Two-year field study shows little evidence that PPO-transgenic rice affects the structure of soil microbial communities

There is global concern about the environmental consequences associated with transgenic crops. Their effects on the soil ecosystem are of special interest when assessing ecological safety and integrity. Although many efforts have been made to develop crops genetically modified to have resistance to protoporphyrin oxidase (PPO)-inhibiting herbicides, little is known about their influence on soil microbial communities. We conducted a 2-year field study and an analysis via terminal restriction fragment length polymorphism (T-RFLP) to assess the impacts of PPO-transgenic rice on bacterial and fungal communities. In the first year we sampled the rhizosphere and surrounding bulk soil, while in the second year we sampled rhizosphere soil only. No differences were observed in the diversity indices and community composition of microbial communities between transgenic rice and its parental non-transgenic counterpart (cultivar Dongjin). Instead, community variation was strongly dependent on growth stage and year. Therefore, we observed no adverse effects by these crops of modified rice on the microbial community composition in paddy soils.     

Transgenic Bt rice does not affect enzyme activities and microbial composition in the rhizosphere during crop development

Use of transgenic crops, including those expressing the insecticidal Cry protein from Bt, is increasing at a rapid rate in worldwide. Field and laboratory studies of transgenic Bt crops have been carried out to detect the persistence and activity of the Cry protein in soil and its effect on soil microorganisms to assess their risks to environment. However, there were few studies that evaluate the seasonal effects of Bt rice on rhizosphere soil microbial communities compared to those of insecticides commonly applied in paddy soil for the control of lepidopteran insects. In this study, seasonal effects of transgenic rice expressing the Cry1Ab insecticidal protein active against lepidoperan pests and the insecticide triazophos [3-(o,o-diethyl)-1-phenyl thiophosphoryl-1,2,4-triazol] on soil enzyme activities and microbial communities were compared under field conditions. During a 2-year field study, rhizosphere soil samples of transgenic-Bt rice (Bt), non-Bt parental rice (Ck) and non-Bt parental rice with triazophos (Ckp) applied were taken at four stages in the rice developmental cycle: seedling, booting, heading and maturing. Microbial processes were investigated by measuring different biochemical activities including those involved in C and P cycling. Denaturing gradient gel electrophoresis (DGGE) and terminal-restriction fragment length polymorphism (T-RFLP) analyses were used to compare rhizosphere microbial compositions. Some occasional and inconsistent effects of the application of triazophos on the bacterial composition in the rhizosphere soil of rice plant were found at the booting and heading stages as compared with that of transgenic-Bt rice. There were no statistically significant differences (P>0.05) in phosphatase activity, dehydrogenase activity, respiration, methanogenesis or fungal community composition in rhizosphere soil between Bt, Ck and Ckp over the rice cropping cycle. However, seasonal variations in the selected enzyme activities and microbial community composition in the rhizosphere soil of Bt, Ck and Ckp were clearly detected. These results suggested that the changes in rhizophere soil microbial community composition associated with the crop growth stage overweighed the application of triazophos and the cry1Ab gene transformation. KMD1 (Bt) rice expressing the cry1Ab gene had no measurable adverse effect on the key microbial processes or microbial community composition in rhizophere soil over 2 years of rice cropping.     

Effects of PAH-Contaminated Soil on Rhizosphere Microbial Communities

Bacterial associations with plant roots are thought to contribute to the success of phytoremediation. We tested the effect of addition of a polycyclic aromatic hydrocarbon contaminated soil on the structure of the rhizosphere microbial communities of wheat (Triticum aestivum), lettuce (Lactuca sativa var. Tango), zucchini (Cucurbita pepo spp. pepo var. Black Beauty), and pumpkin (C. pepo spp. pepo var. Howden) 16S rDNA terminal restriction fragment length polymorphism (T-RFLP) profiles of rhizosphere microbial communities from different soil/plant combinations were compared with a pairwise Pearson correlation coefficient. Rhizosphere microbial communities of zucchini and pumpkin grown in the media amended with highest degree of contaminated soil clustered separately, whereas communities of these plants grown in unamended or amended with lower concentrations of contaminated soil, grouped in a second cluster. Lettuce communities grouped similarly to cucurbits communities, whereas wheat communities did not display an obvious clustering. The variability of 16S rDNA T-RFLP profiles among the different plant/soil treatments were mostly due to the difference in relative abundance rather than presence/absence of T-RFLP fragments. Our results suggest that in highly contaminated soils, the rhizosphere microbial community structure is governed more by the degree of contamination rather than the plant host type.     

