Final Report for GS08-065

Project Type: Graduate Student
Funds awarded in 2008: $10,000.00
Projected End Date: 12/31/2011
Grant Recipient: University of Kentucky
Region: Southern
State: Kentucky
Graduate Student:
Major Professor:
Elisa D'Angelo
University of Kentucky
Major Professor:
Mark Williams
University of Kentucky
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Project Information


This project evaluated the use of organic amendments to foster a soil microbial community profile that favors improved soil structure. The effects of soil amendments on microbial community dynamics and aggregate formation were tested in three different agricultural soils. The native soil structure of experimental soils was destroyed by forcing soils through a 250µm sieve. Soils were treated with organic amendments based on amendment C content at a rate of 0.01g-amendment C/g soil. Amendment treatments included hairy vetch residue, dairy manure, compost, or no-amendment (control). Soils were then incubated at 25°C for 82 days. Soils were destructively sampled on incubation days 0, 5, 12, 30, and 82. A wet sieving method was used to assess the formation of large macroaggregates (LMacAg) which were defined as aggregates >2000 µm. Differences in microbial community structure were assessed through analysis of microbial fatty acid methyl esters (FAME). Incubated soils were also analyzed for the fungal biomarker ergosterol. Relationships between amendment treatments, microbial parameters, and aggregate formation through time were investigated in order to test the hypothesis that amendments that stimulate greater fungal activity will also promote greater macroaggregate formation. In general, the vetch treatment resulted in the greatest formation of LMacAg followed by the manure, control, and compost treatments, respectively. In two of the three soils tested, the vetch treatment showed the greatest fungal/bacterial ratio (F:B) after 82 days of incubation, while the manure treatment resulted in the highest 82 day F:B in the much sandier Yeager soil. Ergosterol content was greatest in the vetch treatment in all soils. Large macroaggregate formation was most positively related to the relative abundance of 18:2?6c, a signature FAME for fungi. Formation of LMacAg was also strongly positively related to soil ergosterol. We conclude that the compost used neither enhanced soil structure nor did it significantly alter the microbial community relative to non-amended soils. In general, vetch is a good amendment for enhancing soil structure because it promotes higher relative abundances of fungi in most soils. Manure also promotes high relative abundance of fungi and may possibly be as effective as vetch in coarser textured soils.


Soil aggregation is the foundation for soil structure in surface horizons, affecting processes such as water infiltration and movement, oxygen diffusion, and plant nutrient availability. Microorganisms play major roles in formation and stabilization of soil aggregates (Oades, 1993). Fungi are thought to be the dominant microbes involved in macroaggregate formation while bacterial activity facilitates microaggregate (< 250µm diameter) formation (Oades, 1993). Fungal hyphae bind macroaggregates by physically enmeshing microaggregates and soil particles (Tisdall and Oades, 1982). Fungi also exude polysaccharides that act as aggregate binding agents (Oades, 1993). Bacteria thrive in micropore zones where they are protected from size-excluded bactivores (Monreal and Kodama, 1997). In these zones, bacteria also facilitate aggregation on a microscopic level by excreting polysaccharides which bind silt and clay particles into microaggregates (Oades, 1993). Monreal and Kodama (1997) found proportions of fungi and bacteria to differ by aggregate size class, with macroaggregates having higher fungi to bacteria ratios than microaggregates. Beare et al. (1997) showed reduced macroaggregate stability when fungicides were applied.

Organic amendments can alter the microbial community (Schutter et al. 2001) and they generally have a positive effect on soil aggregation (Bronik and Lal, 2005). Organic amendments are a carbon source for microbes and their composition can affect microbial community dynamics. Larkin et al. (2006) found that manure inputs caused increased bacterial populations. Schutter et al. (2001) found that cover crops increased molecular biomarkers for fungi. Amendment carbon complexity can also affect the soil microbial community. Schutter and Dick (2001) used fatty acid biomarkers to assess soils amended with cellulose and found these soils had elevated levels of fungal biomarkers relative to soils amended with simpler carbon substrates such as glucose or gelatin. Thus, given their influence on soil microbial community profiles, different amendments may stimulate different microbial aggregation processes. In the interest of building and maintaining good soil structure, understanding which amendments promote aggregation by fostering a favorable microbial community would be useful in developing soil amendment plans for producers.

