Virus indexing and DNA fingerprinting for the international movement and conservation of taro germplasm

• Secretariat of the Pacific Community, Fiji
• Ministry of Agriculture, Forests, Fisheries and Meteorology, Samoa
• University of Queensland, Australia
• University of Technology, Papua New Guinea
• University of the South Pacific, Samoa
• National Agricultural Research Institute, Papua New Guinea

Taro is widely grown in Papua New Guinea (PNG) and many other Pacific Island countries. It also plays an important cultural role. The roots are a source of carbohydrate, and the foliage is also eaten. It is cultivated mainly in gardens for local use, but there is also a domestic and export market. Over the last 20 years there has been a gradual decline in the production of taro because of the effects of pests and diseases. Taro leaf blight, caused by a fungus, is the most serious and widespread disease of the plant in Pacific countries. It has long been present in Micronesia, Papua New Guinea and the Solomon Islands, but in 1993 it spread to American Samoa and Samoa with devastating consequences. Many growers have since abandoned taro cultivation in these countries, causing major social and economic problems. Export earnings in Samoa fell from 9.5 million Tala to 158,000 Tala in just one year after the arrival of the blight.

The genetic diversity of taro is poorly known, but some described varieties are resistant to the fungus. In 1993, a breeding program started up in PNG to develop these varieties but the taro germplasm cannot be moved between countries because of the presence of a lethal virus disease known as alomae. It is now important to characterise this disease (which seems to be associated with the presence of two viruses together) and develop reliable tests for the presence of both viruses within taro germplasm. This should then allow free movement of germplasm and thereby help in combating leaf blight and in developing other features of the plant.

Increased knowledge of alomae will be helpful of itself because this disease is now the main constraint on taro production in PNG and the Solomons. Elsewhere it seems that the two viruses do not occur together; when only one virus is present, disease symptoms are much milder. Characterising the virus diseases of taro, a Pacific Island staple crop, is underway as the first step to developing sensitive specific tests for each virus.

The aim of the project is to characterise the virus diseases of taro, a Pacific Island staple crop, and to develop sensitive specific tests for each virus.

The work will be divided into four strands. The first will classify taro viruses, and identify any previously undescribed ones. The second subproject will develop diagnostic tools and protocols for the detection of characterised viruses.

The third subproject will develop and apply DNA marker technology to fingerprint taro DNA in a bid to characterise the different varieties of the plant in Polynesia and Melanesia as a further aid to developing disease resistance.

The final part of the project will be the construction of a database of DNA profiles resulting from the investigation into genetic diversity.

Characterisation of all taro viruses and development of diagnostics
(a) Taro reovirus (TaRV): Investigation of the sequence variability in the genome of isolates from PNG, Solomon Islands, New Caledonia and Vanuatu TaRV enabled researchers to gather sufficient data to develop a specific PCR-based diagnostic assay for this virus.
(b)Taro vein chlorosis virus (TaVCV): The researchers cloned and sequenced the entire genome of a Fijian TaVCV isolate. A diagnostic PCR test has now been developed for this virus based on a 220 nt region of the L-gene.
(c) Colocasia bobone disease virus (CBDV): The researchers cloned and sequenced approximately half (~5000 nt) of a PNG isolate of CBDV. A PCR-based diagnostic has been developed based on a region of 'gene 3'.
(d) TaBV-like sequence: Preliminary characterisation and analysis studies indicated that the TaBV-like sequences present in taro are integrated sequences. Further work is needed to determine whether these sequences can be activated and cause disease.

Thus the characterisation of TaRV and the two rhabdoviruses has enabled the subsequent development of sensitive diagnostic tests for these viruses. As such, diagnostics tests have now been developed for all known viruses infecting taro. The availability of a suite of taro virus diagnostics will now enable taro germplasm to be virus-indexed, thus facilitating safe international movement of taro germplasm.

Virus survey
Surveys were conducted in Vanuatu, Samoa, American Samoa, Fiji, PNG, Solomon Islands and New Caledonia. Samples were also provided from Micronesia and the Cook Islands. These samples have been indexed for all known viruses using the newly developed molecular-based diagnostic tests. The virus surveys, conducted in countries wishing to share germplasm under the TaroGen project, provided updated information on virus distribution. These data, combined with the results from virus-indexing from the TaroGen germplasm collection, allowed countries to make informed decisions on the importation of taro germplasm.

Virus indexing of TaroGen taro germplasm collection held at SPC, Fiji Approximately 450 tissue-cultured taro lines held in the germplasm collection at SPC have been sent to Brisbane for growing in AQIS-greenhouses and indexing. Of these, 159 have been indexed for each of the taro viruses according to an internationally-recommended schedule. Safe international transfer of indexed taro germplasm will therefore now be possible, allowing countries access to a diverse pool of germplasm with disease resistance and other agronomic qualities.

