Impact of carbon starvation on stress resistance, survival in soil habitats and biocontrol ability of Pseudomonas putida strain 2C8

Soil Biology & Biochemistry 33 (2001) 1155±1162

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Impact of carbon starvation on stress resistance, survival in soil habitats and biocontrol ability of Pseudomonas putida strain 2C8 Yu-Huan Gu, Mark Mazzola* USDA-ARS, 1104 N. Western Avenue, Wenatchee, WA 98801, USA Received 21 March 2000; received in revised form 3 October 2000; accepted 14 November 2000

Abstract Exposure to carbon-limited growth conditions has been shown to induce resistance to a variety of environmental stresses in bacteria. Adaptation to nutrient-limited conditions by repeated culture on soil agar media was found to induce resistance to osmotic tension, oxidative stress and carbon limited culture conditions in Pseudomonas putida strain 2C8. Rifampicin resistant derivatives of strain 2C8 adapted to nutrient-limited conditions (strains 2C8-26A rif and 2C8-28C rif) did not exhibit enhanced survival in bulk soil relative to the parental strain. However, colonization of the apple rhizosphere by 2C8-26A rif and 2C8-28C rif was superior to that of the parental strain, 2C8 rif. Improved colonization of the apple rhizosphere was not the result of an elevated ability to utilize apple root exudates as a sole carbon source. Enhanced colonization of the rhizosphere by carbon starved derivatives of 2C8 rif was associated with improved biological control of Rhizoctonia root rot of apple caused by an introduced strain of Rhizoctonia solani AG 5. These ®ndings demonstrate that inoculum production conditions can signi®cantly alter the survival and ef®cacy of strain 2C8, and should be carefully considered in optimizing the use of this biocontrol rhizobacterium. Published by Elsevier Science Ltd. Keywords: Biocontrol; Rhizoctonia; Pseudomonas; Rhizosphere

1. Introduction Bacteria introduced into soil as agents for the biological control of plant pathogens must be capable of adaptation to a hostile environment in terms of both abiotic stress and competition imposed by the resident soil micro¯ora. Conditions employed in the culture of biological control agents are known to have a signi®cant impact on the production of antimicrobial metabolites (Duffy and DeÂfago, 1999), viability in storage, and biocontrol ability even in non-competitive environments such as a sterilized plant growth substrate (Slininger et al. 1998). The long-term storage and repeated cultivation of these agents may result in altered physiology (Persson et al., 1990) and reduced competitive ability in natural environments (Weller, 1988), such as soil, that are oligotrophic in nature. Such an event is likely to contribute to reduced ef®cacy as survival and competitive ability are generally believed to be linked to the effectiveness of introduced biological control agents. Adaptation of bacterial strains to soil conditions prior to introduction into natural soil environments could be instrumental in their ultimate survival and ef®cacy as biological * Corresponding author. Tel.: 11-509-664-2280; fax: 11-509-664-2287. E-mail address: [email protected] (M. Mazzola). 0038-0717/01/$ - see front matter Published by Elsevier Science Ltd. PII: S 0038-071 7(01)00019-0

control agents. Several studies have demonstrated that carbon starvation mediates bacterial resistance to a number of environmental stresses (van Overbeek et al., 1995; Givskov et al., 1994a,b; Jenkins et al., 1988, 1990). Carbonstarved cells exhibit enhanced survival relative to exponentially growing cells upon subsequent exposure to several stress factors including heat, oxidative stress, acid stress, osmotic stress, and exposure to ethanol (Jorgensen et al., 1994). Although carbon starvation was found to elicit the same stress resistance response in Pseudomonas ¯uorescens strain R2F, the carbon-starved strain did not exhibit altered survival relative to exponentially growing cells of strain R2f when introduced into soil (van Overbeek et al., 1995). Pseudomonas putida strain 2C8 was originally isolated from the rhizosphere of apple (Malus domestica cv. `Fuji' on M.26 rootstock) that was in its second year of growth. Prior to establishment, the orchard site had been planted with continuous wheat monoculture. Strain 2C8 was selected for its ability to suppress Rhizoctonia root rot of apple caused by Rhizoctonia solani AG 5 (Mazzola, 1997), and was subsequently shown to enhance growth of apple in orchard replant soils (Mazzola, 1998a). The wheat root system supports substantial populations of strains possessing the 2C8 phenotype (Mazzola, 1999; Mazzola and Gu, 2000). Although strains possessing the 2C8 phenotype are

