Studies On The Resistance Effect Of Three Mild CTV Isolates Against Severe CT4

Tristeza caused by Citrus tristeza virus (CTV) is an economically important citrus disease in the world. Mild strain cross protection (MSCP) is the most effective method to control the stem pitting tristeza virus. To evaluate the resistance effect of mild CTV isolates on severe ones is essential to gain the mild CTV isolates that can be applied in MSCP. Studies on mild CTV isolates' resistance against a severe one in this paper would facilitate the application of MSCP in Chinese citrus industry to some extent.HinfI/RFLP analysis of RT-PCR products from genes p20, p23 and p25 of three mild CTV isolates CT10, CT12 and CT17 as well as one severe isolate CT4 collected by the National Citrus Virus Exclusion Center (NCVEC) were carried out. The three genes of the four CTV isolates were sequenced and analyzed with HinfI-restriction enzyme map and sequence identity. Four pairs of primers were designed and used to establish the RT-PCR method in order to distinguish the severe and mild isolates. In combination with observation of stem-pitting symptom in different treated citrus plants, the resistance effect of the 3 mild isolates on the severe one CT4 was studied. In addition, the Hinf I RFLP and SSCP of gene p25 of 9 CTV isolates collected from sweet orange were comparatively analyzed in different citrus cultivars by graft-inoculating different CTV isolates and transferring from Xinhui sweet orange to Fenghuang pummelo. The following results are achieved:(1) The severe CTV isolate CT4 was found to be mixture of p25/Hinf I RFLP group I and group III, whereas the mild isolate CT10 and both of the mild isolates CT12 and CT17 belong to p25/Hinf I RFLP groups IV and I , respectively. CT10 was not able to be cut by the restriction enzyme Hinf I on p20 gene while p20/Hinf I RFLP profiles of CT17 and CT12 both had two DNA bands of 180bp and 370bp, and p20/Hinf I RFLP profile of CT4 had four DAN bands of 100bp, 180bp, 280bp and 370bp.(2) The sequence analysis on genes p20, p23 and p25 indicated that CT17 of the three mild CTV isolates had the most identity to the severe isolate CT4.(3) The primer pair T30K17 (f, r) and T30 5' (f, r) was used to identify the severe isolate CT4 from both of the mild isolates CT17 and CT12 by RT-PCR.(4) Molecular monitoring of CT4 has been performed by the RFLP analysis combined with the RT-PCR method established in this study.(5) one, three, five and eight months after the severe CTV isolate CT4 attacked, the percentages of CT17-inocuated seedlings with detection of CT4 were 9.1%, 27.3%, 35.4 % and 45.6%, respectively, whereas that of CT10-inoculated seedlings were 25%, 50%, 87.5%, and 100%, and CT12-inoculated seedling were 40%, 50%, 80%, >80%. All the three mild CTV isolates prevented the severe isolate CT4 from its infection or delaying its infection. Of them, CT17 was the most effective one. This result was consistent with the observation of stem pitting symptoms in eight months CT4-inoculated seedlings.(6) Analyzing the Hint I RFLP and SSCP of gene p25 of 9 CTV isolates collected from sweet orange after those isolates were graft-inoculated and transferred from Xinhui sweet orange to Fenghuang pummelo, it was found that the Hinf I RFLP group of the isolates CT90 and CT20 in Fenghuang pummelo was the same as in Xinhui sweet orange while RFLP groups of other six isolates detected as mixed groups in Xinhui sweet orange changed into single group in Fenghuang pummelo. Likewise, DNA bands of p25/SSCP of the isolate CT90 in Fenghuang pummlo was found to be the same as that in Xinhui sweet orange, whereas DNA bands of the other eight isolates in Fenghuang pummlo were fewer than that in Xinhui sweet orange. It could result from inhibition of the replication of some composition of above eight CTV isolates in Fenghuang pummelo.