Pergamon 003%0717(95)00020-8

Soil Biol. Biochm~. Vol. 27. No. 7. 881-892, pp. 1995 Copyright c, 1995 Elsevier Science Ltd

Printed in Great Britain. All rights reserved 0038-0717/95 $9.50 + 0.00

MATRIC POTENTIAL AND THE SURVIVAL AND ACTIVITY OF A PSEUDOMONAS FLUORESCENS INOCULUM IN SOIL

AUDREY MEIKLE,‘* SOHEILA AMIN-HANJAN&‘* L. ANNE GLOVER,’ KENNETH KILLHAM and JAMES I. PROSSER’t

Departments of ‘Molecular and Cell Biology and lPlant and Soil Science, University of Aberdeen, Marischal College, Aberdeen AB9 1 AS. Scotland

(Accepted 12 January 1995)

Summary-The survival and activity of a strain of Pseudomonasjuorescens, chromosomally marked with lux genes, was studied following inoculation into microcosms containing autoclaved or non-autoclaved soil adjusted to matric potentials of - 30, - 750 and - 1500 kPa and incubated for up to 3 months. Viable cell concentrations were determined by dilution plate counting and population activity was measured by luminometry, radiorespirometry and dehydrogenase activity. Although increased matric stress appeared to reduce survival, measured by viable cell concentration, effects were not statistically significant (P > 0.05) when variability within replicates was taken into account. Similarly, when all data were taken into account, changes in via.ble cell concentration were statistically insignificant (P > 0.05) during the incubation. Survival was, however, significantly (P < 0.05) greater in autoclaved soil at - 30 and - 750 kPa, presumably because of reduced competition and predation. Survival at - 1500 kPa was not affected by autoclaving of soil, possibly due to reduced protozoan mobility at this matric potential. Metabolic activity measured by luminescence decreased significantly with time (P c 0.05) and was a sensitive indicator of differences in matric potential, autoclaving and the duration of the incubation. Luminescence following amendment with substrates (potential luminescence) demonstrated the time required for activation of the starved inoculum. Reactivation decreased greatly with prolonged starvation and was not detectable in non-autoclaved soil at - 1500 kPa after 28 days. Potential luminescence also indicated the amount of biomass capable of reactivation, which decreased with starvation. Luminescence was more sensitive, reproducible and convenient as a measure of population activity than radiorespirometry and dehydrogenase activity and, in non-autoclaved soil, enabled selective assessment of inoculum activity in the presence of the indigenous microflora. In addition, luminescence was the only activity technique which demonstrated the effects of matric potential and time on population activity. Our study demonstrates the use of luminescence-based marker systems in tracking genetically-modified bacteria in soil, and in selective and sensitive assessment of their metabolic activity. It also highlights the importance of measurement of both viable cell concentration and population activity when assessing the potential environmental effects and efficiency of microbial

IINTRODUCITON

Assessment of the efficiency of microbial inocula, and determination of potential risks to the target ecosystem, requires knowledge of the effects of environmental factors on microbial survival, activity and dispersal in the soil. These requirements have increased because of the potential for commercial application of genel:ically modified microorganisms in agriculture and the environment. The majority of risk assessment studies have involved determination of microbial survival by enumeration of viable popu- lations following cell extraction and cultivation on laboratory media. Such techniques do not consider differences in extractability of cells, through differen- tial adhesion to soil particles, cell death associated with extraction an.d the formation of dormant or

*Present address: Horticulture Research International, Worthing Road, Littlehampton, West Sussex BN 17 6LP, England.

tAuthor for correspondence.

non-culturable cells. The use of molecular-based detection techniques and marker genes solves some of these problems. For example, in the absence of gene transfer, PCR amplification and probing for specific marker genes enable detection of non-culturable cells, although quantification by PCR amplification is difficult and clay and humic components interfere strongly with the PCR reaction (Steffan and Atlas, 1991). Enumeration of viable marked cells in the soil is also possible. For example, antibiotic resistance has enabled selective enumeration of Pseudomonas

_&orescens (van Elsas et al., 1986b) and .uylE- marked P. putidu (Macnaughton et al., 1992), while enumeration of IacZY-marked pseudomonads is possible following culture on minimal lactose media supplemented with the chromogenic X-gal (Drahos et al., 1986).

