DANIEL McALPINE MEMORIAL LECTURE 1993

Plant viruses, viroids and virologists of Australasia

 J. W. Randles

Department of Crop Protection, Waite Agricultural Research Institute, Glen Osmond, South Australia 5064

 Introduction 

Daniel McAlpine’s contribution as the ‘father of plant pathology in Australia’ was ‘to attend to any disease that might form the subject of inquiry’ – a definition of the role of a plant pathologist which is still relevant. His activities in Australia started from his appointment as Lecturer in Biology in Ormond College of Melbourne University in 1885, but his important activities as a plant pathologist commenced when he became vegetable pathologist of Victoria in 1890. His most notable contributions were to study wheat rust following the 1889 epidemic, to classify and describe Australian smuts, and to recognise Ophiobolus graminis (now Gaeumannomyces graminis) as the cause of wheat take-all. He also collaborated with Farrer on resistance to rust in wheat. It has been written that he did a difficult pioneering job ‘pushing down deeply the roots of plant pathology in his adopted country and preparing the way for Australian plant pathologists of the future’ (Fish 1976). Plant pathologists continue to provide an invaluable service to Australia and its neighbours.

In this lecture I will attempt to present an overview of the sub-microscopic agents of plant disease which have emerged as a fascinating group of pathogens during the 61 year period since McAlpine’s death. I will summarise the discipline of plant virology, outline aspects of work specific to Australia, and discuss some of the exciting new developments which are arising from an understanding of virus genome organisation. I will make brief mention of viroids in this topic as they share some of the biological properties of viruses (limited literature citations only are included because of the breadth of the topic).

Plant virology as a discipline

The cycle of infection Plant viruses are mutable, intracellular nucleoprotein parasites of the plant’s translation system. The mode of parasitism of viroids is poorly understood, but in contrast to viruses they appear to be parasites of the plant’s transcription system. Viruses replicate and move within the symplast of the plant, and move between plants either passively or via vectors. Their infection cycle outside the plant (Figure 1) is simple compared with the extracellular pathogens because they have an inert phase outside susceptible cells. Thus, the movement, survival, pre-penetration and penetration phases of the classical infection cycle of cellular pathogens are simple, passive, or non-existent for viruses. In contrast, their cycle within the plant is complex, and involves the interplay of the genome of the virus with the nucleic acid and protein metabolism of host cells and tissue.

Importance After the fungi, viruses are regarded as the second most important plant pathogens world-wide. Viruses cause diseases such as citrus tristeza, rice tungro, cocoa swollen shoot, and barley yellow dwarf, all of which result in great absolute losses in certain areas. More subtle losses occur when plants are latently infected with one or more viruses. Thus, even in the absence of symptoms, significant reductions in yield and quality occur. ‘Running out’ of potato seed is one of the earliest examples of the dramatic, but subtle effects of viruses on crop production. Synergistic interactions arising from mixed virus infections have been reported in fruit trees (e.g. cherries), vegetables and ornamental crops.

Tools of trade Virologists require expertise in a range of biological and technical skills, and access to a wide range of facilities. Isolation, purification, characterisation and diagnosis of viruses require access to glasshouses, ultracentrifuges, a transmission electron microscope, serological and molecular facilities. Thus, laboratories are probably best set up in association with research centres and their libraries. Virus diseases, however, generally occur at sites remote from research facilities, and methods of study appropriate for field work often need to be developed. For example, simple and sensitive serological techniques such as the dot-immunobinding assay (DIBA) can replace ELISA (Ligat et al. 1991). Non-radioactive nucleic acid probing techniques (Randles et al. 1992) can now be used in simple laboratories if their particular advantages of specificity and sensitivity are required.

Identification and classification The classification and nomenclature system for viruses is currently in evolution (Francki et al. 1991). The principal criteria used for classification are particle morphology and properties of the nucleic acid genome. A family–genus–species structure is now in place for plant viruses, with around 400 described viruses being placed in ca. 35 genera. Some viruses can also be placed in one of the three established families which include members from both animal and plant hosts. Nevertheless, as most virologists use biological and serological methods for identification of viruses, a level of expertise beyond that offered by the classification system has to be developed by virus pathologists.

