Analyses of air samples for ascospores ofLeptosphaeria maculans andL.biglobosa by light microscopy and molecular techniques

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 ofLeptosphaeria maculans andL. biglobosa — the causal organisms of phoma stem canker and stem lesions ofBrassica 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 fromL. maculans andL. 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.     

Efficient Detection of Leptosphaeria maculans from Infected Seed lots of Oilseed Rape

An efficient DNA extraction protocol and polymerase chain reaction (PCR) assay for detecting Leptosphaeria maculans from infected seed lots of oilseed rape were developed. L. maculans, the causal agent of blackleg, a damaging disease in oilseeds rape/canola worldwide, was listed as a quarantine disease by China in 2009. China imports several millions of tons of oilseeds every year. So there is a high risk that this pathogen will be introduced to China via contaminated seeds. Seed contamination is one of the most significant factors in the global spread of phytopathogens. Detection of L. maculans in infected seed lots by PCR assay is difficult due to the low level of pathogen mycelium/spores on seeds and PCR inhibitors associated with the seeds of oilseed rape. In our study, these two major obstacles were overcome by the development of a two‐step extraction protocol combined with a nested PCR. This extraction protocol (kit extraction after CTAB method) can efficiently extract high‐quality DNA for PCR. Amplification results showed that the detection threshold for conventional PCR and nested PCR was, respectively, 1 ng and 10 fg of DNA per μl in mycelia samples. On contaminated seed lots of oilseed rape, the detection threshold of conventional and nested PCR was 709 fg/μl and 709 ag/μl of DNA, respectively. The DNA extraction protocol and PCR assay developed in this study can be used for rapid and reliable detection of L. maculans from infected seeds of oilseed rape .     

Influence of meteorological parameters on Leptosphaeria maculans and L. biglobosa spore release in central and eastern Poland

Leptosphaeria maculans and L. biglobosa are fungal pathogens able to cause allergic reactions in humans and infect plants of Brassica species. The rate of their development and subsequent spore release depend on weather conditions. The aim of this paper was to pinpoint the exact meteorological conditions triggering the release of L. maculans and L. biglobosa ascospores in central and eastern Poland. Multiple regressions indicated that the frequency and amount of rainfall over short periods were important in mediating spore release. The first ascospore event depended mainly on the number of rainy days during the first 10 days of July and the cumulative precipitation during July and September. The most important variables for maximum spore release were cumulative rainfall in the beginning of July and the end of September, as well as the number of days with precipitation events in the first 10 days of August. The results highlighted for the first time the importance of the days preceding the collection of oilseed rape plants from the field. Higher moisture content of senescing but still living stems play a crucial role in the early start of the ascospore season and the maximum release of ascospores. This was not yet considered to date.     

Effect of climate change on sporulation of the teleomorphs of <Emphasis Type="Italic">Leptosphaeria</Emphasis> species causing stem canker of brassicas

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.     

Impact of a resistance gene against a fungal pathogen on the plant host residue microbiome: The case of the Leptosphaeria maculans–Brassica napus pathosystem

Oilseed rape residues are a crucial determinant of stem canker epidemiology as they support the sexual reproduction of the fungal pathogen Leptosphaeria maculans. The aim of this study was to characterize the impact of a resistance gene against L. maculans infection on residue microbial communities and to identify microorganisms interacting with this pathogen during residue degradation. We used near-isogenic lines to obtain healthy and infected host plants. The microbiome associated with the two types of plant residues was characterized by metabarcoding. A combination of linear discriminant analysis and ecological network analysis was used to compare the microbial communities and to identify microorganisms interacting with L. maculans. Fungal community structure differed between the two lines at harvest, but not subsequently, suggesting that the presence/absence of the resistance gene influences the microbiome at the base of the stem whilst the plant is alive, but that this does not necessarily lead to differential colonization of the residues by fungi. Direct interactions with other members of the community involved many fungal and bacterial amplicon sequence variants (ASVs). L. maculans appeared to play a minor role in networks, whereas one ASV affiliated to Plenodomus biglobosus (synonym Leptosphaeria biglobosa) from the Leptosphaeria species complex may be considered a keystone taxon in the networks at harvest. This approach could be used to identify and promote microorganisms with beneficial effects against residue-borne pathogens and, more broadly, to decipher the complex interactions between multispecies pathosystems and other microbial components in crop residues.     