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occasionally isolated from the rhizosphere of young apple trees grown in soil following wheat, the relative recovery of strains with this phenotype decline dramatically with increasing tree age (Mazzola, 1999). This may result from qualitative and quantitative attributes of root exudates produced by these plants. Compared to the wheat rhizosphere, apple roots appear to be a rather inhospitable environment for sustaining growth of ¯uorescent pseudomonads in general and strain 2C8 in particular. Relative ability of ¯uorescent pseudomonads to utilize carbon substrates can be altered depending upon physiological state of the bacterium. Consumption of nutrients by P. ¯uorescens strain 378 varied depending upon culture conditions (Persson et al., 1990) and the relative ability of a ¯uorescent pseudomonad to grow in nutrient-limited media is dependent upon prior culture conditions. The objective of this study was to examine the impact of adaptation of strain 2C8 to a carbon-limited environment on resistance to environmental stress, and ability to persist in soil habitats and provide biological control of Rhizoctonia root rot of apple. 2. Materials and methods 2.1. Bacterial and fungal strains Pseudomonas putida strain 2C8, which inhibits in vitro growth of a broad spectrum of fungi and provides biological control of apple replant disease (Mazzola, 1998a), was used as the parental strain in this study. Elements of the fungal complex that incites replant disease (Mazzola, 1998b) including Rhizoctonia solani AG 5 strain 5-103, Pythium sylvaticum strain A30-2 and Phytophthora cactorum strain D7-2 were used to evaluate the antagonistic abilities of P. putida 2C8 and its derivatives after adaptation in vitro to a nutrient-limited environment. 2.2. Adaptation of P. putida 2C8 Strain 2C8 was adapted to a carbon-limited environment by repeated culturing on soil agar media amended with a diluted suspension of King's medium B (KMB; King et al., 1954). Soil used in media preparation was obtained from replant sites at the Columbia View Experimental (CV) or the Wenatchee Valley College Auvil Research and Demonstration (WVC) orchard. Soil types are Burch sandy loam (pH 6.5) and Cowiche silt loam (pH 6.9) at the CV and WVC orchards, respectively. Initial selections were made by cultivating strain 2C8 on a medium composed of 17 g agar, 2.4 g proteose peptone #3, 0.18 g K2PO4, 0.18 g MgSO4, 0.24 g dextrose, 1.2 ml glycerol brought to a volume of 1 l by addition of deionized water, and then 400 g of CV or WVC orchard soil was added prior to sterilization (40% soil agar). Strain 2C8 was grown overnight in KMB broth at 288C and 100 ml of the resulting cell suspension was spread onto 40% CV and WVC soil agar.

