-68.

The TristezaDisease

Ary A. Salibe

Many centuriesago a singlemicroscopical entity cried for independenceand freedomto conquer. Built like a chromosome and possessingthe incrediblepower of self multiplication, this thread-likeparticle abandonedits abnormalcell to becomea fa. mousworld-wide virus, responsiblefor the most destructivedisease of citrus. Carriedby men and aphids,it spreadfirst through the SoutheastAsia regions,then to South Africa and from there it reachedSouth America in the first decadesof this century. About this sametime the virus wasintroduced in the United States. It is believedthat with time the tristezavirus will take over all existing citrus treesin the entire world.

Entering many citrus areas,the invaderhas caused extensive destruction to orchardsbudded on the intolerant sour orange rootstock. Certainly, an accumulated total of more than 20 million citrus trees have already been killed and many thousands are declining.

The total number of citrus treespresently existing in the world is around400-500 million plants and perhaps50 percentare carryingthe tristezavirus. Among the healthy trees,over 100 million are buddedon tristezaintolerant rootstocks,mainly sour orange.

In a generalview, the citrus areasof the world may be currently divided today into 3 regionsor classesin relation to tristeza.

RegionI - Areas havingpractically all citrus treesinfected with the virus: Southeast Asia Australia Africa South of Sahara Four South American countries (Argentina, Brazil, Paraguay, and Uruguay)

The black aphid Toxopteracitricidus Kirk, the most efficient vector of tristeza, occursin fairly high populationsapparently in all of theseareas. The total citrus presentlyexisting in this regionis estimatedat around200 million trees,all of them, of course,budded on tristeza tolerant rootstocks.

RegionII - Areas not yet infected by the virus, but wheremost treesare susceptibleto the tristezadisease: Mediterranean countries (except Spain and perhaps Morocco) Mexico and certain areas of the United States Central America South American countries (except Argentina, Brazil, Paraguay, Uruguay and Peru)

Sour orangeis usedextensively in the citrus areasof thesecountries, the number of tristezaintolerant treesamounting to more than 100 million. Isolateddeclining trees have been found infectedwith the virus within this region,but no evidenceof field spreadwas noticed. Black aphid hasnot beenreported here, but other aphidsexist It is totally unpredictablehow much longerthese citrus areaswill remainuntouched by tristeza.

RegionIII Citrusareas where tristeza virus is known to be spreadingslowly: Peru- a largepart of me coastalarea of mis country is alreadyinfected and me virus hasbeen encountered in me Amazonarea of me ChanchamayoRiver. The black aphid is presentin Peru.

United States - slow spreadingof the virus is occurringin Florida and California. Tree losseshave been quite limited, particularly in Florida, becausemost orchards are budded on tolerant rootstocks.

Spain. spreadingof the virus is occuringin the famousorchards of the Valenciaarea. A high proportion of the treesin this country is buddedon sour orangerootstock.

Morocco. a numberof decliningtrees was found recently in this country, exhibiting symptomsresembling those of tristeza. Further studies may indicate a slow spread of tristeza in Morocco (personal communication' -69.

Disseminationof the tristezavirus in theseareas, except for Peru,is madeby Toxoptera aurantii, Aphis gossypii, Myzus persicae and other aphids. Theseare inefficient vectors,but the rate of spreadof the virus frequently accelerateswith the increase of inoculum in an area. Replantings are being made on tolerant rootstocks.

Since the greatpotentialities of the virus for causingdestruction were fully realized,extensive investigations and efforts havebeen dedicated to the study of tristeza and its control. As a result, man haslearned to grow citrus in the presenceof the virus. Sour orangeand severalother citrus types havehad to be abandonedas rootstocksfor buddingmost commercial citrus varieties. To live with tristezais not an easytask. Severalimportant varietiesare affected by the virus independent of the rootstock and the productivity and vigor of the tree are reduced. Thesetrees exhibit more or lesssevere stem pitting symptomsaccording to the virulenceof the infecting virus strain. Fortunately, pre-immunizationof mother treesof partially intolerant varietieswith selectedmild strainsis provingto be successfulin preventinggreater damage in the orchards.