While marker techniques increase risk assessment capability in terms of enumeration of viable and non-culturable cells, they provide little information on the activity of the introduced organisms or the effects

881

882 Audrey Meikle et al.

ofenvironmental factors on this activity. This prevents reliable assessment of the potential effects of inocula on the target ecosystem. Luminescence-based marker systems enable assessment of viable and non-cultur- able cells without the need for extraction of cells, and additionally provide information on their metabolic activity. Viable cell enumeration, in the absence of antibiotic selection enables detection of 1 cell in a background indigenous population of 3 x lo-’ cells (Grant et al., 1991) and sensitivity is increased with simultaneous use of antibiotic selection. Lumines- cence, measured by luminometry, is an indicator of the metabolic activity of the marked population in soil (Meikle et al., 1992, 1994). The technique is rapid, sensitive, correlates closely with other measures of activity and is selective in assessing the activity of the marked population only, against a background of indigenous organisms. In addition, charge-coupled device (CCD) imaging provides information on the spatial distribution of individual cells (Silcock et al., 1992) and of colonies (Shaw et al., 1992; Waterhouse el al., 1993).

Commonly used methods for assessment of the activity of microbial populations in soil include respirometry, adenylate energy charge, heat and substrate disappearance (Killham, 1985) and measure- ment of enzyme activity (Benefield et al., 1977). The activity of individual cells may be determined using microscopic techniques, involving vita1 stains such as acridine orange (Swannell and Williamson, 1988) and fluorescein diacetate (Soderstrom, 1977), and microautoradiography. We report the use of lux- modified cells in determining the influence of matric potential and the indigenous soil microflora on the survival and activity of inoculated populations of P. jfuorescens, using both commonly used and lumines- cence-based techniques. Matric potential was studied as this was considered to be a major factor in survival of microbial inocula in soil. Matric stress has a significant effect on microbial activity (Wilson and Griffin, 1975) and antecedent matric potential is important in determining the pore location of inoculated cells within the soil matrix (Postma and van Veen, 1990).

MATERIALS AND METHODS

Bacterial strains and culture conditions

All experiments involved the use of P. fruorescens 10586s/FAC510 which contains chromosomally en- coded genes for kanamycin resistance and IuxAB, the structural genes for luciferase, originally cloned from Vibriojscheri. Luciferase production by this strain is constitutive and full details of its construction and light emission characteristics are given by Amin- Hanjani et al. (1993). Routine growth and cultivation were carried out at 30°C in L-broth (tryptone, 10 g; NaCl, 5 g; yeast extract, 5 g; glucose, 1 g; distilled water, 1 litre; pH 7).

Soil microcosms

Soil microcosms consisted of Universal bottles containing 12 g (air dried weight) Craibstone soil (Countesswells series; sandy loam; cation exchange capacity, 7.4 cmol kg- ‘; organic matter, 4.25%) amended with Ca(OH)? to give a final pH value of 7. The initial pH of the soil was 6.46 (in water) and 5.38 in CaC12. Soil pH was adjusted to 7 to provide environmental conditions more favourable for growth and survival of P.Jlu0rescen.s. Bottles were sealed with steri-stoppers to facilitate gaseous diffusion and to prevent OX limitation. Soil was sterilized, when required, by autoclaving for 1 h at 121°C on 3 consecutive days. Bacterial inocula were prepared from exponentially-growing cells of P. Jluorescens, washed 3 times and resuspended in sterile phosphate buffer (15 mM, pH 7) and grown overnight at 25°C. I$crocosms were inoculated with volumes of cell suspension, evenly dispersed, to enable equilibration at soil matric potentials of -30, -750 and - 1500 kPa, with a final cell concentration of approximately 108 cells g - ’ soil in all cases. These matric potentials were equivalent to moisture contents of 0.22,0.070 and 0.041 ml water g- ’ air dried soil. A matric potential of - 30 kPa was considered optimal for microbial growth and activity, - 1500 kPa is wilting point and - 750 kPa was chosen as a convenient intermediate. Following inoculation, microcosms were kept for up to 3 months at 25”C, and periodically triplicate microcosms were destructively sampled for each of the three matric potentials for estimation of viable cell concentration, luminescence, dehydrogenase activity and radiorespirometry. Matric potentials were main- tained by additions of water lost through evaporation. Experiments involving sterile soil were terminated if and when soil became contaminated. In a separate parallel experiment, the above procedure was repeated except that soil was sterilized (121”C, 1 h), after addition of Ca(OH)? and 1 ml of distilled water. This procedure was repeated twice, replacing water lost during autoclaving to give a final matric potential of -30 kPa.

