The evaluation of rhizomania resistant sugar beet for the UK

Sugar beet rhizomania disease, caused by Beet necrotic yellow vein virus and transmitted by the soil‐borne parasite Polymyxa betae, was first recorded in the UK in 1987. Recently, breeding lines and cultivars with partial resistance to the virus derived from the ‘Holly’ source of resistance have been developed and their suitability for use under UK conditions is explored in this paper. Virus multiplication in the roots of resistant lines exposed to severe disease pressure in glasshouse tests, when quantified by ELISA, was less than one third of that in susceptible controls. More recently developed resistant lines had a lower virus content, on average, largely due to a reduced frequency of susceptible individuals. There was no evidence for resistance to the vector, P. betae, in virus resistant lines. However, the proportion of viruliferous P. betae resting spores in the roots, estimated using the most probable number (MPN) technique, was reduced by at least one third in resistant lines compared with the most susceptible control. A novel line, containing an additional gene to that in ‘Holly’, was the most effective, reducing the infection level to 3% of that in the susceptible control. In two field experiments on severely infested sites, the rate of infection of a resistant line, when assessed by ELISA, was reduced by half compared with a susceptible cultivar and sugar yields of resistant lines were consistently 2–3 times higher than those of susceptible cultivars. In 41 trials on rhizomania‐free sites, several recently introduced resistant lines exhibited sugar yields and agronomic performance comparable to that of three selected high yielding, susceptible cultivars. Results are discussed in relation to the specific UK requirements for rhizomania resistant cultivars. One resistant line, Beta 805 (cv. Concept), fulfilled the requirements for widespread use to control the disease.     

Evaluation of Trichoderma spp. from central and northern regions of Turkey for suppression of Polymyxa betae as a vector of rhizomania disease

In the current study, 18 Trichoderma spp. isolates were obtained from different provinces in central and northern regions of Turkey. The ability of nine selected isolates to suppress the colonisation of roots by P. betae and the multiplication of BNYVV in sugar beet roots under controlled conditions were tested. Roots of seedlings growing in the P. betae-BNYVV-infested soil were analysed by enzyme-linked immunosorbent assay to test for the presence of BNYVV and checked microscopically for the density of cystosori of P. betae. The numbers of P. betae resting spores in cystosori for each treatment were counted using a light microscope. Except for isolates Tr-1 and Tr-5, the effect of selected Trichoderma isolates on suppressing multiplication of BNYVV varied between 4 and 53%. The total number of resting spores in the roots varied between 14.4 and 25.1 for the different Trichoderma spp. treatments. The lowest number of resting spores in clusters was recorded in T. harzianum Tr-8. In addition, the shapes of resting spores were not normal in the Tr-8 treatments. The cystosori from this treatment were also abnormally dark in colour and had deformed walls.     

Rapid screening for dominant negative mutations in the beet necrotic yellow vein virus triple gene block proteins P13 and P15 using a viral replicon

Point mutations were introduced into the genes encoding the triple gene bock movement proteins P13 and P15 of beet necrotic yellow vein virus (BNYVV). Mutations which disabled viral cell-to-cell movement in Chenopodium quinoa were then tested for their ability to act as dominant negative inhibiters of movement of wild-type BNYVV when expressed from a co-inoculated BNYVV RNA 3-based replicon. For P13, three types of mutation inhibited the movement function: non-synomynous mutations in the N- and C-terminal hydrophobic domains, a mutation at the boundary between the N-terminal hydrophobic domain and the central hydrophilic domain (mutant P13-A12), and mutations in the conserved sequence motif in the central hydrophilic domain. However, only the boundary mutant P13-A12 strongly inhibited movement of wild-type virus when expressed from the co-inoculated replicon. Similar experiments with P15 detected four movement-defective mutants which strongly inhibited cell-to-cell movement of wild-type BNYVV when the mutants were expressed from a co-inoculated replicon. Beta vulgaris transformed with two of these P15 mutants were highly resistant to fungus-mediated infection with BNYVV.     

