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Biological control of bacterial spot of tomato caused by Xanthomonas campestris pv. vesicatoria by Rahnella aquatilis
ARTICLE IN PRESS Microbiological Research 160 (2005) 343—352
www.elsevier.de/micres
Biological control of bacterial spot of tomato caused by Xanthomonas campestris pv. vesicatoria by Rahnella aquatilis Hoda H. El-Hendawy, Mohamed E. Osman, Noha M. Sorour Botany and Microbiology Department, Faculty of Science, Helwan University, Ain Helwan, Cairo, Egypt Accepted 27 February 2005
KEYWORDS Biological control; Rahnella aquatilis; Tomato; Xanthomonas vesicatoria
Summary Xanthomonas campestris pv. vesicatoria strain 2 was isolated from infected tomato seedlings grown in open field in Egypt. This strain produced irregular yellow-necrotic areas on tomato leaves and spotting of the stem. In an attempt to control this disease biologically, four experiments were conducted and tomato seedlings were pretreated, before the pathogen, with either of two antagonistic strains of Rahnella aquatilis through leaves, roots, soil or seeds. In all experiments, seedlings pretreated with R. aquatilis showed reduced susceptibility toward X. c. pv. vesicatoria. They also contained reduced protein concentration and showed reduced number of protein bands in SDS-PAGE analysis as well as increased fresh and dry weight relative to control seedlings inoculated with the pathogen only. This indicates that R. aquatilis reduced the deleterious effect and the stress exerted by X. c. pv. vesicatoria on tomato seedlings. Foliar application of R. aquatilis was the most effective method in disease reduction which could be attributed to the direct effect of the antagonistic bacteria on the pathogen. The highest amounts of fresh and dry weight ere obtained from seed treatment, which might suggest that bacterial seed inoculation provides earlier protection than could be achieved with foliar, soil or root treatment. & 2005 Elsevier GmbH. All rights reserved.
Introduction Bacterial spot of tomato (Lycopersicon esculentum Mill.) caused by Xanthomonas campestris pv. vesicatoria (Doidge), Dye (Xanthomonas axonopo-
dis pv. vesicatoria, Vauterin et al., 1995) is one of the most serious diseases in many areas (Sherf and MacNab, 1986; Scott et al., 1989; Ward and O’Garro, 1992; Uys et al., 1996). The disease affects stems, leaves and fruits (Sherf and MacNab,
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ARTICLE IN PRESS 344 1986; Agrios, 1997) and causes significant losses when environmental conditions are suitable for the pathogen (Pohronezny and Volin, 1983). Different strategies have been employed for controlling the disease including sanitation, the use of pathogen-free seed and other cultural practices (Goode and Sasser, 1980; Sherf and MacNab, 1986; Jones et al., 1991), and also use of tomato cultivars resistant to X. c. pv. vesicatoria (Mew and Natural, 1993; Bouzar et al., 1999). Chemical control by using copper and streptomycin sprays have also been used (Thayer and Stall, 1961; Conover and Gerhold, 1981; Jones and Jones, 1985). Disadvantages of chemical applications such as potential chemical residues on fruit, cost and development of resistant bacterial strains have been reported (Stall and Thayer, 1962; Marco and Stall, 1983; Jones and Jones, 1985; Stall et al., 1986; Ritchie and Dittapongitch, 1991). Furthermore, biological control of the disease by treatment with antagonistic bacteria was also reported. For example, reduction of the disease incidence was obtained by using Pseudomonas putida and P. syringae (Campbell et al., 1998). Also treatment with nonpathogenic mutants of X. vesicatoria was found to be effective in disease suppression (Wilson et al., 1998). In this study, the role of two antagonistic strains of Rahnella aquatilis in protecting tomato of bacterial spots caused by X. c. pv. vesicatoria was tested and also the level of protection resulting from application of R. aquatilis strains to seeds, roots and soil or injection into leaves of tomato seedlings was compared.
Materials and methods Bacterial strains Rahnella aquatilis strain 17 and strain 55 were isolated from soil in Egypt (El-Hendawy et al., 2003). Xanthomonas campestris pv. vesicatoria strain 2 was isolated from diseased tomato seedlings growing in open field in Egypt and identified by consulting Bradbury (1984), Lelliott and Stead (1987) and Vauterin et al. (1995).
H.H. El-Hendawy et al.
Seeds and growth of seedlings Seeds of tomato (L. esculentum Mill) were obtained from the Ministry of Agriculture, Egypt. Plastic pots, each of 20 cm diameter and containing clay soil were used for growth of seedlings, five seeds were sown in each pot at equal distances and watered as required to keep soil moist but not wet, all pots were placed on a bench at room temperature.
Inoculations Inoculum was prepared from overnight shaken cultures incubated at 30 1C. Cultures were centrifuged at 6000g for 20 min at room temperature then the pellet was washed twice in distilled water. After the final wash the pellet was suspended in sterile distilled water (SDW) and the number of cells in the suspension was determined from optical density measurements at OD600. The suspension was diluted to give the required number of cells ml 1 and samples were injected into leaves or stems using a 1 ml sterile disposable syringe fitted with a 25 G needle.
Foliar treatment of tomato seedlings This experiment was carried out as described by El-Hendawy et al. (1998). Three weeks old tomato seedlings were grouped into four sets, each contained 20 identical seedlings, these sets were subjected to the following treatments: The first set was inoculated with R. aquatilis strain 17, three leaves per seedling were inoculated, and each received 1 108 cfu prepared from a 24 h old culture, then the pathogen was inoculated 24 h later with 3 106 cfu of a 24 h old culture of X. c. pv. vesicatoria, the inoculum size of both the antagonistic bacteria and the pathogen was based on the in vitro antibiosis, this treatment was symbolized R1X. The second set was inoculated with R. aquatilis strain 55 and X. c. pv. vesicatoria as described before, and this treatment was symbolized R2X. R. aquatilis strain 17 and R. aquatilis strain 55 each was inoculated to a set of seedlings, these last two treatments were symbolized R1 and R2, respectively.
Antagonistic activity
Seed treatment
The antagonistic activity of R. aquatilis strains toward X. c. pv. vesicatoria was tested by the welldiffusion method as described by Vignolo et al. (1993).
Tomato seeds were soaked for 2 h in bacterial suspension containing 1 108 cfu ml 1 prepared from an overnight cultures of R. aquatilis strain 17 or R. aquatilis strain 55, seeds were air dried for
ARTICLE IN PRESS Biological control of bacterial spot of tomato 24 h then sowed in plastic pots containing clay soil. All pots were watered as required with tap water. Three weeks later, seedlings were grouped into 4 batches of 20 tomato seedlings and subjected to the following treatments: seedlings originated from seeds soaked in R. aquatilis strain 17 and left without inoculation (R1) or inoculated with the pathogen X. c. pv. vesicatoria (R1X), seedlings originated from seeds soaked in R. aquatilis strain 55 and left without inoculation (R2) or inoculated with the pathogen (R2X). Inoculation of the pathogen was carried out through leaves; each leaf received 3 106 cfu.
