CEV) and Related Mild Citrus Viroids (CV) on Field Performance of Washington Navel Orange on Two Rootstocks

Home , Citrus exocortis

The Effect of Citrus Exocortis Viroid (CEV) and Related Mild Citrus Viroids (CV) on Field Performance of Washington Navel Orange on Two Rootstocks

E. M. Nauer, C. N. Roistacher, E. C. Calavan and T. L. Carson

ABSTRACT. Washington navel orange trees were inoculated with one citrus exocortis viroid (CEV) and three citrus viroid (CV) isolates with shoot-tip-grafted viroid-free trees as controls. Two rootstocks were evaluated: sour orange and Troyer citrange. Trees were planted in 1978 in a repli- cated block in the Central Valley of California. CEV significantly reduced fruit yields of trees on Troyer compared to all other treatments and of trees on sour orange when compared to shoot-tip- grafted control trees. CEV also induced significantly poorer color of fruit for trees on sour orange or Troyer rootstocks. Tree size was significantly reduced for trees on sour orange and Troyer in the presence of CEV. The three CV isolates had no significant effect on tree size. There were also no significant effects by CEV or CV on sugar-acid ratio, rind thickness, fruit shape, or juice percentage. When sour orange was compared to Troyer citrange as rootstocks with or without viroids, trees on Troyer produced larger fruit, increased yield, and in three out of four years, produced fruit with significantly better rind color. There was no significant difference in the size of trees on sour orange compared to Troyer citrange. Index words. Dwarfing, sour orange, stunting, Troyer citrange, Washington navel orange, citrus exocortis viroid, viroids, citrus viroids.

