جستجوی مقالات مرتبط با کلیدواژه « phylogenetic analysis » در نشریات گروه « گیاهپزشکی »

Grapevine virus A (GVA) belongs to Vitivirus genus and is one of the most important causes of wood rugose in grapes. During the years 2019 to 2021, a total of 79 leaf samples showing symptoms of leaf yellowing and reddening from vineyards of Khorasan Razavi Province (including Bardaskan, Kashmar, Khalilabad, and Mohammadiyeh) were collected. The initial infection of the samples to GVA was directly checked using an ELISA test, and the final confirmation of results was performed by RT-PCR with specific primers. Total RNA extraction was performed from scraped bark of young stems using CTAB method. GVA was detected in 14 samples through the ELISA test, and a fragment of 238 base pairs from the p10 protein was amplified in these 14 samples using RT-PCR. The amplified products from four samples were ligated into the pTG19-T vector, and the recombinant plasmids were sequenced bidirectionally. The obtained sequences were compared with the other GVA sequences in GenBank using BlastN tool and the nucleotide similarity was 85.7-95%. The phylogenetic tree based on p10 protein showed there are four main groups, and Khorasan Razavi isolates classified within the fourth group. Comparison of p10-protein sequence of Iranian isolates with other isolates showed there is a conserved region in N-terminal region, which includes an arginine-rich motif followed by a zinc-finger motif. There have been many studies on other viruses of grape in this province, but this is the first study on ithe phylogenyof GVA isolates in the vineyards of Khorasan Razavi province.

The Saffron Latent Virus (SaLV) is new species in the Potyvirus genus, which has been recently characterized from Iran. Even though most of the symptoms on infected plants remain hidden, it causes considerable damage to saffron fields. There is no data available on the phylogenetic relationship among different strains of this virus based on its coat protein, despite its high incidence and economic significance. To address this, we collected 152 samples of saffron leaves, flowers, and corms from fields in South Khorasan and Razavi Khorasan provinces, both with and without symptoms like stunting, yellowing, and twisting. After extracting RNA, the presence of SaLV was examined using reverse transcription-polymerase chain reaction (RT-PCR) with specific primers targeting the coat protein. The findings revealed that 92 out of 152 samples (60%) were infected with the virus. Subsequently, seven representative samples, each from a selected region were sequenced. Phylogenetic analysis using MEGAX software showed that the virus strains formed two distinct groups, and the strains studied were placed in the second group. Interestingly, strains from the same geographical region and product were distributed among different groups, suggesting that geographical separation and host type did not significantly impact the viral diversity. This study is the first in detecting of SaLV in saffron flowers and corms and pinpointing the diversity of its new strains based on the coat protein in Iran.

In the visits of the spring, summer, and autumn of 2021 and 2022 to the orchards of stone fruit trees in Golestan province, suspected symptoms of phytoplasma disease such as yellowness, deterioration, reddening, and small leaves were observed, and 28 samples of leaves from different hosts of stone fruit trees were collected. P1/P7 general primers were used for general PCR, and R16F2n/R16R2 primers were used for nested PCR. Infected samples amplified 1800 bp fragments in general PCR and 1250 bp fragment in nested PCR. Out of the 28 samples collected, 3 samples of peach trees had a band in the target area. The nested PCR product after sequencing was registered in the World Gene Bank with accession numbers OQ945143 and OP055811. At the same time, transplanting was done on the benchmark plant, Periwinkle, with the aim of gene storage and observation of phytoplasma symptoms. The comparison of the obtained sequences with the sequences available on the NCBI by the BLAST showed the most similarity to Aster yellows phytoplasmas, and the phylogenetic analysis of the target sequence with the maximum likelihood estimation algorithm in comparison with the different phytoplasma subgroups reported in Iran also showed the results confirmed the blast. Also, the comparison of patterns obtained from virtual RFLP identified the most similar reference pattern of the 16SrI group, subtype AD (GenBank accession: DQ286577), with a similarity coefficient of 0.77, which indicates that this strain may represent a new group. The results of this study confirm the presence of phytoplasma in peach trees in Golestan province for the first time and indicate the presence of a phytoplasma species close to Candidatus phytoplasma asteris in Golestan province.

