The differential interactions of V. longisporum (VL) and V. dahliae (VD) on the root surface and in the root and shoot vascular system of Brassica napus were studied by confocal laser scanning microscopy (CLSM), using GFP tagging and conventional fluorescence dyes, acid fuchsin and acridin orange. VL and VD transformants expressing sGFP were generated by Agrobacterium-mediated transformation. GFP signals were less homogenous and GFP tagging performed less satisfactory than the conventional fluorescence staining when both were studied with CLSM. Interactions of both pathogens were largely restricted to the root hair zone. At 24 h post-inoculation (hpi), hyphae of VL and VD were found intensely interwoven with the root hairs. Hyphae of VL followed the root hairs towards the root surface. At 36 hpi, VL hyphae started to cover the roots with a hyphal net strictly following the grooves of the junctions of the epidermal cells. VL started to penetrate the root epidermal cells without any conspicuous infection structures. Subsequently, hyphae grew intracellularly and intercellularly through the root cortex towards the central cylinder, without inducing any visible plant responses. Colonisation of the xylem vessels in the shoot with VL was restricted to individual vessels entirely filled with mycelium and conidia, while adjacent vessels remained completely unaffected. This may explain why no wilt symptoms occur in B. napus infected with VL. Elevated amounts of fungal DNA were detectable in the hypocotyls 14 days post-inoculation (dpi) and in the leaves 35 dpi. Root penetration was also observed for VD, however, with no directed root surface growth and mainly an intercellular invasion of the root tissue. In contrast to VL, VD started ample formation of conidia on the roots, and was unable to spread systemically into the shoots. VD did not form microsclerotia in the root tissue as widely observed for VL. This study confirms that VD is non-pathogenic on B. napus and demonstrates that non-host resistance against this fungus materializes in restriction of systemic spread rather than inhibition of penetration.
The introduction of high-yielding and hybrid cultivars and the opening of new markets in the food and feed sector have steadily increased rapeseed production since the 1980s in the main production regions, Canada, Europe, China, India, and Australia. Since the 1990s, however, the average growth rate of yields has declined in Europe and Australia, which has been associated with a less effective control of biotic stresses. A global survey including the knowledge of 22 experts from 10 countries revealed a total of 16 diseases, 37 insect pests, several species of nematodes, and slugs currently affecting rapeseed production globally. A ranking of the top 10 most important biotic stresses in the four global regions where Brassica napus is grown (Canada, China, Europe, Australia) indicated an increase in several important stresses and distinct regional differences in the priority of prevailing diseases and pests. A stronger overlap exists among diseases, with Sclerotinia stem rot, Phoma stem canker, and clubroot occurring in all the four global regions on the top 10 list, while the range of prevailing insect pests was more diverse among the regions, with no top 10 insect playing an equally important role worldwide. Management options are substantially broader in disease than in pest control, making the latter the larger challenge. Since common integrated pest management (IPM) tools such as crop rotation, soil management, resistant cultivars or biocontrol are ineffective or not available, insect control largely relies on insecticides. Increasing restrictions on insecticide use, particularly in Europe, and losses in insecticide efficacy threaten the profitability of oilseed rape production and its role as an important break crop in cereal dominated cropping systems. Since the survival time of insects in the absence of their main host is relatively short (<1 year), a regional synchronization of cropping schemes resulting in one or more years without the crop could lead to a substantial disruption of regional insect populations. If rotation schemes were implemented on the landscape instead the farm level, by coordination among growers in zones covering the range distances of insect pests, an efficient and chemical low management strategy could be established and enable a more sustainable rapeseed production in the future.