Soil treatment This method was based on that reported by Molina et al. (1998). Ten milliliter bacterial suspension containing 1 108 cfu prepared from an overnight culture of either R. aquatilis strains were mixed thoroughly with each kg of clay soil. Four sets of pots, each containing 4 pots, were used in this experiment. The first two sets where the soil was mixed with bacterial suspension of R. aquatilis strain 17 (treatment R1) the second two sets in which the soil was mixed with bacterial suspension of R. aquatilis strain 55 (treatment R2). Five seeds were sown in each pot, then watered with tap water and maintained at room temperature. Three weeks after sowing, one set of seedlings of either R1 and R2 treatments were inoculated through leaves with a bacterial suspension of X. c. pv. vesicatoria to obtain treatments R1X, and R2X, each leaf received 3 106 cfu. Two sets of seedlings originated from nonbacaterized seeds and nonbacaterized soil were inoculated through leaves, one with the pathogen (treatment X) and the other with SDW (treatment SDW), and used as a control for the previous three experiments.
Root treatment This method was described by Sivamani and Gwanamanickam (1988), 3 weeks old tomato seedlings were lifted off the soil without injuring the roots, the soil particles were removed with running tap water and root tips were excised, the seedlings were grouped into six groups each of 20 identical seedlings. Group 1: seedlings were dipped for 1 h in 10 ml of a bacterial suspension containing 1 108 cfu ml 1 of R. aquatilis strain 17 (R1), group 2: seedlings were dipped for 1 h in bacterial suspension containing 1 108 cfu ml 1 of R. aquatilis strain 17 then, in bacterial suspension contain-
345 ing 3 106 cfu ml 1 of the pathogen for another 1 h (R1X), group 3: seedlings were dipped for 1 h in a bacterial suspension containing 1 108 cfu ml 1 of R. aquatilis strain 55 (R2), group 4: seedlings were dipped for 1 h in a bacterial suspension containing 1 108 cfu ml 1 of R. aquatilis strain 55 then transferred to a bacterial suspension containing 3 106 cfu ml 1 of the pathogen for another 1 h (R2X), group 5: seedlings were dipped for 1 h in a bacterial suspension containing 3 106 cfu ml 1 of the pathogen (X), group 6: seedlings were dipped for 1 h in SDW. Seedlings were replanted in identical pots with the same amounts of soil, each pot contained five seedlings. All pots were placed on a bench at room temperature and watered as required. In all experiments, symptom developments were recorded daily over 2 weeks and percentage of infection was calculated, each experiment was repeated three times.
Determination of fresh and dry weight Seedlings of different treatments, after recording the symptom development and percentage of infection, were removed, washed with SDW, blotted with tissue paper, and fresh weight was determined. Seedlings were then dried at 60 1C for 72 h and dry weight was recorded.
Preparation of protein extracts and electrophoresis Seedlings were powdered in liquid nitrogen and proteins were extracted as described by Brymgelsson et al. (1988) with 1 ml g 1 84 mM citric acid, 32 mM Na2HPO4, pH 2.8, 14 mM b-mercaptoethanol. After centrifugation, protein were precipitated from the supernatant with nine volumes of ice-cold acetone. The redissolved pellet was dialyzed against 1 mM Tris–HCl pH 6.8 and stored at 20 until required. SDS-PAGE was carried out according to Laemmli (1970). The separating and stacking gels contained 10% and 4.55% (w/v) acrylamide, respectively. Electrophoresis was performed at room temperature at a constant current of 20 mA and a maximum of 450 V. Molecular mass markers (SDS-6H Kit, Sigma) were included in each run. The gels were stained with silver nitrate according to Blum et al. (1987). Protein concentration was determined by the method of Bradford (1976).
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H.H. El-Hendawy et al.
Statistical analysis All data presented are means of 3 replica, the results were analyzed by variance analysis and significance was determined using LSD values at 1% and 5%.
Results In vitro inhibition Two antagonistic strains of R. aquatilis were tested for in vitro antibiosis against X. c. pv. vesicatoria strain 2. The test was carried out by using overnight cultures or culture supernatants of R. aquatilis. The obtained results revealed that either R. aquatilis strains or their supernatants were able to restrict the growth of X. c. pv. vesicatoria strain 2. Maximum inhibition zones were obtained in plates containing 1 108 cfu of either R. aquatilis strains and 3 106 cfu of X. c. pv. vesicatoria strain 2.
Effect of R. aquatilis on the infection of tomato seedlings by X. c. pv. vesicatoria
a bacterial suspension of R. aquatilis (seed application), or the roots of the seedlings were dipped in R. aquatilis suspension (root application). In the four experiments, neither R. aquatilis strains nor SDW induced disease symptoms when inoculated into leaves of 3 weeks-old tomato seedlings (Table 1). In contrast to this result, inoculation of X. c. pv. vesicatoria into leaves of control seedlings, originated from nonbacterized seeds and soil, resulted in disease symptoms in 60% of the inoculated seedlings. In case of root application 50% of the inoculated seedlings showed disease symptoms. In the foliar application, treatment with R. aquatilis strain 17 and strain 55 before inoculation with X. c. pv. vesicatoria reduced the infection up to 50% and 58% , respectively whereas, when applied to seeds, either R. aquatilis strains reduced the infection up to 33%. On the other hand, mixing the bacterial suspension of R. aquatilis strains with the soil, reduced the infection by 16.6% and 25%, treatments R1X and R2X, respectively. When the roots of tomato seedlings were dipped in R. aquatilis suspension, the infection was reduced up to 40%, treatment R1X, and 50%, treatment R2X. Results are presented in Table 1.
Fresh and dry weight of tomato seedlings The in vitro antibiosis of R. aquatilis strains against X. c. pv. vesicatoria raised questions concerning the in vivo interaction between these strains. Four experiments were carried out to investigate the possible role of R. aquatilis strains in reducing the deleterious effect of X. c. pv. vesicatoria on tomato seedlings. R. aquatilis strains were inoculated into tomato leaves in advance to the pathogen (foliar application), or mixed with the soil before sowing of the seeds (soil application), or the seeds were soaked in
Highly significant reduction in fresh and dry weight was obtained when X. c. pv. vesicatoria strain 2 was inoculated into leaves of 3 weeks old tomato seedlings, treatment X, relative to control seedlings inoculated with SDW, treatment SDW. On the other hand, pretreatment of seedlings with either R. aquatilis strains reduced the deleterious effect of X. c. pv. vesicatoria on tomato seedlings. This was revealed by the highly significant increase in fresh and dry weight of seedlings pretreated with
Table 1. Percentage of infection of 3 weeks old tomato seedlings inoculated with X. c. pv. vesicatoria strain 2 and R. aquatilis strains Treatment
SDW R1 R2 X R1X R2X
Foliar
Seed
Soil
Root
Infected seedlings
% of infection
Infected seedlings
% of infection
Infected seedlings
% of infection
Infected seedlings
% of infection
— — — 12 6 5
0 0 0 60 30 25
— — — 12 8 8
0 0 0 60 40 40
— — — 12 10 9
0 0 0 60 50 45
— — — 10 6 5
0 0 0 50 30 25
Each treatment contained 20 seedlings. SDW, seedlings treated with sterile distilled water; R1, seedlings treated with R. aquatilis strain 17; R2, seedlings treated with R. aquatilis strain 55; X, seedlings treated with X. c. pv. vesicatoria strain 2 and the same treatment was used as a control for foliar, seed and soil applications; R1X, seedlings treated with R. aquatilis strain 17 then Xanthomonas; R2X, seedlings treated with R. aquatilis strain 55 then Xanthomonas.
