UC Riverside International Organization of Citrus Virologists Conference Proceedings ( 1957-2010 ) Title Huanglongbing ( Greening ) in Vietnam : Detection of Liberobacter asiaticum by DNA-Hybridization with Probe in 2 . 6 and PCR-Amplification of 16 S Ribosomal DNA Permalink

Leaf samples were collected from 55 citrus trees from north, central and south Vietnam in January-February 1995, and analyzed by PCR amplification, DNA hybridization and immunofluorescence for the presence of the Huanglongbing (HLB) (greening) liberobacter. Fortyeight of the 55 samples hybridized with probe In 2.6 which is specific for Liberobacter asiaticum. No hybridization was obtained with probe As 1.7, specific for L. africanum. Forty-five of 47 samples analyzed by PCR gave the 1,160 bp amplicons expected from the liberobacter-specific primers used, and on Xbal digestion, the amplicons gave the two restriction fragments (640 bp and 520 bp) specific for L. asiaticum. The combined use of hybridization and PCR amplification was able to detect L. asiaticum in 53 of the 55 samples tested. Fourteen of 48 samples tested gave a positive immunofluorescence reaction with monoclonal antibodies 10A6 as well as 1A5, known to react with certain L. asiaticum serotypes only. Liberobacters were detected by electron microscopy in four samples tested. These samples were also found to be positive for L. asiaticum by hybridization and PCR. Finally, Hamlin sweet orange seedlings graft-inoculated with budwood sticks from four trees, which were later found positive for L. asiaticum by hybridization and PCR, developed symptoms. These results show that HLB is widespread throughout Vietnam. Symptoms of cristacortis, previously unrecorded in Vietnam, were also seen. As Huanglongbing (HLB) (greening) is widespread throughout China and South-East Asia, it is not surprising that the symptoms of the disease have been reported from various regions of Vietnam where Diaphorina citri Kuwayama, the psyllid vector of the disease agent is known to be present (5). We have recently developed techniques to detect the Asian HLB agent, "Candidatus Liberobacter asiaticum", in citrus: DNA hybridization with probe In 2.6, specific of L. asiaticum (1, 10, ll), and PCR amplification with L. asiaticum-specific primers 011 and OI2C (6, 7). Similar techniques have also been developed for the specific detection of the African HLB agent, "Candidatus L. africanum" (6, 7, 9). We have applied these methods to citrus leaf samples that we collected during a survey in Vietnam in January and February 1995. Some of these samples were also used for detection of the liberobacter by electron microscopy and immunofluorescence. Budwood sticks, in addition to leaf samples, were collected from some of the trees and used for graft transmission assays of the HLB agent. MATERLALS AND METHODS Leaf samples, designated 1 to 55, were collected from 55 different citrus trees in various regions of north, central and south Vietnam from January 25 to February 14, 1995 (Table 1). All the trees had HLB or HLBlike symptoms, except tree 1. Attention was paid, whenever possible, to collect leaves affected by mottle, as this symptom is one of the most reliable ones for HLB diagnosis. The leaf samples were washed in tap water, surface dried and mailed by express mail to the INRA laboratory in Bordeaux. Before mailing, the leaf samples were kept in a refrigerator at 4°C for not more than 8 days. Some of the first leaf samples collected in northern Vietnam (samples 9,11,13, 14, 18, 19, 23) and one sample from the south (sample 39) underwent ferTABLE 1 2 DNA HYBRIDIZATION WITH PROBE IN 2.6, PCR AMPLIFICATION OF 16s RIBOSOMAL DNA AND IMMUNOFLUORESCENCE FOR THE DETECTION OF LIBEROBACTER ASIATICUM IN CITRUS CULTIVARS FROM VARIOUS PROVINCES OF VIETNAM $ S No. of positive hybridizaNo. positive PCR samNo. positive hybridization or Positive immunofluor2 Sample designation tion samples/Total no. ples/Total no. of samPCR samples/Total no. of escence with MA 1OA6 d Location (sample no.) samples analyzed ples analyzed samples analyzed and 1A5 (sample no.)