Distribution of Cereal Luteoviruses and Molecular Diversity of BYDV‐PAV Isolates in Central and Southern Iran: Proposal of a New Species in the Genus Luteovirus

During a survey , 148 wheat, 70 barley and 24 wild grass samples of plants showing symptoms of yellowing or reddening of leaves and general stunting were collected in central and southern provinces of Iran and tested for Barley yellow dwarf virus (BYDV) and Cereal yellow dwarf virus (CYDV) infection by enzyme‐linked immunosorbent assay (ELISA) and tissue print immunoassay (TPIA). The results showed the presence of the viruses in most regions. Positive reactions to BYDV‐PAV, BYDV‐MAV, CYDV‐RPV and BYDV‐SGV antisera were recorded. BYDV‐PAV was the most prevalent virus. The genetic diversity of BYDV‐PAV isolates in central and southern provinces was studied by analysing ORF1 (903 nt) and read through domain (RTD) (575 nt) of 13 and nine isolates respectively. Sequence analysis of RTD at nucleotide and amino acid levels revealed a high identity (91.8–97.2% and 91.4–100% respectively) between Iranian and other available isolates in the GenBank. However, in regards to ORF1, a high genetic diversity among Iranian and other known PAV isolates at both amino acid (2–16.9%) and nucleotide (4.1–16.5%) levels were detected. Based on phylogenetic analysis of ORF1, two major groups of BYDV‐PAV isolates were distinguished. The Iranian isolates were divided between the two clusters. Our results suggest that the occurrence of two genetically distinct groups of PAV isolates in central and southern Iran, from which according to the ICTV criteria for species demarcation in the family Luteoviridae, four isolates from central parts of the country, qualify for designation as new species.     

Analysis of Coat Protein of Peanut stunt virus Subgroup II Isolates from Alfalfa Fields in West Iran

Alfalfa fields in three western provinces of Iran were surveyed for Peanut stunt virus (PSV) during 2011 and 2012. Forty‐seven of 115 samples tested (41%) were infected with PSV. Phylogenetic analysis using coat protein (CP) gene sequences showed that the Iranian isolates belong to the subgroup II of PSV. Pairwise identity analysis revealed four groups representing four phylogenetic subgroups. PSV strains in subgroups III and IV are closely related to each other, as supported by the lowest nucleotide diversity, high pairwise nucleotide identity and high haplotype diversity as evidence of a recent population expansion after a genetic bottleneck. Using the maximum likelihood method, amino acid 86S in the CP gene of the Iranian PSV isolates was found to be under positive selection, although the likelihood ratio test statistics is not significant. This is the first report of the occurrence and phylogenetic relationships of Iranian PSV isolates in west Iran.     

Detection and molecular characterisation of Potato virus S of weed reservoirs in Iran

Potato virus S causes destructive disease on the plants. In this research, 44 weed samples symptomless were collected during 2015 from Fars, Razavi Khorasan and Kerman provinces of Iran. The coat protein (CP) and 11 K genes from eight PVS isolates were amplified, cloned and sequenced. PVS was detected in eight weed samples including: one sample of Solanum nigrum, two samples of Chenopodium botrytis, three samples of Chenopodium album and two samples of Amaranthus hybridus. Phylogenetic analysis showed that seven Iranian isolates fell into group I, II near to European isolates and one Iranian isolate formed a separate group. Comparison of coat protein and 11 k nucleotide indicated that all Iranian isolates belonged to Ordinary strain and there were 79–100% identity among the eight Iranian isolates and the world isolates of PVS. The highest identity was between Iranian and Ukraine isolates. Recombination analysis identified four recombinant isolates among eight new Iranian isolates.     

