Application of High-Throughput Sequencing for Comprehensive Virome Profiling in Grapevines Shows Yellows in Iran.

A comprehensive study on the whole spectrum of viruses and viroids in five Iranian grapevine cultivars was carried out using sRNA libraries prepared from phloem tissue. A comparison of two approaches to virus detection from sRNAome data indicated a significant difference in the results and performance of the aligners in viral genome reconstruction. The results showed a complex virome in terms of viral composition, abundance, and richness. Thirteen viruses and viroids were identified in five Iranian grapevine cultivars, among which the grapevine red blotch virus and grapevine satellite virus were detected for the first time in Iranian vineyards. Grapevine leafroll-associated virus 1 (GLRaV1) and grapevine fanleaf virus (GFLV) were highly dominant in the virome. However, their frequency and abundance were somewhat different among grapevine cultivars. The results revealed a mixed infection of GLRaV1/grapevine yellow speckle viroid 1 (GYSVd1) and GFLV/GYSVd1 in grapevines that exhibited yellows and vein banding. We also propose a threshold of 14% of complete reconstruction as an appropriate threshold for detection of grapevine viruses that can be used as indicators for reliable grapevine virome profiling or in quarantine stations and certification programs.

Penetration and Development of Meloidogyne Javanica on Four Pistachio Rootstocks and their Defense Responses.

Pistachio yield is annually reduced due to root-knot nematode infections. In order to evaluate its resistance to Meloidogyne javanica, three domestic pistachio rootstocks, namely, Badami, Ghazvini and Sarakhs, and a wild pistachio, Baneh (Pistacia atlantica subsp. mutica), were selected. Their response to the nematode infection was evaluated based on different plant and nematode indexes, 120 days post-inoculation (dpi). The penetration and development rate of nematode in roots of these four pistachio rootstocks were evaluated at different time points by acid fuchsin staining. Based on the measured indexes, Badami, Ghazvini, Sarakhs, and Baneh rootstocks ranked as susceptible, moderately resistant, moderately resistant, and resistant, respectively. The penetration rate of second-stage nematode juveniles (J2) into four rootstocks was discussed. The first "midstage" or swollen juveniles appeared at 4 dpi but to a lesser extent in Ghazvini, Sarakhs, and Baneh cultivars. The first females were seen in Badami at 21 dpi, in Ghazvini and Sarakhs at 35 dpi, and in Baneh at 45 dpi. Three types of defense responses were distinguished and characterized in the examined pistachio rootstocks: (i) a hypersensitive response (HR)-like reaction in the cortex in Ghazvini, Sarakhs, and Baneh root tips at 4 dpi and 6 dpi; (ii) an HR response, degrading J2 which induce giant cells in the vascular cylinder of all rootstocks, at 6 dpi and 10 dpi; and (iii) an HR response, degrading females and giant cells in the vascular cylinder of all rootstocks at 15 dpi onward. These observations open new fields of study in breeding programs of this crop.

Identification of garlic-infecting leek yellow stripe virus through deep-sequencing analyses from Iran.

In the study presented here, the first complete genome sequence of Leek yellow stripe virus (LYSV) designated as isolate LYSV-AE65 from Southwest of Iran, was reported. The small RNA deep sequencing analysis showed that, the Iranian isolate has a full RNA genome of 10,142 nucleotide in length (Except for poly (A) tail) and it was shared 77.91-92.16% nucleotide (nt) and 83.62-96.35% amino acid (aa) sequences identities with other known LYSV isolates. The coat protein (CP) region showed 80.21-95.24% nucleotide identity to those of other isolates, while high degrees of nucleotide sequence identity with G77-LYSV isolate (MN059504) from China. Phylogenetic analysis based on full genome sequence of LYSV-AE65, showed the closest relationship with LYSV isolates from China, Australia, Spain and Mexico. Also, phylogenetic analysis of the 5´-untranslated region (UTR)-P1 gene sequences of 44 isolates, confirmed the formation of two main groups, N-type and S-type, in agreement with the previous studies. Isolate LYSV-AE65 was similar to the members of clade S and has two large deletions in P1 gene. Recombination analysis demonstrated that LYSV-AE65 was a recombinant with most part of its genome was derived from already reported LYSV isolates infecting allium species. To the best of our knowledge, this is the first report of complete genome sequencing of LYSV isolate infecting garlic through small RNA deep sequencing method in Iran. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at (10.1007/s13337-021-00733-z).

