INTRODUCTION

Microcosms are, by design, an environment in miniature. Although microcosms are uncoupled from nature (Schindler, 1980; Heath, 1979) they provide a complex system containing the biological and chemical framework from which they were obtained. Several designs for soil microcosms exist for testing ecological effects, chemical degradation and survival of introduced organisms. The need for standardization of microcosm design for risk assessment of microbial agents has recently been addressed (Krimsky, 1995). The intact soil core (Angle, 1994, 1995) was shown to accurately predict field survival, in a single soil, using Pseudomonas chloroaphis 3732RN-L11 (Barry, 1986, 1988), when held at the mean field growing season high temperature, in a lighted incubator. The intact soil core microcosm accurately predicted field survival in both soil and in wheat rhizospheres.

Validation of the soil core microcosm, before acceptance as a risk assessment tool, requires testing in diverse climates and soils and with other organisms. This study describes testing of the microcosm in diverse ecozones and soil types during year one of a two year trial period. Four sites representing the major ecozone, climate and soil types throughout Canada were selected to test the soil core microcosm with the GEM:

Agriculture and Agri-Foods Canada; Ottawa, Ontario (AG)

University of British Columbia; Vancouver, B. C. (BC)

Carleton University; Ottawa, Ontario (CL)

University of Saskatchewan; Saskatoon, Sask. (SK)

The Ottawa site comprised two soil types.

Year one included a dry-run followed by a concurrent microcosm and field release trial at each site, with simultaneous replication of all cosms at the University of Maryland. Results of this trial are presented here. Year two will include replication of the microcosm and field release trial using Pseudomonas chloroaphis 3732RN-L11 (GEM), and a separate study in microcosms to assay other, dissimilar microorganisms.

MATERIALS AND METHODS

Construction of the soil core microcosm. The cosms are simple and inexpensive to construct. Material requirements include two inch, schedule 40 PVC plumbing pipe, aluminum foil (or polyester mesh, e.g. Silk-screen), and rubberbands (or duct tape). The pipe is cut into seven inch lengths and beveled at one end to forty-five degrees. The cost is $1.00 each if made by a machine shop, $0.50 if homemade. Cores can be autoclaved, upright, but must be allowed to cool afterward.

Use of the soil core microcosm. Insertion. It is necessary to prepare the soil before use. Remove organic matter from the soil surface including grass and debris. Hold the core and drive it into the ground six inches, leaving one inch exposed. We recommend a dead-blow hammer with a face wider than the core. Dead-blow hammers with plastic faces and wide heads are available through Industrial Supply catalogs. If using another type of hammer, a two-by-four or metal plate should be placed over the core while hammering. If resistance is met from rocks, etc., stop and choose another site

Removal. Dig the core out from the side, cutting the soil evenly at the bottom. Cover the bottom with aluminum foil held by a rubber band or with polyester mesh held by duct tape. Weigh each core. Cores are incubated at 28C or 23C (see below) and will lose water through evaporation. Water must be added to each core to return to the original weight periodically to maintain moisture level. Cores that don't allow water to infiltrate or that leak water from the bottom should be discarded.

Sample preparation. Soil. Hold the core top down over a 4mm sieve, hammer on the top (facing down) and on the sides until the soil comes out. Force the soil through the sieve.

Rhizosphere. 1. Remove the contents of the core into a sieve, as above.

2. Carefully separate the bulk soil from the root (rhizosphere), retaining adhering soil on the root. Weigh the entire fresh rhizosphere.

Microbial assay. Soil. Mix the soil thoroughly and weigh out 10 grams of soil. Place soil and 95 ml of diluent sterile distilled water to a glass blender. Blend for 1 minute on high speed, and allow to settle for 1 minute. Plate immediately.

Rhizosphere. Add the rhizosphere to 100 ml sterile distilled water (minus the rhizosphere weight), blend, allow to settle and plate as above. This dilution is 1:100/rhiz. fresh weight, giving plate counts as CFU per gram fresh rhizosphere. (Angle, 1996)

Microorganisms. Pseudomonas chloroaphis 3732RN-L11 (Barry, 1986, 1988) was used throuout the study. The microorganism has been described by Barry (1986, 1988). The micororganism is resistant to rifamycin and naladixic acid (100 µg/L each). It was maintained in Pseudomonas f broth, (Difco, Detroit Mich.) plus 50% glycerol at -55C.

Soil inocuulum was prepared by adding a single loopful to 10 ml of Pseudomonas f broth and incubating (20 hrs, 37C) untill a cell density of approximately 10⁶/ ml was reached. This was used to inoculate 1.5 L of broth, which was incubated as above. Prior to inoculation, the cells were centrifuged (4C; 5000 x g) and resuspended in distilled water.

