Verstärkung der antiphytoviralen Wirkung von 2,4-Dioxohexahydro-l,3,5-triazin durch Kombination mit Verbindungen mit Guanidinstruktur

Intensified antiphytoviral activity of 2,4-dioxohexahydro-l,3,5-triazine by combination with guanidines Combined application of the antiphytoviral compound 2,4-dioxohexahydro-l,3,5 triazine (DHT) and different guanidines (GDs) that were either unsubstituted or substituted only by low-molecular substituents reduced the concentration of potato virus χ (PVX) in leaves or Nicotiana tabacum L. cv. Samsun much more than application of either agent alone. In secondarily PVX-infected leaves, the activity of 2,4-dioxohexahydro- 1,3,5-triazine was increased by a larger number of GDs and to a greater degree than in inoculated ones. The activities of GDs against tobacco mosaic virus (TMV) in Nicotiana tabacum L. cv. Samsun as well as against brome grass mosaic virus (BRV) in Hordeum vulgare L. cv Vogelsanger Gold were only insignificantly increased by combination with DHT. On the other hand, in experiments with so-called identical potato eye cuttings, in which several eye cuttings were obtained from each potato tuber, one serving as a control and the others being treated with DHT, N-cyano guanidine or a combination of these substances, the number of cuttings with symptoms of potato leaf-roll virus (PLRV) could be much more greatly reduced by the combination than by the individual preparations. The number of cuttings with symptoms of potato virus Y and potato virus A was significantly reduced by treatment with the combined preparation, but not by treatment with DHT or N-cyano guanidine alone. Additional investigation with N-cyano-GD, and, beside this, with acetyl-GD and N N′ N triamino-GD indicated a close correlation between the diminution of numbers of potato eye cuttings with virus symptoms and the increase in tuber weight. The greater the reduction in the number of cuttings showing virus symptoms, the greater was the increase in tuber weight. These relationships were observed even in those cases where controls had been treated with ammonium nitrate solutions whose N contents equivalent to the N contents of the preparations. The observed effects of the preparations therefore are not attributable to N-fertilizing effects.     

Hemmung von Pflanzenviren durch Membranlipidanaloga unter besonderer Berücksichtigung von Alkanmonosulfonaten

Inhibition of plant viruses by membrane lipid analogs under special regard of alkane monosulfonates Under investigation was the inhibition of plant viruses by compounds, which could be comprehended as membrane lipid analogs because of some structural similarities to these lipids. Among these analogs, alkane monosulfonates (AMS, preparation “Emulgator E 30”) exhibit distinctly marked antiphytoviral activities, which are reported in detail. The mentioned AMS reduced the concentrations of PVX (potato virus X) as well as of TMV (tobacco mosaic virus) markedly in inoculated leaves of Nicotiana tabacum L. ‘Samsun’ and remarkably in secondarily infected ones. The formation of local lesions on leaves of hypersensitive hosts is influenced strongly by AMS. Likewise, the activity of the free virion is reduced in vitro. The mentioned effectsproved dependent on the length of the alkane chains and the concentration of AMS. Innertherapeutical effects of AMS have been pointed out. Several substances proved excellent coergists of AMS and vice versa, above all DHT (2,4-dioxohexahydro-1,3,5-triazine), several guanidines, a thiourea compound, a thiadiazole compound and ribavirin. In part full synergistic effects were obtained. AMS as well as combinations of AMS with some of the mentioned coergists also influence potato viroses. Using so-called identical potato eye cuttings, it was demonstrated that the mentioned treatments resulted in a significant and in part considerable reduction of the number of potato eye cutting plants with virus symptoms and in a partially high increase in the weight of tubers formed by the eye cutting plants. Likewise, the number of plants with virus symptoms was reduced and the tuber mass was increased by newly developed granulated AMS. A combined treatment with granulated AMS and granulated DHT resulted in a strong reduction of the number of potato eye cutting plants with virus symptoms, but the tuber mass was not augmented.     

