Vol. 59, No. 7APPLIED AND ENVIRONMENTAL MICROBIOLOGY, July 1993, p. 2056-20630099-2240/93/072056-08$02.00/0Copyright © 1993, American Society for Microbiology

Use of an Exotic Carbon Source To Selectively Increase MetabolicActivity and Growth of Pseudomonas putida in Soil

STEPHEN F. COLBERT, THOMAS ISAKEIT,t MARIO FERRI,4 ALBERT R. WEINHOLD,MAVIS HENDSON, AND MILTON N. SCHROTH*

Department ofPlant Pathology, University of California at Berkeley,Berkeley, California 94720

Received 23 October 1992/Accepted 21 April 1993

Respiration and growth ofPseudomonas putida PpG7, containing catabolic plasmid NAH7, was determinedin three agricultural field soils amended with the carbon source salicylate. The addition of salicylate to soilsignificantly increased the population of PpG7. However, there was a lack of relationship between microbialnumbers and activity as determined by evolution of CO2. In soils containing 30 to 1,500 pg of salicylate per g,metabolic activities ofPpG7 peaked between 18 and 42 h and population densities increased approximately 10'-

to 105-fold. However, the metabolic activity ofPpG7 rapidly declined after salicylate was utilized, whereas peakpopulation densities were maintained for the duration of the experiments (5 to 7 days). Thus, elevatedpopulation densities ofPpG7 were represented by inactive cells. Soil type had only minor effects on respirationrates or growth curves of PpG7 when amended with comparable concentrations of salicylate. Respiration andgrowth rates were optimal at concentrations between 300 and 1,000 pg of salicylate per g in the test soils. At1,500 to 2,500 pg/g, respiration and growth of PpG7 were initially suppressed, but after a short lag time bothattained levels similar to or greater than those resulting from the use of lower concentrations of salicylate. Theculturing of PpG7 on a salicylate-amended medium to induce salicylate-degradative enzymes did not affect thelag time before utilization of salicylate in soil. Although PpG7 competed well with fungi for the substrate,suppression of fungal populations with cycloheximide resulted in significantly increased population densities ofPpG7 in two of three soils amended with salicylate. The beneficial activities of bacteria in soil are discussed inrelation to population density, population metabolic activity, and selective carbon source utilization.

A better understanding of the factors that affect thepopulation dynamics of interacting microorganisms and theirtemporal-spatial relationships in natural settings is essentialfor the effective use of microorganisms for such purposes asbioremediation, plant growth promotion, and the biologicalcontrol of plant pests (25). It is becoming increasinglyevident that the application of beneficial microorganisms tosoil and plant parts will not result in their becoming signifi-cant, active components of the microflora for sustainedperiods of time unless the environment is simultaneouslymodified to make it more conducive for their growth andsurvival. One way to effect this would be to provide anenhanced nutritional environment since competition for sub-strates in soils and rhizospheres is intense. This is anattractive strategy because microorganisms in soil are gen-erally carbon source limited, although there are periods ofgreat activity and competition when nutrients are available(29). The outcome of this competition is influenced by therelative abundance of microorganisms, their nutritional char-acteristics, and their specific growth rates under the partic-ular environmental conditions (10, 17, 26).

It should be possible to apply substrates with inoculum orthrough irrigation systems (3) that would preferentially en-able the microorganisms of interest to grow and become animportant population for extended periods of time. How-ever, this is complicated by the fact that many serioussoil-borne pathogens, such as Pythium ultimum, are nutri-

* Corresponding author.t Present address: Department of Plant Pathology, University of

Arizona, Tucson, AZ 85721.i Present address: Enichem Agricoltura, Istituto Donegani, No-

vara, Italy.

tionally versatile and have the ability to germinate fromresting spores and infect plant parts within 3 h when exposedto a suitable substrate (20). This fungus escapes competitionby entering the plant before most microorganisms havebegun to grow. Thus, the use of exogenous substrates couldwell promote the growth of undesirable microorganisms.

