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Brassica napus is an important oil source. Its narrow gene pool can be widened by interspecific hybridization with the Brassicaceae species. One of the agronomically important traits, that can be transferred through the hybridization, is the resistance to blackleg, a dangerous disease mainly caused by Leptosphaeria maculans. Hybrid individuals can be analyzed with various molecular markers, including Simple Sequence Repeats (SSR). We investigated the genetic similarity of 32 Brassicaceae hybrids and 19 parental components using SSR markers to reveal their genetic relationship. Furthermore, we compared the field resistance to blackleg of the interspecific progenies. The tested set of 15 SSR markers proved to be useful in revealing the genetic distances in the Brassicaceae hybrids and species. However, genetic similarity of the studied hybrids could not be correlated with the level of field resistance to L. maculans. Moreover, our studies confirmed the usefulness of the Brassicaceae hybrids in terms of blackleg management.

Species of the genus Plenodomus (Leptosphaeria) are phytopathogens of the Brassicaceae family, which includes oilseed rape. The spores of these fungi spread by airborne transmission, infect plants, and cause crop losses. The secondary metabolism of P. lingam and P. biglobosus was studied and compared, with the main focus being on the ability to produce Extracellular Polymeric Substances (EPS). In spite of the 1.5–2-fold faster growth rate of P. biglobosus on Czapek-Dox and other screening media, the average yield of EPS in this fungus was only 0.29 g/L, compared to that of P. lingam (0.43 g/L). In turn, P. biglobosus showed a higher capacity to synthesise IAA, i.e., 14 µg/mL, in contrast to

Hybridization ofBrassica napuswith various Brassicaceae species can result in obtaining new forms with increased resistance to blackleg, a dangerous disease caused mainly byLeptosphaeria maculans. In this study, we aimed to correlate the field resistance of selectedBrassicahybrids to blackleg with chromosomal structure revealed by Fluorescencein situhybridization. Tested genotypes varied in the number of chromosomes and rDNA signals. The greatest variation was observed for A1-type chromosomes. Field evaluation also revealed significant differences inL. maculansresistance. Performed analyses allowed to distinguish threeB. napus×Brassica fruticulosagenotypes in which variable patterns of chromosomal structure might be connected to field resistance. However, a more thorough study, including the detection of all A-genome chromosomes, is required.

Clubroot, caused by

In contrast to the long-lasting taxonomic classification of Plenodomus lingam and P. biglobosus as one species, formerly termed Leptosphaeria maculans, both species form separate monophyletic groups, comprising sub-classes, differing considerably with epidemiology towards Brassicaceae plants. Considering the great differences between P. lingam and P. biglobosus, we hypothesized their metabolic capacities vary to a great extent. The experiment was done using the FF microplates (Biolog Inc., Hayward, CA, USA) containing 95 carbon sources and tetrazolium dye. The fungi P. lingam and P. biglobosus subclade ‘brassicae’ (3 isolates per group) were cultured on PDA medium for 6 weeks at 20 °C and then fungal spores were used as inoculum of microplates. The test was carried out in triplicate. We have demonstrated that substrate richness, calculated as the number of utilized substrates (measured at λ490 nm), and the number of substrates allowing effective growth of the isolates (λ750 nm), showed significant differences among tested species. The most efficient isolate of P. lingam utilized 36 carbon sources, whereas P. biglobosus utilized 60 substrates. Among them, 25–29 carbon sources for P. lingam and 34–48 substrates for P. biglobosus were efficiently used, allowing their growth. Cluster analysis based on Senath criteria divided P. biglobosus into two groups and P. lingam isolates formed one group (33% similarity). We deduce the similarities between the tested species help them coexist on the same host plant and the differences greatly contribute to their different lifestyles, with P. biglobosus being less specialized and P. lingam coevolving more strictly with the host plant.

