Study On The Mild Strain Cross Protection Against Citrus Tristeza Virus In China

Tristeza caused by Citrus tristeza virus (CTV) is an economically important citrus disease, which is mainly spreaded by aphid and bud-grafting. Various isolates of CTV and the most efficient vector brown citrus aphid (BrCA, Toxoptera citricida Kirkaldy) are widely distributed in China. In the past 20 years, some superior cultivars of pummelo, sweet orange and mandarin hybrid have been developed to some extent in Chongqing and other areas in China, as the result, stem-pitting tristeza problem on pummelos and some susceptible cultivars of sweet oranges has become serious since mid-1990s'. So far, according to the experience obtained in developed countries, mild strain cross protection (MSCP) has been proved to be the most effective way to control this problem where severe CTV isolates and BrCAs co-exist.Nowadays, all effective mild isolates used in MSCP were derived from remained trees with normal appearance in orchards where severe CTV isolates were endemic. CTV exists as a large number of distinct strains differing in biological characterization in citrus. The mixture of CTV strains often occurs in citrus trees, graft-transmission and aphid-transmission may change the composition of CTV isolates. The effectiveness of the mild strains used in MSCP was influenced by regional condition and host factors. As the results, before applying MSCP, mild isolates should be segregated by single aphid transmission and regional condition should be considered. *For better cognition on the CTV population structure and the relationship between molecular and biological characterization, 72 CTV samples collected from five provinces in China were studied by biological indexing, p25/Hinf I restriction fragment length polymorphism (RFLP) groups assay (the 25kDa protein encoded by the p25 gene is the major coat protein which encapsidates most of the CTV genome) , multiple molecular markers (MMM) and bi-directional reverse transcription polymerase chain reaction (BD-PCR) assay. Finally, p25/Hinf I RFLP groups assay was used to demonstrate the composition of CTV isolates in Chongqing, the influence of citrus varieties (cultivars) and single aphid transmission on the composition of CTV isolates. Combining with the biological characterization, p25/Hinf I RFLP groups assay and MMM assay by RT-PCR were used to detect the challenge severe CTV isolates in Symons sweet orange seedlings or Fenghuang pummelo grafted onto Chandler pre-immunised with mild isolates, and challenged by 50 BrCAs transmission. In order to determine the relative effectiveness of various source plants and CTV isolates in the aphid transmission of CTV, as well as whether apterous adults and alate adults differed greatly in their ability as vectors, 10 CTV isolates collected from field were used to evaluate the transmissibility by BrCAs from different citrus host. The study has provided foundation data for better understanding the interaction between citrus hosts and CTV isolates, and for better controlling severe stem-pitting tristeza problem by MSCP in the future .The main results are as fellows:1. Characterization of CTV isolates by indicators and molecular biological methodsSeventy-two CTV isolates collected from five provinces in China were studied, using biological indexing, p25/Hinf I RFLP groups assay, MMM assay and BD-PCR assay. The results suggested that(1) The mixture of severe stem-pitting isolates was found to be dominant in the field. CTV isolates with p25/Hinf I RFLP group 3 and p23/BD-PCR group I , III were the main epidemic ones (p23 gene encode the 23 kDa protein localized predominantly in the cytoplasm of CTV-infected cells, a RNA-binding protein, and associates with the range of host) , and most CTV isolates were with the mixture of T30 and VT genotypes.(2) CTV isolates with single p25/Hinf I RFLP group 4, or 5, or T30 genotype might associate with mild isolates, and single p23/BD-PCR II might correlate with severe CTV isolates.(3) Comparing the three molecular biological methods, p25/Hinf I RFLP groups assay could provide most effective way to distinguish different CTV isolates in the field. More accurate identification of strain mixtures in the field and understanding of the biological traits of the isolates may be achieved by applying the three molecular detection methods together.Eleven CTV isolates and four typical foreign CTV isolates were analyzed by p25/Hinf I RFLP groups assay and the sequences of p23 and p18 genes (encodes the 18kDa protein, needed for the movement of CTV in sour orange), demonstrated that CTV isolates derived from the same location or have similar p25/Hinf I RFLP group(s) have higher sequence identity matrix, and these derived from the same location have closer homogeneity.Two hundreds and forty-one CTV isolates collected from 10 different locations in Chongqingwere analyzed by p25/Hinf I RFLP groups, the results revealed that the mixture of CTV strainsoften occurs in the field trees. In this study, about 90% of the isolates analyzed contain thosestrains with p25/Hinf I RFLP groups 3, 6 and 1. Sweet orange samples contained more p25/HinfI RFLP groups than these of other citrus varieties. CTV isolates with single p25/Hinf I RFLP group 3 or 6, and the mixing of p25/Hinf I RFLP groups 1 and 3, p25/Hinf I RFLP groups 1 and 6 were the epidemic isolates in sweet orange. CTV isolates with single p25/Hinf I RFLP group 6 and the mixing of p25/Hinf I RFLP groups 1 and 6 were dominant types in pummelo. CTV isolates dominant in mandarin