Iris spp. is reported to be affected by several viruses in the family Potyviridae including iris mild mosaic virus (IMMV), iris severe mosaic virus (ISMV), iris fulva mosaic virus,  bean yellow mosaic virus (BYMV), turnip mosaic virus (TuMV), ornithogalum mosaic virus (OrMV), narcissus latent virus (NLV), butterfly flower mosaic virus (BFMV), and gladiolus mosaic virus (tentative name). Narcissus latent virus (NLV) is a member of the genus Macluravirus in the family Potyviridae. It has non-enveloped flexuous filamentous virions of 657 nm long and 13 nm wide, which encapsidate a single-stranded, positive-sense RNA molecule of approximately 8,000 nt long. NLV is distributed widely throughout the major planting areas of Japan, New Zealand, and European countries. It is one of the most common viruses infecting narcissus, iris, gladiolus, and nerine, causing significant yield losses and quality deterioration in their bulbs and flowers. Due to the presence of asymptomatic infection of NLV in iris and narcissus, the relevance of its infection in host plants may be severely underrated. As Khorasan Razavi province is one of the major producing areas of ornamental plants in Iran, identification of this virus is a concern. In this study, we attempted to identify NLV infecting iris plants and compare Iranian NLV isolates with other sequences from different geographical regions to provide the first detailed information of phylogenetic characterization of this virus in Iran.

Iris leaf samples showing virus-like symptoms of leaf chlorosis and mosaic were collected from field-grown plants in Khorasan Razavi province. Total RNA was extracted from the field samples using Promega SV Total RNA Isolation Kit (USA). Reverse-transcription polymerase chain reaction (RT-PCR) was performed using specific primer pair CPU-F (5΄-CATTACACCCGACCTGGAACT-3΄) and CPU-R (5΄-CCATTTCAGGGCATTGGAGGA-3΄), which were designed to amplify a 1066 bp fragment of the 3΄-region of NLV genome (encompassing partial NIb (25 nt), complete CP (894 nt), and partial 3'UTR (147 nt)). PCR products and DNA ladder were separated by agarose gel electrophoresis, visualized using DNA Green viewer staining, and photographed with ultraviolet-illumination. Amplified fragments of the expected size were purified, cloned into pTG19-T vector and bi-directionally sequenced. Obtained sequences were phylogenetically compared with the corresponding isolates available in the GenBank after multiple alignments. The phylogenetic tree was constructed based on the nucleotide sequences of the CP-UTR using the neighbor-joining method by MEGA11.

Amplification product (1066 bp) was obtained from five infected samples, but not from healthy samples. The most typical symptoms in positive samples were mosaic, and interveinal chlorosis. Three selected PCR positive samples were cloned into the pTG19-T vector and sequenced. BLASTn analysis of the sequenced data revealed that the PCR-amplified fragments belonged to NLV. Three selected isolates which are referred to as IR, IR2, and IR3 were deposited in GenBank. The previously identified and conserved amino acid sequence motifs described in CP of macluraviruses were present in Iranian CP sequences. The phylogenetic tree placed the NLV sequences into two distinct phylogroups I and II; the Iranian isolates clustered together with isolates from Poland, New Zealand, and United Kingdom into group II. Phylogenetic analysis showed that Iranian isolates shared 77.47 to 98.12% nucleotide sequence identity and 77.70-99.34% amino acid sequence identity with other isolates of NLV. Also, identity of these three isolates in the nucleotide and amino acid levels ranged between 97 to 97.84% and 97.38 to 99.02%, with each other, respectively. Iranian isolates showed the highest nucleotide sequence identity with NLV5_1 isolate (JX270766) from Poland (between 97.65 to 98.12 %) and the lowest with NLV3 isolate (JX270762) from Poland (between 77.47 to 77.95 %).

NLV is a major constraint to iris and narcissus production worldwide. The phylogenetic analysis showed a low correlation between genetic and geographic distances which further emphasizing the importance of the exchange and use of virus-free propagating organs in preventing the dissemination of this virus. It seems that contaminated vegetative organs from some European countries (e.g. Netherlands), which are the major producer and the largest exporters of flowers and ornamentals in the world, can play a significant role in the worldwide distribution of the virus. Identification and the use of more isolates are recommended for a better understanding of the genetic structure and variation of NLV populations on a large geographical scale. The data obtained in this study will be beneficial to improve control strategies for this virus in Iran.

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.

Cucumber mosaic virus [CMV] is one of the most important viruses infecting cucurbits worldwide. To study CMV infections in cucurbit fields of Kavar region in Fars province, samples were collected from watermelon, melon and squash plants showing symptoms of mosaic, yellowing, necrotic spots and stunting. A total of 85 samples were collected during spring and summer 2014. The infection of samples was examined using DAS-ELISA and RT–PCR. The serological method confirmed the infection of 40 samples with CMV. The coat protein gene of the virus was amplified by RT-PCR, using primers P1 and P2, which confirmed the results of DAS-ELISA. The amplified genes of three isolates were sequenced and compared with 19 corresponding sequences from Genbank. Phylogenetic analysis was performed using MEGA6, by Neighbor-joining method. Sequenced were divided to two separate groups, and all Iranian sequences were placed in group A. The results of this study showed a significant infection with CMV in melon fields of Kavar region which may cause significant yield losses.