Hybridization of

Clubroot, caused by Plasmodiophora brassicae, is a crucial oilseed rape disease worldwide. Information on the virulence of P. brassicae populations is essential to apply disease control with proper clubroot-resistant cultivars. In 2016–2020, 84 isolates of P. brassicae were collected in the Czech Republic (CZ), Germany (DE), Poland (PL), and Sweden (SW). Pathotypes were designated using 17 Brassica hosts, including the European Clubroot Differentials (ECD), Somé set, and clubroot-resistant oilseed rape cv. Mendel. According to the ECD set, virulence analyses differentiated the isolates into 42 pathotypes. The most common pathotypes were 16/31/31 (in DE, PL, and SW) and 16/06/12 (in CZ, DE, and PL). Six pathotypes were found according to the Somé set, including 1–4 pathotypes per country. P1 was most prevalent in DE, PL, and SW, while P3 was abundant in CZ, DE, and PL. The current study provides clear evidence for a shift towards increased virulence in P. brassicae populations compared to previous studies. Several isolates overcame the resistance of cv. Mendel and of Brassica rapa genotypes ECD 01 to ECD 04. Considering all investigated samples, significant negative correlations were found between clubroot incidence and the frequency of oilseed rape in crop rotation, as for clubroot incidence and soil pH.

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).

The fungus, Diaporthe toxica, anamorph Phomopsis sp., previously classified as P. leptostromiformis, is a plant endophyte and occasional pathogen, causing Phomopsis stem blight. This disease is damaging not only to lupins but also to the animals grazing on infected plants, due to the toxic secondary metabolites called phomopsins. The aim of this work was to validate markers for resistance to Phomopsis stem blight in narrow-leafed lupins and identify novel germplasm with increased levels of resistance to the disease. Plant inoculations were performed using ten isolates of D. toxica, originating from Australia and Poland. The European core collection of L. angustifolius was evaluated both in a controlled environment and with field experiments to classify the accessions based on their resistance to the disease. Simultaneously, the accessions were assayed with disease resistance markers to identify donors of hypothetical resistance alleles. We have found that the European lupin germplasm collection preserves wild and domesticated donors of at least two resistance genes to Phomopsis stem blight, including Phr1 and PhtjR. Molecular markers PhtjM7, InDel2, and InDel10, tagging PhtjR gene, were applicable for marker-assisted selection targeting the European gene pool with an expected accuracy of 95%. None of diagnostic markers for the Phr1 locus was found useful for European breeding programs; two existing markers Ph258M1 and Ph258M2 were unreliable, due to a high percentage of false-positive results (up to 58%) and a high recombination rate between markers (~ 30%).

Clubroot is a damaging disease of oilseed rape and vegetable brassicas worldwide, caused by the soil-borne protist Plasmodiophora brassicae Wor. Due to the long life of resting spores, the assessment of the pathogen abundance in agricultural fields can serve as a guideline for disease control at the country-wide level or the regional scale. Between 2013 and 2019, we collected 431 soil samples from fields cultivated with Brassicaceae crops throughout 16 provinces of Poland. The samples were subjected to qPCR based analysis of P. brassicae DNA concentration. From these data, the spore loads and gene copies g&minus, 1 soil were calculated and used to produce an assessment of the current clubroot risk potential at a country-wide and regional scale. The country-wide map, showing the spread of the pathogen in agricultural soils, was made using ArcGis software package implementing the interpolation with the Inverse Distance Weight method. The calculation of gene copies specific to P. brassicae helped to formulate the recommendations for farmers in respect to the cultivation guidelines. It showed a high risk of yield losses in defined regions of north, south-west and central Poland and an urgent need to undertake intensive preventative measures.

