PDF (81 K)

Color profile: Generic CMYK printer profile Composite Default screen

1069

Enhancement of population densities of fluorescent pseudomonads in the rhizosphere of tomato plants by addition of acibenzolar-S-methyl W.D. Fakhouri and H. Buchenauer

Abstract: Fluorescent pseudomonad isolates G309 and CW2, in combination with the resistance inducer acibenzolar-Smethyl (ASM), improved control of fungal and bacterial diseases on tomato plants. The interactions of the bacteria in the presence of ASM showed that in vitro growth of Pseudomonas fluorescens G309 and Pseudomonas sp. strain CW2 was not affected in King’s B broth supplemented with 10 and 20 µM ASM. Also, the bacterial cells were not able to utilize ASM as a nutrient source. In vitro production of the two antimicrobial secondary metabolites phenazine-1carboxylic acid and 2-OH-phenazine by the isolate CW2 was not affected within 3 days from incubation. In contrary, addition of ASM at a concentration of 20 µM to King’s B liquid medium significantly increased production of salicylic acid by isolate G309. When roots of tomato plants were treated with G309 or CW2 cell suspensions containing 20 µM ASM, the number of bacterial cells recovered from the rhizosphere was significantly higher in the combined treatments than in the single applications 5, 10, and 15 days after inoculation. However, ASM at a higher concentration (50 µM) did not appreciably enhance the population sizes of either bacterial isolate in the rhizosphere. Enhanced bacterial cell densities in the rhizosphere of tomato plants were also determined following simultaneous treatments of tomato roots with 10 and 20 µM ASM in combination with the transformed isolate G309-384 (mini-Tn5gfp), which encodes the green fluorescent protein. Key words: acibenzolar-S-methyl, fluorescent pseudomonads, green fluorescent protein, tomato. Résumé : Les isolats de pseudomonades G309 et CW2 combinés à l’inducteur de résistance acibenzolar-S-methyl (ASM) ont amélioré le contrôle de maladies de la tomate causées par des champignons ou des bactéries. Les interactions de la bactérie en présence d’ASM ont indiqué qu’un bouillon de culture King’s B enrichi avec 10 et 20 µM d’ASM n’a pas affecté la croissance in vitro de la souche G309 de Pseudomonas fluorescens et de la souche CW2 de Pseudomonas sp. De plus, les bactéries ont été incapables de consommer l’ASM comme nutriment. La production in vitro des deux métabolites secondaires antimicrobiens, l’acide phenazine-1-carboxylique et la 2-OH-phenazine, par l’isolat CW2 n’a pas été influencé au cours des 3 jours d’incubation. En revanche, l’ajout d’ASM à un milieu liquide King’s B à une concentration de 20 µM a augmenté significativement la production d’acide salicylique par l’isolat G309. Lorsque des racines de plants de tomate ont été traitées avec des suspensions de cellules G309 ou CW2 renfermant 20 µM d’ASM, le nombre de bactéries isolées de nouveau à partir de la rhizosphère à la suite d’un traitement combiné fut significativement supérieur à celui obtenu à la suite d’applications simples, 5, 10 ou 15 jours après l’inoculation. Cependant, une concentration d’ASM plus élevée (50 µM) n’a pas substantiellement accru les tailles des populations des deux isolats bactériens dans la rhizosphère. Des densités accrues de bactéries dans la rhizosphère de plants de tomate ont également été décelées à la suite de traitements simultanés de racines de tomates avec 10 et 20 µM d’ASM combinés avec l’isolat transformé G309-384 (mini-Tn5gfp), qui code la protéine verte fluorescente. Mots clés : acibenzolar-S-methyl, pseudomonades fluorescentes, protéine verte fluorescente, tomate. [Traduit par la Rédaction]

Fakhouri and Buchenauer

Introduction Fluorescent pseudomonads have the potential to play an important role in suppressing soilborne plant pathogens, proReceived 5 June 2002. Revision received 31 October 2002. Accepted 5 November 2002. Published on the NRC Research Press Web site at http://cjm.nrc.ca on 18 January 2003. W.D. Fakhouri1 and H. Buchenauer. Institute of Phytomedicine, University of Hohenheim, Otto-Sander Str. 5, 70593-Stuttgart, Germany. 1

Corresponding author (e-mail: [email protected]).

