= 1 µm

1. Relationship of Glyphosate Application and Foliar Amendment on IAA-Producing Bacteria and Microbial Activity in the Rhizosphere of Glyphosate-Resistant Soybean = 1µm Su-Jung Kim Department of Soil, Environmental, and Atmospheric Sciences

2. Outline • Introduction - Characteristics of Herbicide Glyphosate - Foliar Amendments - Deleterious Rhizobacteria (DRB) - Indole-3-acetic acid (IAA) Effects of DRB on Weed Growth • Hypotheses and Objectives • Methods and Materials (Experimental Design) • Results • Conclusions

3. Glyphosate or N-(phosphonomethyl)glycine • A broad spectrum, non-selective postemergence herbicide of grasses and broadleaf weeds • The mode of action of glyphosate - inhibition of 5-enolpyruvylshikimic acid-3-phosphate (EPSP) synthase • Roundup®is the most widely used herbicide (glyphosate is the active ingredient), produced by Monsanto corporation.

4. Glyphosate in Plants • Glyphosate is not readily metabolized by plants; it is translocated and may accumulate in meristematic regions including roots and nodules (Duke, 1988; Hernandez et al., 1999, Reddy et al., 2000). • Glyphosate that accumulates in the roots of treated susceptible plants is eventually released into the rhizosphere (Coupland and Casely, 1979; Rodrigues et al., 1982).

5. Effects of Glyphosate on Soil Microorganisms • Glyphosate increases soil bacteria and fungi populations (Wardel and Parkinson, 1992; Abdel-Mallek et al., 1994; Lévesque et al., 1987; Haney et al., 2000; Busse et al., 2001). • Glyphosate may be toxic to some bacteria and fungi possibly due to inhibition of microbial metabolic pathways (Mekwatanakarn and Sivasithamparam, 1987; Kawate et al., 1992; Abdel-Mallek et al., 1994; Busse et al., 2001).

6. Foliar Amendments • Biostimulants increase plant growth through various mechanisms - Establishment of selected microorganisms in soil - Enhancement of soil microbial activity - Promotion or augmentation of the activities of critical soil enzymes, providing chelating substances and plant growth hormones, or supplementation of micronutrients (Kinnersley, 1993).

7. Foliar Amendments • Benefits to soil fertility - Directly soil organic fraction by microorganisms - Indirectly microbially synthesized metabolites such as phytohormones (Yamada and Xu, 2000). • Furthermore, increase water stress resistance in plants (Yamada et al., 1997; Huilian et al., 1998).

8. Foliar Amendments • PT-21® (AgSpectrum, DeWitt, Iowa) - A nutritional supplement (21.0% total nitrogen in the form of urea) - Designed for foliar application to increase crop yield. • Grozyme® (AgSpectrum, DeWitt, Iowa) - A biostimulant (boric acid, cobalt sulfate, copper sulfate, ferric nitrate, manganese nitrate, sodium molybdate, and zinc nitrate) - Effects on rates of organic matter decomposition, soil microbial activity and mineralization.

9. Importance of Soil Microorganisms • In nutrient cycling, decomposition, and plant growth. - Management practices influencing soil microorganisms can affect crop yields and soil and environmental quality. • However, foliar application of Roundup®, Grozyme® and PT-21® and the potential impact on rhizosphere bacteria have not been studied.

10. Deleterious Rhizobacteria (DRB) • Naturally-occurring soil bacteria inhabiting rhizosphere • Toxigenic but not parasitic for plants • Species specific • Suppress weed growth; not affect crop growth Biological weed control (Aldrich and Kremer, 1997; Kremer, 1987)

11. Rhizosphere (Sylvia et al., 1998)

12. Biological Weed Control • Intentional use of living organisms for control of weeds (Quimby and Birdsall, 1995) • Biotic agents: foliar or stem fungal pathogens, foliar bacterial pathogens and non-pathogenic soil-borne fungi and bacteria (Kremer, 2002) - Production of antibiotics, siderophores, and volatile compounds - Parasitism, competition for nutrients and ecological niche - Production of plant growth-promoting compounds, such as IAA, gibberellin, and cytokinin-like substances

13. An auxin, Indole-3-acetic acid (IAA), produced by Bacteria • Indole-3-acetic acid - Compounds that stimulate plant growth (coleoptile tissue) in lower concentrations. - In contrast, if the concentration becomes higher, the effect reverses and elongation of root and shoot is inhibited. • Bacteria synthesize IAA (IAA-Producing Bacteria) - Inhibit root growth in sugarbeet, blackcurrant, and morningglory

14. IAA-Producing Bacteriain Relationship of IAA with herbicides • Natural auxins have modes of action similar to many herbicides that interfere with plant growth such as 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) (Patten and Glick, 1996) • The degree of growth inhibition in glyphosate-treated plants correlated with an increase in IAA metabolism (Lee, 1984; Lee and Dumas, 1985).

