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Keywords:

  • BABA;
  • Bion (BTH);
  • chemical plant activators;
  • induced resistance;
  • Zantedeschia aethiopica

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The potential of three externally applied chemical plant activators, Bion, BABA and methyl jasmonate, known to act only through the plant defence system and not on the pathogen directly, to induce resistance against wild-type Pectobacterium carotovorum was examined in white-flowered calla lily (Zantedeschia aethiopica). Following a 24-h induction period, plants were challenge-inoculated with P. carotovorum, originally isolated from calla lily or potato plants, previously transformed using a gfp broad-host-range promoter-probe vector. After another 24 h, Bion treatment (10 µg mL−1, as a drench) reduced disease symptoms more than sixfold and bacterial proliferation by four orders of magnitude. BABA treatment (5–10 µg mL−1, also as a drench) reduced the rate of infection by 75–85%. However, the protection afforded by both inducers did not persist. Also, at higher concentrations both displayed a phytotoxic effect. By contrast, methyl jasmonate (10 mm, applied as a leaf spray) completely inhibited P. carotovorum development in calla lily leaves and afforded a long-lasting effect. It is suggested that the defence response of calla lily against P. carotovorum involves the SA-signalling pathway in the short term, but the jasmonate/ethylene-signalling pathway is required for durable protection.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The use of externally applied chemical stimulators to elicit natural plant defence mechanisms is a well-recognized approach to crop protection (Oostendorp et al., 2001; Walling, 2001; Vallad & Goodman, 2004; Walters et al., 2005). This type of resistance was proven effective in dicots and model monocots, mostly grasses (Poaceae), against a wide spectrum of fungal, bacterial and viral pathogens (Oostendorp et al., 2001; Conrath et al., 2002; Heil & Bostock, 2002; Gozzo, 2003; Durrant & Dong, 2004). Despite this experience and new insights into resistance mechanisms in numerous plant systems, there is hardly any knowledge about these responses in monocotyledonous geophytes, an economically important botanical group, commonly termed ‘flower bulbs’, characterized by specialized underground storage organs (Le Nard & De Hertogh, 2002).

Ornamental geophytes such as Zantedeschia, Ornithogalum, Gladiolus, Iris, Hyacinthus and others, which are cultivated for cut-flower and bulb production, sustain substantial yearly losses from the soft rot bacterial pathogen Pectobacterium carotovorum, previously included in the genus Erwinia (Wright, 1998).

Calla lily (Zantedeschia) (Araceae) is a genus of about eight species native to South Africa, all producing tuberous or rhizomatous storage organs which may be contaminated by P. carotovorum in the field or in storage (Wright, 1998; Snijder & van Tuyl, 2002). The first symptoms of soft rot disease in calla lily plants are poor shoot growth and yellowing of the leaves. Subsequently, small water-soaked lesions appear on leaves, petioles, tubers and stem bases. Under conditions that favour disease spread (warm and wet climate), the lesions rapidly enlarge, resulting in a soft and mushy decay. The rot often begins at the bases of the leaf petioles, below the soil line, and may progress up the stems, causing blighting of leaves, or down into the tubers (Wright, 1998; Snijder & van Tuyl, 2002). Currently, there are no effective control measures against P. carotovorum in ornamental geophytes. The use of common bactericidal formulations such as copper-based compounds to decrease P. carotovorum proliferation is relatively ineffective and is often phytotoxic (Gracia-Garza et al., 2002), while other measures, such as antibiotics, in addition to their high cost, may promote bacterial resistance within a few generations (McManus et al., 2002). Furthermore, the necrotrophic nature of P. carotovorum, i.e. its reliance on nutrients derived from dead or dying cells, makes it a difficult pathogen to control, as programmed cell death in host cells, associated with salicylic acid (SA)-dependent signalling pathways, simply allows the pathogen to thrive (Glazebrook, 2005).

To date, the only way to combat P. carotovorum in calla lily species is through strict sanitation measures and selection for more resistant cultivars (Snijder & van Tuyl, 2002; Snijder et al., 2004a,b).

