interactions of glyphosate and foliar amendments with iaa

1
Increased use of glyphosate-resistant crops has raised concerns regarding the potential environmental impact of glyphosate. Furthermore, other foliar-applied chemicals may impact soil microbial ecology. Biostimulants (products containing plant hormones) and liquid fertilizers affect soil microorganisms by providing additional nutrients or growth factors that alter metabolic activity and improve crop growth and productivity. For example, Grozyme® and PT-21® are foliar amendments used to increase crop yield. Metabolites implicated in growth-suppressive activity include hydrogen cyanide, phytohormones such as indole- 3-acetic acid (IAA), and unidentified phytotoxins. The objectives of this study were to describe changes in IAA synthesis and urease activity of rhizosphere bacteria of glyphosate-resistant soybean (Glycine max , ‘Roundup Ready’ soybean). Introduction Interactions of Glyphosate and Foliar Amendments with IAA Synthesis and Urease Activity by Rhizobacteria of Glyphosate-Resistant Soybean Su-Jung Kim 1 and Robert J. Kremer 1,2 1 University of Missouri-Columbia and 2 USDA-ARS, Cropping Systems and Water Quality Research Unit Glyphosate or N- (phosphonomethyl)glycine References Objectives Figure 2. Average total rhizobacteria population with days after glyphosate application (DAA) and (A) no foliar amendments applied, (B) with biostimulant (Bio) applied, or (C) with urea solution (US) applied during the cropping season. Vertical bars indicated LSD (o.o5). NS is not significant. Busse, M.D., A.W. Ratcliff, C.J. Shestak, and R.F. Powers. 2001. Glyphosate toxicity and the effects of long-term vegetation control on soil microbial communities. Soil Biol. Biochem. 33:1777-1789. Haney, R.L., S.A. Senseman, F.M. Hons, and D.A. Zuberer. 2000. Effect of glyphosate on soil microbial activity and biomass. Weed Sci. 48:89-93. Kim, S.-J. and R.J. Kremer. 2005 Scanning and transmission electron microscopy of root colonization of morningglory (Ipomoea spp.) seedlings by rhizobacteria. Symbiosis 39:117-124. Kremer, R.J., N. Means, S.-J. Kim. 2005. (In Press). Glyphosate affects soybean root exudation and rhizosphere microorganisms. Int. J. Environ. Anal. Chem. Leinhos, v. and O. Vacek. 1994. Biosynthesis of auxins by phosphate- solubilizing rhizobacteria from wheat and rye. Microbiol. Res. 149:31-35. Patten, C.L. and B.R. Glick. 1996. Bacterial biosynthesis of indole-3-acetic acid. Can. J. Microbiol. 42:207-220. Reddy, K.N., R.E. Hoagland, and R.M. Zablotowicz. 2000. Effect of glyphosate on growth, chlorophyll, and nodulation in glyphosate-resistant and susceptible soybean ( Glycine max) varieties. J. New Seeds 2:37-52. Tabatabai, M.A. 1994. Soil enzymes. p. 775-833. In R.W. Weaver et al., (ed) Methods of Soil Analysis, Part 2. Microbiological and Biochemical Properties. Soil Science Society of America. Madison, WI. 64.02 a Bradyrhizobium japonicum isolate GD3 0.28 b Bacillus megaterium isolate GP4 IAA (m M) Treatments To describe changes in IAA synthesis and urease activity of rhizosphere bacteria of glyphosate-resistant soybean (Glycine max , ‘Roundup Ready’ soybean). Days after Inoculation 1 2 3 4 5 6 7 8 Root Elongation (cm) 0 2 4 6 8 10 12 14 16 18 20 22 Check Isolate GP4 Isolate GD3 LSD (P•0.05) Up to 80% of rhizobacteria can produce IAA (Leinhos and Vacek, 1994). Various microbes in the rhizosphere produce an auxin, IAA, identical to that found in plants. Figure 1. (A) Root elongation of morningglory seedlings inoculated by B. megaterium isolate GP4 and B. japonicum isolated GD3 in the hydroponic system, and (B) SEM of root tissue of morningglory seedlings seven days after inoculation. Root surface of B. japonicum isolate GD3. Examples of distinct fibrillar material released by bacteria are denoted by arrows . (Kim and Kremer, 2005) Are IAA metabolites produced by rhizobacteria plant-growth promoting or suppressive ? Why is microbial urease activity important? An indicator to estimate urea hydrolysis involved in C and N mineralization of rhizosphere microorganisms. Catalyze the hydrolysis of