Calavan, E. C.

Christianson, D. W.

Roistacher, C. N.

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UC RiversideInternational Organization of Citrus Virologists Conference Proceedings (1957-2010)TitleDistribution of Stubborn Disease Virus in Trees of Citrus sinensis and C. paradisi at Different SeasonsPermalinkhttps://escholarship.org/uc/item/6jc098xtJournalInternational Organization of Citrus Virologists Conference Proceedings (1957-2010), 4(4)ISSN2313-5123AuthorsCalavan, E. C.Roistacher, C. N.Christianson, D. W.Publication Date1968DOI10.5070/C56jc098xt Peer reviewedeScholarship.org Powered by the California Digital LibraryUniversity of CaliforniaSTUBBORN, GREENING, and RELATED DISEASES 145 Distribution of Stubborn Disease Virus in Trees of Citrus sinensis and C. paradisi at Different Seasons E. C. CALAVAN, C. N. ROISTACHER, and D. W. CHRISTIANSEN THE PER CENT OF transmission and perpetuation of stubborn disease from severely affected mother trees has ranged from 0 to 100 in numer- ous experiments by the authors during the past decade. Other workers also noted normal trees among inoculated plants (5) and among the progeny of diseased trees (4, 7). Calavan and Christiansen (2) suggested that normal trees occur among inoculated plants and bud progeny of stubborn trees because of the irregular distribution of the pathogen within the host. With the advent of a rapid-indexing method for stub- born disease (3), it became possible to determine the distribution of virus within stubborn-diseased trees by the reaction of inoculated indi- cator plants. This paper reports results of grafting sensitive indicator plants at different seasons with tissues from various parts of stubborn trees. Materials and Methods INOCULA.-T~~ main sources of inocula were 3 severely diseased nu- cellar-clone trees: a 6-year-old pink Marsh grapefruit (Citrus paradisi Macf.) on sour orange (C. aurantium L.) rootstock, a 5-year-old Frost Washington navel sweet orange [C. sinensis (L.) Osb.] on Troyer cit- range [C. sinensis x Poncirus trifoliuta (L.) Raf.] rootstock, and an 8- year-old Campbell Valencia sweet orange on Troyer citrange rootstock. These grapefruit, navel, and Valencia source trees (designated G, N, and V, respectively) all had shown fruit, leaf, and shoot symptoms normally associated with stubborn disease (1). They had been graft-inoculated with virus from trees used in previous experiments; the G source from tree 29-36, and the N and v sources from tree (2-189 (2).  Tree v is also infected by tristeza virus. Other inocula used were columellas and an- thers obtained from naturally infected 5-year-old Frost Valencia trees on trifoliate orange [P. trifoliuta (L.) Raf.] rootstock. Inocula were col- lected October 11, 1965, January 19, 1966, May 3, 1966, and August 15, 1966. The portions of trees G, N, and v used as inocula are indicated in Figure 1. Ten branches were cut from each tree on each collection date to ob- tain all the branch parts used for inocula, except the expanding leaves, 146 PROCEEDINGS of the IOCV columella, and anthers. Grafting procedures were as follows: leaf pieces and wood shields were inserted under flaps of bark; trunk bark patches replaced bark pieces removed from the indicator plants; bud grafts, in- ternodal bark shields, and root bark were inserted into T-cuts; and side grafts were inserted into diagonal clefts in the stem of indicator plants. The upper and lower sides of midveins in leaf pieces were scraped. All grafts were wrapped with plastic tape or rubber bands. Side grafts and portions of supporting stems were enclosed in polyethylene sleeves for YOUNG SIDE GRAFT- 4 cm BUD - 8 0  sq mm MATURE SIDE G INTERNODAL BARK SHIELD 'ROOT BARK - 8 0  rq mm OOT SIDE GRAFT- 3 cm FIGURE 1. Sketch showing kinds of  pieces taken from stubborn trees G (grape- fruit), N (navel), and V (Valencia) for graft-inoculation of indicator plants. 