1-aminocyclopropane-1-

carboxylic acid deaminase activity as

a marker for identifying plant-growth

promoting rhizobacteria in cultivated

soil

Researcher: Motlagomang Khantsi

NWU (Mafikeng)

Promoter: Prof O.O. Babalola

HEAT STRESS

MAIZE ROOT

RHIZOBACTERIA

ETHYLENE HORMONE

Introduction

ETHYLENE HORMONE

NH3 + α- ketobutyrate

Study area

�Sample sites-Ngaka Modiri Molema district

municipalities

Soil sampling location & sample codesLocation Plant Scientific name GPS co-ordinates Sample code

Mahikeng Onion Allium cepa 525°53.318´ MO1

Maize Zea mays 525°53.296´ MM2

Cabbage Brassica oleracea 525°53.276´ MC3

Spinach Spinacia oleracea 525°56.946´ MS4

Tswaing Sunflower Helianthus

annuus

526°23.738´ TM4

Peach Prunus persica 526°24.512´ TP3

Pumpkin Cucurbita pepo 526°23.731´ TP2

Ratlou Maize Zea mays 526°04.306´ RM1

Sunflower Helianthus

annuus

526°03.036´ RS3

Ditsobotla Maize Zea mays 526°09.628´ DM2

Sunflower Helianthus

annuus

526°09.633´ DS1

Ramotshere Maize (Dry) Zea mays 525°54.558´ ZM1

Maize (fresh) Zea mays 525°54.289´ ZM2

Aims and objectives

�Isolate rhizobacteria

�Characterize isolates

�Identification of isolates

�Determine the effect of ACCD containing bacteria in plant growth and development

Methodology

� Isolation of rhizobacteria

� ACCD activity assay OD540 (Penrose and Glick 2003)

� Cultural characterization: shape, size, texture, pigmentation , optical property etc.

� Morphological characterization: biochemical tests: catalase, oxidase, starch hydrolysis, Gram stain, motility, etc. (Cappucino and Sherman 2011)

Cultural characteristicsIsolate

no.

Shape Margin Size Texture Appearance pigmentation

Ms4(4) Irregular Undulate Large Smooth Dull White

Tp2(4) Irregular Undulate Large Rough Smooth Cream

Rs3(2) Irregular Undulate Large Smooth Dull White

Rs3(3) Circular Entire Punctiform Smooth Partly shinning White

Rs3(4) Irregular Curled Small Rough Dull Cream

Rs3(5) Irregular Undulate Small Rough Dull Tan

Rs3(10) Irregular Curled Small Rough Dull White

Rs3(11) Irregular Undulate Small Rough Dull White

Rm2(2) Circular Undulate Large Rough Dull White

Rm2(3) Irregular Undulate Small Smooth Partly dull Cream

Rm2(4) Irregular Undulate Punctiform Smooth Shinning Pink

Zm2(7) Irregular Undulate Large Rough Dull White

Mm5(6) Irregular Undulate Large Rough Dull White

Mm5(9) Irregular Lobate Large Rough Dull White

Mc3(8) Irregular Undulate Moderate Smooth Shinny White

Morphological characteristics

Isolate

number

Oxidase Catalase Gram stain Starch

hydrolysis

Motility Gelatine

liquefaction

Ammonia

production

Protease

activity

Citrate

utility

Ms4(4) - + + coccus - + - - + -

Tp2(4) + + + rods + V - + + +

Rm2(2) - + - rods + + - + + -

Rm2(3) + + - rods + + - + + +

Rm2(4) - + - rods + + - + - -

Zm2(7) + + - rods - V - + + -

Mm5(6) + + + rods - + - + + +

Mm5(9) + + + rods

(endospore)

+ - - + + -

Rs3(2) + + - coccus + V - + + +

Rs3(3) + + - rods + + - + + +

Rs3(5) - + - rods + + - + + +

Mc3(8) - + - rods + + - + + -

Phenotypic tests

�Chinolytic and Cellulose activity

�Indole Acetic Acid production

�Phosphate solubilisation

�Antifungal susceptibility

�Siderophore production

�Hydrogen cyanide production

PGPR TraitsIsolate

number

ACCD OD 540 Phosphate Antifungal IAA Siderophore Chitinolytic &

cellulose

activity

HCN

Rs3(3) 0.557 + + + - + +

Rs3(2) 0.896 + + + - + +

Mm5(6) 0.522 + + + + + +

Rs3(4) 0.720 + - + + - -

Rs3(5) 0.979 + + + + + +

Rm2(6) 0.557 + + + - + -

Rm2(9) 0.763 + - - + + +

Tp2(4) 0.542 + + + - + +

Zm2(7) 0.723 + + + + + +

Rm2(4) 0.509 + + + - + -

Rm2(2) 0.621 + - + - + +

Rm2(3) 0.640 - + + _ + +

Mm5(9) 0.796 + + + + - +

Ms4(4) 0.951 + + + - - +

Molecular identification�Extraction of genomic DNA (ZR Soil microbe DNA

MiniPrep)

�PCR amplification of 16S rDNA and ACCD specific

Marker Temperature cycling Primer sequences (5'-3')

PCR 16S rDNA 30 cycles of 5 min @ 94 °C,

30 S @ 95 °C, 1 min @ 54

°C, 2 min @ 72 °C, 5 min @

72 °C

27 Forward

AGA GTT TGA TCC TGG CTC

AG

1492 Reverse

TAC CTT GTT ACG ACT T

PCR ACCD Specific 30 cycles of 3 min @ 94 °C,

30 S @ 94 °C, 30 S @ 54 °C,

1 min 30 S @ 72 °C, 5 min

@ 72 °C

ACC Forward

GGG ACC GGA TCC TCA

AGG AAC AGC GCC ATG

ACC Reverse

GAA CGG AAG CTT CTG

GCG GCG CCA AGC TCA

PCR amplicons

M 1 2 3 4 5 6 7 8 9 Kb

6.0

3.0

1.5

16S rDNA fragments

Agarose gel eletrophoresis of 16S rDNA of ACCD containing isolates. M is a 1 Kb plus

DNA ladder and numbers above are rhizobacterial strains as defined (see table 1). 1.

