Methods of studying soil microbial diversity

Soil microorganisms, such as bacteria and fungi, play central roles in soil fertility and promoting plant health. This review examines and compares the various methods used to study microbial diversity in soil.     

Molecular microbial diversity of an agricultural soil in Wisconsin.

A culture-independent survey of the soil microbial diversity in a clover-grass pasture in southern Wisconsin was conducted by sequence analysis of a universal clone library of genes coding for small-subunit rRNA (rDNA). A rapid and efficient method for extraction of DNA from soils which resulted in highly purified DNA with minimal shearing was developed. Universal small-subunit-rRNA primers were used to amplify DNA extracted from the pasture soil. The PCR products were cloned into pGEM-T, and either hypervariable or conserved regions were sequenced. The relationships of 124 sequences to those of cultured organisms of known phylogeny were determined. Of the 124 clones sequenced, 98.4% were from the domain Bacteria. Two of the rDNA sequences were derived from eukaryotic organelles. Two of the 124 sequences were of nuclear origin, one being fungal and the other a plant sequence. No sequences of the domain Archaea were found. Within the domain, Bacteria, three kingdoms were highly represented: the Proteobacteria (16.1%), the Cytophaga-Flexibacter-Bacteroides group (21.8%), and the low G+C-content gram-positive group (21.8%). Some kingdoms, such as the Thermotogales, the green nonsulfur group, Fusobacteria, and the Spirochaetes, were absent. A large number of the sequences (39.4%) were distributed among several clades that are not among the major taxa described by Olsen et al. (G.J. Olsen, C.R. Woese, and R. Overbeek, J. Bacteriol., 176:1-6, 1994). From the alignments of the sequence data, distance matrices were calculated to display the enormous microbial diversity found in this soil in two ways, as phylogenetic trees and as multidimensional-scaling plots.     

BIPES,a cost-effective high-throughput method for assessing microbial diversity

Pyrosequencing of 16S rRNA (16S) variable tags has become the most popular method for assessing microbial diversity, but the method remains costly for the evaluation of large numbers of environmental samples with high sequencing depths. We developed a barcoded Illumina paired-end (PE) sequencing (BIPES) method that sequences each 16S V6 tag from both ends on the Illumina HiSeq 2000, and the PE reads are then overlapped to obtain the V6 tag. The average accuracy of Illumina single-end (SE) reads was only 97.9%, which decreased from ∼99.9% at the start of the read to less than 85% at the end of the read; nevertheless, overlapping of the PE reads significantly increased the sequencing accuracy to 99.65% by verifying the 3′ end of each SE in which the sequencing quality was degraded. After the removal of tags with two or more mismatches within the medial 40–70 bases of the reads and of tags with any primer errors, the overall base sequencing accuracy of the BIPES reads was further increased to 99.93%. The BIPES reads reflected the amounts of the various tags in the initial template, but long tags and high GC tags were underestimated. The BIPES method yields 20–50 times more 16S V6 tags than does pyrosequencing in a single-flow cell run, and each of the BIPES reads costs less than 1/40 of a pyrosequencing read. As a laborsaving and cost-effective method, BIPES can be routinely used to analyze the microbial ecology of both environmental and human microbiomes.     

The diversity and function of soil microbial communities exposed to different disturbances

To improve understanding of the relationship between the diversity and function of the soil ecosystem, we investigated the effect of two different disturbances on soil bacterial communities—long-term exposure to the heavy metal mercury and transient exposure to the antibiotic tylosin. In the mercury-contaminated soil the diversity (Shannon index) was reduced as assessed from denaturing gradient gel electrophoresis (DGGE) of amplified 16S rDNA sequences from the soil community DNA and from colony morphology typing of the culturable bacterial population. However, analysis of the substrate utilization profiles did not reveal any differences in diversity. In the tylosin-treated soil, DGGE revealed a small difference in the diversity of 16S rDNA compared to the control soil, whereas analysis of the colony morphology typing or substrate utilization results did not reveal any differences in diversity. Soil function was also affected by mercury contamination. The lag time before soil respiration increased following addition of glucose or alfalfa substrate was longer in the mercury-contaminated soil than in the control soil. Moreover, it was markedly prolonged in mercury-contaminated soil subjected to heat treatment prior to substrate addition, thus indicating reduced resistance to a new disturbance in the mercury-contaminated soil as compared to the control soil. Tylosin treatment did not have any significant effect on any of the respiration parameters measured, either with or without prior heat treatment of the soil.     

Reclamation of abandoned saline-alkali soil increased soil microbial diversity and degradation potential

Plant and Soil - Reclamation of saline-alkali soils to grow cotton (Gossypium spp.) is very common in the arid Manas River Basin in Northwest China. However, little is known about the degradation...     