Verticillium longisporum is a vascular fungal pathogen presently threatening oilseed rape production in Europe. Systemic spread and vascular responses were studied in a susceptible ('Falcon') and a resistant genotype (SEM 05-500256) of Brassica napus. Colonization of both genotypes after dip-inoculation of the roots followed by quantitative polymerase chain reaction revealed similarities only in the initial stages of root penetration and colonization of the hypocotyl, while a substantial invasion of the shoot was only recorded in 'Falcon'. It is concluded that the type of resistance represented in SEM 05-500256 does not prevent the plant base from being invaded as it is internally expressed well after root penetration and colonization of the plant base. The morphological and biochemical nature of barriers induced in the hypocotyl tissue upon infection was studied with histochemical methods accompanied by biochemical analyses. Histochemical studies revealed the build-up of vascular occlusions and the reinforcement of tracheary elements through the deposition of cell wall-bound phenolics and lignin. Furthermore, the accumulation of soluble phenolics was observed. Although these responses were found in vascular tissues of both genotypes, they occurred with a significantly higher intensity in the resistant genotype and corresponded with the disease phenotype. In the resistant genotype phenols were differentially expressed in a time-dependent manner with preformed soluble and cell wall-bound phenolics at earlier time points and de novo formation of lignin and lignin-like polymers at later stages of infection. This is the first study identifying a crucial role of phenol metabolism in internal defense of B. napus against V. longisporum and locating the crucial defense responses in the plant hypocotyl.
Ascospores of both A-group and B-group Leptosphaeria maculans germinated at temperatures from 5-20degreesC on distilled water agar or detached oilseed rape leaves. After 2 h of incubation on water agar, some A-group ascospores had germinated at 10-20degreesC and some B-group ascospores had germinated at 5-20degreesC. The percentages of both A-group and B-group ascospores that had germinated after 24 h of incubation increased with increasing temperature from 5-20'C. The observed time (Vo(50)) which elapsed from inoculation until 50% of the spores had germinated was shorter for B-group than for A-group ascospores. Germ tube length increased with increasing temperature from 5-20degreesC for both ascospore groups. Germ tubes from B-group ascospores were longer than germ tubes from A-group ascospores at all temperatures tested, but the mean diameter of germ tubes from A-group ascospores (1.8 mum) was greater than that of those from B-group ascospores (1.2 mum) at 15degreesC and 20degreesC. The average number of germ tubes produced from A-group ascospores (3.8) was greater than that from B-group ascospores (3.1) after 24 h of incubation at 20degreesC, on both water agar and leaf surfaces. Germ tubes originated predominantly From interstitial cells or terminal cells of A-group or B-group ascospores, respectively, on both water agar and leaf surfaces. Hyphae from A-group ascospores grew tortuously with extensive branching, whilst those from B-group ascospores were predominantly long and straight with little branching, whether the ascospores were produced from oilseed rape debris or from crosses between single ascospore isolates, and whether ascospores were germinating on water agar or leaf surfaces
Factors limiting trichothecene contamination of mature wheat grains after Fusarium infection are of major interest in crop production. In addition to ear infection, systemic translocation of deoxynivalenol (DON) may contribute to mycotoxin levels in grains after stem base infection with toxigenic Fusarium spp. However, the exact and potential mechanisms regulating DON translocation into wheat grains from the plant base are still unknown. We analyzed two wheat cultivars differing in susceptibility to Fusarium head blight (FHB), which were infected at the stem base with Fusarium culmorum in climate chamber experiments. Fungal DNA was found only in the infected stem base tissue, whereas DON and its derivative, DON-3-glucoside (D3G), were detected in upper plant parts. Although infected stem bases contained more than 10,000 μg kg⁻¹ dry weight (DW) of DON and mean levels of DON after translocation in the ear and husks reached 1,900 μg kg⁻¹ DW, no DON or D3G was detectable in mature grains. D3G quantification revealed that DON detoxification took mainly place in the stem basis, where ≤ 50% of DON was metabolized into D3G. Enhanced expression of a gene putatively encoding a uridine diphosphate-glycosyltransferase (GenBank accession number FG985273) was observed in the stem base after infection with F. culmorum. Resistance to F. culmorum stem base infection, DON glycosylation in the stem base, and mycotoxin translocation were unrelated to cultivar resistance to FHB. Histological studies demonstrated that the vascular transport of DON labeled with fluorescein as a tracer from the peduncle to the grain was interrupted by a barrier zone at the interface between grain and rachilla, formerly described as "xylem discontinuity". This is the first study to demonstrate the effective control of influx of systemically translocated fungal mycotoxins into grains at the rachilla-seed interface by the xylem discontinuity tissue in wheat ears.
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