Individual colonies were selected after 72 h incubation at 288C and transferred to 60% soil agar (17 g agar, 1.6 g proteose peptone #3, 0.12 g K2HPO4, 0.12 g MgSO4, 0.8 ml glycerol, 0.16 g dextrose, sterile deionized water to a volume of 1 l, and 600 g soil). Resulting colonies were cultured twice more on 60% soil agar. Isolates that possessed a stable colony phenotype and exhibited rapid growth on 60% soil agar were considered to be adapted strains for the two speci®c soil types. Three adapted strains from CV soil agar and from WVC soil agar were selected for evaluation of physiological characteristics. 2.3. Assessment of adaptation of strains to environmental stress Studies to assess the survival of adapted strains in carbonlimited conditions were conducted in a minimal medium [MM; 1 g sucrose, 1 g Ca(NO3)2´4H2O, 0.2 g KH2PO4, 0.25 g MgSO4´7H2O, 0.15 g NaCl, 15 g agar l 21]. Parental and adapted strains were pre-cultured at 288C for 48 h on 1/ 10th strength KMB agar as all strains exhibited equivalent growth rates on this medium. Cells were re-suspended in sterile deionized water and adjusted to a concentration of 5± 6 £ 10 8 cfu ml 21. One microliter aliquots of the cell suspension were spotted onto minimal media with 10 replicates per strain. Linear expansion of each bacterial colony was determined after incubation at 288C for 48 h. Growth rates of adapted and parental strains were also assessed in broth culture. Strains were pre-cultured as described above, and bacteria were adjusted to an initial concentration of 10 8 cfu ml 21 in 50 ml of minimal broth or 1/20th-strength KMB broth in a 250 ml ¯ask. Cultures were incubated at 288C on an orbit environ-shaker (Lab-line Instruments, Inc., Melrose Park, IL) at 200 rev min 21. Bacterial growth was assessed at 5, 10 and 24 h after inoculation by plating serial dilutions of the cell suspensions on KMB amended with chloramphenicol, cycloheximide and ampicillin at 13, 100 and 75 mg ml 21, respectively (KMB 1, Simon and Ridge, 1974). The experiment contained two replicates for each strain in each medium and the experiment was conducted three times. Sensitivity of the parental and adapted strains to various forms of environmental stress was evaluated. Growth studies were conducted in 1/10th-strength KMB, and amended with 1 M NaCl to simulate osmotic stress or 8 mM H2O2 to simulate oxidative stress. Cultures were incubated at 288C on a rotary shaker at 200 rev min 21. Thermotolerance was assessed by incubating strains at 458C for 24 h and monitoring subsequent growth of the heat stressed cultures when transferred to 288C. Bacterial concentrations (cfu ml 21) were determined turbidimetrically at an absorbance of 600 nm at 5, 10 and 24 h after stress induction. Optical density of bacterial suspensions was linearly correlated to CFU and cell density was determined using growth curves developed for P. putida 2C8 and its derivatives.

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2.4. Anti-fungal activities of adapted strains in vitro Growth inhibition, by P. putida 2C8 and its adapted derivatives, of fungal plant pathogens that incite replant disease in Washington apple orchards was assessed using a polycarbonate membrane method (Gu and Ko, 1997). Approximately 5 £ 10 8 cells of an individual strain were placed on a sterile polycarbonate membrane (pore size: 0.2 mm) which was over-layed on the surface of nutrient broth yeast extract agar (NBY; Vidaver, 1967) in a 9.5cm-diameter Petri dish. Bacteria grew on the membrane surface without directly contacting the medium. After 4 days incubation at 288C, bacteria were removed from the plate with the membrane. A 3-mm-diameter plug from the margin of an actively growing culture of R. solani AG 5, P. sylvaticum or P. cactorum on potato dextrose agar (Difco, Detroit, MI) was transferred to the center of an NBY plate, or an NBY plate that had been pre-cultured with one of the respective bacterial isolates. Plates were incubated at 248C and linear growth of fungi was measured after 24 h for P. sylvaticum, 48 h for R. solani AG 5 and 72 h for P. cactorum. Each fungal/bacterial strain combination was replicated three times and the experimented was conducted twice. 2.5. Utilization of root exudates as sole carbon source Wheat (cv. `Rely') seeds were surface disinfested in 1.5% sodium hypochlorite for 60 min and then washed with sterile deionized water six times. Seeds were incubated on moistened ®lter paper in sterile Petri plates at 248C for 48 h. Roots of two-week-old `Gala' apple seedlings grown in sterilized potting soil were immersed in a 1.5% sodium hypochlorite solution for 20 s and then rinsed repeatedly with sterile deionized water. Wheat or apple seedlings were aseptically transferred to a sterile 25-ml capped test tube containing 3 ml of minimal media without sucrose. Tubes were incubated at 208C with a 12 h photoperiod. Media exhibiting any form of microbial contamination was discarded. Seedlings were removed after 7 days and the solution containing root exudate was passed through a membrane (0.2 mm pore size) prior to storing at 48C until use. Aliquots of root exudate were plated on tryptic soy agar (Difco, Detroit, MI) prior to use to check for bacterial contamination. Strain 2C8 and its derivatives adapted to growth on soil agar media were examined for the ability to utilize root exudates as a sole carbon source in minimal media amended with 5% (v/v) root exudate solution. Media (30 ml) was dispensed into 50-ml sterile screw-cap tubes and inoculated with strain 2C8, 2C8-26A or 2C8-28C to establish an initial cell concentration of approximately 10 8 cfu ml 21. Cultures were incubated at 288C on a gyrotory shaker set at 200 rev min 21 for 24 h. Serial dilutions of culture aliquots were plated onto KMB 1 agar and colonies were enumerated after 72 h incubation at 288C.

