Neighbor - Joining phylogenetic analysis. Distribution of the 26 fingerprints. Advances on the biology of CBB. outbreeding

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1 FP1 FP2 FP4 FP5 FP3 FP6 FP7 FP8 FP9 FP14 FP13 FP11 FP15 FP16 FP1 FP17 FP18 FP24 FP19 FP21 FP2 FP22 FP23 FP26 FP12 Neighbor - Joining phylogenetic analysis FP1 ME1-ME15-CR1, CR3, CR4 FP15 BR1, BR3, BR5,CO1, FP2 CR2 CO2, CO8,CO9, CO1, FP3 BR2, BR7, BR14 PE1, PE2, PE5, PE1, SA1 FP4 BR11 FP16 NI2 FP5 CO3 FP17 IA1-IA4 FP6 BR4, BR6, CO6, EC1, EC2 FP18 CA1, CA2 EC4, EC5, HO1, HO4, HO6 FP19 UG4, UG7,UG8,UG9, HO7, NI1, NI3, NI4, JA1, JA2 UG1 FP7 BR9, BR12, BR13 FP2 UG1, UG2, UG11,UG12 FP8 EC3 FP21 UG6 FP9 HO2, HO3, HO5 FP22 KE1 FP1 DR1 FP23 KE2 FP11 IN1-IN6 FP24 UG3 FP12 CO4, CO5, CO7, PE3, PE4,PE7, PE8, PE9 FP25 ET1, ET2 FP13 BR8 FP26 UG5 FP14 BR1 Molecular fingerprints Fingerprint Sample and country FP1 ME1-ME15-CR1, CR3, CR4 FP2 CR2 FP3 BR2, BR7, BR14 FP4 BR11 FP5 CO3 FP6 BR4, BR6, CO6 EC1, EC2, EC4, EC5 HO1, HO4, HO6, HO7 NI1, NI3, NI4, JA1, JA2 FP7 BR9, BR12, BR13 FP8 EC3 FP9 HO2, HO3, HO5 FP1 DR1 FP11 IN1-IN6 FP12 CO4, CO5, CO7 PE3, PE4, PE7, PE8, PE9 FP13 BR8 FP14 BR1 FP15 BR1, BR3, BR5, CO1, CO2 CO8, CO9, CO1, PE1, PE2 PE5, PE1, SA1 FP16 NI2 FP17 IA1-IA4 FP18 CA1, CA2 FP19 UG4, UG7, UG8, UG9, UG1 FP2 UG1, UG2, UG11, UG12 FP21 UG6 FP22 KE1 FP23 KE2 FP24 UG3 FP25 ET1, ET2 FP26 UG5 Distribution of the 26 fingerprints FP25 outbreeding Advances on the biology of CBB September 1988: Tumaco May 1989: Ancuyá October 1989: La Plata March 199:Ansermanuevo June 199: Vegachí July 199: Páez September 199: Pradera 1991: Coffee Central Region Sib-mating Functional Haplo-diploidy High inbreeding Genotyping GABA in artifitial crosses corroborated the origin of the polymorphisms Crosses between maternal lines in natural conditions 5-1% showed multiple infestations from different maternal line founders 6% of the coffee beans showed multiple progenies in field conditions These results indicate out-crossing events in natural conditions in Colombia and would allow the use of a genetic control approach

2 MEIOSIS IN THE CBB EVALUATED BY MEANS OF CYTOLOGICAL ANALYSIS: Meiosis in CBB males indicates no visible recombination and the presence of every stage of the Meiosis II. These results are controversial from what was previously reported Profase 1 The Reproductive Tract of Hypothenemus hampei Metafase 1 Profase Anafase 1 Equational Division Interkinesis No Meiosis II Metafase 2 Spermatogenesi s Anafase 2 Spermiogenesis JD Rubio, AE Bustillo, LF Vallejo, JR Acuña, P Benavides. 