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ARS Home » Northeast Area » Beltsville, Maryland (BARC) » Beltsville Agricultural Research Center » Sustainable Perennial Crops Laboratory » Research » Research Project #432718

Research Project: Developing Pathogen- and Plant-Based Genetic Tools for Breeding Disease Resistance in Theobroma cacao

Location: Sustainable Perennial Crops Laboratory

2018 Annual Report

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.

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
Theobroma cacao (cacao) is the source of cocoa butter and cocoa powder used to make chocolate and many other products. Cacao production around the world is threatened by diseases that reduces yields by 30% to 40% each year. New information on pathogens causing cacao diseases will help us develop tolerant cacao varieties, develop new disease management methods, and modify cultural practices to reduce losses due to disease. Although this project originally was focused on frosty pod rot and black pod rot, opportunities have presented themselves allowing us to expand this work to include most of the major pathogens causing disease on cacao including witches’ broom (caused by Moniliophthora perniciosa), vascular streak die back (caused by Ceratobasidium theobromae), tip die back (caused by Lasiodiplodia theobromae), thread blight (caused by as Marasmiellus scandens) and several others. Efforts continue to define the biology of these pathogens and their genetic diversity along with defining the defense mechanisms and associated genes used by cacao to protect against disease. Most recently the importance of temperature in the biology of Phytophthora megakarya and Phytophthora palmivora was studied in detail. As it turns out, the more aggressive species, P. megakarya, is unable to grow at temperatures of 30°C or above while P. palmivora can grow at temperatures up to 32°C. This temperature difference effects disease development under laboratory conditions and is expected to have similar effects, yet undefined, in the field. This simple difference also serves a low technology tool for differentiating isolates of P. megakarya from P. palmivora, something important for researchers in Africa. Although, it was thought for many years that M. roreri, a hemibiotroph, was haploid in the biotrophic phase and dikaryotic in the necrotrophic phase, we recently proved the fungus lacks a dikaryotic phase and is therefore unlikely to undergo sexual reproduction. This distinguishes the biology of M, roreri from its sister pathogen M. perniciosa and suggests M. roreri has limited ability to produce genetic diversity through recombination. This completely changes our understanding of M. roreri’s biology. We have continued our efforts with 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. This work was initiated through collaborations with the Indonesian Cocoa and Coffee Research Institute the University of Hasanuddin and has been to include collaborators from all major cacao producing countries in Southeast Asia. We continue to refine the C. theobromae draft genome sequence by evaluating its completeness. This is a significant accomplishment since the pathogen is very difficult to grow in pure culture. Through the same collaborations, we have obtained the genome sequence for Lasiodiplodia theobromae, the pathogen causing tip die back and have recently submitted its genome sequence to Genbank. Our most recent efforts have focused on the chararterization of the genome and genetic diversity associated with the thread blight pathogen, Marasmiellus scandens. Working with a PhD student from Ghana, we have acquired all the required DNA and RNA sequence for completing the M. scandens draft genome and are currently carrying out the assembly. We also have cultures and DNA from more than 60 isolates of M. scandens in the laboratory and are assessing their biological and genetic diversity.

1. Radical new understanding of the basic biology of Moniliophthora roreri, causal agent of frosty pod rot of cacao. Frosty pod rot of Theobroma cacao, caused by the fungus Moniliophthora roreri, is the greatest threat to cacao production globally should it escape the Americas and in its initial disease phase the pathogen avoids detection (compatible phase) as it grows throughout cacao fruit tissues before switching to a destructive phase and rapidly destroys the fruits. For many years, it was thought that M. roreri was haploid in the compatible phase, having one nucleus per cell, before converting to a dikaryotic stage, carrying 2 nuclei per cell. Having a dikaryotic phase is critical to sexual reproduction in a fungus like M. roreri and plays a large role in the generation of genetic diversity in the fungus. ARS scientists in Beltsville, Maryland, used nuclear staining to examine the nuclear content of M. roreri throughout its life cycle and discovered that the fungus never forms true dikaryotic cells. This information redefines the lifecycle of M. roreri supporting the idea M. roreri lacks the ability to undergo sexual reproduction; further, these results indicate that frosty pod resistant cacao trees should remain stable in the field with a limited risk of the pathogen overcoming the resistance through genetic recombination. These findings will impact all cacao improvement programs, globally, and as new resistant materials are identified, breeders will be able to supply farmers with cacao trees that will remain productive for extended periods of time despite the presence of this disease.

Review Publications
Bailey, B.A., Ali, S., Strem, M.D., Meinhardt, L.W. 2018. Morphological variants of Moniliophthora roreri on artificial media and the biotroph/necrotroph shift. Fungal Biology. 122(7):701-716.
Puig, A.S., Ali, S., Strem, M.D., Sicher Jr, R.C., Gutierrez, O.A., Bailey, B.A. 2018. The differential influence of temperature on Phytophthora megakarya and Phytophthora palmivora pod lesion expansion, mycelia growth, gene expression, and metabolite profiles. Physiological and Molecular Plant Pathology. 102:95-112.