A method based on restriction analysis of polymerase chain reaction (PCR)-amplified ribosomal DNA was developed for the rapid characterization of large populations of Rhizoctonia solani at the anastomosis group (AG) level. The restriction maps of the internal transcribed spacers (ITS) sequences were compared for 219 isolates of R. solani belonging to AG-1 to AG-12 and AG-BI, representing diverse geographic and host range origins. Four discriminant restriction enzymes (MseI, AvaII, HincII, and MunI) resolved 40 restriction fragment length polymorphism (RFLP) types among the 219 ITS sequences of R. solani. Each RFLP type could be assigned to a single AG except for two RFLP types, which were common to two AG. A fifth enzyme allowed the discrimination of AG-6 and AG-12. In addition, the combination of four enzymes allowed the discrimination of subsets within AG-1, AG-2, AG-3, and AG-4. The efficiency of the typing method was confirmed by analyzing PCR-amplified ITS sequences of 30 reference strains. Furthermore, the PCR-RFLP method was used to characterize at the AG level 307 isolates of R. solani originating from ten sugar beet fields exhibiting patches of diseased plants in France. The PCR-based procedure described in this paper provides a rapid method for AG typing in R. solani.
The syndrome "basses richesses" of sugar beet (SBR) was first observed in 1991 in Burgundy, France. A cixiid planthopper, Pentastiridius beieri, has been proved to be involved in the transmission to sugar beet of a stolbur phytoplasma, which could be detected in some affected plants. In 2000, periwinkle and sugar beet exposed to field-collected cixiids developed symptoms similar to SBR on sugar beet. Use of 4'-6-diamidino-2-phenylindole (DAPI) staining and transmission electron microscopy confirmed the presence of phytoplasma in some of the plants, which were also positive for this pathogen in a polymerase chain reaction (PCR) analysis. A phloem-restricted gram-negative bacteria was seen in all other plants with symptoms but PCR-negative for phytoplasma. Three primer pairs reported as diagnostic for phloem-limited bacteria were tested but only primers specific for 'Candidatus Phlomobacter fragariae' gave a positive signal, which related to the presence of DAPI-stained bacteria-like objects in diseased plants. Although phytoplasma and bacterium-like organisms were associated with the same macroscopic symptoms on sugar beet, histochemical analysis of phloem cells showed that phytoplasma were associated with cell necrosis and cell wall lignification, while bacteria were associated with these same abnormalities as well as deposit of phenolic compounds in the lumen of phloem cells.
The disease syndrome 'Basses richesses' (SBR) affects sugar beet (Beta vulgaris) crops and causes important economic damage in eastern France. Up to now two phloem-restricted prokaryotes which cannot be cultivated, a stolbur phytoplasma and a c-3 proteobacterium (called SBR proteobacterium), have been associated with the disease. The SBR proteobacterium is closely related to endosymbionts of Hemiptera in the genus Arsenophonus. Both the phytoplasma and the proteobacterium are transmitted by the insect vector Pentastiridius sp. (Hemiptera: Cixiidae). In the present work, we developed sensitive PCR tools for routine detection of SBR proteobacteria in sugar beets. The monitoring with PCR since 1997, of both SBR pathogen agents, showed the predominant aetiological role of SBR proteobacteria in SBR disease. Detection of SBR proteobacteria in sugar beet was correlated with development of SBR symptoms and reduction of sugar content in the taproot. Severity of symptoms and sugar content in experimentally inoculated sugar beet plants were a function of the number of Pentastiridius sp. used for transmission or the length of inoculation access period (IAP), suggesting a direct relationship between importance or precocity of populations of inoculative insects in fields and low sugar yield of crops.
Concerns have been raised in Europe about the efficiency, sustainability, and environmental impact of the first genetically modified crops. The committees and regulators in charge of approving procedures have encouraged a field trial approach for safety assessment studies under current agronomic conditions. We describe the gene flow from sugar beet (Beta vulgaris L.) in a multi-year and multi-crop monitoring study on farmers' fields at two locations that has been carried out since 1995. We analyzed two sugar beet lines that have been genetically transformed for herbicide resistance. One sugar beet has resistance to glufosinate and the other to glyphosate. Large differences among lines, years and locations were observed. These differences provided a broad range of situations to estimate the risks. Sugar beet bolters produced the majority (86%) of the herbicide-resistant seeds harvested in the field. Direct pollen flow from sugar beet bolters to weed beets that were growing within the same field as well as in a neighboring field that was left fallow accounted for only 0.4% of the resistant seeds released over the years and locations. Descendants of the hybrids between the sugar beet and the weed beet produced the remaining 13.6% of resistant seeds. Herbicide-resistant seeds from the progeny of the weed beet were recorded up to 112 m away from the closest transgenic pollen donor. Indications were observed of non-randomness of the weed beet producing resistant progeny. We also analyzed pollen flow to male-sterile bait plants located within and outside of the sugar beet field. Herbicide-resistant pollen flow was recorded up to 277 m, and fitted with an inverse power regression. Using sugar beet varieties with no, or very low, sensitivity to bolting and destroying bolters are two necessary measures that could delay gene flow.
Pollen-mediated gene flow has important implications for biodiversity conservation and for breeders and farmers' activities. In sugar beet production fields, a few sugar beet bolters can produce pollen as well as be fertilized by wild and weed beet. Since the crop, the wild beets, and the weed beets are the same species and intercross freely, the question of pollen flow is an important issue to determine the potential dispersal of transgenes from field to field and to wild habitats. We report here an experiment to describe pollen dispersal from a small herbicide-resistant sugar beet source towards male sterile target plants located along radiating lines up to 1,200 m away. Individual dispersal functions were inferred from statistical analyses and compared. Pollen limitation, as expected in root-production fields, was confirmed at all the distances from the pollen source. The number of resistant seeds produced by bait plants best fitted a fat-tailed probability distribution curve of pollen grains (power-law) dependent on the distance from the pollen source. A literature survey confirmed that power-law function could fit in most cases. The b coefficient was lower than 2. The number of fertilized flowers by background (herbicide-susceptible) pollen grains was uniform across the whole field. Airborne pollen had a fertilization impact equivalent to that of one adjacent bolter. The individual dispersal function from different pollen sources can be integrated to provide the pollen cloud composition for a given target plant, thus allowing modeling of gene flow in a field, inter-fields in a small region, and also in seed-production area. Long-distance pollen flow is not negligible and could play an important role in rapid transgene dispersal from crop to wild and weed beets in the landscape. The removing of any bolting, herbicide-resistant sugar beet should be compulsory to prevent the occurrence of herbicide-resistant weed beet, thus preventing gene flow to wild populations and preserving the sustainable utility of the resistant varieties. Whether such a goal is attainable remains an open question and certainly would be worth a large scale experimental study.
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