Detection of Citrus Greening-infected Citrus in South Africa Using a DNA probe and Monoclonal Antibodies

Monoclonal antibodies directed against Indian, Chinese and South African greening originally developed in France, were used to detect the greening organism in South African citrus plants by indirect Enzyme-Linked Immunosorbent Assay and immunofluorescence. In addition, a Taiwanese DNA probe was used to evaluate cross reaction with South African greening by dot blot hybridization. Monoclonal antibody results were variable, but most positive reactions were obtained using the Indian monoclonal 2D12 (Poona strain) which cross-reacted with greening infected material obtained from hotter citrus growingareas in South Africa. No reaction was found between the Taiwanese DNA probe and South African greening. Citrus greening is an economically important disease, not only in South Africa (l7), but also internationally (2). Two forms of greening have been recognised viz. Asian and African greening (8). The former is more widespread and occurs at lower altitudes where ambient average temperatures vary between 30-35C (3), as compared to the more localized African greening which occurs a t higher altitudes with average temperatures between 20-23C (16). Asian greening is transmitted by Dia-phorina citri (Kuwayama) and African greening by Trioxa erytreae (Del Guercio) (2). I t is generally accepted that the two forms of greening are caused by different strains of the same pathogen (23). Currently, control in South Africa focuses mainly on reduction of inoculum by management practices such as vector control, pruning of infected branches, removal of infected trees and using healthy nursery material (4). Early detection of greening in the nursery or in newly planted orchards is therefore essential. Due to the non-specific nature of leaf symptoms, greening can often be confused with mineral deficiency, root rot or other stress-relatedleaf symptoms. Hitherto, only unsuccessful attempts a t isolating and culturing the greening organism (GO) on artificial media (5), have been made. However, numerous indirect molecular approaches to detect the GO in situ have been developed (8, 12, 13, 19, 22, 23). With these indirect approaches, greening-infected material was identified and used, either as antigen to raise monoclonal antibodies (mAbs) (8, 12, 13, 19), or to extract greening-specific DNA for development of DNA probes (20, 23). In 1987 two mAbs were produced in France against Indian greening (8) and later three against South African and five against Chinese greening (7). This paper reports on the preliminary testing of South African greening material by Enzyme Linked Immunosorbent Assay (ELISA) and immunofluorescence (IF) using mAbs produced in France against Indian, Chinese and South African greening. In addition, a Taiwanese DNA probe was also tested with South African greening material collected at various citrus producing areas by dot blot hybridization. MATERIALS AND METHODS Evaluation of Indian greening monoclonal antibody 2D12 by means of ELISA. A total of 70 greening-infected citrus leaf and twig samples were collected from several citrus producing areas in South Africa. This included samples from the severely affected areas of Pretoria (central Transvaal) and Mooinooi (western Transvaal), and from a moderately affected area, Letaba (northern Transvaal) (14). Asymptomatic leaves were collected from Delta Valencia on rough lemon rootstock (DVIRL) maintained in the greenhouse, University of Pretoria which Citrus Greening Disease 225 were originally bud-inoculated with greening-infected material obtained from the Philippines (Table 1). Bark was stripped from twigs, midribs excised from leaves and samples cut into small pieces before 2 g of material per sample were powdered in liquid nitrogen. Ten ml sterile phosphate buffered saline, pH 7.0 (PBS) was added, mixed thoroughly on a vortex and incubated for 1 hr at 37C. The slurry was centrifuged for 1 min at 11 000 g at 4C in a Sorvall superspeed cooled centrifuge. Supernatants were collected and frozen at -20C until required. Extracts were also made from the bark of a symptomatic greening-infected branch collected at Mooinooi from at 5 cm and 10 cm below a fruit with symptoms, and from the fruit columella and the midrib from the leaves above the f i t . Samples were tested three times to verify results. An indirect ELISA (21) was used to evaluate the efficacy of monoclonal 2D12 (Indian greening) obtained from M. Gamier and J. M. Bove (INRA, Bordeaux, France) with above mentioned extracts as solid phase antigens. Samples were considered positive if ELISA signals were greater than the sum of three times background and three times standard deviation with a corresponding coefficient of variation ofless than 15%. Evaluation of Asian and African greening monoclonal antibodies using immunofluorescence. Greening-infected citrus leaf samples were collected from the Pretoria, Mooinooi and Letaba areas, as well as Zebediela (central Transvaal), Nelspruit (moderately affected areas), and Malelane (eastern Transvaal; no natural spread area) (Table 2). Asymptomatic leaves were also collected from greenhouse maintained DVIRL trees inoculated with greening-infected material collected from Mooinooi, Nelspruit, Letaba, Zebediela and Pretoria. Healthy citrus leaves were also collected from DV/RL certified disease-free trees, maintained in the greenhouse, University of Pretoria. These trees were originally obtained from the South African citrus plant improvement program, Uitenhage. Symptomatic leaves with either yellow vein, mottling, green islands or secondary "Zn-deficiency-type" symptoms, were selected for sectioning (Table 2). Eight sections of 15-20 km were cut witha Reichert Jung cryomicrotome and mounted on a &well multitest slide (Sterilin). One well per slide was used as a negative control and was treated with Sp2/0 supernatant instead of monoclonal supernatant. Sections were treated with 70% methanol for 5 min, TABLE 1 EVALUATION OF ANTIBODY 2D12 (INDIA) FOR CROSS-REACTION WITH SOUTH AFRICAN GREENING BY MEANS OF ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA) No. of samples Percentage Cultivar tested Symptomsb positivec Letaba (moderate area) Mooinooi (severe area) Pretoria (severe area) phillipinesd Valencia