黑胫病菌侵染过程中油菜响应基因的表达分析

油菜黑胫病是造成油菜产量损失的病害之一,致病菌为Leptosphaeria biglobosa。该研究采用形态学观察和转录组测序技术,分析油菜接种病原菌Leptosphaeria biglobosa 4、12、24、36、48和96 h后的表型及基因表达变化情况,以探讨响应死体营养型真菌L.biglobosa侵染时油菜的防御反应及抗病机理,为揭示油菜与L.biglobosa互作的分子机制提供理论依据,并为培育油菜抗病品种积累了基因资源信息。结果显示:(1)接种4~96 h,叶片病斑逐渐扩大,病原菌侵染48~96 h后形成菌丝网。(2)通过RNA-Seq测序,在L.biglobosa侵染油菜的不同时间点(4、12、24、36、48和96 h)分别得到3384、2270、3802、5811、6155和7153个差异表达基因。(3)15个油菜差异表达基因的qRT-PCR检测表达水平与转录组测序结果基本一致。(4)利用短时间序列聚类和KEGG富集分析差异表达基因,结果发现植物病原菌互作、蛋白激酶、茉莉酸/乙烯/水杨酸和芥子油苷合成途径中的基因被强烈诱导表达,而且基因表达呈动态变化趋势。     

Distribution of mating‐type alleles and genetic variability in field populations of Leptosphaeria maculans in western Canada

Leptosphaeria maculans is the most important fungal pathogen of canola (Brassica napus, oilseed rape) that causes the devastating stem canker in canola fields of western Canada. The population genetic structure of L. maculans, represented by nine subpopulations from a 6‐year period and three different provinces in western Canada, was determined using ten minisatellite markers. Isolates collected at different locations in six consecutive years had an even distribution of MAT1‐1 and MAT1‐2 across the nine subpopulations. All subpopulations of L. maculans exhibited a moderate gene diversity (H = 0.356–0.585). The majority of the genetic variation occurred within subpopulations. Approximately 8% and 4% of the variations were distributed between sampling year and location, respectively. Genetic distance (F_ST) results, using analysis of molecular variation (AMOVA), indicated that subpopulation pairing within isolates by year ranged from F_ST = 0.010 to 0.109, and the location subpopulation ranged from F_ST = 0.038 to 0.085. Bayesian clustering analyses of multiloci inferred two distinct clusters in all the subpopulations examined. This study indicates a relatively high degree of gene exchange between the different L. maculans isolates. Our results suggest that this can occur in the wide growing areas of canola fields in western Canada. This gene exchange produced different gene allele frequencies and divergence between populations.     

Effects of temperature and rainfall on date of release of ascospores of Leptosphaeria maculans (phoma stem canker) from winter oilseed rape (Brassica napus) debris in the UK

Data from a controlled environment experiment investigating effects of temperature on maturation of Leptosphaeria maculans pseudothecia were used to derive equations describing the times until 30% or 50% of pseudothecia were mature as a function of temperature. A wetness sensor was developed to estimate the oilseed rape debris wetness and operated with debris exposed in natural conditions in 2000 and 2001. The maturation of L. maculans pseudothecia on debris and concentrations of airborne L. maculans ascospores were observed from 1999 to 2004. There were considerable differences between years, with the first mature pseudothecia observed in September in most years. There were linear relationships between the first date when 10% of maximum ascospore release was observed and the dates when 30% or 50% of pseudothecia were mature. By summing the daily temperature‐dependent rate of pseudothecial maturation for days after 1 August with rainfall >0.5 mm, the dates when 30% or 50% of pseudothecia were mature were predicted. There was good agreement between predicted and observed dates when 30% or 50% of pseudothecia were mature. These equations for predicting the timing of L. maculans ascospore release could be incorporated into schemes for forecasting, in autumn, the severity of phoma stem canker epidemics in the following spring/summer in the UK.     

