Sponsoring Institution
National Institute of Food and Agriculture
Project Status
Funding Source
Reporting Frequency
Accession No.
Grant No.
Project No.
Proposal No.
Multistate No.
Program Code
Project Start Date
Jul 1, 2014
Project End Date
Jun 30, 2019
Grant Year
Project Director
Guiltinan, MA.
Recipient Organization
Performing Department
Plant Science
Non Technical Summary
Overwhelmingly, plant pathogens present the major challenge to sustainable cacao farming throughout the world. It is estimated that pests and diseases reduce the potential crop by an estimated 810,000 tons annually (30% of world production) and individual farm losses can approach 100%. In one example, Witches' broom disease resulted in a decrease of production in Southern Bahia, Brazil, from 300,000 tons in 1989 to 130,000 tons 10 years later, for an estimated loss of $220 million each year. This caused a cascade of widespread social disruption among small-holder growers including loss of land and home ownership. Widely dispersed cacao pathogens include several species of Phytophthora. P. megakarya, the most aggressive species, is reported to have entered Ivory Coast, the world's leading cocoa producer. Other important diseases and pests include frosty pod in Central America, the cocoa pod borer in Asia and cocoa swollen shoot virus in West Africa.The project will address societal needs to help alleviate poverty and provide stable incomes to poor under-resourced farmers in developing countries. Increasing farmers incomes will have a wide ranging effect from family health to child education. Economically and environmentally sustainable cropping systems are necessary to enhance the productivity of the cocoa farmers. Developing molecular tools such as DNA markers will help speed breeding programs and development of high yielding varieties. Development of propagation systems will help speed the roll out of the new varieties. This will increase income to small holder farmers and help build micro-enterprises for cacao propagation.
Animal Health Component
Research Effort Categories

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
Goals / Objectives
Using combination of genomic and functional genomic approaches, identify genes involved in plant disease resistance in cacao.Develop molecular markers to disease resistance genes and make these available to cacao breeders in producing countries.Contribute to scientific capacity building in cacao producing countries by supporting scientific exchange visits.
Project Methods
We have developed functional genomics tools for cacao including a stable transformation system44, Agrobacterium infiltration transient leaf expression assay, whole genome microarray and predicted proteome peptide database for mass spectrometry based protein identification (unpublished data). We have used transgenic Arabidopsis, tobacco and tomato to study the function of different cacao genes including genes involved in the defense and flavonoid pathways40,45-49. Perhaps the most powerful method we have adapted for cacao functional studies is a transient expression system that was recently developed with funding from the NSF-BREAD program. Using vacuum infiltrated Agrobacterium, we demonstrated high levels transgene expression in a large proportion of cells in cacao leaves. After several days of expression, the leaves were challenged by placement of a small agar block containing mycelia of Phytophthora sp. (or other pathogens) on one half of the leaf, and a water control on the other half. After 2-4 days they were photographed, lesion size measured, and a disc of material removed for extraction of RNA and DNA. Transgene and endogenous gene expression will be measured using QPCR and the relative amount of replication estimated via QPCR with pathogen specific primers.Summary: We will identify high priority candidate genes based on sequence homology to genes from Arabidopsis and other species, expression patterns associated with plant defense, and colocalization to QTLs for disease resistance. These genes will be tested using the various methods described above. Molecular markers for important genes will be created using standard PCR techniques and validated in collaboration with our collaborators in developing countries.Identification of Candidate Genes: Based on the genome sequence information, along with data from prior expression studies using a microarray approach, we already have identified several hundred interesting candidate genes. We have further prioritized thise by colocalization to QTLs of interest for disease resistance resulting from published reports. Isolation of allelic forms of candidate genes: Allelic versions of prioritized candidate genes will be isolated by PCR amplification and cloned into Agrobacterium transformation vectors we have developed44. We have designed a cassette based series of Ti plasmid cloning vectors capable of over-expression of candidate genes, expression with native promoter or expression of an inverted repeat designed to induce gene silencing. The candidate gene cloning sites are closely linked to a CaMV35S promoter-driven GFP gene that is used for visual observation of transformation efficiency. We will sequence verify each Ti construct via dideoxy sequencing at the PSU Genomics Core lab. After cloning and verification of plasmids replicating in E. coli, plasmids are transferred to Agrobacterium tumefaciens strain AGL1 via electroporation, then structurally verified by PCR to eliminate and recombinants that do occur in Agrobacterium occasionally.Phytophthora Infection Assays, Phenotyping and Tissue Collection: The study will focus on resistance to the Phytophthora disease using the leaf assay method described above (preliminary results). To measure the resistance of each genotype against Phytophthora, we will perform resistance assays on leaves from greenhouse grown plants. At least four replicate experiments will be performed on each of the 16 diversity panel and 6 segregating population parental genotypes. Symptom evaluation will be measured as average lesion size and photographs taken. Relative pathogen biomass amount will be measured using QPCR of DNA extracted from the lesions and primers specific to Phytophthora relative to cacao as previously described40. Tissues samples will be excised using a scalpel and frozen in bar-coded tubes and all data will be stored in a cross referenced database (see data management plan).Cacao Leaf transient expression-Phytophthora resistance assay: We will utlize the transient leaf assay as described in preliminary results and in our prior publications40,41. After 2 days of transient expression, leaves will be inoculated with agar blocks containing either water or Phytophthora mycelium, and incubated for an additional 2-4 days. Lesion size, candidate gene expression, and pathogen replication rates are then measured using photography, RT-QPCR (using candidate gene specific primers) and QPCR (using pathogen nuclear gene specific primers) respectively. Expression levels and pathogen genomic DNA content will be normalized to cacao actin controls. Our current method uses photographic imaging to quantify lesion size at a single time point. Funds requested in this project to acquire a Qubit Systems chlorophyll fluorescence instrument will enable us to increase throughput and accuracy of our assays. Chlorophyll fluorescence parameters are perturbed by infection long before lesions are visible because inhibition of photosynthesis is one of the first effects of pathogen infection. The method is non-invasive, and the spread of inhibition can be observed and quantified with time. This will allow us to examine the dynamics of host-pathogen interactions rather then a single point in time. All candidate gene tests will be performed with 9 replicate inoculations and repeated at least three times on different days.Transgenic complementation in Arabidopsis: When possible, we will take advantage of the massive amount of information related to defense response genes in Arabidopsis. This can help us to develop testable hypotheses related to function of specific candidate genes in cacao. For candidate genes which have a clear ortholog in Arabidopsis and for which there is a mutant phenotype previously described, we will obtain seeds from the TAIR stock center, generate homozygous mutants and transform these lines with the cacao orthologs using the Ti plasmid vectors described above. We will test the function of the candidate genes using standardized pathogen assays including bacterial, fungal and oomycete pathogens. We have successfully used this approach with several cacao genes in the past as discussed in preliminary results40,45,47.Stable Transgenic cacao: For the most promising of candidate genes and combinations of multiple gene, we will create transgenic lines of cacao using methods previously described by the Guiltinan lab44,53-55. In short, primary somatic embryos derived from floral explants are used for co-cultivation with Agrobacterium containing the candidate genes, and transgenic secondary somatic embryos will be regenerated. We will transform a disease susceptible accession of cacao for these tests. After acclimation to greenhouse conditions, the Phytophthora resistance assay can be carried out on leaves from these plants. In addition we can silence the expression of the candidate resistance gene using the transient expression system to further validate the function of the stably introduced transgene. Ultimately, but out of the time frame of this proposal, we will be able to test resistance of developing fruit tissue in greenhouse grown plants.Production of Molecular Markers: One we have validated the importance of specific genes, it is very simple to design primers specific to that gene and specific to a particular allele of a gene based on the SNPs present in the desired allele. These will be generated and the information transferred to our collaborators in Africa who are best positioned to use them in their breeding programs

