Introduction

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This working handbook is designed to facilitate the detection of the major citrus graft-transmissible pathogens (CGTPs) and their associated diseases. Most aspects of detection are included. The primary object of this handbook is to enable users anywhere in the world to "see" or detect CGTPs, thereby assuring their elimination from citrus tissue. The ultimate objective is the elimination of these pathogens from propagative budwood and the establishment and maintenance of disease-free primary or foundation budwood source trees and nurseries to produce healthy, high-yielding citrus groves.

The importance of a good indexing programme as a means of identifying causal agents of transmissible diseases cannot be overemphasized. A prime responsibility of those in charge of a certification programme is the prevention of the distribution of thousands or millions of trees contaminated with pathogens via propagation of infected budwood. This handbook is an aid and guide toward reaching that goal.

Indexing may be defined as any test that will consistently confirm the presence or absence of a transmissible pathogen, or identify a disease. The index test should be specific for the pathogen or disease. Inoculation of plants is the primary means by which most graft-transmissible diseases of citrus are diagnosed (Table 3). Inoculation is primarily by graft-transmission, but can al so be effected by vector or mechanical transmission. Other approaches to indexing are direct observation of the pathogen in plant cells by light or electron microscopy, by microscopic observation of inclusion bodies or gum deposits within the plant cells, by antibody-antigen reaction, by analysis of pathogen-induced chemical changes, by culturing an organism, by observation of pathogen-specific nucleic acids via polyacrylamide gel electrophoresis (PAGE) or by molecular hybridization. Other indexing techniques involve detection of physiological changes, such as in water uptake or mineral content of trees as used in testing for blight.

In general, any consistent, measurable or striking change in the morphology or chemistry of a plant brought about by the direct or indirect presence of a pathogen, or the direct observation of the pathogen, its by-products or constituents, can be considered as an index.

Many diseases can be diagnosed by specific field symptoms. Where these field symptoms are diagnostic, they are presented and illustrated. However, many graft-transmissible diseases are symptomless in commercial citrus, and these can be detected only by indexing.

The majority of the citrus diseases dealt with in this handbook are detected primarily by graft-transmission to indicator plants. Proven indexes are given in Part I. Some new detection techniques are mentioned under the heading of "Miscellaneous". These may be promising and useful but are not yet fully proved.

A listing of the indicator plants needed for a comprehensive indexing programme designed to detect the known major CGTPs reviewed in this handbook are given in Table 1. Other CGTPs not mentioned in this handbook (psorosis-like pathogens, Dweet mottle virus, yellow vein virus and other CGTPs) can also be detected by many of the listed indicator plants.

TABLE 1. The minimum number of indicator plants recommended for a comprehensive indexing programme and the pathogens that can be detected

A controlled-climate greenhouse, i.e. a plant laboratory for the production and maintenance in isolation of indicator plants, is essential to any successful programme for detection and diagnosis of these diseases. Details for the plant laboratory and its requirements, together with grafting techniques and inoculation procedures, are given in Part II.

Detailed procedures and pertinent references for laboratory methods of detection of virus and virus-like diseases are given in Part III.

MAJOR GRAFT-TRANSMISSIBLE DISEASES OF CITRUS

The graft-transmissible pathogens of citrus have two general effects. Either they are destructive and cause sharp decline and death of the tree, or they do not kill the tree but cause severe stunting, loss of vigour, reduced fruit size and yield, and result in extensive long-term losses.

The major graft-transmissible diseases of citrus are presented below, based on economic impact and transmissibility:

•Diseases which induce severe loss and where vector transmission is known, i.e. tristeza, greening and stubborn, or which are suspected to be vector-transmitted, i.e. blight and related diseases.
• Diseases which induce moderate loss, are mechanically transmitted and also bud-transmitted or vectored by unknown means, i.e. exocortis, cachexia, satsuma dwarf, tatterleaf, infectious variegation and ringspot psorosis.
• Diseases which are readily controlled by use of virus-free budwood and are primarily bud-transmitted by humans, i.e. the psorosis-A family and the oak-leaf pattern family, including concave gum, impietratura and cristacortis.
• Diseases which are vector-transmitted but of minor economic impact, such as vein enation.
• Miscellaneous diseases and those of unknown etiology, i.e. gummy bark and abnormal bud-unions.