Existing in a number of million entities in each tree, virus mutants occur rather frequently. One of these mutants particu- larly destructive to sweet orange varieties and to the Rangpur lime rootstock, was recently found in Brazil, in the Capao Boni- to area of the State of Sao Paulo. Mutants capable of severely affecting other scion and rootstock varieties may eventually ~pear in this area or others of the world. Continuous research is the only guarantee that citrus growers can have of a success- ful citrus industry in the areaswhere tristeza virus has become endemic.

The TristezaVirus and DiseaseSymptoms

Scientistsrealized that the declinedisease invading citrus orchardsof the Americaswas caused by an infectious pathogen sometimein the beginningof this century. By 1942 the diseasewas named tristeza by Sylvio Moreira in Brazil. The virus na- ture of the disorderwas revealedin 1946 by Fawcettand Wallacein the United Statesand by Meneghiniin Brazil. The first electronmicrograph of the virus showingits thread-likeparticle about 2,000 m.-.in length and 10-12 mAin diameterwas made by Kitajima and co-workersin 1964 in Brazil.

Researchconducted in variouscitrus areasof the world hasshown that the tristezavirus must exist in a multiplicity of strainsranging from extremely mild to very severe,according to the degreeof symptomexpression on infected test plants. Cross-protectiontests with selectedvirus sourceshas resulted in a certain protectiveeffect. However,virus strain mixtures ~pear to occur within a givenhost plant. Virus strainswith increasingseverity have been named T l' T 2' T 3' , Tn and ..-parently the more severestrains are alwaysaccompanied by the milder strains. For instance,a tree infected with T 3 also yields T 2 and T 1. It hasbeen suggested that thesestrain componentsmay occur in treesin different proportions inducing variabledegrees of symptom expressionamong infected treesand within branchesof a tree. The explanationfor the presence of virus strainswithin a certain tree, accordingto Grant and Higgins,appears to be relatedto the failure of any 1 strain to be. comethoroughly systemic,thus affording susceptiblesites for the developmentof other strains.

All virus strainsof the tristezacomplex probably havebeen introduced in most citrus producingareas. However,strain componentsand diseaseexpression vary from country to country. This behavioris beingattributed mainly to climatic con- ditions, differencesin host reactionand, to a largerextent, differencesin the ability of aphid speciesto extract and transmit strainsof the tristezacomplex. There are 3 major recognizeddisease symptoms caused by the tristezavirus: stem pitting, typical tristezadecline, and seedlingyellows. Tristezastem pitting occurringin grapefruit and many other citrus types is characterizedby the develop- ment of longitudinal pits or furrows in the xylem surfaceof trunk and branches.Diseased trees show poor, bushy growth, decreasedvigor and sometimesdistorted fruits. 'West Indian' lime, extensivelyused as indicator plant for tristeza, develops vein flecking, vein clearingand eventuallyleaf cuppingin addition to stempitting. Stem pitting is inducedin intolerant hosts by very mild, mild and severetristeza strains.

Typical tristezadecline of infectedsweet orange or mandarin treesbudded on sour orangerootstock havemany features. Vein yellowing, weak flushes, dieback, thickening of the bark at the union and honeycombing from the surfac-eof the wood of the sour orange stock are some of the more characteristic symptoms of the disease. According to Schneider, the virus causes sieve-tube necrosis in the sour orange stock just below the union, thereby girdling the tree. In consequence,there is no down- ward passageof carbohydrates, the reservesof starch in the stock are consumed and the roots decay, followed by the decline .70-

and death of the scion. The declinemay progressquickly in 2 to 4 months,affecting all the foliage,and thereafter death of the tree; or it may take 12 months or more to succumb. Mild and severestrains of the virus cause this type of tristeza.

Seedling yellows is a reactionof certain citrus types havingextremely intolerant tissues,to the artificial infection with severestrains of the virus. It doesnot occur naturally in the field. Voung seedlingsof sour orange,lemon and someother citrus, when inoculatedwith severetristeza by budding,develop the yellows reactioncharacterized by severestunting and yellowing of the leaves. New leavesare often small,yellow and distortedwith corky eruptionsalong the midrib and veins. The diseasedseedlings mayor may not recovertheir normal appearance.

