Citrus tristeza virus complex

Preface Based on strictly economic criteria, Citrus tristeza virus (CTV) is often considered one of the most important plant viruses known to date. This alone makes it a prime candidate for a research program. Given the worldwide distribution of citrus, the high value of citrus crops, and the severe damage inflicted by the virus, it comes as no surprise that CTV has been studied by plant pathologists all over the world. However, in addition to these practical considerations, CTV is the largest and most complex virus among all known plant viruses. If one adds a genetically complex woody perennial citrus host to this interesting mix, it becomes clear that to solve the problem of managing CTV infections in various species of citrus grown in diverse environments all over the world, a comprehensive research approach has to be applied.

This book represents an attempt to present such a comprehensive approach, which includes research of the virus itself, its interactions with the host plant and mechanisms of disease induction in different hosts, its interactions with aphid vectors, and research of the factors involved in host resistance to the virus. The book also includes chapters describing the history of CTV, the diseases caused by the virus, and the management of the disease in different countries. CTV was one of the first plant viruses to be subjected to extensive research efforts by plant pathologists and virologists, but because of the complexity of the virus–host pathosystem, it lagged behind some of the more simple viruses as far as virus genome and virus–host interactions are concerned. However, because of this same complexity and the sophistication of the virus, many new virus features and mechanisms were first described while studying CTV. CTV turned out to be one of the most “fertile” scientific objects studied in plant virology and readers are invited to look into interesting, often unexpected, and always exciting findings that came from various studies of the virus.

This book will be of interest to plant pathologists, plant virologists, horticulturists, and graduate students in plant pathology and related sciences.

Alexander V. Karasev Mark E. Hilf Editors November 2009

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Contents Section I: Disease Description Chapter 1 The History of Citrus tristeza virus—Revisited ................................. 3 M. Bar-Joseph, O. Batuman, and C. N. Roistacher Chapter 2 Citrus Tristeza Diseases—A Worldwide Perspective .......................27 P. Moreno and S. M. Garnsey Section II: Molecular Biology of the Pathogen Chapter 3 Molecular Genetics of Citrus tristeza virus......................................53 W. O. Dawson Chapter 4 Interference or Insurance? More Questions than Answers on the Roles of the Multiple Defective RNAs of Citrus tristeza virus ....73 O. Batuman, X. Che, G. Yang, M. Mawassi, and M. Bar-Joseph Chapter 5 Identification and Characterization of Silencing Suppressors Encoded by Citrus tristeza virus....................................................95 S.-W. Ding Chapter 6 The Tail That Wags the Virus: Recombination Defines Two Gene Modules and Provides for Increased Genetic Diversity in a Narrow-Host-Range Plant Virus .......................................... 103 M. E. Hilf Chapter 7 Citrus tristeza virus and the Taxonomy of Closteroviridae................ 119 A. V. Karasev and M. Bar-Joseph

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Section III: Aphid Transmission and Epidemiology of CTV Chapter 8 Concepts in the Epidemiology of Citrus tristeza virus ..................... 133 T. R. Gottwald Chapter 9 Transmission and Spread of Citrus tristeza virus in Central California ............................................................... 151 R. K. Yokomi, M. Polek, and D. J. Gumpf Chapter 10 Nucleotide Sequence-Based Detection, Analysis, and Variation Among California Citrus tristeza virus Isolates.............................. 167 G. Roy, D. E. Ullman, and B. W. Falk Section IV: Resistance to CTV in Citrus spp.

Chapter 11 Toward Positional Cloning of the Citrus tristeza virus Resistance Gene ...................................................................... 187 T. E. Mirkov, Z.-N. Yang, M. Rai, J. J. Molina, M. L. Roose, and X.-R. Ye Chapter 12 Pathogen-Derived Resistance to Citrus tristeza virus in Transgenic Citrus Plants....................................................... 203 L. Peña, C. Fagoaga, C. López, A. Domínguez, R. Ghorbel, A. H. de Mendoza, P. Moreno, L. Navarro, and R. Flores Section V: Management of CTV—A Worldwide Experience Chapter 13 Eradication of Tristeza in the Central Valley of California ............. 219 M. Polek Chapter 14 Citrus tristeza virus Regulation in the State of Florida .................... 233 P. J. Sieburth and M. C. Kesinger Chapter 15 Managing Citrus tristeza virus Losses Using Cross Protection.......... 247 J. V. da Graça and S. P. van Vuuren

1 The History of Citrus tristeza virus— Revisited Moshe Bar-Joseph, Ozgur Batuman, and Chester N. Roistacher The S. Tolkowsky Laboratory, Department of Virology, ARO, Volcani Center, Bet Dagan, 50250 Israel Names of plant viruses are often based on the most noticeable symptom of the host plant in which the disease was first recognized (96). That is why some symptoms manifested on leaves, like mosaic, ringspot, and necrosis, are common among the names of dozens of different plant viruses. Thus, a citrus disease named “tristeza” (134), a Portuguese word that translates to “sadness”, is rather exceptional, and “Citrus tristeza virus” (CTV) stands alone among the names of hundreds of plant virus species. Although the first reference to the disease was as the Spanish “Podredumbre de las raicillas” (28,196,197) and the Portuguese “O Podridao das radicelas” (29)—literally translated as “rotting of the rootlets”—the citrus community preferred “Tristeza”, most probably because of the need to refrain from associating it with the symptoms of citrus root rot diseases resulting from fungal infections. In historical perspective, this choice seems justified as it is a sensitive descriptor of (i) the sad look of a large orange tree grafted on sour orange, with leaves lying on the ground like a carpet with the tree skeleton still loaded with hundreds of good-size fruits that will drop later and change the carpet color first to yellow and finally to black1; (ii) the sadness of growers who lose in a few weeks tens or hundreds of trees, almost ready for harvest; and (iii) the years of frustration of researchers who have had to deal with one of the most complex plant viruses.

