| Viroids are small (246～401nucleotides) covalently closed, single stranded RNAs that replicate in their host plants in which they may elicit diseases. Citrus spp. are natural hosts of seven viroids species of four genera:Citrus exocortis viroid (CEVd) in Pospiviroid; Hop stunt viroid (HSVd) in Hostuviroid; Citrus bark cracking viroid (CBCVd) in Cocadviroid; Citrus bent leaf viroid (CBLVd, including CVd-Ⅰ-LSS, a distinct variant of CBLVd), Citrus dwarfing viroid (CDVd), Citrus viroid V (CVd-Ⅴ) and Citrus viroid Ⅵ (CVd-Ⅵ) in Apscaviroid. Within the viroids that have been found infecting citrus naturally, CEVd and specific sequence variants of HSVd are the causal agents of the exocortis and cachexia diseases of citrus, respectively.(1) Citrus is the most cultivated and highest value crop in the15southern provinces and municipalities in South China. Trifoliate orange (Poncirus trifoliata) and citrange (Citrus sinensis X P. trifoliate) are the main rootstocks for citrus cultivars and are known to be susceptible to citrus viroids. Surveys conducted from1995to2007revealed42symptomatic samples from33cultivars (21from sweet oranges (C. sinensis),6from mandarins (C. reticulata),2from satauma mandarins (C. unshiu),6from lemons (C. lemon), and7from mandarin hybrids). Samples with symptoms of stunting, bark scaling or cracking on the Trifoliate orange rootstock were collected from citrus orchards in the Chongqing municipality, Sichuan, Zhejiang, Jiangxi, Hunan, and Yunnan provinces. Of the42samples,27were cultivars imported from abroad and15were local cultivars. Budwoods from infected trees were grafted onto Arizona861-S1;Etrog’citron (C. medica) on rough lemon (C. jambhiri) rootstock. After more than12months,39of42samples revealed typical viroid symptoms of stunting, epinasty and leaf rolling on the’Etrog’indicator plants.In September2009, total RNA was extracted with TRIZOL Reagent and a one-step multiplex reverse transcription RT-PCR assay was used to detect simultaneously CEVd,CBLVd, HSVd and CDVd. Also, one-step simplex RT-PCR protocols using four primer pairs respectively targeting the four complete genome sequences were used to detect CBCVd, CVd-Ⅴ, CVd-Ⅵ and CVd-Ⅰ-LSS. Of the42samples, CEVd, CBLVd, HSVd, CDVd, CBCVd, CVd-Ⅴ, CVd-Ⅵ and CVd-Ⅰ-LSS were detected in14,13,37, 35,2,3,8and2samples, respectively. Thirty-six harbored more than one viroid species. Some of the symptoms causedby the samples harboring the citrus viroids other than CEVd were as severe as those caused by CEVd. Some source citrus trees showing the severe bark scaling characteristic of exocortis disease in trifoliate orange rootstocks contained several citrus viroids other than CEVd in complex, suggesting that certain exocortis-like diseases in China were caused by some combination of citrus viroids except CEVd.RT-PCR products of each viroids were cloned by standard methods. Four to six of cDNA clones for each viroid isolate were sequenced and deposited in GenBank. BLAST analysis of the CEVd sequences revealed highest nucleotide sequence identity (99%-100%) to a CEVd-E117strain which was severe strain in citrus. One of the CBLVd isolates was CVd-Ⅰb and the other two were CVd-Ⅰa. All of the HSVd sequences belonged to CVd-Ⅱa strain. One of the CDVd isolates was CVd-Ⅲb, but the other two were mix-infected with both CVd-Ⅲa and CVd-Ⅲb.CBCVd, CVd-Ⅴ, CVd-Ⅵ and CVd-Ⅰ-LSS sequences from China, were used to perform the phylogenetic analysis together with all the sequences reported in Genebank.The phylogenetic analysis demonstrated that:(a) CBCVd and CVd-Ⅰ-LSS sequences were clustered into two main groups according to the sequence difference;(b) CVd-Ⅴ sequences were grouped into two main clusters that reflect the geographic origin of samples;(c) CVd-Ⅵ sequences were divided into three main groups according to the host specificity. To our knowledge, this is the first report of CBCVd, CVd-Ⅴ, CVd-Ⅵ and CVd-Ⅰ-LSS in China.