Population Genetics of Mycosphaerella fijiensis Morelet, the causative agent of Black Sigatoka

1. Population Genetics of Mycosphaerella fijiensis Morelet, the causative agent of Black Sigatoka Meghan Wyatt1, Jean Beagle Ristaino1, and Luis Gomez Alpizar2.1North Carolina State University, Raleigh, NC and 2Universitdad de Costa Rica, San Jose, Costa Rica. Discussion Materials and Methods Results Introduction The sequences obtained from the three markers, translation elongation factor EF1 alpha (TEF1a), calmoduline (CaM), and glyceraldehyde-3-phosphate dehydrogenase (G3PD), are consistent with previous population genetics studies on Mycosphaerella fijiensis. Two haplotypes were found in TEF1a, and these are consistent with the two haplotypes found for Costa Rican samples in the paper, Contrasting introduction scenarios among continents in the worldwide invasion of the banana fungal pathogen Mycosphaerella fijiensis (Robert 2012). For the CaM marker, only one haplotype was observed, which is consistent with the previous study. However, Robert et al found one haplotype in the G3PD marker, but in this study two haplotypes were found. This suggests that a haplotype that has not been previously observed was found in Costa Rica. It was originally expected that there would be more genetic diversity in the Costa Rican population due to the pathogen’s ability to reproduce sexually and asexually. This could be due to bottlenecks as the pathogen spread from Papua New Guinea to Central America, and the founder effects that follow. Currently, new isolates from 2013 are being cultivated, and will be added to the data set. Sequences are being acquired that are missing from the data set, and further analysis will be done with the data once it is finished being collected. This is what you discovered by doing this research. Collection: Collections were made from mature lesions found in the leaves of Musa spp. A mature lesion was identified by looking for black streaks in the leaves, a determining characteristic of the pathogen. Leaves were sampled when the lesion is beginning to become necrotic, and psuedothecia are visible, but not when the leaf is completely black and melted. (Image)Figure: This chart shows the conditions and locations of the fields where Mycosphaerella fijiensis samples were collected in the past three years. Leaf tissue was cut from the leaf using a clean razor, and stored in an envelope until it is able to be isolated in the lab. Bananas are among the world’s most important crops, and are particularly important in developing countries such as Costa Rica. Not only is banana production a large part of the country’s economy, bananas are also a staple in the local diet. One of the major limiting factors to banana production is disease. MycosphaerellafijiensisMoreletis the causative agent of Black Sigatoka, or black leaf streak disease. This disease causes up to a 50% loss of fruit yield due to the destruction of the plant’s photosynthetic abilities and premature ripening. Bananas, plantains, and heliconia are the hosts of this pathogen, and it is thought to have originated in Papua New Guinea. The disease is found where ever (common name?) Musa spp is grown. The pathogen infects the banana plant through the stomata, and then forms cholorotic spots, and then brown streaks on the leaf. The streaks become darker until they form large, necrotic lesions. In mature lesions, the fruiting body of the fungus forms, called psuedothecia. These are filled with asci, which release ascospores into the wind. The pathogen is a wind-dispersed ascomycete, and can reproduce both sexually and asexually. The sexual process occurs when sexual structures, called spermagonium, emerge from mature lesions to produce spermatia, the male reproductive cells. These spermatia then fertilize the female hyphae, trichogynes, and produce psuedothecia. The psuedothecia, when hydrated by rain, will expel ascospores into the wind to be carried to other hosts. The asexual process occurs through the conidia, which also infect the plant through the stomata. The conidia produce conidiaphores, and then more conidia. This is a clonal process. Black Sigatoka is one of the most economically important diseases in Central America. Genetic analysis and a thorough understanding of the ecological patterns of the pathogen will lead to more effective methods of control and prevention of this devastating disease. Figure 1 goes here. Figure: This chart shows the sampling locations and conditions for field samples from 2010, 2011, 2012 Figure 1. Title of your figure goes here. References References 1) Robert, S., V. Ravigne, M-F. Zapater, C. Abadie, and J. Carlier. "Contrasting Introduction Scenarios Among Continents in the Worldwide Invasion of the Banana Fungal Pathogen Mycosphaerella fijiensis." Molecular Ecology (2012): 1098-1114 2) Guiliano Garisto Donzelli, Bruno, and Alice C. Churchhill. "A Quantitative Assay Using Mycelial Fragments to Assess Virulence of Mycosphaerella Fijiensis." Phytopathology 97.8 (2007): 916-929. 3) "Mycosphaerella Fijiensis." Genome Portal. US Department Of Energy, n.d. Web. 9 Apr. 2013. <http://genomeportal.jgi-psf.org/Mycfi2/Mycfi2.home.html>. 4) Tamura K, Peterson D, Peterson N, Stecher G, Nei M, and Kumar S (2011) MEGA5: Molecular Evolutionary Genetics Analysis using Maximum Likelihood, Evolutionary Distance, and Maximum Parsimony Methods. Moletular Biology and Evolution 28: 2731-2739 5)Hall, T.A. 1999. A user-friendly Biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucl. Acids. Symp. Ser. 41:95-98. 6) Bennett, R.S, and P.A Arneson. "Black Sigatoka of Bananas and Plantains." APSnet. The American Phytopathological Society, 2003. Web. Apr. 2013. <http://www.apsnet.org/edcenter/intropp/lessons/fungi/ascomycetes/Pages/BlackSigatoka.aspx>. Figure: This image shows infected leaf material attached to paper, ready to be soaked in water to hydrate psuedothecia. Figure: This image illustrates lesions typical of Black Sigatoka. (Photo: Meghan Wyatt) Objectives Isolation: The samples of leaf tissue were cut into 2x2 squares and stapled to sheets of paper, psuedothecia facing up. The paper was soaked in 1 L of deionized water for 10 minutes, to allow the psuedothecia to hydrate. After the 10 minutes, the paper was removed from the water. The devices are placed face down over water agar, so that the psuedothecia are facing the agar, but not touching it. The petri dish was closed over the discharge device. After 1 hour, the devices were removed and the plates were sealed with parafilm. The plates were incubated at room temperature to allow the ascospores to germinate. After 12 hours, a microscope was used to find ascospores. The selected ascospores were then plated onto Mycophil agar. Bioinformatics: The PCR product for the samples was sequenced by Genewiz. For the bioinformatics portion of this project, BioEdit was used to trim and edit the sequences by hand, and ClustaW Multiple Alignment was used to align them. After the sequences were aligned, consensus sequences were created and converted to a .meg file before uploading into MEGA5 for analysis. Maximum likelihood and neighbor joining analyses were performed on the sequences with 1,000 bootstrap replications. To study the population structure of the pathogen, Mycosphaerellafijiensis, in different habitats and geographic regions in Costa Rica. These habitats include a) full sun or shade, b) commercial plantations that have or have not been treated with fungicides, c) private homes and d) roadsides. Acknowledgements This project was funded by the National Science Foundation, as part of the IRES (International Research Experiences for Students) program. A special thank you to Lance McGhee, Mary Lewis, Laura Bostic, and Roslyn Noar for their contributions to this research. ** Presented at the: 22nd Annual Undergraduate Research Symposium, North Carolina State University, July 2013, Raleigh, NC. Figure: This map shows the global distribution of Black Sigatoka. It is found in any location Musa spp is grown. (Photo: APS website, Rebecca S Bennett and Phil A Arneson