Rufus Akinrinlola is a PhD candidate researching plant-microbe interactions. His work has included identifying the black pod pathogen of cocoa in Nigeria (Phytophthora megakarya), surveying nematode populations across Tennessee fields (finding high levels of soybean cyst, lesion, and root knot nematodes), and identifying Bacillus strains that promote corn growth in greenhouse studies (with increases up to 215% in shoot weight). Understanding beneficial and pathogenic plant-microbe relationships can help improve crop productivity to meet rising global food demand.

1. Harnessing plant-microbe interactions for
sustainable agriculture
Rufus Akinrinlola
Research Specialist and PhD Candidate
University of Tennessee
West TN Research and Education Center
Jackson, TN
rakinrin@vols.utk.edu

2. 4 Years Research Experience
 AAUA Nigeria (1)
 University of Nebraska-Lincoln (3)
 University of Tennessee (7 months)
Over 2 Years Teaching Experience
Major Supervisor
Dr. Heather Kelly
Associate Professor and Extension Specialist
Nigeria
Ondo State
BS. Microbiology
Adekunle Ajasin
University
MS. Plant Pathology
University of Nebraska-Lincoln
NE
TN
PhD Cand. Plant Pathology
University of Tennessee-Knoxville
Hometown: Idanre (Pop.~200k)
Major crop: Cacao
Attraction: Rock Climbing festival (Mare)
About
me:

4. Vegetables Grains Beans
Fruits Tubers Roots
PLANT: GLOBAL FOOD SOURCE
 Plants feed the world

5. PLANT: GLOBAL FOOD SOURCE
 Food demand increases daily
 Demand for food is a demand on plants
Source: FAO

6. PHYTOBIOMES: PLANT MICROBIOMES
Handoo,1998
Nematode Virus
Microbes interacting
with plants:
 Bacteria
 Fungi
 Oomycetes
(fungal-like)
 Nematodes
 Virus
Bacteria Fungi

7. PHYTOBIOMES HABITATS
Berg, G., Grube, M., Schloter, M., & Smalla, K. (2014). Unraveling the plant microbiome: looking back and future perspectives. Frontiers in microbiology, 5, 148.
Wang, N., Jin, T., Trivedi, P., Setubal, J. C., Tang, J., Machado, M. A., ... & Wang, X. (2015). Announcement of the international citrus microbiome (Phytobiome) consortium. Journal of Citrus Pathology, 2(1), 1-2.
 Ectosphere: outer part
• Above ground: airborne
o Phyllosphere : leaf and stem surfaces
• Below ground: soil borne
o Rhizosphere: soil around roots
o Rhizoplane: root-soil interface
 Endosphere: inner part

8. PLANT - MICROBE INTERACTIONS
 Pathogenic interaction
• Phytotoxins
• Cell wall degrading enzymes
• EPS- extracellular
polysaccharides
• Modulation of host hormones
 Beneficial interaction
 Nitrogen fixation
 Auxin production
 Phosphate solubilization
 Protect plant from pathogens
Rout, M. E. (2014). The plant microbiome. In Advances in Botanical Research (Vol. 69, pp. 279-309). Academic Press.

9. BACKGROUND SUMMARY
 Plant is a major source of food for the world
 Several microbes interact with plants and plant parts
 Plant-microbe interactions may be pathogenic or beneficial to plants
 Studying these interactions help us to improve crop production

10. Studies on black pod disease of cocoa – Nigeria
Theobroma cacao
Cacao products:
Cocoa powder
Cocoa butter
Chocolate bar
Chocolate drinks
 Study conducted at Adekunle Ajasin University, Nigeria
 Nigeria: World’s 4th largest cocoa producer
 Black pod or Phytophthora pod rot is a major cocoa disease

11. Black pod disease of cocoa
 Pathogen: Phytophthora spp.
 Oomycetes: Fungus-like
 Survival forms:
 Oospore
 Chlamydospores
 Mycelium
 Niches on plants
 Below ground and above ground parts
 Spread by:
Soil, Water, rainsplashes, Wind, Insects Source: http://forestphytophthoras.org

12. Black pod disease of cocoa
Pathogen: Phytophthora spp.
Symptoms:
 Necrotic lesion on pods
 Discolored spot
 Brown or black lesion
 Whole pod blackens and shrivels
 Pod, tissue, pulps and beans rot
 Canker on stems
1 3
2
4
Source: Google

13. Study objective:
To isolate and identify pathogen causing black pod
disease of cacao in Idanre city

14. 1. Prepare tomato juice media
2. Surface sterilized infected pod
3. Plate infected part
4. Incubate at 25 C for 3 to 5
days
5. Transfer pure culture to new
plate
6. View spores under
microscope
1 2
3
46 5
Isolation and identification black pod pathogen
Identified as
Phytophthora megakarya

