Location: Sustainable Perennial Crops Laboratory
2020 Annual Report
Objectives
Objective 1: Develop genomic tools for assessing pathogen genetic diversity and determining the basis for virulence in support of improved cacao breeding. [NP303, C2, PS2A]
Sub-objective 1.A. Identify the genome structure of established and emerging pathogens of cacao to determine the genetic basis for their virulence in cacao.
Sub-objective 1.B. Develop and use genomic tools to characterize the regional distribution of Ceratobasidium theobromae genetic diversity.
Sub-objective 1.C. Combining genotyping by sequencing and whole genome sequencing data to assess the genetic diversity of P. megakarya and evaluate the evolution/selection of virulence-associated genes.
Objective 2: Identify cacao defense genes through transcriptome analysis, and develop tools to exploit these genes for the improvement of cacao breeding. [NP303, C2, PS2A]
Sub-objective 2.A. Identify the cacao defense gene complement by assessing changes in gene expression of elite cacao clones responding to infections by diverse pathogens.
Sub-objective 2.B. Identify components of the cacao defense transcriptome with potential for providing durable tolerance against infections by diverse pathogens.
Approach
Diseases caused by Moniliophthora roreri, Phytophthora megakarya, Ceratobasidium theobromae, and Cocoa Swollen Shoot Virus reduce cacao yields, impacting farmers’ profits, and increase the costs of chocolate products. Emerging pathogens such as Marasmiellus scandens and Lasiodiplodia theobromae further threaten cacao production. While progress is being made in breeding tolerance to some cacao diseases, these efforts are hindered by an undefined pathogen genetic diversity and a limited understanding of cacao genes that confer disease tolerance. Therefore, this project will develop tools including pathogen genome and transcriptome sequences and associated single-nucleotide polymorphism panels characterizing pathogens and their variants pertinent to breeding programs and an annotated cacao transcriptome database identifying cacao genes linked to disease tolerance. The genome and transcriptome sequences of established and emerging cacao pathogens will be acquired and their genetic diversity will be determined. We will also characterize the expression of the cacao transcriptome during interactions between five cacao pathogens and eight genetically diverse clones. Using advanced mathematic approaches, we will identify differences and commonalities in the cacao responses associated with tolerance to pathogens. The most common traits associated with tolerance to cacao diseases relate to polyphenol biosynthesis and cell wall development. Gene expression and other measures associated with these specific traits will be evaluated during cacao developmental processes in leaves and pods and during the disease interactions previously mentioned. Once incorporated into an annotated cacao transcriptome database, these data along with gene sequence variations will provide new markers to help accelerate cacao breeding and increase the likelihood of obtaining sustainable disease tolerance.
Progress Report
We have made progress over the last year on Objective 1: Develop genomic tools for assessing pathogen genetic diversity and determining the basis for virulence in support of improved cacao breeding. [NP303, C2, PS2A], Sub-objective 1.A. Identify the genome structure of established and emerging pathogens of cacao to determine the genetic basis for their virulence in cacao. We have continued our efforts studying vascular streak dieback of cacao in Indonesia, which is one of the main diseases limiting cacao production in Southeast Asia. Vascular streak dieback is caused by the fungus Ceratobasidium theobromae. Working with collaborators (Indonesian Cocoa and Coffee Research Institute and the University of Hasanuddin) we published, for the first time, a draft genome of C. theobromae, the causal agent of vascular streak dieback. Through the same collaborations, we published the genome sequence for Lasiodiplodia theobromae, a pathogen causing tip die back of cacao. Recent efforts have focused on charactering the genome and genetic diversity associated with the thread blight pathogens, including Marasmiellus scandens through collaborations with scientists from Ghana. We recently discovered that five different species of basidiomycete fungi can cause thread blight on cacao. We have obtained genomic sequences for the five species along with their full mitochondrial sequences. In all, the genetic diversity of more than 60 isolates of thread blight associated has been analyzed.
Substantial progress has also been made toward accomplishing Sub-objective 1.C. Combining genotyping by sequencing and whole genome sequencing data to assess the genetic diversity of P. megakarya and evaluate the evolution/selection of virulence-associated genes. Working with our collaborators at the University of California, Davis and MARS Incorporated, we have published the analysis of 21 Phytophthora palmivora genomes using short read Illumina technology and 2 genomes using long read technology. We have obtained 19 P. megakarya genomes using short read technology and 3 genomes using long read technology. We have also obtained 23 genome sequences for Moniliophthora roreri and 28 genome sequences for M. perniciosa using short read technologies. We also have obtained one near complete genome assembly of M. roreri and M. perniciosa using long read technology.