Determination of viable cell concentration

Serial dilutions were prepared from a suspension of 1 g soil in 9 ml phosphate buffer (15 mM, pH 7), shaken for 10 min using a Stuart wrist-action shaker. Samples from appropriate dilutions were spread plated, in triplicate, on L broth containing 25 mg kanamycin, ampicillin and spectinomycin ml- ’ and 0.1 mg cycloheximide ml-‘, solidified by addition of 1.5% (w/v) Technical No. 3 agar (Oxoid). Colonies were enumerated after 48 h at 30’C.

Luminescence and potential luminescence

Luminescence was determined in soil suspensions consisting of either 0.5 g soil suspended in 1 ml phosphate buffer or 1 g soil suspended in 10ml phosphate buffer. N-Decyl aldehyde (1 ~1) was added

Survival of P. juorescens in soil 883

and luminescence was measured after 4 min using an LKB 125 1 Lumirmmeter. Luminescence measure- ments were carried out in triplicate for each of 3 microcosm samples, and light output was integrated over a period of 10 s. Results were expressed as relative light units (RLU). A suspension of un- inoculated soil was used as a blank. Potential luminescence was determined in triplicate 1 g soil samples as describsed by Meikle et al. (1994). Soil suspensions were prepared by shaking (Stuart wrist-action shaker) soil in 9 ml phosphate buffer for 10 min. A sample (D.5 ml) of the soil suspension was then mixed with 1 ml double-strength 523 medium (Kado et al., 1972) supplemented with 10 mg sodium citrate ml-‘. Luminescence was measured every 15 min for 2 h, as described above, to determine the kinetics of luminescence activation, and final potential luminescence (PL,) values were determined as luminescence, measured in RLU, at the end of the 2 h incubation.

Dehydrogenase activity

Dehydrogenase activity was measured using a modification of the method of Benetield et al. (1977). Duplicate 4 g soil samples were placed in sterile Universal bottles containing 4 ml phosphate buffer (20 mM, pH 7.7) and 400 ~1 filter sterilized aqueous INT [2-(4-iodophenyl)-3-(4-nitrophenyl)-5 phenyltetrazolium chloride] solution (0.2%, w/v). Soil suspensions were then kept in the dark for 6 h at 30°C after which, following extraction with I-pentanol, formazan concentration was determined spectro- photometrically at 490 nm.

Radiorespirometry

Respiration was measured as YO2 evolved during utilization of [U-Wlglucose. Soil samples (1 g), and a control of autoclavcd uninoculated soil, were placed in thick walled acid-washed Universal bottles and [Y]glucose (5 &i) was added with sterile unlabelled glucose solution to give a final concentration of 50 pg glucose g- ’ soil. CO2 evolved during incubation for 1 h at room temperature was trapped in 0.1 M KOH and 14C activity measured on an LKB Rackbeta scintillation counter. Counts of CO2 evolution were converted to rates using 14C bicarbonate standards in a 0.1 M KOH matrix.

Statistical analysis

Each experiment was duplicated. Data were analysed statistically by carrying out a three- way ANOVA, with time, matric potential and autoclaving as factors, and three sets of two-way ANOVAs to examine in more detail the effects of individual factors. Lower detection limits for viable cell concentration and luminescence were 0.5 cells g- ’ ovendry soil (0.d.s.) and 0.5 RLU g-’ o.d.s., respectively.

RESULTS

Changes in viable cell concentration and PL,

Autoclaved soil. Following inoculation into auto- claved soil at -30 kPa, viable cell concentration of P. jluorescens decreased to 2.6 and 16% of initial values after 83 and 92 days, respectively, in duplicate experiments [Fig. l(a)]. Viable populations were therefore still large after prolonged starvation in autoclaved soil at this matric potential, with cell concentrations of 6.13 and 503 x lo6 cells g-l soil in duplicate experiments. PLr decreased significantly during starvation in autoclaved soil [Fig. l(a)], from 2.6 x lo4 at day 0 to 14 RLU g-’ soil after 71 days. In samples taken after this time, PLr was undetectable.