During spring and summer of 2018, suspected symptoms of the phytoplasma disease such as leaf shrinkage and yellowing as well as leaves with abnormally-enlarged stipules were observed in apple orchards in Khorasan Razavi province and 33 leaf samples were collected. Samples of symptomatic apple tree were assayed for infection with phytoplasma, using universal primer pairs P1 / P7 and nested PCR using primer pair R16F2n / R16R2, fU5 / rU3 and R16MF2 / R16MR2 in Polymerase Chain Reaction (PCR). The universal PCR products were 1800 bp fragments and the nested-PCR products were 1250, 876 and 1400 bp fragments, respectively, which were amplified by primer pairs. Of 33 leaf samples from apple, 18 samples were tested positive for phytoplasmas. The R16MF2/R16MR2 primed nested PCR products were cloned, sequenced and submitted into GenBank. Comparison of the obtained sequences with the sequences of NCBI using BLAST software and Restriction Fragment Length Polymorphism (RFLP) analysis of the nested PCR products using the enzymes AluI, RsaI and TruI (MseI) indicated that phytoplasma in apple trees in Razavi Khorasan province was most similar to Candidatus Phytoplasma trifolii related to Clover Proliferation (CP) 16SrVI group. Analysis of sequences using iPhyClassifier software, determination of nucleotide similarity, and phylogenetic analysis of full-length 16S rDNA showed that phytoplasma in apple tress of Razavi Khorasan province belongs to 16SrVI-A subgroup. This is the first report on Ca. Phytoplasma trifolii infections in apple trees in Iran and other regions across the world.

In spring 2018, yellowing and wilting symptoms were observed on melon plants, Janna variety, in Dashti county, Bushehr province, southern Iran. In order to identify the causal agent of disease, infected tissues were transferred to the laboratory and five fungal isolates were isolated. Based on the morphological characteristic and sequence data of three different loci (ITS, EF-1α, and IGS), the fungi were identified as Fusarium oxysporum f. sp. melonis . The disease assay and race determination were performed in greenhouse using three melon differential hosts including Cha-T (no resistance gene), Cha-Fom1 (contains Fom1 resistance gene) and Cha-Fom2 (contains Fom2 resistance gene). The results of bio-assay experiment reveald that these isolates are belonged to race 1.2 of Fusarium oxysporum f. sp. melonis. According to the phylogenetic analysis, the isolates from Bushehr and the other Iranian isolates belonging to VCG0134 were clustered in the same linage and there were no nucleotide differences between them based on DnaSp analysis. Although F. oxysporum f. sp. melonis is known as a fungal pathogen of melon in cold and temperate regions, the presence of this pathogen in the tropical region of Iran is reporting for the first time in this study.

Garlic (Allium sativum L.) is one of the medicinal and economically important crop plants belongings to the Amaryllidaceae and is well known for its worldwide applications. It is widely used as a vegetable. In Iran despite the agricultural potential, we are still facing a garlic shortage in the market, mainly due to the high prevalence of plant diseases particularly viral infections. Most garlic plants are infected by several viruses belonging to different taxonomic groups known as “garlic viral complex”. However, Onion yellow dwarf virus (OYDV) acts as a major element of this complex. This virus is an important and damaging potyvirus in garlic which causes mild chlorotic to bright yellow stripes on infected leaves. OYDV survives in bulbs and sets and therefore can be transmitted during vegetative reproduction. Also, the green peach aphid, Myzus persicae, as well as other aphids, spreads the virus from plant to plant in a nonpersistent manner.  OYDV, RT-PCR technique is an effective analytical tool for this purpose. In Iran, for the first time, Shahraeen et al. (2008) were reported OYDV by the serological method in garlic. Afterward, Baghalian et al. (2010), studied the molecular structure of Iranian OYDV based on the coat protein gene analysis. The current study was intended to investigate the partial spreading, genetic diversity and phylogenetic analysis of the different OYDV isolates from garlic and comparing them with other sources of OYDV isolates available in the GenBank. In this study, we present for the first time the genome sequence of Iranian isolates of OYDV based on the CI gene. Besides, the recombination pattern of the CI gene in Iranian isolates was analyzed.