Development of control strategies Plant tissue can be cured of virus infection only by heat therapy and/or meristem culture. Control of the infection of plants by viruses is generally indirect and depends on determining the biological properties of the virus and interfering with its spread by ecological methods. A wide range of novel techniques has been developed for such ‘biological’ control (Matthews 1991). Genetic resistance is available for a few viruses, such as tomato mosaic virus, but the mutability of viruses generally requires the use of multiple major genes for resistance to be durable. Current efforts to produce transgenic plants with selected virus genes to confer resistance to the homologous virus have shown that the approach is effective at the experimental level (Wilson 1993). The gene delivery systems are most commonly based on plant transformation with the Agrobacterium T-DNA, and this requires plant species which are easily regenerated from callus to embryo. This work is slow and expensive, with each plant-virus system having to be developed separately. Commercial enterprise and industry funded research and development organisations are supporting research on these plant transformation systems, and we can expect to see the results of extensive field testing in the next few years.

The role of the virologist Numerous challenges face the virologist in the development and application of control measures. They need to identify and distinguish virus diseases from other disorders by a range of diagnostic techniques. They require skills in transmitting viruses. Loss assessment generally requires knowledge of agronomy and plant physiology. Numeracy is needed for epidemiology and forecasting. As control practices are frequently based on avoidance of infection, an appreciation of virus ecosystems is needed, perhaps together with an ability to integrate control with farm practice. Resistance breeding and virus-gene-mediated resistance require skills in plant breeding and molecular biology, respectively. Difficulties arise from the poor availability of resistance genes in host plants, the mutability of viruses, and inadequate information on virus ecosystems with the consequent inability to forecast epidemics. With this scenario, the molecular biology approach is a particularly attractive addition to the virologist’s repertoire.

The development of virology in Australia The agents of virus diseases were first shown to be sub-bacterial in 1892, and their molecular nature was first demonstrated in 1934, when tobacco mosaic virus (TMV) was crystallised. The nature of viruses was unknown to McAlpine although he was aware of passionfruit woodiness disease which was apparently the first virus disease reported in Australia (Cobb 1901).

Virologists G.G. Samuel, in the first research paper on viruses in Australia, described the movement of TMV in tomato. He became Head of Plant Pathology at the Waite Agricultural Research Institute where he worked from 1922 to 1934. R.J. Best A.O. was also at the Waite Institute from 1927 to 1968. He purified TMV concurrently and independently of the Nobel Laureate W.M. Stanley, and devoted most of the latter part of his career to tomato spotted wilt virus. Table 1 lists the specialist plant virologists who have been active in research in Australia and New Zealand (also see Fenner 1990).

Viruses Some of the most important viruses in Australia are listed in Table 2. Four viruses which cause severe diseases are known to occur only in Australia (Büchen-Osmond et al. 1988) and/or New Zealand and may, therefore, be native to Australia. Of these, passionfruit woodiness virus (PWV) and lettuce necrotic yellows virus (LNYV) occur in native hosts which could be their original hosts. Chloris striate mosaic and subterranean clover stunt viruses both occur in introduced species and no putative native host is known.

The majority are introduced viruses of exotic plant species. It is probable that many were brought in with the original plant propagules but there are also examples of new viruses and strains entering recently (e.g. papaya ringspot virus-type P, Thomas and Dodman 1993; broad bean stain virus, Moghal and Francki 1974; alfalfa mosaic virus, Garran and Gibbs 1982; and pea seed-borne mosaic virus, Ligat et al. 1991). Thus, despite continued application of plant quarantine, regular new introductions of plant viruses occur in Australia.

Control measures for viruses in Australia are generally adaptations of those described elsewhere. In particular, pathogen testing (PT) schemes developed during the 1960s have established foundation material of known virus status, and have contributed greatly to the health and productivity of fruit crops (e.g. citrus, stone, pome, berry and vine crops) and potatoes. Virus control generally contributes more to improved yield and quality than breeding for improvement, yet some breeders still need to be convinced that only virus- or viroid-free germplasm should be used in breeding and selection programs.

A number of other viruses have been found only in Australia (Table 3). They are sufficiently different from type members of current virus taxonomic groups to indicate that they have evolved in Australia. For example, Nicotiana velutina mosaic virus is seedborne at a rate of 50–70% in Nicotiana spp. This possibly explains its survival in an annual species distributed only in the arid zone of South Australia where insects or other potential vectors could be expected to be rare.

Economic effects Virus pathogens are known for almost all exotic crops grown in Australia. For example, severe epidemics are caused in lettuce by LNYV (Stubbs and Grogan 1963), in lupins by cucumber mosaic virus (CMV; Alberts et al. 1985), in bananas by banana bunchy top virus (BBTV; Dale 1987) and in cereals by barley yellow dwarf virus (BYDV; Henry et al. 1992). Viruses of pasture species are more difficult to evaluate economically. Alfalfa mosaic virus (AMV) is probably the most prevalent virus of pasture legumes. In South Australia it reduces annual medic dry matter yield by 30–60%, as well as reducing N fixation, with consequences for overall pasture productivity (Dall et al. 1989). Citrus tristeza virus is aphid-borne and probably ubiquitous in citrus orchards, but apart from direct effects on grapefruit quality, its other effects on yield and quality are not known.