转Bt基因棉花根际细菌与古菌群落结构分析

在一年内棉花的四个生长时期（苗期,蕾期,花铃期,吐絮期）分别采集转Bt基因抗虫棉GK12和非转基因亲本棉花泗棉3号根际土壤,以及未种植棉花的背景土壤,利用末端标记限制性片段长度多态性（T-RFLP）分析技术,分析三种土壤中细菌和古菌的16S rRNA基因片段多态性,结合克隆文库建立和测序,研究了土壤中细菌和古菌群落结构的变化.结果表明：在棉花生长的各个时期,背景土壤中细菌群落结构发生了明显的变化,生物多样性指数明显降低,古菌群落结构也有一定的变化,说明季节性变化对土壤中微生物群落产生了明显的影响.与背景土壤相比,棉花种植后根际土壤中细菌和古菌群落发生显著的变化.转基因棉花与非转基因棉花相比,根际土壤细菌和古菌的种类和种群大小的分布也发生了明显的改变.克隆文库和测序结果表明土壤中主体微生物为目前未培养的、功能特性未知的细菌和古菌,转基因棉花种植对这些细菌和古菌影响的原因、环境危害和生态风险目前尚不清楚.与古菌群落相比,棉花种植对细菌群落结构的影响较小.     

Impact of fresh root material and mature crop residues of oilseed rape (Brassica napus) on microbial communities associated with subsequent oilseed rape

Glasshouse bioassays were conducted to assess the impact of different inputs of oilseed rape plant material on soil and rhizosphere microbial diversity associated with subsequently grown oilseed rape (Brassica napus) plants. The first bioassay focussed on the effect of oilseed rape rhizodeposits and fresh detached root material on microbial communities, in a rapid-cycling experiment in which oilseed rape plants were grown successively in pots of field soil for 4 weeks at a time, with six cycles of repeated vegetative planting in the same pot. Molecular analyses of the microbial communities after each cycle showed that the obligate parasite Olpidium brassicae infected the roots of oilseed rape within 4 weeks after the first planting (irrespective of the influence of rhizodeposits alone or in the presence of fresh detached root material), and consistently dominated the rhizosphere fungal community, ranging in relative abundance from 43 to 88 % when oilseed rape was grown more than once in the same soil. Fresh detached root material also led to a reduction in diversity within the soil fungal community, due to the increased relative abundance of O. brassicae. In addition, rhizosphere bacterial communities were found to have a reduced diversity over time when fresh root material was retained in the soil. In the second glasshouse experiment, the effect of incorporating mature, field-derived oilseed rape crop residues (shoots and root material) on microbial communities associated with subsequently grown oilseed rape was investigated. As before, molecular analyses revealed that O. brassicae dominated the rhizosphere fungal community, despite not being prevalent in either the residue material or soil fungal communities.     

Subtle changes in rhizosphere microbial community structure in response to increased boron and sodium chloride concentrations

