THERAPY AND VARIETY IMPROVEMENT

The Variety Improvement Program in Spain after Eleven Years

L. Navarro, J. Juarez, J. A. Pina, J. F. Ballester and J. M. Arregui

ABSTRACT. The Citrus Variety Improvement Program in Spain (CVIPS) started in 1975 with the following objectives: a) to recover virus-free plants by shoot-tip in vitro from cultivars selected in Spain; b) to release virus-free budwood to citrus nurseries and c) to establish a germplasm bank of healthy plants. In 1983 these objectives were expanded with the importation of citrus bud- wood from other areas through a quarantine station based on tissue culture in vitro techniques. Sixty- three species and varieties have been introduced through this quarantine station so far. Presently the CVIPS has 267 accessions and 112 have been freed of virus and virus-like diseases and are available to citrus nurseries. So far, over 12 million healthy trees from the CVIPS have been planted in commercial orchards. Index words. shoot-tip grafting in vitro, quarantine, viruses, indexing.

Virus and virus-like diseases are 3) Release of healthy budwood to widespread in older Spanish citrus growers. plantings producing very important 4) Establishment of a citrus germ- economic losses to . plasm bank. Tristeza, exocortis, cachexia, concave gum, impietratura, ringspot, cris- RECOVERYOFHEALTHY tacortis, vein enation-woody gall, PLANTS SELECTED IN SPAIN crinkly leaf, and stubborn have been detected in commercial orchards (9), This part of the program is based where most trees are infected with on shoot-tip grafting in vitro (6, 12, more than one disease. 14), and it includes the following steps These diseases posed important (8, 9). limitations to new plantations, be- Selection of mother trees. Selec- cause virus-free budwood of Spanish tion of mother trees of each cultivar varieties was not available and virus is based on their horticultural perfor- and virus-like diseases restricted the mance regardless of virus content. use of many rootstocks (5). Commercial varieties were selected in To solve these problems the Cit- private orchards, whereas other vari- rus Variety Improvement Program in eties were selected in citrus variety Spain (CVIPS) was established in collections. They include varieties 1975 (4, 8, 9) with the general objec- presently grown in Spain, old vari- tive of producing virus-free, true-to- eties not currently grown commer- type plants of a wide range of species cially, new varieties obtained by and varieties for commercial propaga- spontaneous bud mutations in the tion and research purposes. field, and foreign varieties introduced Presently, the CVIPS has the fol- in to Spain before the establishment lowing activities: of the CVIPS (often without adequate sanitary control). Presently, 204 ac- 1) Recovery of bealthy plants cessions have been included in the selected in Spain. program (table 1). 2) Introduction of foreign species Indexing of mother trees. Index- and varieties through the citrus ing is performed by graft inoculation quarantine station. to the following indicator plants: Therapy and Variety Improvement

TABLE 1 ACCESSIONS OF THE CITRUS GERMPLASM BANK OF INSTITUTO VALENCIANO DE INVESTIGACIONES AGRARIAS (IVIA) No. of accessions Selected Introduced Genus Species in Spain by quarantine Total

Citrus amblycarpa aurantifolia aurantium bergamia clementina deliciosa depressa excelsa gmndis halimii hystrix ichangiensis karna latifolia limettioides limon macroptern. madurensis medica meyeri myrtifolia nobilis paradisi shunkokan sinensis sunki tachibana tangerina temple unshiu webberi hybrids Afraegle paniculata Atalantia bilocularis citroides ceylanica Eremocitrus glauca Fortunel la crassifolia hindsii margarita Glycosmis pentaphylla Microcitrus austmlasica Murraya paniculata Pamburus misionis Poncirus trifoliata Severinia buxifolia trifolia TOTAL