1 The word “tristeza” apparently sounds melodically attractive to Portuguese and Spanish songwriters as one need only turn on the radio while visiting any Latin American country to hear this word repeated in almost every second hit, nearly as often as during the tristeza sessions of the international citrus virology meetings. And the Latin American songwriters and poets were not alone in incorporating some forms of tristeza into their art; as the late Nissim Aloni, one of the most famous Israeli playwrights, who as a young immigrant studied in an agricultural high school, also could not resist including in a play from the 1960s a passage in which one of the three gardeners employed by “Aunt Lisa” mentioned this terrifying wilt disease that struck suddenly and killed her large and productive orange trees.

As often happens, art preceded reality and the play Aunt Lisa became an artistic success years before CTV started to cause the decline of citrus trees in Israel.

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What Can Be New in a “Historical” Chapter?

Naturally such an important disease problem has already been addressed by several comprehensive and/or popularly written accounts on various historical aspects of the causal virus and the disease, e.g., Bar-Joseph et al. (17,18,21,26,48,69,100, 105–107,121,123,124,133,135,156,167,168,172,202), while others dealt mainly with the taxonomical properties of the virus (1,15,61,99,118).

THE GLOBAL EXPANSION OF CITRUS PRODUCTION DURING THE LAST TWO CENTURIES AND ITS PHYTOPATHOLOGICAL COSTS Placing CTV in the Context of Citrus History and Taxonomy. With annual citrus production close to 100 million tons with the fruit markets in every corner of the world replete year-round with high-quality citrus fruits, and with ships regularly carrying thousands of metric tons of concentrated orange juice from Brazil and Florida to European and Far Eastern ports, the global citrus industry today is clearly very different from just a few decades ago, and its progress apparently rivals even those industries based on the most advanced technology2. It should, however, be noticed that this prolific output is mostly based on less than a dozen cultivars (184).

These, together with hundreds of other Citrus spp. of little or no immediate commercial value but holding considerable promise for future breeding needs, are systematically grouped in three closely related genera which yield fertile crosses, namely Citrus, Fortunella and Poncirus. These genera belong to the subtribe Citrinae, tribe Citreae, subfamily Aurantioideae, of the family Rutaceae (27,92,184, 198). The classification of the hundreds of different Citrus spp. has undergone considerable changes. Tanaka (190,191) first proposed their division into 145 and later 159 Citrus species and further separated these into groups, sections and two subgenera. Swingle and Reece (187) recognized 16 species, belonging to two subgenera: Papeda (containing six species) and Citrus (formerly Eucitrus) with 10 species. A comparative systematic analysis of 145 morphological characters, affinity relationships, chemical data and breeding behavior conducted by Barrett and Rhodes (24) favored the contention that the Eucitrus group consists only of three species, i.e., Etrog citron (C. medica), pummelo (C. grandis) and mandarin (C. reticulata). Thus,

2 The late Nancy Hardy, one of the most knowledgeable writers of the Floridian growers journal Citrus Industry, started one of her articles during the 1990s with the question, What did change in the Florida citrus industry? and her answer was simple, everything. From tens of thousands of small family citrus growers, that made most of their living from cultivating citrus, just a few large corporations now manage most of today’s citrus industry in Florida and throughout most of the rest of the world. The implications of these economically driven major changes on citrus production, economics and particularly on public interest of funding long-term research projects dealing with complicated disease problems such as CTV are mostly negative.

It seems therefore timely to warn that while the reduced research efforts will not have an immediate impact, they might eventually involve serious drawbacks on the health and productivity of future citrus groves.

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almost all of the citrus species included in Tanaka’s list were probably derived from past natural crosses among the three ancestor species. Phylogenetic analyses using a variety of molecular markers supported this proposition (140). The considerable efforts invested in determining the genetic basis of Poncirus trifoliate resistance to CTV, the attempts to isolate the resistance gene and to transfer it to commercial citrus cultivars offers great potential (2,47,49–51,63,80,129–131,208,209,213). However, there is still a wide gap in our understanding of the genetic basis of the highly diverse reactions to CTV found among the three Citrus ancestor species and their hybrids, rootstocks and cultivars, including the basic question of what causes the decline of the combination of sweet orange on sour orange rootstock (23).

