Predominance of Nonculturable Cells of the Biocontrol Strain Pseudomonas fluorescens CHA0 in the Surface Horizon of Large Outdoor Lysimeters

The persistence of the biocontrol agent Pseudomonas fluorescens CHA0 in the surface horizon of 12 large outdoor lysimeters planted with winter wheat, Phacelia tanacetifolia followed by spring wheat, or maize was monitored for 1 year. Soil was inoculated with a spontaneous rifampin-resistant mutant (CHA0-Rif) of CHA0, and the strain was studied by using colony counts, Kogure's direct viable counts, and total counts (immunofluorescence). The number of culturable cells of the inoculant decreased progressively from 8 to 2 log CFU/g of soil or lower. However, culturable cells of CHA0-Rif accounted for less than 1% of the total cells of the inoculant 8 months after release in autumn. Since viable but nonculturable cells represented less than a quarter of the latter, most cells of CHA0-Rif in soil were thus inactive-dormant or dead at that time. Nonculturable cells of the inoculant were predominant also in the surface horizon of the lysimeters inoculated in the spring, and a significant fraction of them were viable. Results suggest that the occurrence of nonculturable cells of CHA0-Rif was influenced by climatic factors (water availability and soil temperature) and the abundance of roots in soil. The fact that the inoculant persisted as mixed populations of cells of different physiological states, in which nonculturable cells were predominant, needs to be taken into account when assessing the autecology of wild-type or genetically modified pseudomonads released into the soil ecosystem.     

Persistence and cell culturability of biocontrol strain Pseudomonas fluorescens CHA0 under plough pan conditions in soil and influence of the anaerobic regulator gene anr

Certain fluorescent pseudomonads can protect plants from soil-borne pathogens, and it is important to understand how these biocontrol agents survive in soil. The persistence of the biocontrol strain Pseudomonas fluorescens CHA0-Rif under plough pan conditions was assessed in non-sterile soil microcosms by counting total cells (immunofluorescence microscopy), intact cells (BacLight membrane permeability test), viable cells (Kogure's substrate-responsiveness test) and culturable cells (colony counts on selective plates) of the inoculant. Viable but non-culturable cells of CHA0-Rif (106 cells g-1 soil) were found in flooded microcosms amended with fermentable organic matter, in which the soil redox potential was low (plough pan conditions), in agreement with previous observations of plough pan samples from a field inoculated with CHA0-Rif. However, viable but non-culturable cells were not found in unamended flooded, amended unflooded or unamended unflooded (i.e. control) microcosms, suggesting that such cells resulted from exposure of CHA0-Rif to a combination of low redox potential and oxygen limitation in soil. CHA0-Rif is strictly aerobic. Its anaerobic regulator ANR is activated by low oxygen concentrations and it controls production of the biocontrol metabolite hydrogen cyanide under microaerophilic conditions. Under plough pan conditions, an anr-deficient mutant of CHA0-Rif and its complemented derivative displayed the same persistence pattern as CHA0-Rif, indicating that anr was not implicated in the formation of viable but non-culturable cells of this strain at the plough pan.     

Deleterious impact of a virulent bacteriophage on survival and biocontrol activity of Pseudomonas fluorescens strain CHAO in natural soil

Many biotic and abiotic factors affect the persistence and activity of beneficial pseudomonads introduced into soil to suppress plant diseases. One such factor may be the presence of virulent bacteriophages that decimate the population of the introduced bacteria, thereby reducing their beneficial effect. We have isolated a lytic bacteriophage (phi)GP100) that specifically infects the biocontrol bacterium Pseudomonas fluorescens CHA0 and some closely related Pseudomonas strains. phiGP100 was found to be a double-stranded-DNA phage with an icosahedral head, a stubby tail, and a genome size of approximately 50 kb. Replication of phiGP100 was negatively affected at temperatures higher than 25 degrees C. phiGP100 had a negative impact on the population size and the biocontrol activity of P. fluorescens strain CHA0-Rif (a rifampicin-resistant variant of CHA0) in natural soil microcosms. In the presence of phiGP100, the population size of strain CHA0-Rif in soil and on cucumber roots was reduced more than 100-fold. As a consequence, the bacterium's capacity to protect cucumber against a root disease caused by the pathogenic oomycete Pythium ultimum was entirely abolished. In contrast, the phage affected neither root colonization and nor the disease suppressive effect of a phiDGP100-resistant variant of strain CHA0-Rif. To our knowledge, this study is the first to illustrate the potential of phages to impair biocontrol performance of beneficial bacteria released into the natural soil environment.     