Two of the major constraints to grain production in large areas of South-East Australia and cropping soils worldwide are high levels of subsoil boron (B) and excessive salinity (NaCl). Although the effect of these constraints is often studied in plants, the effect on microbially mediated plant-beneficial processes is unclear. To that end, we investigated the impact of B and NaCl on soil microbial community structure (MCS) in the wheat rhizosphere using BIOLOG ecoplates and terminal restriction fragment length polymorphism (T-RFLP). In addition, the effects of B and NaCl on the nitrogen (N) cycle processes of N fixation and ammonia oxidation were assessed by the construction of clone libraries of diazotrophic (nifH) and ammonia oxidising (amoA) rhizobacteria. Analysis of BIOLOG plates using non-metric multidimensional scaling (MDS) revealed addition of both B and NaCl significantly changed MCS, the latter of which was also significant through the analysis of T-RFLP data. Utilisation of several chemical groups of BIOLOG substrates significantly changed in NaCl-amended soil; both B and NaCl affected utilisation of several individual substrates indicative of plant stress including serine and malic acid. A significant decrease in diversity and species richness was observed in high B rhizosphere soil. The community structure of ammonia-oxidising bacteria (AOB), all of which clustered with Nitrosospira-like sequences, did not significantly change in response to addition of B or NaCl, but addition of the latter resulted in a significant increase of diazotroph clones within the α-proteobacteria similar to Azospirillum sp. It appeared that the addition of B and NaCl to soil changed rhizosphere MCS indirectly through increased soil moisture and subtle changes in root exudate patterns, the addition of the latter producing a more distinct change through increased osmotic pressure, leading to a greater increase in rhizodeposition of nutrients, especially carbohydrates. The implications for the current study are that B and NaCl are more likely to affect rhizosphere MCS indirectly through root exudate quantity and/or quality than directly through microbial toxicity, and that plant health is a major determinant in rhizosphere MCS and normal N cycling.     

Rhizosphere microbial community and Zn uptake by willow (Salix purpurea L.) depend on soil sulfur concentrations in metalliferous peat soils

On numerous occasions, rhizosphere microbial activities have been identified as a key factor in metal phytoavailability to various plant species and in phytoremediation of metal-contaminated sites. For soil bioremediation efforts in heavy metal contaminated areas, microbes adapted to higher concentrations of heavy metals are required. This study was a field survey undertaken to examine rhizosphere microbial communities and biogeochemistry of soils associated with Zn accumulation by indigenous willows (Salix purpurea L.) in the naturally metalliferous peat soils located near Elba, NY. Soil and willow leaf samples were collected from seven points, at intervals 18 m apart along a willow hedgerow, on four different dates during the growing season. Soil bacterial community composition was characterized by terminal restriction fragment length polymorphism (T-RFLP) analysis and a 16S clone library was created from the rhizosphere of willows and soils containing the highest concentrations of Zn. Bacterial community composition was correlated with soil sulfate, but not with soil pH. The clone library revealed comparable phylogenetic associations to those found in other heavy metal-contaminated soils, and was dominated by affiliations within the phyla Acidobacteria (32%), and Proteobacteria (37%), and the remaining clones were associated with a wide array of phyla including Actinobacteria, Gemmatimonadetes, Planctomycetes, Verrucomicrobia, Bacteriodetes, and Cyanobacteria. Diverse microbial populations were present in both rhizosphere and bulk soils of these naturally metalliferous peat soils with community composition highly correlated to the soil sulfate cycle throughout the growing season indicative of a sulfur-oxidizing rhizosphere microbial community. Results confirm the importance of soil characterization for informing bioremediation efforts in heavy metal contaminated areas and the reciprocity that microbial communities uniquely adapted to specific conditions and heavy metals may have on an ecosystem.     

Shifts in rhizosphere microbial communities and enzyme activity of Poa alpina across an alpine chronosequence