Epidemiology of Leptosphaeria maculans in relation to forecasting stem canker severity on winter oilseed rape in the UK

In the UK, ascospores of Leptosphaeria maculans first infect leaves of oilseed rape in the autumn to cause phoma leaf spots, from which the fungus can grow to cause stem cankers in the spring. Yield losses due to early senescence and lodging result if the stem cankers become severe before harvest. The risk of severe stem canker epidemics needs to be forecast in the autumn when the pathogen is still in the leaves, since early infections cause the greatest yield losses and fungicides have limited curative activity. Currently, the most effective way to forecast severe stem canker is to monitor the onset of phoma leaf spotting in winter oilseed rape crops, although this does not allow much time in which to apply a fungicide. Early warnings of risks of severe stem canker epidemics could be provided at the beginning of the season through regional forecasts based on disease survey and weather data, with options for input of crop-specific information and for updating forecasts during the winter. The accuracy of such forecasts could be improved by including factors relating to the maturation of ascospores in pseudothecia, the release of ascospores and the occurrence of infection conditions, as they affect the onset, intensity and duration of the phoma leaf spotting phase. Accurate forecasting of severe stem canker epidemics can improve disease control and optimise fungicide use.     

Survival of A-group and B-group Leptosphaeria maculans (phoma stem canker) ascospores in air and mycelium on oilseed rape stem debris

Mycelium of Leptosphaeria maculans survived on oilseed rape stem base debris buried in sand for 2,4, 6, 8,10 or 12 months and produced pseudothecia after subsequent exposure on the surface of the ground under natural conditions for 2–4 months, but did not survive on upper stem debris buried for 2 months. Only A‐group L. maculans ascospores were produced on the stem base debris which had been buried; no B‐group ascospores were produced. Mycelium of L. maculans survived on both stem base and upper stem debris exposed on the sand surface for 2, 4, 6, 8, 10 or 12 months and pseudothecia with viable ascospores were observed at the time of sampling. Both A‐group L. maculans (predominant on stem bases) and B‐group L. maculans (predominant on upper stems) ascospores were produced on unburied stem base and upper stem debris. Thus B‐group L. maculans survived longer on unburied debris than on buried debris. A‐group ascospores which were exposed in dry air in darkness at 5–20°C survived longer than B‐group ascospores; 10–37% of A‐group ascospores, compared with 2–31% of B‐group ascospores, survived after 35 days.     

Molecular and phenotypic characterization of near isogenic lines at QTL for quantitative resistance to Leptosphaeria maculans in oilseed rape (Brassica napus L.)

The most common and effective way to control phoma stem canker (blackleg) caused by Leptosphaeria maculans in oilseed rape (Brassica napus) is by breeding resistant cultivars. Specific resistance genes have been identified in B. napus and related species but in some B. napus cultivars resistance is polygenic [mediated by quantitative trait loci (QTL)], postulated to be race non-specific and durable. The genetic basis of quantitative resistance in the French winter oilseed rape ‘Darmor’, which was derived from ‘Jet Neuf’, was previously examined in two genetic backgrounds. Stable QTL involved in blackleg resistance across year and genetic backgrounds were identified. In this study, near isogenic lines (NILs) were produced in the susceptible background ‘Yudal’ for four of these QTL using marker-assisted selection. Various strategies were used to develop new molecular markers, which were mapped in these QTL regions. These were used to characterize the length and homozygosity of the ‘Darmor-bzh’ introgressed segment in the NILs. Individuals from each NIL were evaluated in blackleg disease field trials and assessed for their level of stem canker in comparison to the recurrent line ‘Yudal’. The effect of QTL LmA2 was clearly validated and to a lesser extent, QTL LmA9 also showed an effect on the disease level. This work provides valuable material that can be used to study the mode of action of genetic factors involved in L. maculans quantitative resistance.     