Spread of beet necrotic yellow vein virus in infected seedlings and plants of sugar beet (Beta vulgaris)

Summary Infection of sugar beet roots by beet necrotic yellow vein virus (BNYVV) was investigated with transmission electron microscopy, immunogold labelling and enzyme linked immuno sorbent assay (ELISA). Here we show that infection of sugar beet roots is very fast, occurring during germination. Seedlings grown directly in infected soil showed higher BNYVV infection than plants transplanted into infected soil after seven days of initial growth in sterilized soil. The earlier the initial infection, the faster was its spread. The study showed that a few differentiated cells of the cortex and of the xylem parenchyma were the preferred sites of viral multiplication. The spread of viral infection was slow through differentiated tissues. Intact virions were frequently found in undifferentiated and mature vessel elements and xylem parenchyma, whereas they were rare in sieve elements. Virus particle number in the differentiating tracheary elements was high, suggesting that infection of the vessel elements preceded their differentiation. This would explain increased infection after early inoculation. Even the xylem tissue of the primary root was highly infected, the seedlings lacked virus particles in their hypocotyls and leaves.     

Strategies for the Detection of Potential Beet necrotic yellow vein virus Genome Recombinations which might Arise as a Result of Growing A type Coat Protein Gene-expressing Sugarbeets in Soil Containing B Type Virus

We have searched for beet necrotic yellow vein virus (BNYVV) populations with a recombined genome which could possibly arise when transgenic sugarbeets expressing the coat protein gene of A type BNYVV are grown in soil containing Polymyxa betae carrying B type BNYVV, in soil samples from previous field release experiments and in a greenhouse model experiment. In order to accelerate the potential evolution of virus populations with recombined genomes in the model experiment, eight successive crops of sugarbeet plantlets were grown in the same soil samples over a period of 3 years. For the sensitive detection of recombined BNYVV genomes, we used nested PCRs with sense primers that are preferentially extended on the A type BNYVV sequence in the region of the coat protein gene and antisense primers which are preferentially extended on the B type BNYVV sequence in a region downstream of the coat protein gene which is not present in the transgene. Controls with mixtures of sap from plants which were singly infected with A or with B type BNYVV only revealed that, unless proper precautions are taken, PCR-mediated recombination artifacts may readily be produced. A method was developed that is able to detect A type/B type recombinant RNA molecules up to dilutions of one to a million in pure B type RNA molecules. Inspite of this high sensitivity we failed to detect any BNYVV with a recombined genome in the transgenic plants of the model experiment or at the sites of the previous field release experiments.     

<Emphasis Type="Italic">Trichoderma</Emphasis> as a potential biocontrol agent for Cercospora leaf spot of sugar beet

Leaf spot disease caused by Cercospora beticola Sacc. (class Ascomycota, ord. Dothideales, fam. Mycosphaerellaceae) is the most destructive foliar disease of sugar beet. Commercial varieties are partially resistant and require repeated fungicide applications to obtain adequate protection levels; this has a high environmental impact and a risk of selecting resistant pathogen strains. A way of reducing chemical inputs could be to use biocontrol agents to replace or supplement fungicide treatments. A well-known class of biological control agents is represented by the fungi belonging to the Trichoderma genus (class Ascomycota, ord. Hypocreales, fam. Hypocreaceae), but there is a lack of information about its behaviour towards C. beticola. This study reports the evaluation of several Trichoderma isolates as possible biocontrol agents of this pathogen. Preliminary in vitro and in vivo assays led to the selection of two Trichoderma isolates characterised by their ability to reduce pathogen sporulation and antagonism towards the pathogen or competence for sugar beet phyllosphere. Repeated foliar applications of the liquid culture homogenate preceded by a single treatment of difenoconazole in 2 year trials under natural inoculum in field reduced the disease incidence and pathogen sporulation from the necrotic spots. An increase in sugar yield was also obtained by means of isolate Ba12/86-based treatments, perhaps due to induced resistance effects.     