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2.6. Survival in soil habitats and suppression of R. solani AG 5 Derivatives of 2C8 were initially adapted to carbonlimited conditions on agar medium containing either CV or WVC orchard soil. Therefore, studies were conducted to determine whether isolates initially adapted to a speci®c soil exhibited differential persistence in these two soils. Bacterial persistence in soil was assessed using spontaneous rifampicin-resistant (Rif r) mutants of P. putida strain 2C8 and the stress-adapted derivatives 2C8-26A and 2C8-28C. Rifampicin resistance was induced through serial culture of the strains on 1/10th±strength KMB amended with rifampicin at 100 mg ml 21. Growth rate of selected Rif r mutants in minimal media,1/20th-strength KMB and on 60% soil agar was comparable to the respective wildtype strains. A suspension of each strain was prepared by culturing on 1/20th-strength KMB agar for 72 h at 248C. Cells were harvested and washed twice in sterile water by centrifugation. Pelleted cells were resuspended in sterile water and adjusted to a concentration of approximately 10 8 cfu ml 21. Individual bacterial isolates were applied to separate CV or WVC orchard soil samples as an atomized mist produced using a chromatography sprayer and compressed air. Soils were mixed thoroughly throughout application and ®nal populations were approximately 10 6 cfu g 21 soil for each introduced bacterium. For each strain, six 1 kg samples were treated for each orchard soil and incubated individually in 3.8 L sealable bags at room temperature (20±228C). Populations of the introduced strains were estimated by resuspending 1 g of soil in 10 ml of sterile water and plating serial dilutions of this suspension on KMB amended with rifampicin (100 mg ml 21) and cycloheximide (75 mg ml 21). Colonies were enumerated after incubation at 288C for 72 h. Soil sampling was conducted at 1, 5, 10, 15, 20 and 30 days after application of bacteria. Colonization of apple roots and suppression of R. solani AG 5 was assessed using the rifampicin-resistant strains 2C8Rif r, 2C8-26ARif r and 2C8-28CRif r. Soil used in Rhizoctonia-suppression assays was collected from noncultivated land adjacent to the Washington State University Tree Fruit Research and Extension Center (WSU-TFREC) in Wenatchee, WA. Oat-bran inoculum of R. solani AG 5 strain 5-103 was prepared as previously described (Mazzola, 1997). Soil was infested with R. solani oat-bran inoculum at a concentration of 0.2% (w/w). Soils were incubated at 248C for 24 h prior to application of bacterial treatments. Bacterial strains were cultured, harvested and washed as described above. Pelleted cells were resuspended in sterile water and adjusted to a concentration of approximately 10 10 cfu ml 21. Individual bacterial isolates were applied to separate samples of R. solani AG 5-infested soils as an atomized mist. Soils were mixed thoroughly throughout application resulting in populations of 1.79, 1.70 and 1.62 £ 10 8 cfu g 21 soil for strains 2C8Rif r,