Clubroot, caused by the protozoan parasite Plasmodiophora brassicae Woronin, is one of the most damaging diseases of Brassica napus worldwide. Resistant plant material is valuable for cultivation in all areas of high incidence of the disease and intensive growth of oilseed rape. We have evaluated clubroot resistance, plant morphology and seed quality in 15 lines of an F4 generation and selected six lines of F5 generation of interspecific hybrids obtained from a cross between a male sterile line of B. napus ‘MS8’, selected from resynthesized oilseed rape (B. rapa ssp. chinensis × B. oleracea var. gemmifera) and an ecotype of B. rapa ssp. pekinensis. Clubroot resistance was evaluated using a bioassay with P1-P5 pathotypes of P. brassicae (according to the classification of Some et al. 1996). The resistance to the pathotype P1 was successfully fixed in the F5 generation, and improved in some lines in respect to the pathotypes P2-P4. The resistance to P1 and the other tested pathotypes was not linked. Characterization of plant material included recent techniques of FISH and BAC-FISH with a special focus on the analysis of ribosomal DNA (rDNA) of selected individuals. Two hybrid lines combined high levels of resistance with appropriate plant morphology, good seed quality traits and a stable chromosome number and arrangement. Recent techniques of ‘chromosome painting’ provided good insight into chromosome organization in the hybrids obtained, and offered opportunities of further improvement of the breeding process.

Genus Trichoderma contains the most powerful agents used in biocontrol. Our study demonstrated that T. harzianum, T. hamatum and T. longibrachiatum can effectively control phytopathogenic fungi Leptosphaeria maculans and L. biglobosa. The effects of Trichoderma on pathogens’ growth (in dual cultures on agar media) and on disease severity (on seedlings in controlled conditions) were also confirmed by field experiments. Additionally, spraying with conidiospores in the autumn was helpful in reducing the incidence and severity of phoma leaf spots. It has also accelerated the degradation of plant stubble and the decomposition of pathogens’ fruiting bodies. Furthermore, all Trichoderma isolates showed higher cellulolytic activity and enhanced resistance to flusilazole treatments as compared to Leptosphaeria spp., which coincided with upregulation of 14α-sterol demethylases and an AbcG5 transporter. The effects we observed justify the use of Trichoderma to enhance the resistance of oilseed rape against pathogens, which in turn may lead to a decrease in the use of pesticides.

Winter oilseed rape (Brassica napus) is an important crop in the Czech Republic and Poland. Clubroot disease caused by the pathogen Plasmodiophora brassicae is a serious and still-growing problem for oilseed rape growers in both countries. The aim of this study was to evaluate the pathotype composition of P. brassicae populations from the Czech Republic and Poland, according to the three evaluation systems, and to determine soil inoculum loads for representative fields via traditional end-point PCR as well as quantitative PCR analysis. There were considerable differences between the populations of P. brassicae from both countries, and the number of pathotypes varied depending on the evaluation system and the threshold used to distinguish susceptible vs. resistant plant reactions. This is the first study comparing the effect of different thresholds. Using an index of disease (ID) of 25 % to distinguish susceptible vs. resistant reactions, there was a total of seven pathotypes identified based on the differentials of Williams, five with the system of Some et al., and 18 with the European Clubroot Differential (ECD) set. However, based on a threshold of 50 %, there were nine pathotypes according to the evaluation system by Williams, four based on the differentials of Some et al., and 15 with the ECD set. Changing of the thresholds led to the reclassification of some pathotypes. Several pathotypes were common in both countries. High amounts of pathogen DNA were found in many of the field soils analysed by quantitative PCR. There was a weak correlation between soil pH and infestation of P. brassicae for the Polish soils.