Curly Top of Cultivated Plants and Weeds and Report of a Unique Curtovirus from Iran

The incidence of curly top disease on cultivated plants and weeds was investigated in Kerman Province (southeastern Iran) from October 2003 to November 2004. A total of 1186 samples were collected in fields of sugar beet and other crops as well as within commercial plastic houses. Curtovirus infection of four field crops, three vegetables and 11 weeds was verified by indirect enzyme‐linked immunosorbent assay (ELISA) using a polyclonal antibody. An undescribed curtovirus, tentatively designated Iranian beet curly top virus (IBCTV), was isolated from three symptomatic beet samples collected randomly in widely separated regions of south‐eastern, southern and central Iran and used for molecular studies. A 672 bp segment of the coat protein (CP) gene of each isolate was amplified by PCR and sequenced. The results showed that the three isolates shared 98.5–98.7% nucleotide homology with each other but only 72.1–76.5% with other members of the genus Curtovirus. IBCTV was also detected by PCR using specific primers in other samples of sugar beet, tomato, spinach, turnip and several weed species collected in different parts of Iran. These results indicated that IBCTV is the dominant curtovirus in Iran.     

Intraspecies diversity of Macrophomina phaseolina in Iran

To study the pathogenic and genetic diversity of the Macrophomina phaseolina in Iran, 52 isolates of the fungus were isolated from 24 host plants across the 14 Iranian provinces. All isolates were confirmed to the species based on the species-specific primers. The aggressiveness of M. phaseolina isolates was evaluated on the common bean. Based on the pathogenicity tests, M. phaseolina isolates from the different hosts displayed different levels of aggressiveness on the common beans. The results showed that there was significant variation in the aggressiveness of the pathogen; however, there was no distinct pattern of differentiation based on the host or geographical origin linked to the virulence of the isolates, as frequently theisolates from the same host or geographical origin had different levels of aggressiveness. Inter-simple sequence repeat (ISSR) markers were used to assess the genetic diversity of the fungus. The unweighted pair-group method, using arithmetic mean clustering of data, showed that isolates did not clearly differentiate to the specific group according to the host or geographical origins; however, usually the isolates from the same host or the same geographical origin tend to group nearly. Our results did not show a correlation between the genetic diversity based on the ISSR and pathogenic patterns on common bean in the greenhouse. Similar to the M. phaseolina populations in the other countries, the Iranian isolates were highly diverse based on the pathogenic and genotypic characteristics.     

Identification of fungal diseases of Rosa damascena in Kerman province of Iran

The oil rose, Rosa damascena Mill. (Rosaceae) is an agricultural crop cultivated in various countries of the northern hemisphere, such as Turkey, Bulgaria, Morocco, Iran, Egypt, France, China and India. Iran, presently, is the largest producer of rose water in world. The major production areas in Iran are Kashan, Fars, Kerman and Azerbaijan. Kerman province with 2297 hectares ha of rose gardens and 6198 tons of flower production is one of the important rose production regions. The productions of this region are organic and do not use anychemical compounds such as pesticides and fertilisers. The major fungal pathogens were studied during 2008–2010 in oil rose production areas in Kerman province, Iran. Verticillium dahlias, Rosellinia necatrix, Alternaria alternata, Seimatosporium fusisporum and Podosphaera pannosa have been detected in the oil rose from different regions in Kerman province. A. alternata has the most isolates and infected plants per cent in the oil rose. This is the first report fromVerticillium dahliae, R. necatrix, A. alternata, S. fusisporum on oil roses (R.damascena) in the world.     