The edaphic quantitative protargol stain: a sampling protocol for assessing soil ciliate abundance and diversity

It has been suggested that species loss from microbial groups low in diversity that occupy trophic positions close to the base of the detrital food web could be critical for terrestrial ecosystem functioning. Among the protozoans within the soil microbial loop, ciliates are presumably the least abundant and of low diversity. However, the lack of a standardized method to quantitatively enumerate and identify them has hampered our knowledge about the magnitude of their active and potential diversity, and about the interactions in which they are involved. Thus, the Edaphic Quantitative Protargol Staining (EQPS) method is provided to simultaneously account for ciliate species richness and abundance in a quantitative and qualitative way. This direct method allows this rapid and simultaneous assessment by merging the Non-flooded Petri Dish (NFPD) method [Prog. Protistol. 2 (1987) 69] and the Quantitative Protargol Stain (QPS) method [Montagnes, D.J.S., Lynn, D.H., 1993. A quantitative protargol stain (QPS) for ciliates and other protists. In: Kemp, P.F., Sherr, B.F., Sherr, E.B., Cole, J.J. (Eds.), Handbook of Methods in Aquatic Microbial Ecology. Lewis Publishers, Boca Raton, FL, pp. 229-240]. The abovementioned protocols were refined by experiments examining the spatial distribution of ciliates under natural field conditions, sampling intensity, the effect of storage, and the use of cytological preparations versus live observations. The EQPS could be useful in ecological studies since it provides both a "snapshot" of the active and effective diversity and a robust estimate of the potential diversity.     

Impact of soil leachate on microbial biomass and diversity affected by plant diversity

Plant and Soil - High plant diversity is usually linked with high soil microbial diversity, which is hypothesized to be attributed to a high diversity of components in the soil leachate, but...     

Correlations between soil microbial and physicochemical variations in a rice paddy: implications for assessing soil health

This study was conducted to test the hypothesis that spatial variations in soil microbial variables in a Thai rice paddy are accurately described by multivariate profiles of the soil bacterial communities. We found that community-level physiological profiles of soil bacterial communities could better describe the population density of Rhizoctonia solani in soil than the physicochemical profiles do. However, soil dehydrogenase levels were closely correlated with soil fertility (P<0.05), and these were better described by the physicochemical profiles. Hence, the hypothesis was rejected, and we suspect that soil microbial variables react differently to the same physicochemical changes. The average population density of R. solani (35 colony-forming units/g dry soil) was relatively high in the soil we studied, and the soil fertility was found to be among the poorest in Thailand. The soil quality was comparable to the most degraded bare ground soil in an adjacent bioreserve in terms of Shannon diversity index based on the communitylevel physiological profile as well as values of soil fertility indices. Overall, the soil microbial and physicochemical indicators showed that the paddy soil needs to be supplemented with soil nutrients. Otherwise, R. solani may cause a significant reduction in rice production.     

Responses of soil microbial catabolic diversity to arbuscular mycorrhizal inoculation and soil disinfection

Although it is usually admitted that arbuscular mycorrhizal (AM) fungi are key components in soil bio-functioning, little is known on the response of microbial functional diversity to AM inoculation. The aims of the present study were to determine the influence of Glomus intraradices inoculum densities on plant growth and soil microflora functional diversity in autoclaved soil or non-disinfected soil. Microbial diversity of soil treatments was assessed by measuring the patterns of in situ catabolic potential of microbial communities. The soil disinfection increased sorghum growth, but lowered catabolic evenness (4.8) compared to that recorded in the non-disinfected soil (6.5). G. intraradices inoculation induced a higher plant growth in the autoclaved soil than in the non-disinfected soil. This AM effect was positively related to inoculum density. Catabolic evenness and richness were positively correlated with the number of inoculated AM propagules in the autoclaved soil, but negatively correlated in the non-disinfected soil. In addition, after soil disinfection and AM inoculation, these microbial functionality indicators had higher values than in the autoclaved or in the non-disinfected soil without AM inoculation. These results are discussed in relation to the ecological influence of AM inoculation, with selected fungal strains and their associated microflora on native soil microbial activity.     