The Viable-but-Nonculturable State Induced by Abiotic Stress in the Biocontrol Agent Pseudomonas fluorescens CHA0 Does Not Promote Strain Persistence in Soil

The effects of oxygen limitation, low redox potential, and high NaCl stress for 7 days in vitro on the rifampin-resistant biocontrol inoculant Pseudomonas fluorescens CHA0-Rif and its subsequent persistence in natural soil for 54 days were investigated. Throughout the experiment, the strain was monitored using total cell counts (immunofluorescence microscopy), Kogure's direct viable counts, and colony counts (on rifampin-containing plates). Under in vitro conditions, viable-but-nonculturable (VBNC) cells of CHA0-Rif were obtained when the strain was exposed to a combination of low redox potential (230 mV) and oxygen limitation. This mimics a situation observed in the field, where VBNC cells of the strain were found in the waterlogged soil layer above the plow pan. Here, VBNC cells were also observed in vitro when CHA0-Rif was subjected to high NaCl levels (i.e., NaCl at 1.5 M but not 0.7 M). In all treatments, cell numbers remained close to the inoculum level for the first 12 days after inoculation of soil, regardless of the cell enumeration method used, but decreased afterwards. At the last two samplings in soil, VBNC cells of CHA0-Rif were found in all treatments except the one in which log-phase cells had been used. In the two treatments that generated high numbers of VBNC cells in vitro, VBNC cells did not display enhanced persistence compared with culturable cells once introduced into soil, which suggests that this VBNC state did not represent a physiological strategy to improve survival under adverse conditions.     

黄土塬区苹果园降雨入渗机制

选取长武黄土塬区20年树龄苹果园,使用4 g·L^-1亮蓝溶液(Brilliant Blue FCF)进行染色示踪试验,分别采用喷洒、倾倒和树干径流模拟3种方式模拟雨量为50 mm的正常降雨入渗、雨量为50 mm的极端性暴雨入渗和树干径流方式的雨水入渗(18 L树干径流量).结果表明: 喷洒方式下染色平均深度为0.28 m,深度较浅,均匀入渗比平均值超过0.65,中位数超过0.7,说明水分入渗过程中均匀部分所占比重较大,活塞流是黄土塬区苹果园降雨入渗的主要机制;倾倒方式下染色平均深度为0.53 m,最大深度为0.59 m,深度较浅,表明在极端暴雨、激活大孔隙等优先路径的条件下,雨水很难短时间补给深层土壤水;树干径流模拟方式下染色最大深度为0.46 m,深度也较浅,表明雨水较难通过树干径流的方式沿着根系补给深层土壤水.活根系与腐烂根系染色比较分析发现,活根系(≥2 mm)不具有良好的导水能力,而腐烂根系(≥2 mm)形成的入渗通道则具有更好的导水能力.在土壤表层果树根系以水平根系为主,当表层根系腐烂时可能发生横向优先流,从而阻碍雨水对深层土壤水的补给.在黄土塬区苹果园,活塞流是深层土壤水降雨补给的主要机制,大孔隙和腐烂垂直根系等通道可能作为雨水补给深层土壤水的途径.     

Impact of biocontrol Pseudomonas fluorescens CHA0 and a genetically modified derivative on the diversity of culturable fungi in the cucumber rhizosphere

