enhanced regional diagnostic protocols, tools and
TRANSCRIPT
Enhanced regional diagnostic protocols, tools and information platforms fit for purpose in cassava systems of Southeast Asia
Wilmer J. Cuellar
Virology and Crop protection, Cassava Program
Crops for Nutrition and Health
(Twitter: @WJCuellar)
Activity 3 Project update
Establishing sustainable solutions to cassava disease in mainland Southeast Asia
Objective 3. Diagnostics and surveillance
Develop, test and deploy diagnostic protocol, tools, and information platforms fit for purpose in monitoring, surveillance, and certification applications
Great team work
CIAT: Jenyfer Jimenez, Ana M. Leiva, Diana Lopez, Maria I. Gomez, Juan M. Pardo, Derlyn Lourido, Rafael Rodriguez
Laos
Cambodia Vietnam
Thailand
Laos
Field sampling
• Field sampling and molecular Dx are expensive activities, we considered the available resources and the level at which we would like to detect the pathogen, setting accordingly the design prevalence and confidence level.
• Cassava: ~10,000 plants/Ha. In collecting 60 observations/Ha we have a 95% probability of detecting as low as 5% CMD. The probability increases if we do molecular Dx (detection of asymptomatic infections).
• If 0/60 there is no 100% certainty that the pathogen is not present. What we can say is that if the pathogen is present its prevalence is below 5%.
• Example: If 4 out of 60 are symptomatic = 6.67% (confidence interval 0.37-12.96%. If 11 out of 60 (counting asymptomatic plants) is infected = 18.33% (confidence interval 8.57 – 28.10%)
Testing protocols –> PDP projects12/17/20206
Field no
LocationNr of plants
% Symptomati
c plant
% PCR positive
% Latent infections
1 13.9386,102.78472 90 43 51 8
2 13.8938,10274472 30 13 16 3
3 13.8844,102.7377 30 3 7 4
4 13.8658,102.7250 30 16 16 0
5 13.9750,102.7827 30 0 0 0
6 13.9300,102.7511 30 17 21 4
7 13.9408,102.7880 30 16 16 -
8 13.7804,102.4569 60 27 76 49
9 13.8244,102.4298 30 33 56 23
10 13.8140,102.5090 30 30 83 53
11 13.7258,102.5547 30 0 17 17
12 13.7177,102.6047 30 0 0 0
13 13.7988,102.5533 30 43 40 3
14 13.8061,102.5958 30 10 10 0
15 13.8275,102.7019 30 23 23 0
Siriwan et al., and Cuellar WJ. 2019. Virus Research
Thailand
Cambodia
CMD in SEA: PestDisPlace project PDP_00063
• Project profile created and running• Maps are updated as soon as the field and lab information is shared• Data available to project participants
CMD in SEA Project: PDP_00063
• Images are organized according to the leaf samples taken
• You can easily find specific fields or samples for a more detailed revision of symptoms
CMD in SEA Project: PDP_00063
• Once you have the Dx lab results, you can update the info for every sample
• You can also update the Dxinformation in batches
CMD in SEA Project: PDP_00063
Molecular diagnostics
• Molecular diagnostics allows us to compare prevalence of infection versus prevalence of visual symptoms. Tests must be affordable, rapid, large scale.
• The probability that test is positive if the sample is infected depends on the ability of the NPPOs officer to recognize the symptoms and the sensitivity of the detection method.
• Need to standardize DNA extraction protocols, primer sets, symptoms identification (CTAB nucleic acid extraction).
• Need to run inter-laboratory tests, once the regional labs are set up.
Confirmation of CMD and SLCMV in Laos (CTAB method)
• More than 1400 dry leaf samples have been sent to CIAT for molecular Dx• ~99.5% of CMD symptomatic samples analyzed so far are positive to SLCMV by standard PCR• Protocols are shared with all partners – we expect partners can implement Dx protocol by 2021
CMD symptoms identification Dx of SLCMV by PCR
Evaluation of homemade DipSticks for rapid detection of SLCMV
• Start with 20 mg of dried top youngest leaves• Processing before PCR: 90 samples/2 hours/person• False negatives due to low virus titers?• DipSticks recommended for rapid confirmation of
SLCMV in symptomatic plants.
