BIOLOGICAL ACTIVITY OF SOIL UNDER ROUNDUP ® READY SOYBEAN PRODUCTION

1. BIOLOGICAL ACTIVITY OF SOIL UNDER ROUNDUP® READY SOYBEAN PRODUCTION Nathan Means Research Assistant – Graduate Student Environmental Soil Science Program University of Missouri

2. WHAT IS GLYPHOSATE? O O OH– C– CH2– NH2–CH2–P–O¯ OH amine + carboxylate phosphonate • Glyphosate N-(phosphonomethyl)glycine active ingredient in Round-Up®, best selling herbicide world-wide • Round-Up® is a nonselective systemic herbicide • In 2002, 75% of the soybeans in the United States were genetically modified for glyphosate resistance (ISAAA, 2002) Round-UP®

3. COO– COO– O H H OH H CH2 OH O P O C— C OO– P H H OH H GLYPHOSATE IS A COMPETITIVE INHIBITOR 5-EPSP SYNTHASE 5-enolpyruvylshikimic acid-3-phosphate (EPSP) synthase PEP Shikimate 3-phosphate Pi 5-Enolpyruvylshikimate 3-phosphate

4. COO- COO- COO- H3N+– C – H H3N+– C – H H3N+– C – H C=CH CH2 CH2 CH2 NH Phenylalanie Tryptophan OH Tyrosine AROMATIC AMINO ACID PRODUCTION IS STOPPED • Phytoalexins Antimicrobial, low-molecular-weight secondary metabolites capable of stopping pathogen development (Hammerschmidt 1999) • Infection by a pathogen induces accumulation of phytoalexins in plants at the infection site (Hammerschmidt 1999)

5. GLYPHOSATE IS SYSTEMIC • Glyphosate is translocated in the symplast and accumulates in roots and meristematic regions (Duke, 1988, Hernandez et al., 1999)

6. GLYPHOSATE IN THE RHIZOSPHERE • Low levels of glyphosate in the rhizosphere of treated plants(Coupland and Caseley 1979 and Rodrigues et al., 1982) Figure from Neumann and Romheld, 2002 in Waisel et al. (eds.) Plant Roots The Hidden Half. Marcel-Dekker.

7. GLYPHOSATE IN SOIL • Low levels of glyphosate appear in the rhizosphere of treated plants (Coupland and Caseley 1979, and Rodrigues et al., 1982) • Glyphosate is rapidly and tightly adsorbed to soil and has shown little potential for runoff and mobility (Vencill 2002) • Inorganic soil materials (clay minerals and metallic cations) impact bioactivity of glyphosate (Weber et al., 1993 and Eberback 1998)

8. O O OH—C—CH2—NH—CH2—P—OH OH FOOD OR POISON FOR THE MICROBES? • Nutrient source 3:1:1 (Haney et al., 2000, Busse et al., 2001, and Vencill 2002) • Glyphosate has been shown to be toxic to bacteria and fungi (Hendricks and Rhodes 1992, Moorman et al., 1992, Wardel et al., 1992, Kawate et al., 1992, Lévesque and Rahe, 1992) Glyphosate

9. SOIL MICROORGANISMS, WHO CARES? • Rhizosphere microorganisms include bacteria, fungi, protozoa, etc…. • These organisms influence • plant-available nutrients and transformations • incorporation of nutrients into biomass • soil structure, reducing leaching and erosion while improving water flow • nitrogen fixation • decompose complex carbon compounds and certain types of pollutants (Myrold 1998) • A healthy soil microbial community is paramount to soil quality

10. IMPACTS OF GLYPHOSATE • Shift in rhizosphere microbial community • nutrient cycling • disease incidence • plant growth and crop yield • Reductions in nodulation, leghemoglobin, chlorophyll in soybean under stress (King & Purcell 1998; Reddy et al. 2000) • Increased severity of “take-all” disease in winter wheat crop following Roundup Ready soybean (Indiana) - caused by soilborne fungal pathogen -(Hickman et al. 2002. Abstr. Weed Sci. Soc. Am., p.7)

11. ROUNDUP READY SOYBEAN, WESTERN ILLINOIS, 2001

12. FOLIAR AMENDMENTS • Used in both horticultural and field crop production • Claim to enhance crop growth and yield by reducing plant stress and increasing nutrient availability (Kinnersley 1993) • Effects on plant growth are thought to be achieved through • inoculation of soil with selected microorganisms • activation of soil microbial activity • promotion or augmentation of the activities of critical soil enzymes, chelating substances, plant growth hormones, or supplementation of micronutrients (Kinnersley 1993)

13. REMEDIATION OF EFFECTS? • Foliar Amendments • PT-21® - 21% Nitrogen in Urea form • Grozyme® - Boron, Cobalt, Copper, Iron, Manganese, Molybdenum, Zinc and “enzyme systems” • PT-21® and Grozyme® have been shown to improve soybean growth and production by affecting soil microbial activity(Chen et al. 2003)

14. OBJECTIVES • Determine effects of glyphosate on root colonization and soil populations of Fusarium spp. and rhizosphere microbial activity on RR soybean. • Determine if foliar amendments can offset the impact of glyphosate on Fusarium spp. populations and microbial activity in the rhizosphere of soybean. • Determine effects of foliar amendments on microbial activity in the rhizosphere of RR soybean with and with out glyphosate application.