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2C8-26ARif r, and 2C8-28CRif r, respectively, at planting. Soil infested with R. solani AG 5 but not treated with one of the bacterial strains served as the control in disease suppression assays. Soils were dispensed into pots (3 L, 16 cm diameter) with a total of ®ve pots per soil treatment. Five 6-week-old `Gala' apple seedlings that had been grown in sterile potting medium as previously described (Mazzola, 1998a,b) were transplanted into each pot and were placed in environmental growth chambers at 188C with a 12 h photoperiod. Soil populations of introduced bacterial strains were determined as described above at 1, 5, 10, 15, 20 and 30 days after planting. Colonization of the apple rhizosphere by the introduced strains and apple root infection by R. solani AG 5 were determined at harvest, 30 days after transplanting into treated soils. Seedlings were removed from pots and shaken to remove loosely associated soil. A composite root sample and associated rhizosphere soil (total weight 0.1 g) was obtained for each pot. Root tissue was placed in a test tube with 10 ml of sterile water and vortexed for 60 s. Serial dilutions of the root wash were plated onto KMB amended with rifampicin (100 mg ml 21) and cycloheximide (75 mg ml 21). Colonies were counted after 72 h incubation at 288C. After completion of sampling for bacterial isolation, the remaining seedling root system was washed under tap water to remove adhering soil. For each plant, 10 0.5±1.0 cm root segments were randomly excised and plated onto 1.5% water agar amended with ampicillin (100 mg ml 21). After incubation at room temperature (20±228C) for 48 h, fungal growth emanating from root segments was examined using a light microscope (100 £ ) and the number of R. solani infected segments was recorded. 2.7. Statistical analysis Data for bacterial growth in various media, colonization of the apple rhizosphere by introduced strains and relative infection by R. solani AG 5 were subjected to analysis of variance and mean separation using Fisher's protected LSD.

3. Results Derivatives of P. putida strain 2C8 that were exposed to carbon-limited conditions by repeated culture on soil agar exhibited enhanced resistance, relative to the parental strain, when cultured in vitro in the presence of various stress conditions. For all strains adapted on either CV or WVC soil agar, growth on MM agar, in MM broth or in 1/20th strength KMB broth was superior to that obtained by wildtype strain 2C8 (Table 1). Likewise, the adapted strains exhibited enhanced tolerance to osmotic tension as evidenced by growth in 1/10thstrength KMB amended with 1 M NaCl (Table 2). Growth of all strains adapted for growth on a nutrient limited soil agar medium was superior to strain 2C8 in 1/10th-strength KMB amended with 8 mM H2O2 indicating that the adapted strains possessed an induced resistance to oxidative stress relative to that of strain 2C8 (Table 3). Tolerance of 2C8 to high (458C) or low (58C) temperature stress was not altered by prior nutrient stress, and all strains exhibited a similar recovery when returned to incubation temperature of 288C (data not shown). Strain 2C8 was not capable of utilizing apple root exudates as a sole carbon source. Adaptation to nutrientlimited conditions by prior cultivation on soil agar did not alter the ability of strain 2C8 to utilize apple root exudates. The parental strain and the strains adapted for growth on soil agar media all exhibited an inability to grow in minimal media containing `Gala' apple root exudates as a sole carbon source (Table 4). In contrast, strain 2C8 was capable of growth in minimal media containing root exudate from the wheat cultivar `Rely' as the sole carbon source. Populations of the 2C8-26A and 2C8-28C were signi®cantly larger than strain 2C8 after 24 h incubation in minimal media amended with `Rely' root exudate. Strain 2C8 produces a volatile compound and a diffusible antibiotic that inhibit growth of fungi in vitro (Mazzola and Gu, unpublished data). The ability of this strain to suppress in vitro growth of R. solani AG 5, P cactorum and P. sylvaticum on NBY agar was unaltered by prior adaptation of the bacterium to nutrient-limited growth conditions (data not shown).

Table 1 Growth of Pseudomonas putida 2C8 and its adapted derivatives in carbon-limited media P. putida strain

2C8 2C8-24A 2C8-25A 2C8-26A 2C8-27C 2C8-28C 2C8-29C

Growth medium a MM agar (colony diameter, mm)

MM broth (cfu £ 10 8 ml 21)

1/20th KMB (cfu £ 10 8 ml 21)

0.3a b 2.5b 2.3b 2.5b 3.3b 2.0b 2.5b

0.5a 3.2c 5.5g 5.2f 1.8b 3.9d 4.5e

14a 40c 58e 56de 28b 52d 48d

a Assessment of bacterial growth was made at 24 and 48 h post-inoculation for broth and agar media, respectively; MM indicates minimal media as de®ned in Materials and methods. b Means in a column followed by the same letter are not signi®cantly (P ˆ 0.05) different based on Fisher's protected LSD test.

