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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.

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.

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.

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.

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.