Double-stranded RNA replicons associated with chloroplasts of a green alga,Bryopsis cinicola

Double-stranded RNAs (dsRNAs) associated with chloroplasts and mitochondria have been found in the coenocytic green alga Bryopsis cinicola. In this study we report molecular properties of the four chloroplast-associated dsRNAs (BDRC1 to BDRC4) The longest dsRNA molecule (BDRC1) was sequenced entirely (1959 bp) and a single large ORF of 1722 bp was found within it. Database searches revealed similarities between the deduced amino acid sequence of this ORF and RNA-dependent RNA polymerase (RdRp) sequences from several RNA viruses. The most similar sequence in the database was the RdRp of beet cryptic virus 3. Phylogenetic analysis revealed that the RdRp-like sequence of BDRC1 can be placed in the Partitiviridae clade. To detect autonomous replication of these dsRNAs, RdRp assays were carried out with actinomycin D, which is an inhibitor of DNA-dependent RNA synthesis. Incorporation of [-³²P]UTP was detected specifically in the chloroplast and mitochondrial dsRNAs, indicating that both the chloroplast dsRNAs (BDRCs) and the mitochondrial dsRNA (BDRM) of B. cinicola are RNA replicons. The green alga B. cinicola harbors different dsRNA replicons in its chloroplasts and mitochondria.     

Characterization of Iranian Pectobacterium carotovorum Strains from Sugar Beet by Phenotypic Tests and Whole-cell Proteins Profile

Thirty isolates of Pectobacterium carotovorum from soft rot‐affected sugar beet plants in the Fars province of Iran were characterized phenotypically and by analysis of whole‐cell protein electrophoresis patterns. The isolates were found to be heterogeneous based on the results of physiological and biochemical tests and protein profiles. The results of numerical analysis of phenotypic characteristics and protein patterns showed that only 27% of the collected isolates (phenon 4) could be identified as P. betavasculorum when compared with reference strains. Strains of the first, second, third and fifth phenon shared similar characters with those of P. carotovorum subsp. carotovorum, P. betavasculorum and P. carotovorum subsp. odoriferum, but were distinct from these subspecies. Inoculation of phenon 4 isolates into wounded sugar beet petioles led to black streaking, root rot and vascular necrosis. Other isolates were incapable of causing systemic symptoms in inoculated plants.     

Development of <Emphasis Type="Italic">Cucumber mosaic virus</Emphasis> as a vector modifiable for different host species to produce therapeutic proteins

We have developed Cucumber mosaic virus (CMV) as a plant virus vector especially for production of pharmaceutical proteins. The CMV vector is a vector modifiable for different host plants and does not require further engineering steps. CMV contains three genomic RNA molecules (RNAs 1–3) necessary for infectivity. With this system, instead of creating different vector constructs for each plant we use, we take advantage of the formation of pseudrecombinants between two CMV isolates by simply reassembling a vector construct (RNA 2 base) and an RNA molecule containing the host determinant (mostly RNA 3). In this study, the gene for acidic fibroblast growth factor (aFGF), one of the human cytokines, was cloned under the control of the subgenomic promoter for RNA 4A of the CMV-based vector, C2-H1. Infected Nicotiana benthamiana plants produced aFGF at levels up to 5–8% of the total soluble protein. The tobacco-produced aFGF was purified, and its biological activity was confirmed. Using this system, which provides a versatile and viable strategy for the production of therapeutic proteins in plants, we also demonstrated a high level of aFGF in Glycine max (soybean) and Arabidopsis thaliana.     

Selection of nuclear polyhedrosis viruses as biological control agents ofSpodoptera exigua [Lep.: Noctuidae]

The virulence of 5 nuclear polyhedrosis viruses infectious for larvae of beet armyworm,Spodoptera exigua, was studied and their potential as biological control agents of this accidentally introduced pest in Dutch greenhouse crops is discussed. Three of the virus isolates were collected from deceased beet armyworm larvae found in Dutch greenhouses. Based on restriction endonuclease patterns of their DNA they appeared to be closely related toMamestra brassicae nuclear polyhedrosis virus (MbMNPV) and therefore were named MbMNPV-NL80, MbMNPV-NL82 and MbMNPV-NL83. These isolates were not related toAutographa californica MNPV (AcMNPV) or toSpodoptera exigua MNPV (SeMNPV), both originating from the USA. Comparison of the oiological activity of these 5 isolates showed that the SeMNPV was more virulent against beet armyworm than the other isolates. There was no significant difference in virulence between MbMNPV-NL80, NL82, NL83 and AcMNPV forS. exigua. The LD-50 values of the 5 isolates for 2nd instar larvae were 3, 26, 14, 17 and 18 polyhedra, respectively. Despite compensating qualities of the other MNPVs, such as a broader host range and potential production in alternate hosts or cell-lines, SeMNPV is considered to be the most suitable candidate as biological control agent of beet armyworm.      