Begomoviruses Associated with Yellow Leaf Curl Disease of Tomato in Iran*

The occurrence of Tomato yellow leaf curl virus (TYLCV; genus Begomovirus, family Geminiviridae) in the major tomato‐growing areas of Iran was determined using TAS‐ELISA and PCR. The nucleotide sequences of the coat protein (CP) gene and intergenic region (IR) of eight Iranian isolates were determined. CP nucleotide identities among the Iranian isolates were 96–98%, and showed 94–96% identity with TYLCV‐IR [IR:Ira:98] and TYLCV‐IL [IL:Reo:86]. However, they showed low identity (68–69%) with ToLCIRV‐[IR:Ira]. Sequence analyses of IR indicated that seven Iranian isolates had sequence identity of 93–100% with each other, and 76% identity with the Jiroft isolate; identities of 75–79% with TYLCV‐IR[IR:Ira:98] were observed in every case, and 59–62% identity with ToLCIRV‐[IR:Ira]. The IR nucleotide sequences of Iranian isolates showed 92–93% identity with TYLCV‐IL[IL:Reo:86], except the Jiroft isolate (75%). The CP and IR sequence analyses suggested that eight Iranian TYLCV isolates probably differ from ToLCIRV‐[IR:Ira]. Based on IR sequence comparisons and phylogenetic analyses, the Iranian isolates were divided into two groups. The first major group (A), consists of seven virus isolates, was most closely related to TYLCV‐IL[IL:Reo:86], and relatively divergent from TYLCV‐IR [IR:Ira:98] and ToLCIRV‐[IR:Ira]. However, the Jiroft isolate from group B did not show high similarity with TYLCV‐IR[IR:Ira:98], ToLCIRV‐[IR:Ira], and TYLCV‐IL[IL:Reo:86], suggesting that the isolate may be a divergent variant. The differences are in a range that suggests different strains or species from TYLCV‐IR[IR:Ira:98] and ToLCIRV‐[IR:Ira] are probably associated with tomato yellow leaf curl disease in Iran.     

Human health risks arising from nitrate in potatoes consumed in Iran and calculation nitrate critical value using risk assessment study

Because of the low amount of nitrogen and organic matter in most soils of Iran, it is recommended to use nitrogen fertilizers in potato fields. Nitrate accumulates in plants naturally and if it enters into the human body it can threaten human health. There is not enough information about nitrate distribution in potatoes in Iran and a scientific value of critical level of nitrate in potatoes in Iran. The objective of this study, then, is to: determine the amount of nitrate in potatoes produced in different parts of Iran, assess the human health risks arising from potatoes nitrate, and calculate the critical concentration of nitrate in potato, using a risk assessment study and Iranian food basket. Two hundred and seventy-seven samples were collected from main provinces producing potatoes in Iran. Concentration of nitrate was measured in all samples. Results showed that Kerman province has more nitrate pollution and non cancer risk arising from nitrate. The most sensitive group to nitrate was boys 7–14 years old residing in Kerman province. Critical value of nitrate in potato for this receptor group, then for Iranian society in conservative conditions, was calculated using intake equations introduced by the USEPA and considering Iranian food basket as 246 mg kg^–1.     

Isozyme Analysis and Soluble Mycelial Protein Pattern in Iranian Isolates of Several formae speciales of Fusarium oxysporum

A total of 13 representative isolates of Fusarium oxysporum f. sp. melonis (FOM) from Iran, USA and France, eight isolates of seven formae speciales from Iran and one isolate of F. oxysporum f. sp. niveum from the USA were compared based on isozyme analysis and soluble mycelial protein pattern. Isozyme analyses of alkaline phosphatase (ALP), catalase (CAT), esterase (EST), malate dehydrogenase (MDH), superoxide dismutase (SOD) and xanthine dehydrogenase (XDH) revealed polymorphism among the F. oxysporum isolates in which 22 electrophoretic phenotypes (EP) were determined. At least 10 putative loci for these six enzymes were detected and they were all polymorphic. Maximum genetic diversity was observed in CAT, EST and XDH loci. Using UPGMA, the 22 isolates were separated into three main groups with one of the groups divided into two subgroups. Group I included isolates belonging to five formae speciales from Iran, whereas group II that included FOM isolates from both Iran and the USA was divided into two subgroups each containing the vast majority of the respective isolates from either country. Group III constituted FOM isolates from France and one pathogenic isolate on pepper from Iran. FOM isolates representing five different geographical regions from Iran belonged to two different races of 1 and 1,2Y and one vegetative compatibility group (VCG)0134 and thus were genetically homologous. Isozyme polymorphism in these isolates was highly correlated with VCG and geographical origins and to a lesser extent with races. Variations in soluble protein profile in FOM isolates were correlated with genetic distances determined in isozyme analysis. This study suggests that isozyme analysis could be a useful tool for identifying genetic diversity not only in FOM but also several formae speciales of F. oxysporum.     

