Survival of κ-carrageenan-encapsulated and unencapsulated Pseudomonas aeruginosa UG2Lr cells in forest soil monitored by polymerase chain reaction and spread plating

Abstract A method was developed for direct extraction, purification and amplification of DNA from forest soil. Eighty-two % of the DNA in Pseudomonas aeruginosa UG2Lr introduced into soil was recovered. The detection limit for the strain was approximately 800 cfu g⁻¹ of dry soil based on the polymerase chain reaction (PCR). Survival of κ-carrageenan-encapsulated and unencapsulated UG2Lr was monitored by antibiotic selective and bioluminescence-based nonselective plating and PCR-amplification of a tnsA fragment. After freeze-thaw treatment of soil samples, the unencapsulated UG2Lr declined from an initial population density of 1 × 10⁹ cfu g⁻¹ of dry soil to below the detection threshold of both selective (14 cfu g⁻¹ of dry soil) and nonselective (1 × 10³ cfu g⁻¹ of dry soil) plating. However, presence of nonculturable UG2Lr cells in the soil was revealed by PCR and resuscitation of the bacteria. Population density of the encapsulated UG2Lr increased from 2.7 × 10⁶ to 2.9 × 10⁸ cfu g⁻¹ of dry soil after a 3-week incubation at 22°C and declined to 6.3 × 10⁶ cfu g⁻¹ of dry soil after the freeze-thaw treatment.     

Influence of woodlot floor topography on microbial decomposer distribution

The distribution of microbial populations that decomposed sugar, cellulose and lignin-related substrates was examined in a beech Fagus grandifolia Ehrh. and maple Acer saccharum Marsh. dominated woodlot developed on glacial till. The topography of the woodlot, characterized by rises, depressions and more extensive level areas about 1 m in diameter with a 0.5 m vertical maximum, produced a mosaic of decomposer habitats designated as high, level and low sites.
In general, populations of sugar, cellulose and lignin decomposing organisms (based on ten estimates made from April to October) were two to four times higher in litter and soil samples from low sites than those from high sites. Sugar decomposing bacteria in litter were most abundant at all topographic sites. 135 × 10⁶ g⁻¹ dry litter at high sites, 396 × 10⁶ g⁻¹ at level sites and 456 × 10⁶ g⁻¹ at low sites; lignolytic fungi were least abundant, 391 × 10² g⁻¹ dry litter at high sites. 700 × 10⁶ g⁻¹ at level sites and 954 × 10² g⁻¹ at low sites. Numbers of microbial decomposers in the topographic sites were correlated with organic matter content. Distribution of fungal genera did not appear to be related to topographic site. Most populations examined showed two numerical peaks, one in late May or June and one in late September or October. It is suspected that these peaks were influenced by the coincident timing of favourable physical conditions and priming by soluble nutrients leached from litter.     

Influence of PCBs on the predator-prey relation between bacteria and protozoa in soil

Abstract This work deals with the impact of a possible accidental pollutant, pyralene (Prodelec, France; PCBs in trichlorobenzene), intoduced into the soil. Its influence on the predator-prey relation between bacteria and amoebae was studied by comparing the population dynamics of (i) an inoculated bacterial population ( A. lipoferum ) chosen as a biological tracer, (ii) the indigenous bacterial microflora, (iii) the infigenous amoebae. In the absence of pyralene the inoculated bacterial population decreased from 10⁷ to 10⁴ bacteria g⁻¹ soil (dw), grazed by the infigenous amoebae whose numbers increased 3-fold. In contrast, in presence of 2500 ppm of pyralene the introduced bacteria survived at a higher level (3·10⁶ bacteria g⁻¹ soil (dw)) while the number of amoebae diminished slightly. No predation occurred with PCB contamination. The indigenous bacterial microflora was not affected quantitatively by pyralene. In pure liquid culture with 500 ppm of pyralene added, bacterial growth was inhibited and an amoebal strain isolated from an inoculated uncontaminated soil was killed. We conclude that the active form of the amoebae were killed, and encystement was inhibited by pyralene in the soil. Hence the protozoa were unable to regulate the introduced A. lipoferum strain as they did in the absence of the pollutant.     