urea to CO2 and NH3 with a reaction mechanism based on the formation of carbamate as an intermediate (Tabatabai, 1994). A broad spectrum, non-selective postemergence herbicide of grasses and broadleaf weeds. The mode of action of glyphosate - inhibition of 5-enopyruvylshikimic acid-3-phosphate (EPSP) synthase. Roundup® is the most widely used herbicide (glyphosate is the active ingredient), produced by Monsanto corporation. Glyphosate in plants Not likely metabolized by plant; it is translocated and may accumulate in meristematic regions including roots and nodules (Reddy et al., 2000). The accumulated glyphosate in the roots which is eventually released into the rhizosphere (Kremer et al., 2005) Effect of glyphosate on soil microorganisms Increases soil bacteria and fungi populations and biomass (Haney et al., 2000). May be toxic to some bacteria and fungi, possibly due to inhibition of microbial shikimic acid pathways (Busse et al., 2001). Results Total rhizobacteria (Figure 2) and IAA-producing bacteria populations (Figure 5) were generally stimulated by application of glyphosate and significantly stimulated at day 20. Urease activity of soils treated with glyphosate was considerably lower than treated glyphosate soils (Figure 3). DAA 0 5 10 15 20 25 30 35 Total bacteria population (Log CFU g -1 fresh root) 7 8 9 10 11 12 13 RR-No Gly RR-Gly NS NS A. DAA 0 5 10 15 20 25 30 35 Total bacteria population (Log CFU g-1 fresh root) 7 8 9 10 11 12 13 RR-No Gly-Bio RR-Gly-Bio NS NS B. DAA 0 5 10 15 20 25 30 35 Total bacteria population Log (CFU g-1 fresh root) 7 8 9 10 11 12 13 RR-No Gly-US RR-Gly-US NS NS C. The physical relationship between a plant and a bacterium takes place via: » Bacterial IAA genes which can be transferred directly into the host plant genome, for example, several Agrobacterium species. » The infecting bacterium which can live within the plant tissues where it can secrete IAA into surrounding tissues. » The bacterium which colonizes at the phyllosphere or the rhizosphere, relying on excreted IAA. Bacterial phytopathogens transform their host plant or colonize their tissues, whereas beneficial bacteria predominantly promote growth while colonizing on the rhizoplane. The outcome of the interaction between a plant and a bacterium varies depending on plant susceptibility to increased levels of auxin (Patten and Glick, 1996). A. B. DAA 0 5 10 15 20 25 30 35 Urease activity (mg NH 4 + -N g -1 dwt 2h r-1 ) 10 20 30 40 50 60 70 RR-No Gly RR-Gly A. NS NS DAA 0 5 10 15 20 25 30 35 Urease activity (mg NH 4 + -N g -1 dwt 2hr -1 ) 10 20 30 40 50 60 70 RR-No Gly-US RR-Gly-US C. NS NS DAA 0 5 10 15 20 25 30 35 Urease activity (mg NH 4 + -N g -1 dwt 2hr -1 ) 10 20 30 40 50 60 70 RR-No Gly-Bio RR-Gly-Bio B. NS NS NS Figure 3. Average urease activity with days after glyphosate application (DAA) and (A) no foliar amendments applied, (B) with biostimulant (Bio) applied, or (C) with urea solution (US) applied during the cropping season. Vertical bars indicated LSD (o.o5). NS is not significant. DAA 0 5 10 15 20 25 30 35 IAA-producing bacteria (Log CFU g-1 fresh root) 6 7 8 9 10 11 12 13 RR-No Gly-US RR-Gly-US C. NS NS NS NS DAA 0 5 10 15 20 25 30 35 IAA-producing bacteria (Log CFU g-1 fresh root) 6 7 8 9 10 11 12 13 RR-No Gly RR-Gly A. NS NS DAA 0 5 10 15 20 25 30 35 IAA-producing bacteria (Log CFU g-1 fresh root) 6 7 8 9 10 11 12 13 RR-No Gly-Bio RR-Gly-Bio B. NS Figure 5. Average IAA-producing bacteria population with days after glyphosate application (DAA) and (A) no foliar amendments applied, (B) with biostimulant (Bio) applied, or (C) with urea solution (US) applied during the cropping season. Vertical bars indicated LSD (o.o5). NS is not significant. A. B. Figure 4. Nitrocellulose membrane assay of rhizobacteria population cultured from glyphosate-resistant soybean and (A) no glyphosate applied and (B) glyphosate applied. Biostimulant application increased total rhizobacteria and IAA-producing bacteria populations from day 20 to 30, irrespective of glyphosate application (Figure 5b). 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. Summary

