Identification and primary distribution of Citrus viroid V in citrus in Punjab, Pakistan.

BACKGROUND: Citrus plants are prone to infection by different viroids which deteriorate their vigor and production. Citrus viroid V (CVd-V) is among the six citrus viroids, belongs to genus Apscaviroid (family Pospiviroidae) which induces symptoms of mild necrotic lesions on branches and cracks on trunk portion. METHODS AND RESULTS: A survey was conducted to evaluate the prevalence of CVd-V in core and non-core citrus cultivated areas of Punjab, Pakistan. A total of 154 samples from different citrus cultivars were tested for CVd-V infection by RT-PCR. The results revealed 66.66% disease incidence of CVd-V. Citrus cultivars Palestinia Sweet lime, Roy Ruby, Olinda Valencia, Kaghzi lime, and Dancy were identified as new citrus hosts of CVd-V for the first time from Pakistan. The viroid infection was confirmed by biological indexing on indicator host Etrog citron. The reported primers used for the detection of CVd-V did not amplify, rather showed non-specific amplification, which led to the designing of new primers. Whereas, new back-to-back designed primers (CVd-V AF1/CVd-V AR1) detected CVd-V successfully and obtained an expected amplified product of CVd-V with 294 bp. Sequencing analysis confirmed the new host of CVd-V showing 98-100% nucleotide sequence homology with those reported previously from other countries while 100% sequence homology to the isolates reported from Pakistan. Based on phylogenetic analysis using all CVd-V sequences in GenBank, two main CVd-V groups (I and II) were identified, and newly identified isolates during this study fall in the group I. CONCLUSION: The study revealed that there are some changes in the nucleotide sequences of CVd-V which made difficult for their detection using reported primers. All isolates of Pakistan showed high sequence homology with other isolates of CVd-V from Iran and USA whereas; the isolates from China, Japan, Tunisia, and Africa are distantly related. It is evident that CVd-V is spreading in all citrus cultivars in Pakistan.

Correction: Path analysis based on genetic association of yield components and insects pest in upland cotton varieties.

[This corrects the article DOI: 10.1371/journal.pone.0260971.].

Toxicological Effects of Sewage Water on Chick Embryonic Development.

For toxicity research, a total of 100 fertilized nonincubated eggs were used for this study. There were two trials in this experiment which were further divided into 2 phases based on a different days of sewage water treatment and observation days. In each trial, 50 eggs were used and divided into 5 groups. Group A, B, and C were treated with three different concentrations of pure and diluted sewage water (100%, 70%, and 30%), respectively. Control group D was given 0.3 ml saline solution (0.9% NaCl) and group E was uninjected. Different parameters such as the embryo's body weight, body length, forelimb length, hindlimb length, and head diameter were determined. In trial 1, eggs were treated with sewage water on 7th day of incubation and opened on 8th day (phase I) and 9th day (phase II). When the trial 1 (phase I) findings were compared to the control groups, it was observed that body weight, body length, forelimb length, and hindlimb length were highly statistically significant differences (p < 0.01), but the head diameter was not significant (p > 0.05). Phase II result showed embryo's head diameter was a highly statistically significant difference (p < 0.01), whereas forelimb length was significant (p < 0.05), and body weight, body length, and hindlimb length were nonsignificant (p > 0.05). In trial 2, eggs were treated with sewage water on 14th day of incubation and opened on 15th day (phase I) and 16th day (phase II). Results of 15th day showed a highly statistically significant (p < 0.01) difference in hindlimb length, while body weight, body length, forelimb length, and head diameter were nonsignificant (p > 0.05). Phase II of trial 2 showed that on 16th day, body weight, body length, forelimb length, hindlimb length, and head diameter showed a nonsignificant (p > 0.05) difference between experimental and control groups. Embryos were observed to be deforming on the 9th day (after 48 hours of exposure to sewage water). Other phases showed no signs of deformation. Except on 8th day of incubation, dose-related mortalities were present in experimental groups, while the control group showed no mortality.

Enumeration of citrus endophytic bacterial communities based on illumine metagenomics technique.