Monitoring the fate of genetically engineered bacteria sprayed on the phylloplane of bush beans and grass

Abstract The fate of a Bacillus amyloliquefaciens with the recombinant plasmid pSB20 sprayed on the phyllosphere of grass, and of a Tn 5 marked Pseudomonas syringae sprayed on the phyllosphere of bush beans was studied in planted soil microcosms. B. amyloliquefaciens showed a decline from 1.5×10⁸ to 3.1×10² cfu g⁻¹ on the phylloplane of grass in the course of the experiment. B. amyloliquefaciens was easy to follow by selective cultivation due to the complete absence of bacterial background growth. Southern blot hybridization of Hin dIII digested genomic DNA showed plasmid restriction patterns identical with pSB20 indicating high plasmid stability. In total DNA extracts from phyllosphere bacteria the recombinant plasmid was detectable by Southern blot hybridization up to 6×10⁴ cfu g⁻¹ (wet weight). Counts of hybridizing colonies showed that P. syringae established on the phyllosphere of bush beans at between 5×10³ and 4×10⁶ cfu g⁻¹ fresh weight. During senescence of the bean plants the strain was no longer detectable by selective cultivation and subsequent colony hybridization. In contrast, Tn5 marked DNA was detected after PCR amplification over the whole period of the experiment.     

Survival of genetically modified Pseudomonas fluorescens introduced into subtropical soil microcosms

Abstract A genetically modified strain of Pseudomonas fluorescens and its parent showed grossly similar decline rates following introduction into subtropical clay and sandy soils. In unplanted clay soit at pH 6.9 and 25°C, population densities declined progressively from about 10⁸ to 10³ colony forming units (cfu) g⁻¹ dry soil over 75 days, but in unplanted sandy soil the introduced populations could not be detected after 25 days. In clay soil at pH 8.7 or 4.7, or at environmental temperature, decay rates were enhanced as compared to those at pH 6.9 and 25°C. Counts of introduced strains in clay bulk soil and in rhizosphere and rhizoplane of maize suggested that the introduced bacteria competed well with the native bacteria, and colonized the roots at about 10⁶ cfu g⁻¹ dry root at 25°C, over 20 days. However, rhizoplane colonization was lower at environmental temperature. The decay rate of both strains was slower in planted than in unplanted sandy soil. The population densities in the rhizosphere and rhizoplane in the sandy soil were significantly lower than those in the clay soil. Both introduced strains colonized the maize roots in both soils, using seeds coated with bacteria in 1% carboxymethyl cellulose. Introduced cells were localized at different sites along the roots of plants developing in clay soil, with higher densities in the original (near the seeds) and root hair zones as compared to the intermediate zones. No significant difference was observed between the extent of root colonization of the genetically modified strain and its parent.     

Quantification of pathogenic micro-organisms in the sludge from treated hospital wastewater

The sludge from hospital waste treatment facilities is a potential source of infectious organisms. The average numbers of micro-organisms in the sludge of hospital wastewater in Taiwan were as follows: total count 8·1 × 10⁷ cfu g⁻¹ (dry weight of sludge), and 1·4 × 10⁶, 3·6 × 10⁵, 1·6 × 10⁵, 2·2 × 10⁵ and 5·5 × 10⁴ cfu g⁻¹ (dry weight of sludge) for total coliforms, faecal coliforms, faecal streptococci, Pseudomonas aeruginosa and Salmonella spp., respectively . Salmonella spp. were detected in 37% (10 of 27) of the sludges from hospital wastewaters. Therefore, the treatment of such sludge to reduce pathogenic micro-organisms should be considered.     

A carbon budget for nematodes, rotifers and tardigrades in a Swedish coniferous forest soil

Nematodes, rotifers and tardigrades from a Swedish pine forest soil were investigated in a monthly sampling programme lasting for one year. The monthly mean values of number, biomass and oxygen consumption were estimated. All groups fluctuated rather much over the year with a summer minimum (1.1 ˙ 10⁶ animals m^-2) and a winter maximum (6.3 ˙ 10⁶ animals m^-2) for the nematodes. The reasons for these fluctuations are discussed in relation to fluctuations of water content and temperature of the soil. On an annual basis a carbon budget was calculated which gave the following values for this fauna; consumption 4.3, production 0.6, respiration 0.9 and defecation 2.8 g carbon m^-2.     