In situ enrichments have often been used to study thebiodegradation activities of specific microorganisms in vari-ous environments (7, 11, 16). However, there is little de-tailed quantitative information available on the relationshipbetween microbial density and activity measurements for aspecific microorganism in natural soil over a range of timeperiods. A specific activity index has been proposed tocorrelate microbial density and activity (15). An understand-ing of activity and the temporal-spatial relationships amongcompeting microorganisms is critical to developing strate-gies to manage microorganisms in the soil, especially in therhizosphere. Too often, microbial activity in soil or on plantparts is related to population size when, in effect, a dormantor relatively quiescent population is merely being observed(10, 18, 25).The concept of selectively enhancing the activities of a

specific microorganism by use of a relatively exotic carbonsource was studied with Pseudomonas putida PpG7 and thesubstrate salicylate. The relationship between activity,growth, and survival was examined by relating evolution ofCO2 to population density in several agricultural soils.

MATERIALS AND METHODS

Bacterial strains and inoculum preparation. P. putida PpG7(ATCC 17485), isolated from soil by W. R. Sistrom, wasobtained from I. C. Gunsalus (Department of Biochemistry,

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TABLE 1. Description of soils used in this study'

% by weight %Name of soil Type 0Mb pH Moisture'oMb ~~Sand Silt Clay osue

Davis Yolo silt loam NDd 26 47 27 7.1 23Tulelake Tulebasin mucky silty clay loam 10-15 8 58 34 7.5 38Southcoast San Emigdio sandy loam 0.5 75.5 12.5 12 7 19Westside Cerini clay loam <1 18 51 31 8 21

a Determinations from USDA Soil Conservation Service.b OM, organic matter.c Percent moisture refers to percentage by weight of water in the soil at a matric potential of -20 kPa.d ND, not determined.

University of Illinois, Urbana) and listed as strain 111 in thetaxonomic study of Stanier et al. (27). PpG7 is the naturalhost of plasmid NAH7, which encodes the enzymes neces-sary for utilization of salicylate in two operons. The upperpathway encodes the enzymes for breakdown of naphtha-lene to salicylate, and the lower pathway encodes theenzymes for breakdown of salicylate to acetaldehyde andpyruvate (6). Both pathways are positively regulated by thepresence of salicylate (30). Strain PpG7 was grown onAyers' minimal medium (2) containing 10 mM sodium sali-cylate (pH 7) (MMsal; Fisher Chemical, Fisher Scientific,Inc., Fairlawn, N.J.). Inocula for experiments were grownas lawns on King's medium B for 24 h at 28°C (13). Bacteriafrom one petri dish (100 by 15 mm) were harvested into 10 mlof 10 mM phosphate buffer (PB), pelleted by centrifugation,and washed in PB three times.Microcosm preparation and treatments. Metabolic activity

was measured in soil microcosms by the induced respirationmethod of Anderson and Domsch (1), by using the auto-mated CO2 measurement system of Brooks and Paul (4).Soils and their characteristics are listed in Table 1. Nativefield soils were stored at 5°C in plastic 20-liter buckets. Oneday prior to use, the soils were air dried and passed througha 2-mm sieve. Soil microcosms consisted of 30 g of air-driedsoil in 225-ml mason jars. Treatments consisted of (unlessotherwise noted) water alone, PpG7 (approximately 104CFU/g of soil), sodium salicylate (1,700 .g/g of soil), strep-tomycin sulfate (3,000 ,ug/g of soil; Sigma Chemical Co., St.Louis, Mo.), cycloheximide (1,000 ,ug/g of soil; Sigma), andcombinations thereof. Materials were added to the soilsurface of triplicate microcosms in sufficient water to attaina gravimetric matric potential of -20 kPa as determined bya pressure plate extraction system (Soil Moisture EquipmentCo., Santa Barbara, Calif.). Each of the soils became evenlywetted by passive movement of water within approximately0.5 to 1 h after addition. Salicylate was added from afilter-sterilized 1 M solution adjusted to pH 7. The concen-tration of carbon added as salicylate was 900 ,ug/g of soil, andthe concentration of sodium in the final salicylate solutionadded to soil was 0.04 M. All experiments were done at 23°Cfor 5 to 7 days.

Induced respiration and growth in soil. (i) Experimentaldesign of respiration measurements. Respiration was mea-sured in microcosms attached by a computer-controlledmultiport valve to a gas chromatograph (4). Every 6 h, thegases from each jar were sequentially flushed with moistenedair to the gas chromatograph for CO2 analysis. The peak ofthe detection signal was measured, converted to percentCO2, and stored on a floppy disk by the computer. Resultswere converted to micrograms of C02-C per g of soil per hafter the average percent background CO2 measured in twoempty control jars was subtracted.