ARTICLE IN PRESS Biological control of bacterial spot of tomato
347
Table 2. Effect of R. aquatilis on fresh and dry weight of tomato seedlings inoculated or non-inoculated with X. c. pv. vesicatoria Treatment
Foliar F.wt
F.wt SDW D.wt
3.520
F.wt
3.350
Seed D.wt
F.wt
Soil D.wt
3.520 0.345
F.wt
Root D.wt
3.520 0.345
4.030
F.wt
LSD D.wt
3.600 0.345
3.432
0.320 3.710
1%
5%
0.270
0.190
0.030
0.020
0.300
0.210
0.100
0.070
0.300
0.220
0.048
0.034
0.270
0.180
0.027
0.019
0.280
0.200
0.060
0.040
0.260
0.180
0.070
0.050
R1 D.wt F.wt
0.342 3.450
0.380 4.160
0.330 3.801
0.360 3.920
R2 D.wt F.wt
0.329 2.520
0.420 2.520
0.359 2.520
0.420 2.370
X D.wt
0.230
0.230 3.240
0.230
F.wt R1X D.wt
3.021
F.wt R2X D.wt
3.200
1% LSD 5%
0.240
0.020
0.350
0.070
0.250
0.100
0.230
0.030
0.170
0.016
0.250
0.050
0.180
0.070
0.160
0.020
0.291
2.610
0.210
0.350 3.670
0.314
3.470 0.242
3.352 0.390
0.340 3.680
0.305
0.420
1
Fresh and dry weights are expressed as g seedling .Only one treatment of SDW and of X was used as a control for foliar, seed and soil application. D.wt, dry weight; F.wt, fresh weight. Results are means of three experiments.
either R. aquatilis strains relative to that inoculated with X. c. pv. vesicatoria only (Table 2). Inoculation of strain 17 (R1) or strain 55 (R2) into tomato leaves (foliar application) reduced fresh and dry weight of the seedlings relative to treatment SDW. In contrast to this result, application of R1 and R2 through seeds and roots increased both fresh and dry weight. In case of soil application, increased fresh and dry weight was obtained only with treatment R2. Significant reduction in fresh and dry weight relative to control seedlings (SDW treatment) was obtained with treatment R1. However, it was observed that R. aquatilis strain 55 is able to enhance the growth of tomato seedlings more than strain 17 (Table 2).
ments. Highly significant increase in protein concentration of seedlings inoculated with X. c. pv. vesicatoria only, treatment X, relative to all other treatments was observed (Table 3). In contrast, highly significant reduction in protein concentration was detected in seedlings treated with either R. aquatilis strains, treatments R1 and R2, relative to control seedlings inoculated with SDW. The obtained results showed that protein concentration of seedlings treated or pretreated with R. aquatilis strain 17, treatments R1 and R1X, is always lower than that of seedlings treated or pretreated with strain 55, treatments R2 and R2X (Table 3).
Protein concentration of tomato seedlings
Sodium dodecyl sulfate polyacrylamide gel electrophoresis
Differences in protein concentrations of tomato seedlings were obtained with the different treat-
Analysis of SDS-PAGE showed that 12 protein bands with molecular weights ranging from 64.97 to
ARTICLE IN PRESS 348
H.H. El-Hendawy et al.
Table 3. Protein concentration of tomato seedlings inoculated and non-inoculated with R. aquatilis strains Treatment
SDW R1 R2 X R1X R2X 1% LSD 5%
Protein concentration Foliar
Seed
Soil
Root
6.00 4.00 5.00 8.00 4.00 5.00 0.25
6.00 3.00 5.00 8.00 5.00 5.50 0.99
6.00 3.50 4.50 8.00 5.00 6.00 0.50
5.00 3.00 4.00 6.00 3.50 4.00 0.75
0.18
0.71
0.36
0.53
Protein concentration was determined as mg g seedlings.
1
dry weight of
17.47 kDa are contained in tomato seedlings inoculated with SDW (Fig. 1a). Also 12 bands were observed by inoculation of SDW into seedlings with excised root tips (treatment SDW). Molecular weights of these bands ranged from 90.95 to 15.01 kDa (Fig. 1b). Different protein profile was also obtained when inoculating the pathogen into seedlings originated from nonbacterized seeds and nonbacaterized soil (treatment X) or seedlings whose roots were excised then transplanted (treatment X). Fifteen bands were detected in treatment X, their molecular weights ranged from 102.75 to 15.01 kDa (Fig. 1a) whereas in treatment XR, 8
Figure 1. (a) SDS-PAGE profile of tomato seedlings treated or pretreated with R. aquatilis through soil and seeds. From left lane 1, molecular weight marker. In soil treatment seedlings were treated with: lane 2, sterile distilled water; lane 3, R. aquatilis 55; lane 4, R. aquatilis 17; ;lane 5, R. aquatilis 55 and Xanthomonas; lane 6, R. aquatilis 17 and Xanthomonas. In seed treatment tomato seedlings were treated with; lane 7, R. aquatilis 55 and Xanthomonas; lane 8, R. aquatilis 17 and Xanthomonas; lane 10, R. aquatilis 55; lane 11, R. aquatilis 17. Lane 9 contains extract of seedlings originated from nonbacterized seed and nonbacterized soil and treated with Xanthomonas only. (b) SDS-PAGE profile of tomato seedlings treated or pretreated with R. aquatilis through leaves and roots. From left lane 1, molecular weight marker. In foliar treatment seedlings were inoculated with: lane 2, sterile distilled water; lane 3, R. aquatilis 55; lane 4, R. aquatilis 17; lane 5, Xanthomonas; lane 6, R. aquatilis 55 and Xanthomonas; lane 7, R. aquatilis 17 and Xanthomonas. In root treatment seedlings were treated with : lane 8, R. aquatilis 55; lane 9, R. aquatilis 17; lane 10, Xanthomonas; lane 11, R. aquatilis 55 and Xanthomonas; lane 12, R. aquatilis 17 and Xanthomonas; lane 13, seedlings with excised roots and treated with sterile distilled water.
protein bands with molecular weights ranging from 90.95 to 15.01 kDa (Fig. 1b). Although identical protein profiles were obtained from tomato seedlings treated with either R. aquatilis strains through roots (treatments R1 and R2), different protein profiles were obtained when inoculating the pathogen into these seedlings (treatments R1X and R2X). Six protein bands with molecular weights ranging from 64.97 to 15.01 were observed in each of R1 and R2. In addition, one more band with molecular weights of 48.42 kDa was observed in either treatments R1X and R2X and two bands with molecular weights of 90.95 and 15.85 kDa in R1X and R2X, respectively.
ARTICLE IN PRESS Biological control of bacterial spot of tomato In the soil and seed application methods, reduced number of protein bands were observed when treating tomato seedlings with R. aquatilis strains only or pretreating before inoculating the pathogen through leaves relative to those obtained from seedlings inoculated with the pathogen only and that inoculated with SDW (Fig. 1a).
Discussion Each of R. aquatilis strains proved to be able to protect tomato seedlings from infection by X. c. pv. vesicatoria. This was revealed by the reduced percentage of infection of seedlings pretreated with either R. aquatilis strains relative to control seedlings inoculated with X. c. pv. vesicatoria. The possible role of R. aquatilis in protecting the Chardonnay vine from galling by five different virulent Agrobacterium vitis species was reported by Bell et al. (1995). However, reduction of infection by plant pathogenic bacteria as a result of pretreatment with antagonistic bacteria has been observed in some diseases. For example, occurrence of bacterial wilt of tomato caused by P. solanacearum was remarkably suppressed when the culture suspension of the antagonistic bacterium Bacillus subtilis NB22 was poured into heavily infested soil. The percentage of dead plants was reduced to one-third that observed in the control (Phae et al., 1992). In another study, pretreatment of melon cotyledons with viable cells of P. fluorescens has been shown to reduce the infection with Erwinia carotovora subspecies carotovora up to 50% (El-Hendawy et al., 1998). Inoculation of tomato leaves with X. c. pv. vesicatoria resulted in significant reduction in fresh and dry weight of the seedlings. Reduction in fresh and dry weight of melon plants as a result of inoculation with the bacterial pathogen has been reported (El-Hendawy et al., 1998). In this study, it is obvious that development of yellow-brown necrotic areas or chlorosis in tomato leaves is attributed to interference of the pathogen with photosynthesis. Nearly all organic nutrients of plants are produced in the leaf cells, following photosynthesis, and are translocated downward and distributed to all the living plant cells (Agrios, 1997). In advanced stages of some diseases, the rate of photosynthesis is no more than one-fourth the normal rate (Agrios, 1997), reduction of photosynthesis will result in reduction in growth and consequently in fresh and dry weight of the plant.
