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Research Project: Development and Application of Genomic-assisted Breeding Strategies to Produce Disease-resistant Cacao Genetic Resources

Location: Subtropical Horticulture Research

2021 Annual Report

1. Evaluate (phenotype) cacao genetic resources for host-plant resistance to the priority diseases black pod, frosty pod, and witches’ broom. Record and disseminate evaluation data via the project’s website, the Cacao Genome Database, the International Cocoa Germplasm Database, and/or other data sources. 1A. Develop artificial inoculation protocols and phenotyping techniques to assess resistance to Phytophthora canker in clones from Ecuador. 1B. Assess Ecuadorian and NPGS cacao germplasm in Puerto Rico for resistance to BP and make data available via cacao databases. 1C. Screen Ecuadorian clones for FP resistance in Colombia and make data available via cacao databases. 1D. Evaluate Ecuadorian clones for resistance to WB in Colombia, Trinidad, and Brazil and make data available via cacao databases. 2. Develop and apply more effective genetic tools, including genetic markers, association mapping, genome-wide selection, and/or experimental populations, for breeding cacao genetic resources with host-plant resistance to the priority diseases black pod, frosty pod, and witches’ broom. 2A. Use exome capture methodology to develop SNP markers from diverse cacao germplasm and utilize them for screening segregating populations and germplasm exhibiting resistance to BP, FP, and WB, as well as desirable horticultural and quality traits. 2B. Utilize genome-wide selection (GWS) models to predict genomic breeding values (GBV) in full-sib family cacao crosses. 3. Breed and release cacao genetic resources with increased host-plant resistance to the priority diseases: black pod, frosty pod, and witches’ broom.

Currently, around thirty percent of the world annual cacao production is lost to pest and diseases. In the Americas, the major losses are caused by three diseases: black pod rot (BPR), caused by several different Phytophthora species of which P. palmivora is the most common, frosty pod rot (FPR), caused by Moniliophthora roreri, and witches’ broom (WB), caused by Moniliophthora perniciosa. Presently over 250 cacao germplasm accessions are available at the USDA-ARS-TARS in Mayaguez, Puerto Rico. However, most of this germplasm has not been evaluated for resistance to these diseases. Screening protocols for selecting germplasm resistance to Phytophthora canker and BPR will be used to select accessions resistant to these diseases. In addition, selected accessions from the USDA-ARS/MARS/INIAP collaborative breeding program will be sent to Colombia for evaluation to BPR, FPR and WB. Also due to the presence of different WB pathotypes in Trinidad and Brazil, these accessions will be also screened for resistance in these countries. Single Nucleotide Polymorphisms (SNPs), both as single base pair substitutions and single base pair insertions/deletions (indels) are the most common sequence differences found between alleles. Due to the sequence of two cacao genomes, Matina 1-6 (Amelonado) and B97-61/B2 (Criollo), methodologies such as whole-exome targeted sequencing have been developed and this has facilitated SNP discovery. Once SNPs have been identified, they can be employed to genotype cacao accessions previously screened for disease resistance as well as populations segregating for disease resistance. Cacao breeding is a slow process, due to the tree’s long reproductive cycle. Therefore, the development of SNP markers and the application of genomic-assisted breeding methodologies will be implemented to strengthen the effectiveness of the selection process. Since FPR and WB are not present in Hawaii and Puerto Rico as well as other cacao producing countries in the Americas, preventive breeding for these diseases will be extremely useful.