Citrus is a valuable crop in Pakistan. It is rich in vitamin C, other nutrients and antioxidants. Huanglongbing (HLB) caused by a bacterium "Candidatus liberibacter asiaticus" (CLas), africanus and americanus has an influence on citrus production around the world. Beside HLB there exist several other bacterial species in citrus groves in Pakistan. The structure and diversity of bacterial species in various ecosystems can be quickly examined using NGS. This approach is considerably quicker and more precise than outdated methods. Healthy or citrus greening infected leaf samples of Grapefruit (Citrus paradisi), C. aurantifolia, and C. reticulata Blanco were used for diversity analysis. In this study high throughput, NGS technique was used to access the population of both cultivable and non-cultivable bacterial endophytes from citrus leaves, by using PCR amplicons of 16S rDNA sequences (V5-V7 regions) with Illumina Hi seq. As a result, a total number of 68,722 sequences were produced from the test samples. According to the NGS-based diversity classification, the most common genera of exploited bacterial endophytes were Proteobacteria, Firmicutes, Bacteroides, Cyanobacteria, and Actinobacteria. C. aurantifolia and C. paradisi showed almost equal diversity, whereas C. reticulata Blanco had a higher proportion of Proteobacteria and Cyanobacteria in their leaves. To determine alpha diversity (AD), additional data was analyzed using statistical indices such as Shannon, Chao1, and Simpson. According to the inverse Simpson diversity index, the abundance of the microbial population in six different citrus samples was 0.48, 0.567, and 0.163, respectively. The metagenomics of microbiota in plant tissues was successfully recorded by NGS technology, which can help us learn more about the interactions between plants and microbes. This research is the first step toward a better understanding of 16SrRNA-based metagenomics from citrus in Pakistan using Illumina (Hi seq) Technology.

Path analysis based on genetic association of yield components and insects pest in upland cotton varieties.

Gossypium hirsutum L. is also called upland cotton or Mexican cotton. It is the most widely cultivated species of cotton in the whole world. Globally, about 90% of all cotton production comes from the cultivars derived from this species. Some genetic parameters like monopodial branches per plant, sympodial branches per plant, sympodial branch length, bolls per plant, boll weight, sympo-boll distance, Ginning Out Turn%, staple length (rg = 0.9199**), and fiber strength along with seed cotton yield were evaluated for their potential utilization via selection in seed cotton yield improvement. Significant positive genetic correlations were estimated for monopodial branches per plant (rg = 0.9722**), sympodial branches per plant (rg = 0.7098**), sympodial branch length (rg = 0.617**), bolls per plant (rg = 0.8271**), boll weight (rg = 0.8065**), sympo-boll distance (rg = 0.6507**), Ginning Out Turn (GOT)% (rg = 0.7541**), staple length (rg = 0.9199**), and fiber strength (rg = 0.7534**) with seed cotton yield. A path analysis of all the yield traits under study revealed strong positive direct effects of monopodial branch length (1.1556), sympo-boll distance (0.8173) and staple length (0.7633), while plant height exerted a highly strong direct negative effect (-1.2096) on yield. It is concluded that a direct selection based on monopodial branch length and sympo-boll distance, and staple length is effective, whereas, monopodial branch length, and sympodial branch length are good selection indicators via bolls per plant for yield improvement in cotton.

Correction: The impact of Fosetyl-Aluminium application timing on Karnal bunt suppression and economic returns of bread wheat (Triticum aestivum L.).

[This corrects the article DOI: 10.1371/journal.pone.0244931.].

New promising high yielding cotton Bt-Variety RH-647 adapted for specific agro-climatic zone.

The Bt-cotton RH-647 was developed by Cotton Research Institute CRI, Khanpur has been acknowledged for its possesses superior plant characteristics and potential to yield out under harsh agro-climatic conditions of cotton productive district of Rahimyar Khan in Bahawalpur Division and southern Punjab in 2016. RH- 647 for its novel plant structure and improved fiber quality heat and drought tolerant to withstand successfully sustain yield out in harsh, highly variable hot and dry climatic conditions of and harsh seasoned. RH-647 was developed through one-way hybridization of elite parental genotypes accompanied by pedigree selection method through gene pyramiding technique for incorporation of excellent combinations of fiber traits and CLCuV disease tolerance with higher yield potential right from F1 population. The superior plant combinations were selected in F2-F6 generations were entirely based on phenotypic plant traits and progeny yield potential in field, plant shape, number of bolls per plant, average boll weight (g) and fiber quality traits over standard varieties. The single plant progenies were selected 56 sister lines were tested for Bt-gene (Cry1 Ac) were evaluated for high yielding performance for this superior cross and finally RH-647 as superior breeding line was bulked in year 2010. The strain was evaluated in Randomized Complete Block Design in preliminary yield trials (PYT) and two years in Advance Yield Trials (AYT) trials and Zonal Varietal trials for two years. The superior line 647/10 was ensued for performance in variety attestation tests as RH-647. RH-647 performed best in two years varietal trials (NCVT and PCCT and DUS) conducted for two successive growing seasons (2014-2015 and 2015-2016). RH-647 yielded out significantly compared with standard varieties MNH-886, FH-142 and CIM 602. After completion of mandatory trials in year 2016, RH-647 was approved as new Bt. cotton variety "RH-647". RH-647 is early in maturity with high yield potential and best suited for wheat-cotton cropping pattern. It has fluffy opening and is easy to pick, strongly tolerant to CLCuV disease, high Ginning out turn GOT% (40.2%) with improved fiber traits; staple length (28.3 mm), fiber strength (4.2ug/inch) is duly capable to fulfill all industrial requisitions.