The efficacy of plant activators to induce resistance against plant pathogens has been well established in numerous plant systems under field and laboratory conditions (Tally et al., 1999; Oostendorp et al., 2001; Walling, 2001; Gozzo, 2003; Durrant & Dong, 2004; Vallad & Goodman, 2004; Walters et al., 2005). Vallad & Goodman (2004) highlighted 32 examples where benzothiadiazole (BTH-Bion®) was found to provide disease control against both bacterial and fungal pathogens in 12 diverse dicot and monocot crops, none of which, however, were ornamental monocots. Another plant activator that induces broad-spectrum resistance in a range of crops is the non-protein amino acid β-aminobutyric acid (BABA) (Cohen, 2001; Kohler et al., 2002; Gozzo, 2003). BABA confers resistance to monocot and dicot plants against several bacterial pathogens, including P. carotovorum (Cohen, 2002). These two chemically different compounds appear to act through different signal transduction pathways, both involving SA (Zimmerli et al., 2000; Cohen, 2001). Jasmonic acid has been implicated as a signal molecule in wound response against feeding insects, as well as in resistance against necrotrophic pathogens (Glazebrook, 2005), including P. carotovorum in Arabidopsis (Norman-Setterblad et al., 2000; Brader et al., 2001). However, none of these compounds appear to have been tested in ornamental monocots.

Here, a system was established to assess the potential of the above dissimilar plant activators to reduce soft rot disease in the ornamental monocot calla lily. A gfp broad-host-range promoter-probe vector (Miller et al., 2000) was used to transform wild-type P. carotovorum, originally isolated from calla lily. This probe allowed comparison of the defence afforded by the different elicitors in the calla lily pathosystem and localization of P. carotovorum in the leaf tissue of calla lily and other ornamental monocots.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Bacterial isolates

Bacterial isolates were obtained from commercial crops showing disease symptoms (Table 1). Pectobacterium carotovorum isolates were obtained from plant samples using common protocols on crystal violet polypectate (CVP) at room temperature (Cuppels & Kelman, 1974). Pectolytic isolates were re-streaked onto Luria-Bertani (LB) medium. Single colonies were cultured and stored in 20% glycerol at –80°C.

Table 1.  Bacterial isolates used in this study
IsolateIdentityHostLocationYearSource
  • a

    Volcani Center, Israel.

  • b

    Gilat Research Center, Israel.

Pc1Pectobacterium carotovorumOrnithogalum dubiumIsrael2004This study
Pc3P. carotovorumSolanum tuberosumIsrael1998S. Manulisa
Pc11P. carotovorumLycopersicon esculentumIsrael1998S. Manulis
Pc13P. carotovorumZantedeschia aethiopicaIsrael2005This study
Pc14P. carotovorumBrassica oleraceaKazakhstan1998S. Manulis
Pc26P. carotovorumS. tuberosumIsrael2005This study
PaP. atrosepticumS. tuberosumIsrael2005S. Manulis
EchDickeya dadantiiS. tuberosumIsrael2006L. Tsrorb
EaErwinia amylovoraPyrus communisIsrael1994S. Manulis
EhgErwinia herbicola pv. gypsophilaeGypsophila paniculataIsrael1991S. Manulis

The green-fluorescent-protein-(gfp)-expressing P. carotovorum isolates Pc3 and Pc13 were generated by introducing, via electroporation, plasmid pPROBE-AT containing a 131-bp nptII promoter fragment from Tn5 fused to the gfp gene, and a replicon from pBR1 carrying ampicillin resistance (Miller et al., 2000).

