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

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

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.

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).

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).

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).

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).

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.

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.

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)

Deleterious Rhizobacteria (DRB)

Rhizosphere

(Sylvia et al., 1998)

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

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

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).

IAA-Producing Bacteriain Relationship of IAA with herbicides

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)

IAA Production of B. japonicum isolate GD3 and B. megaterium isolate GP4 and Suppressive Effect of isolate GD3 on Morningglory Growth

Treatments IAA ( M)

Bacillus megaterium isolate GP4 0.276 b

Bradyrhizobium japonicum isolate GD3

64.015 a

Days after Inoculation

1 2 3 4 5 6 7 8

Ro

ot

Elo

ng

atio

n (

cm)

0

2

4

6

8

10

12

14

16

18

20

22

CheckIsolate GP4Isolate GD3

LSD (P≤0.05)

(Kim and Kremer, 2005 in Press)

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.

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.

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

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

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).

Methods and Materials

No Glyphosate

Glyphosate

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).

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).

Methods and Materials

Truspec® C/N Determinator

Buck Scientific Model 910 gas chromatogr-aphy

Lachat Quikchem Automated ion analyzer

Results

Selected chemical characteristics of Mexico silt loam at the field site

Treatment pHs N.A.Organi

c Matter

P Ca Mg K CEC

meg 100g-

1 % Kg ha-1 meg 100g-1

Glyphosate 6.6 1.0 3.5 27 8130 830 260 22.5

Biostimulant

6.6 1.0 3.3 24 7300 740 270 20.3

Urea Solution

6.6 1.0 3.7 20 7820 820 230 21.8

Cumulative CO2 efflux for 7-d

Days after Herbicide Application

-5 0 5 10 15 20 25 30 35

CO

2 e

fflu

x (

mg

C k

g-1 s

oil)

800

820

840

860

880

900

920

No GlyBio US

NSNS NS

Days after Herbicide Application

-5 0 5 10 15 20 25 30 35

750

800

850

900

950

1000

GlyGly+BioGly+US

NS NS

Vertical bars indicate LSD (P≤0.05).

Total Rhizobacteria and IAA-Producing Bacteria Populations

Days after Herbicide Application

-5 0 5 10 15 20 25 30 35

Lo

g C

FU

g-1

fre

sh

ro

ot

0

2

4

6

8

10

12

14

No Gly - Total BacteriaNo Gly - IAA Producing BacteriaGly - Total BacteriaGly - IAA-Producing Bacteria

NS

NS

NSNS

NSNS

Total Rhizobacteria and IAA-Producing Bacteria Populations

Days after Herbicide Application-5 0 5 10 15 20 25 30 35

To

tal R

hiz

ob

ac

teri

a P

op

ula

tio

n

(Lo

g C

FU

g-1

fre

sh

ro

ot)

4

6

8

10

12

14

No GlyUSBioGlyGly+USGly+Bio

NS

NS

Days after Herbicide Application-5 0 5 10 15 20 25 30 35IA

A-P

rod

uci

ng

Bac

teri

a P

op

ula

tio

n(L

og

CF

U g

-1 f

res

h r

oo

t)

4

6

8

10

12

14

No GlyUSBioGlyGly+USGly+Bio

NS

NS

Total Organic C

Days after Herbicide Application-5 0 5 10 15 20 25 30 35

To

tal o

rga

nic

C (

g C

kg

-1 s

oil)

12

13

14

15

16

17

18

No GlyGlyUSBioGly+USGly+Bio

NS NS

Total N and inorganic N mineralized

Days after Herbicide Application-5 0 5 10 15 20 25 30 35

To

tal N

(g

N k

g-1

so

il)

0

5

10

15

20

25

30

No GlyUSBioGlyGly+USGly+Bio

NS NS

Days after Herbicide Application-5 0 5 10 15 20 25 30 35

So

il in

org

anic

N (

mg

N k

g-1

so

il)

0

5

10

15

20

25

30

No GlyUSBioGlyGly+USGly+Bio

NS NS

Urease Activity

Days after Herbicide Application-5 0 5 10 15 20 25 30 35

Ure

as

e a

cti

vit

y

(g

NH 4

+-

N g

-1 d

ry s

oil 2

hr

-1)

10

20

30

40

50

60

No GlyUSBioGlyGly+USGly+Bio

NS NS

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.

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.

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.

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.

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

Acknowledgements