Effect of host plant on cornucopia of mango fruit flies (Diptera: Tephritidae) and their triumphant management in context of climate change.

A study was performed to assess the preference of fourteen mango cultivars for fruit flies and their management by bagging. So the choice of Tephritid flies to mango cultivars during fruiting phase is crucial. Fourteen different cultivars of mango viz., 'Dusehri', 'Malda', 'Langra' early cultivars, 'Chaunsa', 'Fajri Klan', 'Sensation' medium whereas 'Sanglakhi', 'Retaul-12', 'Mehmood Khan', 'Tukhmi', 'Kala Chaunsa', 'Chitta Chaunsa', 'Dai Wala' and 'Sobey De Ting' late cultivars were assessed for their suitability for fruit flies. The results indicate that the population density of fruit flies was higher on late cultivars like 'Sanglakhi' (20.61 percent), 'Mehmood Khan' (20.22 percent) and 'Reutal-12' (19.92 percent) were proved to be highly susceptible to fruit flies. Among these the cultivar 'Reutal-12' was selected being commercial and future cultivar for the management of fruit flies through bagging. The results reported that the attack of tephritid fruit flies and other insect pests were zero in bagged fruits as compared with control. It was further recorded that the bagged fruits has maximum average fruit weight i.e. 203.50 and 197.83 g per fruit was noted in those treatments where butter paper bag and brown paper bag was wrapped with better coloration as compared with un-bagged fruit with 159.5 g per fruit. Similarly, on an average fruit length were more i.e. 90.17, 91.33 mm in bagged fruit and 85.33 in un-bagged fruits. Furthermore, bagged fruits have zero incidence of disease with reduced fruit crack, fruit sunburn, mechanical damage, bird damage, fruit blemished and agrochemical residues on the fruit. So, it is concluded that the special attention should be given on 'Reutal-12' for the management of fruit flies when devising an IPM program for the control of fruit flies. Further, bagging has proved to be the good agricultural practices for the production of quality mango.

The impact of Fosetyl-Aluminium application timing on Karnal bunt suppression and economic returns of bread wheat (Triticum aestivum L.).

Fungal pathogens exert severe qualitative and quantitative damages to wheat crop. Karnal bunt of wheat caused by Tilletia indica Mitra, Mundkur is a severe threat to global food security. Nonetheless, T. indica is regulated as a quarantine pest in numerous countries, which further aggravates the situation. Tolerant varieties and appropriate management practices for Karnal bunt are imperative to meet the global wheat demands. This two-year study explored the impact of fungicide [Fosetyl-Aluminium (Aliette)] application timing on allometric traits, disease suppression and economic returns of bread wheat. Four bread wheat cultivars differing in their tolerance to Karnal bunt were used in the study. Fungicide was applied as either seed treatment (ST), foliar application at heading (FAH) or ST + FAH, whereas no application (NA) was taken as control. Lasani-08 performed better than the rest of the cultivars in terms of allometric traits (plant height, leaf area, crop growth rate, photosynthesis, and chlorophyll content), yield and economic returns. Nonetheless, minimal disease severity was recorded for Lasani-08 compared to other cultivars during both years. The ST improved allometric traits of all cultivars; however, ST + FAH resulted in higher yield and economic returns. Cultivar Pasban-90 observed the highest disease severity and performed poor for allometric traits, yield and economic returns. It is concluded that ST + FAH of Fosetyl-Aluminium could be a pragmatic option to cope Karnal bunt of wheat. Nonetheless, Pasban-90 must not be used for cultivation to avoid yield and quality losses.

Two Unique Prophages of 'Candidatus Liberibacter asiaticus' Strains from Pakistan.

