This presentation summarizes a study on molecular determination and characterization of phytoplasma 16S rRNA gene in selected wild grasses from western Kenya. The study aimed to detect and identify phytoplasma strains infecting wild grasses and identify host grass species. Over 600 grass samples were collected and tested via PCR and sequencing. The results showed that phytoplasma is widespread in local grass populations, with over half of infections being latent. Several grass species like C. dactylon and D. scalarum had high infection proportions and acted as wild phytoplasma hosts. Two phytoplasma subgroups, 16SrXI and 16SrXIV, were identified infecting the grasses. The study concludes that wild gr

1. PRESENTION BY;
Okinyi J. Adam
I56/10103/08
(Kenyatta University)
SUPERVISORS;
Dr. Runo S. Maina
Department of Biochemistry and Biotechnology
Kenyatta University
Dr. Charles A. O. Midega
Department of Plant Health
International Center of Insect Physiology and Ecology.
Molecular determination and characterization of
phytoplasma 16S rRNA gene in selected wild
grasses from western Kenya

2. HypothesisBackground information
• Napier grass an indigenous tropical African
clumping grass, is the major livestock feed in
zero-grazing systems in western Kenya.
• In East Africa, Napier stunt phytoplasma poses a
serious threat to Napier grass farming.
• The disease symptoms include severe stunted
growth and loss of biomass
• It is mainly transmitted by a leafhopper
Maeistas (=Recilia) banda in Kenya (Obura et al.,
2009)
Photographs illustrating the
comparison between Health (a) &
Diseased (b) Napier grass.
a
b

3. Life cycle of NSD in the region
Recilia banda

4. Statement of the problem
• Napier stunt disease has reduced Napier productivity by 30-90% in
the region
• Phytoplasma attacks other wild grasses, it is likely that several wild
grasses could be infected by specific phytoplasma strains
• These wild grasses might also act as reservoirs for fresh inoculums
• The determination and characterization of phytoplasma 16sr RNA
gene in wild grasses from western Kenya is necessary for precise and
sustainable phytoplasma disease management
BGWL HWLD NSD

5. Hypothesis
There is no diversity of phytoplasmas and wild
grasses hosting phytoplasmas in western
Kenya.

6. Objectives
General Objective
• To characterize phytoplasmas, and describe their host
range among wild grasses in western Kenya.
Specific Objectives
• To detect and identify phytoplasma strains infecting
wild grasses in western Kenya
• To identify wild grass species hosting phytoplasmas in
western Kenya.

7. Study AreaKEY:
 Positive
 Negative

8. Sampling strategy
QuadratTransects
1m
1m
Grass field boundary
1-3m
1-3 m

9. Phytoplasma detection & Characterization
Collection of leaf
samples (300mg)
DNA Extraction
(CTAB)
PCR amplification
P1/P6 then
NapF/NapR
Purification of PCR products
genotypic Sequencing
of PCR products
Sequence editing via
BioEdit softwareComparison of
sequences using BLAST
search at NCBI
Phylogenetic analysis by
neighbour joining method
(Saitou & Nei, 1987)

10. Symptomatic C. dactylon as observed in the field: (A) and (C)
Chlorosis of above ground plant parts. (B) and (D) shortened
internodes, stunted growth and bushy plants.
A
C
B
D

11. Gel electrophoresis results of 1.0L purified PCR
products obtained for direct sequencing
2000
1500
1000
500
700
100
300

12. Unrooted bootstrap consensus tree illustrating the phylogenetic relationships for the 16Sr RNA
genes of the phytoplasma strains derived from wild grasses in western Kenya.

13. Unrooted computed linearized phylogenetic tree assembled by the Neighbour-Joining method
highlighting two major groups of phylogenetic relationships for phytoplasma isolates

14. Phytoplasma 16S rDNA sequences retrieved from the GenBank
and employed in phylogenetic analyses in this study.
Isolate
Phytoplasma
species
16S rRNA
Group-
subgroup
Host species Location
NCBI
Accession No.
Literature
Aster yellows Ca. P asteris 16SrI Cannabis sativa L India EU439257.1 Raj et al, 2008
Napier grass stunt Ca. P oryzae 16SrXI P. purpuruem Kenya, Mbita FJ862999.2 Obura et al, 2009
Napier grass stunt Ca. P oryzae 16SrXI P. purpuruem Kenya, Bungoma FJ862997.2 Obura et al, 2009
Bermuda grass white leaf Ca. P cynodontis 16SrXIV Cynodon dactylon China EU999999.1 Li et al, 2008
Rice yellow Dwarf Ca. P oryzae 16SrXI Oryza sativa Vietnam JF927999.1 Trinh et al, 2011
Peanut witches’ - broom Ca. P aurantifolia 16SrII Citrus araurantifolia Oman, Rumis AB295060.1 Natsuaki & Al-Zadjali, 2007
X-disease Ca. P pruni 16SrIII-A stone fruits, Prunus U.S.A/ canada JQ044393.1 Davies R. E., 2011
Stolbur Ca. P solani 16SrXII-A Solanum tuberosum Romania/ Russia HQ108391.1 Ember et al, 2011
Elm yellows Ca. P ulmi 16SrV-A Ulmus spp Serbia HM038459.1 Jovic et al, 2011
Clover proliferation Ca. P trifolii 16SrVI Calotropis gigantean India: Gorakhpur HM485690.1 Priya et al, 2010
Ash yellows Ca. P fraxini 16SrVIIA Graminella nigrifrons Canada JN563608.1 Arocha-Rosete, 2011
Pigeonpea witches'-broom Ca. P phoenicium 16SrIX Blueberry U.S.A JN791267.1 Lee et al, 2012
Apple proliferation Ca. P mali Gn-16SrXA Graminella nigrifrons Canada JN563610.1 Arocha-Rosete, 2011
Apple proliferation Ca. P pyri 16SrX Cacopsylla pyri Portugal JN644986.1 Sousa et al, 2011
Mexican periwinkle viresc Unidentified 16SrXIII-A Catharanthus roseus U.S.A AF248960.1 Dally et al, 2000
Bermuda grass white leaf Ca. P cynodontis 16SrXIV Dicanthium annulatum India FJ348654.1 Rao et al, 2008
Hibiscus witches'-broom Ca. P brasiliense 16SrXV Prunus persica Azerbaijan FR717540.1 Balakishiyeva et al, 2010

