World Cocoa Foundation Research Update: March 2008
Project II. Sequencing the Genomes of Witches’ Broom and Frosty Pod Rot. Summary: In collaboration with USDA Beltsville (B. Bailey, and L. Meinhardt) and UNICAMP, Brazil (G. Guimaraes), we are sequencing and analyzing the entire genome sequences of these two related cacao plant pathogens, which cause the majority of losses in Latin America.
Progress: At PSU, the DNA sequencing has been completed. We have sequenced 612.3 million base pairs of the monilia genome and 253.2 million base pairs of witches’ broom genome. In short, this represents a multiple fold coverage of both genomes, which enables us to predict the presence of essentially all genes of these two pathogens, and to study their structure and evolution. The annotation and analysis of this data is ongoing but has revealed interesting features and possible leads to better understand the mechanisms of fungal infection. For the witches’ broom sequence, our data has been combined with that of our Brazillian collaborators to constitute a combined sequence. We have also determined the entire mitochondrial genome sequences of both organisms.
Future Impact: This data will accelerate the study of these two pathogens and provide leads into the mechanisms of pathogenicity. These mechanisms are potential targets for efforts to reduce the damage caused by them. The study will also shed light on the evolution of these pathogens. In the long run, this knowledge will help plant breeders and pest management specialists in developing germplasm and agronomic practices to reduce the development of this disease and its spread into new cacao growing areas.
Project III. Development of a Model System to Study Witches’ Broom Disease of Cacao. Summary: Research on the interactions of cacao with witches’ broom disease are difficult at best due to a number of reasons including; difficulty in obtaining reproducible infections, difficulty in growing large number of cacao plants, lack of genomic and other basic resources, limited genetic information on cacao etc. Therefore we have developed a system to study the interactions of the S strain of witches’ broom fungus with tomato. Tomato is a model plant system with none of the limitations mentioned above. This model system will greatly speed up our progress in understanding how witches’ broom infects cacao and causes the symptoms and eventually near total loss of pods.
Progress: We have completed the development and characterization/validation of this model system. In almost every way, the symptoms of witches’ broom disease in tomato are similar to that seen in cacao. We have observed that the swelling seen in WB pathogenesis is caused primarily by increased cell division and increased cell enlargement of the vascular tissues. We have evidence that this may be caused by changes in the plant hormone auxin induced by the fungus. In collaboration with scientists at Cornell University and Nestles Tours, we have identified several regions of the tomato genome containing resistance genes for witches’ broom. We are in the process of verifying this and will begin isolation and characterization of these genes in the near future.
Future Impact: Like project 2 above, this project will contribute to increasing our knowledge of the mechanism of WB disease progress and lead to strategies to fight the disease via breeding and or agronomic practices.
Project IV. Molecular Biology of Defense Response in Cacao. Summary: Using a combination of approaches we are investigating the mechanisms by which cacao responds to pathogen infection and switches on its defense response. We have collaborated with scientists at USDA Beltsville (B. Bailey) and Miami (R. Schnell), the Smithsonian Tropical Research Station (A. Herre), and the University of Sao Paulo, Brazil (A. Figueria) in these studies. We have used several genomics approaches to identify key genes involved in defense pathway in cacao. We have developed microarrays that allow us to measure the expression of tens of thousand cacao genes in a single experiment. We have also made use of the model plant Arabidopsis to isolate and study key cacao genes involved in regulating the defense response.
Progress: A large number of genes that are implicated in the cacao defense pathway have been isolated and sequenced. We have observed the expression of these genes during interactions with pathogens and with endophytic fungi. These studies are now being extended to compare expression of these genes between different cacao genotypes. We have evidence to support the hypothesis that cacao genotypes thought to be resistant to pathogens do so by reacting quicker and to higher levels then do sensitive genotypes. We have also obtained direct evidence supporting the idea that endophytes act in part by inducing the pathways leading to thicker cell walls, thus creating a stronger barrier to fungal infection.
Future Impact: This knowledge will ultimately aid plant breeders in designing crosses aimed at stacking multiple sources of resistance against pathogens that act on different mechanisms.
Project V. Field Testing and Technology Transfer of Cacao Somatic Embryogenesis Summary: Over the past decade, our group, along with others notably at CIRAD and Nestles, Tours, has developed efficient protocols for multiplication of cacao via tissue culture. More recently we have established several field tests of plant produced by these methods. We have made concerted efforts to transfer this technology to cacao producing countries and produced a protocol book freely available on our lab website (http://guiltinanlab.cas.psu.edu/Research/Cocoa/tissue_culture.htm).
Progress: We have obtained detailed data on the growth and development of plants in a field test on St. Lucia during the first years of establishment and have submitted a manuscript based on this data. Our analysis shows that the plants produced by tissue culture perform nearly identically to those produced from seeds or rooted cuttings. We are now in the process of obtaining yield data from these plants. Additional tests in Ecuador and Puerto Rico are in progress and data is being collected for future analysis, but preliminary observations of both sites concur with those of St. Lucia; the plants are growing normally. During 2008, we are hosting three visitors from developing countries who will focus on learning these methods (Venezuela, Nigeria and Ghana) and we taught a workshop of these methods in Ecuador. From this increase in interest of developing country scientists, we feel that the technology is now adopted widely.
Future Impact: As breeders develop improved varieties of cacao, and as the existing aging cacao populations decline, it will become increasingly important to have in place large scale clonal multiplication systems for cacao. Cacao somatic embryogenesis will provide one tool in this and will enable the deployment of the newest, highest yielding and disease resistant plants to farmers in the shortest possible time after they are developed. This method could also be applied to germplasm conservation in that cacao somatic embryos can be frozen indefinitely as a means to safeguard the living collections of cacao germplasm that are at risk from diseases, environmental disasters and in some cases, urban development. We have knowledge that a major lab for cacao tissue culture is being developed in Indonesia, so we believe that this technology has finally come of age and will be important in the large scale propagation of cacao throughout the world in near future.