The history of citrus cultivation and its special role in the cultures of ancient civilizations has been excellently recorded by Tolkowsky (192). The attractive fruits of some citrus species were taken from the regions of origin of citrus (184), which stretch from the southern slopes of the Himalayas to the southern parts of Indochina and to many other areas. In effect, globalization appears to have begun to put down its roots soon after humans began cultivating plants and breeding animals. Through at least two-and-a-half millennia, citron fruits spread first to Media (now Persia, Iran), hence the name C. medica, and later, probably mainly as result of their adaptation as a Jewish sacramental fruit, to the land of Israel and through the Jewish Diaspora. Lemons and sour oranges, also of Indian origin but known in the Mediterranean regions since Roman Times, mainly as spices and as additives to other meals, also had little impact on early horticulture. It was with the return of Vasco de Gamma, the famous explorer (1460–1524), that sweet oranges collected near Mombassa, where they had previously arrived from the Far East, were brought to Portugal from where they eventually spread to other Mediterranean areas. Sweet oranges, also named “golden apples”, “bortucale” in Arabic and several other languages or “Chinese” in others, were attractive to consumers of all classes, and so eventually acquired their present status as commodities. The interest in oranges encouraged their planting in every region of the world with favorable climatic conditions. The early spread of citrus was mainly in the form of fruits and seeds, therefore many citrus diseases, and especially those caused by pathogens such as CTV (21), which are associated with phloem tissues, were not spread, along with the propagation materials, to the new cultivation areas. Grafting, a technique known from antiquity, had little if any importance for citrus cultivation in most areas until the nineteenth century, since trees and groves grown on their own roots as seedlings were prospering (18). Although these practices helped the avoidance of diseases like CTV, new diseases often appeared that were caused by infection with endemic pathogens (21). Generally, however, citrus, like other crops, often performed well in areas distant from its center of origin and in many cases part of this success could be attributed to a lower load of pathogens and/or pests.

The Phytophthora Pandemic and the Advance to Tristeza The beginning of the nineteenth century saw considerable improvements in transportation, which enabled the movement of large cargoes of fruits and con-

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tainer-grown plants over long distances. This resulted not only in easier ingress of citrus fruits to foreign markets, but also in the dissemination of soil pathogen(s) such as Phytophthora spp., the cause of citrus root, crown or collar rots, generally known to growers as gummosis (17). First reported in 1832 in the island of Madeira, the disease rapidly swept through the Mediterranean Basin, resulting in catastrophic losses to citrus growers and eventually to the advance of the tristeza pandemic, which started a century later and 10,000 km away. With the decline of groves planted with sweet orange seedlings and the realization that sour orange plants were tolerant to Phytophthora root rot, this strong and highly adapted rootstock was eagerly adopted for the new citrus plantations. The global interest in fresh oranges that emerged during the second half of the nineteenth century stimulated expansion of the production areas’ occupied trees grafted on sour orange, especially in the Mediterranean region and the United States. However, when sour orange was tried as a rootstock in Australia and South Africa, the grafted trees died, and the problem was thought to be incompatibility (a more comprehensive historical review of this period is provided by Bar-Joseph et al. [17]). The first report of a serious decline and eventual death of sweet orange trees grafted on the sour orange rootstock was by Zeman3 (214) in Argentina, where later it received the Spanish name “Podredumbre de la raicillas”, i.e., “rotting of the rootlets”.

A similar problem, which started in 1937, was reported in Brazil (29). The symptoms of the disease suggested that nematodes or cryptogrammic pathogens were the probable cause of this rootlet-rotting disease. Nevertheless, as often happens, although the early symptomatological observations were accurate and remain relevant even today, most of the early interpretations have long been invalidated. In Java, on the other side of the globe (193), it was reported that combinations involving sweet and sour orange as scion, interstock or rootstock were all failing and it was suggested that infected sweet orange produced a substance toxic to the sour orange4. Anatomical studies by Schneider (180) showed that sieve tubes of the sour orange rootstock just below the bud union were necrotic and degenerate, which

3 In a search for a picture of Victor Zeman, I received a message from Pedro Moreno saying that Palacios and Foguet provided the following biographical data on Victor Zeman (1883–1937). Born in Moravia, studied at the Facultad de Agronomía, Universidad de La Plata, and later became professor of “Histology, anatomy and plant pathology” at the Universidad de Corrientes, his major being citrus diseases. Zeman was apparently an entomologist who described the decline of orange trees propagated on sour orange in Bella Vista (Corrientes), including the rootlets rot symptom. Reading his description, one can be pretty sure that he was describing tristeza decline, but he had no doubt that this decline was due to the nematode “Aphelenchus citri n. sp.”, that according to him was different from “Tylenchus semipenetrans Cobb”.

4 Interestingly, CTV, like every other plant virus, does not produce toxins (although the original meaning of the word “virus” was a toxic substance); however, some translation products of the CTV genome, e.g., p23, will result in CTV-like symptoms even in the absence of the virus (62). While the possibility that any of the CTV genes or their translation products could spread systemically from the sweet orange to the sour orange is remote, it is highly possible that small RNAs resulting from a silencing mechanism (25) could move systemically in the phloem. And it will be interesting to determine whether one of those molecules is able to spread and to cause decline when transported from the sweet orange scion into the sensitive sour orange rootstocks.