Table 2 lists these transmissible diseases of citrus grouped by causal agent (procaryote, virus, viroid etc.) and by primary mode of transmissibility (graft, vector or mechanical).

Vector-transmitted citrus tristeza and greening rank as the most serious and destructive of the citrus diseases. Where sour orange is the predominant rootstock and appropriate vectors are present, tristeza can spread rapidly and destroy an industry. Even where tristeza becomes endemic in an area and is contained by use of other rootstocks and cross-protection techniques, some of the stem-pitting forms can superimpose, decimate and destroy the industry. In many areas of the world, where greening has been introduced, citrus as a viable crop has declined. It is most important to understand the destructive potential of these two diseases and to develop every strategy to prevent, detect and destroy the pathogens before they can become established within an area, country or region. Recently, blight and related declines have inflicted severe damage on the industries of many countries, and some diseases, such as the Misiones disease or fruta bolita in northern Argentina, have destroyed local industries.

Some of the graft and mechanically transmissible diseases of citrus such as tatterleaf, psorosis, ringspot and satsuma dwarf, are considered moderately destructive. Although they may severely limit crop production, these diseases can be readily detected in propagative budwood, are easily eliminated and their rapid spread curtailed. The mechanically transmitted viroid diseases, such as exocortis and cachexia, are destructive to certain varieties and, once introduced and established in a country, can spread relatively rapidly by mechanical means. These diseases can be readily detected by indexing and should not be permitted to enter expanding citrus industries of new or old regions.

Other citrus diseases, such as concave gum, cristacortis, impietratura, infectious variegation and vein enation, are generally less serious compared with the other diseases because they are relatively rare, do not cause severe effects or are spread only by grafting. Once eliminated from propagative budwood, they should not pose further problems.

Certain pathogens, once distributed throughout a region, are difficult to remove and may remain indefinitely with the citrus industry. Therefore, prevention by rigid quarantine should be the first priority. Detection by indexing and a programme for reducing inoculum, by either eradication or a certification programme, may have wide benefits.

BENEFITS AND OBJECTIVES OF INDEXING

The benefits derived from the development of an indexing programme will usually far outweigh the costs of development and maintenance of the programme. In those countries where no organized programme of testing has been established, most old-line citrus cultivars are infected with viruses, viroids or other graft-transmissible agents. Reports from Spain, Brazil, Florida and the Foreign Budwood Importation Program in California have shown that almost all citrus was infected with one or more CGTP.

TABLE 2. A classification of the major graft-transmissible diseases of citrus

¹XXX = The primary mode of transmission
XX = Readily transmitted
X = Secondary mode of transmission
- = Probably not transmitted by this mode
n = Not known
²Certain isolates of psorosis-A have been shown to be mechanically transmissible.

The first benefit of a comprehensive indexing programme is accurate knowledge of the pathogens that are present in citrus within the country or area. This knowledge allows formulation of sound strategies in the development of a programme to avoid pathogens in propagative budwood.

One indirect benefit of establishing a comprehensive indexing programme is its potential for early discovery of introduced pathogens that could be highly destructive to citrus should they become disseminated via vector or budwood. In this world of rapid movement of people and plants by air transportation, budwood from other countries is continually being introduced by uninformed nursery staff and growers. This budwood can readily harbour pathogens potentially destructive to citrus. A well-equipped and properly staffed diagnostic laboratory can be a first line of defence for the rapid identification of these potentially destructive pathogens. If detected early, they may be eliminated before they can spread.

Perhaps the most rewarding aspect of a comprehensive indexing programme is the production of pathogen-free trees derived by shoot-tip grafting in vitro or by thermotherapy. Trees so produced are healthy, more uniform, higher-yielding with better coloured fruit, and ultimately more profitable to the grower. Those countries which have well-established indexing and certification programmes have demonstrated the economic benefits of such programmes to the grower. Production of disease-free trees requires the knowledge of which CGTPs are present and verification of their elimination by thermotherapy and/or shoot-tip grafting.