All isolates were subjected to gas chromatographic analysis of fatty-acid methyl esters (GC-FAME) at the Israeli Plant Protection and Inspection Services (Volcani Center, Bet Dagan, Israel) according to described procedures (Miller, 1982). GC-FAME similarity indexes were determined by statistical comparison to the MIDI TSBA40 library through the MIDI Sherlock Microbial Identification system (Midi Inc.). All isolates were also subjected to intergenic transcribed spacer (ITS)-PCR analysis as described by Toth et al. (2001) to confirm the isolate identification using primer G1 (5′-GAAGTCGTAACAAGG-3′) and L1 (5′-CAAGGCATCCACCGT-3′). Pectobacterium carotovorum was distinguished from other species (detailed in Table 1), by PCR product sizes of 540, 575, and 740 bp (Toth et al., 2001). Pectate lyase amplification was carried out using the primers Y1 (5′-TTACCGGACGCCGAGCTGTGGCGT-3′) and Y2 (5′-CAGGAAGATGTCGTTATCGCGAGT-3′) as described by Darrasse et al. (1994).

Infection experiments and treatment with plant activators

Calla lily (Zantedeschia aethiopica) plants were grown in pots in the greenhouse for two seasons (10–25°C, natural daylight). Fully expanded young leaves were cut off and soaked in 0·7% sodium hypochlorite (20 min) for external disinfection, followed by a double wash in sterile distilled water. Leaf discs (20 mm in diameter) were excised following disinfection and transferred on Petri dishes containing 20 mL of half-strength Murashige and Skoog (MS) mineral sugar-free agar medium. Inoculation was performed with fresh cultures of P. carotovorum at 108 CFU mL−1 (0·1 OD, 600 nm) washed twice in double-distilled water. Leaf discs were pierced at the centre with a sterile tip and inoculated with 10 µL of the P. carotovorum suspension.

Pathogenicity tests were carried out in this way on three monocot crops (calla lily, O. dubium and Hippeastrum) and on potato tubers as a reference dicot. There were three independent experiments using 20 leaf discs for each P. carotovorum isolate. Disease symptoms were recorded 24 h after inoculation and therefore 48 h after treatment with plant activators (see below).

Calla lily plants were grown in pots for two seasons under greenhouse conditions (10–25°C). The youngest fully open leaf was cut at the base of the petiole and suspended for 24 h (25°C) in Bion (Syngenta) or BABA (Sigma-Aldrich) solution at the following concentrations: 0, 2·5, 5, 10 or 25 µg mL−1. Alternatively, methyl jasmonate (MeJ; Sigma-Aldrich) solution (1, 10 or 50 mm) was applied as a foliar spray 24 h before inoculation. Leaf discs were prepared and inoculated with P. carotovorum as described above. Two independent experiments were carried out, using 20 leaf discs for each elicitation treatment.

Quantification of bacterial cells, 24 h after challenge inoculation, was achieved by grinding five leaf discs (1 g) per treatment, followed by serial dilution and plating onto LB agar containing 100 µg ampicillin mL−1 as a selective agent.

Microscopic observation

Leaf sections infected with gfp-Pc13 were excised 24 h after challenge inoculation and viewed using an OLYMPUS IX 81 inverted laser scanning confocal microscope (FLUOVIEW 500) equipped with a 488-nm argon-ion laser. Green fluorescing protein was excited by 488 nm light and the emission was collected through a BA 515–525 filter. For autofluorescence, a BA 660 IF emission filter was used. Images were colour-coded green for gfp and red for autofluorescence.

Statistical analysis

Statistical comparisons were made using one-way anova by prism 3·02 software (GraphPad). Where anova yielded significance (P < 0·05), further analysis was performed using the Tukey-Kramer multiple comparisons test. Data presented were means ± SEM. Overall differences in growth curves were assessed by logistic curve comparison using jmp software.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Isolation of P. carotovorum from calla lily

Soft rot symptoms were easily detected on leaf petioles and bulbs of commercially cultivated calla lily (Fig. 1) and Ornithogalum dubium grown along the Mediterranean coast region of Israel. Diseased plants were collected from several fields during the 2004–5 winter season and pectolytic bacteria were isolated on CVP selective medium. Bacteria were present in all portions of the plants, including bulbs, petioles and leaves. Pectinolytic isolates were confirmed as P. carotovorum using fatty acid analysis. In addition, the presence of the pectate lyase-encoding gene, confirmed by amplifying a 434-bp fragment, supported this identification (Darrasse et al., 1994). The PCR banding pattern of the 16S-23S intergenic transcribed spacer (Toth et al., 2001) fitted the P. carotovorum pattern with primary fragments of 540, 575 and 740 bp, with slightly smaller fragments of 520 and 565 bp for the P. carotovorum isolates from O. dubium and calla lily (data not shown).

image

Figure 1. Calla lily showing bacterial soft rot symptoms in the greenhouse: (a) discoloration of leaves; (b) rotting leaf petioles; (c) leaf base and bulb rot.