Can. J. Microbiol. 48: 1069–1075 (2002)

I:\cjm\cjm48\4812\W02-105.vp Thursday, January 16, 2003 11:38:53 AM

1075

moting plant growth, and induce systemic acquired resistance (Knoester et al. 1999; Weller 1988; Wilson and Lindow 1993). In recent years, there has been an increased interest in improving the biological control of plant diseases to overcome the variability in efficacy and augment the effectiveness of biocontrol agents. One of the important issues to improve the biological control of plant diseases is to prolong the persistence and impact of introduced bacteria. Colbert et al. (1993) stated that amendments of selective feeding of a carbon source (sodium salicylate) to field soils planted with tomato and treated with Pseudomonas putida PpG7 increased the population densities of the bacteria. This bacterial strain (PpG7) was able to utilize the salicylate as a

DOI: 10.1139/W02-105

© 2002 NRC Canada


1070

nutritional carbon source, and the bacterial densities were increased in low-inoculum soils up to 26- and 29-fold in rhizosphere and non-rhizosphere soils, respectively, while the fungal population densities did not increase. Furthermore, addition of L-asparagine and L-proline to an antagonistic Pseudomonas syringae isolate greatly enhanced controlling blue mould disease on ripe apples. It was noticed that both amino acids encouraged the population of the antagonist P. syringae on apple fruits by more than 10-fold (Janisiewicz et al. 1992). Enhancement of resistance activities by a combination of antagonistic microorganisms with resistance inducers could be an additional issue to improve the controlling of plant diseases. Improvement of controlling damping-off and powder mildew of cucumber have been reported elsewhere by combining the bacterial cells of fluorescent pseudomonads with the resistance inducer DL-3-amino-n-butyric acid (Vogt and Buchenauer 1997). Also, other researches mentioned that the resistance of tomato plants against Fusarium infection was triggered by bacterization of tomato roots with Bacillus pumilus and addition of chitosan to the bacterial culture. Combination of B. pumilus with chitosan induced specific alterations in the physiology of the host tissues (Benhamou et al. 1998). Acibenzolar-S-methyl (ASM) is a potent resistance inducer used against plant pathogens and is known under the commercial name Bion®. It has been shown that ASM induces disease resistance and gene expression of a wide spectrum of pathogenesis-related proteins (Friedrich et al. 1996; Stadnik and Buchenauer 2000). Application of ASM at concentrations ranging from 10 to 50 µM in combination with fluorescent pseudomonad isolates G309 and CW2 induced synergistic disease resistance against bacterial and fungal pathogens of tomato plants (Fakhouri et al. 2000). However, the exact mechanisms by which combinations of biocontrol antagonists with nutritional analog or resistance inducers provide enhanced plant protection are not fully understood in previous literatures (Singh et al. 2000; Vogt and Buchenauer 1997). Therefore, the first aim of this study was to explore the influence of ASM on the population densities of the wild types of fluorescent pseudomonads G309 and CW2 in vitro and in the rhizosphere of tomato plants (Lycopersicon esculentum Mill. cv. TipTop). The second aim was to label the bacterial bioagents G309 and CW2 with the marker gene gfp to be able to monitor the colonization behavior of the applied gfp-tagged bacteria alone or in combination with ASM.

Materials and methods Bacterial strains and resistance inducer Pseudomonas fluorescens G309 (isolated from barley (Hordeum vulgare) leaves ) and Pseudomonas sp. CW2 (isolated from radish (Raphanus sativus) rhizosphere) were cultured on King’s B liquid medium (King et al. 1954) for application on tomato plants. The biochemical and antagonistic characteristics of both isolates are described in a previous report (Fakhouri et al. 2001a). The resistance inducer ASM was supplied by K.L. Nau (Novartis Ltd., Frankfurt, Germany).