Citrus exocortis, now known to be tense epinasty, vein - browning and caused by the citrus exocortis viroid, stunting of citron plants and leaves as was first reported to induce stunting does CEV. The general classification of grapefruit, navel, and Valencia of "citrus" viroids has been proposed oranges on trifoliate orange for these low molecular weight RNAs rootstock. (2, 9). Calavan et al. (2) (8). showed that exocortis was responsi- With the advent of shoot tip-graft- ble for reduction in growth and fruit ing in vitro, (14, 16), CEV and other production of Valencia orange trees viroids could be eliminated from old on trifoliate orange rootstock, but line citrus, thus bypassing the long- that in some years it induced larger term nucellar seedling process, the fruit size. Also, the tendency toward only other known method of eliminat- alternate bearing was absent or less ing CEV from infected budlines. It prevalent in infected trees. The con- then became possible to study the ef- cept of using exocortis for dwafing fect of CEV directly on the perfor- trees was first proposed by Cohen (4). mances of sweet orange trees and Since then, other studies using CEV fruit by removing CEV from old line for dwarfing have been made (1, 3, 5, citrus or by inoculating CEV back 6,). into CEV-free lines. For many years exocortis was be- An experiment was designed in lieved to be a virus disease, but was 1977 to study the effect of CEV and later shown to be a pathogenic low mild reacting isolates on citron, now molecular weight RNA or viroid (19). known as CV isolates, on the perfor- Recent studies have shown that the mance of navel orange trees on two milder reacting isolates on citron, rootstocks. In addition, a shoot-tip- classified as mild CEV isolates, repre- grafted virus and a viroid-free control sent distinct viroids with molecular were included for comparison. weights of 311 to 355 nucleotides, MATERIALS AND METHODS smaller than the 371 nucleotide CEV (7, 8, 20). These citrus viroids do not Greenhouse-grown seedlings of multiply in gynura nor induce the in- sour orange and Troyer citrange were Diseases Induced by Viroids and Viroidlike Pathogens 205 budded in March 1977. Scions used D. Inoculated with buds from vir- were an old budline Parent navel oid source E-803 in Frost nucellar orange, an old budline Atwood navel navel orange. This inoculum contains which had been shoot-tip grafted to CV I, IIa, and IIIb (8,20), and reacts eliminate a very mild isolate of a moderately in citron, inducing leaf CEV-like viroid (now identified as cit- epinasty and stem browning, but only rus viroid (CV) IIa). and an Atwood slight stunting. navel, not shoot-tip grafted contain- E. Inoculated with buds from ing CV IIa. The Atwood navel is vir- source E-800 from a field lemon bud- tually indistinguishable in tree and ded on trifoliate stock which shows fruit characteristics from Parent (10, severe exfoliation on the trifoliate and 12). The Parent navel orange selec- severe stunting of the tree. In addition, VI 12, was believed to be free of tion to CEV this inoculum contains viruses and CEV in 1977 based on the CV IIa and IIIb. Reaction on citron best available index procedures at was very severe with intense stunt- that time. However, it was later ing, severe leaf epinasty, and severe found to contain the mild CV IIa by stem cracking and browning. use of a more sensitive citron indi- F. The shoot-tip grafted control. cator (17). Rootstocks were sour An Atwood navel which had been orange and Troyer citrange. shoot-tip grafted and contained no Trees of Parent navel were inocu- viruses or viroids. lated in June 1977 by inserting two G. The Atwood navel that was the buds from a known viroid source source of E-818 which was used as in- plant, one bud each into the scion and oculum in treatment B and contains rootstock. Treatments and viroids CV IIa. used as inoculum are given in table 1. All inoculum sources and all scion Treatments were as follows: sources were tested and found free of A. Control. Inoculated with tristeza, psorosis, concave gum, vein Troyer seedling buds for wounding ef- enation, and cachexia by the methods fect. Later, all Parent navel control employed in the Citrus Clonal Protec- plants were found to contain CV IIa tion Program of the University of and this budline was subsequently California (13). coded as source E-829. Source E-829 These trees were planted in the (CV IIa) induces only a very mild leaf- field at the Lindcove Field Station in tip browning symptom when inocu- the Central Valley of California in the lated into 861 S-1 citron (17) and may spring of 1978. There were five repli- cause mild to moderate bark cracking cates in a randomized block; each re- of trifoliate rootstock. plicate consisting of two trees each of B. Inoculated with buds of At- the seven scion treatments on two wood navel viroid source E-818. rootstocks for a total of 70 trees. Reaction on 861 S-1 citron is mild, but stronger than the E-829 infected Par- ent, even though E-829 and E-818 RESULTS have been found to contain CV IIa To date, after 8 yr in the field, tree (table 1). The viroid from this source size, fruit production, and quality also induces mild definitive bark data show little effect of CV infection, cracking of trifoliate rootstock. regardless of severity of the isolate. C. Inoculated with buds from vir- Several significant differences were oid source E-805, in Bearss lime. This found when sour orange and Troyer viroid from E-805 induces mild to citrange rootstocks were compared, moderate leaf epinasty on citron and and between CEV-infected trees and mild to moderate bark cracking on non-CEV-infected trees. These re- Rangpur lime and trifoliate rootstocks. sults are summarized in table 2. Source E-805 contains CV IIIb (Table Total yield over six harvest sea- 1). sons from 1980181 through 1985186 is TABLE 1. EXPERIMENTAL TREATMENTS SHOWING SCIONS USED, CITRUS EXOCORTIS VIROID (CEV) AND CITRUS VIROIDS USED AS INOCULUM OR PRESENT IN SCION WOOD - Navel Budwood Citrus viroids" ReactionY scion source Inoculum on Treatment used code source CEV I IIa IIIb citron

A Parent Non-inoculated - - + - 0.5 B Parent Atwood navel - - + - 1.5 C Parent Bearss lime - - -X + 3.0 D Parent Frost navel - + + + 4.0 E Parent Eureka lemon + - + + 10.0 F Atwood Non-inoculated (STG)" - - - - 0.0 G Atwood Non-inoculated - - + - 1.5 "See Duran-Vila et al. (7, 8) and Semancik et al. (20) for explanation. YReactionrating on 861-SI citron: 0 = none, 1 = very mild, 2 = mild, 4 = mild-moderate, 6 = moderate, 8 = severe, 10 = very severe. "Treatments B, C, D, and E also contained the citrus viroid IIa which was present in Parent navel scion source E-829. "STG = shoot-tip grafted. TABLE 2 EFFECTS OF VIROID TREATMENTS ON YIELD, RIND COLOR, FRUIT SIZE, AND TREE SIZE OF NAVEL ORANGES

