Janice E. Thies

Analysis of T-RFLP data using analysis of variance and ordination methods: A comparative study

The analysis of T-RFLP data has developed considerably over the last decade, but there remains a lack of consensus about which statistical analyses offer the best means for finding trends in these data. In this study, we empirically tested and theoretically compared ten diverse T-RFLP datasets derived from soil microbial communities using the more common ordination methods in the literature: principal component analysis (PCA), nonmetric multidimensional scaling (NMS) with Sørensen, Jaccard and Euclidean distance measures, correspondence analysis (CA), detrended correspondence analysis (DCA) and a technique new to T-RFLP data analysis, the Additive Main Effects and Multiplicative Interaction (AMMI) model. Our objectives were i) to determine the distribution of variation in T-RFLP datasets using analysis of variance (ANOVA), ii) to determine the more robust and informative multivariate ordination methods for analyzing T-RFLP data, and iii) to compare the methods based on theoretical considerations. For the 10 datasets examined in this study, ANOVA revealed that the variation from Environment main effects was always small, variation from T-RFs main effects was large, and variation from T-RFxEnvironment (TxE) interactions was intermediate. Larger variation due to TxE indicated larger differences in microbial communities between environments/treatments and thus demonstrated the utility of ANOVA to provide an objective assessment of community dissimilarity. The comparison of statistical methods typically yielded similar empirical results. AMMI, T-RF-centered PCA, and DCA were the most robust methods in terms of producing ordinations that consistently reached a consensus with other methods. In datasets with high sample heterogeneity, NMS analyses with Sørensen and Jaccard distance were the most sensitive for recovery of complex gradients. The theoretical comparison showed that some methods hold distinct advantages for T-RFLP analysis, such as estimations of variation captured, realistic or minimal assumptions about the data, reduced weight placed on rare T-RFs, and uniqueness of solutions. Our results lead us to recommend that method selection be guided by T-RFLP dataset complexity and the outlined theoretical criteria. Finally, we recommend using binary or relativized peak height data with soil-based T-RFLP data for ordination-based exploratory microbial analyses.

Diversity of Planctomycetes in Soil in Relation to Soil History and Environmental Heterogeneity

ABSTRACT Members of the Planctomycetes, which were once thought to occur primarily in aquatic environments, have been discovered in soils on five continents, revealing that these Bacteria are a widespread and numerically abundant component of microbial communities in soil. We examined the diversity of Planctomycetes in soil samples obtained from experimental plots at an agricultural site in order to assess the extent of Planctomycetes diversity in soil, to determine whether management effects such as past land cover and compost addition affected the composition of the Planctomycetes community, and to determine whether the observations made could provide insight into the ecological distribution of these organisms. Analysis of Planctomycetes 16S rRNA gene sequences revealed a total of 312 ± 35 unique phylotypes in the soil at the site examined. The majority of these Planctomycetes sequences were unique, and the sequences had phylogenetic affiliations that included all major lineages in the Planctomycetaceae, as well as several novel groups of deeply divergent Planctomycetes. Both soil management history and compost amendment had significant effects on the Planctomycetes diversity, and variations in soil organic matter, Ca2+ content, and pH were associated with variations in the Planctomycetes community composition. In addition, Planctomycetes richness increased in proportion to the area sampled and was correlated with the spatial heterogeneity of nitrate, which was associated with the soil management history at the orchard site examined. This report provides the first systematic assessment of the diversity of Planctomycetes in soil and also provides evidence that the diversity of this group increases with area as defined by the general power law description of the taxon-area relationship.