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suggested that the quick decline was the result of girdling induced by the causal agent of the disease. Electron microscope examination of thin sections from infected sour orange stems showed that not all of the phloem cells contained virions or turned necrotic (12), and ELISA tests of severely damaged sour orange rootlets and roots showed that they only contained low levels of the virus (207), supporting the notion that decline of sweet orange on sour orange trees was the result of a hypersensitive reaction of the sour orange when used as rootstock (a detailed review of CTV–sour orange relationships is provided by Bar-Joseph et al. [23]).

Tristeza and Quick Decline. In the year that Moreira realized that “tristeza” was an appropriate name for this disease, a new ailment called “quick decline” or “sudden decline of citrus” was reported in the Covina district of the San Gabriel Valley in southern California, where, according to the anonymous reporter, the problem had already been noticed in 1939. In 1943, Webber (206) advanced a theory that the quick decline disease was vector-transmitted and was caused by a virus. Bitancourt (30) also suggested the involvement of an infectious agent in the tristeza disease, and he developed a starch-iodine test to detect trees that were declining as a result of tristeza.

A lively historical report on the events leading to the recognition of the CTV problem in Australia was presented by the late Lillian Fraser (65), who was also one of the pioneers in the characterization of the seedling yellows tristeza phenomena (66,122).

Citrus trees were brought to Sydney from Rio de Janeiro and also from other countries along the shipping lane, and from China, Japan and other Eastern ports. The newly planted orange trees were doing well as indicated by the following dramatic description of that period: “The orange trees loaded beautifully with the bright yellow fruits against the thick dark green foliage can scarcely fail to remind the scholar of the gardens of the Hesperides” (66). However, by 1865, the thick dark green foliage of the Australian orange trees had become yellow and sickly, and trees were dying with gummosis. Although orange trees grafted on sour (Seville) orange were doing well initially (1843–1851), four decades later, at a Fruit Growers’ Conference in Sydney, it was stated “never use Seville orange stocks as they have proved a complete failure” and this was to be the experience in many subsequent trials in New South Wales.

That tristeza was in many cases a man-made disease problem is apparent from this account, and a similar conclusion was reached earlier by Wallace et al. (204), who presented evidence that South Africa was probably the source of the South American tristeza epidemics. Records showed that, in 1930, 2,600 trees were exported to South America from South Africa. Later, other trees were shipped to Argentina from South Africa, resulting in almost immediate outbreaks of tristeza (17,18).

THE SEARCH FOR THE CAUSAL AGENT Graft Transmission of Quick Decline and Aphid Transmission of Tristeza The year 1946 was a turning point in CTV research: Fawcett and Wallace (64) showed that trees of sweet orange grafted on sour orange exhibited quick decline

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symptoms after grafting with tissue from quick-decline-infected trees, and Meneghini (127,128) demonstrated transmission of tristeza from tristeza-infected to healthy plants by using the brown citrus aphid Toxoptera citricida. At that time, transmission of a disease, which was not associated with an axenically cultivable agent, either by grafting or an insect vector, was commonly accepted as sufficient evidence to consider it as caused by virus.

One Virus with Many Manifestations. In 1949, Hughes and Lister (95) described a serious dieback disease of lime trees, which had been spreading since 1938 and destroying the prosperous lime industry of the Gold Coast (now Ghana).

They reported that dieback of limes was graft and aphid (T. citricida) transmissible and that infected lime trees also exhibited conspicuous vein-clearing symptoms. Following this observation, acid lime (C. aurantifolia) seedlings from various sources were examined as possible indicator plants for tristeza in Brazil and Argentina (43, 45), for quick decline in California (203), for stem pitting of grapefruit and sweet orange (31,32,107,121,125,132), for sour orange incompatibility in South Africa (120) and Australia (65) and for seedling yellows (200). All the experimentally infected plants exhibited typical vein-clearing symptoms in Mexican lime seedlings (83,85). At that time, these results were considered to be sufficient evidence that all these diverse designations of the pathological manifestations were synonymous and belonged to infection by the virus that caused the “tristeza” disease. As a result of this seminal realization, acid limes, often called “Mexican limes” (202), were introduced as standard indicator plants for detecting tristeza-infected trees.

International Cooperation and the Tristeza Pandemic. Cooperation between Californian, Floridian and Brazilian scientists was initiated shortly after it was realized that tristeza and quick decline diseases presented a threat to citrus production in all these areas. These cooperative efforts advanced the understanding of the etiology and epidemiology of these diseases and yielded information that facilitated the continued production of citrus despite the massive spread of the virus (26, 43,45,68,84,86–91). Within the framework of these cooperative efforts, Costa and Grant (43) showed that a single aphid of T. citricida could transmit the tristeza disease. The discovery of the new indicator plant enabled cooperation to be extended to countries where the disease was still in its hidden stages (161,205) and eventually led the late J. M. Wallace and his many collaborators to establish the International Organization of Citrus Virologists (IOCV). Indeed, CTV research continues to enjoy international cooperation as indicated by numerous joint meetings and projects of Central and North American researchers (111–114,165,166, 212). Over the years, the U.S.-Israel Binational Agricultural Research and Development fund (BARD) has also generously supported several CTV-related projects that greatly contributed to our understanding of this virus. International cooperation had also led to the establishment of a worldwide collection of CTV isolates (77) and to standardized evaluation of the biological properties of CTV isolates (76).

