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Summary From January 2003 to June 2005, samples from 3416 batches of wooden packaging material were inspected for the presence of nematodes in the Ningbo Entry–Exit Inspection and Quarantine Bureau, China. Bursaphelenchus spp. were detected in 202 batches from 25 different countries. The following species were identified on the basis of their morphology and their intergenic transcribed spacer-restriction fragment length polymorphism (ITS-RFLP) patterns: B. xylophilus, B. fungivorus, B. rainulfi, B. hylobianum, B. thailandae, B. mucronatus, B. aberrans, B. lini, B. singaporensis, B. doui, B. conicaudatus, B. vallesianus, B. pinasteri, B. hofmanni and B. arthuri. The most frequently found species were B. mucronatus (57 batches), B. xylophilus (40 batches), B. fungivorus (21 batches), B. rainulfi (19 batches) and B. thailandae (nine batches). The pine wood nematode B. xylophilus was not only found in packaging wood imported from areas where it is known to occur (i.e. The United States of America, Japan, Korea, Hong Kong and Taiwan), but also from countries considered to be free of this dangerous pest (i.e. Brazil, Thailand, Belgium, The Netherlands, Italy and Spain). The occurrence of B. xylophilus in packaging wood from countries regarded as being free of the nematode can most likely be explained by the global circulation of wooden packaging material among infested and non-infested countries. Our findings emphasize the need to fully implement international standards on phytosanitary treatment of packaging wood, in order to prevent further spread of the pine wood nematode, and the need for careful re-examination of the current heat treatment measures. Resume De Janvier 2003 a Juin 2005, des echantillons de 3416 lots de materiel d'emballage en bois ont ete inspectes pour rechercher la presence de nematodes au bureau de quarantaine de Ningbo, en Chine. Des Bursaphelenchus spp. ont ete detectes dans 202 lots en provenance de 25 pays differents. Les especes suivantes ont ete identifiees sur la base de leur morphologie et des profils de restriction de l'ITS: B. xylophilus, B. fungivorus, B. rainulfi, B. hylobianum, B. thailandae, B. mucronatus, B. aberrans, B. lini, B. singaporensis, B. doui, B. conicaudatus, B. vallesianus, B. pinasteri, B. hofmanni, et B. arthuri. Les especes les plus frequemment trouvees sont B. mucronatus (57 lots), B. xylophilus (40 lots), B. fungivorus (21 lots), B. rainulfi (19 lots) et B. thailandae (9 lots). Le nematode du pin, B. xylophilus, a ete trouve dans du bois d'emballage en provenance non seulement de zones ou sa presence est connue (Etats-Unis, Japon, Republique de Coree, Hongkong et Taiwan) mais aussi de pays consideres comme indemnes de ce redoutable pathogene (Bresil, Thailande, Belgique, Pays-Bas, Italie et Espagne). La presence de B. xylophilus dans du bois d'emballage de pays consideres comme indemnes s'explique probablement par la circulation generalisee de materiel d'emballage en bois entre pays infestes ou non. Nos resultats soulignent la necessite d'appliquer pleinement les normes de traitement phytosanitaire du bois d'emballage, afin d’eviter une plus grande dissemination du nematode du pin, et de reexaminer de facon approfondie les mesures actuelles de traitement thermique. Zusammenfassung Von Januar 2003 bis Juni 2005 wurde im Quarantane-Inspektionsburo in Ningbo, China, das Verpackungsholz von 3416 Sendungen nematologisch untersucht. Bursaphelenchus spp. wurden in 202 Sendungen aus 25 verschiedenen Landern festgestellt. Folgende Arten wurden morphologisch und mit Hilfe der ITS-RFLP-Methode identifiziert: B. xylophilus, B. fungivorus, B. rainulfi, B. hylobianum, B. thailandae, B. mucronatus, B. aberrans, B. lini, B. singaporensis, B. doui, B. conicaudatus, B. vallesianus, B. pinasteri, B. hofmanni, und B. arthuri. Die am haufigsten gefundenen Arten waren B. mucronatus (57 mal), B. xylophilus (40), B. fungivorus (21), B. rainulfi (19) and B. thailandae (9). Der Quarantaneschadling B. xylophilus wurde nicht nur in Verpackungsholz aus Gebieten mit bekannter Verbreitung des Nematoden (USA, Japan, Republik Korea, Hongkong, Taiwan) gefunden, sondern auch aus Landern, die als frei vom Kiefernholznematoden gelten (Brasilien, Belgien, Niederlande, Italien, Spanien). Die Feststellung von B. xylophilus in Verpackungsholz aus Nichtbefallslandern kann nur durch Zirkulation des Verpackungsholzes zwischen Befalls- und Nichtbefallslandern erklart werden. Der mittlere prozentuale Anteil der jegliche Nematoden enthaltenden Packholzproben lag im Zeitraum von Januar 2000 bis August 2005 bei 21,3 %; eine klare Tendenz zur Zu- oder Abnahme war wahrend dieser Jahre nicht erkennbar. Die Befunde unterstreichen die Notwendigkeit der strikten Anwendung international erarbeiteter Standards der phytosanitaren Behandlung von Verpackungsholz zur Verhinderung der Verschleppung des Kiefernholznematoden und der Uberprufung der Wirksamkeit der zur Zeit angewandten Masnahmen.

