“ COMPARISON OF METHODS FOR EARLY EVALUATION OF RESISTANCE TO WITCHES BROOM (Crinipellis perniciosa (Stahel)

Singer) IN COCOA (Theobroma cacao L.)" .

Patricio del Pozo & Carmen Suárez­Capello.

Characterístics Characterístics

Clones used for the tests Clones used for the tests

SCA SCA­ ­6 6 SCA SCA­ ­12 12 CCAT CCAT­ ­ 4675 4675 BE BE­ ­10 10 EET EET­ ­95 95

Variety type Variety type Amazónica Amazónica Amazónica Amazónica Nacional Nacional Baja Baja

Amazónica Amazónica Nal x Desc Nal x Desc

Synonym Synonym EET EET ­ ­ 11 11 EET EET­ ­ 110 110 EET EET­ ­ 575 575 ­­­­­­­­­­­­­­­ ­­­­­­­­­­­­­­­

­ ­ Tenguel Tenguel­ ­33 33

Original country Original country Ecuador Ecuador Ecuador Ecuador Ecuador Ecuador Brasil Brasil Ecuador Ecuador

Estimated level of Estimated level of resistance resistance

Resistant Resistant R R R R Susceptible Susceptible Suscep. Suscep.

Actual location Actual location (EETP) (EETP)

Internacional Internacional collection collection Herrera Herrera

Col. Col. “Nacional” “Nacional”

.Col. .Col. Internacional Internacional “Herrera” “Herrera”

Characteristics of the clones used to study early methods to evaluate resistance to M. perniciosa, Ecuador, 2006

Méthods Méthods Variable Variable Description Description

Spore germination in Spore germination in aqueous extract of aqueous extract of cocoa shoots. cocoa shoots. (Umaharam (Umaharam et al et al. . 2002; Da Silva 2002; Da Silva et al. et al. 2001 2001

% of germination % of germination

Infussion of 50 shoots from 5 plants Infussion of 50 shoots from 5 plants (10/plant) from each genotipe. 1 (10/plant) from each genotipe. 1µl µl extract + 10µl of extract on wels of a extract + 10µl of extract on wels of a ELISA platel. Spore counting took place ELISA platel. Spore counting took place 3 hours afterwards. 3 hours afterwards.

Spore germination on Spore germination on leaf disks leaf disks

% de % de germinatión. germinatión.

10 petri dishes/ material, 8 leaves disks/ 10 petri dishes/ material, 8 leaves disks/ plate. plate. Spore counting took place 3 hours Spore counting took place 3 hours afterwards. afterwards.

Description of the methods used to evaluate resistance to C. perniciosa. Ecuador, 2006*

……. CONTINUA

Group 1. Spore germination on two different substrates

Méthods Méthods Variable Variable Description Description

Holliday Test Holliday Test (Holliday, 1954). (Holliday, 1954).

% of infection % of infection Any symptom Any symptom Inmerssion of 4 Inmerssion of 4 ­ ­ 8 days old seedlings. 8 days old seedlings.

10 plants/clon. 5 Repetitions 10 plants/clon. 5 Repetitions

Holliday Test Holliday Test Modified Modified

% of infection % of infection Any symptom Any symptom

Spraying of 1ml of spore suspension to Spraying of 1ml of spore suspension to 4 4 ­ ­ 8 days old seedlings. 10 plants/ 8 days old seedlings. 10 plants/ clon. 5 Repetitions clon. 5 Repetitions

Automated Belt Automated Belt Spray (SAI) Spray (SAI) (Frías, (Frías, Purdy Purdy

Dickstein & Schmidt, Dickstein & Schmidt, 1998) 1998)

15 to 22 days old 15 to 22 days old seedlings seedlings Spray of 1ml/plant Spray of 1ml/plant

Aspersión de 1 ml/ planta of spore Aspersión de 1 ml/ planta of spore suspension to suspension to

Description of the methods used to evaluate resistance to M. perniciosa …..