In summary, the ultimate objective or goal of any indexing programme should be the development of certified pathogen-free budwood to improve production in the industry, and also to have the means for detection of new, serious pathogens.

CONCEPTS IN DETECTION OF CITRUS GRAFT-TRANSMISSIBLE PATHOGENS

The visible diseases

Many CGTPs will induce specific symptoms in the field tree, and many of these symptoms are diagnostic for a specific disease. This handbook illustrates some of these field symptoms. However, the individual responsible for indexing should read and study the literature relative to each of the diseases. Although some pertinent literature is given with each chapter in this handbook, a study of the literature presented in all the volumes of the Proceedings of the International Organization of Citrus Virologists (IOCV) gives a more comprehensive and helpful reference guide. A historical review and bibliography for each of 14 diseases plus shoot-tip grafting is given by Roistacher (1988a). Recommended publications illustrating many of the citrus graft-transmissible diseases are: Bové and Vogel's Description and illustration of citrus virus and virus-like diseases (1980); the fourth volume of The citrus industry, containing articles by Wallace (1978) on citrus virus and virus-like diseases and by Calavan et al. (1978) on registration, certification and indexing of citrus trees; and the Compendium of citrus diseases (Whiteside et al.,1988).

Diseases which may show characteristic and diagnostic symptoms in the field on certain varieties are cachexia, concave gum, cristacortis, exocortis, greening, infectious variegation, satsuma dwarf, psorosis-A, ringspot, stubborn, tatterleaf, tristeza and vein enation-woody gall (Table 3). However, clear diagnostic symptoms are not always obvious on infected trees in the field. It is very important to realize that trees infected with most of these same CGTPs are symptomless in various hosts and may show little or no diagnostic evidence of their presence in the field. Plants showing symptoms of one disease may also be infected with other pathogens. Thus, these CGTPs can unknowingly be spread throughout a country by bud-propagation or by mechanical or insect transmission. These are the "invisible diseases" which can be detected only by indexing.

The invisible pathogens

Most of the CGTPs listed in Table 3 exist in an invisible state in many citrus cultivars and can be detected only by indexing. Unless identified, the invisible pathogen can be readily spread in propagative budwood and can be distributed en masse within a country. The primary sources of infected budwood usually lie within the country, but with the current facility of air transport new, exotic and possibly destructive strains are being imported (often unwittingly) by nursery staff and growers.

TABLE 3.2.Diagnosis of citrus graft- tranmissible disesses

* Preferred indicator or method of detection.
¹ Maximum daytime temperatures.

Here are examples of CGTPs that can be symptomless in citrus:

• Certain isolates of citrus tristeza virus (severe stem-pitting and seedling-yellows strains) do not show symptoms in field trees of mandarin cultivars. However, when these mandarins are planted near sweet orange or grapefruit trees, aphids will transmit the citrus tristeza virus from the symptomless mandarins into reactive hosts (Roistacher,1988b).
• The citrus cachexia viroid, which induces no symptoms in grapefruit or sweet orange cultivars, can induce a severe reaction in tangelo or mandarin if infected buds are used as scions or if the viroid is mechanically transmitted to these cultivars.
• Citrus tatterleaf virus expresses no symptoms in most citrus cultivars including sweet orange, mandarin, sour lemon, Meyer lemon and grapefruit. However, if virus-infected budwood is used as scion wood and grafted to citrange or trifoliate orange or any hybrids of trifoliate used as a rootstock, a brown bud-union crease will usually be evident, and deep pits and grooves may develop in the rootstocks, and trees of these stionic combinations will usually decline.
• Citrus vein-enation virus induces no symptoms in most mature scions and can usually be detected only by indexing.
• The concave gum pathogen induces no symptoms in lemon and grapefruit.
• The psorosis-A pathogen may be present for seven to 15 years without inducing symptomatic bark scaling in sweet orange. In some rare cases psorosis-A-infected sweet oranges may be symptomless for up to 50 years. However, progeny trees propagated with budwood from such symptomless trees will usually show bark scaling symptoms within seven to 15 years. The psorosis-A pathogen induces no symptoms in infected lemon, sour orange or many varieties of pummelo.
• Exocortis and related citrus viroids will induce no symptoms in most sweet orange, mandarin and grapefruit scion cultivars. However, when viroid-infected budwood is grafted on rootstocks of trifoliate orange, hybrids of trifoliate, Palestine sweet lime or Rangpur lime, distinct symptoms may occur on these rootstocks.