'Introduced by seed YIntroduced by budwood

Arizona 861-S-1 for detection of for (CTV) and citrus exocortis viroid (CEV); Par- vein enation; Pineapple sweet son's special mandarin for citrus and Dweet for psorosis, con- cachexia viroid (CCaV); Mexican cave gum, impietratura and cristacor- 402 Tenth ZOCV Conference tis; Eureka or Arizona 861-S-1 plants (psorosis, concave gum, im- citron for crinkly leaf, and Madam pietratura and cristacortis) are very Vinous sweet orange for stubborn. common on mandarins and sweet Inoculated plants of Pineapple sweet oranges, but are rare in . Vein orange and Dweet tangor are chal- enation is widespread in sweet lenge-inoculated with psorosis lesion oranges, and also found in lemons and bark inoculum (13) to determine the mandarins. Citrus tristeza virus was presence of psorosis A. found only in mandarins; although Indicator plants for detection of this disease is widespread in Spain. CEV, CCaV and stubborn are incu- Declining trees were not selected for bated in a warm greenhouse at 27-32 the Program. C, whereas indicator plants for detec- None of the Spanish varieties of tion of the other diseases are incu- the CVIPS were virus-free. Manda- bated in a cooler greenhouse at 18-25 rins had the highest rate of infection, C. followed by sweet oranges and le- Table 2 shows a summary of in- mons. All the mandarins of the dexing results for virus and virus-like CVIPS had at least two diseases, and diseases of mother trees of sweet 87% has three or more diseases. Sixty- oranges, mandarins and lemons of three per cent of the sweet oranges Spanish origin. The exocortis viroids were also infected by three or more (2) are the most common pathogens. diseases. Lemons are usually infected Only two sweet orange mother trees by one or two diseases. These high were found to be free of them. One rates of infection have been found in was a large and productive old Wash- selected trees that, in general, did not ington navel tree, that probably is a show severe disease symptoms in the very early introduction of the Wash- field. Most trees in commercial or- ington Parent navel from California. chards are in worse condition, par- The other one is of Foia Menuda, an ticularly due to tristeza and psorosis. old variety located on the island of These data clearly suggest the very Majorca. All plants found infected important economic losses produced with cachexia also were infected with by virus and virus-like diseases to the exocortis, except a Cajel sour orange Spanish citrus industry. which is the only plant found so far in Shoot-tip grafting (STG) "in Spain infected only with citrus vitro". The technique applied is the cachexia viroid. Cachexia is also quite standard procedure for STG (6, 12). widespread, particularly in manda- The infected varieties are propagated rins. The diseases producing psorosis- on vigorous rootstocks in the green- like young leaf symptoms on indicator house. When the resulting plants are

TABLE 2 INCIDENCE OF VIRUS AND VIRUS-LIKE DISEASES IN THE VARIETIES OF SPANISH ORIGIN INCLUDED IN THE CITRUS VARIETY IMPROVEMENT PROGRAM IN SPAIN (CVIPS)

Group of varieties" Disease Sweet orange Mandarins Lemons

Exocortis 95 Cachexia 32 Psorosis 27 Concave gum Impietratura and/or CristacortisY 57 Vein Enation 75 Tristeza 0

