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Quick and authentic identification of exotic and potentially invasive taxa with capability of causing high economic losses or detriments is essential prerequisite for effective plant quarantine and biological control initiatives. The order Thysanoptera includes several agricultural pest species that, not only because of their minute size but also due to their cryptic behavior, incline to undetected transport through international trade of plants. Identification of thrips, particularly at species level, is pretty demanding and requires expertise in knowledge about Thysanoptera. Moreover, in most cases, identification of larval Thysanoptera to species is impossible without presence of adults. Hence, there is a great desire for a facile, accurate, and highly reliable technique for thrips identification. The present study describes species-specific primers for four pest thrips species, and the use of a multiplex PCR assay to detect and to distinguish between the four target species. Five primers were used to simultaneously amplify a specific region of the mitochondrial DNA and produce species-specific fragments. Results indicated that the primers were capable of detecting these four species and amplifying uniquely sized, species-specific PCR products. Furthermore, using a multiplex PCR assay, the primers maintained specificity and sensitivity, and allowed detection of each of the four species in a single reaction. The stringency of the method was tested using specimens of different developmental stages and consistent results were obtained for all of the examined samples. This method is simple enough to be implemented by non-experts and also can be extended to any organism for which quick and reliable identification is needed.

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Citrus Viroid V (CVdV) is a member of the genus Apscaviroid, in the Pospiviroidae family. It is restricted to citrus species naturally. The herbaceous host range of CVdV was determined using the viroid infectious clone. Several herbaceous plants from the Cucurbitaceae, Solanaceae, Fabaceae, and Asteraceae families were found to be susceptible to CVdV. Also, CVdV could be transmitted to these hosts through rubbing of monomeric DNA plasmids and through mechanical inoculation of infected sap. The accumulation of CVdV in the tomato was monitored up to 28 days after inoculation and a further 56-fold increase of viroid titer was observed. Analysis of sequences of the viroid progenies from herbaceous plants revealed several nucleotide substitutions, which mostly concentrated in the pathogenicity domain on the secondary structure of the viroids.
 

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The common cutworm, Agrotis segetum, is a serious soil pest of many vegetable and field crops all over the world. Morphological identification of Agrotis species is predominantly performed on adults due to the deficiency of adequate identification keys for immature stages. In international trade, the immature life stages are frequently being intercepted at point of inspection, challenging the possibilities of morphological identification. To realize a rapid and reliable identification for all stages of A. segetum, a TaqMan real-time Polymerase Chain Reaction (PCR) was developed based on the mitochondrial Cytochrome Oxidase I (COI) gene. All specimens of A. segetum (including various life stages) were detected and no cross-reactivity was observed with 5 non-target Agrotis species in the specificity tests. The tests showed to be repeatable, reproducible, and robust. The assay performed equally well with crushed insects and purified DNA, so, the efficiency was added by removing DNA extraction step. The method has proven to be suitable tools for routine identification of all life stages of A. segetum considering the speed, specificity, as well as sensitivity of the assay.

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Production of virus-free stocks is crucial for efficient management of plant viruses in cultivation of pome fruits. Regarding the importance of producing the pre-basic stocks of valuable fruit trees, pear cultivar ʽNatanzʼ, an important local pear cultivar in Iran, was selected for virus eradication. In the present study, tissue culture combined with in vitro thermotherapy and thermo-chemotherapy techniques were used for elimination of Apple Stem Pitting Virus (ASPV) and Apple Mosaic Virus (ApMV). In thermotherapy approach, in vitro shoots were initially incubated for 55, 60, 65, and 70 days in alternating temperatures (32/38°C), then, meristems were cultivated on meristem medium. In thermo-chemotherapy approach, in vitro shoots were incubated for 50 days at 32/38°C, and then meristems were cultivated on a medium containing ribavirin. Virus detection by RT-PCR using specific primers was carried out after rooting and adaptation of the regenerated shoots. The percentage of survived shoots and meristem establishment were depended on thermo-duration. After 55 days, 83.33% of shoots survived, while it decreased to 33.33% after 70 days. Both ASPV and ApMV were eliminated after 60 days of thermotherapy. Ribavirin at 10 and 20 mg L^-1 reduced the percentage of meristem establishment to 50 and 37%, respectively, compared to the control (88.88%). Thermo-chemothery was also effective for ASPV and ApMV eradication from pear shoots.

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For the first time, the nearly complete genome sequence of onion yellow dwarf virus (OYDV) was found in Iran from garlic (Allium sativum L.) by deep RNA sequencing. Complete coding sequence of the Iranian isolate of OYDV (MN528769) consists of 10,212 nucleotides (nt), encodes a polyprotein with 3,403 amino acids (aa). Pairwise sequence comparisons showed that IR-Kh2 shares 74.84-97.39% identity at the nt level and 75.67-98% identity at the amino acids level, respectively with other OYDV isolates deposited in the GenBank previously. According to the phylogenetic analysis, the OYDV isolates were divided into two main groups based on the coding sequence of genome and the Iranian OYDV isolate cluster together with the Australian (MS/SW1), Spanish (SG1), Chinese (G78 and G37-2), and Indian (RR1) isolates. Furthermore, a genetic recombination analysis was also performed, in which a putative recombination event was detected in the nuclear inclusion body b (NIb) gene.

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Squash Mosaic Virus (SqMV) is a Comovirus that infects many cucurbit crops worldwide. In this study, the first two complete genome sequences of SqMV (BSQ and TSQ) from Iran were determined. The RNA genomes of isolates BSQ and TSQ were, respectively, 5,754 and 5,755 (RNA1) and 3290 and 3271 (RNA2) nucleotides (nt) in length, excluding the 3'-terminal poly (A) tail. RNA1 of both isolates encodes a single polyprotein of 1858 amino acids (aa). The identity between the two Iranian isolates (BSQ and TSQ) was 94.24% nt and 94.82% aa for RNA1 and 88.80% nt and 89.50% aa for RNA2. In comparison to other SqMV isolates, BSQ and TSQ shared the highest nucleotide sequence identities of 95.12 % to 93.56 % (RNA1), and 87.59 % to 87.19 % (RNA2), respectively, with the Spanish isolate (RZ-SqMV). Phylogenetic analysis based on complete genome sequences reveals that SqMV isolates cluster into three distinct groups. BSQ was clustered alongside a Spanish isolate in one group and TSQ was separately clustered with a Chinese and US isolates in another group. Recombination analysis revealed that BSQ (RNA1, 2) and TSQ (RNA2) were putative recombinants. BSQ had 6 recombination sites within 5'-UTR, helicase, protease, RdRP (in RNA1), SCP and 3'-UTR (in RNA2) regions, whereas TSQ had 4 recombination sites within 5'-UTR, MP (two breaking points) and LCP region.

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