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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

2020 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
Objective 1, artificial inoculation protocols and phenotyping developed by ARS scientists in Miami, Florida, as part of Objective 1a are being used to screen elite Ecuadorian clones for resistance to black pod rot and Phytophthora canker. Pods (4-5 months of age) produced using hand pollination are screened in the Plant Pathology Laboratory by inoculating unwounded pods with Phytophthora palmivora zoospores. This method was 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 as readily as P. megakarya and not all pods will develop lesions following inoculation. Approximately 160 pods from these clones have been screened in Miami, Florida to date. A modified screening protocol was developed for the cacao germplasm collection in collaboration with ARS scientists in Mayaguez, Puerto Rico, and so far, 100 pods have been screened in Miami, Florida. Grafted plants of these clones are also being produced and have begun screening for canker resistance under Objective 1B. Inoculations are 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. This screening will provide the necessary information regarding the level of resistance to Phytophthora canker of each accession and will be used to find single nucleotide polymorphism markers associated with resistance to Phytophthora canker. The resulting linked markers will be used in our genomics-assisted breeding program. Substantial progress was also made by ARS scientists in Miami, Florida, in identifying pathogens affecting cacao in Hawaii, Puerto Rico, Brazil, and Ecuador. Phytophthora spp. and other pathogens isolated from diseased pods and stems were shipped to Miami, Florida for pathogenicity tests or completion of Koch’s postulates. Approximately 100 Phytophthora isolates are now at the USDA-ARS-SHRS Plant Pathology Laboratory in Miami, Florida. DNA extraction and genetic barcoding has been completed for the Internal Transcribed Spacer, Cytochrome Oxidase 1, and Cytochorome Oxidase 2 regions. A subset of P. palmivora isolates from Hawaii and Ecuador had their genomes sequenced as part of the pathogenomics project (University of California-Davis/Mars Inc./USDA-ARS). Although T. cacao originated in the Americas, P. palmivora isolates from the Western Hemisphere have been underrepresented in pathogen diversity analyses. A pathogen collection trip to Puerto Rico was carried out by ARS scientist in Miami, Florida, in August/September 2019 to determine the diseases affecting cacao on the island. Symptomatic plant material was collected at 7 locations around the island: the USDA-ARS-TARS germplasm collection in Mayaguez, a field trial in Corozal, and five commercial farms. In addition to the expected Lasiodiplodia theobromae and Phytophthora palmivora, Cacao Mild Mosaic Virus (CaMMV) was detected on symptomatic RIM 52 plants at a commercial farm. Pathogen identity was confirmed using molecular diagnostics developed by collaborating scientist at the University of Arizona. These findings were published as a First Report and an illustrated document with information on CaMMV as well as management recommendations was created for growers in Puerto Rico. A second collection trip (March 2020) to determine how widespread CaMMV is on the island, and identify the affected clones was cancelled due to the Covid-19 pandemic. The Material Transfer Agreement (MTA) required to send the cacao clones to Colombia was approved and quarantine facilities were completed and certified by the Colombian Phytosanitary Agency. In addition, the rootstocks have been planted and are ready for grafting. However, due to the COVID-19 pandemic, the transfer of the cacao clones has been put on hold. Also, the MTAs for the transferring of the clones to Brazil and Trinidad are still in process. Conversations with these countries are ongoing and will continue once these issues are resolved. Currently, MTA drafts are being designed. All these previously described issues have stalled meeting Subobjectives 1C and 1D. Subobjective 2A, an additional 96 cacao DNA samples, that included accessions from Guiana and selected clones from the Centro Agronómico Tropical de Investigación y Enseñanza (CATIE) breeding program, were recently sent for sequencing. Previously, 1,054 cacao genotypes were sequenced and included germplasm 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 resistance. A completed data analysis of all the sequenced accessions is in progress. In addition, two SNP chips containing a total of 10,633 markers were designed and are being used to screen selected accessions from the 10 cacao genetics groups as well as from breeding populations. The main goal is to identify SNP markers that would allow us to classify genotypes into each one of the previously established genetic groups as well as to validate identification of current newly discovered accessions. In a recent screening using 1060 SNPs loci, preliminary results indicated that two hundred and nineteen of them were able to categorize different accessions into the 10 genetic groups. However, the screening of more accessions as well as the use of additional SNP loci are recommended since a more uniform covering of the cacao genome is desirable. Furthermore, the discovery of SNPs associated with disease resistance, quality and horticultural traits would also allow the development of cacao varieties using genomic-assisted breeding approaches. Objective 3, the collection of cacao yield data at Hawaii and Puerto Rico continued. Based on preliminary data, several clones were selected and used as parents for the development of segregating populations. Currently, several pods from crosses that were made in December 2019 have been harvested and seven segregating populations were planted in the greenhouse and are in the process of being planted in the field in Mayaguez, Puerto Rico, for further phenotypic and genotypic evaluation. Also, the making of the crosses for rootstocks (‘EET-400 × IMC 67’) will begin as soon as the fingerprinting analysis of two versions of IMC 67 present in the collection is completed.