Viable cell concentration decreased at a greater rate during starvation at a matric potential of -750 kPa [Fig. l(b)]. After 21 days, viable cell concentration had decreased by 2 orders of magnitude, with a further lo-fold decrease by 45 days. PLr values in samples taken at day 0 were similar to those at -30 kPa but decreased at a faster rate, reaching 25% of those at the higher matric potential after 27 days [Fig. l(a)]. At this matric potential, viable cell concentration and PLr varied in a similar manner. PLr values in one experiment decreased by 2 orders of magnitude within 21 days, with a further decrease by day 30. A similar decrease in PL[ occurred in the second experiment within 14 days, with a subsequent reduction below the limit of detection. In general, changes in viable cell concentration and PLr were greater at - 1500 kPa than at - 30 and - 750 kPa [Fig. l(c)]. Both viable cell concentrations and PL, decreased by 2 orders of magnitude between days O-2 1. PLr values at day 0 were 1 order of magnitude lower than those at -30 and - 750 kPa and fell below the level of detection after 14 and 27 days in the 2 replicates.

Non-autoclaved soil. At -30 kPa, the viable cell concentration decreased by 3 orders of magnitude by days 27 and 30 in the two experiments [Fig. 2(a)]. In one experiment, the cell concentration subsequently decreased to 1.3 x lo4 cells g - ’ soil by day 71, after which enumeration was not possible due to growth of non-marked organisms on selective media. This growth occurred despite the presence of three selective antibiotics. Naturally-occurring resistant organisms were present at concentrations of approximately 10) cells g- ’ soil and increased the lower limit of detection of the inoculum. PLr values decreased by 2 orders of magnitude by 22 days [Fig. 2(a)]. Luminescence in samples taken subsequently was below the level of detection after incubation in the presence of substrate for 2 h, but was detectable after incubation for a total of 24 h. PLr in autoclaved soil did not decrease below the level of detection until 92 days.

Following inoculation of non-autoclaved soil at - 750 kPa, viable cell concentration and PL( varied in a similar manner [Fig. 2(b)]. Viable cell concentration decreased by 2 orders of magnitude after 21 days, approximately lo-fold less than in autoclaved soil at

884 Audrey Meikle er al.

- 750 kPa and in non-autoclaved soil at - 30 kPa at concentrations in autoclaved soil and similar to this time. Numbers fell below detection limits by days those in non-autoclaved soil at -30 and -750 kPa. 28 and 45 in both experiments. PLr values from Reliable estimation of viable cell concentration samples taken immediately after inoculation were 1 was not possible in subsequent samples due to order of magnitude less than equivalent samples of similar concentrations of marked and non-marked non-autoclaved soil at -30 kPa and decreased by 3 organisms. PLr values in samples taken immediately orders of magnitude by day 14 in one experiment, after inoculation were similar to those in autoclaved subsequent samples falling below the level of soil at - 1500 kPa but less than those in detection. In the second experiment, PLr values non-autoclaved soil at - 30 and - 750 kPa. PLr values became undetectable after 28 days. then decreased by 2 orders of magnitude by 21 days

Viable cell concentration at - 1500 kPa decreased and subsequently fell below the level of detection. by 3 orders of magnitude by 30 days. Concentrations These changes were similar to those observed in at this time were higher than equivalent viable autoclaved soil.

1J3+07

1E+O6

m+05

1E+aI

1E+O3

lE+O2

lE+Ol

lB+oo

04 _ 1E+O6

- lE+O5

- 1E+o4

- lE+O3

- lE+02

- lE+Ol

1E+04t lE+OO 0 20 40 60 80

1B+O7

lE+O6

lE+05

lE+O4

lE+O3

lE+O2

lE+Ol

0 20 40 60 80 lE+OO

Fig. 1. Changes in viable cell concentration (0, 0) and PLI value (0. ??) in autoclaved soil microcosms inoculated with P.j?uorescens 10586s/FAC510 and adjusted to a matric potential of (a) - 30 kPa, (b) - 750

kPa and (c) - 1500 kPa. SEs for viable cell concentration and PLf were < 35% of the mean.

Survival of P. fluorescens in soil 885

lE+ 101 lE+O7

lE+O6

lE+OS

lE+O4 lE+ 03

lE+ 02

lE+ 01

lE+O4t 4 I I , I J 0 20 40 60 80 lE+OO

-750 kPa

6 - lB+03

-KI

hQ

.1E+O2 nt

.1B+01 1s3+04)- L I I I

n 317 dn brt sn lE+OO

lE+O4

lE+O3

lE+O2

ra+Ol

Fig. 2. Changes in viable cell concentration (0,O) and PLrvalue (0, ??) in non-autoclaved soil microcosms inoculated with P.Jluorescens 10586s/FAC510 and adjusted to a matric potential of (a) - 30 kPa, (b) - 750 kPa and (c) -- 1500 kPa. SEs for viable cell concentration and PLr were < 25% of the mean at - 30 kPa

and ~40% of the mean at lower matric potentials.

