This document describes a study evaluating bacillus strains for their ability to promote plant growth on corn, wheat, and soybean. Several bacillus strains were tested in greenhouse experiments and found to significantly increase growth of corn, wheat, and soybean compared to untreated controls. The best performing strains increased crop growth by over 200% in some cases. Further experiments aim to determine if physiological traits expressed by the strains in laboratory assays correlate with and can predict their ability to promote plant growth.

1. Evaluation of bacillus strains for plant
growth-promotion potentials on corn, wheat,
and soybean.
Rufus J. Akinrinlola
MS Student
Advisors:
Gary Yuen
Tony Adesemoye
February 2018.

2. PGPR:
plant growth-promoting
rhizobacteria
 Aggressive root-colonizing bacteria.
 High survival in rhizosphere.
 Increases plant growth.
 Increases crop productivity/yields.
 Includes up to 12 bacteria genera
 Most common PGPR
 Bacillus, Burkoldhera and Pseudomonas
Adesemoye and Kloepper, 2009; Bhattacharyya and Jha, 2012; Burr et al., 1978; Kloepper and Schroth, 1978; Dotaniya et al., 2015.

3.  Soils attached to plant roots.
 2 to 80 mm around root.
 Habitat to rhizobacteria.
 108–1012 bacterial cell per g-soil
 Rich in sugars and amino acids.
 Root exudates.
 Cell lysates.
 Organic matters.
The rhizosphere
Huang, X. F., Chaparro, J. M., Reardon, K. F., Zhang, R., Shen, Q., & Vivanco, J. M. (2014). Rhizosphere interactions: root exudates, microbes, and microbial
communities. Botany, 92(4), 267-275.
Photo credit: Adesemoye Lab

4. How PGPR increase plant growth
 Direct mechanisms.  Indirect mechanisms
PGPR PGPR

5. Direct plant growth-promotion
 Direct supply of nutrients and growth hormones to plant.
 Nitrogen fixation (N).
 Phosphate solubilization (P).
 Iron mobilization (Fe).
 Indole acetic, cytokinin and gibberellic acid.
 ACC deaminase enzyme activity.
Adesemoye and Kloepper, 2009; Bhattacharyya and Jha, 2012)

6.  Control of rhizospheric plant-deleterious microbes
Indirect plant growth-promotion
 Antibiotics production
 Lytic enzymes synthesis
 Siderophores production
 Competition for nutrients
 Induced systemic resistance
Pathogens
PGPR
Kobayashi and Yuen, 2007;

7. Agrochemicals - Alternatives to PGPR
 Chemical fertilizers
 Pesticides
 Great nutrient supply
 Pathogen control
 Improved crop productivity.
Photo credit: Google Image

8. Agrochemical concerns
Photo credit: Google Image
 Nitrate leaching (ground water concerns).
 Phosphorus runoff (surface water concern).
 Accumulation chemical residues (food safety)
 Pesticide resistance concerns.
 Need for alternative options.
Vejan, P., Abdullah, R., Khadiran, T., Ismail, S., & Nasrulhaq Boyce, A. (2016). Role of plant growth promoting rhizobacteria in agricultural sustainability—a review. Molecules, 21(5), 573.

9. Importance of PGPR
 Improves crop productivity.
 No environmental concern.
 Reduces agrochemical use.
 Alternative to agrochemical
 Supplement to agrochemicals
 Replacement ( in organic farming).
 Alleviate agrochemical concerns
Vejan, P., Abdullah, R., Khadiran, T., Ismail, S., & Nasrulhaq Boyce, A. (2016). Role of plant growth promoting rhizobacteria in agricultural sustainability—a review. Molecules, 21(5), 573.

10. Problems with PGPR
Weller, 1988; Bly and Gelderman (2009) study.
 Inconsistent performance in different environments.
 Loss of ecological competence.
 Loss of viability
 Loss of active traits
 Narrow spectrum activity.

11. Inconsistent PGPR products
 Six PGPR products tested.

12.  None of the products increased yield on soybean
Bly and Gelderman (2009) study.

13.  None of the products increased yield on soybean
Bly and Gelderman (2009) study.

14. Focus of this research project.
 To identify bacillus-PGPR for crop production in Nebraska

15. Bacillus as important PGPR
 Spore-forming Gram-positive
bacteria
 Formally Bacillus genera
 Now Bacillus and related genera
 Paenibacillus, Brevibacillus,
Lysinibacillus
 A preferred PGPR.
 Stress-tolerant.
 Multiple species are known
PGPR
 Broad-spectrum activity.
Kumar et al., 2011; Xu and Côte, 2003
Bacillus pumilus
Paenibacillus cineris
Lysinibacillus fusiformis
Photo credit: Rufus