CGTPs can also be masked by heat. For example, certain strains of citrus tristeza virus may be symptomless in sweet orange and grapefruit grown under hot conditions. However, when budwood is taken from these trees and propagated as scions in cooler areas, severe stem pitting and stunting may develop in progeny trees.

Pathogen-free budlines

If a control strategy is adopted based on pathogenfree budwood obtained by shoot-tip grafting in vitro with or without thermotherapy, the questions are often asked: "Why should we index? Won't the shoot-tip grafted budwood automatically be pathogen-free?" Shoot-tip grafting and thermotherapy, though proved excellent as therapeutic methods for eliminating many pathogens from citrus budwood, will not guarantee 100 percent success in eliminating all pathogens (Caravan et al.,1972; Roistacher et al.,1976; Navarro et al..,1976). In fact, 100 percent elimination of pathogens by these techniques is the exception, and indexing is an absolute necessity if freedom from CGTPs is to be assured. Distribution of budwood after therapy, but without indexing, may result in dissemination of infected material.

Once pathogen-free budlines are obtained by therapeutic means, and foundation or mother trees are planted, these foundation-source trees must be periodically reindexed to ensure they have not been contaminated by mechanical transmission or by insect vectors. In countries where vectors are present, insect-proof screenhouses are needed to protect foundation and mother trees as well as primary increase block trees from reinfection.

New developments in indexing

The use of serology, especially ELISA, for the mass detection of certain pathogens is one of the most exciting and helpful developments in recent years in the field of indexing. The rapidity of the test and its accuracy for detection of some CGTPs make it a necessary part of any indexing programme. ELISA has proved useful in mapping the distribution of such viruses as tristeza, satsuma dwarf and infectious variegation over large areas, and its future is bright for the detection of other pathogens affecting citrus. It can also be used in combination with plant indexing for detection of virus strains which are very mild-reacting in plant indicator hosts and otherwise could escape detection. As shown in Table 3, ELISA and other immuno-assay techniques are available for detection of tristeza, infectious variegation, satsuma dwarf and stubborn. It is important to realize that ELISA cannot be used as a substitute for indexing with plants in the critical evaluation for presence of pathogens because, under certain conditions, ELISA can fail to detect pathogens in low titre.

Electrophoretic techniques are being developed for detection of the viroids of exocortis and cachexia and many of the more recently discovered citrus viroids. With additional research, these electrophoretic methods may become the primary index for viroids difficult to index by other methods.

Identification of viral pathogens by nucleic acid hybridization assays is also promising for some applications, especially for detection of specific strains of a given pathogen and for viroid detection. Development of nonradioactive labels is needed to make this approach more useful for practical application.

The stubborn organism, Spiroplasma citri, can now be cultured, and culturing has replaced plant indexing as the primary index for this disease. This is one of the very few laboratory procedures that has replaced the plant itself for indexing of CGTPs.

Other techniques such as ISEM, molecular hybridization, dsRNA, and light microscopy with fluorescent antibodies, are useful for detection of specific pathogens and may ultimately replace the plant index for some applications. However, as shown in Tables 1 and 3, at present plants are still the primary means for the certain detection of most CGTPs, and the need for a well-equipped plant laboratory with skilled personnel is evident. Indexing based on plant indicators will always remain a valuable adjunct to other procedures, especially where absolute verification of virus freedom is essential. The concept should not be to replace all use of plant indicators but to utilize them efficiently in a total programme.

An old proverb expresses the concept of the need for plant indexing while continuing research for new and more rapid techniques:

• Be not the first to take the new astride;
• Be not the last to cast the old aside.