Genetic diversity of the common bacterial blight pathogen of bean, <Emphasis Type="Italic">Xanthomonas axonopodis</Emphasis> pv. <Emphasis Type="Italic">phaseoli</Emphasis>, in Iran revealed by rep-PCR and PCR–RFLP analyses

Xanthomonad-like bacteria that are associated with common bacterial blight of bean in Iran were identified on the basis of their colonial morphology, biochemical and serological properties, presence of a specific DNA fragment using PCR primers and pathogenicity on bean. Xanthomonas axonopodis pv. phaseoli (Xap) strains were further characterized using rep-PCR and restriction fragment length polymorphism (RFLP). RFLP profiles generated by the restriction endonucleases RsaI, TaqI, HaeIII and Sau96I and rep-PCR analysis revealed that Iranian strains were relatively genetically homogenous. The similarity coefficients among the strains ranged from 0.87 to 1. The genetic diversity coefficients among strains from three infected provinces, Isfahan, Markazi and Lorestan, were 0.019, 0.072 and 0.033, respectively. The low overall level of polymorphism within Xap isolates collected from the three Iranian infected regions could suggest that few initial inoculum introductions might have distributed among these different bean-growing areas in Iran.     

Assessment of salinity indices to identify Iranian wheat varieties using an artificial neural network

In arid and semi‐arid regions of the world, including Iran, soil salinity is one of the major abiotic stresses. One of the ways to achieve high performance in these areas is to use salt‐tolerant varieties of wheat. Iran is known as one of the places where the D‐genome originated and evolved. In order to evaluate the salt tolerance of Iranian genotypes based on the eight indices using analysis of variance, regression and an artificial neural network (ANN), 41 Iranian wheat varieties (Trticum aestivum L.) were planted in a randomised complete block design with three replications under two saline irrigation conditions, 0.631 and 11.8 dS m^?1, in Kerman, Iran. Significant differences between the varieties were observed, and the significant two‐way interaction of environment × varieties in combined analysis and non‐significant correlation, 0.07, between the yield in two environments (yield in non‐stress conditions, Yp, and yield in stress conditions, Ys) indicates the existence of genetic variation among varieties and the different responses of the varieties in both the environments. The indices of tolerance, geometric mean product (GMP), mean product (MP), harmonic mean (HM) and stress tolerance index (STI) were calculated based on grain yield evidence of positive significant correlation with Yp and Ys. Based on the ANN results, yield stability index (YSI), MP, GMP and STI were the best indices to predict salinity‐tolerant varieties. The varieties selected based on these indices, such as Bolani, Sistan, Ofogh, Pishtaz, Karchia and Arg, produced high yield in both the environments. These results show that bread wheat originating from Iran has salt tolerance potential and can also be used in studies related to salinity tolerance mechanisms.     

Diversity of Sinorhizobium strains nodulating Medicago sativa from different Iranian regions