Summary We determined the incidence of Bursaphelenchus species in declining Pinus sylvestris stands in the Swiss Alps. A total of 217 trees showing different degree of decline were sampled from various locations in Valais between 2001 and 2004. Bursaphelenchus species were only found in recently dead or dying trees with an overall incidence of 40%. There was a positive relationship between Bursaphelenchus infestation and blue stain in these trees. Five Bursaphelenchus species were identified: Bursaphelenchus vallesianus, Bursaphelenchus mucronatus, Bursaphelenchus sexdentati, Bursaphelenchus leoni and Bursaphelenchus silvestris. The most frequent species were B. vallesianus detected in 75% and B. mucronatus in 20% of all Bursaphelenchus-positive trees. Bursaphelenchus vallesianus is a recently described species within the B. sexdentati group, and could be a contributing factor in the observed pine decline in Valais. Resume Des forets de pins sylvestres (Pinus sylvestris) ont eteetudiees en fonction de leur infestation par des especes du genre Bursaphelenchus. Entre 2001 et 2004, 217 arbres presentant une defoliation plus ou moins marquee ont ete examines dans differentes sites du Valais. 40% des pins sylvestres fortement affaiblis ou deja deperis etaient infestes par des especes de Bursaphelenchus. Pour ces arbres, une relation etroite a pu etre etablie entre l'attaque par Bursaphelenchus et le bleuissement du bois. Cinq especes de Bursaphelenchus ont ete identifiees a l'aide de methodes morphologiques et biomoleculaires: B. vallesianus, B. mucronatus, B. sexdentati, B. leoni et B. silvestris. Les especes les plus frequentes etaient B. vallesianus et B. mucronatus, que l'on a pu determiner respectivement dans 75% et 20% de l'ensemble des arbres contamines par Bursaphelenchus. C'est seulement recemment que B. vallesianus a ete decrit comme nouvelle espece faisant partie du groupe de B. sexdentati (Braasch et al. 2004). Zusammenfassung Kiefernbestande (Pinus sylvestris) wurden hinsichtlich ihres Befalls mit Bursaphelenchus-Arten untersucht. Insgesamt 217 Baume mit unterschiedlich ausgepragter Kronenverlichtung wurden zwischen 2001 and 2004 an verschiedenen Standorten im Wallis beprobt. 40% der stark geschwachten oder bereits abgestorbenen Kiefern waren mit Bursaphelenchus-Arten befallen. In diesen Baumen wurde eine enge Beziehung zwischen dem Bursaphelenchus-Befall und der Holz-Blaue gefunden. Funf Bursaphelenchus-Arten wurden mittels morphologischer und molekularbiologischer Methoden identifiziert: B. vallesianus, B. mucronatus, B. sexdentati, B. leoni und B. silvestris. Die haufigsten Arten waren B. vallesianus und B. mucronatus, die in 75% bzw. 20% aller mit Bursaphelenchus kontaminierten Baume festzustellen waren. B. vallesianus wurde erst kurzlich als neue Art innerhalb der B. sexdentati Gruppe beschrieben (Braasch et al. 2004). Obwohl Nematoden der Gattung Bursaphelenchus in vielen der abgestorbenen oder stark geschwachten Kiefern nachgewiesen wurden, muss deren Rolle beim Fohrensterben in weiteren Studien geklart werden.