A Comparison of the Disease Reactions of Stems and Detached Leaves of Soil and in vitro Grown Plants and Regenerants of Oilseed Rape to Leptosphaeria maculans and Protocols for Selection for Novel Disease Resistance

Protocols for selecting plant tissues of winter oilseed rape (Brassica napus subsp. oleifera) with resistance to Leptosphaeria maculans by either stem or leaf inoculation of both soil and in vitro grown plant material are described. The stem inoculation procedure gave good correlation (r = 0. 92) between the 50 day stem disease scores of eight out of nine cultivars of soil grown winter oilseed rape inoculated with isolate 41A4 of L. maculans and the N. A. B. esistance ratings or resistance data from field trials. The exception was the cultivar Liradonna. Inoculation of stems of five cultivars with isolates 41A4, 433 and 478 indicated a range of isolate virulence 478 > 41A4 > 433. This was the inverse of that observed in leaf inoculations. Application of the stem inoculation procedure to in vitro shoot cultures allowed differentiation of resistant and susceptible cultivars, including the cultivar Liradonna, after 20 days incubation at 20°C. The protocol was also applicable to plantlets regenerated from thin cell layer explants grown in vitro. Inoculations with isolate 433 allowed the differentiation of resistant, intermediately resistant and susceptible leaf material of soil grown plants, when leaf discs from young leaves were incubated on water agar supplemented with BAP (1 × 10^?5 M) at 25°C for 10 days. Intermediately resistant leaves were resistant after 10 days and susceptible after 15 days of incubation. Leaves of shoot cultures grown in vitro were more susceptible than the corresponding soil grown material. However, inoculation of old leaves with isolate 41A4 (an isolate of less virulence on leaves than 433) distinguished the cultivars after 15 days of incubation. These protocols allow the accurate assessment of resistance to L. maculans at the stem or leaf level and are of use in traditional as well as in vitro selection programmes.     

The Use of Ergosterol as a Quantitative Measure of the Resistance of Cultured Tissues of Brassica napus ssp. oleifera to Leptosphaeria maculans

A simplified method for the quantitative assessment of the fungal lipid ergosterol was used to assess the levels of infection in tissue cultures of oilseed rape (Brassica napus ssp. oleifera) inoculated with Leptosphaeria maculans. The growth of L. maculans in liquid culture throughout a 36-day period correlated well (r = 0·92) with the amount of ergosterol extracted from the mycelium. There were significant differences (P < 0·05) in the amount of ergosterol extracted from infected thin cell layer (TCL) explants and callus tissue of two resistant and three susceptible cultivars of oilseed rape. Amounts of ergosterol extracted from resistant cultivars were < 100 (g and from susceptible > 100 (g. The mean amounts of ergosterol extracted from shoot cultures of two resistant and four susceptible cultivars were similar to those for TCL explants and callus tissue, although the values obtained were variable. This technique can be used in in vitro breeding programmes to accurately assess the resistance of tissue cultures of B. napus to L. maculans and could also have value in conventional breeding programmes.     

Assessing Quantitative Resistance against Leptosphaeria maculans (Phoma Stem Canker) in Brassica napus (Oilseed Rape) in Young Plants

Quantitative resistance against Leptosphaeria maculans in Brassica napus is difficult to assess in young plants due to the long period of symptomless growth of the pathogen from the appearance of leaf lesions to the appearance of canker symptoms on the stem. By using doubled haploid (DH) lines A30 (susceptible) and C119 (with quantitative resistance), quantitative resistance against L. maculans was assessed in young plants in controlled environments at two stages: stage 1, growth of the pathogen along leaf veins/petioles towards the stem by leaf lamina inoculation; stage 2, growth in stem tissues to produce stem canker symptoms by leaf petiole inoculation. Two types of inoculum (ascospores; conidia) and three assessment methods (extent of visible necrosis; symptomless pathogen growth visualised using the GFP reporter gene; amount of pathogen DNA quantified by PCR) were used. In stage 1 assessments, significant differences were observed between lines A30 and C119 in area of leaf lesions, distance grown along veins/petioles assessed by visible necrosis or by viewing GFP and amount of L. maculans DNA in leaf petioles. In stage 2 assessments, significant differences were observed between lines A30 and C119 in severity of stem canker and amount of L. maculans DNA in stem tissues. GFP-labelled L. maculans spread more quickly from the stem cortex to the stem pith in A30 than in C119. Stem canker symptoms were produced more rapidly by using ascospore inoculum than by using conidial inoculum. These results suggest that quantitative resistance against L. maculans in B. napus can be assessed in young plants in controlled conditions. Development of methods to phenotype quantitative resistance against plant pathogens in young plants in controlled environments will help identification of stable quantitative resistance for control of crop diseases.     

