Recent developments in diagnostics of Foc: practical applications toward disease management
Miguel Dita – Embrapa, Brazil Gert Kema – PRI-WUR, The Netherlands
FAO, Rome, December 2014
Map
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Razas 1, 2
R4T por confirmar oficialmente
R4T, 1, 2
Map
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avid
Bro
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? ?
Fusarium wilt: Global distribution Possible scenarios for transcontinental spreading
Este MAPA es un intento aproximado de la distribución de
Foc realizado para fines académicos, no está basado en
estudios científicos y no debe ser tomado como referencia
por las autoridades
Symptoms of Foc TR4 are identical to those caused by
Foc R1 and R2
Race 1 – Gros Michel, Costa Rica TR4 –Cavendish ,Taiwan
© M
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© M
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Cortesía Wayne O’Neil - Australia TR4 – Cavendish, Australia Rac 2 – Monthan (ABB), Brazil
Symptoms of Foc TR4 4 are identical to those caused by
Foc race 1 and 2
Foc can not be distinguished morphologically from other Foxys
Sou
rce:
Sm
ith
(2
00
7)
To stop /control something first we need to identify it !
1st Diagnostic tools for Foc – 2008, 2010
ST4 + TR4
~ 140 year after the 1st disease report
VCGs: 012-0, 1, 2, 6, 9, 10, 11
Specific for TR4
18S$ 28S$ 18S$28S$
ITS IGS IGS
VCG01213/16
0 1 2 3 4 5 6 8 9 10 11 13 14 15 18 19 20 22 24 12 M M
242 bp
Lin et al. (2008)- Foc-1/ Foc-2
Primers - FocTR4-F/ FocTR4-F (Dita et al. 2010)
VCG 01213
Foc-1/Foc-2: Positive isolates from Honduras, Brazil and Costa Rica!
Diagnostic tools for Foc – 2008, 2010
2012
… as low as 10−4ng gDNA and 20 conidia, respectively.
The SCAR primer set FocSc-1/FocSc-2 (5′-CAGGGGATGTATGAGGAGGCTAGGCTA /5′-GTGACAGCGTCGTCTAGTTCCTTGGAG)
It is not specific for TR4! – It just detects R4 [7 VCGs]
Diagnostic tools for Foc TR4
2013 LAMP
Validation, improvement and implementation of LAMP for TR4- Needed
• Amplification under isothermal conditions (60-65 ° C); No Thermo cycler needed • Capacity for “reading” and quantification at a glance; No gel/electrophoresis needed • High sensitivity and specificity (3 primers set) – Robust, multi-target • Faster (30 min), low cost, easy application - Directly on field;
Diagnostic tools for Foc TR4
Data base analyses
• Initially 80 unique fragments
• Embrapa data base resulted in 12 unique fragments for TR4
• We’re currently validating three primer sets on all VCGs and various other Fusaria
• We’re extending the data base to ~ 400 isolates
spores/ml log spores Average 1,000,000 6 17.74 100,000 5 16.19 10,000 4 19.09 1,000 3 24.80 100 2 27.68 10 1 27.16 1 0 28.83
Detection threshold
spores/g soil log spores Ct value
100,000 5 21.87
1000 3 31.41
100 2 34.45
10 1 34.88
Detection threshold
qPCR/LAMP - Sensitivity tests
Towards LAMP on-site TR4 diagnosis
Sample collection
Pseudostem, petiole etc.
Place , variety, etc
DNA
Extraction
samples
RT4?
Komada media
RT4?
Foc -PDA
?
