Predicting survival of a genetically engineered microorganism,Pseudomonas chlororaphis 3732RN-L11, in soil and wheat rhizosphere across Canada with linear multiple regression models

Pseudomonas chlororaphis 3732RN-L11 survival rates in soil and wheat rhizosphere were measured using intact soil core microcosms representing 23 sites across Canada. Linear multiple regression (LMR) models were developed to predict the survival rate of this genetically engineered microorganism (GEM) as a function of soil parameters measured at the time of microcosm inoculation. LMR models were tested by comparing their predicted survival rates with observed survival rates from environmental introductions of the GEM by Gagliardi et al. (2001) at five field sites across Canada over two years. No soil parameter (e.g., % clay) was highly correlated with GEM survival rates in soil or wheat rhizosphere. Total fungal colony-forming units (CFUs), % soil titanium (positive correlations), and % soil magnesium (negative correlation) were found to be the best LMR predictors of GEM survival rates in soil over two years. Total soil bacterial CFUs, nitrate, % soil potassium (positive correlations), and exchangeable magnesium (negative correlation) were found to be the best LMR predictors of GEM survival rate in wheat rhizosphere over two years. While LMR models were statistically significant, they were unable to reliably predict the survival rate of the GEM in field trial introductions. The results indicate that there can be considerable uncertainty associated with predicting GEM survival for multi-site environmental introductions.     

The fate of a genetically modified Pseudomonas strain and its transgene during the composting of poultry manure

The fate of the genetically modified (GM) Pseudomonas chlororaphis strain 3732 RN-L11 and its transgene (lacZ insert) during composting of chicken manure was studied using plate count and nested polymerase chain reaction (PCR) methods. The detection sensitivity of the nested PCR method was 165 copies of the modified gene per gram of moist compost or soil. Compost microcosms consisted of a 100-g mixture of chicken manure and peat, whereas soil microcosms were 100-g samples of sandy clay loam. Each microcosm was inoculated with 4 x 1010 CFU of P. chlororaphis RN-L11. In controlled temperature studies, neither P. chlororaphis RN-L11 nor its transgene could be detected in compost microcosms after incubation temperature was elevated to 45 degrees C or above for one or more days. In contrast, in the compost microcosms incubated at 23 degrees C, the target organism was not detected by the plate count method after 6 days, but its transgene was detectable for at least 45 days. In compost bins, the target organism was not recovered from compost microcosms or soil microcosms at different levels in the bins for 29 days. However, the transgene was detected in 8 of the 9 soil microcosms and in only 1 of the 9 compost microcosms. The compost microcosm in which transgene was detected was at the lower level of the bin where temperatures remained below 45 degrees C. The findings indicated that composting of organic wastes could be used to reduce or degrade heat sensitive GM microorganisms and their transgenes.     

Validation of Microcosms for Examining the Survival of Pseudomonas aureofaciens (lacZY) in Soil

Evaluating the safety and efficacy of a recombinant bacterium prior to its release into the terrestrial environment requires that risk assessment data be collected in the laboratory. Much of this information is obtained with the use of microcosms. The design of the microcosm significantly affects the ability of the recombinant microorganism to survive in soil and, thus, complicates the risk assessment process. To standardize microcosms for future use, we evaluated the survival of Pseudomonas aureofaciens 3732 RN-L11 (lacZY Rif(supr) Nal(supr)) in intact soil cores (5.0 by 15 cm; polyvinyl chloride core) and disturbed soil microcosms (50 g of fresh, sieved soil). Survival data were compared with those obtained during a field release. The intact soil core microcosm was shown to closely simulate results obtained in the field. The intact soil core microcosm closely predicts survival in bulk soil and in the rhizosphere of wheat. Data obtained with the microcosm were also similar when evaluated in separate studies in two different years. In 1993, P. aureofaciens survived for approximately 63 days in bulk soil and 96 days in the rhizosphere. The disturbed soil microcosm exhibited a much more rapid decline in population size (34 days to zero) than the intact core microcosm. We speculate that drying and sieving of soil for the disturbed soil microcosm affected the ability of the soil to support the survival of P. aureofaciens. These results demonstrate that a small, inexpensive, and simple intact soil core microcosm may be appropriate for risk assessment.     