Inheritance of resistance to beet necrotic yellow vein virus in Beta vulgaris conferred by a second gene for resistance

Rhizomania is a serious disease of sugar beet, caused by beet necrotic yellow vein virus (BNYVV). The disease can only be controlled by the use of resistant cultivars. The accession Holly contains a single dominant gene for resistance, called Rz. The identification of a locus for resistance that differs from Rz would provide possibilities to produce cultivars with multiple resistance to BNYVV. Inheritance of resistance to BNYVV was studied by screening progenies of crosses between resistant plants of the accessions Beta vulgaris subsp. maritima WB42 and B. vulgaris subsp. vulgaris Holly-1–4 or R104. Observed and expected segregation ratios were compared to elucidate whether the resistance genes in the three accessions are alleles or situated on different loci. STS markers, linked to the genes for resistance, were used to study the segregation in more detail. The results demonstrated that the genes for resistance to BNYVV inHolly-1-4 and WB42 are closely linked. The gene for resistance in R104 is at the same locus as in Holly-1-4, and also closely linked to the gene in WB42. As the Holly resistance gene has been named Rz, the name Rz2 is proposed to refer to the resistance gene in WB42. Consequently, the gene Rz should be referred to as Rz1. Received: 29 October 1998 / Accepted: 12 March 1999     

dsRNA-mediated resistance to Beet Necrotic Yellow Vein Virus infections in sugar beet (Beta vulgaris L. ssp. vulgaris)

Rhizomania, one of the most devastating diseases in sugar beet, is caused by Beet Necrotic Yellow Vein Virus (BNYVV) belonging to the genus Benyvirus. Use of sugar beet varieties with resistance to BNYVV is generally considered as the only way to maintain a profitable yield on rhizomania-infested fields. As an alternative to natural resistance, we explored the transgenic expression of viral dsRNA for engineering resistance to rhizomania. Transgenic plants expressing an inverted repeat of a 0.4 kb fragment derived from the BNYVV replicase gene displayed high levels of resistance against different genetic strains of BNYVV when inoculated using the natural vector, Polymyxa betae. The resistance was maintained under high infection pressures and over prolonged growing periods in the greenhouse as well as in the field. Resistant plants accumulated extremely low amounts of transgene mRNA and high amounts of the corresponding siRNA in the roots, illustrative of RNA silencing as the underlying mechanism. The transgenic resistance compared very favourably to natural sources of resistance to rhizomania and thus offers an attractive alternative for breeding resistant sugar beet varieties.     

Expression of single-chain antibody fragments (scFv) specific for beet necrotic yellow vein virus coat protein or 25 kDa protein in Escherichia coli and Nicotiana benthamiana

The coding sequences for the variable regions of heavy and light chains of monoclonal antibodies (mAbs) to beet necrotic yellow vein virus (BNYVV) coat protein (cp) or the 25 kDa nonstructural protein (P25) were cloned into the pCOCK vector and expressed as single-chain antibody fragments (scFv) in Escherichia coli. For expression in higher plants the scFv were targeted either to the secretory pathway by including the sequences encoding the pectate lyase B (PelB) or the phytohemagglutinin (PHA) signal peptides in the vector constructs or they were targeted to the cytoplasm by omitting a signal peptide-encoding sequence from the constructs. The scFv were detected mainly in plants in which the PHA signal peptide had been used for targeting demonstrating for the first time the usefulness of this peptide for enabling scFv expression in plants. The scFv were not secreted into the culture fluids of suspension cultures, but were retained in the cells. The amount of expression of scFv in the best expressing plants was at least as high as in bacterial culture supernatants. In a dot blot immunoassay, 0.4 ng BNYVV cp or 0.8 ng P25 were detected by the respective scFv either from E. coli or from plants. The majority of the 21 plants expressing cp-specific scFv had near-normal growth whereas the three plants expressing P25-specific scFv grew poorly and did not form roots.     

Significance of lytic enzymes from Trichoderma spp. in the biocontrol of fungal plant pathogens

The use of specific mycolytic soil microorganisms to control plant pathogens is an ecological approach to overcome the problems caused by standard chemical methods of plant protection. The ability to produce lytic enzymes is a widely distributed property of rhizosphere-competent fungi and bacteria. Due to the higher activity of Trichoderma spp. lytic enzymes as compared to the same class of enzymes from other microorganisms and plants, effort is being aimed at improving biocontrol agents and plants by introducing Trichoderma genes via genetic manipulations. An overview is presented of the data currently available on lytic enzymes from the mycoparasitic fungus Trichoderma. This revised version was published online in August 2006 with corrections to the Cover Date.     

