Stem canker is a damaging disease of winter oilseed rape, caused by ascomycetous fungi Leptosphaeria maculans and L. biglobosa. The initial source of plant infection is ascospores, formed in pseudothecia on stubble from the previous growing season. The aim of this work was to evaluate the effectiveness of fungicide treatments at different times of application in the autumn on quality and quantity of seed yield, in relation to the concentration of ascospores of L. maculans and L. biglobosa in the air. Field experiments were done in Lower Silesia, the region of intensive oilseed rape cultivation, located in south-west Poland. We have demonstrated that, in case of high disease pressure, fungicide treatment against stem canker was most efficient when done 4-11 days after the highest concentration of pathogen ascospores in air. The seed yield, oil and protein content increased by 17.7-18.8 %, 1.60-1.72 % and 0.8-0.9 % respectively. In low disease pressure the optimal spray time was more flexible, but the highest yield was always following the maximum ascospore release. In some cases it also coincided with the increase of oil, without adverse effect on protein content. Furthermore, it coincided with the increase of indole glucosinolates, such as glucobrassicine and 4-hydroxyglucobrassicine, and -besides one studied year -it was also connected with decreased amounts of alkenyl glucosinolates. Both Leptosphaeria species were present in air and in infected plants during their vegetation. The ratio between both species depended on seasons; L. maculans prevailed on leaves in the autumn, whereas L. biglobosa dominated on stems before harvest.
A combination of staining, light microscopy and SYBR green- and dual-labelled fluorescent probe-based qPCR chemistries with species- and gene-specific primers was employed to evaluate fluctuations in the aerial biomass of Leptosphaeria maculans spores captured by volumetric spore trappings in Poznan, Poland (2006, 2008) and Harpenden, UK (2002, 2006). Arising from these surveys, DNA samples extracted from Burkard spore-trap tapes were screened for fluctuation patterns in the frequencies of AvrLm1 and AvrLm6, the most prominent of the 15 genes that code for avirulence effectors in this Dothideomycete cause of the destructive phoma stem canker disease of oilseed rape worldwide. In Poznan, very low frequencies of AvrLm1 allele were found in the autumn of both 2006 and 2008, reflecting significantly increased cultivation of rape seed with Rlm1-based resistance. In contrast, at least six folds-higher frequencies of AvrLm6, which were also confirmed by end-point PCR bioassays on phoma-infected leaves from the same region of Poland, were obtained during both years. In the UK, however, relatively higher AvrLm1 allele titres were found in L. maculans spores captured in air samples from the autumn of 2002 on the experimental fields of Rothamsted Research, Harpenden, that were historically sown to genetically heterogeneous B. napus cultivars. In the 2006 screen these levels had plummeted, to a 1:4 ratio, in favour of frequencies of the AvrLm6 allele. Patterns of fluctuations in erg11 (CYP51) fragments coding for sterol 14α-demethylase suggest October as the month with the most viable wind-dispersed L. maculans propagules of each season of the screens.
Leptosphaeria maculans and L. biglobosa are closely related sibling fungal pathogens that cause phoma leaf spotting, stem canker (blackleg) and stem necrosis of oilseed rape (Brassica napus). The disease is distributed worldwide, and it is one of the main causes of considerable decrease in seed yield and quality. Information about the time of ascospore release at a particular location provides important data for decision making in plant protection, thereby enabling fungicides to be used only when necessary and at optimal times and doses. Although the pathogens have been studied very extensively, the effect of climate change on the frequencies and distributions of their aerially dispersed primary inoculum has not been reported to date. We have collected a large dataset of spore counts from Poznan, located in central-west part of Poland, and studied the relationships between climate and the daily concentrations of airborne propagules over a period of 17 years (1998–2014). The average air temperature and precipitation for the time of development of pseudothecia and ascospore release (July–November), increased during the years under study at the rates of 0.1 °C and 6.3 mm per year. The day of the year (DOY) for the first detection of spores, as well as the date with maximum of spores, shifted from 270 to 248 DOY, and from 315 to 265 DOY, respectively. The acceleration of the former parameter by 22 days and the latter by 50 days has great influence on the severity of stem canker of oilseed rape.
A b s t r a c t LAMP is an innovative, simple, rapid, specific and cost--effective nucleic acid amplification method. Due to the use of a special enzyme -GspSSD polymerase, the reaction takes a short time and can be performed at isothermal conditions. The sensitivity and specificity of LAMP technique is significantly higher, than standard PCR techniques, as two or three specific primer pairs are used. The technique is regarded as a useful tool for the detection and identification of plant pathogens. In this work, LAMP was used to study the composition of the population of fungi of the genus Leptosphaeria, causing a damaging disease of oilseed rape, called blackleg or stem canker. The detection concerned DNA present in fungal spores contained in air samples obtained using Hirst-type volumetric trap, in Pomerania (north Poland) in 2010. The results achieved using the LAMP technique were similar to these obtained with previously used, highly specific method of Real-time PCR. Conducting LAMP reaction was much easier and less time-and cost-consuming, due to a simplified method of DNA isolation of pathogens from plant tissues. Then, the LAMP technique was used to assess the composition of the population of Leptosphaeria spp. in plants of oilseed rape collected from the field in the Opole region (south-western part of Poland) in 2013. In contrast to studies conducted in [2002][2003], the analysis of leaf symptoms showed a higher proportion of L. maculans compared to L. biglobosa, what reflects changes in the composition of pathogen population of fungi causing blackleg on oilseed rape in this part of Poland.
Winter oilseed rape (Brassica napus L.) is the main source of domestic oil in central and northern Europe, bringing profits to farmers, but the plants are often damaged by stem canker, caused by two fungal species belonging to the genus Leptosphaeria. Due to environmental concerns, the benefits of fungicide applications must outweigh disadvantages. The aim of this work was to determine the effect of stem canker on seed yield and its quality and find out the best timing of fungicide application. The multi-year field experiments were done at two sites in south-west Poland, where the disease is regarded as a serious problem. The fungicide treatments with the azole-containing preparation followed the same scheme each year; a single application was made at one-week intervals, starting in late September through mid-November for a total of eight treatments. Seed yield, oil and protein content, mass of thousand seeds as well as indole-and alkenyl-glucosinolate contents in seeds were statistically unrelated with the incidence and severity of phoma leaf spotting and stem canker symptoms. The significant decrease of the seed yield was observed in three (site × year combinations) of eight, in which phoma leaf spotting and stem canker were severe. Yield loss was noted only in years with warm and wet autumns, when cumulative mean temperatures between BBCH14 and BBCH19 plant growth stages exceeded 60 °C and precipitation in this period exceeded 110 mm of rain. Under these conditions, fungicide treatments were highly effective when they were done between BBCH15–BBC16 growth stages (5–6 true leaves).
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