During two consecutive growing seasons of 2016-2017 and 2017-2018, a survey was conducted in some of the major areas under the cultivation of garlic in Iran (Khorasan Razavi: Mashhad, Golbahar and kadcan, Mazandaran: Sari and Babolsar, Kerman: Jiroft, Lorestan: Aleshtar and Khuzestan: Shoshtar). Leave samples with yellow striping; crinkling; dwarfing and mosaic symptoms were collected and taken to the laboratory for both sap (mechanical) and molecular tests (RT-PCR/PCR), to identify the disease cause. Total RNA was extracted from the symptomatic leaf samples using the RNeasy Mini Kit (Qiagen, Germany) and used for the reverse transcription (RT-PCR) test. Primary detection was done by RT-PCR using degenerate primer pairs (CIF/R). After performing PCR, products with 700 bp length, separated by electrophoresis in 1% agarose gel extracted and purified with the Qiaquick Gel Extraction Kit (Qiagen, Germany). Then PCR products were cloned in pTG19-T Vector (Vivantis, Malaysia). The recombinant plasmids were extracted using the plasmid DNA isolation kit (Denazist, Iran) followed by sequencing (Macrogene, South Korea). Nucleotide sequence data were analyzed using Clustal Omega, MEGA 6, and RDP 4 software.

Among Iranian OYDV garlic isolates which detected in this study, seven isolates from Golbahar, kadcan, Sari, Jiroft, Aleshtar and Shoshtar with severe mosaic symptom and dwarfing were selected for sequencing and determination of their molecular and biological characteristics. Comparative analyses of the partial CI gene sequences showed that seven Iranian isolates shared 75.04-99.86% and 90.22-100% sequence identities at the nucleotide and amino acid level respectively, with different OYDV isolates available in the GenBank. Phylogenetic tree based on partial CI gene placed Iranian OYDV isolates in two varying evolutionary groups; I and II. Group, I consisted of OYDV isolates from Argentina (KF632714/KF632715), Japan (AB219833/AB219834), Australia (HQ258894/JN127342), China (AJ510223), Spain (JX429964), India (KJ451436) alongside Iranian isolates. This group is geographically heterogeneous. All these isolates in group I are isolated from the garlic host. Three Iranian isolates, IR-Kh1, IR-Kh2, and IR-Kh16, which isolated from garlic placed in subgroup I-II, along with isolates from Spain (JX429964), Australia (HQ258894), and India (KJ451436). It indicates the high degree of genetic relationship among Iranian isolates in this subgroup. However, group II, which is also divided into two subgroups, includes four Iranian garlic isolates, IR-Kh33, IR-Kh54, IR-Kh23, and IR-Kh48, and are closely related to the German (JX433020)  and Argentine (JX433019) onion isolates. Recombination analysis among Iranian and other isolates in the CI gene showed that there was no recombination in this part of the genome.

Based on the results of this study, the presence of OYDV isolates from some garlic areas of Iran was confirmed and for the first time, the molecular characteristics of the virus based on CI gene were determined. Results showed that OYDV is a dominant pathogen in the garlic field, which is highly prevalent in most sampled areas. Considering the importance of garlic in the food, pharmaceutical, and medical industries, determining the molecular characteristics of its viruses is highly important to determine an efficient control program.

Tomato is one of the most important vegetables and Tomato Yellow Leaf Curl Virus (TYLCV) is one of the most important viral disease of tomato in worldwide. In order to study the situation of TYLCV in Bushehr province, sampling of tomato plants with symptoms of virus was done during winter and spring of 2013. Samples were collected from farms of Tang Eram and Dashtestan areas. After DNA extraction , a part of coat protein gene (about 550 pb) was amplified using specific primers. Amplified segments were separated via electrophoresis and sequenced. For comparison and phylogenetic analysis, the sequences Blasted in Gene bank and using CLUSTALW and Mega4 software the phylogenetic tree was drawn via maximum parsimony method. TYLCV Phylogenetic analysis showed that all isolates were divided into two clades named as A and B. In clade A isolates of Dashtestan (KP635447) and Tang Eram (KP635448) were closed to isolates of China (AY594194) and Venezuela (EF625895) and some Iranian isolates including GU076444 from (Shiraz), KC106648 and KC 582150 from (Tehran). In clade B another isolates of this study from Dashtestan was closed to Arabic countries isolates including HM590558 from Saudi Arabia, KF229721 and KF229725 from Ammanand Iranian isolates of GU076450 from Jirof. Results showed variation among Bushehr province. Due to the results of this study can conclude that there is variation among isolates of Bushehr province and requires further studies on the existence of recombinant strains in Iranian samples.

In this study three hundred fifty samples collected from diseased potato, tomato and eggplant plants that showed virus suspected symptoms such as: mosaic and yellowing of leaves, necrotic spots on veins and stunting from Jahrom region of Fars province. The samples were detected by using enzyme linked immunosorbent assay (DAS-ELISA) method with polyclonal antibodies for serological distinguishing of Potato Virus Y (PVY). Almost sixty samples collected from potato were infected by PVY in this phase. For molecular diagnostic of the virus, RT-PCR assay were used for infected samples with primer pairs of P1 and P2. These primers could amplify the coat protein region of virus genome. Eventually PCR products of two samples were sequenced and compared with 19 sequences available in GenBank using Blast search. The MEGA6 software and UPGMA method were used to draw phylogenetic tree. The drawn tree showed that the isolates of jahrom-Iran and the most isolates of Europe were placed in separate clusters.This results showed that in this area, virus infected only found in potato plants so it is possible that crop infections were related to use of infected seed tubers. This is the first report of in Jahrom area.

