Genetic variability and distribution of mating type alleles in field populations of Leptosphaeria maculans from France

Leptosphaeria maculans is the most ubiquitous fungal pathogen of Brassica crops and causes the devastating stem canker disease of oilseed rape worldwide. We used minisatellite markers to determine the genetic structure of L. maculans in four field populations from France. Isolates were collected at three different spatial scales (leaf, 2-m2 field plot, and field) enabling the evaluation of spatial distribution of the mating type alleles and of genetic variability within and among field populations. Within each field population, no gametic disequilibrium between the minisatellite loci was detected and the mating type alleles were present at equal frequencies. Both sexual and asexual reproduction occur in the field, but the genetic structure of these populations is consistent with annual cycles of randomly mating sexual reproduction. All L. maculans field populations had a high level of gene diversity (H = 0.68 to 0.75) and genotypic diversity. Within each field population, the number of genotypes often was very close to the number of isolates. Analysis of molecular variance indicated that >99.5% of the total genetic variability was distributed at a small spatial scale, i.e., within 2-m2 field plots. Population differentiation among the four field populations was low (GST < 0.02), suggesting a high degree of gene exchange between these populations. The high gene flow evidenced here in French populations of L. maculans suggests a rapid countrywide diffusion of novel virulence alleles whenever novel resistance sources are used.     

Genetic diversity and population structure of Leptosphaeria maculans isolates in Western Canada

Leptosphaeria maculans is a serious concern for canola production worldwide. For effective disease management, knowledge of the pathogen's genetic variability and population structure is a prerequisite. In this study, whole-genome sequencing was performed for 162 of 1590 L. maculans isolates collected in the years 2007–2008 and 2012–2014 in Western Canada. DNA variants in genome-wide and specific regions including avirulence (Avr) genes were characterized. A total of 31,870 high-quality polymorphic DNA variants were used to study L. maculans genetic diversity and population structure. Cluster analysis showed that 150 isolates were clustered into 2 main groups and 4 subgroups by DNA variants located in either Avr or small secreted protein-encoding genes and into 2 main groups and 6 subgroups by genome-wide variants. The analysis of nucleotide diversity and differentiation also confirmed genetic variation within a population and among populations. Principal component analysis with genome-wide variants showed that the isolates collected in 2012–2014 were more genetically diverse than those collected in 2007–2008. Population structure analysis discovered three distinct sub-populations. Although isolates from Saskatchewan and Alberta were of similar genetic composition, Manitoba isolates were highly diverse. Genome-wide association study detected DNA variants in genes AvrLm4-7, Lema_T86300, and Lema_T86310 associated with the years of collection.     

Characterization of an opsin gene from the ascomycete Leptosphaeria maculans.

An opsin gene (ops) has been characterized from Leptosphaeria maculans, the ascomycete that causes black-leg disease of Brassica species. This is the second opsin identified outside the archaeal and animal kingdoms. The gene encodes a predicted protein with high similarity (70.3%) and identity (53.3%) to the nop-1 opsin of another ascomycete Neurospora crassa. The L. maculans opsin also has identical amino acid residues in 20 of the 22 residues in the retinal-binding pocket of archaeal opsins. Opsin, on the fourth largest chromosome of L. maculans and 22 cM from the mating type locus, is the first cloned gene to be mapped in L. maculans. Opsin is transcribed at high levels in mycelia grown in the presence and absence of light; this pattern is in contrast with that of the N. crassa opsin, which is transcribed only in the light.     

Genetic and Physical Variability at the Mating Type Locus of the Oomycete, Phytophthora Infestans

Mating type in the oomyceteous fungus, Phytophthora infestans, is determined by a single locus. In a previous study of a few isolates, the locus segregated in a manner genetically consistent with its linkage to a system of balanced lethal loci. To determine the prevalence of this phenomenon within P. infestans, genetic analyses were performed using isolates representative of the diversity within the species that had been selected by DNA fingerprinting using probes linked to mating type. Non-Mendelian segregation of the mating type locus was observed in crosses performed with each isolate. An unusual group of isolates was identified in which the mating type determinants had been rearranged within the genome; these strains also produced an aberrantly large number of self-fertile progeny. Curiously, in all isolates, markers linked to the mating type locus appeared prone to duplication, transposition, deletion, or other rearrangement. This was not observed for loci unlinked to mating type. Data from the crosses and analyses of marker variation were used to erect models to explain the bases of mating type determination and of the unusual segregation of the chromosomal region containing the mating type locus.     

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 .     