349 Treatment of tomato seedlings with R. aquatilis strain 55 (R2) to seeds or soil or roots, significantly enhanced fresh and dry weight in comparison with untreated control (SDW). Whereas, strain 17 increased fresh and dry weight when applied to seeds or roots only. However, seedling treatment or pretreatment of the pathogen with either R. aquatilis strains, by the different application methods, enhanced fresh and dry weight relative to that of seedlings inoculated with the pathogen only. This result agrees with that reported in the literature, where pretreatment of plants with antagonistic bacteria or plant growth promoting rhizobacteria (PGPR) enhanced plant growth and consequently its weight. For example, significant increases in growth and yield of potato plants were achieved by treating seed pieces with suspensions of two Pseudomonas spp. prior to planting (Burr et al., 1978). Also Sivamani and Gwanamanickam (1988) obtained better root growth and enhanced plant weight by pretreatment of the pathogen with P. fluorescens. Furthermore, Wei et al. (1996) tested the capacity of three PGPR to induce systemic resistance against cucumber diseases and found that two of the three tested strains significantly increased runner length, and all three strains significantly increased leaf number per plant compared with the disease control. Also, pretreatment of melon cotyledons with P. fluorescens was able to reduce the deleterious effect of E. carotovora ssp. carotovora on fresh and dry weight of melon seedlings (El-Hendawy et al., 1998). Another report comes from Raupach and Kloepper (1998), where they tested the ability of two bacterial strains, Burkholderia gladioli and Bacillus pumilus, to promote cucumber growth and suppress anthracnose and angular leaf spot and found that either strains significantly allowed protection from diseases and increased the main runner length relative to control. Application of R. auatilis strains onto tomato seedlings by different methods, foliar, seed, soil and root resulted in differences in the level of disease reduction as well as in fresh and dry weight of the seedlings. Foliar application of either R. aquatilis strains was the most effective in disease reduction. This result is in agreement with that reported by Hsieh and Buddenhagen (1974) where they found that foliar application of E. herbicola reduced the infection of rice by X. oryzae more than root application. Protection of seedlings from disease as a result of inoculation with the antagonist and the pathogen through leaves could be attributed to the direct inhibition of the pathogen by the antagonist as they come in contact in the same leaf. Seed inoculation with R. aquatilis strains
ARTICLE IN PRESS 350 enhanced fresh and dry weight more than the other methods. In contrast to this result, no significant difference in the level of induced resistance between seed inoculation and cotyledon injection with two PGPR strains (Liu et al., 1995). Coating of cucumber seeds or spraying with Burkholderia gladioli and Bacillus pumilus showed no significant difference in cucumber growth and disease suppression between each strain in either method of application (Raupach and Kloepper, 1998). However, seed inoculation might provide earlier protection than could be provided with foliar, soil or root treatment. Results from SDS-PAGE showed that tomato seedlings inoculated with the pathogen only contained the largest number of protein bands relative to all other treatments. Fragmentation of host protein might occur by the action of pathogen proteases and this could be responsible for the detection of more bands. In this study, tomato seedlings inoculated with the pathogen only contained the highest protein concentration relative to all other treatments. Based on these results, it can be concluded that more proteins could be accumulated in these seedlings. However, it has been reported that structurally diverse group of plant proteins that are toxic to invading pathogens are widely distributed in plants in trace amounts but are produced in much greater concentration following pathogen attack or stress, these proteins are called pathogenesis-related (PR) proteins (Agrios, 1997; Rep et al., 2002). These proteins may be effective in inhibiting pathogen growth, multiplication and/or spread and be responsible for the state of systemic acquired resistance (Van Loon and Van Strien, 1999). Reduced protein concentration as well as reduced number of protein bands are detected in seedlings treated with R. aquatilis strains relative to control seedlings inoculated with SDW. This could be attributed to reduction in fresh and dry weight of seedlings. Also, reduction in protein concentration as well as the number protein bands was obtained from seedlings pretreated with either R. aquatilis strains relative to that inoculated with the pathogen only. However, Hoffland et al. (1996) demonstrated that the plant growth promoting rhizobacterium (PGPR) P. fluorescens strains WCS417 has been shown to induce systemic resistance against Fusarium oxysporum in several plant species without inducing synthesis of PR proteins. They also found that the absence of PR after induction by P. fluorescens does not lower protection. Other studies have proposed several mechanisms for disease resistance other than accumulation of PR proteins. These mechanisms
H.H. El-Hendawy et al. could be: altered ion fluxes across the plant cell membrane, generation of active oxygen species, changes in the phosphorylation state of regulatory proteins and transcriptional activation of plant defense systems culminate in cell death at the site of infection, local accumulation of phytoalexins and cell wall rigidification as a result of callose, lignin and suberin deposition (Hammond-Kosack and Jones, 1996; Yang et al., 1997). It is likely that excision of root tips induced resistance of tomato seedlings. This might explain the reduction in infection when inoculating the pathogen into tomato seedlings the roots of which were excised before transplanting compared to seedlings originated from nonbacterized soil and seeds and inoculated with the pathogen only. The former seedlings showed reduced protein concentration as well as reduced number of protein bands than the latter. However, defense mechanisms other than protein accumulation could be evoked in this case.
