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Virological Aspects to an Integrated Management of Vector-Borne Virus Diseases of Citrus
Stephen M. Garnsey
U.S. Horticultural Research Laboratory,
ARS, USDA, 2120 Camden Rd., Orlando, Florida, U.S.A., 2002-11-01

Abstract

Citrus production is affected by numerous pests and pathogens. This paper discusses the citrus viruses that are known or suspected to be transmitted by vectors. The most economically important of these is citrus tristeza, transmitted by aphids. The range of variability of viruses strains is discussed, and the relationships between virus and plant hosts. The final section reviews control methods such as improved genetic resistance to virus which can be combined into an integrated management strategy.

Abstracts in Other Languages: 中文(961), 日本語(1219), 한국어(1089)

Introduction

Citrus is a major fruit crop in numerous countries, and is grown for both domestic consumption and for export. Citrus production is affected by numerous pests and pathogens which must be avoided or controlled to maintain a successful industry. A number of graft-transmissible pathogens affect citrus. These include at least eight groups of viruses, five groups of viroids, four types of prokaryotes, and a number of uncharacterized viruslike and decline-inducing agents. Several viruses and all of the prokaryotic agents are vector-borne. The natural spread of other viruses by unknown means has also been reported.

Preventative measures, such as quarantine and the use of virus-free propagating stock, have been used to control some citrus virus diseases, and tolerant or resistant cultivars are used to manage others. However, preventative steps alone do not adequately control vector-borne diseases, and adequate sources of host resistance or tolerance are not available for several important diseases. Moreover, large plantings of susceptible plants exist which must be protected and conserved where possible.

The need for a more integrated approach to controlling citrus virus diseases has been recognized. This has promoted increased efforts to develop the necessary information on biological and molecular characteristics of citrus viruses, and also better detection methods, a better knowledge of virus/host interactions, and an improved knowledge of citrus virus epidemiology.

The purpose of this paper is to provide an overview of the citrus viruses that are known or suspected to be transmitted by vectors, and information from a virology standpoint that is relevant to developing a more integrated approach to citrus virus disease management.

Virus and Viruslike Diseases of Citrus

At least eight groups of citrus viruses have now been described. These vary in distribution, economic impact and degree of characterization.

Citrus tristeza virus (CTV), an aphid-borne closterovirus, is undoubtedly the most famous and economically important citrus virus. CTV poses a continuing problem for citrus production in a large number of countries, and increasingly threatens other regions where it is still either absent or rare.

Vein enation is also aphid-borne. It is widely distributed, but causes little economic damage.

Leprosis, an apparent rhabdovirus, is vectored by a mite and is a significant problem in Brazil.

New vector-borne diseases continue to appear. A significant example is the virus-like disease citrus chlorotic dwarf, recently recognized in Turkey, which is transmitted by the bayberry whitefly ( Parabemesia myricae). Natural spread is suspected for several other viruses, but the vectors remain unconfirmed.

Satsuma dwarf is spread by an unknown, soil-borne vector. A severe form of psorosis ( citrus necrotic ringspot) is spreading in Argentina and is a significant problem there.

Relationships between several citrus viruses and viruses of other crops have been established, and it is possible that some citrus viruses may have moved into citrus from other hosts. For example, tatterleaf virus of citrus is closely related to apple stem grooving virus and a lily latent virus. While natural spread in citrus remains unconfirmed, tatter leaf is widely distributed in many Asian countries. It remains a matter of concern, because most current citrus rootstocks have genetic vulnerability to it.

Citrus viruses with known vectors and those suspected of being naturally disseminated are summarized in Table 1(1145).

Variability of Viruses

An accurate understanding of the variability and the relationships between the strains of a given virus is essential for many aspects of disease management. These include detection, the development of cross protection strategies, and the breeding or genetic engineering of new cultivars for virus resistance. Most citrus viruses have isolates or strains that vary from each other to some degree.

Differences in symptom expression are generally the first recognized indication of variation among strains or isolates of a given virus. Other properties may provide a more precise estimate of genetic relationships between viruses and virus strains, but disease-inducing capacity is the key economic property. CTV provides an excellent illustration of the complexity in disease reactions that may occur. Some isolates are very mild in all hosts. Some cause decline in trees on sour orange rootstocks, and others cause stem pitting in one or more cultivars.