Can. J. Microbiol. Vol. 48, 2002

Effect of ASM on the population sizes of both isolates and analysis of ASM utilization, salicylic acid (SA), and antibiotics The influence of the ASM in vitro on the population size of the bacterial cells of G309 and CW2 was studied in liquid King’s B (KB) medium at 8, 16, and 24 h after incubation. The dilution and plating methods on KB agar medium were used for counting the bacterial colonies. The quantitative assays of ASM utilization, SA, and antimicrobial production by the two bacterial isolates G309 and CW2 were carried out according to the method described by Fakhouri et al. (2001b). One hundred millilitres of liquid KB broth containing 0, 10, 20, and 50 µM of ASM were inoculated with 200 µL of 24-h-old bacterial suspension. The inoculated liquid medium was shaken at 100 rpm at 28°C for 24 h and then acidified with HCl (1 M) to pH 2.0. Acetone (80%) in a ratio of 1:1 (v/v) was then added, and the mixtures were centrifuged at 2600 × g for 20 min. The supernatants were poured into round bottom flasks, and the acetone was removed with a rotary evaporator under vacuum. The aqueous phases were transferred to extraction flasks and extracted again with ethylacetate. The organic phases were separated from the aqueous phase and dried using a rotary evaporator. The residues were resuspended in 1 mL of methanol and centrifuged at 10 600 × g for 2 min to remove the nondissolved substances. The analyses of ASM, SA, and antibiotics were carried out by using HPLC (L-6200, Merck–Hitachi, Darmstadt, Germany) coupled with a photodiode array detector (PAD, Waters 99, Water Associates, Milford, Mass., U.S.A.) and autosampler (Merck-Hitachi 4000A). Separation of substances was done with a RP18 reversed phase column (4 mm i.d. × 250 mm length; 5-µm particle size, GROM-SIL, Herrnberg, Germany) at 35°C with a flow rate of 0.5 mL min–1 using gradient elute phosphate buffer–acetonitrile, maximum pump pressure of 250 bar (1 bar = 100 kPa) and oven temperature of 25°C. KB broths containing only 10, 20, and 50 µM of ASM have been included to determine the recovery rate of ASM during the extraction process as controls. Three different antibiotics (2,4-diacetylphloroglucinol, pyoluteorin, and 2-OH-phenazine (2-OH-Phz)), pure SA, and pure ASM were included as standards. Plant cultivation conditions and population dynamics Tomato seedlings were grown in pots containing Humosoil® (Floragard, Oldenburg, Germany)–sand mix (3:1, v/v) and kept in the greenhouse in randomized blocks under controlled conditions at 25°C in the light for 14 h and at 18°C in the dark for 10 h per day. During the whole experimental period, all tomato plants were fertilized with 0.2% WuxalSuper® (Aglukon, Dusseldorf, Germany) liquid fertilizer (N– P2O5–K2O, 8:8:6 + microelements) twice a week. Population dynamics of the wild-type strains G309 and CW2 were monitored in the rhizosphere of tomato plants. Two-week-old tomato seedlings were treated with either wild-type strain G309 or CW2 (2.1 × 108 CFU/mL) and with 0, 10, 20, and 50 µM of ASM by soil-drenching (50 mL/pot), either alone or in combination. Nontreated tomato plants or a plant treated with ASM served as controls. Five, ten, and fifteen days after inoculation of the tomato roots with either wild-type strain, the whole mass of roots was cleaned gently © 2002 NRC Canada




from soil particles under tap water for short time to prevent damaging and cutting of hairy roots. The tomato roots, including the soil particles sticking on the outer surface, were cut into small pieces and immersed in KB broth. The KB broth containing the root pieces was placed on the shaker for 2 h at 28°C at 200 rpm. The aqueous phase of the broths was filtered through muslin two times to remove the root pieces and the nondissolved particles. Dilution series from the filtrate were prepared up to 1:10–6. From each dilution (1:10–4–1:10–6), 200 µL were spread on each plate of KB and succinic acid–asparagine media (DSMZ; German Collection of Microorganisms and Cell Cultures Catalogue, 1998). Succinic acid–asparagine medium was used as the selective medium for growth of fluorescent pseudomonads. Two plates of each dilution were used. All plates were incubated at 28°C for 1–3 days. After incubation, all the colonies showing blue-green fluorescence under UV light (366 nm) were counted, and the population density of each isolate was calculated. Construction of gfp-tagged vector strain and phenotypes of fluorescent pseudomonad isolates G309 and CW2 Methods and molecular tools for tagging the bacterial strains G309 and CW2 have been conducted according to Sambrook et al. (1989). The plasmid pUTmini-Tn5gfp was transformed into Escherichia coli S17-1λpir (Table 1) by using the CaCl2 (0.1 M) transformation method according to Cosloy and Oishi (1973). The plasmid pUTmini-Tn5gfp was cloned by cultivation of the transformed E. coli S17–1λpir in Luria–Burtani (LB) liquid medium amended with ampicillin (50 µg/mL) and tetracycline (Tc) (12.5 µg/mL) as selective markers. Conjugation of fluorescent pseudomonads Biparental mating was conducted by using the vector strain E. coli S17-1λpir carrying the plasmid pUTminiTn5gfp with the gene gfp–mut3b (Suarez et al. 1997) within the Tn5 cassette. The conjugation of fluorescent pseudomonad isolate G309 and CW2 was done according to the standard protocol of Sambrook et al. (1989). Screening for the transformed bacterial isolates expressing green fluorescent protein (GFP) Approximately 500 transformed colonies of each isolate G309 and CW2 that grew on M9-medium after conjugation were isolated, and colonies were screened for production of high amounts of GFP by using spectrofluorometry (Hitachi F2000, Hitachi Ltd., Tokyo, Japan). The stability of the Tn5 insertions was tested by growing the transformed isolate G309-384 in KB broth for approximately 30 generations, followed by plating on LB medium alone or LB amended with Tc. The stability of gfp gene expression was also examined by fluorescence microscopy. Testing the transformed isolate G309-384 in vitro In in vitro studies, the biochemical and physiological characteristics of the transformed isolate G309-384 were compared with that of the wild-type isolate G309. Production of the extracellular enzymes, namely proteinase, gelatinase, chitinase, and arginine dihydrolase, was qualitatively tested