Enrichment of Bradyrhizobium spp populations in soil due to cropping of the homologous host legume

Host legumes can enrich their immediate soil environment with rhizobia through rhizosphere effects. The extent to which this enrichment occurs, the specificity of the process and its interaction with soil management factors remain poorly described. In a series of field trials, we measured changes in the size of indigenous populations of Bradyrhizobium in response to cropping of host and non-host legumes under two N fertilizer regimes. Uninoculated cowpea (Vigna unguiculata) and soybean (Glycine max) were grown with or without applied urea (900 kg N ha−1) at three field sites on the island of Maui, HI, U.S.A., not previously cropped with legumes. Using the most-probable-number plant infection method and Siratro (Macroptilium atropurpureum) as the host, the population density of Bradyrhizobium in the bulk soil at each site was measured at planting and at grain maturity and compared to the population density in adjacent fallow soil. When the size of the initial indigenous population was low (18 and 580 cells g−1 soil), significant increases in the population density compared to fallow soil were observed only in soils cropped with cowpea receiving no applied urea. When the size of the initial indigenous bradyrhizobial population was high (5.8 × 104 cells g−1 soil), no significant increase in the population density was observed. These results suggested that enrichment of soil bradyrhizobial populations was host-specific, that symbiotic legumes can enrich their soil environment with microsymbionts up to a threshold level and that such enrichment can be curtailed by soil management practices that suppress nodulation.

Rootstock genotype and orchard replant position rather than soil fumigation or compost amendment determine tree growth and rhizosphere bacterial community composition in an apple replant soil

AbstractApple replant disease (ARD) is a complex soilborne disease syndrome that often causes problems when renovating old orchard sites. Soil fumigants sometimes control ARD, but biological and cultural alternatives are needed. In this study the growth of two widely used clonal apple (Malus domestica) rootstocks (M7 and M26) were compared to three new rootstocks from the Cornell-Geneva series (CG16, CG30 and CG210 (a.k.a. CG6210)) in an orchard site with a history of ARD, in Ithaca, NY. Trees were planted in two distinguishable positions – the previous tree rows versus the old inter-row grass lanes. Additionally, we compared the effects of compost amendment and fumigation with dichloropropene plus chloropicrin on tree growth on each replant rootstock. Rhizosphere bacteria and actinobacteria were assessed using PCR-DGGE for the rootstocks M7, M26, CG30 and CG210. Tree growth on the rootstocks M7, M26 and CG16 was suppressed in the old tree rows relative to grass lanes, while trees on CG30 and CG210 rootstocks grew equally well in both positions. The species composition of rhizosphere bacteria and actinobacteria differed significantly between the planting positions and between the rootstocks M7 and M26 compared to CG30 and CG210. In contrast, the preplant compost or fumigation soil treatments had no effect on tree growth and little impact on rhizosphere bacterial community composition. Abbreviations: ARD – apple replant disease; DGGE – denaturing gradient gel electrophoresis; PCR – polymerase chain reaction.

Use of 13C labeling to assess carbon partitioning in transgenic and nontransgenic (parental) rice and their rhizosphere soil microbial communities.

Photosynthetic assimilation of CO2 is a primary source of carbon in soil and root exudates and can influence the community dynamics of rhizosphere organisms. Thus, if carbon partitioning is affected in transgenic crops, rhizosphere microbial communities may also be affected. In this study, the temporal effects of gene transformation on carbon partitioning in rice and rhizosphere microbial communities were investigated under greenhouse conditions using the 13C pulse-chase labeling method and phospholipid fatty acid (PLFA) analysis. The 13C contents in leaves of transgenic (Bt) and nontransgenic (Ck) rice were significantly different at the seedling, booting and heading stages. There were no detectable differences in 13C distribution in rice roots and rhizosphere microorganisms at any point during rice development. Although a significantly lower amount of Gram-positive bacterial PLFAs and a higher amount of Gram-negative bacterial PLFAs were observed in Bt rice rhizosphere as compared with Ck at all plant development stages, there were no significant differences in the amount of individual 13C-PLFA between Bt and Ck rhizospheres at any growing stage. These findings indicate that the insertion of cry1Ab and marker genes into rice had no persistent or adverse effect on the photosynthate distribution in rice or the microbial community composition in its rhizosphere.

