Improved diagnosis and control of peanut stripe virus

• Bogor Research Institute for Food Crops Biotechnology, Indonesia

Peanut stripe virus (PStV), a potyvirus transmitted by seed and aphid vectors, causes severe reductions in yield of peanuts in China, Southeast Asian countries, India and the USA. While the virus is not present in Australia, several of its aphid vectors are widespread.
Peanuts are a major food legume in Indonesia. However, PStV is an important constraint to production, and some peanut products are currently imported. An earlier ACIAR project (8834) demonstrated in field experiments that the virus can cause losses of more than 50%. The most severe yield reductions occur under relatively dry conditions similar to those used for growing peanuts in Australia.
While production of virus-free seed is an ideal way of reducing early infection, this method is limited by the difficulties of clean seed production and by reinfection from other hosts. Project 8834 showed that the use of insecticides to prevent aphid transmission causes the vectors to feed on, and infect, a greater number of plants. Furthermore, conventional breeding methods to select for resistance do not offer a solutionno evidence of PStV-resistance has been found in numerous screenings of peanut plants (Arachis hypogaea), or in genetically compatible material derived from wild Arachis species.
Molecular technologies to provide diagnosis of viral infection and add plant-virus resistance will be applied in this project. To generate genetically engineered transgenic peanut plants resistant to PStV, scientists will propagate and purify a single isolate of PStV from Indonesia and clone its genome RNA. They will then identify complementary DNA clones harbouring the viral coat-protein gene, determine its nucleotide sequence, and introduce a translational start codon and regulatory sequences for gene expression in the plant host into the coat-protein gene construct.
At the same time, the team will develop a gene-transfer and plant-regeneration system using marker genes for the commercial Indonesian peanut cultivar Gajah. The techniques will be used to transfer the gene controlling synthesis of PStV coat protein into peanut cells, and to generate transformed plants. These plants and their progeny will then be assayed for the expression of the viral coat-protein gene and evaluated for PStV resistance.
As PStV does not occur in Australia, this evaluation cannot be performed here. Indonesian scientists will be trained in molecular biological techniques in order to undertake evaluation of the progeny of virus-resistant lines at the BORIF Biotechnology Centre in Bogor. They will challenge the plants with virus and use a nucleic acid probe to test for virus replication.
A nucleic acid probe for the detection of PStV in peanut tissue is an expected output of the cDNA cloning of the PStV RNA genome. Ultimately, nucleic acid probes will be labelled and viral RNA detected using an enhanced chemiluminescence or colorimetric assay. An easy-to-use PStV detection procedure will be developed for testing of peanut seed lots in developing countries and within the Australian Quarantine and Inspection Service.
In view of the experience of the research team in molecular engineering, the project has a good chance of success. Productivity will rise with resistance of peanuts to PStV, and commercial release of resistant peanuts could be expected within 510 years. The availability of a gene-transfer system for peanuts will make other genetic improvements in peanuts possible. A possible broad-spectrum resistance to other potyviruses that cause losses in peanut crops (e.g. peanut mottle virus, which is seed-borne) could follow. In the short term, the use of a sensitive and reliable non-radioactive nucleic acid probe will allow the testing of seed lots for infection and ease the movement of peanut seeds between countries.
In the long term, the research will benefit Australia by helping to prevent possible accidental introduction of PStV in imported peanuts. Peanut production under Australian conditions would not be viable if yield losses typical of those produced by PStV were to occur.