Expression vectors were constructed from 35S promoter-containing full-length cDNA clones of Zygocactus virus X (ZVX). The expression of foreign genes was driven by the ZVX coat protein (cp) subgenomic promoter. It was successful only when the variable region downstream of the conserved putative promoter region GSTTAAGTT(X12–13)GAA was retained. Most of the ZVX cp gene, except for a short 3′ part, was replaced by the corresponding sequence of the related Schlumbergera virus X (SVX) and its cp subgenomic promoter to enable encapsidation of the transcribed RNA by an SVX/ZVX hybrid cp. Vector-expressed cp of Beet necrotic yellow vein virus (BNYVV) assembled in Chenopodium quinoa, Tetragonia expansa and Beta vulgaris leaves into particles resembling true BNYVV particles. The virus produced from these constructs retained its ability to express BNYVV cp in local infections during successive passages on C. quinoa. This ability was lost, however, in the rarely occurring systemic infections.

Bursaphelenchus leoni was found in wood of a severely wilted pine tree (Pinus radiata) in South Africa. Morphological studies and morphometric measurements showed the South African population to be conspecific with B. leoni which is widespread in southern Europe and indigenous in the Mediterranean region. In addition, molecular biological tests (RFLP analysis of amplified ribosomal DNA) confirmed this diagnosis and allowed comparisons with other Bursaphelenchus spp. from pine and other trees, i.e. B. xylophilus, B. mucronatus, B. fraudulentus and B. sexdentati. This is the first record of a Bursaphelenchus species in South Africa as well as in the southern hemisphere. P. radiata is recorded as a new host for B. leoni.

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. Zusammenfassung Virusgehalt und Virulenz von Polymyxa betae Keskin-Isolaten aus verschiedenen Regionen Europas Polymyxa betae-Isolate wurden mit Hilfe von Fangpflanzen aus einer grosen Anzahl Bodenproben gewonnen, die in Ostdeutschland gesammelt worden waren. Weitere P. betae-Isolate wurden von mehreren Institutionen in Westdeutschland und dem Ausland erhalten. Die Isolate wurden an Zukerrubensamlingen kultiviert und auf ihren Gehalt an beet necrotic yellow vein virus (BNYW) und beet soilborne virus (BSBV) getestet, BNYVV war nur in Isolaten aus Westdeutschland und dem Ausland enthalten und fehlte in allen ostdeutschen Isolaten. Im Gegensatz dazu wurde BSBV geographisch gleichmasiger verteilt in 14 von 33 getesteten Isolaten nachgewiesen. Die Virulenz der P. betae-lsolate wurde anhand der Starke des Dauersporenbefalls im Wurzelsystem eingeschatzt. Virulenzunterschiede wurden sowohl innerhalb der virusfreien als auch innerhalb der virustragenden P. betae-lsolate gefunden. Der Mittelwert der Virulenz war bei den BNYVV-tragenden Isolaten deutlich geringer und bei den BSBV-tragenden Isolaten etwas geringer als bei den virusfreien Isoiaten.

An interactive key is described for larvae and adults of economically important species of thrips. This is based on computerized photographs (Automontage), video clips and an interface to a molecular key for larvae based on ITS-RFLP analysis.

Beet necrotic yellow vein virus (BNYVV) inocula with different RNA compositions were prepared from infectious transcripts of RNAs 3 and 4 and the Rg 1 isolate, which has a genome consisting only of RNAs 1 and 2. The recombinant viruses were inoculated on 6- to 8-day-old sugarbeet seedlings by 'vortexing'. Inocula containing RNAs 1 and 2 or 1, 2 and 4 produced some growth reduction, but the most dramatic effects, with yield reductions of about 95% in a highly susceptible variety, were seen when RNA 3 was also present in the inoculum. Under these conditions the side roots were brown and brittle and often deteriorated, but 'root beardedness' was not observed. This might be due to the fact that our experiments were done in the absence of Polymyxa betae. Alternatively, the heavy inoculation at a very young age may either have weakened the plants to such an extent that extensive root proliferation was impaired or it may have led to rapid deterioration of the proliferating rootlets, which would therefore be lost prior to or during removal of the tap roots from the soil. In the presence of RNA 3 the virus concentrations in tap roots were markedly increased suggesting that this RNA facilitates the multiplication and/or spread of the virus in root tissues.