Three-way ANOVAs indicated that matric poten- which significantly affected viable cell concentration tial had no effect on viable cell concentration when the was autoclaving soil prior to inoculation which, factors of time and soil autoclaving were combined as indicated in Figs 1 and 2, led to a significantly (Table 1) (5% level of significance). Data in Figs 1 and greater decrease in viable cell concentration. None of 2 suggest a greater decrease in viable cell concentration the interaction terms was significant for viable cell at - 750 and - 1500 kPa than at - 30 kPa, and the concentration. Two-way ANOVAs (Table 2) indi- lack of statistical significance may have been due to cated significant effects of time and autoclaving at variability between duplicate experiments and vari- -750 kPa, but not at the other matric potentials ability within replicate viable cell counts. Similarly, studied. Viable cell concentrations did not vary time did not affect viable cell concentration when data significantly with time or matric potential when data from all matric potentials and autoclaved and on autoclaved soil were combined and, in non- non-autoclaved soil were combined. The only factor autoclaved soil, only showed significant variation with

886 Audrey Meikle et al.

Table I. Valuesofprobabilitiescalculated using three-way ANOVAs toassess theeffectsoftime. matric potential and autoclaving soil on viable cell concentration, PLr and luminescence in soil microcosms

inoculated with P.~uorescerrs 10586s/FAC510

Viable cell concentration PLI Luminescence

Time 0.034 <O.OOl 0.001 Matric potential 0.604 0.004 <O.OOl Autoclaving 0.013 0.410 0.035 Time x matric potential 0.881 0.263 0.002 Time x autoclaving 0.986 0.866 0.891 Matric potential x autoclaving 0.723 0.192 0.098 Time x matric potential x autoclaving 0.995 0.465 0.917

time. When data from all times were combined, autoclaving soil had a significant effect on viable cell concentration, but there was no significant effect of matric potential.

Changes in population activity

Population activity was assessed by luminescence in the absence of substrate amendment, dehydrogenase activity and radiorespirometry. In addition, changes in luminescence during incubation with substrate for 2 h provided information on the kinetics of activation of the starved population. In autoclaved soil, all three techniques measured the activity of the marked population. In non-autoclaved soil, luminescence measured the activity of the marked population while dehydrogenase activity and radiorespirometry measured the activity of the total microbial population.

Autoclaved soil. In autoclaved soil, inoculated at - 30 kPa, luminescence decreased gradually with time, values at 92 days being approximately 2 orders of magnitude lower than those in samples taken immediately after inoculation [Fig. 3(a)]. Both respiration and dehydrogenase activity increased immediately after inoculation and decreased to a minimum at 42 days, although other trends in behaviour were difficult to discern. At -750 kPa, values obtained from all three techniques immediately after inoculation were not significantly different (P > 0.05) from those at -30 kPa [Fig. 3(a,b)]. Respiration and dehydrogenase activity did not show

initial increases, decreased to low rates by 14 days, increased up to 28 days and subsequently decreased. Luminescence also decreased rapidly, falling below 1 RLU g- ’ soil between days 28 and 45, but was still detectable at 0.3 RLU g - ’ soil after 70 days. At - 1500 kPa, luminescence, respiration and dehydrogenase activity were low at day 0 and remained low until day 30 when they became undetectable, except for 1 replicate where respiration was still detectable at day 50 [Fig. 3(c)].

Non-autoclaved soil. Experiments in non-auto- claved soil were terminated after 48, 70 and 49 days for - 30, - 750 and - 1500 kPa, respectively. At - 30 kPa, luminescence declined rapidly and was not detectable after day 12 [Fig. 4(a)], whereas in autoclaved soil at -30 kPa, activity was detectable after 92 days. Similar decreases in luminescence were observed in non-autoclaved soil at the lower matric potentials [Fig. 4(b, c)], although low but measurable amounts of luminescence were maintained until days 70 and 30 at -750 and - 1500 kPa, respectively. Dehydrogenase activity in non-autoclaved soil at - 30 kPa increased initially and then decreased to the initial rate for the remainder of the experiment [Fig. 4(a)]. This general pattern was also observed at the lower matric potentials (i.e. drier soil) [Fig. 4(b, c)]. Similar changes were observed in respiration [Fig. 4(a-c)], although initial increases were less than those in dehydrogenase activity.

