytopathology 60, 969–76. , ,
- 1983. Systemic movement of Pseudomonas syringae pv. lachrymans in the stem, leaves, fruits, and seeds of cucumber. Canadian Journal of Plant Pathology 5, 273–8. , ,
- 1985. Detection of Xanthomonas campestris pv. carotae in carrot seed. Plant Disease 69, 758–60. , , ,
- 1979. Bacterial blight of Pelargonium: movement, symptom production and distribution of Xanthomonas pelargonii (Brown) Starr and Burkholder in Pelargonium hortorum Bailey, following artificial inoculation. In: Proceedings of the Fourth International Congress on Plant Pathogenic Bacteria, 1978. Angers, France: INRA, 943–956. , ,
- 1921. Stewart's disease of corn. Journal of Agricultural Research 21, 263–4. , ,
- 1997. Identification of the bacterial leaf spot pathogen of lettuce, Xanthomonas campestris pv. vitians, in Ohio, and assessment of cultivar resistance and seed treatment. Plant Disease 81, 1443–6. , ,
- 1996. Association of Xanthomonas campestris pv. vitians with lettuce seed (abstract). Phytopathology 86, S3. , , ,
- 1984. Agar medium for selective isolation of Xanthomonas campestris pv. carotae from carrot seeds (abstract). Phytopathology 74, 114. , ,