Progress 07/01/14 to 06/30/19

Target Audience:Plant science community: scientific manuscripts and conference presentations Cacao genetics and breeding community: linking functional genomics to marker assisted selection Graduate students from developing countries: MS and Ph.D. students from Colombia and Peru Visiting scientists: currently hosting one scientist from France and an undergraduate from the Netherlands Chocolate industry: through collaborative sponsored research programs The US government through funded research agreements with USDA FAS, USDA NIFA and NSF. Changes/Problems:Methods for genomics have continued to rapidly evolve and fall in cost. In particular DNA sequencing costs have fallen dramatically changing what is now feasibile to conceive within a given budget. The Illumina Novaseq platform, available to us at Penn State Hershey Medical School, enables very large sequencing projects to be run at significant cost savings. Together with other new capabilities we have moved from the genome era into the genetic diversity era, exploring the differences between individual genomes to discover genes for important traits. We continue to monitor the technological changes in this field and to test and develop new methods and adopt them to cacao research. The rapidly evolving research landscape requires us to also form interdisciplinary collaborations with bioinformaticians populations geneticists, evolutionary and ecological biologists and others to meaningfully interpret and utilize this new data resource. The need for this integration alone is a challenge that will require institutional level support to fully accomplish. What opportunities for training and professional development has the project provided?Experience documented by authorship on manuscripts includes: 1 undergraduate, 4 technical assistants, 6 graduate students 3 postdocs and 8 visiting scientists/collaborators. Developed the Penn State Cacao and Chocolate Research Network, (CCRN) which brings together about 50 faculty and graduate students. CCRN has sponsored training workshops for all project participants in leadership, communication and grant writing. Through our NSF funded research project, we have offered two summer research fellowships to undergraduate minority students for the past two years and will host a third program in 2020. These students experience research at Penn State, in our field sites in Costa Rica and take classes and gain field experience at EARTH University in Costa Rica.Students and postdocs in our program are supported for research conferences and presentations about 1 time per year.Mentored several graduate students to write graduate fellowships, one of which was successful last year (NIFA Fellowship to Noah Winters) How have the results been disseminated to communities of interest?Our results and activities are documented and distributed via peer review publications, scientific presentations and conferences, poster presentations at conferences and articles and interviews in the public press. We maintain an active internet presence via a lab website, and active Facebook group. Guiltinan - Maximova Labs Members, Alum, Collaborators and Friends What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

