Metabolic activity of Flavobacterium strain P25 during starvation and after introduction into bulk soil and the rhizosphere of wheat

MICROBIOLOGY ECOLOGY

EISEVIER

FEMS Microbiology

Ecology

I8 (1995) I29- 138

Metabolic activity of Flauobacterium strain P25 during starvation and after introduction into bulk soil and the rhizosphere of wheat C.E. Heijnen

*, S. Page I, J.D. van Elsas

IPO-DLO. P.O. Bo.x 9060. 6700 GW Wn,qeningen.The Netherlands Received 20 July 1994: revised 17 May 1995: accepted 3 July I995

Abstract The physiological state of introduced Fkwobucterium strain P25 cells was determined in starvation cultures, in bulk soil, and in the rhizosphere of wheat using direct viable counts (DVC: based on cell elongation after use of nalidixic acid and substrate addition, resulting in a potential activity measurement) and the redox dye 5-cyano-2, 3-ditolyl tetrazolium chloride (CTC; based on respiration without substrate additions, resulting in an in situ activity measurement). Both methods clearly demonstrated that the metabolic activity of Fla~obacrerium P25 cells decreased during starvation, followed by increased activity after amendment with substrate. This confirmed the applicability of DVC and CTC methods to Flu~obucterium P25. Both DVC and CTC methods showed that the percentage of active cells in an introduced Fkwobacterium F25 population in rhizosphere soil was lower than that in bulk soil in the first 1-2 weeks after planting wheat seedlings. After two weeks. the percentage of metabolically active cells in the P25 population in rhizosphere soil was higher than in bulk soil. Since different aspects of cellular physiology are measured when applying DVC and CTC. the impact of variations in environmental factors on the metabolic state of introduced strains may be monitored closely by these methods. Keytnrds:

Introduced

bacteria:

Bacterial

activity:

Nalidixic

acid; 5-Cyano-2.3-ditolyl

1. Introduction Bacteria

have frequently

been introduced

into soil

of purposes, e.g. for the biological control of soil-borne plant pathogens or for the degradation of xenobiotics [l]. The effectiveness of introduced bacteria is variable, primarily since their survival in soil is often poor and unpredictable. Survival of introduced bacteria in soil may be infor

a variety

* Corresponding author. ’ Present address: Biological Laboratory, Canterbury. Kent CT2 7NJ. UK. 0168-6496/95/$09.50 0 1995 Federation SSDf 0168.6496(95)00049-6

University

of European

of Kent,

Microbiological

tetrazolium

chloride (CTC): Bulk; Rhizosphere

creased by amending soil with bentonite clay [2], or with organic material [3]. When applying introduced bacteria for the control of soil-borne plant pathogens, root colonization may be improved by various methods, as extensively reviewed by Stephens [4]. Apart from (increased) survival of introduced bacteria, it is important to consider the metabolic state of these introduced bacteria in soil, since this will largely determine whether the introduced populations will be effective in the afore-mentioned application or not. Many methods have been described for determining the physiological state of the bacterial population present in various environments. Some of the many examples are: (1) the determination of cell size Societies.

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in relation to culturability [5]; (2) the determination of cells capable of forming microcolonies on solid media [6]; (3) the determination of substrate responsiveness in the presence of nalidixic acid inhibiting cell division. often designated the direct viable count (DVC, [7]); (4) the use of tetrazolium salts as redox indicators [8,9]; and (5) the rate of accumulation of rhodamine 123 in cells [IO]. When focusing on the physiological state of a specific bacteria1 strain in a mixed community, e.g. in soil, the measurement of the amount of light produced by bacteria containing lux genes (reviewed by Prosser [ 1 I]), or of carbon respiration resulting from the addition of an exotic substrate which is metabolized by the introduced strain only [ 121, appear to be useful methods. In this paper we report on studies on the physiological state (metabolic activity) of an introduced FlaL~obacteriurn P25 population in bulk and rhizosphere soil and in starvation cultures using the DVC and the redox dye CTC [13]. Flarsobacterium P25 was chosen, since this strain was described as being a representative of the ‘zymogenous’ bacteria by Thompson et al. [l4], which may grow rapidly when simple nutrient sources become available [14]. To make the techniques specific for the introduced strain in a mixed soil community. DVC and CTC methods were combined with the use of a specific monoclonal antiserum. This combination was previously shown to be successful for the detection of metabolically active Enterobacter cloacae cells on leaves and in soil using the DVC method [15]. In this paper we compare DVC and CTC methods and discuss their applicability to bacteria in the soil environment.