The aim of this study was to explore the species diversity of Trichoderma obtained from samples of wood collected in the forests of the Gorce Mountains (location A), Karkonosze Mountains (location B) and Tatra Mountains (location C) in Central Europe and to examine the cellulolytic and xylanolytic activity of these species as an expression of their probable role in wood decay processes. The present study has led to the identification of the following species and species complex: Trichoderma atroviride P. Karst., Trichoderma citrinoviride Bissett, Trichoderma cremeum P. Chaverri & Samuels, Trichoderma gamsii Samuels & Druzhin., Trichoderma harzianum complex, Trichoderma koningii Oudem., Trichoderma koningiopsis Samuels, C. Suárez & H.C. Evans, Trichoderma longibrachiatum Rifai, Trichoderma longipile Bissett, Trichoderma sp. (Hypocrea parapilulifera B.S. Lu, Druzhin. & Samuels), Trichoderma viride Schumach. and Trichoderma viridescens complex. Among them, T. viride was observed as the most abundant species (53 % of all isolates) in all the investigated locations. The Shannon’s biodiversity index (H), evenness (E), and the Simpson’s biodiversity index (D) calculations for each location showed that the highest species diversity and evenness were recorded for location A—Gorce Mountains (H′ = 1.71, E = 0.82, D = 0.79). The preliminary screening of 119 Trichoderma strains for cellulolytic and xylanolytic activity showed the real potential of all Trichoderma species originating from wood with decay symptoms to produce cellulases and xylanases—the key enzymes in plant cell wall degradation. Electronic supplementary material The online version of this article (doi:10.1007/s13353-015-0326-1) contains supplementary material, which is available to authorized users.

The genus Leptosphaeria contains numerous fungi that cause the symptoms of asthma and also parasitize wild and crop plants. In search of a robust and universal forecast model, the ascospore concentration in air was measured and weather data recorded from 1 March to 31 October between 2006 and 2012. The experiment was conducted in three European countries of the temperate climate, i.e., Ukraine, Poland, and the UK. Out of over 150 forecast models produced using artificial neural networks (ANNs) and multivariate regression trees (MRTs), we selected the best model for each site, as well as for joint two-site combinations. The performance of all computed models was tested against records from 1 year which had not been used for model construction. The statistical analysis of the fungal spore data was supported by a comprehensive study of both climate and land cover within a 30-km radius from the air sampler location. High-performance forecasting models were obtained for individual sites, showing that the local micro-climate plays a decisive role in biology of the fungi. Based on the previous epidemiological studies, we hypothesized that dew point temperature (DPT) would be a critical factor in the models. The impact of DPT was confirmed only by one of the final best neural models, but the MRT analyses, similarly to the Spearman’s rank test, indicated the importance of DPT in all but one of the studied cases and in half of them ranked it as a fundamental factor. This work applies artificial neural modeling to predict the Leptosphaeria airborne spore concentration in urban areas for the first time. Electronic supplementary material The online version of this article (10.1007/s00484-018-1500-z) contains supplementary material, which is available to authorized users.

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.

Ramularia collo-cygni is the biotic factor responsible for the disease Ramularia leaf spot (RLS) of barley (Hordeum vulgare). Despite having been described over 100 years ago and being considered a minor disease in some countries, the fungus is attracting interest in the scientific community as a result of the increasing number of recorded economically damaging disease epidemics. New reports of disease spread and fungal identification using molecular diagnostics have helped redefine RLS as a global disease. This review describes recent developments in our understanding of the biology and epidemiology of the fungus, outlines advances made in the field of the genetics of both the fungus and host, and summarizes the control strategies currently available.