Alfalfa is believed to have originated in north-western Iran and has a long history of coexistence with its bacterial symbiont Sinorhizobium in soils of Iran. However, little is known about the diversity of Sinorhizobium strains nodulating Iranian alfalfa genotypes. In this study, Sinorhizobium populations were sampled from eight different Iranian sites using three cultivars of Medicago sativa as trap host plants. A total of 982 rhizobial strains were isolated and species were identified showing a large prevalence of Sinorhizobium meliloti over Sinorhizobium medicae. Analysis of salt tolerance demonstrated a great phenotypic diversity. The genetic diversity of the Sinorhizobium isolates was analysed using BOX-PCR and enterobacterial repetitive intergenic consensus (ERIC)-PCR. Patterns ofBOX-PCR fingerprinting were statistically analysed with AMOVA to evaluate the role of plant variety and site of origin in the genetic variance observed. Results indicated that most of the total molecular variance was attributable to divergence among strains isolated from different sites and cultivars (intrapopulation, strain-by-strain variance). Moreover, the analysis showed the presence of two geographic populations (west and northwest), indicating that the effect of the site of origin could be more relevant in shaping population genetic diversity than the effect of cultivar or individual plant.     

Occurrence and Genome Analysis of Cucurbit chlorotic yellows virus in Iran

In 2011 and 2012, several cucurbit‐growing regions of Iran were surveyed and samples with symptoms similar to those induced by Cucurbit chlorotic yellows virus (CCYV) were collected. The pathogen was transmitted to cucumber and melon under greenhouse conditions by whiteflies (Bemisia tabaci). RT‐PCR using designed CCYV‐specific primer pair (CCYV‐F/CCYV‐R) resulted in amplification of the predicted size DNA fragment (870 bp) for the coat protein (CP) gene in samples collected from Boushehr, Eyvanakay and Varamin. Nucleotide sequences of the CP of the three Iranian CCYV isolates were compared with five CCYV isolates obtained from GenBank and analysed. Phylogenetically, all CCYV isolates clustered in two groups; Group I is composed of five non‐Iranian isolates from China, Lebanon, Japan, Sudan and Taiwan, and the three Iranian isolates formed Group 2. Among Iranian isolates, the Eyvanakay isolate clustered in a distinct clade with the Boushehr and Varamin isolates. A phylogenetic tree based on amino acid identity of CP showed that CCYV was closely related to Lettuce chlorosis virus (LCV), Bean yellow disorder virus (BnYDV) and Cucurbit yellow stunting disorder virus (CYSDV). This is the first report of CCYV in Iran.     

Assessment of genetic diversity of Iranian Ascochyta rabiei isolates using rep‐PCR markers

Genetic diversity and population structure among 29 isolates of Ascochyta rabiei (AR) obtained from diseased chickpea plants in six different geographical origins in Iran was characterized by MAT and rep‐PCR (BOX/ERIC/REP) markers. Both mating types were found in all six populations, and the frequencies of mating types were variable between populations. The majority of the isolates belonged to Mat1‐1 (58.12%) with the remainder (41.88%) being Mat1‐2. A dendrogram was calculated with Jaccard's similarity coefficients with unweighted pair group method clustering (UPGMA) for the combination of rep‐PCR results, AR strains were differentiated into four clusters (A–D) at 60% similarity level. ERIC, REP and BOX showed a total of 19, 37 and 24 alleles per locus, respectively. Gene diversity (He) and Shannon's information index (I) were the highest in the REP (He = 0.82; I = 2.11), while the lowest values were estimated for the ERIC (He = 0.42; I = 1.3). Our result showed that among the three techniques studied, REP‐PCR produced the most complex amplified banding patterns, which reflected a high degree of diversity among the Iranian AR strains. ERIC‐PCR was the least discriminating method, and BOX‐PCR was intermediate. To the best our knowledge, this is first study of assessment of genetic diversity of AR isolates by rep‐PCR markers.     

