جستجوی مقالات مرتبط با کلیدواژه « آنالیز فیلوژنتیک » در نشریات گروه « گیاهپزشکی »

Diapause is a physiological adaptation that allows an organism to survive adverse environmental conditions. Sunn pest, Eurygaster integriceps (Heteroptera: Scutelleridae), is a key pest of wheat and barley and univoltine species and its life cycle has two different phases, reproductive phase, and adult stage diapause. In this study, the gene expression patterns of Hexamerin storage protein were evaluated by quantitative Real-Time PCR assay in diapause and non-diapause female insects, in the Sunn pest’s fat body. The Hexamerin gene expression in diapausing females of sunn pest was 6.4 times higher than in non-diapausing females. The results of subcellular localization and signal peptide prediction suggested that the protein was categorized as an extracellular protein. According to phylogenetic analysis and multiple alignment analysis of the Sunn pest’s Hexamerin amino acid sequence and other bugs belonging to the Hemiptera order, the amino acid sequence was placed at clade belonging to the Hemiptera order (with 100 bootstrap number) and revealed high similarity and closeness to brown marmorated stink bug, Halyomorpha halys Hexamerin gene amino acid sequence (identity 62.6%). Identifying the Hexamerin gene as a reliable biochemical indicator for Sunn pest diapause can be a proper and valuable candidate for further studies at the cellular and molecular levels in this pest.

Among legume crops, common bean (Phaseolus vulgaris L.) is one of the most important worldwide crops, because of its cultivation area and nutritional value. The closely related potyviruses Bean common mosaic virus (BCMV) and Bean common mosaic necrosis virus (BCMNV) are the most common and most destructive viruses that infect common beans throughout the world. The viruses induced similar symptoms in numerous bean genotypes, including mosaic, leaf distortion, stunting, and lethal necrosis. Like all potyviruses, BCMV and BCMNV have non-enveloped flexuous filamentous virions of 750 nm long and 11–13 nm wide, which encapsidate a single-stranded, positive-sense RNA molecule of approximately 10,000 nt long. Both are naturally transmitted by aphids in a non-persistent manner and by seed, which explains their worldwide distribution. These viruses are major constraints on bean production and can cause serious crop losses. Mazanadaran province in north of Iran is one of the major producing areas of legumes, so identification of these viruses is a concern. However, so far, no studies have been done with these viruses in this province. The aim of this research was to study the existence of BCMV and BCMNV in research areas and determining of their phylogenetic relationship. Polymerase chain reaction (PCR) with degenerate primers for conserved sequences of the viral genomes has facilitated the rapid detection of many potyviruses and enabled partial genomic sequencing. In the absence of complete genomic sequences of potyviruses, CI-coding region is more suitable for diagnostic and taxonomy purposes, rather than the coat protein (CP) usually used. The CI gene most accurately reflects the taxonomic status according to the complete ORF.
From July to September 2013 and 2014, a total of 50 leaf samples of beans showing virus symptoms were collected from different bean fields in Mazandaran province. Total RNA was extracted from all samples. The RT-PCR assay was performed using potyvirus degenerate primers corresponding to the virus CI gene. Expected PCR products of 680 bp were purified from 1% agarose gels using the Gel Recovery kit, then cloned into the pTG19-T vector and sequenced. Sequences were compared to data available in GenBank. Phylogenetic tree for grouping based on nucleotide sequences was constructed by MEGA 5.1 software program using neighbor-joining method. Multiple alignments of the nucleotide and amino acid sequences were carried out using the Clustal W and DNAMAN7 software.
Using potyvirus degenerate primers CI F/R, an amplicon of the expected size (680 bp) was generated from 13 plant samples. Specific amplification using the potyvirus degenerate primers in infected samples, but not from healthy samples, confirmed the presence of a potyvirus. The most typical symptoms in positive samples were mosaic, mottling, rugosity, leaf distortion and necrosis. Two selected PCR positive samples were cloned into the pTG19-T vector, sequenced and submitted to BLASTn to identify the best matching sequences recorded in GenBank. BLASTn analysis of the sequenced data revealed that the PCR-amplified fragments belonged to Bean common mosaic virus (Cowpea) and Bean common mosaic necrosis virus (White bean). Phylogenetic tree based on multiple sequence alignment of 680 nt of CI gene divided all BCNMV isolates into two groups: I and II. Members of each group were divided into two subgroups: A, B. Isolates in subgroup IA included three isolates from China and two isolate from Indonesia. Iranian isolate (BCMV-MAZ) was classified in the group IB with RU1M isolate (USA). Group II included a wide range of Chinese isolates and also one isolate from USA, Germany, India and South Korea. Phylogenetic analysis by comparing the 680 bp of CI gene sequences showed that all BCMNV sequences can be placed into two groups: Only TN1 isolate (USA) was classified in group I. Group II included 2 subgroups A, B. Iranian isolate (BCMNV-MAZ) with NL8 isolate (USA) were classified in the subgroup IIA. Isolates in group IIB included a number of USA isolates and one isolate from the UK. Isolate of BCMV-MAZ (from Sari) showed the highest (96.8% - 98.7%) and the lowest (79.5%-91.6%) nucleotide and amino acid sequence identity with RU1M isolate (USA) and Habin1 (Korea), respectively. Also BCMNV-MAZ (from Jouybar) displayed the highest (97.8%) and the lowest (96.9%) nucleotide sequence identity with NL-3 K, NL5 and NL8, respectively. This isolate was 97.7 % identical with other isolates of the BCMNV at the amino acid identity level.
BCMV and BCMNV are widespread in almost all bean growing areas of Iran and often present in the mixture. In this study, for the first time we reported the occurrence of BCMV and BCMNV in common beans in Mazandaran province based on the RT-PCR, and CI gene analyses, and determining their phylogenetic relationship with other isolates of these viruses available in the GenBank. Primary detection was performed by using CI F/R degenerate primer based on the potyvirus CI gene motifs I and V. Since the sequence identity of CI gene is higher when compared to that of the CP gene and is involved in helicase activity during replication, the use of CI is more accurate in defining orders in potyvirus taxonomy and in evolutionary relationships. Due to ease in the spread of these viruses by seed and vectors, detection of such viruses has a crucial role in the control of these diseases. The data obtained in this study will be beneficial to improve control strategies for these viruses in Iran. Study on the distribution of BCMV and BCMNV will be useful for breeders to incorporate virus resistance into bean cultivars, where any or both of the two viral species occur.