16. Test strains
 Bacillus acidiceler R228
 B. megaterium strains R181 and R232
 B. pumilus strains R174, R183, and R190
 B. safensis strains R173 and R176
 B. simplex R180
 Lysinibacilus fusiformis R198
 Paenibacillus cineris R177
 P. graminis R200
 Wheat Rhizosphere.
 Nebraska soils.
 Adesemeoye Lab, North Platte, NE

17. I. To identify strain that can promote growth in corn, wheat, and
soybean.
II. To determine whether the physiological traits expressed in
vitro by the strains can. predict their plant growth promotion
efficacy.
 Mechanisms exhibited by the strains.
 Presence of numerous/ specific set of in vitro traits.
Study objectives

18. POTENTIAL PGPR
For future field tests
Research studies plan
Best strains
Test on soybean
and wheat.
Identify modes of
actions
Compare strain’s
traits with efficacy
on corn
In vitro assays for
physiological traits
Greenhouse test
on corn
Test strains
Best strains on
corn
Objective I
ObjectiveII

19. Culture Inoculum
Lab procedures for Objective I
 Cultured on 10%
TSA medium
 Incubation:28 °C, 2
days.
 Cells washed and
diluted with sterile
PB to 108 cfu/mL.
 Seeds treated with
inoculum by
soaking for 30 or
60 minutes.
Treatment

20. Sowing
Greenhouse procedures for Objective I
 2 to 1; sands
to soil.
 8 to 5
replicates.
 Once daily
without
fertilizer.
 Roots washed
and separated
from shoots.
Soil mix Data collectionWatering

21. What was observed.
Roots of
untreated
plants.

22. Data analysis
 Dunnett’s test.
 Compare strain against
control
 Analysis of variance
 Determine treatment
effects.
 Mean separation with LSD
(α ≤ 0.05).
 Determine differences
between strains.
Photo credit: Google image

23. Results for strains’ effect on corn
 Eleven out of twelve strains increased sweetcorn growth
significantly compared to control.
 Corn growths were frequently increased over 200%
compared to control plants.

24. 0
1
2
3
4
5
6
7
8
9
10
B. megaterium
R181
B. pumilus
R183
B. safensis
R173
B. simplex
R180
Lysinibacillus
fusiformis R198
P. graminis
R200
Control
Shootweight(g)
Treatment
AB (137%) B (118%) B (122%)
A (215%)
B (103%)
AB (140%)
a (59%)
b
C
Strains increased corn shoot weight significantly
a (37%)

25. 0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
B. megaterium
R181
B. pumilus
R183
B. safensis R173B. simplex R180 Lysinibacillus
fusiformis R198
P. graminis
R200
Control
Rootweight(g)
Treatment
b
ab (75%)
AB
(121%)
AB (167%)
AB (135%)
C
Strains increased corn root weight significantly
a (206%)
b (12%)
AB (122%)
B (112%)
A (203%)

26. Strain’s consistency in promoting growth.
Strain
% increase compared to control a Frequency
of growth
stimulation
(%)
Shoot height Shoot weight Root weight
Trial 1 Trial 2 Trial 3 Trial 1 Trial 2 Trial 3 Trial 1 Trial 2 Trial 3
B. simplex R180 - 41*** 30*** - 118** 68** - 112*** 206** 100
B. safensis R176 - 34*** 20** - 111*** 42** - 124** 110 83
B. megaterium R181 19*** 45*** 28** 40 140*** 59** 36** 121*** 132 78
Paenibacillus cineris
R177
9 51*** 20** 20 155*** 42 3 168*** -8 67
P. graminis R200 - 54*** 18** - 215*** 37 - 203*** 75 67
B. safensis R173 3 44*** 15** -15 137*** 51** -14 167*** 222 56
Lysinibacillus
fusiformis R198
5 47*** 17** 6 122*** 33** -25 135*** 147** 56
B. pumilus R190 - d 38*** 13 - 77*** 32 - 93*** 104 50
Bacillus acidiceler
R228
7 42*** C 28** 7 118*** 66 -11 155*** 92 44
B. megaterium R232 17 45*** 12 24 144*** 32** -6 107*** 173 44
B. pumilus R183 12 40*** 28** 33 103*** 62 0 122*** 12 44
B. pumilus R174 13 41*** 7 30 126*** 24 -3 117*** 91 33
Mean e 18 43 23 - 131 48 36 135 177

