Versuche zur Ausarbeitung einer Laborprüfmethode zwecks Feststellung der Knollenresistenz gegenüber Phytophthora infestans

Beim routinemäßigen Nachweis mechanisch übertragbarer Viren in Bäumen des Kern‐ und Steinobstes kann die Probeentnahme das Testergebnis u. U. nachhaltig beeinflussen. Die Kenntnis der Verteilung dieser Erreger in der Baumkrone ist deshalb von entscheidender Bedeutung. Folgende Viren wurden in die Untersuchungen einbezogen: Chlorotisches Apfelblattfleckungs‐Virus (apple chlorotic leaf spot virus, CLSV), Apfelstammfurchungs‐Virus (apple stem grooving virus, SGV) und Apfelmosaik‐Virus (apple mosic virus, ApMV) (Kernobst) bzw. Nekrotisches und Chlorotisches Kirschenringflecken‐Virus (Prunus necrotic ringspot virus, PNRV; prune dwarf virus, PDV), ApMV, CLSV, Scharka‐Virus der Pflaume (plum pox virus, PPV), Petunia asteroid mosaic virus (PAMV) und Kirschenblattroll‐Virus (cherry leaf roll virus, CLRV) (Steinobst). In der Regel kam der ELISA, nur in Einzelfällen der Latextest, zur Anwendung. Die genannten Viren lassen sich hinsichtlich der Verteilung in der holzigen Wirtspflanze 3 Gruppen zuordnen:

• Viren mit systemischer Verteilung: CLSV, SGV, PNRV, PDV, PPV in hochanfälligen Pflaumensorten und Pfirsich

• Viren mit teilsystemischer Verteilung: ApMV, PPV in weniger anfälligen Pflaumensorten

• Viren mit sporadischer Verteilung: PAMV, CLRV

     

Characterisation of cucumber leaf spot virus isolated from recycled irrigation water of soil-less cucumber cultures

Greenhouse‐grown cucumber plants showed mosaic‐type symptoms and irregular yellow spots on their leaves. The disease did not affect plant growth and the fruits remained symptom free. A virus having isometric particles, 30 nm in size, was isolated from the infected tissues and from recycled drainage water collected from tuff (volcanic rock) raised beds on which plants were grown. The virus was identified as a variant of cucumber leaf spot virus (CLSV) that has a host range similar but not identical to that of a previously described CLSV isolate. The overall nucleotide sequence identity between the RNAs of the Israeli isolate and the type isolate virus (accession numbers: DQ227315 and AY571334, respectively) amounts to 96%.     

A yellow mosaic disease of horse chestnut (Aesculus spp.) caused by apple mosaic virus

A severe foliar yellow mosaic disease was observed in horse chestnut trees (Aesculus carnea and A. hippocastanum). Reactions in woody indicator plants grafted with diseased horse chestnut suggested the presence of an ilarvirus. Virus isolates obtained by mechanical inoculation of herbaceous test plants reacted with antisera to apple mosaic virus but not with antisera to its serotype prunus necrotic ringspot virus, or to prune dwarf virus. Yellow mosaic was induced in horse chestnut seedlings grafted with tissues from herbaceous hosts infected with horse chestnut isolates or with the European plum line pattern isolate of apple mosaic virus. Virus was detected by enzyme-linked immunosorbent assay (ELISA) in embryo and endosperm of immature seed from infected trees but not in mature seed, or progeny seedlings. Strawberry latent ringspot virus was detected in one of six A. hippocastanum trees with a leaf vein yellows disease.     

The Particle Length of an Isolate of Apple Chlorotic Leafspot Virus from Prunus domestica

The modal length of an isolate of apple chlorotic leafspot virus (CLSV) from Prunus domestica was determined by serologically specific electron microscopy. Particles adsorbed from leaf sap extracts (modal length c. 825 nm) were longer than those adsorbed from purified virus preparations (modal length c. 795 nm). Particles of a CLSV isolate from apple, however, had a modal length of only c. 700 nm.     