A new avirulence gene of Leptosphaeria maculans,AvrLm14, identifies a resistance source in American broccoli (Brassica oleracea) genotypes

In many cultivated crops, sources of resistance to diseases are sparse and rely on introgression from wild relatives. Agricultural crops often are allopolyploids resulting from interspecific crosses between related species, which are sources of diversity for resistance genes. This is the case for Brassica napus (oilseed rape, canola), an interspecific hybrid between Brassica rapa (turnip) and Brassica oleracea (cabbage). B. napus has a narrow genetic basis and few effective resistance genes against stem canker (blackleg) disease, caused by the fungus Leptosphaeria maculans, are currently available. B. rapa diversity has proven to be a valuable source of resistance (Rlm, LepR) genes, while B. oleracea genotypes were mostly considered susceptible. Here we identified a new resistance source in B. oleracea genotypes from America, potentially effective against French L. maculans isolates under both controlled and field conditions. Genetic analysis of fungal avirulence and subsequent cloning and validation identified a new avirulence gene termed AvrLm14 and suggested a typical gene-for-gene interaction between AvrLm14 and the postulated Rlm14 gene. AvrLm14 shares all the usual characteristics of L. maculans avirulence genes: it is hosted in a genomic region enriched in transposable elements and heterochromatin marks H3K9me3, its expression is repressed during vegetative growth but shows a strong overexpression 5–9 days following cotyledon infection, and it encodes a small secreted protein enriched in cysteine residues with few matches in databases. Similar to the previously cloned AvrLm10-A, AvrLm14 contributes to reduce lesion size on susceptible cotyledons, pointing to a complex interplay between effectors promoting or reducing lesion development.     

Besides stem canker severity,oilseed rape host genotype matters for the production of Leptosphaeria maculans fruit bodies

For fungal cyclic epidemics on annual crops, the pathogen carry-over is an important but poorly documented step. Plant resistance affects the pathogen development within the epidemics but we lack data on the inter-annual transmission of inoculum. For Leptosphaeria maculans on 15 oilseed rape genotypes in field during 4 growing seasons, stem canker severity was visually scored at harvest. The number of fruit bodies produced on incubated stubble was quantified using an automated image analysis framework. Our results confirm that fruit body production increases with disease severity and is significantly affected by host genotype and nitrogen supply. Tracking individual stems through incubation, we confirm for the first time that the oilseed rape genotype has a direct effect on inoculum production, not only disease severity. This major effect of genotype on inoculum carry-over should be taken into account in models of varietal deployment strategies.     

Association mapping of quantitative resistance for <Emphasis Type="Italic">Leptosphaeria maculans</Emphasis> in oilseed rape (<Emphasis Type="Italic">Brassica napus</Emphasis> L.)

Stem canker caused by the fungus Leptosphaeria maculans is a major disease of Brassica napus. Quantitative resistance factors appear to be important components for effective and durable control of this pathogen. Quantitative trait loci (QTL) for stem canker resistance have previously been identified in the Darmor variety. However, before these QTL can be used in marker-assisted selection (MAS) to breed resistant varieties, they must be validated in a wide range of genetic backgrounds. We used an association mapping approach to confirm the markers located within the QTL previously identified in Darmor and establish their usefulness in MAS. For this, we characterized the molecular diversity of an oilseed rape collection of 128 lines showing a large spectrum of responses to infection by L. maculans, using 72 pairs of primers for simple sequence repeat and other markers. We used different association mapping models which either do or do not take into account the population structure and/or family relatedness. In all, 61 marker alleles were found to be associated with resistance to stem canker. Some of these markers were associated with previously identified QTL, which confirms their usefulness in MAS. Markers located in regions not harbouring previously identified QTL were also associated with resistance, suggesting that new QTL or allelic variants are present in the collection. All of these markers associated with stem canker resistance will help identify accessions carrying desirable alleles and facilitate QTL introgression.