Expression of the beet necrotic yellow vein virus (BNYVV) coat protein (CP) gene in transgenic sugar beet hairy roots was accomplished as a step towards CP-mediated virus resistance. A cDNA for the CP gene and its 5' terminal untranslated leader sequence was prepared from BNYVV RNA, using two oligodeoxynucleotides to prime the synthesis of both strands. Second-strand synthesis and amplification of the cDNA were done by Taq DNA polymerase chain reactions. Run-off transcripts of the cloned cDNA sequence were obtained and translated in vitro, yielding immunoreactive CP. A binary vector construction containing the CP gene under the control of the 35S promoter of cauliflower mosaic virus was prepared and used for Agrobacterium rhizogenes-mediated transformation of sugar beet tissue. Stable integration and expression of the CP gene in sugar beet hairy roots was demonstrated by Southern, Northern, and Western blot analysis, respectively.

A small genomic library from DNA of G. pallida population Delmsen (pathotype 3) was constructed. A number of clones were tested as hybridization probes with 21 G. pallida and 6 G. rostochiensis populations. The two species were easily distinguished by their hybridization patterns. A total of 32 RFLP markers were identified which permitted the differentiation of all G. pallida populations. By means of cluster analysis of the RFLP data the genetic distances between the 21,G. pallida populations were calculated and a dendrogram was constructed. The arrangement of the 21 G. pallida populations in the dendrogram suggests a partial correlation between genetic distance and virulence according to biological pathotyping.

The genome properties of three potexviruses which previously had been isolated from different genera in the family Cactaceae and had been found to be only distantly related serologically have been studied. The sequence of the 3040 3′ terminal nucleotides of the genomic RNA of isolate K11 from Schlumbergera bridgesii and the complete RNA sequences of isolates B1 and CC10 from Zygocactus sp. and Opuntia sp., respectively, were determined. Starting sequences were obtained by means of immunocapture reverse transcription PCR using primers derived from highly conserved sequences in other potexviral RNAs. The known parts of the sequences were extended by means of random-primed cDNAs and specific primers derived from the known parts of the sequences. The genome structure of the three viruses resembles that of other potexviruses. The conserved motifs typical for replication-associated proteins, triple gene block (TGB) proteins and coat proteins of potexviruses were readily identified in the translation products of the five open reading frames. The 3′ untranslated regions of the three RNAs are folded into secondary structures containing three characteristic hairpins. Rather low percentages of amino acid sequence identities ranging from 62% to 76% for the coat proteins and 41% to 49% for TGB proteins 3 suggest that these viruses should be regarded as distinct virus species for which the names Zygocactus virus X, Schlumbergera virus X and Opuntia virus X are proposed. It is also suggested that the name Cactus virus X which originally was coined for all three virus isolates should no longer be used.

cDNAs of beet necrotic yellow vein virus RNAs 3 and 4 could be rendered biologically active when they were placed under the control of the cauliflower mosaic virus 35S promoter and polyadenylation signal. Although the 35S in vivo transcripts should have contained up to forty 5′ and several hundred 3′ nonviral nucleotides, the progeny viral RNAs had the same sizes as in naturally infected sugarbeets. The progeny RNAs did not hybridize with the nonviral sequences indicating that they were apparently not replicated. Deletion and insertion mutants of RNA 3 cDNA clones were also biologically active in plants but a plasmid which contained the cDNA of RNA 3 in antisense orientation was not. The biological activity of plasmid DNAs compared with the corresponding synthetic transcripts is discussed.

