Tentative Species Research Articles

White yam (Dioscorea rotundata) plants exhibiting virus-like symptoms are co-infected with a new potyvirus and a new crinivirus in Ethiopia.

White yam (Dioscorea rotundata) plants collected from farmers' fields and planted at the Areka Agricultural Research Center, Southern Ethiopia, displayed mosaic, mottling, and chlorosis symptoms. To determine the presence of viral pathogens, an investigation for virome characterization was conducted by Illumina high-throughput sequencing. The bioinformatics analysis allowed the assembly of five viral genomes, which according to the ICTV criteria were assigned to a novel potyvirus (3 genome sequences) and a novel crinivirus (2 genome sequences). The potyvirus showed ~ 66% nucleotide (nt) identity in the polyprotein sequence to yam mosaic virus (NC004752), clearly below the demarcation criteria of 76% identity. For the crinivirus, the RNA 1 and RNA 2 shared the highest sequence identity to lettuce chlorosis virus, and alignment of the aa sequence of the RdRp, CP and HSP70h (~ 49%, 45% and 76% identity), considered for the demarcation criteria, revealed the finding of a novel virus species. The names Ethiopian yam virus (EYV) and Yam virus 1 (YV-1) are proposed for the two tentative new virus species.

Genome analysis of gyroviruses identified in waterfowl in Arizona (USA).

Gyroviruses are small single-stranded DNA (ssDNA) viruses that are largely associated with birds. Chicken anemia virus is the most extensively studied gyrovirus due to its disease impact on the poultry industry. However, we know much less about gyroviruses infecting other avian species. To investigate gyroviruses infecting waterfowl, we determined six complete genome sequences that fall into three gyrovirus groups, referred to as waterfowl gyrovirus 1 (n = 3), 2 (n = 2), and 3 (n = 1), in organs from hunter-harvested waterfowl from Arizona (USA). The waterfowl gyrovirus 1 variants were identified in multiple organs of a single American wigeon and represent a tentative new species. The waterfowl gyrovirus 2 variants were identified in the livers of two American wigeons and share >70% VP1 nucleotide sequence identity with gyrovirus 9, previously identified in the spleen of a Brazilian Pekin duck (MT318123) and a human fecal sample (KP742975). Waterfowl gyrovirus 3 was identified in a northern pintail spleen sample, and it shares >73% VP1 nucleotide sequence identity with two gyrovirus 13 sequences previously identified in Brazilian Pekin duck spleens (MT318125 and MT318127). These gyroviruses are the first to be identified in waterfowl in North America, as well as in American wigeons and northern pintails.

Freshwater trematodes differ from marine trematodes in patterns connected with division of labor.

Prior research suggests that trematode rediae, a developmental stage of trematode parasites that reproduce clonally within a snail host, show evidence of division of labor (DOL). Single-species infections often have two morphologically distinct groups: small rediae, the 'soldiers', are active, aggressive, and do not appear to reproduce; large rediae, the 'reproductives', are larger, sluggish, and full of offspring. Most data supporting DOL come from trematodes infecting marine snails, while data from freshwater trematodes are more limited and generally do not supported DOL. The shorter lifespan typical of freshwater snails may partially explain this difference: defending a short-lived host at the expense of reproduction likely provides few advantages. Here, we present data from sixty-one colonies spanning twenty species of freshwater trematode exploring morphological and behavioral patterns commonly reported from marine trematodes believed to have DOL. Trematode rediae were obtained from sixty-one infected snails collected in central Vermont, USA. A portion of the COI gene was sequenced to make tentative species identifications ('COI species'). Samples of rediae were photographed, observed, and measured to look for DOL-associated patterns including a bimodal size distribution, absence of embryos in small rediae, and pronounced appendages and enlarged pharynges (mouthparts) in small rediae. Additional rediae were used to compare activity levels and likelihood to attack heterospecific trematodes in large vs. small rediae. Many of the tests for DOL-associated patterns showed mixed results, even among colonies of the same COI species. However, we note a few consistent patterns. First, small rediae of most colonies appeared capable of reproduction, and we saw no indication (admittedly based on a small sample size and possibly insufficient attack trial methodology) that small rediae were more active or aggressive. This differs from patterns reported from most marine trematodes. Second, the small rediae of most colonies had larger pharynges relative to their body size than large rediae, consistent with marine trematodes. We also observed that colonies of three sampled COI species appear to produce a group of large rediae that have distinctly large pharynges. We conclude that these freshwater species likely do not have a group of specialized non-reproductive soldiers because small rediae of at least some colonies in almost every species do appear to produce embryos. We cannot rule out the possibility that small rediae act as a temporary soldier caste. We are intrigued by the presence of rediae with enlarged pharynges in some species and propose that they may serve an adaptive role, possibly similar to the defensive role of small 'soldier' rediae of marine trematodes. Large-pharynx rediae have been documented in other species previously, and we encourage future efforts to study these large-pharynx rediae.