This study quantifies the influence of Poa alpina on the soil microbial community in primary succession of alpine ecosystems, and whether these effects are controlled by the successional stage. Four successional sites representative of four stages of grassland development (initial, 4 years (non-vegetated); pioneer, 20 years; transition, 75 years; mature, 9500 years old) on the Rotmoos glacier foreland, Austria, were sampled. The size, composition and activity of the microbial community in the rhizosphere and bulk soil were characterized using the chloroform-fumigation extraction procedure, phospholipid fatty acid (PLFA) analysis and measurements of the enzymes β-glucosidase, β-xylosidase, N-acetyl-β-glucosaminidase, leucine aminopeptidase, acid phosphatase and sulfatase. The interplay between the host plant and the successional stage was quantified using principal component (PCA) and multidimensional scaling analyses. Correlation analyses were applied to evaluate the relationship between soil factors (C_org, N_t, C/N ratio, pH, ammonium, phosphorus, potassium) and microbial properties in the bulk soil. In the pioneer stage microbial colonization of the rhizosphere of P. alpina was dependent on the reservoir of microbial species in the bulk soil. As a consequence, the rhizosphere and bulk soil were similar in microbial biomass (ninhydrin-reactive nitrogen (NHR-N)), community composition (PLFA), and enzyme activity. In the transition and mature grassland stage, more benign soil conditions stimulated microbial growth (NHR-N, total amount of PLFA, bacterial PLFA, Gram-positive bacteria, Gram-negative bacteria), and microbial diversity (Shannon index H) in the rhizosphere either directly or indirectly through enhanced carbon allocation. In the same period, the rhizosphere microflora shifted from a G⁻ to a more G⁺, and from a fungal to a more bacteria-dominated community. Rhizosphere β-xylosidase, N-acetyl-β-glucosaminidase, and sulfatase activity peaked in the mature grassland soil, whereas rhizosphere leucine aminopeptidase, β-glucosidase, and phosphatase activity were highest in the transition stage, probably because of enhanced carbon and nutrient allocation into the rhizosphere due to better growth conditions. Soil organic matter appeared to be the most important driver of microbial colonization in the bulk soil. The decrease in soil pH and soil C/N ratio mediated the shifts in the soil microbial community composition (bacPLFA, bacPLFA/fungPLFA, G⁻, G⁺/G⁻). The activities of β-glucosidase, β-xylosidase and phosphatase were related to soil ammonium and phosphorus, indicating that higher decomposition rates enhanced the nutrient availability in the bulk soil. We conclude that the major determinants of the microflora vary along the successional gradient: in the pioneer stage the rhizosphere microflora was primarily determined by the harsh soil environment; under more favourable environmental conditions, however, the host plant selected for a specific microbial community that was related to the dynamic interplay between soil properties and carbon supply.     

From the micro-scale to the habitat: Assessment of soil bacterial community structure as shown by soil structure directed sampling

Natural structural units of a luvisol under maize crop were studied to assess if soil structure directed sampling could improve the understanding of arrangements of bacteria in spatially constraint location. Three habitats were defined: (i) soil around fine lateral roots (rhizo-aggregates), (ii) soil close to basal roots (core clods) and (iii) unplanted soil between rows (bare soil clods). These habitats were also investigated with maize plants resulting from Azospirillum lipoferum CRT1 inoculated seeds as a model of enhanced fine root system. Rhizo-aggregates were clearly separated from each other (disconnected habitat) in contrast to micro-samples (fragments) from clods, which belong to cohesive macro-structures. Genetic fingerprints on metagenomic extracts were used to characterize the structure of bacterial communities on 95 micro-samples from the three habitats. For eubacteria, automated RISA (Ribosomal Intergenic Spacer Analysis) of ITS (Internal Transcribed Spacer) profiles were performed. PCR-RFLP on nifH gene were used to describe the N-fixer guilds. Exploratory multivariate analyses (PCA and MDS) revealed bacterial community patterns in the sampled habitats. On the basis of ITS profiles, rhizo-aggregates harboured closely related communities, distant from those of the unplanted soil, and each sampled rhizo-aggregate could therefore be considered as a sub-unit of the whole macro-habitat, comprising all the fine roots. The observed low dissimilarity of disconnected rhizo-aggregates is likely to result from the direct influence of maize root tips on the recruitment of rhizosphere bacteria. Molecular fingerprints of nifH from basal root clods (core) were more similar to bare soil than to rhizo-aggregates, indicating similar ecological conditions without, or with, at least, poor maize exudating root influence. Although our study was performed on a limited number of situations, the distribution of bacteria was revealed to be patterned by soil structure units, which is a first step to improve the modelling of microbial ecology in soils.     

