GRAPEVINE LEAFROLL is one of the most important virus diseases of GRAPEVINE in all grape growing countries. This disease causes poor pigmentation, delayed maturity and up to 40% yield reduction of grapes. These symptoms are produced by a closterovirus and a few ampeloVIRUSES which are called GRAPEVINE leaf-roll ASSOCIATED VIRUSES ((GLRAVS)). Nine filamentous VIRUSES referred to as (GLRAVS) 1 to 9 (all belonging to the family Closteroviridae) have been found ASSOCIATED with the disease. Furthermore, GRAPEVINE virus A (GYA), a vitivirus, is also ASSOCIATED with LEAFROLL syndrome as well as with rugose wood complex. Although GLRaV-1 and GYA have already been reported from Iran, little information is available on the incidence and prevalence of these VIRUSES as well as the presence of other GRAPEVINE LEAFROLL-ASSOCIATED VIRUSES in this country. In this work the presence and prevalence of GRAPEVINE LEAFROLL-ASSOCIATED VIRUSES (GLRav-1, -2, -3, -4, -5 and -9) and GYA in vineyards of four Iranian provinces, namely, Fars, Eastern Azarbaijan, Western Azarbaijan and Kohgiluieh-Boyerahmad were investigated. in addition, the rate of incidence of these VIRUSES in vineyards of the four provinces was determined. For this purpose total RNA was extracted from scraped bark tissues of GRAPEVINEs and used in one-tube RT-PCR with specific primers to analyze the samples the expected products of GVA and (GLRAVS) -1. -2. -4 and -5 were obtained from diseased samples collected from all provinces. However in regard to the GLRaV-9 which was found only in Fars and Kohgiluieh-Boyerahmad the size of PCR fragments was smaller than expected. No PCR product was found in healthy control plants. The results from analysis of 609 samples revealed the wide distribution of GVA and some of the (GLRAVS) in vineyards of the four provinces. GVA was the most prevalent one (18.22%) followed by GLRaV-1 (13.46%). Representing 38.7 and 24.5% of the total infected samples, respectively certain samples contained GLRaV-1 +GV A, GLRaV-2+GV A or GLRaV-4, -5, -9 at the same time. Among the Iranian GRAPEVINE varieties the lowest infection rate was recorded in Ghezelozom variety in W-Azarbaijan province while Rishbaba variety in fars province had the highest infection rate.

Background and Aims: The genetic variability and population structure of GRAPEVINE LEAFROLL-ASSOCIATED virus 3 (GLRaV-3) have not already been studied in mid-Eurasia Iran. The investigations will contribute to developing efficient and durable control strategies of latent and graft-transmissible virus. Materials and Methods: During the winter of 2018-2019, a total of 229 dormant cutting samples were collected from 15 vineyards in West Azarbaijan province. All the samples were analyzed by serological and RT-PCR assays. The phylogenetic tree and two-dimensional nucleotide diversity plot were constructed using CP sequences by the SplitsTree4 v.4.12.6, and SDTv1.2 soft wares, respectively. Dnasp v.6.10.04 was used for genetic diversity and demographic analysis. We provide analyses of the codon usage and composition of GLRaV-3 based on 517 nucleotide sequences of the CP gene including 11 full CP sequences from Iran.  Results: Using serological assay 31.87% GLRaV-3 infection was detected. Neighbor-Net method analysis of the virus complete CP gene showed that the Iranian isolates fell into phylogroup I (GI) “as it is dominant in the rest of the world”. High haplotype diversities indicate the recent expansion of GLRaV-3. No clustering was found according to where the GLRaV-3 was isolated. Using dN/dS values it was found that the different populations of this virus is under negative (purifying) selection. The highest gene flow was determined between Europe and East Asia. Moderate or low genetic differentiation, and frequent gene flow (FST < 0.33 and Nm > 1) also confirmed with Ks*, Kst* Z, Z* and Snn statistics values. The frequency of amino acid coded by A/G ended optimal codon indicates the overlapping influences of natural selection and mutational pressure on the codon preferences in the CP gene. Codon bias of the CP gene was strongly affected by natural selection rather than mutation according to the effective number of codons-ENC vs. GC3s plot. Principal component plot analysis (PCA), illustrated the possible origin of GLRaV-3 in the Old World.  Conclusions: This analysis is the first demonstration of the population structuring of GLRaV-3 in mid-Eurasian Iran. Indeed, these consequences explain selectively driven codon bias in GLRaV-3 species; and reveal the potential importance of expression-mediated selection in shaping the genome evolution of this virus.

