Spread of Citrus Tristeza Virus in a Heavily Infested Citrus Area in Spain P. Moreno, J. Piquer, J. A. Pina, J. Juarez and M. Cambra ABSTRACT. Spread of Citrus Tristeza Virus (CTV) in a heavily infested citrus area in Southern Valencia (Spain) has been monitored since 1981. Two adjacent plots with 400 trees each were selected and tested yearly by ELISA (enzyme-linked immunosorbent assay). One of them was planted to 4-yr-old Newhall navel orange on Troyer citrange and the other to 8-yr-old Marsh seedless grapefruit on the same rootstock. Both had been established using virus-free budwood. In 1981, 98.7% of the Newhall navel plants indexed CTV-positive and by 1984 all of them were infected, whereas only 17.8% of the Marsh grapefruit indexed CTV-positive in 1981, and 42.5% were infected in 1986. This is an indication that grapefruit is less susceptible than navel orange to tristeza infection under the Spanish field conditions. Wild plants of 66 species collected in the same heavily tristeza-infested area were also tested by ELISA to find a possible alternate non-citrus host. CTV was not found in any of the more than 450 plants analyzed. Index words. virus spread, ELISA, noncitrus hosts. Tristeza was first detected in virus diffusion under various environ- Spain in 1957 and since then has mental conditions. In this paper we caused the death of about 10 million present data on CTV spread in a trees grafted on sour orange and the heavily infested area. A survey progressive decline of an additional among wild plants growing in the several thousand hectares of citrus on same citrus area was also undertaken this rootstock. The disease was ini- to search for some noncitrus natural tially observed at the Ribera Alta hosts of CTV. area (Southern Valencia), but new foci, probably originating through un- MATERIALS AND METHODS controlled movement of infected bud- wood, successively appeared in other In 1981, two adjacent plots with spots. At present, tristeza can be 400 trees each were selected at El found with variable incidence in most Realengo, a citrus farm located at the of the citrus areas (2). Ribera Alta (Southern Valencia). This Three aphid species (Aphis gos- farm is within the area where tristeza sypii Glover, Aphis citricola Van der was first detected. One of them was Goot and Toxoptera aurantii (Boyer planted to 4-yr-old Newhall navel de Fonscolombe)) have enabled trans- orange on Troyer citrange and the mission of CTV in Spain, with vari- other to &yr-old Marsh seedless able efficiency depending on virus iso- grapefruit on the same rootstock. late and vector species (4). These Both of them had been established aphids, particularly A. gossypii and using virus-free budwood. A. citricola, are thought to be respon- Tristeza spread was monitored by sible for natural spread of tristeza (4) yearly indexing all trees by ELISA. from the new foci established through Each tree was sampled taking two infected budwood. young shoots of the last flush from op- Decline of trees in tristeza-in- posite sides of the tree. Shoots were fested orchards has been occurring at trimmed, the clippings placed into a variable rate depending on years plastic tubes and homogenized in ca. and citrus areas (6). Nevertheless, no 10 volumes of an extraction buffer data on the actual rate of virus spread using a Polytron Kinematics homog- under field conditions in Spain are enizer. Extracts were tested by a available. standard ELISA double antibody In 1981 several plots were selected sandwich procedure (1,ll) using CTV in different citrus areas to monitor antisera (879 or R4, kindly provided Tenth ZOCV Conference by Dr. S. M. Garnsey and Dr. D. J. plots were also sampled and tested by Gumpf, respectively) or monoclonal ELISA in the same conditions as cit- antibodies specific to CTV obtained as rus trees. In some cases, extracts of described elsewhere (11, 12). wild plants were also examined by im- The rate of CTV increase was munoelectron microscopy (IEM) as analyzed using the expressions Ln (XI described by Garnsey et al. (3). 1-x) or Ln (111-x) (lo), where x is the per unit incidence of the disease at a RESULTS given time. Tristeza incidence around the ex- The amount and distribution of in- perimental plots was estimated by oculum around the monitored plots is random sampling and testing by shown in figure 1. At the beginning of ELISA 2.5% of the citrus trees grown the experiment, the proportion of within a band 150 m wide around the CTV-infected trees in the 150 m band plots (120 trees in each case). CTV surrounding the grapefruit plot was incidence in citrus plantings of differ- 69 * 9% (P 5 0.05). Corresponding ent age or scion-rootstock combina- values for the navel plot were 70 r tion was also estimated separately. 