27. Brun et al., model, 1995 Berrío & Benavides, model, 28 Dispersal of Hypothenemus hampei During Coffee Plant Renovation Castaño et al. 22 Mean coffee berries per site (,5m 2 ) and CBB infestation, according to the ripe stage of the coffee berries during coffee plant renovation Coffee berry Total coffee Infested coffee CBB Infestation level stage berries berries Unriped 538,8 a 56,4 a 1,5 a Riped 4,3 b 18, b 44,7 b overripped 19,3 b 7,8 c 4,4 c Mean followed by different letters indicates statistical difference under a 5% Tukey comparison Test Mean CBB individuals per berry per site (,5m 2 ) at different coffee berry ripeness during the renovation of coffee plantations CBB individuals Coffee berry stage Eggs Larvae Pupae Adults Unriped 1,5a 1,5a 1,3ª 1,5a Riped 1,6a 2,3b 1,6b 1,6a Overripped 1,9b 1,7a 1,5ab 1,6a Means followed by different letters indicates statistical difference under a 5% Tukey comparison test Mean CBB per coffee berry CBB individuals into coffee berries on the ground after coffee plant renovation Days after coffee plant renovation Alive CBB farm 1 Alive CBB farm 2 Dead CBB farm 1 Dead CBB farm 2 2'575,667 y 3'617, CBB adults trapped per hectare Dispersal of Hypothenemus hampei During Coffee Plant Renovation total CBB trapped per site Mean adult CBB trapped per site Castaño et al Days after coffee plant renovation La Catalina S. Barbara Impact of The CBB Infested Coffee Berries on the Ground Over the CBB Levels in the Trees TREATMENTS T1: 1 infested coffee berry on the ground (TTO 1) T2: 5 infested coffee berry on the ground (TTO 5) T3: 1 infested coffee berry on the ground (TTO 1) T4: 15 infested coffee berry on the ground (TTO 15) T5: 2 infested coffee berry on the ground (TTO 2) T: (TTO ) 15 replications per treatment LOCATION Paraguaicito, Quindío, 1218 m.a.s.l. Naranjal, Caldas, 1381 m.a.s.l. La Bella, Quindío, 147 m.a.s.l. Santacruz, Risaralda, 17 m.a.s.l. CBB trapped every 1 days using sticky traps CBB infestation level and total number of infested coffee berries every 3 days Total mean CBB infested berries per tree during a six month period in four locations Total Mean of CBB infested coffee berries per tree Location (Altitude) TTO TTO 1 TTO 5 TTO 1 TTO 15 TTO 2

3 Alcohol traps to monitor CBB in the field: Ethological approach Alcohol traps to monitor CBB in the field: Ethological approach CBB trapped Nov Dic Ene Feb Mar Abr May Jun Jul Ago Sep Cap 4 tram Infe 4 tram Total adult CBB trapped and tree infestation level Infestation level (%) CBB Trapped A 5 4 B 3 2 Feb 6 Feb 21 Mar 7 Mar 21 Abr 9 Abr 25 May 7 May 22 Jun 7 Jun 21 Jul 7 Jul 23 Ago 7 Ago 23 Sep 7 Sep Mean CBB trapped in alcohol hand-made traps at two positions in the trees : 1.5m (A) and.4m (B) throughout a calendar year A:125 ± 8 B: 247 ± 13 Infestation Feb 21 Mar 21 Abr 21 May 22 Jun 21 Jul 21 Ago 21 Sep 24 ALTA BAJA LOTE 27 CBB infestation level in trees containing traps at two positions: 1.5m and.4m and CBB infestation level in the experimental plot Contribution of Cultural, Chemical and Biological Approaches in the off CBB Molecular techniques to develop CBB markers in advancing biology, genetics and ecology CBB LEVEL IN THE FIELD 3 25 T: (traditional harvest) T1:Cultural control CBB populations Developing molecular markers Genotyping techniqes INFESTATION (%) 2 15 T2: Chemical control T3:Cultural + Chemical T4:Cultural + B. bassiana T5:Cultural + Chemical + B. bassiana Locations Generation F3 Alelle 2 Alelle 