In the Search of Hidden Foci. Field surveys in California (185) and the use of Mexican lime indicators for large surveys of latent infections revealed that trees in San Diego, Ventura and counties in central California were still tristeza-free (186),

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whereas tristeza had already spread through Florida, although apparently in a milder form since many sweet/sour grafted trees did not show decline (84,105,186).

This early survey cleared the way for the Central Valley Tristeza Eradication Program and efforts to make the Central Valley of California a tristeza-free area (the history of tristeza in California is provided by Roistacher and Gumpf [171]). In 1956, Knorr (104) confirmed tristeza was widespread in Florida and suggested that the disease had been present there for 50 years, probably as the result of cultivating exotic Meyer lemon trees that were infected. In 1959, Grant (83) and Grant and Costa (84) described the presence of many isolates of CTV in Florida in diverse citrus cultivars; with isolates causing different symptoms ranging from mild to quite severe. In 1954, Olson and Sleeth (149) found tristeza infections among old Satsuma and Meyer lemon trees that had been imported into Texas, and the trees were eradicated. Similarly, in California (203), Arizona (42) and Florida (104), Meyer lemon trees were found to be infected with a seedling yellows strain of tristeza, although there was no natural spread from the infected Meyer lemon to the surrounding trees.

Meyer Lemon and the Cost of Exotic Attractions. The finding of CTV in many Meyer lemon trees, originally introduced to the United States from Beijing, China in 1908 by the famous botanical hunter Frank N. Meyer and subsequently spread to many other countries, turned attention to many other cases of unintentional CTV dissemination. These were found in infected budwood imported from China, Japan, South America, South Africa, India and Australia, mainly during the years 1920 to 1940, when collecting exotic citrus and other fruit trees was highly fashionable worldwide. It is almost certain that, whereas the horticultural benefits derived from most or all of these imported exotic cultivars were only marginal, the epidemiological consequences of spreading CTV and other diseases to the established citrus production areas were considerably greater and were detrimental (171, 172).

Virus Vector Relationships The finding that tristeza was transmitted by T. citricida (127,128) was a landmark in CTV research and led to intensive collaborative investigations into CTV host range and vector efficiency. The absence of T. citricida from North America, where quick decline was spreading naturally, was indicative of the existence of other possible CTV vectors. Transmission by the aphid Aphis gossypii varied considerably between cases and between geographical locations (53–56,94,145–148, 173,211). Single A. gossypii individuals were significantly less efficient vectors than were those of T. citricida. However, some naturally spreading CTV isolates were transmitted with high efficiency by populations of about 100 aphids (6,13, 169,210). These results, first noted for the VT isolate from the Hibbat Zion area in Israel (13,157,159,160) and later confirmed in California, explained accounts and observations of high infection rates in regions of the world where A. gossypii was the dominant vector. Additional information was gathered on the epidemiology of CTV (103,113), including CTV–vector relationships (181,182) and the effects of

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CTV strains, host plants and environmental conditions on transmissibility5 (13,158, 160,169,170,210).

THE VIRAL ASPECTS OF CTV From Associated Threadlike Particles to Full-Length Sequence to an Infectious cDNA Clone A major development that paved the way to present-day CTV research was the finding of Kitajima et al. (102) of threadlike particles (TLPs) in tristeza-infected trees. The association of TLPs with diverse hosts infected with tristeza, including Passiflora gracilis (138), and with infected trees from various localities and geographical regions strongly supported the hypothesis that TLPs were the causal virus particles of the tristeza disease. The unusually long (2,000 nm) and thin (10–12 nm in diameter) TLPs presented a challenging problem for isolation (purification), which was an essential step toward virus characterization and specific antibody production. Several purification methods were developed for TLPs6 (4,8–10,82, 116,155,195). The infectivity of TLP-enriched preparations, inoculated mechanically by the slashing of citrus stems, was first demonstrated by Garnsey et al. (70, 74), but the unequivocal completion of the Koch postulates for TLPs was completed only in 2001, with the mechanical infection of citrus plants with TLPs obtained from RNA transcripts of an infectious CTV cDNA clone amplified through serial passaging in Nicotiana benthamiana protoplasts (179). Developments in virus purification CTV paved the way to antibody preparations (71), to virus diagnosis by enzyme-linked immunosorbent assay (ELISA) (14,16) and to estimating the size of the unusually large RNA genome of CTV (20). RNA extracts of CTV-enriched particles were also used for molecular cloning of CTV cDNA molecules, which were used as probes to differentiate CTV isolates (174,175). Although these early results

5 In June 2005, the HortScience and the Phytopathology (supplement) issues of that month contained four articles dealing with CTV transmission by T. citricida. The last decade had seen considerable progress in understanding the molecular biology of the virus and the genetic basis of the resistance of trifoliate oranges to CTV, yet we know little about the CTV–vector interaction and it will be interesting to gain a better understanding of the vector component of the CTV pathology triangle.