Three-way ANOVAs indicated luminescence to be the most sensitive indicator of effects of time, matric

Table 2. Values of probabilities calculated from three sets of two-way ANOVAs to assess the effects of time, matric potential and autoclaving soil on viable cell concentration and

PL, in soil microcosms inoculated with P. hmww~s 10586s/FAC510

Non-autoclaved

-30 kPa

Effect of

Time

Effect on Viable cell concentration Luminescence

0.957 0.006

- 750 kPa

- 1500 kPa

Autoclaved

Non-autoclaved

All times

Autoclaving 0.122 Time 0.033 Autoclaving 0.001 Time 0.409 Autoclaving 0.259 Time 0.691 Matric potential 0.954 Interaction 0.760 Time 0.197 Manic potential 0.472 Interaction 0.849 Autoclaving 0.006 Matric potential 0.923

0.073 0.024 0.008 0.005 0.567 0.180

<0.001 0.095 0.028 0.001 0.049 0.027

<O.OOl 0.070 Interaction 0.773

Survival of P. fluorescens in soil 887

I O-l0

I 20 40 60 80 ‘All

6 1 I 20 40 60 80 ‘AI

arO 1 20 40 1 60 I 80 I I ‘Ol Time (days)

Fig. 3. Changes in luminescence (A), dehydrogenase activity (*) and respiration rate (A) in autoclaved soil microcosms inoculated with P.JIuorescens 10586s/FAC510 and adjusted to a matric potential of (a) -30 kPa, (b) -750 kPa and (c) - 1500 kPa. SEs for luminescence, dehydrogenase activity and respiration rate

were <30%, ~58% and ~75% of the respective means.

potential and autoclaving of soil on the marked organism (Table 1). Significant (P < 0.005) effects on luminescence were found with all three factors, when all data were combined, and interaction effects were also observed with time and matric potential. Three sets of two-way ANOVAs (Table 2) indicated significant (P < 0.05) effects for all but four combinations. These were the effect of autoclaving at - 1500 kPa, the effect of time and the time-matric potential interaction in autoclaved soil and the interactive term when data from all times were combined. Data on dehydrogenase activity and

respiration were analysed using two ANOVAs for autoclaved and non-autoclaved, as these treatments examined activity of different populations (Table 3). In autoclaved soil, dehydrogenase activity and respir- ation were significantly affected by matric potential but not time. In non-autoclaved soil, dehydrogenase activity was affected significantly by time and matric potential, while respiration was only affected by matric potential.

Potential luminescence was measured for 2 h following nutrient amendment and changes in final luminescence values have been discussed. Information

888 Audrey Meikle et al.

40 60 80 ‘Ob

Time (days)

Fig. 4. Changes in luminescence (A), dehydrogenase activity (*) and respiration rate (A) in non-autoclaved soil microcosms inoculated with P.@orescens 10586s/FAC510 and adjusted to a matric potential of (a) - 30 kPa, (b) - 750 kPa and (c) - 1500 kPa. SEs for luminescence, dehydrogenase activity and respiration

rate were < 30%, < 29% and < 56% of the respective means.

on the rate of activation of luminescence-marked on days 0,4,9 and 28. In all cases, luminescence was populations can be obtained from changes in greater for cells subjected to lower matric stress and luminescence during the incubation period and data was higher in autoclaved than in non-autoclaved soil. are presented in Fig. 5 for representative samples taken Initial luminescence values for samples taken on day

Table 3. Values of probabilities calculated from two sets of two-way ANOVAs to assess the effects of time and matric potential on dehydrogenase activity and respiration in autoclaved and non-auto&wed soil microcosms

inoculated with P.~7uorescens 10586s/FACSIO Autoclaved Non-autoclaved

E&t of Dehydrogenase activity Respiration Dehydrogenase activity Respiration

Time 0.865 0.844 <O.OOl 0.597 Matric potential 0.004 <O.OOl 0.010 <o.OOt Interaction 0.685 0.399 0.040 0.166

Survival of P. Juorescens in soil

Fig. 5. Changes in luminescence in soil samples following nutrient amendment. Samples were taken after (a) 0, (b) 4, (c) 9 and (d) 28 days from autoclaved soil at - 30 kPa (O), - 750 kPa (A) and - 1500 kPa (0) and from non-autoclaved soil at -30 kPa (W), - 750 kPa (A) and - 1500 kPa (a). SEs for

luminescence were ~25% of the mean.