Extraction Washing Elution
Not infected Asymptomatic Symptomatic
Sam
ple
sP
roce
ss
Dipsticks
0
5
10
15
20
25
30
Non-Infected InfectedAsympomatic
InfectedSymptomatic
PCR results (3 replicates)/31 samples per treatment
CTAB
Not infected Asymptomatic Symptomatic
CWBD symptoms and molecular test for phytoplasma 16sr1
M 1 2 3 4 5 6 7 8 9 10 C 11 12 13 14 15 16 17 18 19 20 C 21 22 23 24 25 26N=120 CWBD
symptomsNo CWBD symptoms
PCR(+) 4 23
PCR (-) 13 80
• More than 1400 dry leaf samples have been sent to CIAT for molecular Dx• Only 23.5% of CWBD symptomatic samples analyzed so far are positive to phytoplsama 16sr1
CMD in southern Laos
Village: PindongDistrict: SanumxayProvince: AttapeuCountry: Lao PDRF67: 14.4142058, 106.2537766(but all fields labelled in Attapue-Laos, fall within Cambodia)
We still have adjustments to make
Confirm mixed symptoms by molecular Dx
Improve the accuracy of GPS location data
More than one field with the same GPS location data
Genome surveillance
• Changes in the genetics and biology of the virus can be triggered by infecting different hosts, by climate variations and recombination with related virus species (there are arounf 12 diff species of CMD-related geminiviruses
• We started carrying on virus genome surveillance for early detection of genetic variations in the virus and rapid ID of newly introduced geminiviruses in SEA
• Portable sequencing technology has been standardized and multiplexed to make it affordable. First results are encouraging, prices can be reduced 10 times/per virus genome.
• Lab training in SEA on this technology was part of a 2020-EM project funded by RTB, but had to be cancelled due to COVID-19 travel restrictions.
Genome surveillance of SLCMV
• Project maps inform us on the new occurrence of the disease and the virus
• Specific samples are selected for virus full genome analysis
Genome surveillance of SLCMV (using Nextstrain)
https://nextstrain.org/community/pestdisplace/CMDASIA
Virus population in SEA split into 2 phylogenetic groups
SampleTotal
Reads
Total
Reads-
Virus
Begom
ovirusACMBFV ACMV EACMCV EACMKV EACMMV EACMV EACMZV EACMUV SACMV SLCMV ICMV
B1-59 10806 9090 99% 0% 0% 0% 0% 0% 0% 0% 0% 0% 40% 5%
B2-61 10251 4792 98% 0% 0% 0% 0% 0% 0% 0% 0% 0% 43% 7%
B3-44 2454 1899 99% 0% 0% 0% 0% 0% 0% 0% 0% 0% 41% 5%
B4-51 30604 21668 99% 0% 0% 0% 0% 0% 0% 0% 0% 0% 39% 5%
B5-64 2120 523 95% 0% 0% 0% 0% 0% 0% 0% 0% 0% 45% 6%
B6-H18 13396 4334 95% 0% 0% 0% 0% 0% 0% 0% 0% 0% 37% 5%
Surin1 57762 12638 79% 0% 0% 0% 0% 0% 0% 0% 0% 0% 24% 7%
Generic detection of cassava-infecting begomoviruses
Po
rtab
le s
eq
ue
nci
ng
tech
no
logy
Amplification using random hexamers + phi29 Whole genome assembly Considers coverage and depth to account for mis-identification of ICMV
SLCMV
Cassava Common Mosaic DiseaseThe Americas (and Asia?)
Cassava Mosaic DIseaseAfrica, Asia
Similar symptoms, different pathogens
B
Lozano et al. 2017. Virus Research.Tuo et al., 2020. Plant Disease.
Recent report of cassava common mosaic (an RNA virus) in China
A
Cassava Common Mosaic DiseaseThe Americas (and Asia?)
Cassava Mosaic DIseaseAfrica, Asia
Similar symptoms different pathogens
B
Lozano et al. 2017. Virus Research.Tuo et al., 2020. Plant Disease.
Recent report of cassava common mosaic (an RNA virus) in China
A
Communication
Thank you!
Wilmer J. Cuellar
Virologist
Twitter: @WJCuellar