Plant virome ecology in African farming systems:
assessing food security
“Alternate hosts for the viruses in weeds and non-maize crops”
MLND Workshop, Nairobi 21-23 August 2013
Francesca Stomeo / Mark Wamalwa
Post Doctoral Scientist - Genomics, BecA- ILRI Hub
(f.stomeo@cgiar.org)
(http://hub.africabiosciences.org) – (http://www. Ilri.org)
Plant virome ecology in African farming systems:
assessing food security
Donor:
•
Swedish Ministry for Foreign Affairs through SIDA
Partners:
•
BecA – ILRI Hub: Dr. Appolinaire Djikeng – Dr. Jagger Harvey
Dr. Francesca Stomeo - Dr. Mark Wamalwa
• KARI: Dr. Douglas W. Miano / Dr. Gichuki
• FERA: Dr. Neil Boonham – Dr. Ian Adams
• UoN: Dr. Dora Kilalo
Project Overview
Small Farming Systems
 Small farms (2 hectares, FAO 2001) are estimated to produce four-fifths of the developing
world’s food
 They are home to approximately two-thirds of the world’s three billion rural residents
 Key role in meeting the future food demands
 Are not homogenous
 Worldwide about half a billion farms are smaller than two hectares and they are getting
smaller due to: growing rural population, urban growth, formal and informal barriers to ruralurban migration, distortionary land policies and climate change
Project Overview
Rationale
 Small farm ecosystems:
• Complex mix of crop, non-crop plants
• Vectors and pathogens (Bacteria, Fungi, Virus)
 Estimated $60 billion global annual losses due to crop diseases
 Maize mixed farming system (maize plus pulses, banana, rice, sorghum,
cassava, yams, minor crops like groundnuts, pigeon pea, vegetables,
intercropped fruit trees and squash)
 Continuous cropping; lack breaks; replicated in different AEZs
 Great pathogens and vector diversity
 Most studies tend to focus on single pathogens affecting a single crop
Project Overview
Aims and Objectives
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Multi pathogens detection – hosts and potential vectors
Emerging diseases
SSA small-scale (mixed) farming systems
Focus on maize (main staple food in East and Southern Africa, 20% gross farm outputs
for small scale farmers)
 3 different Kenyan AEZs (climate change)
Project Overview
Sampling Strategy
 Trans Nzoia/ Uasin Gishu
 Major maize zones
 Upper midland and Lower highlands
 1800-200m asl
 Bomet
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Rainfall bimodal throughout the year with peaks in May, Aug, Nov
Maize grown on relay
Lower highlands, Upper midlands and lower midlands
Transition between the best maize growing areas and
pastoralist grazing land where farmers are moving
 Narok
 500-1800mm rainfall; unreliable rains
 Occupied by pastoralists with a lot of uncultivated land
 Some areas too dry and soil shallows unable to support crop growth
3-4 farms randomly selected will be samples from each area
Selected crops (and vectors) to be sampled:
maize and beans, grass and any other 3-4 (or more)
crop species
Trans Nzoia
Bomet
Narok
Project Overview
Maize Lethal Necrosis
• Maize Chlorotic Mottle Virus
• Sugarcane Mosaic Virus
MLN
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•
•
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MCMV
SMV
Symptoms: leaf, stem and ears
Mottling and chlorosis progressing to deformity and
necrosis
Foliar infection often associated with the leading
growth with early formed leaves remaining green
For plants with foliar or stem infection, grain filling
was shown to be markedly reduced
•
New disease first identified in the autumn of 2011
•
South Rift region (Narok North, Narok South,
Chepalungu, Sotik), Eastern Province
(Embu, Meru and Kibwezi) and Central Province
(Murang’a, Kirinyaga and Nyeri) of Kenya, Bomet
Project Overview
Methods
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•
•
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NGS sequencing (MiSeq)
Total RNA/dsRNA/small RNAs (viral diversity)
DNA
16S rRNA gene Metagenomics
(Plants, Soils, Vectors)
 Bioinformatics analysis
• Plant Virus Database/Pipelines
• Explore viral diversity through
automatically constructed
time-measured phylogenies
• Viromes comparison
Project Overview
Outputs
 Confirmation of known diseases/pathogens
 Pathogen Discovery / Emerging diseases
 Host range and vector information
 Risk analysis based on AEZs and dynamics of disease spread/climate change
 Policy makers, donors and researchers
Preliminary Analysis and Results
First Sampling
Bomet Farm 1
Trans Nzoia
Bomet
Narok
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•
•
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•
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• Mixed cropping system (maize, cabbage,
pumpkin, Irish potato, grasses, etc.)