The effects of viruses range from death of the host to minor reductions in yield. Losses arise from replacement costs, lost time, costs of control, reduced quality and reduced yield. Loss assessment is not a glamorous activity, and it is both difficult and time-consuming. However, it is important, as one of the most frequently asked questions put to virologists is ‘what losses are caused by this virus (or viroid)?’

Equipping for research At a time when our research base in Australia is being whittled away, it is worth remembering that during the early 1960s, L.L. Stubbs called for each State to develop minimum facilities for virus research; glasshouse, ultracentrifuge, spectrophotometer and electron microscope. This was largely achieved and has allowed Australian virologists to develop an independent approach to their research problems. We still must endeavour to develop and maintain modern research laboratories, as it is vital to preserve our research base for studying viruses in the Australasian-Pacific region. Viruses and their genes are still waiting to be discovered, and some may provide future advantages for the genetic manipulation of plants.

Tales of the unexpected Australia and its neighbouring regions offer a unique botanical and geographical area for research. In this section I wish to describe some of the intriguing features of viruses and viroids which have been revealed in the course of my research with colleagues and students in the Australia-Pacific-South East Asian region. This segment also can be taken as a plea to preserve a well-equipped research base and a stimulating environment for curiosity-guided study by students and scientists alike in Australia.

1 Nicotiana velutina mosaic virus (NVMV) (Table 4) As already mentioned, this Australian virus has a high rate of seed transmission which may confer a survival advantage. It also defies classification into any of the known virus groups. Its genome organisation could not be determined by virus purification and infectivity assay because the particles are fragile, and numerous RNA components can be isolated which are probably only breakdown products of the virus genome. Cloning of the virus RNA allowed mapping of the genome, and provided probes which showed that the genome has two RNAs, of about 8000 and 3000 nucleotides, respectively. Sequencing of the 3’ end of the 3 kilobase RNA showed an arrangement of open reading frames similar to, but different from, other rod-shaped viruses from other parts of the world (Randles and Rohde 1990). Another aspect of interest would be the identification of the virus gene responsible for seed transmission. Such a gene could have application in molecular biology. Moreover, if RNA viruses are ever used as expression vectors, seed transmission could allow vertical transmission of exogenous genes inserted in the virus genome.

2 Velvet tobacco mottle virus (VTMoV) (Table 5) This icosahedral virus was first isolated from the arid zone of South Australia in the same host species as NVMV. It was the first virus found to have a circular satellite RNA and the satellite is so similar in size and structure to viroids that it was thought originally to be a viroid in evolution. Sequencing has since shown that it is unlike viroids in primary structure, and it is also incapable of independent replication. Similar satellites were found in Solanum nodiflorum mottle, subterranean clover mottle, and lucerne transient streak viruses. These satellite RNAs are used as models for specific self-cleavage of RNA, and have led to the synthesis of tailor-made ribozymes which are being developed as ‘gene shears’. A further intriguing aspect of VTMoV is that it is the only virus known to be transmitted by mirids. Moreover, the mode of transmission has features of non-persistent, semi-persistent and persistent transmission. An acquisition-regurgitation or defecation-probing model is the favoured hypothesis for the observed mode of transmission (Gibb and Randles 1991).

3 Cucumber mosaic virus strain B_SA (Table 6) A series of serious but spasmodic epidemics of CMV in lupins commenced in South Australia in 1983. The CMV isolated from these plants had an unusually high rate of seed-transmission. It seems likely that this high rate was the main engine driving the epidemics as a high primary incidence of CMV was found in crops. A seed-testing scheme developed in South Australia and Western Australia for use by growers has greatly reduced the risk of future epidemics. Good and rapid liaison between virologists in both states led to CMV being controlled in breeders lines, thus ensuring that newly released cultivars are not also a source of this highly pathogenic virus. The CMV-lupin system has been a good model for studying virus epidemics in field crops (Geering 1992).

