Written by Chamini Kanatiwela from the University of Colombo, Sri Lanka. This is the report from a BSPP Junior Fellowship. Click here to read more/apply for one yourself.
Coconut is one of the most important plantation crops in Sri Lanka, providing livelihood for nearly 0.5 million people. Weligama Coconut Leaf Wilt Disease (WCLWD), a major threat to the crop, was first reported in the Weligama area in the southern province of Sri Lanka.
Although this disease is considered non-lethal, it will cause a drastic effect if it spreads to the Coconut Triangle (a triangular area formed by joining the districts of Colombo, Kurunegala and Puttalam) which consists of more than 70% of the total land under coconut cultivation. So far no reliable therapeutic agent has been discovered for this disease. Therefore a sensitive, specific and quick diagnostic test would be highly desirable for routine detection, mainly to avoid using infected planting material. Molecular biological and immunological diagnostic techniques would provide excellent diagnostic tools.
My PhD research project is carried out under the supervision of Prof Preethi Udagama (the principal investigator) at University of Colombo, Sri Lanka. It is based on developing immunological diagnostic reagents and molecular biological methods against the phytoplasma associated with the WCLWD, which is of national importance as the coconut industry produces an annual turnover of £480,000 sterling. Under the phase I of the research project polyclonal antibodies have been developed against WCLWD and this will be used in screening of monoclonal antibodies which are going to be produced under phase II.
My one month fellowship study which was funded by BSPP was hosted by Prof Matthew Dickinson at the Sutton Bonington Campus of the University of Nottingham. The objective of the programme was to get hands on experience of new rapid DNA extraction methods and loop mediated isothermal amplification (LAMP) and to establish a LAMP assay to diagnose WCLWD.
I learnt about a rapid DNA extraction technique which can even be done in the field. It involves a small amount of plant material being shaken in a small tube containing buffer and metal balls to break some of the tissues. This technique is in place of a previous method which took 24 hours, and only taking 5 minutes, it enables hundreds of samples a day to be processed. DNA extraction by this rapid method was successfully achieved for fresh and preserved samples.
Confirmation of the presence of WCLWD causing phytoplasma was achieved by sequencing Polymerase Chain Reaction (PCR) amplicons. DNA extracted using a CTAB method was subjected to conventional PCR of the SecA gene utilising nested PCR. The sequence results proved that WCLWD phytoplasma is closely related Bermuda Grass White Leaf Disease (BGWLD) phytoplasma which belongs to 16Sr XIV group.
Loop-mediated isothermal amplification (LAMP) is a rapid and novel molecular diagnostic tool which has been successfully established for rapid detection of certain phytoplasmas. Since performing LAMP does not require well equipped laboratories, such a technique would be more appropriate for a developing country with agriculture based economy such as Sri Lanka, where the diagnosis will be carried out in the field with minimum facilities. Therefore as an initial step I experienced the technique of LAMP using DNA samples of different types of phytoplasma available at the University of Nottingham. The products of the assay were visualized by agarose gel electrophoresis, but it was more accurately and rapidly achieved using the OptiGene Genie® II machine. To design LAMP primers, it is necessary to obtain the sequence of 16SrDNA and the spacer region of 16SrDNA and 23SrDNA. This is done by conventional PCR and this work will be continued at my home University in Sri Lanka.
Gaining experience in novel techniques has boosted my knowledge and confidence enabling me to handle my research work much more easily. Other than the laboratory exposure, during my stay I was lucky enough to visit special places with historic and natural value in London and Nottingham. I could experience the beauty of the onset of autumn and it will remain in my mind forever. Nottingham is a very scenic and beautiful city with extremely friendly people who made my stay very enjoyable.
I would like to thank everyone in the Department of Plant Sciences of Sutton Bonington Campus, University of Nottingham. Particularly Prof Matt Dickinson for hosting me and for his hospitality, and to Naofel and Praphat for taking care of many of little things that made my study in the lab comfortable and good. I am very much grateful to BSPP for selecting me for this Junior Fellowship programme, which has helped to strengthen my research career and to the advancement of the scientific field of Sri Lanka.
University of Colombo, Sri Lanka
IMAGE: Chamini Kanatiwela with Prof Matthew Dickinson at Sutton Bonington Campus, University of Nottingham