Citrus Rootstock Improvement

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w. P. Bitters

By their very nature, rootstock experimentstake a long time. I feel most rootstock workerswould like a 20 year period to draw their conclusionsand maketheir recommendationsto the industry. A citrus tree takesa long time in coming into full bearingbut oncein full bearingmay havea long productivelife. A rootstock is a very important part of any orchard and is not changedovernight like a cultural practice,a fertilizer program,or an irrigation schedule.Th~refore, it is important to choosethe right rootstock. What rootstock to userequires the right answer. The principal problem confronting the rootstock worker is that the answershe obtainsfrom his plots today are the answersto questionsthe plot was designed to answer20 yearspreviously. Theseanswers may not fit today's needs,and under today's conditions the answerswe need tomorrow arestill 20 yearsaway. It is urgent, therefore,something be done to closethis gapfrom 20 yearsto 15, or even 10 or less. If reliableinformation upon wtlich dependablerecommendations can be madeto the industry can be obtained in a shorter interval than previously,then we owe this responsibilityto the industry. To someextent we needto anticipate ~at a few of theseproblems might be and havesome answers waiting.

I amhappy to sayprogress is beingmade with Citrus rootstocksand evenmore progresslies ahead.This has come about through new ideas,new tools, new techniques,new equipment,and new facilities. It hascome about through better collabo- ration and cooperation with those in associated fields of interest suchas the nematologist,the plant pathologist,the nutri- tionist, the plant breeder,the plant physiologist,the antomist, and others. Eachcan and hascontributed something. In a sense,the rootstock researchermust be the key to the rootstock puzzle. It is up to him to bring togetherthe knowledge contributed separatelyor cooperatively by the other investigatorsand try to interpret meaningfullythe overall rootstock picture. For example,what good is gummosisresistant if the rootstock is suaptible to tristeza? What good is tristeza re- sistantif the stock is susceptibleto burrowing nematode?What good is resistanceto the burrowing nematodeif the tree is too susceptibleto frost? The nematologistmay not be interestedin cold hardinessand the plant pathologistmay not be interestedin fruit quality, but the rootstock man is. In a sense,the rootstock man must be the hub aroundwhich the total rootstock programrevolves. He must not be subservientto the other groups,and they must not be a servicegroup for him. He must either work cooperativelywith his associatesor borrow techniquesthey haveperfected and which are useful to his cause. Both may be to his advantageand to the advantageof rootstock progress. Progress is beingmade at Riversideas well aselsewhere, I'm sure,and I'd like to cite a few examplesto illustrate somenew horizonswhich I think havebeen attained.

Oneof the most important stepsis the availability of cleanbud lines for rootstock test purposes.Many a rootstock trial hasbeen a sulVivaltest to disease,rather than a matter of measuringtrue rootstock increments. Cleanbud lines may arise from severalsources. They may arisefrom nucellarlines in thosevarieties in which this is possible,and they of coursein due time can be recontaminatedby mechanicalmeans, by propagation,or by insectvectors. It is important to know at all times if and what virusesare present In many instances,nucellar lines are not availableand virus-freeold lines are a necessity.These may be ascertainedby virus indexingprocedures which havebeen perfected by the plant pathologists. At Riversidewe havea Citrus Variety ImprovementCommittee, which consistsbasically of plant pathologists,horticulturists, and plant breeders.The solepurpose of this committee is to provide to the industry and researchpersonnel thru a budwoodcertification programvirus- free budwood which is true to type. It is not only important to provide it initially, but it is equally important that it continues to be maintainedas such through budwoodfoundation blocks and a budwoodcertification programwhich verifies the freedom from recognizedviruses. Certified rootstocksmust be recognizedas well. The effectivenessof this programis basedon the use of indicator plants,plants which, when inoculatedto a particularvirus to which it is extremelysensitive, express key symptoms of the diseasein a very short period of time. I cite, for example,the useof the 'West Indian' lime asan indicator plant for tris- tela, the 'Etrog' citron asan indicator plant for exocortis,and Citrus excelsaas an indicator plant for tatter-leaf. As horticulturists, we can assistthe plant pathologistby supplyingnew plant materialto be tested. With thesetechniques, it is possible to determinein monthsinformation which would take yearsto obtain under field conditions. We usethese techniques to fol. low virus progressionin our rootstock blocks in the field, althoughvirus-free bud lines may havebeen used initially. Other pathologicalproblems will be referredto later.