Characterization of Fusarium oxysporum Isolates from Common Bean and Sugar Beet Using Pathogenicity Assays and Random-amplified Polymorphic DNA Markers

Fusarium wilt is an economically important fungal disease of common bean and sugar beet in the Central High Plains (CHP) region of the USA, with yield losses approaching 30% under appropriate environmental conditions. The objective of this study was to characterize genetic diversity and pathogenicity of isolates of Fusarium oxysporum obtained from common bean and sugar beet plants in the CHP that exhibited Fusarium wilt symptoms. A total of 166 isolates of F. oxysporum isolated from diseased common bean plants were screened for pathogenicity on the universal susceptible common bean cultivar ‘UI 114’. Only four of 166 isolates were pathogenic and were designated F. oxysporum f.sp. phaseoli (Fop). A set of 34 isolates, including pathogenic Fop, F. oxysporum f.sp. betae (Fob) isolates pathogenic on sugar beet, and non‐pathogenic (Fo) isolates, were selected for random‐amplified polymorphic DNA (RAPD) analysis. A total of 12 RAPD primers, which generated 105 polymorphic bands, were used to construct an unweighted paired group method with arithmetic averages dendrogram based on Jaccard's coefficient of similarity. All CHP Fop isolates had identical RAPD banding patterns, suggesting low genetic diversity for Fop in this region. CHP Fob isolates showed a greater degree of diversity, but in general clustered together in a grouping distinct from Fop isolates. As RAPD markers revealed such a high level of genetic diversity across all isolates examined, we conclude that RAPD markers had only limited usefulness in correlating pathogenicity among the isolates and races in this study.     

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.     

The effectiveness of commercial antimicrobial compounds against saccharolytic microorganisms isolated from a beet sugar production line

The antimicrobial activities of five commercial disinfectants containing quaternary ammonium compound-isopropanol (D1), sodium methyl dithiocarbamate (D2), sodium thiocarbamate (D3), sodium dimethyl dithiocarbamate (D4) and formaldehyde (D5) were studied against three main saccharolytic indigenous isolates (Bacillus cereus, Lactobacillus plantarum and Leuconostoc mesenteroides) from a beet sugar extraction line. Preliminary studies suggested that although all the disinfectants were effective against those isolates, the high economic cost in combination with large amounts of the disinfectants D2, D3 and D4 weaken their possibility for industrial use. Therefore, the minimum inhibitory concentration (MIC) of the other two examined disinfectants D1 and D5 was determined and survivor curves were obtained, for a period of 7 days. Bacterial counts against time (h) suggested that D1 was more effective than D5 against the microbial population. In particular, D1 was bacteriolytic above 7 mg/l for B. cereus and bactericidal above 80 mg/l for Lc. mesenteroides and above 100 mg/l for L. plantarum. The disinfectant D5 was bacteriolytic above 25 mg/l for B. cereus and bactericidal above 500 mg/l for Lc. mesenteroides and L. plantarum. Taking into consideration both features, i.e. high concentration and very low cost, the use of D5 (formaldehyde) appeared more suitable to the concerned beet sugar processor. This revised version was published online in August 2006 with corrections to the Cover Date.     

Long‐term preservation of a collection of Rhizoctonia solani using cryogenic storage

Rhizoctonia solani is an important plant pathogen for a number of crops and maintaining an extensive collection of reference isolates is important in understanding relationships of this pathogen with multiple hosts. Current long‐term storage methods typically call for frequent transfer increasing the risk of changes in morphological, physiological or virulence characteristics. Cryopreservation using storage in liquid nitrogen (LN) was evaluated to examine the potential for storage of a R. solani culture collection containing 106 isolates (primarily from sugar beet). Cultures were stored on autoclaved barley grains in the vapour phase of LN. After 60 days, 5 years and 10 years in storage, all isolates were tested for viability by calculating the percentage of barley grains from which R. solani mycelia grew. Five years after initial storage, all isolates except one had no change in viability. After 10 years in storage, 67 of 106 isolates had no significant decrease in viability, 39 of 106 isolates had a significant decrease in viability but only 9 isolates had less than 10% growth, with 4 having no growth. A subset of isolates stored for 10 years were tested for pathogenicity on a susceptible (FC901) and resistant (FC703) sugar beet germplasm. All isolates tested maintained approximately the same level of virulence that they had prior to storage on both germplasms. This indicates that cryogenic methods are suitable for the preservation or storage of R. solani culture collections, although efficacy may vary with individual isolates.     