6-yr total Rind color Fruit diameter Tree height Tree width yield rating (em) (m) (m) (kgltree) Nov. '85 Nov. '85 Nov. '85 Nov. '85 Treatmentz Sour Troyer Sour Troyer Sour Troyer Sour Troger Sour Troyer

A B C D E F G Average

"See Table I. YMeanseparation within measurements by Duncan's multiple range test, 5% level. Tenth IOCV Conference shown in table 2. All treatments on the fact that trees on Troyer pro- Troyer rootstock, except for treat- duced more total fruit than trees on ment E (trees inoculated with CEV), sour orange, fruits from trees on bore significantly larger crops than Troyer were slightly larger in most those on sour orange rootstock. There seasons. In 1985-86 (table 2), trees on were no significant yield differences Troyer produced larger fruits in six among viroid-free and CV-inoculated out of seven treatments, and this dif- trees on Troyer, whereas CEV-inocu- ference was significant in two treat- lated trees bore significantly less fruit ments. There was no apparent effect than all other treatments. On sour of CV inoculations on fruit size. orange, Parent navel trees with CEV Neither was there any apparent ef- bore significantly less fruit than trees fect of CEV on fruit size with sour infected with mild CV. Trees of shoot- orange rootstock. Trees on Troyer in- tip-grafted, viroid-free Atwood navel oculated with CEV produced slightly, on sour orange yielded significantly but not significantly, smaller fruit in larger crops than the original Atwood 1983-84 and 1984-85, and significantly budline containing the very mild CV. larger fruit in 1985-86 than CV-inocu- Fruit rind color was evaluated lated trees; however, in 1985-86, early in the harvest season by com- these trees produced only about 60% paring laboratory fruit samples with of the crop average of the other Par- a color chart and by evaluating color ent navel treatments, so large-size in the field. In four seasons, rind color fruit could be accounted for by low of fruit from trees on Troyer citrange production. was slightly better (darker orange, Tree height and width were meas- less green) than that of fruit from ured in November 1985. CEV infec- trees on sour orange. In 1985-86, tion produced significantly smaller color was evaluated by walking trees on sour orange and Troyer through the orchard and rating color rootstocks (table 2). Trees on Troyer on two sides of the tree on a scale inoculated with a moderate CV (treat- of 1 to 5 with 1 being the greenest ment D) were significantly smaller in and 5 the best colored fruit. The width measurement than viroid-free evaluator did not know which treat- trees and very mild CV-infected trees ment was being rated. In this case, on Troyer (treatments A, B, F, G). direct comparisons between Troyer There is a slight indication of reduc- and sour orange showed Troyer pro- tion of tree size by inoculation with ducing slightly better color than sour mild and moderate CV isolates, but, orange in six of seven treatments, but to date, this reduction is too small to this difference was statistically signif- be promising on a commercial basis. icant only with treatment G (table 2). Future tree size measurements will We noted a poorer color with in- be taken on this block to further creasing severity of CV isolates on evaluate the possibility of using CV sour orange in 1982-83 and on both inoculation to reduce tree size in com-. rootstocks in 1985-86, but this trend mercial orchards. was not significant. The effect of CEV Analysis of juice samples showed on rind color varied. Generally, CEV- slightly higher sugar-acid ratios in inoculated trees on sour orange and fruit from trees on Troyer when com- Troyer produced fruit with poorer pared with trees on sour orange for color than CV-infected trees, but in all three seasons when such analyses 1983-84, trees on Troyer with severe were made. This result was statisti- CEV produced the best colored fruit. cally significant in o$ly one season. Fruit size effects are difficult to There were no effects of CEV or of evaluate because of interactions be- CV inoculation on juice sugar-acid tween numbers of fruit and fruit size; ratio. generally, trees producing larger Other measurements taken were crops produce smaller fruit. Despite rind thickness, fruit shape, and juice