Mm5(6), 2. Rs3(4), 3. Rs3(5), 4. Rm2(9), 5. Tp2(4), 6. Zm2(7), 7. Rm2(3), 8. Mc3(8),

9. Ms4(4)

Genbank nucleotide sequences

Isolate

number

Assigned

accession

number

Sequence

length

Description Accession

number

% similarity

Zm2 (7) JX971510 1380 Bacillus

pumilus

JX293286 100

Rs3 (5) JX971507 1376 Bacillus

cereus

HE821233 100

Tp2 (4) JX971509 1382 Bacillus

sonorensis

JN013186 99

Rs3 (2) JX971506 1377 Bacillus

thuringiensis

JX283457 99

Rm2 (3) JX971513 1387 Bacillus

mojavensis

JN585852 99

Rm2 (2) JX971512 1403 Bacillus

aryabhattai

JX312579 99

Greenhouse screening

�Inoculation of bacterial strains to tomato

seedlings (Solanum lycopersicum)

�Seeds were grown on a suitable soil substrate

�Monitoring and watering

�After 40 days, plants were uprooted and data

was recorded

Tomato plants at 40 days after

planting

Does ACCd- containing bacteria enhance the development of tomato plants?

The second leave stage of the inoculated pot (B) shows more development over

the control (A) at 40 days after planting (DAP). The tomato pots were treated

with (A) 5 mL water or (B) 5 mL 1.79 x 106 CFU/mL of Bacillus pumilus for 5 days

uninterrupted.

A B

Plant growth parameter records

c o n tro l Z m 2 (7 ) R s 3 (5 ) T p 2 (4 ) R s 3 (2 ) R m 2 (3 )

0

2

4

6

8

Roo

t le

ngth

(cm

)

c o n tro l Z m 2 (7 ) R s 3 (5 ) T p 2 (4 ) R s 3 (2 ) R m 2 (3 )

0

1

2

3

4

5

6

7

Sh

oo

t le

ngth

(cm

)

c o n tro l Z m 2 (7 ) R s 3 (5 ) T p 2 (4 ) R s 3 (2 ) R m 2 (3 )

0

2

4

6

8

Fre

sh m

ass (

gm

pla

nt-1

)

T re a tm e n ts

c o n tro l Z m 2 (7 ) R s 3 (5 ) T p 2 (4 ) R s 3 (2 ) R m 2 (3 )

0 .0

0 .1

0 .2

0 .3

0 .4

0 .5

0 .6

0 .7

0 .8

Sh

oo

t d

ry m

ass (

mg

pla

nt-1

)

T re a tm e n ts

Research key findings

�Research show that PGPR containing ACCdeaminase activity are effective for improvinggrowth of tomato seedlings

�PGPR containing ACC deaminase activity couldprove to be cost effective and environmentallyfriendly to ensure sustainable agriculture

�Potential candidates for biofertilizers andbiocontrol (Lee et al. 2012)

�Microbial technology is expanding to greaterlengths and thus proved to be useful to theenvironment, like nature intended

Future perspectives

�ACC deaminases have been shown to protectplants from deleterious effects (Belimov et al.,2005; Mayak et al., 2004b). Accordingly, findingACC deaminase-containing bacteria could be ofgreat interest for field application and especiallyunder stress conditions (high temperatures anddrought).

�ACCD plant growth properties should be exploredfurther in the screen house and field conditionsespecially for its ability to compete with theendogenous soil and rhizosphere microbialcommunities.

References

� Arshad , M; Saleem, M and Hussain, S (2007).Perspectives of bacterial ACC deaminase inphytoremediation. Trends in Biotechnology 25:356-362.

� Babalola, OO (2010). Beneficial bacteria of agriculturalimportance. Biotechnology Letters 32: 1559-1570.

� Belimov, AA; Hontzeas, N; Safronova, VI; et al. (2005).Cadnium-tolerant plant growth-promoting bacteriaassosiated with the roots of indian mustard (Brassica

juncea L.Czern). Soil Biology and Biochemistry 37:241-250.

� Cappucino, JG and Sherman, N (2011). Microbiology: Alaboratory manual. Benjamin-Cummings, San Fransisco,USA.

References cont.� Lee, S; Ka, JO and Song, HG (2012). Growth promotion of

Xanthium italicum by application of rhizobacterial isolatesof Bacillus aryabhattai in microcosm soil. Journal ofMicrobiology 50:45-49.

� Mayak, S; Tirosh, T; Glick, BR (2004b). Plant growth-promoting bacteria that confer resistance to water stress in tomatoes and peppers. Plant Science 166:525-530.

� Penrose, DM and Glick, BR (2003). Methods for isolating and characterizing ACC deaminase-containing plant-growth promoting rhizobacteria. Physiologia Plantarum118: 10-15.

Acknowledgements

�Dr Hamid Igbal Tak and Ms Mobolaji Felicia

Adegboye

� DST-National Research Foundation SA

�NWU Postgraduate bursary

“He who lives in harmony with himself, lives in harmony with the world”.

Marcus Aurelius

Thank you