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Apple stem grooving virus is associated with leaf yellow mottle mosaic disease on Citrus grandis cv. Huangjinmiyou in China

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Journal of Integrative Agriculture 2022年第7期21卷 2031-2041页

作者： XUAN Zhi-you ZHANG Song LI Ping YANG Fang-yun CHEN Hong-ming LIU Ke-hong zhou Yan LI Zhong-an zhou chang-yong CAO Meng-ji National Citrus Engineering and Technology Research Center Citrus Research InstituteSouthwest UniversityChongqing 400712P.R.China State Cultivation Base of Crop Stress Biology for Southern Mountainous Land Academy of Agricultural SciencesSouthwest UniversityChongqing 400715P.R.China

Although it is usually latent on citrus, apple, and pear, apple stem grooving virus(ASGV) poses a great risk to many sensitive cultivars. Since severe leaf yellow mottle mosaic(LYMM) symptoms have been observed on Huangjinmiyou(HJY) pummelos(Citrus grandis cv. Huangjinmiyou), a commercial variety that is widely cultivated in South China, high throughput sequencing(HTS) was used to find potential pathogens and only three divergent ASGV variants were identified. The three ASGV variants shared 81.03–82.34% genome-wide pairwise identities with each other, and were separately closest to other ASGV variants from different hosts and/or geographical regions, as indicated by viral phylogenies. However, these new variants may have developed from viral interstrain interactions, based on the results of recombination analysis. A large-scale survey using reverse transcription-PCR(RT-PCR) protocols designed for the three ASGV variants revealed a high incidence(92.7–100%) of ASGV in symptomatic HJY trees from 11 major citrusproducing regions in China. None of ASGV were detected in asymptomatic trees. Temperature treatments applied to the symptomatic HJY plants showed that ASGV is sensitive to high temperatures(30–35°C), at which not only the plants recovered, but also the viruses were not detected by RT-PCR, while at low temperatures(20–24°C), both the symptoms and viruses remained detectable. These data show that ASGV is associated with the LYMM disease prevalent on HJY in China, and this is the significant basis especially of taking appropriate measures timely to manage the disease.

关键词： citrus pummelo apple stem grooving virus genome sequence thermotherapy

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A rapid multiplication system for'Candidatus Liberibacter asiaticus'through regeneration of axillary buds in vitro

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Journal of Integrative Agriculture 2022年第6期21卷 1683-1693页

作者： LEI Tian-gang HE yong-rui ZOU Xiu-ping WANG Xue-feng FU Shi-min PENG Ai-hong XU Lan-zhen YAO Li-xiao CHEN Shan-chun zhou chang-yong Citrus Research Institute Southwest University/National Citrus Engineering Research CenterChongqing 400712P.R.China

‘Candidatus Liberibacter asiaticus(CLas)’,which causes citrus Huanglongbing(HLB)disease,has not been successfully cultured in vitro to date.Here,a rapid multiplication system for CLas was established through in vitro regeneration of axillary buds from CLas-infected‘changyecheng’sweet orange(Citrus sinensis Osbeck).Stem segments with a single axillary bud were cultured in vitro to allow CLas to multiply in the regenerating axillary buds.A high CLas titer was detected in the regenerated shoots on an optimized medium at 30 days after germination(DAG).This titer was 28.2-fold higher than in the midribs from CLas-infected trees growing in the greenhouse.To minimize contamination during in vitro regeneration,CLas-infected axillary buds were micrografted onto seedlings of‘changyecheng’sweet orange and cultured in a liquid medium.In this culture,the titers of CLas in regenerated shoots rapidly increased from 7.5×10^(4)to 1.4×10^(8)cellsμg^(-1)of citrus DNA during the first 40 DAG.The percentages of shoots with>1×10^(8)CLas cellsμg^(-1)DNA were 30 and 40%at 30 and 40 DAG,respectively.Direct tissue blot immunoassay(DTBIA)indicated that the distribution of CLas was much more uniform in regenerated plantlets than in CLas-infected trees growing in the greenhouse.The disease symptoms in the plantlets were die-back,stunted growth,leaf necrosis/yellowing,and defoliation.The death rate of the plantlets was 82.0%at 60 DAG.Our results show that CLas can effectively multiply in citrus plantlests in vitro.This method will be useful for studying plant-HLB interactions and for rapid screening of therapeutic compounds against CLas in citrus.