Watermelon mosaic virus is the most prevalent potyvirus infecting cucurbits in Iran. In order to determine the distribution and frequency of the virus, 326 watermelon, melon, cucumber, winter and summer squash samples were collected from 22 villages around Urmia during summer 2010 and tested by DAS-ELISA with a polyclonal antibody. Ninety four samples were detected as infected by ELISA. To determine host range of the virus, different plants from 4 familieas were tested. Cucurbita pepo, Cucumis melo L. Cv. Flexus, C.sativus L.cv. Peto Seed and Nicotiana tobaccum cv. Burley were detected as the virus hosts. One strain of squash that was positive in ELISA was used for IC-RT-PCR. A 958 bp product was amplified by IC-RT-PCR using specific primers and was sent for sequencing directly. Phylogenetic trees were constructed by Maximum likelihood, Maximum parsimony and Neighbour joining. Urmian isolate of WMV was not classified in any of the 6 groups which were shown by previous researches.

To determine the prevalence of Potato virus Y (PVY) in tobacco fields of Golestan province, a total of 182 samples showing virus type symptoms PVY infection in collected samples was assessed using a polyclonal antiserum in DAS-ELISA test. Of all collected samples 90 were infected by PVY. Viral RNA was extracted using RNX-Plus kit. The extracted RNA was reverse transcribed using oligo dT primer to create the respective cDNA, wich was amplified by PCR using a PVY-specific primer pair designed on CP region. After verification on 1% agarose gel in electrophoresis, the expected band of ~1000 bp size was amplified. PCR products were directly sequenced and the sequences of about 850 nucleotides were aligned with 30 sequences of the GenBank. Multiple alignment and phylogenetic analyses were performed by ClustalX and BLAST programs in BioEdit software and phylogenetic dendrogram was produced using maximum parsimony method. The results showed that all PVY sequences can be placed into 4 groups. Aliabad and Fazelabad isolates were grouped with isolates from Italy, Spain and Boushehr, while Minoodasht isolate was grouped with isolates from the Netherlands, United Kingdom and Taiwan.

Zucchini yellow mosaic virus (ZYMV (was isolated from squash samples with symptoms of stunting, leaf blistering and distortion of leaf and fruits, collected in cucurbit fields in Kaftarak area (Shiraz) and verified by ELISA test, designated Fars isolate (ZYMV-F). The virus was propagated in squash plants under greenhouse conditions and purified from infected tissues. The antiserum obtained from purified virus was used in serological tests. Complete sequence of the virus genome was determined using mRNA capture kit and PCR with specific primers followed by cloning and sequencing. Following the sequence walking technique a contig of 9591 bp (JN183062) representing the whole genome of the virus was generated. Phylogenetic analysis based on the nucleotide and amino acid sequence of the whole genome and individual genes showed that ZYMV-F is closest to Israel and Slovakia isolates. Analysis of partial sequence of CP gene showed that ZYMV isolates in Iran are very similar, with Fars and Karaj isolates clustering in the same clade.

During 2008-2009, 323 samples showing mosaic, yellowing, vein banding, leaf malformation and blistering were collected from different Cucurbitaceous plant fields and cucumber greenhouses in Razavi and Northern Khorasan provinces. Through doing DAS-ELISA, 95 samples showed infection with WMV. WMV in samples testing positive by ELISA was confirmed by RT-PCR. RT-PCR assay with specific primers corresponding to the virus coat protein gene led to the amplification of the expected DNA fragment with a length of approximately 822 bp. PCR product from two isolates from Zucchini squash (Shirvan) and melon (Torghabe-Shandiz) were sequenced. The sequenced fragments after multiple alignments using ClustalW2 program compared with other GeneBank isolates. Phylogenetic tree was drawn using Neighbor-joining method of MEGA4.1 program. Phylogenetic analysis showed that Iranian isolates of WMV with 92-99 % nucleotide sequence identity and 96-100% amino acid identity with other isolates of WMV. The nucleotide and amino acid sequence homologies of 2 isolates were found to be 99.1% and 99.6%, respectively. The two new isolates were not similar in symptoms on some indicator plants including Cucumis sativus, C. melo var. reticulates, Cucurbita pepo and Citrullus lanatus. These results showed that the two new isolates have high homology with isolates of WMV that previously reported from Iran.