Allantoin-induced changes of microbial diversity and community in rice soil

The effects of slurry application method and weather conditions after application on ammonia volatilisation are well documented, however, the effect on slurry N recovery in herbage is less evident due to large variability of results. The objective of this field experiment was to determine the recovery of cattle slurry NH₄-N in herbage and soil in the year of application as affected by application method (trailing shoe versus broadcast) and season of application (spring versus summer), using ¹⁵N as a tracer. In 2007 and 2008, ¹⁵N enriched slurry was applied on grassland plots. N recovery in herbage and soil during the year of application was determined. Both spring and trailing shoe application resulted in significantly higher herbage DM yields, N uptake and an increased recovery of ¹⁵NH₄-N in herbage. Additionally, the recovery of slurry ¹⁵NH₄-N in the soil at the end of the growing season was increased. Spring and trailing shoe application reduced the losses of slurry ¹⁵NH₄-N by on average 14 and 18 percentage points, respectively, which corresponded closely to ammonia volatilisation as predicted by the ALFAM model. It was concluded that slurry N recovery in temperate pasture systems can be increased by adjusting the slurry application method or timing.     

Distinct soil microbial diversity under long-term organic and conventional farming

Low-input agricultural systems aim at reducing the use of synthetic fertilizers and pesticides in order to improve sustainable production and ecosystem health. Despite the integral role of the soil microbiome in agricultural production, we still have a limited understanding of the complex response of microbial diversity to organic and conventional farming. Here we report on the structural response of the soil microbiome to more than two decades of different agricultural management in a long-term field experiment using a high-throughput pyrosequencing approach of bacterial and fungal ribosomal markers. Organic farming increased richness, decreased evenness, reduced dispersion and shifted the structure of the soil microbiota when compared with conventionally managed soils under exclusively mineral fertilization. This effect was largely attributed to the use and quality of organic fertilizers, as differences became smaller when conventionally managed soils under an integrated fertilization scheme were examined. The impact of the plant protection regime, characterized by moderate and targeted application of pesticides, was of subordinate importance. Systems not receiving manure harboured a dispersed and functionally versatile community characterized by presumably oligotrophic organisms adapted to nutrient-limited environments. Systems receiving organic fertilizer were characterized by specific microbial guilds known to be involved in degradation of complex organic compounds such as manure and compost. The throughput and resolution of the sequencing approach permitted to detect specific structural shifts at the level of individual microbial taxa that harbours a novel potential for managing the soil environment by means of promoting beneficial and suppressing detrimental organisms.     

The effects of mineral fertilizer and organic manure on soil microbial community and diversity

The effects of mineral fertilizer (NPK) and organic manure on phospholipid fatty acid profiles and microbial functional diversity were investigated in a long-term (21-year) fertilizer experiment. The experiment included nine treatments: organic manure (OM), organic manure plus fertilizer NPK (OM + NPK), fertilizer NPK (NPK), fertilizer NP (NP), fertilizer NK (NK), fertilizer N (N), fertilizer P (P), fertilizer K (K), and the control (CK, without fertilization). The original soil was extremely eroded, characterized by low pH and deficiencies of nutrients, particularly N and P. The application of OM and OM + NPK greatly increased crop yields, soil pH, organic C, total N, P and K, available N, P and K content. Crop yields, soil pH, organic C, total N and available N were also clearly increased by the application of mineral NPK fertilizer. The amounts of total PLFAs, bacterial, Gram-negative and actinobacterial PLFAs were highest in the OM + NPK treatment, followed by the OM treatment, whilst least in the N treatment. The amounts of Gram-positive and anaerobic PLFAs were highest in the OM treatment whilst least in the P treatment and the control, respectively. The amounts of aerobic and fungal PLFAs were highest in the NPK treatment whilst least in the N and P treatment, respectively. The average well color development (AWCD) was significantly increased by the application of OM and OM + NPK, and the functional diversity indices including Shannon index (H ^′ ), Simpson index (D) and McIntosh index (U) were also significantly increased by the application of OM and OM + NPK. Principal component analysis (PCA) of PLFA profiles and C source utilization patterns were used to describe changes in microbial biomass and metabolic fingerprints from nine fertilizer treatments. The PLFA profiles from OM, OM + NPK, NP and NPK were significantly different from that of CK, N, P, K and NK, and C source utilization patterns from OM and OM + NPK were clearly different from organic manure deficient treatments (CK, N, P, K, NP, NK 6 and NPK). Stepwise multiple regression analysis showed that total N, available P and soil pH significantly affected PLFA profiles and microbial functional diversity. Our results could provide a better understanding of the importance of organic manure plus balanced fertilization with N, P and K in promoting the soil microbial biomass, activity and diversity and thus enhancing crop growth and production.     

