Cryotherapy: A New Method to Eliminate Pathogens from Sweetpotato Propagation Materials

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1 Cryotherapy: A New Method to Eliminate Pathogens from Sweetpotato Propagation Materials Margaret Worthington Graduate Group in Horticulture and Agronomy University of California, Davis April 14,

2 Basics of Sweetpotato Propagation In tropical areas, sweetpotatoes can grow yearround and propagation is by cuttings from plants of the previous crop. In temperate areas, however, roots are stored during the winter months to serve as a source of 'seed' for sprout production. Sprouts are transplanted into the production field. This practice is inefficient, but practical alternatives have not been developed. Sweetpotato plants can be cultivated from meristem tissue, but this is done to remove viruses from seed stock. Planting sweetpotato root pieces directly in the field does not result in sufficiently uniform storage roots. True seed is not a viable option because sweetpotatoes are genetically complex and plants growing from true seed are extremely variable. As with all vegetatively propagated crops control of pathogens in planting materials is of supreme importance

3 Phytoplasmas Phytoplasmas are intracellular, cell wall-less bacteria Live as obligate parasites in insect vectors and sieve elements of plants Sieve elements are elongated cells which transport carbohydrates in the phloem tissue Phytoplasmas are restricted to the phloem cannot colonize the meristem very well Symptoms include: Green flowers (phyllody) Development of floral organs into leaf like structures Sterility of flowers Witches broom (bushy form) General stunting Dieback Chlorosis Necrosis Pathogenic mechanisms unknown

4 Sweetpotato Little Leaf Phytoplasma (SPLL) SPLL is a major disease in the Pacific Islands and Australia (major producers) Causes malformed leaves and stunted tuber development Little leaf disease can cause up to 50% loss in sweetpotato yields Clean propagation materials are necessary to prevent the spread of the disease

5 Basics of Cryotherapy Pathogen tested plant stocks obtained from other methods including shoot tip culture, thermotherapy, leaf tissue derived somatic embryogenesus, organogenesis, stem culture, micrografting, and treatment with antibiotics Cryotherapy is a new technique for producing virus free plants (used for production of virus free Prunus, banana, grapevine, and potato) But can it be used to eliminate bacterial pathogens like phytoplasma? Cryotherapy: freezing tissue in liquid Nitrogen to kill pathogens/viruses and then regenerating the treated tissue

6 Methods Pathogen free plantlets graft-inoculated with scions from an Australian cultivar infected with SLLP Shoot tips (1 mm long) from the inoculated plants were frozen in liquid Nitrogen for 1 hour Frozen shoot tips were warmed in a water bath at 40 C Treated tissue regenerated in a group medium for 2 months In vitro plantlets transferred to soil in a greenhouse and evaluated for phytoplasma symptoms Basic shoot tip culture and dehydration were used as the untreated and treated controls

7 Success! Survival (88%) and regrowth (85%) were slightly lower in cryotherapy treated shoot tips than the control and dehydrated treatments. BUT 100% of regenerated cryotherapy treated shoots were phytoplasma-negative! (Only 7% for Dehydration and 10% for Tissue culture) Confirmed with the adult plants in the greenhouse and PCR (polymerase chain reaction amplifies the bacteria DNA for easy detection) Source: Wang and Valkonen, 2008

8 Probable Reasons for Success Cryotherapy is usually lethal for cells located more than 0.5 mm below the apical dome at the 3rd leaf promordium No phytoplasma live near the tip of the plant (0.5 mm to the apical dome) Phytoplasma establishment at the shoot tip could be inhibited by plant hormones produced there or prevented by the small, undeveloped sieve pores at the tip Cryotherapy kills all the cells where phytoplasma can live, leaving only the clean cells for regeneration and propagation Source: Wang and Valkonen, 2008

9 Conclusions SPLL phytoplasma can be easily removed from sweetpotato plants by cryotherapy of in-vitro grown shoot tips Another advantage of cryotherapy is that it can simultaneously be used to prepare shoot tips for long term germplasm storage Use of cryogenically frozen tissue for international transport and trade can prevent disease spread Control of pathogens in vegetatively propagated crops like sweetpotato is a challenge, but we can manage disease with new techniques like cryotherapy

10 References Wang, Q.C. and J.P.T Valkonen Efficient elimination of sweetpotato little leaf phytoplasma from sweetpotato by cryotherapy of shoot tips. Plant Pathology 57: