Control of gemini virus diseases of cotton and tomato in Pakistan and Australia

• The World Vegetable Center, Taiwan
• National Institute for Biotechnology and Genetic Engineering, Pakistan

Over the past decade geminiviruses have emerged as devastating disease agents infecting major crops in many regions of the world. The epidemics are linked to the spread of a new form of the whitefly, Bemisia tabaci, a particularly efficient vector of geminiviruses. The resulting diseases of concern to this project were leaf curls of cotton and tomato. The recent rate of spread and the scale of damage caused by geminiviruses worldwide now rank them among the most serious plant diseases on record. Geminivirus epidemics in Pakistan during the 1990s were severe, with combined losses to cotton and related industries estimated to exceed 162 billion Pak rupees (over A$5 billion)

In Australia tomato geminivirus has been recorded in the Northern Territory for many years, however it has not previously been a serious problem because the indigenous form of Bemisia tabaci is a relatively poor vector (virus carrier). But a new more efficient biotype (B) of the virus vector is spreading through northern and eastern Queensland and NSW. At the outset of this project the geminivirus in the Northern Territory and the biotype B vectors in Queensland did not overlap, but scientists were preparing for this to happen and cause crop losses well in excess of $100 million annually. Meanwhile the damage caused by geminiviruses in Pakistan was having an impact of national magnitude on the country's economy. Scientists in Pakistan had attempted without success to control the disease through the use of pesticides and change of agronomic practices.

This project sought to produce disease-resistant lines of cotton and tomato, focusing on developing transgenic resistance that would offer a higher likelihood of success than the conventional means that had so far proven ineffective. The project also sought natural geminivirus resistance in tomato genotypes produced through an existing breeding program.

The first component of the research undertook field surveys in the Punjab province of Pakistan to establish virus diversity of the cotton leaf curl virus and to identify the overwintering host and whitefly biotypes. As well, field surveys in Queensland, Northern Territory and the Ord River area of Western Australia established the extent of geminivirus spread in Australia.

The second component then focused on establishing viral genome characteristics of the cotton leaf curl virus in Pakistan and tomato leaf curl virus in Australia. The information generated formed the basis for designing synthetic gene constructs.

The final focus was on developing virus-resistant cultivars - of cotton in Pakistan and of tomato in Australia. Transgenic plant lines produced were assessed for their resistance, and the desirable plant lines selected for field evaluation in a later project.

The project, initially planned for 3 years, was extended for a further 18 months to enable completion of the research.

Between 2002 and 2006 field surveys and complementary work on virus characterisation found that cotton leaf curl disease (CLCuD) in Pakistan can be caused by several different viruses - including cotton leaf curl Alabad virus, cotton leaf curl Multan virus (CLCuMV), cotton leaf curl Khokhran virus (CLCuKV) or papaya leaf curl virus in combination with a satellite DNA. The DNA is responsible for the observed disease symptoms. CLCuD is a major constraint to cotton production, with a resistance-breaking strain (the Burewala strain) appearing during the course of the project and spreading through the Punjab. Similarly, virus disease complexes were identified from leaf curl diseases of tomato, chilli, cucurbits, okra and papaya, as well as the yellow vein disease of the weed Ageratum conyzoides in Pakistan (which acts a reservoir of infection for cotton crops).

The function of C1 protein in virus movement was investigated. Tomato leaf curl New Delhi virus is a bipartite begomovirus in which both DNA A and DNA B are required for infection. DNA B is required for viral spread in plants. The project team demonstrated that DNA can substitute the DNA B component in infection. Given the presence of a large number of DNA satellites and their promiscuity for helper viruses, then the ability of DNA to substitute for DNA B raises the probability that new diseases such as cotton leaf curl may emerge from bipartite begomoviruses. The DNA associated with begomovirus diseases of cotton, tomato and chilli in Pakistan was not detected in Australian surveys of tomato crops. If it does appear, the threat to tomato, chilli, cotton and other crops in Australia would increase significantly.

In parallel with virus surveys, studies of viral genome and satellite DNA characterisation effects on cotton strains have enhanced understanding of strain variability, pathogenicity and the regulation of symptom development, and underpinned the development of virus-specific (targeting DNA A) and DNA-targeting strategies for transgenic resistance. Transgenic resistance strategies have been developed and trialled in cotton. Transgenic cotton lines containing gene constructs targeting replication genes of cotton leaf curl Alabad virus showed good resistance against viral infection in glasshouse tests and have subsequently shown promising results in field trials in CLCuD hot spots. Best performing selections are undergoing further evaluation with release of transgenic lines to farmers feasible within five years. Alongside the molecular approaches to disease management, a cotton genotype (NIBGE-115) that was found to be resistant to the resistance-breaking Burewala strain of CLCuD in Pakistan is being used in the National Breeding Program. Release of resistant lines to farmers in Pakistan is likely within three years.

In parallel with the cotton work, understanding of the tomato leaf curl disease and its spread has been enhanced in Pakistan, at AVRDC (The World Vegetable Centre) and in Australia, while chilli leaf curl studies have been restricted to Pakistan and AVRDC. AVRDC has led broader tomato and chilli work across the region, and Pakistan is now well placed to capitalise on the project findings and AVRDC germplasm/breeding lines to seek natural and transgenic resistance in both crops. Parallel work funded by Horticulture Australia has identified a natural source of resistance to tomato leaf curl virus.