Evidences of biological control capacities of Streptomyces spp. against Sclerotium rolfsii responsible for damping-off disease in sugar beet (Beta vulgaris L.)

Ten antibiotic-producing Streptomyces spp. isolated from Moroccan soils were evaluated for their ability to inhibit in vitro Sclerotium rolfsii development. Four isolates having the greatest pathogen inhibitory capabilities were subsequently tested for their ability to inhibit sclerotial germination in sterile soil. This test was carried out by using biomass inoculum, culture filtrate, and spore suspension of the isolates as treatment. Treatment with biomass inoculum and culture filtrate gave the highest inhibition of sclerotia. Biological control tests against Sclerotium rolfsii damping-off of sugar beet seeds showed that the selected Streptomyces isolates reduced significantly the disease severity, the J-2 isolate being the more potent. In addition, treatment with the isolate J-2 resulted in a significant increase (P ≤ 0.05) in seedling development compared to the control. All antagonistic Streptomyces selected here were able to grow in the rhizosphere soil from infected sugar beet culture.     

The in vitro screening of 118 Trichoderma isolates for antagonism to Rhizoctonia solani and an evaluation of different environmental sites of Trichoderma as sources of aggressive strains

Trichoderma isolates were collected from different sources and screened for their in vitro parasitism of Rhizoctonia solani. They were grouped according to the different sources and each group compared statistically. 74% of the total isolates collected were regarded as antagonistic to R. solani in vitro. Isolates associated with pine bark source were very aggressive. The most aggressive strains were isolated from soil samples collected under the Speedling^® trays of a commercial seedling nursery.     

Agrotis segetum granulosis virus as a control agent against field populations ofAgrotis ipsilon andA. Segetum [Lep.: Noctuidae] on tobacco,okra, potato and sugar beet in northern pakistan

Agrotis segetum Schiff granulosis virus (AsGV) propagated in Denmark was supplied against naturally occurring cutworm populations (A. ipsilon and to a less extentA. segetum) in experimental field plots of tobacco, okra, potato and sugar beet in northern Pakistan. AsGV doses varied between 4 × 10⁷ and 4 × 10¹¹ capsules per m² plot, and no. of applications between 1 and 3. One treatment with AsGV did not reduce cutworm damage significantly to tobacco seedlings and potato plants. Two treatments with AsGV reduced cutworm damage significantly. In tobacco, reduction was 64–82%, in okra and potato 85% and 77% respectively. Damage in sugar beet was reduced 78%. Three treatments with AsGV dis not reduce damage significantly better than two treatments. AsGV and the chemical insecticides Tamaran and Dieldrin, andBacillus thuringiensis (Thuricide) were about equally effective, reducing damage by 85%, 79%, 87% and 69%, respectively. No difference was found between the efficiency of highly purified AsGV to which activated charcoal was added and partially purified AsGV without charcoal.      

Analysis of DNA from a Beta procumbens chromosome fragment in sugar beet carrying a gene for nematode resistance

Summary We have begun to apply techniques for the preparation and anaylsis of large DNA segments from sugar beet (Beta vulgaris) addition lines carrying a mitotically stable chromosome fragment from B. procumbens that confers monogenic resistance to the nematode Heterodera schachtii, with a view towards isolating the resistance gene. DNA probes specific for this chromosome fragment were selected, and various methods for cloning genome-specific fragments, including probes from megabase DNA separated in pulsed-field slab gels, are compared. Probes that display high homology to B. procumbens have been used for hybridization of a representative genomic library and for initial step in mapping the chromosome fragment via pulsed-field gel electrophoresis after restriction with infrequently cutting enzymes. Our data indicate that DNA molecules from the entire chomosome fragment can be separated from protoplast DNA lysates.     