15. A dendogram of partial 16S rRNA gene sequences from 33 wild grass
phytoplasmas from western Kenya

16. Grass species
PCR status Percentage
of infection
Total
0 1
Brachiaria brizantha 71(0.8452) 13(0.1548) 13(16.0000) 84
Cenchrus ciliaris 0 1(1.0000) 1(1.2000) 1
Cymbopogon nardus 2(1.0000) 0 0 2
Cynodon dactylon 55(0.6395) 31(0.3605) 31(38.3000) 86
Digitaria scalarum 286(0.9533) 14(0.0467) 14(17.3000) 300
Echinichloa pyramidalis 2(1.0000) 0 0 2
Eleusine indica 6(0.7500) 2(0.2500) 2(2.5000) 8
Eragrostis curvula 4(1.0000) 0 0 4
Hyparrhenia pilgerama 6(1.0000) 0 0 6
Other 65(0.8784) 9(0.1216) 9(11.1000) 74
Panicum maximum 28(0.8750) 4(0.1250) 4(4.9000) 32
Pennisetum polystachion 5(1.0000) 0 0 5
Pennisetum purpureum 1(1.0000) 0 0 1
Poverty grass 24(0.8000) 6(0.2000) 6(7.4000) 30
R. cochinchinensis 1(1.0000) 0 0 1
Setaria incrassata 2(0.6667) 1(0.3333) 1(1.2000) 3
Sorghum versicolor 2(1.0000) 0 0 2
Sporobolus pyramidalis 4(1.0000) 0 0 4
Themeda triada 1(1.0000) 0 0 1
Total 565 81 81(100) 646
Chi square test 75.787(a)
df 18
Likelihood Ratio 68.054
P Value (≤0.05) 0.001
Total grass species, their phytoplasma statuses and the proportions of
infection

17. grass species collected and their associated 16S rRNA sub-group
Grass species 16SrXI 16SrXIV Not done Total
B. brizantha 4(57.14%) 3(42.86%) 77 84
C. ciliaris 0 0 1 1
C. nardus 0 0 2 2
C. dactylon 2(18.18%) 9(81.81%) 75 86
D. scalarum 6(100%) 0 294 300
E. pyramidalis 0 0 2 2
E. indica 1(100%) 0 7 8
E. curvula 0 0 4 4
H. pilgerama 0 0 6 6
Other 2(100%) 0 72 74
P. maximum 3(100%) 0 29 32
P. polystachion 0 0 5 5
P. purpureum 0 0 1 1
Poverty grass 3(100%) 0 27 30
R. cochinchinensis 0 0 1 1
S. incrassata 0 0 3 3
S. versicolor 0 0 2 2
S. pyramidalis 0 0 4 4
T. triada 0 0 1 1
Total 21 12 613 646

18. Association between Phytoplasma strains and Grass species

19. Association between Phytoplasma strains and Locations of survey

20. Inferences
• There is great diversity of wild grasses in Busia and Bungoma districts with D.
scalarum, C. dactylon, B. brizantha grasses being most abundant: (72.5%)
• 63% of all sampled phytoplasma positive grasses had latent infections
• C. dactylon, D. scalarum and B. brizantha had the highest proportions of
infections at 38%, 17.3% and 16% respectively
• C. dactylon and B. brizantha grass species were the only grasses that
registered phytoplasma sub-group 16SrXIV infections
• 16SrXI group of phytoplasma infects wide range of grasses; D. scalarum, P.
maximum, E. indica, poverty grass and to a lesser extent C. dactylon and B.
brizantha.

21. Conclusions
1. Phytoplasma is widespread in many local wild grass populations in Busia
and Bungoma counties
2. Approximately more than half of phytoplasma infections are latent/
assymptomatic
3. C. dactylon, B. brizantha, D. scalarum, P. maximum and poverty grass act
as wild phytoplasma hosts and are abundantly distributed
4. Phytoplasma sub-groups 16SrXI and 16SrXIV are the only groups infecting
wild grasses in western Kenya. (No novel strain)

22. Recommendations
• Establish a buffer zone clear of wild grasses around Napier fields to reduce
the risk of phytoplasma inoculation
• Conduct back-transmission studies to confirm transmissibility of Ns
phytoplasma to Napier grass wild grasses
• Carry out a wider survey with a larger sample size to fully understand the
dynamics of Ns disease
• Need to genetically engineer napier grass for the production of transgenic
tolerant clones against Ns disease

23. Acknowledgement
• KENYATTA UNIVERSITY
• SUPERVISORS
• I.C.I.PE

24. Thank you