Greatprogress has also beenmade through increasesin the genebank of material availablefor rootstock testing. This has resultedfrom increasedimportations and exchangeof geneticmaterial, increased interest in the Citrusrelatives, and increased efforts by the plant breeders.During the 1940's,citrus variety collectionshad somewhatstagnated. The inroadsof tristeza, .93.

if it did nothing else,stimulated interest in rootstock and variety collectionsand really awakenedinterest in this field. Since d1e1940's, there hasbeen a tremendousexchange of citrus materialbetween many of the citrus producingcountries of the world, and varietiesand speciesthat were onceextremely localized are now worldwide in their distribution. The researcher can try not only selectionsfrom his own nativecountry but from many other countriesas well.

Citrus relativesare relativelyunexplored as rootstocks. Within the subfamilyAurantioideae there are 33 genera,of which Citros is one. This means32 other generaand their speciesare potential rootstocks. The successof the trifoliate orange,Pon- ciros trito/iata, and one of its hybrids, the 'Troyer' citrange,are well established.Precursory investigations indicate severalof the relativesmay alsohave a potential rootstock value. I cannot agreewith someof the conclusionsdrawn by other investiga- tors about non-congenialityof someof thesecombinations. Perhapssome of the reactionswere basedon diseasedmaterial or under improperecological conditions. The fact that citrus may not do well with 1 speciesof a genusdoes not meanit won't do well with another. Interestingresults have been obtained with specieswithin someof the generaof Severinia,A ta/antia, Citropsis,Hesperethusa, Microcitrus, C/ymeniaand P/eiospermium, to mention a few. It should also be pointed out that Citros hybridizesreadily with someof theseother genera,as for examplethe hybrids, the citranges,the limequats,the faust- rimes,etc. At Riverside,we havenot succeededin creatingnew bigenerichybrids other than with Poncirus,although we havetried, but arehopefully looking forward to followingsuch crosses and testing their progeny.

The citrus breederhas also become aware of the importanceof breedingcitrus for other purposesthan edible fruits. Here- tofore, if existing hybrids had no commercialvalue and servedno useful purpose,they were as a last resort,tried as a rootstock. I am happy to say that the plant breedersat the Citrus ResearchCenter at Riversideand the USDA Dateand Citrus Field Sta- tion at Indio havemade crosses with specific rootstock purposesin mind. At Indio, accentis primarily on rootstockstolerant to salinity and to Phytophthora. At Riverside,nematode resistance has been a primary factor. In someinstances they have taken the best rootstock performanceof somespecies and crossedwith the best rootstock performanceof other speciesin hopesof improvingthe beneficialcharacters of both in the hybrids. Examplesof suchcrosses are Poncirus trifoliata crossed vlftthrough lemon, rangpurlime, cleopatramandarin, Citrus macrophylla,and others. Someof the first generationseedlings havecome into bearingand progenyplants are in variousstages of testing.