Wisteria vein mosaic virus (WVMV) is a member of the Potyvirus genus (Potyviridae) with flexuous filamentous particles. The disease was first reported by Brierley & Lorentz (1957) in the USA from W. floribunda. They cited reports of similar cases dating back to the 1940s and suspected that a virus was responsible for the disease. Afterward the virus has been reported in Wisteria spp. in Australia, New Zealand, China, Germany, Czech Republic, Italy and in the Netherland. In comparison to the majority of potyviruses, WVMV has a very narrow host range, naturally infecting only Wisteria spp. (W. sinensis, W. floribunda, and W. venusta). This virus can be introduced into the landscapes on new plants that were infected during their production. Other wisteria can become infected by mechanical inoculation if infected sap is moved on cutting tools or by aphids during feeding from sap of these plants. In terms of symptoms induction, the virus can cause systemic, local chlorotic (sometimes necrotic) lesions or mottling and mosaic on the leaves of wisteria spp. plants and other experimental hosts of Chenopodium spp., Nicotiana megalosiphon and several legumes. The virus is serologically related to Bean common mosaic virus (BCMV), Bean yellow mosaic virus (BYMV), Clover yellow vein virus (ClYVV) and Watermelon mosaic virus (WMV). WVMV is transmitted by aphids, grafting or mechanical means with no significant impact on ornamental host plant except inducing an unfavorable leaf appearance in the landscape. In August 2017, a plant of Wisteria sinensis in the campus of Ferdowsi University of Mashhad in Iran was observed to have chlorotic, mottling and mosaics on some leaves, which resembled the symptoms caused by wisteria mosaic disease. RNA was extracted from the symptomatic plant with an RNeasy Plant Mini Kit (Qiagen, Germany) and tested by RT-PCR using potyvirus degenerate primers CIF/R. Amplicons of the expected size, 680 bp were obtained. The PCR product was cloned and then sequenced. The partial sequence of Nib gene was also amplified (350 bp) by degenerate primer for potyvirus Nib gene and the obtained fragment were cloned and then sequenced. A BLASTn search in the GenBank showed 90.64% nucleotide sequence identity with WVMV Chinese isolate (AY656816) followed by Watermelon mosaic virus (80.67%) and Soybean mosaic virus (80.46%). The least identity was with an isolate of Lettuce mosaic virus (56.34%). It seems that the Chinese and Iranian isolates of this virus are closer to each other but due to lack of nucleotide sequence from other part of the world in CI and Nib genes it is hard to conclude that Iranian isolate is just close to Chinese isolate and it should be considered that may other isolates exist which are closer to Iranian WVMV isolate than Chinese isolate. From this research, it can be concluded that this virus is present in wisteria spp. plant in Khorasan Razavi province of Iran. In terms of sequence similarity based on partial nt sequence of CI gene (680 nt), it is most similar to a Chinese isolate of WVMV (90.64 %) followed by WMV (80.67 %) and SMV (80.46 %) in nt level and within BCMV subgroup of potyviruses. It seems likely that the disease is spread primarily through vegetative propagation rather than by aphid or mechanical transmission. Although the disease does not markedly reduce the vigour of infected plants, the foliage of such plants is chlorotic and mottled, rendering them unsaleable or can cause the ornamental plants as unfavorable host.

In order to study molecular identification and genetic diversity of Prunus necrotic ringspot virus (PNRSV), 149 leaf samples from 37 orchards of pome and stone fruit trees of Kurdistan province were collected and tested by RT-PCR using a PNRSV-F3/PNRS-R3 specific primer pair. Results showed that 20.8% of the samples were infected by PNRSV.In the next stage of the study, 13 isolateswere selected based on the host and geographic region and their amplified fragments were ligated into the pTG19-T plasmid, transformed to E. coli DH5α, and sequenced. Phylogenetic analysis showed that PNRSV isolates formed three clades PV96, PV32, and PE5, and all new-sequences from Kurdistan (in this research) were categorized in PV96 phylogenetic clade, which is close to other Iranian isolates. The new isolates shared 99 ± 0.002 identities together and 94/9 ± 0.005 with other previously PNRSV reported isolates at the nucleotide level. Pairwise comparisons of sequences showed that isolates peach from Kamyaran (KH10), pear from Sanandaj (SZ93), and peach from Dehgolan (D7) had the highest nucleotide similarity (98%) with Iranian isolate nectarine (KX353935) whereas isolate pear from Sanandaj (SZ26) had the lowest nucleotide identity with isolate plum from Poland (DQ983499). The low dN/dS ratio in all populations of the virus showed that negative selection plays important role in PNRSV-CP evolution and recombination. There is no recombination event in this domain of PNRSV genome of these isolates.