6 It is interesting to note the considerable difficulties and frustration experienced in the early attempts to purify and characterize this extremely large virus. This was partly the result of CTV restriction to the phloem tissue, low titers and its tendency to break and/or aggregate. Besides, there were the great technical difficulties that arose mainly from the dependence on electron microscopy for assessing the outcome of the purification efforts. Electron microscopy with negative staining was only useful at a very late stage of the purification procedure. It is not surprising that it took almost 5 years (1967– 1972) to obtain sufficient concentrated and purified TLPs to enable partial characterization of the molecular composition of TLPs. However, the adaptation of these methodologies to the isolation of Beet yellows virus (BYV) and of Apple chlorotic leaf spot virus (ACLSV) was achieved in about 8 and 3 months, respectively (4,10), almost in inverse relation to the 2,000-, 1,400- and 700-nm lengths of their respective viral particles.

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did not enable the association of the cDNA molecule with a specific biological character of these differentially reacting isolates, they did indicate the considerable genomic variation among some of the tested CTV isolates. The finding that plants infected with CTV and other closteroviruses (57) contained large amounts of doublestranded (ds) RNA of the CTV genome enabled the size to be estimated correctly.

The availability of these molecules suggested their use for diagnosis (19,58–60) and their stability turned them as preferential templates for cDNA synthesis. Indeed, cDNA sequences of many CTV isolates and of most other closteroviruses were obtained mostly by using dsRNA as templates. The sequencing of the CTV coat protein gene (183) and a large genome fragment from the 3′ end of the CTV genome led eventually to the full-length sequence of CTV T36 (101). This was followed with the finding of CTV defective RNAs (119), as described in detail in Chapter 4.

CTV Detection and Diagnosis. For a virus like CTV, which manifests itself differently on different citrus cultivars and with different scion/rootstock combinations and which gives rise to many differentially reacting isolates and combinations of isolates and to defective RNAs, the availability of an effective means of specific diagnosis is of the utmost importance. The progress in biological indexing was described above, and the discovery of the association of TLPs with CTV led to the development of new methods of CTV diagnosis. The first of these new means, introduced in 1970, was based on electron microscopic (EM) observation of partially purified particles (5). This method enabled the diagnosis of about 100 suspected samples per week, but it was expensive. However, it was of considerable value for locating new infection loci in Israel (11) The EM method was later enhanced by combining it with biological indexing of composite samples (5–10 samples per indicator) and rapid EM observations of the individual infected donor sources. Antibodies were developed in Florida (71,82) and other locations that reacted specifically with TLP-enriched preparations. However, it was only with the introduction of ELISA for CTV diagnosis (14,72) that rapid and large-scale diagnosis of CTV became practical for a variety of purposes, including certification and eradication programs, breeding programs, and as a research tool (73,75,78). There have been many modifications to this technology, such as means of obtaining recombinant antigens, immunogen injection schemes, the animals used for antibody preparation and the means of tissue preparation (7,22,37,38,41,78,93,98,117,141–143,150,162, 163,178,195). Monoclonal antibodies were first introduced by Vela et al. (199). In certain specific cases, as with the CTV situation in Florida (152), these monoclonal antibodies enabled the differentiation between mild and severe local isolates. Other diagnostic methods, such as Northern blot hybridization (139,174), which allows differentiation between certain CTV isolates, are still not specific enough to identify CTV isolates according to some specific biological characteristic. The introduction of polymerase chain reaction (PCR) and combinations of immunocapture reverse transcription (RT)-PCR (79,97,110,144,176) have further increased our ability to differentiate isolates and to indicate genetic identities or variations between CTV isolates, but these techniques are also unable to predict the biological characteristics. It is interesting to note that although the PCR technology has considerably advanced our ability to obtain genomic information on part or complete CTV genomes, it is

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of little value for practical diagnosis, and in many situations, it is still most probably far more practicable and reasonable to use ELISA for CTV detection.

Living with Tristeza. Horticulturists have always been alert and constantly in search of effective means to avoid the costly consequences of diseases. Thus, it was natural that, in the face of the threat of tristeza, all available citricultural and phytopathological resources were mobilized, in keeping with the famous quotation of the prophet Isaiah: “What could have been done more to my vineyard that I have not done in it?” (Isaiah 5:4). Naturally, the attempted control strategies varied at different periods and in different geographic regions, and the choice depended mainly on the extent to which CTV was already spreading within these areas and the sensitivity of the specific local cultivars. Thus, countries that were still mostly free of CTV were employing programs aimed at preventing the spread of the virus, mainly by focusing on certification schemes that ensured that all the propagative material was CTV-free. Other areas infected with CTV were attempting far more costly and ambitious programs of eradication7 (21). The outcomes of these efforts differed considerably and are covered in Chapter 13. Countries in which CTV was widely spread developed alternative strategies to enable continued commercial citrus production despite the presence of CTV. Indeed, long before the viral nature of the tristeza disease was realized, Japanese citrus growers were grafting CTV-tolerant Satsuma mandarins on the cold-tolerant and CTV-resistant trifoliate orange (Poncirus trifoliata) rootstocks, thus enabling them to produce high-quality fruits throughout their CTV-infested areas (189). Similarly, the change of rootstocks from the failing sour orange to Rough lemon and mandarins allowed the South African growers to produce good crops of oranges despite infection with severe CTV strains and the presence of the most efficient aphid vector. Forced by the killing of trees grafted on sour orange, Brazilians found that the Rangpur lime was the most drought- and CTV-tolerant rootstock for Brazilian conditions. In Brazil, with almost 200 million orange trees grafted on Rangpur lime and mostly preimmunized with protective CTV isolates, CTV was apparently of little concern to Brazilian producers during the last quarter century. In California, following the outbreak of quick decline and the move of citrus cultivation to the central valley, most of the new plantings were grafted on Troyer citrange, a hybrid rootstock developed half a century earlier and which was shown experimentally to be highly tolerant to CTV (33–36).