Persistence of Escherichia coli O157:H7 in soil and on plant roots

Soil microcosms were inoculated with Escherichia coli O157:H7 to test persistence in fallow soil, on roots of cover crops and in presence of manure. In fallow soils, E. coli O157:H7 persisted for 25-41 days, on rye roots for 47-96 days and on alfalfa roots, in a silt loam soil, for 92 days whereas on other legumes persistence ranged from 25-40 days, similar to fallow soil. Manure did not seem to affect the persistence of E. coli O157:H7 in these soils. Indigenous and manure-applied coliform populations often decreased faster when E. coli O157:H7 was applied, indicating possible competition between microflora. Coliform populations in microcosms not inoculated with E. coli O157:H7 decreased more slowly or increased. Microbial community analyses showed little effect for E. coli O157:H7 inoculation or addition of manure. Microbial community metabolic activity was enhanced from rye roots after 14 days and by 63 days from alfalfa roots. Microbial community lactose utilization increased over time on rye roots in all soils and on alfalfa roots in a silt loam soil when E. coli O157:H7 was inoculated. Lactose utilization also increased for uninoculated rye roots, soil around rye roots and in some fallow soils. Our data suggest that clay increases persistence and activity of E. coli O157:H7 and other coliforms. In frozen soil stored for over 500 days, E. coli O157:H7 was viable in 37% of tested samples. In summary, E. coli O157:H7 persisted longer and activity was enhanced with some cover crops in these soils due to plant roots, the presence of clay and freezing.     

Field calibration of soil-core microcosms: Ecosystem structural and functional comparisons

Microcosms containing intact soil-cores are a potential biotechnology risk assessment tool for assessing the ecological effects of genetically engineered microorganisms before they are released to the field; however, microcosms must first be calibrated to ensure that they adequately simulate key field parameters. Soil-core microcosms were compared with the field in terms of ecological response to the introduction of a large inoculum of a rifampicin-resistant rhizobacterium,Pseudomonas sp. RC1. RC1 was inoculated into intact soil-core microcosms incubated in the laboratory at ambient temperature (22°C) and in a growth chamber with temperature fluctuations that mimicked a verage field values, as well as into field lysimeters and plots. The effect of the introduced bacterium on ecosystem structure, including wheat rhizoplane populations of total and fluorescent pseudomonads, total heterotrophic bacteria, and the diversity of total heterotrophic bacteria, was determined. Fluorescent pseudomonads were present on the rhizoplane in significantly lower numbers in soil inoculated with RC1, in both microcosms and the field. Conditions for microbial growth appeared to be most favorable in the growth chamber microcosm, as evidenced by higher populations of heterotrophs and a greater species diversity on the rhizoplane at the three-leaf stage of wheat growth. Ecosystem functional parameters, as determined by soil dehydrogenase activity, plant biomass production, and¹⁵N-fertilizer uptake by wheat, were different in the four systems. The stimulation of soil dehydrogenase activity by the addition of alfalfa was greater in the microcosms than in the field. In general, growth chamber microcosms, which simulated average field temperatures, were better predictors of field behavior than microcosms incubated continuously at 22°C.     

Development of nitrogen mineralisation gradients through surface soil depth and their influence on surface soil pH

Following mixing of the surface soil to about 7.5 cm depth in the field, soil layers (0–2.5, 2.5–5, 5–10 and 10–15 cm) were separately incubated in the laboratory to determine the rate of development of net N mineralisation gradients through surface soil depth under fallow, wheat and subterranean clover plots. Gradients in net N mineralisation were compared with those observed in the field, and their contribution to the observed pH changes was investigated.Heterotrophic activity, and thus net N mineralisation, decreased only slightly with depth immediately after soil mixing. This pattern persisted over time in soil layers sampled from fallow plots. In contrast, within 1 growing season after soil mixing, heterotrophic activity and net N mineralisation decreased significantly with depth in soil sampled from wheat and clover plots. In 0–15 cm soil sampled from under senescing plants, 32–38% of CO₂-C produced and net N mineralised originated from the surface 2.5 cm, while 52–56% originated from the surface 5 cm of soil. This resulted from an increase of pH and organic substrate concentration within the surface 2.5 cm of soil following plant residue return. Limitations of the in situ measurement of net N mineralisation in fallow soil was identified.Laboratory incubation studies showed that since most net N mineralisation occurred within the surface 2.5 cm of soil under senescing plants, nitrification and acidification were also concentrated at this depth. Despite this, compared to fallow soil, high potential acidification rates of 0–2.5 cm soil under senescing plants were not realised in the field due to the exposure to prolonged dry periods and moist-dry cycles. As a consequence, in the field the large magnitude of surface soil pH gradient which resulted from the return of alkaline plant residues was maintained over summer and autumn.     