What was accomplished under these goals? Goals 1 and 2: To explore the role of salicylic acid in the regulation of defense in cacao, we demonstrated that SA treatment decreased susceptibility to a pod rot pathogen, Phytophthora tropicalis in two genotypes, Scavina 6 and Imperial College Selection 1, which differ in their resistance to several agriculturally important pathogens. These experiments revealed critical insights regarding the molecular differences between cacao varieties, which will allow a better understanding of defense mechanisms to help guide breeding programs. The pathogenesis-related (PR) group of proteins are operationally defined as polypeptides that increase in concentration in plant tissues upon contact with a pathogen. To date, 17 classes of highly divergent proteins have been described that act through multiple mechanisms of pathogen resistance. Characterizing these families in cacao, an economically important tree crop, and comparing the families to those in other species, is an important step in understanding cacao's immune response. We describe candidate genes that are likely to be involved in cacao's defense against Phytophthora and Colletotrichum infection and could be potentially useful for marker-assisted selection for breeding of disease resistant cacao varieties. The data presented here, along with existing cacao--omics resources, will enable targeted functional genetic screening of defense genes likely to play critical functions in cacao's defense against its pathogens. We developed the first application of genome editing technology in cacao, using Agrobacterium-mediated transient transformation to introduce CRISPR/Cas9 components into cacao leaves and cotyledon cells. As a first proof of concept, we targeted the cacao Non-Expressor of Pathogenesis-Related 3 (TcNPR3) gene, a suppressor of the defense response. After demonstrating activity of designed single-guide RNAs (sgRNA) in vitro, we used Agrobacterium to introduce a CRISPR/Cas9 system into leaf tissue, and identified the presence of deletions in 27% of TcNPR3 copies in the treated tissues. The edited tissue exhibited an increased resistance to infection with the cacao pathogen Phytophthora tropicalis and elevated expression of downstream defense genes. As part of a long-term goal to define key disease resistance genes in cacao we used a transcriptomic analysis of the disease-resistant cacao clone SCA6 and the susceptible clone NA32 to characterize basal differences in gene expression, early responses to infection, and polymorphisms in defense genes. Gene expression measurements by RNA-seq along a time course revealed the strongest transcriptomic response 24 h after inoculation in the resistant genotype. We observed strong regulation of several pathogenesis-related genes, pattern recognition receptors, and resistance genes, which could be critical for the ability of SCA6 to combat infection. These classes of genes also showed differences in basal expression between the two genotypes prior to infection, suggesting that prophylactic expression of defense-associated genes could contribute to SCA6's broad-spectrum disease resistance. We used a 90 SNP array to verify genetic identity of a set of clones in the International Cocoa Collection at CATIE, Costa Rica, and assign the clones into known genetic groups. We also used a detached leaf inoculation technique to measure the susceptibility of 60 genotypes to Phytophthora palmivora, a major cacao pathogen with global importance. We identified 24 genotypes with disease tolerance statistically similar to a standard tolerant variety (SCA6) and another 24 which performed similarly to a standard susceptible variety (ICS1). Our results indicate that each of the four included genetic groups show variability for quantitative resistance to P. palmivora. These results provide a foundation for future genomic and transcriptomic analysis of disease tolerance and susceptibility in the field at CATIE and provide guidelines for breeders searching for novel sources of tolerance that can be introduced into breeding programs. We have transferred this information and gene sequences to our collaborators at USDA ARS and we are working together to develop molecular makers for marker assisted selection in the future. Goal 3: Since 2015 we have hosted a number of short and long term visiting scientists from developing countries who have performed research on cacao molecular biology and have learned many different techniques and how to use various genomics resources we have developed. For example, Adriana Gallego from the University of Antioquia, Colombia was hosted for about one year and performed metabolomic and gene expression studies with tissue cultured cacao cells resulting in manuscripts.


