A New Graft Transmissible Disease Found in Nagami Kumquat L

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A New Graft Transmissible Disease Found in Nagami Kumquat L. Navarro, J. A. Pina, J. F. Ballester, P. Moreno, and M. Cambra

ABSTRACT. An undescribed graft transmissible disease has been found on Nagami kumquat. Three types of symptoms have been observed: 1) vein clearing on Pineapple sweet orange, Troyer citrange, sour orange, Marsh grapefruit, Orlando tangelo, Dweet tangor and Alemow, but not on Mexican lime, Etrog citron, Cleopatra mandarin, rough lemon, Eureka lemon, Volkamer lemon, trifoliate orange, Nules clementine and Parson's special mandarin; 2) stem pitting on Etrog citron, but not on the other species and hybrids; and 3) graft incompatibility of Nagami kumquat on Troyer citrange, but not on rough lemon. Vein clearing symptoms were more severe in seedlings grown at 18- 25OC than at 27-32OC. Stem pitting was induced only at 18-25OC. Some kumquat plants obtained by shoot-tip grafting in vitro were compatible with Troyer citrange, and did not induce vein clearing, but still produced stem pitting. These data suggest the presence of more than one pathogen on the original plants. Preliminary electron microscopy studies have shown the presence of some virus-like particles about 800 nm long in extracts of infected Troyer citrange and sweet orange plants. Diseased kumquats gave negative reactions by ELISA using four different tristeza antisera.

A Citrus Variety Improvement cortis, but it also induced vein Program (CVIPS) was started in clearing on Pineapple sweet orange. Spain in 1975 to recover virus-free This type of vein clearing symp- plants from all commercial varie- toms on sweet orange are usually ties and other citrus species, valu- not induced by tristeza in Spain. able for research work or for a Furthermore, SRA-153 kumquat germplasm bank (1, 2, 3). In this indexed negative for tristeza on program, Nagami kumquat from Mexican lime and by ELISA. Thus, two origins was selected and in- the possibility existed that vein dexed for virus content. One of the clearing induced on Pineapple by kumquat sources was selected from the two kumquat sources was due a private variety collection at Al- to a new pathogen. In addition, hama, Murcia, Spain, (Alhama both kumquats grew well on rough kumquat), and the other was lemon but were apparently incom- introduced from the Station des patible with Troyer citrange. Recherches Agrumicoles at Saint In this paper we describe ex- Giuliano, Corsica, France, (SRA- periments directed to determine 153, kumquat), kindly provided by the characteristics of the agent or Dr. Vogel). Both kumquats were agents inducing vein clearing on propagated on rough lemon and Pineapple and to establish a Troyer citrange in the greenhouse possible relationship between those and graft-inoculated to Mexican agents and the incompatibility be- lime, Pineapple sweet orange, tween Nagami kumquat and Troy- Etrog citron and Parsons Special er citrange. In most experiments mandarin to test for tristeza, pso- described below we used SRA-153 rosis and/or concave gum, exocortis kumquat to avoid possible inter- and xyloporosis, respectively. Al- ference from tristeza and exocortis hama kumquat indexed negative which were present in Alhama for xyloporosis, positive for tris- kumquat. teza and exocortis and gave con- spicuous vein clearing on Pineapple. EXPERIMENTS AND RESULTS SRA-153 kumquat indexed negative Citrus species and varieties sus- for xyloporosis, tristeza and exo- ceptible to Nagami kumquat in- Other Graft-Transmissible Agents oculum. Four seedlings of each of the citrus species and varieties listed in table 1 were graft-inoculated with two bark patches from SRA-153 kumquat and incubated in a greenhouse at 18-25°C. New leaves were observed in each flush for symptoms, and eight months after inoculation plants were peeled to look for stem pitting. Vein clearing symptoms, similar to those induced by tristeza on Mexican lime, were induced not only on Pineapple (fig. I),but also on Comuna sweet orange, Troyer citrange (fig. 2), Alemow, Marsh grapefruit, Orlando tangelo, Dweet tangor, sour orange, and Cleo- patra mandarin. Stem pitting symptoms, similar to those caused by tristeza, were induced only in Etrog citron (fig. 3). Vein clearing usually appeared Fig. 1. Pineapple sweet orange leaf in half to fully expanded leaves in showing vein clearing induced by SRA- the first flush (one month or less 153 kumquat inoculum. after grafting) and sometimes later disappeared. In subsequent flushes, TABLE 1 SYMPTOMS OBSERVED ON DIFFER- ENT CITRUS SPECIES AND VARIE- TIES GRAFT-INOCULATED WITH NAGAMI KUMQUAT SRA-153.

