Molecular Identification Of Mild And Severe Isolates Of Citrus Tristeza Virus From Pummelos

Citrus tristeza virus (CTV), a member of the genus Closterovirus, is one of the most destructive and economically important virus pathogens of citrus in the world. CTV and its efficient vector, brown citrus aphid (Toxoptera citricida Kirkaldy), are widely distributed in China. China is the number 1 pommelo producer both in yield and in planting areas in the world, and will perhaps produce even more in the near future due to higher economic returns. Unfortunately, most pummelo cultivars and their rootstocks are susceptible to CTV, especially the stem pitting tristeza virus, which has emerged to be a threat to the development of pommelo industry since mid-1990s' when pommelo cultivation became increasingly popular in China. Mild strain cross protection (MSCP),developed in western countries, has been proven to be one of the most effective ways in preventing sensitive citrus varieties from CTV damage where both severe CTV strains and brown citrus aphid co-exist. It is a high priority to investigate, isolate and identify the severe and mild CTV strains from infected pommelos in order to facilitate the application of MSCP in Chinese pommelo industry.In this paper, 450 pummelo samples, including many varieties collected from different areas, were first detected for the existence of CTV by Direct Tissue Blot Immuno-Assay(DTBIA). Reverse transcription polymerase chain reaction (RT-PCR), restriction fragment length polymorphism (RFLP), and Bi-Directional reverse transcription polymerase chain reaction (BD-PCR) were then used to primarily identify the 126 CTV isolates from DTBIA positive samples. Six typical CTV isolates were further characterized by single aphid transmission and biological indexing. Twenty nine subisolates obtained from single aphid transmission were then analyzed by RFLP, BD-PCR, and single strain conformation polymophism (SSCP). Six of them and the 2 isolates failed in single aphid transmission were sequenced for their p23 and p25 genes. Their sequences were then compared with those of the mild CTV isolate PB61 from Australia and the completely sequenced CTV isolates T30, T36, T385, SY568, VT, NUAGA in the world. The following conclusions were drawn:1. The single p25/Hinf1 RFLP groups were dominant in all CTV-positive samples, accounting for 83.3% of the total CTV infected samples, among which CTV isolates with p25/Hinf1 RFLP group 6 occupied 69.8%, indicating that is the predominant CTV strain infecting pummlos.2. BD-PCR analysis showed that: 84.1% of CTV isolates yielded a 612 bp DNA fragment, which is characteristic of the atypical groups. Whereas the rest of the isolates yielded 239 bp or 450 bp DNA fragment, characteristic bands of the mild and severe groups respectively.3. CTV isolate S051, S052 were close to the mild CTV strainsT30, T385, and PB61, according to the results of p25/Hinf 1 RFLP,BD-PCR assay and the p23, p25 sequence analyses. In addition, biological indexing by indicator showed that S051, S052 were probably mild strains too.4. S053, S054, S055, S056, S057, and S058 might be new CTV strains since their p25/Hinf1 RFLP analysis, BD-PCR assay and sequences of p25 and p23 were different to CTV isolates T30, T36, T385, SY568, VT, NUAGA and PB61 as shown in the phylogenetic tree .