Multigene Sequence Analysis of Aster Yellows Phytoplasma Associated with Primrose Yellows

Primula acaulis (L.) Hill. plants showing stunting, leaf‐yellowing and virescence were first discovered in the Czech Republic. Polymerase chain reactions with subsequent restriction fragment length polymorphism analyses and sequencing enabled classification of the detected phytoplasmas into the aster yellows group, ribosomal subgroup 16SrI‐B, tufI‐B, rpI‐B, groELIB‐III and SecY‐IB subgroups. Phylogeny of the 16S rRNA gene sequences as well as sequence analysis of several chromosomal regions, such as the 16S‐23S ribosomal operon, ribosomal proteins, spc ribosomal protein operon, genes for elongation factor EF‐Tu, molecular chaperonin large subunit GroEL, immunodominant membrane protein, ribosome recycling factor, urydilate kinase, ATP‐ and Zn²⁺‐dependent proteases not only confirmed its affiliation with the ‘Candidatus Phytoplasma asteris’ species but also enabled its detailed molecular characterization. The less researched regions of phytoplasma genome (amp, adk, hflB, pyrH‐frr genes) could be valuable as additional markers for phytoplasma through differentiation especially within the 16SrI‐B ribosomal subgroup.     

Detection and Molecular Identification of Aster Yellows Phytoplasma in Date Palm in Egypt

Phytoplasma‐like symptoms were detected in date palm trees (Phoenix dactylifera L.) in Al‐Giza Governorate in Egypt. Symptoms varied from leaf chlorotic streaks, stunting and marked reduction in fruit and stalk sizes. Direct and nested PCR of symptomatic samples using P1/P7 and R16F2n/R16R2n primers, respectively, of the 16S rRNA gene, resulted in a DNA amplification product of c. 1.3 kbp. Symptomless samples collected from the same location and the healthy control produced no product upon amplification. Products were cloned into TOPO TA vector for sequencing. Data generated were deposited in the GenBank (Accession KF826615 ). A BLAST search showed that the sequence of the 16SrRNA gene shared ‘Candidatus Phytoplasma asteris’ (16SrI group) with other isolates. Phylogenetic analysis revealed that the isolate clustered with the date palm phytoplasma causing Al‐Wijam disease in Saudi Arabia.     

Leafhopper transmission of a virus causing maize wallaby ear disease

A virus causing maize wallaby ear disease was transmitted experimentally by Cicadulina bimaculata to fourteen species of monocotyledonous plants. It was also transmitted by Nesoclutha pallida, and by grafting. The symptoms obtained resemble closely those reported for maize leaf gall disease in the Philippines and maize rough dwarf virus in Italy and Israel. About 85% of C. bimaculata caught in the field carried maize wallaby ear virus (MWEV), and many of their progeny were viruliferous even when not allowed access to infected plants. The proportion of infective individuals in clones bred for nine generations from selected non-transmitting adults decreased from 85% in the first nymphs to less than 1%; such individuals were difficult to rear, as their fecundity and longevity decreased greatly. N. pallida transmitted MWEV after injection with partially purified extracts of infected plants. Spherical particles c. 85 nm in diameter were found in the salivary glands of viruliferous C. bimaculata, but not in those of non-transmitting individuals. The particles occurred in tubules in the cytoplasm and each had a densely stained core c. 50 nm in diameter. Particles similar in size to the core were found in extracts of infected but not uninfected maize, and in extracts of viruliferous but not in non-viruliferous C. bimaculata and N. pallida.     

Aster Yellows Phytoplasma Identified in Scentless Chamomile by Microscopical Examinations and Molecular Characterization

Scentless chamomile (Matricaria perforata Mérat) plants were commonly found infected with a yellows-type disease caused by phytoplasma in several fields in Alberta, Canada. Typical phytoplasmas were detected in the phloem cells in ultrathin sections from leaf, stem, root and flower petiole tissues examined by electron microscopy. Application of 4′6-diamidino-2-phenylindole- 2HCl (DAPI) staining techniques confirmed the presence of the phytoplasma in these tissues. These observations were supported by polymerase chain reaction (PCR) assays, using two primer pairs, P1/P6 and R16(1)F1/R1, derived from phytoplasma rDNA sequences. Aster yellows and potato witches′-broom (PWB) DNA phytoplasma samples served as positive controls and were used to study group relatedness. In a direct PCR assay, DNA amplification with universal primer pair P1/P6 gave the expected PCR products of 1.5 kb. Based on a nested-PCR assay using the latter PCR products, as templates, and a specific primer pair R16(1)F1/R1 designed on the basis of AY phytoplasma rDNA sequences, a PCR product of 1.1 kb was obtained from each phytoplasma-infected chamomile and AY samples but not from PWB phytoplasma and healthy chamomile controls. DNA amplification with specific primer pair R16(1)F1/R1 and restriction fragment length polymorphism indicated the presence of AY phytoplasma in the infected scentless chamomile sample.     

