Recent developments in diagnostics of Foc: practical applications toward disease management

Transcription

1 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

2 Fusarium wilt: Global distribution Possible scenarios for transcontinental spreading Mapa: David Brown Mapa: D. Brown?? Razas 1, 2 R4T, 1, 2 R4T por confirmar oficialmente 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

3 Symptoms of Foc TR4 are identical to those caused by Foc R1 and R2 Miguel Dita Miguel Dita TR4 Cavendish,Taiwan Race 1 Gros Michel, Costa Rica

4 Symptoms of Foc TR4 4 are identical to those caused by Foc race 1 and 2 Cortesía Wayne O Neil - Australia TR4 Cavendish, Australia Rac 2 Monthan (ABB), Brazil

5 Source: Smith (2007) To stop /control something first we need to identify it! Foc can not be distinguished morphologically from other Foxys

6 1 st Diagnostic tools for Foc 2008, 2010 ~ 140 year after the 1 st disease report ST4 + TR4 VCGs: 012-0, 1, 2, 6, 9, 10, 11 Specific for TR4 28S$ IGS VCG01213/16 18S$ ITS

7 Diagnostic tools for Foc 2008, 2010 Lin et al. (2008)- Foc-1/ Foc-2 VCG M M 242 bp Primers - FocTR4-F/ FocTR4-F (Dita et al. 2010) Foc-1/Foc-2: Positive isolates from Honduras, Brazil and Costa Rica!

8 Diagnostic tools for Foc TR 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]

9 Diagnostic tools for Foc TR4 LAMP 2013 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; Validation, improvement and implementation of LAMP for TR4- Needed

10 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

11 qpcr/lamp - Sensitivity tests spores/ml log spores Average 1,000, , , , Detection threshold spores/g soil log spores Ct value 100, Detection threshold

12 Towards LAMP on-site TR4 diagnosis

13 Stages Sample collection Pseudostem, petiole etc. Place, variety, etc Send samples to a lab with information about plantation RT4? RT4? samples 30 mins - Field 6 hours - Lab DNA Extraction LAMP Samples processing Komada media Komada media DNA Extraction PCR Fungus Isolation Purification PDA media? DNA Extraction 3-5 days - Lab Foc -PDA

14 Linking Diagnostic to Epidemiology & Management 1 Planting material Soil Irrigation 3 Weeds Disease center 2 Adapted from Hwang & Ko 2004 Dita et al. (2013)

15 Fusarium TR4 Inoculum Level at Different layers of soil and shoes (preliminary results) calculated using the equation y= x (standard curve for pure spore suspension)

16 Foc TR4 Detection in soil from shoes 1 Amplification curve Melting curve Blue positive control Green soil sample Ave. Ct value a Calculated # of spores/g soil b a average Ct value of 10 independent extractions b calculated using the equation y= x (standard curve for pure spore suspension) 1 Shoes with soil and rice bran (used in the Foc infested farm in the Philippines)

17 TR4 Quantification Field Water Irrigation water Test positive 75 spores/ml 1 calculated using the equation y= x (standard curve for pure spore suspension)

18 Key biological and epidemiological caracteristics for Foc management vs. Diagnostic Disease symptoms of TR4 are similar to R1, R2 Chlamydospores can survive in the soil for long periods Latent period is long - the disease coud be only detected after a considerable period Foc can survive as a non pathogenic endophyte in weeds and other hosts Foc can be dissminated in symptomless, but infected planting material It is easily disseminated in soil particles and water

19 K.meters 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

20 1. Quarantine Services Contingency plan for Foc TR4 A regional template for National Contingency Plans Free-online knowledge source for LAC

21 A. Molina

22 A. Molina According to Horticulture Plant Protection Department (2007) reported that epidemic rates of Foc race 4 in Sumatra and other province reach 100 km/year

23 2. Supporting eradication practices Next victim Future victim? RIP - Killed by TR4

24 2. Supporting eradication practices Early diagnostic of Fusarium wilt vs. management en a plant is detected cted how many ghbors we should troy around? When a plant is detected infected how many neighbors we should destroy around? ers think more on che$ they don t care much on inoculum ssure, epidemiology? ut at the end they loose ba le 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 Area%A%( 15%m) Area%B%( 40%m) Infected Symptomless, but infected? 1. Erradicate plants in the zone A mandatory 2. Erradicate plant in zone B - Recommended

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26 3. Certification of planting material Healthy plant? Farmer leave this plant in the field or use suckers as planting material? Infected plant Farmer may not be aware about Foc infestation and use (even sell) symptomless, but infected suckers

27 3. Surveillance and monitoring preventive actions Colombia Valle del Cauca

28 3. Surveillance and monitoring risk analyses Data: C. Roman/ Map: D. Brown ~ 150 Farms mapped ~ 80 % Foc Collection established

29 Early Detection: A Key Action Area%A%( 15%m) Activate contingency plans Containment of the first outbreak Management at different levels Farm, Multi-farm, Landscapes, Watershed, Territory, Country Region Area%B%( 40%m) Surveillance, Monitoring, Eradication Certification of planting material Symptomless, Infected Symptomless, but infected? but Infected Diagnostic capacities Responsiveness

30 Strengthening capacities and increasing preparedness for Foc TR4 in LAC Bioversity, MUSALAC & National Plant Protection Organizations Trained In the pipeline

31 Weak points on productios systems vs. Foc Management Farms with open access with intern walking routes No fences Farmer Introduction of planting material and soil from infected sites Low levels of information on epidemology & management Lack of erradication measures No application (or lower) of organic ammendements Pathogen Host Susceptible varieties High virulent Wide host range Monocrop systems Low genetic diversity Environment Acid soils Desqilibrium (Chemical, Phisical, Biological) Inadequated topography- enabling flooding Poor drainage; Adequate temperatures

32 LAC already has serious problems with Foc R1/R2 Miguel Dita

33 Rome, December, 2014