3 Alelle 1 Alelle 2 Alelle 3 Alelle 1 PASA RT-PCR SSCP 1 5 ENE FEB MAR ABR MAY JUN JUL AGO SEP OCT NOV DIC TTO TTO 1 TTO 2 TTO 3 TTO 4 TTO 5 There are eight molecular markers being used in addressing biological and genetic issues of the coffee berry borer, and two being developed to address dispersal patterns in the field MORTALITY % Beauveria bassiana de d Bb 91 Bb 95 IPM approaches: Biological control bcd d Bb 91 Bb 92 Grupo 1 Bb 911 cde Bb 916 Bb 9119 STRAINS AND MIXES ab e Bb 925 Grupo 2 Bb 923 Bb 924 Grupo 3 Mezcla A e a Mezcla B testigo.185 CBB Mortality by B. bassiana Treatment Mean (%) C. V. (%) Bb b 8.7 Bb ab 16.2 Bb ab Bb ab Bb ab Bb ab Mix A EXPERIMENT I 6.22 ab (Bb 92 + Bb Bb 925) Cárdenas et al. 27 Mix B a (Bb 91 + Bb Bb 924) CBB Mortality in the field by B. bassiana (2 x 1 espores/branch) Commercial strain ab within the plot outside the plot CBB Mortality Treatment x SE Prepared 3 25,5bc 3,6 EXPERIMENT II Prepared 4 32,3b 3,9 Bastidas et al. 27 Commercial control (on rice) 62,2a 4,9 CBB Mortality in the field by B. bassiana (2 x 1 7 Pure spores 15c 3,3 espores/branch) within the plot 4,2d,8 outside the plot 4d,9 EXPERIMENT III Bernal et al % mortality of adult CBB from infested coffee berries on the ground

4 Parasitoids as a CBB control strategy Number of released individuals in the field in Colombia stephanoderis P. nasuta P. C. coffea Municipality Oct Sep 2 Nov Sep 2 Oct Sep 2 Antioquia Boyacá Caldas Caquetá Cauca Cesar Cundinamarca Huila Magdalena Meta Nariño Quindío Risaralda Santander Norte de Santander Tolima Valle Total Norte de Santander 8. ind Caldas ind Risaralda ind Quindío ind Nariño 6. ind ind Genetic Variability and Biological Performance of Prorops nasuta in samples collected in Colombia State Municipality present/total sampled Parasitism Pamplonita 2/2,74% - 3,78% Chinácota 2/3 1,2% - 3,25% Bochalema /1 % Total 4/6 66,67% Palestina 6/8,49% - 1,2% Chinchiná 1/1 1,85% Manizales 1/2 1,2% San José 1/3,25% Risaralda /3 % Total 9/18 5,% Pereira 2/7,5% - 1,38% Marsella /5 % Total 2/12 16,67% Pueblo /1 Tapao % Quimbaya 4/1,5% - 3,5% Buenavista 1/2,5% Montenegro 1/1 3,75% Total 6/14 42,86% Sandoná 11/11 13,43% - 29,9% Consacá 1/1 1,5% - 5% Ancuyá 1/1 2,36% - 21,63% Total 31/31,% Biological performance Predatorism Longevity Locality Parasitis Eggs adults Larvae Adults m Sandoná Low High High High Middle Consacá Low Middle Middle Low Middle Quindío Low Low High Middle Low Caldas Low Low High Middle Middle Norte de Santander Middle Middle Low Middle Low Cenicafe High Middle High High High Molecular fingerprints of P. nasuta sampled in Colombia Valle Pance 1/1 - Cesar Jagua de Ibirico 2/2 - Gran Total 65,6% Maldonado C, Benavides P 27 Correspondence analysis Collecting parasitized CBB infested coffee berries 1992 México Ecuador Uganda Togo 1996 Kenya 1987 Brasil : 3 granos, 5 adultos, Ancuyá 1996: 42 adultos, Chinchiná Ensure the establishment of the parasitoids in the field: Prorops nasuta Maldonado C, Benavides P 27 Monanus sp. (Col: Cucujidae) Native Predators Chemical Biorational insecticides for the of Coffee Berry Borer in Colombia Main characteristics of biorational insecticides Two native predator species with potential as CBB control strategy Crematogaster sp. (Hym: Formicidae) NEXT: Biology aspects Lethal effect under low dosis Very specific Decompossed quickly Low levels of toxicity Low environmental impact Vera L, Gil Z, Benavides P 27

