The Citrus tristeza virus (CTV) uses 3 silencing suppressor genes, p20, p23 and p25, to resist the attacks from its Citrus hosts. Inactivating these genes is therefore obviously a potential defensive option in additio...The Citrus tristeza virus (CTV) uses 3 silencing suppressor genes, p20, p23 and p25, to resist the attacks from its Citrus hosts. Inactivating these genes is therefore obviously a potential defensive option in addition to the current control strat-egies including aphid management and the use of mild strain cross protection. In this study, we cloned partial DNA frag-ments from the three genes, and used them to construct vectors for expressing hairpin RNAs (hpRNAs). To facilitate the formation of hpRNAs, the constructs were introduced in a loop structure. Fol owing transformation of sour orange (Citrus aurantium) with these constructs, 8 p20 hpRNA (hp20) and 1 p25 hpRNA (hp25) expressing lines were obtained. The 7 hp20 transgenic lines were further characterized. Their reactions to CTV were tested fol owing inoculation with CT14A and/or TR-L514, both of which are severe strains. Results showed that 3 lines (hp20-5, hp20-6 and hp20-8) were completely resistant to TR-L514 under greenhouse conditions for no detectable viral load was found in their leaves by PCR. However, they exhibited only partial suppression of TR-L514 under screen house conditions since the virus was detected in their leaves, though 2 months later compared to non-transgenic controls. Further tests showed that hp20-5 was tolerant also to CT14A under screen house conditions. The growth of hp20-5 was much better than others including the controls that were concurrently chal enged with CT14A. These results showed that expressing p20 hpRNA was sufifcient to confer sour orange with CTV resistance/tolerance.展开更多
Citrus tristeza virus （CTV） exists in citrus as a large number of distinct strains differing in biological characters. The control strategies such as mild strains cross protection （MSCP） require a clear understand...Citrus tristeza virus （CTV） exists in citrus as a large number of distinct strains differing in biological characters. The control strategies such as mild strains cross protection （MSCP） require a clear understanding of the characterization of CTV. For better understanding of the structure of CTV population and the relationship between molecular and biological characterization, 72 CTV samples collected from five provinces in China were studied, using biological indexing, p25/Hinf I restriction fragment length polymorphism （RFLP）, multiple molecular markers, and bidirectional RT-PCR assay. 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 cause of epidemics, and most CTV isolates were found to be the mixture of T30 and VT genotypes. More accurate identification of strain mixtures in the field and better understanding of the biological traits of the isolates may be achieved by applying the three molecular detection methods simultaneously.展开更多
Citrus tristeza virus （CTV） causes economically important losses to the citrus industry worldwide. Mild strain cross protection （MSCP） against tristeza has hardly been practised due to mixed infection of different...Citrus tristeza virus （CTV） causes economically important losses to the citrus industry worldwide. Mild strain cross protection （MSCP） against tristeza has hardly been practised due to mixed infection of different CTV-strains and little background of its molecular biology in China. For better cognition on CTV, 192 sweet orange samples collected from eight provinces （Chongqing, Sichuan, Fujian, Hunan, Guangxi, Yunnan, Guangdong and Jiangxi） were tested by direct tissue blot immuno-assay （DTBIA）, and 158 of them were tested positively, which therefore were subjected to coat protein gene （CPG）/Hinf Ⅰ restriction fragment length polymorphism （RFLP） analysis. Sample bulks were compared between Chongqing and Fujian by some statistical data, including ratios of single infection and mixed infection to local samples, proportions of CTV isolates with single RFLP groups, and rates of each RFLP group. The simplified analysis of samples from the other six provinces were then conducted. This study suggests that CTV isolates with CPG/Hinf Ⅰ RFLP groups Ⅲ and Ⅰ are the main epidemic ones in China, and mixed infection of CTV in fields are popular. Based on observation of severity of stem-pitting symptom in field trees, CTV isolates with CPG/Hinf Ⅰ RFLP groups Ⅲ and Ⅰ caused severe stem-pittings in sweet oranges in China.展开更多