Over the past year we have carried out the preliminary work required for accomplishing Objective 2: Identify cacao defense genes through transcriptome analysis and develop tools to exploit these genes for the improvement of cacao breeding. [NP303, C2, PS2A]. Sub-objective 2.A. Identify the cacao defense gene complement by assessing changes in gene expression of elite cacao clones responding to infections by diverse pathogens. Scientist at the International Cocoa Quarantine Centre (ICQC), Reading, UK and the Cocoa Research Centre (CRC), the University of the West Indies, Trinidad and Tobago have prepared plant material and learned extraction techniques required for the work. These institutions will provide RNA for sequencing isolated from diverse cacao clones after infection with the Cocoa Swollen Shoot Virus or Moniliophthora perniciosa, causal agent of witches’ broom disease. In addition, we have carried out leaf inoculations on 8 genetically diverse cacao clones with zoospores of P. megakarya and P. palmivora. We are currently analyzing the transcriptome sequences acquired from these samples. We have also contracted services to carry out metabolite analysis of cacao tissues as part of completing Objective 2.B. Identify components of the cacao defense transcriptome with potential for providing durable tolerance against infections by diverse pathogens and will begin sending samples for analysis over the next few weeks.
Accomplishments
1. Public release of the genome sequences of multiple pathogens of Theobroma cacao. Knowledge of the genetic makeup of pathogens is needed to manage diseases in the field. Cacao disease reduce cacao production by 25% annually. ARS scientists from Beltsville, Maryland, with collaborators from the Indonesian Cocoa and Coffee Research Institute; the University of Hasanuddin, Indonesia, the University of California, Davis, and MARS Incorporated, published the genome sequences of four important cacao pathogens: We sequenced, analyzed, and released the first genome sequences for the difficult to grow pathogen C. theobromae, causal agent of vascular streak dieback in Southeast Asia and the emerging pathogen L. theobromae, causal agent of tip dieback and pod rot globally, and in another collaborative effort released greatly improved genome sequences of P. palmivora, causal agent of black pod rot globally and P. megakarya causal agent of black pod rot in Africa. The nature of the pathogen causing vascular streak dieback was unknown and establishing its genetic makeup is critical to understand this disease. Lasiodiplodia species are emerging as pathogens of cacao in many regions and the published genome provides a foundation for future research. Resequencing of the Phytophthora genomes led to the new discovery that P. megakarya underwent a whole genome duplication and high levels of retroelement activity reorganized its genome with an extensive composition of virulence related genes. This work expands our knowledge of the genetic makeup of multiple cacao pathogens providing new knowledge for scientists developing sustainable disease management practices in cacao, globally, and for scientist working with similar pathogens in other crops. These publicly released genomes establish the basic foundation for in-depth analysis of these disease-causing pathogens and will be the basis for studying the genetic diversity of these pathogens and their interactions with cacao by scientists around the globe.
Review Publications
Ali, S., Shao, J.Y., Adi, A., Balidion, J., Strem, M., Puig, A.S., Meinhardt, L.W., Bailey, B.A. 2019. Genome and transcriptome analysis of the latent pathogen lasiodiplodia Theobromae, an emerging threat to cacao industry. Genes, Genomes, and Genomics. 63:37–52.
Adi, A., Rosmana, A., Amin, N., Bailey, B.A., Ali, S. 2019. Survival capacity of cacao top grafted with scions infected by vascular streak dieback pathogen: potential source of ceratobasidium theobromae long-distance spread. Archives of Phytopathology and Plant Protection. 52:13-14.
Jaimez, R.E., Vera, D.I., Mora, A., Loor, R.G., Bailey, B.A. 2020. Disease and production Index (DPI) for selection of Cacao (Theobroma cacao) genotypes highly productive and tolerant to frosty pod rot (Moniliophthora roreri). Plant Pathology. 69(4), p.698-712.
Ali, S., Asman, A., Shao, J.Y., Firmansyah, A., Susilo, A., Rosamana, A., Mcmahon, P., Junaid, M., Guest, D., Kheng, T., Meinhardt, L.W., Bailey, B.A. 2019. Draft genome sequence of fastidious pathogen Ceratobasidium theobromae, which causes vascular-streak dieback (VSD) in Theobroma cacao. Fungal Biology and Biotechnology. 6:14.