Intercropping with aerobic rice suppressed Fusarium wilt in watermelon

Watermelon is susceptible to Fusarium wilt in successively mono-cropped soil. Pot experiments were carried out to investigate the effect of intercropping with aerobic rice on Fusarium wilt in watermelon. The tested soil was classified as a loam soil, previously planted with watermelon and collected from Hexian county, Anhui province, China. The results obtained are listed as follows: (1) 66.7% of watermelon plants were infected with wilt disease and 44.4% died on 40 days after transplanting in mono-cropped soil, but plants were much less susceptible to infection when intercropped with rice; (2) the density of Fusarium oxysporum f. sp. niveum decreased by 91% in soil from the intercropped watermelon rhizosphere when compared with that from the mono-crop 40 days after transplanting; (3) densities of bacteria and actinomycetes increased, but fungal density decreased in rhizosphere soil from the intercrops in comparison with the mono-crop control; (4) compared to the control, the germinated Fusarium spores were decreased by 41.0% in the treatment with addition of 1.5 ml rice root exudates. Adding 20 ml of root exudates decreased Fusarium spore production by 76.4%; and (5) the activities of defense enzymes in the leaves and roots of watermelons in the intercropped system were significantly lower than those in the mono-cropped system. It is suggested that intercropping with aerobic rice alleviated Fusarium wilt in watermelon, by restraining the spore production of Fusarium and by changing the microbial communities in rhizosphere soil through the production of rice root exudates.     

Influence of grass species and soil type on rhizosphere microbial community structure in grassland soils

A number of studies have reported species specific selection of microbial communities in the rhizosphere by plants. It is hypothesised that plants influence microbial community structure in the rhizosphere through rhizodeposition. We examined to what extent the structure of bacterial and fungal communities in the rhizosphere of grasses is determined by the plant species and different soil types. Three grass species were planted in soil from one site, to identify plant-specific influences on rhizosphere microbial communities. To quantify the soil-specific effects on rhizosphere microbial community structure, we planted one grass species (Lolium perenne L.) into soils from three contrasting sites. Rhizosphere, non-rhizosphere (bulk) and control (non-planted) soil samples were collected at regular intervals, to examine the temporal changes in soil microbial communities. Rhizosphere soil samples were collected from both root bases and root tips, to investigate root associated spatial influences. Both fungal and bacterial communities were analysed by terminal restriction fragment length polymorphism (TRFLP). Both bacterial and fungal communities were influenced by the plant growth but there was no evidence for plant species selection of the soil microbial communities in the rhizosphere of the different grass species. For both fungal and bacterial communities, the major determinant of community structure in rhizospheres was soil type. This observation was confirmed by cloning and sequencing analysis of bacterial communities. In control soils, bacterial composition was dominated by Firmicutes and Actinobacteria but in the rhizosphere samples, the majority of bacteria belonged to Proteobacteria and Acidobacteria. Bacterial community compositions of rhizosphere soils from different plants were similar, indicating only a weak influence of plant species on rhizosphere microbial community structure.     

Rhizosphere bacterial community composition in natural stands of Carex arenaria (sand sedge) is determined by bulk soil community composition

The relative importance of specific plant properties versus soil characteristics in shaping the bacterial community structure of the rhizosphere is a topic of considerable debate. Here, we report the results of a study on the bacterial composition of the rhizosphere of the wild plant Carex arenaria (sand sedge) growing at 10 natural sites in The Netherlands. The soil properties of the sandy soils at these sites were highly disparate, most notably in pH, chloride and organic matter content. Rhizosphere and bulk soil bacterial communities were examined by culture-independent means, namely, 16S rDNA-directed PCR-DGGE profiling. Large differences were observed between the bacterial communities of the different sites for both bulk and rhizosphere soil. Cluster analysis of bacterial profiles revealed that the rhizosphere community of each site was generally more closely related to the bulk soil community of that site rather than to rhizosphere communities of other sites. Hence, bacterial community structure within the rhizosphere of C. arenaria appeared to be determined to a large extent by the bulk soil community composition. This conclusion was supported by a reciprocal planting experiment, where C. arenaria shoots of different sites yielded highly similar rhizosphere communities when planted in the same soil.     

