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
Infection of plum bark necrosis stem pitting associated virus(PBNSPaV)has been reported in many Prunus species in several countries,causing significant economic losses.The very small proteins encoded by plant viruses ...Infection of plum bark necrosis stem pitting associated virus(PBNSPaV)has been reported in many Prunus species in several countries,causing significant economic losses.The very small proteins encoded by plant viruses are often overlooked due to their short sequences and uncertain significance.However,numerous studies have indicated that they might play important roles in the pathogenesis of virus infection.The role of small hydrophobic protein P6,encoded by the open reading frame 2 of PBNSPaV,has not been well explored.In this study,we amplified the P6 fragment from a PBNSPaV isolate by RT-PCR using specific primers and found that it is 174 bp long and encodes a protein of approximately 6.3 kD with a transmembrane domain.Subcellular localization analysis of P6 proteins in tobacco leaves showed that P6 localizes to the cytomembrane and nuclear membrane.To further clarify the pathogenicity of P6 proteins,we constructed a PVX-P6 expression vector by inserting the p6 fragment into a potato virus X(PVX)-based vector and transformed it into Agrobacterium tumefaciens GV3101.Infiltration of Nicotiana benthamiana(N.benthamiana)with the PVX vector-transformed A.tumefaciens led to slight mosaic symptoms at 14 days of post-inoculation.Meanwhile,infiltration with the PVX-P6 vector-transformed A.tumefaciens resulted in no significant symptoms.These results demonstrated that heterologous expression of P6 in N.benthamiana could not enhance the pathogenicity of PVX.Our study indicates that P6 may not be a potential pathogenic factor associate with the causing of symptoms,and the mode of action of PBNSPaV-P6 protein remains to be further studied.展开更多
<p style="text-align:justify;"> <span><i><span style="font-family:""><span style="font-family:Verdana;">Citrus </span><span style="font-fa...<p style="text-align:justify;"> <span><i><span style="font-family:""><span style="font-family:Verdana;">Citrus </span><span style="font-family:Verdana;">tristeza</span><span style="font-family:Verdana;"> virus</span></span></i></span><span><span><span style="font-family:""><span style="font-family:Verdana;"> (CTV) outbreaks have been reported in the main citrus growing region of Mexico in the past four years. Recently, in eastern Mexico (the major citrus-growing region in the country), severe CTV isolates have been detected. However, the molecular identity of </span><span style="font-family:Verdana;">observed</span><span style="font-family:Verdana;"> isolates remains unestablished. This research was undertaken to elucidate the molecular characterization of CTV populations spreading in this region and to compare it with </span><span style="font-family:Verdana;">phylogeny</span><span style="font-family:Verdana;"> of existing isolates. Genotyping of 32 collected isolates was performed using reverse-transcription polymerase chain reaction (RT-PCR) with sequence analysis of the coat protein (CP) gene, putatively associated with pathogenicity. This protein is a 25 kDa major capsid protein, which forms a long virion body coating 95% of the particle length. </span><span style="font-family:Verdana;">A comparative</span><span style="font-family:Verdana;"> sequence </span><span style="font-family:Verdana;">analys</span></span></span></span><span style="font-family:Verdana;"><span style="font-family:Verdana;"><span style="font-family:Verdana;">is</span></span></span><span style="font-family:Verdana;"><span style="font-family:Verdana;"><span style="font-family:Verdana;"> was performed using CTV sequences from different geographical origins already published and deposited in the GenBank databases. Phylogenetic analysis showed that the degree of sequence divergence among isolates correlated with their pathogenicity. Based on the sequencing results, the collected isolates were categorizedn as mild or severe phylogenetic clusters展开更多
Partial sequence(314 bp) of ASPV was cloned and used as a probe labelled with digoxigenin-11dUTP.The total RNA extracted from samples with Apple stem pitting virus and a series of dilutions of plasmid with ASPV-cDNA w...Partial sequence(314 bp) of ASPV was cloned and used as a probe labelled with digoxigenin-11dUTP.The total RNA extracted from samples with Apple stem pitting virus and a series of dilutions of plasmid with ASPV-cDNA were detected by dot blot hybridization.The results showed that the probe was sensitive and specific.The probe couldn’t hybridize with total RNA of Apple stem grooving virus,Apple mosaic virus and Apple chlorotic leaf spot virus samples as well as negative control,only hybridized with that extracted from dormant shoot infected with ASPV.The sensitivity for detection of plasmid contained ASPV-cDNA was 1.64 μg.展开更多
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funded by the National Natural Science Foundation of China(32102143)Shandong Province Natural Sciences Foundation of China(ZR2019PC011 and ZR2020QC122)+1 种基金Scientific Research Foundation for Ph.D.Programs of Zaozhuang University(2018BS040 and 2018BS042)Science and Technology Program of Zaozhuang(2019NS03).