2. Materials

and methods

2.1. Bacterial strain A derivative of Flauobacteriurn sp. strain P25 (isolated from soil), resistant to kanamycin (Km), rifampicin (Rp) and streptomycin (Sm). and described by Thompson et al. [ 141, was employed. The strain was cultured for 20 h to obtain exponentially growing cells in Nutrient Broth (Oxoid), supplemented with 100 mg/l Km, 10 mg/l Rp and 60 mg/l Sm at 26°C on a gyratory shaker (150 rpm). Prior to use. cells were washed once for soil experi-

ments and 3 times for the starvation experiment. after which they were harvested by centrifugation (7000 X g. 15 min) and resuspended in sterile demineralized water to obtain a concentration of approximately 10” cells ml-‘. Stock cultures were maintained at - 80°C in the presence of 15-20% glycerol . 2.2. Soil A loamy sand, obtained from the vicinity of Ede, the Netherlands, and described by Heijnen et al. [16] was applied throughout the experiments. 2.3. Plants and plant growth conditions Wheat seedlings (Triticum aestir,utn cv. Sicco) were allowed to germinate for approximately 4 days on moist, sterile filter paper and were then used in root colonization experiments. Plants were grown in a climate chamber at 17°C with a 14 h day/ IO h night rhythm and a relative humidity of 65%. The light intensity was 10000 lux. produced by lamps situated 25 cm above the plants. Plants were watered daily, thereby adjusting the soil moisture content to 17% (w/w), which corresponds to a matric potential of - 10 kPa. Watering was done by hand, adding sufficient water so as to account for the weight loss which occurred within 24 h. 2.4. Bacterial

enumeration

In order to determine the total number of FlaLtobacterium P25 cells and the number of colony forming units (cfu) in bulk soil. wheat roots and adhering rhizosphere soil were first separated from bulk soil by careful, manual shaking. The bulk soil was then thoroughly homogenized by mixing, and a 10 g subsample was transferred into a 250-m] bottle containing 195 ml sterile demineralized water and 10 g gravel (2-4 mm diameter). Bottles were shaken (200 rpm) for IO min at room temperature. For determination of the number of cfu of P25, an aliquot of the soil suspension was serially diluted and plated on nutrient agar (Oxoid), supplemented with 100 mg/l Km, 10 mg/l Rp and 60 mg/l Sm and 100 mg/l cycloheximide (the latter to inhibit fungal growth). Plates were incubated at 27°C until colonies