Symptoms* Citrus species Vein Stem and varieties clearing pitting

Pineapple sweet orange Comuna sweet orange Troyer citrange Alemow Marsh grapefruit Orlando tangelo Dweet tangor Sour orange Cleopatra mandarin Etrog citron Volkamer lemon Mexican lime Rough lemon Eureka lemon Parson Special mandarin Nules clementine Trifoliate orange Fig. 2. Troyer citrange leaf showing vein clearing induced by SRA-153 kum- *+, positive; -, negative. quat inoculum. 236 Ninth IOCV Conference

kumquat on Troyer citrange were incubated in the cool and warm greenhouses respectively. Vein clearing symptoms on Pineapple were more intense at cool than at warm temperature (table 2). Stem pitting on Etrog citron only appeared in plants incubated at the cool greenhouse. Incompatibility symptoms of kumquat on Troyer citrange were similar in plants incubated at both temperatures. Transmission experiments. a. Graft transmission. Vein clearing of sweet orange and stem pitting of citron were readily transmitted by grafting. All inoculated citron plants showed pitting, and about 75 percent of sweet orange plants, inoculated in different experiments, showed vein clearing. Fig. 3. Stem pitting on etrog citron b. Dodder transmission. A stems. Left, uninoculated control; center, citron plant inoculated with SRA- inoculated with SRA-153 kumquat; right, 153 kumquat and showing stem inoculated with tristeza. pitting, was connected by Cuscuta vein clearing was rarely observed. subinclusa Dur. and Hilg. to eight Stem pitting on Etrog citron some- healthy citron plants for two times appeared as early as two months. Five months later none of months after grafting. the eight citron plants showed Influence of temperature on stem pitting. symptom expression. Four plants c. Mechanical transmission of Pineapple sweet orange and four from citrus to citrus. Mechanical of Etrog citron, were graft-inocu- transmission from citron to citron lated with SRA-153 kumquat and was assayed by cutting citron incubated in a cool greenhouse stems 100 times with a knife con- (18-25°C). A similar group of in- taminated each time by cutting inoculated plants was incubated in a fected plants. To inoculate the nega- warm greenhouse (27-32°C). Two tive control a healthy instead of an groups of ten plants of SRA-153 infected citron plant was cut with TABLE 2 INFLUENCE OF INCUBATION TEMPERATURE OF INDICATOR PLANTS ON SYMPTOM EXPRESSION INDUCED BY INOCULUM FROM NAGAMI KUMQUAT SRA-153

Symptoms* Incubation Vein Stem temperature Indicator clearing pitting

18-25OC Pineapple ++ - 27-32OC Pineapple + - 18-25OC Etrog Citron +++ 27-32OC Etrog Citron - -