'Candidatus Liberibacter asiaticus' (CLas) is a pathogen causing Huanglongbing (HLB, yellow shoot disease), which is highly destructive to citrus production. The CLas strains harbor prophages. We identified two unique prophages, designated as P-PA19-1 and P-PA19-2, in CLas strain PA19 from Pakistan using next-generation sequencing analysis. P-PA19-1 prophage has high sequence similarity (identity: 78.23%) at the early-gene region of prophage SC1 (Type 1), but it is significantly divergent in the late-gene region (identity: 62.03%). P-PA19-2 was highly similar to SC2 (Type 2) in the late gene region (identity: 97.96%), and also in the early gene region except for a deletion of a 7,179-bp nucleotide sequence that contains a CRISPR/cas system in SC2. Both P-PA19-1 and P-PA19-2 had circular plasmid forms, and only P-PA19-2 was found integrated in the PA19 chromosome. The two new prophages were only found in Pakistani samples. Identification of prophages enhances our understanding of CLas genomic diversity and also the biology and evolution of CLas prophages.

The impact of adjacent habitats on population dynamics of red cotton bugs and lint quality.

Red cotton bugs [Dysdercus spp. (Hemiptera: Pyrrhocoridae] are among the most destructive pests of cotton and many other crops. Red cotton bugs (RCBs hereafter) damage cotton plants by sucking sap and deteriorate lint by staining. The incidence of RCBs causes boll injury along the field margins neighboring with various peripheral areas. The adjacent habitat/crops strongly mediate the population dynamics of RCBs. However, limited is known about the impact of adjacent habitat on population dynamics of RCBs and lint quality. This two-year field study evaluated the impact of adjacent habitat (okra, unpaved road, water channel and Eucalyptus trees) on population dynamics of RCBs and lint quality of cotton. The RCBs were sampled weekly from margins to 4 meter inside the cotton field. The RCBs' populations were monitored and plucked cotton bolls were examined for internal damage. The highest incidence of RCBs was recorded for cotton field adjacent to okra and water channel. Similarly, the highest number of damaged bolls were observed for the field side neighboring with okra and water channel. Furthermore, the highest number of unopened bolls were recorded for okra and water channel sides with higher percentage of yellowish lint. Field sides bordering with Eucalyptus trees and unpaved road had lower RCBs incidence and lint staining. Nonetheless, RCBs incidence was higher at field margins compared to field center indicating that population was strongly affected by adjacent habitat. It is concluded that sowing okra and weedy water channels adjacent to cotton would support RCBs population and subsequent lint staining. Therefore, water channels must be kept weed-free and okra should not be sown adjacent to cotton. Nonetheless, detailed studies are needed to compute monetary damages caused by cotton pests to the crop. Furthermore, effective management strategies must be developed to manage RCBs in cotton to avoid lint-staining problem.

Genome Sequence Resources of Two 'Candidatus Liberibacter asiaticus' Strains from Pakistan.

'Candidatus Liberibacter asiaticus' (CLas) is an unculturable, phloem-restricted αProteobacteria, associated with citrus Huanglongbing (HLB), which is one of the most destructive diseases in citrus production worldwide. Here, we present the genome sequences of CLas strains PA19 and PA20 from HLB-affected kinnow trees in Multan, Punjab Province, Pakistan. The CLas genomes of PA19 and PA20 comprise 1,224,156 bp and 1,226,225 bp, respectively, with an average GC content of 36.4%. Both harbored the Type 2 prophage. In this study, we report two CLas genomes from Pakistan, which extends the sequence database of CLas and will contribute to CLas biology and HLB management.

Discovery and Survey of a New Mandarivirus Associated with Leaf Yellow Mottle Disease of Citrus in Pakistan.

During biological indexing for viruses in citrus trees, in a collection of Symons sweet orange (SSO) (Citrus sinensis L. Osbeck) graft inoculated with bark tissues of citrus trees from the Punjab Province in Pakistan, several SSO trees exhibited leaf symptoms of vein yellowing and mottle. High-throughput sequencing by Illumina of RNA preparation depleted of ribosomal RNAs from one symptomatic tree, followed by BLAST analyses, allowed identification of a novel virus, tentatively named citrus yellow mottle-associated virus (CiYMaV). Genome features of CiYMaV are typical of members of the genus Mandarivirus (family Alphaflexiviridae). Virus particles with elongated flexuous shape and size resembling those of mandariviruses were observed by transmission electron microscopy. The proteins encoded by CiYMaV share high sequence identity, conserved motifs, and phylogenetic relationships with the corresponding proteins encoded by Indian citrus ringspot virus (ICRSV) and citrus yellow vein clearing virus (CYVCV), the two current members of the genus Mandarivirus. Although CYVCV is the virus most closely related to CiYMaV, the two viruses can be serologically and biologically discriminated from each other. A reverse-transcription PCR method designed to specifically detect CiYMaV revealed high prevalence (62%) of this virus in 120 citrus trees from the Punjab Province, Pakistan, where the novel virus was found mainly in mixed infection with CYVCV and citrus tristeza virus. However, a preliminary survey on samples from 200 citrus trees from the Yunnan Province, China failed to detect CiYMaV in this region, suggesting that the molecular, serological, and biological data provided here are timely and can help to prevent the spread of this virus in citrus-producing countries.