A novel approach to establish virus resistance in plants is based on recent progress to clone regions of immunoglobulin genes which encode the antigen binding domains of the variable regions of the heavy (VH) and the light chains (VL) of antiviral monoclonal antibodies (mAb) and to express these polypeptide chains connected by a linker peptide as single-chain fragments (scFv).

Summary Northern blot hybridization experiments with cDNAs to the four RNA species of a Yugoslavian isolate of beet necrotic yellow vein virus (BNYVV) revealed identical virus RNA patterns in root extracts from field-grown sugarbeets and sugarbeet seedlings grown in soil from rhizomania-affected fields in various regions in Germany and abroad. In contrast, in leaf extracts from mechanically infected Chenopodium quinoa, Tetragonia expansa and sugarbeet and from a naturally infected sugarbeet we observed great variations in the number, size and relative concentration of the small BNYVV RNAs, which suggests that they may undergo deletion mutations when the virus is propagated in leaf tissues.

The coat proteins of tymo-, tombus-, como-, nepo-, tobamo-, potex-, carla- and potyviruses were subjected to sodium dodecylsulphate (SDS)-polyacrylamide gel electrophoresis, electro-blotted onto nitrocellular membranes and reacted with homologous and heterologous antisera to intact plant viruses. Immune complexes were detected after reaction with alkaline phosphatase-labelled goat anti-rabbit antibodies. SDS coat proteins of comoviruses reacted only with antisera which were homologous for their intact particles. In all other groups, however, a rather broad range of serological relationships was detected. With a number of viruses, serological relations to other members of the same group were demonstrated for the first time. SDS coat proteins of maclura mosaic virus, a possible potyvirus, and of beet necrotic yellow vein virus, a possible tobamovirus, did not react with antisera to definitive members of the respective groups. Several unexpected reactivities of SDS coat proteins of viruses from one group with antisera to viruses in other groups were, however, also observed. This indicated that electro-blot immunoassay, like other serological methods, cannot be used reliably to assign a virus to a certain group. The SDS coat proteins of several tombusviruses were able to mimic serological reactions by a direct binding of enzymelabelled antibodies which occurred also without serum treatment. Zusammenfassung Electro-blot Immunoassay—eine Methode zum Studium serologischer Verwandtschaften zwischen Pflanzenviren? Nach Elektrophorese in SDS-Polyacrylamidgelen wurden die Hullproteine („SDS”-proteine) von verschiedenen Tymo-, Tombus-, Como-, Nepo-, Tobamo-, Potex-, Carla- und Potyviren elektrophoretisch auf Nitrozellulosemembranen ubertragen und mit Seren behandelt, die gegen intakte Viren hergestellt worden waren. Immunkomplexe wurden durch Ziegen-Anti-Kaninchen-Antikorper nachgewiesen, die mit alkalischer Phosphatase markiert waren. Die „SDS”-proccine der Comoviren reagierten nur mit den fur die intakten Viren homologen Antiseren. In allen anderen Gruppen wurden daruber hinaus auch Reaktionen mit Seren gegen andere Viren der gleichen Gruppe beobachtet. Eine Reihe von entfernten Verwandtschaften zwischen Gliedern einer Gruppe wurden zum ersten Mal festgestellt. Die „SDS”-proteine von Maclura mosaic virus, einem muglichen Potyvirus, und beet necrotic yellow vein virus, einem muglichen Tobamovirus, reagierten nicht mit Seren gegen definitive Viren der entsprechenden Gruppen. Unerwarteterweise reagierten die „SDS”-proteine von mehreren Viren auch mit Seren gegen Viren in anderen Gruppen. Electro-blot Immunoassay kann daher, ebenso wie andere serologische Methoden, nicht zuverlassig zur Bestimmung der Gruppenzugehorigkeit eines Virus benutzt werden. Die „SDS”-proteine einiger Tombusviren tauschten serologische Reaktivitaten durch eine direkte Bindung von enzymmarkierten Antikorpern vor, die auch ohne Serumbehandlung beobachtet wurde.