Whole genome characterization of wisteria vein mosaic virus from Iran and its relationship to other members of bean common mosaic virus group.

To date, the complete genome of two wisteria vein mosaic virus (WVMV) has been sequenced worldwide. Here, the genomic sequence of WVMV isolated from Wisteria sinensis in Iran was determined for the first time, using deep RNA sequencing and RT-PCR followed by Sanger sequencing. The sequence was 9694 nucleotides in length; excluding the 3'-poly(A) tail and contained a single open reading frame of 9279 nucleotides encoding a large polyprotein of 3092 amino acids and predicted molecular weight of 35,368 KDa. The genome contained nine putative proteolytic cleavage sites and motifs conserved in homologous proteins of other potyviruses. Sequence analysis suggested that WVMV-Ir sequence shared 76.37-86.01% nucleotide (nt) identity and 82.45-91.91% amino acid (aa) identity with two other isolates (Beijing and JEBU-p) available in the GenBank, the highest with the Chinese isolate Beijing (86.01% nt identity, 91.91% aa identity). Sequence identities over most of the genome were within the range 80-86% and 85-95% at the nt and aa levels, respectively; however, high variability was observed in the 5'-UTR (51.62%), P1 (62.03% nt identity, 50.78% aa identity) and P3 (79.82%nt identity, 78.67% aa identity) regions, suggesting that Ir, Beijing, and JEBU-p are three different strains. These variabilities may be due to different mutation phenomena of a common ancestor virus or mutations caused by different selection pressures in different agro-ecological regions. The results of the phylogenetic analysis indicated that WVMV was most closely related to soybean mosaic virus and watermelon mosaic virus and less closely related to the zantedeschia mild mosaic virus and dasheen mosaic virus. In the greenhouse, WVMV-Ir caused severe symptoms in Phaseolus vulgaris, Vicia faba, W. sinensis, Chenopodium quinoa, C. amaranticolor, and Nicotiana benthamiana. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02957-8.

Analysis of the molecular and biological variability of Zucchini yellow mosaic virus isolates from Iran and Iraq.

During the growing season of 2018, several field-grown cucurbit plants in different parts of Iraq and Iran were surveyed for the presence of zucchini yellow mosaic virus (ZYMV), using two degenerate primer pairs (CIF/Rev and NIb2F/3R) targeting the two separated partial regions of the potyvirus genome (CI and NIb respectively). 7 out of 20 samples were confirmed to be infected with ZYMV. Phylogenetic analyses based on the CI gene grouped all Iranian and two Iraqi (ZYMV1 and ZYMV2) isolates together with isolates from the Middle East in the subgroup (AI), whereas the other Iraqi (ZYMV3 and ZYMV4) isolates were clustered in the subgroup (DI), which was only consisted of American isolates. The highest and lowest identity between the studied isolates and the GenBank isolates showed that the two genes (CI, NIb) of each isolate particularly the Iraqi isolates were more similar to a specific and geographically scattered mosaic of worldwide isolates, suggestive of mixed infection might have occurred between different worldwide isolates in Iraq. Furthermore, the first complete nucleotide sequence of an Iraqi ZYMV (ZYMV-Iq) isolate was done, using the Illumina sequencing technique. The complete nucleotide sequence of ZYMV-Iq isolate was 9650 nt, excluding the 3'poly (A) tail. ZYMV-Iq isolate shared the highest nt identity of 98.8% with an American (KC665630) isolate. Phylogenetic analysis based on the full genome sequence placed ZYMV-Iq in subgroup A of group I alongside 18 isolates from the US and two isolates from Australia. In addition, recombination analysis detected lone significant recombination between ZYMV-Iq and South Korean (AY279000) isolate. Moreover, the results showed that symptom intensity was varied across experimental host plants.