This research examined changes in microbial community dynamics and aggregate dynamics in response to soil amendment with different organic materials. The primary hypothesis being investigated is that, relative to other amendments, amendments that stimulate greater fungal activity will also lead to greater levels of stable macroaggregates.

Project Objectives:
  1. 1) Determine the effects of organic soil amendments on the dynamics of microbial community composition and on soil aggregate formation. 2) Determine if amendment input influenced microbial community composition can be related to soil aggregate formation.


Click linked name(s) to expand
  • Elisa D'Angelo
  • Shawn Lucas
  • Mark Williams


Materials and methods:
Soil incubation

Three agricultural soils from Kentucky were used in this experiment: 1) Maury silt loam; 2) Salvisa silty clay loam; 3) Yeager sandy loam. Soils were collected to a depth of 15cm and air dried. Native soil structure was destroyed by forcing soils through a 250 µm sieve, effectively reducing all soil structure to microaggregate size. Sand particles were mixed back into the sieved soil.

Four amendment treatments were used: 1) hairy vetch (Vicia villosa Roth); 2) dairy manure; 3) vegetable compost; 4) non-amended (control). Soils were amended based on amendment C content at a rate of 0.01g amendment C / g soil.

Amended soils were incubated in microcosms at 25°C for 82 days. Moisture content was maintained to approximate field capacity (matric potential = -33kPa). Soils were sampled on incubation days 0, 5, 12, 30, 82. Upon sampling soils were immediately subjected to aggregate analysis (see below) and a subsample was stored at -20°C for microbial analyses.

Soil aggregation

Water stable aggregates were assessed via the wet sieving method of Elliot (1986). This yields 4 aggregate size classes: 1) large macroaggregates (> 2000µm), 2) small macroaggregates (250-2000µm), microaggregates (53-250µm), and the silt + clay fraction (<53µm). Formation of large macroaggregates (LMacAg) is the primary focus of this research.

Microbial parameters

Fatty acids are integral components of cell membranes. They are rapidly degraded after cell death and certain fatty acids are signature biomarkers for specific microbial groups (Table 1). These characteristics make fatty acids good indicators of the living constituents of soil microbial communities. Fatty acids were extracted from soils as ester-linked fatty acid methyl esters (FAME) using the mild alkaline saponification method described in Schutter and Dick (2000). FAMEs were analyzed via gas chromatography. A fungi to bacteria ratio (F:B) was calculated using the fungal biomarker 18:2?6c and the sum of all bacterial FAME biomarkers as described in Frostegård and Bååth (1996).

Ergosterol is a major sterol component in cell membranes of filamentous fungi and it is generally absent from higher plants. Like fatty acids, ergosterol degrades rapidly on cell death making it a useful biomarker for quantifying living fungi in an ecosystem. Ergosterol was extracted using the microwave-assisted method of Zhang et al. (2008) and analyzed via HPLC.

Research results and discussion:
Soil aggregation

Significant amendment effects on LMacAg formation were seen as early as incubation day 5 (Fig. 1). Vetch stimulated the greatest LMacAg formation in all soils. In general, manure also stimulated elevated LMacAg formation relative to the control, but this effect was consistently significant only in the Salvisa soil. The compost treatment trend (only significant on day 30 in the Yeager soil) of less LMacAg formation relative to the control is interesting. Electrical conductivity tests of the compost will be carried out to determine if it has high levels of dispersive salts.

Microbial parameters

Amendment treatments showed significant effects on soil F:B in all soils (Fig. 2). Significant effects due to amendments were seen in individual soils as early as day 5 (data not shown), however significant soil*treatment interactions were seen in the analysis of amendment effects on F:B across all soils. This indicates that amendment effects on microbial communities differ from soil to soil. In general, manure and vetch significantly increased F:B relative to the control and compost treatments. Vetch generally stimulated greater F:B in finer textured soils while the manure had the greatest F:B on days 30 and 82 in the Yeager sandy loam. The Yeager soil was a much sandier soil than the others investigated and it was a floodplain soil rather than an upland soil. The inherent members of the fungal population in this soil may be somewhat different than those found in the other soils. Prior management history may also have played a role in the difference in F:B response to amendments in this soil.