A 3-year field investigation of impacts of Monsanto’s transgenic Bt-cotton NC 33B on rhizosphere microbial communities in northern China

Monsanto’s Bt-cotton NC 33B, planted in northern China for more than one decade, effectively controls cotton bollworms and decreases the use of chemical insecticides. Because of the concern about undesirable ecological side-effects of transgenic Bt-cottons, it is important to assess Bt-cotton NC 33B’s effects on soil microorganisms in this zone. Microbial communities in the rhizosphere soil of Bt and non-Bt cottons were monitored under field conditions by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) fingerprints of eubacteria, fungi and actinomycetes at six growth stages after three-year cultivation. Results showed that the population sizes and community structures of eubacteria, fungi and actinomycetes in rhizosphere soil were markedly affected by natural variations in the environment related to cotton growth stages. However, there was no significant difference in eubacterial, fungal and actinomycete population size and community structures in rhizosphere soil between NC 33B and its non-transgenic parent. In general, Bt-cotton NC 33B did not show evident effects on microbial communities in the rhizosphere soil under field conditions after three-year cultivation. This study provides a theoretical basis for environmental impact monitoring of transgenic Bt cottons.     

A DGGE analysis shows that crop rotation systems influence the bacterial and fungal communities in soils

A better understanding of the relationships among different cropping systems, their effects on soil microbial ecology, and their effects on crop health and productivity is necessary for the development of more efficient, sustainable crop production systems. We used denaturing gradient gel electrophoresis (DGGE) to determine the impacts of crop rotations and crop types on bacterial and fungal communities in the soil. The communities of bacterial 16S rRNA genes and fungal 18S rRNA genes were analyzed in experimental field plots that were kept under 4 different crop rotation systems from 1999 to 2008 (continuous cabbage (Brassica oleracea var. capitata L.), cabbage–lettuce (Lactuca sativa L.) rotation, cabbage–radish (Raphanus sativus L. var. longipinnatus L.H. Bailey) rotation, and a 3-year crop rotation). A principal component analysis (PCA) and a canonical correspondence analysis (CCA) revealed that both the bacterial and fungal communities in bulk soils were influenced by the crop rotation systems. However, the primary factors influencing each community differed: bacterial communities were most affected by soil properties (especially carbon content), while fungal communities were influenced most strongly by rotation times. To elucidate factors that may cause differences in crop rhizosphere microbial communities, the microbial communities in the harvested cabbage rhizospheres were also analyzed. The results suggest that the fungal communities in bulk soil are related to the rhizosphere fungal communities. Our present study indicates that the microbial communities in bulk and rhizosphere soils could be managed by crop rotation systems.     

Persistence of Cry1Ac Protein from Transgenic Bt Cotton Cultivation and Residue Returning in Fields and Its Effect on Functional Diversity of Soil Microbial Communities

The persistence of Cry1Ac protein in the soil and its effect on soil microbial communities are a core issue in assessing the ecological risk of transgenic Bacillus thuringiensis (Bt) cotton. In this study a field experiment was conducted on the cultivation of transgenic Bt cotton (Jin 26 and BtJi 668) with the immediate returning of residues to the fields, in order to quantify the Cry1Ac protein content in the fields and investigate its effects on the functional diversity of soil microbial communities. Cry1Ac protein in the residue-soil mixture was gradually degraded in the transgenic Bt cotton fields. After transgenic Bt cotton straw was returned to the fields for 30 d, 63.73% and 58.33% of the initial amounts of Cry1Ac protein were degraded in the Jin 26 and BtJi 668 fields, respectively. Before the crops were sown in the following year (180 d after returning the straw), no Cry1Ac protein was detected in the fields. After returning the cotton straw to the fields for 30 d, the Shannon-Wiener and McIntosh indices of soil microbial communities in the transgenic Bt cotton fields were significantly higher than those in the non-transgenic cotton fields. Meanwhile, the utilization of carbon sources including amino acids, amines, and carbohydrates by the soil microbial communities significantly increased. Both the McIntosh index and the utilization of carbohydrates increased until 180 d. Principal component analysis revealed that amino acids, amides, and carbohydrates were the main carbon sources distinguishing the two principal component factors. These findings indicated that Cry1Ac protein did not accumulate in the fields after transgenic Bt cotton was planted for one year and the residues were immediately returned to the fields; however, the original functional diversity of soil microbial communities was affected continuously.     

