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.

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.

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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.

Pakistan is among the top 10 citrus-producing countries of the world and the leader in Kinnow mandarin production with production concentrated in the province of Punjab, which produces more than 96% of the total citrus crop. To evaluate the presence and distribution of citrus viroids in this area, 34 samples were collected in September 2008 from citrus orchards in the Sargodha, Bhalwal, and Faisalabad areas of Punjab, including 15 ‘Mosambi’ and two ‘Bloodred’ sweet oranges (Citrus sinensis), eight ‘Kinnow’ and four ‘Feutrell Early’ mandarins (C. reticulata), three ‘Jatti Khatti’ rough lemon (C. jambhiri), and two grapefruit (C. paradisi), which showed stunting, bark scaling, and cracking symptoms on the rootstock which was either citrange (Poncirus trifoliata × C. sinensis) or sweet lime (C. limetta). Infected budwood from these trees was grafted onto indicator plants of Arizona 861-S-1 ‘Etrog citron’ (C. medica) budded on rough lemon rootstock, and after 3 months, the citron showed typical viroid symptoms of mild epinasty and leaf roll with 23 of the 34 samples. A one-step multiplex reverse transcription (RT)-PCR assay (3) was used to detect simultaneously Citrus exocortis viroid (CEVd), Citrus bent leaf viroid (CBLVd), Hop stunt viroid (HSVd), Citrus dwarfing viroid (CDVd), and Citrus bark cracking viroid (CBCVd). On the basis of amplification of the appropriately sized DNA, CEVd, CBLVd, HSVd, and CDVd were detected in 12, 8, 31, and 17 samples, respectively, whereas CBCVd was not detected. Twenty-three of 34 infected samples harbored more than one viroid species and one had four viroids. Budwood from 11 trees did not induce viroid symptoms on Etrog citron. Two of these trees were infected with CBLVd only and nine with HSVd only. Four primer pairs were used to amplify the full sequences of CEVd, CBLVd, HSVd, and CDVd by RT-PCR (2), which were cloned by standard methods. Sequences of three cDNA clones each of CEVd (Nos. FJ773253, FJ773254, and FJ773255), CBLVd (Nos. FJ773262, FJ773263, and FJ773267), HSVd (Nos. FJ773268, FJ773269, and FJ773271), and CDVd (Nos. FJ773274, FJ773275, and FJ773276) were deposited in GenBank. BLAST analysis showed that these nucleotide sequences had greater than 97% nucleotide identity to the most similar genome sequences in GenBank. One of the HSVd sequences, FJ773271, presented the cachexia determinants. To our knowledge, this is the first report of CBLVd and CDVd in Pakistan (1). These results indicate the need for proper indexing of mother trees and a virus-free propagation scheme to create healthy budwood sources in Pakistan. References: (1) M. Arif et al. Pak. J. Bot. 37:407, 2005. (2) L. Bernard and N. Duran-Vila. Mol. Cell. Probes 20:105, 2006. (3) X. F. Wang et al. Eur. J. Plant. Pathol, 124:175, 2009.