Location: Sustainable Perennial Crops Laboratory
2022 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
Sustained progress was made in the completion of research laid out in 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. Efforts studying vascular streak dieback of cacao, one of the main diseases limiting cacao production in in Indonesia, were continued. Vascular streak dieback is caused by Ceratobasidium theobromae, a very difficult to isolate fungus. Working with collaborators (Indonesian Cocoa and Coffee Research Institute and the University of Hasanuddin) we obtained a small DNA sample of the fungus along with infected cacao tissue samples and were able to sequence the pathogens genome and transcriptome. This resulted in the first published draft genome of C. theobromae, the causal agent of vascular streak dieback marking a significant advance toward understanding this cacao/pathogen interaction. Through the same collaborations, we published the first genome/transcriptome sequence for cacao isolates of Lasiodiplodia theobromae, an emerging pathogen causing tip die back of cacao. This and related species have been repeatedly cited as causing significant disease on cacao in recent years. Working with collaborators from Ghana in support of a PhD student (completed in 2021), we were able to identify a disease complex causing thread blight symptoms on cacao. Thread blight was originally attributed to the little studied fungal pathogen Marasmiellus scandens. We discovered that four different species of basidiomycete fungi can cause thread blight on cacao, the most common of which was Marasmius tenuissimus. M. tenuissimus was also recently identified in South America also causing thread blight, which indicates this pathogen is a problem in areas outside Ghana also. We published the mitochondrial genome sequences for the four species in the thread blight complex and demonstrated their close genetic relationship to Moniliophthora species causing frosty pod rot and witches’ broom of cacao in South America. We have obtained high-quality genome sequences for M. tenuissimus, which is being prepared for public release, and are pursuing high quality genome sequences for the remaining 3 species.
Sub-objective 1.B. “Develop and use genomic tools to characterize the regional distribution of Ceratobasidium theobromae genetic diversity”, was not completed due to the unexpected loss of a support scientist due to a medical emergency and its dependence on international collaborators impacted by COVID19. The sequencing of the pathogen’s genome described above did provided molecular markers, including single nucleotide polymorphisms, critical to accomplishing this objective in the future.
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 also obtained 19 P. megakarya genomes using short read technology and 3 genomes using long read technology. Using these sequence data, we established an informal collaboration with the University of Florida to dissect the effector profiles of P. megakarya and P. megakarya as part of a PhD program. We had further opportunity to expand approaches associated with this subobjective to 3 other major cacao pathogens. We obtained 23 genome sequences for Moniliophthora roreri (causal agent of frosty pod rot) and 28 genome sequences for M. perniciosa (causal agent of witches’ broom disease) using short read technologies. We also obtained 5 complete/near complete genome assembly of M. roreri and M. perniciosa using long read technology. As a result, we are continuing to evaluate the evolution of these 2 Moniliophthora species, the primary pathogens of cacao in its center of origin. We also rapidly sequenced the genome of Phytophthora theobromae, a new species causing black pod rot of cacao in Brazil and was only recently identified (2020-2021).
Although in-house components of Objective 2, Identify cacao defense genes through transcriptome analysis and develop tools to exploit these genes for the improvement of cacao breeding [NP303; C3; PS 3A], were carried out, other aspects of this objective dependent on international collaborations were postponed due to collaborators not being able to provide required samples due to the COVID pandemic. 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, sought to characterize the cacao transcriptome using tissues from an overlapping set of cacao clones (ranging from tolerant to susceptible) after infection with multiple cacao pathogens. We obtained transcriptome data on cacao leaves and pods from multiple cacao clones grown in our greenhouses after infection by Phytophthora palmivora and are currently completing the full transcriptome characterization of these cacao clones. The Phytophthora studies are relevant to Objective 2 of the proposed new project as they will identify genes involved in the resistance response to Phytophthora infection. We have also produced pod samples from multiple cacao clones infected with M. roreri and will be extracting them this fall. Although we did obtain RNA from one replication of grafted seedlings of multiple cacao clones infected with M. perniciosa (part of a PhD program in Trinidad), the additional replications required have been delayed due to COVID. Collaborations with the University of Reading, United Kingdom to produce Cacao swollen shoot virus (CSSV) infected cacao were also initiated but were also shut down due to COVID. We were able to provide transcriptome data characterizing the expression of genes associated with quantitative trait loci providing resistance to frosty pod rot and black pod rot using previously acquired data.
Although initial samples were collected and analyzed for Sub-objective 2.B. Identify components of the cacao defense transcriptome with potential for providing durable tolerance against infections by diverse pathogens, they have not been followed up on due to the unexpected loss of a support scientist due to a medical emergency. Samples supplied by international collaborators were also required to complete this objective.
Several additional studies concerning cacao diseases were also carried out during the project. Studies on the influence of temperature on growth and infection of cacao pods by P. megakarya and P. palmivora were carried out identifying keys differences. In collaboration with Indonesia scientists, it was demonstrated that cacao scions are a source for long distance spread of Ceratobasidium theobromae. An index for selection of cacao clones both highly productive and tolerant to frosty pod rot diseases was developed through collaborations with Ecuador scientists. In a unique in-house study, we discovered that M. roreri spores germinate and maintain the biotrophic phase of growth for extended time periods before converting to necrotrophic mycelia, fundamentally changing our understanding of the pathogen’s biology.
Accomplishments
1. Resequencing of fungal pathogens of Theobroma cacao. Pathogen genome sequencing provided valuable information on the genetic diversity of the pathogen and the basis of disease virulence which is used to support and improve cacao breeding programs. In collaboration with scientists from the University of California, Davis, and MARS Incorporated, ARS scientists in Beltsville, Maryland, have analysis 97 genomes for cacao pathogens that cause black pod, witches’ broom and frosty pod diseases of cacao (24 genomes of Phytophthora palmivora, 22 genomes of P. megakarya, 23 genomes of Moniliophthora roreri and 28 genomes of M. perniciosa). These genomes and their analysis have allowed full lengthen genome assemblages for M. roreri and M. perniciosa. As a result, we are continuing to evaluate the evolution of these 2 Moniliophthora species, the primary pathogens of cacao in its center of origin. The methods developed here allowed for rapidly sequencing of the genome of Phytophthora theobromae, a new species causing black pod rot of cacao in Brazil that was recently identified in 2020-2021. This information will be used by cacao researchers, and plant breeders, around the world, to improve cacao plant breeding improvement programs and to develop disease control methods.
Review Publications
Barauh, I., Ali, S., Shao, J.Y., Lary, D., Bailey, B.A. 2022. Identification of cacao genes responsive to P. palmivora infection in resistant and susceptible cacao clones. Frontiers in Plant Science. 12:780805. https://doi.org/10.3389/fpls.2021.780805.
Ali, S., Amoako-Attah, I., Shao, J.Y., Meinhardt, L.W., Bailey, B.A. 2021. Mitochondrial genomics of six cacao pathogens from the basidiomycete family Marasmiaceae. Frontiers in Microbiology. 12:752094. https://doi.org/10.3389/fmicb.2021.752094.