Detection of Double-Stranded RNA (DSRNA) in Crude Extracts of Virus-Infected Plants by Indirect ELISA

An antiserum against polyinosinic-polycytidylic acid (In-Cn) was used to detect double-stranded RNA (dsRNA) by indirect ELISA (ELISA-I). DsRNA from cucumber mosaic virus (CMV) and plum pox virus (PPV)-infected plants was detected using different types of extracts. The pH of the extraction buffer was very important in dsRNA detection, the highest optical density values being obtained at pH 6 or in aqueous extracts. Extracts heated at 80°C for 2 min showed increased optical density values compared with unheated extracts. DsRNA from Nicotiana benthamiana plants infected with each of six PPV isolates was readily detected by ELISA-I 50 days after inoculation. ELISA values then obtained with the In-Cn antiserum were generally higher than those obtained by double antibody sandwich ELISA using an antiserum to virus coat protein. Purified dsRNA from the same infected plants showed no visible band, but it produced a fluorescent background when analysed by polyacrylamide gel electrophoresis.     

Strains of Prunus necrotic ringspot virus in hop (Humulus lupulus L.)

Purified preparations of Prunus necrotic ringspot virus (NRSV) from hop plants formed two light-scattering zones when centrifuged in sucrose density gradients; the upper and lower zones contained particles 25 mμ and 31 mμ in diameter respectively whose sedimentation coefficients were 79 S and 107 S. NSRV isolates from hop were of two distinct serological types: ‘A’ strains, serologically very closely related to NRSV isolates from apple; and ‘C’ strains more nearly related to NRSV from cherry. The variety Fuggle is tolerant to hop mosaic (not related to NRSV) and different selections of apparently healthy female plants usually contained A strains; but C strains were usually isolated from nettlehead-diseased plants. Either A or C strains occurred in male plants grown with the hop-mosaic tolerant varieties. In mosaic-sensitive varieties (Goldings and Bramlings) apparently healthy female plants tested were usually infected with C strains; either A or C types occurred in mosaic-sensitive male plants. NRSV was not detected in the seventy-four hop seedlings obtained from virus-infected plants. Some varieties developed nettlehead when infected with NRSV (A) or (C) + the hop form of arabis mosaic virus, but not with NRSV (A) or (C) alone. Others developed nettlehead when infected with arabis mosaic virus + NRSV (C) but not with arabis mosaic + NRSV (A). A and C strains can multiply together in the same hop plant. There is evidence of partial antagonism, however, and the fluctuating behaviour of the nettlehead syndrome probably reflects changes in the relative concentration of the two serotypes.     

Identification of Tomato black ring virus from tomato plants grown in greenhouses in Saudi Arabia

A survey was conducted in Al-Kharj governorate, Riyadh region to identify viruses causing variety of virus-like symptoms on tomato plants. A total of 135 samples were collected from symptomatic tomato plants. Symptoms included mottling, deformation, necrosis of leaves and fruits. Eighteen viruses were tested by DAS-ELISA. Tomato black ring virus (TBRV) was the virus of concern as it was not detected in Saudi Arabia before and was detected in 52.6% of the collected samples in this study. RT-PCR was used to confirm detection of TBRV and to sequence the amplified products to determine molecular characteristics of this virus. In the host range test study that was performed using a purified isolate of TBRV, sixteen out of the twenty two tested plants showed symptoms. Brassica oleracea was not infected by this virus. Gel electrophoreses (2% agarose) yielded fragments of 978 bp of coat protein gene of TBRV. Nucleotide sequences of purified RT-PCR products for three TBRV Saudi isolates were deposited in the GenBank with the following accession numbers MT274656, MT274657, and MT274658. These isolates of TBRV indicated a close Phylogenetic relationship of (99–100%) among themselves and with five isolates from Poland (95–98%) but a distant relationship of 85% with isolates from England and Lithuania deposited in the GenBank. This is the first report for detection and molecular characterization of TBRV infecting tomato plants in Saudi Arabia.     