Airborne sexual spores of Leptosphaeria maculans and L. biglobosa are primary inoculum of fungi causing phoma stem canker of oilseed rape. In this study ascospore release of these two species was compared between Lithuania (of autumn 2010 and 2011) and Poland (autumns of 2009 till 2012), using identical equipment and methods. Dynamics of L. maculans and L. biglobosa ascospore dispersal was investigated using volumetric samplers followed by microscopic and molecular methods of detection. In Lithuania, the earliest detection of Leptosphaeria ascospores, using microscopy was on 1 st September (2010) and on 2 nd September (2011), whereas in Poland the earliest detection was on 5 th September (2009 and 2010) and the latest on 11 th October (2011), which demonstrates that differences between the seasons may exceed a month (36 days). In the case of molecular detection performed for the Polish samples, the dates of the earliest and the latest release of the first ascospores ranged from 5 th September to 15 th October (40 days). The number of days with ascospores detected in the air samples ranged from 6 (2012) to 25 (2009). At earliest, the detection of the highest concentration of ascospores was on 6 th September (2010) and the latest – on 28 th October (2009). These results demonstrate big differences between the results of spore monitoring between the years and the importance of aerobiological studies to follow the development of the pathogen in natural infection. The fluctuations in quantities of spores and fungal deoxyribonucleic acid (DNA) strongly depended on the weather conditions, mainly rainfall, air temperature and relative humidity. Ascospore release was observed immediately after the rainfall events, but not later than two days after the rainfall. On days without rain, spores were present in the air samples when average relative air humidity exceeded 90% and day temperature was below 15°C. In 2009, molecular detection revealed the presence of both fungal species in the air samples, whereas in 2010–2012 only L. biglobosa DNA was found. Molecular detection with a quantitative real-time polymerase chain reaction (qPCR) was identified as a fast and accurate method of pathogen detection and identification from air samples. The highest coefficient of correlation between microscopic and molecular detection of Leptosphaeria species was 0.828 (2009) and the lowest was 0.483 (2011); in all years both techniques of detection were statistically correlated. We have demonstrated that the correlation coefficient highly depended on the number of ascospores in air samples. Based on the studies performed in Lithuania and Poland, we have shown that climatic conditions in the northern part of Central Europe are favourable for the spread of Leptosphaeria spp. by ascospores; however, the patterns of ascospore release greatly differ between the both countries.

There has been a rapid, worldwide increase in oilseed rape production that has resulted in enormous intensification of oilseed rape cultivation, leading to tight rotations. This in turn, has caused an accumulation of pests as well as foliar and soil-borne diseases. Recently, clubroot has become one of the biggest concerns of oilseed rape growers. Clubroot is caused by the soil-borne protist Plasmodiophora brassicae Woronin. The pathogen may be present in groundwater, lakes, and irrigation water used in sprinkling systems. It can be easily transmitted from one field to another not only by water, but also by soil particles and dust transmitted by wind and on machinery. The aim of our overall study was to check for P. brassicae infestation of Polish agricultural soils. This paper presents the 2012 results of a study performed along the Polish-Ukrainian border in two provinces: Lublin (Lubelskie Voivodeship) and the Carpathian Foothills (Podkarpackie Voivodeship), in south-east Poland. Monitoring was done in 11 counties, including nine rural and two municipal ones. In total, 40 samples were collected, out of which 36 were collected from fields located in rural areas and four from municipal areas, with two per municipal region. Each sample was collected at 8-10 sites per field, using a soil auger. The biotest to detect the presence of P. brassicae was done under greenhouse conditions using seedlings of the susceptible Brassicas: B. rapa ssp. pekinensis and the Polish variety of oilseed rape B. napus cv. Monolit. Susceptible plants grown in heavily infested soils produced galls on their roots. A county was regarded as free from the pathogen, if none of the bait plants became infected. The pathogen was found in three out of 40 fields monitored (7.5%) in the Carpathian Foothill region. The fields were located in two rural counties. The pathogen was not found in Lublin province, and was also not detected in any of the municipal counties. The detection with a biotest was fully confirmed by PCR-based molecular detection of P. brassicae DNA in soil samples.

The aim of this study was to determine the relationship between ascospore release and autumn fungicide spray time as well as, the incidence and severity of stem canker and seed yield. The concentration of ascospores in air was determined using Hirst-type volumetric samplers located in the region of Pomerania in central-north Poland, in the zone of intensive cultivation of oilseed rape, over three consecutive seasons from 2009/2010 to 2011/2012. A fungicide spray was most efficient, when applied 4 to 14 days after the highest concentration of Leptosphaeria ascospores was found in air samples. An application of fungicide at this time reduced the incidence of phoma leaf spotting by 12.5–24.7%, stem canker symptoms by 11.3–33.0% and decreased yield loss by 2.7–5.0 dt ha−1. The results demonstrate that knowledge about the concentration of pathogen inoculum in the air allows more efficient protection of oilseed rape against stem canker.