1071

on agar plates containing the substrates of each enzyme. In addition, production of other secondary metabolites such as salicylic acid, cyanide, and antimicrobial substances was tested as described in a previous reports (Fakhouri and Buchenauer 1998; Fakhouri et al. 2001b). The influence of the ASM (10 and 20 µM) in vitro on the population size of the bacterial cells of G309-384 was conducted as described above. Population dynamics of G309-384 in the rhizosphere of tomato Two-week-old tomato seedlings were inoculated with the transformed bacterial isolate G309-384 either alone or in combination with 10 and 20 µM ASM. The bacterial cells were applied to the tomato roots by soil-drenching (2.1 × 108 CFU/mL, 50 mL/pot). At 2, 3, and 4 days after treatment, seedlings were carefully removed from the pots, and the roots were adequately washed under tap water. The roots of the seedlings were cut into small pieces and placed in KB medium amended with Tc (20 µg L–1) on the shaker for 2 h. The KB broth containing the root pieces was filtered through layers of muslin, and four samples from each flask were taken to measure the fluorescent intensity of the GFP produced by the transformed isolate G309-384. The fluorescence intensity was measured using a spectrofluorometer (Hitachi F2000) with a filter set including a band pass excitation light source at 489 nm via a fluorescence detector set with a long pass emission light at 520 nm. Counting the number of transformed bacterial cells having the Tc marker gene was performed by cultivation of the diluted bacterial suspensions on KB agar medium amended with Tc (20 µg mL–1). The same experiment was repeated four times. Plants treated with the wild-type strain G309 or without any treatment served as controls. Data analysis All experiments were repeated at least two times. The results were analysed using Tukey’s test after a significant F test (P ≤ 0.05) of the analysis of variance (ANOVA). Each value presented in the results is the mean of five replicates of an individual treatment. The variation among the treatments was tested for homogenicity using Bartlett’s test.

Results Effect of ASM on the population densities of bacterial strains, ASM utilization, production of SA, and antimicrobial secondary metabolites Addition of ASM at concentrations of 10, 20, and 50 µM to KB liquid medium did not show any significant effect on bacterial populations of G309 and CW2 after 8, 16, and 24 h of incubation (data not shown). G309 and CW2 were not able to utilize ASM as a nutrient source regardless of the applied concentrations after 24 h of incubation. The same results have been obtained after incubation of the bacterial cells either in poor or in rich media containing different concentrations of ASM for 3 days (data not shown). ASM at concentrations of 10 and 20 µM did not significantly reduce the production of the antimicrobial secondary metabolites phenazine-1-carboxylic acid and 2-OHPhzof the isolate CW2 in liquid KB medium (data not © 2002 NRC Canada



1072

Can. J. Microbiol. Vol. 48, 2002 Table 1. Bacterial strains and plasmid used in this study.