  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Fister, A.S., Landherr, L., Perryman, M., Zhang, Y., Guiltinan, M.J., and Maximova, S.N. (2018). Glucocorticoid receptor-regulated TcLEC2 expression triggers somatic embryogenesis in Theobroma cacao leaf tissue. PLOS ONE 13, e0207666
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Pokou, D.N., Fister, A.S., Winters, N. et al. Plant Mol Biol (2019) 99: 499.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Tuomas H�m�l�, Mark J. Guiltinan, James H. Marden, Siela N. Maximova, Claude W. dePamphilis, and Peter Tiffin (2019), Gene Expression Modularity Reveals Footprints of Polygenic Adaptation in Theobroma cacao, Molecular Biology and Evolution,

Progress 10/01/17 to 09/30/18

Target Audience:General public, scientific community, policy makers. Changes/Problems:Significant Change in Approach Overview: Project is proceeding as planned as discussed in last progress report.It has progressed through the germplasm selection and phenotyping phase and is entering the high throughput sequencing phase. This report summarizes several changes made in the experimental plan of our project that together, are significant enough to warrant this interim report. All changes have been made by consensus with discussion of all project coPIs and project participants in a number of lab meetings and other communications. During the time period after project approval, the technical capabilities and costs of high throughout genome sequencing have continued to evolve rapidly. The largest factor we are taking into consideration is the Illumina Novaseq platform, which we have available through a new instrument located at Penn State Hershey Medical School. This instrument enables very large sequencing projects to be run at significant cost savings.We have discussed the specifications of the Novaseq planform in detail with the Illumina technical representatives and our sequencing facility director (Dr. Craig Praul), and examined manuscripts and datasets made using this platform. The new platform produces sequence data with error rates roughly the same as instrument we originally specified in our proposal (Illumina Hiseq 2500) but with some possible differences in the spectrum of error types and frequencies. These errors will be overcome primarily with the very high sequence coverage rates (100x). Summary of Experimental Plan Changes 1.All sequencing to be performed on the Novaseq platform 2.Added small pilot study with Arabidopsis 3.Added new cacao genotypes 4.Added basal transcriptomes Specific Changes to Experimental Plan and Justification 1.All high-throughput genome and transcriptome sequencing will be performed on Illumina Novaseq platform This will reduce costs with little significant change is sequence quality. 2.Added small pilot study with Arabidopsis: Co-PI dePamphillis has pointed out that the Novaseq technology exhibits small changes in error which is not yet fully characterized specifically the impact on the quality of de novo assembled genomes and transcriptomes relative to data from other platforms. To address this concern, we will perform a small pilot study at the same time as the main sequencing runs to compare the different platform performances within our experimental design using control Arabidopsis RNA and DNA. 3.Number of Genomes Expanded: With the reduction in cost and as the project moved forward, we decided to add a set of additional diverse genomes to the project design. We have added 13 new genotypes to our sequencing project. These include 8 more in our diversity collection and 5 additions of elite hybrids produced in breeding programs.These new samples will strengthen the power of our main objective by adding a fourth genetic group, and provide resources for exploring the domestication of cacao as spin off projects. 4.Added new objective: Cacao basal transcriptomes for de novo assemblies. We will run one leaf RNA sample from each of our targeted varieties to allow creation of variety specific de novo transcriptome assemblies that will be useful in guiding the genome assemblies and gene annotations. We will run an additional set of 9 RNAs to test tree to tree variation in transcriptome data. Total of 46 added transcriptomes. Our scientific plan specifies the importation of cacao plants to PSU for phenotypic and genomic analysis. For reasons specified below, we have changed our approach to access plants growing in Costa Rica (CATIE) and Ivory Coast West Africa (CNRA) for phenotyping and genomic analysis. All other goals and objectives remain as planned. The changed results in a one-year delay in the original strategy. An alternative plan discussed below was developed to address this issue. This change impacts the project positively through increased interactions with our collaborators at CATIE, increased access to cacao genetic resources, and access to pathogen assays we did not previously have. Our strategy to utilize plants in Costa Rica and West Africa not only provided us with a solution to the delay in issue of plant importation permits, it opened some new interesting potentials for our team to explore the genetic diversity of the large collections at CATIE and INRA, and to work closely with scientists in the field. Reasons for change New USDA Aphis Regulations During the review period of this project new USDA regulations for importation of plant materials for research activities were put into place. During a routine renewal of our import permits, we were told that the new regulation required us to develop a quarantine facility as defined by USDA Aphis regulations. Design, funding and construction of the facility upgrade caused a one-year delay in our ability to import the plant materials selected for the project. As of June 2017, we are still waiting on the final permit authorization from USDA Aphis. Actual or Anticipated problems or delays and actions or plans to resolve them Actual Delays 1 year for plant importation. Most other activities not impacted. Plans to Resolve Delays Greenhouse Upgrade PI Guiltinan raised funds (PSU), designed GH modifications to meet USDA Aphis Quarantine requirements and initiated a work order in 2016. The plans specify a screening and ventilation system and a new airlock entry chamber that would be installed in section 109 of Greenhouse J, a research greenhouse funded by NSF Academic Infrastructure program in 1994 (Guiltinan, PI).Modifications were installed and tested in Spring 2017.USDA Aphis performed inspections May 2017. We anticipate shipping first plants in summer of 2017. Alternate Plan 1 Because of the time delay we developed a new plan for genotype selection. We took advantage of our network of cacao scientists and institutions to develop collaborative agreements (MOUs) with our partners in Costa Rica (CATIE) and Ivory Coast (CNRA). We developed plans to screen diverse sets of cacao genotypes using materials growing in our partners' field stations, rather then in our greenhouse. We are taking advantage of specific populations found at these two excellent research sites. In retrospect, despite the time loss, Plant B is a large improvement to our experimental plan; 1) we have access to a much larger set of genetic diversity; 2) we can apply historical data and look at multiple pathogens; 3) we have further engaged two young developing country Ph.D. scientists in cacao research to participate in our project. What opportunities for training and professional development has the project provided?Three graduate students (one received his Ph.D. this year). Three postdocs were mentored in research and career development. 3 undergraduate students received research experience via independant studies credits. How have the results been disseminated to communities of interest?Scientific manuscripts, lab website and facebook pages, seminars, news articles and radio interviews We have developed a new network of scientists engaged in cocoa and chocolate research at Penn State What do you plan to do during the next reporting period to accomplish the goals?We will continue the analysis of genetic diversity of disease resistance genes and of other key genes inolved in added value traits in cacao. Additionally we will be hosting several visiting scientists from developing countries and mentoring 5 graduate students from Peru and Colombia as part of our scientifi capacity building goals. We will continue to work with Cacao for Peace fundinded by USAID to develop research capacity in Colombia and expore the genetic variability of cacao growing in various areas of the country. We will continue publishing high quality manuscripts with 6 papers currently in progress. We will continue our public outreach in the areas of general education oon topics surrounding plant genetics and ag innovation.

