Jeffrey S. Buyer

Interpreting Ecological Diversity Indices Applied to Terminal Restriction Fragment Length Polymorphism Data: Insights from Simulated Microbial Communities

ABSTRACT Ecological diversity indices are frequently applied to molecular profiling methods, such as terminal restriction fragment length polymorphism (T-RFLP), in order to compare diversity among microbial communities. We performed simulations to determine whether diversity indices calculated from T-RFLP profiles could reflect the true diversity of the underlying communities despite potential analytical artifacts. These include multiple taxa generating the same terminal restriction fragment (TRF) and rare TRFs being excluded by a relative abundance (fluorescence) threshold. True community diversity was simulated using the lognormal species abundance distribution. Simulated T-RFLP profiles were generated by assigning each species a TRF size based on an empirical or modeled TRF size distribution. With a typical threshold (1%), the only consistently useful relationship was between Smith and Wilson evenness applied to T-RFLP data (TRF-Evar) and true Shannon diversity (H′), with correlations between 0.71 and 0.81. TRF-H′ and true H′ were well correlated in the simulations using the lowest number of species, but this correlation declined substantially in simulations using greater numbers of species, to the point where TRF-H′ cannot be considered a useful statistic. The relationships between TRF diversity indices and true indices were sensitive to the relative abundance threshold, with greatly improved correlations observed using a 0.1% threshold, which was investigated for comparative purposes but is not possible to consistently achieve with current technology. In general, the use of diversity indices on T-RFLP data provides inaccurate estimates of true diversity in microbial communities (with the possible exception of TRF-Evar). We suggest that, where significant differences in T-RFLP diversity indices were found in previous work, these should be reinterpreted as a reflection of differences in community composition rather than a true difference in community diversity.

Comparison of substrate utilization assay and fatty acid analysis of soil microbial communities

Two methods for the characterization of microbial communities in field soil samples were compared. Experimental subplots were established in the Farming Systems Trial at the Rodale Institute Research Center in Kutztown, PA, USA. In the legume system, plots receiving green manure (hairy vetch) were compared to those receiving corn stover and a small amount of rye residue. In the conventional system, samples were collected in plots where corn stover was incorporated. Carbon source utilization profiles were developed using Biolog plates, while total soil fatty acids were determined by fatty acid methyl ester analysis. Principal component analysis and canonical discriminate analysis were used to analyze the data. The two methods gave similar but not identical results. Management history had more effect than specific crop residue, but temporal effects were greater than treatment effects. Certain shifts in microbial communities were detected by fatty acid analysis but not by carbon utilization assay, suggesting that changes in microbial species composition occurred that were not accompanied by changes in microbial community function.

Soil microbiological properties 20 years after surface mine reclamation: spatial analysis of reclaimed and undisturbed sites

Spatial characteristics of soil microbial community structure and selected soil chemical factors were analyzed in soil surrounding Agropyron smithii (Western wheatgrass) and Artemisia tridentata (Wyoming big sagebrush) plants in sites reclaimed after surface mining and adjacent undisturbed sites in Wyoming. Microbial biomass C (MBC) and fatty acid methyl ester (FAME) biomarkers for total biomass, bacteria, and fungi were used as indicators of soil microbial community abundance and structure. In soil 20 years after reclamation FAME total microbial biomass, bacterial and fungal biomarkers, MBC and soil organic matter (SOM) averaged only 20, 16, 28, 44 and 36% of values found in undisturbed soils. In contrast to undisturbed soils, FAME biomarkers and MBC of reclaimed soils exhibited spatial correlation up to 42 cm. Reclaimed soils also exhibited localized enrichment of bacterial, fungal, and total microbial biomass, as well as depletion of inorganic N concentrations, around plant bases (, 10 cm), suggesting relatively poor soil exploration by roots and microorganisms compared to the undisturbed ecosystem. Strong spatial stratification of undisturbed SOM and soil NH4 pools was found with highest concentrations on the leeward side of shrubs, likely due to localized changes in microclimate and plant litter deposition. This indicates that shrub cover plays a central role in the establishment of site heterogeneity and regulation of ecological processes, such as C and N mineralization and immobilization, which has important implications for reclamation. q 2002 Elsevier Science Ltd. All rights reserved.