Antiphytovirale Wirkungen von 1,5-Diacetyl-2,4-dioxohexahydro-1,3,5-triazin (DA-DHT)*

Antiphytoviral activities of 1,5-diacetyl-2,4-dioxohexahydro-1,3,5-triazine Substitution of 2,4-dioxohexahydro-1,3,5-triazine (DHT) by two acetyl groups resulted in an antiphytoviral compound, 1,5-diacetyl-2,4-dioxohexahydro-1,3,5-triazine (DA-DHT), which inhibits PVX, PVY, ToMV, TMV and TRV better than DHT, but only after, application at relatively high concentrations (10^?2 mol/1). The antiphytoviral activity of DA-DHT is enhanced by combined treatments with DHT as well as with cyanoguanidine, sodiumalkanemonosulfonate and 2-anilino-5-adamantyl-1,3,4-thiadiazole., DA-DHT reduced the number of symptoms of PLRV bearing potato eye cutting plants to a higher percentage than DHT. But the highest reduction was brought about by combined treatments with DA-DHT, cyanoguanidine, sodiumalkanemonosulfonate and 2-anilino-5-adamantyl-1,3,5-thiadiazole. Moreover, treatments with DA-DHT increased more than treatments with DHT the mass of potato tubers produced by potato eye cutting plants. Combined treatments with DA-DHT and the above mentioned substances reduced the natural infection with PLRV of a completely healthy potato stock for about 70 % and that of PVY for about 40 %. Thus, the DA-DHT containing preparations may be capable of keeping potatoes in a good state of health even in regions with a high infection pressure.     

The Mode of Inhibition of TMV- and PVX-Induced RNA-Dependent RNA Polymerases by some Antiphytoviral Drugs

Six nucleobase and nucleoside analogues (6-aminouracil [6-AU], 6-aminothymin [6-AT], 6-fluorothymin [6-FT], 2,4-dioxohexahydro-1,3,5-triazine [DHT], 9-(2,3-dihydroxypropyl) adenine [DHPA] and 1-β-D-ribofuranosyl-1,2,4-triazol-3-carboxamid [ribavirin]) with potential antiphytoviral activity were tested in an in vitro replication system for their effect on tobacco mosaic virus (TMV)-and potato virus X(PVX)-induced RNA dependent RNA polymerase. 6-AU, 6-AT, 6-FT, and DHPA inhibited both polymerases which may be a target of these compounds. A Putative noncompetitive mode of inhibition is discussed. Whereas ribavirin did not affect the in vitr RNA synthesis in the established systems, the uracil-derivative DHT inhibited only the PVX-specific enzyme.     

Nutzung von In-vitro-Pflanzen der Kartoffel zur Prüfung der antiphytoviralen Wirkung von 2,4-Dioxohexahydro-1,3,5-triazin (DHT) in Kombination mit N-Cyanoguanidin

Suitability of in vitro potato plantlets for testing of antiphytoviral effect of a combination of 2,4-dioxo-hexahydro-1,3,5-triazine (DHT) and N-cyano guanidine The suitability of a semiquantitative determination of virus content for the detection of antiphytoviral effects of chemicals was demonstrated by ELISA directly applying at in vitro potato cultures systemically infected and chemotherapeutically treated. The combination of 2, 4-dioxohexahydro-1,3,5-triazine (DHT) and N-cyanoguanidine both applied at a concentration of 0.03% to the culture medium resulted in a high significant reduction of the relative concentration of the potato virus S in the, potato genotype ‘M-812820’ of 44–73 %. A phytotoxic influence of the substances was not observed. The combination of the two antiphytoviral substances had no effect against potato virus Y in explants of the variety ‘Ackersegen’. Differences in the susc, eptibility of various genotypes against biologically active substances were indicated.     

Obtaining a High Percentage of Explants with Negative Serological Reactions against viruses by Combining Potato Meristem Culture with Antiphytoviral Chemotherapy

A high percentage of plantlets, in which PVX, PVM, and PVS were no longer detectable by precipitin tests was obtained from 100% virus-infected stocks of the potato varieties Rosa and Priobskij rannija by combining meristem (tip) culture with antiphytoviral chemotherapy. When 2,4 - dioxohexahydro 1,3,5-triazinc (DHT) was added to the nutrient media, no virus was detectable serologically in 72% of the corresponding explants of the variety Rosa and in 66% of that of the variety Priobskij rannija, respectively. Contrary to this, all untreated control explants proved virusinfected. Treatment with 2-thiouracil resulted in 46.6%, with cyanoguanidine in 28.6% of plantlets, in which no virus was detectable serologically.     