Anaerobic Biodegradation of Alkylbenzenes in Laboratory Microcosms Representing Ambient Conditions

A microcosm study was performed to document the anaerobic biodegradation of benzene, toluene, ethylbenzene, m- xylene, and/or o-xylene in petroleum-contaminated aquifer sediment from sites in Michigan (MI) and North Carolina (NC) and relate the results to previous field investigations of intrinsic bioremediation. Laboratory microcosms, designed to simulate ambient conditions, were constructed under anaerobic conditions with sediment and groundwater from source, mid-plume, and end-plume locations at each site. The general patterns of biodegradation and electron acceptor utilization in the microcosms were consistent with field data. At the MI site, methane was produced after a moderate lag period, followed by toluene degradation in all sets of microcosms. At the NC site, biodegradation of the target compounds was not evident in the source area microcosms. In the mid-plume microcosms, toluene and o-xylene biodegraded first, followed by m-xylene and benzene, a pattern consistent with contaminant decay along the plume length. Chemical extraction of microcosm sediment at the beginning and end of me incubation indicated that iron-reducing conditions were dominant and iron reduction occurred on a sediment fraction not extracted by 0.5N HC1. In the end-plume microcosms, degradation of benzene, toluene, and xylene isomers occurred but was variable between replicates. Consistent with field data, dissolved concentrations of the target contaminant(s) persisted at low but detectable levels (0.05 to 0.25 μM) in microcosms from both sites where biodegradation was measured.     

Population Changes and Persistence of Rhizobium phaseoli in Soil and Rhizospheres

The impact of legume cultivation on the establishment and persistence of an inoculant strain of Rhizobium phaseoli and its ability to compete with a resident population of R. phaseoli for nodule occupancy was examined utilizing strain-specific fluorescent antibodies. The soil (Hubbard loamy sand) was inoculated homogeneously with 5 × 10⁵ cells per g of soil and confined in plastic cylinders kept in field plots. Inoculated and uninoculated cylinders were either left fallow or planted to two seeds of legumes. Two hosts, navy bean (Phaseolus vulgaris L.) cv. Seafarer and snap bean cv. Picker, as well as a nonhost, soybean (Glycine max (L.) Merr.) cv. Wilkin, were used. Inoculant Viking 1 was highly stimulated in all three rhizospheres sampled at 6 (flowering), 10 (podfill), and 17 (decay) weeks and in the following spring, whereas counts in fallow soil decreased rapidly. Although the overwintering population remained highest in the vicinity of decaying host roots, Viking 1 persisted, even in fallow soil, to produce abundant nodulation of host plants the following spring. Viking 1 was an excellent competitor for nodulation sites on the roots of the hosts; it thoroughly outcompeted the resident population of R. phaseoli, occupying virtually 100% of the nodules under inoculated conditions in all experiments.     

Slow arbuscular mycorrhizal colonisation of field-grown cotton caused by environmental conditions in the soil

 Slow arbuscular mycorrhizal colonisation is characteristic of a growth disorder of cotton occurring in crops in northern New South Wales, Australia. To determine whether or not slow colonisation is caused by poor survival of mycorrhizal fungi between crops, we examined colonisation of cotton in field crops and in a series of pot bioassays. Cotton roots were sampled at sites with or without severe symptoms of the growth disorder in each of three fields in 1991 and two fields in 1993. The bioassays were at intervals over the winter fallow prior to the crops in both years. In each bioassay, soil was collected from the field sites and sown with cotton in pots in a controlled environment cabinet. Colonisation was assessed at 14, 28 and 42 days after sowing. In the bioassay series, colonisation at 14 days, which was representative of primary infections of roots and hence propagule density in soil, tended to decline over the winter fallow. In contrast, colonisation at 42 days, which included secondary spread of infection, first declined and then returned to its original level or higher. In the field, plants affected by the growth disorder were colonised slowly, while healthy plants were colonised rapidly. In the bioassays, however, colonisation in the soil from sites with the growth disorder equalled or surpassed that in soil from sites with healthier cotton. Thus, the slow colonisation and growth of field-grown cotton did not result from a lack of mycorrhizal inoculum and was most likely caused by soil factors. Accepted: 21 August 1998     

