Masters Theses

Date of Award

5-1998

Degree Type

Thesis

Degree Name

Master of Science

Major

Entomology and Plant Pathology

Major Professor

Kimberly D. Gwinn

Committee Members

Brad Reddick, Bonnie Ownley

Abstract

Tall fescue (Festuca arundinaceae) infected with the endophyte Neotyphodium coenophialum is correlated with increased resistance to damping-off. The objective of this research was to determine the mechanism for endophyte-mediated resistance (EMR) to Pythium aphanidermatum. In experiments using Kentucky 31 seed differing only in endophyte infestation levels (endophyte-infected (95%) was defined as 95E+ and endophyte-free was defined as 95E-), 95E+ seed were more resistant to damping-off than 95E- seed. However, when seed were presoaked in sterile deionized water (SDW) for 24 h before planting, no resistance was observed. Embryo infection and seed colonization of 95E-t- and 95E- seed were compared in P. aphanidermatum-infested soilless medium. Seed were incubated in infested medium for 20 h, rinsed, placed on selective medium, and observed at 20 and 30 h. Embryos of 95E- seed developed necrosis at twice the rate of 95E-I- seed. However seed coat colonization was not different and did not affect embryo viability. Radicle emergence was assessed fi-om seed incubated in rotating flasks of SDW for 24, 48, 72, or 96 h. At 24 h the germination rate of 95E-^ seed was 13% and 95E- seed was 34%; at 96 h germination rates of 95E+ and 95E- seed were equivalent (ca. 80%). To determine the effect of presoaking on germination three seed treatments were compared: 1) nonscarified, 2) scarified, presoaked for 3 h, 3) scarified, presoaked for 24 h. Seeds were planted in soilless mix and incubated for 7 d. No differences in germination were observed between 95E-I- and 95E- seed when presoaked for 24 h, but 95E-I- shoot lengths were 15% less than those of 95E-. Germination of 95E-I- seed was reduced by 30% and 40% when nonscarified or presoaked for 3 h, respectively, but 95E seed was not affected by seed treatment. Similar germination rates for all seed treatments were observed when incubated for 3 weeks. Antifungal activity of 95E+ seed against P. aphanidermatum mycelium was not observed. The influence of endophyte on the spermosphere was measured using pH, conductivity, carbohydrate content, and a Pythium bioassay. Seeds were placed in 24-well plates with 1 ml SDW. Using iodine tests of 24 h steep water, it was determined that wells with 95E- seed contained more carbohydrate. Conductivity and pH were measured at 3 and 24 h. Rates of pH decline and conductivity increase were greater in 95E- wells than 95E+ wells. In wells containing 2 seeds/ ml SDW the pH decreased to 4.0 at 3 h. The effects of pH, molarity, and endophyte status on sporangia of P. aphanidermatum were tested in sterile sodium citrate buffer with 95E+ and 95E- seed. Buffers tested ranged from pH 4.0 to pH 6.5, and concentrations ranged from 0.05 to 0.00IM. Seeds were placed in tissue culture dishes with buffer then a Py/Zz/M/n-infested grass piece was placed in each well. Controls with no seeds were included. Cultures were incubated at 28 C with continuous fluorescent light for 24 h and observed for presence of zoospores. Zoospore production was evident in wells with high pH and low molarity. No zoospores were observed in wells with 0.05M buffers; no zoospores were observed in wells with pH of 4.0. At low buffer concentration (0.00IM and 0.005 M) and medium pH levels (5.5 and 6.0) zoospore production was less in wells with 95E+ seed than 95E- seed or controls. Thus differences in pH and conductivity in the spermosphere as a result of delayed germination may partially explain EMR. These results were inconsistent with the response of P. aphanidermatum propagules to 95E+ and 95E- seed in SDW, where 95F+ seed differentially inhibited zoospore production. Based on these data, factors other than a reduction in 95E+ seed exudation may be involved in EMR. However, it was concluded that a primary factor of reduced infection of 95E+ seed by P. aphanidermtum is believed to be escape through germination delay.

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