1. The study isolated and characterized rhizobacteria from cultivated soil samples in Ngaka Modiri Molema district municipalities that exhibited 1-aminocyclopropane-1-carboxylic acid deaminase (ACCD) activity, a marker for plant growth promoting rhizobacteria. 2. 16S rDNA sequencing identified the isolated bacteria, which included Bacillus pumilus, Bacillus cereus, Bacillus sonorensis, Bacillus thuringiensis, and Bacillus mojavensis. 3. Greenhouse screening found that inoculation of tomato seedlings with the ACCD-containing rhizobacteria, particularly B. pumilus, enhanced plant growth parameters like

1. 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

2. HEAT STRESS
MAIZE ROOT
RHIZOBACTERIA
ETHYLENE HORMONE
Introduction

3. ETHYLENE HORMONE
NH3 + α- ketobutyrate

4. Study area
Sample sites-Ngaka Modiri Molema district
municipalities

5. Soil sampling location & sample codes
Location 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

6. Aims and objectives
Isolate rhizobacteria
Characterize isolates
Identification of isolates
Determine the effect of ACCD containing
bacteria in plant growth and development

7. 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)

8. Cultural characteristics
Isolate
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

9. 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 + + - + + -

10. Phenotypic tests
Chinolytic and Cellulose activity
Indole Acetic Acid production
Phosphate solubilisation
Antifungal susceptibility
Siderophore production
Hydrogen cyanide production

11. PGPR Traits
Isolate
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 + + + - - +

12. 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

13. PCR amplicons
M 1 2 3 4 5 6 7 8 9Kb
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)

14. 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

15. 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

16. 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

17. 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
Rootlength(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
Shootlength(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
Freshmass(gmplant
-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
Shootdrymass(mgplant
-1
)
T re a tm e n ts

18. Research key findings
Research show that PGPR containing ACC
deaminase activity are effective for improving
growth of tomato seedlings
PGPR containing ACC deaminase activity could
prove to be cost effective and environmentally
friendly to ensure sustainable agriculture
Potential candidates for biofertilizers and
biocontrol (Lee et al. 2012)
Microbial technology is expanding to greater
lengths and thus proved to be useful to the
environment, like nature intended

19. Future perspectives
ACC deaminases have been shown to protect
plants from deleterious effects (Belimov et al.,
2005; Mayak et al., 2004b). Accordingly, finding
ACC deaminase-containing bacteria could be of
great interest for field application and especially
under stress conditions (high temperatures and
drought).
ACCD plant growth properties should be explored
further in the screen house and field conditions
especially for its ability to compete with the
endogenous soil and rhizosphere microbial
communities.

20. References
Arshad , M; Saleem, M and Hussain, S (2007).
Perspectives of bacterial ACC deaminase in
phytoremediation. Trends in Biotechnology 25:356-362.
Babalola, OO (2010). Beneficial bacteria of agricultural
importance. Biotechnology Letters 32: 1559-1570.
Belimov, AA; Hontzeas, N; Safronova, VI; et al. (2005).
Cadnium-tolerant plant growth-promoting bacteria
assosiated with the roots of indian mustard (Brassica
juncea L.Czern). Soil Biology and Biochemistry 37:241-
250.
Cappucino, JG and Sherman, N (2011). Microbiology: A
laboratory manual. Benjamin-Cummings, San Fransisco,
USA.

21. References cont.
Lee, S; Ka, JO and Song, HG (2012). Growth promotion of
Xanthium italicum by application of rhizobacterial isolates
of Bacillus aryabhattai in microcosm soil. Journal of
Microbiology 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 Plantarum
118: 10-15.

22. Acknowledgements
Dr Hamid Igbal Tak and Ms Mobolaji Felicia
Adegboye
DST-National Research Foundation SA
NWU Postgraduate bursary

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

24. Thank you