INDEXING: FACILITIES, METHODS AND CONSIDERATIONS

How does one get started in an indexing programme? This will depend on objectives. If the objective is to survey for presence or extent of infestation of a particular pathogen or disease within a region (i.e. tristeza, seedling-yellows tristeza, stem-pitting tristeza, tatterleaf or satsuma dwarf, etc.), then certain facilities and methods may be needed for this survey. The use of ELISA, if antiserum is available, would be the preferred strategy. Where antiserum is not available and plants must be used for indexing field trees over a large area, then other strategies must be used, i.e. multiple indexing of a number of trees to larger indicator plants.

If the objective is to establish a comprehensive indexing programme, the information contained in this handbook would be pointedly applicable. Of immediate concern would be the training personnel to develop and lead a certification programme, to develop a system for growing index plants, to obtain seed, to obtain positive controls etc.

The decision to develop a certification programme, including a foundation block with its supportive laboratory, must be well supported and adequately financed as a long-term project. The programme should include policies for education and publicity to inform growers and the public of the dangers of introduced pathogens and the benefits of pathogen-free stock.

This handbook includes a number of methods that can be used to detect CGTPs and is designed to be self-explanatory. Although the methods are given in considerable detail, they should be used as guidelines. These are methods and procedures that have worked effectively for indexing at certain laboratories, but they should not be considered as absolute standards. Modifications are desirable and encouraged, and should be shared. Table 3 gives a summary of various diagnostic techniques that can be used for detecting a number of the major citrus graft-transmissible diseases. This table can be used as an aid in developing an indexing strategy.

Each country must establish its goals and priorities depending on the seriousness of the immediate problem, the size of the industry, government support and funding available. An important consideration is the selection of a suitable site for the plant laboratory and the provision of appropriate temperature controls. The minimum needs, as proposed in Tables 1 and 3, are a plant-growth structure and a workplace equipped for laboratory-oriented procedures such as ELISA, PAGE and nucleic acid hybridization.

Perhaps the most important ingredients for a successful indexing programme are the quality, education, "mining, dedication and integrity of the personnel responsible for the conduct of the programme. A well-trained, well-paid and dedicated person may mean the difference between success and failure of a programme. Training can be arranged with institutions that have established certification programmes. Courses in indexing similar to those presented in the past are projected for the future under the auspices of FAO in cooperation with various research institutes. Comprehensive workshops are being planned at meetings of the International Organization of Citrus Virologists.

REFERENCES

Bové J.M. & Vogel, R., eds.1980. Description and illustration of virus-and virus-like diseases of citrus. A collection of colour slides. Paris, I.R.F.A. SETCO-FRUITS.

Calavan, E.C., Mather, A.M. & McEachern, E.H.1976. Registration, certification and indexing of citrus trees. In Reuther, W.,Calavan, E.C. & Carmen, G.E. eds. The citrus industry, Vol. 4, p. 185-222. Univ. Calif. Div. Agric. Sciences.

Calavan, E.C., Roistacher, C.N. & Nauer, E.M.1972. Thermotherapy of citrus for inactivation of certain viruses. Plant Dis. Rep., 56(11): 976-980.

Navarro, L., Roistacher, C.N. & Murashige, T.1976. Effect of size and source of shoot tips on psorosis-A and exocortis content of navel orange obtained by shoot-tip grafting in vitro. In Proc. 8th Conf: IOCV, p.194-197. Riverside, lOVC.

Roistacher, C.N.1988a. Observation on the decline of sweet orange trees in coastal Peru caused by stem pitting tristeza. FAO Plant Pror. Bull.36(1):19-26.

Roistacher, C.N.1988b. Directory of major citrus virus and virus-like diseases. Historical review and bibliography. Mediterranean Fruit Crop Improvement Council.. Rome, FAO.

Roistacher, C.N., Navarro, L. & Murashige, T.1976. Recovery of citrus selections free of several viruses, exocortis viroid, and Spiroplasma citri by shoot-tip grafting in vitro. In Proc . 8th Conf. IOCV, p. 786-793. Riverside, IOCV.

Wallace,J.M.1978. Virus and virus-like diseases. In Reuther W. Calavan, E.C. & Carmen, G.E., eds. The citrus industry, Vol.4, p.67- 184. Univ. Calif. Div. Agric. Sciences,

Whiteside, J.O., Garnsey, S.M. & Timmer, L.W.1988. Compendium of citrus diseases. St Paul, MN 55121, APS Press.

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