Microbial community development in the rhizosphere of apple trees at a replant disease site

Apple replant disease (ARD) is a complex syndrome that affects young trees in replanted orchard sites causing necrotic lesions on feeder roots, stunted tree growth and reduced cumulative yields. Use of ARD-tolerant rootstocks is an emerging control strategy. We studied the bacterial, fungal, and oomycetes populations in the rhizosphere of five rootstock cultivars (M.7, M.26, G.16, G.30 and CG.6210) planted into the old tree row or grass lanes of a previous orchard in Ithaca, NY, to better understand the role of rhizosphere microbial communities in the prevalence and control of ARD. The possible involvement of antagonistic Pseudomonas species, Pythium spp., Phytophthora spp. and rhizosphere cyanide concentrations in ARD were also examined. The rootstocks M.7, M.26 and G.16 were susceptible to ARD, while G.30 and CG.6210 were more tolerant. Tree growth on the rootstocks M.7, M.26 and G.16 was reduced by 10% when planted in the old tree rows, but this did not significantly reduce yields in the first fruiting year. The susceptible rootstocks, M.7 and M.26, supported higher densities of culturable rhizosphere fungi and bacteria than G.16, G.30 and CG.6210. Over 2 years, microbial densities were highest in July, lower in May and lowest in September. The composition of bacterial and fungal communities in the rhizosphere was highly variable and changed over seasons and years, as assessed by terminal restriction fragment length polymorphism (T-RFLP) analyses. Initial differences in fungal rhizosphere communities between the two planting positions converged 2 years after the trees were replanted. In contrast, the bacterial rhizosphere community composition still differed significantly between the two planting positions 3 years after the orchard was replanted. The bacterial and fungal rhizosphere community compositions of susceptible rootstocks, M.7 and M.26, differed from those of the tolerant rootstocks, G.30 and CG.6210; G.16, differed from all the other rootstocks. The observed effects of rootstocks, planting positions and time on microbial community composition were small relative to the high variability observed overall. Pythium spp. and Phytophthora spp. infestations were high and similar for all rootstocks and planting positions. Neither potentially antagonistic Pseudomonas nor rhizosphere cyanide concentrations appeared to be involved in the ARD-complex at the studied site. Avoiding replanting into the old tree rows coupled with use of tolerant rootstocks appear to be the best strategies for reducing ARD in replanted orchards. Changes in rhizosphere microbial communities are among the many factors that contribute to improved tree growth when these management strategies are used.     

Ecological Risk Assessment of Alfalfa Medicago Varia L.) Genetically Engineered to Express a Human Metallothionein (hMT) Gene

The objectives of these studies were two-fold: (1) to determine efficacy of low and high expression hMT gene constructs by assessing accumulation of Cu in shoots of parental and transgenic plants of alfalfa (Medicago varia L.) exposed to different concentrations of CuSO₄ by addition of CuSO₄ solutions to soil and (2) to identify potential unintended effects of the genetic engineering on root and shoot biomass, shoot nutrient content, arbuscular mycorrhizal infection and on the metabolic functions of microbial communities in the rhizosphere. In the absence of exogenous CuSO₄ additions to soil shoot biomass and the macronutrient (C, P, K, Ca, Mg and N) content of plants expressing hMT were not significantly different from the parental control. In the 0.5 mM and 1.0 mM CuSO₄ treatments transgenic plants expressing the commonly used transgenic β-glucuronidase (GUS) marker had significantly higher Fe content than the parental genotype. Significant differences were observed in the carbon substrate utilization patterns of rhizosphere microbial communities among the transgenic plants; no significant differences were observed in the percent mycorrhizal infection of parental and transgenic plants. Shoot biomass increased significantly in all genotypes treated with 0.5 mM CuSO₄ and decreased in all genotypes at CuSO₄ concentrations of 1.5 mM and 2.0 mM. Root dry weights decreased significantly in all genotypes at concentrations of 1.0 mM, 1.5 mM and 2.0 mM CuSO₄. The largest decreases in root dry weight were observed in hMT genotypes grown in soil treated with 1.5 and 2.0 mM CuSO₄. In plants treated with 1.5 mM CuSO₄, shoots of transgenic plants expressing the hMT gene accumulated nominally, but not statistically significantly higher levels of Cu in shoot tissue. Our results were surprising with regard to lack of sufficient efficacy of the current hMT constructs for significant accumulation of Cu from soil treated with CuSO₄. However, our results suggest the utility of applying adverse levels of CuSO₄ or other environmental stressors to identify potential unintended effects of genetic engineering that may not be apparent under typically more optimal plant growth test conditions.     