Troyer and especially its close relative Carrizo citrange were also used widely in Spain and California along with other trifoliate hybrids. The CTV panic often led to large-scale plantings of new groves on CTV-tolerant rootstocks, which were later found to suffer from various disease and cultural problems. Thus, the change to the better (CTV-tolerant) rootstocks was not free of penalties, and many growers in

7 Eradication, considered as one of the most promising means to manage the disease, was attempted with great hope and persistence in many places, but, with a few exceptions, it was effective for only a decade or so, and in most places it was either abandoned or curtailed, almost always after considerable discussion and amid the frustration of the growers and of the technical and research people who were involved.

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many areas would have been happy to continue with the production on sour orange, mainly because of its adaptability to heavy and calcareous soils and also its better tolerance to blight, a disease that severely affects production on Rough lemon and citrange rootstocks. Although the change to CTV-tolerant rootstocks prevented the manifestation of CTV-caused quick decline, it did not save trees of cultivars that were sensitive when infected by isolates causing stem pitting (39). The CTV-associated stem pitting, although it does not normally kill infected trees, is mostly associated with reduced yields and small, poor-quality fruits, so that the economic damage often equaled that inflicted by quick decline/tristeza. In many of these cases, adapting the cross protection8 technology helped growers.

In Brazil, where the Pera cultivar was in most demand in the market, the trees were declining shortly after planting on tolerant rootstock and the desperate CTV problem was only solved through the use of selected mild strains (44,115,136,137, 151). In South Africa (Chapter 15) and Australia (40,46,194), growers continued the production of the highly stem-pitting-sensitive grapefruit trees by selecting mother trees infected with protective mild isolates. Mild isolates (86) were also reported to provide protection against the severe decline of lime trees in India (3) and stem pitting and Hassaku dwarf of sweet orange in Japan (108,109,177). It is, however, important to note that to date, cross protection has not been widely applied for protecting combinations of sweet orange on sour orange against decline-inducing CTV isolates, although some reports indicated this may be possible (153,154,164) others were very contradictory (188,201). Most of the Israeli isolates that provided protection against decline, induced severe stem-pitting symptoms on Star Ruby grapefruit trees but their field use was not practicable (Bar-Joseph, 1990, unpublished data). An extensive summary and discussion of the use of mild-strain cross protection for solving CTV disease problems is provided in Chapter 15.

CONCLUSIONS The Complicated CTV Economy The economic costs of CTV epidemics, which have caused the loss of millions of productive citrus trees, were estimated to be of the order of tens and even hun-

8 Cross protection, one of the most challenging virological phenomena, was first described by McKinney in 1929 (126), almost coincidentally with the outbreak of CTV in South America. When this phenomenon was recognized, before the development of serological and molecular plant virology, cross protection or preimmunization was mainly used as a research tool to distinguish between closely related isolates of viruses. Although it was also considered as a potentially practical means to protect plants against viruses, it was rarely applied in practice, probably because breeding for resistance, using a variety of genetic resources, was usually found more attractive, was considered safer and was less labor intensive.

There were some exceptions, e.g., the use of mild Tobacco mosaic virus (TMV) isolates to protect greenhouse-grown tomatoes; but even this practice was soon discontinued, apparently because the protection was not efficient at high temperatures (67). Similarly, the use of mild selections of Papaya ringspot virus (PRSV) to protect papaya against PRSV infections was also discontinued and was replaced with mechanical protection with white nets and, more recently, with the cultivation of transgenic papaya (81).

14 / The History of Citrus tristeza virus—Revisited

dreds of millions of dollars (21). Although there were considerable variations9 in the estimates of the actual costs of losing productive trees, depending on a variety of factors, such as the market value of the lost production capacity, alternative uses of the land and the time needed for the CTV-tolerant replants to enter production, the costs of CTV were immense. There were also indirect costs incurred because growers in many areas were forced to replace the well-adapted sour orange rootstock with CTV-tolerant rootstocks, which either performed poorly or produced fruits of low quality. Other indirect costs resulted from the sensitivity of some CTVtolerant rootstocks to diseases such as citrus blight and citrus sudden death (52).

