2013 Annual Report
1a.Objectives (from AD-416):
There are three primary objectives of this research. The first objective is to sequence the Moniliophthora roreri genome and partially sequence the Moniliophthora perniciosa genome using equipment available at Penn State University. The genomes of the two related species, both severe pathogens of Theobroma cacao, are to be compared once the sequencing is completed. The second objective is to support laboratory and field studies of Bacillus species for selective control of diseases of cacao, specifically, Moniliophthora roreri and Moniliophthora perniciosa. The identification of native bacterial endophytes of cacao with disease biocontrol activities in the field are of special interest. The third objective is to exploit genetic transformation technologies for the purpose of carrying out functional analysis of genes of potential importance in the resistance of cacao to biotic and abiotic stresses, including plant disease. An additional objective will be to sequence the genomes and transcriptomes of Phytophthora species pathogenic on cacao. Our primary species of interest are P. megakarya and P. plamivora but additional species may be sequenced.
1b.Approach (from AD-416):
Pyrosequencing sequences small DNA fragment but is capable of sequencing very large numbers of small sequences in a very short time. Bioinformatic techniques allow for the assembling of these short sequences in to larger sequences and ultimately into linkage groups. Compared to traditional sequencing this can be done at a much lower cost. The sequence of M. roreri will be determined using this technique. At the same time, we have access to the nearly complete sequence of the related genome of M. perniciosa. Once the sequence of M. roreri has been collected, it will be overlaid onto the M. perniciosa to aid in assembling the sequence. In addition, we will carry out limited sequencing of the M. perniciosa to filling gaps in that sequence.
In addition to the Trichoderma species we are considering, Bacillus species have good potential as biocontrol agents for control of cacao disease. Bacillus species have some advantages in that they are easy to produce and very stable once formulated. There use in a tropical perennial crop like Theobroma cacao has not been studied in detail. We propose to support the continued study of Bacillus species isolated from Theobroma cacao for the control of cacao disease. The isolates have been collected from Ecuador and are ultimately to be screened in the field in Ecuador.
Also, the functional analyses of specific genes involved in plant defense against stress are critical as the genomics capabilities available for cacao research expand. Identifying the exact function of genes considered important in plant defense will allow their exploitation in traditional and advanced breeding efforts to develop cacao materials resistant to the negative effects of stress. Cacao will be both constitutively and transiently transformed to over express genes of potential importance to plant defense. The transformed materials will be evaluated under conditions of stress, including disease to validate their importance in the defense process. The proposed research parallels objective within the associated main project.
In order to sequence the genomes and transcriptomes of Phytophthora species pathogenic to cacao we will grow the Phytophthora species in pure culture. DNA and RNA will be isolated from the biomass produced. The DNA will be sequenced and the genomes assembled and annotated using current advanced sequencing technologies. RNA will be isolated from Phytophthora cultures grown under varying conditions. The RNA will be sequenced and the transcriptomes assembled and annotated.
The major objectives of our work are to discover and characterize the main mechanisms of disease resistance in cacao, and to utilize this knowledge to develop strategies to protect cacao from its major plant pathogens. Towards these goals, we have made major progress during this period. Our collaborative work has been accelerated greatly as a result of the completion of the cacao genome sequence. Other synergistic projects have been funded by the National Science Foundation that have further enhanced this project.
Genome Sequencing of Cacao Pathogens: Determining the sequences of plant pathogens reveals clues to the mechanisms of pathogenesis and allows scientists to study the genetic diversity of different pathogen strains. In collaboration with USDA Beltsville, MD, along with collaborators at Penn State and Brazil, we have sequenced the genomes of two of the most devastating cacao plant pathogens, Moniliothplora perniciosa (witches' broom) and Moliniophthora roreri (frosty pod). These two fungi are responsible for the majority of cacao crop losses throughout Central and South America. Sequencing was performed at Penn State University with genomic DNA from both fungi. The completion of the genome sequences of these pathogens opens up new frontiers in understanding the mechanisms of pathogenesis and in developing strategies for increased resistance.
We have recently embarked on a new project to completely sequence the genomes of two more cacao pathogens, Phytophthora palmivora (black pod) and Phytophthora megakarya. We have obtained the sequences of both pathogens and are now in the process of analyzing the sequences and preparing them for release to the public. This project has overlap with the follow-up project, Project 1245-21220-252-01S, so the details on the progress after DNA sequencing will be summarized in a separate progress report for this project.
Functional Genomics: During the course of this project experimentation in both of the collaborating labs has identified a number of candidate genes involved in defense responses. These have been identified as either: induced by pathogens, induced by endophytic microorganisms, used for biological control, induced by salicylic acid, a defense signaling molecule, induced by NEP1, a fungal protein involved in pathogenicity, or identified by homology to known defense regulators previously identified in model plants such as Arabidopsis. Furthermore, through analysis of the cacao genome sequence, we have identified a large number of candidate resistance genes of various classes. We have begun to explore the function of some of these genes using several different approaches. A major breakthrough was achieved in 2010-2011 with the development of a transient expression/pathogenicity assay. Using this assay it is possible to test the involvement of any given gene in disease resistance in a matter of weeks. In short, the method involves infiltration of cacao leaves with Agrobacterium (a specific type of bacteria) which harbor a Ti plasmid (a DNA fragment that moves into the plant cells) containing the gene of interest. After several days, a large number of leaf cells express high levels of the inserted genes. We have been using the transient expression assay to identify genes that provide resistance to the cacao pathogens Phytopthora and Colletotrichum. Using this method we have demonstrated that several key cacao genes do in fact play a role in disease resistance. We continue to test additional resistance genes and novel gene expression systems.
We have also used this method to compare the defense response between two cacao genotypes, Sca6 and ICS1, which are model systems for witches’ broom resistance. We have shown that these two genotypes show strong differences in resistance in both the degree of susceptibility and the speed of response to pathogen infection. This evidence is providing us with a new understanding of the major mechanisms of defense response in cacao.
We continue to develop and utilize transformed cacao plants for functional genomics. While this process is very difficult and inefficient, we have generated new plants containing several test genes and will be testing them with pathogens in the near future. In addition, we have tested several novel antifungal genes to demonstrate the potential of transgenic resistance in cacao.
Biological Control: Work in collaboration with Penn State University Plant Pathology Lab has focused on the potential of bacterial endophytes from cacao as protective agents against witches' broom and frosty pod. Working with collaborators in Ecuador, several promising strains have been identified and field-tested. We have completed a nearly two year study on the biological control of witches’ broom which demonstrates that the Bacillus pumilis isolate ET reduces this disease. An additional field trial was established between Penn State University, Ecuador, and USDA-ARS Sustainable Perennial Crops Laboratory on the endophytes’ ability to reduce pod diseases and cherelle wilt. This research could lead to deployment of biological control of these diseases on a commercial basis. This is the final report for this project.