(ii) Experimental design of population density measure-ments. Population densities of organisms utilizing salicylatein soil microcosms were monitored at the same time respi-ration measurements were made. Separate microcosms pre-pared as described above were loosely sealed with mason jarlids, and approximately 1-g samples of soil were removeddaily and weighed. The soil samples were placed into 9 ml ofPB, agitated with a vortex mixer for 1 min, and seriallydiluted in 9-ml PB blanks. Fifty-microliter subsamples fromeach dilution were spotted in duplicate onto MMsal andincubated at room temperature (25 + 5°C) or in a 28°Cincubator. The numbers of CFU were determined for ap-proximately 5 days. Soil samples from the microcosms wereoven dried (110°C) at the conclusion of the experiment, andcounts were converted to CFU per gram of oven-dried soil.

Respiration and growth of native microflora. Utilization ofsalicylate by native soil microflora was assayed in Davis soil.Salicylate was applied to soil in water to detect inducedrespiration of native organisms. This was compared withrespiration caused by the addition of water alone. Strepto-mycin or cycloheximide was added with salicylate to ob-serve the effects of suppressing bacterial and fungal popula-tions, respectively. Population densities of organismsutilizing salicylate in soil were assayed after 46 h of incuba-tion (peak of respiration). In parallel experiments, naturalsoil amended with salicylate was examined over a 140-h timeperiod for indigenous bacteria that could utilize salicylate.

Respiration and growth of PpG7. Utilization of salicylateby PpG7 was monitored in microcosms containing eitherDavis, Tulelake, or Southcoast soil. Fungal competition forsalicylate detected in preliminary studies was suppressed insome treatments with cycloheximide. PpG7 alone and PpG7with cycloheximide were added to soils to determine back-ground respiration rates. Salicylate and salicylate with cy-cloheximide were added to determine respiration of indige-nous microorganisms and PpG7. Population densities ofsalicylate-utilizing bacteria were monitored daily in treat-ments containing strain PpG7, and all treatments weresampled at the end of the experiment to determine growth ofbacteria other than strain PpG7. Bacteria that morphologi-cally resembled PpG7 were screened on a number of differ-ent carbon and nitrogen sources which distinguish Pseudo-monas fluorescens biovars (12, 27). One thousand isolatesincluding those from a field study (5) were examined byreplica plating techniques.The effect of salicylate concentration on respiration and

growth of PpG7 was determined by application of 0- to5,000-jig amounts of salicylate per g of Davis, Southcoast,and Westside soils. All treatments included strain PpG7 andcycloheximide. Respiration rates and population increaseswere monitored as described previously.

Effect of pregrowing PpG7 on media containing salicylate on

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its ability to utilize salicylate in soil. Inocula of PpG7 grownon King's medium B alone or with 10 mM salicylate (toinduce the salicylate degradation enzymes) and cyclohex-imide were applied to Davis soil with or without salicylate(1,700 ,ug/g). Microcosms were attached to the respirationmeasurement apparatus, sealed with a mason jar lid, andallowed to incubate. Microcosms were sampled for CO2production and population growth of PpG7 as describedpreviously.

Suppression of glucose utilization in soil by salicylate.14C-labeled glucose was used to determine the effect ofsalicylate on the metabolism of indigenous organisms andPpG7 in Davis soil. Sterile distilled water (0.8 ml) was addedto three replicates of 10 g of Davis soil in petri dishes (60 by15 mm) with or without PpG7 (approximately 104 CFU/g ofsoil). A 0.2-ml mixture of unlabeled and labeled glucosesolution (1.8 ,Ci/mg of glucose) and 0.2 ml of salicylatesolution or 0.2 ml of sterile distilled water instead of salicy-late were added to the soil after 18 h and thoroughly mixed.The final concentrations of glucose and salicylate in the soilwere 100 and 1,700 ,ug/g, respectively. The petri dishes wereplaced in sealed 250-ml mason jars fitted with plastic septa.The atmospheres in the mason jars were sampled at 2, 6, and8 h after the start of incubation. Air samples (20 cm3) wereremoved with a syringe from each mason jar and transferredto 0.5 ml of 0.5 N NaOH in vacuum-evacuated scintillationvials sealed with plastic septa. The scintillation vials weregently swirled to mix the introduced gases and NaOH. Aftersampling, the mason jars were opened in a laboratory fumehood to reduce 14CO2 to background levels. At 1 h aftersampling, septa on scintillation vials were removed and 10ml of Ecolume (ICN Biomedicals Inc., Costa Mesa, Calif.)was added before the vials were recapped with scintillationvial lids. Counts per minute for each sample were deter-mined with a Tri-Carb 4530 liquid scintillation counter(United Technologies, Packard Instrument Co., DownersGrove, Ill.). A 0.5-ml volume of 5 N NaOH in 10 ml ofEcolume was used as a background control. Counts perminute were converted to micrograms of glucose utilized pergram of soil per hour.Data analysis. The mean log1o CFU of PpG7 per gram of