Progresshas also been made in the selectionof Phytophthora resistantstocks. Underfield conditions, it may take years for an epidemicto develop. Underlaboratory conditions,considerable information may be developedin a rather shQrtperiod of time. Two techniquesdeveloped by pathologistshave proved very useful. Oneconsists of developinga heavyaqueous sporesuspension of the Phytophthora speciesin question. Immersingthe roots of test speciesin the sporesuspension for a givenperiod of time and estimatingthe percentageof rotten feederroots providesa fairly good index of susceptibility. Some- times the techniqueis modified to a soil type pest hole, but againthe degreeof survivalis determined. The secondtechnique consistsof insertingmycelium of the test organismunder the bark of a host plant. The surfaceof the bark may be surfacester- ilized, a plug of bark removedwith a cork borer, and a small bit of mycelium insertedunderneath. The organismis permitted to incubatefor a r/lvenperiod of about 60 days. The lesionarea to which it hasspread can then be calibratedand the susceptibility determinedin this way. Both tests aresomewhat indicative, in a short period of time, of the relativetolerance of the host plant The soil pest hole may be too severe,and othersargue that the bark itself may contain the resistancefactor and inserting the organismunderneath it doesn'tanswer the completepicture. Resistancemust be structural, or chemical,or both, and an in- sight into the actual nature of resistancewould be most helpful. Wemust know the why's and the wherefore'sof thesecomplex problems. The Citrus replant problem is a good exampleof excellentcooperation between departments and individuals. Workersat Riverside were among the first to notice that a citrus tree planted v.A1erea citrus tree previously grew never grow as well as the original rootstock. Investigation of this problem revealed a number of causes,and the complete picture is finally emerging. The work of Dr. Baines has indicated the citrus nematode as one of the causal factors. However, the work of Dr. Martin indicated the reduction in growth could be just as great in the absenceof the CitlUs nematode. Martin suggestedthe possible role that other soil organisms might play and suggestedFusarium spp. and Thialeviopsis basicolaas possible suspects. Further searchinto this problem by Tsao and others has demonstrated that the presence of T. basicola in soil does retard growth of citrus seedlings and that an extract made from it also retarded growth. I had the pleasure of working with Dr. Baines and checking the resistance of a large number of Citrus speciesand Citrus relatives to the Citrus nematode. Resistancewithin Citrus speciesdoes not exist, tol- erance is only relative and true resistance is to be found only in the CitlUs relatives. Apparently, it is a dominant character in the .94-

genusPoncirus and is inherited to a degreein the bigenericcross with Citrus. Kirkpatrick, Van Gundy, and Bitters, examining the effect of bark extractsfrom resistantgenera, found the extractscontained a nematocidalcompound effective at almost the sameconcentration as chemical compounds on the commercialmarket This compound(or compounds)needs to be isolated and identified so that it might serveas the marker in resistanthybrids. Kirkpatrick and Van Gundy alsoinvestigated the ana- tomical resistanceof the 'Troyer' citrangeand its 2 parents,Poncirus trifoliata and Citrus sinensis.They found resistancewas directly relatedto the host plant's ability to form a wound peridermin the areaof infection by the gravoidfemale. The resis- tant plants reactedrapidly to the invasionof the nematodeand formed the wound peridermrapidly enoughto slough off the nematodebefore it had causedserious damage. In the susceptiblehosts there is a rudimentarywound peridermlaid down which is inadequateto stop the penetrationof the nematode. Onecan look at the infected roots of the trifoliate orangeor the 'Troyer' citrangeand quite clearly seethe little pock markswhere the tissueand the nematodehave been sloughed off. This is makingprogress. In the replant programI alsoworked closelywith Dr. Martin. Our studiesin the absenceof the citrus nematode showedalso that there wasonly a relativeresistance of citrus speciesto this problem and, again,only good replant growth camewhen citrus wasfollowed by other genera.Martin and I conducteda rootstock replant experimentin which 6 different rootstockswere used. Eachwas replanted following itself and reciprocallywith the others. Eachrootstock had the greatest depressedgrowth following itself and, asidefrom this, the 'Troyer' citrangehad the greatestoverall depressing effect As a result of theseexperiments, Martin and I suggestedrootstock rotation might be a good practice. While soil fumigation can pre. vent this problem, it would be better to havea built-in resistance.It hasbeen our experiencethat, following soil fumigation, the microflora of the soil may be upset.as the incidenceof gummosisusually increasestremendously.

