Date of Graduation

5-2018

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Cell & Molecular Biology (PhD)

Degree Level

Graduate

Department

Biological Sciences

Advisor

Burt H. Bluhm

Committee Member

Terry Kirkpatrick

Second Committee Member

Ken Korth

Third Committee Member

Vibha Srivastava

Fourth Committee Member

Ned French

Keywords

Biocontrol, Cercospora zeae-maydis, Forward genetic screen, Nematodes, Probe capture

Abstract

Plant-parasitic nematodes pose a major threat to crop yield worldwide. Discontinued use of harmful chemicals has prompted the search for alternative management strategies that are effective yet environmentally friendly. Harpin proteins, which are derived from bacteria, and nematophagous fungi, natural predators of nematodes, are ideal for biological control of plant-parasitic nematodes. However, research on the efficacy of harpin proteins on nematodes, and biology of nematophagous fungi is minimal. Previously, a taxonomically uncharacterized nematophagous fungus designated ARF18 effectively suppressed nematode populations in soil. The overarching goal of this dissertation is to utilize applied and functional genomic approaches to augment management of plant-parasitic nematodes with the following objectives: 1) To test the efficacy of harpin protein treatments in different host-nematode interactions, 2) Utilize genome of ARF18 to provide taxonomic placement and establish it as a biocontrol agent against plant-parasitic nematodes, 3) Identify genes regulated by harpin during reniform pathogenesis, and 4) Develop target enrichment sequencing method using Cercospora zeae-maydis as model system to accelerate functional genomics research. Evaluation of harpin protein on soybean and cotton showed a consistent reduction in reniform populations in soybean without significant effects on plant growth. Similarly, ARF18 parasitized cysts in-vitro, and based on the ITS region, ARF18 grouped within Brachyphoris. ARF18 genome assembled into 412 scaffolds, indicating a size of 45.6 Mb with 14,461 putative protein-encoding genes. Transcriptome of soybean using Ion Torrent PGM identified numerous genes from soybean roots albeit lower sequencing depth hindered the identification of differentially expressed transcripts between different treatments. Subsequently, a target enrichment method was developed in C. zeae-maydis to dissect cercosporin regulation. Ease of genetic manipulation, availability of near complete genome, and presence of easily screen able phenotype facilitated the method development in C. zeae-maydis. The method identified more than 80 genes that altered cercosporin production in C. zeae-maydis. Additionally, RNAi lines created for six genes confirmed the linkage of the phenotypes to the mutation identified. The method could be easily adapted to different organisms, especially in nematophagous fungi to accelerate gene discovery and function to advance research towards the management of different plant parasitic nematodes.

Available for download on Sunday, May 03, 2020

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