Rapid progress is now being made in the molecular characterization of several citrus viruses, and several, including CTV, have been completely sequenced. Recent molecular studies on CTV have also provided information on gene organization and its expression. These have revealed the presence of defective RNA species, and indicate that the degree of sequence homology between strains differs in different areas of the viral genome.

More work is needed, however, to define relationships among strains at the molecular level, and to correlate biological and molecular properties. The development of infectious clones that can be genetically manipulated has become feasible, and provides a means of systematically identifying portions of the viral genome that control disease expression and other functions.

Accurate characterization of pure strains is often difficult. Field isolates often consist of a mixture of several strains. Even after these have been separated by vector passage, host passage, single lesion transfer etc., a single strain may still contain a population of several RNA species. A single cloning and sequencing study may not accurately depict the consensus sequence. Use of PCR has greatly speeded studies on molecular characterization of viruses and virus comparisons, but a bias also exists for amplification of the target species most homologous to the primers used and this may not be the dominate species present in the template used.

Current results with CTV suggest that the issue of strains and strain relationships is much more complex than recently envisioned, and that it will take extensive effort to clarify these relationships.

Virus-Host Relationships

Most citrus is propagated by budding or grafting a scion cultivar onto a nucellar rootstock seedling. Citrus viruses may affect the scion, the rootstock, or the bud-union between stock and scion.

Citrus plants respond to infection in a variety of ways. In a broad sense, plants can be considered either susceptible or resistant to infection. Susceptible plants may show a variety of symptoms which include foliar, fruit, and scaffold symptoms and a reduction in vigor, but frequently they are quite tolerant and show few symptoms, even though the virus replicates to a high titer. Although tolerant plants may not suffer yield loss, they are an important reservoir of inoculum and a significant problem in disease management.

Resistant citrus cultivars do not support systemic infection by a given virus. Resistance may result from a hypersensitive response when infection occurs, so that the virus fails to spread, or for other reasons. Plants are considered immune when there is no evidence of virus replication. Trifoliate orange, for example, is often considered immune to CTV, although immunity in a strict sense at the cellular level has not been demonstrated.

The virus-host interaction is complex in grafted plants and the term "disease tolerant" or "disease resistant" does not indicate the precise relationship of each component of the plant to the virus. For example, Valencia sweet orange grafted on Carrizo citrange is considered tolerant to tristeza decline. More precisely, the sweet orange scion is susceptible to infection, but tolerant, and the Carrizo rootstock is highly resistant (immune) to infection. Selective resistance to specific CTV isolates has been identified in pummelo and some pummelo hybrids.

The distribution of the virus in infected plants, and the association of the virus with specific tissues and its titer, are also important for disease management, since they affect detection of the virus, acquisition of the virus by vectors, and cross protection. Some citrus viruses, such as CTV, are restricted to the phloem. They can only be transmitted by the vector when it acquires the virus in the phloem from the donor plant and then inoculates it into susceptible cells in the receptor host. In general, most citrus viruses become well distributed in citrus hosts soon after infection occurs. Others remain irregularly distributed for long periods after infection occurs, and may remain erratically distributed throughout the life of the plant. Virus titer in citrus plants can vary remarkably over time, and is affected by temperature and the stage of growth. The net virus content present in the plant reflects the difference between the synthesis of new virus and the degradation of existing virus, and can be expected to be highest when there is extensive activity in the meristem (shoot tip).

Detection of Citrus Viruses

Access to rapid, sensitive, accurate, and inexpensive assays is important in most disease management strategies. The methods of detecting citrus viruses have continued to evolve as new information on viruses and new technology has become available, and the process is continuing.

Inoculation of citrus varieties especially reactive to certain viruses was the first step to rapid detection. This remains the only reliable method to detect some diseases, and to distinguish strains of others. Herbaceous hosts have been used to detect citrus viruses that are easily transmitted mechanically. Electron microscopy was the first detection tool based on a specific property of the virus.