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Calibration of disease symptoms on calla lily leaf discs

In order to quantify induced resistance response against P. carotovorum, a quantitative technique to inoculate calla lily was first established to measure and compare disease symptoms. Several inoculation techniques with varying infection efficiencies were evaluated in this work. Once the leaf disc assay was developed, it was tested at different inoculum concentrations and with different P. carotovorum isolates. The use of sugar-free MS agar medium for the leaf disc assay allowed up to 7 days for detection of disease development.

Generally, isolates from different plant sources diverged in their pathogenicity towards calla lily (Fig. 2a). The two P. carotovorum isolates from O. dubium and calla lily (monocots) were more aggressive than those from the dicots (potato, cabbage and tomato) on calla lily and O. dubium (Fig. 2b,c). The most aggressive isolate was Pc1, from an O. dubium bulb, which was significantly more aggressive than all other isolates on the three crops tested (P < 0·05). Calla lily leaf discs were more sensitive to P. carotovorum than O. dubium leaf discs (Fig. 2b,c). The differences among the P. carotovorum isolates on O. dubium were not statistically significant, with the exception of the O. dubium isolate and the tomato isolate. Hippeastrum, another monocot bulb belonging to the family Amaryllidaceae, was generally resistant to P. carotovorum, with the exception of the O. dubium isolate. In a pathogenicity test on potato tubers, there was no significant difference among all isolates (data not shown). Growth curves of all isolates in LB broth showed similar growth patterns for all isolates tested in this study.

image

Figure 2. (a) Leaf discs of calla lily inoculated with five Pectobacterium carotovorum (Pc) isolates from different host plants. Necrotic area (mm2) produced by 10 µL of the corresponding Pc isolates (108 CFU mL−1) on (b) calla lily or (c) Ornithogalum dubium. Bars represent average of necrotic area (mm2) ± standard errors. Each Pc isolate was tested on 20 leaf discs taken from four different plants. All infection experiments were repeated twice.

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Expression of pProbe-At gfp in P. carotovorum

Varying transformation efficiency rates were obtained for P. carotovorum isolates that originated from different plants. The highest transformation rates were obtained in P. carotovorum isolates from tomato, potato and calla lily, in that order (data not shown). Some P. carotovorum isolates, such as those from O. dubium and cabbage, failed to express the plasmid using common electroporation procedures.

Two wild-type P. carotovorum isolates, Pc3 from potato and Pc13 from calla lily, were selected for further work along with their corresponding pProbe-At gfp-expressing isolates Pc3+ and Pc13+. The two isolates were used to calibrate and correlate bacterial concentrations and disease symptoms on calla lily leaf discs and to study the effect of pProbe-At gfp expression on pathogenicity of the isolates. Direct correlation between bacterial concentrations and disease development on the leaf discs was observed (Fig. 3a). The gfp-expressing isolates revealed similar patterns of pathogenicity in the leaf disc assay as their corresponding wild-type isolates. Isolate Pc13 from calla lily was more aggressive towards calla lily than Pc3 (the potato isolate). The leaf disc assay demonstrated that the gfp-expressing isolates (Pc3+ and Pc13+) were slightly less aggressive than their corresponding wild-type isolates (Pc3 and Pc13). Growth curves in LB broth of wild-type and gfp-expressing isolates did not reveal significant differences (P < 0·05) in growth dynamics among all isolates (Fig. 3b).

image

Figure 3. (a) Calibration of disease symptoms on calla lily leaf discs infected with Pectobacterium carotovorum isolates (Pc3 and Pc13) or the gfp-expressing strains (Pc3+ and Pc13+) from potato and calla lily, respectively, using increasing concentrations of P. carotovorum (0·05, 0·1, 0·5, 1·0 OD, 600 nm); data represent average necrotic area (mm2) determined in two independent experiments. Standard deviations are represented by the error bars. (b) Growth curves of the above isolates in LB.