Table 1. Soil characteristics.

ID	pH	Text	% Sand	% Silt	% Clay	% OM	CEC (meq)
MD	5.8	L	43.3	42.0	14.7	2.63	5.33
AG	6.2	LS	81.3	16.0	2.7	3.57	5.57
BC	6.1	LS	86.3	9.0	4.7	5.67	5.77
CL	7.5	SL	59.3	28.7	12.0	3.57	46.90
SK	7.5	L	34.3	44.0	21.7	5.93	29.97

Field trials. Field plots. Each plot is 1 meter square (500 x the core surface area). Six inch continuous plastic edging is buried 3 inches deep around each plot. Four replicates are sampled at each time, from four randomized split-plots, at 8 sampling times.

Inoculum. The inoculum is 5 x 10⁹ CFU/ml, as above. Cores receive 5 ml inoculum on the seeds, and field plots receive 2.5 L inoculum, spread over plot.

The trials were begun in May and continued through October. Each trial required approximately 100 microcosms at each site with an additional 100 shipped to Maryland from each site. Thus almost 1000 microcosms, each assayed in quadruplicate, were utilized. Preliminary investigation of conditions at the sites led to a temperature selection of 23C for Canadian site microcosms and 28C for the Maryland microcosms.

Sampling times were chosen arbitrarily at first, based on previous work in the Maryland soil, these were: 0, 7, 14, 28 and 42 days after inoculation for soil and 14, 28 and 42 days for rhizosphere samples. As the experiment progressed, data was submitted electronically to the Maryland site for analysis after each time point. Further sampling was based on predicted survival patterns specific to each site.

RESULTS AND DISCUSSION

Soil microcosms. Soil microcosm data obtained from samples shipped to Maryland were similar to on-site samples (Tables 2, 3). Although the regresion analysis indicated differences at 0.95P, these were significant by values ranging from 0.001-0.004 0.95P and would be nonsignificant at 0.90P. Die-off of the GEM in soil was more rapid in the field plots than in microcosms for all sites. The differences in days to Y intercept averaged 104 days, or 31% of the average total time. Thus, the laboratory data indicate that the cosms can be used to indicate die off rate and will behave similarly in different labs. In addition, the cosms yield a conservative estimate of time to extinction, with three of the four locations indicating a longer than actual extintion time.

A pattern emerged with two of the soils. The soils at the SK and CL sites, in both the field and the lab microcosms, supported higher populations of the introduced GEM - longer projected survival, or 'days to zero' - compared to the other sites. Soils from the AG and BC sites closely paralleled survival in the MD soil for both soil and rhizosphere samples (Figures 1, 2).

Survival, or die-off, of the GEM thus fell into two categories that appear related to the cation exchange capacity or CEC (see Table 1) of the soil. The amount of clay present did not affect survival, though the type of clay present, namely less-weathered or smectite clays may be significant. Survival in two soils with the highest clay content was approximately 4 fold longer, with laboratory data mimicking the field results.

The presence or absence of plant roots greatly affected survival/persistence. When the plants were present in the microcosms, the difference between soil types was not as great. Survival/persistence in high clay soils was only approximately 2 fold greater than low clay. Rhizosphere microcosm data obtained from samples shipped to Maryland were identical to on site samples. In AG and BC soils, survival in the field closely paralleled microcosm survival and the microcosm served as an accurate predictive tool. Results in soil and rhizosphere for the AG and BC sites closely paralleled results obtained during a 1993 field release in the MD soil (Angle, 1995). Field survival for the CL and SK rhizosphere was greater toward the end of the growing season (140 days). Indications are that wheat plants in the field, which tend to have about ten times more above and below ground biomass than in microcosms at these sites, enhance survival of the GEM to a greater extent than plants in a microcosm.

SUMMARY

The microcosm protocol can be used to produce reliable, conservative, estimates if microbial persistence/survival.

Data indicate that there are differences in survival and persistence due to soil type and climate. However, in all climates and with all soil types, the microcosm protocol is able to generate laboratory data which is comparable to field data. In addition, trials conducted in Maryland, with samples shipped from the four locations, indicate that off site data, generated using the microcosm protocol, is comparable to field data from any site.

Presence or absence of the plant greatly affects the persistence and survival of both the GEM and parent microbe. However, the microcosm reflected field data at each site, and provided a consistent, conservative, estimate of microbial survival/persistence.

REFERENCES

Angle, J.S., Gagliardi, J.V., McIntosh, M.S. and Levin, M.A. 1996. Enumeration and Expression of Bacterial Counts in the Rhizosphere. In: Soil Biochemistry, Volume 9. Stotzky, G. and Bollag, J.M. eds. Marcel Dekker, Inc. pp233-251.