Little is known about the effects of Pseudomonas biocontrol inoculants on nontarget rhizosphere fungi. This issue was addressed using the biocontrol agent Pseudomonas fluorescens CHA0-Rif, which produces the antimicrobial polyketides 2,4-diacetylphloroglucinol (Phl) and pyoluteorin (Plt) and protects cucumber from several fungal pathogens, including Pythium spp., as well as the genetically modified derivative CHA0-Rif(pME3424). Strain CHA0-Rif(pME3424) overproduces Phl and Plt and displays improved biocontrol efficacy compared with CHA0-Rif. Cucumber was grown repeatedly in the same soil, which was left uninoculated, was inoculated with CHA0-Rif or CHA0-Rif(pME3424), or was treated with the fungicide metalaxyl (Ridomil). Treatments were applied to soil at the start of each 32-day-long cucumber growth cycle, and their effects on the diversity of the rhizosphere populations of culturable fungi were assessed at the end of the first and fifth cycles. Over 11,000 colonies were studied and assigned to 105 fungal species (plus several sterile morphotypes). The most frequently isolated fungal species (mainly belonging to the genera Paecilomyces, Phialocephala, Fusarium, Gliocladium, Penicillium, Mortierella, Verticillium, Trichoderma, Staphylotrichum, Coniothyrium, Cylindrocarpon, Myrothecium, and Monocillium) were common in the four treatments, and no fungal species was totally suppressed or found exclusively following one particular treatment. However, in each of the two growth cycles studied, significant differences were found between treatments (e.g., between the control and the other treatments and/or between the two inoculation treatments) using discriminant analysis. Despite these differences in the composition and/or relative abundance of species in the fungal community, treatments had no effect on species diversity indices, and species abundance distributions fit the truncated lognormal function in most cases. In addition, the impact of treatments at the 32-day mark of either growth cycle was smaller than the effect of growing cucumber repeatedly in the same soil.     

Inactivation of the regulatory gene algU or gacA can affect the ability of biocontrol Pseudomonas fluorescens CHA0 to persist as culturable cells in nonsterile soil

Rifampin-resistant Pseudomonas fluorescens CHA0-Rif and mutants in which the regulatory gene algU (encoding sigma factor sigma(E)) or gacA (encoding a global regulator of secondary metabolism) was inactivated were compared for persistence in three nonsterile soils. Functional algU and (particularly) gacA were needed for CHA0-Rif to maintain cell culturability in soil.     

Impact of Biocontrol Pseudomonas fluorescens CHA0 and a Genetically Modified Derivative on the Diversity of Culturable Fungi in the Cucumber Rhizosphere

Little is known about the effects of Pseudomonas biocontrol inoculants on nontarget rhizosphere fungi. This issue was addressed using the biocontrol agent Pseudomonas fluorescens CHA0-Rif, which produces the antimicrobial polyketides 2,4-diacetylphloroglucinol (Phl) and pyoluteorin (Plt) and protects cucumber from several fungal pathogens, including Pythium spp., as well as the genetically modified derivative CHA0-Rif(pME3424). Strain CHA0-Rif(pME3424) overproduces Phl and Plt and displays improved biocontrol efficacy compared with CHA0-Rif. Cucumber was grown repeatedly in the same soil, which was left uninoculated, was inoculated with CHA0-Rif or CHA0-Rif(pME3424), or was treated with the fungicide metalaxyl (Ridomil). Treatments were applied to soil at the start of each 32-day-long cucumber growth cycle, and their effects on the diversity of the rhizosphere populations of culturable fungi were assessed at the end of the first and fifth cycles. Over 11,000 colonies were studied and assigned to 105 fungal species (plus several sterile morphotypes). The most frequently isolated fungal species (mainly belonging to the genera Paecilomyces, Phialocephala, Fusarium, Gliocladium, Penicillium, Mortierella, Verticillium, Trichoderma, Staphylotrichum, Coniothyrium, Cylindrocarpon, Myrothecium, and Monocillium) were common in the four treatments, and no fungal species was totally suppressed or found exclusively following one particular treatment. However, in each of the two growth cycles studied, significant differences were found between treatments (e.g., between the control and the other treatments and/or between the two inoculation treatments) using discriminant analysis. Despite these differences in the composition and/or relative abundance of species in the fungal community, treatments had no effect on species diversity indices, and species abundance distributions fit the truncated lognormal function in most cases. In addition, the impact of treatments at the 32-day mark of either growth cycle was smaller than the effect of growing cucumber repeatedly in the same soil.     

Inactivation of the Regulatory Gene algU or gacA Can Affect the Ability of Biocontrol Pseudomonas fluorescens CHA0 To Persist as Culturable Cells in Nonsterile Soil

Rifampin-resistant Pseudomonas fluorescens CHA0-Rif and mutants in which the regulatory gene algU (encoding sigma factor σ^E) or gacA (encoding a global regulator of secondary metabolism) was inactivated were compared for persistence in three nonsterile soils. Functional algU and (particularly) gacA were needed for CHA0-Rif to maintain cell culturability in soil.     