Regulation of Fusarium oxysporum population introduced into soil: the amoebal predation hypothesis

Abstract Inoculation of fungi into soil has been suggested for biological control of plant diseases. The aim of our work was to test the ability of protozoa to reduce the density of introduced fungal populations. The survival of Fusarium oxysporum in non-sterile soil was studied after introduction at densities of: 1 × 10⁴, 1 × 10⁶ and 5 × 10⁷ cfu/g soil. The dynamics of protozoa were also followed. The fungal populations remained close to the initial inoculation densities and did not induce the growth of indigenous protozoa. A bacterial population ( Enterobacter aerogenes ) was used to promote and stimulate the predatory activity of amoebae. Then, after simultaneous inoculation with bacteria and fungi, the density of protozoa increased but this had no effect on the fungal population, although some amoebae are able to feed on small fungal propagules such as conidia. The physiological state of Fusarium in soil and intraspecific competition seem to be more important in regulating introduced fungal populations than amoebal predation. We conclude that the regulation of bacterial and fungal populations in soil depend on different mechanisms.     

Transgenerational marking of freshwater fishes with enriched stable isotopes: a tool for fisheries management and research

A promising new method of marking larval freshwater fishes with enriched stable isotopes by means of injecting the maternal parent with the marking agent was investigated. The ¹³⁸Ba:¹³⁷Ba ratios in the otoliths of larval golden perch Macquaria ambigua were compared to determine the effect of injecting female broodstock with different dosages of enriched ¹³⁷Ba at various times before spawning. There was 100% mark success in the progeny of fish injected with 20 μg g⁻¹ of enriched ¹³⁷Ba 24 h before inducing spawning with hormones and 40 μg g⁻¹ administered at the same time as inducement of spawning. Injection of 40 μg g⁻¹ enriched ¹³⁷Ba 21 days before spawning resulted in only 81% mark success and suggests rapid elimination of barium in M. ambigua . Injection with enriched ¹³⁷Ba did not significantly affect the fertilization rate, number of fertilized eggs or hatching rate compared with long-term hatchery records. These results suggest that transgenerational marking is an effective and affordable means of mass-marking larval fishes. Thousands of larval fishes can be permanently marked with a unique artificial isotopic mark via a single injection into the maternal parent, thus avoiding the handling of individual fishes or having to deal with chemical baths. Because no single mark or tagging method is suitable for all situations, transgenerational marking with enriched stable isotopes provides another method for researchers and managers to discriminate both hatchery-reared and wild fishes.     

In vitro analysis of intestinal absorption of cadmium and calcium in rainbow trout fed with calcium- and cadmium-supplemented diets

The protective effects of dietary Ca²⁺ supplementation against Cd accumulation in rainbow trout Oncorhynchus mykiss fed with Cd-contaminated food were evaluated in relation to chronic changes in intestinal absorption rates. The changes were measured ' in vitro '. The control diet contained c. 20 mg Ca²⁺ g⁻¹ food and 0·25 μg Cd g⁻¹ food; the experimental diets were supplemented with CaCO₃ and Cd(NO₃)₂·4H₂O to levels of 50 mg Ca²⁺ g⁻¹ food and 300 μg Cd g⁻¹ food, alone and in combination. The Ca²⁺ and Cd absorption rates were measured using radiotracers (⁴⁵Ca, ¹⁰⁹Cd) at total Ca²⁺ and Cd concentrations of 3·0 and 0·12 mmol l⁻¹, respectively in the intestinal saline. Chronically elevated dietary Cd caused a significant increase in Cd absorption rate by up to 10-fold at 30 days in the mid-intestine. The high Ca²⁺ diet prevented this up-regulation of Cd transport rate. Conversely, intestinal Ca²⁺ absorption was significantly increased by two- to five-fold by the Ca²⁺-supplemented diet at 30 days in both the mid- and posterior intestine, and this effect was eliminated when Cd was simultaneously elevated in the diet. Ca²⁺ and Cd probably interact at common pathways and transport mechanisms in the intestine, though independent pathways may also exist.     