Most .thors regardtristeza as a singlevirus with numerousstrains, the differencein symptomatologyattributed to dif- ferent host re.:tions to the variousstrains, as explained above. However,some authors consider that "2 distinct virusesare associatedwith the tristezavirus complex", but no one hasbeen able so far to separatethese 2 components.

Host Reaction to Infection

Reactionsof the variousspecies and varieties of citrus and citrus relativesto tristezainfection dependlargely on the c. pacity of the plant to allow multiplication of the virus and to the degreeof toleranceof the tissueto presenceof the virus. Accordingly,citrus types and relativesmay belongto 1 of the following classes:

,. Extremely intolerant types

Citrus types and relativesincluded in this classhave tissues extremely sensitive to infection by the tristezavirus. Common virus strainscannot establishthemselves in the plant, probably becauseinvaded cells die readily beforemultiplication can take place. Grown asseedling plants or scionsthey behaveas resistanttypes, but a few cells arecertainly killed everytime an aphid inoculatesthe virus. Thus the hypersensitivityof thesecells protectsthe whole plant.

Extremelymild strainsmay, however,become systemic in plants of this classand then slight stem pitting may occur.

If extremely intolerant types are usedas a rootstcxk, and largeamounts of the virus descendsfrom the top with the sap, many phloem cells are killed below the union and tristezasymptoms are shown by the tree. Young seedlingsof thesevarie. ties exhibit seedlingyellows reactionwhen bud-inoculatedwith severestrains of the virus, providedenough inoculum is in- troducedto causesieve-tube blockage.

Sour orange and lemons are the best representatives of this class. Citremon, certain varieties of shaddock (C. grandis) and possibly Severinia buxifolia also belong to this class.

They can be successfully used as rootstock only for citrus types belonging to this same class.

2. Intolerant types

Types of this classhave tissues intolerant to the virus, particularly to severestrains. Mild and very mild strainsof the virus inducestem pitting symptomson them, while more severeones kill the cells and do not becomesystemic in plants of these types. If usedas rootstocksand inoculatedwith largeamounts of severestrains of the virus, treesdevelop typical tristezasymp- toms. This type of behavioris shownby grapefruit,most shaddockcultivars, some tangelos and possiblyother citrus types. Severityof stempitting varieswithin varietiesof thesetypes, suggesting genetic differences in the degreeof susceptibility to the virus. Someof the more intolerant onesmay alsoshow seedlingyellows symptoms.

3. Partially intolerant types

Citrus types includedhere have tissues that allow multiplication of the tristezavirus complex and are affected by the virus. They all show stempitting symptomsthat aremore or lessnumerous according to the severityof the infecting virus strain and the degreeof susceptibilityof their tissues.There is a wide rangeof susceptibilityamong types belongingto this class. Trees ooddedon someof the more intolerant types of this classdecline and die. Among types representativeof this class,according -71-

to decreasingdegree of intoleranceare Acid limes,Papeda group, certain tangelos,several lemon hybrids, Limequatsand kumquats,calamondin, citrons, sweetlimes, certain citranges,some tangors, and certainsweet oran~ varieties.

Some of the types includedin this classhave a good perfonnanceas rootstocks in spite of a certain amount of stem pitt- ing, for example 'Morton' citrange.

4. Toleranttypes

Plantsof citrus types belongingto this class allow the multiplication of the tristezavirus strainsand are little or not affec- ted by their presence. Examples of this class are tangerines, most sweet oranges, most tangelos, most tangors, rough lemon, 'Rangpur'lime and other mandarin limes, and Citrus volkameriana.

Tangerinesare amongthe more tolerant types and rarely exhibit stem pitting. Sweetorange cultivars havevarying degrees of toleranceand may show stem pitting in the presenceof severestrains of the virus. Citrus types listed in this classwhen used as rootstock, generallyshow good performance.