As citrus viruses have been purified, serological detection techniques have been developed; ELISA is now widely used for CTV and other citrus viruses. Molecularly based probes, such as PCR and nucleic acid hybridization assays, are now being developed as information on the sequence of citrus viruses is becoming available.

Probes are needed for a general or "universal" detection of all strains or isolates of a given virus, and also to identify specific strains. Broad serological probes are generally based on the use of polyclonal antisera, or the use of mixtures of monoclonal antibodies to well-conserved epitopes. Monoclonal antibodies, which selectively recognize only specific isolates, have also been developed. The ability to express various virus genes or portions of virus genes in vectors, and to obtain proteins from these for use as immunogens, is providing a new approach to serological detection of citrus viruses.

Sequence-based probes for general and strain-specific detection are also being developed. For example, PCR is being adapted for general and selective identification of CTV by designing specific primers for different genome areas. Immunocapture PCR could also be very useful for surveys where the incidence of virus infection is low. This would be a key component in preventative management strategies.

Making practical use of these new detection technologies for disease management, however, requires extensive testing to determine how to apply the technology accurately, and comparison with biological testing. The development of appropriate controls for each type of assay is also a challenging problem.

Ecology and Epidemiology

Up till now, accurate information on the rates and patterns of virus spread has been lacking. This is expecially true for the early stages of an epidemic, where management activities might be most effective. The development of rapid serological assays, such as ELISA, have allowed widespread surveys to determine the distribution and sources of citrus viruses, and monitoring of spatial and temporal changes in virus incidence.

Data on the progress of infection early in the epidemic cycle has now been obtained for CTV. Data has now been gathered and analyzed from Spain, Florida and California, where natural spread is by the melon aphid, a moderately effective vector, and from Costa Rica, the Dominican Republic, Puerto Rico, and Taiwan, where the brown citrus aphid, a highly efficient vector, is present.

Choice of Management Strategies

The management of virus diseases of citrus involves protecting existing orchards which have genetic susceptibility to one or more vector-borne viruses, and developing new cultivars for future planting with improved virus resistance.

Host resistance is probably the single most attractive approach to control of virus diseases in long-lived horticultural crops such as citrus, where short-term measures do not give any benefit. However, the development of new citrus cultivars is difficult. The breeding cycle for citrus is usually 8-10 years. Because most citrus cultivars are highly heterozygous, the chances of combining virus resistance with all other essential horticultural features are low.

Genetic engineering offers a means of incorporating virus resistance into existing desirable cultivars. The introduction of viral genes into plant genomes to confer resistance has now been demonstrated for a number of viruses via coat protein mediated cross protection, and other genes may be even more effective. This work is already under way in citrus for CTV, and has great potential. Although transformation can be accomplished and resistance demonstrated in a relatively short period, the horticultural evaluation of transformed plants for fruit production still requires many years. Genetic engineering does not dramatically shorten the time frame for genetic improvement, but greatly increases the options. The genetic variability of individual viruses is an important consideration when choosing sources of genes, and when making evaluations of resistance once transformation is achieved.

While genetic improvement offers good prospects for future plantings, it does not address disease management in existing ones. However, several other approaches are available. Prevention and avoidance are basic components for the management of citrus viruses. Many of the current virus disease problems in citrus could have been prevented by eliminating the distribution and propagation of infected propagating materials, and by wiser use and movement of germplasm. Shoot-tip grafting is a powerful tool to free existing cultivars of virus infection. Continued improvement in indexing procedures also makes verification of virus freedom much easier, and augments other regulatory approaches to control. Even once a virus is well established, selective certification or regulation to curtail movement of the more severe isolates can be an effective component of management. Suppression of inoculum sources is also useful in some situations.

Mild strain cross protection has been used successfully in several locations to reduce losses from CTV stem pitting, but has failed in others. The difficulty involved in finding isolates which are both mild and protective and then showing that these are not harmful to other cultivars or crops has limited the wider application of mild strain cross protection. Our inability to predict or control all potential hazards have also precluded the preventative use of cross protection.

Recent studies with CTV indicate that the successful application of mild strain cross protection will be more complex than originally believed. At the same time, as our knowledge of citrus viruses increases at the molecular level, the options to engineer isolates with desirable properties, and to eliminate liabilities such as vector trans-missibility, are rapidly increasing. The development of "designed" protective isolates could provide creative solutions for a number of citrus virus problems.