Detection and Identification of Aster Yellows Phytoplasma Associated with Lipstick Yellow Frond Disease in Malaysia

Yellowing symptoms similar to coconut yellow decline phytoplasma disease were observed on lipstick palms (Cyrtostachys renda) in Selangor state, Malaysia. Typical symptoms were yellowing, light green fronds, gradual collapse of older fronds and decline in growth. Polymerase chain reaction assay was employed to detect phytoplasma in symptomatic lipstick palms. Extracted DNA was amplified from symptomatic lipstick palms by PCR using phytoplasma‐universal primer pair P1/P7 followed by R16F2n/R16R2. Phytoplasma presence was confirmed, and the 1250 bp products were cloned and sequenced. Sequence analysis indicated that the phytoplasmas associated with lipstick yellow frond disease were isolates of ‘Candidatus Phytoplasma asteris’ belonging to the 16SrI group. Virtual RFLP analysis of the resulting profiles revealed that these palm‐infecting phytoplasmas belong to subgroup 16SrI‐B and a possibly new 16SrI‐subgroup. This is the first report of lipstick palm as a new host of aster yellows phytoplasma (16SrI) in Malaysia and worldwide.     

First Report of Aster Yellows Phytoplasma in Goldenrain Tree (Koelreuteria paniculata) in Korea

Aster yellows phytoplasma was detected for the first time in goldenrain tree (Koelreuteria paniculata) growing in Sinpyeong‐myeon, Jeollabuk‐do, South Korea. DNA was extracted from the infected leaf samples and part of the 16S rDNA, rp operon and tuf gene were amplified using R16F2n/R2 and gene‐specific primers. The sequence analysis showed that the phytoplasma was closely related (99%) to members of the Aster Yellows (AY) group, and belonging to 16Sr I, subgroup B. Moreover, the 16S rDNA sequences of the isolate showed 88–96% identity with members of other 16Sr and undesignated groups. Based on the sequence identity and phylogenetic studies, it was confirmed that phytoplasma infecting goldenrain tree in South Korea belongs to the AY group.     

Molecular Characterization of Aster Yellows Phytoplasma Associated with Valerian and Sowthistle Plants by PCR–RFLP Analyses

In Alberta, Canada, valerian grown for medicinal purposes and sowthistle, a common weed, showed typical aster yellows symptoms. Molecular diagnosis was made using a universal primer pair (P1 / P7) designed to amplify the entire 16S rRNA gene and the 16 / 23S intergenic spacer region in a direct polymerase chain reaction (PCR) assay. This primer pair amplified the DNA samples from valerian and sowthistle and reference controls (AY‐27, CP, PWB, AY of canola, LWB). They produced the expected PCR products of 1.8 kb, which were diluted and used as templates in a nested PCR. Two primer pairs R16F2n / R2 and P3 / P7 amplified the DNA templates giving PCR products of 1.2 and 0.32 kb, respectively. No PCR product was obtained with either set of primers and DNA isolated from healthy plants. Restriction fragment length polymorphism (RFLP) was used to analyse the partial 16S rDNA sequences (1.2 kb) of all phytoplasma DNA samples after restriction with four endonucleases (AluI, HhaI, MseI and RsaI). The restriction patterns of these strains were found to be identical with the RFLP pattern of the AY phytoplasma reference control (AY‐27 strain). Based on the RFLP data, the two strains are members of subgroup A of the AY 16Sr1 group. We report here the first molecular study on the association of AY phytoplasmas with valerian and sowthistle plants.     