Citrus tristeza virus （CTV） is one of the most economically important citrus viruses and harms the citrus industry worldwide. To develop reliable and effective serological detection assays of CTV, the major capsid p...Citrus tristeza virus （CTV） is one of the most economically important citrus viruses and harms the citrus industry worldwide. To develop reliable and effective serological detection assays of CTV, the major capsid protein （CP） gene of CTV was expressed in Escherichia coli BL21 （DE3） using the expression vector pET-28a and purified through Ni＊-NTA affinity chromatography. The recombinant protein was used to immunize BALB/c mice. Four hybridoma cell lines （14B10, 14Hll, 20D5, and 20G12） secreting monoclonal antibodies （MAbs） against CTV were obtained through conventional hybridoma technology. The titers of MAb-containing ascitic fluids secreted by the four hybridoma lines ranged from 10-6 to 10.7 in indirect enzyme-linked immunosorbent assay （ELISA）. Western blots showed that all four MAbs could specifically react with CTV CP. Using the prepared MAbs, dot-ELISA, Tissue print-ELISA, and triple antibody sandwich （TAS）-ELISA were developed to detect CTV in tree nurseries and epidemiological studies. The developed dot-ELISA and TAS-ELISA methods could detect CTV in crude extracts of infected citrus leaves with dilutions of 1：2560 and 1：10, 240 （w/v, g/mL）, respectively. Tissue print-ELISA was particularly useful for large-scale field sample detection, mainly owing to its simplicity and lack of sample preparation requirements. The field survey revealed that CTV is prevalent on citrus trees in the Chongqing Municipality, Jiangxi Province, and Zhejiang Province of China. The coincidence rate of serological and RT-PCR test results reached more than 99.5%. The prepared MAbs against CTV and established sensitive and specific serological assays have a significant role in the detection and prevention and control of CTV in our country.展开更多
基金supported by the International Science & Technology Cooperation Program of China (2012DFA30610)the National Natural Science Foundation of China (30571291)the Special Fund for Agro-Scientific Research in the Public Interest, China (201203075-07)
文摘The Citrus tristeza virus (CTV) uses 3 silencing suppressor genes, p20, p23 and p25, to resist the attacks from its Citrus hosts. Inactivating these genes is therefore obviously a potential defensive option in addition to the current control strat-egies including aphid management and the use of mild strain cross protection. In this study, we cloned partial DNA frag-ments from the three genes, and used them to construct vectors for expressing hairpin RNAs (hpRNAs). To facilitate the formation of hpRNAs, the constructs were introduced in a loop structure. Fol owing transformation of sour orange (Citrus aurantium) with these constructs, 8 p20 hpRNA (hp20) and 1 p25 hpRNA (hp25) expressing lines were obtained. The 7 hp20 transgenic lines were further characterized. Their reactions to CTV were tested fol owing inoculation with CT14A and/or TR-L514, both of which are severe strains. Results showed that 3 lines (hp20-5, hp20-6 and hp20-8) were completely resistant to TR-L514 under greenhouse conditions for no detectable viral load was found in their leaves by PCR. However, they exhibited only partial suppression of TR-L514 under screen house conditions since the virus was detected in their leaves, though 2 months later compared to non-transgenic controls. Further tests showed that hp20-5 was tolerant also to CT14A under screen house conditions. The growth of hp20-5 was much better than others including the controls that were concurrently chal enged with CT14A. These results showed that expressing p20 hpRNA was sufifcient to confer sour orange with CTV resistance/tolerance.
基金supported by National Natural Science Foundation of China(30471205).
文摘Citrus tristeza virus （CTV） exists in citrus as a large number of distinct strains differing in biological characters. The control strategies such as mild strains cross protection （MSCP） require a clear understanding of the characterization of CTV. For better understanding of the structure of CTV population and the relationship between molecular and biological characterization, 72 CTV samples collected from five provinces in China were studied, using biological indexing, p25/Hinf I restriction fragment length polymorphism （RFLP）, multiple molecular markers, and bidirectional RT-PCR assay. 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 cause of epidemics, and most CTV isolates were found to be the mixture of T30 and VT genotypes. More accurate identification of strain mixtures in the field and better understanding of the biological traits of the isolates may be achieved by applying the three molecular detection methods simultaneously.