Group 2. Symptom evaluation on seedlings

Group 2. Symptom evaluation on seedlings

Holliday tests

Belt Spray method

Inoculación de brotes Inoculación de brotes tiernos en campo tiernos en campo ( (Sreenivasan, 1996; Sreenivasan, 1996; Surujdeo Surujdeo­ ­ Maharaj, Maharaj,

2003; Umaharam et al. 2003; Umaharam et al. 2002; Da Silva et 2002; Da Silva et

al.2001) al.2001)

Brotes tiernos de Brotes tiernos de cacao cacao

Colocación de 0.1 ml de una Colocación de 0.1 ml de una suspensión de 100000 suspensión de 100000

basidiosporas / ml al 0.3 % de basidiosporas / ml al 0.3 % de agar agua sobre el brote. agar agua sobre el brote.

Description of the methods used to evaluate resistance to M. perniciosa …..

Group 3. Field inoculations

FIRST RESULTS FIRST RESULTS

CLON CLON

S U S T R A T E S S U S T R A T E S

Leaves disks Leaves disks Aquous extracts Aquous extracts

Chi Chi 2 2 : 10.67 ** : 10.67 ** Chi Chi 2 2 : 18.43 ** : 18.43 **

SCA6 SCA6 4,83 4,83 89 89

SCA12 SCA12 4.87 4.87 90 90

CCAT4675 CCAT4675 7,91 7,91 93 93

BE10 BE10 5,58 5,58 95 95

EET95 EET95 5,19 5,19 95 95

Control Control ­ ­ 96 96

Spore germination on two different substrates taken from five Cocoa clones. INCOPED, Costa Rica, 2006

* Mean values. ns=no significance for Chi2 Test from Kruskal­Wallis

Incubation period of M. perniciosa in days for three methods of early evaluation of resistance for five cocoa clones. INCOPED, Costa Rica, 2006

CLON CLON

Inoculation Methods* Inoculation Methods*

Holliday Holliday Holliday Holliday Modified Modified

Belt Belt Spray Spray

Chi Chi 2 2 : : 2.70 2.70 ns ns Chi Chi 2 2 : : 2.30 2.30 ns ns

Chi Chi 2 2 : 2.27 : 2.27 ns ns

SCA 6 SCA 6 17.86 17.86 17.71 17.71 18.39 18.39 SCA 12 SCA 12 16.60 16.60 17.75 17.75 19.43 19.43 CCAT CCAT 4675 4675

17.25 17.25 18.34 18.34 18.55 18.55

BE 10 BE 10 18.80 18.80 17.52 17.52 19.79 19.79 EET 95 EET 95 16.34 16.34 17.14 17.14 17.72 17.72 * Mean values. ns=no significance for Chi2 Test from Kruskal­Wallis

CLON CLON

Number of Number of inoculated inoculated Active buds/ Active buds/ branch/plant branch/plant

% DE % DE INFECCIÓN INFECCIÓN Number of Number of

inoculated inoculated shoots/plant shoots/plant

%of %of INFECtIÓN INFECtIÓN

Incubation Incubation period period

(days) (days)

Chi Chi 2 2 : : 10.64 10.64 * *

Chi Chi 2 2 : :15.33 15.33 * * * *

Chi Chi 2 2 : :9.95 9.95 * ** *

SCA6 SCA6 37 37 0.45 0.45 21 21 0,16 0,16 71 71

SCA12 SCA12 140 140 3.16 3.16 28 28 2,07 2,07 42,39 42,39

CCAT4675 CCAT4675 54 54 3.17 3.17 16 16 4,11 4,11 45,38 45,38

EET95 EET95 102 102 3.40 3.40 18 18 3,59 3,59 44,63 44,63

BE10 BE10 122 122 4.91 4.91 27 27 5,09 5,09 49,04 49,04

Porcentaje de infección por ramas y por árbol y período de incubación en cinco clones de cacao Inoculados con C. perniciosa en campo. INCOPED, Costa Rica, 2006

Range of symptoms presented on seedlings when inoculated with the SAI

CLON CLON

Inoculation methods Inoculation methods

Holliday Holliday Control Control

Holliday Holliday Modified Modified

Control Control SAI SAI

Control Control Chi Chi 2 2 : : 1.73 ns 1.73 ns

Chi Chi 2 2 : 2.88 : 2.88 ns ns

Chi Chi 2 2 : 18.97 : 18.97 ** **

SCA 6 SCA 6 84.40 84.40 45.74 45.74 68.38 68.38 45.74 45.74 42.67 42.67 33.79 33.79 SCA 12 SCA 12 81.31 81.31 47.60 47.60 78.14 78.14 47.60 47.60 41.36 41.36 41.16 41.16 CCAT 4675 CCAT 4675 81.84 81.84 39.45 39.45 79.34 79.34 39.45 39.45 54.69 54.69 42.24 42.24 BE 10 BE 10 69.92 69.92 48.57 48.57 84.20 84.20 48.57 48.57 49.36 49.36 37.30 37.30 EET 95 EET 95 80.74 80.74 54.94 54.94 74.94 74.94 54.94 54.94 70.48 70.48 47.88 47.88