hybrids were with single p25/Hinf I RFLP group 3, and with the mixing of p25/Hinf I RFLP groups 1 and 3.2. Influence of citrus varieties (cultivars) on the polymorphism of CTV isolates2.1 Influence of six citrus varieties on the composition of CTV isolate TR-L514Eighteen su-bisolates derived from CTV isolate TR-L514 were obtained by graft inoculation from Jin cheng sweet orange to six citrus varieties. Biological indexing was used to detect the pathogenicity of the sub-isolates and TR-L514. RFLP and single-strand conformation polymorphism (SSCP) assay on p25 gene were conducted, and p23 gene was sequenced. In Jin cheng, TR-L514 was detected to contain p25/Hinf I both groups 1 and 6, and showed more complex SSCP patterns than those in other four citrus varieties. Sub-isolates in Ponkan and Shatian pumello have single p25/Hinf I RFLP group and more simple SSCP patterns than TR-L514 has. However, similar polymorphism to TR-L514 occured, after they were graft-inoculated back in Jin cheng again. The results indicated that citrus variety would change the composition of CTV isolate, and Jin cheng might be more suitable to CTV propagation. The sequence diversity of p23 gene might result in the adaptation to CTV in different citrus hosts.2.2 Influence of 28 sweet orange cultivars on the polymorphism of CTV isolates CT14 and CT36CTV isolates CT14 and CT36 were graft-inoculated in28 sweet orange cultivars respectively. CT14 with single p25/Hinf I RFLP group 3, didn't change its p25/Hinf I RFLP group or p25/SSCP patterns in different sweet orange cultivars .However, as CT36 with the mixture of p25/Hinf I RFLP groups 1,3 and 6 was graft-inoculated in different sweet orange cultivars, its sub-isolates had different composition of p25/Hinf I RFLP groups or p25/SSCP patterns.3 Segregation of CTV by single BrCAFour severe isolates and six mild isolates were used to evaluate the transmission efficiency of CTV from Jin cheng, Feng-huang pummelo and Mexican lime to Jin cheng plants by single BrCA, respectively. Genes p20 (involved in the formation of inclusion bodies in the infected cells, and may associate with aphid transmission) and p25 were sequenced to determine the possibility of them as CTV-BrCA specific interacters during the transmission. And p25/Hinf I RFLP groups assays of 10 CTV isolates and 161 sub-isolates obtained via single aphid transmission were conducted, the results indicated that(1) Citrus varieties markedly influenced the transmissibility of CTV by BrCA, and Jin cheng was more likely to be the best host for CTV acquisition than Mexican lime and Feng-huang pummelo.(2) The pathogenicity of CTV isolate might not absolutely correlate with its transmissibility by BrCA. And alatae and apterae of BrCA had no significant effect on transmission rates.(3) CTV isolates with p25/Hinf I RFLP group 3 had higher aphid transmissibility than those with other p25/Hinf I RFLP groups.(4) p25/Hinf I RFLP groups assay and p25/SSCP patterns both demonstrated that single aphid transmission was an effect way to separate CTV isolates from the mixed infection.4 Distribution of CTV isolates CT13, CT37 and TR-L514 in Mexican lime on trifoliate orangeTwo virus-free buds of Mexican lime were grafted onto the stem of trifoliate orange seedlings 10cm and 15cm above the soil surface of containers, respectively. Inoculum buds from the Jin cheng seedlings, with CTV positive tested by direct tissue blot immuno-assay (DTBIA), were graft-inoculated between the buds of Mexican lime. Days past-inoculation, DTBIA, RT-PCR and p25/Hinf I RFLP groups assay were used to detect CTV in trifoliate orange and in the shoots of Mexican lime periodically. The results showed that(1) CTV isolates were not detected by DTBIA in the trifoliate orange, but their movement was detected to be acropetally and basipetally in both Mexican lime shoots and new shoots of trifoliate oranges by RT-PCR.(2) The composition of CT13 with single p25/Hinf I RFLP group was not changed after graft-inoculated in trifoliate orange. But TR-L514 and CT37 with mixture of p25/Hinf I RFLP groups graft-inoculated in trifoliate oranges had less complex p25/Hinf I RFLP groups than these in Mexican limes.(3) Inoculum buds graft-inoculated onto indicators directly could achieve more apparent symptoms rather than onto triloliate oranges.5 Effectiveness of seven mild CTV isolates in cross protection experiments p25/Hinf I RFLP groups assay, MMM assay and biological characterization were approached to evaluate the effectiveness of five Chinese mild CTV isolates and two overseas protective isolates in cross protected Symons sweet orange seedlings after challenged with severe stem-pitting isolate CT4, and the effectiveness of one mild isolate in cross protected Fenghuang pummelo on Chandler pummelo rootstock after challenged with severe stem-pitting isolate CT3 via 50 BrCAs per plant. The results suggested that(1) p25/Hinf I RFLP groups assay and MMM assay could provide effective ways to distinguish the composition of CTV isolates in the pre-immunized plants. Application of these two methods and biological characterization to analyze the mild CTV isolate together could result in accurately understanding the effectiveness of cross protection.(2) Seven CTV mild isolates used in this study were all experimentally proven to have somehow protective effectiveness against severe stem-pitting CTV isolares. CTV isolates CT9 and CT11 could partially protect sweet oranges against severe stem-pitting isolate CT4, and CT9 could partially protect pummelos against severe stemp-pitting isolate CT3.