Persistence and Suppressiveness of Pasteuria penetrans to Meloidogyne arenaria Race

The long-term persistence and suppressiveness of Pasteuria penetrans against Meloidogyne arenaria race 1 were investigated in a formerly root-knot nematode suppressive site following 9 years of continuous cultivation of three treatments and 4 years of continuous peanut. The three treatments were two M. arenaria race 1 nonhost crops, bahiagrass (Paspalum notatum cv. Pensacola var. Tifton 9), rhizomal peanut (Arachis glabrata cv. Florigraze), and weed fallow. Two root-knot nematode susceptible weeds commonly observed in weed fallow plots were hairy indigo (Indigofera hirsuta) and alyce clover (Alysicarpus vaginalis). The percentage of J2 with endospores attached reached the highest level of 87% in 2000 in weed fallow, and 63% and 53% in 2002 in bahiagrass and rhizomal peanut, respectively. The percentage of endospore-filled females extracted from peanut roots grown in weed fallow plots increased from nondetectable in 1999 to 56% in 2002, whereas the percentages in bahiagrass and rhizomal peanut plots were 41% and 16%, respectively. Over 4 years, however, there was no strong evidence that endospores densities reached suppressive levels because peanut roots, pods, and pegs were heavily galled, and yields were suppressed. This might be attributed to the discovery of M. javanica infecting peanut in this field in early autumn 2001. A laboratory test confirmed that although the P. penetrans isolate specific to M. arenaria attached to M. javanica J2, no development occurred. In summary, P. penetrans increased on M. arenaria over a 4-year period, but apparently because of infection of M. javanica on peanut at the field site root-knot disease was not suppressed. This was confirmed by a suppressive soil test that showed a higher level of soil suppressiveness than occurred in the field (P ≤ 0.01).     

Watershed liming effects on the forest floor N cycle

The forest floor was expected to play a major role in determining the total ecosystem response to watershed liming because of its high concentration of nutrients and its high level of activity. Net N mineralization and net nitrification were estimated in a field survey using the buried-bag approach. In a laboratory incubation experiment, forest floor humus was mixed with 6 doses of lime to determine the sensitivity of N mineralization and nitrification to lime dose. Forest floor microcosms with and without live tree roots were used to calculate a N budget for the system.The pH of the forest floor increased from 3.6 to 4.9 in the Oe and to 4.0 in the Oa two years after liming. The extractable ammonium pool in both the field survey and microcosm study was substantially smaller after liming and was probably a result of the 36% to 55% lower net N mineralization rate in limed plots than in reference plots. The laboratory incubation results agreed with the field survey results and further demonstrated that at higher lime doses (pH 5 to 6), N mineralization increased above controls. Net nitrification in limed humus in both the buried bags and laboratory incubation was as much as three times higher than controls, which could explain why nitrate leaching in limed microcosms was greater than in control microcosms. However, nitrate leaching from microcosms with live. roots was not affected by liming, suggesting that roots in the forest floor may prevent excess nitrate leaching. Reductions in N mineralization had no effect on N leaching or N uptake, but reduced the extractable ammonium pool.     

Phosphorus in a model pond study: I sediment selection and preparation

We chose two surface soils with contrasting textures as model sediments for a model pond study. One soil, a calcareous clay, had a relatively high natural phosphate content and a large phosphate adsorption capacity. The second soil, a non calcareous loam, had a relatively low natural phosphate content and a small phosphate adsorption capacity. Chemical characteristics of both soils were roughly proportional to mineral surface area.Pasture sites of each soil were tilled to a depth of 15 cm and two plots at each site were fertilized by hand with triple superphosphate. A third plot at each site was left unfertilized. After fertilization the plots were mechanically mixed and left fallow for 2 to 3 months. Then the plots were resampled and equilibration phosphate concentrations were determined again. Results showed significant phosphate fixation by the clay soil but no fixation by the loam soil.Research Soil Scientist, Botanist, Physical Science Technician and Physical Science Technician, respectively, USDA-ARS Agric. Water Quality Management Lab.Contribution from the USDA-ARS agricultural Water Quality Management Lab., Durant, OK 74701.     