C.E. Heijnen et al./ FEMS Microbio1og.v Ecology 18 (1995) 129-138

were clearly visible (approximately 3 days). The soil suspension was also used for determinations of the physiological state of the cells (described below) and for total (immunofluorescence) FlaL~obacterium P25 counts. The latter were performed according to Postma et al. [ 171, and involved a floculation of the soil particles for 30 min after the addition of a 20% CaClz solution at a ration of 19: 1. Cells present in the supematant were concentrated (using 1 ml of supematant) on polycarbonate membrane filters (Nucleopore, 0.4 pm, 25 mm diameter) which had previously been stained with Irgalan black [18]. Filters were rinsed with 20 ml sterile 0.85% NaCl, and non-specific staining was reduced by applying gelatin-rhodamine isothiocyanate [ 191. Flatiobacterium P25 cells were then stained according to Mason and Bums [20] by indirect immunofluorescence using a highly specific monoclonal antibody raised in mice [20] combined with the secondary fluorescein-isothiocyanate (FITC) labelled anti mouse serum (Sigma F-0257). After staining, the filters were rinsed with sterile 0.85% NaCl. Flar>obacterium P25 cells were enumerated according to Postma et al. [17], using a Zeiss epifluorescence microscope. For total (immunofluorescence) and cfu counts of Flavobacterium P25 in the rhizosphere, the wheat roots and adhering rhizosphere soil (approximately 10 g, if sufficient root material was available) were transferred to 250-ml bottles containing 195 ml sterile demineralized water and 10 g gravel (2-4 mm diameter). All further treatments were similar to those described for the bulk soil. For determining survival of Flacobacterium P25 in starvation cultures, the number of cfu was determined by serially diluting an aliquot of the starvation culture. followed by plating on selective media as described above. For total immunofluorescence P25 counts, l-ml aliquots of the starvation culture were concentrated on filters and stained as described for bulk soil samples. 2.5. Determination

of the physiological

state of cells

The physiological state of FlaLlobacterium P25 was determined using DVC and CTC methods. The DVC method involved pre-incubation of a sample with substrate in the presence of nalidixic

131

acid (Nx). Nx is a specific inhibitor of DNA synthesis in Gram-negative bacteria, preventing cell division in metabolically active cells, hence causing the appearance of elongated cells [7]. Thus, the DVC method shows whether cells are substrate responsive (potentially active) and may thus be used in the determination of the potential activity of introduced bacteria. Applying the DVC to bulk and rhizosphere soil involved taking an aliquot (I ml) of the soil suspensions, prepared for the bacterial enumerations. This subsample was incubated with an equal volume of a substrate in the presence of 20 mg 1-j Nx for 24 h (rotary shaker, 150 rpm; 27°C). To decide which substrate was most suitable for determining substrate responsiveness of FlaLlobacterium P25 cells extracted from soil, we tested the following: 0.05% yeast extract (YE), 0.2% calcium malate (M), nutrient broth (NB), soil extract (SE, prepared according to Wollum [21]), nutrient broth with 0.2% calcium malate (NBM) and soil extract with 0.2% calcium malate (SEMI. After the 24 h incubation period in the presence of Nx, metabolic activity of cells in the soil suspension was inhibited by adding 0.025% NaN,. The soil suspension was then allowed to flocculate followed by concentration and staining of the cells on filters (as described for total immunofluorescence counts). Controls (also taken from the soil suspensions) were treated with 0.025% NaN, before adding substrate and Nx. It was noted that substrate responsiveness was similar whether soil was flocculated before or after the 24-h incubation with substrate and Nx. The DVC method was applied to the starvation cultures in a similar way, incubating aliquots of these cultures with NB or NBM. Concentration on filters, staining and microscopic observations were performed as described for total immunofluorescence P25 counts in the soil experiments. Cell lengths were determined microscopically using an ocular micrometer. For each sample, 100 cells were picked randomly and measured. Since cells in the control samples were generally shorter than 2 pm, a cell was defined to be substrate responsive when it was longer than 2 pm ( > 2 pm). Data were corrected for cells found in the controls longer than 2 pm. The use of the tetrazolium salt CTC is based on the fact that CTC will compete with molecular oxygen as an electron acceptor [ 131. Hence, in metaboli-

tally active cells, CTC is reduced to (fluorescent red) CTC-formazan crystals, which accumulate intracellularly. Application of the CTC method in bulk and rhizosphere soil involved incubation of an aliquot ( I ml) of the soil suspensions prepared for bacteria1 enumerations with CTC (5 mM). No substrate was added, resulting in an in situ activity measurement of the introduced cells. Controls (also taken from the soil suspensions) were treated with 0.0258 NaN, before adding CTC. Samples were incubated for 1 h (rotary shaker. 1.50 t-pm; 27°C). after which metabolic activity was inhibited by adding 0.025% NaN,. After incubation, the soil suspension was allowed to flocculate followed by concentration and immunofluorescence staining of the cells on filters (as described for total immunofluorescence P25 counts). For microscopic observation of CTC-formazan crystals, filter sets applied for FITC stained cells [ 171 were used. enabling us to specifically enumerate the CTC-formazan stained Flac,obacterium P25 cells. and to compare them with the total number of FITC stained cells. Increased CTC concentrations (> 5 mMl or longer incubation time (> 1 h) resulted in similar numbers of CTC stained cells. For the starvation experiment, aliquots of the cell culture were incubated with CTC and further treated as described for the soil experiments. 2.6. Experimental