• The MLND has been a threat to the farm.
As a result the farmer decided to plant
maize only in a small area
December 2012
• The farmer uses commercial seeds and
Bomet County
Consent Form, Questionnaire he does not control the disease
Botanist
Two Farms were sampled
Pilot Sampling
Bomet Farm 2
• Mixed cropping system
• The diseases present are: Northern Leaf
Blight, Common Rust, Maize Streak
Disease
• The farmer uses his own plant materials
and he does not control the diseases.
Preliminary Analysis and Results
First Sampling
Bomet Farm 1
Sample Type
Number
Bomet Farm 2
Sample Type
Description
Number
Description
10
6 SYM (1 RUST) - 4 ASYM (1 PROB
ASYM)
MAIZE
9
6 SYM - 3 ASYM
MAIZE
SORGHUM
5
TEA
5
5 ASYM (5 PROB ASYM)
PUMPKIN
8
1 SYM - 4 ASYM
1 SYM - 7 ASYM (6 PROB
ASYM)
ONION
6
5 SYM - 1 ASYM
SUGARCANE
5
5 ASYM (4 PROB ASYM)
IRISH POTATO
2
2 PROB ASYM
SOLANUM TUBEROSUM
9
4 SYM - 5 ASYM
CLIMBING BEAN
2
2 PROB ASYM
SWEET POTATO
5
PUMPKIN
1
1 ASY
PHASEOLUS VULGARIS
6
5 ASYM (4 PROB ASYM)
3 SYM (1 FUNGAL DESEASE)
- 3 ASYM
KALES
5
2 SYM - 3 ASYM
CABBAGE
2
2 SYM
NAPIER GRASS
4
4 PROB ASYM
NAPIER GRASS
CROTON MEGALOCARPUS
(TREE)
4
4 PROB ASYM
WEED
12
1
1 ASYM
MAIZE
10
12 PROB ASYM
6 SYM (1 RUST) - 4 ASYM (1 PROB
ASYM)
LANTANA CAMARA
2
2 ASYM
TEA
5
5 ASYM (5 PROB ASYM)
WEED
11
1 SYM - 10 ASYM
ONION
6
5 SYM - 1 ASYM
Total: 67
4 Soil samples and 3 Vectors (2 aphids, 1 whitefly)
Visual
Characterization!
Total: 47
4 Soil samples and 6 Vectors (5 aphids, 1 beetle)
Preliminary Analysis and Results - NGS
FERA – June 2013 – MiSeq Sequencing
Sample Type
Sample
ID
Sample Status
Sample Type
Sample
ID
Sample Status
Maize
M1
Symptomatic
Phaseolus Vulgaris
PV3
Asymptomatic
Maize
M2
Symptomatic
Phaseolus Vulgaris
PV3
Symptomatic
Maize
M3
Asymptomatic
Cabbage
C1
Symptomatic
Maize
M5
Symptomatic
Lantana Camara
LC1
Symptomatic
Sorghum
S1
Symptomatic
Amaranthus Hybridus
W6
Asymptomatic
Sorghum
S2
Asymptomatic
Weed
(Acantachaceae)
W1
Symptomatic
Pumpkin
P1
Symptomatic
Gyanadropis
W3
Asymptomatic
Pumpkin
P3
Asymptomatic
Datura Stramonium
W7
Asymptomatic
Sugarcane
SC1
Asymptomatic
Megalocarpus
CM1
Asymptomatic
Sugarcane
SC2
Asymptomatic
Napier Grass
NG4
Asymptomatic
Solanum
Tuberosum
ST7
Asymptomatic
Sweet Potato
SP1
Asymptomatic
Solanum
Tuberosum
ST8
Symptomatic
Sweet Potato
SP2
Asymptomatic
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Bomet Farm 1
RNA Extraction
Libraries Prep
Illumina TruSeq RNA Kit
No mRNA!