GRAPEVINEs in north-eastern provinces of Iran (North Khorasan, Razavi Khorasan, Semnan and Golestan) were randomly surveyed for presence of GRAPEVINE fanleaf virus (GFLV), GRAPEVINE LEAFROLL ASSOCIATED virus 3 (GLRaV-3) and GRAPEVINE virus A (GVA). Symptoms of mosaic, abnormal double nodes, Z shaped, abnormal shoot development and reduced growth were observed in few of the plants. 588 samples of GRAPEVINE were tested for presence of three VIRUSES in dormant canes, young leaves, shoot tips and petioles by double antibody sandwich-enzyme linked immunosorbent assay (DAS- ELISA). All three VIRUSES were found to be present in vineyards of north-eastern provinces of Iran. From the 588 samples tested, 78 vines were found to be infected with at least one virus. ELISA tests showed presence of GFLV, GLRaV-3 and GVA in 7%, 6.6% and 3% of samples respectively. In addition, reverse transcription-polymerase chain reaction (RT-PCR) was used to verify ELISA tests for GFLV in vine tissues. RT-PCR using specific primers directed to the GFLV coat protein gene region at the 3′ end of RNA2 amplified DNA of the expected size (321bp). Also the results showed presence of an unexpected 150 bp band in some samples.These results indicated that RT-PCR as a rapid and sensitive detection method is necessary to confirm healthy plant material.

GRAPEVINE VIRUSES cause significant losses in the yield of grape. This study describes applying silver nanoparticles (AgNPs) to produce virus-free GRAPEVINE plants and compares it with chemo and thermotherapy. Preliminary molecular analysis proved the presence of GRAPEVINE fanleaf virus (GFLV) and GRAPEVINE LEAFROLL-ASSOCIATED virus-1 (GLRaV-1) in the ʻAsgariʼ, ʻPeykaniʼ, and ʻShahaniʼ cultivar samples, then single node explants were cultivated in the MS medium. Thermotherapy at 35 ± 1 ºC and cycles of 35/38 ± 1 ºC, chemotherapy with ribavirin 0, 20, 25, and 30 μg.ml-1 and using AgNPs at 0, 10, 15, and 20 ppm in medium and 40, 50, and 60 ppm sprayed during acclimatization stage were applied to obtain virus-free explants. The results indicated that using 20 ppm AgNPs in medium and AgNPs combined treatment (15 ppm AgNPs in medium and sprayed with 50 ppm AgNPs in the acclimatization stage) were the most effective treatments for the elimination of VIRUSES. The best treatment led to 100% eradication of GLRaV-1 and 67% of GFLV in ʻAsgariʼ, 100% eradication of GLRaV-1 and GFLV in ʻPeykaniʼ and 100% eradication of GLRaV-1 and 67% of GFLV in ʻShahaniʼ. Furthermore, applying of AgNPs improved plant growth parameters, including plant height, which in infected plantlets was (18.06, 12.36, and 14.92 cm in ʻAsgariʼ, ʻPeykaniʼ, and ʻShahaniʼ, respectively) less than virus-free plantlets. Leaf number was 45, 34, and 27 in virus-free plantlets of ʻAsgariʼ, ʻPeykaniʼ, and ʻShahaniʼ, respectively, but in infected plantlets, it was 24.40, 19.80, and 12. Leaf area increased from 5.34, 5.50, and 5.94 cm2 in infected plantlets to 9.56, 11.43, and 12.33 cm2 in virus-free plantlets of ʻAsgariʼ, ʻPeykaniʼ, and ʻShahaniʼ, respectively. Complementary results proved that chlorophyll content in virus-free is significantly higher than in virus-infected plantlets, which explains and confirms the change in growth parameters after virus removal.

During 2004–2007 various own rooted vineyards were inspected to study the fungi ASSOCIATED with vine trunk diseases in Iran. Samples from declining vines showing yellowing and reduced growth and different symptoms in wood, including browning of the wood, necrosis, brown and black streaking and white rot were collected. Fungal isolations were made from affected tissues onto Malt Extract Agar (MEA) supplemented with 1 g l–1 streptomycin sulphate (MEAS). Based on morphological and molecular characteristics, the following species were identified: Phaeoacremonium (Pm.) aleophilum, Phaeomoniella (Pa.) chlamydospora and less frequently Pm. parasiticum, Pm. inflatipes, Pm. cinereum, Cylindrocarpon liriodendri, Diplodia seriata and Neofusicoccum parvum. Results of the pathogenicity tests under field conditions showed that Pa. chlamydospora and Phaeoacremonium spp. caused large wood discoloration 10 months after inoculation without any external foliar symptoms. Phaeomoniella chlamydospora caused larger lesions than Phaeoacremonium spp. All inoculated species were re-isolated from the margin of the lesions completing Koch’s postulates. This study represents the first comprehensive work that investigates the molecular and morphological identification and pathogenicity of Phaeoacremonium spp. and Pa. chlamydospora ASSOCIATED with vine decline in Iran. This is also the first report of Pm. inflatipes, N. parvum and D. seriata ASSOCIATED with GRAPEVINE decline in this country.