*-, negative ; + ,mild ; + + , moderate ; + + + ,severe. Other Graft-Transmissible Agents the knife. Positive control was a hama and SRA-153 kumquats on citron plant graft-inoculated from Troyer citrange. In all cases grafted the same source of inoculum. plants showed incompatibility None of the four citron plants symptoms. Growth of kumquat mechanically inoculated showed buds was very poor, with small symptoms, whereas the positive sized and yellow leaves (fig. 4) controls showed stem pitting. that usually dropped. Budunions d. Mechanical transmission to were abnormally weak. herbaceous hosts. Plants of Rut- To elucidate if graft incom- gers tomato, Blackeye Cowpea, patibility of Nagami kumquat on Long vert maraichere cucumber, Troyer citrange was due to the Physalis floridana Rydb. and presence of pathogens or was of Xanthi tobacco were dusted with genetic origin, a few plants were carborundum and then mechanical- obtained from SRA-153 by shoot- ly inoculated with leaf extracts of tip grafting (4). Graft success, Troyer citrange, Pineapple sweet growth in vitro of shoot-tip grafted orange or Dweet tangor with vein plants and growth of those plants clearing symptoms. Extracts were after transplanting to soil were prepared by mortar and pestle normal and no symptoms of in- using three different buffers (0.1 M compatibility were observed. Three citrate, pH 5.8; 0.1 M phosphate, of the micrografted plants were in- pH 6.5; and 0.1 M Tris-HC1, pH dexed by graft inoculation to 4 7.9) to each of which was added 2 Troyer citrange, 4 Pineapple sweet per cent mercaptoethanol. Nine to orange, 4 Dweet tangor and 4 twenty plants were inoculated with Etrog citron plants. All inoculated the extract from diseased plants citron plants showed stem pitting in each case, and five to ten more were inoculated with similar extracts from healthy citrus plants. No difference was found between plants inoculated with extracts from healthy or diseased citrus plants. Movement of the infectious agents within citrus plants. Twelve Pineapple seedlings were graft- inoculated with SRA-153 kumquat bark patches and immediately girdled above or below the inoculum graft. None of six plants girdled above the inoculum showed vein clearing in leaves from flushes produced above the girdle, whereas three of them did show vein clearing in new leaves produced below the girdle. Two of the six Pineapple seedlings girdled below the inoculum showed vein clearing in upper leaves but none of them showed symptoms in leaves produced below the girdle. Fig. 4. Growth of SRA-153 kumquat Incompatibility studies. Several on Troyer citrange 7 months after graft- trials were done to propagate Al- ing. Ninth IOCV Conference whereas none of the Troyer cit- duced stem pitting on Etrog range, Pineapple sweet orange or citron. Dweet tangor plants showed vein Since incompatibility between clearing. kumquat and Troyer citrange was To confirm the compatibility of associated with the presence of the kumquat plants obtained by shoot- agent inducing vein clearing, an tip grafting on Troyer citrange, ten experiment was done to find out buds from a micrografted plant whether this agent was also able and ten more from the original to induce incompatibility between SRA-153 were propagated on Troy- Pineapple sweet orange and Troy- er citrange. All plants from the er citrange. micrografted source grew vigor- Healthy Pineapple sweet orange ously whereas plants from the buds were propagated on ten seed- original source showed severe in- lings of Troyer citrange previously compatibility symptoms, (fig. 5). graft-inoculated with bark patches Total growth of micrografted of SRA-153 kumquat. Ten healthy plants 7 months after propagation seedlings of Troyer citrange were averaged 200 cm per plant whereas grafted with Pineapple buds in- growth of plants from the original fected with the vein clearing agent source averaged only 0.5 cm per and ten more were budded with plant. healthy Pineapple. Another Nagami kumquat Growth of healthy and in- found later in Sevilla, that had oculated plants was similar and no been introduced from California, incompatibility symptoms were ob- was compatible on Troyer citrange. served. Indexing of this kumquat dis- Electron microscopy studies. closed that it was free of tristeza, Formvar-coated grids were floated it did not induce vein clearing on for 10 minutes on a drop of leaf Pineapple sweet orange but it in- extract (200 mg of leaf homogen- ized in 1ml of 0.1 M Tris-HC1 buffer pH 7.6) or, alternatively, leaf pieces were dipped for 1 min in one drop of 0.1 M phosphate buffer pH 7.8. Fully expanded leaves with vein clearing symptoms and equiva- lent leaves from healthy control plants were used. Grids were washed by floating them for 1 min on a drop of distilled water, stain- ed for 4 min with 2% phospho- tungstic acid adjusted to pH 7, washed for 1 min with distilled water and blotted with a filter paper. Grids were observed in a JEOL JEM 100s electron microscope at 80 KV. Four to six openings of each grid were thoroughly scanned at a magnification of 21,000X and at least the same number of observa- Fig. 5. Nagami kumquat on Troyer tions were made on negative citrange 7 months after grafting. Left, controls. original SRA-153 kumquat; right, SRA- 153 kumquat obtained by shoot-tip A few virus-like particles about graft in^ in vitro. 