What was accomplished under these goals? In a breakthrough made possible by steeply dropping sequencing costs and through funding from NSF, 34 new cacao genomes were sequenced and a large number of transcriptomes acquired. This data will soon be mined to help us eplore the evolution and function of disease resistance genes. Additionally we are also studying genes for important traits isolated and functionally tested (flower development, flavonoid biosynthesis, cadmium uptake). Phenotyping for disease resistance completed with collaborations at CATIE Costa Rica. Several new genes implicate d in disease resistance are currently under functional analysis and may be promising candidates for plant breeding. A specific flavonoid important for disease resistance was identified in cacao and the genes encoing enzymes involved in its biosynthesis were isolated and functionally verified. A patent disclosure is in preparation related to this. We have explored the metabolomic diversity of a set of 56 cacao genotypes in relation to seed storage triglycerides. Significant results: Target genotypes validated by SNP analysis:To validate the genotypes of each of our target clones which are growing in the field in CATIE, Costa Rica, we isolated genomic:DNA and performed SNP ancestry analysis using a set of 90 SNP markers. The genotyping PCA is a representation of genetic distance between clones in the four selected genetic groups based on our set of 90 SNPs (figure 1). From this data we can see clear separation of the four different genetic groups and identified one misidentified tree which will be eliminated from future analysis. Target genotypes phenotyped for disease resistance:We completed our phenotyping of our genetic diversity collection for resistance to Phytopthora. The phenotyping graph summarizes the defense response of the clones from the four genetic groups as well as two control varieties (Sca6 and ICS1) and two special interest clones (R4 and R6) (Figure 2). Each genotype in the graph was sampled at least 10 times (biological reps) and on at least three days. Pilot sequencing projects to test new sequencing technologies:We compared the efficacy of gene expression measurements using two commercial platforms, Illumina Truseq vs Novagen Quantseq. We compared the changes in gene expression between several different samples by high depth sequencing using the two methods. In short, our analysis shows that with only 5 million reads of Quantseq, we can obtain basically identical data compared to 20 million reads of Illumina Trueseq (r2>0.98). This is because the Quantseq method only reads 3'UTR thus removing a lot of redundant sequencing of identical mRNAs that do not provide additional gene expression measurement power. Thus, this method can be used for gene expression experiments at a reduction of about 75% of the actual sequencing cost. We will still use Trueseq libraries to obtain full length cDNA sequences to assist in gene annotation. Development of Rooted Cutting Capacity at CATIE Costa Rica:Initially our collaborator at CATIE had difficulty to make rooted cuttings of the genotypes that will be sent to PSU for future experiments and to form the foundation of a permanent germplasm collection representing all genotypes sequenced in this project. After working with several modifications of their protocol, the rooting systems appears to be functioning well now. We are anticipating shipping of germplasm to PSU in Fall 2018. Scientific Capacity Building:Summer Internship for Minority Undergraduate College Students in Cacao Research and International Agricultural Development: Two undergraduates participated with a summer internship sponsored by NSF spending 8 weeks at psu and 6 weeks in Costa Rica participating in research programs and learning about plant biology research. The interns will participate in plant genetics, molecular biology, biotechnology or bioinformatics research on Theobroma cacao, the chocolate tree. During the first 2 weeks of the internship period at Penn State University Park campus in Pennsylvania, the interns will join research teams and will be mentored by a postdoctoral and/or graduate student researchers. Students will also participate in an international agricultural development orientation presented by International Agriculture and Development Graduate Programs (INTAD) graduate students at Penn State. For the following 4-week period the interns will join members of the research team to travel to Costa Rica to participate in a 2-week summer session at Earth University, focusing on sustainable development and a 2-week training at the Tropical Agricultural Research and Higher Education Center (CATIE) in Costa Rica. The internship will conclude with 2 weeks back at Penn State during which each participant will prepare a presentation and present a summary of their experiences at a project meeting involving all participants. The two students are Gabriela Silva Pumarada and Fernando Prieto Santiagofrom University of Puerto Rico. The students are participating in 8-week PennState/Earth/CATIE internship program for minority undergraduate students.The students will be at CATIE from June 30th to July 14th and willwork closely with Mariela Leandro one of our key collaborators.The proposed students activities include: • Greenhouse work: seed preparation and sowing, and cacao grafting. • Cacao flowers artificial pollinations. • Artificial inoculations and evaluation of Moniliophthora roreri and Phytophthorapalmivora. • Pruning, chupon elimination, soil and leave fertilization. • Weed and insect control. • Pod harvest and fermentation. • Cacao root cuttings. Two visitors from Peru spending October at PSU working in a collaboratingsoil science lab as part of cadmium project. As a result in a scientific exchange visitof Dr. Adriana Gallego from Colombia, last year a manuscript was published:Adriana M. Gallego, Luisa F. Rojas, Oriana Parra, Héctor A. Rodriguez, Juan C. Mazo Rivas, Aura Inés Urrea, Lucía Atehortúa, Andrew S. Fister, Mark J. Guiltinan, Siela N. Maximova & Natalia Pabón-Mora Scientific Reports Volume 8, Article number: 13575 (2018) Two additional collaborative manuscripts are in review resulting from visiting scientist exchanges.


