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

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

Global Transcriptomic Analysis Reveals Insights into the Response of ‘Etrog’ Citron (<i>Citrus medica</i> L.) to <i>Citrus Exocortis Viroid</i> Infection

Citrus exocortis viroid (CEVd) is the causal agent of citrus exocortis disease. We employed CEVd-infected &#8216;Etrog&#8217; 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

Rainfall Prediction System Using Machine Learning Fusion for Smart Cities

Precipitation in any form—such as rain, snow, and hail—can affect day-to-day outdoor activities. Rainfall prediction is one of the challenging tasks in weather forecasting process. Accurate rainfall prediction is now more difficult than before due to the extreme climate variations. Machine learning techniques can predict rainfall by extracting hidden patterns from historical weather data. Selection of an appropriate classification technique for prediction is a difficult job. This research proposes a novel real-time rainfall prediction system for smart cities using a machine learning fusion technique. The proposed framework uses four widely used supervised machine learning techniques, i.e., decision tree, Naïve Bayes, K-nearest neighbors, and support vector machines. For effective prediction of rainfall, the technique of fuzzy logic is incorporated in the framework to integrate the predictive accuracies of the machine learning techniques, also known as fusion. For prediction, 12 years of historical weather data (2005 to 2017) for the city of Lahore is considered. Pre-processing tasks such as cleaning and normalization were performed on the dataset before the classification process. The results reflect that the proposed machine learning fusion-based framework outperforms other models

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. Keywords: Pollinators, Assemblage, Forests, Punjab, Pakista

Transcriptome sequencing reveals novel <i>Citrus bark cracking viroid</i> (CBCVd) variants from citrus and their molecular characterization

<div><p><i>Citrus bark cracking viroid</i> (CBCVd), previously called <i>Citrus viroid IV</i>, belongs to the genus <i>Cocadviroid</i> within the family <i>Pospiviroidae</i>. 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.</p></div

Blast results of contigs for samples (PCV-I, PCV-II, respectively) against viroid sequences available in NCBI.

<p>Blast results of contigs for samples (PCV-I, PCV-II, respectively) against viroid sequences available in NCBI.</p

Transcriptome sequencing reveals novel <i>Citrus bark cracking viroid</i> (CBCVd) variants from citrus and their molecular characterization - Fig 1

<p><b>Transcriptome reads mapped onto sequences of CBCVd-P3-1 (A) and P5-1 (B) in PCV-I and PCV-II libraries.</b> Dots represent mismatches of reads.</p

Nucleotide diversity of <i>citrus bark cracking viroid</i> (CBCVd) sequences from Pakistan, China, and other countries^a.

<p>Nucleotide diversity of <i>citrus bark cracking viroid</i> (CBCVd) sequences from Pakistan, China, and other countries^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198022#t003fn001" target="_blank">a</a>}.</p