set-up

Survival and physiological status of introduced cells in bulk and rhizosphere soil were studied over a period of 3 weeks. Triplicate soil portions (100 g dry weight; bulk density 1.2 g cmm3: matric potential - 10 kPa) were inoculated with Flarwbacterium P25 cells at levels of approximately lo8 cells gg’ dry soil, thoroughly mixed with a spatula, planted with three wheat seedlings. and incubated in the climate chamber. On sampling days. soil portions were sampled in order to determine survival and physiological state (metabolic activity) of introduced bacteria1 populations. In total, two soil experiments were performed. The first one focused on selecting the substrate which would be most suitable for assessing the substrate responsiveness (DVC) of an introduced FZar:obacterium P25 cell population in soil. In the second soil experiment we compared potential activity measurements (DVCI with in situ activity mea-

surements (CTC). For the DVC, we used the substrates which had shown to be most suitable in the first soil experiment. Survival and physiological status of Flavobacterium P25 cells during total nutrient and energy starvation were studied in triplicate in 100 ml Erlenmeyer flasks. containing 25 ml of l/4 strength Ringer’s solution (2.17 g 1-l NaCl; 0.11 g I- ’ KC]; 0.12 g II’ CaCl,: 0.05 g I- ’ NaHCO,) inoculated with approximately IO6 cells ml-‘. Flasks were incubated on a rotary shaker (100 rpm> at 27°C. At regular time intervals, samples were taken and numbers of surviving cells and their physiological state (metabolic activity) were determined. After a starvation period of approximately 4 weeks. the cell cultures were amended with nutrient broth for reactivation, using equal volumes of starved culture and nutrient broth. Samples for determining physiological status and cell numbers were taken with short time intervals (h) directly after reactivation. as well as daily for several days following this. 2.7. Statistical analysis All data (from starvation cultures, bulk and rhizosphere soil) were obtained by destructively harvesting triplicate samples. An analysis of variance. using Genstat 5 (Lawes Agricultural Trust, Rothamsted Experimental Station, UK). was performed on survival data and on the data obtained using the CTC or DVC methods. Differences were considered to be significant at P < 0.05.

3. Results Survival of Flalwbacterium P25 after introduction into soil resulted in similar trends for the first (Fig. 1) and second (results not shown) soil experiments. In the first soil experiment, a significant decrease in cfu numbers in bulk soil (3.3 log units) was observed over a period of three weeks (Fig. 1I. This was significantly more than the decrease observed in the rhizosphere in the same time period (1.7 log units). Numbers of cfu were significantly lower in bulk soil than in the rhizosphere as from (immunofluorescencel of day 6. Total counts Flalvbacterium P25 also showed a significant de-

C. E. Heijnen

et al. / FEMS Microhiolog~

Fig. I. Log number of total cells (immunofluorescence. IF). log number of cfu (plate counts, PC) and log number of substrate responsive (DVC) cells after incubation with nalidixic acid (20 mg I-’ ) for 24 h in the presence of nutrient broth (NB) of Fladxwrerium P25 per g of dry bulk and rhizosphere soil during a period of 21 days. LSD = 0.27.