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Page 1: Interactions of Glyphosate and Foliar Amendments with IAA

• Increased use of glyphosate-resistant crops has raisedconcerns regarding the potential environmental impact ofglyphosate. Furthermore, other foliar-applied chemicalsmay impact soil microbial ecology.

• Biostimulants (products containing plant hormones) andliquid fertilizers affect soil microorganisms by providingadditional nutrients or growth factors that alter metabolicactivity and improve crop growth and productivity. Forexample, Grozyme® and PT-21® are foliar amendmentsused to increase crop yield.

• Metabolites implicated in growth-suppressive activityinclude hydrogen cyanide, phytohormones such as indole-3-acetic acid (IAA), and unidentified phytotoxins. Theobjectives of this study were to describe changes in IAAsynthesis and urease activity of rhizosphere bacteria ofglyphosate-resistant soybean (Glycine max, ‘RoundupReady’ soybean).

Introduction

Interactions of Glyphosate and Foliar Amendments with IAA Synthesis andUrease Activity by Rhizobacteria of Glyphosate-Resistant Soybean

Su-Jung Kim1 and Robert J. Kremer1,21University of Missouri-Columbia and 2USDA-ARS, Cropping Systems and Water Quality Research Unit

Glyphosate or N-(phosphonomethyl)glycine

References

Objectives

Figure 2. Average totalrhizobacteria population with daysafter glyphosate application (DAA)and (A) no foliar amendmentsapplied, (B) with biostimulant (Bio)applied, or (C) with urea solution(US) applied during the croppingseason. Vertical bars indicatedLSD (o.o5). NS is not significant.

Busse, M.D., A.W. Ratcliff, C.J. Shestak, and R.F. Powers. 2001. Glyphosatetoxicity and the effects of long-term vegetation control on soil microbialcommunities. Soil Biol. Biochem. 33:1777-1789.

Haney, R.L., S.A. Senseman, F.M. Hons, and D.A. Zuberer. 2000. Effect ofglyphosate on soil microbial activity and biomass. Weed Sci. 48:89-93.

Kim, S.-J. and R.J. Kremer. 2005 Scanning and transmission electronmicroscopy of root colonization of morningglory (Ipomoea spp.) seedlingsby rhizobacteria. Symbiosis 39:117-124.

Kremer, R.J., N. Means, S.-J. Kim. 2005. (In Press). Glyphosate affectssoybean root exudation and rhizosphere microorganisms. Int. J. Environ.Anal. Chem.

Leinhos, v. and O. Vacek. 1994. Biosynthesis of auxins by phosphate-solubilizing rhizobacteria from wheat and rye. Microbiol. Res. 149:31-35.

Patten, C.L. and B.R. Glick. 1996. Bacterial biosynthesis of indole-3-aceticacid. Can. J. Microbiol. 42:207-220.

Reddy, K.N., R.E. Hoagland, and R.M. Zablotowicz. 2000. Effect ofglyphosate on growth, chlorophyll, and nodulation in glyphosate-resistantand susceptible soybean (Glycine max) varieties. J. New Seeds 2:37-52.

Tabatabai, M.A. 1994. Soil enzymes. p. 775-833. In R.W. Weaver et al., (ed)Methods of Soil Analysis, Part 2. Microbiological and BiochemicalProperties. Soil Science Society of America. Madison, WI.

64.02 aBradyrhizobium japonicum isolate GD3 0.28 bBacillus megaterium isolate GP4IAA (µ M)Treatments

• To describe changes in IAA synthesis and urease activityof rhizosphere bacteria of glyphosate-resistant soybean(Glycine max, ‘Roundup Ready’ soybean).

Days after Inoculation1 2 3 4 5 6 7 8

Roo

t Elo

ngat

ion

(cm

)

0

2

4

6

8

10

12

14

16

18

20

22

CheckIsolate GP4Isolate GD3

LSD (P•0.05)

– Up to 80% of rhizobacteria can produce IAA (Leinhosand Vacek, 1994).

– Various microbes in the rhizosphere produce an auxin,IAA, identical to that found in plants.

Figure 1. (A) Root elongation of morningglory seedlings inoculated by B.megaterium isolate GP4 and B. japonicum isolated GD3 in thehydroponic system, and (B) SEM of root tissue of morninggloryseedlings seven days after inoculation. Root surface of B. japonicumisolate GD3. Examples of distinct fibrillar material released by bacteriaare denoted by arrows . (Kim and Kremer, 2005)

Are IAA metabolites produced byrhizobacteria plant-growth promoting orsuppressive ?

Why is microbial urease activity important?– An indicator to estimate urea hydrolysis involved in C

and N mineralization of rhizosphere microorganisms.

– Catalyze the hydrolysis of urea to CO2 and NH3 with areaction mechanism based on the formation ofcarbamate as an intermediate (Tabatabai, 1994).

• A broad spectrum, non-selective postemergence herbicideof grasses and broadleaf weeds.

• The mode of action of glyphosate - inhibition of 5-enopyruvylshikimic acid-3-phosphate

(EPSP) synthase.

• Roundup® is the most widely used herbicide (glyphosateis the active ingredient), produced by Monsantocorporation.

Glyphosate in plants• Not likely metabolized by plant; it is translocated and may

accumulate in meristematic regions including roots andnodules (Reddy et al., 2000).