Influence of soil fauna and habitat patchiness on plant (Betula pendula) growth and carbon dynamics in a microcosm experiment

We tested (1) how the presence of a diverse soil faunal community affects ecosystem carbon balance and (2) whether habitat patchiness modifies the influence of soil fauna on plant growth and carbon dynamics. We constructed cylindrical microcosms that contained coniferous forest humus and different litter materials either mixed or in separate patches, and in the presence or absence of diverse soil mesofauna. A birch seedling was planted in the centre of each microcosm. The experiment continued for two growing periods during which net carbon assimilation was measured continuously. At the end of the experiment, the microcosms were destructively sampled for plant biomass, soil fauna, and soil physical and chemical properties. All systems, independently of treatment, were net CO₂ producers in the beginning. In the presence of a diverse fauna, the plant growth was drastically increased, and the mixed-litter systems respired more than the patchy ones. During the second season, the patch effect disappeared, while the birch seedlings and mosses continued to grow better in the microcosms with diverse fauna. In the long term, patchiness did not modify the effect of fauna on plant growth or carbon balance. By the end of the experiment, the carbon balance approached zero in the refaunated microcosms, while it remained negative in the "simple" systems. The weak impact of patchiness in comparison to the faunal effect may be due to a homogenising role of plant roots and progressive decay of the substrates.     

Yield Loss Caused by Pratylenchus thornei on Wheat in South Australia

A two-year field trial with 130 plots was conducted at Tanunda, South Australia. Ten cereal cultivars differing in susceptibility to Pratylenchus thornei, two poor host crops (non-leguminous), and a bare fallow treatment were used to manipulate the numbers of nematodes in the plots in the first year. Initial and final densities were determined for each plot and varied from 0 to 9,400 nematodes/200 g oven-dried soil at the beginning of the second year. A highly susceptible wheat cultivar, Warigal, and two wheat lines known to have some resistance to P. thornei, GS50A and AUS4930, were planted in the second year. High densities of P. thornei caused more extensive lesions and severe cortical degradation in roots of Warigal than in GS50A or AUS4930. There was a significant linear relationship between initial density of P. thornei and Warigal grain yield (t/ha), with the estimated regression equation Y = 1.86 - 0.0000557x, where Y is the grain yield in t/ha and x is the number of P. thornei/200 g oven-dried soil. High initial densities (9,000 P. thornei/200 g oven-dried soil) caused up to 27% yield loss of this commercial Australian wheat. In contrast, the yield of the two resistant lines was not affected by initial density, suggesting that both were tolerant as well as resistant in the field.     

Effects of Soil Temperature and Planting Date of Wheat on Meloidogyne incognita Reproduction,Soil Populations,and Grain Yield

Wheat cultivars Anza and Produra grown in winter in California were planted in Meloidogyne incognita infested and noninfested sandy loam plots in October (soil temperature 21 C) and November (soil temperature 16 C) of 1979. Meloidogyne incognita penetrated roots of mid-October planted Ataza (427 juveniles/g root), developed into adult females by January, and produced 75 eggs/g root by harvest in April. Penetration and development did not occur in late plantings. Anza seedlings grown in infested soil in pots buried in field soil in early spring were not invaded until soil temperature exceeded 18 C. Meloidogyne incognita juveniles can migrate through soil and penetrate roots at temperatures above 18 C (activity threshold), however development can occur at lower temperatures. Grain yields were not significantly different between nematode infested (3,390 kg/ha) and noninfested (2,988 kg/ha) plots. Winter decline of eggs and juveniles in two late plantings anti in fallow soil were 69, 72, and 77%, respectively, but egg and juvenile decline was only 40% in the early Anza plots that supported nematode reproduction in the spring. Delay of planting date until soil temperature is below 18 C is suggested to maximize the use of wheat in rotation as a nematode pest management cultural tactic for suppressing root-knot nematodes.     