0 from soil at -30 and -750 kPa were close to the maximum. There was little subsequent change in luminescence at -30 kPa, but increases occurred during the incubation period at -750 kPa. In autoclaved and non-autoclaved soil at - 1500 kPa and in non-autoclaved sloil at - 750 kPa, populations had been inactivated immediately following inoculation and luminescence increased reaching a maximum at 90 min. After starvation for 4 days, the effects of matric potential and the indigenous microflora were more apparent. At .- 30 kPa in autoclaved soil, initial luminescence was lower than that at day 0 but activation occurred rapidly, reaching a maximum, equal to that at day 0, by 30 min. In samples of autoclaved soil at -750 kPa and non-autoclaved soil at -30 and -750 kPa, initial luminescence was significantly lower than at day 0, but increases occurred at -30 klPa during the incubation period, indicating activation of the population. No activation was observed in samples of autoclaved soil at - 1500 kPa, although initial luminescence values were higher than those in non-autoclaved soil. In samples of autoclaved soil at - 1500 kPa, luminescence increased from negligible levels within 15 min and then increased slowly, although all values were close to the detection limit. Similar effects were seen at all matric potentials after starvation for 9 days, with further decreases in initial luminescence. At 28 days, luminescence could only be measured in autoclaved soil at - 30 and - 750 kPa and non-autoclaved soil at -750 kPa and activation was only detectable in soil at - 30 kPa.

Soil sterility

During the course of our experiments, it became apparent that the soil sterilization procedure was not completely effective, as a bacterial contaminant appeared on antibiotic selective medium following plating of samples from autoclaved soil microcosms. Although the contaminant was present at low initial concentrations, a study was carried out to eliminate it completely and to determine whether survival and activity were unchanged in its absence. The original sterilization procedure involved autoclaving air-dried soil at 12 1 “C for 1 h on three separate occasions, with subsequent addition of water to achieve the required matric potential. It was presumed that the contami- nant was surviving through incomplete penetration of heat due to the low water content of the soil and poor diffusion of steam into the soil. To correct this problem, water was added to the soil prior to autoclaving, and water lost during autoclaving was replaced, either as distilled water (between successive autoclavings) or by addition of the inoculum cell suspension. This study was carried out at -30 kPa only and changes in viable cell concentration and luminescence are shown in Fig. 6. Viable cell concentration increased initially, through growth on material released during autoclaving, and then decreased at a similar rate to that described above [Fig. l(a)]. Luminescence also decreased as described above for autoclaved soil, reaching low but detectable values until the study was terminated at 130 days.

890 Audrey Meikle et al.

6. Changes in viable cell concentration (0) and luminescence (A) in sterile soil microcosms inoculated I P. fruorescens 10586s/FAC510 and adjusted to a matric potential of - 30 kPa. SEs for viable cell

concentration and PLr were ~25% of the mean.

Fig. with

Neither the rates of decrease in viable cell concen- tration nor luminescence values were different to those observed in autoclaved soil and the amount of contamination is therefore considered to have been too low to have affected survival or activity of the inoculum.

DISCUSSION

Comparison of techniques used to measure activity

Viable cell enumeration is the major technique used for assessment of survival and persistence of microbial inocula in soil. The use of molecular marker systems has enabled selective enumeration and tracking of inocula but such systems still suffer from the many disadvantages of techniques requiring cell extraction and subsequent laboratory culture (Prosser, 1994). Of particular significance for risk assessment studies is the inability of most marker systems, with the exception of bioluminescence-based systems, to provide any indication of the environmental effects of inocula, mediated through population activity. In our study, three activity techniques were compared, lumines- cence, dehydrogenase activity and radiorespiration. None involved amendment of soil with significant quantities of nutrients, and all were therefore designed to measure activity through utilization of nutrients available in the soil, or endogenous metabolism, as opposed to potential activity. Comparison of these techniques was facilitated in autoclaved soil microcosms, where the inoculated microorganism, P.Jluorescens, dominated. Luminescence was the most

rapid, most convenient and cheapest method. It did not always correlate with dehydrogenase activity and respiration, which frequently did not correlate with each other. This could be due to their measuring different types of activity. Luminescence showed less variability between replicates than dehydrogenase and respiration, and was more sensitive. The last two techniques were approaching their lower limits of detection, despite use of high cell concentrations, because population activities were low. Correlations of, for example, matric potential with luminescence, could therefore be considered most reliable and highlighted effects of environmental factors which were not detected by the other activity techniques.

