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Conservation of Citrus Germplasm 2 страница






Scientists shipping plant germplasm to other scientists may be expected to be more aware of potential phytosanitary issues than commercial interests, but there are times when these issues may not be com- pletely known. There is also a risk when exchanging cultures of pathogens, and in exchanging insects that may harbour viruses. Although generally there are regu-


 


 

lations regarding handling of these types of materials both pre- and post-entry, no system is perfect. For scientists, there is a higher standard for careful and safe intro- ductions of plant material than there is for the general public.

 

Introduction of accessions from other countries: the quarantine process

There are two different concepts for citrus quarantine procedures. The classical approach involves propagation of imported material, followed by observations and indexing for the presence of pests and pathogens; infected material can be destroyed or subject to a therapy proce- dure. The biotechnology-based approach (Navarro et al., 1984a, 1991) involves cul- turing in vitro the imported budwood, the recovery of plants by shoot-tip grafting in vitro, and observations and indexing for the presence of pests and pathogens. This tech- nique is covered in detail in Chapter 17. The material is released from quarantine only when no pests and pathogens are pres- ent. This sometimes involves repeated cycles of testing, therapy and re-testing. It is important to note that the concept of ‘releasing’ also includes the planting of the imported material in the fi eld collec- tion of the germplasm bank. Citrus seeds are not quarantine items in the USA and Spain. Although usually not strictly required from a regulatory standpoint, accessions received as seed are in some cases also routinely pathogen tested before release.

 

Introduction of new accessions from the same country: sanitation

Usually there are no legal restrictions on the introduction of new accessions from the same country where the germplasm bank is located. However, an uncontrolled intro- duction of pathogen-infected accessions may cause serious problems for the man- agement, characterization, evaluation and utilization of the genotypes included in the repository. To avoid these problems, it is


 

advisable that all materials introduced from the same country will be subject in germplasm bank facilities to a sanitation programme for pathogen elimination (Navarro, 1993). Even if there are no regula- tions for within-country movement of prop- agative material, sometimes the risk of introducing new severe pathogens in the areas where the collection is located, or new and more severe strains of existing pathogens, is even higher with local mate- rial than with imported material. This is the case in countries or areas with severe endemic diseases. In some cases, obtaining propagative materials from a reliable source of pathogen-tested material outside the country would result in less risk than obtaining material from within the country. For these reasons, and despite the higher cost, some citrus germplasm banks, like those of the USDA-ARS and IVIA, have among their objectives to maintain only healthy genotypes, even for those acces- sions selected in the country.

 

Quarantine and sanitation technology: pathogen elimination

The quarantine and sanitation programmes share practically the same technical needs and facilities. The relationships between these various areas of plant health are dia- grammed in Fig. 4.2. The two main areas are pathogen testing (see following section) and pathogen elimination.

Pathogens can be eliminated from citrus by nucellar embryony in vivo or in vitro (Weathers and Calavan, 1959; Navarro and Juá rez, 1977, 1981a, b; Roistacher, 1977), by thermotherapy (Roistacher, 1977) and by shoot-tip grafting in vitro (Navarro et al., 1975; Navarro, 1992). This last tech- nique is recommended because it is effec- tive in eliminating all citrus pathogens, and recovered plants do not have juvenile char- acters.

Thermotherapy is the older of these pathogen elimination techniques. Initial attempts to use high temperatures to elimi- nate viruses from citrus materials were unsuccessful. Finally, Grant (1957) was


 

 

Fig. 4.2. Phytosanitary considerations in the management of citrus genetic resources.

 


able to eliminate tristeza and psorosis from infected budwood using hot air chambers. Later, various other viruses were elimi- nated using similar techniques (see a review in Roistacher, 1995). Calavan et al. (1972) further developed the use of ther- motherapy for elimination of viruses by identifying appropriate rootstocks that were better able to withstand the high tempera- tures, and Roistacher and Calavan (1972) demonstrated the importance of pre-condi- tioning prior to heat treatment. There are many variations in thermotherapy tech- niques, but a commonly used diurnal tem-


perature regime is 40/30˚ C (16/8 h). While thermotherapy has proven to be very effec- tive in eliminating viruses from citrus, the work of Calavan et al. (1972) and that of various other workers (summarized in Roistacher, 1977, 1995) indicated that in many instances non-viral infectious agents (e.g. viroids and mycoplasmas) were not eliminated using this technique. This short- coming led, in part, to the development of the other major method for pathogen elimi- nation used with citrus, shoot-tip micro- grafting, which is at this time more widely used (see Chapter 17).