文摘Infection of plum bark necrosis stem pitting associated virus(PBNSPaV)has been reported in many Prunus species in several countries,causing significant economic losses.The very small proteins encoded by plant viruses are often overlooked due to their short sequences and uncertain significance.However,numerous studies have indicated that they might play important roles in the pathogenesis of virus infection.The role of small hydrophobic protein P6,encoded by the open reading frame 2 of PBNSPaV,has not been well explored.In this study,we amplified the P6 fragment from a PBNSPaV isolate by RT-PCR using specific primers and found that it is 174 bp long and encodes a protein of approximately 6.3 kD with a transmembrane domain.Subcellular localization analysis of P6 proteins in tobacco leaves showed that P6 localizes to the cytomembrane and nuclear membrane.To further clarify the pathogenicity of P6 proteins,we constructed a PVX-P6 expression vector by inserting the p6 fragment into a potato virus X(PVX)-based vector and transformed it into Agrobacterium tumefaciens GV3101.Infiltration of Nicotiana benthamiana(N.benthamiana)with the PVX vector-transformed A.tumefaciens led to slight mosaic symptoms at 14 days of post-inoculation.Meanwhile,infiltration with the PVX-P6 vector-transformed A.tumefaciens resulted in no significant symptoms.These results demonstrated that heterologous expression of P6 in N.benthamiana could not enhance the pathogenicity of PVX.Our study indicates that P6 may not be a potential pathogenic factor associate with the causing of symptoms,and the mode of action of PBNSPaV-P6 protein remains to be further studied.
文摘<p style="text-align:justify;"> <span><i><span style="font-family:""><span style="font-family:Verdana;">Citrus </span><span style="font-family:Verdana;">tristeza</span><span style="font-family:Verdana;"> virus</span></span></i></span><span><span><span style="font-family:""><span style="font-family:Verdana;"> (CTV) outbreaks have been reported in the main citrus growing region of Mexico in the past four years. Recently, in eastern Mexico (the major citrus-growing region in the country), severe CTV isolates have been detected. However, the molecular identity of </span><span style="font-family:Verdana;">observed</span><span style="font-family:Verdana;"> isolates remains unestablished. This research was undertaken to elucidate the molecular characterization of CTV populations spreading in this region and to compare it with </span><span style="font-family:Verdana;">phylogeny</span><span style="font-family:Verdana;"> of existing isolates. Genotyping of 32 collected isolates was performed using reverse-transcription polymerase chain reaction (RT-PCR) with sequence analysis of the coat protein (CP) gene, putatively associated with pathogenicity. This protein is a 25 kDa major capsid protein, which forms a long virion body coating 95% of the particle length. </span><span style="font-family:Verdana;">A comparative</span><span style="font-family:Verdana;"> sequence </span><span style="font-family:Verdana;">analys</span></span></span></span><span style="font-family:Verdana;"><span style="font-family:Verdana;"><span style="font-family:Verdana;">is</span></span></span><span style="font-family:Verdana;"><span style="font-family:Verdana;"><span style="font-family:Verdana;"> was performed using CTV sequences from different geographical origins already published and deposited in the GenBank databases. Phylogenetic analysis showed that the degree of sequence divergence among isolates correlated with their pathogenicity. Based on the sequencing results, the collected isolates were categorizedn as mild or severe phylogenetic clusters
文摘Partial sequence(314 bp) of ASPV was cloned and used as a probe labelled with digoxigenin-11dUTP.The total RNA extracted from samples with Apple stem pitting virus and a series of dilutions of plasmid with ASPV-cDNA were detected by dot blot hybridization.The results showed that the probe was sensitive and specific.The probe couldn’t hybridize with total RNA of Apple stem grooving virus,Apple mosaic virus and Apple chlorotic leaf spot virus samples as well as negative control,only hybridized with that extracted from dormant shoot infected with ASPV.The sensitivity for detection of plasmid contained ASPV-cDNA was 1.64 μg.