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351 in out door assays. Appl. Environ. Microbiol. 64, 2072–2078. Phae, C.G., Shoda, M., Kita, N., Nokana, M., Ushiyama, K., 1992. Biological control of crown and root rot and bacterial wilt of tomato by Bacillus subtilis NB22. Ann. Phytopathol. Soc. Jpn. 58, 329–339. Pohronezny, K., Volin, R.B., 1983. The effect of bacterial spot on yield and quality of fresh market tomatoes. HortScience 18, 69–70. Raupach, G.S., Kloepper, J.W., 1998. Mixtures of plant growth promoting rhizobacteria enhance biological control of multiple cucumber pathogens. Phytopathology 88, 1158–1164. Rep, M., Dekker, H.L., Vossen, J.H., de Boer, A.D., Houterman, P.M., Speijer, D., Back, J.W., deKoster, C.G., Cornelissen, B.J.C., 2002. Mass spectrometric identification of isoforms of PR proteins in xylem sap of fungus-infected tomato. Plant Physiol. 130, 904–917. Ritchie, D.F., Dittapongitch, V., 1991. Copper- and streptomycin-resistant strains and host differentiated races of Xanthomonas campestris pv.vesicatoria in North Carolina. Plant Dis. 75, 733–736. Scott, J.W., Somodi, G.C., Jones, J.B., 1989. Resistance to bacterial spot fruit infection in tomato. HortScience 24, 825–827. Sherf, A.F., MacNab, A.A., 1986. Vegetable Diseases and Their Control. Wiley, New York. Sivamani, E., Gwanamanickam, S.S., 1988. Biological control of Fusarium oxysporum f.sp. cubense in banana by inoculation with Pseudomonas fluorescens. Plant Soil 107, 3–9. Stall, R.E., Thayer, P.L., 1962. Streptomycin resistance of the bacterial spot pathogen and control with streptomycin. Plant Dis. Rep. 46, 389–393. Stall, R.E., Loschke, D.C., Jones, J.B., 1986. Linkage of copper resistance and a virulence loci on a selftransmissible plasmid in Xanthomonas campestris pv. vesicatoria. Phytopathology 76, 240–243. Thayer, P.L., Stall, R.E., 1961. A survey of Xanthomonas vesicatoria resistance to streptomycin. Proc. Fla. State Hortic. Soc. 75, 163–165. Uys, M.D.R., Thompson, A.H., Holz, G., 1996. Diseases associated with tomato in the main tomato growing of South Africa. J. S. Afr. Soc. Hort. Sci. 6, 78–81. Van Loon, L.C., Van Strien, E.A., 1999. The families of pathogenesis-related proteins, their activities, and comparative analysis of PR-1 type proteins. Physiol. Mol. Plant Pathol. 55, 85–97. Vauterin, L., Hoste, B., Dersters, K., Swings, J., 1995. Reclassification of Xanthomonas. Int. J. Syst. Bacteriol. 45, 472–489. Vignolo, M.G., Suriani, F., de Ruiz, A.P., Oliver, G., 1993. Antibacterial activity of Lactococcus strains isolated from dry fermented sausages. J. Appl. Bacteriol. 75, 344–349. Ward, H.P., O’Garro, L.W., 1992. Bacterial spot of pepper and tomato in Barbados. Plant Dis. 76, 1046–1048.
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Biological control of bacterial spot of tomato caused by Xanthomonas campestris pv. vesicatoria by Rahnella aquatilis Hoda H. El-Hendawy, Mohamed E. Osman, Noha M. Sorour Botany and Microbiology Department, Faculty of Science, Helwan University, Ain Helwan, Cairo, Egypt Accepted 27 February 2005
KEYWORDS Biological control; Rahnella aquatilis; Tomato; Xanthomonas vesicatoria
Summary Xanthomonas campestris pv. vesicatoria strain 2 was isolated from infected tomato seedlings grown in open field in Egypt. This strain produced irregular yellow-necrotic areas on tomato leaves and spotting of the stem. In an attempt to control this disease biologically, four experiments were conducted and tomato seedlings were pretreated, before the pathogen, with either of two antagonistic strains of Rahnella aquatilis through leaves, roots, soil or seeds. In all experiments, seedlings pretreated with R. aquatilis showed reduced susceptibility toward X. c. pv. vesicatoria. They also contained reduced protein concentration and showed reduced number of protein bands in SDS-PAGE analysis as well as increased fresh and dry weight relative to control seedlings inoculated with the pathogen only. This indicates that R. aquatilis reduced the deleterious effect and the stress exerted by X. c. pv. vesicatoria on tomato seedlings. Foliar application of R. aquatilis was the most effective method in disease reduction which could be attributed to the direct effect of the antagonistic bacteria on the pathogen. The highest amounts of fresh and dry weight ere obtained from seed treatment, which might suggest that bacterial seed inoculation provides earlier protection than could be achieved with foliar, soil or root treatment. & 2005 Elsevier GmbH. All rights reserved.
Introduction Bacterial spot of tomato (Lycopersicon esculentum Mill.) caused by Xanthomonas campestris pv. vesicatoria (Doidge), Dye (Xanthomonas axonopo-
dis pv. vesicatoria, Vauterin et al., 1995) is one of the most serious diseases in many areas (Sherf and MacNab, 1986; Scott et al., 1989; Ward and O’Garro, 1992; Uys et al., 1996). The disease affects stems, leaves and fruits (Sherf and MacNab,
Corresponding author.
E-mail address: [email protected] (H.H. El-Hendawy). 0944-5013/$ - see front matter & 2005 Elsevier GmbH. All rights reserved. doi:10.1016/j.micres.2005.02.008
ARTICLE IN PRESS 344 1986; Agrios, 1997) and causes significant losses when environmental conditions are suitable for the pathogen (Pohronezny and Volin, 1983). Different strategies have been employed for controlling the disease including sanitation, the use of pathogen-free seed and other cultural practices (Goode and Sasser, 1980; Sherf and MacNab, 1986; Jones et al., 1991), and also use of tomato cultivars resistant to X. c. pv. vesicatoria (Mew and Natural, 1993; Bouzar et al., 1999). Chemical control by using copper and streptomycin sprays have also been used (Thayer and Stall, 1961; Conover and Gerhold, 1981; Jones and Jones, 1985). Disadvantages of chemical applications such as potential chemical residues on fruit, cost and development of resistant bacterial strains have been reported (Stall and Thayer, 1962; Marco and Stall, 1983; Jones and Jones, 1985; Stall et al., 1986; Ritchie and Dittapongitch, 1991). Furthermore, biological control of the disease by treatment with antagonistic bacteria was also reported. For example, reduction of the disease incidence was obtained by using Pseudomonas putida and P. syringae (Campbell et al., 1998). Also treatment with nonpathogenic mutants of X. vesicatoria was found to be effective in disease suppression (Wilson et al., 1998). In this study, the role of two antagonistic strains of Rahnella aquatilis in protecting tomato of bacterial spots caused by X. c. pv. vesicatoria was tested and also the level of protection resulting from application of R. aquatilis strains to seeds, roots and soil or injection into leaves of tomato seedlings was compared.
Materials and methods Bacterial strains Rahnella aquatilis strain 17 and strain 55 were isolated from soil in Egypt (El-Hendawy et al., 2003). Xanthomonas campestris pv. vesicatoria strain 2 was isolated from diseased tomato seedlings growing in open field in Egypt and identified by consulting Bradbury (1984), Lelliott and Stead (1987) and Vauterin et al. (1995).
H.H. El-Hendawy et al.
Seeds and growth of seedlings Seeds of tomato (L. esculentum Mill) were obtained from the Ministry of Agriculture, Egypt. Plastic pots, each of 20 cm diameter and containing clay soil were used for growth of seedlings, five seeds were sown in each pot at equal distances and watered as required to keep soil moist but not wet, all pots were placed on a bench at room temperature.
Inoculations Inoculum was prepared from overnight shaken cultures incubated at 30 1C. Cultures were centrifuged at 6000g for 20 min at room temperature then the pellet was washed twice in distilled water. After the final wash the pellet was suspended in sterile distilled water (SDW) and the number of cells in the suspension was determined from optical density measurements at OD600. The suspension was diluted to give the required number of cells ml 1 and samples were injected into leaves or stems using a 1 ml sterile disposable syringe fitted with a 25 G needle.
Foliar treatment of tomato seedlings This experiment was carried out as described by El-Hendawy et al. (1998). Three weeks old tomato seedlings were grouped into four sets, each contained 20 identical seedlings, these sets were subjected to the following treatments: The first set was inoculated with R. aquatilis strain 17, three leaves per seedling were inoculated, and each received 1 108 cfu prepared from a 24 h old culture, then the pathogen was inoculated 24 h later with 3 106 cfu of a 24 h old culture of X. c. pv. vesicatoria, the inoculum size of both the antagonistic bacteria and the pathogen was based on the in vitro antibiosis, this treatment was symbolized R1X. The second set was inoculated with R. aquatilis strain 55 and X. c. pv. vesicatoria as described before, and this treatment was symbolized R2X. R. aquatilis strain 17 and R. aquatilis strain 55 each was inoculated to a set of seedlings, these last two treatments were symbolized R1 and R2, respectively.