Potential luminescence provided a valuable indi- cation of the biomass of the marked population capable of activation following nutrient amendment and the rate at which activation occurs. The 2 h incubation was found to be optimal on the basis of studies in liquid culture (Meikle et a/., 1994), longer incubation periods leading to growth, but was also thought to be appropriate for environmental studies. For a starving organism to utilize nutrients when they become available, it must be capable of rapid activation to prevent utilization by competitors. If activation takes longer than 2 h, under optimal growth conditions, the organism is unlikely to benefit from the increased nutrient supply. Potential luminescence measured under these conditions is therefore likely to be of relevance to studies in which the response time is important. Activation of cells starved for prolonged periods was shown to continue well beyond 2 h. Values of PLr should therefore not be considered directly

Survival of P. @orescens in soil 891

equivalent to the viable biomass or viable cell concentration of the marked organism and did not always correlate with viable cell concentrations determined by growth on media for 48 h.

Effects of matricpotential and the indigenous microflora

Survival of P. juorescens, measured as viable cell concentration, was not greatly affected by matric potential, despite the known effects of matric stress on cell physiology and nutrient diffusion. This contrasts with studies on effects of matric stress on survival of Escherichia cob in 1:he soil (Rattray et al., 1990) in which significant loss in cell viability was observed. This may be due to dlifferences in soil properties, as the soil used in our study had a greater clay content, which has been implicated in increased survival of P.

JItlorescens (van Elsas et al., 1986a; Wessendorf and Lingens, 1989). In a’ddition, tolerance to matric stress is known to vary between microorganisms (Wilson and Griffin, 1975) and E. colt’, whose natural habitat is not the soil, may be more susceptible to matric stress than P.fluorescens. The major factor affecting viable cell concentration was the presence of the indigenous microflora, presumably through competition and predation. In addition, decreases in viable cell concentration during long-term studies may be due to a reduction in the efficiency of techniques for extraction of cells from soil as populations become established.

In autoclaved soil, matric potential reduced activity measured by all three techniques, with additional reductions caused by the indigenous microflora in non-autoclaved soil. The most marked effects were on the activity of P.Jluorescens assessed by luminescence and potential luminescence. Luminescence was to some extent correlated with viable cell concentration, but generally decrea,sed at a greater rate, indicating the ability of cells to survive when their metabolic activity is low. Although reduced matric stress has been shown to reduce microbial activity (Wilson and Griffin, 1975), ours is the lirst study to quantify the effect on activity of a single component of the microflora. Our findings have important implications for risk assessment of genetically-modified microbial inocula and the environmental impact of inocula in general. The data indicate that inocula will rapidly lose activity, particularly if introduced into soil at low matric potential. This will reduce their influence on the target environment, which may have favourable implications for environmental safety, but may also reduce their ability to carry out the function for which they were designed. These modifications to environmental effects and inoculum efficiency will not be detected by viable plate counts, even if problems concerning cell extraction and cultivation on laboratory media can be overcome. In addition, potential luminescence data demonstrate that populations starved for even a short period do not recover rapidly when provided with ideal growth conditions. After starvation in non- autoclaved soil for 4 days, recovery during incubation

with nutrient for 2 h was slow and after 28 days was not detectable. Reactivation was also slower at lower matric potentials. This suggests that repeated introductions would be required for efficient use of inocula. The ability of the luminescence-based marker systems to assess both viable cell concentrations and the activity of inocula therefore provides essential information both for assessment of risks and benefits associated with the use of genetically-modified or non-recombinant microbial inoculants which cannot be obtained using traditional techniques. The bioluminescence system also provides the ability to study basic environmental factors controlling micro- bial growth, survival and activity in the soil which may lead to a greater understanding of soil microbial ecology.

Acknowledgements-Our research was supported by PROSAMO (Planned Release of Selected and Modified Organisms), g programme established by IO industrial companies and the U.K. Department of Trade and Industry and the Agricultural and Food Research Council, to investigate the environmental safety of the genetic modification of plants and microorganisms for agricultural benefit.

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