Diseases Induced by Viroids and Viroidlike Pathogens 209 percentage. None of these charac- A significant effect of the presence teristics were affected by viroid in- of CEV was the stunting of all trees oculation or rootstock treatments. on sour orange as well as those on Troyer citrange. Trees on sour DISCUSSION orange showed a 19% reduction in The mild CV initially selected for height and width compared to the this experiment were believed to be shoot-tip-grafted controls. Similarly, isolates of CEV. They were known to trees on Troyer citrange infected with induce mild bark cracking in trifoliate CEV showed a 29% reduction in rootstocks (17), and some induced height and 25% reduction in tree stunting of trees on trifoliate. How- width. The effects of CEV on trees ever, recent studies show that these on citrange, and trifoliate orange are are distinct citrus viroids,and must be well documented (2, 3, 4, 5, 6, 15). considered as separate pathogens. However, our studies show the effect Therefore, the results of this study of a specific CEV isolate plus pure not only reflect the effect of CEV but and mixed CV isolates in a tristeza- also include the effects of these newly free environment on trees growing in discovered citrus viroids (7, 8, 20) on the absence of any other known virus tree and fruit production and quality. or pathogen. The stunting effect of One of the most significant results CEV in trees on sour orange is prob- obtained thus far was the compara- ably a new finding. . tive reaction of navel orange on sour In general, the citrus viroids orange vs. Troyer citrange for the alone, or in combination, had little ef- particular soil and enviromental con- fect on yield, rind color, fruit size, or ditions in the Central Valley of juice quality when present in navel California. Trees of all treatments on orange trees on either sour orange or Troyer citrange stock (except those Troyer citrange stocks. There was inoculated with CEV), bore signific- some slight indication of tree size re- antly more fruit, had better rind duction by the citrus viroids but to color, and larger fruit than treat- date differences appear too small for ments on sour orange stock. commercial usage. The CEV source inoculum came The use of CEV for stunting of cit- from a severely affected lemon on rus has been widely discussed and is trifoliate and when indexed induced a somewhat controversial. There is a very severe reaction in citron indi- worldwide need for a dwarfed citrus cator plants. The presence of this in- tree primarily for economic reasons. oculum in trees on Troyer citrange re- The use of dwarfing rootstocks such duced yield significantly when com- as Flying Dragon trifoliate (18) ap- pared to all other treatments. At this pear promising where trifoliate can be time, no comparative evaluation or successfully grown. The ethics of projection is given for the yield per using or introducing any viroid that is unit area where the CEV-infected highly transmissible by cutting tools trees were close planted. In general, into a grove, region, or country where and with one year's exception, the it is not established are, in the judg- presence of CEV in trees on Troyer ment of these authors, questionable. and sour orange resulted in fruit color The potential movement of the viroid inferior to those without CEV. The to other existing or future scion-stock effect of CEV on fruit size could not combinations might result in unfore- be effectively analyzed since crops seen problems; once introduced and were significantly reduced by CEV, present, the pathogen may be virtu- and smaller crops tend to produce ally impossible to eliminate from the large fruit. However, there appeared area. to be no apparent effect of CEV on This is the first report, 8 yr after fruit size on trees on sour orange planting in the field, and further stock. evaluations may in time substantiate 210 Tenth IOCV Conference or modify these results. However, trees on Troyer citrange. Also, the some results appear significant, i.e., stunting effect of CEV on trees of the effect of sour orange rootstock on navel orange on sour orange stock as fruit production, rind color, and fruit well as trees on Troyer citrange stock size of navel oranges compared to appears to be significant.

