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Hop Powdery Mildew (Podosphaera macularis) Spore Cryopreservation
1Department of Horticulture, Oregon State University Corvallis, OR, USA. Sugae.Wada@ Oregonstate.edu.
Background:
Hop powdery mildew (HPM), Podosphaera macularis, an important disease organism for hops, is an obligate parasite, requiring constant culture on living plant tissue for strain maintenance.
Objective:
This study determined the parameters required to successfully cryopreserve HPM spores for the first time and reduce the need for constant culture.
Materials And Methods:
Spores of an Oregon HPM strain, OSU C-100 were desiccated over silica gel for 2-10 h to determine the spore moisture content (MC). Regrowth of the hyphae before and after drying and liquid nitrogen exposure was determined on glass slides and leaf discs of several susceptible hop cultivars. A second mixture of strains was later tested with the protocol.
Results:
Desiccation to an optimal 2-3 percent MC produced hyphal growth on slides and infection of leaf discs. The OSU C-100 HPM spore strain required 8-10 h desiccation to reach 2-3 percent MC while the mixed strains required 6-8 h due to slightly different MC when collected.
Conclusion:
HPM strains should be placed in cryovials, dried to 2-3 percent MC over silica gel, cryopreserved by direct immersion in liquid nitrogen. They can be rewarmed for 1 min each in 45C and 20C water and the viability tested on isolated leaf discs.
Insights
Cryopreservation of hop powdery mildew (HPM) spores is now possible. Drying spores to 2-3% moisture content and immersing them in liquid nitrogen allows for long-term storage and reduced need for continuous cultivation.
Area of Science:
- Plant Pathology
- Mycology
- Cryobiology
Background:
- Hop powdery mildew (HPM), caused by *Podosphaera macularis*, is a significant pathogen of hops.
- HPM is an obligate parasite, necessitating continuous cultivation on living hop plants for strain maintenance.
Purpose of the Study:
- To establish cryopreservation parameters for HPM spores.
- To reduce the labor-intensive requirement of maintaining HPM cultures on living plant tissue.
Main Methods:
- HPM spores (OSU C-100 strain and a mixed strain) were desiccated over silica gel to determine optimal moisture content (MC).
- Spore viability was assessed by observing hyphal regrowth on glass slides and successful infection of hop leaf discs after cryopreservation in liquid nitrogen.
- Regrowth was evaluated before and after desiccation and liquid nitrogen exposure.
Main Results:
- Optimal spore desiccation to 2-3% MC was determined to be crucial for successful cryopreservation.
- The OSU C-100 strain required 8-10 hours of desiccation, while mixed strains needed 6-8 hours to reach the target MC.
- Successful hyphal growth and leaf disc infection confirmed spore viability post-cryopreservation.
Conclusions:
- A protocol for cryopreserving HPM spores was successfully developed.
- The recommended method involves drying spores to 2-3% MC over silica gel, followed by direct immersion in liquid nitrogen.
- Rewarming involves sequential 1-minute incubation in 45°C and 20°C water, with viability confirmed on hop leaf discs.
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