Microbial biomarkers as an indicator of ecosystem recovery following surface mine reclamation

Increased disturbance of terrestrial ecosystems in recent years for purposes of mineral extraction has created interest in development and optimization of reclamation methodologies for these lands. Currently, criteria for judging surface mine reclamation success, or progress toward reclamation goals, predominantly rely on aboveground indicators that fail to account for the abundance and composition of soil microbiota, an essential aspect of soil health. To test the utility of fatty acid methyl ester (FAME) biomarkers as indicators of reclamation progress, FAME bacterial, fungal, and total biomass biomarkers extracted from soil of surface mine reclamation sites of different ages and an adjacent undisturbed site were compared with other indicators of reclamation progress and ecosystem stability. Our results indicate that FAME microbial biomarkers and soil organic matter (SOM) contents were greatly impacted by disturbance. Discriminant analysis of FAME bacterial, fungal and total microbial biomass biomarkers, although clearly able to discriminate between disturbed and undisturbed ecosystems, indicated a trend towards the undisturbed condition with reclamation age. The ratio of FAME bacterial to fungal biomarkers reflected changes in other indicators of soil health (SOM, inorganic N concentration), suggesting that this ratio is a useful indicator of reclamation progress.

Phylum- and Class-Specific PCR Primers for General Microbial Community Analysis

ABSTRACT Amplification of a particular DNA fragment from a mixture of organisms by PCR is a common first step in methods of examining microbial community structure. The use of group-specific primers in community DNA profiling applications can provide enhanced sensitivity and phylogenetic detail compared to domain-specific primers. Other uses for group-specific primers include quantitative PCR and library screening. The purpose of the present study was to develop several primer sets targeting commonly occurring and important groups. Primers specific for the 16S ribosomal sequences of Alphaproteobacteria, Betaproteobacteria, Bacilli, Actinobacteria, and Planctomycetes and for parts of both the 18S ribosomal sequence and the internal transcribed spacer region of Basidiomycota were examined. Primers were tested by comparison to sequences in the ARB 2003 database, and chosen primers were further tested by cloning and sequencing from soil community DNA. Eighty-five to 100% of the sequences obtained from clone libraries were found to be placed with the groups intended as targets, demonstrating the specificity of the primers under field conditions. It will be important to reevaluate primers over time because of the continual growth of sequence databases and revision of microbial taxonomy.

Isolation and partial characterization of Bacillus subtilis ME488 for suppression of soilborne pathogens of cucumber and pepper

Environmentally friendly control measures are needed for suppression of soilborne pathogens of vegetable crops in the Republic of Korea. In vitro challenge assays were used to screen approximately 500 bacterial isolates from 20 Korean greenhouse soils for inhibition of diverse plant pathogens. One isolate, Bacillus subtilis ME488, suppressed the growth of 39 of 42 plant pathogens tested. Isolate ME488 also suppressed the disease caused by Fusarium oxysporum f. sp. cucumerinum on cucumber and Phytophthora capsici on pepper in pot assays. Polymerase chain reaction was used to screen isolate ME488 for genes involved in biosynthesis of 11 antibiotics produced by various isolates of B. subtilis. Amplicons of the expected sizes were detected for bacD and bacAB, ituC and ituD, and mrsA and mrsM involved in the biosynthesis of bacilysin, iturin, and mersacidin, respectively. The identity of these genes was confirmed by DNA sequence analysis of the amplicons. Bacilysin and iturin were detected in culture filtrates from isolate ME488 by gas chromatography coupled with mass spectroscopy and by thin layer chromatography, respectively. Detection of mersacidin in ME488 culture filtrates was not attempted. Experiments reported here indicate that B. subtilis ME488 has potential for biological control of pathogens of cucumber and pepper possibly due to the production of antibiotics.

Yeasts associated with nectarines and their potential for biological control of brown rot