Rapid identification of Bactrocera zonata (Dip.: Tephritidae) using TaqMan real-time PCR assay.

Tephritid fruit flies are ranked as one of the most damaging groups of insect pests. Morphological identification of fruit flies is mainly performed on adults due to the lack of adequate identification keys for immature stages. The peach fruit fly, Bactrocera zonata (Saunders), infests some of the principal commercial fruits and vegetables. It is, therefore important to avert its global dispersal, particularly by accurately identifying this species at ports of entry. In this study, a TaqMan real-time polymerase chain reaction (PCR) was developed for the accurate identification and sensitive detection of the peach fruit fly. A novel set of primers and probe were designed to specifically identify the mitochondrial cytochrome oxidase I (COI) gene. All specimens of peach fruit fly (including various life stages) were detected, and no cross reactivity with other tested tephritids were observed. Since this assay performed equally well with crushed insects and purified DNA, we note added efficiency by eliminating DNA extraction step. Considering the speed, specificity as well as sensitivity of the assay, Taqman real-time PCR can be used as a swift and specific method for pest species at ports of entry.

Iranian johnsongrass mosaic virus: the complete genome sequence, molecular and biological characterization, and comparison of coat protein gene sequences.

Iranian johnsongrass mosaic virus (IJMV) is one of the most prevalent viruses causing maize mosaic disease in Iran. An IJMV isolate, Maz-Bah, was obtained from the maize showing mosaic symptoms in Mazandaran, north of Iran. The complete genomic sequence of Maz-Bah is 9544 nucleotides, excluding the poly(A) tail. It contains one single open reading frame of 9165 nucleotides and encodes a large polyprotein of 3054 amino acids, flanked by a 5'-untranslated region (UTR) of 143 nucleotides and a 3'-UTR of 236 nucleotides. The entire genomic sequence of Maz-Bah isolate shares identities of 84.9 and 94.2 % with the IJMV (Shz) isolate, the lone complete genome sequence available in the GenBank at the nucleotide (nt) and deduced amino acid (aa) levels, respectively. The whole genome sequences share identities of 51.5-69.8 and 44.9-74.3 % with those of other Sugarcane mosaic virus (SCMV) subgroup potyviruses at nt and aa levels, respectively. In phylogenetic trees based on the multiple alignments of the entire nt and aa sequences, IJMV isolates formed a separate sublineage of the tree with potyviruses infecting monocotyledons of cereals, indicating that IJMV is a member of SCMV subgroup of potyviruses. IJMV is most closely related to Sorghum mosaic virus and Maize dwarf mosaic virus and less closely related to the Johnsongrass mosaic virus and Cocksfoot streak virus. To further investigate the genetic relationship of IJMV, 9 other isolates from different hosts were cloned and sequenced. The identity of IJMV CP nt and aa sequences of 11 Iranian isolates ranged from 86.4 to 99.8 % and 90.5 to 99.7 %, respectively, indicating a high nt variability in CP gene. Furthermore, in the CP-based phylogenetic tree, IJMV isolates were clustered together with a maize potyvirus described as Zea mosaic virus from Israel (with 86-89 % nt identity), indicating that both isolates probably are the strains of the same virus.

The complete genome sequences of two naturally occurring recombinant isolates of Sugarcane mosaic virus from Iran.