1. Virus detected in commercial cacao in Puerto Rico. Cacao production is a rapidly expanding industry in Puerto Rico (PR); new farmers have planted ~20,000 trees in the past few years. Symptomatic pods and leaves were sampled and analyzed by ARS scientists (Miami, Florida and Mayaguez, Puerto Rico), in collaboration with cacao farmers in Puerto Rico and scientists from the University of Arizona. Recently developed molecular diagnostic tools confirmed the presence of Cacao mild mosaic virus (CaMMV), previously known as Cacao Trinidad virus strain A. This is the first report of CaMMV in Puerto Rico and believed to be the first report outside of Trinidad. This discovery is significant to the global cacao industry as it indicates CaMMV is more widespread than originally thought and highlights the importance of molecularly indexing germplasm before releasing from quarantine. An illustrated document was created for farmers to help them recognize CaMMV and its insect vectors in the field, along with management recommendations to prevent disease spread.

2. Discovery of pathogen genes that contribute to disease development in cacao. Lasiodiplodia theobromae can be an endophyte of Theobroma cacao, but under certain conditions it causes disease and can kill trees. A phylogenetic tree containing 52 Lasiodiplodia isolates obtained by ARS scientists in Beltsville, Maryland, and Miami, Florida, from symptomatic material from southeast Asia and the United States, indicated the presence of at least three different Lasiodiplodia spp. To understand the disease at the molecular level, the fungal genome was sequenced and fungal gene expression during the disease cycle was examined. More than 13,000 genes were predicted, of which around 2,800 are unique to Lasiodiplodia theobromae when compared to other closely related fungi. The gene expression data show that the fungus expresses genes that interfere with the plant defense mechanisms and degrade the plant cell-walls. These findings expand our knowledge on L. theobromae genes involved in disease development in cacao and will be used by researchers to improve disease management practices and develop improved cacao varieties.

3. Independent whole-genome duplications in the primary black pod pathogens, Phytophthora megakarya and Phytophthora palmivora. Phytophthora megakarya and P. palmivora are oomycete pathogens that cause black pod rot, the most economically important disease on cacao globally. The genomes and transcriptomes of 31 isolates of these two species were compared by ARS researchers in Miami, Florida, using new single-molecule sequencing techniques to understand their evolution and to identify factors determining their virulence on cacao. It was discovered that both species underwent a genome duplication, with the genome of P. megakarya is nearly twice the size of the P. palmivora genome (222 Mbp vs 135 Mbp, respectively). However, the P. megakarya transcriptome was further increased by gene duplication processes involving transposable elements, resulting in it having more virulence related genes than any other species of Phytophthora. This study showed that, P. megakarya has an extreme potential to adapt, overcoming the plant host defenses, which is of great concern to cacao breeders. It is critical to limit the spread of P. megakarya, and in areas where it already occurs, disease management practices must be optimized to limit pressures that would drive pathogen adaptation.

4. Rapid molecular identification of beetles affecting Theobroma cacao. Ambrosia and bark beetles bore into branches, stems, and pods of T. cacao and may be increasing disease incidence in the field by wounding plants and spreading inoculum. To enable more rapid screening for high priority insects and detect new invasions, ARS scientists at Miami, Florida, collaborated with Mars, Inc. to develop a molecular identification approach that requires only basic molecular biology skills and equipment. This sequence-based protocol provides faster identification of ambrosia and bark beetles, and new introductions can be detected within days of capture. Identifying insects affecting healthy and diseased T. cacao, will provide valuable information by determining whether any genera may be actively transporting Phytophthora sp. inoculum to healthy plants. This will directly benefit cacao breeders because the presence of insect vectors will influence how disease incidence data from the field is analyzed.

5. A complex of badnavirus species associated with cacao swollen shoot disease in West Africa: evidence for high genomic variability and interspecific recombination. West Africa is responsible for 70% of the cacao production in the world. However, cacao swollen shoot virus disease (CSSD) has been severely affecting cacao production. Also, a genetic characterization of the complex of badnavirus species associated viruses causing CSSD in West Africa has not been completed. Researchers at the University of Arizona collaborated with ARS scientists at Miami, Florida, to characterize genomic variability and genetic diversity of the badnavirus infecting cacao in Cote d’Ivoire and Ghana and causing CSSD in cacao producing countries in West Africa. Genome sequencing and phylogenetic analysis results indicated the existence of thirty new genomes that included previously described species such as Cacao swollen shoot virus (CSSV), Cacao swollen shoot CD virus (CSSCDV), and Cacao red vein virus (CRVV). Recombination among badnavirus genomes was observed in the phylogenetic results which also provided evidence of viral subpopulations geographically structured among Cacao red vein banding virus (CRVBV) isolates. These discoveries will help with the development of new molecular diagnostic tools that will identify all CSSD causing strains in West African countries and help to reduce the spread of CSSD.

Review Publications
Engbersen, N., Gramlich, A., Lopez, M., Schwarz, G., Gutierrez, O.A., Schulin, R. 2019. Cadmium accumulation and allocation in different cacao cultivars. Science of the Total Environment. 678, 660-670.
Ramos-Sobrinho, R., Chingandu, N., Gutierrez, O.A., Marelli, J., Brown, J.K. 2020. A complex of badnavirus species infecting cacao reveals mixed infections, extensive genomic variability, and interspecific recombination. Viruses. 12, 443-461.