Survival ofalginate-ecapsulated Pseudomonas fluorescens cells in soil

Alginate-encapsulated and unencapsulated cells of Pseudomonas fluorescens Rsf were introduced into soil microcosms with and without wheat plants to evaluate bacterial survival and colonization of the rhizoplane and rhizosphere. Encapsualtion of cells in alginate amended with skim milk or with skim milk plus bentonite clay significantly enchanced long-term survival of the cells. There was a negligible effect on long-term bacterial survival when cells were encapsulated in alginate amended with TY medium or soil extract, as compared to water. Drying of beads resulted in a significant reduction in bacterial viability. After addition to soil, cells in dried beads increased in numbers and exhibited stable population densities, whereas cells added in moist beads showed stable dynamics at a higher level. Cells encapsulated in dried beads or fresh beads survived better than unencapsulated cells added to soil. Both cells in moist and dried alginate beads also survide a dry/wet cycle in soil, whereas unencapsulated cells were sensitive to these moisture fluctuations. Shortly after inoculation and 63 days after this, cells from moist beads colonized wheat roots at significantly higher levels than unencapsulated cells, whereas cells in dried beads did so at levels similat to unencapsulated cells. Cells in beads initially placed at different distance from developing root mat were able to move towards and colonize the rhizosphere, at levels of roughly 10⁴ to 10⁶ colony-forming units fo P. fluorescens R2f per gram of dry soil. Correspondence to: J. T. Trevors or J. D. van Elsas     

Survival and proliferation of alginate encapsulated Trichoderma spp. in Egyptian soil in comparison with allyl alcohol soil fumigation

Conidia of Trichoderma harzianum and T. pseudokoningii (Rifai) were formulated to make alginate pellets withor without 10% cellulose as a food-base material. The formulations were compared for their ability for survival and proliferation of Trichoderma spp. in clay-loamy soil (50% moisture content)with allyl alcohol fumigation (0.05, 0.1 and 0.2 ml/1000 ml space). Trichoderma medium E (TME) containing 100 g/ml pentachloronitrobenzene (PCNB) was valuable for isolation and counting of Trichoderma spp. from the tested soil than the Glucose-Czapek's agar medium containing 1:15000 Rose-bengal. The promotive effect of Trichoderma by different doses of allyl alcohol fumigationstill enhanced after two-month incubation period. Conidia entrapped in alginate with or without cellulose and introduced into the soil survived better than conidia added directly to the same soil after three months incubation period. Sterile soil provided a more favorable environment for the proliferation and survival of immobilized conidia than the non-sterile soil, and the addition of 10% cellulose increased the survival of the entrapped conidia more than those prepared without cellulose. Soil fumigation inhibited the occurrence of other fungal species; however, inoculation of the soil with alginate immobilized conidia or conidial suspension had such inhibitory effect but in a less extent.This revised version was published online in October 2005 with corrections to the Cover Date.     

Comparisons of ectomycorrhizae on pinyon pines (Pinus edulis; Pinaceae) across extremes of soil type and herbivory

Using field and greenhouse studies, we examined the relationships among pinyon pines (Pinus edulis), their ectomycorrhizal mutualists, and their moth herbivores as a function of soil fertility. We studied two soil types—the ash and cinder soils of the San Francisco volcanic field and nearby sandy loam soils. In the field, pinyons growing in cinders suffered from reduced moisture, negative nitrogen mineralization rates, low phosphate levels, reduced growth, and high moth herbivory relative to pinyons growing in sandy loam. Pinyons growing in cinders also had twofold higher levels of ectomycorrhizal colonization than their noncinder counterparts. Similarly, in the greenhouse, seedlings grown in cinders had higher levels of ectomycorrhizal colonization and greater numbers of ectomycorrhizae than seedlings grown in sandy loam. Seedling shoot growth was significantly enhanced by ectomycorrhizae in both soils. These patterns support three conclusions. First, field and greenhouse studies demonstrated that trees growing in nutrient-poor soils had higher levels of ectomycorrhizal colonization than trees growing in better soils. Second, across soil types, variation in ectomycorrhizal colonization was better predicted by soil fertility than by herbivory. However, herbivory negatively affected ectomycorrhizae in the stressful cinder environment. Third, although mycorrhizae can be parasitic under some conditions, ectomycorrhizae had mutualistic impacts on pinyon seedlings across the environmental extremes we studied.     