EVALUATION OF LABORATORY-MEASURED SOIL PROPERTIES AS INDICATORS OF SOIL PHYSICAL QUALITY

Routine soil analyses provide an approach for assessment and monitoring of soil quality and targeted implementation of management practices, but suitable indicators are mostly undefined. We used three long-term experiments on several soil types where maize (Zea mays L.) was grown under different tillage (no till and plow till), rotation (continuous maize and maize after grass), and harvesting (silage and grain) methods to identify suitable indicators for evaluating soil physical quality. Disturbed and undisturbed soil samples were collected, and laboratory-based analyses were performed for water-stable aggregation, saturated hydraulic conductivity, several pore size parameters, penetration resistance at &PSgr; = −10 MPa, and bulk density. Sensitivity to management, expense of measurement, measurement consistency, and relevance to critical physical soil processes were used as criteria to evaluate indicator suitability. Indicators varied significantly seasonally and by soil type, and several showed significant differences and trends between management treatments. Small water-stable aggregates (0.25-2 mm) showed the most consistent and significant treatment differences. Bulk density, available water capacity, and air-filled pores at field capacity (PO > 30) were also related to treatment effects and had low variability. Penetration resistance and effective porosity (PO > 0.2) were not sensitive to management practices, whereas aeration pores and saturated hydraulic conductivity were too variable to use as indicators. Several indicators measured on undisturbed cores may be predicted from those measured from disturbed samples using pedotransfer functions. Small water-stable aggregates (0.25-2 mm), available water capacity, bulk density, and PO > 30 appear most promising as indicators for routine evaluation and monitoring of soil physical quality.

An ecological assessment of transgenic crops

Abstract Since the first commercial release of a transgenic crop in 1994, the land area planted to these crops has expanded to over 90 million ha worldwide, with approximately 8.5 million farmers in 21 countries cultivating transgenic crops. Public apprehension has mounted apace. Concerns include: (i) the potential for gene flow into wild plant populations or soil organisms; (ii) adverse effects on non-target organisms; (iii) gene products or crop residues persisting in the environment with deleterious effects and, for insecticidal crops; (iv) resistance developing in target pest populations. Numerous studies on the environmental risks of transgenic crops are published. Gene flow to a crops wild relatives has been demonstrated in the field; hence, the use of these crops is restricted to regions where wild relatives are not endemic. Gene flow to soil organisms is yet to be demonstrated under field conditions and is unlikely given the safeguards employed, but not impossible. The weight of the evidence suggests that there is little risk to non-target soil organisms, but reduced numbers of non-target beneficial insects have been reported with the use of insecticidal crops in some systems. Population effects on non-target insects associated with the use of insecticidal crops are significantly less extensive than those experienced using chemical pesticides, and it has yet to be determined if observed population changes are ecologically significant in these cropping systems. Resistance of target pests to insecticidal crops is possible and eventually likely, but after nearly a decade of use has yet to be detected under field conditions. Several strategies to reduce potential ecological impacts are either under development or near release. Ecological risks posed by new technologies under development and the need for in-country risk assessment and post-release monitoring are discussed.

Diversity and community structure of Archaea inhabiting the rhizoplane of two contrasting plants from an acidic bog.

Plant root exudates increase nutrient availability and influence microbial communities including archaeal members. We examined the archaeal community inhabiting the rhizoplane of two contrasting vascular plants, Dulichium arundinaceum and Sarracenia purpurea, from an acidic bog in upstate NY. Multiple archaeal 16S rRNA gene libraries showed that methanogenic Archaea were dominant in the rhizoplane of both plants. In addition, the community structure (evenness) of the rhizoplane was found markedly different from the bulk peat. The archaeal community in peat from the same site has been found dominated by the E2 group, meanwhile the rhizoplane communities on both plants were co-dominated by Methanosarcinaceae (MS), rice cluster (RC)-I, and E2. Complementary T-RFLP analysis confirmed the difference between bulk peat and rhizoplane, and further characterized the dominance pattern of MS, RC-I, and E2. In the rhizoplane, MS was dominant on both plants although as a less variable fraction in S. purpurea. RC-I was significantly more abundant than E2 on S. purpurea, while the opposite was observed on D. arundinaceum, suggesting a plant-specific enrichment. Also, the statistical analyses of T-RFLP data showed that although both plants overlap in their community structure, factors such as plant type, patch location, and time could explain nearly a third of the variability in the dataset. Other factors such as water table, plant replicate, and root depth had a low contribution to the observed variance. The results of this study illustrate the general effects of roots and the specific effects of plant types on their nearby archaeal communities which in bog-inhabiting plants were mainly composed by methanogenic groups.