Axoplasmic RNA species synthesized in the isolated squid giant axon

Isolated squid stellate nerves and giant fiber lobes were incubated for 8 hr in Millipore filtered sea water containing [³H]uridine. The electrophoretic patterns of radioactive RNA purified from the axoplasm of the giant axon and from the giant fiber lobe (cell bodies of the giant axon) demonstrated the presence of RNA species with mobilities corresponding to tRNA and rRNA. The presence of labeled rRNAs was confirmed by the behavior of the large rRNA component (31S) which, in the squid, readily dissociates into its two constituent moyeties (17S and 20S). Comparable results were obtained with the axonal sheath and the stellate nerve. In all the electrophoretic patterns, additional species of radioactive RNA migrated between the 4S and the 20S markers, i.e. with mobilities corresponding to presumptive mRNAs. Chromatographic analysis of the purified RNAs on oligo(dT)cellulose indicated the presence of labeled poly(A)⁺ RNA in all tissue samples. Radioactive poly(A)⁺ RNA represented approximately 1% of the total labeled RNA in the axoplasm, axonal sheath and stellate nerve, but more than 2% in the giant fiber lobe. The labeled poly(A)⁺ RNAs of the giant fibre lobe showed a prevalence of larger species in comparison to the axonal sheath and stellate nerve. In conclusion, the axoplasmic RNAs synthesized by the isolated squid giant axon appear to include all the major classes of axoplasmic RNAs, that is rRNA, tRNA and mRNA.Special Issue dedicated to Prof. Holger Hydén.     

Selection of natural isolates of Trichoderma spp. for biocontrol of Polymyxa betae as a vector of virus causing rhizomania in sugar beet

The soil fungus Polymyxa betae, Keskin, besides being a root parasite, plays a role of a vector in dissemination of Beet necrotic yellow vein virus (BNYVV) causing rhizomania in sugar beet. An alternative to its chemical control is the application of antagonistic microorganisms suppressing proliferation of the fungal vector. In the present work, 66 Trichoderma isolates have been obtained from sugar beet plantations from diverse locations in Slovakia. The ability of the selected isolates to grow at low temperature (10 °C) and to suppress the colonization of roots with P. betae and the multiplication of BNYVV in roots under glasshouse conditions were tested. The roots of sugar beet seedlings growing in the BNYVV-infested soil were analyzed by serological ELISA test using monoclonal and polyclonal antibodies for the presence of BNYVV and checked microscopically for the occurrence of cystosori of P. betae. The efficacy of the selected strains to suppress the proliferation of BNYVV varied on the average between 21 and 68%. On the basis of these tests, candidate strains for practical application in biocontrol of sugar beet rhizomania were selected.     

Biocontrol of Rhizoctonia crown and root rot of sugar beet by binucleate Rhizoctonia spp. and Laetisaria arvalis

Two isolates of Laetisaria arvalis and 10 of binucleate Rhizoctonia spp. (BNR) from the Ohio sugar beet production area, were tested in the greenhouse and field for biocontrol of Rhizoctonia crown and root rot of sugar beet, caused by Rhizoctonia solani anastomosis group 2, type 2. L. arvalis was ineffective in standard greenhouse tests, and the single isolate used in the field was generally ineffective. Seven of 10 BNR isolates effectively controlled crown and root rot in greenhouse tests. Delayed application of biocontrol agents to plants 5 – 10 wk old was generally more effective than applications made at planting. A BNR isolate significantly reduced % plant loss and disease ratings and increased yield in a 1985 field test as compared with the control infested with R. solani alone. Two BNR isolates were effective in a 1986 field test and increased yields c. 22% in comparison to a L. arvalis treatment, which did not differ from the R. solani-infested control. The Ohio binucleate Rhizoctonia isolates appear to have considerable potential as applied biocontrol agents and may play a role in the natural ecology of R. solani in the sugar beet production area of Ohio.     