• The accumulated glyphosate in the roots which iseventually released into the rhizosphere (Kremer et al.,2005)

Effect of glyphosate on soil microorganisms• Increases soil bacteria and fungi populations and biomass

(Haney et al., 2000).

• May be toxic to some bacteria and fungi, possibly due toinhibition of microbial shikimic acid pathways (Busse et al.,2001).

Results• Total rhizobacteria (Figure 2) and IAA-producing bacteria

populations (Figure 5) were generally stimulated by applicationof glyphosate and significantly stimulated at day 20.

• Urease activity of soils treated with glyphosate wasconsiderably lower than treated glyphosate soils (Figure 3).

DAA0 5 10 15 20 25 30 35

Tota

l bac

teri

a po

pula

tion

(Log

CFU

g-1

fres

h ro

ot)

7

8

9

10

11

12

13

RR-No GlyRR-Gly

NS NS

A.

DAA0 5 10 15 20 25 30 35

Tota

l bac

teri

a po

pula

tion

(Log

CFU

g-1

fres

h ro

ot)

7

8

9

10

11

12

13

RR-No Gly-BioRR-Gly-Bio

NS NS

B.

DAA0 5 10 15 20 25 30 35

Tota

l bac

teri

a po

pula

tion

Log

(CFU

g-1

fres

h ro

ot)

7

8

9

10

11

12

13

RR-No Gly-USRR-Gly-US

NS NS

C.

– The physical relationship between a plant and abacterium takes place via:

» Bacterial IAA genes which can be transferreddirectly into the host plant genome, for example,several Agrobacterium species.

» The infecting bacterium which can live within theplant tissues where it can secrete IAA intosurrounding tissues.

» The bacterium which colonizes at the phyllosphereor the rhizosphere, relying on excreted IAA.

– Bacterial phytopathogens transform their host plant orcolonize their tissues, whereas beneficial bacteriapredominantly promote growth while colonizing on therhizoplane.

– The outcome of the interaction between a plant and abacterium varies depending on plant susceptibility toincreased levels of auxin (Patten and Glick, 1996).

A. B.

DAA0 5 10 15 20 25 30 35

Ure

ase

activ

ity(m

g N

H4+ -

N g

-1 d

wt 2

hr-1

)

10

20

30

40

50

60

70

RR-No GlyRR-Gly

A.

NS NS

DAA0 5 10 15 20 25 30 35

Ure

ase

activ

ity(m

g N

H4+ -

N g

-1 d

wt 2

hr-1

)

10

20

30

40

50

60

70

RR-No Gly-USRR-Gly-US

C.

NSNS

DAA0 5 10 15 20 25 30 35

Ure

ase

activ

ity(m

g N

H4+ -

N g

-1 d

wt 2

hr-1

)

10

20

30

40

50

60

70

RR-No Gly-BioRR-Gly-Bio

B.

NS NS NS

Figure 3. Average urease activitywith days after glyphosateapplication (DAA) and (A) no foliaramendments applied, (B) withbiostimulant (Bio) applied, or (C)with urea solution (US) appliedduring the cropping season.Vertical bars indicated LSD (o.o5).NS is not significant.

DAA0 5 10 15 20 25 30 35

IAA

-pro

duci

ng b

acte

ria

(Log

CFU

g-1

fres

h ro

ot)

6

7

8

9

10

11

12

13

RR-No Gly-USRR-Gly-US

C.

NSNSNSNS

DAA0 5 10 15 20 25 30 35

IAA

-pro

duci

ng b

acte

ria

(Log

CFU

g-1

fres

h ro

ot)

6

7

8

9

10

11

12

13

RR-No GlyRR-Gly

A.

NS NS

DAA0 5 10 15 20 25 30 35

IAA

-pro

duci

ng b

acte

ria

(Log

CFU

g-1

fres

h ro

ot)

6

7

8

9

10

11

12

13

RR-No Gly-BioRR-Gly-Bio

B.

NS

Figure 5. Average IAA-producingbacteria population with days afterglyphosate application (DAA) and (A)no foliar amendments applied, (B)with biostimulant (Bio) applied, or (C)with urea solution (US) appliedduring the cropping season. Verticalbars indicated LSD (o.o5). NS is notsignificant.

A. B.

Figure 4. Nitrocellulose membrane assay of rhizobacteriapopulation cultured from glyphosate-resistant soybean and(A) no glyphosate applied and (B) glyphosate applied.

• Biostimulant application increased total rhizobacteria andIAA-producing bacteria populations from day 20 to 30,irrespective of glyphosate application (Figure 5b).

• Glyphosate-resistant soybean may modify the bacterialcomposition and activity in the rhizosphere to a limited extent.

• These changes may impact crop productivity and soilbiological processes.

Summary