Conclusion

Vector-borne citrus viruses will be a steadily increasing problem in citrus production. Solutions to existing problems will be more complex than we thought, and new problems are steadily arising. Conventional approaches used in the past will not meet anticipated needs. Genetic improvement can address some problems in the future, but will not protect existing orchards, and may not be fully effective unless also incorporated with other management strategies. The rapid recent progress in the characterization of citrus viruses and access to new technologies creates new opportunities to combine several approaches into an integrated management strategy.

References

  • Bar-Joseph, M., Marcus, R., and Lee, R.f. 1989. The continuous challenge of citrus tristeza virus control. Ann. Rev. Phytopath. 27: 291-316.
  • Gonsalves, D., Garnsey, S.M. 1989. Cross-protection techniques for control of plant virus diseases in the tropics. Plant Disease 73: 592-597.
  • Korkmas, S.A., Cinar, A., and Kersting, U. 1995. Citrus Chlorotic Dwarf: A new whitefly-transmitted viruslike disease of citrus in Turkey. Plant Disease 79: 1074.
  • Lee, R.F., Baker, P.S., and Rocha-Pena, M.A. 1994. The Citrus Tristeza Virus (CTV). Intern. Inst. of Biol. Control, Ascot, Berks, UK. 197 pp.
  • Navarro, L. 1993. Citrus sanitation, quarantine and certification programs. In: P. Moreno, J.V. da Graca, and L.W. Timmer, (eds.). Proc. 12th Conf. Intern. Organ. Citrus Virol., 10CV, Riverside, pp. 383-391.
  • Pappu, H.R., Niblett, C.L., and Lee, R.F. 1995. Application of recombinant DNA technology to plant protection: Molecular approaches to engineering virus resistance in crop plants. World Jour. MicroBiol. and Biotechn. 11: 426-437.
  • Rolstacvher, C.N. 1991. Graft-transmissible Diseases of Citrus: Handbook for Detection and Diagnosis. FAO, Rome. 286 pp.
  • Whiteside, J.O., Garnsey, S.M., and Timmer, L.W. 1988. Compendium of Citrus Diseases. APS Press, St. Paul, MN. 80 pp.

Discussion

Dr. Garnsey was asked whether a mild strain used for cross protection may sometimes be a severe strain in other varieties. Dr. Garnsey agreed that this could be the case, and emphasized that the identification of mild strains is host specific. Furthermore, the term "mild is a relative one - what is considered a severe strain of the disease in California might be considered a mild strain by Australian standards. He suggested that the best solution would be to design a mild isolate from which the transmissibility had been omitted. Otherwise a mild strain has to be tested with all varieties, and this is in practice very difficult.

One participant was interested in what factors are related to transmissibility. Dr. Garnsey explained that research is now being carried out in Florida on the molecular characteristics, but we still do not know where transmissibility is controlled in the genetic structure, or whether it is a helper type component. Interest was expressed in the recent discovery of citrus chlorotic dwarf (CCD) virus in Turkey, and Dr. Garnsey was asked whether the virus recorded is a geminivirus. Dr. Garnsey thought it was not, and explained that he had been unable to get hybridization with virus probes. The virus cannot be transmitted by leaf inoculation, although it can be transmitted by stem dashing.

International Seminar on the

MANAGEMENT OF INSECT-BORNE VIRUS AND VIRUS-LIKE DISEASES OF FRUIT TREES IN THE ASIAN AND PACIFIC REGION

Held at the Taiwan Banana Research Institute, December 10-17

Final Discussion

Summary of Closing Remarks by DR. Stephen M. Garnsey

It is clear that the insect-borne virus diseases of fruit are likely to continue. Diseases such as citrus tristeza, greening and papaya ringspot have been damaging the region's production for several decades, and new diseases are appearing. These problems are not going to be easy to solve.

Variability of plant viruses

Recent developments in the laboratory have greatly improved our ability to identify and characterize plant viruses. As our ability to detect and describe viruses has increased, so has the complexity of the picture. Plants which show severe symptoms of virus disease often do not have a single type of virus, but multiple infections with several different viruses.