 


 

Following therapy, the material is indexed (see below). If the results indicate that one or more graft-transmissible pathogens are still present, the material must be subjected to another cycle of therapy. The alternate cycling of therapy and indexing continues until the material tests negative for all known graft-transmissible pathogens. Infected materials from previous cycles are destroyed by incineration or autoclaving.

 

Quarantine and sanitation technology: pathogen testing

Pathogen testing of citrus for graft-transmissi- ble pathogens is one of the central points of the entire quarantine process. Detection of graft-transmissible pathogens is based prima- rily upon biological indexing on specifi c citrus indicator plants, supplemented with laboratory tests. Specifi c graft-transmissible pathogens have been reported from certain countries. Indexing for these pathogens should be added to the normal indexing pro- gramme when germplasm is introduced from these countries. New citrus pathogens are reported periodically. As they are identifi ed and assay procedures developed, these dis- eases should be added to the indexing pro- gramme. In addition, as additional diseases are newly reported from different countries or geopolitical areas, indexing for these pathogens should be added to the normal indexing programme.

Biological indexing is based upon dis- tinctive pathogen-specifi c symptoms (usually foliar) which develop in specifi c plants (indi- cators) when infected tissue is inoculated therein. This is generally done by the grafting of bark tissue (phloem). Biological indexing is done by comparing symptom expression in indicators inoculated with tissue from the plants to be tested with the symptoms devel- oped in indicators inoculated with tissue from plants known to be infected with spe- cifi c pathogens and with uninoculated, virus- free asymptomatic controls. Most graft-transmissible pathogens produce symp- toms best at cool temperatures, whereas a lesser number produce symptoms best at warm temperatures. Growth chambers are


 

inadequate for indexing, and therefore a well- constructed, properly functioning green- house facility is critical (see the following section).

The fi rst report of the use of seedling indicator plants for the diagnosis of citrus diseases was apparently the report of Wallace (1945) on the use of sweet orange for the detection of psorosis. Previous to this, diag- nosis was made either on the basis of symp- toms on fi eld trees or inoculation and long-term observations. Since then, a number of additional citrus diseases have been reported, demonstrated to be caused by graft- transmissible pathogens, and biological indexing methods developed. For a historical perspective on these matters for most major graft-transmissible diseases of citrus, see Roistacher (1995).

A comprehensive set of protocols for biological indexing of graft-transmissible pathogens is beyond the scope of this chap- ter. Rather than listing a complete list of pub- lished reports, it is better to refer to the published compendiums or handbooks in this area: International Organization for Citrus Virologists (1968, n.d.), Calavan et al. (1978), Frison and Taher (1991), Roistacher

(1991, 1998) and Lee et al. (1999). Additional information on the application of indexing to importation and certifi cation programmes can be found in Navarro (1993), Roistacher (1977) and Roistacher et al. (1977).

More recently, various types of labora- tory tests have become available for graft- transmissible pathogens. These include serologically based tests, such as enzyme- linked immunosorbent assay (ELISA), dot immunobinding assay (DIBA) and immunospecifi c electron microscopy (ISEM); nucleic acid-based assays, such as sequential polyacrylamide gel electrophoresis (sPAGE) and polymerase chain reaction (PCR)-based assays; culture of pathogens on media; and microscopy. A complete discussion of the theoretical bases behind these techniques is beyond the scope of this chapter, and the reader should refer to such works as Hampton et al. (1990), Dhingra and Sinclair (1995), Singh and Singh (1995) and Schaad et al. (2001).


 


In some cases, laboratory-based assays offer advantages over biological indexing. They are more rapid, require fewer human resources and greenhouse space, and are therefore generally cheaper, and a large number of samples can be processed in a short time. A disadvantage is the need for a well-equipped laboratory with specialized equipment and additional training for the technicians. In some cases, there is the poten- tial for the production of false-positive and false-negative reactions, particularly when using PCR methods on a large scale with dif- ferent genotypes.

In some cases, laboratory tests can pro- vide additional information, such as titre levels or (to some extent) isolate identity. However, laboratory tests do not so far pro- vide information as to the severity of the iso- late, the reaction of the indicator to the isolate, and other biological characteristics. Furthermore, if a disease is of unknown aeti- ology, it may be possible to detect it with an indicator but not with a biological assay. Only analysis of double-stranded RNA (dsRNA) will detect unknown viruses that produce dsRNA during their replication cycle. In this way, greenhouse and laboratory tests complement but do not replace each other. As mentioned, in some cases, labora- tory tests can be performed when it is not possible to perform biological indexing for some reason. For the purpose of a complete index, if a biological test is available for a pathogen, a laboratory test can be used as an adjunct to the biological test but should not entirely replace it. In many cases, laboratory- based tests are not accepted by the regulatory authorities, and the ‘index of record’ is the biological index. On the other hand, for pur- poses of re-testing a large number of pathogen-tested germplasm accessions, labo- ratory tests are often the only feasible method.