Bis-thiourea Metal Complexes with Enhanced Antiphytoviral Activity

The activity of bis-thiourea, diallyl-bis-thiourea and in part of diphenyl-bis-thiourea against potato virus X (PVX) can be augmented considerably by complexation of the mentioned compounds with Cu^±±, Ni^±±, and Co^±±. Contrary to this, complexation with Fe^±± and Cd^±± does not augment or reduce the antiphytoviral activity. Most antiphytoviral active are the diallyl-bis-thiourea complexes with Cu^±±, Ni^±± and Co^±±. The antiphytoviral activity of the Cu^±± and Cd^±± complexes with bisthiourea can be additionally augmented by combined treatment with small amounts of 2,4-dioxohexahydro-l,3,5-triazine (DHT).     

Beziehungen zwischen dem Wirkungsgrad antiphytoviraler Substanzen und dem Niveau der quantitativen Sortenresistenz sowie der Virulenz von Virusisolaten an Virus/Wirt-Systemen der Kartoffel

Relationship between the efficiency of antiphytoviral substances, the degree of quantitative resistance of cultivars, and the virulence of virus isolates on virus/host-systems of potato. In vitro systematically infected with potato virus S, potato virus M or potato virus X stem cuttings of different potato varieties were treated with 2,4-dioxohexahydro-1,3,5-triazine and Ribavirin. The reduction of virus replication by chemotherapy differed between varieties depending on their level of quantitative resistance and the virulence of virus isolates. An increasing resistance level of cultivars and a decreasing virulence of the virus isolates resulted in a relative enhancement of the inhibitory of the antiphytoviral substances.     

Effect of DHT (2,4-Dioxohexahydro-1,3,5-triazine) as Well as a Combination of DHT, Alkane Monosulfonate and N-Phenyl-N'-p-carboxyphenyl Thiourea on Tomato Mosaic Virus in Infected Glasshouse Tomato Plants

In two consecutive trials, three treatments of tomato plants of the variety Rivermoon with the antiphytoviral substance DHT (2,4-dioxohexahydro-1,3,5-triazine, 0.15%) 2 d a.i. and 2 and 7 d p.i. reduced the concentration of ToMV (tomato mosaic virus) by 62.3 and 60.5%. The average tomato yields increased by 40 and 26 % compared with the virus diseased, untreated controls, but the yields of the healthy controls were not achieved. A combined treatment of DHT, alkane monosulphonate and N-phenyl-N'-p-carboxyphenyl thiourea only resulted in a minor reduction in the ToMV titre and only a slight increase in yield, compared with the DHT-solo treatment.     

Genetically engineered resistance to potato virus X in four commercial potato cultivars

The genes for the capsid protein (CP) and the 8K movement protein of PVX were introduced into potato (Solanum tuberosum L.) and expressed under the control of CaMV 35S promoter using a binary vector andAgrobacterium tumefaciens. Four commercial potato cultivars (Russet Burbank, Shepody, Desirée and Bintje) have been efficiently transformed. Eleven independent transgenic clones, with CP expression levels higher than 0.05% of the soluble leaf proteins, were analyzed for resistance to inoculation with PVX (5 and 50µg/ml). The resistance of the transgenic plants to PVX was observed with the lower titer of virus inoculation (5 µg/ml) but not with higher titer (50 µg/ml). A significant reduction in the accumulation of virus in the inoculated transgenic potato plants has been observed under greenhouse and field conditions. Furthermore, the CP gene is very stable and is transferred to new plants originated from stem cuttings or from tubers. The transgenic plants appeared to be phenotypically identical to the nontransformed controls.Abbreviations BAP benzyl-aminopurine - BCIP 5-bromo-4-chloro-3-indolylphosphate p-Toluidine salt - CaMV cauliflower mosaic virus - CP capsid protein - GA₃ gibberellic acid - Kbp kilobase pair - NAA naphthalene acetic acid - NBT nitroblue tetrazolium chloride - NOS nopaline synthase - NPT II neomycin phosphotransferase II - PMSF phenyl methyl sulfonyl fluoride - PVX potato virus X - PVY potato virus Y     