Molecular Characterization of Phytoplasmas Related to Apple Proliferation and Aster Yellows Groups Associated with Pear Decline Disease in Iran

Pear trees showing pear decline disease symptoms were observed in pear orchards in the centre and north of Iran. Detection of phytoplasmas using universal primer pair P1A/P7A followed by primer pair R16F2n/R16R2 in nested PCR confirmed association of phytoplasmas with diseased pear trees. However, PCR using group‐specific primer pairs R16(X)F1/R16(X)R1 and rp(I)F1A/rp(I)R1A showed that Iranian pear phytoplasmas are related to apple proliferation and aster yellows groups. Moreover, PCR results using primer pair ESFYf/ESFYr specific to 16SrX‐B subgroup indicated that ‘Ca. Phytoplasma prunorum’ is associated with pear decline disease in the north of Iran. RFLP analyses using HaeIII, HhaI, HinfI, HpaII and RsaI restriction enzymes confirmed the PCR results. Partial 16S rRNA, imp, rp and secY genes sequence analyses approved that ‘Ca. Phytoplasma pyri’ and ‘Ca. Phytoplasma asteris’ cause pear decline disease in the centre of Iran, whereas ‘Ca. Phytoplasma prunorum’ causes disease in the north of Iran. This is the first report of the association of ‘Ca. Phytoplasma asteris’ and ‘Ca. Phytoplasma prunorum’ with pear decline disease worldwide.     

The Distribution of Grapevine Yellows Disease Associated with the Buckland Valley Grapevine Yellows Phytoplasma

Three blocks of Chardonnay in one vineyard in the Buckland Valley of Victoria, Australia, were surveyed over 4 years for grapevine yellows disease (GYd). Buckland Valley grapevine yellows phytoplasma (BVGYp) was the only phytoplasma detected by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis in GYd affected grapevines. GYd affected many grapevines and was characterized by remission of disease, some recurrence and occurrences in previously unaffected grapevines. A regional survey of the Buckland Valley indicated that both GYd and BVGYp occurred in the same restricted grape growing area. Within this area, BVGYp was detected in two vineyards that had been established using planting material from different sources. One could therefore speculate that BVGYp was present in these grapevines as a result of aerial transmission and was not present in the original planting material.     

1-beta-D-arabinofuranosylcytosine inhibits borna disease virus replication and spread

Borna disease virus (BDV) is a nonsegmented, negative-strand RNA virus that causes neurological diseases in a variety of warm-blooded animal species. There is general consensus that BDV can also infect humans, being a possible zoonosis. Although the clinical consequences of human BDV infection are still controversial, experimental BDV infection is a well-described model for human neuropsychiatric diseases. To date, there is no effective treatment against BDV. In this paper, we demonstrate that the nucleoside analog 1-beta-D-arabinofuranosylcytosine (Ara-C), a known inhibitor of DNA polymerases, inhibits BDV replication. Ara-C treatment inhibited BDV RNA and protein synthesis and prevented BDV cell-to-cell spread in vitro. Replication of other negative-strand RNA viruses such as influenza virus or measles virus was not inhibited by Ara-C, underscoring the particularity of the replication machinery of BDV. Strikingly, Ara-C treatment induced nuclear retention of viral ribonucleoparticles. These findings could not be attributed to known effects of Ara-C on the host cell, suggesting that Ara-C directly inhibits the BDV polymerase. Finally, we show that Ara-C inhibits BDV replication in vivo in the brain of infected rats, preventing persistent infection of the central nervous system as well as the development of clinical disease. These findings open the way to the development of effective antiviral therapy against BDV.     

Surface glycoprotein of Borna disease virus mediates virus spread from cell to cell

Borna disease virus (BDV) is a non‐segmented negative‐stranded RNA virus that maintains a strictly neurotropic and persistent infection in affected end hosts. The primary target cells for BDV infection are brain cells, e.g. neurons and astrocytes. The exact mechanism of how infection is propagated between these cells and especially the role of the viral glycoprotein (GP) for cell–cell transmission, however, are still incompletely understood. Here, we use different cell culture systems, including rat primary astrocytes and mixed cultures of rat brain cells, to show that BDV primarily spreads through cell–cell contacts. We employ a highly stable and efficient peptidomimetic inhibitor to inhibit the furin‐mediated processing of GP and demonstrate that cleaved and fusion‐active GP is strictly necessary for the cell‐to‐cell spread of BDV. Together, our quantitative observations clarify the role of Borna disease virus‐glycoprotein for viral dissemination and highlight the regulation of GP expression as a potential mechanism to limit viral spread and maintain persistence. These findings furthermore indicate that targeting host cell proteases might be a promising approach to inhibit viral GP activation and spread of infection.     