Bacteria Escherichia coli S17-1λpir Pseudomonas fluorescens G309 Pseudomonas sp. CW2 Plasmid pUTmini-Tn5gfp

Characteristics of phenotype and genotype

Reference

Smr, pro, thi, hsdR–M+, RP4-2-Tc:Mu-Km:Tn7, λpir SA+, Prot.+, Gel+, Pch+, Chi–, Tc– 2-OH-Phz+, PCA+, HCN+, SA+, Prot.+, Pch+, Chi–, Tc–

Matthysse et al. 1996 Fakhouri et al. 2001a Fakhouri et al. 2001a

AmpR; TcR and gfp reporter on mini-Tn5; mob+

Matthysse et al. 1996

NOTE: Sm, streptomycin; SA, salicylic acid; Prot., proteinase; Gel, gelatinase; Pch, pyochelin; Chi, chitinase; Tc, tetracycline; 2OH-Phz, 2-OH-phenazine; PCA, phenazine-1-carboxylic acid; Amp, ampicillin.

shown). Isolate G309 produced no antimicrobial substances. On the contrary, a concentration of 20 µM ASM in KB liquid medium significantly increased production of SA by the isolate G309 up to 80.5%, as well as by the isolate CW2 up to 17.3% (Fig. 1). Effect of ASM on the population densities of G309 and CW2 strains in the rhizosphere Roots of tomato seedlings inoculated with bacterial suspensions of G309 and CW2 isolates either alone or in combination with ASM were evaluated for population sizes after 5, 10, and 15 days inoculation (Table 2). Recovery of the bacterial cells from the rhizosphere of tomato plants revealed positive effects of ASM on the population sizes of both isolates. The addition of 20 µM of ASM to liquid medium containing G309 and CW2 significantly increased the size of the bacterial cells by 6.0- and 10-fold in the rhizosphere of tomato roots, respectively, 5 days after application compared with application of the bacterial isolates alone. Ten days after application, population densities of isolate CW2 in combination with 20 µM ASM were increased by 4.8-fold compared with treatment of roots of tomato plants with CW2 alone. Similarly, the populations of isolates G309 and CW2 were both significantly higher in the presence of 20 µM ASM 15 days after application compared with the control (Table 2). Transformation of the fluorescent pseudomonad isolates G309 and CW2 with the marker gene gfp The conjugation method revealed that the plasmid carrying the gfp gene could be successfully transferred into the cells of both isolates G309 and CW2. Three gfp-tagged strains, G309-384, G309-378, and CW2-213, showed notable accumulation of GFP, and the fluorescence intensities were 180, 80, and 35 units, respectively. The transformed isolate G309-384, producing the highest amount of GFP, was used for further studies. Biochemical and physiological characteristics of the transformed isolate G309-384 The gfp-tagged isolate G309-384 proved to be very stable. Plating on LB medium alone or on medium amended with Tc showed no loss in producing the GFP and no loss in stability of Tc resistance. This isolate was used in our experiments for one year without any alteration in production of GFP or viability. Biochemical and physiological characteristics of the transformed isolate G309-384 were compared with the wild-type isolate G309. The phenotypic characteris-

Fig. 1. Total amount of salicylic acid (SA) in liquid King’s B (KB) medium produced by fluorescent pseudomonad isolates G309 and CW2 alone and by addition of acibenzolar-S-methyl (ASM; 10 and 20 µM) to KB medium.

tics of the transformed strain were not changed compared with the wild-type isolate G309. Population dynamics of the transformed isolate G309-384 Addition of ASM at concentrations of 10 and 20 µM to KB liquid media did not significantly affect the bacterial densities per millilitre compared with the control. The population of G309-384 was 2.80 × 109 and 2.50 × 109 CFU/mL in the presence of 10 and 20 µM ASM, respectively, compared with the population size (1.2 × 109 CFU/mL) in the absence of ASM 24 h after cultivation. In the rhizosphere of tomato plants treated with the gfptagged G309-384 isolate in combination with 10 and 20 µM ASM, the population densities significantly increased compared with G309-384 alone 2 and 3 days after inoculation (Table 3). By day 4, the number of bacterial cells in the treatment (G309–gfp + ASM 10) was significantly higher compared with the other treatments (Table 3). The fluorescence intensity of the recovered, transformed isolate G309-384 from root was correlated with the bacterial cell densities in the cultivation medium, as determined by dilution and plating analysis. A standard curve of bacterial cell densities of the transformed isolate G309-384 and the fluorescent intensity was calculated and the following equation was produced: Y = 8.4483 + X × 0.01559 where Y = Log10 CFU/mL and X = fluorescence intensity. © 2002 NRC Canada




1073

Table 2. Effect of acibenzolar-S-methyl (ASM; 10, 20, and 50 µM) on the population size of fluorescent pseudomonad isolates G309 and CW2 in the rhizosphere of tomato roots 5, 10, and 15 days after application at bacterial density of 2.1 × 108 CFU/mL. In vivo (CFU/g root) 5 days

10 days

15 days

Treatments

G309

CW2

G309

CW2

G309

CW2

Control ASM 10 µM ASM 20 µM ASM 50 µM

3.5×106b 6.8×106b 1.2×107a 3.2×106b

2.5×106b 7.6×106b 2.2×107a 4.5×106b

1.43×105b 2.5×105b 4.3×105b 1.93×105b

1.33×105b 2.03×105b 6.39×105a 1.7×105b

1.1×104b 5.5×104ab 7.8×104a 3.1×104b

2.6×104b 5.1×104ab 8.6×104a 3.3×104b

Note: Values represent an average of five replications of each treatment. Means accompanied by a different letter are significantly different (P ≤ 0.05).

