Improved diagnosis and control of peanut stripe virus

• Institut Pertanian Bogor, Indonesia
• Research Institute for Food Crops Biotechnology, Indonesia
• Chinese Academy of Agricultural Sciences, China

Peanut stripe virus (PStV) is a major cause of yield reductions in peanut (Arachis sp.) crops in many countries. Naturally occurring infections have been reported in China, Japan, Thailand, the Philippines, Malaysia, Indonesia and Myanmar, and the virus has entered India and the USA with germplasm introductions. Yield losses due to infection under dry season, broadacre peanut production are frequently as high as 75-80%.
Australian peanut crops are free of the virus, and it is important that quarantine remains effective in keeping it out. Peanut production under Australian conditions would not be viable if yield losses typical of those produced by PStV were to occur. The risk of accidental introduction of the virus in imported raw peanuts is considered high, and aphids capable of transmitting it are widespread in Australian peanut crops.
Previous work using the classical breeding approaches incorporating host plant resistance had not produced any sources of PStV resistance despite exploring the entire world peanut germplasm collection (over 11,000 accessions). Resistance to PStV had been identified among wild Arachis relatives, but crosses to introduce this trait had not been successful, due to incompatibility between species. Therefore, genetic engineering offered the best opportunity to improve existing cultivars by selectively adding specific new traits such as virus resistance.

The main aims of this project were to develop further the gene transfer system developed in ACIAR project CS1/1990/017 (Improved diagnosis and control of peanut stripe virus) and to produce PStV-resistant peanuts by inserting a coat protein gene from the virus into commercial cultivars. Expression of coat protein genes in genetically transformed plants had yielded protection against a range of polyviruses (the family to which PStV belongs), but systems for producing transgenic peanut plants had yet to be developed.

The project scientists sought to:
generate and assess genetically engineered commercial peanut cultivars with resistance to peanut stripe virus;
develop a nucleic acid-based diagnostic kit for the identification of PStV and other viruses infecting peanuts;
transfer the new technology to collaborating institutions in Indonesia and China.
The research teams from the Queensland Agricultural Biotechnology Centre and University of Queensland Botany Department, together with their collaborators in Indonesia and China, continued to investigate a range of approaches to the production of transgenic peanuts. They applied systems under development to various commercial Australian and Asian cultivars to test their effectiveness.
Once successful transfer of the coat protein gene had been achieved, the scientists planned to assess the transgenic plants for resistance to PStV and evaluation of their field performance. Other research further refined the virus detection approach developed in project CS1/1990/017, with the aim of making available a reliable and sensitive diagnostic kit. In addition, the group studied the genetic diversity of PStV strains in the Asia-Pacific region.

This project developed a practical and efficient genetic transformation and regeneration system for cultivars in both botanical types of peanut. Using particle bombardment technology in Australia and China, reporter and viral resistance genes were introduced into peanut. As well, an alternative Agrobacterium-mediated transformation system was investigated.

Four modified versions of PStV coat protein (CP) sequences were engineered and assessed in transgenic Nicotiana benthamiana plants for protection against PStV infection. The scientists achieved immunity to infection in about 70 per cent of the lines following mechanical challenge inoculation. Immune lines were protected even against isolates with the highest sequence divergence in the CP gene. Seven transgenic peanut lines of cultivar Gajah (Spanish market type) carrying CP2 or CP4 variations were identified as resistant or immune to infection to the virus in glasshouse trials in Australia.

Scientists working in Australia, China and Indonesia developed diagnostic methods for detecting the presence of modified viruses (a non-radioactive probe hybridisation for PStV, and reverse transcriptase-PCR assays for detecting and identifying a range of seed-borne peanut viruses).

A study of the biological and genetic variability of PStV isolates in Indonesia, Thailand and China found geographically related groups with wide symptom diversity. Indonesian isolates of PStV were identified as intra-species recombinants, while Chinese strains of peanut stunt virus were shown to differ sufficiently
from strains in other locations, suggesting that they may form a third taxonomic subgroup of the species. This information is significant for future diagnostic tests.

Scientists in collaborating institutes in Indonesia and China working on peanut transformation and regeneration and diagnostics benefited from the project through the provision of equipment and the opportunity to participate in training with the Australian team. The scientists also took part in project coordination visits and a two-week workshop in Indonesia. Since the project's final review, further transgenic resistance in the Gajah and Kelinci peanut cultivars was demonstrated using the technologies developed during the project.