Effects of soil frost on nitrogen net mineralization, soil solution chemistry and seepage losses in a temperate forest soil

Freezing and thawing may alter element turnover and solute fluxes in soils by changing physical and biological soil properties. We simulated soil frost in replicated snow removal plots in a mountainous Norway spruce stand in the Fichtelgebirge area, Germany, and investigated N net mineralization, solute concentrations and fluxes of dissolved organic carbon (DOC) and of mineral ions (NH₄⁺, NO₃⁻, Na⁺, K⁺, Ca²⁺, Mg²⁺). At the snow removal plots the minimum soil temperature was −5 °C at 5 cm depth, while the control plots were covered by snow and experienced no soil frost. The soil frost lasted for about 3 months and penetrated the soil to about 15 cm depth. In the 3 months after thawing, the in situ N net mineralization in the forest floor and upper mineral soil was not affected by soil frost. In late summer, NO₃⁻ concentrations increased in forest floor percolates and soil solutions at 20 cm soil depth in the snow removal plots relative to the control. The increase lasted for about 2–4 months at a time of low seepage water fluxes. Soil frost did not affect DOC concentrations and radiocarbon signatures of DOC. No specific frost effect was observed for K⁺, Ca²⁺ and Mg²⁺ in soil solutions, however, the Na⁺ concentrations in the upper mineral soil increased. In the 12 months following snowmelt, the solute fluxes of N, DOC, and mineral ions were not influenced by the previous soil frost at any depth. Our experiment did not support the hypothesis that moderate soil frost triggers solute losses of N, DOC, and mineral ions from temperate forest soils.     

Transformation of Methanococcus maripaludis and identification of a Pst I-like restriction system

Abstract An optimized polyethylene glycol (PEG) method of transformation was developed for Methanococcus maripaludis using the pKAS102 integration vector. The frequency of transformation with 0.8 μg of plasmid and 3×10⁹ cells was 4.8×10⁻⁵ transformants cfu⁻¹, or 1.8×10⁵ transformants μg⁻¹, which was four orders of magnitude greater than with the natural transformation method. A Pst I restriction activity in M. maripaludis was also identified. Methylation of the plasmid with Pst I methylase increased the methanococcal transformation frequency at least four-fold. Also, chromosomal DNA from M. maripaludis was resistant to digestion by the Pst I endonuclease.     

Clostridia spore formation during aerobic deterioration of maize and sorghum silages as influenced by Lactobacillus buchneri and Lactobacillus plantarum inoculants

Aims:  The effect of the inoculation of maize and sorghum silages with Lactobacillus plantarum (LP) and Lactobacillus buchneri (LB) on the clostridia spore formation during aerobic deterioration has been studied.
Methods and results:  The crops were ensiled in 30 l jars, without a lactic acid bacteria inoculant (C), and with an LP or LB inocula (theoretical rate of 1 × 10⁶). After 90 days of conservation, the silages were analysed for the chemical and microbiological characteristics and subjected to an aerobic stability test, during which pH, temperature, nitrate, yeast, mould and clostridia spores were measured. Compared to the C and LP silages, yeasts were reduced in the LB silages, resulting in an increased aerobic stability. Clostridia spores, determined by most probable number (MPN) procedure, increased to 6 log₁₀ MPN g⁻¹ in the C and LP maize silages, whereas they reached 3 log₁₀ MPN g⁻¹ in C and LP sorghum silages.
Conclusions:  Clostridia spore count only slightly increased in the LB maize silages after 342 h (2·59 log₁₀ MPN g⁻¹), whereas it did not show any increase in the LB sorghum silages for the whole period of air exposure.
Significance and impact of the study:  The data indicated that clostridia spore outgrowth can take place during silo feedout in aerobic-deteriorated silages and that LB inoculation reduces the risk of clostridia outgrowth after silage opening by increasing the aerobic stability.     