800 nm long were found in some Other Graft-Transmissible Agents preparations from Troyer citrange Troyer citrange, Cleopatra man- and sweet orange with vein clear- darin and Comuna and Pineapple ing and from kumquat obtained by sweet oranges. shoot-tip grafting from SRA-153 These data indicate that the kumquat. No such particles were kumquat disease is not caused by observed in negative controls. common Spanish tristeza strains. ELISA tests. To confirm the ab- Results of ELISA tests confirm sence of tristeza virus in SRA-153 that the causal agent of this new kumquat the following groups of disease is not related to tristeza. plants were tested by ELISA using Compatibility between kumquat 4 antisera (873 and 879 provided by obtained from shoot-tip grafting S. M. Garnsey, R4 provided by and Troyer citrange suggests that D. J. Gumpf and M1 obtained in incompatibility of SRA-153 and our laboratory) : a) SRA-153, Al- Alhama kumquats was not genetic, hama, Sevilla, SRA obtained by but was due to the presence of shoot-tip grafting, and SRA-153 some pathogen. The ability of seedling kumquats ; b) Mexican micrografted kumquat to induce lime, Etrog citron Arizona 861-S1, stem pitting on Etrog citron, but alemow and Pineapple sweet not vein clearing on Pineapple and orange plants inoculated with bark other hosts is an indication that at patches of SRA-153 kumquat or least two pathogens were present tristeza T-300 and c) noninoculated in the original kumquat and that controls. 'the incompatibility between Na- Only Alhama kumquat and gami kumquat and Troyer is as- plants of group b inoculated with sociated with the agent inducing tristeza gave positive reactions. vein clearing. The later discovery No difference was observed be- of a different kumquat source tween the rest of the plants and which was compatible with Troy- the negative controls with any of er citrange and also induced stem the four antisera. pitting on Etrog citron, but not vein clearing, supports the associ- DISCUSSION ation between the incompatibility A new graft transmissible dis- and the vein clearing agent. ease inducing incompatibility be- The pathogens inducing vein tween Nagami kumquat and Troyer clearing and stem pitting were citrange has been found. Inoculum readily transmitted from citrus to from affected kumquats induced citrus by grafting. Dodder and me- stem pitting on Etrog citron and chanical transmission from citron vein clearing in several citrus to citron were unsuccessful; how- species and varieties (table 1). Al- ever, these experiments were though symptoms are similar to carried out before there was any those induced by tristeza the host evidence of the presence of two range of both diseases are differ- pathogens in kumquat. Thus, the ent. Common Spanish tristeza absence of symptoms on inoculated strains and kumquat inoculum in- citron plants only indicates that duce vein clearing on C. rnacrophyl- the stem pitting agent was not la and stem pitting on Etrog citron, transmitted. No data is available but only tristeza strains induce on dodder and mechanical trans- stem pitting on Mexican lime and missibility of the vein clearing vein clearing on Mexican lime and agent. All attempts to mechanical- Etrog citron. Only kumquat in- ly transmit the agents of the kum- oculum induced vein clearing on quat disease to a limited range of sour orange, Orlando tangelo, herbaceous hosts were also unsuc- Dweet tangor, Marsh grapefruit, cessful. Failure to transmit those 240 Ninth IOCV Conference agents to noncitrus hosts may indi- different indicators and observa- cate that, like most citrus virus and tion of virus-like particles on dis- virus-like pathogens, they have a eased but not on healthy plants is narrow host range. an indication that at least one virus Results of the girdling experi- may be implicated in the disease ments indicate that the agent in- complex. ducing vein clearing on Pineapple So far, incompatibility induced is restricted to the outer tissues of by this complex has been found the stems of infected plants. These affecting Nagami kumquat on experiments were also carried out Troyer citrange, but experiments before evidence of the presence of underway show that infected kum- two pathogens in kumquat was quat may also be incompatible on available. Thus, no conclusion can other rootstocks. Although no in- be drawn about the localization of compatibility was observed on Pine- the stem pitting agent within the apple sweet orange grafted on infected plant. Troyer citrange inoculated with A few virus-like particles were SRA-153 kumquat, other com- observed in extracts of infected mercial varieties could become in- plants that were not found in compatible. In this case, the disease healthy plants. The presence of could become a serious problem these particles in different hosts for the citrus industry. may be an indication that they are The agents inducing vein clear- associated with the disease. In ad- ing and stem pitting have been dition, finding of those particles in found in Nagami kumquat from micrografted plants suggests that different origins. This is an indica- they may be related to the agent tion that these pathogens may be inducing stem pitting on citron but present in several countries and it not with the vein clearing or in- could be an explanation why certain compatibility agents (s) . lines of kumquat have been con- The fact that the kumquat dis- sidered incompatible on Troyer ease is readily graft-transmitted, citrange. the type of symptoms produced on

LITERATURE CITED 1. NAVARRO, L. 1976. The Citrus Variety Improvement Program in Spain, p. 198-203. In: Proc. 7th Conf. IOCV. IOCV, Riverside. 2. NAVARRO, L., J. F. BALLESTER, J. JUAREZ, J. A. PINA, J. M. ARREGUI, R. BONO, L. FERNANDEZ DE C6RDOVA, and C. ORTEGA 1980. The Citrus Variety Improvement Program in Spain (CVIPS) after four years, p. 289-294. In: Proc. 8th Conf. IOCV. IOCV, Riverside. 3. NAVARRO, L., J. F. BALLESTER, J. JUAREZ, J. A. PINA, J. M. ARREGUI, and R. BONO 1981. Development of a program for disease-free citrus budwood in Spain. 1981 Proc. Int. Soc. Citriculture 1: 70-73. 4. NAVARRO, L.. C. N. ROISTACHER, and T. MURASHIGE 1975. Improvement of shoot-tip grafting in vitro for virus-free citrus. J. Amer. Soc. Hort. Sci. 100: 471-479.