2 weeks. Except for side grafts, 2 contiguous and similar sections from a source tree were used to inoculate each indicator plant. No plant was inoculated with grafts from more than 1 branch or root. Side grafts were limited to 1 per plant; the side graft from each shoot was supplemented by a bud graft from that shoot. JNDICATOR P L A N T S . - M ~ ~ ~ ~  Vinous sweet orange was selected as the indicator variety because it consistently gave a strong and typical reac- tion to stubborn virus. Indicator plants for the October and January series were produced by propagating 1 bud from a Madam Vinous seed- ling on each Rough lemon (C. jambhiri Lush.) seedling at the time of inoculation. Ten replications were made for each type and major source STUBBORN, GREENING, and RELATED DISEASES 147 of inoculum on the October and January dates. Replications of May and August inoculations were changed to 5 inoculations in Rough lemon seedlings budded with Madam Vinous and 5 inoculations in Madam Vinous seedlings. Grafts were examined for vitality after 3-4 weeks. After 4 weeks, plants in the October and January series were bent to force the Madam Vinous buds; after 12 weeks the Rough lemon tops were cut off about 3 cm above the upper grafts. Plants in the May and August series were bent at 3 weeks and cut off at 4 weeks after inocula- tion. A single Madam Vinous shoot was grown near an inoculation site on each indicator plant until it showed symptoms of stubborn disease or attained a height of 1 m. Symptomless shoots 1 m tall were cut back to 20 cm to force growth of a new shoot. Ten non-inoculated control plants for each series were similarly treated. A side graft from each ma- ture shoot used as inocula from trees N and v was grafted into a Rough lemon seedling and grown to serve as an indicator of disease perpetua- tion. All plants were examined weekly for symptoms. The plants were grown in sterile U.C. mix, to which minor elements had been added, in a glasshouse free of insects and Phytophthora spp. harmful to citrus. Temperature ranges in the glasshouse for the 3-month period following inoculation averaged 21-29, 20-31, 20-34, and 19-33'C, respectively, for the October, January, May, and August series. A supplemental experiment compared the effectiveness of bark and wood grafts from an Atwood navel orange tree infected with stubborn disease, vein-enation, and tristeza viruses. Separate wood and bark por- tions of 50 internodal shields from 25 branches were inserted into T-cuts in Hinckley sweet orange seedlings, using one piece of inoculum per plant. Results Survival of bud, bark, and side grafts was nearly 100 per cent. How- ever, both grafts died in 12 per cent of the plants inoculated from ex- panding leaves, and in 25 per cent of those inoculated from mature leaves. All pieces of wood and most columella grafts seemed dead. The possibility of tissue union between indicator plants and grafts that died was not verified; nor was the fate of the anthers determined. REACTION TO VARIOUS INOCULA.--Results are listed by type, source, and date of graft in Table 1. With few exceptions, positive reactions were severe. All inoculated normal plants cut back to 20 cm remained symptomless. Type and source of inocula influenced the probability of reaction but not the incubation period or symptomatology. Grafts from TABLE 1. REACTION OF MADAM VINOUS SWEET ORANGE SEEDLINGS AND BUDLINGS TO INOCULA FROM VARIOUS PARTS OF STUBBORN-DIS- EASED GRAPEFRUIT, NAVEL, AND VALENCIA ORANGE TREES COLLECTED IN OCTOBER, JANUARY, MAY, AND AUGUST Kind of graft Number of indicator plants with stubborn symptomsa Source of inoculum and month grafted Grapefruit Navel Valencia 0ct.b Jan.b MayC Aug.C 0ct.b J a n . V a p  Aug.c 0ct.b Jan.b M a p  Aug.C Leaf patch, young 3 0 0 5 2 7 4 3 0 0 3 5 Leaf patch, mature 0 0 0 0 0 0 0 0 0 0 0 0 Bud 1 0 8 0 1 2 4 1 0 1 5 0 Bark shield, internodal 0 0 4 2 2 1 3 0 0 0 2 0 Wood shield, internodal 0 0 0 0 0 0 0 0 0 0 0 0 Side graft + bud, young 9 4 7 8 7 9 9 2 8 2e 8e 1 Side graft + bud, mature 8 6 9 ge 7 6 10 9e 1 2 10 4 Side graft, rootd 0 0 0 0 2 0 0 0 0 0 0 1 Bark patch, trunk 0 0 0 0 7 0 4 9 0 0 0 4 Bark, rootd 0 0 0 0 0 0 0 0 0 0 0 0 TOTALS 21 10 28 24 28 25 34 30 2 5 28 16 a. Of 10 plants inoculated in each category. b. Indicator plants: Madam Vinous budded on Rough lemon, 10 per treatment. c. Indicator plants: Madam Vinous seedlings, 5 per treatment, and Madam Vinous budded on Rough lemon, 5 per treatment. d. Sour orange rootstock for grapefruit; Troyer citrange for navel and Valencia. e. Only 9 indicator plants grew. STUBBORN, GREENING, and RELATED DISEASES 149 grapefruit transmitted the virus from 36 (90 per cent) of the branches used; those from navel, 37 (93 per cent) ; and those from Valencia, 21  (53 per cent). Combined results from grapefruit, navel, and Valencia inocula show that symptoms developed in leaves and shoots (Fig. 2) of 65 per cent of all plants inoculated with a side graft plus a bud. Symp- FIGURE 2. A .  