4 Pea seed-borne mosaic virus (PSbMV) (Table 7) PSbMV has reached incidences as high as 90% in breeding lines around the world over the last 20 years. In the USA, a major effort has been made to eliminate the virus from the pea breeding program (Hampton et al. 1993). There has been some doubt about the seriousness of the virus, as reports of symptomless infections counter evidence that the virus induces severe disease in plants and major yield losses of pea seed. PSbMV was found by D. Cartwright in South Australia in about 1985. A study of the isolates of this virus by J. Ligat led to the development of an improved dot immuno-binding assay for serological detection of the virus in small tissue samples (Ligat et al. 1991). In the pea cv. Dundale, chosen for its high sensitivity, Ligat noted that in the third sequential generation raised from the seed of a symptomatic second generation, many of the plants were symptomless. Symptomless plants also did not have detectable antigen, yet the seeds were positive for virus by DIBA. He maintained four strains through five generations, and showed that the virus was vertically transmitted at 90–100% in the seed right through, yet the plants remained symptomless and antigen was undetectable in all leaves (Ligat and Randles 1993). The plants were also partially protected against reinfection by inoculation. Virus was detected by DIBA and infectivity in immature and mature seed, and the pod. This showed that PSbMV can be passed vertically through plants which are subliminally infected, to repeated generations of seed in which the virus reaches high concentrations.

These results could explain why breeders can obtain high levels of virus in seed without seeing symptoms on the plants. As a corollary, disease epidemics may be the result of secondary spread of infection by aphids. More work on this is urgently required to ensure that Australian pea breeders are not perpetuating PSbMV infection in their lines. It will be necessary to test whether this phenomenon applies to other hosts of PSbMV, and to other seed-borne viruses.

5 Coconut foliar decay virus (CFDV) (Table 8) CFDV is the first plant virus to be reported to have a small, single-stranded circular DNA resembling that of the animal circoviruses, and smaller than that of the geminiviruses. It was found to be associated with a lethal disease of introduced coconut palms in Vanuatu, whereas the local palms are disease-free. Its unique features are its vector, small particle size, and virion DNA. Structurally it most closely resembles porcine circovirus, but considerable work needs to be done to fully characterise it and compare it with the similar subterranean clover stunt and banana bunchy top viruses. It has a DNA with six possible open reading frames represented in a 1291 nucleotide single-stranded circle (Rohde et al.1990). In addition, part of the DNA has promoter activity which can be used to express a reporter gene in the phloem of tobacco (W. Rohde and J.W.Randles, unpublished). Such a promoter has potential value for plant molecular biology.

6 Coconut cadang-cadang viroid (CCCVd) (Table 9) You may be aware of my long-term interest in cadang-cadang disease of coconut palm in the Philippines. This has been revived recently by the discovery that about half of the coconut palms in about 28 tropical countries contain nucleic acids related by sequence homology and size to CCCVd. No epidemics of cadang-cadang have been reported from these countries, but most of the countries concerned realise that germplasm movement should occur only after an indexing scheme for viroids is put in place. Breeders also do not wish to proceed with breeding and selection programs when they may be comparing material with or without the viroid-like sequences.

A recent sequence analysis of CCCVd cloned from palms with a very severe form of cadang-cadang disease has shown that the clones differ from CCCVd by only one or two mutations at only three sites in the molecule (Rodriguez and Randles 1993). These minor changes show that there is a significant risk that the CCCVd-like sequences found in coconuts outside the Philippines could mutate to increase pathogenicity. This is added incentive to rapidly develop safe practices for the exchange of germplasm of the ‘tree of life’.

The new era  When presenting the 4th McAlpine lecture, Matthews (1983) correctly predicted ‘that the next decade will see new ideas and techniques of molecular biology and molecular genetics successfully applied for the benefit of man in the control of plant disease’. The tools of molecular biology are indeed allowing us to develop a precise understanding of the genomes of plant viruses and to use this information for crop protection.

Recent advances in control Molecular techniques are now allowing viruses to be turned against themselves, with their isolated genes being incorporated into plants to achieve transgenic resistance. This, and acquired resistance are discussed here.

Transgenic resistance The ability to introduce specific virus genes into plants offers the most direct means of control available. The coat protein genes of around 14 viruses, including tobacco mosaic virus, alfalfa mosaic virus, cucumber mosaic virus, potato virus X, potato virus Y, and potato leafroll virus, confer specific resistance to virus infection in plants in which they are inserted, and in which they are expressed (Sturtevant and Beachy 1993). The effectiveness of the resistance is generally strain specific. In addition, there are recent examples of the use of incomplete replicase genes for protecting against viruses such as tobacco mosaic virus and cucumber mosaic virus (Anderson et al. 1992).