Use of the GFP reporter as a vital marker for Agrobacterium-mediated transformation of sugar beet (Beta vulgaris L.)

Molecular approaches to sugar beet improvement will benefit from an efficient transformation procedure that does not rely upon exploitation of selectable marker genes such as those which confer antibiotic or herbicide resistance upon the transgenic plants. The expression of the green fluorescent protein (GFP) signal has been investigated during a program of research that was designed to address the need to increase the speed and efficiency of selection of sugar beet transformants. It was envisaged that the GFP reporter could be used initially as a supplement to current selection regimes in order to help eliminate “escapes” and perhaps eventually as a replacement marker in order to avoid the public disquiet associated with antibiotic/herbicide-resistance genes in field-released crops. The sgfp-S65T gene has been modified to have a plant-compatible codon usage, and a serine to threonine mutation at position 65 for enhanced fluorescence under blue light. This gene, under the control of the CaMV 35S promoter, was introduced into sugar beet via Agrobacterium-mediated transformation. Early gene expression in cocultivated sugar beet cultures was signified by green fluorescence several days after cocultivation. Stably transformed calli, which showed green fluorescence at a range of densities, were obtained at frequencies of 3–11% after transferring the inoculated cultures to selection media. Cocultivated shoot explants or embryogenic calli were regularly monitored under the microscope with blue light when they were transferred to media without selective agents. Green fluorescent shoots were obtained at frequencies of 2–5%. It was concluded that the sgfp-S65T gene can be used as a vital marker for noninvasive screening of cells and shoots for transformation, and that it has potential for the development of selectable marker-free transgenic sugar beet.     

Effect of <Emphasis Type="Italic">Trichoderma</Emphasis> spp. isolates for biological control of tan spot of wheat caused by <Emphasis Type="Italic">Pyrenophora tritici-repentis</Emphasis> under field conditions in Argentina

Trichoderma spp. have been used as biocontrol agents to protect plants against foliar diseases in several crops, but information from field assays is scarce. In the present work, experiments were carried out to determine the effect of six isolates of Trichoderma harzianum and one isolate of T. koningii on the incidence and severity of tan spot, caused by Pyrenophora tritici-repentis (anamorph: Drechslera tritici-repentis) under field conditions. Significant differences between years, wheat cultivars and treatments were found. In 2003, two of the isolates assayed (T5, T7) showed the best performance against the disease applied as seed treatments or sprayed onto wheat leaves at different stages. The application of six of the treatments on wheat plants significantly reduced disease severity by 16 to 35% in comparison with the control. Disease control provided by isolate T7 was similar to that provided by the fungicide treatment (56% reduction). This is the first report on the efficacy of Trichoderma spp. against tan spot under field conditions in Argentina.     

Inheritance of resistance to potyviruses in Phaseolus vulgaris L. II. Linkage relations and utility of a dominant gene for lethal systemic necrosis to soybean mosaic virus

Summary A single dominant factor, Hss, that conditions a rapid lethal necrotic response to soybean mosaic virus (SMV) has been identified in Phaseolus vulgaris L. cv. Black Turtle Soup, line BT-1. Inoculated plants carrying this factor developed pinpoint necrotic lesions on inoculated tissue followed by systemic vascular necrosis and plant death within about 7 days, regardless of ambient temperature. BT-1 also carries dominant resistance to potyviruses attributed to the tightly linked or identical factors, I, Bcm, Cam, and Hsw, so linkage with Hss was evaluated. No recombinants were identified among 381 F₃ families segregating for potyvirus susceptibility, thus if Hss is a distinct factor, it is tightly linked to I, Bcm, Cam, and Hsw. BT-1 was also crossed reciprocally with the line Great Northern 1140 (GN 1140) in which the dominant gene, Smv, for systemic resistance to SMV was first identified. Smv and Hss segregated independently and are co-dominant. The (GN 1140 x BT-1) F₁ populations showed a seasonal shift of the codominant phenotype. Evaluation of the (GN 1140 x BT-1) F₂ population under conditions where Smv is partially dominant allowed additional phenotypic classes to be distinguished. Pathotype specificity has not been demonstrated for either Smv or Hss. Genotypes that are homozygous for both dominant alleles are systemically resistant to the virus and in addition show undetectable local viral replication or and no seed transmission. This work demonstrates that a gene which conditions a systemic lethal response to a pathogen may be combined with additional gene(s) to create an improved resistant phenotype.     