soil or mean respiration rates were compared betweentreatments by randomized complete-block analysis of vari-ance (CoStat Statistical Software, Cohort Software, Berke-ley, Calif.). Population counts from the last sample timewere used for statistical analysis. Reported and plottedpopulation densities are means of the transformed data +standard errors of the means.

RESULTS

Authentication of strain PpG7 in soil isolations. Strain PpG7was identified by the growth pattern and distinctive white,opaque, raised, entire colony type on MMsal isolation me-dium. The strain identification was further authenticated byreplica plating 1,000 isolates on media to determine theircarbon and nitrogen utilization characteristics. No indige-nous salicylate-utilizing bacteria were isolated from any ofthe soils during the 140-h time period of the experiments,including those amended and nonamended with salicylate.

Effect of salicylate on respiration and growth of indigenousmicroflora. Some components of the indigenous microflorain Davis soil utilized salicylate as indicated by increasedrespiration (Fig. 1). This response was delayed several hoursby the addition of cycloheximide, indicating that the re-sponse was due to fungi. Respiration of fungi induced by

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FIG. 1. Effect of salicylate on respiration of indigenous microor-ganisms in Davis soil. Mean respiration rates in soil amended withwater (0), salicylate (1,700 pg/g; x), salicylate and streptomycin(3,000 tLg/g; A), and salicylate and cycloheximide (1,000 ,g/g; 0) areshown. Bars represent standard errors of the means where variationwas great enough to be presented.

addition of salicylate (1,700 p,g/g of soil) was detected after14 h of incubation, and the peak rate, 11.5 ,ug of C02-C perg of soil per h, occurred after 56 h of incubation. In contrast,suppression of fungal populations by the addition of cyclo-heximide with salicylate delayed the onset of induced respi-ration for approximately 38 h, with a peak of 9 ,g of C02-Cper g of soil per h after 98 h of incubation. Respirationinduced by salicylate with streptomycin was similar to thatinduced by salicylate alone, indicating lack of bacterialactivity.The mean population density of salicylate-utilizing fungi

after 46 h of incubation was significantly increased (P = 0.01)from 5.6 x 102 CFU/g in nonamended soil to 1.45 x 104CFU/g in salicylate-amended soil. When cycloheximide wasadded with salicylate, the mean population density of salic-ylate-utilizing fungi was initially suppressed (< 100 CFU/g ofsoil). However, by about 98 h, fungal utilization of salicylatewas detected.

Respiration and growth of strain PpG7 versus indigenousmicroflora in salicylate-amended soils. Respiration rates insoils amended with salicylate (1,700 ,ug/g) and PpG7 weresimilar to rates detected in soils amended with salicylatealone because of the masking effect of fungal respiration(Fig. 2A, B, and C). When cycloheximide was added tosuppress fungal utilization of salicylate, induced respirationof PpG7 was easily detected. In the absence of fungalcompetition for salicylate, induced respiration rates of PpG7peaked at 16.2, 16.4, and 19.2 P,g of C02-C per g of soil perh after 54, 51, and 66 h of incubation in Davis, Tulelake, andSouthcoast soils, respectively.The addition of salicylate significantly increased (P =

0.01) the final mean population densities of PpG7 in each ofthe soils, even when fungal activity was not suppressed withcycloheximide (Table 2). Population densities were in-creased 4.57 x 102-, 8.0 x 101-, and 1.7 x 106-fold insalicylate-amended Davis, Tulelake, and Southcoast soils,respectively. In addition, suppression of fungal activity insalicylate-cycloheximide-amended soils resulted in signifi-

