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Fungal endophytes from apple (Malus domestica) roots as biological control agents of apple replant disease

Document
Call Number
LE3 .A278 2023
Date Issued
2023
Degree Name
Master of Science
Degree Level
Masters
Degree Discipline
Affiliation
Abstract

When new young apple trees (Malus domestica) are planted in an orchard soil that was previously used to grow apples or related plant species, young trees can experience Apple Replant Disease (ARD). ARD results in root necrosis, reduction in plant growth and vigour, as well as reduced fruit yield and delayed fruit bearing, all of which harm growers’ return. Conventionally, chemical fumigants have been used to disinfect soil between plantings as a treatment for ARD, but the deregistration of harmful, chemical fumigants due to environmental and public health concerns has created a demand for eco-friendly alternative treatments. I explored fungal endophytes from healthy, mature apple roots as potential biocontrol agents to treat ARD and reduce the need for chemical fumigants.

48 fungal endophytes were isolated from apple root and tested in dual culture competition assays for antagonistic activity against Pythium ultimum and Rhizoctonia solani; two pathogens associated with ARD. In dual culture competition assays, all endophytes were able to inhibit pathogens to some extent and different types of endophyte-pathogen interactions were observed. From the results of these assays, five biocontrol candidates were selected for further investigation, including two Trichoderma species, a Talaromyces sp., a Mortierella sp. and a Gongronella sp. The cell-free culture filtrates and extracts of these five biocontrol candidates were tested for antagonistic activity against ARD pathogens and for broad-spectrum antimicrobial activity. These five biocontrol candidates were also tested for phosphate solubilization ability, optimal growth temperature, fungicide tolerance, and initial growth promotion and anti-ARD trials were performed with apple rootstocks in the greenhouse.

The cell-free culture filtrate from liquid cultures of five selected biocontrol endophytes demonstrated a range of inhibition potential against four ARD pathogens, with an endophyte identified as Mortierella NS-01 inhibiting all four pathogens to the greatest extent. We can infer that the antagonistic activity demonstrated in the dual culture assays of the isolates with antimicrobial culture filtrates was likely due to secreted non-volatile compounds and could occur independently of direct contact. Further studies could seek to identify specific antimicrobial molecules and test them in a similar assay. Conversely, ethyl acetate extracts did not generally support the results of the culture filtrate assays, perhaps due to antimicrobial molecules that were not extractable by ethyl acetate. Endophyte culture filtrates also demonstrated a range of inhibition against bacteria, yeast and fungi in liquid culture. Trichoderma isolates were tolerant to tested fungicides, whereas Talaromyces NS-01 showed sensitivity. Generally, the fungal endophytes grew optimally between 20-30℃. In vivo, inoculation of Talaromyces NS-01 and Mortierella NS-01 in combination resulted in a greater apple rootstock height in comparison to controls, indicating potential growth promoting effects. In ARD soil, there was generally not a statistically significant difference in size of inoculated apple plants compared to uninoculated after 8 weeks. Even so, in G202 rootstock, there tended to be an increase in size in inoculated plants in ARD soil, although the reverse was observed in M26 rootstock, and neither of these effects was statistically significant. Some of these inconsistencies could be addressed by developing a protocol that optimizes colonization of the apple roots.

In conclusion, five biocontrol candidates were selected and particularly, Trichoderma NS-01, Trichoderma NS-02, Talaromyces NS-01 and Mortierella NS-01 inhibited ARD pathogens through secreted molecules. No clear conclusions were made on the potential for endophyte inoculations to work against ARD in vivo, although Talaromyces NS-01 and Mortierella NS-01 in combination have demonstrated growth promotion in non-ARD conditions.

Rights
The author retains copyright in this thesis. Any substantial copying or any other actions that exceed fair dealing or other exceptions in the Copyright Act require the permission of the author.
Publisher
Acadia University

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