Two Novel Betarhabdovirins Infecting Ornamental Plants and the Peculiar Intracellular Behavior of the Cytorhabdovirus in the Liana Aristolochia gibertii.

Two novel members of the subfamily Betarhabdovirinae, family Rhabdoviridae, were identified in Brazil. Overall, their genomes have the typical organization 3'-N-P-P3-M-G-L-5' observed in mono-segmented plant-infecting rhabdoviruses. In aristolochia-associated cytorhabdovirus (AaCV), found in the liana aristolochia (Aristolochia gibertii Hook), an additional short orphan ORF encoding a transmembrane helix was detected between P3 and M. The AaCV genome and inferred encoded proteins share the highest identity values, consistently < 60%, with their counterparts of the yerba mate chlorosis-associated virus (Cytorhabdovirus flaviyerbamate). The second virus, false jalap virus (FaJV), was detected in the herbaceous plant false jalap (Mirabilis jalapa L.) and represents together with tomato betanucleorhabdovirus 2, originally found in tomato plants in Slovenia, a tentative new species of the genus Betanucleorhabdovirus. FaJV particles accumulate in the perinuclear space, and electron-lucent viroplasms were observed in the nuclei of the infected cells. Notably, distinct from typical rhabdoviruses, most virions of AaCV were observed to be non-enclosed within membrane-bounded cavities. Instead, they were frequently seen in close association with surfaces of mitochondria or peroxisomes. Unlike FaJV, AaCV was successfully graft-transmitted to healthy plants of three species of the genus Aristolochia, while mechanical and seed transmission proved unsuccessful for both viruses. Data suggest that these viruses belong to two new tentative species within the subfamily Betarhabdovirinae.

Alfalfa vein mottling virus, a novel potyvirid infecting Medicago sativa L.

BackgroundWe have recently identified a novel virus detected in alfalfa seed material. The virus was tentatively named alfalfa-associated potyvirus 1, as its genomic fragments bore similarities with potyvirids. In this study, we continued investigating this novel species, expanding information on its genomic features and biological characteristics.MethodsThis research used a wide range of methodology to achieve end results: high throughput sequencing, bioinformatics tools, reverse transcription-polymerase chain reactions, differential diagnostics using indicator plants, virus purification, transmission electron microscopy, and others.ResultsIn this study, we obtained a complete genome sequence of the virus and classified it as a tentative species in the new genus, most closely related to the members of the genus Ipomovirus in the family Potyviridae. This assumption is based on the genome sequence and structure, phylogenetic relationships, and transmission electron microscopy investigations. We also demonstrated its mechanical transmission to the indicator plant Nicotiana benthamiana and to the natural host Medicago sativa, both of which developed characteristic symptoms therefore suggesting a pathogenic nature of the disease.ConclusionsConsistent with symptomatology, the virus was renamed to alfalfa vein mottling virus. A name Alvemovirus was proposed for the new genus in the family Potyviridae, of which alfalfa vein mottling virus is a tentative member.

First report of Fig virus B in Ficus carica in New Zealand.