A small water sample of only 60 ml from the Oker Aue canal near Braunschweig was ultracentrifuged. The resuspended pellet was rubbed on Chenopodium quinoa leaves which responded with the formation of almost 200 local lesions. The virus causing these lesions was identified as carnation ringspot virus (CarRSV) by means of dot blot hybridization using random-primed cDNAs to the viral nucleic acids and by means of serology. Northern blot analysis revealed that the two RNA species of the virus which consisted of c. 3.7 and 1.5 Kb, respectively, have little or no base sequence homology. In immunoelectrophoresis at pH 7.0 the virus migrated towards the cathode. The isometric particles were 33 nm in diameter, round to slightly angular in outline, and showed a distinct granular or knobly surface structure. Virus particles occurred in the cytoplasm, nuclei, and vacuoles of infected cells which in addition contained amorphous granules in the cytoplasm and/or proliferated endoplasmic reticulum. Heavily, affected cells were necrotized and contained large virus particle aggregates which sometimes were crystallized. CarRSV is the third carnation virus, after carnation mottle and carnation Italian ringspot viruses, which was identified in a natural water.

Complementary deoxyribonucleic acids (cDNAs) to the four ribonucleic acid (RNA) species of isolate YU2 of beet necrotic yellow vein virus (BNYVV) were prepared by reverse transcription. The duplexes of these cDNAs were incorporated in the plasmid pBR322 and cloned into E. coli strain 5K. The insert sizes of the cDNA clones ranged from 280 to 2100 base pairs. In Southern blot hybridizations not all cDNAs specific for a particular RNA of BNYVV cross-reacted. This suggests that they are complementaryto different regions of the respective RNA. In dot blot hybridization experiments sixteen out of eighteen cDNA clones detected all thirteen BNYVV isolates which were obtained from sugar beets originating from Yugoslavia, Italy, Switzerland, France and various regions in Germany and which were maintained in Chenopodium quinoa. Two cDNA clones specific for RNA-3 failed to detect one German isolate. Pronounced differences between the thirteen virus isolates were seen when the electrophoretic migration of their RNAs was compared in Northern blot hybridizations. RNA-1 and RNA-2 of all isolates had the same sizes of 7100 and 5200 bases, respectively. The size, number and relative concentration of the smaller RNAs, however, varied greatly with different, isolates.

Two photosensitive iodocyanopindolol derivatives, 1-(4-azidobenzimidyl)-3,3-dimethyl-6-hydroxy-7-(2-cyano-3-iodoindol-4-yloxy)-1,4-diazaheptane (ICYP-azide-1) and 1-(4-azidobenzoyl)-3,3-dimethyl-6-hydroxy-7-(2-cyano-3-iodoindol-4-yloxy)-1,4-diazaheptane (ICYP-azide-2) have been prepared. [125I]ICYP-azide-1 and -2 (specific radioactivity up to 2.2 Ci/mumol) bind specifically and with very high affinity (KD = 40-45 pM) to beta-adrenergic receptors of turkey erythrocyte membranes. When [125I]ICYP-azide-1 or -2 were incubated with membranes and UV-irradiated, two polypeptides (Mr = 40,000 and 50,000) were specifically photolabeled as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These polypeptides may represent subunits of the beta-adrenergic receptor. The yield of specific covalent label incorporation into both polypeptides was up to 17.2% with [125I]ICYP-azide-2 when expressed as fraction of total beta-receptor binding sites. Since the Mr = 40,000 polypeptide was labeled predominantly and since covalent incorporation had the same concentration dependence as reversible specific binding, this polypeptide could contain a beta-adrenergic ligand binding site. Due to the low working concentration (10-100 pM) of [125I]ICYP-azide-1 and -2, nonspecific labeling of membrane proteins was extremely low. The new photoaffinity labels should therefore become valuable tools for probing beta-receptor structure.