(2) Pakistan is among the top10citrus-producing countries of the world and the leader in ’Kinnow’ mandarin production with production concentrated in the province of Punjab, which produces more than96%of the total citrus crop. To evaluate the presence and distribution of citrus viroids in this area,34samples were collected in September2008from citrus orchards in the Sargodha, Bhalwal, and Faisalabad areas of Punjab, including15’Mosambi’and two’Bloodred’sweet oranges (C. sinensis), eight ’Kinnow’ and four’Feutrell Early’mandarins (C. reticulata), three’Jatti Khatti’rough lemon (C jambhiri), and two grapefruit (C. paradisi). A one-step multiplex reverse transcription RT-PCR assay was used to detect simultaneously CEVd, CBLVd, HSVd, CDVd and CBCVd. On the basis of amplification of the appropriately sized DNA, CEVd, CBLVd, HSVd, and CDVd were detected in12,8,31, and17samples, respectively, whereas CBCVd was not detected. Twenty-three of34infected samples harbored more than one viroid species and one had four viroids.Four primer pairs were used to amplify the full sequences of CEVd, CBLVd, HSVd, and CDVd by RT-PCR, which were cloned by standard methods. Five to six of cDNA clones for each viroid were sequenced and deposited in GenBank. BLAST and multiple alignment analysis showed that all the CBLVd sequences were CVd-Ⅰa. Most of the HSVd were CVd-Ⅱa. However, one of the HSVd sequences, presented the cachexia determinants, was characterized to be CVd-Ⅱb. CVd-Ⅲb variants were isolated from selected samples and one possible new strain of CVd-Ⅲ were identified. To our knowledge, this is the first report of CEVd, HSVd, CBLVd and CDVd in Pakistan.(3) Citrus viroid V (CVd-Ⅴ) was recently characterized and belongs to the genus Apscaviroid within the family Pospiviroidae.334CVd-Ⅴ isolates were identified from Punjab, Pakistan, where CVd-Ⅴ had not been reported. Budwoods from seven random selected trees of different cultivars were grafted onto’Etrog’citron for biological indexing. Then RT-PCR results showed that the seven samples were infected by most of citrus viroids except CBCVd and CVd-Ⅵ.To our knowledge, this is the first report of CVd-Ⅴ and CVd-Ⅰ-LSS in Pakistan. A total of68independent CVd-Ⅴ cDNA clones were sequenced from11infected trees of different cultivars, ranging from292to295nucleotides. The nucleotide diversity estimated from the nucleotide distances of the CVd-V Pakistan population was similar to that reported from other countries. Based on genetic diversity and phylogenetic analysis, two main CVd-Ⅴ groups were identified indicating that Pakistan might be one of the geographic origins of CVd-V worldwide. We demonstrated that this viroid has not emerged recently and it is more widespread than previously expected.(4) Citrus are natural hosts of seven viroid species of the family Pospiviroidae. Although there is little information about the’pathogenesis of citrus viroids, several experiments indicated that viroid-derived small RNAs (sRNAs) are key effectors of viroid pathogenesis. To understand the host RNA silencing defence induced by citrus viroids, deep sequencing of the sRNAs from three viroid-infected and one healthy citron leaves using Solexa-Illumina platform were determined. Each infected citron plant contained4to6citrus viroids and displayed typical viroid symptoms. Sequencing data showed that:(a) Citrus viroid sRNAs represented about7%of the total sRNAs in each sample,(b) Citrus viroid sRNAs were predominantly of21-and22-nt, with different viroids yielding a distinct biased distribution of their5’nucleotide and different accumulation of both RNA polarities,(c) Citrus viroid sRNAs derived mostly from specific regions (hot spots) of their RNAs. Most of the (-) polarity viroid-derived sRNAs of CEVd, CBCVd, CVd-V, CVd-VI and CVd-I-LSS were from downside of the TR region. CBLVd, HSVd and CDVd had the same hot spot region in different viroid combinations,(d) The plant sRNAs profile, dominated by the24-nt sRNAs in the mock-inoculated control, exhibited a significant reduction of the24-nt sRNAs and increased of the21-nt sRNAs in two viroid-infected citrons. We also determined how viroid influences21-nt miRNAs accumulation and24-nt rasiRNAs reduction in citron.(5) This study verified the’Progressive Filtering of Overlapping small RNAs"(PFOR) program to predict citrus viroids in the three viroid-infected citron samples. Not all of the citrus viroids can be predicted by PFOR. PFOR can not discriminate viroids which have high sequence similarity. However, PFOR is still a really powerful tool to discovery new viroids after overcome these defects,... |
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