Molecular characterization and RSV Co-infection of Nicotiana benthamiana with three distinct begomoviruses.

Geminiviruses constitute a family of plant viruses with characteristic twinned quasi-icosahedral virions and a small circular DNA genome. Geminiviruses, especially begomoviruses, cause substantial economic losses in tropical and subtropical regions globally. Geminiviruses use the host's transcriptional mechanisms to synthesize their mRNAs. They are considered as an attractive model to understand the transcription mechanism of their host plants. Experiments were conducted to identify transcriptional start sites (TSSs) of the three begomoviruses, i.e., Cotton leaf curl Multan virus (CLCuMuV), Corchorus yellow vein virus (CoYVV), and Ramie mosaic virus (RamV). We first rub-inoculated Rice stripe tenuivirus (RSV), a segmented negative-sense RNA virus that uses cap-snatching to produce capped viral mRNAs, into N. benthamiana. After the inoculation, RSV-infected N. benthamiana were super-infected by CoYVV, CLCuMuV, or RamV, respectively. The capped-RNA leaders snatched by RSV were obtained by determining the 5'-ends of RSV mRNA with high throughput sequencing. Afterwards, snatched capped-RNA leaders of RSV were mapped onto the genome of each begomovirus and those matching the begomoviral genome were considered to come from the 5' ends of assumed begomoviral mRNAs. In this way, TSSs of begomoviruses were obtained. After mapping these TSSs onto the genome of the respective begomovirus, it was found very commonly that a begomovirus can use many different TSSs to transcribe the same gene, producing many different mRNA isoforms containing the corresponding open reading frames (ORFs).

Application of biological and single-strand conformation polymorphism assays for characterizing potential mild isolates of Citrus tristeza virus for cross protection.

Citrus tristeza virus (CTV) by killing millions of citrus cultivars grown on sour orange rootstock worldwide has become one of the most dangerous viral pathogen. Characterization of 12 CTV isolates was analyzed by biological indexing. Infected samples of citrus were collected from citrus growing areas of Pakistan and CTV was detected by symptoms on indicator plants and confirmed by direct tissue blot immunoassay (DTBIA). CTV positive samples were graft inoculated on six biological indicator hosts in the study. A standardized protocol was deployed to study biological characteristics of these isolates. All biological indicators induced mild and from mild to moderate reactions against all of the CTV isolates tested. About two isolates produced stem-pitting symptoms from moderate to severe on Mexican lime. CTV strains were further characterized and confirmed by the analysis of p25 gene of CTV isolates using single-strand conformation polymorphism (SSCP) assay. SSCP analysis revealed that most isolates confined only one predominant sequence variant. SSCP profiles of PCR amplified products from CTV isolates showed bands patterns corresponding to mild and sever strain. Three isolates (4MF, 8KBS and 10GS) from different regions and cultivars were identified as potential source of mild strains for cross protection. These results are the best base for mild strain cross protection (MSCP) in the country.

Assemblage of pollinator communities in four widely isolated nature reserves of southern Punjab, Pakistan.

Pollinators are key components of the global biodiversity, providing vital ecosystem services of pollination to crops and wild plants. Understanding pollinator assemblage and how it changes spatially is important in order to device effective ecosystem management planning. This is the first empirical study to explore pollinator community structure in four widely isolated nature reserves of southern Punjab, Pakistan, i.e., the central agricultural plain (Pirowal Forest), the Indus delta plain (Ghazi Ghat Wetland), hilly mountains (Fort Munro), and a sandy desert (Lal Suhanra Forest). Six assemblage parameters, i.e., abundance, richness, dominance, evenness, Simpson index, and Shannon-Wiener index, were studied over seven consecutive months (February to August 2012). Maximum abundance and richness of the pollinators were recorded at Ghazi Ghat (1354 individuals and 84 species), followed by Pirowal (1331 individuals and 80 species), Lal Suhanra (1197 individuals and 77 species), and Fort Munro (808 individuals and 65 species). The highest values of Simpson and evenness indexes were recorded at Fort Munro, while the Shannon-Wiener and dominance indexes were highest at Ghazi Ghat and Pirowal. Species richness was highest for bees, while it was lowest for flies, yet the latter were the highest in abundance among the four pollinator groups. The current study is a first account of pollinator assemblage structure in four widely isolated forest reserves of Punjab, Pakistan. This will support ecosystem management planning, and opens doors for further research in order to reveal complexities in pollinator assemblage structure especially in relation with plant communities.

