Location: Subtropical Horticulture Research
2023 Annual Report
Objectives
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.
Approach
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
Advancement is being attained during FY2023 on all three objectives and their sub-objectives. Under Sub-objective 1a. Develop artificial inoculation protocols and phenotyping techniques to assess resistance to Phytophthora canker in clones from Ecuador, the resulting procedures and methodologies obtained here were used in Subobjective 1. B. Assess Ecuadorian and NPGS cacao germplasm in Puerto Rico for resistance to BP and make data available via cacao databases. A total of 58 accessions (837 pods) from the local collection and 235 accessions (859 pods) from the USDA-ARS collection in Mayaguez, Puerto Rico, were screened in Miami, Florida. Phenotypic data analysis is in process and DNA is being extracted from all these accessions and fingerprinted with 11K SNPs markers. Genome wide association methodology will be used to find SNP markers associated with genes associated with resistance to black pod rot and Phytophthora canker. The resulting information will be used in our cacao breeding program and made available to the cacao research community worldwide.
Badnaviruses have been reported affecting cacao production in West Africa since the early 1930s. Also, around the same time, they were observed infecting in cacao in the Americas. Mealybugs are responsible for the transmission these viruses. Trinidad was the first place where Badnaviruses were found producing losses in cacao. Badnaviruses species currently known as Cacao Mild Mosaic Virus and Cacao Yellow Vein-Banding Virus (CaMMV and CYVBV) were the target of an eradication program by the Trinidad government in the 1950s. Recently, CaMMV, CYVBV have been located and recognized in Puerto Rico, Brazil, Florida, Indonesia and at the International Cocoa Quarantine Centre-Reading in Reading, United Kingdom. ARS researchers in Miami, Florida, and Mayaguez, Puerto Rico, have been collecting leaf samples at their locations and sending them to collaborators at the University of Arizona where the virus identification assays are being developed and tested. At least six CaMMV clades have been identified after analyzing eighty genome sequences. Genomic tools optimization for virus detection is currently ongoing as well as genomic variability and genetic diversity of CaMMV and CYVBV. The detection of the presence of these viruses in cacao collections worldwide and in cacao farms in Puerto Rico and Hawaii is an important priority since Badnaviruses are widespread in the cacao growing regions of the Americas and the application of molecular diagnostic tools is mandatory to guarantee safe movement of cacao germplasm among producing countries. Due of the discovery of these viruses in our cacao collections in Puerto Rico and Miami quarantine facilities, the transferring of the Ecuadorian clones to Colombia, Trinidad, and Brazil as part of Subobjective 1C and 1D was postponed.
In reference to Subobjective 2A, single nucleotide polymorphism (SNP) markers obtained using exome capture methodology and filtered with the cacao variety Matina 1-6 V1.1 and V 2.1 assemblies are in the process of being refiltered with three new assemblies developed by collaboration between USDA-ARS in Beltsville, Maryland, Miami, Florida, and the University of Nebraska. These new genomic resources embody the three most significant genetic clusters of wild cacao trees from the Upper Amazon region where genes for resistance to important diseases have been obtained. Also, this process is conducted using the Galaxy bioinformatic tools that is available from SciNet. These SNPs will be used to screen segregating populations and identify SNPs associated with disease resistance, quality, and horticultural traits. Two SNP panels comprehending a total of 10,633 SNP markers are being used to screen segregating populations and over 1,5000 germplasm accessions. The purpose of these screening is to identified markers that could be used in the discovery of markers associated with disease resistance genes and other important horticultural and quality traits as well as in populations structure determination, identification of population admixture levels and in. This is going to allow the development of cacao varieties using genomic- assisted breeding methodologies.
Concerning Objective 3, field data collection is in process in trials in Hawaii and Puerto Rico. Furthermore, six segregating populations were planted in Mayaguez, Puerto Rico, in 2021 and already initiated the flowering process and production of pods. The populations were developed from crosses made by ARS researchers in Mayaguez, Puerto Rico, in 2020 using high-yielding and disease-tolerant parents selected based on preliminary yield trial data. Leaf collection and lyophilization process have been completed. DNA extraction and SNP fingerprinting is being conducted. Regarding Objective 3, field data collection is ongoing in trials in Hawaii and Puerto Rico. In addition, segregating populations have been planted in Mayaguez, Puerto Rico, in 2021. The populations were developed from crosses made by ARS researchers in Mayaguez, Puerto Rico, in 2020 using high-yielding and disease-tolerant parents selected based on preliminary yield trial data. Leaf collection, DNA extraction and SNP fingerprinting are currently in process.