We are finding a great deal of variability in viruses such as Papaya ringspot virus. This makes it more difficult to develop reliable indexing methods for virus identification and disease control. In order to certify planting material as virus free, we have to be sure that we are able to detect all strains of the virus. Similarly, any control measures must take into account this variability of strains, and their different patterns of symptom expression.

Need for epidemiological information

In the absence of adequate pest resistance, the control of insect-borne virus diseases often depends on avoiding or controlling the insect vector. People are already using epidemiological information to good effect. For example, planting dates of crops such as papaya, passionfruit and banana in Taiwan have been adapted so that the young seedlings are planted at a time when populations of the insect vector are low.

However in the case of greening and other virus and virus-like diseases, little is known about the range of vector action or the sources of inoculum. How do these pathogens spread? To control the spread of the pathogen, it is essential to understand how the pathogen spreads, not only over time but also spatially. Study of the spatial and temporal spread of virus pathogens should include long-distance movements. Most studies of virus transmission are carried out in small localized areas, but we also need to understand disease spread on a national, regional and global level. This may need a different structure of research to gather data, involving large multinational teams working in coordination.

Need for entomologists

Most participants at this conference on plant virus diseases vectored by insects were virologists and plant pathologists. However, an important key to controlling these diseases is understanding the insect vectors. It would be a good idea for more entomologists also to be present at these conferences, and work together with plant pathologists in developing control measures.

Management and prevention of virus diseases

"The key to management is prevention". Every country can identify highly destructive pathogens that exist elsewhere. The best way to control such pathogens is to avoid them, by maintaining strict precautions to prevent their entry. Unfortunately, it is difficult to find funding for preventative pathology. Funding is easier to find when a new disease problem appears, but by then it is too late.

Virus free planting materials

The use of virus free planting materials is a key component in the control of insect borne virus diseases. Many successful programs have been developed to propagate seedlings which are free of virus for distribution to farmers. The use of virus-free motherstock, and techniques such as heat treatment and meristem culture, are effective in producing planting materials which are free of virus disease. However, there is less understanding of how to keep them free of virus after they are planted.

In development programs, often a lot of money is spent in developing virus free material, but these programs tend to be short-term, and funds are not set aside to continue this work after the development program ends. Any program related to virus free germplasm needs permanent funding. Otherwise, a lot of money is wasted in setting up a system to produce virus free germplasm, which is not utilized after the program and its funding come to an end.

Extending information to farmers

Scientists have the tendency to regard their work as over when a scientific question has been answered. In fact, this is only the first step: the information must then be given to growers so that they can make use of it. I was impressed with the work of Dr. C.A. Chang of Taiwan on passionfruit diseases. Dr. Chang used his scientific expertise to find what could be done to control passionfruit virus diseases, and then worked with farmers to help them apply these methods in the field. The result was a highly successful control program.

While virus detection has made great progress, and can be a very powerful and useful tool in the hands of those who know how to use it, it can give very misleading results if misapplied or badly used. Those who have developed these methods have often not made enough effort to see that they are put to practical use by people who are aware of the difficulties and limitations of the technology they are using. For example, if PCR is not used properly it can give either false negative or false positive results, either of which might be disastrous if used as the basis for a virus control program.

Host resistance

In many cases, it is difficult to use conventional breeding methods to develop resistance to virus diseases. Many techniques have been used to get around this difficulty, especially for banana. Molecular biology can genetically transform plants in ways which were impossible only five years ago. I was interested in the recent work in Taiwan to create transgenic papaya plants, which contain genes from papaya ringspot virus and are resistant to infection by papaya ringspot virus in the field.

8. Promotion of biological control

At an international level, there has been a reduction in funds for biological pest and disease control, while at the same time we are being called on to make less use of chemicals, and replace them with biological inputs. How can this be done? We tend to blame politicians for this situation, but I feel that we scientists are partly to blame for not being more involved in educating the public and politicians on the needs and benefits of agricultural research.

Index of Images

  • Table 1 Summary of Citrus Viruses

    Table 1 Summary of Citrus Viruses

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