Progress Report
Progress is being attained by ARS researchers in Miami, Florida, during FY2021 on all three objectives as well as their sub-objectives which are under NP301. Advances are also being made on all sub-components of oOjective 1. Artificial inoculation protocols and phenotyping developed as part of Objective 1a are being used to screen elite Ecuadorian clones for resistance to black pod rot and Phytophthora canker. This method is selected because it evaluates both types of resistance (resistance to infection and resistance to colonization), which makes it representative of what happens in the field. P. palmivora doesn’t produce appressoria (specialized fungi cell used to infect host) as readily as P. megakarya and not all pods are developing lesions following inoculation. However, all screening previously done used high rates of inoculum to achieve 100% infection. These excessively high levels of inoculum bypass the plants’ resistance to colonization and only portray resistance to colonization. Approximately 40 accessions (180 pods) have been screened in Miami to date. A modified screening protocol developed for the cacao germplasm collection in collaboration with Cacao Curator in Mayaguez, Puerto Rico, is being used for the USDA-ARS collection in Mayaguez, Puerto Rico, and so far, 182 accessions (780 pods) have been screened in Miami, Florida. Grafted plants of selected Ecuadorian clones are being screened for canker resistance as part of Objective 1B. Inoculations are being done with plugs of actively growing mycelia placed over a site wounded with a 1mm diameter probe, with lesions being measured 5 weeks post-inoculation. Approximately 30 trees are being screened in Miami, Florida, to date. This screening is going to provide the information necessary to estimate each accession’s level of resistance to Phytophthora canker and is going to be employed to find single nucleotide polymorphism markers associated with resistance. The resulting linked markers are going to be utilized in our genomics-assisted breeding program. Pathogens affecting cacao in Hawaii, Puerto Rico, Brazil, and Ecuador are being identified using molecular markers, and Koch’s postulates are being carried out to determine whether novel organisms are pathogens or endophytes. Approximately 100 Phytophthora isolates and 30 fungal isolates are being maintained at the USDA-ARS-Subtropical Horticulture Research Plant Pathology Laboratory in Miami, Florida, and used in experiments to determine their biology, epidemiology, and genetics. Leaf, pod, and seedling inoculations are being done with P. theobromicola, as part of a RNAseq study for the pathogenomics project (UC Davis/Mars/USDA-ARS) to identify host resistance genes. P. theobromicola is a newly described species of Phytophthora that causes pod rot on cacao in Brazil. It is more prevalent and aggressive than P. palmivora. Badnaviruses have been mainly reported affecting cacao production in West Africa. Mealybugs are responsible for the spread of these viruses. In the early 1930s, Trinidad was the only place in the Americas where Badnaviruses have been reported causing losses in cacao. They have been identified as Cacao Trinidad virus (CTV) strains A and B. CTV was later classified as Cacao mild mosaic virus and Cacao yellow vein-clearing virus, respectively. More recently, Cacao mild mosaic virus have been found in Puerto Rico, Brazil, and the quarantine and breeding greenhouses in Miami, Florida, and it has not been determined if the two discovered strains are indistinguishable. Researchers from the University of Arizona, University of Brasília, MARS and USDA-ARS scientists at Miami, Florida, have been collaborating to investigate genomic variability and genetic diversity of the Cacao mild mosaic virus samples that have been collected at the three locations. The main purpose of this study has been to sequence and describe the Cacao mild mosaic virus genomes of the samples gathered in Brazil, Puerto Rico, and Florida because this information is necessary to develop diagnostic tools to accurately identify the presence of the virus in the collection as well as in the commercial production farms. Genome sequencing and phylogenetic analysis results have shown that the collected virus samples are Cacao mild mosaic virus at all locations. However, a recombination event has been observed in the samplein a grower’s field from Puerto Rico and this is indicating that the samples collected are not alike. Also, the obtained sequencing information has been used to increase the precision of new diagnostic tools to identify the presence of the viruses. Furthermore, these results have pointed out that Badnaviruses are prevalent in the cacao growing regions of the Americas and that the implementation of quarantine facilities in combination with molecular diagnostic tools is a requisite for the transferring and exchange of cacao germplasm among producing countries. As a consequence of this discovery, the transferring of the Ecuadorian clones to Colombia, Trinidad, and Brazil as part of Subobjective 1C and 1D is being suspended until the testing of all clones is completed. Cacao production in the Americas has been severely reduced by Black Pod and Frosty Pod Rot diseases. Due to high costs and environmental concerns, the application of fungicides to control these diseases is not realistic. The development of resistance varieties is the most environmentally sound solution. However, the breeding of resistant germplasm is a slow process due to the tree’s long reproductive cycle. Scientists from the Centro Agronómico Tropical de Investigación y Enseñanza (CATIE) in Turrialba, Costa Rica, MARS Inc., Indiana University and researchers from USDA-ARS in Beltsville, Maryland, and Miami, Florida, using a segregating population developed by crossing the Black Pod Rot resistant parent Pound 7 with UF 273 a Frosty Pod Rot resistant parent and have identified SNP markers associated with Black Pod and Frosty Pod Rot resistance in cacao. These markers will be used by MARS Inc. and cacao breeders in Latin America to accelerate the development of cacao varieties resistant to Frosty Pod and Black Pod Rot. Regarding sSbobjective 2A, a total of 98,179 SNP markers previously developed using exome capture methodology have been converted from the Matina V1.1 assembly version to the new version Matina 2.1 using bioinformatic tools. These new SNPs are being developed using 1,150 DNA samples belonging to germplasm from the 10 genetic groups that are coming from Ecuador, the USDA-ARS cacao germplasm collection in Mayaguez, Puerto Rico, accessions from Mexico, Honduras, Trinidad, a random mating population from Hawaii, and a CATIE population segregating for black pod and frosty pod rot resistance. Linkage disequilibrium analysis is in progress analysis. Also, population structure analysis of the USDA-ARS cacao germplasm collection is ongoing. Furthermore, results of two newly designed SNP chips containing a total of 10,633 SNP markers are indicating that these can be used to develop a core set of primers. The main objective of these two SNP development projects is to select markers that are going to help us in the selection of reference genotypes and in the classification of the accessions by putting each of them in specific genetic groups and to determine the level of population admixture present in them. Moreover, the identification of SNPs associated with disease resistance, quality and horticultural traits is going to allow the improvement of cacao varieties using genomic-assisted breeding approaches. Regarding Objective 3, field data collection is ongoing in Hawaii and Puerto Rico. In addition, segregating populations have been planted in Mayaguez, Puerto Rico. The populations were developed from crosses that were made in December 2019 using parents selected based on preliminary yield trial data. Leaf collection, DNA extraction and SNP fingerprinting are going to start in FY2021.