禾谷多粘菌对小麦梭条斑花叶病毒的传播及其土壤侵染潜力的测定

从大田侵染小麦梭条斑花叶病毒的小麦病根中挑取禾谷多粘菌休眠孢子堆,接种受侵染小麦品种扬麦４号,经砂培养纯化,获得５个禾谷多粘菌分离物,但都为无毒。无毒多粘菌休眠孢子堆接种表现ＷＳＳＭＶ症状的小麦,经培养可饲获病毒,并可经接咱后将病毒传播给无病小麦,供试的４个大小麦禾谷多粘菌分离物都可对大小进行交叉侵染,产生同样数量的游动孢子产量。供试５个病土和２个无病土样品,都具有强大持多粘菌侵染潜力,即使稀释放     

Arabis mosaic and Prunus necrotic ringspot viruses in hop (Humulus lupulus L.)

Purified virus preparations made from nettlehead-diseased hop plants, or from Chenopodium quinoa, to which the virus was transmitted by inoculation of sap, contained polyhedral virus particles of 30 mμ diameter which were identified serologically as arabis mosaic virus (AMV). There were serological differences between AMV isolates from hop and from strawberry, and also differences in host range and in symptoms caused in C. quinoa and C. amaranticolor. AMV was always associated with nettlehead disease. The nematode Xiphinema diversicaudatum occurred in small numbers in most hop gardens, but was numerous where nettlehead disease was spreading rapidly. Preparations from nettlehead-affected hops also contained a second virus, serologically related to Prunus necrotic ringspot virus (NRSV), in mild and virulent forms which infected the same range of test plants but showed some serological differences. Mild isolates did not protect C. quinoa plants against infection by virulent isolates. Hop seedlings inoculated with virulent isolates of NRSV developed symptoms indistinguishable from those of split leaf blotch disease. Latent infection with NRSV was prevalent in symptomless hop plants. Nettlehead disease is apparently associated with dual infection of AMV and virulent isolates of NRSV. An unnamed virus with rod-shaped particles 650 mμ long was common in hop and was transmitted by inoculation of sap to herbaceous plants. Cucumber mosaic virus was obtained from a single plant of Humulus scandens Merr.     

Confirmation of the transmission of barley yellow mosaic virus (BaYMV) by the fungus Polymyxa graminis

Resting spores (cystosori) of Polymyxa graminis, selected from roots of barley plants infected with barley yellow mosaic virus (BaYMV), were used to start mono-fungal sand cultures. Out of 20 attempts using over 800 cystosori, P. graminis became established in 12, and in two of these BaYMV symptoms also occurred. BaYMV was detected by ELISA in extracts of dried roots heavily infected with cystosori and in zoospores of P. graminis. Calculations suggested that, on average, each zoospore carried less than 100 virus particles. In two virus acquisition experiments, non-viruliferous isolates of P. graminis failed to acquire BaYMV from roots of mechanically-inoculated plants. In two further experiments, non-viruliferous isolates were grown on rooted tillers produced from healthy plants and those infected with BaYMV by either vector or mechanical inoculation. Zoospores and cystosori of P. graminis subsequently transmitted the virus, but only from plants where it had been introduced by the vector. Repeated mechanical transmission appeared to have selected a strain of virus that could not be acquired and/or transmitted by the vector. The results provide convincing evidence that P. graminis is a vector of BaYMV but suggest that, in natural populations, only a small proportion of spores may be viruliferous.     