Permanence of cotyledons in days after infection for three early evaluation methods for resistance against Witches Broom in cacao seedlings. INCOPED, Costa Rica, 2006

* Valores Medios. Abreviatura ns muestra la no significancia en la prueba del Chi2 del test de Kruskal­Wallis

DHPGER 1 EXPGER 0,6 1 HMPI 0,1 0,7 1 HMPCI 0,1 0,7 0,3 1 HPI 0,2 0,4 0,1 0,5 1 HPCI ­1 ­0,6 ­0,1 ­0,1 ­0,2 1 SAIPI ­0,3 0,1 0,4 0,1 0,7 0,3 1 SAIPC 0,7 0,9 0,5 0,6 0,7 ­0,7 0,3 1 CPI 0,1 0,3 ­0,3 0,8 0,6 ­0,1 0 0,4 1 CPIR 0,7 0,9 0,6 0,5 0 ­0,7 ­0,3 0,7 0,1 1 CPIPLT 0,9 0,7 0,3 0,2 ­0,1 ­0,9 ­0,5 0,6 0 0,9 1

SAIPI SAIPC CPI CPIR CPIPLT DHPGER EXPGER HMPI HMPCI HPI HPCI

Análisis de correlación entre variables de los métodos de evaluación temprana de resistencia a C. perniciosa. INIAP. Pichilingue. Quevedo­ Los Ríos.2005.

Variables measure in the leaf disk and plant extract correlate with field infection

Variable permanence of cotyiledons (SAI) correlate as well with field data

The SAI was one the method that allowed discrimination

between cultivars, however all clones presented acute

symptoms of the disease and the first factor to consider is

the virulence of the ecuadorian strain and the

concentration of the inoculum.

Resistants ?

Susceptible

Conclusions: Conclusions:

n n The reaction of the plants in the five methods The reaction of the plants in the five methods compared were of a magnitude that did not compared were of a magnitude that did not allow proper discrimination between the clones; allow proper discrimination between the clones;

n n Variables to measure vary with the method; Variables to measure vary with the method;

n n The permanence of cotyledons in the belt spray The permanence of cotyledons in the belt spray method; spore germination on both methods method; spore germination on both methods used, and field infection were the variables and used, and field infection were the variables and methods that allowed to appreciate differences. methods that allowed to appreciate differences.

Conclusions: Conclusions:

n n Although differences found with the different Although differences found with the different methods were very short, clones SCA, 6 y 12 did methods were very short, clones SCA, 6 y 12 did showed its resistant conditions and the EET 95 showed its resistant conditions and the EET 95 maintained as well its susceptible condition. maintained as well its susceptible condition.

n n Efforts should be addressed to overcome factors Efforts should be addressed to overcome factors and conditions that affect the efficiency of the and conditions that affect the efficiency of the methods; methods;

n n A “calibration” of the stage of the substrate to A “calibration” of the stage of the substrate to use and the spore concentration seems to be a use and the spore concentration seems to be a condition to fulfill wherever and whenever one condition to fulfill wherever and whenever one of this methods will be used. of this methods will be used.

Conclusions: Conclusions:

n n The modified Holliday test, the belt spray The modified Holliday test, the belt spray and the agar drop methods can be the and the agar drop methods can be the tool breeders and pathologist require for tool breeders and pathologist require for WB resistant material selection, once the WB resistant material selection, once the adjustments sugested in this comparative adjustments sugested in this comparative work be completed. work be completed.

Patricio and me want to Patricio and me want to acknowledge to Bertus Eskes, acknowledge to Bertus Eskes,

Erik Rosenquists, Raymond Erik Rosenquists, Raymond Schnell and J.C. Motamayor for Schnell and J.C. Motamayor for their support to the Ecuadorian their support to the Ecuadorian Cocoa Program, being this one Cocoa Program, being this one of the outcomes that are now of the outcomes that are now

flowing flowing. .