BIOLOGICAL ACTIVITY OF BIOLOGICAL ACTIVITY OF SOIL UNDER ROUNDUPSOIL UNDER ROUNDUP®®

READY SOYBEAN READY SOYBEAN PRODUCTIONPRODUCTION

Nathan MeansResearch Assistant – Graduate StudentEnvironmental Soil Science Program

University of Missouri

WHAT IS GLYPHOSATE?WHAT IS GLYPHOSATE?

• 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®

O O

OH– C– CH2– NH2–CH2–P–O¯

OH

+

carboxylate

amine

phosphonate

GLYPHOSATE IS A GLYPHOSATE IS A COMPETITIVE COMPETITIVE

INHIBITOR 5-EPSP INHIBITOR 5-EPSP SYNTHASESYNTHASE

OH

OH

COO–

H

HH

OP

Shikimate 3-phosphate

O

OH

COO–

H

HH

OP C— C OO–

CH2

5-Enolpyruvylshikimate 3-phosphate

PEP

Pi

5-enolpyruvylshikimic acid-3-phosphate (EPSP) synthase

AROMATIC AMINO ACID AROMATIC AMINO ACID PRODUCTION IS PRODUCTION IS

STOPPED 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)

H3N+– C – H

COO-

CH2

Phenylalanie

H3N+– C – H

COO-

CH2

OH

Tyrosine

H3N+– C – H

COO-

CH2C=CH

NH

Tryptophan

GLYPHOSATE IS GLYPHOSATE IS SYSTEMICSYSTEMIC

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

GLYPHOSATE IN THE GLYPHOSATE IN THE RHIZOSPHERERHIZOSPHERE

• 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 RootsThe Hidden Half. Marcel-Dekker.

GLYPHOSATE IN SOILGLYPHOSATE 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)

FOOD OR POISON FOR FOOD OR POISON FOR THE MICROBES?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)

O O

OH—C—CH2—NH—CH2—P—OH

OH

Glyphosate

SOIL SOIL MICROORGANISMS, MICROORGANISMS,

WHO CARES?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

IMPACTS OF IMPACTS OF GLYPHOSATEGLYPHOSATE

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)

ROUNDUP READY ROUNDUP READY SOYBEAN, WESTERN SOYBEAN, WESTERN

ILLINOIS, 2001ILLINOIS, 2001

FOLIAR AMENDMENTSFOLIAR 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)

REMEDIATION OF REMEDIATION OF EFFECTS?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)

OBJECTIVESOBJECTIVES

Determine effects of foliar amendments on microbial activity in the rhizosphere of RR soybean with and with out glyphosate application.

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 glyphosate on root colonization and soil populations of Fusarium spp. and rhizosphere microbial activity on RR soybean.

HYPOTHESESHYPOTHESES

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.

FIELD EXPERIMENTFIELD EXPERIMENT

• DeKalb DKB38-52 (RRDeKalb DKB38-52 (RR®®) planted in 76 cm rows on a Mexico silt ) planted in 76 cm rows on a Mexico silt loam (fine, montmorillonitic, mesic Mollic Endoaqualf) prepared loam (fine, montmorillonitic, mesic Mollic Endoaqualf) prepared by light tillage with a disk harrow, fertilized and managed by light tillage with a disk harrow, fertilized and managed consistent to practices common in Missouri. consistent to practices common in Missouri.

• Experimental design - randomized complete block with a split-Experimental design - randomized complete block with a split-block arrangement for herbicide application and four block arrangement for herbicide application and four replications. replications.

TREATMENTSTREATMENTS

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

SAMPLINGSAMPLING

• 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.

ANALYSESANALYSES• 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

WEATHER DATA AND WEATHER DATA AND RESULTSRESULTS

2002 2003

Precipitation 21.3 cm 7.5 cm

Day 0 July 22 July 1

SOIL FUSARIUM -2002SOIL FUSARIUM -2002C

FU

·g d

ry s

oil

-1

0

3000

6000

9000

0 5 10 15 20 25

DAYS AFTER HERBICIDE APPLICATION

HERBRU

0

3000

6000

9000

0 5 10 15 20 25

HERBRUPT21+RUPT21+HERBGRO+RUGRO+HERBGRO+PT21+RUGRO+PT21+HERB

HERB = Herbicide Tank-mixRU = Roundup®

ROOT FUSARIUM - 2002ROOT FUSARIUM - 2002

0

50

100

150

0 5 10 15 20 25

HerbicideRUGro+PT21+HerbGro+PT21+RUGro+HerbGro+RUPT21+HerbPT21+RU

0

50

100

150

0 5 10 15 20 25

DAYS AFTER HERBICIDE APPLICATION

HerbicideRU

CFU

·100cm

root

-1

Herb = Herbicide Tank-mixRU = Roundup®

FUSARIUM FUSARIUM COLONIZATION ON COLONIZATION ON

ROOTS DAY 25 2003ROOTS DAY 25 2003

RoundUp® Tank-mix Herbicide

TOTAL SOIL FUNGI - TOTAL SOIL FUNGI - 20022002

0

5000

10000

15000

20000

25000

0 5 10 15 20 25

DAYS AFTER HERBICIDE APPLICIATION

HerbicideRU

CFU

·g d

ry s

oil -

1

0

5000

10000

15000

20000

25000

0 5 10 15 20 25

GRO+PT21+HERB

GRO+PT21+RU

PT21+HERB

PT21+RU

GRO+HERB

GRO+RU

HERB

RU

Herb = Herbicide Tank-mixRU = Roundup®

SOIL FUNGI DAY 25 SOIL FUNGI DAY 25 20032003

PT-21® RU® PT-21® Tank-mix Herbicide

DEHYDROGENASE DEHYDROGENASE ACTIVITYACTIVITY

0

1

2

3

0 5 10 15 20 25

Herb

RU

Gro+PT21+Herb

Gro+PT21+RU

Gro+Herb

Gro+RU

PT21+Herb

PT21+RU

0

1

2

3

0 5 10 15 20 25

DAYS AFTER HERBICIDE APPLICATION

HerbRU

TP

F ·

g d

ry s

oil -

1

Herb = Herbicide Tank-mixRU = Roundup®

ß-GLUCOSAMINIDASEß-GLUCOSAMINIDASE

0

50

100

150

200

0 5 10 15 20 25

HerbRUGro+PT21+HerbGro+PT21+RUGro+HerbGro+RUPT21+HerbPT21+RU

0

50

100

150

200

0 5 10 15 20 25

DAYS AFTER HERBICIDE APPLICATION

HerbRU

Ρ-n

itro

ph

enol·g d

ry s

oil-

1

Herb = Herbicide Tank-MixRU = Roundup®

SUBSTRATE INDUCED SUBSTRATE INDUCED RESPIRATIONRESPIRATION

0

200

400

600

800

0 5 10 15 20 25

Gro+PT21+HerbGro+PT21+RUHerbRUPT21+HerbPT21+RUGro+HerbGro+RU

0

200

400

600

800

0 5 10 15 20 25

DAYS AFTER HERBICIDE APPLICATION

HerbRU

Carb

on

Dio

xid

e (

pp

m)

Carb

on

Dio

xid

e (

pp

m)

RU=Roundup®

Herb=Herbicide Tank-mix

PRELIMINARY PRELIMINARY CONCLUSIONSCONCLUSIONS

• 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.

FUTURE WORKFUTURE WORK

• Similar trials with Roundup Ready® corn

• Greenhouse study focusing on soil moisture interactions with Roundup® application and soil microorganisms

ACKNOWLEDGEMENTSACKNOWLEDGEMENTS

• 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