"Data expressed as percentage of infected trees. These diseases are placed together because they produce similar symptoms on indicator plants. Therapy and Variety Improvement used for STG, they are completely de- characters between virus-free plants foliated by hand and placed in a and the original virus-infected plants growth chamber at a constant 32 C. of each cultivar. So far, the available Flushes produced after 10 to 15 days data indicate that the plants freed of are used as source of shoot tips. These disease-agents are true-to-type. are composed of the apical meristem and three leaf primordia, and meas- INTRODUCTION OF ure 0.1-0.2 mm. Troyer is FOREIGN VARIETIES used as the rootstock for all species Although many citrus cultivars except lemons, which are grafted on are currently grown in Spain, intro- citron. Routinely, 60-70% duction of new species and cultivars grafting success is achieved and over from other areas is necessary to span 90% survival is usually obtained upon the production period. In addition, it transplanting. is also desirable to introduce many Indexing of micrografted plants. citrus species and varieties and citrus Plants obtained by STG are indexed relatives, to increase the germplasm by the same methods as described bank necessary for research work. above. Tests for pathogens producing However, the introduction of vegeta- psosoris-like young leaf symptoms are tive material may lead to the intro- repeated, because sometimes they duction of new pests and diseases. produce very mild symptoms which To solve this problem, a Citrus are difficult to detect. These tests are Quarantine Station was legally estab- usually conducted in spring, when lished in Spain in 1982 and a new tis- symptom expression is best. sue culture procedure was developed Following the pretreatment at 32 for citrus introduction (11). This pro- C previously indicated, more than cedure basically consists of culturing 90% of the plants recovered by STG the imported budwood in vitro at 32 were free of virus and virus-like C to induce formation of flushes, from pathogens, including psorosis, con- which shoot-tips are isolated and cave gum, cristacortis and impietrat- micrografted in vitro to obtain plants ura. These diseases were more dif- that are grown and indexed in a ficult to eliminate when flushes were quarantine greenhouse. produced in field trees or in plants This STG method has not been growing in greenhouses at 18-25 C or adapted yet for many citrus relatives, 27-32 C (10, 14). and these are introduced as seed. To Ninety accessions have been freed enhance seed germination, and to of virus and virus-like pathogens. exclude possible soilborne pathogens, They include all the cultivars pre- seed are germinated in vitro, accord- sently grown in Spain, and the most ing to the procedure used to grow promising new cultivars for commer- seedling rootstock for STG (6, 12). cial propagation, except for a few The resulting plants are also grown which have been very recently intro- and indexed in the quarantine green- duced. The other accessions are in dif- house. ferent stages of the program. Sixty-three species and varieties Horticultural studies. Horticul- have been introduced from California, tural evaluation of virus-free plants is Argentina, Italy, France, Morocco, carried out to ascertain their fruit and Cuba and Japan (Table 1). Both in growth characteristics. Three types vitro procedures have been success- of observations are made: a) leaf and fully applied to a wide range of citrus fruit morphology on plants grown in species and citrus relatives (Table 1). greenhouses and screenhouses; b) Indexing of introduced plants is fruit characterization on healthy done by the same procedure as de- plants topworked on adult trees; c) scribed above. In addition, indicator comparison of growth and fruit plants of Troyer citrange, C. excelsa 404 Tenth ZOCV Conference and Arizona 861-S-1 citron are inocu- and the initial multiplication blocks lated and incubated in the greenhouse for budwood increase. Only 16 legal at 18-25 C for possible detection of citrus nurseries are operating in other pathogens not present in Spain. Spain, and they are associated in two All indicators are observed for leaf groups, one composed of 14 nurseries symptoms and stem pitting. and the other of two. Each group has For additional safety, we have re- a foundation block. Inspection and cently included enzyme-linked im- control of sanitary status, trueness- munosorbant assay (ELISA) testing to-type and plant quality are easily for tristeza (3, 16), electrophoresis of achieved because of the small num- purified RNA extracts for detection bers of nurseries and foundation of viroids (2, 15) and ds-RNA assays blocks. for unknown viruses (1). Table 3 shows the number of Indexing has been finished for 22 plants of different varieties recovered introductions and it is underway for through the CVIPS that have been the others. In all cases the resulting planted in commercial orchards since plants were free of virus and virus- 1982. The total number of plants is like pathogens. over 12.7 million and represent al- most 12% of the Spanish citrus indus- RELEASE OF try. HEALTHY BUDWOOD All varieties propagated by legal Virus-free cultivars recovered in citrus nurseries in Spain are free of Spain or introduced through the known virus and virus-like diseases. Quarantine Station are available to This is a great change, because before citrus nurseries for commercial prop- 1982 all the Spanish varieties com- agation through a Certification Pro- mercially propagated were infected gram (7, 8). with one or more pathogens. Budwood is released in small The CVIPS has made available to amounts to establish the foundation growers many new varieties that

TABLE 3 NUMBER OF VIRUS-FREE PLANTS, ORIGINALLY RECOVERED THROUGH THE CITRUS VARIETY IMPROVEMENT PROGRAM IN SPAIN, PLANTED IN COMMERCIAL ORCHARDSz Year Variety 1982 1983 1984 1985 1986 TOTAL Foyos Washington navel Navelina navel Newhall navel Navelate navel Nules Oroval clementine Fina clementine Hernandina clementine Arrufatina clementine Esbal clementine Fortune mandarin Clausellina satsuma Fino lemon Verna lemon Others TOTAL .. . . "Source: Instituto Nacional de Semillas y Plantas de Vivero YEstimatedamounts "This year there was a severe freeze that affected nursery plants, especially lemons. Therapy and Variety Improvement 405 were not previously propagated, par- in containers in an insect-proof screen- ticularly in the mandarin group. The houses. The principal objective of this program is having a very important block is to prevent reinfection of heal- impact on the Spanish citrus industry thy material. It is the primary source and most of the problems posed by of material for commercial propaga- virus and virus-like diseases to new tion and for international exchange of plantations are being solved. citrus germplasm. CITRUS GERMPLASM BANK CONCLUSION The species and varieties selected The CVIPS has made it possible in Spain and introduced through the for all the plants propagated in com- quarantine station are included in the mercial citrus nurseries in Spain to be citrus germplasm bank. Presently it virus-free. A wide range of Spanish includes 207 accessions (table 1). The and foreign varieties are available to recovered virus-free plants are grown growers, who can select the most ap- in a field block and in a protected propriate according to the orchard block. characteristics and the international The field block is used for horticul- market demand. This situation will tural evaluation and characterization. produce very important economic It is composed of three trees of each benefits for the Spanish citrus indus- accession, propagated whenever pos- try. sible on Troyer citrange. For com- mercially important varieties, three ACKNOWLEDGMENTS additional trees propagated on Cleo- patra mandarin are included. The technical assistance of C. The protected block is composed Ortega, V. Ortega, S. Cardo, A. Con- of two plants of each accession propa- chilla and E. Cardo is gratefully gated also on Troyer citrange, grown acknowledged.

LITERATURE CITED

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