cline in time, both in bulk and rhizosphere soil. These counts were significantly lower in bulk than in rhizosphere soil as from day 10. In bulk soil, total P25 immunofluorescence counts were significantly higher than plate counts as from day 13. When comparing different substrates for their effectiveness in determining substrate responsiveness (DVC) in the soil experiment, NB and NBM consistently resulted in significantly more elongated cells than YE, SE, SEM or M. With YE, at least 50% fewer cells were elongated than with NB and NBM. The application of SEM and M resulted in an even lower frequency of cell elongation. SE gave very variable results. The percentage of cells of the Flauobacterium P25 cell populations showing substrate responsiveness in bulk soil decreased significantly during the 3 week incubation period (Fig. 2). In rhizosphere soil, there was also a significant decrease of substrate responsiveness during this incubation period. This decrease, however, was much smaller than in bulk soil (Fig. 2). On days 2 and 6 the percentage of substrate responsive cells in bulk soil was significantly higher than in rhizosphere soil; on days 13 and 2 1 rhizosphere soil contained a significantly higher percentage of substrate responsive ceils than bulk soil (Fig. 2). Finally, the log number of substrate responsive Flar~obacterium cells also declined significantly both in bulk and rhizosphere soil (Fig. I). This decrease was significantly

Ecology

18 f 1995) 129- 138

133

stronger in bulk soil than in rhizosphere soil. No significant differences were detected between the log number of cfu and the log number of DVC-positive cells except for in the bulk soil on day 21 (Fig. I), where significantly more cells showed substrate responsiveness than were capable of forming visual colonies on plates. In the second soil experiment, potential (DVC, using NB and NBM as substrates) and in situ (CTC) activity were determined simultaneously (Fig. 3; only NB and CTC are presented). As in the first experiment, the percentage of cells of the introduced Flacobacterium P25 population responding to substrate in the DVC-assay declined in time. Also, substrate responsiveness was initially lower in rhizosphere than in bulk soil. Thus, the results from the first soil experiment were generally confirmed. The use of CTC for determining the in situ activity of the introduced Flauobacterium P25 population in bulk soil (Fig. 3) revealed that about 70% of the cells was capable of forming CTC formazan crystals on day 2 (80% of the cells in the inoculum showed CTC reduction). This amount was reduced significantly to 9% by day 2 1. In rhizosphere soil, however, only 5% of the cells of the Ffnuobacterium P25 population contained CTC-formazan crystals on day 2, suggest-

Fig. 2. Percentage of elongated cells (> 2 pm) of Flmobacrerium P25 cells extracted from bulk and rhizosphere soil and subsequently incubated with nalidixic acid (20 mg I- ’ ) for 24 h in the presence of nutrient broth (NB). Controls were treated with 0.025% NaN,. LSD = 12.8.

134

C.E. Heijnen

et al. / FEMS Microbiology

Ecology

18 f lYY5,51129-138

tween total P25 counts and the number of cfu on day 3 1. After amendment with nutrient broth, numbers of cfu and total counts increased; however, after 24 h total counts were still one log unit higher than cfu counts. Substrate responsiveness of the Flawbacterium P25 population (using NB as a substrate) was fairly stable during the first 16 days of starvation (Fig. 4). Then. a significant reduction in the percentage of elongated cells (relative to the controls) was observed on day 24. which continued until day 3 1. One day after amendment of the starved cultures with nutrients (Fig. 4), substrate responsiveness had increased significantly. The in situ metabolic activity of the cell population was also determined using

0'

0

1

1

5

10

/

/

15

20

days

100 ,-,

IW

NB, bulk NB, rhsl CTC, bulk CTC, rhst -_c ._o_. -_e .-Q_.