15. Summary of black pod disease studies
 Pathogen causing blackpod disease of cacao in Idanre was identified as
Phytophthora megakarya
 The study provided information for cocoa farmers and stakeholders in
Idanre Published 2014:

16. Studies on nematodes - Tennessee
 Interact with different plant parts  Several kinds plant nematodes

17. Studies on nematodes - Tennessee
Handoo,1998
 A global threat to crop
 Wide plant host range
 Can damage all plant
parts
 Huge financial loss
Forest plantsOrnamentalsFood crops
$80 billionGalls Lesions Patches

18. .
1. Survives as cyst (dead female) and eggs in
winter
2. Spring: Juvenile worms hatch from eggs
(24 °C), burrow into roots to feed and
develop.
3. Female juvenile worms stay in the root and
continue to feed.
4. Females make ~50 eggs outside their body
and fills up with another 200+ internally.
5. Fall: Females bodies harden to form cyst
after they die, and overwinter till next
season. Source: TheSCNcoalition.com
The Soybean Cyst Nematode Life Cycle

19. From TheSCNcoalition.com

20. Managements of nematodes
 Cultural
practices
Handoo,1998; Jones et al. 2013.
 Crop rotation Resistant varieties  Chemical/ nematicides  Biocontrol
Knowledge of nematode type and population levels would
enable better management decision.
Study objective:
To determine the population distributions and densities of
plant parasitic nematodes in row crop fields in Tennessee

21. Field Crop Pathology Lab Team
SAMPLING METHOD
 J2 and eggs extracted
from 100 cc of soil
 Row crop fields
 Corn
 Cotton
 Soybean
 1 in. diameter probe
 6 – 8 inches deep
 20 cores /20 acre area
1
32 4

22. SAMPLING AREA
 Number of samples: 173
 Number of Counties: 23
Kentucky
(KY)
Sampled County
Map of Tennessee and Kentucky, showing sampling area. Counties where samples were collected are painted blue.
Rufus Akinrinlola, January 31, 2018, from the website: ttp://www.herbarium.unc.edu/atlasmaps/USA-Can-clickable.html
Tennessee
(TN)
Not sampled

23. RESULTS
Nematode Infested
Not tested
 Sample infestation:
97 % of samples infested
167 out of 173
 Plant parasitic nematodes were found
in all tested counties.

24. SOYBEAN CYST NEMATODE
Infested
Non-infested
POPULATION DISTRIBUTION AND DENSITIES.
Map of Tennessee and Kentucky, showing sampling area. Counties infested with soybean cyst
nematode are painted orange. Blue is non-infested. Recreated by Rufus Akinrinlola.
Counties infested with soybean cyst nematode
40%
60%
SCNNO SCN
Percentage of fields infested
with SCN
83%

25. SOYBEAN CYST NEMATODE
Nematode
Eggs/ 100 cc of soil Soybean Thresholds
Min Max Average Low Moderate High
Soybean cyst
nematode
115 4,569** 847 200-2,000 2,000-5,000 5,000+
POPULATION DISTRIBUTION AND DENSITIES.
Soybean cyst nematode population densities in samples
Threshold modified from VA publication https://pubs.ext.vt.edu/content/dam/pubs_ext_vt_edu/spes/spes-15/SPES-15.pdf
SCN eggs
SCN cyst
SCN Juvenile

26. Infested
Non-infested
Map of Tennessee and Kentucky, showing sampling area. Counties infested with lesion nematode are painted
green. Blue is non-infested. Credit: Rufus Akinrinlola.
LESION NEMATODE
POPULATION DISTRIBUTION AND DENSITIES.
Counties infested with lesion nematode
57%
25%
75%
Lesion No Lesion
Percentage of fields infested with
lesion nematode

27. Nematode
J2/ 100 cc of soil Soybean Thresholds
Min Max Average Low Moderate High
Lesion
nematode
7 62* 15 0-18 20 – 58 60+
LESION NEMATODE
POPULATION DISTRIBUTION AND DENSITIES.
Lesion nematode population densities in samples
Threshold modified from VA publication https://pubs.ext.vt.edu/content/dam/pubs_ext_vt_edu/spes/spes-15/SPES-15.pdf

28. Non-infested
ROOT KNOT NEMATODE
POPULATION DISTRIBUTION AND DENSITIES.
Infested
Counties infested with root knot nematode
22%
4%
96%
Root-knot No Root-knot
Percentage of fields infested
with root knot

29. Nematode
J2/ 100 cc of soil Soybean Thresholds
Min Max Average Low Moderate High
Root-knot 23 446*** 187 0-8 10-32 34+
Threshold modified from VA publication https://pubs.ext.vt.edu/content/dam/pubs_ext_vt_edu/spes/spes-15/SPES-15.pdf
ROOT KNOT NEMATODE
POPULATION DISTRIBUTION AND DENSITIES.
Root knot nematode population densities in samples