基金supported by the National Natural Science Foundation of China(30471205)under the program"Comparison Research on Strains of Citrus tristeza virus."
文摘Citrus tristeza virus （CTV） causes economically important losses to the citrus industry worldwide. Mild strain cross protection （MSCP） against tristeza has hardly been practised due to mixed infection of different CTV-strains and little background of its molecular biology in China. For better cognition on CTV, 192 sweet orange samples collected from eight provinces （Chongqing, Sichuan, Fujian, Hunan, Guangxi, Yunnan, Guangdong and Jiangxi） were tested by direct tissue blot immuno-assay （DTBIA）, and 158 of them were tested positively, which therefore were subjected to coat protein gene （CPG）/Hinf Ⅰ restriction fragment length polymorphism （RFLP） analysis. Sample bulks were compared between Chongqing and Fujian by some statistical data, including ratios of single infection and mixed infection to local samples, proportions of CTV isolates with single RFLP groups, and rates of each RFLP group. The simplified analysis of samples from the other six provinces were then conducted. This study suggests that CTV isolates with CPG/Hinf Ⅰ RFLP groups Ⅲ and Ⅰ are the main epidemic ones in China, and mixed infection of CTV in fields are popular. Based on observation of severity of stem-pitting symptom in field trees, CTV isolates with CPG/Hinf Ⅰ RFLP groups Ⅲ and Ⅰ caused severe stem-pittings in sweet oranges in China.
基金supported by Public Science and Technology Research Funds Projects of Agriculture (20120307605)
文摘Citrus tristeza virus （CTV） is one of the most economically important citrus viruses and harms the citrus industry worldwide. To develop reliable and effective serological detection assays of CTV, the major capsid protein （CP） gene of CTV was expressed in Escherichia coli BL21 （DE3） using the expression vector pET-28a and purified through Ni＊-NTA affinity chromatography. The recombinant protein was used to immunize BALB/c mice. Four hybridoma cell lines （14B10, 14Hll, 20D5, and 20G12） secreting monoclonal antibodies （MAbs） against CTV were obtained through conventional hybridoma technology. The titers of MAb-containing ascitic fluids secreted by the four hybridoma lines ranged from 10-6 to 10.7 in indirect enzyme-linked immunosorbent assay （ELISA）. Western blots showed that all four MAbs could specifically react with CTV CP. Using the prepared MAbs, dot-ELISA, Tissue print-ELISA, and triple antibody sandwich （TAS）-ELISA were developed to detect CTV in tree nurseries and epidemiological studies. The developed dot-ELISA and TAS-ELISA methods could detect CTV in crude extracts of infected citrus leaves with dilutions of 1：2560 and 1：10, 240 （w/v, g/mL）, respectively. Tissue print-ELISA was particularly useful for large-scale field sample detection, mainly owing to its simplicity and lack of sample preparation requirements. The field survey revealed that CTV is prevalent on citrus trees in the Chongqing Municipality, Jiangxi Province, and Zhejiang Province of China. The coincidence rate of serological and RT-PCR test results reached more than 99.5%. The prepared MAbs against CTV and established sensitive and specific serological assays have a significant role in the detection and prevention and control of CTV in our country.
文摘应用限制性片段长度多态性（RFLP）、双向逆转录-聚合酶链式反应（BD-PCR）、序列分析等技术对我国部分地区柑橘衰退病毒（Citrus tristeza virus，CTV）柚类分离株进行了鉴定分析。结果表明：柚类以受相对单一的CTV株系侵染为主；柚类上的优势流行株系属于p25／Hin f ⅠRFLP6组群（占79．4％）和p23／BD-PCRⅢ组群（占84．1％）；在对柚类CTV分离株S051～S058及国外CTV分离株PB61、T30、T36、T385、SY568、VT、NUAGA的p23、p25基因序列分析中，S051与弱毒株PB61的同源性最高；S052与弱毒株T30、T385的同源性最高；S053、S054、S055、S056、S057、S058与其它CTV分离株的同源关系均较远，并在系统发生树上形成了独立的分枝。