Isolation and characterization of Azotobacter chroococcum from the roots of Zea mays

Abstract Bacteria showing rapid growth on a nitrogenfree medium and acetylene-reducing activity were isolated from maize roots collected from agricultural soils in Spain. The isolates were Gram-negative motile rods and were identified as Azotobacter chroococcum . Acetylene-reducing activity and microbial counts were determined on root segments from 7- and 30-day-old plants. Rates obtained were in the range of 0.0053–0.848 nmol C₂H₂· g⁻¹· h⁻¹. Root populations were 1.4–6.0 × 10⁴ micro-organisms · g⁻¹. These results showed that there was an association between A. chroococcum strains and roots of maize planted in some Spanish soils.     

Population dynamics of a motile and a non-motile Azospirillum lipoferum strain during rice root colonization and motility variation in the rhizosphere

Abstract: Azospirillum lipoferum 4B and non-motile A. lipoferum 4T have been simultaneously isolated from rice rhizosphere at the same frequency. A. lipoferum 4T showed stable morphological and metabolic traits which are atypical for A. lipoferum species such as lack of motility, carbohydrate metabolism and laccase activity. Inoculation experiments showed that A. lipoferum 4T, but not A. lipoferum 4B, needed rice roots to stabilize in sterile soil. Both strains were able to colonize efficiently rice roots (10⁸ cfu g⁻¹ fresh roots) but motile form 4B remained dominant. In spite of their phenotypical differences, A. lipoferum 4B and 4T co-existed without exclusion in sterile soil (planted or not) and rice rhizosphere. Inoculation of rice roots with A. lipoferum 4B showed that rice rhizosphere enhanced the frequency of appearance of stable non-motile forms (40%). This percentage was weaker in plantlet growth medium (4%). However, these non-motile bacteria kept the same biochemical traits than the motile parental strain 4B (carbohydrates metabolism, laccase activity).     

Production of amylase by the intestinal microflora in cultured freshwater fish

The amylase-producing ability of the intestinal microflora in cultured specimens of ayu, carp, channel catfish, Japanese eel and tilapia was determined. Mean viable counts of aerobes and anaerobes ranged from 1·1×10⁶ to 3·7×10⁸ cfu g⁻¹ and from 1·3×10³ to 1·6×10⁸ cfu g⁻¹, respectively. Aeromonas spp. and Bacteroidaceae were predominant in four to five fish species. Of 206 strains examined, 65 (31·6%) produced ≥0·01 U amylase ml⁻¹. The percentage of producers differed among families and genera of bacteria and fish species. While 56% of the anaerobes produced amylase, only 20% of the aerobes did. More than 50% of Aeromonas , Bacteroidaceae and Clostridium strains produced amylase efficiently while Acinetobacter , coryneforms, Enterobacteriaceae, Moraxella , Plesiomonas and Streptococcus strains did not. High amylase production (≥0·05 U ml⁻¹) was found in 12 strains, 11 from Aeromonas and one Pseudomonas . The percentage of high amylase producers in Japanese eel was lower than the other four fish (2–30%). These results strongly suggest that the amylase produced by the intestinal microflora play an important role in the digestion of starch in freshwater fish to some extent.     

Persistence of Shiga toxin-producing Escherichia coli O26 in various manure-amended soil types

Aims:  To evaluate the behaviour of Shiga toxin-producing Escherichia coli (STEC) O26 strains inoculated in manure-amended soils under in vitro conditions.
Methods and Results:  Four green fluorescent protein (GFP)-labelled STEC O26 strains were inoculated in duplicate (at 10⁶ CFU g⁻¹) in three different manure-amended soil types, including two loam soils (A and B) and one clay loam soil (C), and two incubation temperatures (4 and 20°C) were tested. STEC counts and soil physical parameters were periodically monitored. STEC O26 cells were able to persist during extended periods in soil even in the presence of low moisture levels, i.e. less than 0·08 g H₂O g⁻¹ dry soil. At 4 and 20°C, STEC could be detected in soil A for 288 and 196 days, respectively, and in soils B and C for at least 365 days postinoculation at both temperatures. The ambient temperature (i.e. 20°C) was significantly associated with the highest STEC count decline in all soils tested.
Conclusions:  The temperature and soil properties appear to be contributory factors affecting the long-term survival of STEC O26 in manure-amended soils.
Significance and Impact of the Study:  This study provides useful information regarding the ecology of STEC O26 in manure-amended soils and may have implications for land and waste management.     