Fig (Ficus carica) has been cultivated since ancient times, and is now grown worldwide, both for its fruit and as an ornamental plant. Several viruses and viroids are associated with Fig mosaic disease (FMD), a disease complex occurring worldwide (Preising et al. 2021). Fig mosaic virus (FMV), fig leaf mottle-associated virus 1 (FLMaV-1), fig mild mottle-associated virus (FMMaV), and fig badnavirus 1 (FBV-1) are known to infect fig in New Zealand (Minafra et al. 2012; Veerakone et al. 2015). In December 2020, leaf samples from a fig tree growing on the roadside at St Heliers, Auckland, showing dieback with foliar chlorotic mosaic symptoms, was received for virus testing. Total nucleic acid was extracted from the symptomatic leaves using a KingFisher™ mL Purification System (Thermofisher Scientific, Waltham, MA) with an InviMag Plant DNA Mini Kit (Invitek Molecular GmbH, Germany) and subjected to high-throughput sequencing on an Oxford Nanopore Technologies MinION device using the method described in Liefting et al. 2021. All sequence analysis was performed using Geneious Prime 2021.1.1 (https://www.geneious.com). A total of 355,858 reads that passed quality check were subjected to BLASTn search against the NCBI nt database as described in Liefting et al. 2021. The following viruses produced hits: FMV, FBV-1, FMMaV and a fig closterovirus. The presence of FMV, FBV-1 and FMMaV were confirmed by species specific RT-PCRs. To identify the closterovirus, reads were mapped to closteroviruses reported in fig including the recently identified tentative species fig virus A (FiVA; GenBank accession no MN817232) and fig virus B (FiVB; GenBank accession no. MN817233). Five viral contigs ranging from 939 to 2,340 nucleotides (nt) were obtained from mapping to FiVB. Subsequently, a 6.4 kb sequence (GenBank accession no. OQ968551) from the 3' region of the NZ isolate was amplified by overlapping RT-PCR using primers designed from the contig sequences. The sequence shared 79.5% nucleotide (nt) identity with FiVB The original sample and a further 25 symptomatic and 10 asymptomatic fig samples, collected from the Auckland area between 2016 and 2021, were tested using FiVB specific RT-PCR and Sanger sequencing using primers FiVB-F1 (5'-GAGGGAGAGATGTAGATGC-3') and FiVB-R2 (5'-TGTCGTCGATATCGTTGTGT-3'), designed to amplify a 725 nt fragment in the 70 kDa heat shock protein (HSP70) ORF. Products of the expected size were amplified from the original sample and three symptomatic samples and their sequences found to be identical. BLAST searches showed that the sequence (GenBank accession no. ON553403) shared 82.7% nt and 87.3% amino acid (aa) identity to an isolate of FiVB (GenBank accession no MN817233). These additional positive samples were collected from a small home nursery where the plants were propagated from cuttings and have been distributed locally, suggesting the virus is very likely to have a limited spread throughout the Auckland area. All three FiVB infected samples were also positive for FMV. However, the association of FiVB with FMD symptoms is unknown. FiVB was first identified from a latex sample exuded from a fig tree collected from Japan (Park et al. 2021) and is the only report of FiVB in the world to date. Although an identical sequence from Argentina, named fig closterovirus 1, was submitted to GenBank, the origin of this isolate is not known. To our knowledge, this is the first report of FiVB in New Zealand.

First report of barley virus G infecting wheat (Triticum aestivum) in Japan.