Madame Vinous sweet orange seedlings 17 weeks after bud-graft in- oculation from stubborn tree N (navel); one seedling is symptomless, the other severely diseased and stunted. B. Immature leaf of inoculated Madame Vinous seedling with typical marginal mottling reaction to stubborn virus. tom expression ranged from 10 per cent of plants inoculated from Valen- cia in October to 95 per cent of plants inoculated from navel orange in May. The per cent of indicator plants reacting to the various types of grafts was as follows: side graft plus bud from mature shoot, 69;  side graft plus bud from young shoot, 63;  bud, 19; expanding-leaf patch, 27; in- ternodal bark shield of mature shoot, 12;  bark patch from scion trunk, 20; and side graft from root section, 3. Inoculation by grafts of columella PROCEEDINGS of the IOCV from green Valencia fruits in October caused reactions in 2 plants (20 per cent) ; no reaction resulted from columella grafts made in January, May, or August. No symptoms appeared in the plants inoculated with mature leaf patches, root bark, wood shields, or anthers. All non-inocu- lated control plants remained normal. These data show that side grafts from shoots were the most effective method of inoculation and that stubborn virus was graft-transmitted from many parts of the tree, but not from wood, mature leaves, or anthers. The failure of many surviving grafts to transmit the virus to indicator plants suggests that the stubborn virus was not present in the graft pieces. PROPAGATIONS.-The shoots produced by side grafts derived from navel branches collected in October, January, May, and August showed symptoms in 7/10, 7/10, 10/10, and 10/10, respectively, and those from Valencia showed symptoms in 2/10, 2/10, 6/10, and 7/10, respectively. Stubborn shoots were produced by side grafts from 34/37 (92 per cent) of the navel branches and from 12/21 (57 per cent) of the Valencia branches from which the virus was concurrently transmitted to Madam Vinous indicators (see above). Stubborn shoots were also produced from side grafts of 5/19 (26 per cent) of Valencia branches from which no transmission to Madam Vinous indicators was apparent. These results confirm the irregular distribution of the virus indicated by the inocula- tion data and discount the possibility that graft-union barriers inter- fered with virus movement into inoculated plants. SEASONAL VARIATION.- -S~~SO~~~ differences in transmission from tree N were small, except that January inoculations with bark from the trunk were ineffective whereas those with young-leaf patches were highly effec- tive. The number of transmissions from grapefruit were similar in Octo- ber, May, and August, but were much lower in January. The number of transmissions from the Valencia varied from 2 in October to 28 in May. Total transmissions from each of the 3 source trees were highest in May and next highest in August. Seasonal ranges in transmission by certain types of grafts, from trees G, N, or V, were as follows: side graft of young shoot plus bud, 50 per cent in January to 80 per cent in May; side graft of mature shoot plus bud, 47 per cent in January to 97 per cent in May; patch of expanding leaf, 17 per cent in October to 43 per cent in August; bud graft, 3 per cent in August to 57 per cent in May; internodal bark shield, 3 per cent in January to 30 per cent in May; bark patch from scion trunk, 0 per cent in January to 43 per cent in August; side graft from roots, 0 per STUBBORN, GREENING, and RELATED DISEASES 151 cent in January and May to 7 per cent in October. These results indi- cate considerable seasonal variation in virus distribution, especially in the Valencia tree, and suggest that distribution within shoots is more general in May than in October or January, that possibly there is more virus in trunks and roots in August and October than in January or May, and that the virus builds up seasonally in young leaves and shoots. SEASONAL EFFECT ON INCUBATION P E R I O D . - A ~ ~ ~ ~  inoculation in Octo- ber, January, May, and August, definite symptoms were first noted at 15, 11, 8, and 8 weeks, respectively. Of the plants reacting in these respective series, 50 per cent developed symptoms within 20, 20, 12, and 12 weeks after inoculation. The rapid development of symptoms during late spring and early summer was associated with high temperatures and extended daylight. The total hours above 25, 30, and 3S°C, respectively, during the 3-month periods following inoculation were 604, 89, and 1 6  hours in October to January; 751, 135, and 14 hours in January to April; 1,019, 618, and 102 hours in May to August; and 907, 415, and 41 hours in August to October. SUPPLEMENTAL E X P E R I