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FIG. 2. Effect of salicylate on respiration rates (A, B, and C) and population densities (D, E, and F) of PpG7 in different soils. Mean valuesfrom Davis soil (A and D), Tulelake soil (B and E), and Southcoast soil (C and F) amended with PpG7 (approximately 104 CFU/g; 0), PpG7and cycloheximide (1,000 p.g/g; x), salicylate (1,700 Fg/gg; A), salicylate and cycloheximide (0), PpG7 and salicylate (C), and PpG7,cycloheximide, and salicylate (V) are shown. Bars represent standard errors of the means where variation was great enough to be presented.Background respiration was constant at approximately 0.5, 1.5, and 0.4 p,g of C02-C per g of soil per h in Davis, Tulelake, and Southcoastsoils, respectively.

cantly increased mean population densities of PpG7 by4-fold in Davis soil (P = 0.05) and 28-fold in Tulelake soil (P= 0.01) compared with amended soils not receiving cyclo-heximide. Although PpG7 population densities were in-creased to 108 CFU/g of soil or greater after completeutilization of salicylate (Fig. 2D, E, and F), the populationswere represented by inactive cells, as evidenced by the rapidreduction of respiration to near basal levels (Fig. 2A, B, andC).

Effect of concentration of salicylate on respiration andpopulation densities of PpG7 in amended soils. Similar in-creases in respiration rates and population densities of PpG7were obtained in all three soil types tested when comparableconcentrations of salicylate were added (Fig. 3). At 30 to 50,ug/g of soil, respiration and population densities increasedcompared with those of nonamended soils in Westside andDavis soils but not in Southcoast soil. Optimal concentra-tions of salicylate in all soils appeared to range from 300 to1,000 ,ug/g of soil. At these concentrations, population sizesof PpG7 increased approximately 102- to 104-fold by 48 h ofincubation, at approximately the same time of greatest CO2

TABLE 2. Population densities of P. putida PpG7 in soilsamended with salicylate and cycloheximide

Log10 CFU/g of soilaSoil amendments'

Davis Tulelake Southcoast

Salicylate NDc ND NDSalicylate + cycloheximide ND ND NDPpG7 5.15 cd 5.42 c 2.26 cPpG7 + cycloheximide 5.25 c 5.55 c 4.53 bPpG7 + salicylate 7.81 b 7.32 b 8.50 aPpG7 + salicylate + cyclo- 8.42 a 8.77 a 8.96 a

heximideLSD' at P = 0.05 0.57 0.31 0.49

a Mean of six replicates sampled after 168, 138, and 148 h of incubation inDavis, Tulelake, and Southcoast soils, respectively.

b Salicylate (1,700 Fig/g of soil), cycloheximide (1,000 A.g/g of soil), andPpG7 (approximately 104 CFU/g of soil) were applied to 30 g of air-dried soilmoistened to a matric potential of -20 kPa.

c No indigenous bacteria capable of utilizing salicylate were detected (< 100CFU/g of soil).

d Values within a column followed by the same letter are not significantlydifferent at P = 0.05.

' LSD, least significant difference.

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FIG. 3. Comparison of effects of increasing concentrations of salicylate on respiration rates (A, B, and C) and population densities (D, E,and F) of PpG7 in different soils. Mean values in Davis soil (A and D), Southcoast soil (B and E), and Westside soil (C and F) amended withdifferent concentrations of salicylate are shown. All soils received PpG7 (approximately 104 CFU/g), cycloheximide (1,000 pLg/g), andsalicylate. Concentrations of salicylate (in micrograms per gram of soil) are indicated by the number associated with each curve. Curvesrepresent triplicate samples at 6-h intervals. Standard errors are not included.

evolution. Respiration and population density increaseswere initially suppressed at approximately 1,500 to 2,500 ,ugof salicylate per g of soil, but, after an initial lag phase, bothrespiration and population densities attained levels similar toor greater than those attained when lesser amounts ofsalicylate were used. Concentrations of salicylate of >2,500,ug/g of soil were very suppressive.

Effect of pregrowing PpG7 on media amended with salicy-late on its ability to utilize salicylate in soil. Prior exposure ofPpG7 to salicylate in culture media did not affect suppressionof respiration when it was subsequently exposed to a highconcentration (1,700 ,ug/g) of the substrate in soil (Fig. 4A).Induction also did not affect population growth of PpG7 insalicylate-amended soil (Fig. 4B).

Suppression of glucose utilization in soil by salicylate.Utilization of [14C]glucose (100 ,ug/g of soil) by indigenousmicroflora and PpG7 was suppressed during 8 h of incuba-tion in Davis soil amended with 1,700 ,ug of salicylate per gof soil (Fig. 5). The amount of 14C02 released from thesesoils was less than half that of nonamended soils 2 h after thestart of incubation, and the difference increased over thetime course of the experiment.