5. Resistantor immune types

Trifoliateorange and someof its hybrids havebeen found to havetissues apparently resistant or immuneto tristeza. The virus cannot multiply in the tissuesof theseplants and it is not recoveredfrom seedlingssometime after inoculation. Usedas rootstocks,they are not affected by the virus coming down with the sapto any visible degree.

Rootstocks for Tristeza Infected Areas

Sour orange and other intolerant rootstock varietieshave had to be abandonedin the tristeza-infestedareas. They havebeen replacedmainly by rough lemon, 'Rangpur' lime, sweetorange, trifoliate orangeand trifoliate orangehybrids and 'Cleopatra' mandarin. Noneof theserootstocks have, however, all the desirablecharacteristics shown by sour orange. In addition, mutants or variant strainsof the virus may affect trees on theseso-called tolerant rootstocks. The declineof sweetorange trees on trifo. liate orangein Argentinais attributed to a new strain of the tristezavirus. In Brazil, the CapaoBonito variant affectstrees of sweetorange and tangerineson 'Rangpur' lime rootstock. The searchfor new rootstock types that aretolerant to tristezais a continuousneed in the areaswhere the virus hasbecome endemic.

Several hundredcitrus types and relativeshave been tested as rootstocks for sweetorange in Brazil. Table 1 lists 301 of these types, of which nearly 60 percent have been found intolerant to the virus. They include several species and varieties of Citrus and other genera, grouped as follows:

Type Tolerant Intolerant Lemonsand lemon hybrids 1 34 Acid limes 1 16 Sweetlimes 0 9 Citrons 0 5 Roughlemons 3 1 Mandarin-limes 14 0 Tangors 9 1 Tangerines 34 0 Tangelos 17 11 Grapefruits 0 13 Shaddocks 0 22 Sour oranges 0 21 Sweetoranges 35 0 Trifoliate orangeand trifoliate hybrids 18 3 Miscellaneous -' z.~ TOTAL ' 140 161 -72-

Most of these types exhibit stem pitting in variableamounts except for sour orangesand true lemonswhich are extremely intolerant to the virus, the tangerines which are highly tolerant and trifoliate orange and some of its hybrids which are resis- tant The presence of stem pitting indicates that the vigor and productivity of the plant is affected to a certain degree by the presenceof the virus. However,certain types that areslightly to moderatelypitted will givefairly good performanceas a rootstock, such as 'Morton' citrange and Citrus taiwanica (Florida selection).

New citrus hybrids, especiallythose beingproduced and testedin California,may be found very valuablefor the future of the citrusindustry in the tristeza-infestedareas.

FINAL REMARKS

A tremendousamount of effort and researchwork hasbeen dedicated to the study of the tristeza disease.Rapid progress toward a comprehensiveknowledge of the disorderhas resulted from this intensiveresearch on an internationalscale. Many scientistsdeserve mention for their greatcontribution to the understandingand control of the tristeza disease.Among them, I ask permissionto point out the namesof H. J. Webber,C. W. Bennett,T. J. Grant, H. Schneider,J. M. Wallace,E. O. Olson, W. P. Bitters, P. A. Norman,W. C. Price, L. C. Knorr, E. P. DuCharme,M. Cohen,and J. F. L. Childsfrom the United States; s. Moreira, A. S. Costa,M. Meneghini,A. A. Bitancourt, V. V. Rossetti,E. W. Kitajima, D. C. Giacometti,and G. W. Muller from Brazil; Lilian Fraserand L. L. Stubbsfrom Australia;A. P. D. McClean,R. E. Schwarz,and P. C. J. Oberholzerfrom South Africa; and K. Mendeland I. Reichertfrom Israel.

The tristezaproblem is not comingto an end. On the contrary, much more work will be necessaryfrom theseand many other scientiststhroughout the world to control the greatinvader, the tristezavirus. .73.

Table1.

Reaction of citrus types and citrus relativesto tristezavirus infection when usedas rootstock for sweetorange and their stem pittingexpression.

Reaction* Citrus types and Reaction* Stem Citrus types and Stempitting** citrus relatives pitting** citrus relatives Lemons and LemonHybrids Acid Limes (cont.)

