1992 Annual Report for AW92-008
Development and Evaluation of Indicators for Agroecosystem Health
Summary
1. Develop critical microbial tests and the Random Amplified Polymorphic DNA (RAPD) technique for determining the diversity and activity of microbial communities as affected by cropping system management, and to evaluate the use of bulked segregant analysis for the identification of microbial sequences linked to soil quality.
2. Correlate critical biological and chemical tests with the microbial indices for assessing soil quality in long-term research sites and farmer fields as affected by cropping system management.
The project on ecosystem health and soil quality indicators focused on how new approaches using molecular indicators and more conventional measurements could be used to determine how farming practices influence microbial populations and microbial diversity. Paired farms in Idaho and Washington comparing conventional rotations of wheat-pea with more complex rotations containing forage legumes and grass showed that soil from more complex rotations was generally higher in microbial indicators, microbial activity, soil organic matter, water infiltration, and pH. Cemetery sites were included to simulate native grassland sites. These same sites were used to evaluate a new technique, Random Amplified Polymorphic DNA (RAPDs), which attempt to look at diversity of extracted soil DNA to estimate the diversity of microbial communities. This technique showed that the microbial communities were different between the paired farms and the native grassland.
Findings
We have evaluated the use of Random Amplified Polymorphic DNA (RAPD) fingerprinting as a means of comparing soil microbial communities beneath various cropping systems. RAPD fingerprinting is a polymerase chain reaction (PCR) based technique that yields DNA products ranging in size from 200 bp to 2000 bp. When these products are separated on an electrophoretic gel, a banding pattern is observed that is very similar to a marketing bar code used in many stores. The RAPD fingerprint is characteristic of the target DNA and has been used to differentiate between closely related cultivars of wheat and barley, and to differentiate between closely related bacterial species. RAPD fingerprints would also be expected to be characteristic of different microbial communities, such as soil microbial communities exposed to different cropping practices. Before this technique may be applied with confidence to the evaluation of systems as complex as soil microbial communities, however, a number of preliminary studies must be conducted. With funds including those supplied by ACE, we have improved existing techniques for the isolation of DNA from soils, evaluated the composition of DNA as to the presence of prokaryotic and eukaryotic DNA, evaluated the technique in a simple model community (compost), and evaluated the technique in soils exposed to a variety of management practices.
DNA Isolation
We have found that RAPD fingerprinting is very sensitive to the presence of humic contaminants in
DNA (much more sensitive than many other PCR based techniques), and have improved protocols developed in our laboratory to reproducibly yield high molecular weight DNA that is suitable for
RAPD fingerprinting.
We are primarily interested in characterizing the bacterial, or prokaryotic, segment of soil microbial communities. The presence of fungal DNA would therefore confound our analyses. We believe that the protocol that we have developed isolates DNA primarily from prokaryotes, with little or no significant amounts of fungal DNA. The composition of the DNA was assessed by screening DNA isolated from soils with PCR primers specific to fungal DNA. No fungal DNA was detected by this approach.
Compost as A Model System
Soil microbial communities are extremely complex in nature, with potentially as many as 10,000 bacterial species per gram of soil. Before proceeding to a system this complex, we evaluated the technique in a model compost system present at Procter & Gamble, Cincinnati, OH (partial funding for this project was also supplied by Procter & Gamble). We developed the first procedure for isolation of microbial DNA from compost, and evaluated the use of RAPDs to detect shifts in the microbial community as it shifted from a thermophilic to a mesophilic community. The RAPD fingerprints were highly reproducible, attained a limit of detection of individual species of approximately 0.4 percent, and identified the predicted shifts in the microbial community.
Evaluation of RAPDs in Soils
We have shown that RAPD fingerprinting in soils can be very reproducible, yielding between 5 and 25 distinct bands. We have also shown that the limit of detection in soils is approximately 1 percent. The limit of detection in soils is higher than in the compost system and is likely to be a function of the complexity of the system; the more complex the system, the higher the limit of detection. RAPD fingerprints were generated that were characteristic of a variety of different soils, including those from a golf course, a natural grassland, and a conventional farm. In paired farm studies, specific RAPD bands were detected that were characteristic of conventional farms and of sustainable farms.
Limitations of the Use of RAPD Fingerprints to Characterize Soil Communities
The generation of RAPD fingerprints from DNA isolated from soil is technically difficult and this is the primary limitation in the technique. Students have been hired to conduct this research but have proven to have great difficulty in reproducibly obtaining useable fingerprints from soil DNA. Well-trained molecular biologists appear to be required to obtain consistently usable results.
Interpretation of data obtained by RAPD fingerprints is limited. Since these fingerprints are random, we do not know what organisms the individual bands arise from. As in the case mentioned earlier where characteristic bands were observed in conventional versus adjacent sustainable farms, we can not say what microorganisms are different in the different soils. We can only say that there are observable differences. This is not to say that RAPD fingerprinting does not have value in characterizing soil communities. In the right hands, it is a rapid and highly reproducible means of identifying differences between different soils, and perhaps may be very useful at identifying similarities or differences between sustainable operations.
Another possible limitation is the great complexity of soil. We have occasionally had difficulty obtaining reproducible fingerprints that is attributable to the great complexity of some systems (this has also been observed in fingerprinting barley chromosomes). It may be that the greatest utility of RAPD fingerprinting lies in characterizing relatively simple communities, such as compost. RAPD fingerprints could be used to identify properly versus improperly functioning compost facilities, as well as to characterize the maturity of compost.
Potential Contributions
Farmers who use green manures and more diverse rotations do so because they believe that there are long-term benefits. One of the growers who uses plow-down of clover believes that there are benefits, but current farm policies do not provide much incentive as one often loses wheat base. this particular grower also believes that the moldboard plow is the best way to turn under the residue which aids in improving soil tilth and water intake. However, current Natural Resource Conservation Service regulations normally do not allow farmers to use the moldboard as the regulations are based almost solely on surface residue. This one grower has attempted to use other options such as no-till, but the amount of green manure residue makes these practices difficult to use.
New Hypotheses
The feasibility of using soil DNA to determine microbial community structure has shown differences between farming practices. The challenge now is to determine what microbial communities these represent at the genus or species level.
Farmer Adoption
The lack of farmer adoption to using more diverse rotations, cover crops, and green manures is largely economic. Changes in the farm program which include more incentives would be a start.
Operational recommendations are to encourage more applied research and on-farm research to document the benefits of longer term rotations.
Reported in 1995