  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Mej�a, L., Herre, E., Sparks, J., Winter, K., Garc�a, M., Van Bael, S., Stitt, J., Shi, Z., Zhang, Y., Guiltinan, M. & Maximova, S. Pervasive effects of a dominant foliar endophytic fungus on host genetic and phenotypic expression in a tropical tree (reprint). Recent Advances in Symbiosis Research: Integrative Approaches, 52-67 ISBN: 9782889450152 (2017).
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Fister, A. S., Landherr, L., Maximova, S. N. & Guiltinan, M. J. Transient Introduction of CRISPR/Cas9 Machinery Targeting TcNPR3 Enhances Defense Response in Theobroma cacao. Plant Biotechnology Frontiers in Plant Science, March 2, 2018.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Gallego AM, Rojas LF, Parra O, Rodriguez HA, Mazo Rivas JC, Urrea AI, Atehort�a L, Fister AS, Guiltinan MJ, Maximova SN et al: Transcriptomic analyses of cacao cell suspensions in light and dark provide target genes for controlled flavonoid production. Scientific Reports 2018, 8(1):13575.

Progress 10/01/16 to 09/30/17

Target Audience:Plant science community, cacao research community, international development agencies, chocolate industry, governmental agencies in Colombia and Peru. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Training of 2 graduate students and 2 undergraduate students. Two staff have attended various workshops and lectures. Two postdocs are gaining experience in research and associated experience with planning, writing grants and supervising other scientists. How have the results been disseminated to communities of interest?Through publications, radio interviews, newspaper articles, seminars and poster presentations. We have been highlighted in Penn State Research, the Penn State Homepage, gave presentations at the community library and elsewhere, and several news articles were published in various worldwide papers and websites covering our work. What do you plan to do during the next reporting period to accomplish the goals?We have developed new research piplelines for genome resequencing which will further accelerate our gene discovery pipeline. Two new graduate students are working on this project. We have established with NSF funding a new team also focused on related goals.

What was accomplished under these goals? We continue to identify disease resistance genes in cacao. We have yet to reach the goal of providing markers to breeders. We have contributed to training of scientists from developing countries by hosting visiting scientists from Colombia and Ivory Coast. Our work this year resulted in phenotyping of 70 accessions of cacao in Costa Rica for resistance to Phytopthora. We have done genotyping with 96 SNP of these same accessions. We are preparing methods to resequence the gneomes of 27 of these genotypes. As part of this project we are working on the Cacao for Peace project. This is funded by USDA FAS and targeted at helping farmers in Colombia. We have organized an extension workshop, a research workshop, and a two week training workshop all which were deliverd in Colombia. We have organized a graduate training program and started 4 Colombian students at Penn State all working on cacao related topics. We have developed a PSU cacao research network cosisting of about 10 faculty members all interested in this area of work. We have also obtained major funding from an international food company to start a new project. Thus we have leveraged the endowned program to new funding and new projects, and involved a grwoing number of psu faculty and students.