关键词： Citrus ‘Candidatus Liberibacter asiaticus’ multiplication in vitro citrus plantlets

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Genetic Evolution Analysis on Wild Isolates of Citrus Tristeza Virus Originated in China Based on Coat Protein Genes Sequences

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Agricultural Sciences in China 2010年第11期9卷 1623-1629页

作者： YI Long zhou chang-yong zhou Yan LI Zhong-an Citrus Research Institute Southwest University~Citrus Research Institute Chinese Academy of Agricultural Sciences Chongqing 400712 P.R. China Jiangxi Navel Orange Engineering and Technology Research Center Gannan Normal University Ganzhou 341000 P.R.China

The coat protein （CP） genes were cloned and sequenced from viral particles of 11 isolates of citrus tristeza virus （CTV） collected from wild citrus plants in China and 4 Chinese isolates from cultivated sweet orange and pummelo varieties, respectively. By analyzing and comparing the nucleotide and amino acid sequences of CP genes, the 11 wild CTV isolates were found over 92% identical with 4 Chinese CTV isolates and 21 exotic CTV isolates from cultivated citrus. From 91 to 100% of the CTV CP gene sequences in wild type citrus plants were generally well conserved. Genetic evolution analysis indicated that the GC% of the CP gene was less than AT%, and more transition were found in the CP genes than transversion with the transition/transversion ratio ranging from 6.3 to 7.0 among species. The substitution frequency was the highest at the third codon, followed by the first and second codon. The ratio of non-synonymous mutations （du） to synonymous mutations （ds） was far lower than 1, suggesting that the CP gene might have experienced purifying selection in the evolution. Phylogenetic analysis revealed that the 11 CTV isolates in Chinese wild type citrus belonged to different phylogenetic clusters, and shared higher homology and closer relationships with other cultivated citrus CTV isolates from different countries, which indicated complicated genetic relationships among the CTV isolates. In addition, CTV isolates with similar biological characteristics usually located into the same clusters. Therefore, the conclusion was drawn that pathogenicity was critical to evolution and origin of CTV.

关键词： wild type citrus citrus tristeza virus sequence comparison phylogenetic evolution

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High-Level Accumulation of Exogenous Small RNAs Not Affecting Endogenous Small RNA Biogenesis and Function in Plants

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Journal of Integrative Agriculture 2014年第5期13卷 1017-1023页

作者： SHEN Wan-xia Neil A Smith zhou chang-yong WANG Ming-bo College of Plant Protection Southwest University CSIRO Plant Industry National Citrus Engineering Research Center Citrus Research Institute Chinese Academy of Agricultural Sciences

RNA silencing is a fundamental plant defence and gene control mechanism in plants that are directed by 20-24 nucleotide （nt） small interfering RNA （siRNA） and microRNA （miRNA）. Infection of plants with viral pathogens or transformation of plants with RNA interference （RNAi） constructs is usually associated with high levels of exogenous siRNAs, but it is unclear if these siRNAs interfere with endogenous small RNA pathways and hence affect plant development. Here we provide evidence that viral satellite RNA （satRNA） infection does not affect siRNA and miRNA biogenesis or plant growth despite the extremely high level of satRNA-derived siRNAs. We generated transgenic Nicotiana benthamiana plants that no longer develop the specific yellowing symptoms generally associated with infection by Cucumber mosaic virus （CMV） Y-satellite RNA （Y-Sat）. We then used these plants to show that CMV Y-Sat infection did not cause any visible phenotypic changes in comparison to uninfected plants, despite the presence of high-level Y-Sat siRNAs. Furthermore, we showed that the accumulation of hairpin RNA （hpRNA）-derived siRNAs or miRNAs, and the level of siRNA-directed transgene silencing, are not significantly affected by CMV Y-Sat infection. Taken together, our results suggest that the high levels of exogenous siRNAs associated with viral infection or RNAi-inducing transgenes do not saturate the endogenous RNA silencing machineries and have no significant impact on normal plant development.