It hasbeen observed through the yearsthat rootstockshave an outstandingeffect on the nutrition of the scion. Rootstocks havethe ability to differentially absorbcations and anionsfrom the soil water solution. In somecases this may be to excessor deficiencyto the ultimate detriment of the plant. In othersit is within the optimum rangeof the needsof the plant and favor- ablefor ideal plant growth. Not a great deal is known about nutritional statusas affectedby rootstocksand particularly with rootstockswhich may be termed experimental. It is known, however,that a rootstock suchas Citrus macrophylla can partially excludethe uptake of boron and maintain it at an optimum level in leavesof the scion;whereas, the samescion on other stocks may be showing2 or 3 times the boronconcentration and showingsevere leaf symptoms. A stock such as YUlU can absorb greateramounts of iron from the soil but, on the other hand, it is very sensitiveto sodiumuptake. What mechanismsare invol- ved are unknown but at presentit is important to know that they arefunctioning. The old techniquesof long drawn out chemical tissueanalysis would greatly retard suchinvestigations. Progress was made possible with the introduction of the flame pho- tometer to the laboratory and today thesehave improved to atomic absorptionand infrared spectrophotometerswhich do in minuteswhat the old laboratory manualtechniques did in days.

Of specificinterest will be to fully evaluatethe approachinitiated in Israelto the physiologicaland chemicalselections of rootstocks. Feelingthat rootstocksof the future will comefrom a plannedrootstock breedingand selectionprogram they are aimingat screeningmethods which will makesuch a programeffective. Sucha programwould be basedon biochemicaland physiologicaltests for the rapid evaluationof thousandsof potential rootstocks. In this approachthey havedemonstrated, for example,that the flavonones,which are phenolic compoundsfound in citrus tissue,act asgrowth inhibitors. Varietieswhose tissuescontain largeamounts of thesecompounds are associatedas beingweak vegetative growers and may make poor root. stocks. An index of the growth rate of seedlingsin the field, basedon the total phenoliccontent of the bark of rootstock seedlings,correlates quite well. They hope to developindicators for growth rate and duration of the adult phase,as they teRn it, aswell asthe juvenile stageand agefor cominginto bearing. Ultimately, they hope to be ableto predict growth vigor, life span,compatibility, fruit quality, level of yield, degreeof resistanceto diseasesand pestsby usingsuch techniques. This work hascontinued with studiesof perioxidaseactivity, effects of salinizationupon nucleicacids and ribosomes,ion exchangein citrus roots, etc., and theseapproaches must and will be watchedwith great interest

Tissueculture offers a new approachto old but interestingproblems. It hasbeen effectively demDnstratedwith many plants that eachplant cell possessestotipotentiality and by proper techniquesit may be possibleto reconstitutean entire new plant from a singlecell. The plant tissueculture techniquesmay be capableof producingvirus-free bud lines,either from singlecell ~olatesof virus-freecitrus plant tissuesor the culture of apicalmeristems which may not asyet be virus infected. This hasnot asyet beenaccomplished with Citrus except thru micrograftingthe shoot apexunto a healthy seedling. Organculture is much simpler. The effects of toxins and metabolicby-products also can be studiedas they diffuse acrossthe mediafrom an infected transplantto a healthy transplant The approachgrafting of tissuesfrom 2 distinct citrus speciesor varietiescan be tried bv usingadjacent transplants and observingwhat type of tissueunion takesplace. Perhapssuch studies would provide a short -95-

term index for predictingcompatibilities or incompatibilitiesbetween 2 individuals. Also, someCitrus speciesand varieties, aswell ascertain CItrus relatives,are characterizedby havingvery small or only partially developedseeds. Under normal conditions, they areweak and a high percentageare lost in germination. Usingthe tissueculture techniquehas been useful in this regard. It is alsopossible to culture citrus roots in either liquid or solid media,and this offers an opportunity to fully study root growd1under completely controlled conditions. I am happy to announcethat the tissueculture techniquehas just proved effectivein producingnucellar embryos in what are normally consideredas monoembryonic lines. With this tool clonal lines of rootstocks,either monoembryonicor polyembryonic,can be readily maintained.