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Disease reduction following application of plant activators

Pectobacterium carotovorum was inoculated onto leaf discs 24 h post-elicitation with either Bion, BABA or MeJ. Disease development of necrotic area and percentage infection were recorded 24 h later (48 h post-elicitation). A dose-dependent reduction of disease symptoms was observed with each plant activator. All inducers displayed an optimum curve. The Bion treatment reduced disease symptoms significantly at all concentrations (P < 0·05), but was most efficient at 10 µg mL−1 (P < 0·001), where lowest disease symptoms and bacterial cell counts were observed (Figs 4a and 5). The BABA treatment displayed a similar pattern at 5 and 10 µg mL−1 (P < 0·001), but a significant phytotoxic effect (more so than with Bion) was observed at 25 µg mL−1 (Figs 4a and 5). The BABA treatment halted almost completely the progression of disease symptoms during the first 24 h, yet the phytotoxic response to the inducer was stronger than that to Bion and disease symptoms exceeded the level of the control treatment. Both inducers failed to express resistance on a longer time scale. Once symptoms developed, disease progress could not be delayed (Fig. 7). Unlike Bion or BABA, MeJ halted disease completely at a concentration of 10 mm (Figs 4a,c and 7) and provided long-lasting protection without further development of disease symptoms up to 7 days post-infection.

image

Figure 4. (a) Necrotic area and (b) percentage infection on calla lily leaf discs following application of plant activators Bion, BABA or MeJ and challenge inoculation with 10 µL of Pectobacterium carotovorum (Pc) suspension (1 × 108 CFU mL−1). Disease symptoms were recorded 24 h after challenge inoculation. Bion and BABA were applied as drenches, MeJ as a spray. (c) Leaf disc assay showing the protection afforded by MeJ 120 h after inoculation with 10 µL of Pc suspension (1 × 108 CFU mL−1). At 1 mm MeJ the leaf discs displayed accelerated senescence, while 10 mm did not cause any visible symptoms on the leaf discs. Bars represent average of two independent experiments including 20 leaf discs for each treatment ± standard error.

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image

Figure 5. Bacterial proliferation of Pectobacterium carotovorum (Pc) isolate Pc13+ in calla lily leaf discs, following pre-treatment with Bion, BABA or MeJ. Inoculation was carried out 24 h post-elicitation with 10 µL of Pc suspension (1 × 108 CFU mL−1). Plant samples were ground in sterile dH2O 24 h post-inoculation. Bacterial populations were calculated from five leaf discs (1 g) for each treatment. Bars represent the average CFU g−1 plant tissue ± standard errors based on two independent experiments of plating serial dilutions on LB ampicillin plates (100 µg mL−1).

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image

Figure 7. Dose-dependent development of disease symptoms on calla lily leaf discs with time, following treatment with Bion applied as a drench or methyl jamonate applied as a leaf spray and subsequent (24 h later) challenge inoculation with Pectobacterium carotovorum (10 µL per disc; 1 × 108 CFU mL−1). Values represent average of total necrotic area (mm2) calculated from 10 leaf discs for each time point.

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At 1 mm MeJ, 120 h post-inoculation with P. carotovorum, disease development was halted, yet a rapid development of senescence was apparent (Fig. 4c). At 10 mm MeJ did not cause any visual symptoms on calla lily leaf discs (Fig. 4c).