Most laboratory tests specifi c for citrus pathogens have been developed since the 1970s. The earliest true ‘laboratory-based’ tests for citrus pathogens were serological tests (ELISAs) developed in the middle to late 1970s for CTV (Bar-Joseph et al., 1979, 1980; Garnsey et al., 1979, 1981). Serological tests


offer the advantage of permitting the process- ing of large amounts of samples in a rela- tively short time. As such, this technique is often preferred for routine re-testing of acces- sions that have tested ‘clean’ and for large- scale surveys of productions areas. One agency in California routinely performs over 400, 000 ELISA tests a year for CTV. This is the pathogen most commonly assayed in these types of large-scale surveys. The ELISA test for CTV, particularly the immunoprint- ing ELISA modifi cation, remains one of the standard and most widely accepted tests for a citrus pathogen. It is accepted in many instances where all other pathogens must be assayed by biological indexing. Probably the only other serological test that has been proved to be of real value for diagnosis at this point is that developed recently for psorosis (Garcí a et al., 1997; Martí n et al., 2002).

More recently, assays based upon nucleic acid analysis have been developed. The fi rst of these were developed in the 1980s for the characterization and later detec- tion of various viroids of citrus. In particular, the sPAGE assay for citrus viroids (Rivera- Bustamante et al., 1986; Duran-Vila et al., 1991, 1993) is widely used and accepted by regulatory agencies.

Since the development of PCR-based techniques in the early 1990s, these have become more common as they are simpler to perform than hybridizations. PCR-based techniques have the advantages of being more sensitive than serological techniques; however, they are more complicated to per- form and require a more expensively equipped laboratory and personnel, and sometimes they have problems of producing false-negative and false-positive results. However, PCR-based technologies are in con- stant development, and performing these types of assays has become easier in recent years due to the development of kits and more automated instruments. At this point, however, nucleic acid-based techniques are less suited to large-scale and rapid testing than are serological techniques.

Other laboratory techniques include hybrid serological–nucleic acid techniques, classical culturing and electron microscopy.


 


 

The culture technique for detection of Spiroplasma citri, the causal agent of stub- born disease, is probably the most widely used and accepted assay for this pathogen, although biological assays and other labora- tory-based techniques are available. Appropriate culturing is also useful for detection of other bacterial diseases.

 

Facilities

Quarantine and sanitation programmes for citrus have some very specifi c requirements with regard to facilities. Any facility used for these purposes must be inspected and approved by the regulatory authorities. A thorough discussion of the technical aspects of design for these facilities is beyond the scope of this chapter, but a few general comments will be made.

In classical quarantine procedures, the basic facilities are greenhouses for propaga- tion and indexing, and laboratories for pathogen diagnosis, although facilities for therapy are highly advisable. In the biotech- nology quarantine procedure, a tissue cul- ture laboratory is also a requirement. These facilities are also used for elimination of pathogens from local accessions. The design of facilities for quarantine pro- grammes is very important. General consid- erations in the design of quarantine facilities are reviewed by Mears and Kahn (1999) and Roosjen et al. (1999), while some considerations specifi c to quarantine of citrus are discussed by Roistacher (1991, 1998) and Gumpf (1999).

Ideally, the facilities should be located in an area with a climate suitable for grow- ing citrus, but remote from areas of com- mercial production. This is almost imperative when the classical procedure is used, but as a practical matter is not always possible. It is therefore important that the facilities are designed and built in a manner which minimizes the risk of pathogens ‘escaping’ from the quarantine facilities and infecting commercial (or even dooryard) citrus. This may be extremely expensive. The biotechnology procedure has the advantage that pathogens (particularly


 

pests, fungus and bacteria which are the more diffi cult organisms to contain) are eliminated at the initial step of introduc- tion. This allows the quarantine facility to be located in citrus research stations where trained personnel and facilities are avail- able. Of course, facilities design is insuffi - cient for this purpose unless personnel are well trained in handling plants, soil, etc., so as to minimize this risk. This includes sup- port personnel as well as the scientists actively engaged in the indexing.

The size of facilities will vary depend- ing upon the size and scope of the quaran- tine or sanitation programme. However, certain general principles apply whether the facilities are large or small. Most of these concern exclusion of pests, preven- tion of the ‘escape’ of pathogens, and main- tenance of environmental conditions suitable for indexing. In some cases, regula- tory agencies will have specifi c require- ments for facilities design. In general, the facilities belong to federal or state agencies involved in plant protection; alternatively, these agencies may have an agreement with a research institution. The facilities, processes and records must be available for inspection by the phytosanitary authorities at any time.