The viable-but-nonculturable state induced by abiotic stress in the biocontrol agent Pseudomonas fluorescens CHA0 does not promote strain persistence in soil

The effects of oxygen limitation, low redox potential, and high NaCl stress for 7 days in vitro on the rifampin-resistant biocontrol inoculant Pseudomonas fluorescens CHA0-Rif and its subsequent persistence in natural soil for 54 days were investigated. Throughout the experiment, the strain was monitored using total cell counts (immunofluorescence microscopy), Kogure's direct viable counts, and colony counts (on rifampin-containing plates). Under in vitro conditions, viable-but-nonculturable (VBNC) cells of CHA0-Rif were obtained when the strain was exposed to a combination of low redox potential (230 mV) and oxygen limitation. This mimics a situation observed in the field, where VBNC cells of the strain were found in the water-logged soil layer above the plow pan. Here, VBNC cells were also observed in vitro when CHA0-Rif was subjected to high NaCl levels (i.e., NaCl at 1.5 M but not 0.7 M). In all treatments, cell numbers remained close to the inoculum level for the first 12 days after inoculation of soil, regardless of the cell enumeration method used, but decreased afterwards. At the last two samplings in soil, VBNC cells of CHA0-Rif were found in all treatments except the one in which log-phase cells had been used. In the two treatments that generated high numbers of VBNC cells in vitro, VBNC cells did not display enhanced persistence compared with culturable cells once introduced into soil, which suggests that this VBNC state did not represent a physiological strategy to improve survival under adverse conditions.     

Conjugative Transfer of Chromosomal Genes between Fluorescent Pseudomonads in the Rhizosphere of Wheat

Bacteria released in large numbers for biocontrol or bioremediation purposes might exchange genes with other microorganisms. Two model systems were designed to investigate the likelihood of such an exchange and some factors which govern the conjugative exchange of chromosomal genes between root-colonizing pseudomonads in the rhizosphere of wheat. The first model consisted of the biocontrol strain CHA0 of Pseudomonas fluorescens and transposon-facilitated recombination (Tfr). A conjugative IncP plasmid loaded with transposon Tn5, in a CHA0 derivative carrying a chromosomal Tn5 insertion, promoted chromosome transfer to auxotrophic CHA0 recipients in vitro. A chromosomal marker (pro) was transferred at a frequency of about 10(sup-6) per donor on wheat roots under gnotobiotic conditions, provided that the Tfr donor and recipient populations each contained 10(sup6) to 10(sup7) CFU per g of root. In contrast, no conjugative gene transfer was detected in soil, illustrating that the root surface stimulates conjugation. The second model system was based on the genetically well-characterized strain PAO of Pseudomonas aeruginosa and the chromosome mobilizing IncP plasmid R68.45. Although originally isolated from a human wound, strain PAO1 was found to be an excellent root colonizer, even under natural, nonsterile conditions. Matings between an auxotrophic R68.45 donor and auxotrophic recipients produced prototrophic chromosomal recombinants at 10(sup-4) to 10(sup-5) per donor on wheat roots in artificial soil under gnotobiotic conditions and at about 10(sup-6) per donor on wheat roots in natural, nonsterile soil microcosms after 2 weeks of incubation. The frequencies of chromosomal recombinants were as high as or higher than the frequencies of R68.45 transconjugants, reflecting mainly the selective growth advantage of the prototrophic recombinants over the auxotrophic parental strains in the rhizosphere. Although under field conditions the formation of chromosomal recombinants is expected to be reduced by several factors, we conclude that chromosomal genes, whether present naturally or introduced by genetic modification, may be transmissible between rhizosphere bacteria.     