Application of free and Ca-alginate-entrapped Glomus deserticola and Yarowia lipolytica in a soil-plant system

This study was performed to investigate the applicability of microbial inoculants entrapped in alginate gel. Glomus deserticola (AM) was inoculated into soil microcosms, enriched with rock phosphate, as either free form or entrapped in calcium alginate alone or in combination with a P-solubilizing yeast culture (Yarowia lipolytica). Plant dry weight, soluble P acquisition, and mycorrhizal index were equal in treatments inoculated with free and alginate-entrapped AM. Dual inoculation with entrapped G. deserticola and free cells of Y. lipolytica significantly increased all analyzed variables. Highest rates of the latter were obtained when both fungal microorganisms were applied co-entrapped in the carrier. The yeast culture behaved as a 'mycorrhiza helper microorganism' enhancing mycorrhization of tomato roots. These results indicate that dual inoculation with an AM fungus and a P-solubilizing microorganism co-entrapped in alginate can be an efficient technique for plant establishment and growth in nutrient deficient soils.     

Preparation of gel-entrapped mycorrhizal inoculum in the presence or absence of Yarowia lipolytica

Two arbuscular mycorrhizal fungi (Glomus deserticola and Glomus fasciculatum) were entrapped in calcium alginate, alone or in combination with a phosphate-solubilizing yeast (Yarowia lipolytica) and, after storage for 60 days, were inoculated into soil microcosms with tomato as the test plant. The average extent of root colonization by gel-entrapped G. deserticola and G. fasciculatum were 32 ± 5.6 and 24 ± 12.1%, respectively. Improved infective potential and colonization efficiency were observed when Y. lipolytica was co-entrapped with the mycorrhizal fungi. The best value, 49%, of mycorrhizal colonization was in roots of plants inoculated with G. deserticola co-entrapped with Y. lipolytica.     

Survival of free and alginate-encapsulated Pseudomonas aeruginosa UG2Lr in soil treated with disinfectants

Pseudomonas aeruginosa UG2Lr, a rifampicin-resistant strain possessing the luxAB on a chromosomal Tn5 insert, was inoculated into soil microcosms as either free cells or encapsulated in dry alginate beads. A 100-fold increase in cell number g^-1 dry soil was observed in microcosms inoculated with alginate-encapsulated UG2Lr after 3 weeks incubation at 22°C compared to microcosms inoculated with free cells. After 98 d, microcosms inoculated with free UG2Lr cells contained 10⁴ cfu g^-1 dry soil compared to 10⁷ cfu g^-1 dry soil in microcosms inoculated with alginate-encapsulated UG2Lr cells. The effects of disinfectants on both the free and alginate-encapsulated UG2Lr cells were also examined. 1·0% (w/g dry soil) calcium hypochlorite, formaldehyde and Spectrum Clear Bath, were added to microcosms each week for 4 weeks. Formaldehyde killed both free and alginate-encapsulated UG2Lr cells within 14 d after only two amendments. Calcium hypochlorite reduced free UG2Lr cell numbers 10-fold 2 d after initial application; however, the introduced population recovered and was unaffected by subsequent treatments at 7, 14 and 21 d. Alginate-encapsulated UG2Lr cells were not affected by calcium hypochlorite treatment. Spectrum Clear Bath did not kill either free or alginate-encapsulated UG2Lr cells in soil. Alginate encapsulation improved survival of introduced bacteria in soil except in the presence of formaldehyde. Killing genetically-engineered bacteria in soil may be difficult unless a powerful disinfectant such as formaldehyde is used or the genetically-engineered micro-organism is allowed to become non-viable over time.     

Soil types with different texture affects development of Rhizoctonia root rot of wheat seedlings

The effect of different soil textures, sandy (97.5% sand, 1.6% silt, 0.9% clay), loamy sand (77% sand, 11% silt, 12% clay) and a sandy clay loam (69% sand, 7% silt, 24% clay), on root rot of wheat caused by Rhizoctonia solani Kühn Anastomosis Group (AG) 8 was studied under glasshouse conditions. The reduction in root and shoot biomass following inoculation with AG-8 was greater in sand than in loamy sand or sandy clay loam. Dry root weight of wheat in the sand, loamy sand and sandy clay loam soils infested with AG-8 was 91%, 55% and 28% less than in control uninfested soils. There was greater moisture retention in the loamy sand and sandy clay loam soils as compared to the sand in the upper 10–20 cm. Root penetration resistance was greater in loamy sand and sandy clay loam than in sand. Root growth in the uninfested soil column was faster in the sand than in the loamy sand and sandy clay loam soils, the roots in the sandy soil being thinner than in the other two soils. Radial spread of the pathogen in these soils in seedling trays was twice as fast in the sand in comparison to the loamy sand which in turn was more than twice that in the sandy clay loam soil. There was no evidence that differences among soils in pathogenicity or soil spread of the pathogen was related to their nutrient status. This behaviour may be related to the severity of the disease in fields with sandy soils as compared to those with loam or clay soils. This revised version was published online in June 2006 with corrections to the Cover Date.     