Antagonistic activity
Seed treatment
The antagonistic activity of R. aquatilis strains toward X. c. pv. vesicatoria was tested by the welldiffusion method as described by Vignolo et al. (1993).
Tomato seeds were soaked for 2 h in bacterial suspension containing 1 108 cfu ml 1 prepared from an overnight cultures of R. aquatilis strain 17 or R. aquatilis strain 55, seeds were air dried for
ARTICLE IN PRESS Biological control of bacterial spot of tomato 24 h then sowed in plastic pots containing clay soil. All pots were watered as required with tap water. Three weeks later, seedlings were grouped into 4 batches of 20 tomato seedlings and subjected to the following treatments: seedlings originated from seeds soaked in R. aquatilis strain 17 and left without inoculation (R1) or inoculated with the pathogen X. c. pv. vesicatoria (R1X), seedlings originated from seeds soaked in R. aquatilis strain 55 and left without inoculation (R2) or inoculated with the pathogen (R2X). Inoculation of the pathogen was carried out through leaves; each leaf received 3 106 cfu.
Soil treatment This method was based on that reported by Molina et al. (1998). Ten milliliter bacterial suspension containing 1 108 cfu prepared from an overnight culture of either R. aquatilis strains were mixed thoroughly with each kg of clay soil. Four sets of pots, each containing 4 pots, were used in this experiment. The first two sets where the soil was mixed with bacterial suspension of R. aquatilis strain 17 (treatment R1) the second two sets in which the soil was mixed with bacterial suspension of R. aquatilis strain 55 (treatment R2). Five seeds were sown in each pot, then watered with tap water and maintained at room temperature. Three weeks after sowing, one set of seedlings of either R1 and R2 treatments were inoculated through leaves with a bacterial suspension of X. c. pv. vesicatoria to obtain treatments R1X, and R2X, each leaf received 3 106 cfu. Two sets of seedlings originated from nonbacaterized seeds and nonbacaterized soil were inoculated through leaves, one with the pathogen (treatment X) and the other with SDW (treatment SDW), and used as a control for the previous three experiments.
Root treatment This method was described by Sivamani and Gwanamanickam (1988), 3 weeks old tomato seedlings were lifted off the soil without injuring the roots, the soil particles were removed with running tap water and root tips were excised, the seedlings were grouped into six groups each of 20 identical seedlings. Group 1: seedlings were dipped for 1 h in 10 ml of a bacterial suspension containing 1 108 cfu ml 1 of R. aquatilis strain 17 (R1), group 2: seedlings were dipped for 1 h in bacterial suspension containing 1 108 cfu ml 1 of R. aquatilis strain 17 then, in bacterial suspension contain-
345 ing 3 106 cfu ml 1 of the pathogen for another 1 h (R1X), group 3: seedlings were dipped for 1 h in a bacterial suspension containing 1 108 cfu ml 1 of R. aquatilis strain 55 (R2), group 4: seedlings were dipped for 1 h in a bacterial suspension containing 1 108 cfu ml 1 of R. aquatilis strain 55 then transferred to a bacterial suspension containing 3 106 cfu ml 1 of the pathogen for another 1 h (R2X), group 5: seedlings were dipped for 1 h in a bacterial suspension containing 3 106 cfu ml 1 of the pathogen (X), group 6: seedlings were dipped for 1 h in SDW. Seedlings were replanted in identical pots with the same amounts of soil, each pot contained five seedlings. All pots were placed on a bench at room temperature and watered as required. In all experiments, symptom developments were recorded daily over 2 weeks and percentage of infection was calculated, each experiment was repeated three times.
Determination of fresh and dry weight Seedlings of different treatments, after recording the symptom development and percentage of infection, were removed, washed with SDW, blotted with tissue paper, and fresh weight was determined. Seedlings were then dried at 60 1C for 72 h and dry weight was recorded.
Preparation of protein extracts and electrophoresis Seedlings were powdered in liquid nitrogen and proteins were extracted as described by Brymgelsson et al. (1988) with 1 ml g 1 84 mM citric acid, 32 mM Na2HPO4, pH 2.8, 14 mM b-mercaptoethanol. After centrifugation, protein were precipitated from the supernatant with nine volumes of ice-cold acetone. The redissolved pellet was dialyzed against 1 mM Tris–HCl pH 6.8 and stored at 20 until required. SDS-PAGE was carried out according to Laemmli (1970). The separating and stacking gels contained 10% and 4.55% (w/v) acrylamide, respectively. Electrophoresis was performed at room temperature at a constant current of 20 mA and a maximum of 450 V. Molecular mass markers (SDS-6H Kit, Sigma) were included in each run. The gels were stained with silver nitrate according to Blum et al. (1987). Protein concentration was determined by the method of Bradford (1976).
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Statistical analysis All data presented are means of 3 replica, the results were analyzed by variance analysis and significance was determined using LSD values at 1% and 5%.
Results In vitro inhibition Two antagonistic strains of R. aquatilis were tested for in vitro antibiosis against X. c. pv. vesicatoria strain 2. The test was carried out by using overnight cultures or culture supernatants of R. aquatilis. The obtained results revealed that either R. aquatilis strains or their supernatants were able to restrict the growth of X. c. pv. vesicatoria strain 2. Maximum inhibition zones were obtained in plates containing 1 108 cfu of either R. aquatilis strains and 3 106 cfu of X. c. pv. vesicatoria strain 2.
Effect of R. aquatilis on the infection of tomato seedlings by X. c. pv. vesicatoria
a bacterial suspension of R. aquatilis (seed application), or the roots of the seedlings were dipped in R. aquatilis suspension (root application). In the four experiments, neither R. aquatilis strains nor SDW induced disease symptoms when inoculated into leaves of 3 weeks-old tomato seedlings (Table 1). In contrast to this result, inoculation of X. c. pv. vesicatoria into leaves of control seedlings, originated from nonbacterized seeds and soil, resulted in disease symptoms in 60% of the inoculated seedlings. In case of root application 50% of the inoculated seedlings showed disease symptoms. In the foliar application, treatment with R. aquatilis strain 17 and strain 55 before inoculation with X. c. pv. vesicatoria reduced the infection up to 50% and 58% , respectively whereas, when applied to seeds, either R. aquatilis strains reduced the infection up to 33%. On the other hand, mixing the bacterial suspension of R. aquatilis strains with the soil, reduced the infection by 16.6% and 25%, treatments R1X and R2X, respectively. When the roots of tomato seedlings were dipped in R. aquatilis suspension, the infection was reduced up to 40%, treatment R1X, and 50%, treatment R2X. Results are presented in Table 1.
