Detection of the Asian Strains of the Greening BLO by DNA-DNA Hybridization in Indian Orchard Trees and Malaysian Diaphorina citri Psyllids

A BLO DNA fragment (In 2.6) from a Poona (India) strain of the greening organism (GO) has been cloned and used as a probe for the detection of the BLO in field trees and psyllids in India, in February 1992. Fifty three samples from Orissa, Rajasthan, Uttar Pradesh, and Myssore areas, were tested by dot hybridization using a2P labelled probes. Most of the samples were also examined by electron microscopy (EM) for the presence or absence of the BLO. The results show that DNA-DNA hybridization is able to reliably detect the GO in all regions of India with a sensitivity at least equivalent to that of EM. The probe was also used to detect the GO in individual Diaphorina citri insects collected on infected trees in Delhiand Bangalore. Avery low percentage of the psyllids (less than 1%) was found contaminated with the GO. This was also the case with psyllids collected in Malaysia in May 1992 (5% infection). On the contrary, when D. citri was collected in Malaysia in September 1991,39% of the insects were found infected with the GO, indicating a variation in the percentage of contaminated D. c i te insects during the year. These studies show that probe In 2.6 is a powerfull reagent to detect the GO in field trees as well as in individual insects, and will be most useful in epidemiology studies. Following the report of Fraser et al. (3) and the demonstration by Capoor et al. (2) that the greening pathogen was vectored in India by the Asian citrus psylla, Diaphorina citri, it became generally admitted that greening was widespread throughout India. This belief was essentially based on symptomatology, particularly foliar zinc deficiency symptoms. Leaf mottle, a better symptom of greening, was generally overlooked. In the early 1970s, Lafleche and Bove (9) showed by electron microscopy (EM) that the greeningpathogen was not a virus but a prokaryotic organism, first described as mycoplasma-like, but later recognized as bacterium-like (1,5,12). The first EM visualization of this newly discovered bacterium-like organism (BLO) in leaf material from India was reported in 1971 (12), following the visit of a group of citrus virologists to S. P. Capoor in Poona after the IOCV conference in Japan in 1969. A Mosambi sweet orange seedling experimentally infected with a Poona strain of the greening pathogen, was taken to Bordeaux, France, where it was shown to contain a phloem-restricted BLO morphologically identical to that seen in greening-affected leaves from South Africa, Reunion island and the Philippines (1). Later, Indian workers in Bangalore also detected the greening BLO by EM in Indian citrus leaves (11). The Mosambi sweet orange seedling infected with the Poona strain of the BLO was used in Bordeaux as a source of inoculum. Through periodic graft transmissions to healthy sweet orange seedlings, the Poona BLO strain has been maintained in the Bordeaux glasshouse, ever since 1969. These graft inoculated seedlings carry only the greening BLO, as the initial Mosambi sweet orange seedling was infected with the BLO through D. citrivector transmission. In particular, they are free of citrus tristeza virus. In 1983, African and Asian strains of the BLO were transmitted experimentally to periwinkle plants by dodder (4), and BLO-infected periwinkle leaves rather than citrus material, have been used thereafter for the development of BLO-specific detection reagents. Since 1987, monoclonal antibodies (MAS) have been obtained, first against the Poona strain of the BLO, then against a BLO strain from Fujian (ChiCitrus Greening Disease na) and one from Nelspruit (South Africa) (6,7,8). Their use resulted in the discovery that different serotypes of the BLO occurred, even in nearby orchards. However, each MA reacted almost exclusively with the BLO strain used for immunization (homologous strain) and, hence, these MAS, even when combined in mixtures, were too strain specific fordiagnosticpurposes. More recently, DNA restriction fragments of the Poona BLO strain have been cloned and sequenced (14, 15). A 2.6 kbp fragment was identified as the rplKAJLrpoBC operon, a wellknown operon in eubacteria, coding for four ribosomal proteins of the large ribosomal subunit, and for proteins P and p-' of RNA polymerase. As ribosomal proteins as well as their genes are phylogenetically conserved, it was not surprising that the 2.6 kbp fragment, when used as a probe in Southern and dot-blot hybridizations, was able to detect not only the homologous Poona BLO strain, but also the seven other Asian strains tested (14). The probe was even able to detect the Nelspruit strain, the only South African strain tested, when hybridization was carried out at a stringency lower than that for the Asian strains (15). These results were obtained with greenhouse-grown plants maintained a t the most favorable conditions for good symptom expression. We wanted to evaluate the adequacy of the 2.6 kbp DNA probe to detect the BLOin Indian orchard trees often growing under suboptimal conditions. This evaluation required that field trees unambiguously known to be infected by the BLO be available as positive control trees. At the beginning of this work, no such trees were known in India. As EM was the only technique by which infection of a tree by the BLO could be relatively quickly established, we have analyzed many of the orchard trees by both EM and dot-blot hybridization (dbH). Previous EM work in our laboratory has shown that the best material to detect the greening BLO consists of midveins from leaves showing blotchy mottle as described by McClean and Schwarz (10). This symptom has been found on greening-affected trees in all geographical areas where African or Asian greening occurs, and it is shown by all cultivars affected, even though mottle is sometimes less pronounced on mandarin than on sweet orange leaves. Therefore, occurrence of leaf mottle was an important factor in selecting orchard trees for BLO detectionby EM