Interactions between a genetically markedPseudomonas fluorescens strain and bacteriophage ΦR2f in soil: Effects of nutrients, alginate encapsulation, and the wheat rhizosphere

The introduction of bacteriophages could potentially be used as a control method to limit the population size of engineered bacteria that have been introduced into soil. Hence, the ability of a species-specific phage, R2f, to infect and lyse its host, a Pseudomonas fluorescens R2f transposon Tn5 derivative, in soil, was studied. Control experiments in liquid media revealed that productive lysis of host cells by phage R2f occurred when cells were freely suspended, whereas cells present in alginate beads resisted lysis. The presence of nutrients enhanced the degree of lysis as well as the production of phage progeny, both with the suspended cells and with cells escaped from the alginate beads. Experiments in which host cells and phage R2f were introduced into two soils of different texture revealed that host cells were primarily lysed in the presence of added nutrients, and phage reached highest titres in these nutrient-amended soils. Encapsulation of the host cells in alginate beads inhibited lysis by the phage in soil. Populations of free host cells introduced into soil that colonized the rhizosphere of wheat were not substantially lysed by phage R2f. However, P. fluorescens R2f populations colonizing the rhizosphere after introduction in alginate beads were reduced in size by a factor of 1,000. Cells migrating from the alginate beads towards the roots may have been in a state of enhanced metabolic activity, allowing for phage R2f infection and cell lysis. Correspondence to: J.D. van Elsas     

Selenium uptake by plants as a function of soil type,organic matter content and pH

In pots containing sandy soils at two levels (pH 5 and 7) to which 0.5 mg Se L^-1 soil had been added, an increase in the proportion of clay soil or peat soil led to a decrease in the uptake of Se by spring wheat grain (Triticum aestivum L., var. Drabant) and winter rape plants (Brassica napus L., var. Emil). The effect was most pronounced for the smallest additions of clay and peat soils. Differences in Se uptake between the two pH levels were greatest in treatments where the additions of clay and peat soils were small. At the high pH, an increase in clay content from 7% to 39% resulted in a decrease in Se uptake of 79% for wheat and 70% for rape. At the low pH, the uptake decreased by 72% and 77%, respectively. At the higher pH, an increase in the content of organic matter from 1.4% to 39% resulted in decreases in Se uptake of 88% for wheat grain and 69% for rape. At the low pH, Se uptake decreased by 63% and 48%, respectively. Adding peat soil to clay soil had little effect on Se uptake. Among the limed, unmixed clay, sand and peat soils to which Se had not been added, uptake was highest from the sandy soil, i.e. 8.3 ng Se/g wheat grain and 42 ng Se/g rape. The lowest uptake rates were obtained in the clay soil, i.e. 3.0 ng Se/g for wheat grain and 9.0 ng Se/g for rape.     

Saprophytic colonization ofFusarium oxysporum f. sp.ciceri in soil

Competitive saprophytic colonization (CSC) of soybean and chickpea stem pieces byFusarium oxysporum f.sp.ciceri increased with the increase in inoculum density in inoculum soil mixtures. The colonization was higher even at loeer concentration of inoculum. Progressive dilutions of autoclaved soils with unsterilized soil decreased the CSC. Lower temperatures favoured the colonization in both red sandy loam and black soils. Maximum colonization occurred at 40°C indicating an inverse relation between colonization and temperature.     