Barley virus G (BVG) is a single stranded, positive sense RNA virus and tentative species of the genus Polerovirus in the family Solemoviridae. BVG was first identified in barley (Hordeum vulgare) in Korea with symptoms that were similar to those of barley yellow dwarf disease (Zhao et al. 2016). It has also been identified from proso millet (Park et al. 2017), barley (Erickson and Falk, 2021; Nancarrow et al. 2019; Svanella-Dumas et al. 2022), maize (Gavrili et al. 2021), wheat (Nancarrow et al. 2019), and oats (Nancarrow et al. 2019) in various countries. In the spring of 2019, wheat (Triticum aestivum) plants that exhibited symptoms of yellow leaves, necrosis, and stunting were observed in a few fields of the Chugoku region (western main island) in Japan. The four soil-borne viruses, wheat yellow mosaic virus (WYMV), Chinese yellow mosaic virus (CWMV), Japanese soil-borne wheat mosaic virus (JSBWMV), and soil-borne wheat mosaic virus (SBWMV), that often occur in winter wheat in Japan, were not detected by DAS-ELISA for WYMV, CWMV, and JSBWMV (Netsu et al. 2011) and ELISA Reagent Set for SBWMV (Agdia, IN, USA). To identify the pathogen, total RNA was isolated from the leaves and petioles using the PureLink RNA Mini kit (Thermo Fisher Scientific, MA, USA), and RT-PCR was performed using the PrimeScript One Step RT-PCR Kit Ver.2 (Dye plus) (Takara Bio Inc, Shiga, Japan). Based on the symptoms, luteoviruses and poleroviruses, which are transmitted by aphids, were suspected and RT-PCR was performed using known primers (Malmstrom and Shu, 2004; Mustafayev et al. 2013). This resulted in an amplicon of approximately 300 bp being obtained from RT-PCR using the Luteo2F/YanR-new primers (Mustafayev et al. 2013). The amplicon was directly sequenced by Sanger sequencing and a nucleotide BLAST search of the database revealed that the sequence was highly similar (99% identity, query coverage 95%) to the genome of BVG. In samples of a single field, four out of six plants that were showing necrosis and stunting were positive by RT-PCR using the primers BVG-CP-F (5´- GCGGGAAACATTTGTATTTTCG-3´)/BVG-CP-R (5´- GATTTTGGGTTAGAACATCCATCG-3´). In addition, five out of six plants with some yellowing of the leaves in the same field were also positive. Other luteoviruses and poleroviruses were not detected by RT-PCR using known primers. The full-length genome sequence of the Chugoku isolate was amplified using the primers BVG-F (5´-ACAAAAGGGACCCAGAGGG-3´)/BVG-R (5´-TACCAAGGATACTAGAGAGAGA-3´), which were designed from the 5´ and 3´ end sequences of the known BVG sequence. The resultant amplicon was directly sequenced by Sanger sequencing and the sequence was deposited into the DNA Data Bank of Japan (Chugoku isolate, LC649801). The sequence comprised of 5,620 bp and the genomic structures were consistent with that of BVG. The sequence showed more than 97% nucleotide identity with the BVG Gimji (KT962089), Uiseong (LC259081), NL1 (MF960779), and California (LC259081) isolates by pairwise comparisons. This is the first report of BVG in wheat in Japan to the best of our knowledge. The correlation between BVG and the observed symptoms and the impact of BVG on wheat production in Japan requires further investigation. Reference Erickson, A. C. and Falk, B. 2021. Plant Dis. doi:10.1094/PDIS-03-21-0478-PDN Gavrili, V. et al. 2021. J. Plant Pathol. 103:1331. doi:10.1007/s42161-021-00903-4 Malmstrom, C. M. and Shu, R. 2004. J. Virol. Methods. 120:69. doi:10.1016/j.jviromet.2004.04.005 Mustafayev, E. S. et al. 2013. Plant Dis. 97:849. doi:10.1094/PDIS-07-12-0656-PDN Nancarrow, N. et al. 2019. Plant Dis. 103:1799. doi: 10.1094/PDIS-01-19-0166-PDN Netsu, O. et al. 2011. Plant Dis. 58:13. doi.org/10.11337/ktpps.2011.13 Park, C.Y. et al. 2017. Plant Dis. 101:393. doi: 10.1094/PDIS-07-16-0952-PDN Svanella-Dumas, L. et al. 2022. Plant Dis. doi: 10.1094/PDIS-06-22-1294-PDN Zhao, F. et al. 2016. Arch. Virol. 161: 2047. doi:10.1007/s00705-016-2881-0.

Diversity of Rickettsiales bacteria in five species of ticks collected from Jinzhai County, Anhui Province, China in 2021-2022.

The order Rickettsiales in the class Alphaproteobacteria comprises vector-borne pathogens of both medical and veterinary importance. Ticks, as a group, are second only to mosquitoes as vectors of pathogens to humans, playing a critical role in the transmission of rickettsiosis. In the present study, 880 ticks collected from Jinzhai County, Lu'an City, Anhui Province, China in 2021-2022 were identified as belonging to five species from three genera. DNA extracted from individual ticks was examined using nested polymerase chain reaction targeting the 16S rRNA gene (rrs), and the gene fragments amplified were sequenced to detect and identify Rickettsiales bacteria in the ticks. For further identification, the rrs-positive tick samples were further amplified by PCR targeting the gltA and groEL gene and sequenced. As a result, 13 Rickettsiales species belonging to the genera Rickettsia, Anaplasma, and Ehrlichia were detected, including three tentative species of Ehrlichia. Our results reveal the extensive diversity of Rickettsiales bacteria in ticks from Jinzhai County, Anhui Province. There, emerging rickettsial species may be pathogenic and cause under-recognized diseases. Detection of several pathogens in ticks that are closely related to human diseases may indicate a potential risk of infection in humans. Therefore, additional studies to assess the potential public health risks of the Rickettsiales pathogens identified in the present study are warranted.