Chemotherapeutical Elimination of Potato Virus X from Potato Stem Cuttings

Potato virus X was completely eliminated from all infected potato stem cuttings grown in nutrient media containing 0.02 or 0.03 % 2,4-dioxohexahydro-1,3,5-triazine (DHT). When DHT was added to the media in concentrations of 0.01 and 0.005% the efficiency by which the virus was eliminated differed between the varieties tested. The method is less time-consuming than the generally used meristem (axillary-) tip culture in combination with chemo- or thermotherapy.     

Complete Genome Sequence,Phylogenetic Relationships and Molecular Diagnosis of an Indian Isolate of Potato Virus X

The complete genome of a Potato virus X (PVX) isolate from India (ptDel‐9), which occurred symptomlessly in potato but induced ringspots on Nicotiana tabacum cv. Xanthi and necrotic mosaic on Nicotiana benthamiana, was sequenced. The genome was 6435 nucleotides long ( JF430080 ) and contained five open reading frames. The isolate was closely related to those reported from the Eurasian region (95.1–97.1% sequence similarity) and distantly related to those reported from South America (77.2–77.9%). The CP gene was expressed in Escherichia coli as a 76‐kDa fusion protein with maltose‐binding protein and used to generate polyclonal antibodies, which successfully detected PVX in field samples of potato by ELISA. In 20% of field samples, for which ELISA failed, the virus was successfully detected by RT‐PCR. This is the first report of molecular characterization of PVX occurring in India.     

Potato virus X-induced gene silencing in leaves and tubers of potato

Virus induced gene silencing (VIGS) is increasingly used to generate transient loss-of-function assays and has potential as a powerful reverse-genetics tool in functional genomic programs as a more rapid alternative to stable transformation. A previously described potato virus X (PVX) VIGS vector has been shown to trigger silencing in the permissive host Nicotiana benthamiana. This paper demonstrates that a PVX-based VIGS vector is also effective in triggering a VIGS response in both diploid and cultivated tetraploid Solanum species. We show that systemic silencing of a phytoene desaturase gene is observed and maintained throughout the foliar tissues of potato plants and was also observed in tubers. Here we report that VIGS can be triggered and sustained on in vitro micropropagated tetraploid potato for several cycles and on in vitro generated microtubers. This approach will facilitate large-scale functional analysis of potato expressed sequence tags and provide a noninvasive reverse-genetic approach to study mechanisms involved in tuber and microtuber development.     

Field and glasshouse experiments on the control of potato mop-top virus

Field observations during 3 yr on a stock of potato cv. Red Craigs Royal partially infected with potato mop-top virus (PMTV) confirmed that the virus was passed by an infected mother plant to only a proportion of its progeny tubers, and showed that in this cultivar symptomless plants gave rise only to symptomless progeny. The elimination of PMTV from stocks can therefore be greatly accelerated by removing symptom-bearing plants. Infected potato tubers were not freed from PMTV by treating them at 37 °C for up to 8 wk. Treating ‘seed’ tubers bearing powdery scabs that contain PMTV-carrying resting spores of Spongospora subterranea with formaldehyde or organo-mercurial fungicide greatly decreased PMTV establishment when the tubers were planted in previously uninfective soil, but fumigation with 2-aminobutane was ineffective. Decreasing the pH of infective soil to 5-0 by applying sulphur greatly decreased the infection of potato cv. Arran Pilot with PMTV and S. subterranea in field experiments, but this treatment did not eliminate either; when the pH of treated soil was raised the transmission of PMTV resumed. Treating infective soil with a range of fungicides greatly decreased the infection of Nicotiana debneyi bait seedlings in glasshouse experiments but only calomel at 75 kg/ha controlled spread of PMTV and 5. subterranea to potato in field experiments. In other field experiments, applying zinc frit, zinc sulphate or zinc oxide to infective soil greatly decreased the spread of both to potato. The amount of zinc required increased with increase in clay content of the soil. However, treatment with zinc compounds did not eliminate PMTV-carrying vectors from soil, and when treated soil was diluted with autoclaved soil many of the bait seedlings planted in the mixture became infected. The zinc frit was phytotoxic because of its boron content but zinc sulphate and zinc oxide caused little or no decrease in tuber yield. The zinc content of potato tubers was increased but not doubled in zinc-treated plots, and during the first year after treatment the zinc content of topsoil decreased greatly. The zinc content of ryegrass grown after potatoes was greater than of potato tubers but did not reach a level considered dangerous to livestock. Treatment of soil with sulphur, zinc oxide or calomel may be useful for small plots used in the early stages of propagation of virus-tested potato clones where there is risk of infection with PMTV.     