Global Transcriptomic Analysis Reveals Insights into the Response of 'Etrog' Citron (Citrus medica L.) to Citrus Exocortis Viroid Infection.

Citrus exocortis viroid (CEVd) is the causal agent of citrus exocortis disease. We employed CEVd-infected 'Etrog' citron as a system to study the feedback regulation mechanism using transcriptome analysis in this study. Three months after CEVd infection, the transcriptome of fresh leaves was analyzed, and 1530 differentially expressed genes were detected. The replication of CEVd in citron induced upregulation of genes encoding key proteins that were involved in the RNA silencing pathway such as Dicer-like 2, RNA-dependent RNA polymerase 1, argonaute 2, argonaute 7, and silencing defective 3, as well as those genes encoding proteins that are related to basic defense responses. Many genes involved in secondary metabolite biosynthesis and chitinase activity were upregulated, whereas other genes related to cell wall and phytohormone signal transduction were downregulated. Moreover, genes encoding disease resistance proteins, pathogenicity-related proteins, and heat shock cognate 70 kDa proteins were also upregulated in response to CEVd infection. These results suggest that basic defense and RNA silencing mechanisms are activated by CEVd infection, and this information improves our understanding of the pathogenesis of viroids in woody plants.

Role of pollination in yield and physicochemical properties of tomatoes (Lycopersicon esculentum).

Very little is known about pollination and its effects on the yield and physicochemical properties of flowering plants in tropical countries. Wind and insect pollinators are among our natural resources because pollination is the most important ecosystem service performed by wind and insects, and is vital to the socio-economic status of human beings. In this experiment, different pollination methods for tomato plants were examined. Self-pollination was encouraged by covering the plants with a plastic sheet. Wind and insects were excluded from these plants, and thus only self-pollination was possible. The experiment occurred during the flowering stage. Wind-pollinated plants were covered with a muslin cloth, which excluded insects, and only wind could pass through the cloth. For insect pollination, plants remained uncovered, allowing free access to insects to pollinate the flowers. At fruit maturity, when fruits were completely red, fruits from each treatment were harvested on the same date and under the same conditions. Results illustrated the substantial importance of insects as pollinators of tomato crops. Open field had greater tomato yield and positive effects on physicochemical properties on fruit than under self and wind pollination.

Transcriptome sequencing reveals novel Citrus bark cracking viroid (CBCVd) variants from citrus and their molecular characterization.

Citrus bark cracking viroid (CBCVd), previously called Citrus viroid IV, belongs to the genus Cocadviroid within the family Pospiviroidae. CBCVd has been identified as an important causative agent in citrus and hops. In this study, we obtained the full-length genomes of different variants of all detected citrus viroids from Pakistan through transcriptome sequencing. Different CBCVd variants were first found in Pakistan. These newly discovered Pakistani CBCVd variants were provisionally called "CBCVd-LSS" for their low sequence similarity (80.9%-88.9%) with the CBCVd RefSeq sequence (NC_003539). The two most predominant CBCVd sequences from Pakistan had the closest identity, 90.6% and 87.9%, with two CBCVd sequences isolated from hops. Identification and molecular characterization of CBCVd from citrus in Pakistan and China were also reported. The length of CBCVd from China ranged from 282 to 286 nucleotides, while that of the one from Pakistan ranged from 273 to 277 nucleotides. Based on genetic diversity and phylogenetic analysis, two main CBCVd clades were identified. CBCVd sequences from Pakistan, China, and other countries were further divided into six sub-clades. Sequence alignment revealed some nucleotide changes between these sub-clades, and analysis indicated that several mutations could significantly affect the primary and secondary structure of the viroid. Our results indicated that the CBCVd sequences from Pakistan and China were significantly different with respect to genome and secondary structure and Pakistan might be one of the independent geographical origins of CBCVd worldwide.