Accessing the soil metagenome for studies of microbial diversity

Soil microbial communities contain the highest level of prokaryotic diversity of any environment, and metagenomic approaches involving the extraction of DNA from soil can improve our access to these communities. Most analyses of soil biodiversity and function assume that the DNA extracted represents the microbial community in the soil, but subsequent interpretations are limited by the DNA recovered from the soil. Unfortunately, extraction methods do not provide a uniform and unbiased subsample of metagenomic DNA, and as a consequence, accurate species distributions cannot be determined. Moreover, any bias will propagate errors in estimations of overall microbial diversity and may exclude some microbial classes from study and exploitation. To improve metagenomic approaches, investigate DNA extraction biases, and provide tools for assessing the relative abundances of different groups, we explored the biodiversity of the accessible community DNA by fractioning the metagenomic DNA as a function of (i) vertical soil sampling, (ii) density gradients (cell separation), (iii) cell lysis stringency, and (iv) DNA fragment size distribution. Each fraction had a unique genetic diversity, with different predominant and rare species (based on ribosomal intergenic spacer analysis [RISA] fingerprinting and phylochips). All fractions contributed to the number of bacterial groups uncovered in the metagenome, thus increasing the DNA pool for further applications. Indeed, we were able to access a more genetically diverse proportion of the metagenome (a gain of more than 80% compared to the best single extraction method), limit the predominance of a few genomes, and increase the species richness per sequencing effort. This work stresses the difference between extracted DNA pools and the currently inaccessible complete soil metagenome.     

Effect of an introduced inoculum on soil microbial diversity

Abstract A laboratory study was carried out to evaluate the impact of the introduction of genetically modified microorganisms into soil, in terms of effect on the diversity of the indigenous microflora, and at the process level. The impact on microbial phenotypic diversity, and on soil denitrification of an inoculum of a lux -modified denitrifier, Pseudomonas fluorescens , was examined using two different soil types in re-packed soil microcosms. The effect on diversity was found to depend on the soil pore size class into which the modified inoculum was introduced. The introduction of lux -modified cells into the 15–30 μm pore neck size class caused a short-term reduction in the overall microbial diversity. There was no significant change in the diversity of the indigenous microbial community, however, when cells were introduced into the 40–60 μm pore class. Partial chloroform fumigation proved useful in differentiating cell populations with respect to pore location. No change in diversity was observed when dead cells (either heat killed or glutaraldehyde fixed) were introduced into either pore size class. At the process level, the effect on soil denitrification of introduction of lux -modified P. fluorescens was not significantly different from introduction of the equivalent inoculum of the parental wild-type, although denitrification was found to be dependent upon both soil structure and pore size location of the introduced inoculum.     

Maintenance of soil functioning following erosion of microbial diversity

The paradigm that soil microbial communities, being very diverse, have high functional redundancy levels, so that erosion of microbial diversity is less important for ecosystem functioning than erosion of plant or animal diversity, is often taken for granted. However, this has only been demonstrated for decomposition/respiration functions, performed by a large proportion of the total microbial community, but not for specialized microbial groups. Here, we determined the impact of a decrease in soil microbial diversity on soil ecosystem processes using a removal approach, in which less abundant species were removed preferentially. This was achieved by inoculation of sterile soil microcosms with serial dilutions of a suspension obtained from the same non-sterile soil and subsequent incubation, to enable recovery of community size. The sensitivity to diversity erosion was evaluated for three microbial functional groups with known contrasting taxonomic diversities (ammonia oxidizers < denitrifiers < heterotrophs). Diversity erosion within each functional group was characterized using molecular fingerprinting techniques: ribosomal intergenic spacer analysis (RISA) for the eubacterial community, denaturing gradient gel electrophoresis (DGGE) analysis of nirK genes for denitrifiers, and DGGE analysis of 16S rRNA genes for betaproteobacterial ammonia oxidizers. In addition, we simulated the impact of the removal approach by dilution on the number of soil bacterial species remaining in the inoculum using values of abundance distribution of bacterial species reported in the literature. The reduction of the diversity of the functional groups observed from genetic fingerprints did not impair the associated functioning of these groups, i.e. carbon mineralization, denitrification and nitrification. This was remarkable, because the amplitude of diversity erosion generated by the dilution approach was huge (level of bacterial species loss was estimated to be around 99.99% for the highest dilution). Our results demonstrate that the vast diversity of the soil microbiota makes soil ecosystem functioning largely insensitive to biodiversity erosion even for functions performed by specialized groups.     

PCR-DGGE技术在土壤微生物多样性研究中的应用

DGGE是一种有效的微生物多样性研究技术。本文简要介绍了DGGE(denaturing gradient gel electrophoresis)的基本原理,及其在研究土壤微生物类群多样性中的应用,并对该技术自身存在的缺陷进行了评价。     

The microbial DNA cycle in soil.

Upon microbial cell death and lysis in soil, the free or naked DNA is exposed to the dynamic environment of the soil. The DNA can be enzymatically degraded by nucleases (DNases), bind to soil components, genetically transform competent bacterial cells and be a nutrient for other microorganisms. In this article we discuss the dual role of DNA as genetic material and as a nutrient source in the soil environment.