Toufic Elbeaino

Deep sequencing of pigeonpea sterility mosaic virus discloses five RNA segments related to emaraviruses.

The sequences of five viral RNA segments of pigeonpea sterility mosaic virus (PPSMV), the agent of sterility mosaic disease (SMD) of pigeonpea (Cajanus cajan, Fabaceae), were determined using the deep sequencing technology. Each of the five RNAs encodes a single protein on the negative-sense strand with an open reading frame (ORF) of 6885, 1947, 927, 1086, and 1,422 nts, respectively. In order, from RNA1 to RNA5, these ORFs encode the RNA-dependent RNA polymerase (p1, 267.9 kDa), a putative glycoprotein precursor (p2, 74.3 kDa), a putative nucleocapsid protein (p3, 34.6 kDa), a putative movement protein (p4, 40.8 kDa), while p5 (55 kDa) has an unknown function. All RNA segments of PPSMV showed the highest identity with orthologs of fig mosaic virus (FMV) and Rose rosette virus (RRV). In phylogenetic trees constructed with the amino acid sequences of p1, p2 and p3, PPSMV clustered consistently with other emaraviruses, close to clades comprising members of other genera of the family Bunyaviridae. Based on the molecular characteristics unveiled in this study and the morphological and epidemiological features similar to other emaraviruses, PPSMV seems to be the seventh species to join the list of emaraviruses known to date and accordingly, its classification in the genus Emaravirus seems now legitimate.

Deep-sequencing analysis of an apricot tree with vein clearing symptoms reveals the presence of a novel betaflexivirus

Deep-sequencing technology applied on double stranded RNA recovered from an apricot tree with vein clearing symptoms allowed the identification of a novel virus with a single-stranded RNA genome, for which the provisional name apricot vein clearing-associated virus (AVCaV) is proposed. Its genome comprises 7315nt, excluding the poly(A) tail, covering four open reading frames (ORFs). The putative virus-encoded proteins, i.e., replicase (REP), movement protein (MP), coat protein (CP) and nucleic acid-binding protein (NB), had an estimated molecular weight of 192.5, 32.15, 25.5 and 16.1kDa, respectively and shared the highest identity (ca. 40%) with citrus leaf blotch virus (CLBV) and with orthologs of other known members of the family Betaflexiviridae. The phylogenetic trees constructed with the sequences of the entire replication-associated polyproteins and the putative CP showed incongruent allocations of AVCaV within the genus Citrivirus or as an outgroup species close to the genus Vitivirus, respectively. The peculiar organization of its genome (four ORFs), different from that typical of members of Citrivirus (three ORFs) and Vitivirus (five ORFs) genera, makes likely AVCaV a novel member of an unassigned genus of the family Betaflexiviridae. In RT-PCR assays, AVCaV was found to infect only one out of 39 varieties of apricot tested; thus, suggesting to be limitedly spread.

OCCURRENCE AND WIDESPREAD DISTRIBUTION OF GRAPEVINE VIRUS D IN TUNISIAN GRAPEVINES

Rugose wood is a complex disease affecting grapevine worldwide. At present, at least six different viruses belonging to the family Betaflexiviridae are known to be associated with the disease (Saldarelli, 2014). Previous studies had shown that Grapevine virus A (GVA), Grapevine virus B (GVB) and Grapevine rupestris stem pitting associated virus (GRSPaV) are common in Tunisian grapevines (Martelli, 2014), whereas no information is available on the presence of Grapevine virus D (GVD). Therefore, a total of 140 samples from several cultivars, were collected from major Tunisian viticultural regions and tested for the presence of this virus by RT-PCR using the GVD-specific primers CP471C and CP7V (Abou-Ghanem et al., 1997). A 474 bp product corresponding to a fragment of the coat protein gene was amplified from 58 samples, accounting for an infection rate of 41.42%. The virus was present in all the surveyed areas and grape cultivars tested. The infection rate ranged from 11.7% (cv. Red Globe) to 88.8% (cv. Muscat RafRaf). To confirm the identity of the target virus, three RT-PCR products were cloned and sequenced. The Tunisian isolates shared 86-91% common nucleotides with the other GVD isolates from GenBank, and 72-73%, 60-62% and 53-55% nucleotide identity with GVA (JF754577), GVB (NC003602.1) and GVE (JX402759.1), respectively. To our knowledge, this is the first report on the occurrence of GVD in Tunisian grapevines. The infection rate of GVD (41.42%), which is close to that of GVA (47.2%) in the same samples, suggests the presence of common vectors for both viruses in Tunisian vineyards.

Viruses and Hop stunt viroid of fig trees in Syria.