Aims We propose and test an efficient and rapid protocol for the detection of toxigenic Fusarium isolates producing three main types of Fusarium-associated mycotoxins (fumonisins, trichothecenes and zearelanone). Methods and Results The novel approach utilizes partially multiplexed markers based on genes essential for mycotoxin biosynthesis (fumonisin—fum6, fum8; trichothecenes—tri5, tri6; zearalenone, zea2) in Fusarium spp. The protocol has been verified by screening a collection of 96 isolates representing diverse species of filamentous fungi. Each Fusarium isolate was taxonomically identified through both molecular and morphological techniques. The results demonstrate a reliable detection of toxigenic potential for trichothecenes (sensitivity 100%, specificity 95%), zearalenone (sensitivity 100%, specificity 100%) and fumonisins (sensitivity 94%, specificity 88%). Both presence and identity of toxin biosynthetic genes were further confirmed by direct sequencing of amplification products. Conclusions The cross-species-specific PCR markers for key biosynthetic genes provide a sensitive detection of toxigenic fungal isolates, contaminating biological material derived from agricultural fields. Significance and Impact of the Study The conducted study shows that a PCR-based assay of biosynthetic genes is a reliable, cost-effective, early warning system against Fusarium contamination. Its future use as a high-throughput detection strategy complementing chemical assays enables effective targeted application of crop protection products.

Knowledge of seasonal and diurnal fluctuations of allergenic and phytopathogenic fungal spores in the air and the determination of the influence of weather and environmental parameters on spore concentrations are of great practical use in control of both human and animal health as well as plant diseases. The aim of this study was to evaluate the dynamics of the occurrence of Alternaria spores in the air at three sites located in different geographical regions of Poland. The study was done from 15 April to 30 September in 2010–2011 in Rzeszów (Carpathian Foothills, south-east Poland), Lublin (Lublin Upland, central east), and Poznań (central west); the distance between the experimental sites ranged from 138 to 441 km. To assess the concentration of Alternaria spores, the volumetric method was used. In both years, the highest concentration of spores was observed in Poznań and the lowest in Rzeszów. In 2010 the annual total, monthly total and maximum spore concentration were higher than in 2011 at all monitoring sites. The greatest concentration of Alternaria spores was recorded in August, followed by July. High spore concentrations were also noted at the beginning of September. The differences in daily concentrations, average concentrations and cumulative numbers of spores were statistically significant. The reasons for this can be related not only to weather parameters, which is often raised in the literature, but also to the type of landscape. Landscape and geo-botanical conditions of Great Poland and Carpathian Foothills greatly differ. In both years, the highest number of Alternaria spores was found in Poznań – the city with the highest urbanization factor and the capital of the region with large-scale farming, connected with the intensification of agricultural practices.

Following a sudden outbreak of light leaf spot disease in Polish winter oilseed rape crops at the beginning of the last decade, volumetric spore traps installed around the country were used for the detection of ascospores, which constitute the primary inoculum of Pyrenopeziza brassicae, the causal organism. In 2004–2008, weather conditions that were adverse to the development of the pathogen appeared to have hindered the macroscopic signs of light leaf spot disease symptoms on oilseed rape, even in Pomerania, a region in north Poland, which cultivates the crop intensively and is favourable for the disease. Recently, however, the development of β-tubulin-based primers and dual labelled fluorescent probes has enabled the detection of minute quantities of P. brassicae DNA that correlated highly and positively with low abundance of inoculum of the pathogen in air samples. Depending on the season, the Spearman's correlation rank between spore abundance obtained by light microscopy and P. brassicae DNA...