No Pools!
• Quality control
• Trimming
• Assembly of ORF
Preliminary Analysis and Results -NGS
Maize
M1 - Symptomatic
MCMV
M2 - Symptomatic
MCMV
Maize
M3 - Asymptomatic
M5 - Symptomatic
SCMV
MCMV
MCMV
MLND = MCMV + SCMV?
Sorghum
S2 -Asymptomatic
S1 -Symptomatic
MCMV
MYDV
SCMV
MCMV
Sweet Potato
SP1 - Asymptomatic
SP2 - Asymptomatic
MCMV
MCMV
Sugarcane
SC1 - Asymptomatic
SC2 - Asymptomatic
MCMV
MCMV
Napier Grass
Weed
Weed 1 - Symptomatic
NG4 - Asymptomatic
MCMV
MCMV
SCMV
Maize Chlorotic Mottle Virus
Genome Coverage
ST, PV, W
(Potato, Beans,
Weeds)
Sugarcane Mosaic Virus
Genome Coverage
Community Reconstruction and Sample Diversity
Community reconstruction and sample diversity
Discussion
 The results show greater estimates of viral richness
 MCMV and SGMV detected
 High coverage
 Is the combination of the two viruses (MCMV and SGMV) a paramount
requisite for the appearance of the Maize Lethal Necrosis?
Future Activities
Bomet Farm 2
 Deep Data Analysis / Paper Writing
 Second Run FERA
 TruSeq RNA MiSeq Sequencing
 Second sampling September 2013
 RNA (smallRNA) Seq, MiSeq, BecA – ILRI Hub
 16S Metegenomics
 Vectors
Sample Name
M1
M2
M3
M6
M7
M9
M4
M8
O1
O2
O4
O5
O6
O3
K2
K3
K4
T1
T4
IP2
CB1
P1
NG3
W5
Sample Type
Maize
Maize
Maize
Maize
Maize
Maize
Maize
Maize
Onion
Onion
Onion
Onion
Onion
Onion
Kale
Kale
Kales
Tea
Tea
Irish potato
Climbing Bean
Pumpkins
Napier Grass
Weed
Sample Status
Symptomatic
Symptomatic
Symptomatic
Symptomatic
Symptomatic
Symptomatic
Asymptomatic
Asymptomatic
Symptomatic
Symptomatic
Symptomatic
Symptomatic
Symptomatic
Asymptomatic
Symptomatic
Symptomatic
Asymptomatic
Asymptomatic
Asymptomatic
Asymptomatic
Asymptomatic
Asymptomatic
Asymptomatic
Asymptomatic
Capacity Building Related Activities
ABCF Fellow: Jane Wamaitha M., PhD, NAIST, Japan
• Understanding the genomics of Maize Lethal
Necrotic Virus and other potyviruses
• Based at Kenya Agricultural Research InstituteNARL, Biotechnology section
Concept note submitted to Africa Biosciences
Challenge Fund (ABCF) - 2013
Acknowledgment
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Appolinaire Djikeng
Mark Wamalwa
Neil Boonham
Ian Adams
Douglas W. Miano
Dora Kilalo
Joyce Njuguna
Jagger Harvey
Martina Kyalo
BecA Hub Team!
 Swedish Ministry for Foreign Affairs through SIDA