Sugarcane Mosaic Virus (SCMV) is one of the important diseases of the sugarcane. In this study, the infection of sugarcane mosaic virus in imported and existing cultivars was screened based on nucleic acid-based molecular methods. During 2014–15, samples showing disease symptoms were collected from 90 different cultivars grown in six sugarcane agro-industries and their partial blades were separated. The samples were freeze-dried and powdered in liquid nitrogen. Suitable primers were designed to amplify 1040 bp from the nuclear inclusion B and coat protein gene sequences using the RT-PCR method. Ten out of 90 surveyed samples were SCMV-infected and produced expected-size fragments. These samples belonged to the cultivars of IRC99-06, V58-4, and Q58 from Sugarcane Research and Training Institute for the Development of Industries in Khuzestan, IRC99-09, IRC00-21, and 4380-3 from Salman Farsi agro-industry, CP80-1557 and V68-74 from Imam Khomeini agro-industry and two uncertain cultivars. Four out of 10 detected samples were directly sequenced and deposited in GenBank. A BLASTn search of the four detected isolates, including Kh10, Kh41, Kh44, and Kh28, showed the highest identity to isolates from Iran and Argentina. Phylogenetic tree constructed using the maximum likelihood algorithm showed that the Iranian isolates were probably originated from Argentina and China.

In this study, 12 native strains and 80 strains from the known species of Allophoma, Didymella, and Neodidymelliopsis were selected. The ITS, D1/D2 LSU rDNA, tub2 and rpb2 regions of the mentioned strains were compared for species delimitation using phylogenetic analysis. The genomic regions of the native strains were amplified using DNA extracted from freeze-dried mycelia and sequenced. The phylogenetic analysis was performed using the maximum likelihood algorithm. The regions of the D1/D2 LSU rDNA, ITS, tub2, and rpb2 delimited 0, 20, 43, and 46 out of 61 species studied, respectively. In the phylogenetic analysis based on combined regions, all datasets (ITS-tub2, ITS-rpb2, tub2-rpb2, ITS-tub2-rpb2, and ITS-28S-tub2-rpb2) could delimit 49 out of the 61 species under survey.Results showed that the sequencing and phylogenetic analysis of the ITS, tub2, and rpb2 regions in combination with morphological studies are necessary for species delimitation of the Allophoma, Didymella and Neodidymelliopsis genera. In a single-locus phylogeny, tub2 or rpb2 genes are the best markers among the genomic regions used.