DISCUSSION

This study shows the importance of using an "ecologicalkinetic approach" (15) when studying the population dynam-ics of specific organisms. By examining the quantitativerelationships between the population sizes of strain PpG7and its activity over many time periods, it was clear thatthere is not a good correlation between population densityand activity as measured by evolution of CO2. Both metab-olism and growth of PpG7 were markedly increased innatural soil by the addition of the selectively utilized carbonsource salicylate. However, an examination of the data inFig. 2 reveals that population densities of PpG7 sampled atabout 30 to 40 h of incubation in salicylate-amended soilwere relatively low, yet highly active. In contrast, samplestaken 48 h later (after salicylate had been consumed) con-tained larger but inactive populations. Although it is impor-tant to be able to increase the population size of organisms ofinterest such as a biocontrol agent, the critical factor iswhether the organism is active at the same time and locationas the pathogen. Other studies using methods to detectrespiring bacteria from environmental samples, such as with

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FIG. 4. Effect of induction of salicylate-degradative enzymes on

respiration rates (A) and population densities (B) of PpG7 insalicylate-amended (1,700 ±g/g) or nonamended Davis soil. Degra-dative enzymes were induced by growth of PpG7 on culture mediumcontaining 10 mM sodium salicylate. Mean values are shown forinduced PpG7 in nonamended soil (El), induced PpG7 in salicylate-amended soil (-), noninduced PpG7 in nonamended soil (0), andnoninduced PpG7 in salicylate-amended soil (0). Bars representstandard errors of the means where variation was great enough to bepresented.

redox dyes, also indicate that the active portion of a bacterialcommunity may vary markedly from the total or recoverableportion (17, 21).The initial suppressive effects of salicylate in soil at

concentrations of >1,500 ,ig/g were unexpected since 1,700,g/g was the optimum concentration for growth on culturemedia (data not shown). Suppression of activity in soil wasevident as a lag in respiration and population increase andreduced utilization of [14C]glucose by PpG7 and indigenousmicroorganisms in Davis soil. Altnough it has been sug-gested that induction of carbon-source-degradative enzymesmay reduce the incubation time before onset of utilization ofsome substrates (22), this strategy was not effective inreducing the lag time before utilization of salicylate by PpG7.Since the solution of salicylate added to soil was adjusted topH 7 and was only 0.04 M with respect to sodium, thereshould not have been a direct effect of pH or sodium. Mostlikely, the suppression of activity at higher concentrationswas caused by the salicyl structure itself since salicylate hasbeen shown to affect the structure and function of cellmembranes (9, 22). This initial suppression at high salicylateconcentrations may be detrimental when the target bacte-rium is intended to interact with a rapidly infecting plantpathogen such as P. ultimum, which can infect germinatingseeds within 3 h (20).

2 4 6 8Incubation time (hours)

FIG. 5. Mean rate of glucose utilization in Davis soil amendedwith ['4C]glucose (100 ptg/g; 0), [14C]glucose and salicylate (1,700,ug/g; A), [14C]glucose and PpG7 (104 CFU/g; x), and [14C]glucose,salicylate, and PpG7 (E). Bars represent standard errors of themeans where variation was great enough to be presented.

The utilization of selective substrates by bacteria has beenstudied by researchers in the field of bioremediation (16, 23),but the emphasis has usually been on the removal of acarbon substrate rather than on following the populationdynamics of a specific strain. Focht and Shelton (7) demon-strated that growth of Pseudomonas alcaligenes in cultureand soil was similar in the presence of the 3-chlorobenzoatesubstrate. They concluded that a nonindigenous bacterialstrain could successfully occupy an open metabolic niche,even when introduced at very low initial inoculum densities(100 cells per g of soil). Although growth of PpG7 in vitro andthat in vivo were not directly compared in our experiments,growth supported by salicylate in soil (1,700 ,ug/g) and onsolid media (1,700 ,ug/ml) occurred during approximatelyequal time periods (about 2 to 3 days). PpG7 exploited theopen metabolic niche caused by the addition of salicylate tosoil, despite fungal competition.The benefit of selective feeding of rhizosphere bacteria

was reported by Lam et al. (14), who used combinations ofisogenic mannopine-producing and nonproducing tobaccoplants and mannopine utilizing and nonutilizing P. putida.The mannopine-utilizing pseudomonad was selectively fa-vored over the nonutilizer in the rhizosphere of the man-nopine-producing tobacco plant. The addition of salicylate tothe rhizospheres of tomato plants had a similar effect on theintroduced bacterium P. putida PpG7 in field experiments(5).

