1. 1. Diseased plant identification and coppicing
Investigating the “recovery phenotype” phenomenon in Aster yellows phytoplasma-infected Vitis vinifera
A.E. van der Vyver1*, H.J. Maree1,2, J.T. Burger1
1Department of Genetics, Stellenbosch University, Matieland 7602, South Africa;
2Agricultural Research Council, Infruitec-Nietvoorbij: Institute for Deciduous Fruit, Vines and Wine, Stellenbosch, 7599, South Africa. *
Introduction
Phytoplasmas are obligate intracellular parasites which are associated with diseases in over 100 economically significant plant species. Grapevine Yellows, which is the focus of this study, is caused by Aster Yellows Phytoplasma (AY; Candidatus Phytoplasma asteris). AY symptomatology includes the dieback of growth-points and abortion of immature grape bunches. Although no effective treatment or cure exist, a number of studies describe the induction of a remission state, or ‘recovery phenotype’ (RP), in infected plants by means of physiologically or chemically induced stresses in infected plants. Coppicing, which entails cutting down the grapevine plant to just above the graft union and allowing a single shoot to regrow, is one such physiological stress. The underlying mechanism(s) that result in the recovery phenotype are unknown. This study investigates this phenomenon in a local vineyard, by following both AY and grapevine virus populations over a number of growing seasons.
1. Diseased plant identification and coppicing
Materials and Methods
Results
Future Work
Determined AY status 
Coppiced half of identified plants
Second round of sampling 
DNA extracted for AY screening 
Continuously monitor AY status
Determine different endophytic communities
Process phloem
Extract DNA
Screen using triple-nested PCR assay
Identify 40 AY+ and 40 AY-
Coppice 20 AY+ and 20 AY-
Continuously monitor over 2 years
Sample 100 canes
2. Determining Aster yellows phytoplasma genetic variants
Extract DNA from 40 AY+ plants
Nested PCR assay for rp, groEL, amp and secY genes
Digest with AluI, separate on 3% agarose gel
Digest with Tru1I, separate on 8% polyacrylamide gel
Materials and Methods
Results 1 2 D C B A
AluI(1) and Tru1I(2) RFLP of groEL(A), amp(B), rp(C) and secY (D).
Only single variant group was observed.
3. Screening for grapevine viruses
Extract RNA from 80 plants
Synthesize cDNA
Screen for 12 grapeviine viruses using established RT-PCR assays
Separate PCR products on 2% agarose gels
Materials and Methods
Results
Future Work
Screen coppiced plants for viruses to see if any change in viral status occurred
Screen for additional viruses
Possibly perform RNAseq to remove PCR bias
All AY- negative plants free of 12 grapevine viruses
Acknowledgements
References
The authors would like to aknowledge Winetech for funding the project, as well as Stellenbosch University for providing the necessary infrastructure and personal funding. The authors also extends a thank you to Coela Smith for allowing samples to be collected from his vineyard.
Engelbrecht M., J. Joubert, and J.T. Burger, 2010 First report of aster yellows phytoplasma in grapevines in South Africa. Plant Diseases. 94(3): 373
Lee I-M., R.E. Davis, and D.E. Gundersen-Rindal, 2000 Phytoplasma: phytopathogenic mollicutes. Annual Review of Microbiology. 54: