De novo Sequencing of Novel Mycoviruses From Fusarium sambucinum: An Attempt on Direct RNA Sequencing of Viral dsRNAs

An increasing number of viruses are continuously being found in a wide range of organisms, including fungi. Recent studies have revealed a wide viral diversity in microbes and a potential importance of these viruses in the natural environment. Although virus exploration has been accelerated by short-read, high-throughput sequencing (HTS), and viral de novo sequencing is still challenging because of several biological/molecular features such as micro-diversity and secondary structure of RNA genomes. This study conducted de novo sequencing of multiple double-stranded (ds) RNA (dsRNA) elements that were obtained from fungal viruses infecting two Fusarium sambucinum strains, FA1837 and FA2242, using conventional HTS and long-read direct RNA sequencing (DRS). De novo assembly of the read data from both technologies generated near-entire genomic sequence of the viruses, and the sequence homology search and phylogenetic analysis suggested that these represented novel species of the Hypoviridae, Totiviridae, and Mitoviridae families. However, the DRS-based consensus sequences contained numerous indel errors that differed from the HTS consensus sequences, and these errors hampered accurate open reading frame (ORF) prediction. Although with its present performance, the use of DRS is premature to determine viral genome sequences, the DRS-mediated sequencing shows great potential as a user-friendly platform for a one-shot, whole-genome sequencing of RNA viruses due to its long-reading ability and relative structure-tolerant nature

Phylogeny of Egyptian isolates of Cucumber mosaic virus (CMV) and Tomato mosaic virus (ToMV) infecting Solanum lycopersicum

Four Cucumber mosaic virus (CMV) (CMV-HM 1\u20134) and nine Tomato mosaic virus (ToMV) (ToMV AH 1\u20139) isolates detected in tomato samples collected from different governorates in Egypt during 2014, were here characterized. According to the coat protein gene sequence and to the complete nucleotide sequence of total genomic RNA1, RNA2 and RNA3 of CMV-HM3 the new Egyptian isolates are related to members of the CMV subgroup IB. The nine ToMV Egyptian isolates were characterized by sequence analysis of the coat protein and the movement protein genes. All isolates were grouped within the same branch and showed high relatedness to all considered isolates (98\u201399%). Complete nucleotide sequence of total genomic RNA of ToMV AH4 isolate was obtained and its comparison showed a closer degree of relatedness to isolate 99\u20131 from the USA (99%). To our knowledge, this is the first report of CMV isolates from subgroup IB in Egypt and the first full length sequencing of an ToMV Egyptian isolate. \ua9 2017 Koninklijke Nederlandse Planteziektenkundige Verenigin

Mycoviruses infecting Fusarium culmorum (W.G. Smith) Sacc.

Fusarium culmorum is a plant pathogenic fungus that causes foot and root rot and ‘Fusarium head blight’ diseases in cereals and particular on wheat and barley. The fungus is also responsible for mycotoxins accumulation on grain. In the last years several studies identified mycoviruses in many fungal species that may cause hypovirulence and hence can be of value in the biocontrol of crop diseases. We analyzed the virome of 40 F. culmorum isolates from Italy, Iran and Syria and the resulting different dsRNA profiles have been associated with several species of mycoviruses. Each dsRNA fragment of the chosen strains was isolated and amplified through a random RT-PCR. PCR amplicons were cloned into plasmid vectors and sequenced. The overall rate of viral infection appears to be 60% and in particular we observed 18% of infection among Italian isolates (3 out of 16), 70% among Syrian isolates (11/16) and 87% among Iranian isolates (7/8). Sequencing results indicate the presence of single and mixed mycoviral infections by members of families Endornaviridae, Narnaviridae and Partitiviridae. Moreover a bipartitic viral species from an Italian fungal isolate has been characterized by virions isolation and full genome sequencing. Observing the low similarity of the genome with other previously described mycoviruses, we have proposed the name of Fusarium culmorum virus 1 (FcV1) for this new viral species. Further studies will focus on the effect of FcV1 on F. culmorum in order to determine its role in the biology and in particular in the fungal pathogenicity

Microscopic ossicle analyses and the complete mitochondrial genome sequence of Holothuria (Roweothuria) polii (Echinodermata; Holothuroidea) provide new information to support the phylogenetic positioning of this sea cucumber species

Sea cucumbers (Holothuroidea) are ecologically important organisms for their bioturbation and alkalinization activities of the seabed. These species are extensively fished as they are considered luxury food. Sea cucumbers are also relevant for biomedical studies and the production of bioactive compounds. A few initiatives are recently evaluating sea cucumbers as novel aquaculture species. The aim of this study was to provide morphological and genetic information useful for the identification of Holothuria polii, the white spot sea cucumber (a common species of the Mediterranean Sea). We generated the complete sequence of the mitochondrial DNA (mtDNA) genome of this species and combined it with a detailed ossicle characterization of the sequenced specimen by scanning electron microscopic analysis. Ossicles (known also as sclerites) are anatomical features that can discriminate Holothuroidea species, including the closely related ones of the genus Holothuria. The complete mitochondrial genome was assembled, functionally annotated and then used to evaluate the phylogenetic relationship of H. polii against the other few Holothuroidea species for which the whole mtDNA was available. The 15,907 bp H. polii mtDNA sequence has the same gene order already reported for H. scabra, H. forskali and other species of the same class. Cox1 and 16S gene sequences were informative for species identification across the genus and could be used for the authentication of commercialized Holothuria spp. The mitochondrial genome sequence presented here provides the basis to a future analysis of the variability of H. polii populations in the Mediterranean region

A simple and reliable method to assess the effects of antimicrobial compounds on phytoplasmas

The only active compounds able to control phytoplasmas are the antibiotic oxytetracyclines and the direct and fast screening of new antimicrobials compounds on media is very complicated due to the difficulty to culture phytoplasmas. A simple and reliable screening method to evaluate the effects of antimicrobial compounds on phytoplasmas by an ex-vivo approach was developed. By the use of scanning electron microscopy in parallel with molecular tools the direct activity of tetracyclines on phytoplasmas was verified

First detection of Tomato Ringspot Virus in pomegranate plants.

Cuttings of Punica granatum for fruit production and ornamental usage have been introduced in accordance with EC directives in the Emilia-Romagna region from the USDA/ARS clonal germplasm repository (California) due to the increasing of pomegranate production all over the Italian territory. This planting material, before its delivering throughout the territory, was subjected to specific analysis in order to ensure a proper sanitary selection. Samples from symptomatic (yellowing, deformation or discoloration of leaves) and symptomless pomegranate trees were tested by ELISA assay using antibodies specific for several viruses affecting fruit crops. In many samples Tomato Ringspot Virus (ToRSV, EPPO A2 list) was detected and its infection confirmed by RT-PCR analysis. Biological characterization of the viral isolates was carried out. Buds were grafted into indicator peach rootstocks and sap from symptomatic tissues was mechanically inoculated onto herbaceous test plants on which typical symptoms of chlorotic ringspot, necrotic lesions, wilting and leaf curling were observed. Phylogenetic analysis revelead high sequence identity of RNA1 and RNA2 3'UTR regions of 4 pomegranate isolates. Official phytosanitary measures have been taken to eradicate the disease. All pomegranate plants of US origins have been uprooted and destroyed. These measures were aimed to avoid the ToRSV diffusion, with the intent to prevent the virus establishment in our territory where it would be harmful not only to pomegranates but also to other economically important fruit crops, as it is happening in USA

Not the one, but the only one: about Cannabis cryptic virus in plants showing ‘hemp streak’ disease symptoms

Interveinal chlorosis and leaf margin wrinkling are widespread symptoms of Cannabis sativa. They are traditionally attributed to the so-called hemp streak virus (HSV), but its existence has not been demonstrated yet. To our knowledge, no molecular investigation has so far been performed in order to identify the causal agent of this symptomatology, we therefore decided to use traditional and molecular virology techniques to better characterize symptoms and pursue the etiological agent. No pathogenic virus was found by using targeted PCR reactions and by RNA sequencing, whereas we were able to detect the Cannabis cryptic virus (CanCV) with both techniques. We, therefore, developed an RT-qPCR assay based on a CanCV-specific TaqMan probe and applied it to a wide range of symptomatic and symptomless plants, using a two-step (for quantification), or a one-step (for fast detection) protocol. Both symptoms and the virus were only shown to be transmitted vertically and did not pass via mechanical inoculation or grafting, though we could not find any cause-effect correlation between them. In fact, the virus was found in all the tested hemp samples, and its abundance varied greatly between different accessions and individuals, independently from the presence and severity of symptoms. The suggestion that hemp streak is caused by a virus is therefore questioned. Some abiotic stresses seem to play a role in triggering the symptoms but this aspect needs further investigation. For breeding purposes, a selection of parental plants based on the absence of symptoms proved to be efficient in containment of the disease