Angle, J.S., Levin, M.A., Gagliardi, J.V. and McIntosh, M.S. 1995. Validation of Microcosms for Examining the Survival of Pseudomonas aureofaciens (lacZY) in Soil. Appl. Env. Micb. 61:2835-2839.

Angle, J.S., Levin, M.A., Gagliardi, J.V., McIntosh, M.S. and Glew, J.G. 1994. Pseudomonas aureofaciens in soil: survival and recovery efficiency. Microb. Releases 2:247-254.

Barry, G.F. 1986. Permanent Insertion of Foreign Genes into the Chromosomes of Soil Bacteria. Biotechnol. 4:446-449.

Barry, G.F. 1988. A Broad-Host-Range Shuttle System for Gene Insertion into the Chromosomes of Gram Negative Bacteria. Gene. 71:75-84.

Heath, R.T. 1979. Holistic studies of an aquatic microcosm. Int. J. Env. 1:87-93.

Krimsky, S., Wrubel, R.P., Naess, I.G., Levy, S.B., Wetzler, R.E. and Marshall, B. 1995. Standardized Microcosms in Microbial Risk Assessment: Uses and limitations for prerelease risk assessment of genetically engineered soilborne bacteria. Bioscience. 45:590-599.

Schindler, J.E., Waide, J.B., Waldron, M.C., Hains, J.J., Schreiner, S.P., Freedman, F.L., Benz, S.L., Pettigrew, D.R., Schissel, L.A. and Clark, P.J. 1980. A microcosm approach to the study of biogeochemical systems. In: Microcosms in Ecological Research. Geisy, J.P., ed. USDOE Symposium Ser. 52 NTIS, Springfield, VA. pp. 192-203.

Table 2. Soil regression data

Sample	Y-int	Slope	Slope SE	Slope CL (upper 95%)	Slope CL (lower 95%)	Days to 0 (X-int)
AG core-lab	5.95	- 0.036	0.002	- 0.032	- 0.040	166
AG field	5.63	- 0.068	0.005	- 0.058	- 0.078	83
AG core-MD lab	5.66	- 0.051	0.005	- 0.041	- 0.061	111
BC core-lab	6.12	- 0.039	0.001	- 0.037	- 0.041	157
BC field	6.29	- 0.075	0.003	- 0.069	- 0.081	84
BC core-MD lab	6.41	- 0.048	0.003	- 0.042	- 0.054	134
MD core-lab	5.98	- 0.073	0.006	- 0.061	- 0.085	82
CL core-lab	6.06	- 0.026	0.001	- 0.024	- 0.028	233
CL field	6.78	- 0.055	0.002	- 0.051	- 0.059	123
CL core-MD lab	5.89	- 0.016	0.002	- 0.012	- 0.020	368
SK core-lab	5.25	- 0.029	0.004	- 0.021	- 0.037	181
SK field	3.33	- 0.019	0.004	- 0.011	- 0.027	175
SK core-MD lab	6.15	- 0.015	0.002	- 0.011	- 0.019	410

Table 3. Rhizosphere regression data

Sample	Y-int	Slope	Slope SE	Slope CL (upper 95%)	Slope CL (lower 95%)	Days to 0 (X-int)
AG core-lab	6.46	- 0.034	0.005	- 0.029	- 0.039	190
AG field	4.86	- 0.035	0.008	- 0.019	- 0.051	139
AG core-MD lab	6.64	- 0.034	0.004	- 0.026	- 0.042	195
BC core-lab	6.38	- 0.022	0.002	- 0.018	- 0.026	290
BC field	5.92	- 0.028	0.003	- 0.022	- 0.034	211
BC core-MD lab	8.05	- 0.049	0.004	- 0.041	- 0.057	164
MD core-lab	7.72	- 0.042	0.004	- 0.034	- 0.050	184
CL core-lab	7.72	- 0.021	0.002	- 0.017	- 0.025	368
CL field	6.84	- 0.016	0.003	- 0.010	- 0.032	428
CL core-MD lab	7.11	- 0.021	0.003	- 0.011	- 0.023	339
SK core-lab	6.17	- 0.017	0.003	- 0.011	- 0.023	363
SK field	6.36	- 0.011	0.002	- 0.007	- 0.015	578
SK core-MD lab	7.15	- 0.019	0.003	- 0.013	- 0.025	376

Figure 1. Persistence of GEM in soil cores at Maryland laboratory.

Figure 2. Persistence of GEM in rhizospheres at Maryland laboratory.