Fig. 3. Percentage of elongated cells (> 2 pm) of Fkwobacterium P25 cells extracted from bulk or rhizosphere soil. subsequently incubated with nalidixic acid (20 mg I- ’ ) for 24 h in the presence of nutrient broth (NB) and the percentage of cells extracted from bulk or rhizo.sphere soil, containing CTC-formazan crystals after an incubation with CTC (5 mM) for I h. Controls using nalidixic acid and CTC were treated with 0.025’7~ NaN,. LSD = 14. I (DVC); LSD = I I .9 (CT0

M

30

-

days WC

CTC

*-

ing a low level of in situ activity. The amount of CTC-formazan positive cells then increased significantly to levels between 20 and 40% during the rest of the incubation period. The in situ metabolic activity was found to be significantly higher in rhizosphere soil than in bulk soil as from day 13. In all cases. no CTC-formazan crystals were observed in cells in the control samples (treated with NaN,). Starvation of Flacobacterium P25 cells in l/4 strength Ringers solution was performed in two separate experiments and both resulted in similar trends for survival and the physiological state of the cell population. Only one set of data is presented here. Total (immunofluorescence) P25 cell counts remained stable for a period of at least 31 days (approx. 3 X lo6 cells/ml). Total immunofluorescence P25 cell counts and numbers of cfu were similar until day 9 of the starvation period. As from day 16 plate counts showed a steady decrease in time, resulting in a significant difference of 0.6 log units be-

Fig. 4. Percentage of elongated cells ( > 2 pm) of Flut,obnctrrium P25 cells in starvation cultures and after amendment with nutrient broth, both after incubation with nalidixic acid (20 mg I-’ ) for 24 h in the presence of nutrient broth. The percentage of cells extracted from these cell cultures containing CTC-formazan crystals, after an incubation with CTC (5 mM) for I h. Controls using nalidixic acid and CTC were treated with 0.025% NaN,. LSD = 14.0 (DVC): LSD = 9.8 (CTC).

C.E. Heijnen et al. / FEMS Microbiology

CTC. In all cases, no CTC-formazan positive cells were found in the controls (cells treated with NaN, before adding CTC). At the beginning of the starvation period, about 80% of the cells of the Flauobacterium P25 population were able to form CTC-formazan crystals (Fig. 4). This number increased slightly during the first week of starvation, followed by a strong, significant decrease on day 9, when only 20% of the cells were able to form CTC-formazan crystals. Number of CTC-formazan positive cells then decreased steadily to almost negligible levels by day 3 1. One day after amendment of the cell cultures with nutrient broth, again approximately 80% of the cells of the Flauobacterium P25 population were capable of forming CTC-formazan crystals (Fig. 41.

4. Discussion The use of the DVC-method is a widely accepted technique for determining the substrate responsiveness of bacteria in various environments [7,15,22,23]. DVC is based on elongation of cells relative to control samples. Kogure et al. [7] found that substrate responsive Pseudomonas sp. cells increased from 2-3 pm to about 15 pm after a 5 h incubation in the presence of nalidixic acid. Brayton and Colwell [22] suggested that cells of Vibrio cholerae should be recorded as being substrate responsive when “they appeared as long and/or fat rods possessing a peripheral green band just below the cell wall”. Pedersen and co-workers [15,23] only enumerated cells 2 4 ,um and shorter swollen cells with a width 2 2 pm as being substrate responsive when studying Enterobacter cloacae in soil and on leaves. With these criteria [7,15,22,23] it is possible to determine the % of substrate responsive cells in samples under observation. The length of FlaLlobacterium P25 cells in controls generally ranged from 1 to 2 pm. We therefore decided to use the length of 2 pm as the boundary between substrate responsive and non-responsive cells. However, a problem arises when, due to their presence in a heterogeneous and oligotrophic environment such as soil, cells in samples not treated with nalidixic acid (controls) have variable lengths. In a pleomorphic population a cell of, e.g., 3 pm could be substrate responsive (having increased in