Bioremediation of Crude Oil–Contaminated Soil By Immobilized Bacteria on an Agroindustrial Waste—Sunflower Seed Husks

This study reports the immobilization and performance of a hydrocarbon-degrading Rhodococcus sp. strain (designated as QBTo) on sunflower seed husks (SH) for the bioremediation of soils polluted with crude oil. The SH performance as inoculants carrier was compared with peat, which is a vegetal material traditionally used in carrier-based inoculants production. The stability of the immobilized culture under storage conditions was assessed by viability at different times when stored at 25°C and 10°C. The catabolic activity of immobilized and free QTBo cells introduced into sandy loam soil, freshly contaminated with crude oil, was studied in microcosms. A higher number of viable QTBo cells were recovered from the inoculants formulated with SH (QTBo-SH) after prolonged storage at 10°C and 25°C. The microcosms amended with QTBo-SH inoculants showed a removal of about 66% of total petroleum hydrocarbons (TPH), whereas in those inoculated with QTBo-peat inoculants, the decrease was of about 47%. In the control microcosms (noninoculated) and liquid culture–amended soils, the TPH removal was about 28%. SH is a waste of edible oil industry, nontoxic, and biodegradable and has demonstrated to confer to the immobilized cultures greater potential to survive not only during storage but also in the soil environment, improving bioremediation process.     

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.     

Growth and survival of cowpea rhizobia in bauxitic silt loam and sandy clay loam soils

Survival of 4 cowpea Rhizobium strains, IRC291, MI-50A, JRW3 and JRC29, in two soil types (bauxitic silt loam and sandy clay loam) undergoing drying at 30°C and 37°C was examined. While all strains except JRW3 showed a general pattern of increase in their numbers during the first 3 weeks in sterile soils, none of the strains showed any increase in their population in non-sterile soils. Cowpea rhizobia showed better survival in non-sterile bauxitic silt loam than in clay loam soils at 30°C. However, the long-term survival (examined up to 6 months) of rhizobia in both soils was poor at 37°C as compared to 30°C. We also found that cowpea rhizobia survived better in soils undergoing drying than in moist soils at 30°C. Our results suggest that (a) cowpea rhizobia survived better in bauxitic silt loam than in clay loam soil and (b) the low indigenous cowpea rhizobial population in Jamaican soils may be due to their poor long-term survival and weak saprophytic competence.     

Immobilization of Saccharomyces uvarum cells in porous beads of polyacrylamide gel for ethanolic fermentation

Summary A procedure which does not involve the use of an immiscible organic solvent phase is described for the entrapment of yeast cells in porous beads of polyacrylamide gel. The cells are rapidly dispersed at 4° C in an aqueous solution containing sodium alginate and acrylamide-N,Nmethylene-bis-acrylamide monomer, and the suspension is immediately dropped into a solution of calcium formate to give calcium alginate coated beads. Polyacrylamide gel forms within the bead. The calcium alginate is subsequently leached out of the composite bead with either sodium citrate or potassium phosphate buffer solution. Cells of Saccharomyces uvarum ATCC 26 602 entrapped in such polyacrylamide beads ferment cane molasses in batch mode at higher specific ethanol productivity than a free cell suspension. Their volumetric productivity in continuous fermentation is higher than that of Ca²⁺-alginate immobilized cells.NCL Communication No. 4383     

Transport of a genetically engineered Pseudomonas fluorescens strain through a soil microcosm.

Vertical soil microcosms flushed with groundwater were used to study the influence of water movement on survival and transport of a genetically engineered Pseudomonas fluorescens C5t strain through a loamy sand and a loam soil. Transport of cells introduced into the top 1 cm of the vertical soil microcosms was dependent on the flow rate of water and the number of times microcosms were flushed with groundwater. The presence of wheat roots growing downward in the microcosms contributed only slightly to the movement of P. fluorescens C5t cells to lower soil regions of the loamy sand microcosms, but enhanced downward transport in the loam microcosms. Furthermore, the introduced P. fluorescens C5t cells were detected in the effluent water samples even after three flushes of groundwater and 10 days of incubation. As evidenced by a comparison of counts from immunofluorescence and selective plating, nonculturable C5t cells occurred in day 10 soil and percolated water samples, primarily of the loamy sand microcosms. Vertical soil microcosms that use water movement may be useful in studying the survival and transport of genetically engineered bacteria in soil under a variety of conditions prior to field testing.     

Transport of a genetically engineered Pseudomonas fluorescens strain through a soil microcosm

Vertical soil microcosms flushed with groundwater were used to study the influence of water movement on survival and transport of a genetically engineered Pseudomonas fluorescens C5t strain through a loamy sand and a loam soil. Transport of cells introduced into the top 1 cm of the vertical soil microcosms was dependent on the flow rate of water and the number of times microcosms were flushed with groundwater. The presence of wheat roots growing downward in the microcosms contributed only slightly to the movement of P. fluorescens C5t cells to lower soil regions of the loamy sand microcosms, but enhanced downward transport in the loam microcosms. Furthermore, the introduced P. fluorescens C5t cells were detected in the effluent water samples even after three flushes of groundwater and 10 days of incubation. As evidenced by a comparison of counts from immunofluorescence and selective plating, nonculturable C5t cells occurred in day 10 soil and percolated water samples, primarily of the loamy sand microcosms. Vertical soil microcosms that use water movement may be useful in studying the survival and transport of genetically engineered bacteria in soil under a variety of conditions prior to field testing.     