DNA fingerprinting of national taro collections
Taro collections from nine Pacific Island Countries were DNA fingerprinted using radiolabelled SSR (Simple Sequence Repeat) markers. These markers were developed as a resource for taro research both within the region and internationally. Taro collections were made in most cases as part of the TaroGen germplasm collection. Entire collections from Fiji, Samoa, Tonga, Niue, Palau, Cook Islands were fingerprinted. A 20% sample of the country collection was fingerprinted from Papua New Guinea, Solomon Islands, Vanuatu and New Caledonia, as these collections were too large to fingerprint all accessions. There were some delays in receiving collections from some countries. Civil unrest, disease and cyclones prevented the Solomons' collection from being received until 2002. Even then, this was not a representative country collection, rather it was separate samples from three provinces: Choiseul, Malaita and Temotu. The Guadalcanal collection was lost to virus and other diseases before samples were taken. Researchers received two collections representing Samoa, one from the Botanic Gardens of the University of Hawaii, and one from MAFF in Samoa. In the case of Solomon Islands, Fiji and the Polynesian countries, collections were received from the Regional Germplasm Collection at SPC as tissue cultures. In other cases, they received leaf samples directly from the country.

From the overall collection of 2206 accessions, 527 were DNA fingerprinted with SSR markers, which were used to assess within- and between-country taro genetic diversity, and to identify a DNA fingerprint of accessions. It was evident that most (if not all) of the genetic diversity within South Pacific taros could be sampled from PNG and Solomon Islands. Interestingly, two SSR alleles were found only in the Solomons. This was somewhat unexpected, and is a tantalising hint at a localised adaptation or separate introduction within taros in the Solomons. It is worth noting that none of the PNG taros fingerprinted were from Bougainville, which is geographically closer to the Solomons than it is to other islands of PNG. There were some quite diverse taros in the small sample of accessions from Palau, probably representing some more Asian taro types. Evidence from our study and that of the EU-TANSAO study suggest that there are two 'Centres of Diversity', one in PNG/Solomons and the other in Indonesia/Malaysia.

Rationalisation of taro germplasm to form a core collection
Based on DNA fingerprints, researchers selected a core collection for each country. The aim of the core collection is to reduce the size of the collection to about 10% of the total accessions while attempting to maintain at least 85% of the genetic diversity available. The core collection can then be conserved more easily and utilised more effectively, as these accessions can be more extensively characterised (for example for pest and disease resistances, corm attributes and response to abiotic stresses). Once core collections were selected for each country, the combined all-country data set was analysed to improve the level of overall genetic variation for the region. The core collection is stored as in vitro tissue cultures, primarily at the Regional Germplasm Collection (RGC) at SPC in Suva, Fiji. Duplicate collections are kept at USP Alafua Campus, Samoa, with plans to maintain a sample at the International Potato Centre (CIP) in Peru, and negotiations are under way to maintain a duplicate collection in PNG.

Regional expertise
This has been boosted with the training at Queensland University of Technology of Mr Macquin Maino from University of Technology, Lae, PNG, who completed his Master of Science Degree within the Centre for Molecular Biotechnology. He has returned to UniTech, Lae, to play a key role in the continued development of the Agricultural Biotechnology Centre, and training of new scientists. Also at QUT, and Mr Apaitia Macanawai from USP (Samoa) who completed his Master of Agriculture Degree. Mr Macanawai investigated the epidemiology of taro small bacilliform virus and examined alternative hosts, vectors and seed transmission. At University of Queensland Mr Tom Okpul from NARI, Bubia spent 3 months in 2000 learning the techniques of DNA fingerprinting. He applied these techniques to a diverse set of germplasm from the NARI breeding program. He has since moved to Vudal University in Rabaul, where he has instigated taro breeding. Also at UQ Mr Robert Plak Pawilnga from Unitech, Lae visited for 2 weeks in 2001 and learnt techniques involved in database maintenance and analysis of DNA fingerprint data. He returned in 2003 where he is currently completing his Masters of Agricultural Studies. His research is focused on tissue culture and genetic transformation of taro.

A virus diagnostics workshop held at USP provided both theoretical and hand-on training in plant virus characterisation and diagnosis, using taro viruses as an example. SPC has been proactive in the development of an in-house virus indexing capacity in Fiji - this workshop provided both technology transfer and an opportunity to discuss issues such infrastructure, equipment and logistics.