Finally, this is the last report for the project 6038-21000-025-000D which concluded on June 20, 2023. This project has identified emergent pathogens such as Neofusicoccum parvum and Lasiodiplodia theobromae affecting cacao production. In addition, the optimization of screening methodologies for the screening of resistance to Phytophthora canker and Black Pod Rot caused by Phytophthora spp. have been important accomplishments of this project. In addition, the detection of presence Badnavirus (CaMMV and CYVBV) in field and quarantine facilities conditions in Puerto Rico and Florida as well as finding Poleroviruses infecting cacao under quarantine conditions. Detection assays have been developed for both Badnavirus and Polerovirus species. Regarding the application of molecular makers, this project found SNP markers associated with resistance to Frosty Pod Rot and Black Pod Rot in cacao. These markers can be used to select trees with resistance to these diseases. Finally, this project has developed a core set of SNP markers for cacao population identification and selection of reference genotypes. The resulting SNP panel and the chosen reference germplasm are appropriate for cacao germplasm preservation, identification as well as their application in breeding programs.
Accomplishments
1. Polerovirus infecting Theobroma cacao germplasm. Cacao production worldwide is severely affected by diseases and pests. The exchange of germplasm across production areas is needed to introduce new sources of disease resistance. However, the detection of pathogens during the germplasm quarantine process is of paramount importance to avoid the introduction of diseases not previously present in production areas. In 2020, leaves with virus-like symptoms that consist of leaf discoloration and distortion with downward rolling at leaf edges, and light-yellow speckling or mottling were observed on cacao grafted trees of the CCN 51, CATIE-R1, CC-137, and CATIE-R4 genotypes that were undergoing quarantine process at the Subtropical Horticulture Research Station of the USDA-ARS in Miami, Florida. Due to the previous detection on this site of Cacao mild mosaic virus (CaMMV), ARS scientists in Miami, Florida, collected leaf samples of symptomatic plants and shipped them to the University of Arizona for further analysis. DNA and RNA was extracted from the samples and tests for the presence of cacao Badnaviruses were conducted and resulted negative. Subsequently, RNA sequencing was conducted, and results compared with plant virus databases. Sequencing results indicated that a Polerovirus was responsible for the observed symptoms, and it was named Cacao Leafroll Virus since it was different from other Poleroviruses that affect other crops. This is the first report of a Polerovirus in cacao and underscores the importance of the development of molecular tools for the detection of viruses before releasing the germplasm from quarantine and to prevent the movement and propagation of virus-infected cacao plants. Further sampling of leaf material at cacao germplasm collection and quarantine stations are needed to determine the presence of Cacao leafroll virus.
2. RNA sequencing analysis of resistance and susceptible cacao pods in response to the infection of the frosty pod pathogen. Frosty pod rot, caused by Moniliophthora roreri, is a damaging disease of Theobroma cacao in the Americas and it has been recently reported in Jamica and Brazil. Resistance to the pathogen has been determined using artificial inoculation techniques. ARS researchers Miami, Florida, Beltsville, Maryland, and CATIE, Costa Rica used RNA-sequencing to identify differences in pathogen-induced gene expression in cacao pods from trees resistant and susceptible to Frosty pod rot (FPR), caused by Moniliophthora roreri. Ten differences in constitutive gene expression were located within QTL on chromosomes 2, 7, and 9 previously associated with FPR resistance in this population. Results of this study provides the first analysis of constitutively expressed genes contributing to FPR resistance and identifies significant genes within known disease resistance QTL. This discovery will accelerate the development of cacao germplasm resistance Frosty Pod Rot.
3. Utilization of imaging technologies to capture three - dimensional variation in cacao floral morphology at micro - and nano – resolutions. Cacao pollination is a very important process since is directly related to cacao production. Theobroma cacao flowers present structural barriers between anthers and stigmas representing bottlenecks that restrict pollinator size and access to reproductive organs. To study the unresolved pollination biology of cacao, researchers from the University of Miami and ARS scientists at Miami, Florida, utilized a novel application of micro - computed tomography (micro - CT) using floral dimensions to quantify pollinator functional size limits. Using this technology, a petal side door (opening between petal hoods and filament) as the main bottleneck for pollinator access was identified. Also, three levels of likelihood for putative pollinators were identified and floral reward microstructures whose presence and location were previously unclear were found. Using micro - CT and GMM to study 3D pollination biology of cacao provides new evidence for predicting unknown pollinators. Incorporating geometry and floral rewards will strengthen plant - pollinator trait matching models for cacao and other species. These results will also allow to further investigate which insects are visiting the cacao flowers and how their sizes relate to determined floral dimensions. Finally, also these methods will be employed to characterize cacao accessions from genetically diverse populations.
Review Publications
Puig, A.S., Cohen, S.P., Koch Bach, R.A., Arciniegas Leal, A., Gutierrez, O.A., Bailey, B.A. 2023. Comparative RNA-seq analysis of resistant and susceptible Theobroma cacao fruits in response to infection with frosty pod pathogen (Moniliophthora roreri). PhytoFrontiers. https://doi.org/10.1094/PHYTOFR-09-22-0101-R.
Adegbola, R.O., Keith, C.V., Gutierrez, O.A., Goenaga, R.J., Brown, J.K. 2022. A previously undescribed polerovirus (solemoviridae) infecting theobroma cacao germplasm. Plant Disease. 107(3). https://doi.org/10.1094/PDIS-06-22-1449-PDN.