LITERATURE CITED Bevington, K. B. and P. E. Bacon 1977. Effect of rootstocks on the response of navel orange trees to dwarfing inoculations, Proc. Int. Soc. Citriculture 2: 567-580. Calavan, E. C. and L. G. Weathers 1961. Evidence for strain differences and stunting with exocortis, p. 26-33. In Proc. 2nd Conf. IOCV. Univ. Florida Press, Gainesville. Calavan, E. C., L. G. Weathers, and D. W. Christiansen 1968. Effect of exocortis on production and growth of Valencia orange trees on trifoliate orange rootstocks, p. 101-104. In Proc. 4th Conf. IOCV. Univ. Florida Press, Gainesville. Cohen, M. 1968. Exocortis virus as a factor in producing dwarf citrus trees. Proc. Florida State Hort. SOC.81: 115-119. Cohen, M. 1974. Effect of exocortis inoculation on performance of Marsh grapefruit trees on various rootstocks, p. 117-121. In Proc. 6th Conf. IOCV. IOCV, Riverside. Cohen, M., W. S. Castle, R. L. Phillips, and D. Gonsalves 1980. Effect of exocortis viroid on citrus tree size and yield in Florida, p. 195-200. Proc. 8th Conf. IOCV. IOCV, Riverside. Duran-Vila, N., R. Flores, and J. S. Semancik 1986. Characteristics of viroid-like RNAs associated with the citron exocortis viroid. Virol- ogy 150: 75-84. Duran-Vila, N., J. A. Pina, J. F. Ballester, J. Juarez, C. N. Roistacher, R. Rivera-Bustamente, and J. S. Semancik 1988. The citrus exocortis disease: a complex of viroid-RNAs. p. 152-164. In Proc. 10th Conf. IOCV. IOCV. Riverside. Fraser, L. R., E.'c. Levitt, and J. Cox 1961. Relationship between exocortis and stunting of citrus varieties on Poncirus trifoliata rootstocks, p. 34-39. In Proc. 2nd Conf. IOCV. Univ. Florida Press, Gainesville. Hodgeson, R. W. 1967. Horticultural varieties of citrus, p. 431-588. In W. Reuther, H. J. Webber, and L. D. Batchelor (eds.), The Citrus Industry, Vol. 1, Univ. California, Div. Agr. Sci., Berkeley. Long, L. J., L. R. Fraser, and J. E. Cox 1972. Possible value of close planted, virus dwarfed orange trees, p. 262-267. In Proc. 5th Conf. IOCV, 1969. Univ. Florida Press, Gainesville. Nauer, E. M., W. P. Bitters, D. Cole, D. R. Atkin, and T. L. Carson 1985. Lindcove navel strain trials after nine years. Citrograph 71: 31-37. Nauer, E. M., E. C. Calavan, C. N. Roistacher, and D. R. Atkins 1980. Update on the CCPP budwood program. Citrograph 65: 207-209. Navarro, L., C. N. Roistacher, and T. Murashige 1974. Improvement of shoot-tip grafting in vitro for virus free citrus. J. Amer. Soc. Hort. Sci. 100: 471-479. Pompeu, J., Jr., 0. Rodriguez, J. Teofilo Sob, J. P. Neves Jorge, and A. A. Salibe 1976. Behavior of nucellar and old clones of Hamlin sweet orange on Rangpur lime rootstocks, p. 96-97. In Proc. 7th Conf. IOCV. IOCV, Riverside. Roistacher, C. N., L. Navarro, and T. Murashige 1976. Recovery of citrus selections free of several viruses, exocortis viroid and Spiroplasma citri by shoot tip grafting in vitro, p. 186-193. In Proc. 7th Conf. IOCV. IOCV, Riverside. Roistacher, C. N., E. C. Calavan, R. L. Blue, L. Navarro, and R. Gonzales 1977. A new more sensitive citron indicator for detection of mild isolates of citrus exocortis viroid (CEV). Plant Dis. Rev. 61: 135-139. Roose, M. L. 1986. Dwarfing rootstocks for citrus. a. 2-8. In Proc. 2nd Conf. Int. Soc. Citrus Nursew- men, Aug. 3-9: 1986. Riverside (in press). Semancik, J. S. and L. G. Weathers 1972. Exocortis disease: evidence for a new species of "infectious" low weight RNA in plants. Nature New Biol. 237: 242-244. ~ernancik,J. S. 1988. Citrus exocortis viroid-a review, p. 136-151. In Proc. 10th Conf. IOCV. IOCV, Riverside.