Determination of the races of potato virus X and its host rang in Karaj, Damavand and Ardabil, Iran

Potato virus x (PVX) is found commonly in potato-growing areas, worldwide. It is an economically important virus which causes losses in tuber yield of approximate 5 to 15 percent. In a 2 year survey, potato leaf and tuber samples were collected from various fields in Damavand and Karaj. The initial isolations from potato were made by mechanical inoculation first to Gomphrena globosa L. and later to Dartura stramonium L. It was not transmitted by 2 species of Cuscuta but transmitted mechamically. The isolates were inoculated to Nicotiana glutinosa L. in which they were maintained throughout the work. Sap from infected N. glutinosa was ineffective after dilution to 10-6, after 10 minutes at 70 degrees C and after 10 weeks at room temperature. The virus had filamentous and slightly flexuous particles with a normal length of about 490-500 nm and 12 nm width. According to the symptoms, TIP results and serological comparisons, the compared isolates showed no difference and they belong to XN group. In order to estimate disease incidence, 773 tubers from Damavand area were tested and compared with that in Ardabil area. Disease incidence in Damavand ranged from 1.1-20.9 percent and was lower than disease incidence in Ardabil. In 8 genera of collected weeds from fields of potato and tomato samples by using test plants and serological methods, they didn't show existence of the potato virus x.     

Characterization of Viruses Infecting Potato Plants from a Single Location in Shetland,an Isolated Scottish Archipelago

Sequence data were obtained from 29 isolates of Potato virus A (PVA), Potato virus S (PVS), Potato virus V (PVV) and Potato virus X (PVX) infecting nine tubers from Shetland, one of the most remote inhabited islands in the United Kingdom. These isolates were sequenced in the coat protein region, as were 29 Scottish mainland isolates of the same four potato virus species, and these 58 isolates were compared to previously published sequence data. This has allowed the characterization of viruses from a relatively isolated location, where there is little production of ware potatoes and no seed potato production. Phylogenetic homogeneity of the Shetland isolates of PVS and PVV was apparent. PVX was more heterogeneous, and Shetland isolates cluster with the Scottish isolates in a group which includes Asian and European isolates. For PVA, the majority of the Shetland and Scottish mainland isolates formed a predominantly Scottish grouping, with the remaining Shetland and Scottish mainland isolates clustering with a previously characterized Scottish isolate. There were three main groups of PVA, of which the Scottish grouping was the only one which did not have a fully characterized representative. To extend the characterization of PVA, the nucleotide sequence of the full polyprotein region encoding all the gene products of an isolate from Shetland was determined.     

Purification and Characterization of an Inducible s-Triazine Hydrolase from Rhodococcus corallinus NRRL B-15444R