Acid 2 Key 3 American 3 Kirk Amber I 0 Marfim 2 Armstrong 0 Philipine I Bernia I 0 Rio Claro T 1 Camargo 2 Seda 2 Cidra 2 Selvagem 2 Cowgill 0 Tahiti 1 De ba Ahmed Sweet Limes DehraDun Deodoro 0 Columbia 1 Des4 saisons 0 Dourada 1 Doce 0 Francana 1 Eureka 0 PalestineThornless 3 Genoa 0 Persia 1 Gigante 2 Sweetlemon 1 Harris 3 Teheran 1 Harvey 2 Umbigo 1 Kulu 2 Vermelhade Goias 1 Kusner (slow decline) lisbon Thornless 0 Citrons lisbon Tetraploid 0 Morocco Commercial I 2 Meyer I Comprida 3 Periforme Q 0 Diamante 2 Perrine 2 Redonda 2 Ponderosa I 2 Sweet 2 Punjab RoughLemons Siciliano 0 Vicosa 0 Brazilian T 0 Villafranca 0 Florida T 0 Woglum I 0 Oeodoro PI.10785 South Africa T 0 PI.126539 MandarinLimes PI.136469 Cravo T 0 Acid Limes Cravodace T 0 Abacaxe 3 Japanshecitroen T. 0 Kusaie T : Beledy 3 0 Cristal 2 Ling Ming T 0 Galego 3 Otaheitelime T 1 0 GalegoThornless 1 Philippine Red T :" 0 Galegodo Norte 2 Pook Ling Ming T 0 Ingles 2 Rangpur T 0 KaduMul 0 RangpurIndia C.26.1 T 0 Kalpi 3 RangpurOtaheite T 0 .74-

Table 1. (cont)

Reaction* Citrus types and Stem Citrustypes and Reaction. Stem citrusrelatives pitting** citrus relatives pitting** Mandarin Limes (cont. Tangerines (cont)

Rangpur 0-33(40) T 0 Weshart T 0 RangpurRed Ling Wilking T 0 Mung T 0 Willow leaf tetraploid T 0 RangpurRose T 0 Tangelos Tangon Minneola T 0 Baiax Mexirica T 0 Drlando T 0 DocinhaS.J.R. 3 Pina 2 Mo T 1 Sampson T 0 Murcon Honey T 1 SanJ.:into T Reticulata T 1 Seminole T 0 Sabara T 0 Sunshine T 0 Tangerona T 0 Suwanee T 0 Temple T 0 Thornton 0 Umatilla T 0 Umatilla T 653 T 0 Yalaha T 2 Watt 1 Tangerines Webber T 3 Batangas T 0 Wekiwa Campiona T 0 Williams T 0 ChaoChou Tien Chieh T 0 TrescaOft x Dancy Clementine T 0 126-5-17 T Cleopatra T 0 TresCHoft x Dancy Cr8Vo T 0 126-5-19 Dancy T 0 TrescaOft x Dancy Kara (Frost) T 0 126-6-18 I King T 0 PI-18-H-6 T King tetraploid T 0 PI-18-1-8 T (7) King of Siam T 0 PI-18-D-14 Kinnow T 0 PI-18-E-7 Kumembo T 0 PI-18-T-2 Mandarin10630 T 0 PI.16-1-4 Mandarin117477 T 0 PI-18-1-13 T Mandarin114212 T 0 PI-16-A-1 T Mandarin114412 T 0 PI-16-N-7 T Mexirica do Rio T 0 PI-52018-W-2F Nobilis (CPB.10642) T 0 oneco T 0 Grapefruits Osceola T 0 Duncan 3 Pau T 0 Foster 3 Ponkan T 0 Leonardy 2 Satsuma,owari T 0 MKCarty 3 Satsuma,Wise T 0 Marshseedless I 3 SuenKut T 0 Pernambuco I 2 Sun Chu Shu Kat T 0 Red Blush I 3 Sunki T 0 Red Mexican I 3 Swatow 10031 T 0 Royal I 3 Swatow 10032 T 0 Ruby Red 3 Swatow 14054 T 0 Thompson I 3 .75.