Resident fruit microflora has been the source of biocontrol agents for the control of postharvest decay of fruits and the active ingredient in commercialized biocontrol products. With the exception of grapes and apples, information on the resident microflora of other fruits is only fragmentary, but greater knowledge in this area can be very helpful in developing biocontrol strategies. We characterized the yeast microflora of nectarines (‘Croce del Sud’) from the early stages of fruit development until harvest. The fruit samples were collected from trees in an unmanaged orchard. The resident fruit microflora was separated from the occasionally deposited microorganisms by discarding initial fruit washings before the final wash, followed by sonication and plating on NYDA medium. The isolated yeasts were identified by BIOLOG and by sequencing the D1/D2 domain of a large subunit of the rRNA gene and, where available, the ITS sequence. BIOLOG identified 19 and the genetic analysis 23 species of yeasts. Although the identification by these two systems was not always the same, the predominant yeasts were Rhodotorula spp., Sporodiobolus spp., Cryptococcus spp., Pichia spp., Candida spp. and yeast‐like Aureobasidium pullulans. Several of the taxa appear to represent new species. The preliminary biocontrol tests against brown rot of nectarine fruit caused by Monilinia fructicola indicates significant decay control potential of some of the identified yeast species, namely Cryptococcus magnus, Cryptococcus sp. nov., Sporidiobolus pararoseus, A. pullulans and Rhodotorula sp. nov. Copyright

Analysis of fungal communities by sole carbon source utilization profiles.

A simple method for characterization of fungal communities in environmental samples was developed. Dilute suspensions of samples in 0.2% agar containing three different antibiotics were pipetted into 96-well plates (Biolog SF-N) containing a diverse collection of 95 different carbon sources. The plates were incubated for 4-12 days at 22 degrees C and the absorbance measured at 650 nm. Canonical variates analysis was then used to analyze the multivariate data. This method allowed fungal communities in rhizosphere soil of corn and soybean to be distinguished according to soil and plant type. Data taken at a single time-point, which varied greatly in total absorbance of the plate, separated rhizosphere samples primarily by soil type. When multiple time-points were combined to keep the total absorbance constant, differences in substrate utilization patterns due to different plant types could be distinguished. The method was also applicable to analysis of phylloplane and compost fungal communities. This method is readily applied to large numbers of samples and should be useful for community analysis in a variety of agricultural and ecological studies.

Microbial diversity in the rhizosphere of corn grown under conventional and low-input systems

Abstract Microbial processes within the rhizosphere of crop plants are crucial to agriculture. The relation of soil microbial community diversity to cropping system, yield, and soil quality are unclear at present. The Farming Systems Trial at the Rodale Institute Research Center, Kutztown, PA, is a 15-year study in which a conventional corn-soybean rotation has been compared with low-input systems (i.e. animal manure or legumes as nitrogen sources). The effects of the cropping systems on diversity of fast-growing, aerobic, culturable bacteria and fungi were conducted in 1994 by taking 84 cores in between corn plants in June, July, and August. Rhizosphere bacteria and fungi were extracted, plated, and counted. Approximately 6000 bacteria were identified by fatty acid methyl ester analysis, while 18000 fungi were identified by microscopic examination of spores. Microbial diversity and evenness were calculated using several different methods. Total counts, diversity, and evenness were not significantly different for the three cropping systems. These results suggest that conventional agricultural practices may maintain high indices of microbial diversity in the rhizosphere. The functional significance of this needs to be investigated.

Structural and functional analysis of whole-soil microbial communities for risk and efficacy testing following microbial inoculation of wheat roots in diverse soils

The increasing use of genetically engineered or modified microorganisms (GEMs) has led to regulations regarding the safety of their use. Intended (target) effects and unintended (non-target) effects of GEMs must currently be evaluated prior to field testing or commercial use. We present soil and rhizosphere microbial community effects testing of two GEMs, Pseudomonas chlororaphis 3732RN-L11 and Pseudomonas fluorescens 2-79RN-L3, parental strains of these organisms and an uninoculated treatment using five diverse soils planted to wheat. An assay using BIOLOG w GN plates measured microbial community functional responses on wheat roots with adhering soil. Overall differences using multivariate statistical methods were highest at inoculation, and these effects persisted while the inoculated organisms were detectible on selective media. Differentiation based on lacZY genes engineered to the chromosome of both GEMs was significant for the 3732 GEM in all five soils tested, but not for the 2-79 GEM in a single soil. Lactose utilization in uninoculated microbial communities varied around a low baseline value. Direct fatty acid extraction and analysis of soil from around wheat roots was also performed using a novel method. Fatty acid analysis differentiated the 3732 GEM from all other treatments, but did not distinguish the 3732 parent inoculated from uninoculated treatments. As with the BIOLOG assay, multivariate statistical differences from fatty acid analysis decreased between GEM inoculated and uninoculated populations as viable counts of the GEM declined. Neither assay showed measurable community-level effects when inoculated organisms declined below detection, though three of six soils with surviving GEM populations still had significant effects after 105 days. Published by Elsevier Science Ltd.