Highlight: The level of pathogen alpha-amylase and plant beta-amylase activities could be components of plant-pathogen interaction associated with the resistance of triticale to Fusarium head blight. Triticale was used here as a model to recognize new components of molecular mechanism of resistance to Fusarium head blight (FHB) in cereals. Fusarium-damaged kernels (FDK) of two lines distinct in levels of resistance to FHB were applied into a proteome profiling using two-dimensional gel electrophoresis (2-DE) to create protein maps and mass spectrometry (MS) to identify the proteins differentially accumulated between the analyzed lines. This proteomic research was supported by a measurement of alpha- and beta-amylase activities, mycotoxin content, and fungal biomass in the analyzed kernels. The 2-DE analysis indicated a total of 23 spots with clear differences in a protein content between the more resistant and more susceptible triticale lines after infection with Fusarium culmorum. A majority of the proteins were involved in a cell carbohydrate metabolism, stressing the importance of this protein group in a plant response to Fusarium infection. The increased accumulation levels of different isoforms of plant beta-amylase were observed for a more susceptible triticale line after inoculation but these were not supported by a total level of beta-amylase activity, showing the highest value in the control conditions. The more resistant line was characterized by a higher abundance of alpha-amylase inhibitor CM2 subunit and simultaneously a lower activity of alpha-amylase after inoculation. We suggest that the level of pathogen alpha-amylase and plant beta-amylase activities could be components of plant-pathogen interaction associated with the resistance of triticale to FHB.

Recent advances in molecular detection of living organisms facilitate the introduction of novel methods to studies of the transport of fungal spores over large distances. Monitoring the migration of airborne fungi using microscope based spore identification is limited when different species produce very similar spores. In our study, DNA-based monitoring with the use of species-specific probes allowed us to track the aerial movements of two important fungal pathogens of oilseed rape (Brassica napus L.), i.e., Leptosphaeria maculans and Leptosphaeria biglobosa, which have identical spore shape and size. The fungi were identified using dual-labelled fluorescent probes that were targeted to a β-tubulin gene fragment of either Leptosphaeria species. Spore identification by Real-Time PCR techniques capable of detecting minute amounts of DNA of selected fungal species was combined with back-trajectory analysis, allowing the tracking of past movements of air masses using the Hybrid Single Particle Lagrangian Integrated Trajectory model. Over a study period spanning the previous decade (2006–2015) we investigated two specific events relating to the long distance transport of Leptosphaeria spp. spores to Szczecin in North-West Poland. Based on the above mentioned methods and the results obtained with the additional spore sampler located in nearby Szczecin, and operating at the ground level in an oilseed rape field, we have demonstrated that on both occasions the L. biglobosa spores originated from the Jutland Peninsula. This is the first successful attempt to combine analysis of back-trajectories of air masses with DNA-based identification of economically important pathogens of oilseed rape in Europe. In our studies, the timing of L. biglobosa ascospore dispersal in the air was unlikely to result in the infection of winter oilseed rape grown as a crop plant. However, the fungus could infect other host plants, such as vegetable brassicas, cruciferous weeds, spring rapeseed and winter rapeseed growing as a volunteer plant.

Spores of many fungal pathogens are dispersed by wind. Detection of these airborne inocula is important in forecasting both the onset and the risk of epiphytotics. Species-specific primers targeted at the internal transcribed spacer (ITS) region of Leptosphaeria maculans and L. biglobosa - the causal organisms of phoma stem canker and stem lesions of Brassica spp., including oilseed rape - were used to detect DNA extracted from particles deposited on tapes obtained from a spore trap operated in Rarwino (northwest Poland) from September to November in 2004 and 2006. The quantities of DNA assessed by traditional end-point PCR and quantitative real-time PCR were compared to microscopic counts of airborne ascospores. Results of this study showed that fluctuations in timing of ascospore release corresponded to the dynamics of combined concentrations of DNA from L. maculans and L. biglobosa, with significant positive correlations between ascospore number and DNA yield. Thus the utilization of PCR-based molecular diagnostic techniques enabled the detection, identification, and accurate quantification of airborne inoculum at the species level. Moreover, real-time PCR was more sensitive than traditional PCR, especially in years with low ascospore numbers.