One of the most prKtical, yet one of the most elusiveof citrus rootstock effectswould be that of dwarfing. Citrus growers and othersassociated with the harvestingoperations are well awareof the fact that citrus treesjust'grow too large. Anyone growingmature citrus treesis very cognizantof the limitations they placeon the cultural operationsand removalof the fruit from the tree. With recognitionof growinglabor problemsand the pendingneed for mechanicalharvesting interest in growing smaller,more compactbut productivetrees becomes more acute. The successof dwarf treesin the apple,peach and pearindus- tries only serveas a constantreminder of why not with citrus. While tree sizesvalY betweenstock and scioncombinations, none of the healthy treesappear to show much in the way of dwarfing tendencies.Sub-standard in size,yes, but not dwarfing. I say this becausein the courseof my investigationsI havetested over 400 rootstocksin many combinationsand none havedemon- stratedtrue dwarfing. Substandardtrees are reducedin size by 25 to 30 percent. Dwarfedtrees should be reducedin sizeby 50 and preferably75 per cent. A truly dwarfedtree should be no more than 8 feet in height at full maturity. This would enableall the fruit to be picked from the groundwithout the aid of picking ladders.

Some combinationsare dwarfed becauseof geneticdwarfing othersare the result of viral dwarfing. Within citrus, there appears to be no geneticdwarfing but substandardsized trees are common. Evena natural dwarf like the chinotto doesnot producedwarf tree but rather substandardtrees with the standardscions. In addition, orangetrees budded on it arevery susceptibleto tristeza.A good exampleof viral dwarfing isexamplified by treeson Palestinesweet lime. Orangeand grapefruittrees budded on it were 1/4 natural sizebut were infectedwith cachexia.They are alsosusceptible to tristeza. This possibility of usingviral dwarfing in com. mercialorchards has long beenrecommended by Or. Cohenof Fort Pierce. Examplesof geneticdwarfs were observedwhen other generawere used as rootstocks. Grapefruittrees on trifoliate orangein an old rootstock block were stunted to between8 and 10 feet in height at 33 yearsof age. No evidenceof exocortiswas present and unfortunately thesetrees were pulled before the etrog citron test wasavailable. This is not to say that all grapefruit treeson trifoliate orangeare dwarfedsince some of the largestgrape- fruit trees I haveobserved have been on trifoliate root Whethersome genetic factor was involvedin the particular casecited or whetherviral dwarfing was involvedis unknown. Grapefruittrees on Severinia buxifolia, the Chinesebox orange,at 30 yearsof a~ were about 8 feet high and yielding 200-250I.bs of fruit. The treeswere healthy but today could not be usedin the presence of tristeza. The 'Thomasville'citrangequat also exhibits somedwarfing characters. From theseexperiences it would 2ppearthat dwarfing is more apt to be found usingsome genus other than citrus or by usingbigeneric or trigenerichybrids in which citrus is 1 of the parents. The useof citrus relativesas interstocksmust not be overlooked. Many of thesehave been tried with rather dis- couragingresults. One of these,however, is encouraging,an interstock of Clymeniapolyandra on 'Troyer' citrangewith a navel top. This combinationis about 50 percentof normal sizeand yet appearshealthy in all respects.In the past, rootstock plantings havenot beendesigned with any accenton dwarfing aspectsin mind. The efforts of Dr. R. L. Phillips from Lake Alfred are per- hapsthe first directedspecifically to this phaseof investigation. Thereis no questionin my mind that progresswill be madein this field in the future and that dwarf citrus rootstocksare just aroundthe corner.

I think it has been clearly pointed out that we are making some advanceswith Citrus rootstocks. Other advances are being made in the knowledge gained in the sphere of still different rootstock effects. I may mention cold hardiness, and drought resistance, as a few. The citrus tree is being considered more and more in its entirety. It is the complete response of the rootstock and the scion to its total environment which is the final consideration. Of these, the rootstock influence has been 1 of the hard- est to evaluate. There has always been a large number of rootstocks to evaluate, and it appears that breeding programs will bring more. Greater rootstock progress is being made now than ever before. Continued citrus rootstock improvement may be expected. -96-

LfTERA TURF CITED

1. Baines, R. C., W. P. Bitters, and O. F. Clarke. Susceptibility of some speciesand varieties of citrus and some rutaceous plants to the citrus nematode. Plant DiseaseReporter 44(4):281-285. 1960.