Microcosm study for revegetation of barren land with wild plants by some plant growth-promoting rhizobacteria

Growth promotion of wild plants by some plant growth-promoting rhizobacteria (PGPR) was examined in the microcosms composed of soils collected separately from a grass-covered site and a nongrass-covered site in a lakeside barren area at Lake Paro, Korea. After sowing the seeds of eight kinds of wild plants and inoculation of several strains of PGPR, the total bacterial number and microbial activity were measured during 5 months of study period, and the plant biomasses grown were compared at the end of the study. Acridine orange direct counts in the inoculated microcosms, 1.3-9.8 x 10(9) cells x g soil(-1) in the soil from the grass-covered area and 0.9-7.2 x 10(9) cells x g soil(-1) in the soil from the nongrass-covered site, were almost twice higher than those in the uninoculated microcosms. The number of Pseudomonas sp., well-known bacteria as PGPR, and the soil dehydrogenase activity were also higher in the inoculated soils than the uninoculated soils. The first germination of sowed seeds in the inoculated microcosm was 5 days earlier than the uninoculated microcosm. Average lengths of all plants grown during the study period were 26% and 29% longer in the inoculated microcosms starting with the grass-covered soil and the nongrass-covered soil, respectively, compared with those in the uninoculated microcosms. Dry weights of whole plants grown were 67-82% higher in the inoculated microcosms than the uninoculated microcosms. Microbial population and activity and growth promoting effect by PGPR were all higher in the soils collected from the grass-covered area than in the nongrass-covered area. The growth enhancement of wild plants seemed to occur by the activities of inoculated microorganisms, and this capability of PGPR may be utilized for rapid revegetation of some barren lands.     

Persistence and colonization strategies of playa wetland invertebrates

Wetland invertebrates have evolved numerous means of inhabiting spatially and temporally flooded wetland environments. The ability of invertebrates to either colonize from other sources and/or to persist in dry wetlands through diapause has seldom been simultaneously studied. We compared strategies of colonization and persistence by invertebrates in variable environments (playa wetlands on the Southern High Plains of Texas). We also examined emergence response time, following flooding, of taxa that persist in playa soil using field experiments and microcosms. At least 26 of 87 invertebrate taxa survive seasonal drying of playas through aestivation in soil. More invertebrate taxa only colonized flooded playas (70.1%) than only persisted in dry soil (29.9%) (P < 0.05). Of the invertebrate taxa that persisted in dry soil, more (P < 0.05) of these were active colonists or relied strictly on diapause rather than a combination of aestivation and colonization. Invertebrate densities were not statistically different among taxa that practiced colonization and persistence (5.2 invertebrates/m², SE = 2.0) or that only persisted (1.5 invertebrates/m², SE = 0.5) in playas (P=0.918). The average amount of time for a taxon to first appear in a microcosm was about 3 weeks less in 1995–96 than 1994–95, which was likely due to greater precipitation during 1995–96. We found that both colonization and persistence was practiced more often than a single strategy for those invertebrates sampled in microcosms. Conservation efforts for playa invertebrates should be implemented at the landscape level and focus on playas with intact watersheds, because these playas have relatively undisturbed hydroperiods.     

Changes in microbial nutrient status during secondary succession and its modification by earthworms

Summary Microbial biomass, nutrient (N and P) status, and carbon and nutrient limitation of the microflora were investigated in soils from five different sites (field, 5-, 12-, and about 50-year-old fallow, beechwood), which represent different stages of a secondary succession from a wheat field to the climax ecosystem of a beechwood on limestone. In addition, the effect of faeces production by the substrate feeding earthworm species Octolasion lacteum (Örley) on the nutrient status of the soil microflora of these sites was studied. Humus had accumulated in the soil of the third fallow site, with an enhanced biomass of microflora. However, in the beechwood soil, which had the highest humus content, microbial biomass was lower than in the soil of the third fallow site and similar to that of the field and the two younger fallow sites. In general, soil microbial biomass was little affected by the passage of soil through the gut of O. lacteum. The soil microflora of the field, the 5-, 12-, and about 50-year-old fallow was limited by carbon, whereas in the beechwood soil phosphorus limited microbial growth. NItrogen availability to the soil microflora was low in the two younger fallow sites and high in the field and the third fallow. In the beechwood soil nitrogen supply did not affect microbial carbon utilization. Application of phosphorus stimulated glucose mineralization in the soil of the field, the third fallow, and the beechwood, but not in the two younger fallow sites. Therefor, the nutrient status of the soil microflora seems to have changed during secondary succession: presumably, during the first phase the availability of nitrogen decreased, whereas during the second phase microbial phosphorus supply became more important, which resulted in phosphorus limitation of the soil microflora in the climax ecosystem. The passage of soil through the gut of O. lacteum caused an alteration in the microbial nutrient status. Generally, microbial growth in earthworm casts was limited by carbon. The relative effect of the gut passage of the soils on microbial carbon utilization seems to increase during succession. Therefore, the effect of decomposer invertebrates on microbial nutrient supply seems to increase during secondary succession. In general, nitrogen did not limit microbial carbon utilization in earthworm casts. Phosphorus requirements of the soil microflora were lowered by the gut passage of the soil of the third fallow site and the beechwood, which indicates an increased phosphorus supply in earthworm casts. Howerver, this additional supply was not sufficient to enable optimal carbon utilization by the soil microflora. The results indicate that the effect of decomposer invertebrates on the soil microflora depends on the nutrient status of the ecosystem.     