1. Uniqueness of Cacao Criollo From Honduras. Cacao is becoming an important crop in Central America. Crop production area has increased in Honduras due to the demand for fine flavor cacao such as Criollo. A premium price for this product is being paid to Criollo cacao producers in Honduras because its white or pale pink seeds are employed in the making of premium chocolate. Since mainly phenotypic characterization is being used to identify cacao criollo cultivars, researchers from Honduras, Italy, and the USDA-ARS in Miami, Florida, have fingerprinted 89 cacao trees collected in four regions of Honduras (Copán, Santa Bárbara, Intibucá, and Olancho) using simple sequence repeat (SSR) molecular markers. Results indicated that 30 cacao accessions are members of the Criollo genetic group. The remaining accessions belong to admixture populations of Criollo with other genetic groups such as Amelonado, Guiana, and Marañon genetic groups. The Criollo accessions are being placed in gene banks for in-situ and ex-situ conservation, as well as for further evaluation and characterization. After the process is completed, accessions would be distributed to farmers interested in planting Criollo accessions.

2. Virulence tests of a new pathogen affecting cacao in Hawaii. Neofusicoccum parvum has been discovered causing disease on cacao pods on Oahu and Hawaii Islands. However, due to its novelty, the potential impact on the Hawaii cacao industry is currently unknown. USDA-ARS scientists from Miami, Florida, and Hilo, Hawaii, as well as MARS Inc. have been conducting inoculations with the pathogen on unwounded and wounded stems and pods to determine the infection routes and virulence of N. parvum. Also, they have added pathogens such as Lasiodiplodia theobromae and Phytophthora palmivora for comparison purposes. Results are indicating that on unwounded stems only one stem that is being inoculated with an isolate of N. parvum developed symptoms. In addition, half of the unwounded stems that are being inoculated with P. palmivora. L. theobromae do not present any lesions on any unwounded stems. In contrast, disease symptoms due to N. parvum isolates are being observed on 40 to 100 percent of unwounded pods of GNV 164 and GNV 360. However, almost no lesions are noticed on ICS 95, SHRS 21 and SHRS 33. The effects of N. parvum, L. theobromae, and P. palmivora on wounded pods is similar. The variation in infection rates of N. parvum among unwounded pods from different clones indicates that clones have differing levels of resistance to this pathogen. This is the first report of L. theobromae causing lesions in unwounded cacao pods and P. palmivora causing infections in unwounded stems. This research improves our understanding of the infection cycle of three economically important cacao pathogens and may potentially lead to the development of effective disease management strategies.