Table 1. (cont)

Citnls typesand Reaction* Stem Citrus types and Reaction- Stem citrus relatives pitting** citrus relatives pitting**

Grapefruits SweetOranges Triumph 3 Baianinha T 2 Vicosa 2 Barao T 2 Blood Oval T 2 Shaddocks BuckeyeNavel T 1 Asia Cadenera T Chinesa 3 Caipira T 1 Cuban 2 Enterprise T Flemmings Florida sweetsdlg T 1 Hawaiian GoldenNugget Navel T India Red 3 Hamlin T 1 Inerme 1 Hart's late T 1 Kao Panne 1 Homosassa T 1 Melancia 2 Jaffa T 2 Nakorn lamb Summer T 3 lemon lue Gim Gong T 1 Dgami 2 MediterraneanSweet T 3 Periforme 2 Mortera T 2 Pink 2 Natal T 1 RangpurxSour Navelencia T 3 Shatenyan 3 ParsonBrown T 2 Siam 2 Pera T 3 Siamese 3 Pineapple T 2 Tau Yau 0 Piralima T 2 Thong Dee Rehovot Tl 1 Vermelha 2 Robertsonnavel T1 1 Zamboa 3 Rubi Blood T 1 2 Saint Michel T ~ 1 SourOranges Sanguinea T AlgiersSeville I 0 SeletaAmarela T 1 Arancia Bizzaria I Shamouti T 2 Azedathornless I 0 Surprisenavel T 2 AzedaSaD Paulo I 0 Trovita ~T, 2 Bergamia I 0 Valencialate T Bigaradier I 0 Washingtonnavel T 1 Stow BitterMeet I 0 Westin T, 3 Daidai(PI.117471) I Dummitt Bittersweet I 0 Trifoliate Orangeand Trifoliate Hybrids Dummitt I 0 Carrizocitrange T 0 Egyptian I 0 CitrumeloPI-4200 T LancetaAmarga I 0 CitrumeloPI-4475 T 0 Viradouro I 0 CitrumeloPI-4477 T; 0 Myrtifolia I 0 CitrumeloPI-4482 T, Oklawaha I 0 CitrumeloPI-4604 T Paraguay I 0 EEL.203 Citremon I 2 RehovothPalestine I 0 EEL.150 Citrangequat I 2 Sauvage I 0 Morton citrange T 2 Sour (U.S.D.A.) I 1 Rusk citrange T 2 Spain I 0 Sacatoncitrumelo T 0 Tunis I 0 Saunderscitrange T 2 -76.

Table 1. (cont)

Reaction* Citrus types and Stem citrus relatives pitting**

Trifoliate Orange and Trifoliate Hybrids (cont.) Savagecitrange T 3 Thompsoncitrangequat I 2 Trifoliate (small flower) T 0 Trifoliate (large flower) T 0 Trifoliate tetraploid T 0 Troyer citrange T 0 Uvaldecitrange T 0 Winter Haven citrumelo T 0 Cunninghamcitrange T 0

Miscellaneous Citruscelebica I 2 c.excelsa I 3 C. hystrix I c. ichangensis I 2 c. junos T 3 C.karna T C. longispina I C. macroptera I C. mitis I 3 (Calamondin) C. macrophylla I 3 C. tachebanaSkek T 0 C. taiwanica(Florida) T 2 C. volkameriana T 1 c. webberii I 3 Citropsissp. I Severiniabuxifolia I 0 Severiniasp. I 0 Microcitrus $p. I 3 Micromelumtephrocarpa I Poormanorange (grapefruit?) I Calashu T * Reactionof stocksto tristezavirus: I = Intolerant Kansu I T = Tolerant CowgillMarcott T Q = Questionable Limue Khargi I Misri Batabi I ** Stempitting expressedin the scionsof the variouscitrus Natsumikan I types,was rated: Satsumelo10-V-3 T 0 = none Nagamikumquat I 1 = slightly pitted Meiwakumquat I 2 = moderatedpitted Nippon kumquat I 3 = severelypitted Lakelandlimequat I Tavareslimequat I Lemonquat I