Since the first reports of coat-protein-mediated resistance to viruses (Powell et al. 1986) there has been an explosion in efforts to develop methods for producing transgenic resistance to viruses in a range of host species. They include strategies such as the use of satellite RNA, antisense RNA, ribozymes, suicide genes, and antibody expression in plants (Wilson 1993). Many questions remain to be answered, and field assessment will provide the final test of durability and effectiveness. Both plant pathologists and environmentalists have a vested interest in these developments. Pathologists will be interested in crop performance, and will need to monitor the appearance of new virus strains which may overcome the resistance. Environmentalists will probably debate the risks of releasing transformed plants containing either the antibiotic resistance or herbicide resistance genes. The next five years will see a surge in these activities in plant virology.

Acquired resistance Certain chemicals and pathogens can induce an effect termed systemic acquired resistance in plants which are later challenge inoculated with a virus. This effect has formerly been something of a curiosity without examples of practical application. However, it has been shown by W. Wahyuni that infection with the beneficial symbiont, Rhizobium, can greatly reduce the susceptibility of barrel medic to infection with CMV(Wahyuni and Randles 1993). The effect should be studied further, particularly in relation to the time of nodulation, and the range of viruses affected. It will be important to determine whether the beneficial effects from Rhizobium due to N fixation will be complemented by its induction of resistance to viruses.

Viruses as benefactors DNA plant viruses contain promoters which are responsible for initiating and controlling the transcription of their DNA to mRNA by viral replicase. The most valuable promoter so far discovered is the cauliflower mosaic virus 35S promoter which is now an essential component of plant expression vectors containing inserted genes. The promoter is placed directly upstream of the gene to be expressed. Geminiviruses and plant viruses allied to the tentatively named circoviruses also have promoter activity in their genomes which have potential use for controlling the level and site of expression of introduced genes in plants. Thus, as mentioned previously, promoter activity in coconut foliar decay virus is phloem specific in tobacco. Such promoters can be expected to play an important role in advancing plant molecular biology.

The future The current literature shows that molecular virology is entering a rapid growth phase, whereas economic virology is moving ahead more slowly. Both have to move together to realise the benefits of one to the other. Of course, the trend towards molecular virology also reflects the curiosity engendered by the availability of new tools to scientists who can attract research funds from non-industrial sources.

As plant pathologists, we have to work not only with the specifics of virus, vector, host and environment, but we also have to address broader issues such as the viability and long term welfare of particular industries, and the most efficient ways to service them; the relative importance of viruses in relation to other constraints on production; and the integration of crop protection practices with farming systems.

It is to be expected that molecular virology will provide a better understanding of the virus life cycle in the plant, and will probably also provide through transgenic plants a ‘magic bullet’ solution to the control of viruses. However, given the number of viruses and crops, biological control measures will remain as the basic strategy for many years. ‘Traditional’ plant virology still has a vital role, but it must continue to evolve through the application of new knowledge.

More than a century after Daniel McAlpine commenced his work as a plant pathologist in Australia, the challenge is still ‘to attend to any disease that might form the subject of inquiry’. Whereas the spectrum of pathogens and the tools available have changed immensely in 100 years, plant pathologists still need the training and the motivation to act appropriately.

Acknowledgements

Many have contributed to the results and ideas presented here. I particularly wish to thank the following: D. Coleman, C. Davies, R. Francki, D. Graddon, A. Geering, K.S. Gibb, M.R. Hajimorad, D. Hanold, B. Ingham, K. Jayasena, J.S. Ligat, D.C. Miller, J.P. Morin, M.J.B. Rodriguez, W. Rohde and W. Wahyuni.

References

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Figure 1 The infection cycle of plant viruses outside the plant.

Table 1 Major contributors to plant virus research in Australasia, 1922–1993 

*Currently active

Table 2 The major pathogenic viruses in Australia

^ACoat protein mediated resistance under test.

Table 3 Viruses found in Australia, and not reported outside Australia (also see Table 2)

Cardamine yellow mosaic Tymovirus

Cassia yellow blotch Bromovirus

Chara australis virus

Glycine mosaic Comovirus

Glycine mottle Carmovirus

Kennedya yellow mosaic Tymovirus

Lucerne Australian latent Nepovirus

Nicotiana velutina mosaic virus

Paspalum mosaic Geminivirus

Solanum nodiflorum mottle Sobemovirus

Subterranean clover mottle Sobemovirus

Velvet tobacco mottle Sobemovirus

Table 4 Nicotiana velutina mosaic virus

Table 5 Velvet tobacco mottle virus

Table 6 Cucumber mosaic virus strain B_SA

Table 7 Pea seed-borne mosaic virus

Table 8 Coconut foliar decay virus

Table 9 Coconut cadang-cadang viroid