27. Strain
Shoot height (cm) Shoot weight (g) Root weight (g) Top 3
frequency
(%)
Trial 1 Trial 2 Trial 3 Trial 1 Trial 2 Trial 3 Trial 1 Trial 2 Trial 3
Bacillus
megaterium R181
51 48ab 40a 8.0 6.5abc 3.0ab 4.9a 3.2ab 1.1 55
Paenibacillus
graminis R200
- 53a 37abcd - 8.4a 2.6abcd - 4.3a 0.9 50
B. megaterium
R232
50 48ab 35cde 7.0 6.6abc 2.5abcd 3.4b 3.1b 1.3 44
P. cineris R177 47 50ab 37abc 7.0 6.9ab 2.7abcd 3.7b 3.9ab 0.5 44
B. pumilus R183 48 47b 40ab 7.0 5.5bc 3.1ab 3.6b 3.2ab 1.1 33
B. simplex R180 - 47b 41a - 5.9bc 3.2a - 3.2b 1.5 33
Bacillus
acidiceler R228
46 47ba a 40a 6.0 6.0bc 3.2ab 3.2b 3.5ab 0.9 22
B. safensis R173 44 48ab 36abcd 6.0 6.4bc 2.9ab 3.1b 3.9ab 1.6 22
B. pumilus R174 49 47b 33cde 7.0 6.0bc 2.4bcd 3.5b 3.1b 0.9 11
B. pumilus R190 - 46b 35bcde - 4.8c 2.5abcd - 2.8b 1.0 0
B. safensis R176 - c 47b 37abc - 5.7bc 2.7abc - 3.3ab 1.0 0
Lysinibacillus
fusiformis R198
45 48ab 36abcd 6.0 6.0bc 2.5abcd 2.7b 3.5ab 1.2 0
Control 43 33c 31e 5 2.7d 1.9d 3.6b 1.5c 0.5
P- value 0.0807 <.0001 0.0015 0.2245 0.0006 0.0419 0.0145 0.0052 0.614
Strain’s consistency in “top 3” category

28. 1.Bacillus megaterium R181
2.B. safensis R173
3.B. simplex R180
4.Paenibacillus cineris R177
5.P. graminis R200
 Selected strains tested on wheat and soybean

29. Results for soybean and wheat tests
 Four out of five strains increased soybean and wheat
 Paenibacillus cineris R177 did not promote growth on both
crops.

30. 0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
Bacillus megaterium
R181
B. safensis R173 P. graminis R200 Control
Shootweight(g)
Treatment
a (31%)
B
A(36%)
ab (9%)
a (16%)
A(46%)
b
Strains increased soybean shoot growth significantly
AB (18%)

31. 0
0.5
1
1.5
2
2.5
3
Bacillus megaterium
R181
B. safensis R173 P. graminis R200 Control
RootWeight(g)
Treatment
a (144%)
B (9%) B
AB (35%)
b (67%)
ab (89%)
A (91%)
c
Strains increased soybean root growth significantly

32. 0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0.5
Bacillus megaterium
R181
B. safensis R173 P. graminis R200 Control
Shootweight(g)
Treatment
b
B
A (43%)A (40%)
a (30%)
ab (10%)
Wheat shoot growth increased consistently.
a (30%)
A (47%)

33. 0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
Bacillus megaterium
R181
B. safensis R173 P. graminis R200 Control
Rootweight(g)
Treatment
A (123%)
A (154%)
B
A (115%)
b
Wheat root growth increased consistently.
a (57%)

34. Summary of results for Objective I
 Eleven strains increased corn growth significantly.
 Growth were frequently increased over 200% on corn.
 Strains - R173, R180, R181, and R200 - increased
growths on all test crops.
 B. safensis R173 - highest growth on soybean.
 B. megaterium R181- highest growth on wheat.
 Soybean shoot and root weights were increased up to
46% and 144% resp.
 Wheat shoot and root weights were increased up to 43%
and 154% resp.

35. Objective II
 To determine whether the physiological
traits expressed in vitro by strains can
predict their plant growth promotion
efficacy.
 Mechanisms exhibited strains.
 Presence of numerous/ specific set
of in vitro traits.
In vitro assays for
physiological
traits
Identify modes of
actions
Compare strain’s
traits with
efficacy on corn
Test strains

36. Assays for direct mechanisms
Phosphate solubilization Siderophores
Indole acetic acid
Nitrogen-fixation
assay
Growth pouch assay

37. Assays for direct mechanisms
Nitrogen-fixation assay
 Strains inoculated onto the
Nitrogen-free medium.
 Incubation: 28 °C for 7 days
 Culture flooded with BTB
solution.
 Observed for color change
from green to dark blue or
bluish green.