  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Ali, S. S., Shao, J., Lary, D. J., Kronmiller, B. A., Shen, D., Strem, M. D., Amoako-Attah, I., Akrofi, A. Y., Begoude, B. A. D., ten Hoopen, G. M., Coulibaly, K., Kebe, B. I., Melnick, R. L., Guiltinan, M. J., Tyler, B. M., Meinhardt, L. W. & Bailey, B. A. Phytophthora megakarya and Phytophthora palmivora, Closely Related Causal Agents of Cacao Black Pod Rot, Underwent Increases in Genome Sizes and Gene Numbers by Different Mechanisms. Genome Biology and Evolution 9, 536-557, doi:10.1093/gbe/evx021 (2017).
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Leandro-Mu�oz, M. E., Tixier, P., Germon, A., Rakotobe, V., Phillips-Mora, W., Maximova, S. & Avelino, J. Effects of microclimatic variables on the symptoms and signs onset of Moniliophthora roreri, causal agent of Moniliophthora pod rot in cacao. PLOS ONE 12, e0184638, doi:10.1371/journal.pone.0184638 (2017).

Progress 10/01/15 to 09/30/16

Target Audience:Plant science community, cacao research community, international development agencies, chocolate industry, governmental agencies in Colombia and Peru Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?See above re scientific exchange visitors. In addition, we have worked with 4-6 undergraduates and 4 graduate students. Two graduate students received Ph.D.s this year affiliated with this project. How have the results been disseminated to communities of interest?Yes: We held a major symposium this year to mark the 30th anniversary of the program hosting 175 scientists from 25 countries and industry partners. During this meeting an entire session was devoted to presenting our progress over the years. We have further communicated via publications, our website, a facebook page, presentation at national and international meetings and by hosting visitors from around the world. What do you plan to do during the next reporting period to accomplish the goals?We are organizing a cacao workshop at the Plant and Animal genome meeting. We continue to communicate as stated above for last year. We will develop new websites for the Cacao for Peace and for the new NSF projects. We have integrated facebook and twitter into our communication platforms.

What was accomplished under these goals? A major paper which chracterized most of the genes in the PR families (pathogenesis related) was published. This is a major benchmark in our analysis of the cacao genome. Visiting scientists from Ivory Coast, Colombia (2) and Trinidad spent 1 year, 1.5 years and 3 months respectively in the lab learning new techniques and performing cacao research. Our team became integrated into a new project: Cacao for Peace Colombia, working with the USAID, UADA FAS and other partners. We have also been granted a new 4 year project by the NSF to study the immune system of cacao which will significantly synergize with this project.


  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Fister, A., Mejia, L., Zhang, Y., Herre, E., Maximova, S., and Guiltinan, M. (2016). Theobroma cacao L. pathogenesis-related gene tandem array members show diverse expression dynamics in response to pathogen colonization. BMC Genomics 17, 363. doi:10.1186/s12864-016-2693-3.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Fister, A.S., Shi, Z., Zhang, Y., Helliwell, E.E., Maximova, S.N., and Guiltinan, M.J. (2016). Protocol: transient expression system for functional genomics in the tropical tree Theobroma cacao L. Plant Methods 12, 19. DOI 10.1186/s13007-016-0119-5

Progress 10/01/14 to 09/30/15

Target Audience:Cacao scientists, general plant science community, general public, chocolate industry Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?We have trained 4 undergraduate and three graduate students during this period. Two students were on a special scientific exchange program from Brazil. The PI and codirector attended a week long workshop in leadership skills. Three graduate students have received graduate coursework and lab based research training. How have the results been disseminated to communities of interest?Publicatioin in peer reviewed journals and presentations at scientific meetings. Several newspaper articles. What do you plan to do during the next reporting period to accomplish the goals?We are seeking collaborators to continue our work and preparing research proposals to various agencies to obtain funding. We will continue with the same work but are adding new direction of postharvest processing. We are expanding our collaborating network to a group of scientists in Colombia.

What was accomplished under these goals? We have continued to explore the functional genomics of traits of interest for cacao crop improvement and methods for mass propagation of cacao. We have discovered new knowledge of the mechanism of disease resistance in cacao. We have explored the possibility of engineering novel sources of disease resistance in cacao. As documented in our publications, we have defined the major genes controlling lipd and flavonoid biosynthesis in cacao at a genome wide level. We have also gained a deeper understanding of the factors controlling embryogenesis in cacao and documented the molecular changes occurring at the gene and protein levels. We have also continued to publish evidence that somatic embryogenesis produces trees with excellent field performance, and have invented methods to increase the efficiency of the SE process. We hosted the visits of 2 undergraduate students from Brazil.


