Identification of Cowpea Aphid Borne Mosaic Virus in Diseased
Passionfruit (Passiflora edulis Sims) and Diagnostic Test Development
Kilalo, Dora1,4, Elijah Ateka2, Hanu Pappu3, Rob Skilton4 and Jagger Harvey4
1Dept.of Plant science and Crop Protection, University of Nairobi; 2Dept. of Horticulture, Jomo Kenyatta University of Agriculture and
Technology; 3Dept. of Plant Pathology, Washington State University; 4 Biosciences eastern and central Africa Hub, ILRI, Nairobi, Kenya
Importance of passionfruit
I f i f i Expected Outputs
•Passionfruit is a major export fruit crop in Kenya (US $375,000/year with supply not yet • Virus pathogen(s) causing passionfruit woodiness disease in Kenya identified.
meeting total demand). • A test for virus diagnosis in passionfruit developed and passed to Kenya Plant Health
•Its production creates employment and provides raw material for processing. Inspectorate Service (KEPHIS) and other key organizations to help in management of
•The fruits are nutritious, containing Vitamins A, C, D and potassium. the disease.
•This crop is capable of helping to alleviate poverty and malnutrition among small scale • Trained personnel that will identify viral pathogens in passionfruit and certify
farmers. nurseries as having virus free planting materials for farmers.
•Pests and diseases have been a major hindrance in the production of passionfruit. • Improvement of crop quality and yields for smallholder and larger scale farmers alike.
•Passionfruit woodiness disease is a critical disease affecting the crop in Kenya and
regionally. However, the causal agent is unknown, complicating management efforts.
Objectives Results
Broad objective: •Virus symptom incidence averaged 65% (figure below)
To contribute knowledge toward developing a certification system of •Severity average score was 3 of 5
planting materials and improve passionfruit production in Kenya •There was no difference in severity and incidence between the two zones
•ELISA confirmed virus incidence was 70%, (CABMV (46%), CMV (4%)
Specific objectives other potyviruses (21%)
• To identify viral pathogens infecting passionfruit in Kenya •All 48 CABMV ELISA positive samples amplified a ~658bp DNA fragment
• To develop and validate a detection tool for viruses infecting passionfruit using primers specific to the CABMV coat protein gene
Incidence of passionfruit viruses in infected samples BLAST results from CABMV E Value
sequences (% identity)
Methodology
40
35
SAPV (98%) 0.0
% incidence
incidence (%)
30
25 Zone 1 Zone 2
20
CABMV‐F 144 (92%) 0.0
15
10 Sesame mosaic potyvirus 1e‐143
5 (86%)
0
CMV CABMV Gen. potyvirus
Viruses
Mosaic symptoms
Sample areas A Conclusions
(stratified according to agroecological zones; elevation and temperature differences): D
B
Zone 1 = A, B C
• CMV, CABMV and other potyviruses are affecting passionfruit in Kenya
, p y gp y
>2000m above sea level (asl)
• These are widely distributed in all the growing areas
Zone 2 = C, D
<2000m asl • CABMV Kenyan isolates are comparable to that of South African passiflora
7 farms per area, 4 plants/farm randomly selected, virus (SAPV) and Brazil (CABMV-Br), where woodiness disease has been
young leaves from 5 different growing points per
vine. reported and is comparable to CABMV found affecting Sesame in Georgia,
USA
Future Directions
Healthy leaf •Diagnostic test to be validated and passed to the Kenya Plant Health
Mottling of
passionfruit leaves Inspectorate Service (KEPHIS) and other key partners.
•454 next generation sequencing to be used for pathogen discovery in
454
diseased passionfruit samples (have we missed other viruses or other
microbes?); see figure below.
Protein extraction (for ELISA)
ELISA RNA extraction (for RTPCR) •PhD to be conferred to Dora, pending submission and defense of
(blue = positive)
Used to initially
screen for positives
dissertation.
All positives, some negatives and internal control
(for virus identification and false negative ELISA Virus/pathogen discovery by 454 sequencing
frequency estimation)
RNA
Healthy and infected
passionfruit plants 454 sequencing
RTPCR result of cloned sRNAs
( C diagnostic
(PCR d ag ost c test
development)
Gel purification of small
All positives sequenced RNA (sRNA) fraction
Virus coat protein gene sequence
Adaptor barcoding of healthy 1 1
and infected samples and
cDNA synthesis 2 2
Sequence database searches
Multiple sequence alignment Clonal amplification
(virus identity and diversity) of cloned sRNAs
Pathogen identification by
sequence analysis
Your ins/
PU