Molecular Evidence for Recombination in <Emphasis Type="Italic">Prunus</Emphasis> Necrotic Ringspot Virus

Genetic RNA recombination plays an important role in viral evolution. The evolutionary history of Prunus necrotic ringspot virus (PNRSV) has been extensively studied, but knowledge of recombination in its genome is still lacking. To investigate the recombination events in this virus, 67 accessions composed by 62 isolates retrieved from the databanks and five Tunisian isolates described in this study were analyzed. The use of RECCO algorithm which is based on cost minimization allowed us to detect several breakpoints in the coat protein gene (CP) of three out of five isolates from Tunisia and one from Poland. Moreover, a recombination signal was also detected in the putative cell-to-cell movement protein-encoding gene of an isolate from the USA. Tajima Neutrality test implemented in MEGA4 program indicated the occurrence of a high level of deletion/insertion events in the sequences. The evolutionary historical relationships were determined by constructing a dendrogram using neighbor joining, minimum evolution, maximum parsimony, maximum likelihood, and unweighted pair group method with arithmetic mean (UPGMA). The first four analyses gave similar results. Three classical groups (PE 5, PV 32, and PV 96) were delineated. The recombinant isolates from Tunisia clustered in a distinct clade except for one nonrecombinant (Ghernghezel) which revealed to be a member of PV 32 group. In contrast, UPGMA algorithm divided the Tunisian isolates in three distinct subgroups. Apart from recombination, reassortment is still an open question among many others and may also represent another way to explore the genetic diversity of PNRSV.     

Acquisition and transmission of potato mop-to furovirus by a culture of Spongospora subterranea f.sp. subterranea derived from a single cystosorus

Potato mop-top virus (PMTV) was detected by ELISA in primary zoospores from four out of six isolates of Spongospora subterranea f.sp. subterranea. One virus-free isolate (N) of S. subterranea was used to acquire PMTV from potato roots and to transmit the virus to healthy plants. A mono-fungal culture of S. subterranea (isolate N) was derived by infecting tomato plant roots with a single cystosorus. The culture was used successfully to acquire PMTV from the roots of infected Nicotiana debneyi plants that had been manually inoculated with virus isolates, and subsequently to transmit the virus to healthy bait plants. These experiments confirm that S. subterranea is a vector of PMTV. Two PMTV isolates that had been maintained by manual inoculation for 19 and 21 passages were also acquired and transmitted by the fungus culture.     

An eclipse of pea seed-borne mosaic virus in vegetative tissue of pea following repeated transmission through the seed

Four isolates of pea seed-borne mosaic virus (PSbMV) representing pathotypes P1 (isolates US and Q) and P4 (isolates S4 and S6), and groups III (US and Q) and V (S4 and S6) have been used in a study of the survival and partitioning of PSbMV under conditions of continuous seed transmission in the commercial pea cultivar Dundale. Assays suitable for detecting virus in small tissue samples were developed, and included dot-immunobinding assay with antisera to both PSbMV and cytoplasmic inclusion body (CIB) protein, and dot hybridisation assay (DHA) with cDNA transcribed from virus RNA. Under the conditions of our experiments, seed transmission occurred at rates exceeding 90% for all virus isolates. Virus was detectable by serology and symptoms in inoculated plants, and in all vegetative tissue of second generation plants raised from seed of the inoculated plants. However, in the third, fourth and fifth sequential generations raised from seed, all plants were symptomless. Neither virus nor CIB were detectable in leaf, stem or roots by serology, but both were readily detectable in some floral parts, and in immature and mature seed. Mature seed contained virus and CIB antigen in the testa, cotyledon and embryo. Inoculum prepared from whole seeds was infectious. The testa was shown not to be involved in transmission between generations, thus implicating the embryo alone in vertical transmission. Virus antigen could not be detected in the emerging cotyledons of germinating seed and all true leaves by serology, but the leaves contained PSbMV RNA detectable by DHA. These results show that PSbMV infection can be transferred through the vegetative phase at a subliminal level, and reaches relatively high concentrations in floral parts and seeds. Thus PSbMV may be maintained at a high level of infection in seed in the absence of any apparent symptoms in the plant, and without a requirement for horizontal transmission between plants by vectors. Such a mechanism may explain the high levels of infection commonly reported in pea breeding lines.     