干湿交替对红河干旱河谷区土壤优先流形成特征的影响

为明确干旱河谷气候区干湿交替作用对土壤优先流形成的影响,本研究以红河干旱河谷区的荒草地为对象,通过模拟干湿交替的方法,基于染色示踪和水分穿透曲线试验并利用图像处理技术,对比分析模拟前后土壤优先流特征的差异性规律。结果表明: 模拟干湿交替条件下基质流发生区在0~10 cm土层,染色深度高达35 cm,其优先路径的水平宽度仅为3～10 cm,且染色面积曲线波动小。模拟干湿交替条件导致土壤稳定出流速率、大孔隙数量和大孔隙率明显增加,在0~20 cm土层,实施干湿交替后的土壤稳定出流速率较非干湿交替条件高约0.27 cm³·s^-1,染色区的大孔隙数量增加约1.4倍,大孔隙率则高13.4%。大孔隙数量与稳定出流速率呈极显著正相关,模拟干湿交替后大孔隙数量从大到小依次为: 0.6～0.8 mm＞0.8～1.0 mm＞1.0～1.5 mm＞1.5～2.0 mm＞2.0～3.7 mm,非干湿交替条件下为: 0.8～1.0 mm＞0.6～0.8 mm＞1.0～1.5 mm＞2.0～3.7 mm＞1.5～2.0 mm。各孔径范围的大孔隙数量与染色面积比呈极显著相关关系,经过模拟干湿交替处理后,其相关性增大,且影响优先流发生的主导因素由孔径1.5～2.0 mm的大孔隙数量变为孔径0.8～1.0 mm的大孔隙数量。干湿交替作用会通过影响大孔隙特征进而导致土壤更易发生优先流且程度增强。     

四面山阔叶林土壤大孔隙特征与优先流的关系

为研究土壤大孔隙数量、分布特征与优先流发生之间的关系,在使用亮蓝染色法划分林地优先流发生区域基础上,利用穿透曲线理论方法,对重庆四面山典型亚热带阔叶林土壤剖面染色和未染色区域内的土壤大孔隙进行了量化分析.结果表明：研究林地土壤剖面内大孔隙半径多在0.3～3.0 mm,大孔隙率为6.3%～10.5%,随着土壤深度的增加,大孔隙呈现出聚集态的分布特征.各孔径范围内,染色区域的土壤大孔隙数量较未染色区域高出约1个数量级.半径＞0.3 mm,尤其是半径＞1.5 mm的大孔隙数量,是影响林地优先流发生的主要通道.森林土壤0.3～3.0 mm孔径范围内,大孔隙数量与其对应的土壤水分稳定出流速率呈显著的正相关关系,其中在0.7～1.5 mm和1.5～3.0 mm孔径范围内大孔隙数量与稳定出流速率相关程度最大,相关系数分别为0.842和0.879.发生优先流的染色区内大孔隙联通状况优于未染色区,两区中1.5～3.0 mm孔径范围内的联通大孔隙数量差异最大,相差78.3%.染色区内大孔隙数量随土壤深度的增加逐渐减少,“漏斗”状的孔隙分布特征可以形成有效的水压梯度,有利于水分优先运移.     

Impact of roots on ground water quality

Preferential flow is perhaps the major chemical transport process influencing the rapid and typically unexpected movement of agricultural chemicals to ground water. Plant roots are a major contributor to preferential flow mechanics as they form spatial voids which can be used as preferential flow pathways. Chemical transport of atrazine, deethylatrazine, and bromide solutions concentrations under tilled and no-tilled corn fields was evaluated below the active root zone. Additionally, the impact of roots on flow pathways was visualized using a soluble dye (Brilliant Blue FCF). Pictures of the dye-stained pattern were subsequently digitized to determine the cross-sectional area used for transport as a function of depth. Bromide transit times through the field soils were not influenced by tillage practice, whereas atrazine transport was strongly influenced by tillage practice. Under no-till field conditions, atrazine was rarely detected but deethylatrazine concentrations were greater than those observed under tilled field conditions. Visual observation indicated that the dye under no-tillage was more predominant in the corn row, indicative of transport through void root channels. No-tillage practices may decrease the likelihood of ground water contamination through leaching due to the formation of stable root channels where an organic carbon source and microbial population are preferentially located to degrade pesticides.     