Citrus Bright Spot Virus: A New Dichorhavirus, Transmitted by Brevipalpus azores, Causing Citrus Leprosis Disease in Brazil

Citrus leprosis (CL) is the main viral disease affecting the Brazilian citriculture. Sweet orange (Citrus sinensis L. Osbeck) trees affected by CL were identified in small orchards in Southern Brazil. Rod-like particles of 40 × 100 nm and electron lucent viroplasm were observed in the nucleus of infected cells in symptomatic tissues. RNA extracts from three plants, which proved negative by RT-PCR for known CL-causing viruses, were analyzed by high throughput sequencing and Sanger sequencing after RT-PCR. The genomes of bi-segmented ss(-)RNA viruses, with ORFs in a typical organization of members of the genus Dichorhavirus, were recovered. These genomes shared 98-99% nt sequence identity among them but <73% with those of known dichorhavirids, a value below the threshold for new species demarcation within that genus. Phylogenetically, the three haplotypes of the new virus called citrus bright spot virus (CiBSV) are clustered with citrus leprosis virus N, which is a dichorhavirus transmitted by Brevipalpus phoenicis sensu stricto. In CiBSV-infected citrus plants, B. papayensis and B. azores were found, but the virus could only be transmitted to Arabidopsis plants by B. azores. The study provides the first evidence of the role of B. azores as a viral vector and supports the assignment of CiBSV to the tentative new species Dichorhavirus australis.

Near-Complete Genome Sequence of Lötschberg Virus (Mononegavirales: Filoviridae) Identified in European Perch (Perca fluviatilis Linnaeus, 1758).

We obtained the near-complete genome sequence of a novel virus, Lötschberg virus (LTBV), from a European perch metatranscriptome. Genome organization and pairwise sequence comparison indicated that LTBV represents a tentative new species and genus of the mononegaviral family Filoviridae.

Identification and reproductive isolation of Euborellia species (Insecta, Dermaptera, Anisolabididae) from East and Southeast Asia.

Euborellia (Anisolabididae: Anisolabidinae) is one of the most speciose genera of earwigs (Dermaptera), and its species-level classification is difficult. To settle the classification of brachypterous species with abbreviated tegmina recorded from East and Southeast Asia, we examined the morphology and reproductive isolation of three tentative Euborellia species, and analyzed the DNA barcoding region of the mitochondrial cytochrome oxidase subunit I (COI) gene. The observed complete reproductive isolation among the three Euborellia taxa and considerable differentiation in the COI sequences clearly show that each should be treated as a separate species. Based on morphology, distribution and the DNA sequence, we identify Euborellia sp. 1 of Malaysia as E.annulata (Fabricius), a circumtropical cosmopolitan with no records of a fully winged form. Samples from Ioto Island (= Iwo-jima Island: Ogasawara Islands, southern Japan) were also identified as this species. Euborellia sp. 3, from the main islands of Japan, was generally larger and lacked a Y-shaped pigmented area on the penis lobe, which is characteristic of Euborellia sp. 1. We propose reinstating E.pallipes (Shiraki) as the oldest name for this taxon. Euborellia sp. 2, even the brachypterous form, can be distinguished from these two species by its paler coloration (particularly the femora), ecarinate post-abdomen, and the shape of the male genitalia (parameres). We tentatively identify this species as E.philippinensis Srivastava based on the morphology of the brachypterous form, although the macropterous form cannot be distinguished from E.femoralis (Dohrn).