M E N T . - S ~ U ~ ~ O ~ ~  symptoms appeared in 35 (70 per cent) of the plants inoculated with internodal bark of Atwood navel. Vein enation was found in 45 (90 per cent) and tristeza in 45 (90 per cent) of the plants. Many plants inoculated with bark grafts de- veloped symptoms of stubborn disease in 6-7 weeks, but none of the 50 plants inoculated with wood shields showed symptoms of any virus dis- ease in 5 months. This evidence indicates that the stubborn virus is absent from or is not readily transmitted from young wood of infected branches. Discussion This work supports the conclusion of Fawcett (6) that stubborn dis- -. ease is a virosis and suggests that the virus multiplies in young leaves and shoots. The possibility that the pathogen might be an obligate fungus is not eliminated, but is not supported by the failure of patches from mature leaves and of wood shields with cambium to transmit the disease. The failure of many inoculated indicator plants and propagations from diseased trees to develop symptoms in six months does not, by itself, prove they have escaped infection, because stubborn-disease symptoms are variable ( I ) ,  and the possibility of mild or delayed reactions exists. However, in previous experiments by the authors, plants that remain normal in the glasshouse four months after inoculation rarely, if ever, develop stubborn symptoms in two to three years in the orchard. In one PROCEEDINGS of the IOCV experiment, four inoculated sweet orange seedlings that were symp- tomless in the glasshouse are vigorous and indistinguishable from three non-inoculated seedlings after 43 months in the orchard. Conversely, eight seedlings that reacted within four months in the glasshouse are stunted and continue to show stubborn symptoms in the field. From time to time, relatively mild reactions have been noted in plants inoculated from trees having severe symptoms. However, all attempts to transmit stubborn disease virus from non-inoculated plants, from normal inocu- lated plants, and from apparently normal branches of diseased trees have failed to produce even mild symptoms. The overall results of inoculation and indexing work from 1957 through 1966, involving several thousand indicator plants and about 100 stubborn source trees, clearly indicate that stubborn virus is distributed irregularly in many trees and may be lost, at least temporarily, from many portions of the tree. The very low percentage of transmission from buds grafted in August, October, and January contrasts sharply with the May results, implying that stubborn virus is inhibited or lost in most buds during much of the year. Conclusions and Suggestions The extensive replication and seasonal repetition, including springtime collection of graft pieces, is very important if not required for indexing budwood trees for stubborn disease. Side grafts appear to be the most dependable method of inoculation for indexing work. The possibility of transmitting or perpetuating stubborn virus is reduced during the winter months. Literature Cited 1. CALAVAN, E. C., and CARPENTER, J. B. 1965. Stubborn disease of citrus trees re- tards growth, impairs fruit quality and decreases yields. Calif. Citrograph 50: 86-87, 96, 98-99. 2. CALAVAN, E. C., and CHRISTIANSEN, D. W. 1961. Stunting and chlorosis induced in young-line citrus plants by inoculations from navel orange trees having symptoms of stubborn disease, p. 69-76. In W. C. Price red.], Proc. 2nd Conf. Intern. Organization Citrus Virol. Univ. Florida Press, Gainesville. 3. CALAVAN, E. C., and CHRISTIANSEN, D. W. 1965. Rapid indexing for stubborn disease of citrus. Phytopathology 55: 1053. 4. CARPENTER, J. B. 1959. Present status of some investigations on stubborn dis- ease of citrus in the United States, p. 101-107. In J. M. Wallace Ced.1, Citrus Virus Diseases. Univ. Calif. Div. Agr. Sci., Berkeley. 5. CASSIN, J. 1%5. Research on stubborn disease in Morocco, p. 2M-206. In W. C. Price red.], Proc. 3d Conf. Intern. Organization Citrus Virol. Univ. Florida Press, Gainesville. STUBBORN, GREENING, and RELATED DISEASES 153 6. FAWCETT, H. S. 1946. Stubborn disease of citrus, a virosis. Phytopathology 36: 675-677. 7. HILGEMAN, R. H. 1961. Response of stubborn-infected trees to iron chelates, p. 84-92. In W. C. Price [ed.], Proc. 2nd Conf. Intern. Organization Citrus Virol. Univ. Florida Press, Gainesville. 

Distribution of Stubborn Disease Virus in Trees of Citrus sinensis and C. paradisi at Different Seasons

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