Herpes simplex virus type 1 glycoprotein E domains involved in virus spread and disease

Herpes simplex virus type 1 (HSV-1) glycoprotein E (gE) functions as an immunoglobulin G (IgG) Fc binding protein and is involved in virus spread. Previously we studied a gE mutant virus that was impaired for IgG Fc binding but intact for spread and another that was normal for both activities. To further evaluate the role of gE in spread, two additional mutant viruses were constructed by introducing linker insertion mutations either outside the IgG Fc binding domain at gE position 210 or within the IgG Fc binding domain at position 380. Both mutant viruses were impaired for spread in epidermal cells in vitro; however, the 380 mutant virus was significantly more impaired and was as defective as gE null virus. gE mutant viruses were inoculated into the murine flank to measure epidermal disease at the inoculation site, travel of virus to dorsal root ganglia, and spread of virus from ganglia back to skin to produce zosteriform lesions. Disease at the inoculation and zosteriform sites was reduced for both mutant viruses, but more so for the 380 mutant virus. Moreover, the 380 mutant virus was highly impaired in its ability to reach the ganglia, as demonstrated by virus culture and real-time quantitative PCR. The results indicate that the domain surrounding amino acid 380 is important for both spread and IgG Fc binding and suggest that this domain is a potential target for antiviral therapy or vaccines.     

Detection and Identification of Aster Yellows Group Phytoplasma (16SrI‐C) Associated with Peach Red Leaf Disease

In 2011, typical symptoms suggestive of phytoplasma infection such as reddening of leaves were observed in peach trees in Fuping, Shaanxi Province, China. Phytoplasma‐like bodies were observed by transmission electron microscope in the petiole tissues of symptomatic peach trees. Products of c. 1.2 kb were generated from all symptomatic peach leaf samples by a nested polymerase chain reaction using phytoplasma universal primer pairs P1?P7 and R16F2n?R16R2, whereas no such amplicon was obtained from healthy samples. Results of phylogenetic analysis and restriction fragment length polymorphism suggested that the phytoplasma associated with such peach red leaf disease was a member of subgroup 16SrI‐C. To our knowledge, this is the first record of 16SrI‐C subgroup phytoplasma occurred in peach tree in China.     

The multiplication and spread of sacbrood virus of bees

Sacbrood virus multiplied without causing symptoms in adult bees when it was injected into them or when it was fed to young individuals. More virus accumulated in heads of infected bees than elsewhere in their bodies, and much was in their hypopharyngeal glands. The extract of each infected head contained about 10²medial lethal doses (LD 50s) of sacbrood virus for larvae when given in their food. The infective dose of sacbrood virus by injection for adults was about 10^-4of the LD 50 in food for larvae. The infective dose by mouth for adults was about 10²LD 50s for larvae, but bees older than 4–8 days could not be infected this way. Infection did not spread between adults but is probably transmitted in nature from infected adults to larvae and back to young adults that ingest remains of larvae killed by sacbrood. The youngest adult bees infected with sacbrood ate little pollen and lived only about 3 weeks in the laboratory, as did bees receiving no pollen, whereas bees fed with ample pollen lived 9 weeks.     

Molecular Characterization of Aster Yellows Subgroup 16SrI‐B Phytoplasma in Verbena × hybrida

Symptoms resembling phytoplasma disease were observed on Verbena × hybrida in Alanya, Turkey, during October 2013. Infected plants were growing as perennials in a flower border and showed symptoms of discoloured flowers, poor flower clusters, inflorescences with a small number of developed flowers and thickened fruit stalks. Electron microscopy examination of the ultra‐thin sections revealed polymorphic bodies in the phloem tissue of leaf midribs. The phytoplasma aetiology of this disease was confirmed by polymerase chain reaction of the 16S rRNA gene, the 16–23S rRNA intergenic spacer region and the start of the 23S rRNA gene using universal phytoplasma‐specific primer pair P1A/P7A, two ribosomal protein (rp) genes (rpl22 and rps3) (the group‐specific primer pair rp(I)F1A/rp(I)R1A) and the Tuf gene (group‐specific fTufAy/rTufAy primers) generating amplicons of 1.8 kbp, 1.2 kbp and 940 bp, respectively. Comparison of the amplified sequences with those available in GenBank allowed classification of the phytoplasma into aster yellows subgroups 16SrI‐B, rpI‐B and tufI‐B. This is the first report about molecular detection and identification of natural infection of the genus Verbena by phytoplasma and occurrence of the aster yellows group phytoplasma on an ornamental plant in Turkey.