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.     

Widespread distribution of Beet necrotic yellow vein virus in Iranian sugar beet industry

Beet necrotic yellow vein virus (BNYVV) is the most devastating pathogen of sugar beet worldwide. This virus has been reported in the majority of sugar beet growing regions of Iran as well. For the present study, we collected samples from different sugar beet varieties with suspected symptoms of BNYVV from the main important sugar beet growing regions in eight provinces of Iran. Infection of collected samples to BNYVV was tested by ELISA and RT-PCR. Upon testing of 167 collected samples of BNYVV suspected through ELISA and RT-PCR, 115 (68.9%) were infected. Different incidences of BNYVV through surveyed provinces may represent the presence of diverse infective viral sources or resistance genes in tested sugar beet varieties which need further attempts to develop control strategies. Results also showed that BNYVV has been recently distributed throughout some surveyed regions. Otherwise, trace infection or resistance to BNYVV infection in some varieties of distinct regions may represent proper sources of resistance to BNYVV.     

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.     

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.     

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.     

Analysis of gene inheritance and expression in hybrids between transgenic sugar beet and wild beets

Reciprocal gene exchange between cultivated sugar beet and wild beets in seed production areas is probably the reason for the occurence of weed beets in sugar beet production fields. Therefore, when releasing transgenic sugar beet plants into the environment, gene transfer to wild beets ( Beta vulgaris ssp. maritima ) has to be considered. In this study the transfer of BNYVV- (beet necrotic yellow vein virus) resistance and herbicide-tolerance genes from two transgenic sugar beet lines that were released in field experiments in 1993 and 1994 in Germany to different wild beet accessions was investigated. In order to evaluate the consequences of outcrossing, manual pollinations of emasculated wild beet plants with homozygous transgenic sugar beet plants were performed. In the resulting hybrids the transgenes were stably inherited according to Mendelian law. Gene expression in leaves and roots of the hybrids was in the same range as in the original transgenic sugar beet plants. Moreover, it was found that in one of the wild beet accessions, transfer and expression of the BNYVV resistance gene did considerably increase the level of virus resistance.     

Development of coupling-repulsion-phase SCAR markers diagnostic for the sugar beet Rr1 allele conferring resistance to rhizomania

In sugar beet genotypes with the Holly type of resistance to rhizomania, a disease due to infection of the beet necrotic yellow vein virus (BNYVV), the major gene rrl is responsible for resistance. Twelve RAPD markers linked to rrl were selected by BSA and mapped on linkage group IV using a segregating population previously analysed by the same group. Markers F61050 and N9600 were tightly linked, respectively in coupling and repulsion, to the Rrl allele (recombination values of 1.4 cM for both markers). After sequencing the products amplified by F61050 and N9600, new PCR primers were used to generate the two SCAR markers F6 and N9. The simultaneous use of these markers in a PCR reaction allows the correct fingerprinting of rrl rrl, Rrl rrl and Rrl Rrl sugar beet plants in populations segregating for the Holly resistance. In a group of sugar beet elite lines containing the Holly type of rhizomania resistance, SCAR F6 is always present whereas the SCAR N9 fragment is absent. Thus, in marker-assisted selection with coupling-repulsion-phase markers, SCAR F6 can be used in combination with N9, or together with any other RAPD marker linked in repulsion to the Rrl allele.