Table 3. Effect of acibenzolar-S-methyl (ASM) on the population densities of transformed Pseudomonas fluorescens G309-384 recovered from the rhizosphere of tomato plants 2, 3, and 4 days after application of bacterial cell suspensions applied at a concentration of 2.1 × 108 CFU/mL containing 0, 10, and 20 µM ASM. Treatment

2 days (CFU/g FRW)

3 days (CFU/g FRW)

4 days (CFU/g FRW)

Control G309–gfp G309–gfp + ASM 10 G309–gfp + ASM 20

1.15±0.005d 3.2×107±0.02c 8.7×107±0.013b 4.3×108±0.007a

1.2±0.003d 2.1×107±0.004c 6.3×107±0.02b 1.2×108±0.015a

1.02±0.004c 6.6×106±0.008b 8.2×106±0.005a 6.1×106±0.007b

Note: Values represent an average of five replications of each treatment. Means accompanied by a different letter are significantly different (P ≤ 0.05). FRW, fresh root weight (g).

Discussion Results of the present study demonstrate the effect of ASM on the population densities of bacterial isolates G309 and CW2 in vitro and in the rhizoplane of tomato roots. ASM did not significantly affect the in vitro growth of bacterial cells of G309 and CW2 after 24 h of incubation in KB medium. Moreover, HPLC analysis showed that the bacterial cells of G309 and CW2 were not able to utilize ASM as a nutrient source within 3 days of incubation. These results could explain why the ASM did not interfere with the bacterial growth of both isolates in vitro. Additionally, the presence of 10 and 20 µM ASM in KB medium also did not affect the production of the antimicrobial substances phenazine-1-carboxylic acid and 2-OH-Phz by isolate CW2. Interference of ASM with production of antimicrobial substances by CW2 in the rhizosphere of tomato plants was not determined. The influence of ASM in vitro on production of SA was also studied. Addition of ASM at concentrations of 10 and 20 µM to KB liquid medium during the cultivation of G309 and CW2 isolates increased production of SA in KB liquid medium 24 h after incubation. This result is very interesting because SA has been described to function in signal transduction for triggering defense responses and as an inducer of pathogenesis-related proteins (De Meyer et al. 1999). Also, SA is the precursor for the bacterial siderophore pyochelin, which mediates the capturing of Fe ions and suppressiveness activity against other microorganisms (Leong 1986). Root-drenching of ASM increased the population densities of the two isolates G309 and CW2 in the rhizosphere of tomato plants 5 and 10 but not 15 days after application. We assume that ASM might influence the root exudates qualita-

tively and quantitatively, and these play a very important role in determining the extent of root colonization by rhizobacteria. It has been shown that ASM led to physiological changes in different crops (Friedrich et al. 1996; Stadnik and Buchenauer 2000). Therefore, it could be expected that ASM would affect the constituents of root exudates of plants. At the higher concentration of ASM, the bacterial populations were not appreciably increased under in vivo conditions compared with the lower concentration 20 µM of ASM. In vitro results have shown that ASM at 50 µM has no negative effect on the population densities of bacteria, but the higher concentrations of ASM caused phytotoxic effects to tomato plants under greenhouse conditions. Such phytotoxic effects of ASM on crops have been mentioned before (Tosi et al. 1999), and this may influence the constituents of root exudates that play a determinant factor on the efficiency of bacterial colonization (Molina et al. 2000). Previous reports showed that amendments of amino acids or other nutritional carbon sources to cultures of fluorescent pseudomonads encouraged the population size of bacteria in field soils or on apple fruits (Colbert et al. 1993; El-Ghaouth et al. 2000; Janisiewicz et al. 1992). Furthermore, Benhamou et al. (1998) reported that resistance of tomato plants against Fusarium wilt was enhanced by a combined application of chitosan and the antagonistic bacterial culture compared with single treatments. This study found that ASM not only enhanced the population densities of the bacterial cells but also encouraged the resistance of tomato plants against fungal pathogens (Fakhouri et al. 2001a). The results reported by Colbert et al. (1993), Janisiewicz et al. (1992), and Benhamou et al. (1998), as well as the findings of our studies, indicate an important strategy to improve the sustainability and efficacy of biological control agents (BCA) in © 2002 NRC Canada



