(Crinipellis perniciosa (Stahel) Singer) IN COCOA (Theobroma

“ 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.
Characteristics of the clones used to study early methods to evaluate resistance to M. perniciosa, Ecuador, 2006
Clones used for the tests Characterístics Variety type SCA­ SCA ­6 6 Amazónica SCA­ SCA ­12 12 Amazónica CCAT ­ CCAT­ 4675 BE­ BE ­10 10 EET­ EET ­95 95 Nacional Baja Amazónica Nal x Desc Tenguel­ Tenguel ­33 33 Synonym EET ­ EET ­ 11 11 EET­ EET ­ 110 110 EET­ EET ­ 575 575 ­­­­­­­­­­­­­­­ ­ Original country Ecuador Ecuador Ecuador Brasil Ecuador Estimated level of resistance Resistant R R Susceptible Suscep. Herrera Col. “Nacional” .Col. Internacional “Herrera” Actual location (EETP) Internacional collection Description of the methods used to evaluate resistance to C. perniciosa. Ecuador, 2006* ……. CONTINUA Group 1. Spore germination on two different substrates
Méthods Spore germination in aqueous extract of cocoa shoots. (Umaharam et al (Umaharam et al. 2002; Da Silva et al. 2002; Da Silva et al. 2001 Variable % of germination Spore germination on % de leaf disks germinatión. Description Infussion of 50 shoots from 5 plants (10/plant) from each genotipe. 1µl (10/plant) from each genotipe. 1 µl extract + 10µl of extract on wels of a ELISA platel. Spore counting took place 3 hours afterwards. 10 petri dishes/ material, 8 leaves disks/ plate. Spore counting took place 3 hours plate. Spore counting took place 3 hours afterwards. Description of the methods used to evaluate resistance to M. perniciosa ….. Group 2. Symptom evaluation on seedlings
Méthods Holliday Test Holliday Test Variable (Holliday, 1954). (Holliday, 1954). % of infection Any symptom Holliday Test Modified % of infection Any symptom Automated Belt Spray (SAI) Spray (SAI) (Frías, Purdy (Frías, Purdy Dickstein & Schmidt, 1998) 1998) 15 to 22 days old seedlings Description Inmerssion of 4 ­ 8 days old seedlings. Inmerssion of 4 ­ 8 days old seedlings. 10 plants/clon. 5 Repetitions Spraying of 1ml of spore suspension to 4 ­ 4 ­ 8 days old seedlings. 10 plants/ 8 days old seedlings. 10 plants/ clon. 5 Repetitions Spray of 1ml/plant Aspersión de 1 ml/ planta of spore suspension to Group 2. Symptom evaluation on seedlings Belt Spray method
Holliday tests Description of the methods used to evaluate resistance to M. perniciosa ….. Group 3. Field inoculations
Inoculación de brotes tiernos en campo (Sreenivasan, 1996; ( Surujdeo ­ Maharaj, Surujdeo­ Maharaj, 2003; Umaharam et al. 2002; Da Silva et al.2001) al.2001) Brotes tiernos de cacao Colocación de 0.1 ml de una suspensión de 100000 basidiosporas / ml al 0.3 % de agar agua sobre el brote. FIRST RESULTS
FIRST RESULTS Spore germination on two different substrates taken from five Cocoa clones. INCOPED, Costa Rica, 2006 S U S T R A T E S CLON Leaves disks Chi 2 : 10.67 ** Aquous extracts Chi 2 : 18.43 ** SCA6 4,83 89 SCA12 4.87 90 CCAT4675 7,91 93 BE10 5,58 95 EET95 5,19 95 Control ­ 96 * 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 Inoculation Methods* CLON Holliday Holliday Modified Belt Spr ay Chi 2 : 2.70 ns Chi 2 : 2.27 Chi 2 : : 2.30 ns ns SCA 6 17.86 17.71 18.39 SCA 12 16.60 17.75 19.43 CCAT 4675 17.25 18.34 18.55 BE 10 18.80 17.52 19.79 EET 95 16.34 17.14 17.72 * Mean values. ns=no significance for Chi2 Test from Kruskal­Wallis