Potato virus Y (PVY) is the type member of the genus Potyvirus, which is responsible for serious diseases in a wide range of plant species, mostly from the family Solanaceaesuch as potato, tomato, tobacco and pepper worldwide. The virus is transmitted by at least 40 aphid species (family Aphididae) in a nonpersistent manner. The level of damage to crop is determined by the strain of PVY infecting the plants, the viral load, the time at which infection occurs as well as the tolerance the host possesses toward the virus. Infection of a potato field with PVY may ultimately result in 10-100% loss in yield. Several distinct strains of PVY have been identified according to their biological and serological properties and their genome sequences. These include the ordinary strain (PVYO), the tobacco veinal necrosis strain (PVYN), the stipple streak strain (PVYC). In addition, two most prominent recombinant strains, PVYNTN and PVYN-Wi (PVYN:O) have been generated by recombination between PVYO and PVYN. Like other potyviruses, PVY has non-enveloped flexuous filamentous virions of 730-740 nm long and 11–12 nm wide, which encapsidate a single-stranded, positive-sense RNA molecule of approximately 10,000 nt long. PVY causes severe crop losses and consequently economic damage in the world as well as in Iran. Hence, identification of the virus in different regions of the country is a concern. The aim of this research was to study the existence of PVY in tomato fields of Mazandaran province and determining the type of the strain and finally finding 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. According to previous studies comparisons using the CI-coding region most accurately reflected those for the complete genome, and this region was deemed to be the most suitable for diagnostic and taxonomy purposes if the complete sequence could not be obtained. From July to September 2013, a total of 38 leaf samples of tomato showing virus symptoms were collected from different tomato fields in Mazandaran province. Total RNA was extracted from all samples. RT-PCR assay was performed using potyvirus degenerate primers corresponding to the virus CI gene. Expected PCR products were purified from 1% agarose gels, cloned into the pTZ57R/T vector and then sequenced. Sequences were compared to data available in GenBank. Multiple alignments of the nucleotide (nt) and amino acid (aa) sequences were performed with Clustal W implemented in MEGA6 or in BioEdit v.7.2.5. Phylogenetic tree for grouping was constructed by MEGA6 using neighbor-joining method. An amplicon of the expected size (680 bp) was generated from 9 out of 38 plant samples. Specific amplification using the potyvirus degenerate primers in infected samples, but not from healthy sample, confirmed the presence of a potyvirus. All PCR-positive samples were cloned, sequenced and submitted to BLASTn to identify the best matching sequences recorded in GenBank. BLASTn analysis showed that the PCR-amplified fragments of four samples belonged to Potato virus Y strainC. The most typical symptoms in PVY-positive leaf samples were mosaic, mottling, distortion and rugosity. Among them two isolates namely GB and GRA were selected for further analyses. Phylogenetic tree based on multiple sequence alignment of 680 nt of CI gene divided 117 PVY isolates into three main groups: I, II and III. Group I included a wide range of isolates from Europe, Asia, Australia, America and Africa. Members of group I were divided into six subgroups. Iranian isolates (GB and GRA) were classified in the group IF with isolates nnp (Italy), Foggia (Italy), CN1 (Australia), PRI-509 (Netherland), LYE84.2 (Spain) and Tannat (Uruguay), which all were belonging to strain C. Isolate of GB showed the highest (96.5%) nt sequence identity with isolate LYE84.2 and the lowest (81.3%) with isolates T13 (Japan), Tu_660 (Canada), ME162 (China) and ID20 (USA). Also, GRA displayed the highest (95.7%) nt sequence identity with LYE84.2 and the lowest (80.9%) withT13 and Tu_660 isolates. The two Iranian isolates had the highest (99.1%) aa sequence identity with isolates LYE84.2, Foggia and Tannat and the lowest (93.4%) with T13. The identities between isolates GB and GRA were 98.4% and 100% at nt and aa levels, respectively. PVY is one of the most destructive and widespread plant pathogen. In this study, for the first time we reported the occurrence of PVY in tomato fields in Mazandaran province and determining its phylogenetic relationship with other isolates of the virus available in the GenBank. Also, to our knowledge this is the first report of PVY strain C in tomato in Iran. Because the use of CI is more accurate in defining orders in potyvirus taxonomy and in evolutionary relationships, detection of the virus was performed by RT-PCR using potyvirus degenerate primers based on CI coding region. Phylogenetic analyses based on CI gene indicates that the Iranian PVYC isolates are much close to European isolates plus one Australian (CN1) and one Uruguayan (Tannat) isolates. One possible explanation for such sequence similarities is that most of seed potatoes in Iran originated from seed tubers imported from Europe, and not directly from its original habitat, South America. So, it is possible that the Iranian C strain originated potentially from an ancestral European PVY strains and then spread in the country via aphid vectors from infected to healthy plants. Resistance to PVY infection by hosts is low in many cases. On the other hand, PVV is transmitted by aphids in a non-persistent manner, which makes the control of the virus vectors inefficient. Therefore, cultivation of resistant varieties is the most suitable approach for control of the virus. The data obtained in this study will be beneficial to improve control strategies for this virus in Iran. Further studies on PVY isolates from different geographic regions and hosts of Iran and genetic diversity evaluation of the virus will be useful for breeders to make more efficient and durable resistant cultivars.

Fig mosaic emaravirus (FMV)is considered as one of the main causal agents of Fig Mosaic Disease complex (FMD). In order to detection and identification of FMV in different regions of Iran and better understanding of the phylogenetic relationships between isolates, a number of 54 symptomatic fig leaves with chlorosis and mosaic symptoms were collected from different fig-growing areas in the center, north and south of Iran. Primary detection for all collected samples performed by DAS-ELISA using polyclonal AP-conjugated antibody which was raised against nucleocapsid protein of FMV positive samples in DAS-ELISA were checked by RT-PCR using NP gene specific primers. The amplified fragments of 14 isolates were cloned and sequenced. DAS-ELISA results indicated to a 55.5%FMV infection of collected isolates. Phylogenetic analysis on the basis of nucleotide sequences categorized the isolates in two main groups in which isolates from the center and northern regions of Iran placed in a separate subgroup beside other isolates from other countries which their complete coding sequences were available in GenBank of NCBI whereas the isolates from south of Fars province (Estahban and Jahrum districts) clustered in a separate phylogenetic group distinct from other Iranian and the world isolates which may show that genetic makeup of FMV may be affected by geographical isolation. A significant correlation between symptoms severity and phylogenetic groups observed that may put forward the probability of having a new viral strains in Fars province.