Pectobacterium carotovorum cells in the infected plant tissues were quantified using the ampicillin-resistance marker of the pProbe-At gfp vector (Fig. 5). The results revealed a reduction in bacterial cell proliferation of up to four orders of magnitude in the induced tissues 24 h after P. carotovorum inoculation. Since the infected control discs were completely macerated at 48 h, bacteria were not quantified any further. In induced leaves treated with Bion, the lowest percentage infection (50%) was obtained at 5 or 10 µg mL−1 (Fig. 4b). With BABA, the 5- and 10-µg mL−1 treatments also gave the lowest percentage infection (25 and 15%, respectively) (Fig. 4b). Bacterial proliferation in the MeJ treatments was not reduced to a greater extent than this. Bacterial cells were present on the leaf discs at all inducer concentrations, but MeJ was able to maintain low bacterial populations for a longer time period (Figs 4c and 7).

The gfp-expressing P. carotovorum isolates allowed visual assessment of the plant's response to the elicitors (Fig. 6a,b), as demonstrated by both bacterial proliferation (Fig. 6, lower panels) and the visible damage to the plant tissue (Fig. 6, upper panels).

image

Figure 6. Calla lily leaf discs after induction with (a) Bion or (b) methyl jasmonate and subsequent challenge inoculation with Pectobacterium carotovorum (Pc; 10 µL of 1 × 108 CFU mL−1). Disease development was recorded using confocal microscopy 24 h later. Images show Pc containing pPROBE-At gfp plasmid around the penetration spot. Red tissue corresponds to chlorophyll luminescence in healthy leaf tissue. Upper panel shows Pc damage to leaf tissue at increasing concentrations of the inducer, as demonstrated by darker shade of macerated tissue around the penetration spot. Lower panel shows the spread of Pc containing pPROBE-At gfp plasmid in the same plant tissue.

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Bacterial proliferation in the induced tissues correlated with the data obtained via the assessment of disease symptoms. However, gfp expression using the confocal microscope and bacterial cell quantification obtained via serial dilutions on LB ampicillin agar plates did not correlate.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Growers of high-value ornamental bulb crops are in urgent need of management strategies against soft rot disease for both plant protection and postharvest uses. Loss of the soil fumigant methyl bromide presents a unique challenge to bulb growers, with special pressure on producers of propagation material (Schneider et al., 2003). A genetically defined defence to soft rot has not yet been devised and related works suggest that the interaction is very complex (Vidal et al., 1997, 1998; Aguilar et al., 2002). Induced resistance pathways against P. carotovorum were studied in Arabidopsis and tobacco plants using elicitors derived from the pathogen, including cell-wall-degrading enzymes and harpin (Vidal et al., 1998; Kariola et al., 2003). Both elicitors triggered a systemic resistance response in Arabidopsis, indicating the potential of externally applied elicitors as a tool to combat soft rot disease. Harpin triggered a systemic response involving SA-dependent and jasmonate/ethylene (JA/ET)-dependent pathways (Kariola et al., 2003), while cell-wall-degrading enzymes (pectic enzymes) seemed to act in a SA-independent pathway (Vidal et al., 1998; Kariola et al., 2003).

In this study, a system was developed for exploring the potential of exogenous chemical inducers to reduce soft rot disease in P. carotovorum-sensitive ornamental bulb crops. The three inducers used, Bion, BABA and MeJ, were ones proven to act only through the plant defence system and not on the pathogen directly (Gozzo, 2003). The system allowed colonization patterns of P. carotovorum in calla lily and two other ornamental bulb crops, O. dubium and Hippeastrum (amaryllis), to be studied and the defence responses afforded by the elicitors to be quantified. The pPROBE-AT gfp broad-host-range plasmid used was designed and tested in Erwinia herbicola (Miller et al., 2000), but not in P. carotovorum. The plasmid was highly stable and transformed P. carotovorum cells were detectable in the host more than 5 days post-inoculation without further antibioitic selection. Pectobacterium carotovorum isolates expressing the plasmid were slightly less infective in all three plant systems than their wild-type counterparts in this study and for all bacterial concentrations. In LB broth, bacteria expressing the plasmid did not show any significant variation in proliferation rates, suggesting that the expression of the plasmid affected infectivity, but not growth. Importantly, specificity of the P. carotovorum isolates towards different plant hosts was maintained in the gfp-expressing isolates, supporting their suitability for studying infection, specificity and plant host resistance during infection.