Background and objective: The live attenuated oral poliovirus vaccine (OPV), used for many years resulted in obviously reducing the poliomyelitis cases world wide. Most RNA VIRUSES have highly mutable genomes and polioVIRUSES are among the most rapidly evolving of all RNA VIRUSES. Mutations naturally occur during their replication in recipients or their contacts and in rare circumstances it can be caused reversion to neorovirulance (vaccine-ASSOCIATED paralytic poliomyelitis). For many years the role of some critical nucleotides in 5 non-coding region in vaccine-ASSOCIATED paralytic poliomyelitis was known but these years investigators have focused on the importance of other nucleotides in capsid regions such as VP1. So, mutations in this region of polioVIRUSES isolated from acute flaccid paralysis cases are analyzed. In Iran the WHO eradication program has been done since 1373 and all acute flaccid paralysis has been studied.Materials and methods: During the years 1380-1381 from all AFP cases, we found 5 patients with residual paralysis which 5 vaccine-like polioVIRUSES were isolated from them by using micronutralization test and intratypic differentiation tests (ELISA, probe-hybridization and RTPCR). Three isolates(no:1,3 and4) were vaccine-like polioVIRUSES type 1 and 2 other isolates (no:2 and 5) were vaccine-like polioVIRUSES type 2.The VP1 region of theses isolates was sequenced and analyzed.Results: Sequence analysis showed that 3 vaccine-like polioVIRUSES type 1(no:1,2,4) had 4,2 and1nucleotide substitutions in VP1 region in comparison with reference Sabin strain 1 and in vaccine-like polioVIRUSES type2(no:2,5) there were 1 and 2 nucleotide substitutions in this region in comparison with reference Sabin strain 2 respectively.Conclusion: Eradication of wild polioVIRUSES is near and it is time to identify any mutations in all three Sabin strains and improve our knowledge about molecular pathogenesis of theses VIRUSES.

To study of fungi and symptoms ASSOCIATED with vine trunk diseases, various vineyards in Kerman province were inspected during spring and summer of 2012. Samples showing decline symptoms were collected from trunk and branches of vines. Fungal isolation from affected tissues were done on malt extract agar medium (MEA) supplemented with 1 g 1–1 streptomycin sulphate (MEAS). A total of 476 fungal isolates from diseased GRAPEVINEs were obtained. Fungal isolates were identified based on their morphological and molecular characteristics. Morphological identifications of Phaeoacremonium spp. were confirmed by sequence analysis of partial b-tubulin gene (BT) sequences amplification using primers T1 and Bt2b. Botryosphaeria isolates were also confirmed by amplification and sequence of the internal transcribed spacer (ITS) region using the primers ITS4 and ITS5. The most common fungi isolated from vineyards were Phaeoacremonium aleophilum and Phaeomoniella chlamydospora with 103 and 30 isolates, respectively. This is the first report of molecular identification of Pm. aleophilum, Pa. chlamydospora, Pm. parasiticum and Botryosphaeria dothidea ASSOCIATED with different internal symptoms of vine decline in Kerman province.

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During 2004-2007, a field survey was conducted in different vineyards to determine the fungal pathogens ASSOCIATED with GRAPEVINE decline in different provinces of Iran including Fars, Hamedan, Isfahan, and Kohgiluyeh and Boirahmad. Twenty-seven isolates of Botryosphaeriaceae were recovered from vines showing decline and dieback symptoms only in Fars Province. Based on morphological and cultural characters along with molecular analysis [partial sequences of the nuclear ribosomal internal transcribed spacer (ITS), beta-tubulin (BT), and elongation factor 1-a (EF)], two species of Botryosphaeriaceae, namely, Diplodia seriata and Neofusicoccum parvum were identified. Both species were pathogenic on rooted cuttings of cv. Cabernet Sauvignon and caused the dieback and drying-out of leaves as well as extended lesions on the shoots 4 months after inoculation. Results showed that N. parvum isolates were the most virulent and produced significantly longer lesions than those caused by D. seriata. Both species were reisolated from the margin of the lesions completing Koch’s postulates.