Ideally, facilities should include green- houses, a screenhouse and a laboratory. The greenhouses are used for the production of indicator plants, rootstocks, etc., and for biological indexing; the screenhouse is used for protected maintenance of plants; and the laboratory is used for tests such as ELISA, PCR, culturing, etc.

The greenhouse facilities are a very important part of the quarantine facilities. However, a detailed discussion of green- house facilities for indexing of citrus pathogens is outside the scope of this chap- ter. A few general points will be made below. If a more thorough treatment is needed, the reader is referred to Roistacher (1991, 1998) and Gumpf (1999).

Greenhouses used for indexing must have at least two and preferably three chambers with independently controlled tempera- tures. A room maintained at cool tempera-


 


tures is used for the detection of most graft- transmissible pathogens. A room main- tained at higher temperatures is used mainly for the detection of viroids and S. citri. If at all possible, a third room of inter- mediate temperature should be used for the production of indicator plants and propaga- tion of trees for other purposes.

Because of the importance of the main- tenance of these temperatures, the heating and cooling systems are vital to a properly designed greenhouse. The cooling system is particularly critical due to the great effect that a higher than optimal temperature can have on symptom expression. In some cases, cooling systems will not be able to maintain the appropriate temperatures during the hottest months of the year, and this can dictate the time of the year in which indexing takes place. The construc- tion of the greenhouse is less critical than the environmental control systems. Greenhouses should be constructed so that they are insect proof, and should have double doors with vestibules for this reason.

Screenhouses may be constructed of metal or wooden frames and screened with nylon or stainless steel. It should be empha- sized that ‘screen’ refers to anti-insect screen, not shade screen. The roof of the screenhouse should be at least 3 m from the ground (preferably 4.0–5.0 m) for adequate headroom for vigorously growing trees. Screenhouses should also have a vestibule with double doors.

The design and construction of the lab- oratory depends on the extent of its use. If only a limited number of laboratory tests are carried out (e.g. ELISA and sPAGE), a fairly small laboratory may be suitable. If larger numbers of materials are handled, or a larger number of laboratory tests carried out, a larger laboratory will be needed. The types of tests carried out will determine the equipment needed, as well. A minimalist indexing laboratory would include an ELISA plate reader and the apparatus for sPAGE. If PCR-based tests are utilized, a thermal cycler and perhaps a gel imaging system will be necessary. Culturing tests for


bacteria and fungi necessitate the appropri- ate supplies and incubators. The biotech- nology approach also requires a facility or portion of the facility devoted to tissue cul- ture. This would include a laminar fl ow hood, incubation facility, etc.

In addition to the construction of the facilities, measures should be taken to min- imize the phytosanitary risks such as escape of pathogens that may be present in the quarantine material; the access of insects to the interior of the facilities; and the spread of fungal pathogens within the facilities. Except under controlled situa- tions, plants should not be introduced into the quarantine facilities from other facilities and especially from the fi eld; this is partic- ularly true of other citrus plants of unknown disease status. Normal phytosan- itary precautions regarding sterilization of soil and equipment, avoiding contact with soil, maintaining the overall cleanliness of the facilities, etc. are also important.

Certain steps concerning access to the facilities can also reduce phytosanitary risk. These basically include restricting access to the facilities to all except those who are authorized and trained in proper phytosanitary conduct. The facilities should be kept locked as much as possible, and should also be behind a locked fence. Appropriate behaviour regarding ingress and egress is also vital.

 

 

Maintenance of citrus germplasm

The germplasm of citrus, as well as of many other perennial crops, is generally main- tained in the form of living trees. This is in contrast to germplasm of most fi eld crops and vegetables, which is generally main- tained in a seed bank, distributed as seed, and regenerated by grow-outs as needed. The term ‘maintenance’ should be con- trasted with ‘preservation’, which refers to long-term conservation and is discussed below.

The accessions of living trees are the source of propagative materials for distribu- tion as well as often being able to serve as


 


 

resources for a limited amount of character- ization and evaluation. As such, these col- lections of living trees are often very similar to working or active collections of germplasm. However, depending on the cir- cumstances and use of the collections, they may also serve as base or even inactive col- lections. Citrus materials which are pre- served cryogenically or in vitro are not generally used as active collections since they are costly to regenerate, but embryo- genic callus is being exchanged after thaw- ing and regrowth.