Infection trials indicated that P. carotovorum isolates from monocot hosts were more aggressive in monocot systems than were isolates from dicot hosts. While all P. carotovorum isolates were equally infective on potato tubers, the monocot isolates were more aggressive toward the three monocot plants. The tomato isolate was incapable of infecting any of the monocots tested. Similar growth curve dynamics of all P. carotovorum isolates in LB suggested that the degree of infection on calla lily and O. dubium was a consequence of an as-yet-unknown mechanism of affinity to the host, rather than differences in growth rates among the isolates.

The potential of plant activators to induce resistance against P. carotovorum in calla lily was studied with isolate Pc13+ from calla. All elicitors tested here were able to reduce disease symptoms and bacterial proliferation in calla lily leaves 24 h after challenge inoculation with the pathogen. BABA was more efficient than Bion in decreasing the proportion of infected leaf discs when given as a drench at a concentration of 5 or 10 µg mL−1. At 25 µg mL−1, BABA treatment amplified disease symptoms and bacterial proliferation was greater than in non-induced plants. Application of Bion delayed bacterial proliferation, but once leaves were infected, soft rot developed within the next 48 h. Bion treatment reduced infection by 50% at 5 or 10 µg mL−1 (as a drench), but at 25 µg mL−1 the necrotic area on leaves was larger and the reduction in bacterial number was negligible. Both inducers displayed an optimum curve apparently derived from their mode of action involving responses that may become phytotoxic at higher concentrations. Such responses were described in many other plant systems (Vallad & Goodman, 2004). The optimum concentration pattern also supports the indirect nature of the mode of action of the inducers.

In the literature, Bion, unlike BABA, is considered to present an inherent difference between monocots and dicots, with greater longevity of resistance elicited in monocots (Oostendorp et al., 2001; Vallad & Goodman, 2004). The system used here did not allow the development of long-term resistance, but preliminary data with O. dubium plantlets displayed the development of long-term resistance (not shown).

Jasmonate has been implicated as a signal molecule in wound response against feeding insects as well as in resistance against necrotrophic pathogens, including the bacterial pathogen P. carotovorum (Brader et al., 2001). A recent review (Glazebrook, 2005) distinguished between biotrophic and necrotrophic pathogens on the grounds that programmed cell death in the host regulated by SA-dependent pathways is not predicted to limit necrotrophic pathogen growth. Necrotrophs like P. carotovorum or the fungus Botrytis cinerea are restricted mainly by resistance mechanisms involving the JA/ET-signalling pathway.

The results of the present study support this model by indicating that the JA/ET-signalling pathway is a key player in the development of resistance against P. carotovorum in calla lily. MeJ was able to reduce disease symptoms dramatically relative to the other inducers and for a longer period. In contrast to Bion or BABA, the SA-pathway-dependent elicitors, MeJ (10 mm) completely inhibited the development of disease symptoms. The results agree with previous studies displaying the involvement of SA or its analogue, benzothiadiazole (BTH) in B. cinerea resistance in Arabidopsis, where treatments with exogenous SA or BTH prior to infection also reduced lesion size, but could not halt the disease (Zimmerli et al., 2001). Kariola et al. (2003) demonstrated that harpin purified from E. carotovora elicits lesion formation in Arabidopsis accompanied by the expression of SA-dependent, but also JA/ET-dependent, marker genes PR1 and PDF1·2, suggesting that both pathways are activated.

The gfp-reporter-based approach provides an improved means to detect and follow the bacterium in planta and to quantify its proliferation using the ampicillin- resistance marker.

The gfp-broad-host-range vector used in this study offered an efficient tool to study the potential of plant activators to induced resistance against P. carotovorum.

Further work is currently underway to elucidate resistance mechanisms and pathways in this diverse group of ornamental monocot plants.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We acknowledge financial support of the Israeli Chief Scientist grant no. 256-0717, and S. Manulis, Agricultural Research Organization, Volcani Ccenter, Israel, for kindly providing some of the Pectobacterium and Erwinia isolates used in this study.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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