A virus survey was carried out in spring and summer 2010 in Syrian fig orchards and gardens in 9 cities and in a varietal collection at Idleb. A total of 90 fig samples were collected and tested by RT-PCR for the presence of Fig leaf mottle-associated virus 1 (FLMaV-1), Fig leaf mottle-associated virus 2 (FLMaV-2), Fig mild mottleassociated virus (FMMaV), Fig mosaic virus (FMV), Fig latent virus 1 (FLV-1), Fig cryptic virus (FCV), Fig fleck-associated virus (FFkaV), and Hop stunt viroid (HSVd) using sets of specific primers. PCR results showed that about 84% of the trees were infected with at least one virus. FMV was the prevailing virus (56.7%), followed by FFkaV (36.7%), FLMaV-2 (31.1%), FMMaV (12.2%), FLV-1 (11.1%) and FLMaV-1 (4.4%), whereas FCV was not found. HSVd was detected in 13.3% of the samples. In a phylogenetic tree, the nucleotide sequences of most Syrian HSVd-fig isolates grouped with those reported in Lebanon from the same host and from mulberry, forming a distinct clade (M-group). This is the first report of FMMaV, FMV, FLV-1 and HSVd in fig trees in Syria.

Incidence and distribution of viruses in Tunisian fig orchards.

A survey for viruses was carried out in the main fig-growing areas of Tunisia and in a fig germplasm collection at the Institut Superieure Agronomique de Chatt Mariem of Sousse. A total of 232 leaf samples were collected randomly from symptomatic and symptomless fig trees of 26 cultivars, and tested by RT-PCR for the presence of Fig mosaic virus (FMV), Fig leaf mottle-associated virus 1 (FLMaV-1), Fig leaf mottle-associated virus 2 (FMMaV-2), Fig mild mottle-associated virus (FMMaV), Fig cryptic virus (FCV), Fig fleck-associated virus (FFkaV) and Fig latent virus 1 (FLV-1), using specific sets of primers. About 62.1% of the samples tested were found to be infected by at least one virus. FMV was the prevailing virus with a 34.5% incidence, followed by FLV-1 (32.4%), FMMaV (10.7%), FLMaV-1 (10.3%), FFkaV (10.3%), FCV (9.9%) and FLMaV-2 (4.3%). The highest infection rate was observed in Sfax (100%), followed by Takelsa (73.4%), Djebba (70.2%), Sousse (66.6%), Mornag (54.4%) and Rafraf (50%), whereas it was moderate in Sidi Fraj (23.3%). Among all viruses detected, FMV was the most widespread, especially in Djebba (51.3%), Sfax (50%) and Takelsa (40.6%). According to the fig varieties, the highest infection rates were in the commercial cvs. Hamouri (87.5%), Bayoudhi (75%), Bouhouli (70.3%), Zidi (54.2%) Bither (53.8%), Bither Abyath (66.6%) and Soltani (69.2%). The results show the need to develop a clean stock program to prevent the dissemination of fig-infecting viruses associated with Fig mosaic disease in Tunisia.

OCCURRENCE OF FIG MOSAIC VIRUS IN TUNISIAN FIG ORCHARDS

The symptomatology of fig mosaic disease is extremely vari- able. The causal agent of the disease was unknown for a long time (Martelli, 2011), until enveloped round to ovoid bodies known as double membrane bodies (DMBs) were found consis- tently associated with mosaic symptoms. DMBs were successfully transmitted by the eriophyd mite Aceria ficus (Martelli, 2011), and eventually identified as the putative particles of Fig mosaic virus (FMV) (Elbeaino et al., 2009). In spring 2011 a survey of fig orchards was carried out in four different areas of north Tunisia (Cap Bon, Mornag, Beja, Rafraf) to assess the presence and prevalence of FMV. A range of foliar symptoms, including vari- ous types of chlorotic mottling and blotching, crinkling, vein clearing, vein banding, vein feathering, ring spots, line patterns and malformations, were observed in all the fields surveyed. Symptomatic leaf samples were collected from fig trees and test- ed by RT-PCR using FMV-specific primers (E5-s CGGTAG- CAAATGGAATGAAA) and (E5-a-AACACTGTTTTTGC- GATTGG). A 302 pb DNA fragment of RNA-1 (Elbeaino et al., 2009) was obtained from 35% (62 out of 175) of the samples tested. The virus was widely distributed in cvs Zidi (54%), Bouhouli (52%), Bayoudhi (25%) and Bither (20%). Mosaic-like symptoms were also present in 63 of 143 samples that were PCR- negative for FMV. This last finding further supports the complex nature of fig mosaic disease in whose aetiology FMV plays a sig- nificant but likely not exclusive role (Martelli, 2011). To our knowledge, this is the first record of FMV in Tunisia.

ICTV Virus Taxonomy Profile: Fimoviridae

Members of the family Fimoviridae, order Bunyavirales are plant viruses with segmented, linear, single-stranded, negative-sense RNA genomes. They are distantly related to orthotospoviruses and orthobunyaviruses of the families Tospoviridae and Peribunyaviridae, respectively. The family Fimoviridae includes the genus Emaravirus, which comprises several species with European mountain ash ringspot-associated emaravirus as the type species. Fimoviruses are transmitted to plants by eriophyid mite vectors and induce similar characteristic cytopathologies in their host plants, including the presence of double membrane-bound bodies in the cytoplasm of the virus-infected cells. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Fimoviridae, which is available at www.ictv.global/report/fimoviridae.