15. IAA-Producing Bacteria in Relationship of IAA with Rhizobacteria • Up to 80% of rhizobacteria can produce IAA (Loper and Schroth, 1986). • The inhibitory effect of some DRB (e.g., Enterobacter taylorae, a transgenic rhizosphere pseudomonad, and Pseudomonas putida) has been related to excretion of high amounts of IAA (Dubeikovsky etal, 1993; Sarwar and Kremer, 1995; Xie et al., 1996)

16. LSD (P≤0.05) IAA Production of B. japonicum isolate GD3 and B. megaterium isolate GP4 and Suppressive Effect of isolate GD3 on Morningglory Growth (Kim and Kremer, 2005 in Press)

17. Hypotheses • Glyphosate released from glyphosate-resistant soybean may be toxic for rhizosphere microorganisms including IAA-producing bacteria. • Accordingly, microbial activity may be suppressed in the rhizosphere. • Foliar amendments may change in microbial activity in combination with glyphosate application. • Total C and N, soil respiration (CO2 efflux), and soil inorganic N mineralized may be influenced.

18. Objectives • To describe changes in IAA-producing bacteria populations in the rhizosphere of glyphosate-resistant soybean (Glycine max, ‘Roundup Ready’). • To determine microbial activity through assessing C and N mineralization in the rhizosphere of glyphosate-resistant soybean.

19. Methods and Materials - Field • At Bradford Agronomy Center of the University of Missouri-Columbia • Soil classified as a Mexico silt loam (fine, smectitic, mesic, aeric, Vertic Epiaqualf) • Roundup Ready soybean (Pioneer 94B01, RR soybean) planted, Roundup (RU) applied at prebloom stage, and 2 foliar amendments (Urea Solution and Biostimulant) applied at 10 days after RU application

20. Experimental Design - Field • Spilt-split block design arranged in completely randomized blocks with 4 replications • Soil and plant samples taken prior to glyphosate application and 10, 20 and 30 days after glyphosate application

21. Methods and Materials • Culture conditions for IAA-Producing Bacteria - Rhizobacteria from RR soybean cultured on half strength King’s B medium for 24-h and colonies counted. - All counted plates were screened for IAA production using an in situ membrane assay (Bric et al., 1991).

22. Methods and Materials No Glyphosate Glyphosate

23. Methods and Materials • Total C and N were measured with a Truspec® C and N Determinator. • Soil Respiration (CO2 efflux) - The incubation was conducted for 7 days with 5g of soil sample added 1ml of 5% glucose solution. - CO2 efflux was measured with a Buck Scientific Model 910 gas chromatography via thermal conductivity detector (TCD).

24. Methods and Materials • Soil inorganic N mineralized (NO3- and NH4+) was measured with Lachat ion analyzer (Zellweger Analytics, 1992, 1993). • Urease activity was estimated (Kandeler and Gerber, 1988).

25. Methods and Materials Truspec® C/N Determinator Buck Scientific Model 910 gas chromatogr-aphy Lachat Quikchem Automated ion analyzer

26. Results • Selected chemical characteristics of Mexico silt loam at the field site

27. NS NS NS NS NS Cumulative CO2 efflux for 7-d Vertical bars indicate LSD (P≤0.05).

28. NS NS NS NS NS NS Total Rhizobacteria and IAA-Producing Bacteria Populations

29. NS NS NS NS Total Rhizobacteria and IAA-Producing Bacteria Populations

30. NS NS Total Organic C

31. NS NS NS NS Total N and inorganic N mineralized

32. NS NS Urease Activity

33. Summary • Glyphosate application followed by urea solution decreased soil CO2 efflux; however, glyphosate only application increased soil CO2 efflux from 20 to 30 days. • Urea solution without glyphosate application was higher than any other treatments from day 20 to 30; however, not significantly different from no glyphosate treatment.

34. Summary • Total rhizobacteria and IAA-producing bacteria populations were generally inhibited by application of glyphosate and significantly inhibited at day 20. • Biostimulant application increased total rhizobacteria and IAA-producing bacteria populations from day 20 to 30, irrespective of glyphosate application.

35. Summary • The lowest levels of total N and inorganic N were in soils treated with glyphosate. • Glyphosate and urea application considerably decreased total N; however, soil inorganic N was higher than any other applications. • Urease activity of soils treated with glyphosate was considerably lower than non-treated glyphosate soils.

36. Conclusions • Glyphosate-resistant soybean may modify the bacterial composition and activity in the rhizosphere to a limited extent. • These changes may impact crop productivity and soil biological processes.

37. Acknowledgements • Dr. Robert J. Kremer • Dr. Mark Ellersieck • Neal Bailey • USDA Special Grant-SCN