Date of Award

5-2011

Level of Access

Campus-Only Thesis

Degree Name

Master of Science (MS)

Department

Botany and Plant Pathology

Advisor

Jody Jellison

Second Committee Member

Seanna Annis

Third Committee Member

David Lambert

Abstract

Brown rot fungi cause the most destructive decay to wooden structures accounting for almost 80% of the damage to in-service wood. During decay, brown rot fungi secrete large amounts of oxalate which has been theorized to assist in cell wall degradation by mobilizing iron, destabilizing pit membranes, and detoxifying copper. Due to the micro environments that occur inside wood during degradation and the limitation of current analytical techniques, the role of oxalate has not been fully characterized. This work aims to elucidate the role of oxalate by studying the gene expression of the model brown rot fungus Postia placenta via quantitative reverse transcription PCR (qRT-PCR). Levels of mRNA were quantified through the development of qRT-PCR primers to oxaloacetase (oah), glyoxylate oxidase (glx), isocitrate lyase (icl), and ^-tubulin (p-tub). Methods used to control for genomic DNA contamination were RNA specific primer design and examination of product quality by electrophoretic and spectophotometric methods. To establish optimal harvest times, liquid cultures of P. placenta were evaluated for pH, oxalate production, glucose consumption, biomass, and gene expression. It was determined that over time, biomass and oxalate levels increased, and pH and glucose levels decreased. The results of the qRT-PCR assay indicate a high level of oah, glx, and icl mRNA during early growth with a decline as the culture ages. The relationship between oxalate production and pH was explored using nutrient limiting as well as alkali treatment and metal treatment experiments. As nutrient content was adjusted, oxalate levels were inconsistent with pH change. To examine the effects of pH on oxalate production, culture pH was adjusted to 7 with NaOH or Na2CO3. Cultures treated with NaOH exhibited no change in gene expression, while those exposed to Na2CO3 demonstrated a decrease in oah and glx expression and a decrease in measurable oxalate in liquid culture. The effects of the metals, iron, manganese, and copper, on P. placenta in liquid culture were assessed using metal sulfate treatments. These studies suggest the role of oxalate may be more complex than previously theorized. Results show that 1 mM manganese, iron, and copper had no effect on gene expression of oah, glx, and icl while 100 mM copper treatments decreased the oah and glx transcript levels. This research identifies possible avenue of copper based fungicide resistance in P. placenta and should be followed up with future studies.

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