1074

plant disease control, especially against soilborne pathogens. BCA can be used to control plant pathogens, activate immunity, and also promote plant growth. Searching for substances that can be used to improve colonization and sustainability of bacterial cells, as well as enhancing induction of acquired resistance of plants, will encourage the application of BCA in biological control. Results of the fluorescent microscopic studies concerning the colonization patterns on the rhizoplane of tomato roots by the gfp-tagged isolate of G309-384 when applied either alone or in combination with ASM are consistent with our previous scanning and transmission electron microscopic studies using the wild-type strains G309 and CW2 (Fakhouri et al. 2001a) (data not shown). The transformed strain G309384 enabled us to determine only the green fluorescent bacterial cells in the rhizosphere of tomato roots and to easily monitor in situ the colonization behavior of isolate G309384. Furthermore, the gfp-tagged bacteria present an excellent candidate for studying both short- and long-term mobility and colonization patterns in the rhizoplane, as well as the endophytic colonization of the interior parts of the roots. These results are in agreement with the findings of other researchers (Normander et al. 1999; Tombolini et al. 1999), who mentioned that GFP is a suitable marker for studying the colonization patterns of pseudomonads because of the stability, minimum requirements of sample preparation, and avoidance of possible disturbance of the natural cell colonization. Thus, combined root treatment with ASM and fluorescent pseudomonads might result in greater advantages in integrated pest management of tomato.

Acknowledgements The authors thank A. Matthysse and his colleagues for supplying the pUTmini-Tn5gfp vector. We also thank Mrs. G. Moll for her excellent photographic work and Dr. P. Karlovsky for technical assistance. The financial support of the German Academic Exchange Service (DAAD, Bonn) for the first author is gratefully acknowledged.

References Benhamou, N., Kloepper, J.W., and Tuzun, S. 1998. Induction of resistance against Fusarium wilt of tomato by combination of chitosan with an endophytic bacterial strain: ultrastructure and cytochemistry of the host response. Planta, 204: 153–168. Colbert, S.F., Schroth, M.N., Weinhold, A.R., and Hendson, M. 1993. Enhancement of population densities of Pseudomonas putida ppg7 in agricultural ecosystems by selective feeding with the carbon source salicylate. Appl. Environ. Microbiol. 59: 2064–2070. Cosloy, S.D., and Oishi, M. 1973. Genetic transformation in Escherichia coli K12. Proc. Natl. Acad. Sci. U.S.A. 70: 84–87. De Meyer, G., Capieau, K., Audenaert, K., Buchala, A., Metraux, J.-P., and Höfte, M. 1999. Nanogram amounts of salicylic acid produced by the rhizobacterium Pseudomonas aeruginosa 7NSK2 activate the systemic acquired resistance pathway in bean. Mol. Plant–Microbe Interact. 12: 450–458. El-Ghaouth, A., Smilanick, J.L., Wisniewski, M., and Wilson, C.L. 2000. Improved control of apple and citrus fruit decay with a combination of Candida saitoana and 2-deoxy-D-glucose. Plant Dis. 84: 249–253.