The effect of soil pH on rhizosphere carbon flow of Lolium perenne

Perennial rye-grass plants were grown at 15°C in microcosms containing soil sampled from field plots that had been maintained at constant pH for the last 30 years. Six soil pH values were tested in the experiment, with pH ranging from 4.3–6.5. After 3 weeks growth in the microcosms, plant shoots were exposed to a pulse of ¹⁴C-CO₂. The fate of this label was determined by monitoring ¹⁴C-CO₂ respired by the plant roots/soil and by the shoots. The ¹⁴C remaining in plant roots and shoots was determined when the plants were harvested 7 days after receiving the pulse label. The amount of ¹⁴C (expressed as a percentage of the total ¹⁴C fixed by the plant) lost from the plant roots increased from 12.3 to 30.6% with increasing soil pH from 4.3 to 6. Although a greater percentage of the fixed ¹⁴C was respired by the root/soil as soil pH increased, plant biomass was greater with increasing soil pH. Possible reasons for observed changes in the pattern of ¹⁴C distribution are discussed and, it is suggested that changes in the soil microbial biomass and in plant nitrogen nutrition may, in particular be key factors which led to increased loss of carbon from plant roots with increasing soil pH.     

Dynamics, spread, and persistence of a single genotype of Pseudomonas syringae relative to those of its conspecifics on populations of snap bean leaflets.

A rifampin-resistant strain of Pseudomonas syringae (R10) was introduced onto bean plants grown in field plots to examine the processes of growth, spread, and survival of a single genotype relative to the dynamics of its conspecifics on populations of individual leaflets. R10 was applied to four plots (400, 200, 100, and 50 m2), each of which was centered in a quadrant of a bean field (90 by 90 m). Population sizes of the species P. syringae and of R10 were determined on each of 25 individual leaflets collected from the largest plot (A) at approximately weekly intervals during a 10-week period following application. The spread of R10 from all plots was monitored by leaf imprinting of individual leaflets collected at sites along four transects, each of which bisected two of the plots. The introduced strain was a dominant component of the species for about 5 weeks postinoculation on leaflets from plot A. Although the population sizes of R10 remained at about 5.0 to 5.5 log10 CFU per leaflet, the strain became a progressively minor component of the species as the population sizes of its conspecifics continued to increase during the latter part of the growing season. In general, a positive correlation was found for the population sizes of R10 and its conspecifics on individual leaflets collected throughout the growing season. This result suggests that large numbers of R10 early in the growing season did not exclude the colonization of bean leaflets by its conspecifics. It is apparent that the species pool comprised genotypes that were more fit than R10 under the conditions that prevailed during the latter part of the growing season. By 6 weeks postinoculation, R10 was detected at all sites sampled within the unsprayed areas of the field. However, it was present as a minor component of the species. The persistence of R10 throughout the winter and into the following growing season was monitored in plot A, which was plowed and replanted with wheat in the fall. R10 was detected on some of the samples (wheat seedlings or soil) taken at approximately monthly intervals from November to June of the following year. In June, the field was plowed and replanted with beans. We could not detect R10 on emerging bean seedlings in plot A. The results demonstrate that the successful spread and persistence of an introduced bacterium do not necessarily lead to the establishment of large populations of the bacterium in adjacent untreated areas or on its host plant in subsequent growing seasons.