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

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Razas 1, 2

R4T por confirmar oficialmente

R4T, 1, 2

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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

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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

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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

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miguel.dita@embrapa.br

Rome, December, 2014