Viruses infecting purple passion fruit (Passiflora edulis f. edulis) in southwestern Antioquia, Colombia

Purple passion fruit is one of the most important crops in Colombia, but unfortunately, viral diseases have started to affect its productivity. Five viruses are known to infect this crop in Colombia: Soybean mosaic virus (SMV), Cowpea aphid-borne mosaic virus (CABMV), Passion fruit yellow mosaic virus (PFYMV), Cucumber mosaic virus (CMV), and the tentative species Gulupa bacilliform virus A (GBVA). In addition, emerging diseases caused by begomoviruses were reported on other Passiflora species in Colombia. The incidence of these viruses in adult plants, plantlets, and seeds from southwestern Antioquia was investigated based on molecular approaches. PFYMV and GBVA scored the highest incidence (>90%). Sequence analyses of the partial genomes confirmed the presence of PFYMV, SMV and GBVA variants in purple passion fruit plants grown in commercial fields at Antioquia.

A Novel Lineage of Cile-Like Viruses Discloses the Phylogenetic Continuum Across the Family Kitaviridae.

An increasing number of plant species have been recognized or considered likely reservoirs of viruses transmitted by Brevipalpus mites. A tiny fraction of these viruses, primarily those causing severe economic burden to prominent crops, have been fully characterized. In this study, based on high-throughput sequencing, transmission electron microscopy analyses of virions in plant-infected tissues, viral transmission experiments, and the morphoanatomical identification of the involved Brevipalpus mites, we describe molecular and biological features of viruses representing three new tentative species of the family Kitaviridae. The genomes of Solanum violifolium ringspot virus (SvRSV, previously partially characterized), Ligustrum chlorotic spot virus (LigCSV), and Ligustrum leprosis virus (LigLV) have five open reading frames (ORFs) > 500 nts, two distributed in RNA1 and three in RNA2. RNA1 of these three viruses display the same genomic organization found in RNA1 of typical cileviruses, while their RNA2 are shorter, possessing only orthologs of genes p61, p32, and p24. LigCSV and LigLV are more closely related to each other than to SvRSV, but the identities between their genomic RNAs were lower than 70%. In gene-by-gene comparisons, ORFs from LigCSV and LigLV had the highest sequence identity values (nt sequences: 70–76% and deduced amino acid sequences: 74–83%). The next higher identity values were with ORFs from typical cileviruses, with values below 66%. Virions of LigLV (≈ 40 nm × 55 nm) and LigCSV (≈ 54 nm × 66 nm) appear almost spherical, contrasting with the bacilliform shape of SvRSV virions (≈ 47 nm × 101 nm). Mites collected from the virus-infected plants were identified as Brevipalpus papayensis, B. tucuman, and B. obovatus. Viruliferous B. papayensis mites successfully transmitted LigCSV to Arabidopsis thaliana. SvRSV, LigCSV, and LigLV seem to represent novel sub-lineages of kitaviruses that descent on parallel evolutionary branches from a common ancestor shared with the tentative cile-like virus hibiscus yellow blotch virus and typical cileviruses. Biological and molecular data, notably, the phylogenetic reconstruction based on the RdRp proteins in which strong support for monophyly of the family Kitaviridae is observed, mark an advance in the understanding of kitavirids.

Genomic Characterisation of an Isolate of Brassica Yellows Virus Associated with Brassica Weed in Tasmania

Brassica yellows virus (BrYV), a tentative species in the genus Polerovirus, of the Solemoviridae family, is a phloem-restricted and aphid-transmitted virus with at least three genotypes (A, B, and C). It has been found across mainland China, South Korea, and Japan. BrYV was previously undescribed in Tasmania, and its genetic variability in the state remains unknown. Here, we describe a near-complete genome sequence of BrYV (genotype A) isolated from Raphanus raphanistrum in Tasmania using next-generation sequencing and sanger sequencing of RT-PCR products. BrYV-Tas (GenBank Accession no. OM469309) possesses a genome of 5516 nucleotides (nt) and shares higher sequence identity (about 90%) with other BrYV isolates. Phylogenetic analyses showed variability in the clustering patterns of the individual genes of BrYV-Tas. Recombination analysis revealed beginning and ending breakpoints at nucleotide positions 1922 to 5234 nt, with the BrYV isolate LC428359 and BrYV isolate KY310572 identified as major and minor parents, respectively. Results of the evolutionary analysis showed that the majority of the codons for each gene are evolving under purifying selection, though a few codons were also detected to have positive selection pressure. Taken together, our findings will facilitate an understanding of the evolutionary dynamics and genetic diversity of BrYV.

