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Development of a cryopreservation protocol for the microcyclic rust-fungus Puccinia spegazzinii
Matthew J Ryan1, Carol A Ellison
1CABI Bioscience, Bakeham Lane, Egham, TW20 9TY, UK. m.ryan@cabi.org
Abstract:
The rust fungus Puccinia spegazzinii (Basidiomycotina: Uredinales) has been identified as a potential classical biological control agent for the invasive weed Mikania micrantha (Asteraceae). Long-term, live storage of this pathogen is required for reference. As biotrophs, almost all rusts species cannot be preserved by traditional cryopreservation protocols, which rely on in vitro culture techniques. In addition, the embedded teliospores and delicate basidiospores of this microcyclic rust are not amenable to direct plunge freezing. Continuous culture of the rust on living plants is both laborious and expensive, so a variety of approaches for cryopreservation and storage were tested. These methods included traditional approaches to fungal cryopreservation such as variation of cooling rate regime and alginate encapsulation techniques. However, an in situ cryopreservation technique was the only method identified as having any potential for the long-term cryopreservation of the 10 isolates tested. Material from either petiole or stem tissue remained viable after cryopreservation, determined by the ability of the material to produce basidiospores. However, despite great progress being made in developing an optimal cryopreservation method, infection of the host plant by basidiospores produced from previously cryopreserved teliospores, embedded in leaf petioles, was not achieved.
Insights
Long-term storage of the rust fungus Puccinia spegazzinii, a biological control for Mikania micrantha, was challenging. An in situ cryopreservation method showed promise for preserving rust isolates, but subsequent host infection was unsuccessful.
Area of Science:
- Plant Pathology
- Biological Control
- Cryopreservation
Background:
- Puccinia spegazzinii is a rust fungus identified as a biological control agent for the invasive weed Mikania micrantha.
- Long-term storage of this pathogen is crucial for its use in classical biological control programs.
- Rust fungi, being biotrophs, are difficult to preserve using traditional cryopreservation methods due to their reliance on in vitro culture and delicate spore structures.
Purpose of the Study:
- To evaluate various cryopreservation techniques for long-term storage of Puccinia spegazzinii isolates.
- To determine the viability of cryopreserved rust pathogen material.
- To assess the potential of cryopreserved Puccinia spegazzinii for biological control of Mikania micrantha.
Main Methods:
- Tested traditional cryopreservation methods including variations in cooling rates and alginate encapsulation.
- Investigated an in situ cryopreservation technique using infected plant tissues (petioles and stems).
- Assessed viability of cryopreserved material by observing basidiospore production.
Main Results:
- Traditional cryopreservation methods were unsuccessful for preserving Puccinia spegazzinii.
- The in situ cryopreservation technique demonstrated potential for long-term storage of 10 tested isolates.
- Cryopreserved material from petioles and stems remained viable, producing basidiospores.
Conclusions:
- In situ cryopreservation is a promising method for the long-term storage of Puccinia spegazzinii.
- While viability was maintained, successful infection of the host plant (Mikania micrantha) using basidiospores from cryopreserved teliospores was not achieved.
- Further research is needed to overcome challenges in achieving successful host plant infection after cryopreservation.