Ecology 18 f 1995) 129-138

13.5

length from, e.g., 1 to 3 pm), or could have been a large, non-responsive cell. In that case we suggest that the average cell length increase of the population (relative to control samples) is used as a measure for substrate responsiveness (metabolic activity) rather than the % of cells which were, e.g., longer than 2 pm. However, when using cell length increase relative to control samples, it must be determined whether cell length and activity are correlated in a simple manner. Also, a careful interpretation of the results is required when only a small percentage of the population accounts for a major cell elongation. With the Flavobacterium strain used here, trends were similar whether average cell length relative to the control or % of cells > 2 pm were used as measures for substrate responsiveness. When determining the physiological state of bacteria, it is important to clearly state which method was used for determining this activity, since each method is based on different aspects of cellular physiology. For example, DVC performed with nalidixic acid is based on the inhibition of DNA synthesis, preventing cell division in metabolically active cells, causing cells responsive to substrate to elongate [7]. Since elongation will only occur in the presence of substrate, the method requires that cells are capable of showing an initial growth response on the substrate used. Many authors have applied DVC using yeast extract as a substrate (the substrate originally applied by Kogure et al. [7]), e.g. in activity measurements of Pseudomonas sp. in seawater [7], Vibrio cholerae in water [22], P. syringae in epiphytic populations [24], total bacterial populations in water [25], and Enterobacter cloacae on leaves [ 151. However, our results clearly show that yeast extract was not an optimal substrate for determining the substrate responsiveness of Flauobacterium P25 in soil, but that nutrient broth and nutrient broth amended with malate are more suitable. Also, various Pseudomonas strains tested showed only a minor response to yeast extract (results not shown). Since the optimal substrate is clearly dependent on the strain, contrary to other authors [25], we think it doubtful that DVC are applicable to total bacterial populations. Our experiments clearly demonstrate that DVC provides meaningful results as to the physiology of Flar:obacterium P25, by the fact that a reduced capacity for cell elongation occurred during

136

C.E. Heijnen et al./ FEMS Microbiolog?: EC&Q

starvation. This suggested that average cell activity was decreasing. Applying DVC to Flacobacterium P25 in soil revealed a steady decrease in the percentage of potentially active cells both in bulk and rhizosphere soil, probably due to the fact that accessible substrate was becoming limited. It was surprising, however, to find that the fraction of potentially active cells in the rhizosphere was lower than the fraction of potentially active cells in the bulk soil during the first 9 days of plant growth (Fig. 21, since higher amounts of substrate, resulting from root exudation, are supposedly available for bacteria in the rhizosphere. A possible explanation for this observation might be that Flarobacterium P2.5 only uses substrates exuded in a later stage of plant development, or that Flauobacterium P25 was initially outcompeted by the indigenous bacterial population in the rhizosphere with regard to the use of root exudates as a substrate for increasing bacterial activity. This corresponded with results obtained by Thompson et al. [14], suggesting that Flauobacterium P25 might be a poor competitor for root exudates since this strain was isolated from bulk soil. Plate counts obviously also give information on the substrate responsiveness of cells, because only cells capable of growing and forming colonies on the substrate used are counted. Plate counts are frequently applied in combination with total (immunofluorescence) count, thereby assuming that the total counts represent live cells, since dead cells will have been largely metabolized by other organisms. The combination of total counts:plate counts results in information on the rate of culturability [26]. However, cells only capable of forming microcolonies would not be counted in regular plate counts and would therefore be designated as non-culturable. The difference between cells capable of growing to visual colonies or only to microcolonies was recently used by Binnerup et al. [6] to describe the metabolic state of bacterial populations in soil. Cells not even capable of forming microcolonies but which are still substrate responsive. could then be enumerated using the DVC method. A bacterial population may now, using culturability as a measure, be divided into ‘total cells’ (immunofluorescence counts), ‘culturable cells’ (plate counts), ‘slightly culturable cells’ (cells with a reduced culturability forming microcolonies), ‘substrate responsive cells’ (cells only ca-

