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)
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®
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