Take-off capacity as a criterion for quality control in mass-produced predators,Rhizophagus grandis (Col.: Rhizophagidae) for the biocontrol of bark beetles,Dendroctonus micans (Col.: Scolytidae)

Time spent by adult beetles in cold storage at 3–7°C accounted for 81% of the loss of take-off capacity inRhizophagus grandis Gyllenhal in windtunnel experiments. At the age of three weeks, the insects were at their highest take-off capacity at about 80%. This was followed by a steady decrease, 7% of the insects failing to take-off each month. Changes in the fat reserves during cold storage could explain at least partly this reduction of flight capacity although there was no significant difference in fresh weight between insects that were able/unable to take-off. Sex had a significant influence on take-off rates, with an 8.7% higher take-off rate in females. Take-off capacity was further reduced when the insects were mass-produced in cultures using parent beetles submitted to a blend of synthetic oviposition stimulants instead of live prey larvae. Response to synthetic attractants by those insects which took-off, however, was not influenced by cold storage or by the use of synthetic stimulants in the culture medium.

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Résumé Le temps passé en conservation à basse température (3–7°C) depuis la métamorphose est intervenu pour 81% dans la perte de la capacité d'envol chez le coléoptère prédateurRhizophagus grandis Gyll. lors d'expériences en tunnel de vol. A l'age de trois semaines, les insectes sont à leur plus haut niveau d'envol (envol d'environ 80% des insectes). Par la suite, il y a un déclin constant de la capacité d'envol, à raison de 7% des insectes chaque mois. Des changements dans les réserves lipidiques peuvent partiellement expliquer cette réduction, bien qu'il n'y ait pas eu de différence entre le poids frais d'insectes capables de s'envoler et celui d'individus qui en étaient incapables. Le sexe a une influence sur le taux d'envol, avec un taux d'envol significativement plus élevé de 8.7% chez les femelles. La capacité d'envol est encore réduite chez des insectes qui ont été produits dans des élevages de masse où les parents étaient soumis à un mélange de stimuli de ponte de synthèse au lieu d'être mis en présence de larves deD. micans vivantes. Chez les insectes qui prennent leur vol, cependant, la réponse aux attractifs de synthèse est indépendante de l'age ainsi que des conditions d'élevage.

     

Salt stress alleviation in transgenic Vigna mungo L. Hepper (blackgram) by overexpression of the glyoxalase I gene using a novel Cestrum yellow leaf curling virus (CmYLCV) promoter

A reproducible and efficient transformation system utilizing the nodal regions of embryonal axis of blackgram (Vigna mungo L. Hepper) has been established via Agrobacterium tumefaciens. This is a report of genetic transformation of Vigna mungo for value addition of an agronomic trait, wherein the gene of interest, the glyoxalase I driven by a novel constitutive Cestrum yellow leaf curling viral promoter has been transferred for alleviating salt stress. The overexpression of this gene under the constitutive CaMV 35S promoter had earlier been shown to impart salt, heavy metal and drought stress tolerance in the model plant, tobacco. Molecular analyses of four independent transgenic lines performed by PCR, Southern and western blot revealed the stable integration of the transgene in the progeny. The transformation frequency was ca. 2.25% and the time required for the generation of transgenic plants was 10–11 weeks. Exposure of T1 transgenic plants as well as untransformed control plants to salt stress (100 mM NaCl) revealed that the transgenic plants survived under salt stress and set seed whereas the untransformed control plants failed to survive. The higher level of Glyoxalase I activity in transgenic lines was directly correlated with their ability to withstand salt stress. To the best of our knowledge this is the only report of engineering abiotic stress tolerance in blackgram. Prasanna Bhomkar, Chandrama P. Upadhyay are contributed equally. An erratum to this article can be found at