Multigene Phylogeny Reveals Endophytic Xylariales Novelties from Dendrobium Species from Southwestern China and Northern Thailand.

Xylariales are common endophytes of Dendrobium. However, xylarialean species resolution remains difficult without sequence data and poor sporulation on artificial media and asexual descriptions for only several species and old type material. The surface-sterilized and morph-molecular methods were used for fungal isolation and identification. A total of forty-seven strains were identified as twenty-three species belonging to Apiosporaceae, Hypoxylaceae, Induratiaceae, and Xylariaceae. Five new species—Annulohypoxylon moniliformis, Apiospora dendrobii, Hypoxylon endophyticum, H. officinalis and Nemania dendrobii were discovered. Three tentative new species were speculated in Xylaria. Thirteen known fungal species from Hypoxylon, Nemania, Nigrospora, and Xylaria were also identified. Another two strains were only identified at the genus and family level (Induratia sp., Hypoxylaceae sp.). This study recorded 12 new hosts for xylarialean endophytes. This is the first report of Xylariales species as endophytes from Dendrobium aurantiacum var. denneanum, D. cariniferum, D. harveyanum, D. hercoglossum, D. moniliforme, and D. moschatum. Dendrobium is associated with abundant xylarialean taxa, especially species of Hypoxylon and Xylaria. We recommend the use of oat agar with low concentrations to induce sporulation of Xylaria strains.

Phlebotomine sandflies and associated pathogens in Algeria: update and comprehensive overview.

Being the only established vectors of the protozoan parasites of the genus Leishmania, sand flies have become very important in all countries where leishmaniasis exists. It is caused by a variety of species, each one having specific mammalian reservoir hosts and vectors. Leishmania and sand fly classification has always been a controversial matter, and the increasing number of sand fly species described world complicates the task. Until recently, sand flies distributed in the Old World were known as vectors for few Phleboviruses including two known species (Sandfly fever Naples virus, and alehebad virus), and tentative species such as sand fly fever Sicilian virus and Corfou virus. These infections are emerging in the Mediterranean region and will likely spread in forthcoming decades, posing a complex threat to public health. Here, we reviewed the current literature on phlebotomine sand flies fauna and epidemiology of sand fly‑borne infections in Algeria.

Characterization of grapevine fungal canker pathogens Fatty Acid Methyl Ester (FAME) profiles

ABSTRACT Fatty acid methyl ester (FAME) analyses can be useful for distinguishing microbial species. This study conducted FAME analyses on 14 fungal species known to cause grapevine trunk diseases. FAME profiles were dominated by oleic acid, albeit profiles were characteristic enough to separate species. Discriminant analyses suggested that palmitoleic acid/sapienic acid, pentadecylic acid, and an unsaturated 17-carbon fatty acid (17:1ω8 c)could explain 79.8% of the variance in the profiles among species in the first three discriminant functions. FAME profile libraries were created for use in a commercialized software, which was able to accurately identify isolates to the species level, with a low rate (9.4%) of samples to be reassessed. Dendrograms created using neighbor-joining cluster analyses with data from FAME profiles were compared with those using internal transcribed spacer (ITS) region sequences. This revealed that FAME profiles, albeit useful for tentative species identification, should not be used for determining phylogenetic relationships because the dendrograms were significantly unconcordant. Regardless, these results demonstrated the potential of FAME analyses in quickly and initially identifying closely related fungal species or confirming conclusions from other species identification techniques that would require independent validation.

Speciation of a subterranean amphipod on the glacier margins in South Eastern Alps, Europe