Nevertheless, the consequences of the spread of CTV to new areas did not always fulfill the dreadful predictions of the outcome of CTV epidemics. Furthermore, in some areas, infected trees grafted on sour orange continued to perform well for years after they were diagnosed as infected with the virus. Such situations naturally caused confusion, and growers were questioning the methodology with the comment that these trees did not suffer from tristeza but from ELISA. Growers were also confused and tended to lose faith in the diagnostic procedure when the results of a first assay were not exactly reproduced in a later test. Although such conflicting results could be easily and objectively explained as the natural outcome of the uneven distribution of CTV within infected trees, nevertheless they had a considerable negative impact on growers’ readiness to cooperate with the eradication authorities. In such cases, the feeling of many involved were as bitter as tristeza itself.

The Lessons of History. Because of the time scale of global CTV infections, this chapter has naturally touched not only on “histories” but also on our own experiences, including some anecdotal accounts of various aspects of CTV. Generally, tristeza infections had considerably varied effects, not only on production but also on management and on citrus virus research throughout most major citrus-growing areas worldwide. It is difficult to recall some of the early effects of this virus on citrus growing in areas such as Japan, China, India and Indonesia, but in many other areas, including South Africa, Australia and, especially, South, Central and North America, Spain and Israel, there is ample information that enables us to follow the progress of the early and later effects of this virus on citrus production. Other areas, such as Italy and Greece, seem to be currently in the early phases of the natural spreading of CTV and could probably benefit from the experience, both positive and negative, accumulated in other areas. The recent FAO announcement of the appearance of T. citricida (brown citrus aphid), the most effective vector of the disease, in Portugal could provide a further example of a situation in which experience gained previously in Central America and Florida is expected

9 The quick decline epidemic in the Los Angeles neighborhood during the 1940s coincided with the big construction boom in that area, and it is reasonable to assume that the spread of CTV had only accelerated growers’ plans to sell their property and to move to new production areas. Similarly, many of the millions of citrus trees in the CTV-infected areas of Spain, where many of the orchards were producing poorly because of a variety of illnesses, and the threat of CTV had forced the rejuvenation of the industry by means of new plantings of high-quality budwood and greatly improved varietal combinations.

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to provide valuable information and to enable us to forecast the possible future expected for the citrus-producing Mediterranean countries. It will be interesting to follow how quickly T. citricida spreads through the Mediterranean Basin, how much damage it causes and how much of the previously gained experience of dealing with CTV outbreaks will be incorporated into the new efforts to deal with the problem.

Although learning from areas where the disease spread previously represents the accepted wisdom, it should be kept in mind that the common experience with CTV is that each place will need to adapt a specific strategy, appropriate for its particular combination of grafted trees, cultivars and edaphic conditions. The folk story of the shepherd who repeatedly troubled his fellows with false warnings of attacks by wolves was unintentionally reproduced throughout the Mediterranean region with respect to CTV, and in these countries it will probably be difficult today to rouse growers to the necessary level of attention to the present reasonable forecast that CTV will seriously affect production if and when the highly effective vectors of CTV, the T. citricida aphids, will continue their spread along the Mediterranean Basin.

On the other hand, it should also be admitted that, when these highly effective CTV-transmitting aphids do spread some highly destructive CTV isolates, there will be little, if anything, to be done by way of immediately protecting the existing sensitive groves of this region. This situation was boldly expressed to one of us when visiting a grove on sour orange rootstock that was rapidly declining after the spread of the brown citrus aphids in Florida: “It will be the value of the boxes produced on the remaining trees (it’s the economy…) that will dictate when/if this grove will have to be replanted with tolerant combinations” (Steve Futch, 1999, personal communication).

LITERATURE CITED 1. Agranovsky, A. A. 1996. Principles of molecular organization, expression, and evolution of closteroviruses: Over the barriers. Adv. Virus Res. 47:119-158.

2. Asins, M. J., Bernet, G. P., Ruiz, C., Cambra, M., Guerri, J., and Carbonell, E. A. 2004.

QTL analysis of citrus tristeza virus-citradia interaction. Theor. Appl. Genet. 108:603-611.

3. Balarman, K., and Ramakrhishnan, R. 1980. Strain variation and cross protection in citrus tristeza virus on acid lime. Pages 60-75 in: Proc. Conf. Int. Organ. Citrus Virol., 8th. E. C. Calavan, S. M. Garnsey, and L. W. Timmer, eds. IOCV, Riverside, CA. 4. Bar-Joseph, M., and Hull, R. 1974. Purification and partial characterization of sugar beet yellows virus. Virology 62:552-562.

5. Bar-Joseph, M., and Loebenstein, G. 1970. Rapid diagnosis of the citrus tristeza disease by electron microscopy of partially purified preparations. Phytopathology 60:1510-1512.

6. Bar-Joseph, M., and Loebenstein, G. 1973. Effects of strain, source plant, and temperature on the transmissibility of citrus tristeza virus by the melon aphid. Phytopathology 63:716-720.

7. Bar-Joseph, M., and Malkinson, M. 1980. Hen egg yolk as a source of antiviral antibodies in the enzyme-linked immunosorbent assay (ELISA): A comparison of two plant viruses. J. Virol. Methods 1:179-183.

8. Bar-Joseph, M., Loebenstein, G., and Cohen, J. 1970. Partial purification of viruslike particles associated with the citrus tristeza disease. Phytopathology 60:75-78.