Location: Sustainable Perennial Crops Laboratory
2021 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. Having already published the genomes of several pathogens as part of this objective, our recent efforts focused on characterizing the mitochondrial genomes and genetic diversity associated with the four species of fungi causing thread blight disease on cacao in Ghana: Marasmius crinis-equi, Marasmius tenuissimus, Marasmiellus palmivorus, and Marasmiellus scandens through collaborations with scientists from Ghana. The published literature cites only M. scandens as causing thread blight of cacao. This is the first time M. tenuissimus, the most commonly isolated of the four species, has been reported as cacao pathogen. Surprisingly, the four species causing thread blight disease of cacao to share a close genetic relationship with the Moniliophthora species causing frosty pod rot and witches broom disease on cacao in the Western hemisphere.
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. Having previously published a detailed analysis of the Phytophthora palmivora and P. megakarya genomes, we established a collaboration with the University of Florida to dissect the effector profiles of P. megakarya and the identify genes contributing to cacao resistance to P. megakarya infection as part of a Ph.D. program. We also sequenced the genome of Phytophthora theobromae, a new species causing block pod rot of cacao in Brazil and only recently identified (2020-2021). In collaborations with the University of California and Mars Incorporated, we have dissected 23 genome sequences for Moniliophthora roreri, identifying unique genetic aspects associated with effector pools, important in the disease process, and its asexual lifestyle while causing frosty pod rot of cacao. These results are currently being summarized. We have also initiated a new genetic diversity analysis of M. roreri isolates obtained from in addition to the 28 genome sequences previously sequenced for M. perniciosa, the causal agent of witches’ broom of cacao. We have begun the sequencing of M. perniciosa biotypes pathogenic on species other than cacao for comparison.
Over the past year, work has accelerated as required for accomplishing Objective 2: Identify cacao defense genes through transcriptome analysis and develop tools to exploit these genes to improve 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. Despite COVID restrictions, we were able to produce and process cacao leaves and pods infected with Phytophthora palmivora for 8 genetically diverse cacao clones for transcriptome analysis. The transcriptome analysis for the leaf samples is being completed while the transcriptomes for the pod samples are being sequenced. After a 1-year delay due to COVID, scientists at the Cocoa Research Centre (CRC), the University of the West Indies, Trinidad and Tobago have recently sent samples for 10 cacao clones infected with Moniliophthora perniciosa, the causal agent of witches’ broom. We have successfully extracted RNA from a subset of those samples recently sent off for transcriptome sequencing. We also carried out preliminary 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.
Accomplishments
1. New cacao pathogens identified. Crop losses due to diseases threaten the chocolate industry in the United States. Ghana is the second-largest cacao producer globally, and Thread blight is a new concern for producers in Ghana. ARS researchers in Beltsville, Maryland, working with researchers at the Cocoa Research Institute in Ghana and the University of Ghana, used DNA sequencing to detect four fungi causing thread blight of cacao. In this study, the fungus, Marasmius tenuissimus, which had never been found on cacao before, was the species most often isolated from infected plants. Now, management tools can be developed for these thread blight pathogens. Successful disease management will improve conditions in the field, helping farmers, and maintain cacao supplies for the chocolate industry.