AbstractAimClimatic oscillations altered distributions through migration, extinction, adaptation or speciation. Their joint effect has been rarely studied. Here, we tested how Pleistocene climatic oscillations shaped the evolutionary history and distribution of a subterranean amphipod. We tested four predictions for extant populations: (i) populations of good dispersers moving along the ice‐sheet margin should have homogenous genetic structure and broad tolerance to temperatures; (ii) adaptation to local temperatures evolved in poor dispersers with spatially structured populations; (iii) Pleistocene extinctions in the range of historical glaciers should reflect founder effects and high sensitivity to low temperatures; (iv) para‐ or allopatric speciation could unfold through diverse scenarios as a response to local adaptation and constrained dispersal.LocationNorth‐Western part of the Balkan Peninsula, Europe.TaxonNiphargus stygius, Amphipoda, Crustacea.MethodsWe used time‐calibrated multilocus phylogenies and multilocus delimitations to test independently evolving lineages. For each lineage, separately we assessed historical population dynamics and dispersal potential, modelled climatic niches and mapped them onto palaeoclimatic maps. Additionally, we conducted enzymatic and respiratory tests of thermal tolerance, and reconstructed historical distribution range using phylogeographical diffusion analyses.ResultsWe identified four independently evolving tentative species, dated to the Pleistocene, with no evidence of extinction events. Three main lineages are parapatric with few pairwise syntopies and no traces of hybridization. They evolved in allopatry and possibly maintained constant effective population sizes. The lineages are phylogeographically substructured, indicating low recent dispersal. They differ in their climatic niche envelopes and physiological responses to unfavourable temperatures.Main conclusionsWe infer that periodic drops of groundwater in the Pleistocene caused two vicariant events and the onset of two allopatric speciation events, followed by phases of dispersal leading to secondary contact and occasional overlap of ranges. Speciation was seemingly fostered by eco‐physiologically differentiation due to adaptation to local climates.

Comparative Analysis of Biological Characteristics among P0 Proteins from Different Brassica Yellows Virus Genotypes.

Simple SummaryPolerovirus P0 proteins are multifunctional proteins. Besides their viral suppressor of RNA silencing (VSR) functions, several P0 proteins can induce a cell death phenotype within the infiltrated region of Nicotiana benthamiana or Nicotiana glutinosa. Recently, the Brassica yellows virus (BrYV) genotype A P0 protein (P0BrA) was identified as a strong viral suppressor of RNAi. In this study, we compared the features of the P0 proteins encoded by different genotypes of BrYV and revealed their difference in inducing cell death in N. benthamiana. Key residues in P0BrA for inducing cell death were also identified. We also showed that all three BrYV genotypes had synergistic interaction with PEMV 2 in N. benthamiana. This study provides theoretical guidance for controlling the viral disease caused by poleroviruses in the future.Brassica yellows virus (BrYV) is a tentative species of the genus Polerovirus, which has at least three genotypes (A, B, and C) in China. The P0 protein of BrYV-A (P0BrA) has been identified as a viral suppressor of RNA silencing (VSR), which can also induce cell death in infiltrated Nicotiana benthamiana leaves. In this study, we demonstrated that the cell death induced by P0BrA was accompanied by the accumulation of reactive oxygen species (ROS) and increased Pathogenesis-related protein genes-1 (PR1) expression. Meanwhile, this cell death phenotype was delayed by salicylic acid (SA) pretreatment. Biological function comparison of the three P0 proteins showed that transiently expressed P0BrB or P0BrC induced a significantly delayed and milder cell death response compared with P0BrA. However, like P0BrA, they also suppressed local and systemic RNA silencing. Six residues of P0BrA essential for inducing cell death were identified by comparative analysis and amino acid substitution assay. We also show that all three BrYV genotypes have synergistic interactions with pea enation mosaic virus 2 (PEMV 2) in N. benthamiana. This study provides theoretical guidance for controlling the viral disease caused by poleroviruses in the future.

A Simple Method for the Acquisition and Transmission of Brassica Yellows Virus from Transgenic Plants and Frozen Infected Leaves by Aphids.

Brassica yellows virus (BrYV) is a tentative species of the genus Polerovirus, which occurs widely, and mostly damages Brassicaceae plants in East Asia. Because BrYV cannot be transmitted mechanically, an insect-based transmission method is required for further virus research. Here, a reliable and unrestricted method is described, in which non-viruliferous aphids (Myzus persicae) acquired BrYV from transgenic Arabidopsis thaliana, harboring the full-length viral genome germinated from seeds and its frozen leaves. The aphids then transmitted the virus to healthy plants. There was no significant difference in acquisition rates between fresh and frozen infected leaves, although the transmission rate from frozen infected leaves was lower compared to fresh infected leaves. This simple novel method may be used to preserve viral inocula, evaluate host varietal resistance to BrYV, and investigate interactions among BrYV, aphids, and hosts.