Attempts were made to introduce three different isolates of beet necrotic yellow vein virus (BNYW) into the roots of sugarbeets by means of mechanical inoculation. In Chenopodium quinoa these isolates had produced different patterns of viral RNA. The RNA pattern of isolate P was similar to that found in sugarbeets infected naturally by means of the vector Polymyxa betae. RNA 3 and 4 were partially deleted in isolate Y and apparently absent in isolate R. About 20% of the sugarbeet roots inoculated either with isolate P or Y became infected systemically, but with isolate R no systemic infections were recorded. In tap and side roots of plants infected with isolate P the virus concentration remained high for at least ten months and the tap roots of these plants showed a very poor growth. In tap and side roots of plants infected with isolate Y the virus concentration gradually dropped until it reached the limit of detectability. The tap roots of these plants became about five times as big as those infected with isolate P. Our results indicate that BNYW with full length RNA 3 and 4 can cause heavy damage to sugarbeets also in the absence of Polymyxa betae and that RNA 3 and/or 4 — in addition to a recently suggested involvement in virus transmission by P. betae — apparently also facilitate the persistent spread of the virus in sugarbeet roots. Zusammenfassung Mechanische Inokulation von Zuckerrubenwurzeln mit Rizomaniavirus-Isolaten unterschiedlicher RNA-Zusammensetzung Es wurde versucht, durch mechanische Inokulation Isolate des Rhizomaniavirus (BNYVV) unterschiedlicher RNA-Zusammensetzung in Zuckerruben-Keimlingswurzeln einzubringen. Das Isolat P hatte in Chenopodium quinoa ein RNA-Muster gezeigt, das von dem in naturlich infizierten Ruben nicht zu unterscheiden war, beim Isolat Y waren RNA 3 und 4 in verkurzter Form aufgetreten, beim Isolat R fehlten sie vollstandig. Systemische Wurzelinfektionen wurden 7 Wochen p.i. bei ca. 20% der entweder mit Isolat P oder Y inokulierten Pflanzen festgestellt, aber nicht bei den mit Isolat R inokulierten. Die BNYW-ELISA-Werte blieben in den Wurzeln der mit Isolat P beimpften Pflanzen wahrend der gesamten einjahrigen Versuchsdauer hoch, in den Wurzeln der mit Isolat Y beimpften Pflanzen wurden sie dagegen immer niedriger, etwa 5 Monaten p.i. war das Virus in der Mehrzahl dieser Pflanzen nicht mehr nachweisbar. Die Rubenkorper dieser Pflanzen wurden etwa funf Mal groser als die mit Isolat P infizierten. Daraus ergibt sich, das BNYW mit intakter RNA 3 und 4 auch in Abwesenheit von Polymyxa betae zu einer schweren Schadigung der Ruben fuhren kann. Unsere Ergebnisse deuten auserdem darauf hin, das intakte RNA 3 und/oder 4 die Ausbreitung des Virus in der Rube erleichtern und nicht nur, wie kurzlich vermutet wurde, seine Ubertragung durch P. betae.

In attempts to use dot-blot hybridization tests for the identification of viruses or for assigning them to a certain taxonomic group we found that hybridization signals may be given not only by the homologous virus, but also by heterologous viruses belonging to the same or different taxonomic groups. Possible reasons for this phenomenon, which was observed with uncloned as well as with cloned cDNAs, are discussed. Quantitative dot-blot hybridization tests with extracted viral RNAs proved to be very sensitive in differentiating closely related viruses which were barely distinguishable in serological tests. Estimates on the degree of homology between the RNAs of different viruses may be influenced by a number of experimental parameters, such as competition for the available cDNA between homologous and heterologous RNAs or homologous RNAs in different concentrations on the same sheet of nitrocellulose, saturation phenomena due to close packaging of highly concentrated RNA on the blot and, of course, stringency conditions during washing procedures. Taking these parameters into account we have reestimated the degree of homology between the RNAs of 5 tombusviruses. Our new data suggest that the order of sequence for the relationships among these 5 tombusviruses is similar to that proposed by Koenig and Gibbs (1986) on the basis of serological data.

Hormonally regulated adenylate cyclase is a complex membrane-bound and vectorially oriented enzyme system of which at present three components are fairly well characterized: The hormone receptor, the guanylnucleotide binding subunit, and the catalytic moiety. The stoichiometry is assumed to be 1:1:1 but this is not proven (Fig. 1) (see Helmreich and Bakardjieva 1980). In the case of β-catecholamine-stimulated adenylate cyclase it became apparent from recent kinetic and biochemical evidence that activity is controlled by reversible interactions between the receptor, the guanylnucleotide binding protein, and the enzyme. Recent data which bear on these interactions are discussed here. Although the presently available information is quite persuasive, it is not yet conclusive.