Y.-H. Gu, M. Mazzola / Soil Biology & Biochemistry 33 (2001) 1155±1162 Table 2 Tolerance to osmotic stress among Pseudomonas putida 2C8 and its carbon starved derivatives as determined by growth at 288C in 1/10th-strength KMB broth amended with 1 M NaCl P. putida strain

2C8 2C8-24A 2C8-25A 2C8-26A 2C8-27C 2C8-28C 2C8-29C

8

21

CFU ( £ 10 ml ) after incubation time 5h

10 h

24 h

5.7a b 5.7a 5.8a 5.6a 5.6a 4.5b 5.6a

5.6a 5.7a 5.7a 5.6a 5.6a 5.3a 5.6a

5.6a 6.5b 7.2c 6.7b 6.7b 6.6b 6.7b

a

a

Strains were established at an initial population of 6.0 £ 10 8 cfu ml 21. Means in a column followed by the same letter are not signi®cantly (P ˆ 0.05) different based on Fisher's protected LSD test. b

Table 3 Tolerance to oxidation stress among Pseudomonas putida 2C8 and its carbon starved derivatives as determined by growth at 288C in 1/10thstrength KMB broth amended with 8 mM H2O2 P. putida strain

CFU ( £ 10 8 ml 21) after incubation time a 5h

2C8 2C8-24A 2C8-25A 2C8-26A 2C8-27C 2C8-28C 2C8-29C

5.6a 5.6a 7.0a 6.1a 6.7a 7.8a 6.2a

b

10 h

24 h

5.8a 10.4b 12.5b 23.0c 24.3c 26.2c 32.4d

10.0a 24.5b 26.6bc 31.3c 25.4bc 28.0bc 27.4bc

a

Strains were established at an initial population of 5.6 £ 10 8 cfu ml 21. Means in a column followed by the same letter are not signi®cantly (P ˆ 0.05) different based on Fisher's protected LSD test. b

Survival of strain 2C8 and its derivatives adapted to carbon-limited conditions was similar in soil when established at an initial population of 10 6 cfu g 21. Populations of each strain exhibited a steady decline after introduction into soil and approached 10 5 cfu g 21 at the ®nal sampling, 30 days after application. There were no signi®cant differences among soil populations of the introduced strains at any sampling and survival of strains 2C8-26ARif r and 2C828CRif r was similar in both CV and WVC orchard soils. As observed in CV and WVC orchard soils, when introduced into soil from the WSU-TFREC no signi®cant differences in populations of 2C8 and its derivatives were observed at any sampling date (data not shown). However, derivatives of strain 2C8 adapted to growth on soil agar media exhibited an enhanced ability to colonize the rhizosphere of apple seedlings when applied to this soil prior to planting. Populations of strains 2C8-26ARif r and 2C828CRif r in the rhizosphere of `Gala' apple seedlings were signi®cantly larger than that of strain 2C8Rif r at 30 days

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Table 4 Growth of Pseudomonas putida 2C8 and its carbon starved derivatives in minimal media containing root exudates as a sole carbon source P. putida strain

2C8 2C8-26A 2C8-28A 2C8 2C8-26A 2C8-28A 2C8 2C8-26A 2C8-28A

Carbon source

None None None Apple Apple Apple Wheat Wheat Wheat

CFU ( £ 10 8 ml 21) after incubation time 0h

24 h

1.3 a 1.3 1.3 1.3 1.3 1.3 1.3 1.3 1.3

0.5a 0.8a 0.5a 1.1a 0.7a 0.9a 2.3b 4.2c 5.7d

a

Values are means of three experiments with two replicates in each trial. Means followed by the same letter are not signi®cantly (P ˆ 0.05) different based on Fisher's protected least signi®cant difference test. Table 5 Colonization of the apple rhizosphere and suppression of Rhizoctonia solani AG 5 root infection by Pseudomonas putida strain 2C8 and its carbon starved derivatives at 30 days after planting P. putida strain a

CFU( £ 10 5) g 21 root

% R. solani root infection b

Control 2C8 2C8-26A 2C8-28C

± 1.8a 11.6b 13.2b

54.3c c 42.3b 16.7a 17.9a

a Strains were applied to separate soil samples to obtain an initial population of 108 cfu g 21. b Soils were infested with R. solani AG 5 by incorporation of pathogencolonized oat bran at a rate of 0.2% (w/w). c Means in a column followed by the same letter are not signi®cantly (P ˆ 0.05) different based on Fisher's protected least signi®cant difference test.

