Since 1962, the public has become familiar with the phenomenon of nontarget organisms being injured by pesticides. Publication of Rachel Carson's "Silent Spring" (4) in that year led eventually to bans on DDT, which was believed partly responsible for the near extinction of such species as bald eagles and California condors.

While sympathy for eagles, condors and bluebirds is great, less public attention is paid to smaller and less-spectacular organisms. Ecologists and other biologists, however, are interested in even microscopic nontarget organisms, such as bacteria.

We have found that one fungicide product in particular has a negative effect on nontarget bacteria found in putting-green root zones at The Meadows Golf Course in Allendale, Mich. From one standpoint, this may be good news to superintendents, because some nontarget bacteria can cause disease. But other bacteria are favorable to plant growth, and inhibiting them inadvertently may have important implications for the general health of golf greens.

The value of bacteria
Bacteria are well-known recycling agents for nitrogen, sulfur, carbon and other elements in water and soil (1). As turf managers we depend, in part, on microorganisms -- including bacteria -- to break down fertilizers and soil nutrients into forms available to plants.

Microbes also play important roles in degradation of other complex molecules, including pesticides. They do all this while competing with other microbes for energy, water and nutrients.

Each type of organism has its own niche -- its own set of complex and poorly understood interactions with other organisms. Evidence suggests that lack of established bacterial populations may play a role in Pythium root rot on first-year greens (8). The prevalence of take-all patch on new golf courses and its subsequent decline over three to five years suggests that microbial populations are out of balance in disrupted soils. Necrotic ring spot has been closely associated with young Kentucky bluegrass sod, but it declines in severity over three to five years. Over time, other microbes repopulate the soil and regain a sustainable level, thus suppressing causal agents of root rot, take-all patch and necrotic ring spot.

The Meadows study
At The Meadows GC, which is affiliated with Grand Valley State University, we found the nontarget effects of fosetyl al fungicide (Aliette) while analyzing the bacterial diversity of the course's putting, green root zones.

The greens are constructed of 100 percent sand that falls within USGA particle-size recommendations. Built in 1992-93, they were treated with a commercially prepared granular sea plant meal before seeding. The greens have developed a 1-centimeter layer of (mostly) Penncross creeping bentgrass, (Agrostis palustris); this includes the verdure, mat and thatch layers.

Although roots extend into the sand, there is no appreciable organic material below the top centimeter. This sparse root zone caused us to wonder what grows there besides turfgrass roots. Our initial investigation concerned the effects of microclimate (shady vs. sunny) on the number and type of bacteria in the root zone. We wondered whether there would be differences in the bacteria found in sunnier (drier) and shadier (moister) greens. Disproving our original hypothesis, our data suggest each of the greens' root zones have similar numbers and types within a fluctuating range of 10,000 to 100,000 microorganisms per cubic centimeter of sample.

Total microbe populations in the 2- to 3-year-old greens increased to 10 million to 100 million per cubic centimeter by spring of 1995, which is within the "typical soil" range of 1 million to 100 million microorganisms per cubic centimeter.

Bacterial inhibition
What drew our attention was the significant drop in bacterial counts on June 15, 1994. Course records revealed that fosetyl al had been applied to the greens three days before the sample was taken.

After we learned that fosetyl al also is used to control some bacterial plant diseases, we wondered whether it had altered the bacterial population of the greens. We tested the most commonly isolated bacteria from the greens (using two separate and independent test methods) against fosetyl al and three other fungicides used at The Meadows. This "broth test" revealed that fosetyl al inhibits the growth of the bacteria tested. (The Streptomyces sp. was not inhibited.)

We then attempted to match field conditions more closely by placing the bacteria in a soil-sand mixture and exposing them to fosetyl al at approximately the concentration used on the golf course (4 ounces per 1,000 square feet). We sterilized a mixture of potting soil and sand, added a known bacterial mixture, incubated it for 24 hours and sprayed on the chemical. Quantitative culturing showed a decrease in population of bacteria in the soil treated with fosetyl al; however, the magnitude of inhibition was not as great as had been demonstrated in the broth test.

Statistical analysis confirmed significant differences in bacterial populations after treatment with fosetyl al, both from the greens and from the soil cultures in the laboratory.

Inhibition of bacteria is not the same as elimination of bacteria. Bacteria have a built-in guarantee that they will replenish themselves. They can reproduce very rapidly, sometimes as quickly as every half-hour. This rapid growth coupled with their ability to pick up genetic materials from other organisms allows bacteria to develop resistance to many agents. Fungicide-resistant Sclerotinia homeocarpa, the causal agent of dollar spot, is an example of the adaptability of microorganisms. With its short half-life (one day), we do not see the effect from one fosetyl al treatment to be a long-term threat to soil bacteria.

Pesticides and nontarget organisms
According to its label, fosetyl al is a systemic fungicide. It controls Pythium blight and yellow tuft (Sclerophthora macrospora) on turfgrasses. The label allows a mix with another fungicide, mancozeb, for control of the fungi that cause summer stress decline. We are attempting to determine whether treatment of greens with fosetyl al may have a beneficial side effect in suppressing Xanthomonas campestris, the agent of bacterial wilt in bentgrass. It's possible that treatment for one plant pathogen, Pythium, may actually control another, X. campestris, without lasting disturbance to the immediate ecosystem.