9% respectively. Wild plants of a number of species Variation of CTV incidence with growing around the experimental time in both plots is summarized in SALUSTIANA/TROYER 100% CTV GRAPEFRUIT/TROYER - 45% CTV NEWHALL/TROYER 92% CTV Monitored plot NEWHALL/TROYER Grapft. /Troyer 100 % CTV Monitored plot Newh./Troyer VEGETABLES 0% CTV GRAPEFRUIT/SOUR ORANGE 43% CTV I Fig. 1. Scion-rootstock combinations and tristeza incidence in citrus plantings around the experimental plots. Tristeza and Related Diseases 73 TABLE 1 in both cases. Thus, natural spread of RATE OF CITRUS TRISTEZA VIRUS tristeza in grapehit, in our condi- SPREAD IN SOUTHERN VALENCIA (SPAIN) tions, may be explained by the expo- nential or compound interest model Newhall Marsh (transformation Ln (xll-x)) as well as navel orangesz grapefruitz by the linear or single interest model Years (% infe~ted)~ (% infe~ted)~ (transformation Ln (Ill-x) (10). CTV incidence in the Newhall navel plot was too high from the be- ginning as to make any mathematical consideration about the rate of dis- ease increase in the following years. The species tested in the survey "Each plot contained 400 trees. of CTV among vegetation in the cit- YAsdetermined by ELISA. rus orchards are listed below with the number of plants sampled in brackets. table 1. All navel plants were infected AMARANTHACEAE: Amaranthus by tristeza seven years after being sp. [2], A. blitoides [21, A. planted in the field and more than gracilis [29], A. hybridus [I]; 98% were already infected in the APOCY NACE AE : Nerium oleander fourth year. In contrast, less than [3]; ARACE AE : Arisamm vulgare 18% of the grapefruit trees were in- [70]; CARIOPHYLLACEAE: Dian- fected in the eighth year and more thus valentinus [4], Stellaria media than a half of the trees were still [I]; CHENOPODIACEAE: Beta sp. healthy 13 yr after planting. [22], Chenopodium album [71, Vibo Figure 2 shows the tristeza in- spinosum [7]; COMPOSITAE: An- crease in the grapefruit plot (data dryala integrifolia [Z], Calendula ar- from table I), using two logarithmic vensis [5], Centaurea aspera [8], transformations of the per unit inci- Chondrilla juncea 121, Conyxa am- dence. Our data can be reasonably bigua [2], Cychorium intibus [Z], well fitted to a straight line with Inula viscosa [6], Phagnalon either of the two transformations mpestre 131, Senecio vulgaris [31, with a correlation coefficient of 0.97 Sonchus oleraceus [4], S. tenerrirnus Fig. 2. Progress of tristeza in a grapefruit plot in Southern Valencia (Spain). Per unit inci- dence of tristeza (x) plotted as Ln xll-x (*) and as Ln 111-x (+). Tenth ZOCV Conference [7]; CONVOLVULACEAE: Convol- p-nitrophenyl phosphate in dieth- vulus althaeoides [I], C. arvensis anolamine buffer, pH 9.6) the solution [141; CRUCIFERAE: Diplotaxis turned yellow in a few minutes, erucoides [18], Lepidium draba 151, suggesting that the positive ELISA L. gramingolium [5], Lobularia reaction observed was probably due mritima [lo], Sinapis nigra [3]; to unspecific binding of enzyme pres- DIPSACACEAE: Cephalaria leuc- ent in Arisarum extracts that con- antha [I], Scabiosa mritima [I]; verted the substrate to nitrophenol SCROPHULARIACEAE: Linaria and caused a false positive reaction. viscosa [I], Verbascum sinnuatum [7], Veronica persica [I]; EUPHOR- DISCUSSION AND BIACEAE: Euphorbia sp. [3], Eu- CONCLUSIONS phorbia segetalis [I]; GERANI- The estimated incidence of ACE AE: Erodium cicutarium [I], E. tristeza in the citrus plantings close malacoides [6], E. moschatum [4]; to the monitored plots is similar to GRAMINEAE: Cynodon dactylon that obtained for this area in a general [I], Poa annua [I], Setaria sp [23]; survey throughout the Valencian HYPERICACEAE: Hypericum per- Community (2). About 95% of citrus foratum [I]; LABIATAE: Sideritis trees (mainly oranges and mandarins) angustgolia [I], Thymus vulgaris were estimated to be infected in the, [I]; LEGUMINOSAE: Glycirhixa Rafelguaraf district, where our ex- glabra [ll], Ononis minutissima perimental plots are located. At El [I], Psoralea bituminosa [36]; Realengo farm almost all orange or LILIACEAE: Allium sp. [2], Allium mandarin trees are CTV infected. In ampeloprasum [6], Asparagus sp. addition, the aphid species vectoring [2]; MALVACEAE: Malva sp. tristeza feed mainly on these species [lo], M. sylvestris [17]; OXALIC- and much less on grapefruit. Thus, ACEAE: Oxalis corniculata [3]; the inoculum potential of CTV around POLYGONACEAE: Polygonum av- the experimental plots was very high. iculare [2], Rumex crispus [9]; POR- The different rates of CTV spread TULACACEAE: Portulaca oleracea measured suggest that navel trees [6]; RANUNCULACEAE : Clematis (and probably any sweet orange) are jlammula [14]; ROSACEAE: Rubus much more susceptible than grape- ulmijlolius [lo], Sanguisorba minor fruit trees to CTV infection, at least [5]; RUBIACEAE: Gallium ap- under our field conditions (virus purine [lo]; RUTACEAE: Ruta strains and vector species present). angustgolia [I]; SOLANACEAE: Additional support for this conclusion Solanum nigrum [lo]; UMBEL- comes from the fact that CTV inci- LIFERAE: Foeniculum vulgare [I]; dence in orange trees around the ex- URTICACEAE: Urtica urens [3].