Sugarcane mosaic virus (SCMV) is the most prevalent virus causing sugarcane mosaic and maize dwarf mosaic diseases. Here, we presented the first two complete genomic sequences of Iranian SCMV isolates, NRA and ZRA from sugarcane and maize. The complete genome sequences of NRA and ZRA were, respectively, 9571 and 9572 nucleotides (nt) in length, excluding the 3'-terminal poly(A) tail. Both isolates contained a 5'-untranslated region (UTR) of 149 nt, an open reading frame of 9192 nt encoding a polyprotein of 3063 amino acids (aa), and 3'-UTR of 230 nt for NRA and 231 nt for ZRA. SCMV-NRA and -ZRA genome nucleotide sequences were 97.3 % identical and shared nt identities of 79.1-92 % with those of other 21 SCMV isolates available in the GenBank, highest with the isolate Bris-A (AJ278405) (92 and 91.7 %) from Australia. When compared for separate genes, most of their genes shared the highest identities with Australian and Argentinean isolates. Phylogenetic analysis of the complete genomic sequences reveals that SCMV can be clustered to three groups. Both NRA and ZRA were clustered with sugarcane isolates from Australia and Argentina in group III but formed a separate sublineage. Recombination analysis showed that both isolates were intraspecific recombinants, and represented two novel recombination patterns of SCMV (in the P1 coding region). NRA had six recombination sites within the P1, HC-Pro, CI, NIa-Vpg, and NIa-pro coding regions, while ZRA had four within the P1, HC-Pro, NIa-Pro, and NIb coding regions.

Molecular variability in the cysteine rich protein of potato virus M.

The potato virus M (PVM), belonging to the genus Carlavirus, is a worldwide endemic pathogen in potato fields. p11 is an 11-16 kDa protein encoded by the last open reading frame of PVM which contains cysteine rich proteins (CRPs) motif. CRPs have been identified as suppressors of gene silencing. In this study the p11 gene from 28 PVM isolates, including 16 new isolates from Iran, were used to determine the global genetic structure of PVM populations. Pairwise nucleotide sequence identity scores showed that global PVM CRP sequence similarity was between 69.3 and 100 %. This genetic diversity divided the 28 isolates into two main divergent phylogenetic clades. The rate of genetic diversity and non-synonymous to synonymous mutations (dN/dS) were significantly different between these two clades. Analysis showed that PVM CP is under significant negative selection pressure with the global ω value of 0.260.

Genetic structure and molecular variability of potato virus M populations.

To investigate the genetic diversity of potato virus M (PVM; genus Carlavirus, family Betaflexiviridae), the complete nucleotide sequence of the coat protein gene of 30 PVM isolates from a major potato-growing region in Iran were determined. Phylogenetic analysis of these Iranian PVM isolates together with those available in the GenBank database suggested two divergent evolutionary lineages that did not reflect the origin of the isolates, and these were designated as PVM-o and PVM-d. Examination of the genetic variability of the coat protein of Iranian isolates and their counterparts whose sequences are available in the Genbank database revealed 16 genotype groups in the PVM population. Analysis of the synonymous-tononsynonymous ratio showed strong purifying selection in the CP gene in the genotype groups of divergent clades.

Diversity of beet curly top Iran virus isolated from different hosts in Iran.

Beet curly top Iran virus (BCTIV) is a major pathogen of sugar beet in Iran. In order to study diversity of BCTIV, we sampled 68 plants in Iran during the summer of 2010 with curly top disease symptoms on beans (Phaseolus vulgaris), cowpeas (Vigna unguiculata), tomatoes (Solanum lycopersicum L.), sea beets (Beta vulgaris subsp. maritima), and sugar beets (Beta vulgaris). Plant samples showing leaf curling, yellowing, and/or swelling of veins on the lower leaf surfaces were collected from various fields in Khorasan Razavi, Northern Khorasan (north-eastern Iran), East Azarbayejan, West Azarbayejan (north-western Iran), and Fars (southern Iran) provinces. Using rolling circle amplification coupled with restriction digests, cloning, and Sanger sequencing, we determined the genomes of nine new BCTIV isolates from bean, cowpea, tomato, sea beet, and sugar beet in Iran. Our analysis reveals ~11 % diversity amongst BCTIV isolates and we detect evidence of recombination within these genomes.