after planting (Table 5). There was no signi®cant difference in rhizosphere populations of 2C8-26ARif r and 2C828CRif r. Soil application of strain 2C8Rif r signi®cantly reduced subsequent apple root infection by R. solani AG 5 relative to that observed for seedlings grown in pathogen infested control soil. However, strains 2C8-26ARif r and 2C828CRif r provided enhanced disease protection resulting in signi®cantly lower root infection by R. solani AG 5. The enhanced disease control achieved with these strains was directly associated with enhanced colonization of the apple rhizosphere by the derivative strains relative to that achieved by the parental strain 2C8.

4. Discussion Upon introduction into soils, exponentially growing cells of Pseudomonas ¯uorescens strain R2f were found to

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develop resistance to speci®c stress factors in a manner equivalent to that obtained by exposing the strain to carbon starvation conditions in liquid culture (van Overbeek et al., 1995). In this study, derivatives of strain 2C8 were generated by repeated cultivation on a soil agar medium and used in studies to evaluate the impact of prior exposure to nutrient-limited conditions on relative competitive ®tness of the strain in soil habitats. Adaptation of P. putida 2C8 to nutrient limited conditions on soil agar medium elicited changes in tolerance to stress similar to those previously observed in other bacterial systems in response to carbon starvation È stling et al., 1993; Givskov et al., (Jenkins et al., 1988; O 1994a; van Overbeek et al., 1995). Derivatives of strain 2C8 adapted for growth on soil agar exhibited resistance to osmotic and oxidative stress, and an enhanced ability to grow in carbon-limited liquid culture relative to the parental strain. Enhanced resistance to high-temperature stress was not observed in response to prior adaptation of strain 2C8 on soil agar medium. Interestingly, van Overbeek et al. (1995) observed that starvation of P. ¯uorescens strain R2f in liquid medium induced thermal resistance, but that cells starved through incubation in soil did not exhibit such a response. Our results suggest that the adaptation of strain 2C8 on soil agar medium elicited a response similar to that which would be obtained in native soil. Although cultivation of bacteria in a nutrient-limited medium characteristically induces stress resistance, adaptation to such an environment has yielded varying results with regards to survival of the strain upon introduction into its native habitat. Survival of carbon starved or exponentially growing cells of P. ¯uorescens R2f over a ten-day period was similar when introduced into soil, with both strains exhibiting a consistent decline in numbers during the course of the study (van Overbeek et al., 1995). Likewise, in this study survival of strain 2C8Rif r and its adapted derivatives 2C8-26ARif r and 2C8-28CRif r was similar when strains were introduced into any of three different soils. However, strains 2C8-26ARif r and 2C8-28CRif r exhibited an enhanced ability to colonize the rhizosphere of apple relative to strain 2C8Rif r. Studies conducted in minimal media revealed no differences in the ability of strain 2C8 and its derivatives to grow when apple root exudate was supplied as the sole carbon source. Therefore, it is unlikely that the enhanced survival of 2C8-26ARif r and 2C8-28CRif r in the apple rhizosphere relative to the parental strain resulted from an altered ability to metabolize apple root exudates. Adaptation to the limited availability of other essential nutrients may have played a direct or indirect role in enhanced survival of these strains. Physiological changes induced in response to nutrient limitation can alter nutritional consumption by ¯uorescent pseudomonads (Persson et al., 1990), which may be of bene®t in a soil environment. Nutrient limitations also can alter expression of genes that have a role in bacterial survival. For instance, siderophores have a role in both biocontrol ef®cacy and survival of

