ESA Magazine October 2002

By Dagmar Hanold, Mike Stukely & John Randles

Extract of the article 'Mundulla Yellows - a new tree die-back threat'. Reprinted with permission of Landscope, Winter 2002, Department of Conservation and Land Management, WA.

Buckingham Old Coach Road, 1999, with tree death due to Mundulla Yellows. Photo courtesy The Border Chronicle. Insert: Top leaf showing interveinal yellowing compared with a healthy one. Photo, Dagmar Hanold.

A little-known and only recently described disease, Mundulla Yellows, has the potential to seriously affect a number our native plant species, as well as revegetation plantings on farms and possibly some eucalypt plantations. Mundulla Yellows is a progressive slow dieback and yellowing disease of many varieties of eucalypts, now suspected of being caused by a virus-like agent. It has been reported in trees of all ages. Once symptoms appear, the affected trees do not recover, and die within a few years.

Where is it?

Mundulla Yellows was first reported in the late 1970s in the south-east of South Australia, by bee-keeper Geoff Cotton. There is evidence that the disease also occurs in other states, including Western Australia, but typical Mundulla Yellows symptoms have not been reported outside Australia. In South Australia, Mundulla Yellows is present in scattered sites throughout an area estimated to exceed 25,000 square kilometres - and it is spreading! Many of the sites show high incidence of the disease. Mundulla Yellows is named after the town of Mundulla in South Australia. Its symptoms have been observed in a wide range of eucalypts growing in modified landscapes, as well as in remnant natural vegetation. Sheoaks (Allocasuarina sp.), banksias (Banksia sp.) and wattles (Acacia sp.) show similar symptoms, which suggests that the disease may not be confined to eucalypts. Mundulla Yellows has so far been observed mainly in sites that have undergone significant disturbance. However, it has been seen occasionally in natural forest in eastern Australia.

An Environmental Concern

Mundulla Yellows is now acknowledged as a threat of national significance to natural biodiversity and ecological sustainability. It causes the irreversible loss of natural vegetation, including ancient gum trees. Given that it appears to affect a wide range of eucalypts and other species of native flora which comprise natural Australian ecosystems, it poses potential danger to natural wildlife habitats and, thus, the conservation of threatened plant and animal species. It is also potentially detrimental to a number of industries and parties, including commercial tree growing, bee-keeping, tourism, native cut-flower nurseries, public amenity, land and forest management, and local government. It could jeopardise revegetation programs, salinity and groundwater level control, input on the international carbon credits policy, and could impact on quarantine practices. The symptoms of Mundulla Yellows differ from those of previously reported diseases of eucalypts. To distinguish Mundulla Yellows from yellowing due to other factors, symptoms have been described in A Field Guide to Mundulla Yellows by D Hanold and J W Randles (1999), published by the University of Adelaide.

Finding the Cause

When investigations into the cause of Mundulla Yellows using molecular methods began, in January 2000, a living organism was thought to be the cause. This was because affected trees occurred in mixed stands with, or immediately adjacent to, unaffected trees. If environmental factors (eg nutrient imbalances, herbicide spraying and high soil salinity) were the cause, they would be more likely to affect most trees in an area. Also, some disease symptoms had developed in previously healthy plants grafted with patches of bark taken from trees affected by Mundulla Yellows. This method is used widely by plant pathologists to see if a contagious agent is present. Unlike Phytophthora-induced dieback, there was no evidence to suggest that disease-causing organisms (eg fungi, bacteria, or nematodes) were associated with Mundulla Yellows. So, researchers began to investigate the hypothesis that Mundulla Yellows was caused by a micro-organism that could not live or replicate outside the host cells. Plant pathogens in this category can belong to the virus, viroid or phytoplasma groups. Phytoplasmas and viroids do not vary much in their structure, but viruses can be very diverse. They can vary in the structure and biochemical properties of their genome (consisting of nucleic acids, ie DNA or RNA), and the composition, size and shape of their particles (consisting of proteins). Consequently, there is a vast range of potential candidates for this investigation. Intracellular plant pathogens cannot spread independently, but need either biological vectors or mechanical means of transmission. Sap-sucking insects, nematodes, fungi, pollen, seed and plant sap carried on tools are examples identified in the past as possible modes of spread.