38.  Phosphate solubilization assay
 Spot inoculation
 Pikovskaya agar medium
 Incubation: 7 days at 28 °C
 Observed for Clear halo
Assays for direct mechanisms

39. Assays for direct mechanisms
Indole acetic acid activity
 Indole acetic acid assay:
 Cultured in tryptophan
supplemented nutrient
broth.
 Fluid mixed with
Salkowski's reagent.
 Observed for color change
from yellow to pink.

40. Assays for direct mechanisms
 Siderophores assay:
 Chrome Azurol S
(CAS) agar medium
 Incubated for 5 days at
28 °C.
 Observed for pink
color halo around
colony.

41. Assays for direct mechanisms
 Growth pouch assay
 Seeds disinfected
 Seeds treated with
strains.
 Treated seeds sown in
pouch for 10 days.
 Data collected for;
 Shoot height
 Shoot weights
 Root numbers.

42. Assays for direct mechanisms
Growth pouch assay- observation

43. Assays for direct mechanisms
Growth pouch assay- observation

44. Chitinase
Assays for indirect mechanisms
Antibiosis
Biosurfactants Protease

45. Assays for indirect mechanisms
 Antibiosis assay
 Growth inhibition assay
 Bacteria
 Fungi
 Oomycetes
 Halo zone indicates
growth inhibition by
strains

46. Biosurfactant activity
 Biosurfactant assay
 Fluid hydrophobicity test.
 Measurement of fluid
droplet spread/diameter.
 High fluid spread
indicated biosurfactant
activity.
Assays for indirect mechanisms

47.  Chitinase assay
Assays for indirect mechanisms
 Chitinase assay
 Colloidal chitin medium.
 Yeast cell medium.
 Incubation: 28 °C for 5
days
 Presence of halo clear
zoneChitinase activity

48.  Chitinase assay
Protease enzyme activity
Assays for indirect mechanisms
 Protease enzyme assay
 Strain spot-inoculation.
 Milk agar medium.
 2 days incubation at 28 C.
 Clear halo zone represent
protease enzyme activity.

49.  Direct mechanisms exhibited by most strains.
Strain
Indirect mechanisms Direct mechanism
Anti
fungal
Anti
bacterial
Protease Chitinase Biosurfactant
Side-
rophore
Side-
rophore
Pho
-sphate
IAA
Nitrogen
fixation
Pouch
assay
Bacillus
acidicelerR228
B.
megateriumR181
B.
megateriumR232
B. pumilusR174
B. pumilusR183
B. pumilusR190
B. safensisR173
B. safensisR176
B. simplexR180
Lysinibacillus
fusiformisR198
Paenibacillus
cinerisR177
P. graminisR200
Results for in vitro assays

50.  Number of trait found in a strain did not predict its efficacy
in pot tests.
Strain
No of trait
exhibited
Strain’s
efficiency
Bacillus megaterium R181 7 High
B. safensis R173 3
B. simplex R180 4
Lysinibacillus fusiniformis R198 2
Paenibacillus graminis R200 1
B. megaterium R232 5 Low
B. pumilus R174 3
B. pumilus R183 5
B. pumilus R190 5
Paenibacillus cineris R177 3

51. Strain
Anti
fungal
Anti
bacterial
Protease Chitinase
Bio-
surfactant
Siderophore Phosphate IAA
Nitrogen
fixation
Pouch
assay
Strain
efficacy.
R173
High
R176
R177
R180
R181
R198
R200
R174
Low
R183
R190
R228
R232
No set of traits was predictive for high PGPR effect

52. Summary of results for Objective II
 Direct mechanisms were exhibited by most of the strains.
 B. pumilus strains mostly exhibited indirect mechanisms.
 Presence of diverse or specific set of traits did not predict
strain’s efficacy/consistency.
 Indole acetic acid, protease enzyme and phosphate
solubilization exhibited by many strains.

53. Conclusions
 Bacillus exhibit great potentials for plant growth promotion.
 Bacillus can stimulate growth on different crop varieties.
 Bacillus exhibits different plant growth-promotion mechanisms.
 Bacillus possess higher tendency for direct mechanisms.
 Corn have high responsivity to bacillus-PGPR.
 Selection of potential PGPR based on in vitro traits is not
recommended.
 Potential PGPR specie (Paenibacillus graminis) identified.
 Field studies are needed to further evaluate the strains.

54. Acknowledgements
 Advisors and Committee member
 Gary Yuen
 Tony Adesemoye
 Rhae Drijber
 Plant Pathology Department.
 Sydney Lab.
The Yuen Team

55. Thanks!