Amendment treatment showed significant effects on soil ergosterol treatment in all three soils. Soils were analyzed individually because, as seen in the F:B analysis, there were significant soil*treatment interactions. However, unlike with F:B, the same general ergosterol response trends were seen in all three soils (Fig. 3). In general, ergosterol concentrations were highest in the vetch treatments. The manure treatment also showed significantly greater ergosterol levels, relative to the control and compost treatments on day 12. In all three soils, by day 82, only the vetch treatment showed ergosterol levels that were significantly different from the other treatments. It is interesting to note that on incubation day 0 the manure treatment showed significantly greater ergosterol levels in two of the soils. This may indicate that fungi are being introduced into the soils with the amendment.

MRPP analysis indicated that microbial community FAME profiles differed significantly between amendment treatments (P<0.0001; within group agreement = 0.38). NMS ordination was able to account for 97% of the variation in microbial community structure (Fig. 4). Axis 1 of the NMS ordination explained 79% of the variability and seemed to capture the amendment treatment effect on microbial community structure, with manure and vetch at the high end of the axis and compost and the control at the low end. Axis 2 captured 18% of the variation and seemed to be loosely determined by soil texture. Generally, on axis 2 a specific amendment treatment was ordinated at the higher end of the axis in the Yeager sandy loam and at the lower end in the Salvisa silty clay loam, with the Maury silt loam falling in between.

Relationships between amendments, microbial communities, and aggregation

The distribution of signature FAMEs on the NMS ordination confirmed what was seen in the analysis of F:B and ergosterol (Fig. 4). The fungal biomarker 18:2?6c was associated with the vetch and manure treatments, as was ergosterol. All bacterial biomarkers, as well as 18:1?9, and the arbuscular mycorrhizal fungus biomarker 16:1?5 were distributed near the center of the ordination and did not show any obvious associations. When the joint plot containing the aggregation dependent variables was overlaid on the NMS ordination LMacAg was strongly related to the manure and vetch treatments as well as the 18:2?6c and ergosterol fungal biomarkers. Pearson’s correlations indicated that LMacAg was significantly related to soil 18:2?6c content (R=0.64) and ergosterol content (R=0.54).

Participation Summary

Educational & Outreach Activities

Participation Summary

Education/outreach description:

Lucas, S.T., E.M. D’Angelo, and M.A. Williams. 2014.  Improving soil structure by promoting fungal abundance with organic soil amendments. Applied Soil Ecology 75:13-23.

Poster Presentation at Soil Science Society of America Annual Meetings: Effects of Organic Amendments On Aggregation and Microbial Community Dynamics in Soils. Poster No. 104-23, Monday 1 November 2010, Long Beach Convention Center, Exhibit Hall BC, Lower Level.

Project Outcomes

Project outcomes:

These results confirm the hypothesis that amendments that stimulate greater fungal activity also promote greater large macroaggregate formation. This work indicates that vetch generally fosters the greatest levels of fungal activity and concurrently promotes the greatest amount of macroaggregate formation. Manure also promotes fungal activity and macroaggregate formation relative to non-amended soils and those amended with compost. Macroaggregate formation is strongly related to fungal activity which is driven by the amendment treatments. These results would be useful to producers who wish to build soil structure and ultimately soil quality through use of organic amendment inputs.


Areas needing additional study

Future research needs to be conducted to assess differences in microbial response to amendments in different soils. Assessments of a wider range of amendments would be beneficial as well as would comparisons within specific amendment categories (ie. Rye cover crops vs. vetch cover crops or swine manure vs. dairy manure). Field studies should be conducted to determine if the results of this highly controlled laboratory-based research carry over to the highly variable environments managed by producers.

Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture or SARE.