Witches' Broom Disease of Lime Trees: Monoclonal Antibody and DNA Probes for the Detection of the Associated MLO and the Identification of a Possible Vector

A total of 18 monoclonal antibodies and 17 DNA probes specific for the witches'broom disease of lime (WBDL)-MLO have been produced. MA 1E2 and probes IlH, IlOH and I28H were used for the detection of the MLO in plants and/or insects in the Sultanate of Oman and the United Arab Emirates. Among 30 different leafhopper species tested, only one species, Hishimonus phycitis, reacted positively both with the MA and the DNA probe (IlOH) used. In addition, leafioppers of this species were captured exclusively on lime trees. This leafhopper is known as the vector of the MLO of eggplant little leaf disease in India. H. phycitis is therefore a candidate insect-vector of WBDL-MLO, although, experimental transmission of the disease has not yet been obtained. Witches' broom disease of lime (WBDL) appeared in the Northern coastal plain of the Sultanate of Oman, near the border with the United Arab Emirates (UAE)probablyin the 1970s. Since then, the disease has spread within Oman and extends all along the coastal plain from the border with the UAE to Muscat. Since 1989 it has also appeared in the oases of the coastal mountain range, but it has not yet been observed in the southern part of the country near Salalah. The disease appeared in the UAE in 1988-1989 (10) (1993 data in Note Added in Proof). In 1986, we showed that a mycoplasmalike organism (MLO) was associated with the disease and that the MLO could be transmitted to periwinkle plants and back to lime by dodder (2, 3). Experimental graft-transmissions of the MLO to lime and Troyer citrange were also successful but not to sweet or sour orange seedlings (11). Recently, transmissions of WBDL-MLO to lemon, rough lemon, and trifoliate orange seedlings have also been obtained (unpublished data). WBDL-infected periwinkle extracts were used to produce monoclonal antibodies (MAS) specific for the WBDL-MLO. In two lymphocyte fusions, a total of 18 WBDL-MLO-specific MAS were obtained (10,ll). In addition, partial purification of WBDL-MLODNA resulted in the production of 17 recombinant plasmids containing an insert of WBDL-MLO-DNA (10). The nature of the causal agent and the rapid spread of the disease strongly suggest that the disease is actively transmitted by an insect vector. In this paper, we report the use of WBDL-specific MAS and DNA probes for the detection of the MLO in plant and insects in the Sultanate of Oman and in the UAE. MATERIALS AND METHODS Plant samples. The specificity of MAS and DNA probes used in this work was determined by testing them against a collection of MLOs maintained in periwinkle plants in a greenhouse in Bordeaux as well as against the MLOs of sesame phyllody and sunhemp phyllody (kindly provided by E. Seemuller, Biologische Bundensanstalt, Heidelberg, Germany). Possible cross reactions with three citrus pathogens, Spiroplasma citri, the greening BLO and tristezavirus, were also examined. Plant samples from more than a hundred trees were collected in the Sultanate of Oman and tested by ELISA for the presence of WBDLMLO as described below. Leaf samples from nine trees were also received from Stubborn Witches Broom 343 the UAE and tested by ELISA and DNA-DNA hybridization. Collection of leafhoppers. Leafhoppers were captured in the Sultanate of Oman during a mission from April 29 to May 13,1991 with a D-Vac aspirator in lime orchards, fields cultivated with other crops and non-cultivated areas. The insects were separated into species and tested either individually by DNA-DNA hybridization or in batches of five to ten insects by double antibody sandwich ELISA (DAS-ELISA). DNA-DNA hybridization. Individual leafhoppers were crushed with a glass rod directly onto a Nylon N + membrane previously soaked into 5 SSC. The DNA was denatured by incubating the membrane for 30 min with 0.4N NaOH. The membrane was air dried, sealed in a plastic bag and carried back to Bordeaux, France for hybridization. Hybridization was performed as described by Garnier et al. (11) with probe IlOH labeled with 32Pa dCTP. This probe was selected because it gave the strongest hybridization signals. DAS-ELISA was done as previously described (12). Five to teninsects (according to their size and availability) were crushed with 300~1 of PBS buffer in a glass homogenizer. The liquid phase was collected and added to the wells of an ELISA plate previously coated with MA 1E2 at 10p.glml. The alkaline phosphatase conjugate of MA 1E2 was used at a 111000 fold dilution. Plant extracts were prepared bygrinding leaf midribs in two volumes of PBS in a Polytron homogenizer. The homogenate was filtered through four layers of cheese cloth and deposited in the wells of the ELISA plate as before. The ELISA reactions had to be read visually, as no ELISA plate reader was available in Oman.