All citrus germplasm banks have a fi eld collection that is needed for characteriza- tion and preliminary evaluation. A few germplasm banks have a duplicate pro- tected collection that is mainly used to maintain healthy genotypes and to dimin- ish risks of losses of plants due to biotic or abiotic stresses. On rare occasions the germplasm bank also holds a cryostored collection for long-term maintenance.

There are certain principles that apply to both protected and field collections. Collections should include at least two copies of each accession. This creates at least one replicate for observations made directly from trees in the collections as well as providing a back-up in the event that one of the trees dies. However, for genotypes of low use, only one plant can be maintained in the protected collection. This reduces cost, and trees in the fi eld collection can be used as back-up. Accurate maps or lists of locations should be maintained and updated in a timely manner. Trees should be observed periodically for signs of pests or pathogens, abiotically induced problems, injuries, off-types or budsports, etc. Problems should be rectifi ed or the tree repropagated and replaced if appropriate. Proper phytosanitary precautions should be taken. This includes the use of a bleach solution on instruments or tools used on all trees of the collections.

Accuracy of identifi cation is critical. Despite best efforts, accessions are occa- sionally misidentifi ed. Sometimes this is immediately apparent and sometimes not. This points out the need for constant


 

review of records and checking them for accuracy. Misidentifi ed accessions should be removed from the collection and either discarded (if identity is known and is duplicated) or re-propagated for further study and (it is hoped) identifi cation.

 

Field collection

Most collections of germplasm of citrus (and, for that matter, most perennial crops) have as their base a fi eld collection or plant- ing. These are necessary for characteriza- tion and evaluation activities, and may or may not serve as sources of germplasm for distribution purposes. From the conserva- tion point of view, the fi eld collections have the risk of losing genotypes due to diseases and abiotic stresses such as freezes, fl oods or strong winds. Because of these threats, but more because of the possibility of becoming infected with a graft-transmissi- ble disease, field collections of citrus germplasm do not normally serve as sources of vegetative propagative material.

Propagation of trees for fi eld collec- tions generally follows practices for pro- duction of commercial trees for use in the area. The same is true of cultural practices in most instances. Generally, these prac- tices were developed in order to produce a healthy and thrifty tree, and this type of tree is desirable in a fi eld collection. However, there are some differences regarding fi eld collections of citrus germplasm as com- pared with commercial plantings.

Commercial spacings are not necessar- ily needed, but there should be suffi cient space between and within rows that a rela- tively normal or natural form of growth may be observed and documented. Cultural practices which may affect fruit set, quality, etc. should be avoided if one of the goals is to observe or characterize fl owering, fruit- ing and other similar characteristics. Likewise, pruning and other manipulations should be avoided if one goal is to charac- terize normal growth habit. Commercial harvesting practices are often not possible or desirable. In many instances, fruit is left on the trees as long as possible so that it is


 


 

available for observation.

Cross-pollination is a common feature of these collections and this does not allow proper characterization of the production of seeds, particularly for self-incompatible but fertile genotypes. Similarly, proper charac- terization of parthenocarpic genotypes is not possible in these collections. The only way to solve these problems is to exclude pollination insects (mainly bees), for exam- ple by covering trees with nets during the fl owering period.

Some type of fenced enclosure is often desirable to prevent both theft of fruit and taking of possibly contaminated budwood. Access to the fi eld collection, like access to quarantine facilities and protected collec- tions, should be restricted to those having legitimate business in the collection. Visitors should be accompanied by scien- tists or workers.

When possible, fi eld collections should be replicated in more than one location. This helps ensure the perpetuation of the accessions since they are backed up at more than one location. In addition, establish- ment of fi eld collections in locations with different environmental or climatic condi- tions can help with the evaluation of geno- type ´ environment interactions.

Even if fi eld collections are periodi- cally re-tested for diseases, as a general rule budwood should not be distributed from fi eld trees unless the recipient has an ade- quate phytosanitary programme in place to prevent introduction of pathogens from this source. Seeds and pollen may be distrib- uted in most instances. Propagative mate- rial for replacement trees should if at all possible be taken from a pathogen-tested source of budwood rather than directly from a fi eld tree. If such a source is not available, budwood may be taken from the fi eld tree. Trees should not be placed in a collection when propagated from non-local budwood unless the phytosanitary health of the budwood is acceptable.

 

Protected collection

A protected collection is maintained under


 

screen or (rarely) in a greenhouse. The pro- tected and fi eld plantings may or may not be located near each other. Ideally, a pro- tected collection, particularly one con- nected with a certification programme, would be located away from commercial areas with their associated higher risk of contamination or infection. However, this is not always possible in practice.






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