3. Phytophthora palmivora in Hawaiian Cacao. Black pod rot disease has been reported on cacao in Hawaii. However, this disease is caused by several species of Phytophthora and at the present time, the causal agent has not been precisely identified using molecular tools. To address this issue, USDA-ARS scientists from Miami, Florida, and Hilo, Hawaii, as well as MARS, Inc., have been collecting symptomatic material from cacao farms on Hawaii Island and Oahu. According to their results, the pathogen has been detected on less than half of the sites visited, none of which are located on Oahu. In addition, based on sequences from two different genes, all collected isolates have been identified as Phytophthora palmivora. Even though no genetic variation has been detected, their temperature-growth responses and virulence are indicating that isolates are different among each other. Also, their sensitivity to commonly used fungicides have been also tested in the laboratory, and it has been discovered that they are more sensitive to mefenoxam, chlorothalonil, and fosetyl-Al, than P. palmivora strains from parts of the globe where cacao production is more well-established. Since cacao production is increasing in Hawaii, farmers are considering black pod rot as the as the most import issue facing the industry.

4. A new species of Phytophthora. Brazil is the world’s sixth largest cacao producer and black pod rot disease, caused by Phytophthora spp., is among the main limiting production factors. Currently, only P. palmivora, P. capsici, P. citrophthora, and P. heveae have been described infecting cacao in Brazil. To estimate the diversity of Phytophthora species affecting cacao in Brazil, scientists from Brazil and the USDA-ARS in Miami, Florida, have been collecting isolates that were obtained from cacao pods exhibiting symptoms of black pod disease across different smallholder farms in 2017. After using molecular tools, they determined the presence of a previously undescribed species of Phytophthora and named as P. theobromicola. In addition to being more prevalent than P. palmivora, (the most common black pod pathogen), the new species is also more aggressive. The biology and pathogenicity of P. theobromicola, and its genetic relationship to other members of the Phytophthora genus have been evaluated. These results are going to be used by scientists and breeders to develop cacao material resistant to infection. The new species is going to be incorporated into disease resistance screening to improve the effectiveness of breeding programs. Ultimately this research benefits producers and consumers by stabilizing cacao yields and commodity supplies.

Review Publications
Puig, A.S., Quintanilla, W.E., Matsumoto Brower, T.K., Keith, L.M., Gutierrez, O.A., Marelli, J. 2021. Phytophthora palmivora causing disease on theobroma cacao in hawaii. Phytopathology. 11(5). Article 396.
Puig, A. S., Keith, L. M., Matsumoto, T. K., Gutierrez, O. A., & Marelli, J. P. (2021). Virulence tests of Neofusicoccum parvum, Lasiodiplodia theobromae, and Phytophthora palmivora on Theobroma cacao. European Journal of Plant Pathology. 159:851–862.
Decloquement, J., Ramos-Sobrinho, R., Galvao Elias, S., Santos Britto, D., Puig, A.S., Reis, A., Batista Pinho, D., Marelli, J., Fernandes Da Silva, R., Martins Newman Luz, E., Honorato-Junior, J. 2021. Phytophthora theobromicola sp. nov. a new species causing black pod disease on cacao in brazil. Frontiers in Microbiology. 12: 486.
Lopez, M., Gori, M., Bini, L., Orodonez, E., Duran, E., Gutierrez, O.A., Masoni, A., Giordani, E., Biricolti, S., Palchetti, E. 2021. Genetic purity of cacao criollo from honduras is revealed by ssr molecular markers. Agronomy Journal. Volume 11, Issue 2.
Gutierrez, O.A., Puig, A.S., Phillips-Mora, W., Bailey, B.A., Ali, S.S., Mockaitis, K., Schnell, R.J., Livingstone, D., Mustiga, G., Royaert, S., Motamayor, J.C. 2021. SNP markers associated with resistance to Frosty Pod and Black Pod diseases in a F1 population of Theobroma cacao L. . Tree Genetics and Genomes. 17, 28.