Evaluating the physical capture method of terminal restriction fragment length polymorphism for comparison of soil microbial communities

Terminal restriction fragment length polymorphism (T-RFLP) is a popular method of comparative microbial community analysis which is normally accomplished by tagging terminal restriction fragments (T-RFs) with a fluorescent primer. Here, we evaluate an alternative method of T-RFLP where T-RFs are physically captured using a biotinylated primer and streptavidin-coated beads. This eliminates one of the primary criticisms of T-RFLP, namely that T-RFs cannot be identified by sequence analysis, and also represents an alternative method for collecting T-RFLP profiles. Microbial communities from forest, agricultural, and turf soils were investigated using several sets of primers specific for different microbial groups. The physical capture method of T-RFLP resulted in similar profiles to those generated by fluorescent T-RFLP. The relationships among ecosystem types captured by both methods and revealed by ordination were virtually identical. The total variance in the profiles that was attributed to ecosystem type was approximately equal, or greater, when generated by the physical capture method, depending on the primers used. However, physical capture T-RFLP resolved fewer T-RFs than fluorescent T-RFLP, and this may reduce the sensitivity to changes in non-dominant populations within the community. Direct cloning and sequencing of physical capture T-RFs revealed that most bands were not comprised of sequences related to those in the database that would generate T-RFs of similar size. T-RFs should therefore be identified by sequencing, rather than by comparing the sizes of T-RFs to computer digests of database sequences. Physical capture T-RFLP should be a useful tool to identify T-RFs by sequencing, and for laboratories without economical access to equipment required to perform fluorescent T-RFLP.     

Above- and below-ground interactions with agricultural management: Effects of soil microbial communities on barley and aphids

Recent research has shown that agricultural management affects microbial biomass and community composition. We investigated the functional implications of such effects in terms of barley biomass production and nutrient acquisition, and whether changes in barley nutrient status affected aphid fecundity. Soils were collected from conventional, ley and organic arable fields and used as inocula in a glasshouse experiment. We determined microbial biomass and community composition using PLFA. We investigated barley growth and nutrient responses to the different soil inoculums, and the impact of excluding arbuscular mycorrhizal fungi (AMF). Aphids were applied to plants within clip cages and numbers of offspring counted. Microbial biomass and community composition were unaffected by agricultural management. The microbial communities altered root and shoot biomass and nutrient allocation, but had no effect on grain yield. Exclusion of AMF significantly increased shoot biomass but reduced grain yield. Aphid fecundity was not significantly affected by the microbial community or shoot nitrogen. We conclude that agricultural intensification does not necessarily have negative consequences for above- and below-ground interactions, and microbial communities from conventionally managed soils may offer equal benefit to crop productivity and nutrition as those from organically managed soils.     

Eubacterial community structure and population size within the soil light fraction, rhizosphere, and heavy fraction of several agricultural systems

The hypothesis that soil light fraction and heavy fraction harbor distinct eubacterial communities and have differing numbers and sizes of bacterial cells was tested in three agronomic cropping systems. This hypothesis would imply that these soil fractions are distinct microbial habitats. Shoot residue and rhizosphere soil were also included in the analysis. Terminal restriction fragment length polymorphism (T-RFLP) of 16S ribosomal DNA was used to assay eubacterial community structure. T-RFLP profiles were affected by both soil fraction and cropping system, accounting for 35-50% of the variance in the profiles. T-RFLP profiles separated samples into two distinct eubacterial habitats: soil heavy fraction, which includes the mineral particles and associated humified organic matter, and soil light fraction/shoot residue and rhizosphere, which includes particulate soil organic matter. Differences were not based on organic C content of fractions alone; T-RFLP profiles were also differentiated by cropping system and by rhizosphere versus light fraction/shoot residue. Heavy fraction communities had the least amount of random variability in T-RFLP profiles, resulting in the clearest cropping system effects, while rhizosphere and shoot residue communities were the most variable. Profiles from organically managed corn soil were more variable than for either conventionally managed corn or alfalfa. The log number of bacterial cells per gram fraction was affected by soil fraction but not cropping system, being highest in the light fraction. The percentage of cells >0.18 μm³ was also greater in the light fraction than in other fractions. While bacterial cell density was generally correlated with C content of the soil fraction, heavy fraction did have a significantly greater number of cells per μg C than other soil fractions. The results show that habitat diversity in soil, related both to the amounts and types of organic matter, as well as other potential factors, are important in maintaining the high soil bacterial species diversity and evenness that is found in soil.     