Fresh and dry weight of tomato seedlings The in vitro antibiosis of R. aquatilis strains against X. c. pv. vesicatoria raised questions concerning the in vivo interaction between these strains. Four experiments were carried out to investigate the possible role of R. aquatilis strains in reducing the deleterious effect of X. c. pv. vesicatoria on tomato seedlings. R. aquatilis strains were inoculated into tomato leaves in advance to the pathogen (foliar application), or mixed with the soil before sowing of the seeds (soil application), or the seeds were soaked in
Highly significant reduction in fresh and dry weight was obtained when X. c. pv. vesicatoria strain 2 was inoculated into leaves of 3 weeks old tomato seedlings, treatment X, relative to control seedlings inoculated with SDW, treatment SDW. On the other hand, pretreatment of seedlings with either R. aquatilis strains reduced the deleterious effect of X. c. pv. vesicatoria on tomato seedlings. This was revealed by the highly significant increase in fresh and dry weight of seedlings pretreated with
Table 1. Percentage of infection of 3 weeks old tomato seedlings inoculated with X. c. pv. vesicatoria strain 2 and R. aquatilis strains Treatment
SDW R1 R2 X R1X R2X
Foliar
Seed
Soil
Root
Infected seedlings
% of infection
Infected seedlings
% of infection
Infected seedlings
% of infection
Infected seedlings
% of infection
— — — 12 6 5
0 0 0 60 30 25
— — — 12 8 8
0 0 0 60 40 40
— — — 12 10 9
0 0 0 60 50 45
— — — 10 6 5
0 0 0 50 30 25
Each treatment contained 20 seedlings. SDW, seedlings treated with sterile distilled water; R1, seedlings treated with R. aquatilis strain 17; R2, seedlings treated with R. aquatilis strain 55; X, seedlings treated with X. c. pv. vesicatoria strain 2 and the same treatment was used as a control for foliar, seed and soil applications; R1X, seedlings treated with R. aquatilis strain 17 then Xanthomonas; R2X, seedlings treated with R. aquatilis strain 55 then Xanthomonas.
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347
Table 2. Effect of R. aquatilis on fresh and dry weight of tomato seedlings inoculated or non-inoculated with X. c. pv. vesicatoria Treatment
Foliar F.wt
F.wt SDW D.wt
3.520
F.wt
3.350
Seed D.wt
F.wt
Soil D.wt
3.520 0.345
F.wt
Root D.wt
3.520 0.345
4.030
F.wt
LSD D.wt
3.600 0.345
3.432
0.320 3.710
1%
5%
0.270
0.190
0.030
0.020
0.300
0.210
0.100
0.070
0.300
0.220
0.048
0.034
0.270
0.180
0.027
0.019
0.280
0.200
0.060
0.040
0.260
0.180
0.070
0.050
R1 D.wt F.wt
0.342 3.450
0.380 4.160
0.330 3.801
0.360 3.920
R2 D.wt F.wt
0.329 2.520
0.420 2.520
0.359 2.520
0.420 2.370
X D.wt
0.230
0.230 3.240
0.230
F.wt R1X D.wt
3.021
F.wt R2X D.wt
3.200
1% LSD 5%
0.240
0.020
0.350
0.070
0.250
0.100
0.230
0.030
0.170
0.016
0.250
0.050
0.180
0.070
0.160
0.020
0.291
2.610
0.210
0.350 3.670
0.314
3.470 0.242
3.352 0.390
0.340 3.680
0.305
0.420
1
Fresh and dry weights are expressed as g seedling .Only one treatment of SDW and of X was used as a control for foliar, seed and soil application. D.wt, dry weight; F.wt, fresh weight. Results are means of three experiments.
either R. aquatilis strains relative to that inoculated with X. c. pv. vesicatoria only (Table 2). Inoculation of strain 17 (R1) or strain 55 (R2) into tomato leaves (foliar application) reduced fresh and dry weight of the seedlings relative to treatment SDW. In contrast to this result, application of R1 and R2 through seeds and roots increased both fresh and dry weight. In case of soil application, increased fresh and dry weight was obtained only with treatment R2. Significant reduction in fresh and dry weight relative to control seedlings (SDW treatment) was obtained with treatment R1. However, it was observed that R. aquatilis strain 55 is able to enhance the growth of tomato seedlings more than strain 17 (Table 2).
ments. Highly significant increase in protein concentration of seedlings inoculated with X. c. pv. vesicatoria only, treatment X, relative to all other treatments was observed (Table 3). In contrast, highly significant reduction in protein concentration was detected in seedlings treated with either R. aquatilis strains, treatments R1 and R2, relative to control seedlings inoculated with SDW. The obtained results showed that protein concentration of seedlings treated or pretreated with R. aquatilis strain 17, treatments R1 and R1X, is always lower than that of seedlings treated or pretreated with strain 55, treatments R2 and R2X (Table 3).
Protein concentration of tomato seedlings
Sodium dodecyl sulfate polyacrylamide gel electrophoresis
Differences in protein concentrations of tomato seedlings were obtained with the different treat-
Analysis of SDS-PAGE showed that 12 protein bands with molecular weights ranging from 64.97 to
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H.H. El-Hendawy et al.
Table 3. Protein concentration of tomato seedlings inoculated and non-inoculated with R. aquatilis strains Treatment
SDW R1 R2 X R1X R2X 1% LSD 5%
Protein concentration Foliar
Seed
Soil
Root
6.00 4.00 5.00 8.00 4.00 5.00 0.25
6.00 3.00 5.00 8.00 5.00 5.50 0.99
6.00 3.50 4.50 8.00 5.00 6.00 0.50
5.00 3.00 4.00 6.00 3.50 4.00 0.75
0.18
0.71
0.36
0.53
Protein concentration was determined as mg g seedlings.
1
dry weight of
17.47 kDa are contained in tomato seedlings inoculated with SDW (Fig. 1a). Also 12 bands were observed by inoculation of SDW into seedlings with excised root tips (treatment SDW). Molecular weights of these bands ranged from 90.95 to 15.01 kDa (Fig. 1b). Different protein profile was also obtained when inoculating the pathogen into seedlings originated from nonbacterized seeds and nonbacaterized soil (treatment X) or seedlings whose roots were excised then transplanted (treatment X). Fifteen bands were detected in treatment X, their molecular weights ranged from 102.75 to 15.01 kDa (Fig. 1a) whereas in treatment XR, 8
Figure 1. (a) SDS-PAGE profile of tomato seedlings treated or pretreated with R. aquatilis through soil and seeds. From left lane 1, molecular weight marker. In soil treatment seedlings were treated with: lane 2, sterile distilled water; lane 3, R. aquatilis 55; lane 4, R. aquatilis 17; ;lane 5, R. aquatilis 55 and Xanthomonas; lane 6, R. aquatilis 17 and Xanthomonas. In seed treatment tomato seedlings were treated with; lane 7, R. aquatilis 55 and Xanthomonas; lane 8, R. aquatilis 17 and Xanthomonas; lane 10, R. aquatilis 55; lane 11, R. aquatilis 17. Lane 9 contains extract of seedlings originated from nonbacterized seed and nonbacterized soil and treated with Xanthomonas only. (b) SDS-PAGE profile of tomato seedlings treated or pretreated with R. aquatilis through leaves and roots. From left lane 1, molecular weight marker. In foliar treatment seedlings were inoculated with: lane 2, sterile distilled water; lane 3, R. aquatilis 55; lane 4, R. aquatilis 17; lane 5, Xanthomonas; lane 6, R. aquatilis 55 and Xanthomonas; lane 7, R. aquatilis 17 and Xanthomonas. In root treatment seedlings were treated with : lane 8, R. aquatilis 55; lane 9, R. aquatilis 17; lane 10, Xanthomonas; lane 11, R. aquatilis 55 and Xanthomonas; lane 12, R. aquatilis 17 and Xanthomonas; lane 13, seedlings with excised roots and treated with sterile distilled water.
protein bands with molecular weights ranging from 90.95 to 15.01 kDa (Fig. 1b). Although identical protein profiles were obtained from tomato seedlings treated with either R. aquatilis strains through roots (treatments R1 and R2), different protein profiles were obtained when inoculating the pathogen into these seedlings (treatments R1X and R2X). Six protein bands with molecular weights ranging from 64.97 to 15.01 were observed in each of R1 and R2. In addition, one more band with molecular weights of 48.42 kDa was observed in either treatments R1X and R2X and two bands with molecular weights of 90.95 and 15.85 kDa in R1X and R2X, respectively.