Expression of resistance to Leptosphaeria maculans in Brassica napus double haploid lines in France and Australia is influenced by location

Blackleg, caused by Leptosphaeria maculans, is a major disease of oilseed rape (Brassica napus), worldwide, including Australia and France. The aims of these studies were first, to determine if higher levels of resistance to L. maculans could be generated in double haploid (DH) lines derived from spring‐type B. napus cv. Grouse, which has a good level of field resistance to blackleg; and second, to determine whether the resistance to blackleg disease of individual DH lines responds differentially to different L. maculans field populations within and between the two countries. DH lines were extracted from cv. Grouse and tested in field experiments carried out in both France and Australia against natural L. maculans populations. Extracting and screening DH lines were an effective means to select individual lines with greatly improved expression of resistance to blackleg crown canker disease in comparison with the original parental population. However, relative disease resistance rankings for DH lines were not always consistent between sites. The higher level of resistance in France was shown to be because of a high expression level of quantitative resistance in the French growing conditions. Big differences were observed for some DH lines between the 2004 and the 2005 field sites in Australia where the L. maculans populations differed by their virulence on single dominant gene‐based resistant lines derived from Brassica rapa ssp. sylvestris. This differential behaviour could not be clearly explained by the specific resistance genes until now identified in these DH lines. This investigation highlights the potential to derive DH lines with superior levels of resistance to L. maculans compared with parental populations. However, in locations with particularly high pathogen diversity, such as in southern Australia, multiyear and multisite evaluations should be performed to screen for the most efficient material in different situations.     

Transmission of Leptosphaeria maculans from a cropping season to the following one

Current modelling of inoculum transmission from a cropping season to the following one relies on the extrapolation of kernels estimated on data at short distances from punctual sources, because data collected at larger distances are scarce. We estimated the dispersal kernel of Leptosphaeria maculans ascospores from stubble left after harvest in the summer previous to newly sown oilseed rape fields, using phoma stem canker autumn disease severity. We built a dispersal model to analyse the data. Source strengths are described in the spatial domain covered by source fields by a log‐Gaussian spatial process. Infection potentials in the following season are described in the space consisting of the target fields, by a convolution of sources and a power‐exponential dispersal kernel. Data were collected on farmers' fields considered as sources in 2009 and 2011 (72 and 39 observation points) and as targets in 2010 and 2012 (172 and 200 points). We applied the Bayesian approach for model selection and parameter estimation. We obtained fat tail kernels for both data sets. This estimation is the first from data acquired over distances of 0 to 1000 m, using several non‐punctual inoculum sources. It opens the prospect of refining the existing simulators, or developing disease risk maps.     

Hidden Genetic Variability within Electromorphs in Finite Populations

The amount of hidden genetic variability within electromorphs in finite populations is studied by using the infinite site model and stepwise mutation model simultaneously. A formula is developed for the bivariate probability generating function for the number of codon differences and the number of electromorph state differences between two randomly chosen cistrons. Using this formula, the distribution as well as the mean and variance of the number of codon differences between two identical or nonidentical electromorphs are studied. The distribution of the number of codon differences between two randomly chosen identical electromorphs is similar to the geometric distribution but more leptokurtic. Studies are also made on the number of codon differences between two electromorphs chosen at random one from each of two populations which have been separated for an arbitrary number of generations. It is shown that the amount of hidden genetic variability is very large if the product of effective population size and mutation rate is large.     

Inheritance of chromosomal length polymorphisms in the ascomycete Leptosphaeria maculans

Pulsed field gel electrophoresis experiments show that chromosomal length polymorphisms are produced during meiosis in the ascomycetous plant pathogen Leptosphaeria maculans. Homologues in tetrads of L. maculans were identified on the basis of their binding to chromosome-specific probes that included β-tubulin, nitrate reductase, 18S ribosomal DNA and two Random Amplified Polymorphic DNA (RAPD) markers. Changes in size of homologues were followed during meiosis. Significant karyotype variation was evident due to the random assortment of parental homologues of different sizes. In most cases, the progeny had the same-sized homologues as the parents; however, in some instances novel-sized homologues were detected that varied in size from those of the parents by up to 50 kb. Our results are consistent with the hypothesis that these novel chromosomal length polymorphisms are produced by reciprocal recombination between parental homologous chromosomes of unequal sizes.     

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.     

Frequency Distribution of Esterase-5 Alleles in Two Populations of DROSOPHILA PSEUDOOBSCURA

Statistical tests comparing allele frequencies in natural populations with those predicted by various theories of genic variation depend critically on the accurate enumeration of alleles. This study used a series of five sequential electrophoretic conditions to characterize the allele frequency distributions of esterase-5 in two large population samples of Drosophila pseudoobscura from California. In Standard chromosome lines 12 electromorphs were discriminated using a single electrophoretic condition. When four additional criteria were used, the number of electromorphs increased to 41, 33 in one population and 22 in the other. Both populations had the same two alleles in high frequency, with other alleles present in frequencies of 6% or less. Although each population had a number of unique alleles, a χ² contingency test demonstrated no significant genetic divergence between them. A statistical comparison of allele frequencies in both populations with that predicted by neutral models suggests that the individual and combined distributions deviate from neutrality in the direction of purifying selection.—Sex-Ratio chromosomes differed markedly from Standard chromosomes in both allelic content and diversity. In 32 Sex-Ratio chromosomes from one population only three alleles were found, all of which were detected under the initial "standard" electrophoretic conditions. Moreover, none of these alleles was found in the Standard chromosome lines.     