Several potyviruses infecting gramineous plants have been reported from Iran. Mosaic is the most widespread viral disease affecting sugarcane and maize production. Sugarcane mosaic virus (SCMV) is one of the causal agents of mosaic disease, which was first described in Ohio in 1963. SCMV belonging to the genus Potyvirus, family Potyviridae, is a single-stranded positive sense RNA virus which can infect gramineous plants such as sugarcane, maize and sorghum. Like all potyviruses, SCMV has 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. SCMV subgroup in the genus Potyvirus consists of seven different species viz., SCMV, Sorghum mosaic virus (SrMV), Maize dwarf mosaic virus (MDMV), Johnsongrass mosaic virus (JGMV); Sorghum mosaic virus (SrMV), Zea mosaic virus (ZeMV), Pennisetum mosaic virus (PenMV) and Cocksfoot streak virus (CSV). These viruses have been transmitted by aphids in a non-persistent manner, and cause mosaic disease and yield loss in sugarcane, maize, sorghum, and other gramineous plants. Among these viruses, only SCMV and SrMV are known to infect sugarcane under natural conditions and are considered as the causal agents of sugarcane mosaic disease. SCMV causes yield losses in many countries, and losses of maize production have been estimated at 30–80%, making it the most economically important problem in world maize production. The first report of SCMV in Iran was from Khuzestan province in 1993. There are also partial sequences, mostly from the 3'-UTR and coat protein regions, of some isolates of this virus in Iran. As Mazandaran province in North of Iran is one of the major producing areas of maize and sugarcane, identification of this virus is a concern. The purpose of this research was to study the existence of SCMV in research areas and determining of its phylogenetic relationship.
From July to September 2013 and 2014, a total of 45 leaf samples of maize and sugarcane showing virus symptoms were collected from different maize and sugarcane fields in Mazandaran province. Total RNA was extracted from all samples. First-strand cDNA and PCR amplification were carried out with an SCMV-specific primer F4/R3 corresponding to the virus CP gene. Expected PCR products of 900 bp were purified from 1% agarose gels using Gel Recovery kit, then cloned into the pTG19-T vector and sequenced. Sequences were compared to data available in the GenBank. Phylogenetic tree for grouping based on nucleotide sequences was constructed by MEGA 6 software program using the neighbor-joining method. Multiple alignments of the nucleotide and amino acid sequences were carried out using the ClustalW and DNAMAN7 software.
Amplification product (900 bp) was obtained from 35 infected samples, but not from healthy samples. The most typical symptoms in positive samples were mosaic, mosaic strips, and yellow chlorotic stripes. Four selected PCR positive samples were cloned into the pTG19-T vector and sequenced. BLASTn analysis of the sequenced data revealed that the PCR-amplified fragments belonged to CP gene of SCMV. Four selected isolates named Maz-SCR1 and Maz-SCR2 from sugarcane (Ghaemshahr), Maz-MR from maize (Ghaemshahr) and Maz-MB from maize (Babolsar). Phylogenetic tree based on multiple sequence alignment of CP gene divided all SCMV isolates into two groups: I and II. Members of each group were divided into two subgroups: A, B. Mazandaran isolates of SCMV were grouped with isolates of Khuzestan and Egypt in a subclade in subgroup IA. Phylogenetic analysis showed that Mazandaran isolates share 78 to 99 % nucleotide sequence identity and 79.5-100 % amino acid sequence identity with other isolates of SCMV. Also, the identity of these four isolates in the nucleotide and amino acid levels ranged between 97 to 98.9 % and 99.3 to 100 %, with each other, respectively. Isolate of Mazandaran showed the highest nucleotide sequence identity with khzL66 and khzQ86 isolates from Khuzestan (between 96.6 to 98.8 %) and EGY7-1 isolate from Egypt (between 96.7 to 99 %) and the lowest nucleotide sequence identity with BD8 isolate from China (between 78 to 78.8 %). The previously identified and conserved amino acid sequence motifs described in CP of potyviruses were present in Mazandaran isolates CP gene.
SCMV is a widespread cereal potyvirus in the world and often occurs in a mixed infection with SrMV. In this study, for the first time, we reported the occurrence of SCMV in maize and sugarcane fields in Mazandaran province based on CP gene analyses and determined its phylogenetic relationship with other SCMV isolates available in the GenBank. Iranian isolates of SCMV were grouped with the isolate of Egypt in a subclade in subgroup IA. It is speculated that the Iranian isolates have been introduced into this country with sugarcane cuttings. The data obtained in this study will be beneficial to improve control strategies for this virus in Iran. Identification and the use of more isolates and analysis of genes in addition to CP are recommended for a better understanding of the genetic structure and variation of SCMV populations on a large geographical scale.