¯uorescent Pseudomonas spp. and production of these compounds is induced under iron-limited conditions (Loper and Buyer, 1991; Raaijmakers et al, 1995b). The apparent difference in contribution of stress resistance to survival in soil and the apple rhizosphere of 2C8 derivatives adapted to growth on soil agar medium may be related to the relative competitive pressures encountered in the two environments. Mazzola et al. (1992) observed that the relative contribution of phenazine antibiotic production to survival of ¯uorescent pseudomonads in soil habitats was more pronounced in the rhizosphere of wheat than in bulk soil. This was attributed to the higher level of microbial competition that is experienced in the rhizosphere relative to bulk soil. Substantial reduction in competitive pressure achieved through soil pasteurization eliminated the bene®t of antibiotic production to the survival of these bacteria in soil habitats. It is plausible that in the absence of signi®cant microbe±microbe competition, the bene®ts of induced resistance to stress resulting from carbon starvation are not realized. Alternatively, resistance of bacteria to a speci®c stress condition may be more critical to survival in the rhizosphere than bulk soil due to an enhanced exposure to a speci®c stress factor in that environment. For instance, among root-colonizing ¯uorescent Pseudomonas strains an apparent relationship exists between colonization potential and in vitro osmotolerance (Loper et al., 1985). Since water matric potential is likely to be lower in the rhizosphere of an actively transpiring plant than in the bulk soil, this attribute would likely provide a greater advantage to survival in the rhizosphere than in bulk soil. The same argument could be proposed for oxidative stress tolerance. Colonization of plant roots would expose bacterial cells to root surface peroxidases that result in the production of superoxide anion and hydrogen peroxide (Albert et al., 1986). Resistance to these oxidizing agents may facilitate root colonization by ¯uorescent pseudomonads (Katsuwon and Anderson, 1989). Persistence of biological control strains in the host rhizosphere has long been regarded as a key element to their successful use in the management of soilborne plant diseases (Schippers et al., 1987; Weller, 1988; O'Sullivan and O'Gara, 1992). As a result, numerous studies have been conducted that have sought to enhance the ef®cacy of biocontrol rhizobacteria through modi®cation of the organism or its environment to promote survival after introduction into the soil environment. These approaches include genetic modi®cation to enhance expression of traits or introduction of novel attributes that contribute to root colonization (Colbert et al., 1993; Roberts et al., 1998). Formulation systems including micro-encapsulation have been developed to improve shelf life of biocontrol strains and to enhance their survival upon introduction into natural environments (Fravel et al., 1985; van Elsas et al., 1992; Lumsden et al., 1995). However, in some instances these studies failed to document the impact of enhanced survival

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or colonization by the introduced strain on disease suppression (van Elsas et al., 1992; Colbert et al., 1993). Recently, Fuchs et al. (2000) reported that composition of the laboratory medium used to prepare inoculum signi®cantly in¯uenced the ef®cacy of P. ¯uorescens strain Pf153 in controlling black root rot of cucumber. The difference in ef®cacy was observed in natural soil, but not an arti®cial soil (Keel et al., 1989) possessing low microbial activity. The difference in disease suppression was not associated with differences in population of the biocontrol strain in the experimental microcosm, however no attempt was made to differentiate soil from rhizosphere populations of the introduced strain. The results obtained in this study also support the premise that laboratory cultivation conditions can impact ef®cacy of biocontrol ¯uorescent pseudomonads as derivatives of strain 2C8 adapted for growth in a nutrientlimited soil agar medium provided disease control that was superior to the wild-type strain cultured on KMB. Results from this study suggest that application of procedures to enhance colonization of the rhizosphere by biocontrol ¯uorescent pseudomonads can lead to improved management schemes for control of soilborne plant pathogens. Although several studies have demonstrated a dose response relationship in the ef®cacy of biological control agents used in the control of soilborne plant pathogens (Raaijmakers et al., 1995a,b; Raaijmakers and Weller, 1998), few studies have documented enhanced disease control through the manipulation of bacterial attributes that promote root colonization. Our ®ndings documented an association between enhanced apple root colonization by stress-adapted derivatives of strain 2C8 and reduced root infection by R. solani AG 5. These results suggest that exposure in vitro to conditions that induce resistance to stress conditions encountered in natural soil environments may be a means to enhance root colonization and ef®cacy of biocontrol ¯uorescent pseudomonads. Preliminary evidence suggests that improved root colonization is not necessarily the result of an increased ef®ciency in utilization of root exudates. Further studies will be necessary to elucidate the physiological factors that contribute to enhanced root colonization and disease control.

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