The widespread use and relative persistence of s-triazine compounds such as atrazine and simazine have led to increasing concern about environmental contamination by these compounds. Few microbial isolates capable of transforming substituted s-triazines have been identified. Rhodococcus corallinus NRRL B-15444 has previously been shown to possess a hydrolase activity that is responsible for the dechlorination of the triazine compounds deethylsimazine (6-chloro-N-ethyl-1,3,5-triazine-2,4-diamine) (CEAT) and deethylatrazine (6-chloro-N-isopropyl-1,3,5-triazine-2,4-diamine) (CIAT). The enzyme responsible for this activity was purified and shown to be composed of four identical subunits of 54,000 Da. Kinetic experiments revealed that the purified enzyme is also capable of deaminating the structurally related s-triazine compounds melamine (2,4,6-triamino-1,3,5-triazine) (AAAT) and CAAT (2-chloro-4,6-diamino-1,3,5-triazine), as well as the pyrimidine compounds 2,4,6-triaminopyrimidine (AAAP) and 4-chloro-2,6-diaminopyrimidine (CAAP). The triazine herbicides atrazine and simazine inhibit the hydrolytic activities of the enzyme but are not substrates. Induction experiments demonstrate that triazine hydrolytic activity is inducible and that this activity rises approximately 20-fold during induction.     

The coat protein of potato virus X is a strain-specific elicitor of Rx1-mediated virus resistance in potato

The Rx1 gene in potato confers extreme resistance to potato virus X (PVX). To investigate the mechanism and elicitation of Rx resistance, protoplasts of potato cv. Cara (Rx1 genotype) and Maris Bard (rx1 genotype) were inoculated with PVX and tobacco mosaic virus (TMV). At 24 h post-inoculation in Maris Bard protoplasts there was at least 100-fold more PVX RNA than in protoplasts of Cara. TMV RNA accumulated to the same level in both types of protoplast. However, when the TMV was inoculated together with PVX the accumulation of TMV RNA was suppressed in the Cara (Rx1 genotype) protoplasts to the same extent as PVX. The Rx1 resistance also suppressed accumulation of a recombinant TMV in which the coat protein gene was replaced with the coat protein gene of PVX. It is therefore concluded that Rx1-mediated resistance is elicited by the PVX coat protein, independently of any other proteins encoded by PVX. The domain of the coat protein with elicitor activity was localized by deletion and mutation analysis to the structural core of a non-virion form of the coat protein.     

Transient expression of<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis> ascorbate peroxidase 3 in<Emphasis Type="Italic"> Nicotiana benthamiana</Emphasis> plants infected with recombinant potato virus X

We have explored the transient over-expression of Arabidopsis thaliana ascorbate peroxidase 3 (APX3) in Nicotiana benthamiana using a viral vector based on the potato virus X (PVX). Plants infected with a PVX:APX3 hybrid had a similar progression of viral particles compared to control plants infected with a PVX:GFP hybrid, indicating that infection was not affected by the over-expression of heterologous APX3. Our results also showed that in PVX:APX3-infected plants, the hybrid virus directed a high level of APX3 expression and the recombinant protein was functional, as inferred from the higher APX activity compared to mock and PVX:GFP hybrid-infected plants. The PVX recombinant expression system used is a simple and quick method for transient expression of heterologous APXs, which are expected to suffer specific processing in plant cells.     

Occurrence of potato virus X strain group 1 in seed stocks of potato cultivars lacking resistance genes

Potato virus X (PVX) isolates were obtained from a simple seed potato production scheme or from ware potatoes produced by seed potatoes obtained from it. In this scheme, PVX infection is widespread in seed stocks and most of the potatoes grown lack PVX resistance genes. Thirteen PVX isolates were typed to strain group by inoculation to potato cultivars containing different combinations of hypersensitivity genes Nx and Nb. Six failed to overcome either gene and therefore belonged to strain group 1, four overcame Nb only and were placed in strain group 3 and three were mixtures of the two. All 13 isolates failed to overcome extreme resistance/immunity gene Rx. Naturally infected cultivars of genotype nx.nb contained strain group 1 alone or strain groups 1 and 3, while those of genotype nx:Nb contained only strain group 3. The widespread occurrence of strain group 1 contrasts with the predominant occurrence of strain group 3 in potatoes in the UK. However, it resembles the UK situation before sophisticated seed potato production schemes were introduced and before PVX hypersensitivity genes Nx and Nb were deliberately exploited in potato breeding. Prior infection with potato leafroll virus (PLRV) did not affect expression of hypersensitivity to PVX in inoculated leaves of an nx:Nb genotype.