Local Crops and Wild Relative Plants

1. Dissemination Seminar Project on the Study and Sustainable Conservation of Local Plants20th October, 2015 Plant Health DirectorateDiagnosis and Control of Pests and Diseases in Local VarietiesPrepared by Mr Roberto Lagana / Mr Darren VellaPresented by Ms Moira Pace - Chief Scientific Officer

2. Local Crops and Wild Relative Plants A number of plant species were chosen to be tested for certain pathogens. These plant species will all be species that are included in the project and the aim of these laboratory tests is to check for harmful plant pathogens so as to eventually have disease-free plants planted in the public garden.

3. Plant Pathogens A list of pathogens was compiled for this project, and the presence of these pathogens in the local plant species were tested. The tests were carried out by using the DAS-ELISA technique.

4. Citrus Mother Plants • 7 local varieties of citrus trees were identified to be used as citrus mother plants, from which several young citrus plants could be propagated.

5. Citrus Sanitised Plants • After, in-vitro propagation of these mother plants, which had mixed results, the successfully propagated plantlets were sent for acclimatization in the Plant Health Department’s glasshouse. The plantlets that survived this process were the following: • L1  (Lemon, Buskett Gardens) – 344 plants • L4  (Lemon, San Anton Gardens) – 62 plants • L6  (Lemon, San Anton Gardens) – 16 plants

6. Citrus Pathogens/Diseases • Both the citrus mother plants and the young sanitised propagated plantlets had to be tested for a variety of citrus diseases caused by viral or viral-like pathogens.

7. Testing Techniques Serological Techniques • ELISA - Enzyme-linked immunosorbent assay • DAS-ELISA (Double Antibody Sandwich ELISA) • Immunoprinting-ELISA • Biological Indexing • Mechanical Transmission on Herbaceous Indicator Plants • Graft Transmission of Pathogens on Citrus Indicator Rootstock

8. DAS-ELISA

9. DAS-ELISA Plant Samples in ELISA plate Primary Antibody (IgG) Pipetting Absorbance Microplate Reader

10. Immunoprinting-ELISA • Nitrocellulose membrane printing • Addition of blocking agent • Addition of antibodies • Washing with washing buffer • Addition of substrate • Observation for colour change If colour changes to bluish purple the sample is POSITIVE. No colour change means that the sample is NEGATIVE.

11. Immunoprinting-ELISA Printed Immunoprinting-ELISA membrane

12. ELISA testing kits Plant Print Diagnostics Immunoprinting-ELISA test kit BIOREBA DAS-ELISA test kit

13. Mechanical Transmission on Herbaceous Indicator Plants Each sample was extracted using the inoculation buffer solution prepared earlier, a sharp scalpel and a pestle and mortar. The extract (inoculum) was applied to the leaves by dipping the pestle in freshly prepared extract and then very gently applied on the leaves.

14. Graft Transmission of Pathogens on Citrus Indicator Rootstock There are several ways to graft one plant onto another and which grafting technique is used usually depends on: • The purpose of the graft (different grafting techniques used for horticultural purposes than for pathogen identification purposes) • The sizes of the rootstock and scions (different techniques used when stock and scion are of similar diameter) • The ability and experience that the person doing the graft has.

15. Grafting Techniques Graft transmission of virus infected plants to susceptible rootstocks and infection in the recipient plant will be already realized when the grafted plants (scion and rootstock) have reached a vascular connection(cambium) for only a short period.

16. Results Local Crops – DAS-ELISA • From the results obtained for local crops and their wild relatives, all samples were negative except for the ‘cikkarda’ plant species, which tested positive for the viral pathogen ZYMV. • The majority of plant species tested were free from the pathogens tested and can be introduced in the public garden without any further harmful consequences. • The infected species will be replaced and tested, once obtained, before being planted with other plant species. • This will ensure that no plant species cultivated will introduce any of the pathogens tested into the public garden and risking other plant species’ health and survival.

17. Results Citrus Mother Plants – Immunoprinting-ELISA Pathogen tested: CTV Sample Collection Date: 21-05-2014 Sample Printing Date: 22-05-2014 Sample Testing (Developing) Date: 05-06-2014

18. Results Citrus Mother Plants – Immunoprinting-ELISA Pathogen tested: CTV Immunoprinting-ELISA membrane after development. No bluish-purple colour observed, therefore NO positive samples.

19. Results Citrus Mother Plants – Mechanical Transmission on Herbaceous Plants

20. Results Citrus Mother Plants – Mechanical Transmission on Herbaceous Plants

21. Results Citrus Sanitised Plants – CTV DAS-ELISA A number of samples at random from the sanitised plants (shown in these photos) found in a PBC glasshouse were obtained and tested for CTV using the DAS-ELISA technique. Samples were grouped according the citrus species and age.

22. Results Citrus Sanitised Plants – CTV DAS-ELISA ELISA microtitre plates, showing NO positive results. Positive samples will convert the substrate into a yellow coloured solution, just like the positive controls.

23. Results Citrus Mother Plants – Graft Transmission of Pathogens on Citrus Indicator Rootstock For each virus/disease tested a table like this (table on the right) was constructed, including the rootstock needed and the amount. Symptoms generally take some time to show and be observed, results are still not available.

24. Results Citrus Sanitised Plants – Graft Transmission of Pathogens on Citrus Indicator Rootstock For each virus/disease tested a table like this (table on the right) was constructed, including the rootstock needed and the amount. Symptoms generally take some time to show and be observed, results are still not available.

25. Thank You