Project Number: 1245-21220-252-05
Project Type: Specific Cooperative Agreement

Start Date: Aug 17, 2012
End Date: Sep 30, 2013

Objective:
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.

Approach:
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.