2013 Annual Report
1a.Objectives (from AD-416):
The goal of this project is to investigate the mechanisms involved in the interactions between cacao, pathogens of cacao, and biocontrol agents in order to develop effective disease management strategies leading to sustainable yield improvements of cacao. Over the next 5 years we will focus on the following objectives:
Objective 1: Discover and characterize key disease-inciting genes and genetic pathways of Moniliophthora species pathogenic on cacao.
Objective 2: Discover and characterize genes and genetic pathways of endophytic biological control organisms critical to limiting diseases caused by Moniliophthora species in cacao.
Objective 3: Optimize and deploy biological agents and formulations for managing cacao diseases caused by Moniliophthora species.
1b.Approach (from AD-416):
Pathosystem/biocontrol interactions will be studied at the molecular level through transcriptome anaylsis using deep sequencing technologies which will exploit recently acquired plant and fungal genomes, and by microscopic analysis of florescent protein tagged microbes. The primary microbes targeted for study will be Trichoderma spp. and Moniliophthora roreri (frosty pod rot) but will also include Phytophthora species (black pod), Moniliophthora perniciosa (witches’ broom), and beneficial Bacillus spp. Specific microbial genes identified in the expression studies will be subjected to functional analysis to reveal their importance to pathogen/biocontrol/host interactions using transformation protocols and advanced RNAi techniques. Transcriptome sequences will be used to develop genetic markers (single nucleotide polymorphisms) for M. roreri to characterize its diversity throughout Latin America and to monitor population changes as they respond to the establishment of partially resistant cacao clones in Central America. Building on molecular knowledge and recent successes with biocontrol in the field, combinations of previously screened Bacillus and Trichoderma isolates will be evaluated in the field in Ecuador for synergy in managing cacao diseases. Biocontrol formulations will be optimized to reduce the cost of application by using low-cost, locally available components and minimizing application volumes. Ultimately, the best available biological agents and formulation combinations will be tested along with optimum management practices on large plots of elite cacao clones.
Cacao clones, tolerant to the disease frosty pod rot, have been developed and are being planted throughout Central America. These clones yield well under severe disease pressure but sometimes develop disease. Knowledge of the mechanisms involved in the tolerance to disease is critical to the proper use of these clones in the field and should contribute to the development of new tolerant clones. Knowledge of the mechanisms used by Moniliophthora roreri, the fungal pathogen causing frosty pod rot, in causing disease should also help guide the distribution of tolerant clones in the field and the development of new cacao clones. Over the past year progress has been made identifying cacao genes associated with tolerance to frosty pod rot. The genes were identified by isolating and sequencing the RNA being expressed by tolerant cacao clones during infection by Moniliophthora roreri. At the same time, candidate genes of the pathogen have been identified that are associated with its ability to cause disease in disease tolerant cacao clones. Understanding the genetic diversity associated with a pathogen’s population in the field is critical to developing disease management tools, whether considering use of tolerant planting materials, or other disease management techniques such as fungicides or microbe based biological methods. We obtained RNA sequences from 15 isolates of Moniliophthora roreri to be used in developing single nucleotide polymorphic (SNP) markers for accessing the pathogen’s genetic diversity. SNP markers identify variations in the DNA sequence of an organism and are useful in identifying an organisms genetic diversity. In associated research, a large collection of Moniliophthora roreri isolates from throughout South and Central America was made and the DNA from each isolate is being extracted. A limiting factor in the use of beneficial microbes to reduce yield losses due to diseases is the cost of their application in the field. We are continuing research that demonstrated invert oil formulations of the beneficial fungus Trichoderma were effective in reducing disease losses due to frosty pod rot. Alternative formulations of lower cost have been developed for applying Trichoderma to cacao trees in the field where frosty pod rot occurs. The new formulations replace the relatively expensive corn oil, being used in formulations during previous proof of concept studies, with palm oil and other lower cost oils. These formulations are being tested on cacao trees in Ecuador. At the same time, we are trying to identify the mechanisms used by Trichoderma in protecting plants from disease. RNA was obtained for sequencing from cacao tissues colonized by Trichoderma, the same fungus being used in the field for managing cacao diseases. The RNA will be used to identify Trichoderma genes important in cacao tissue colonization allowing optimization of the processes involved. Colonization is critical for establishing the Trichoderma in the field and limiting the need for and cost of repeated applications.
Identification of an alternative vegetable oil replacing corn oil in invert oil formulation of Trichoderma for use in managing cacao diseases. The beneficial fungus Trichoderma is being studied for management of diseases in cacao. Past field experiments have demonstrated that Trichoderma spores applied to cacao trees in an invert corn oil formulation reduced disease losses and increased the yield of healthy cacao fruit. Although effective in reducing disease, corn oil can be expensive in areas where cacao is grown. Experiments were carried out to find a less expensive vegetable oil to replace corn oil in water-in-oil invert oil formulations of Trichoderma. Trichoderma spore germination and survival in palm oil and corn oil formulations was similar when applied to cacao fruit in the field. Palm oil, which is less expensive than corn oil is a suitable replacement for corn oil in invert oil formulations and should greatly reduce the cost of formulation applications in the field.
Identification of cacao genes potentially important in tolerance against Frosty Pod Rot and pathogen genes potentially important in the overcoming cacao field tolerance. RNA was extracted from tolerant and highly susceptible cacao clones after infection by Moniliophthora roreri, the causal agent of frosty pod rot. RNA sequencing techniques were used to obtain both plant and fungal RNA sequences. By studying the expression of the plant and fungal RNAs, candidate genes were identified. Candidate genes contributing to tolerance against frosty pod rot were identified for cacao, and candidate genes used in overcoming the tolerance were identified for the Frosty Pod Rot pathogen. Understanding the interactions between cacao and its pathogens at the genetic level will aid in the continued development of cacao tolerant to Frosty Pod Rot.
Pereira, G.A., Ambrosio, A.B., Nascimento, L.C., Oliveira, B.V., Teixeira, P.J., Tiburcio, R.A., Thomazella, D.P., Leme, A.F., Carazzolle, M.F., Vidal, R.O., Mieczkowski, P., Meinhardt, L.W., Garcia, O. 2013. Global analyses of Ceratocystis cacaofunesta mitochondria: from genome to proteome. Biomed Central (BMC) Genomics. 2013(14):91.
Oliveira, B., Teixeira, G., Reis, O., Barau, J., Teixeira, P.J., Rio, M.S., Domingues, R.R., Meinhardt, L.W., Leme, A.F., Rincones, J., Pereira, G.A. 2012. A potential role for an extracellular methanol oxidase secreted by Moniliophthora perniciosa in Witches' broom disease in cacao. Fungal Genetics and Biology. 49:922-932.
Bailey, B.A., Crozier, J., Strem, M.D., Melnick, R.L., Zhang, D., Maximova, S., Guiltinan, M., Meinhardt, L.W. 2013. Dynamic changes in pod and fungal physiology associated with the shift from biotrophy to necrotrophy during the infection of Theobroma cacao by Moniliophthora roreri. Physiological and Molecular Plant Pathology. 81:84-96.