关键词： Nicotiana benthamiana transformation satellite RNA RNA silencing small RNA

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Complete genome sequence analysis of two Citrus tatter leaf virus(CTLV) isolates from China

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Journal of Integrative Agriculture 2015年第5期14卷 984-987页

作者： SONG Zhen LI Zhong-an LIU Ke-hong zhou chang-yong Citrus Research Institute Southwest University/Chinese Academy of Agricultural Sciences

In order to understand molecular characterization of Citrus tatter leaf virus（CTLV） isolated from China,full-length cDNAs of CTLV-MTH and CTLV-XHC from Citrus reticulata and Citrus sinensis were cloned and sequenced based on whole-genome amplification by RT-PCR.The complete nucleotide sequences of CTLV-MTH and CTLV-XHC were determined to be 6497 nucleotides in length and shared 79.9-91.0%and 78.8-98.0%nucleotide sequence identity,respectively,with other Apple stem grooving virus（ASGV） or CTLV strains available in GenBank.Unexpectedly,CTLV-MTH showed the highest nucleotide sequence identity（91%） with an apple isolate of ASGV,followed by 86.5%with ASGV-HH and 85.7%with ASGV-CHN.Furthermore,CTLV-MTH and three ASGV strains were grouped to a separate cluster in the phylogenetic tree,suggesting it has a closer relationship to ASGV than to CTLV.Therefore,it can be concluded roughly that CTLV may be not a distinct strains of ASGV.We proposed that Citrus tatter leaf virus should be renamed Apple stem grooving virus.

关键词： Citrus isolates Apple apple citrus sequenced length phylogenetic cloned orange

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Limited infection by ‘Candidatus Liberibacter asiaticus’ in ‘Valencia’ sweet orange trees in the presence of Citrus tristeza virus

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Journal of Integrative Agriculture 2019年第10期18卷 2284-2293页

作者： FU Shi-min Cristina Gouin zhou chang-yong John S. Hartung National Citrus Engineering Research Center Citrus Research InstituteChinese Academy of Agricultural Sciences/Southwest UniversityChongqing 400712P.R.China Molecular Plant Pathology Laboratory United States Department of Agriculture-Agricultural Research ServiceBeltsvilleMaryland 20705USA Biotechnology and Regulatory Services United States Department of Agriculture-Animal and Plant Health Inspection ServiceRiverdaleMaryland 20705USA

Huanglongbing (HLB) is the most destructive disease of citrus and is associated with ‘Candidatus Liberibacter asiaticus’(CLas), a member of the α-proteobacteria. Citrus tristeza virus (CTV) is another pathogen of citrus with very great historic as well as current importance. Both CLas and CTV are phloem-restricted pathogens. A severe CTV isolate, CTV-B6, and CLas-B232 induce a group of symptoms of phloem dysfunction that overlap, but the mild isolate CTV-B2 does not cause any loss to commercial trees. Prior inoculation and establishment of CLas-B232 did not affect subsequent establishment of either CTV-B2 or CTV-B6, while super infection by CLas-B232 was reduced by prior establishment of CTV-B2 and to a lesser extent by prior infection with CTV-B6. Trees co-infected with CTV-B6 and CLas-B232 developed more severe symptoms, typical of CTV-B6, than either of the two pathogens co-infected with CTV-B2. In this study, we confirmed that CLas established in the rootlets earlier and with higher concentration than in leaves. The distribution of CLas in the plant infected by CLas-B438 alone and with CTV-B2 fits a previously proposed model but CLas was more sporadically distributed in a plant co-infected by CLas and CTV-B2 than in a plant infected by CLas alone. These biological phenomena are aligned with previously analyzed transcriptome data and the study provides a novel idea that mild CTV strains may provide some protection against CLas by limiting its multiplication and spread. The protective effect may be due to opposite regulation of key host defense pathways in response to CTV-B2 and CLas-B438.

关键词： huanglongbing plant defense plant protection

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Development of a sensitive and reliable droplet digital PCR assay for the detection of ‘Candidatus Liberibacter asiaticus'

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Journal of Integrative Agriculture 2018年第2期17卷 483-487页

作者： ZHONG Xi LIU Xue-lu LOU Bing-hai zhou chang-yong WANG Xue-feng National Citrus Engineering Research Center Citrus Research Institute Chinese Academy of Agricultural Sciences/Southwest University Guangxi Key Laboratory of Citrus Biology Guangxi Academy of Specialty Crops

Citrus Huanglongbing（HLB, yellow shoot disease） is one of the most serious citrus diseases worldwide. To better improve the detection sensitivity, a droplet digital PCR（ddPCR） assay was developed for the rapid detection of ‘Candidatus Liberibacter asiaticus＇（Las）, the putative causal agent of HLB. The detection of sensitivity comparison using positive plasmid indicated that dd PCR was superior to quantitative PCR（qPCR） for detecting and quantifying Las at low concentrations. The Las detection of 40 field samples also showed that six of 13 asymptomatic samples（46.15%） with high Ct value（〉35） were positive by dd PCR. This methodology showed great potential for early HLB infection diagnosis.

