Date of Award

5-2012

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Legacy Department

Plant and Environmental Science

Advisor

Agudelo, Paula

Committee Member

Knap , Halina T

Committee Member

Jeffers , Steven N

Committee Member

Abbott , Albert G

Abstract

The foliar nematode Aphelenchoides fragariae is an economically important and frequent pest of ornamental crops. In nurseries and landscapes in the United States, foliar nematodes can be a serious problem affecting hostas (Hosta spp.), a common perennial ornamental plant that thrives in shady environments. Fundamental research on the biology and ecology of this nematode provides the basis for development of sound management strategies. The general objective of this dissertation was to study two behaviors that make foliar nematodes very successful as plant pests: desiccation tolerance and alternative feeding on fungi (or facultative feeding on the plant). To study the molecular basis of these behaviors, I used a transcriptomic approach complemented with in vitro and greenhouse experiments. As part of the greenhouse studies, I developed a standard protocol for the assessment of resistance to Aphelenchoides fragariae on hosta cultivars. I evaluated the effects of inoculum type (nematodes maintained on fungus vs. maintained on plants), inoculation method (with injury vs. without injury), and harvesting method to select the best procedures, and estimated the correlations between nematode reproduction and symptom severity. The variability of the correlation between symptoms and nematode reproduction highlighted the importance of measuring both parameters when evaluating cultivar resistance. More importantly, I demonstrated that there is a loss of virulence for inoculum maintained and increased on fungus cultures. For the molecular studies, I used Illumina technology and de novo assembly to complete a transcriptome of the nematode (a non-model organism lacking a reference genome) to generate data corresponding to nematode mRNAs from mixed life stages under four different treatments (fungus diet, plant diet, diet changed from plant to fungus, and desiccation stress). This transcriptome is the first comprehensive sequence resource available for foliar nematodes. I present an annotated overview of the Aphelenchoides fragariae transcriptome and provide information on gene families, gene structure, potential gene functions, and gene pathways (molecular, cellular and biological). I identified and characterized a ß-1,4-endoglucanase, which I named Afr-ENG-1, that is differentially expressed when the nematode feeds on fungi or plants. When individuals from hosta plants were transferred to a fungus culture, expression of the enzyme decreased 1,812- fold after five generations on the fungus diet. Diet was also associated with changes in nematode body size and in the severity of symptoms caused on hosta leaves. Plant-diet nematodes caused larger lesions and were longer and thinner than fungus diet nematodes. Full length sequences of Afr-eng-1 were obtained and found to encode a glycosyl hydrolase family 5 (GHF5) protein. This is the first ß-1,4-endoglucanase and plant-parasitism related gene described in the genus Aphelenchoides. For the desiccation tolerance experiments, Aphelenchus avenae, a model nematode for anhydrobiosis studies, was used as a reference. Aphelenchoides fragariae showed higher survival rates than A. avenae under desiccation and osmotic stress conditions. Two glutaredoxin (glx) and three trehalose phosphate synthase (tps) genes were identified in the A. fragariae transcriptome of nematodes subjected to desiccation conditions. Phylogenetic analysis showed that the Af-TPS was closely related to TPS of A. avenae. Expression of Af-tps and Af-glx suggests participation of anti-oxidation and cellular membrane protection mechanisms in the desiccation tolerance mechanisms of foliar nematodes. These observations on the feeding behavior and desiccation tolerance of the foliar nematode improve our knowledge of the basic biology of this nematode and will enhance our ability to increase the arsenal of weapons to fight damage to plants from these parasitic nematodes.

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