The location of five SDS-stable epitopes on the coat protein (CP) of beet necrotic yellow vein virus was determined by reacting Escherichia coli-expressed free CP, as well as fusion proteins (FP) containing fragments of the CP, with polyclonal and monoclonal antibodies on Western blots. Epitope 1, which has previously been found to be exposed on only one extremity of the virus particle, was located in the region between amino acids (aa) 1 and 7, i.e. on the N terminus of the CP. It was blocked when the N terminus of the CP was linked to a portion of the beta-galactosidase sequence in an FP. Epitope 3, which has previously been found to be exposed on the opposite extremity of the particle, was located in the region between aa 37 and 59. Epitope 4, which is exposed along the entire length of the particle, occurs on the C terminus of CP (aa 183 to 188). Two previously unknown epitopes were identified in the regions between aa 115 and 125 and 125 and 140, respectively. The former was located on the same extremity of the particle as epitope 3, the latter became accessible only after denaturation of the particle. Nothing is known about the probably non-adjacent aa sequences that participate in the formation of the two SDS-labile epitopes (epitopes 2 and 5) which are found on one extremity and along the entire length of the particle, respectively.

Beet necrotic yellow vein virus (BNYVV)-infected sugarbeets were obtained from many parts of Europe and also from some sites in Asia and the U.S.A. Reverse transcription (RT)-PCR products of more than 1 kbp were obtained for four different regions of the viral genome which may be particularly important with respect to the pathogenic properties of the virus, i.e. for the coat protein and the 42K protein-encoding regions on RNA 2 and for major parts of RNAs 3 and 4. Restriction fragment length polymorphism (RFLP) patterns obtained with these PCR products revealed the existence of two major strain groups of BNYVV, named type A and type B. The A type was detected in Greece, the former Yugoslavia, Slovakia, parts of Austria, Italy, Spain, parts of France, Belgium, The Netherlands and England as well as in Asia (Turkey, Kazachstan, China and Japan) and the U.S.A. The B type occurs in Germany and parts of France. Mixed infections were detected at the borderline regions between areas of the A and B types. Comparisons of published and newly determined nucleotide sequences of the respective parts of the BNYVV genome indicate that the percentage of nucleotide differences between the A and the B type is approximately 3% for the respective regions of RNAs 2 and 3 and approximately 1·5% for RNA 4. Nucleotide sequences appear to be remarkably stable within each of the two strain groups. The majority of the nucleotide differences between the A and B types occur in the third triplet position. The amino acid changes in the coat protein area are outside the four previously determined antigenic regions that are accessible on the surface of the virus particles and are involved in the formation of continuous and presumably also discontinuous epitopes. This may explain why serological differences between the two strain groups have not been found.

By means of monoclonal antibodies (MAbs), five (groups of) epitopes were identified on particles of beet necrotic yellow vein virus (BNYVV). Epitopes 1 and 2, which were located on the opposite extremities of virus particles, are discontinuous (SDS-labile) epitopes which were destroyed when the particles were treated with trypsin. Epitope 3 is a continuous (SDS-stable) epitope located at the same extremity as epitope 2. It was not destroyed when the particles were treated with trypsin and was present on an Escherichia coli-expressed fusion protein containing amino acids (aa) 1 to 103 of the BNYVV coat protein. The continuous epitope 4, which was located along the entire length of the particles, was found to be present on a fusion protein containing aa 104 to 188 of the BNYVV coat protein but not on trypsin-treated virus particles. In Western blots, these treated particles yielded two slightly smaller coat proteins which failed to react with MAbs specific for epitope 4 but did react with polyclonal antisera and MAbs specific for epitope 3. BNYVV coat protein has a trypsin cleavage site on the carboxyl side of arginine in position 182, so it is therefore suggested that epitope 4 is located on the exposed C terminus, which is composed of aa 183 to 188. Epitope 5 was also located along the entire length of the particles but in a more uneven distribution than epitope 4. This may be because it is a discontinuous epitope that is very sensitive to subtle changes in protein conformation.