Zucchini Yellow Fleck Virus is an Emergent Virus on Melon in Sicily (Italy)

Since 2006, winter melon plants (Cucumis melo L. var inodorus) showing symptoms of pin‐point yellow spots were noticed in Sicily (Italy). Leaf samples were tested by enzyme‐linked immunosorbent assay to the most important viruses‐infecting cucurbits. Zucchini yellow fleck virus (ZYFV, genus Potyvirus) was the only virus detected. Surveys in 2007 and 2008 revealed an increasing number of sites in Sicily with ZYFV‐infected winter melon plants. To confirm the identity of the virus as ZYFV, two isolates from different locations were sequenced and shown to be approximately 85% identical to the published sequences of isolates previously identified in Italy and France. This is the first report of ZYFV occurring on melon in Italy.     

Genetic Variability and Evolutionary Implications of RNA Silencing Suppressor Genes in RNA1 of Sweet Potato Chlorotic Stunt Virus Isolates Infecting Sweetpotato and Related Wild Species

Background

The bipartite single-stranded RNA genome of Sweet potato chlorotic stunt virus (SPCSV, genus Crinivirus; Closteroviridae) encodes a Class 1 RNase III (RNase3), a putative hydrophobic protein (p7) and a 22-kDa protein (p22) from genes located in RNA1. RNase3 and p22 suppress RNA silencing, the basal antiviral defence mechanism in plants. RNase3 is sufficient to render sweetpotato (Ipomoea batatas) virus-susceptible and predisposes it to development of severe diseases following infection with unrelated virus. The incidence, strains and gene content of SPCSV infecting wild plant species have not been studied.

Methodology/Principal Findings

Thirty SPCSV isolates were characterized from 10 wild Ipomoea species, Hewittia sublobata or Lepistemon owariensis (family Convolvulaceae) in Uganda and compared with 34 local SPCSV isolates infecting sweetpotatoes. All isolates belonged to the East African (EA) strain of SPCSV and contained RNase3 and p7, but p22 was not detected in six isolates. The three genes showed only limited genetic variability and the proteins were under purifying selection. SPCSV isolates lacking p22 synergized with Sweet potato feathery mottle virus (SPFMV, genus potyvirus; Potyviridae) and caused severe symptoms in co-infected sweetpotato plants. One SPCSV isolate enhanced accumulation of SPFMV, but no severe symptoms developed. A new whitefly-transmitted virus (KML33b) encoding an RNase3 homolog (<56% identity to SPCSV RNase3) able to suppresses sense-mediated RNA silencing was detected in I. sinensis.

Conclusions/Significance

SPCSV isolates infecting wild species and sweetpotato in Uganda were genetically undifferentiated, suggesting inter-species transmission of SPCSV. Most isolates in Uganda contained p22, unlike SPCSV isolates characterized from other countries and continents. Enhanced accumulation of SPFMV and increased disease severity were found to be uncoupled phenotypic outcomes of RNase3-mediated viral synergism in sweetpotato. A second virus encoding an RNase3-like RNA silencing suppressor was detected. Overall, results provided many novel and important insights into evolutionary biology of SPCSV.     

Virus Content and Virulence of Polymyxa betae Keskin Isolates Obtained from Different Regions in Europe

Polymyxa betae isolates were obtained by means of bait plants from a large number of soil samples collected in eastern Germany. Additional P. betae isolates were received from several institutions in western Germany and abroad. Isolates were grown on sugarbeet seedlings and tested for the presence of beet necrotic yellow vein virus (BNYVV) and beet soilborne virus (BSBV). BNYVV was only present in isolates from western Germany and abroad but absent in all isolates from eastern Germany., In contrast, BSBV was detected in more uniform geographic distribution in 14 out of 33 P. betae isolates tested. The virulence of P. betae isolates was estimated on the basis of the extent of resting spore formation in the root system of sugarbeet seedlings. Differences in virulence were found among virus-free as well as virus-carrying P. betae isolates. The mean value of virulence ratings was distinctly lower with BNYVV-carrying isolates and slightly lower with BSBV-carrying isolates as compared to virus-free isolates.