Effect of Cucumber mosaic virus (CMV) infection on antineoplastic alkaloids from periwinkle (Catharanthus roseus L.) cultured in the Mecca region and resistance induction by plant-growth-promoting rhizobacteria (PGPR)

ABSTRACT More than 100 alkaloids have been found in periwinkle, of which vincristine and vinblastine are the most notable for the treatment of diseases such as leukaemia. In this study, characterization of naturally occurring Cucumber mosaic virus (CMV) infection showed mosaic, leaf deformation and stunting of plants. Viral identification was confirmed by double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) with a specific CMV polyclonal antibody and reverse transcription PCR with a specific primer for the CMV-RdRp-gene, which yielded a 513 base pair DNA fragment. The effect of CMV infection on the antineoplastic alkaloids in periwinkle leaves was determined. We also studied the effect of using plant-growth-promoting rhizobacteria (PGPR) isolates against virus infection to stimulate resistance induction in host plants. Bacillus subtilis 281 and B. pumilus 293 were examined individually (B1 and B2) and in a mixture (B1&B2) for their effectiveness against infection with CMV. The results of greenhouse experiments were confirmed by artificial mechanical inoculation. The PGPR strain treatments and results were reinforced by analysing the protein patterns as well as determining the total phenol, total flavonoid and total alkaloid contents. PGPR treatment evidently lowered the virus concentrations, the percentage of infected plants and the disease severity compared with healthy and infected controls. Seedlings treated with the B1&B2 strain mixture yielded significantly lower levels of virus infection than B1 or B2 individually in all experiments compared to controls. Our findings demonstrate the possibility of using selected Bacillus spp. strains to induce systemic resistance for CMV infection control.

Inhibitory activity of different medicinal extracts from Thuja leaves, ginger roots, Harmal seeds and turmeric rhizomes against Fig leaf mottle-associated virus 1 (FLMaV-1) infecting figs in Mecca region

Fig leaf mottle-associated virus-1 (FLMaV-1) is a closterovirus newly identified in fig trees, in the Mecca region, suffering from mosaic disease symptoms and apparently is compromising the fig plantation in the country. In the present study, we demonstrated the efficiency of two in vivo experiments including pre and post treatments using Thuja leaf, ginger roots, Harmal seeds and turmeric rhizome extracts on symptoms expression of rooted cuttings infected with FLMaV-1- and their impact on virus multiplication. Results showed that individual treatments with ginger roots and turmeric rhizomes in pre-grafting experiments and Thuja extract following Harmal seeds in post grafting experiments were efficient against symptom development. In addition, results showed that the total photosynthesis pigments; total soluble intracellular proteins and total phenol contents were higher in infected treated cuttings compared with healthy ones, thus it was taken as evidence on a mutual interaction between these extracts and virus multiplication.

DETECTION OF FIG MOSAIC VIRUS, FIG LEAF MOTTLE-ASSOCIATED VIRUS 1 AND FIG MILD MOTTLE-ASSOCIATED VIRUS IN BOSNIA AND HERZEGOVINA

During May 2015, a small scale survey was conducted on 27 fig trees situated in three locations (Mostar, Trebinje and Ljubuski) in Bosnia and Herzegovina, to investigate the presence of Fig mosaic virus (FMV), Fig leaf mottle-associated virus 1 (FLMaV-1), Fig leaf mottle-associated virus 2 (FLMaV- 2) and Fig mild mottle-associated virus (FMMaV). Samples consisted of leaves singularly taken from trees with symptoms of mosaic, vein yellowing, ring spots, necroses and leaf malformations (23 plants) and one asymptomatic tree, all situated in a fig germplasm collection plot. Additional three samples were taken from symptomatic fig plants of three outdoor gardens. Total RNAs extracted from the leaf midrib using RNeasy Plant Mini Kit (QIAGEN), were used in RT-PCR assays with specific primer pairs for each virus following protocols described in Elbeaino et al. (2006, 2009, 2010). RT-PCR results revealed 19 FMV- (70%), 25 FLMaV-1- (92%) and 4 FMMaV- (15%) positive samples. Double infections with FLMaV-1 and FMV were detected in 74% samples, while triple infections with FLMaV- 1, FMV and FMMaV affected 15% of tested figs. After cloning and bi-directional sequencing of 2 clones from random PCR amplicons for each virus, nucleotide BLASTn sequence analyses for FLMaV- 1 (350 bp) (GenBank Accession Nos. KU198378-KU198382), FMMaV (311 bp) (KU198388) and FMV (302 bp) (KU198367-KU198373) showed identity levels of 84-95%, 89-92% and 83-97%, respectively, with homologues available in GenBank. To our knowledge, this is the first report of three fig-infecting viruses (FLMaV-1, FMMaV and FMV) in Bosnia and Herzegovina and the results obtained, even if limited, reflect a precarious sanitary status of fig that needs a clean stock program in this country.