Send samples to a lab
with information
about plantation
Samples processing
Komada media
Fungus Isolation
Purification
PDA media
DNA
Extraction
DNA
Extraction
PCR
Stages
LAMP 30 mins - Field
6 hours - Lab
3-5 days - Lab
Weeds
Pla
ntin
g m
ater
ial
Diseasecenter
Irrigation
Soil
1
2
3
AdaptedfromHwang&Ko2004
Dita et al. (2013)
Linking Diagnostic to Epidemiology & Management
calculated using the equation y=-4.9117x+37.229 (standard curve for pure spore suspension)
Fusarium TR4 Inoculum Level at Different layers of soil and shoes (preliminary results)
1 Shoes with soil and rice bran (used in the Foc infested farm in the Philippines)
a average Ct value of 10 independent extractions
b calculated using the equation y=-4.9117x+37.229 (standard curve for pure spore suspension)
Ave. Ct valuea Calculated # of spores/g soilb
36.487
142
Amplification curve Melting curve
Blue – positive control
Green – soil sample
Foc TR4 Detection in soil from shoes 1
1 calculated using the equation y=-4.9117x+37.229 (standard curve for pure spore suspension)
TR4 Quantification Field Water
• Irrigation water
• Test positive
• 75 spores/ml
Key biological and epidemiological caracteristics for Foc management vs. Diagnostic
Foc can be dissminated in symptomless, but infected planting material
Latent period is long - the disease coud be only detected
after a considerable period
It is easily disseminated in soil particles and water
Foc can survive as a non pathogenic
endophyte in weeds and other hosts
Chlamydospores can survive in the soil for
long periods
Disease symptoms of TR4 are similar to R1, R2
Diagnostic vs. Management
1. Quarantine Services - Exclusion
2. Supporting eradication practices – Prevencion/Eradication
3. Certification of planting material – Prevention/Exclusion
4. Surveillance and monitoring – Preventive actions, risk analyses
5. Support decision making process – Cultivar deployment, land use
Geografic distribution (GS +)
Kmeters
K.m
ete
rs
Contingency plan for Foc TR4 • A regional template for National Contingency Plans • Free-online knowledge source for LAC
1. Quarantine Services
A. M
olin
a
According to Horticulture Plant Protection Department (2007) reported that epidemic rates of Foc race 4 in Sumatra and other
province reach 100 km/year
A. M
olin
a
2. Supporting eradication practices
RIP - Killed by TR4
Next victim
Future victim?
Early diagnostic of Fusarium wilt vs. management
When a plant is detected infected how many ‘neighbors’ we should destroy around?
Farmer and companies think more on bunches and boxe$… they don’t care too much on inoculum pressure, epidemiology ? ….but at the end of the day the scenario just becomes worst
1. Erradicate plants in the zone “A” – mandatory 2. Erradicate plant in zone B - Recommended
Whenaplantisdetectedinfectedhowmany‘neighbors’weshoulddestroyaround?
Farmersthinkmoreonbunche$…theydon’tcaretoomuchoninoculumpressure,epidemiology?….butattheendtheyloosetheba le
Area%A%( 15%m)
Area%B%( 40%m)
Infected Symptomless,butinfected?
2. Supporting eradication practices
• Infected plant
• Farmer may not be
aware about Foc
infestation and use
(even sell)
symptomless, but
infected suckers
• Healthy plant ?
• Farmer leave this
plant in the field or
use suckers as
planting material ?
3. Certification of planting material
Valle del Cauca
Colombia
3. Surveillance and monitoring – preventive actions
Dat
a: C
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man
/ M
ap: D
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~ 150 Farms mapped ~ 80 % Foc Collection established
3. Surveillance and monitoring – risk analyses
Early Detection: A Key Action
Whenaplantisdetectedinfectedhowmany‘neighbors’weshoulddestroyaround?
Farmersthinkmoreonbunche$…theydon’tcaretoomuchoninoculumpressure,epidemiology?….butattheendtheyloosetheba le
Area%A%( 15%m)
Area%B%( 40%m)
Infected Symptomless,butinfected?
• Activate contingency plans • Containment of the first outbreak • Management at different levels
• Farm, • Multi-farm, • Landscapes, Watershed,
Territory, • Country • Region
Symptomless, but Infected
Infected
• Surveillance, • Monitoring, • Eradication • Certification of planting material
• Diagnostic capacities • Responsiveness
Trained
In the pipeline
Bioversity, MUSALAC
& National Plant
Protection Organizations
Strengthening capacities and increasing preparedness for Foc TR4 in LAC
Farmer
Host
Environment
Pathogen
Weak points on productios systems vs. Foc Management
Susceptible varieties
Monocrop systems
Low genetic diversity
Acid soils
Desqilibrium (Chemical, Phisical, Biological)
Inadequated topography- enabling flooding
Poor drainage; Adequate temperatures
High virulent Wide host range
No application (or lower)
of organic ammendements
Introduction of planting material
and soil from infected sites
Lack of erradication
measures Farms with open
access with intern
walking routes
No fences Low levels of information on
epidemology & management
LAC already has serious problems with Foc R1/R2
© M
igu
el D
ita
Rome, December, 2014