关键词： citrus Huanglongbing early diagnosis droplet digital PCR

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Small RNA deep sequencing reveals full-length genome of Citrus yellow vein clearing virus in Chongqing,China

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Journal of Integrative Agriculture 2017年第2期16卷 503-508页

作者： YU Yun-qi WU Qiong SU Hua-nan WANG Xue-feng CAO Meng-ji zhou chang-yong College of Plant Protection Southwest University National Citrus Engineering and Technology Research Center Citrus Research InstituteSouthwest University/Chinese Academy of Agricultural Sciences National Navel Orange Engineering and Technology Research Center Gannan Normal University

To identity the potential pathogen associated with the yellow vein clearing symptom on lemon trees, the profiles of virus-de- rived small interfering RNAs from citrus samples were obtained and analyzed by deep sequencing method in this study. Twenty-seven contigs almost cover the full length genome of Citrus yellow vein clearing virus （CYVCV） isolate YN were obtained using the small RNA deep sequencing technology. Analysis showed that this isolate CQ shared the highest nucleotide identity with isolate Y1 （JX040635） and YN （KP313242）, both of which belong to the genus Mandarivirus in the family Alphaflexiviridae. Mapping analysis of viral-derived siRNA （vsiRNA） profiles revealed an uneven distribution pattern of their generations along both positive and negative genome strands, and 22- and 21-nt vsiRNAs ranked the majority. BLAST against viroids and other viral databases confirmed that this sample was single-infected only by CYVCV, which indicated that CYVCV was the exact causal agent for the yellow clearing symptom occurring on lemon. This is the first CYVCV isolate detected in Chongqing and the second in China. This result could provide a molecular basis for the investigation of citrus viral diseases to protect citrus health in this region.

关键词： citrus Citrus yellow vein clearing virus （CYVCV） small RNA deep sequencing

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The complete genome sequence of Citrus vein enation virus from China

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Journal of Integrative Agriculture 2015年第3期14卷 598-601页

作者： HUANG Ai-jun SONG Zhen CAO Meng-ji CHEN Hong-ming LI Zhong-an zhou chang-yong Citrus Research Institute Chinese Academy of Agricultural Sciences Southwest University College of Plant Protection Southwest University

The complete nucleotide sequence of an isolate of Citrus vein enation virus（CVEV-XZG） from China has been determined for the first time. The genome consisted of 5 983 nucleotides, coding for five open reading frames（ORFs）, had a similar genomic organization features with Pea enation mosaic virus（PEMV）. Nucleotide and deduced amino acid sequence identity of the five ORFs compared to isolate CVEV VE-1 range from 97.1 to 99.0% and 97.4 to 100.0%, these values compared to isolate PEMV-1 range from 45.2 to 51.6% and 31.1 to 45.2%. Phylogenetic analysis based on the complete genome sequence showed that the isolate CVEV-XZG had close relationship with Pea enation mosaic virus. The results supports CVEV may be a new member of genus Enamovirus. The full sequence of CVEV-XZG presented here may serve as a basis for future study of CVEV in China.

关键词： Citrus vein enation virus（CVEV）,genome sequence,Enamovirus

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Preliminary Studies on CPG/Hinf Ⅰ RFLP Groups of Citrus tristeza virus Infected Sweet Oranges in China

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Agricultural Sciences in China 2006年第1期5卷 39-44页

作者： XU Xiao-feng zhou chang-yong SONG Zhen YANG Fang-yun Citrus Research Institute Chinese Academy of Agricultural Sciences Chongqing 400712 P.R.China College of Plant Protection Southwest University Chongqing 400716 P.R.China

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.

关键词： Citrus tristeza virus （CTV） CPG/Hinf Ⅰ RFLP groups stem-pitting sweet orange

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