The scientific literature, however, includes reports of pesticides inhibiting both beneficial and disease-causing bacteria:

• Thiram, captan, benomyl and carboxin have inhibited Cyanobacteria, which are beneficial soil bacteria (3).
• Captafol has had an adverse effect on the nitrogen-fixing bacterium Rhizobium trifolii (13).
• Fourteen pesticides are inhibitory for Bacillus popillae, the agent of milky spore disease in Japanese beetle larvae (6).
• Decreases in soil bacteria treated with captan were found at high concentrations, although bacteria were less affected than fungi and actinomycetes (2).
• Propiconazole in low dosages in the field has produced side effects on microbial respiration that lasted for eight weeks (7).

The scientific literature also contains reports of bacteria inhibiting fungi. Some strains of the bacterium Pseudomonas fluorescent produce a phenazine antibiotic that inhibits the fungus Gaeumannomyces graminis tritici in laboratory tests. Some data suggest that enough is produced in natural soils to inhibit the pathogen in concentrations as low as 1 millionth of a gram per milliliter (14). Also, a study of the direct effect of metalaxyl on Phytophthora cinnamomi showed that efficacy was greater in soil containing bacteria than in sterilized soil (11). This may suggest that bacteria play a role in fungal inhibition.

Bioindicators
Use of nontarget organisms as biological indicators of environmental health is viewed as a promising alternative to chemical analysis. Biological indicators have been compared to canaries in the coal mine. One authority discusses such invertebrates as fly larvae, bees and earthworms, and such vertebrates as fish and birds as biological indicators (12). A study analyzed the influence of a single aldicarb treatment on the succession of Collembola, total mites and predacious mites and earthworms (10). Pesticide residues were not detectable after 37 weeks; however, changes in the succession of mesofauna were observed over a four-year period.

For small field research projects we do not recommend using fluctuations in bacterial populations in soil as a means of assessment of environmental impact. Enumeration of soil bacteria is, at best, an imprecise procedure, one fraught with frustrations and difficulty in controlling for variables.

Perhaps with more research, golf course superintendents will someday be able to monitor levels of bacteria and decide whether to apply chemicals with a fuller understanding of all the benefits and problems that might result.

1. Atlas, R.M., and R. Bartha. Microbial ecology: fundamentals and applications. 3rd ed. The Benjamin/Cummins Pub. Co. Inc., N.Y.
2. Banerjee, A., and A.K. Banerjee. 1987. Influence of captan on some microorganisms and microbial processes related to the nitrogen cycle. Plant and Soil 102:239-245.
3. Cameron, H.J., and G.R. Julian. 1984. The effects of four commonly used fungicides on the growth of Cyanobacter. Plant and Soil 78:409-415.
4. Carson, R. 1962. Silent Spring. Houghton Mifflin, N.Y.
5. Casida, L.E. 1968. The ecology of soil bacteria. University of Toronto Press. Toronto, Ont.
6. Dingman, D. 1994. Inhibitory effect of turf pesticides on Bacillus popillae and the prevalence of milky disease. Applied and Environmental Microbiology 60:2343-2349.
7. Elmholt, S. 1992. Effects of propiconazole on substrate amended soil respiration following laboratory and field application. Pesticide Science 34:139-146.
8. Hodges, C.F. 1994. The persistent puzzle of Pythium root rot. Golf Course Management 62(5):62-95.
9. Kenna, M.P. 1995. What happens to pesticides applied to golf courses? USGA Green Section Record 33:1-9.
10. Koehler, H.H. 1992. The use of soil mesofauna for judgment of chemical impact on ecosystems. Agriculture, Ecosystem and Environment 40:193-205.
11. Malajczuk, N., T.J. Boughton, N.A. Campbell, and R.T. Litchfield. 1983. Interaction between the fungicide metalaxyl and soil microorganisms on survival of Phytophthora cinnamomi. Ann. Appl. Biol. 103:57-61.
12. Root, M. 1990. Biological monitors of pollution. Bioscience 40:83-86.
13. Ruiz-Sainz, J.E., J.E. Beringer, and A.M. Gutierrez-Navarro. 1984. Effect of the fungicide captafol on the survival and symbiotic properties of Rhizobium trifolii. J. Appl. Bacteriol. 170:3499-3508.
14. Thomashow, L.S., and D.M. Weller. 1988. Role of a phenazine antibiotic from Pseudomonas fluorescens in biological control of Gaeumannomyces graminis var. tritici. Journal of Bacteriology 170:3499-3508.

We wish to thank our students who assisted in specimen collection and processing, and Timothy Lesnick, Ph.D., Grand Valley State University, for the statistical analysis. Special gratitude is expressed to Joseph Vargas, Ph.D., Michigan State University, for his advice, support and assistance in pointing our investigation in a new and interesting direction.

Kathy Antaya is golf course superintendent at The Meadows GC in Allendale, Mich., which is affiliated with Grand Valley State University. Johnine Callahan, Ph.D., is professor of microbiology at Grand Valley State University.