5 Chemical Mean CBB Mortality in Field Conditions Porcentaje de mortalidad Treatment Chemical Efficacy of the ENTBIO 6 Insecticide for the control of CBB Mean Mortality Days after spraying No. Code Treatment No. Code Mortality days after spraying ENTBIO A 95.4 AB B C 2 ENTBIO A C A BC PROYECTIONS Dosis Residual effect ENTBIO 1 ENTBIO 2 ENTBIO 3 ENTBIO 4 ENTBIO A 97.1 A 96.6 A 61. B 54. B 57.8 C 98.2 A 98.6 A 96.1 A 66.6 B 69.4 B 74. B 6.4 C 13. D 5.78 D 1.3 C 1.54 E 1.44 D 3 ENTBIO A AB A 88.3 AB 4 ENTBIO A BC A AB 5 ENTBIO A 99.8 A 97. A AB 6 ENTBIO A A A A 7 Lorsban 4 EC 96.3 A A AB ABC Commercial Plots Residues Side effects Villalba D B 3.42 D 1.4 C 2.52 D Dosis: cc/ha Villalba D, Yandar S 27 Traits of interest in Colombian Coffee breeding Coffee Berry Borer resistance Genomic Strategies To Detect Genes Involved In Resistance To The Coffee Berry Borer Hypothenemus hampei Yield Cup quality Evaluation of resistant sources in Coffea >9 accessions were field evaluated. Coffee Berry Borer control Seven wild accessions of Coffea liberica and C. arabica showed antibiosis against the insect (Decreased fecundity 27% - 35% vs. control). Similar results have been obtained using artificial diets developed by CENICAFE. Resistance to Colletotrichum kahawae Resistance to Coffee Rust CBB Gene Expression to Antibiosis Compounds from coffee seeds Coffee Berry Borer resistance FISH on CBB chromosomes indicating the presence of Mannanase in H. hampei and it is absence in H. obscurus Treatments 35% oviposition Reduction Total eggs laid Carbohidrates metabolism Lipid Metabolism Protein metabolism Trasnport mechanism Protein destination Cellular structure Cellular Transcription development

6 Plant disease resistance response protein Proteins with differential expression C. liberica and C. arabica Farm Management Glycoside hydrolase, family 16 BURP Leucoanthocyanidin dioxygenase Tyrosine aminotransferase Glycoside hydrolase, family 18 Lipoxygenase Peptidase A1, pepsin Lectins Heat shock protein Hsp7 S-adenosylmethionine synthetase PAR1 Heat shock protein Hsp9 O-methyltransferase 1 Barwin, pathogenesis related 1-aminocyclopropane-1-carboxylate oxidase SAM dependent carboxyl methyltransferase Class III acidic chitinase Homologue function on other species defense genes related to pathogen and insects Candidate genes C. arabica var. Caturra C. liberica Agricultural Approaches Biological (B. bassiana, parasitoids) Genetical IPM Coffee Insect-Pests Cultural, Re-Re, Re, Coffee Process Chemical, Ethological Proteinase inhibitor I3, Kunitz legume Absolute frequency Farm Management Thank you