Purification and Some Properties of an Isolate of Beet Yellows Virus from Ukraine

A purification procedure, which yielded up to 15–30 mg of beet yellows virus (BYV) per 100 g of infected Tetragonia expansa leaves, has been developed. The procedure included sap clarification with Triton X-100, and two cycles of ultracentrifugation through sucrose cushion, which contained PEG-6000 and NaCl. A specific antiserum was prepared, and BYV infection was successfully detected by the double-antibody sandwich (DAS) ELISA in infected sugar beet leaves and roots diluted up to 1 × 10⁵ and 1 × 10⁴, respectively. The virus concentration was demonstrated to decrease in infected sugar beet roots slowly during 7 months, thus allowing successful diagnosis of planting material in winter storage. BYV presence in Myzus persicae aphids was also reliably detectable using the DAS-ELISA. In a competitive DAS-ELISA test, the Ukraine and the British BYV isolates were found serologically indistinguishable.     

Preferential spreading of RNA silencing into the 3’ downstream region of the transgene in tobacco

The amplification mechanism of short interfering RNAs (siRNAs) along the transgene sequence exists in RNA interference (RNAi). The RNA-dependent RNA polymerase synthesizes complementary RNAs by using the transgene mRNA as a template, and the secondary siRNAs are generated from the outside of primary RNAi target. Four independent RNAi vectors which produced primary siRNAs against distinct regions of the tobacco endoplasmic reticulum ω-3 fatty acid desaturase gene (NtFAD3) were transiently expressed in leaves of theNtF4D3-overexpressed transgenic plants. Regardless of the RNAi vector used, the secondaryNtFAD3 siRNAs were generated preferentially from the 3’ downstream region of the transgene. Secondary siRNAs from the 5’ upstream region adjacent to the annealing site of primary siRNAs accumulated under the detection level. Our results suggest that different regulatory mechanisms are involved in the spreading of RNA silencing into 5’ upstream and 3’ downstream regions of the target sequence, respectively.     

Suppression of sugar beet damping-off caused by Rhizoctonia solani using bacterial and fungal antagonists

Rhizoctonia damping-off caused by Rhizoctonia solani Kühn, is one of the most damaging sugar beet diseases. It causes serious economic damage wherever sugar beets are grown. Biological control is an efficient and environmentally friendly way to prevent damping-off disease. Suppression of damping-off disease caused by R. solani was carried out by four isolates of Bacillus subtilis (Ehrenberg) Cohn as well as three isolates of each of Trichoderma harzianum Rifai and Trichoderma hamatum (Bonord.) Bainier. The effect of Bacillus and Trichoderma isolates against R. solani was investigated in vitro and tested on sugar beet plants under greenhouse conditions. Isolates of Bacillus and Trichoderma were able to inhibit the growth of R. solani in dual culture. Furthermore, Trichoderma isolates gave high antagonistic effect than isolates of B. subtilis. Under greenhouse conditions, coating seeds by T. harzianum and B. subtilis separately, reduced seedling damping-off significantly. However, applications of T. harzianum increased the percentage of surviving plants more than B. subtilis in comparison to control. The obtained results indicate that T. harzianum and B. subtilis are very effective biocontrol agents that offer potential benefit in sugar beet damping-off and should be harnessed for further biocontrol applications.     

Rapid vascular movement of tobraviruses does not require coat protein: evidence from mutated and wild-type viruses

Early studies of the tobravirus Tobacco rattle virus (TRV) described two types of virus isolate with apparently different disease characteristics. M‐type isolates, which contain both viral genomic RNAs and form virus particles, could be passaged by mechanical inoculation and produced rapid but shortlived systemic symptoms. In contrast, NM‐type isolates, which contain only RNA1 and do not form virus particles, were difficult to passage by mechanical inoculation and were very slow to produce systemic symptoms. From the early observations on such isolates made in the 1960s, it has become accepted that M isolates with encapsidated TRV particles move rapidly through the vascular system whereas NM isolates containing only unencapsidated TRV RNA1 move only slowly via plasmodesmata from cell to cell and take many weeks to reach the upper parts of plants. However, we show that NM isolates of TRV and another tobravirus Pea early‐browning virus (PEBV) move into systemic tissue of TV. benthamiana and N. clevelandii by 6 days post inoculation, suggesting that this rapid movement occurs via the vasculature. The systemic movement of TRV and PEBV mutants lacking functional coat protein that have been modified to express the green fluorescent protein were examined by confocal microscopy. This confirmed that the tobraviruses do not require the CP for long distance movement via the phloem, a property that is shared with only a small group of plant viruses.