Eubacterial communities in different soil macroaggregate environments and cropping systems

Different positions within soil macroaggregates, and macroaggregates of different sizes, have different chemical and physical properties which could affect microbial growth and interactions among taxa. The hypothesis that these soil aggregate fractions contain different eubacterial communities was tested using terminal restriction fragment length polymorphism (T-RFLP) of the 16S ribosomal gene. Communities were characterized from two field experiments, located at the Kellogg Biological Station (KBS), MI, USA and the Ohio Agricultural Research and Development Center (OARDC), Wooster, OH, USA. Three soil management regimes at each site were sampled and management was found to significantly affect T-RFLP profiles. The soil aggregate erosion (SAE) method was used to isolate aggregate regions (external and internal regions). Differences between eubacterial T-RFLP profiles of aggregate exteriors and interiors were marginally significant at KBS (accounting for 12.5% of total profile variance), and not significant at OARDC. There were no significant differences among macroaggregate size classes at either site. These results are in general agreement with previous studies using molecular methods to examine microbial communities among different soil macroaggregate size fractions, although further study of communities within different aggregate regions is warranted. Analysis of individual macroaggregates revealed large inter-aggregate variability in community structure. Hence the tertiary components of soil structure, e.g. arrangement of aggregates in relation to shoot residue, roots, macropores, etc., may be more important than aggregate size or intra-aggregate regions in the determination of the types of microbial communities present in aggregates. Direct microscopic counts were also used to examine the bacterial population size in aggregate regions at KBS. The proportion of bacterial cells with biovolumes >0.18 μm³ was higher in aggregate interiors than in exteriors, indicating potentially higher activity in that environment. This proportion was significantly related to percent C of the samples, while total bacterial cell counts were not.     

Woodland trees modulate soil resources and conserve fungal diversity in fragmented landscapes

Resource islands around woody plants are thought to define the structure and function of many semiarid and arid ecosystems, but their role in patterning of soil microbial communities remains largely unexamined in dry environments. This study examined soil resource distribution and associated fungal communities in two Allocasuarina luehmannii (buloke) remnants of semiarid north-western Victoria, Australia. These savannah-like woodlands are listed as endangered due to extensive clearing for agriculture. We used the DNA-based profiling technique T-RFLP and ordination-based statistical methods to compare fungal community compositions in surface soils from two remnants (located 1.6 km apart) and three sampling positions (beneath individual buloke canopies; grassy inter-canopy areas; and adjoining cleared paddocks). Resource island formation beneath buloke trees was clearly evident in soil physicochemical properties (e.g. threefold concentrations of total carbon and nitrogen in canopy versus non-canopy soils). This heterogeneity of resources was moderately correlated with soil fungal community compositions, which were distinct for each sampling position. We argue that fungal composition patterns reflected multiple roles of fungi in dryland ecosystems, namely: responses of saprotrophic fungi to tree organic matter inputs; specificity of ectomycorrhizal fungi to tree rooting zones; and fungal involvement in biological soil crusts that variably covered non-canopy soils. Our data did not indicate that buloke canopy areas were particular hotspots of soil fungal diversity, but that they increased landscape-level diversity by supporting a distinct suite of fungi. In addition, we provide evidence of phylogenetic differentiation of soil fungal communities between our two remnants, which adds to growing evidence of fungal genetic structure at localised scales. These findings highlight the importance of remnant trees in conserving both soil resources and microbial genetic diversity. In addition, evidence of differentiation of soil fungal phylogenetics between nearby but isolated remnants suggests that conserving soil fungal diversity requires conservation of host habitats over their entire (remaining) range, and indicates previously unseen consequences of tree loss from extensively cleared landscapes.     

Soil properties associated with organic matter-mediated suppression of bean root rot in field soil amended with fresh and composted paper mill residuals

The ability of an organic amendment to suppress soil-borne disease is mediated by the complex interactions between biotic and abiotic soil factors. Various microbiological and physicochemical soil properties were measured in field soils with histories of receiving 4 or 5 years of spring additions of paper mill residuals (PMR), PMR composted alone (PMRC), PMR composted with bark (PMRB), or no amendment under a conventionally managed vegetable crop rotation. The objectives of this study were to (i) determine the residual and re-amendment effects of the organic materials on root rot disease severity; (ii) determine the influence of amendment type on the structure of bacterial communities associated with snap bean roots grown in these soils; and (iii) quantify the relative contributions of microbiological and physicochemical properties to root rot suppression in the field and greenhouse. While all amendment types significantly suppressed root rot disease compared to non-amended soils in both environments, only soils amended with PMR or PMRB sustained suppressive conditions 1 year after the most recent amendment event. Disease severity was inversely related to microbial activity (fluorescein diacetate assay) in recently amended soils only. Terminal restriction fragment length polymorphism (T-RFLP) analysis of the 16s rRNA gene was performed to obtain bacterial profiles. Principal component analysis (PCA) of terminal restriction fragments (TRFs) revealed general differences in bacterial community composition (PC1) among amendment types, and specific TRFs contributed to these differences. Correlation and multiple regression analyses of the measured soil variables revealed that the composition of root-associated bacterial communities and the amount of particulate organic matter—carbon in bulk soils imparted independent and relatively equal contributions to the variation in disease severity documented in the field and greenhouse. Together, our findings provide evidence that disease suppression induced by annual PMR inputs was mediated by their differential effects on bacterial communities and the amount and quality of organic matter in these soils.