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
CLON Number of inoculated Active buds/ branch/plant %of INFECtIÓN % DE INFECCIÓN Number of inoculated 2 :15.33 2 15.33 Chi : : 10.64 10.64 shoots/plant Chi : * Incubation period
period (days) * * * * Chi 2 : :9.95 9.95 ** * * SCA6 37 0.45 21 0,16 71 SCA12 140 3.16 28 2,07 42,39 CCAT4675 54 3.17 16 4,11 45,38 EET95 102 3.40 18 3,59 44,63 BE10 122 4.91 27 5,09 49,04 Range of symptoms presented on seedlings when inoculated with the SAI
Permanence of cotyledons in days after infection for three early evaluation methods for resistance against Witches Broom in cacao seedlings. INCOPED, Costa Rica, 2006 Inoculation methods CLON Holliday Chi 2 : 1.73 ns Control Holliday Modified Chi 2 : 2.88 ns Control SAI Chi 2 : 18.97 ** Control SCA 6 84.40 45.74 68.38 45.74 42.67 33.79 SCA 12 81.31 47.60 78.14 47.60 41.36 41.16 CCAT 4675 81.84 39.45 79.34 39.45 54.69 42.24 BE 10 69.92 48.57 84.20 48.57 49.36 37.30 EET 95 80.74 54.94 74.94 54.94 70.48 47.88 * Valores Medios. Abreviatura ns muestra la no significancia en la prueba del Chi2 del test de Kruskal­Wallis
Análisis de correlación entre variables de los métodos de evaluación temprana de resistencia a C. perniciosa. INIAP. Pichilingue. Quevedo­ Los Ríos.2005. DHPGER EXPGER HMPI HMPCI HPI HPCI SAIPI SAIPC CPI CPIR CPIPLT DHPGER EXPGER HMPI HMPCI HPI HPCI SAIPI SAIPC CPI CPIR CPIPLT 1 0,6 0,1 0,1 0,2 ­1 ­0,3 0,7 0,1 0,7 0,9 1 0,7 0,7 0,4 ­0,6 0,1 0,9 0,3 0,9 0,7 1 0,3 1 0,1 0,5 1 ­0,1 ­0,1 ­0,2 0,4 0,1 0,7 0,5 0,6 0,7 ­0,3 0,8 0,6 0,6 0,5 0 0,3 0,2 ­0,1 1 0,3 1 ­0,7 0,3 ­0,1 0 ­0,7 ­0,3 ­0,9 ­0,5 1 0,4 1 0,7 0,1 1 0,6 0 0,9 1 Variables measure in the leaf disk and plant extract correlate with field infection Variable permanence of cotyiledons (SAI) correlate as well with field data
Resistants ? 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. Susceptible
Conclusions: n The reaction of the plants in the five methods n
compared were of a magnitude that did not allow proper discrimination between the clones; n Variables to measure vary with the method; n
n The permanence of cotyledons in the belt spray n
method; spore germination on both methods used, and field infection were the variables and methods that allowed to appreciate differences.
methods that allowed to appreciate differences. Conclusions: n Although differences found with the different n
methods were very short, clones SCA, 6 y 12 did showed its resistant conditions and the EET 95 maintained as well its susceptible condition. n Efforts should be addressed to overcome factors n
and conditions that affect the efficiency of the methods; n A “calibration” of the stage of the substrate to n
use and the spore concentration seems to be a condition to fulfill wherever and whenever one of this methods will be used.
of this methods will be used. Conclusions: n The modified Holliday test, the belt spray n
and the agar drop methods can be the tool breeders and pathologist require for WB resistant material selection, once the adjustments sugested in this comparative work be completed.
work be completed. Patricio and me want to
acknowledge to Bertus Eskes,
Erik Rosenquists, Raymond
Schnell and J.C. Motamayor for
their support to the Ecuadorian
Cocoa Program, being this one
of the outcomes that are now
flowing. flowing
.