30. Summary of nematode studies
 Soybean cyst, Lesion and root knot nematodes are found in many Tennessee field
 Soybean cyst nematode is most abundant, followed by lesion and root knot
nematodes
 The nematodes populations are very high in many of the fields
 Appropriate actions are needed immediately to avoid damages in the infested fields

31. Studies on beneficial microbes - Nebraska
Bacillus PGPR - plant growth-promoting-rhizobacteria
Yuen Lab Team
 PGPR:
 Root colonizing bacteria
 Increase plant growth
 Protect plants from pathogens
 Includes up to 12 bacteria genera
 Most common PGPR
 Bacillus, Burkoldhera and Pseudomonas

32. Bacillus as plant beneficial bacteria
Kumar et al., 2011; Xu and Côte, 2003
Source: Rufus Akinrinlola
 Microscopic and omnipresent in soils
 Niche: Different plant parts
 Survival traits:
 Multilayered cell wall structure,
stress-resistant endospores,
antibiotics, peptide signal
molecules, and extracellular
enzymes
 A preferred PGPR
 Stress-tolerant
 Broad-spectrum activity
 Protectant to plants
 Increase nutrients availability to
plants.

33. Bacillus as plant beneficial bacteria
Study objective:
To identify bacillus strains that can promote plant growth in
greenhouse.
Bacillus pumilus
Paenibacillus cineris
Lysinibacillus fusiformis
Photo credit: Rufus
Test strains
9 Bacillus strains
2 Peanibacillus spp
1 Lysinibacillus spp
North Platte NE

34. Lab procedures
 Bacteria isolated and preserved
in 80% glycerol at -75 ⁰C
 Stock culture prepared 10%
TSA for experiments
 Spread on TSA medium and
incubated at 28 °C for 48 hrs.
 Cells washed to make inoculum
suspension (108 cfu/mL)
 Seeds soaked in suspension for
30 to 60 min before sowing
 Control treatment soaked in
sterile phosphate buffer
Bacterial culture. Inoculum preparation. Seeds treatments
1 32
Akinrinlola, 2018.

35. Greenhouse procedures
 Non-sterile soil mix
 Ratio 2 to 1; Sands to
soil
 Seeds sown into potted
soil in greenhouse
 Complete randomized
design
 Watered appropriately
and grown for 20 days
 Roots separated from
shoot after washed
 Shoot height and fresh
weights measured
Soil mix Seed sowing Watering Data collectionData collection
1 32 3
Akinrinlola, 2018.

36. What was observed.
Untreated plants.
Published 2018:
Akinrinlola, 2018.

37. What was observed.
Untreated plants.
Published 2018:
Akinrinlola, 2018.

38. 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%)
C
Strains increased corn shoot weight significantly
 Shoot growth increased as high as 215% by strain R200Akinrinlola, 2018.

39. 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
AB
(121%)
AB (167%)
C
Strains increased corn root weight significantly
AB (122%) B (112%)
A (203%)
 Root growth increased as high as 203%Akinrinlola, 2018.
AB (135%)

40. Assays for for mechanisms of action

41. Strain
Anti
fungal
Anti
bacterial
Protease Chitinase
Bio-
surfactant
Siderophore Phosphate IAA
Nitrogen
fixation
Pouch
assay
R173
R176
R177
R180
R181
R198
R200
R174
R183
R190
R228
R232
Results for mechanisms of action

42. Summary of beneficial microbes studies
 Ten bacterial strains identified for plant-growth promotion ability
 Bacillus megaterium, B. simplex, and Paenibacillus graminis were among the most effective
in increasing plant growth
 The bacteria increased corn shoot and root growth at a high percentage
 Indo-acetic acid, protease and biosurfactant production were most frequent modes of
action
 More studies will test the bacteria for efficacy in field environment
Akinrinlola, 2018.

43. Take home message
 Many microbes interact with plants in several ways
 Some interactions are pathogenic while some are beneficial
 Phytophthora - and nematodes –plant interactions are pathogenic interactions
 Bacillus strains reported here are examples of beneficial plant-microbe interaction
 Our understanding of these interactions will help to improve plants/crop productivity
to meet global food demand

44. ACKNOWLEDGEMENTS
Dr. Heather Kelly
Dr. Zach Hansen
Rachel Guyer
Field Crop Pathology Lab Team
Arkansas Nematode Diagnostic Lab
Dr. Terry Kirkpatrick and Cathy Howard

45. Thank you!!
Real . Life. SolutionReal . Life. Solution
rakinrin@vols.utk.edu