PHENOLOGY, GROWTH, AND YIELD OF FIELD-GROWN SOYBEAN AND BUSH BEAN AS A FUNCTION OF VARYING MODES OF N NUTRITION

Abstract In field trials conducted at four sites in Hawaii, soybean (Glycine max) and bush bean (Phaseolus vulgaris) were either inoculated with homologous rhizobia, fertilized at high rates with urea, or left unamended. Crop phenology was assessed every few days. Rates of biomass and N accumulation and components of yield were measured five times during each crop cycle to assess the extent to which: (i) crops relying on soil, symbiotic, or fertilizer N differed in their growth characteristics; (ii) mode of N nutrition affected the timing of developmental stages; and (iii) effects of N nutrition on crop growth and development were related to final yield. While all measured variables differed significantly between sites, the effect of changing N source on these variables, in N limited environments, was consistent across sites. Rate and extent of node production, crop growth and yield were increased in symbiotic and N-fertilized crops as compared to unamended, non-fixing crops, while reproductive development was protracted. Extended time required to reach reproductive maturity was attributable to an increase in seed fill duration as time to flowering was not affected. Development and yield of N2-fixing crops were similar but not equivalent to those of N-fertilized crops. To produce reliable yield estimates, legume growth simulation models must be able to accurately simulate crop growth and phenology. The present data indicate that information relating to source and supply of N must be incorporated before such models can be used to generate reliable yield estimations. Results of these trials also provide a valuable dataset for calibrating model subroutines for inorganic nitrogen uptake and nitrogen fixation in soybean and bush bean growth under field conditions and adjusting model coefficients for tropical environments.

Decomposition rates and residue-colonizing microbial communities of Bacillus thuringiensis insecticidal protein Cry3Bb-expressing (Bt) and non-Bt corn hybrids in the field.

ABSTRACT Despite the rapid adoption of crops expressing the insecticidal Cry protein(s) from Bacillus thuringiensis (Bt), public concern continues to mount over the potential environmental impacts. Reduced residue decomposition rates and increased tissue lignin concentrations reported for some Bt corn hybrids have been highlighted recently as they may influence soil carbon dynamics. We assessed the effects of MON863 Bt corn, producing the Cry3Bb protein against the corn rootworm complex, on these aspects and associated decomposer communities by terminal restriction fragment length polymorphism (T-RFLP) analysis. Litterbags containing cobs, roots, or stalks plus leaves from Bt and unmodified corn with (non-Bt+I) or without (non-Bt) insecticide applied were placed on the soil surface and at a 10-cm depth in field plots planted with these crop treatments. The litterbags were recovered and analyzed after 3.5, 15.5, and 25 months. No significant effect of treatment (Bt, non-Bt, and non-Bt+I) was observed on initial tissue lignin concentrations, litter decomposition rate, or bacterial decomposer communities. The effect of treatment on fungal decomposer communities was minor, with only 1 of 16 comparisons yielding separation by treatment. Environmental factors (litterbag recovery year, litterbag placement, and plot history) led to significant differences for most measured variables. Combined, these results indicate that the differences detected were driven primarily by environmental factors rather than by any differences between the corn hybrids or the use of tefluthrin. We conclude that the Cry3Bb corn tested in this study is unlikely to affect carbon residence time or turnover in soils receiving these crop residues.