Pathogenicity studies on isolates of Leptosphaeria maculans from brassica seed production crops in south-east England

Out of 117 isolates of Leptosphaeria maculans collected from a range of brassica seed crops in south-east England, 26 were virulent when tested on cabbage cv. January King. Of these, 24 were isolated from oil-seed rape (Brassica napus) and the remainder from a swede (B. napus) and a cabbage (B. oleracea) seed crop. Virulent strains were derived from infected stems and from single ascospores of the sexual state present on diseased stubble. In glasshouse tests virulent isolates were not host-specific and caused severe cankers on cabbage, kale, swede, turnip and oil-seed rape; under field conditions, ascospores originating from diseased oil-seed rape stubble produced severe cankers and high levels of seed-borne infection in eight horticultural and vegetable brassica hosts (B. napus, B. campestris and B. oleracea). Virulent and non-virulent types could be distinguished by certain cultural characteristics. On nutrient agar virulent types grew slowly, irregularly and quickly staled whereas non-virulent types grew rapidly, regularly and did not stale. The latter also produced a yellow-brown pigment in liquid culture. The potential for cross-infection of virulent strains originating from oil-seed rape to other brassica seed crops has serious implications for disease incidence on forage and vegetable brassica seed crops.     

Presence of Leptosphaeria maculans Group A and Group B Isolates in Sweden

Leptosphaeria maculans isolates have been assigned to one of two groups, A or B, on the basis of differences in their characteristics. Group A can further be divided into pathogenicity groups (PG) 2, 3 and 4 and group B into PG1. To determine if isolates belonging to the aggressive canker forming group A are present in Sweden, physiological and genetic characterisation of 120 isolates collected in the year 2000 were performed. Thirty‐seven isolates were classified as belonging to pathogenicity group PG3 and 63 isolates as PG4, based on a cotyledon assay. Twenty isolates did not cause any symptoms at all, and were classified as PG1. When comparing two geographical regions, Skåne and Östergötland, equal numbers of PG3 and PG4 isolates were found. By analysing the isolates by PCR, the collection was further classified into 100 group A isolates and 20 group B isolates. A corresponding classification of the isolates was observed when the ability to produce pigments in Czapek Dox broth was examined. The results showed a clear predominance of group A. This was also the case for the isolate collection from 2001. In a detailed survey of disease development in a L. maculans infected winter oilseed rape field in southern Sweden (Skåne), basal stem canker was not observed until early June Although the disease index value increased from 8.4 in June to 18.0 in July, few severely damaged plants were observed before harvest in mid‐July, despite infection with group A isolates.     

Resistance to Leptosphaeria maculans in Hybrids of Brassica oleracea and Brassica insularis

The inheritance of hypersensitive resistance to Leptosphaeria maculans in a cross between B. oleracea var. alboglabra and B. insularis was studied. Analyses of F₁ and F₂ progeny suggested that resistance is determined by two dominant, independently-segregating genes. F₁ hybrids were semifertile but normal levels of fertility were restored in a proportion of the F₂ progeny.     

Morphological and molecular identification of Leptosphaeria maculans in canola seeds and flowers collected from the North Iran

Blackleg disease, caused by Leptosphaeria maculans, is one of the most important diseases of rapeseed Brassica napus in Iran as in other regions of the world. The samples including canola petals and seeds were collected during 2014–2015 from canola field in North Iran. Isolates characteristics of fungus were assessed based on the colony growth rate and pycnidia in Potato Dextrose Agar. The pycnidia of the fungus were black, globose to subglobose in shape, the single-celled conidia, hyaline and fusiform with diameters of 4–5 × 1.5–2 μm. Most of the isolates were produced pigment in the liquid culture in variable color brown to black. Thirteen isolates were then separated into pathogenicity groups based on the interactions on B. napus differential cultivars. For the direct detection of seed contamination with L. maculans, PCR was developed using specific primers pair (LmacF, LmacR) which can amplify ITS1 and ITS2 along with the 5.8S rRNA region of L. maculans genome. Based on the followed information and sequence analysis, the fungal isolates from these samples were identified as L. maculans. The findings of this research showed that the disease was aggressive and highly distributed from infested seeds to oilseed rape fields.     

4个HLA等位基因在5个民族中分布的多样性研究

应用ARMS-SSP结合的方法调查与AIDS相关的HLA等位基因,即HLA-A*02,B*35,B*27,B*57四个基因在五个民族群体（云南汉族、彝族、傣族、新疆维吾尔族、广西壮族）中的分布情况,并进行了相应的遗传学分析。结果显示,HLA-A*02在壮族和汉族中检出的阳性样本频率非常高,在另外几个民族中的分布频率偏低;B*35基因在各民族中分布差异不大;B*27在傣族中的分布频率较高,汉族、彝族和壮族中分布的频率差异不大;B*57则在各民族中的分布差异不大;保护性的等位基因即A*02,B*27,B*57的基因型频率在5个民族中有极显著的差异。此课题增进了对我国部分AIDS流行地区AIDS相关HLA等位基因型遗传背景的了解,是对HLA与AIDS相关性研究资料的有益补充。