Breakthrough

Thanks to advances in biotechnology, there is a range of methods available for testing whether such a pathogen is responsible for Mundulla Yellows. Tissue samples from normal eucalypts were compared with those affected by Mundulla Yellows using different means. Tests were carried out that detected phytoplasmas in eucalypts; however, they were found with similar frequency in trees affected by Mundulla Yellows and healthy trees, so phytoplasmas did not appear to be a cause of Mundulla Yellows. Virus-like particles could be observed occasionally in leaf tissue by means of electron microscopy, but their role as a cause could not be confirmed by this method. As no viruses or viroids of eucalypts had previously been characterised, a molecular test for virus-like agents in eucalypt tissue first had to be developed to test trees affected by Mundulla Yellows. A breakthrough was made with this approach. Unusual nucleic acids (MY-RNAs) were found in Mundulla-Yellows-affected river redgums (E. camaldulensis) in the south-east of South Australia. Indications from the size and biochemical characteristics of these nucleic acids pointed to a possible association with a virus or viroid. MY-RNAs were detected in the leaf tissue of affected trees in South Australia before symptoms developed. This suggests that MY-RNAs are more likely to be associated with a cause rather than an effect of the disease. It also indicates that there may be a significant lag period between infection and appearance of disease symptoms. MY-RNAs have been detected in trees within revegetation sites and plantations less than a year after they were planted into areas previously free of Mundulla Yellows. They have also been found in nursery-grown seedlings in the glasshouse. This suggests that the widely used practice of raising seedlings in one area for planting elsewhere may carry a risk of spreading Mundulla Yellows.

Survey

A survey conducted in the southern Australian states in 2000-2001 detected MY-RNAs in more than 30 species of eucalypts, as well as in sheoaks (Allocaurina sp.) and bottlebrushes (Callistemon sp.), at a number of locations. However, it appears that symptoms may vary, perhaps due to environmental or host factors, or possibly due to variations in the MY-RNAs. There is preliminary evidence that insects may spread Mundulla Yellows. Field sites are being monitored for the development of symptoms and spread of MY-RNAs. Because symptoms take time to develop, and the spread of the disease appears to be slow, samples need to be collected regularly over at least five years. With the above evidence, we are now investigating the hypothesis that Mundulla Yellows is caused by a virus-like agent. MY-RNAs now need to be characterised to test their association with a potential virus-like pathogen. A sensitive, specific and fast routine test -; suitable for screening large numbers of samples from a wide range of plant species - needs to be developed. This will be essential for testing nursery material and plant stock for infected material, and to identify possible sources of genetic disease resistance for use in breeding programs. It will also be essential for investigating mechanisms of disease expression, modes of spread (such as different insect species) and other features of the disease cycle.

The Future

In the case of Phytophthora dieback in WA, it took more than 40 years from the first records of symptoms to the discovery of the cause - and, by then, huge areas of vegetation had been devastated. Groundbreaking advances in Mundulla Yellows research, during the past two years, have opened the way towards identifying the cause of this disease. Only when the disease cycle is known can specific strategies be designed to disrupt it and thus control the spread of the disease. Until more specific knowledge is available, general plant hygiene practices will help to minimise the risk, through human activity, of spreading diseases (including Mundulla Yellows) from plant to plant and, most importantly, into new areas. An integrated research program has been devised towards nationwide control of Mundulla Yellows, but funding still needs to be secured.

Dagmar Hanold is a Molecular Biologist, specialising in plant viruses and viroids of trees and John W Randles is a Plant Virologist, with a special interest in viroids and the characterisation and epidemiology of plant viruses. Both work at the Waite Institute (University of Adelaide), Department of Applied and Molecular Ecology.

Contact details (08) 8303 7307 or email: dagmar.hanold@ adelaide.edu.au Reprints available from authors. Full article on website: www.waite.adelaide.edu.au/AME/virology/research.html

Mike Stukely is a Research Scientist at CALM (Department of Conservation & Land Managment), WA.

Further reading, Diseases and Pathogens of Eucalypts, published by CSIRO (Commonwealth Scientific & Industrial Research Organisation), edited by Keane, Kile, Podger and Brown.

This article first appeared in Environment South Australia, Vol 9 No 2 - October 2002. The whole or part of this journal may be reproduced without permission provided that acknowledgement is made and provided the reproducer agrees to provide gratis a right of reply in the publication or medium in which the reproduction was published or broadcast, and in a form similar to the reproduction should the Conservation Council of SA or its agents desire to make such a reply. Views expressed in this publication are not necessarily those of the Conservationn Council of South Australia. Non-sexist and non-racist language is a policy of Environment South Australia.