ARTICLE IN PRESS Biological control of bacterial spot of tomato In the soil and seed application methods, reduced number of protein bands were observed when treating tomato seedlings with R. aquatilis strains only or pretreating before inoculating the pathogen through leaves relative to those obtained from seedlings inoculated with the pathogen only and that inoculated with SDW (Fig. 1a).
Discussion Each of R. aquatilis strains proved to be able to protect tomato seedlings from infection by X. c. pv. vesicatoria. This was revealed by the reduced percentage of infection of seedlings pretreated with either R. aquatilis strains relative to control seedlings inoculated with X. c. pv. vesicatoria. The possible role of R. aquatilis in protecting the Chardonnay vine from galling by five different virulent Agrobacterium vitis species was reported by Bell et al. (1995). However, reduction of infection by plant pathogenic bacteria as a result of pretreatment with antagonistic bacteria has been observed in some diseases. For example, occurrence of bacterial wilt of tomato caused by P. solanacearum was remarkably suppressed when the culture suspension of the antagonistic bacterium Bacillus subtilis NB22 was poured into heavily infested soil. The percentage of dead plants was reduced to one-third that observed in the control (Phae et al., 1992). In another study, pretreatment of melon cotyledons with viable cells of P. fluorescens has been shown to reduce the infection with Erwinia carotovora subspecies carotovora up to 50% (El-Hendawy et al., 1998). Inoculation of tomato leaves with X. c. pv. vesicatoria resulted in significant reduction in fresh and dry weight of the seedlings. Reduction in fresh and dry weight of melon plants as a result of inoculation with the bacterial pathogen has been reported (El-Hendawy et al., 1998). In this study, it is obvious that development of yellow-brown necrotic areas or chlorosis in tomato leaves is attributed to interference of the pathogen with photosynthesis. Nearly all organic nutrients of plants are produced in the leaf cells, following photosynthesis, and are translocated downward and distributed to all the living plant cells (Agrios, 1997). In advanced stages of some diseases, the rate of photosynthesis is no more than one-fourth the normal rate (Agrios, 1997), reduction of photosynthesis will result in reduction in growth and consequently in fresh and dry weight of the plant.
349 Treatment of tomato seedlings with R. aquatilis strain 55 (R2) to seeds or soil or roots, significantly enhanced fresh and dry weight in comparison with untreated control (SDW). Whereas, strain 17 increased fresh and dry weight when applied to seeds or roots only. However, seedling treatment or pretreatment of the pathogen with either R. aquatilis strains, by the different application methods, enhanced fresh and dry weight relative to that of seedlings inoculated with the pathogen only. This result agrees with that reported in the literature, where pretreatment of plants with antagonistic bacteria or plant growth promoting rhizobacteria (PGPR) enhanced plant growth and consequently its weight. For example, significant increases in growth and yield of potato plants were achieved by treating seed pieces with suspensions of two Pseudomonas spp. prior to planting (Burr et al., 1978). Also Sivamani and Gwanamanickam (1988) obtained better root growth and enhanced plant weight by pretreatment of the pathogen with P. fluorescens. Furthermore, Wei et al. (1996) tested the capacity of three PGPR to induce systemic resistance against cucumber diseases and found that two of the three tested strains significantly increased runner length, and all three strains significantly increased leaf number per plant compared with the disease control. Also, pretreatment of melon cotyledons with P. fluorescens was able to reduce the deleterious effect of E. carotovora ssp. carotovora on fresh and dry weight of melon seedlings (El-Hendawy et al., 1998). Another report comes from Raupach and Kloepper (1998), where they tested the ability of two bacterial strains, Burkholderia gladioli and Bacillus pumilus, to promote cucumber growth and suppress anthracnose and angular leaf spot and found that either strains significantly allowed protection from diseases and increased the main runner length relative to control. Application of R. auatilis strains onto tomato seedlings by different methods, foliar, seed, soil and root resulted in differences in the level of disease reduction as well as in fresh and dry weight of the seedlings. Foliar application of either R. aquatilis strains was the most effective in disease reduction. This result is in agreement with that reported by Hsieh and Buddenhagen (1974) where they found that foliar application of E. herbicola reduced the infection of rice by X. oryzae more than root application. Protection of seedlings from disease as a result of inoculation with the antagonist and the pathogen through leaves could be attributed to the direct inhibition of the pathogen by the antagonist as they come in contact in the same leaf. Seed inoculation with R. aquatilis strains
ARTICLE IN PRESS 350 enhanced fresh and dry weight more than the other methods. In contrast to this result, no significant difference in the level of induced resistance between seed inoculation and cotyledon injection with two PGPR strains (Liu et al., 1995). Coating of cucumber seeds or spraying with Burkholderia gladioli and Bacillus pumilus showed no significant difference in cucumber growth and disease suppression between each strain in either method of application (Raupach and Kloepper, 1998). However, seed inoculation might provide earlier protection than could be provided with foliar, soil or root treatment. Results from SDS-PAGE showed that tomato seedlings inoculated with the pathogen only contained the largest number of protein bands relative to all other treatments. Fragmentation of host protein might occur by the action of pathogen proteases and this could be responsible for the detection of more bands. In this study, tomato seedlings inoculated with the pathogen only contained the highest protein concentration relative to all other treatments. Based on these results, it can be concluded that more proteins could be accumulated in these seedlings. However, it has been reported that structurally diverse group of plant proteins that are toxic to invading pathogens are widely distributed in plants in trace amounts but are produced in much greater concentration following pathogen attack or stress, these proteins are called pathogenesis-related (PR) proteins (Agrios, 1997; Rep et al., 2002). These proteins may be effective in inhibiting pathogen growth, multiplication and/or spread and be responsible for the state of systemic acquired resistance (Van Loon and Van Strien, 1999). Reduced protein concentration as well as reduced number of protein bands are detected in seedlings treated with R. aquatilis strains relative to control seedlings inoculated with SDW. This could be attributed to reduction in fresh and dry weight of seedlings. Also, reduction in protein concentration as well as the number protein bands was obtained from seedlings pretreated with either R. aquatilis strains relative to that inoculated with the pathogen only. However, Hoffland et al. (1996) demonstrated that the plant growth promoting rhizobacterium (PGPR) P. fluorescens strains WCS417 has been shown to induce systemic resistance against Fusarium oxysporum in several plant species without inducing synthesis of PR proteins. They also found that the absence of PR after induction by P. fluorescens does not lower protection. Other studies have proposed several mechanisms for disease resistance other than accumulation of PR proteins. These mechanisms
H.H. El-Hendawy et al. could be: altered ion fluxes across the plant cell membrane, generation of active oxygen species, changes in the phosphorylation state of regulatory proteins and transcriptional activation of plant defense systems culminate in cell death at the site of infection, local accumulation of phytoalexins and cell wall rigidification as a result of callose, lignin and suberin deposition (Hammond-Kosack and Jones, 1996; Yang et al., 1997). It is likely that excision of root tips induced resistance of tomato seedlings. This might explain the reduction in infection when inoculating the pathogen into tomato seedlings the roots of which were excised before transplanting compared to seedlings originated from nonbacterized soil and seeds and inoculated with the pathogen only. The former seedlings showed reduced protein concentration as well as reduced number of protein bands than the latter. However, defense mechanisms other than protein accumulation could be evoked in this case.
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