Ogunseitan et al. (19) reported that 160 ,ug of salicylate perg of soil sustained population densities of PpG7 for 30 dayswhen applied at 10 CFU/g in polluted soil. They alsodetected the presence of nahAB transcripts after 30 days ofincubation. However, the level was low since a 6-dayautoradiographic exposure was required to detect a signal.Although the presence of mRNA indicates microbial activ-ity, no quantitative measurements were made between ac-tivity and densities. It is difficult to compare their resultswith ours with respect to metabolic activity and populationnumbers since they did not specifically distinguish betweeninoculated strains and indigenous naphthalene degraders ormeasure activity over a series of time periods. Our dataresulting from the use of nonpolluted agricultural soils indi-cated that 160 ,ug of salicylate per g of soil would have beenconsumed within 24 to 48 h by an initial bacterial population

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4 orders of magnitude smaller than that used by Ogunseitanet al. (19) and that any significant amount of metabolicactivity would have stopped by then. A limiting factor of theuse of mRNA transcripts to measure metabolic activity of abacterium in soil is the relatively high density of cells pergram of soil that is presently required for detection (about1.6 x 106) (28). Also, with respect to examining and com-paring the population dynamics of individual organisms,recognition should be given to the importance of the initialinoculum density. For example, leaves, seeds, and soil havea finite carrying capacity. There is little room to detect thedynamics of growth or to determine the effects of variousenvironmental parameters on growth patterns when veryhigh inoculum densities are used in the experimentation.The effect of salicylate on population densities and meta-

bolic activities of PpG7 was proportional to its concentrationover a range of 30 to 1,500 ,ug/g of soil, with the resultingpopulation densities ranging from 101- to 105-fold greaterthan those in nonamended control soil. Although the re-sponses varied somewhat with soil type, it was particularlyencouraging that the addition of even small amounts ofsalicylate (30 to 50 ,ug/g of soil) caused significant increasesin population densities of the target bacterium. This suggeststhat the practical use of selective substrates to affect theactivities of specific groups of organisms in commercial andnatural settings is possible. The small increase in bothrespiration and population sizes that occurred in soilamended with water alone were expected because of therelease of nutrients associated with the drying and rewettingof soil (8).

Salicylate is an excellent selective carbon source for PpG7because it supports rapid population increases and inducesenzymes for its own degradation (30) and its utilization is notsuppressed by other simple carbon source compounds (24).In addition, we did not detect indigenous bacterial utilizersof salicylate in the soils that were tested, although fungalcompetition for salicylate was evident. Fungal competitiondid not preclude population increases by PpG7 in responseto salicylate addition. The genera of salicylate-utilizing fungiencountered were Fusarium, Aspergillus, and Penicillum(data not shown). While no known plant pathogens weredetected, there is a need for further study to ensure thatpopulations of deleterious fungi will not increase in the field.

Strain PpG7 was easily identified in the population studiesby the distinctive growth pattern and morphology of itscolonies on minimal medium. Phenotypic tests further con-firmed its identity. The common procedure of using rifampinresistance as a marker was not employed since all markedstrains differed from the wild type in growth. There also wasno evidence for the occurrence of plasmid transfer. AlthoughNAH7 is a conjugative plasmid, we have found that thefrequency of transfer to a number of different recipientsunder various conditions was low (>10-8 per donor) or didnot occur (results not shown). Similar results were obtainedby Dunn and Gunsalus (6; personal communication).The strategy of providing a food base which can be

selectively utilized by organisms appears to be a practicalway of increasing their activities and population sizes in soil.However, the results of this study show that it will benecessary to provide a continuous source of substrate toprolong activity since population size alone may be of littleimportance. Thus, it is envisioned that in the future, espe-cially in the field of biological control of pests, inoculum willbe accompanied with formulations of slow-release com-pounds to extend activity for longer periods of time. Al-though many beneficial organisms do not have known car-

bon source utilization characteristics that could beexploited, such traits could conceivably be genetically engi-neered into them.

ACKNOWLEDGMENTSWe thank Paul Brooks and Mary Firestone (Department of Soil

Science, University of California at Berkeley) for technical assis-tance and advice.

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