Effects of simulated fire on vesicular-arbuscular mycorrhizae in pinyon-juniper woodland soil

Effects of fire on vesicular-arbuscular mycorrhizal fungi were tested using microcosms constructed from soil, litter, and duff collected beneath canopies of pinyon pine, Utah juniper, and in the open space (interspace). Burning was conducted over wet and dry soils. Soil temperatures were monitored continuously throughout the microcosms during burning. Plants grown in soils burned when dry had a lower VAM colonization than soils burned when wet. Juniper soils demonstrated the greatest reduction, over 95%, compared to their respective controls. Plants grown in interspace soils burned when wet were least affected. There was a positive correlation (r²=0.90) between the decrease in VAM colonization and the soil temperature as a result of the fire. Temperature effects, and associated reductions in VAM, were related to amount of litter burned in each microcosm and the moisture content of the soils.     

Survival of Bifidobacterium longum Immobilized in Calcium Alginate Beads in Simulated Gastric Juices and Bile Salt Solution

Bifidobacterium longum KCTC 3128 and HLC 3742 were independently immobilized (entrapped) in calcium alginate beads containing 2, 3, and 4% sodium alginate. When the bifidobacteria entrapped in calcium alginate beads were exposed to simulated gastric juices and a bile salt solution, the death rate of the cells in the beads decreased proportionally with an increase in both the alginate gel concentration and bead size. The initial cell numbers in the beads affected the numbers of survivors after exposure to these solutions; however, the death rates of the viable cells were not affected. Accordingly, a mathematical model was formulated which expressed the influences of several parameters (gel concentration, bead size, and initial cell numbers) on the survival of entrapped bifidobacteria after sequential exposure to simulated gastric juices followed by a bile salt solution. The model proposed in this paper may be useful for estimating the survival of bifidobacteria in beads and establishing optimal entrapment conditions.     

Transport and survival of alginate-encapsulated and free lux-lac marked Pseudomonas aeruginosa UG2Lr cells in soil

Transport and survival of alginate-encapsulated and unencapsulated Pseudomonas aeruginosa UG2Lr through soil microcosms was examined. Bacterial cells encapsulated in alginate beads or mixed with soil were introduced into soil microcosms. Microbial cell survival and cell transport were monitored by destructive sampling and selective plating of the microcosms over a 9-week period. Survival rates were greatest when using encapsulated P. aeruginosa UG2Lr cells. Water flow increased microbial cell dispersal from the site of inoculation. After 3 weeks, encapsulated and free cells showed similar distribution patterns. However, after 9 weeks microbial cell distribution was more extensive throughout the soil in the encapsulated treatments under all conditions. Therefore, alginate encapsulation is a suitable method to enhance survival and distribution of microbial inocula in the soil environment.     

Growth and survival of Verticillium chlamydosporium goddard,a parasite of nematodes,in soil

Selective media for the isolation of the nematophagous fungus Verticillium chlamydosporium, are described. These enabled densities > 500 colony forming units (CFU) g^‐1 soil to be reliably estimated. However, there was little relationship between estimates of the Verticillium biomass in a sterilized soil and the numbers of CFU which developed on the selective media. The growth and survival of the fungus infield soils were studied and estimates of the numbers of CFU in soils in which cyst‐nematode multiplication was suppressed were greater than in those in which the nematode multiplied. Isolates of the fungus differed in their ability to proliferate in soil, but some increased rapidly from applications of chlamydospores or a mixture of hyphae and conidia in alginate granules containing wheat bran. The energy source (wheat bran) was essential for the establishment of the fungus from granular applications. Numbers of CFU greatly exceeded those of chlamydospores, but there was considerable variation in the relationship in different soils. Some isolates of V. chlamydosporium proliferated in soil and survived in considerable numbers for at least 3 months. Hence, pre‐cropping applications of the fungus should survive long enough to kill nematode eggs and females that develop on roots of spring‐sown crops.