  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Goenaga, R., Guiltinan, M., Maximova, S., Seguine, E. & Irizarry, H. Yield Performance and Bean Quality Traits of Cacao Propagated by Grafting and Somatic Embryo-derived Cuttings. HortScience 50, 358-362 (2015).
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Zhang Y, Maximova SN, Guiltinan MJ. Characterization of a stearoyl-acyl carrier protein desaturase gene family from chocolate tree, Theobroma cacao L. Frontiers in Plant Science 2015, 6(DOI 10.3389/fpls.2015.00239).
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Helliwell EE, Vega-Arreguin J, Shi Z, Bailey B, Xiao S, Maximova SN, Tyler BM, Guiltinan MJ. Enhanced resistance in Theobroma cacao against oomycete and fungal pathogens by secretion of phosphatidylinositol-3-phosphate-binding proteins. Plant Biotechnology Journal 2015. 10.1111/pbi.12436
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Niemenak, N., Kaiser, E., Maximova, S. N., Laremore, T. & Guiltinan, M. J. Proteome analysis during pod, zygotic and somatic embryo maturation of Theobroma cacao. Journal of Plant Physiology 180, 49-60, doi: (2015).
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Florez, S. L., Erwin, R. L., Maximova, S. N., Guiltinan, M. J. & Curtis, W. R. Enhanced somatic embryogenesis in Theobroma cacao using the homologous BABYBOOM transcription factor. BMC Plant Biol 15, 121, doi:10.1186/s12870-015-0479-4 (2015).
  • Type: Book Chapters Status: Published Year Published: 2015 Citation: Guiltinan M, Maximova S. Applications of Genomics to the Improvement of Cacao. In: Chocolate and Health : Chemistry, Nutrition and Therapy. Edited by Wilson P, Hurst WJ: The Royal Society of Chemistry; 2015.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Liu, Y., Shi, Z., Maximova, S., Payne, M. J. & Guiltinan, M. J. Tc-MYBPA an Arabidopsis TT2-like transcription factor and functions in the regulation of proanthocyanidin synthesis in Theobroma cacao. BMC Plant Biology 15, doi:10.1186/s12870-015-0529-y (2015).
  • Type: Other Status: Published Year Published: 2015 Citation: Maximova, S. N., Guiltinan, M. J., Chapter 3 - Tissue Culture, I. & Lalibert�, B., End, M., eds. Supplying new cocoa planting material to farmers: a review of propagation methodologies. pp. 67-87 (Bioversity International, Rome, Italy, 2015).
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Fister, A. S., O'Neil, S. T., Shi, Z., Zhang, Y., Tyler, B. M., Guiltinan, M. J. & Maximova, S. N. Two Theobroma cacao genotypes with contrasting pathogen tolerance show aberrant transcriptional and ROS responses after salicylic acid treatment. J Exp Bot, doi:10.1093/jxb/erv334 (2015).

Progress 07/01/14 to 09/30/14

Target Audience: General plant sciences research community. Cocoa research community. Chocolate industry specialists. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Three graduate students, one postdoc one technician all received training in plant science research. How have the results been disseminated to communities of interest? Through publications and presentations at professional and industry meetings. What do you plan to do during the next reporting period to accomplish the goals? We continue to isolate and study candidate genes of interest and to provide training to scientists form the deveoping world.

What was accomplished under these goals? We have isolated and functionally tested a number of the genes controlling somatic embryogenes, cocoa butter biosynthesis and disease resistance. We have hosted the visit of two west African scientists who were trained in the molecular methods and are involved in the main projects of our lab.


  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Mej�a LC, Herre EA, Sparks JP, Winter K, Garc�a MN, Van Bael SA, Stitt J, Shi Z, Zhang Y, Guiltinan MJ, Maximova SN: Pervasive effects of a dominant foliar endophytic fungus on host genetic and phenotypic expression in a tropical tree. Frontiers in Microbiology 2014, 5. DOI 10.3389/fmicb.2014.00479.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Maximova S, Florez S, Shen X, Niemenak N, Zhang Y, Curtis W, Guiltinan M: Genome-wide analysis reveals divergent patterns of gene expression during zygotic and somatic embryo maturation of Theobroma cacao L., the chocolate tree. BMC Plant Biology 2014, 14(1):185.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Zhang Y, Smith P, Maximova SN, Guiltinan MJ: Application of glycerol as a foliar spray activates the defence response and enhances disease resistance of Theobroma cacao. Molecular Plant Pathology doi:10.1111/mpp.12158 (2014).
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Zhang, Y., Clemens, A., Maximova, S. N. & Guiltinan, M. J. The Theobroma cacao B3 domain transcription factor TcLEC2 plays a duel role in control of embryo development and maturation. BMC Plant Biol 14, 106, doi:10.1186/1471-2229-14-106 (2014).
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Meinhardt, L. W., Costa, G. G., Thomazella, D. P., Teixeira, P. J., Carazzolle, M. F., Schuster, S. C., Carlson, J. E., Guiltinan, M. J., Mieczkowski, P., Farmer, A., Ramaraj, T., Crozier, J., Davis, R. E., Shao, J., Melnick, R. L., Pereira, G. A. & Bailey, B. A. Genome and secretome analysis of the hemibiotrophic fungal pathogen, Moniliophthora roreri, which causes frosty pod rot disease of cacao: mechanisms of the biotrophic and necrotrophic phases. BMC genomics 15, 164, doi:10.1186/1471-2164-15-164 (2014).