Effects of triploidy on rainbow trout myogenesis in vitro

To examine the effects of triploidy on rainbow trout myogenesis in vitro , mononuclear cells were liberated enzymatically from the lateralis muscles of diploid and triploid trout. The muscle of diploids yielded 1 × 10⁶± 1 × 10⁵ (± s.e.m. ) mononuclear cells g⁻¹ muscle compared to 0.7 × 10⁶± 8 × 10⁴ cells g⁻¹ from triploids ( P <0.01). The plating efficiencies of diploid and triploid mononuclear cells on Matrigel™ following 18 h of culture in Leibovitz's L-15 + 10% foetal bovine serum were not significantly different, 35.0 ± 3.5% and 33.0 ± 2.9%, respectively. For most time points examined, the proportion of nuclei in multinucleated cells and the proportion of nuclei in myosin positive cells were not significantly different for diploid and triploid trout. Taken together, these data suggest that diploid and triploid myogenic cells will differentiate similarly when compared under identical, in vitro conditions.     

Detection and quantification of Desulforhabdus amnigenus in anaerobic granular sludge by dot blot hybridization and PCR amplification

A species-specific 16S rRNA oligonucleotide probe (ASRB1) was developed for the detection of Desulforhabdus amnigenus in anaerobic granular sludge. The presence of nucleic acids from cells of D. amnigenus in granular sludge was determined using ASRB1 as a specific primer for polymerase chain reaction (PCR) amplification or as a probe for dot blot hybridizations. The detection threshold and the reproducibility of these two methods were determined with sludge amended with 10⁴–10¹⁰ D. amnigenus cells per gram of volatile suspended solids (VSS). For D. amnigenus cells with a ribosomal RNA content of 15 fg cell⁻¹, the lowest number of target cells detected by hybridization was 1 × 10⁸ cells g⁻¹ VSS. With the PCR amplification method the lowest number of target cells which could be detected was 1 × 10⁷ g⁻¹ VSS. This corresponds to a threshold level for hybridization of 0·1–0·001‰ of the total bacterial sludge population, while the threshold level obtained with the PCR approach amounted to 0·01–0·0001‰. The rRNA content of D. amnigenus was found to be affected by the growth rate and the growth phase, and it ranged from 19 fg cell⁻¹ in slow-growing cultures to 90 fg cell⁻¹ in fast-growing cultures. Therefore, the detection threshold of the dot blot hybridization method for fast-growing cells is lower than for slow-growing cells.     

The effects of water hardness upon the uptake, accumulation and excretion of zinc in the brown trout, Salmo trutta L.

The effects of water hardness (9 and 220 mgl⁻¹ as CaCO₃) upon zinc exchange in brown trout exposed to 0.77 μmol Zn 1⁻¹ have been investigated using artificial soft water (<49.9 μmol Ca ^l-1, <40.1 μmol Mg 1⁻¹) and mains hard water (1671.7 μmol Ca 1⁻¹, 493.6 μmol Mg 1⁻¹) of known composition. Both hard and soft water-adapted fish exhibited a bimodal pattern of net zinc influx. Net zinc influxes during both fast and slow uptake phases were significantly greater ( P <0.001) in soft (82.9 and 6.2 μmol Zn 100 g⁻¹ h⁻¹) than in hard water (46.3 and 2.4 μmol Zn 100 g h⁻¹). Zinc efflux (- 0.2 μmol Zn 100 g⁻¹ h⁻¹) was enhanced only in hard water during the slow net influx phase.
Brown trout exposed to zinc in hard water and placed in metal-free media exhibited a greater net efflux (- 25.6 μmol Zn 100 g⁻¹ h⁻¹) of the metal than did fish in soft water (-4.2 μmol Zn 100 g⁻¹ h⁻¹) treated in the same manner. Tissue ⁶⁵Zn activities reflected both the differences in uptake and excretion rates of the metal between hard and soft water fish. During zinc exposure (0.77 μmol Zn 1⁻¹) high water hardness reduced tissue burdens of the metal by reducing net branchial influx, and enhancing efflux of the metal in hard water fish.