Can. J. Microbiol. Vol. 48, 2002 Fakhouri, W., and Buchenauer, H. 1998. Establishment in tomato plants and mechanism of action of fluorescent pseudomonads antagonistic to the Fusarium wilt pathogen. In Molecular approaches in biological control. Edited by B.K. Duffy, U. Rosenberger, and G. Defago. International Organisation of Biological Control (IOBC/wprs) Bulletin, 21: 61–64. Fakhouri, W., Neemann, M., Atia, M., Walker, F., and Buchenauer, H. 2000. Combination of fluorescent pseudomonads with benzothiadiazole (BTH) effectively controls bacterial and fungal pathogens on tomato and cucumber. Phytopathology 90: 22A [Abstr.] Fakhouri, W., Kang, Z., and Buchenauer, H. 2001a. Scanning and transmission microscopic studies on the mode of action of fluorescent pseudomonads alone and in combination with acibenzolar-S-methyl effective against Fusarium oxysporum f.sp. lycopersici in tomato plants. J. Plant Dis. Prot. 108: 513–529. Fakhouri, W., Walker, F., Vogler, B., Armbruster, W., and Buchenauer, H. 2001b. Isolation and identification of Nmercapto-4-formylcarbostyril, an antibiotic produced by Pseudomonas fluorescens. Phytochemistry, 58: 1297–1303. Friedrich, L., Lawton, K., Ruess, W., Masner, P., Specker, N., Rella, M.G., Meier, B., Dincher, S., Staub, T., Uknes, S., Metraux, J.-P., Kessmann, H., and Ryals, J. 1996. A benzothiadiazole derivative induces systemic acquired resistance in tobacco. Plant J. 10: 61–70. Janisiewicz, W.J., Usall, J., and Bors, B. 1992. Nutritional enhancement of biocontrol of blue mold on apples. Phytopathology, 82: 1364–1370. King, E.O., Ward, M.K., and Raney, D.E. 1954. Two simple media for the demonstration of pyocyanin and fluorescin. J. Lab. Clin. Med. 44: 301–307. Knoester, M., Pieterse, C.M.J., Bol, J.F., and van Loon, L.C. 1999. Systemic resistance in arabidopsis induced by rhizobacteria requires ethylene-dependent signaling at the site of application. Mol. Plant–Microbe Interact. 12: 720–727. Leong, J. 1986. Siderophores: their biochemistry and possible role in the biocontrol of plant pathogens. Annu. Rev. Phytopathol. 24: 187–209. Matthysse, A.G., Stretton, S., Dandie, C., McClure, N.C., and Goodman, A.E. 1996. Construction of GFP vectors for use in Gram-negative bacteria other than Escherichia coli. FEMS Microbiol. Lett. 145: 87–94. Molina, L., Ramos, C., Duque, E., Ronchel, M.C., Garcia, M., Wyke, L., and Ramos, J.L. 2000. Survival of Pseudomonas putida KT2440 in soil and in the rhizosphere of plants under greenhouse and environmental conditions. Soil Biol. Biochem. 32: 315–321. Normander, B., Hendriksen, N.B., and Nybroe, O. 1999. Green fluorescent protein-marked Pseudomonas fluorescens: localization, viability and activity in the natural barley rhizosphere. Appl. Environ. Microbiol. 65: 4646–4651. Sambrook, J., Fritsch, E.J., and Maniatis, T. 1989. Molecular cloning: a laboratory manual. 2 ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Singh, U.P., Prithiviraj, B., Singh, K.P., and Sarma, B.K. 2000. Control of powdery mildew (Erysiphe pisi) of pea (Pisum sativum) by combined application of plant growth-promoting rhizobacteria and Neemazal™. Z. Pflanzenkr. Pflanzenschutz, 107: 59–66. Stadnik, M.J., and Buchenauer, H. 2000. Inhibition of phenylalanine ammonia-lyase suppresses the resistance induced by benzothiadiazole in wheat to Blumeria graminis f.sp. tritici. Physiol. Mol. Plant Pathol. 57: 25–34. © 2002 NRC Canada



Fakhouri and Buchenauer Suarez, A., Güttler, A., Strätz, M., Staendner, H.L., Timmis, K.N., and Guzman, C.A. 1997. Green fluorescent protein-based reporter systems for genetic analysis of bacteria including monocopy applications. Gene, 196: 69–74. Tombolini, R., van der Gaag, D.J., Gerhardson, B., and Jansson, J.K. 1999. Colonization pattern of the biocontrol strain Pseudomonas chlororaphis MA 342 on barley seeds visualized by using green fluorescent protein. Appl. Environ. Microbiol. 65: 3674–3680. Tosi, L., Luigetti, R., and Zazzeriui, A. 1999. Benzothiadiazole induces resistance to Plasmopara helianthi in sunflower plants. J. Phytopathol. 147: 365–370.

1075 Vogt, W., and Buchenauer, H. 1997. Enhancement of biological control by combination of antagonistic fluorescent Pseudomonas strains and resistance inducers against damping-off and powdery mildew in cucumber. Z. Pflanzenkr. Pflanzenschutz, 104: 272–280. Weller, D.M. 1988. Biological control of soil-borne plant pathogens in the rhizosphere with bacteria. Annu. Rev. Phytopathol. 26: 379–407. Wilson, M., and Lindow, S.E. 1993. Release of recombinant microorganisms. Annu. Rev. Microbiol. 47: 913–944.

© 2002 NRC Canada