Toxic aspergilli from pistachio nuts

Pistachio nut samples taken during various stages of development from orchards in Iran, showed that contamination with fungi occurred mainly during the later stages of nut development. Members of the genera Aspergillus and Penicillium occurred most frequently. Of the Aspergilli, the species A. niger, A. flavus and A. fischeri var. spinosus occurred most frequently, followed by A. terreus, A. tamarii and A. nidulans. Twenty-two isolates comprising 13 species were tested for toxicity to ducklings. Isolates of known toxic fungi included A. flavus, A. niger, A. parasiticus, A. ochraceus, A. versicolor, A. nidulans and A. terreus. The toxicity of A. fischeri var. spinosus is reported. Chemical analysis showed that all isolates of A. flavus and A. parasiticus produced aflatoxin B₁, the isolates of A. versicolor and A. nidulans produced sterigmatocystin while the toxic isolate of A. ochraceus did not produce ochratoxins.Toxic fungi have been shown to occur in a variety of nuts (4), (5), (11), (12), (13), (15), (18), (20), (21). Aflatoxin contamination of pistachio nuts has been reported and has in the past led to the rejection of consignments of Iranian pistachio nuts (1). In Iran, pistachio nuts are produced mainly in the south eastern provinces (Kerman & Zahedan) and to a limited extent in the Northern part (Kazvin & Damghan). In 1975 it was estimated (7) that there were some 24 million pistachio trees in Iran, of which 60% were situated in Rafsanjan, Kerman (Table 1). Economic considerations as well as the potential health hazard posed by aflatoxin-contaminated nuts, prompted the University of Isfahan to initiate a study of various aspects of the mycotoxin problem in pistachio nuts.     

Diversity and characterization of Candidatus Liberibacter asiaticus strains causing huanglongbing disease in Iran,based on two prophage loci

Huanglongbing (HLB), also known as citrus greening, is a destructive disease of citrus; it is considered a newly emerging disease which has spread to the Middle East and North Africa (MENA). In Iran, the disease was first found in 2009. In this study, two hypervariable prophage and phage‐related loci, bacteriophage repressor protein C1 (CLIBASIA_ 01645 locus) and prophage terminase gene (CLIBASIA_05610 locus), were used to determine the diversity and characterization of Candidatus Liberibacter asiaticus ( CLas) strains associated with HLB samples. Analyses of the CLIBASIA_01645 locus, characteristic of variable tandem repeat numbers (VTRNs), revealed the homogeneity of Iranian CLas isolates: However, this result showed two distinct genotypes (TRN < 10 and TRN > 10) of CLas in Iran. This is the first report documenting the presence of two differentially distributed genotypes of CLas in Iran. Sequence analysis of prophage terminase revealed the presence of two putative prophages (prophage I and prophage II) in the genome of CLas isolates of Iran. Frequency analysis of these two prophages by specific loci revealed the association between prophages populations, the development HLB symptoms and CLas genotypes and their interactions with another obligate symbiontic, HLB phytoplasma.     

Genetic diversity and natural selection at the domain I of apical membrane antigen-1 (AMA-1) of Plasmodium falciparum in isolates from Iran

The apical membrane antigen-1 (AMA-1) of Plasmodium falciparum is a prime malaria asexual blood-stage vaccine candidate. Antigenic variation is one of the main obstacles in the development of a universal effective malaria vaccine. The extracellular region of P. falciparum AMA-1 (PfAMA-1) consists of three domains (I-III), of which the domain I is the most diverse region of this antigen. The objective of our study was to investigate and analyze the extent of genetic diversity and the effectiveness of natural selection at the AMA-1 domain I of P. falciparum in isolates from Iran. A fragment of ama-1 gene spanning domain I was amplified by nested PCR from 48 P. falciparum isolates collected from two major malaria endemic areas of Iran during 2009 to August 2010 and sequenced. Genetic polymorphism and statistical analyses were performed using DnaSP and MEGA software packages. Analysis of intrapopulation diversity revealed relatively high nucleotide and haplotype diversity at the PfAMA-1 domain I of Iranian isolates. Neutrality tests provided strong evidence of positive natural selection acting on the sequenced gene region. The findings also demonstrated that, in addition to natural selection, intragenic recombination may contribute to the diversity observed at the domain I. The results obtained will have significant implications in the design and the development of an AMA-1-based vaccine against falciparum malaria.