Influence of introduced potential biocontrol agents on maize seedling growth and bacterial community structure in the rhizosphere

Two species of Pseudomonas chromosomally tagged with gfp, which had shown antagonistic activity against the tomato pathogen Ralstonia solanacearum in a previous study, were assessed for their impact in the rhizosphere of maize. Plant growth characteristics, numbers of indigenous heterotrophic bacteria, changes in the bacterial community structure according to the r/K strategy concept, and shifts in MIDI-FAME profiles of culturable bacterial fractions as well as total rhizosphere microbial communities were determined in relation to seed and soil treatment with the exogenous pseudomonads. The maize rhizosphere proved to be a suitable habitat for the introduced P. chlororaphis IDV1 and P. putida RA2, which showed good survival after introduction. However, both inoculants showed a small growth-reducing effect towards maize, which might have been caused by the high densities of inoculants used (i.e. competition for nutrients and action of metabolites produced) and/or changes in microbial community structure (both culturable bacterial fraction and the total microflora). Probably, an altered balance among the indigenous maize rhizosphere populations occurred. Thus, the culturable bacteria, as well as the total microflora in the rhizosphere, changed in response to the introduced pseudomonads, and their development was dependent on the growth stage of the plant. The FAME analyses showed that these microbial communities comprised different populations, and were separated according to, first, the method used (direct versus cultivation-based), second, sampling time, and, finally, inoculation level.     

In situ dynamics of soil fungal communities under different genotypes of potato, including a genetically modified cultivar

Fungi are key to the functioning of soil ecosystems, and exhibit a range of interactions with plants. Given their close associations with plants, and importance in ecosystem functioning, soil-borne fungi have been proposed as potential biological indicators of disturbance and useful agents in monitoring strategies, including those following the introduction of genetically modified (GM) crops. Here we report on the impact of potato crop varieties, including a cultivar that was genetically modified for its starch quality, on the community composition of the main phyla of fungi in soils, i.e. Ascomycota, Basidiomycota and Glomeromycota in rhizosphere and bulk soil. Samples were collected at two field sites before sowing, at three growth stages during crop development and after the harvest of the plants, and the effects of field site, plant growth stage and plant cultivar (genotype) on fungal community composition assessed using three phylum-specific T-RFLP profiling strategies and multivariate statistical analysis (NMDS ordinations with ANOSIM test). In addition, fungal biomass, arbuscular mycorrhizal colonization of roots and activities of extracellular fungal enzymes (laccases, Mn-peroxidases and cellulases) involved in degradation of lignocelluloses-rich organic matter were determined. Fungal community compositions, densities and activities were observed to differ significantly between the rhizosphere and bulk soil. The most important factors determining fungal community composition and functioning were plant growth stage for the rhizosphere communities and location and soil properties for the bulk soil communities. The basidiomycetes were the most numerous fungal group in the bulk soils and in the rhizosphere of young plants, with a shift toward greater ascomycete numbers in the rhizosphere at later growth stages. There were no detectable differences between the GM cultivar and its parental cultivar in terms of influence on fungal community structure of function. Fungal community structure and functioning of both GM- and parental cultivars fell within the range of other cultivars at most sampling moments.     

Effects of the introduction of a biocontrol strain of Trichoderma atroviride on non target soil micro-organisms

The main objective of this study was to assess the impact of the application of an antagonistic strain of Trichoderma atroviride on the native microbial soil communities. The structures of the fungal and bacterial communities were assessed by T-RFLP (terminal restriction fragment length polymorphism) method, based on T-RFLP analysis of 18S and 16S rRNA genes, respectively. Results showed that the introduction of the strain I-1237 into two soils slightly modified the microbial diversity, only for a short period of time. Nine months post-inoculation resilience took place, resulting in similar structures of the fungal and bacterial communities in the inoculated and control soils.