18 (199.51 129-138

pable of elongation in the presence of substrate and nalidixic acid) and ‘non-culturable cells’ (the difference between total counts and the total of microcolony- . elongatedand plate-counts). It is now interesting to note that total immunofluorescence counts, cfu counts and DVC count in rhizosphere soil are very similar (Fig. 1). In bulk soil, however, a divergence between the three methods occurred from day 10. Probably due to the fact that cells were becoming more and more deprived of nutrients, they were becoming less culturable and substrate responsive. By day 21 the ability to react to substrate had decreased even further, resulting in a significant number of cells which were still substrate responsive, but unable to grow to visible colonies on agar plates. The CTC method is based on the detection of metabolically active cells by their reduction of CTC to the red fluorescent insoluble CTC-formazan. Due to the fact that no substrate was added (contrary to the DVC method), the in situ activity of bacterial cells was measured. However, the usefulness of CTC as a metabolic dye is dependent on the strain as was clearly demonstrated by Thorn et al. [27] for various tetrazolium salts. A prerequisite for the strain under observation could be that the cell envelope is permeable for the tetrazolium salt (e.g. hydrophillic components may constitute a significant barrier to lipophilic tetrazolium salts [27]). As with DVC. the CTC method is therefore not applicable when the composition of the microbial population is variable and unknown [27]. Our work, however, clearly shows that CTC is a useful metabolic dye for Flar:obacterium strain P25. In starvation cultures, the percentage of CTC-formazan stained cells declined in time (corresponding to a reduced metabolic activity), whereas freshly grown cultures show a high percentage of CTC-formazan positive cells. Also, as with DVC, the CTC method showed a reduction in metabolic activity of P25 in time in bulk soil, again probably resulting from a lack of substrate. However, it was surprising to find a low percentage of CTC positive cells in the rhizosphere on day 2, which can be translated into a low in situ activity of the introduced cells. at the beginning of the plant growth period. Corresponding to the DVC results, this would also suggest the inability of Flacobacterium P25 to use the substrates exuded during the

C.E. Heijnen et al. / FEMS Microhio1og.v Ecolo~.v 18 f 19951 129-138

early phases of plant growth, as well as poor competition of this strain with the indigenous population. CTC and DVC results do not necessarily correspond (Figs. 3 and 4). Differences in metabolic activity measurements found by DVC and CTC methods may largely be explained by the fact that CTC is an in situ determination of metabolic activity, and that DVC is determining potential activity. Thus, the low percentage of cells accumulating CTC formazan crystals in the initial stages of plant growth probably reflected the fact that Flapobacterium was incapable of exploiting root exudates produced, and/or P25 could not compete for substrate with the indigenous population. However, the Flavobacterium cells were still substrate responsive, as demonstrated by DVC (and plate counts) which involved the availability of an ideal substrate as well as reduced competition due to the presence of nalidixic acid. The starvation experiment also clearly demonstrates the difference between potential and in situ activity. Here, the CTC-method revealed a reduced metabolic activity between days 6 and 9. whereas substrate responsiveness remained fairly stable as determined by DVC until day 16. The early response, albeit with a lag phase, measured using CTC after reactivation in comparison to the much slower response measured by DVC was similar to results found by Kaprelyants and Kell [28], who described a resuscitation experiment with Micrococcus luteus. They found that, in the early phase of increasing viable cell counts, the percentage of cells that accumulated CTC-formazan was higher than the percentage of viable cells that could be determined by plating. They suggested that membrane energization is necessary but not sufficient for resuscitating cells of M. luteus in order for them to be able to multiply on solid media [28]. A similar explanation could be used for the reactivation of FlaL!obacterium P25; membrane energization occurred in early stages of reactivation, but this was not yet sufficient to obtain 100% DVC positive cells. It may be concluded from these experiments that both DVC and CTC methods are useful for describing the metabolic state of Flarobacterium P25, both in soil and in liquid cultures. Since different aspects of cellular physiology are measured, the impact of variations in environmental factors, such as root exudation by young plant roots, competition for nu-

137

trients by the indigenous bacterial population, substrate additions to starvation cultures, or other physiological adaptations on the metabolic state of introduced bacteria may be monitored closely.

Acknowledgements We thank C.H. Hok-A-Hin for technical assistance, J. de Bree for help with statistical analysis and E. Smit and J.A. van Veen for useful discussions and for critically reading the manuscript.

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