Estimating hydraulic conductivity of a sandy soil under different plant covers using minidisk infiltrometer and a dye tracer experiment

The objective of this study was to estimate the hydraulic conductivity of sandy soil under different plant cover at the locality Mláky II at Sekule (southwest Slovakia). Two sites were demarcated at the locality, with mainly moss species at glade site, and pine forest at forest site. The estimation of unsaturated hydraulic conductivity was conducted by (a) minidisk infiltrometer and (b) the analysis of a dye tracer total resident concentration. The latter approach assumed the applicability of the stochastic—convective flow theory in the sandy soil. In the dye tracer experiment, two plots (1 × 1 m each) were established in both sites, and 100 mm of dye tracer (Brilliant Blue FCF) solution was applied on the soil surface. Similar results were obtained in both plots, with more than 70 % area of horizons stained in the depth of 30–50 cm. In some cases, the predicted and measured hydraulic conductivity were found within an order of magnitude, thus revealing similar impact of different plant cover on hydraulic properties of sandy soil studied. In contrast to sandy soils used for agriculture, the influence of the plant/surface humus and topsoil interface extended in the form of a highly heterogeneous matrix flow to the depth of 50–60 cm, where it was dampened by horizontal layering.     

Improvement of Arbuscular Mycorrhiza Development by Inoculation of Soil with Phosphate-Solubilizing Rhizobacteria To Improve Rock Phosphate Bioavailability ((sup32)P) and Nutrient Cycling

The interactive effect of phosphate-solubilizing bacteria and arbuscular mycorrhizal (AM) fungi on plant use of soil P sources of low bioavailability (endogenous or added as rock phosphate [RP] material) was evaluated by using soil microcosms which integrated (sup32)P isotopic dilution techniques. The microbial inocula consisted of the AM fungus Glomus intraradices and two phosphate-solubilizing rhizobacterial isolates: Enterobacter sp. and Bacillus subtilis. These rhizobacteria behaved as "mycorrhiza helper bacteria" promoting establishment of both the indigenous and the introduced AM endophytes despite a gradual decrease in bacterial population size, which dropped from 10(sup7) at planting to 10(sup3) CFU g(sup-1) of dry rhizosphere soil at harvest. Dual inoculation with G. intraradices and B. subtilis significantly increased biomass and N and P accumulation in plant tissues. Regardless of the rhizobacterium strain and of the addition of RP, AM plants displayed lower specific activity ((sup32)P/(sup31)P) than their comparable controls, suggesting that the plants used P sources not available in their absence. The inoculated rhizobacteria may have released phosphate ions ((sup31)P), either from the added RP or from the less-available indigenous P sources, which were effectively taken up by the external AM mycelium. Soluble Ca deficiency in the test soil may have benefited P solubilization. At least 75% of the P in dually inoculated plants derived from the added RP. It appears that these mycorrhizosphere interactions between bacterial and fungal plant associates contributed to the biogeochemical P cycling, thus promoting a sustainable nutrient supply to plants.     

An innovative brilliant blue FCF method for fluorescent staining of fungi and bacteria

Abstract

Most natural and synthetic dyes currently used for microbial fluorescent staining are toxic or carcinogenic and are harmful to animals, humans and the environment. A food dye for microbial staining, brilliant blue FCF, was used as an alternative to lactofuchsin and lactophenol blue. Brilliant blue FCF shows pronounced microbial cell fluorescence staining of an array of pathogenic/toxigenic (Fusarium granunearum 3- and 15-acetyldeoxynivalenol chemotypes, and Escherichia coli O157:H7) and beneficial fungi and bacteria (Trichoderma harzianum and Bacillus subtilis). Brilliant blue FCF has no toxic effects on the microbes tested and is inexpensive.     

Survival of Ralstonia solanacearum in Soil under Irrigated Rice Culture and Aquatic Weeds

The survival of Ralstonia solanacearum in naturally infested sandy loam soil under irrigated rice culture was investigated in Sankhu village (1400 m above sea level) in central Nepal. The experimental plot had a previous history of bacterial wilt and a range of 1.5 × 10⁴–3 × 10⁴ colony-forming units (CFU) per g soil was present. The survival of R. solanacearum was monitored in roots of naturally growing aquatic weeds in the rice plot and in soil before and after rice harvest. The incidence of the bacterial infection in the weeds, Dopatrium sp. and Monochoria vaginalis , were 57.5 and 10%, respectively. The bacterial population detected in soil before rice harvest was 1.5 × 10⁴ CFU per g soil whereas a range of 7.5 × 10²–1.5 × 10³ CFU per g was detected after the rice harvest. Biovar typing of R. solanacearum isolated from potato plants, potato tubers, aquatic weeds, and the soil from the experimental plot yielded the diverse biovars 2 A, 3 and 4. This is the first report of the survival of these biovars in soil, which was under continuous flow of irrigation water for 3 months during rice culture.     