15. HYPOTHESES • Glyphosate applied to soybean has no effect on rhizosphere microorganisms. • There is no interaction between foliar amendments and glyphosate on soybean rhizosphere microorganisms. • Glyphosate and foliar amendments do not effect Fusarium spp. populations in the rhizosphere of soybean.

16. FIELD EXPERIMENT • DeKalb DKB38-52 (RR®) planted in 76 cm rows on a Mexico silt loam (fine, montmorillonitic, mesic Mollic Endoaqualf) prepared by light tillage with a disk harrow, fertilized and managed consistent to practices common in Missouri. • Experimental design - randomized complete block with a split-block arrangement for herbicide application and four replications.

17. TREATMENTS Split Block Treatments • Roundup Ultra Max® (0.84 kg a.e.·ha rate-1) • Reflex 2LC® (0.42 kg·ha -1) + Select 2EC® (0.175 kg·ha-1) with 1.101 L crop oil concentrate Treatments • PT-21® - 21% N in Urea form (9.2 kg·ha-1) • Grozyme® (Gro) - enzymes, micronutrients (33.5 mL·ha-1) • PT-21® (9.2 kg·ha-1) + Grozyme® (33.5 mL·ha-1) • Control

18. SAMPLING • Plant roots and attached soil were collected. Loosely adhering soil on the roots was used for enzyme assays and soil fungi counts. Roots were used for determination of colonization of Fusarium spp. • Three samples (roots + soil) per plot were taken 0, 5, 10, 15, 20 and 25 days after glyphosate application and mixed to generate a composite sample.

19. ANALYSES • Soil dehydrogenase activity - activity of viable microorganisms (Bauer et al. 1991, Casida et al. 1964, and Tabatabai 1994) • Soil β-Glucosaminidase - enzymatic indicator of soil enzyme activity, C and N cycling (Parham and Deng 2000) • Fusarium spp. rhizosphere and root populations were estimated using Komada medium (Mekwatanakarn, P. and K. Sivasithamparam 1987) • Rhizosphere fungal populations were estimated using Martin’s Rose Bengal Agar • Soil Respiration - substrate induced CO2 production

20. WEATHER DATA AND RESULTS

21. SOIL FUSARIUM -2002 CFU·g dry soil-1 HERB = Herbicide Tank-mix RU = Roundup®

22. ROOT FUSARIUM - 2002 CFU·100cm root -1 Herb = Herbicide Tank-mix RU = Roundup®

23. FUSARIUM COLONIZATION ON ROOTS DAY 25 2003 RoundUp® Tank-mix Herbicide

24. TOTAL SOIL FUNGI - 2002 Herb = Herbicide Tank-mix RU = Roundup® CFU·g dry soil -1

25. SOIL FUNGI DAY 25 2003 PT-21® RU® PT-21® Tank-mix Herbicide

26. DEHYDROGENASE ACTIVITY Herb = Herbicide Tank-mix RU = Roundup® TPF · g dry soil -1

27. ß-GLUCOSAMINIDASE Ρ-nitrophenol·g dry soil-1 Herb = Herbicide Tank-Mix RU = Roundup®

28. SUBSTRATE INDUCED RESPIRATION RU=Roundup® Herb=Herbicide Tank-mix Carbon Dioxide (ppm)

29. PRELIMINARY CONCLUSIONS • Roundup® application increased soil Fusarium spp. populations and root colonization. • PT-21® and Grozyme® reduced Fusarium spp. populations and root colonization associated with Roundup® application. • Roundup® application affects enzymatic soil microbial activity.

30. FUTURE WORK • Similar trials with Roundup Ready® corn • Greenhouse study focusing on soil moisture interactions with Roundup® application and soil microorganisms

31. ACKNOWLEDGEMENTS • Dr. Robert Kremer • Ag Spectrum • USDA Special Grant - SCN • Soil Microbiology Lab at the University of Missouri - Heidi, Atim, Michael, Sara • Tim Reinbott and Staff at Bradford Farm