Molecular phylogeny, diagnostics, and diversity of plant-parasitic nematodes of the genus Hemicycliophora (Nematoda: Hemicycliophoridae)

The genus Hemicycliophora (Nematoda: Hemicycliophoridae) contains 132 valid species of plant-parasitic nematodes, collectively known as ‘sheath nematodes’. Hemicycliophora spp. are characterized morphologically by a long stylet with rounded basal knobs and a cuticular sheath, present in juvenile and adult stages. Populations of 20 valid and 14 putative species of Hemicycliophora and Loofia from several countries were characterized morphologically using light (LM) and scanning electron microscopy (SEM) and molecularly using the D2-D3 segments of 28S rRNA and internal transcribed spacer (ITS) rRNA gene sequences. LM and SEM observations provided new details on the morphology of these species. PCR-restriction fragment length polymorphisms (PCR-RFLPs) of the D2-D3 of 28S rDNA were proposed for identification of the species. Phylogenetic relationships within populations of 36 species of the genus Hemicycliophora using 102 D2-D3 of 28S rDNA and 97 ITS rRNA gene sequences as inferred from Bayesian analysis are reconstructed and discussed. Ancestral state reconstructions of diagnostic characters (body and stylet length, number of body annuli, shape of vulval lip and tail), using maximum parsimony and Bayesian inference, revealed that none of the traits are individually reliable characters for classifying the studied sheath nematode. The Shimodaira–Hasegawa test rejected the validity of the genus Loofia. This is the most complete phylogenetic analysis of Hemicycliophora species conducted so far.Fil: Subbotin, Sergei A.. California Department of Food and Agriculture; Estados Unidos. Institute of Ecology and Evolution of the Russian Academy of Sciences; RusiaFil: Chitambar, John J.. California Department of Food and Agriculture; Estados UnidosFil: Chizhov, Vladimir N.. Institute of Ecology and Evolution of the Russian Academy of Sciences; RusiaFil: Stanley, Jason D.. Florida Department of Agriculture and Consumer Services; Estados UnidosFil: Inserra, Renato N.. Florida Department of Agriculture and Consumer Services; Estados UnidosFil: Doucet, Marcelo Edmundo. Universidad Nacional de Cordoba. Facultad de Cs.exactas Fisicas y Naturales. Centro de Zoologia Aplicada; Argentina. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Cordoba. Instituto de Diversidad y Ecologia Animal; ArgentinaFil: Mcclure, Michael. University Of Arizona; Estados UnidosFil: Ye, Weimin. North Carolina Department of Agriculture & Consumer Services; Estados UnidosFil: Yeates, George.Fil: Mollov, Dimitre S.. University Of Minnesota; Estados UnidosFil: Cantalapiedra Navarrete, Carolina. Consejo Superior de Investigaciones Científicas. Instituto de Agricultura Sostenible; EspañaFil: Vovlas, Nicola. Istituto per la Protezione delle Piante; ItaliaFil: Van Den Berg, Esther. ARC-Plant Protection Research Institute; SudáfricaFil: Castillo, Pablo. Consejo Superior de Investigaciones Científicas. Instituto de Agricultura Sostenible; Españ

Alfalfa virus S, a new species in the family Alphaflexiviridae.

A new species of the family Alphaflexiviridae provisionally named alfalfa virus S (AVS) was discovered in alfalfa samples originating from Sudan. A complete nucleotide sequence of the viral genome consisting of 8,349 nucleotides excluding the 3' poly(A) tail was determined by high throughput sequencing (HTS) on an Illumina platform. NCBI BLAST searches revealed that the virus shares the greatest degree of sequence identity with members of the family Alphaflexiviridae, genus Allexivirus. The AVS genome contains six computationally-predicted open reading frames (ORF) encoding viral replication protein, triple gene block protein 1 (TGB1), TGB2, TGB3-like protein, unknown 38.4 kDa protein resembling serine-rich 40 kDa protein characteristic for allexiviruses, and coat protein (CP). AVS lacks a clear 3' proximal ORF that encodes a nucleic acid-binding protein typical for allexiviruses. The identity of the virus was confirmed by RT-PCR with primers derived from the HTS-generated sequence, dot blot hybridization with DIG-labeled virus-specific RNA probes, and Western blot analysis with antibodies produced against a peptide derived from the CP sequence. Transmission electron microscopic observations of the infected tissues showed the presence of filamentous particles similar to allexiviruses in their length and appearance. To the best of our knowledge, this is the first report on the identification of a putative allexivirus in alfalfa (Medicago sativa). The genome sequence of AVS has been deposited in NCBI GenBank on 03/02/2016 as accession № KY696659

Phylogenetic relationship between alfalfa virus S (highlighted), classified allexiviruses and unassigned members of the family <i>Alphaflexiviridae</i>.

<p>The tree was built based on the available complete nucleotide sequences using MEGA 7 software (version 7.0.21) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178222#pone.0178222.ref025" target="_blank">25</a>] and the Neighbor-Joining method. The optimal tree with the sum of branch length = 3.10976014 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches.</p

Electron micrographs of viral particles observed in the AVS-infected alfalfa samples 98.3A, 98.1A and 98.3A(0).

<p>Scale bars represent 100 nm and 200 nm (left panel and right panel, respectively).</p

Adjusted open reading frame finder output (ORFfinder, NCBI, https://www.ncbi.nlm.nih.gov/orffinder/) showing genome organization of alfalfa virus S.

<p>ORF1, encoding RNA-dependent RNA polymerase (RdRp); ORF 2,3, and 4, encoding triple gene block proteins (TGB) 1, 2 and TGB3-like protein, respectively; ORF5, encoding p38.4, an unknown protein; ORF6, encoding AVS coat protein (CP).</p

Primers used for RT-PCR.

<p>Primers used for RT-PCR.</p

Dot blot hybridization assay with DIG-labeled RNA probes.

<p>RNA probes derived from the AVS CP. (A) and p38.4 (B) ORFs. Total RNA was extracted from the following alfalfa samples: 1, 98.3A; 2, 89.2AS; 3, 89.3H; 4, 89.1H; 5, 89.1AS; 6, 98.1A; 7, 98.3A (0); 8, 98.2RR and 9, 97.2A.</p