Plasmid Transfer between Spatially Separated Donor and Recipient Bacteria in Earthworm-Containing Soil Microcosms

Most gene transfer studies have been performed with relatively homogeneous soil systems in the absence of soil macrobiota, including invertebrates. In this study we examined the influence of earthworm activity (burrowing, casting, and feeding) on transfer of plasmid pJP4 between spatially separated donor (Alcaligenes eutrophus) and recipient (Pseudomonas fluorescens) bacteria in nonsterile soil columns. A model system was designed such that the activity of earthworms would act to mediate cell contact and gene transfer. Three different earthworm species (Aporrectodea trapezoides, Lumbricus rubellus, and Lumbricus terrestris), representing each of the major ecological categories (endogeic, epigeic, and anecic), were evaluated. Inoculated soil microcosms, with and without added earthworms, were analyzed for donor, recipient, and transconjugant bacteria at 5-cm-depth intervals by using selective plating techniques. Transconjugants were confirmed by colony hybridization with a mer gene probe. The presence of earthworms significantly increased dispersal of the donor and recipient strains. In situ gene transfer of plasmid pJP4 from A. eutrophus to P. fluorescens was detected only in earthworm-containing microcosms, at a frequency of (symbl)10(sup2) transconjugants per g of soil. The depth of recovery was dependent on the burrowing behavior of each earthworm species; however, there was no significant difference in the total number of transconjugants among the earthworm species. Donor and recipient bacteria were recovered from earthworm feces (casts) of all three earthworm species, with numbers up to 10(sup6) and 10(sup4) bacteria per g of cast, respectively. A. trapezoides egg capsules (cocoons) formed in the inoculated soil microcosms contained up to 10(sup7) donor and 10(sup6) recipient bacteria per g of cocoon. No transconjugant bacteria, however, were recovered from these microhabitats. To our knowledge, this is the first report of gene transfer between physically isolated bacteria in nonsterile soil, using burrowing earthworms as a biological factor to facilitate cell-to-cell contact.     

Microbiological and chemical properties of soil associated with macropores at different depths in a red-duplex soil in NSW Australia

Some agricultural soils in South Eastern Australia with duplex profiles have subsoils with high bulk density, which may limit root penetration, water uptake and crop yield. In these soils, a large proportion (up to 80%) of plant roots maybe preferentially located within the macropores or in the soil within 1–10 mm of the macropores, a zone defined as the macropore sheath (MPS). The chemical and microbiological properties of MPS soil manually dissected from a 1–3 mm wide region surrounding the macropores was compared with that of adjacent bulk soil (>10 mm from macropores) at 4 soil depths (0–20 cm, 20–40 cm, 40–60 cm and 60–80 cm). Compared to the bulk soil, the MPS soil had higher organic C, total N, bicarbonate-extractable P, Ca⁺, Cu, Fe and Mn and supported higher populations of bacteria, fungi, actinomycetes, Pseudomonas spp., Bacillus spp., cellulolytic bacteria, cellulolytic fungi, nitrifying bacteria and the root pathogen Pythium.In addition, analysis of carbon substrate utilization patterns showed the microbial community associated with the MPS soil to have higher metabolic activity and greater functional diversity than the microbial community associated with the bulk soil at all soil depths. Phospholipid fatty acids associated with bacteria and fungi were also shown to be present in higher relative amounts in the MPS soil compared to the bulk soil. Whilst populations of microbial functional groups in the MPS and the bulk soil declined with increasing soil depth, the differentiation between the two soils in microbiological properties occurred at all soil depths. Soil aggregates (< 0.5 mm diameter) associated with plant roots located within macropores were found to support a microbial community that was quantitatively and functionally different to that in the MPS soil and the bulk soil at all soil depths. The microbial community associated with these soil aggregates thus represented a third recognizable environment for plant roots and microorganisms in the subsoil.