2. Bitters, W. P. Citrusvariety planting at Riverside.Citrus Leaves35(7):8,9,32. 1955.

3. Bitters, W. P., J. A. Brusca,and D. A. Cole. The searchfor new citrus rootstocks. Calif; Citrog. 49(11):443-448. 1964.

4. Bitters, W. P. and Toshio Murashige.A placefor tissueculture in citrus research.Calif. Citrog. 52(5):226,228. 1966. 52(6):270-272. 1966. 52(7):304; 306. 1966.

5. Embleton,T. W., C. K. Labanauskas,and W. P. Bitters. The influenceof certain rootstockson the concentrationof boron, iron, manganese,and other elementsin lemon leaves,and on boron toxicity symptoms. Proc. Amer. Soc. Hart. Sci. 80: 285-290. 1962.

6. Jones,Winston W., JamesP. Martin, and W. P. Bitters. Influenceof exchangeablesodium and potassiumin the soil on the growth and compositionof young lemon treeson different rootstocks. ProtoAmer. Soc. Hart. Sci. 69:189-196. 1957.

7. Kessler,B. The physiologicalbasis of die toleranceof evergreentrees to lime and salinesoil and water conditions,with specialreference to the selectionof rootstocksof avocadoand citrus by physiologicaltests. The VolcaniInstitute of Agricultural Research.Rehovot, Israel. 99 pp. 1966.

8. Kirkpatrick, J. D., S. D. Van Gundy, and W. P. Bitters. A factor in rootsapof citrus rootstocksassociated with resistance to citrus nematode.Phytopath. 52(8):738. 1962.

9. Klotz, l. J., W. P. Bitters, and T. A. DeWolfe. Effect of different rootstockson growth of Valenciaand lisbon treesin in- festedsoil. Calit Citron. 50(4):143,144,146,147. 1965.

10. Klotz, L. J., W. P. Bitters, and T. A. DeWolfe. Resistanceto Phytophthorarootrot. Calit Citrog. 51(6):257-258. 1966.

11. Martin, J. P., W. P. Bitters, and J. O. Ervin. Replantstudies with citrus seedlings.Calif: Citrog.41{2):70.72. 1955.

12. Martin, J. P., R. B. Harding,and M. J. Garber. Relation of soil propertiesand plant compositionto growth of citrus seedlings in 100 nonfumigatedand fumigatedold citrus soils. Soil Sci. 91:317-323. 1961.

13. Martin, J. P., and W. P. Bitters. Greenhousecitrus replant studieswith rootstock seedlingsand rootstock.scioncombinations. Proc. Amer. Soc. Hart. Sci. 80:274-284. 1962.

14. Mendal,K. and A. Cohen. Methodsfor the r.id evaluationof rootstocksfor citrus. Spec.bull. no. 46. Project No. P-III 6-7. Report submitted to the Ford Foundation.

15. Phillips, R. L. Dwarfingrootstocks for citrus. Proc. First Int. CitrusSymp. (Ed.) H. D. Chapman.Univ. of Calif., Riverside. 1:401-406. 1969.

16. Tsao,Peter H. and S. D. Van Gundy. Thielaviopsisbasicola as a citrus root pathogen.Phytopathology 52(8):781-786. 1962.

17. Ts80,Peter H. The relativesusceptibility of certainvarieties and hybrids of citrus speciesand relativesto Thielaviopsis b8sicola.Plant DiseaseReporter 47(5):437-439. 1963.

54(4):419-427.Van Gundy, S. D., 1964. and J. D. Kirkpatrick. Natureof resistancein certain citrus rootstocksto citrus nematode.Phytopathology -