MBC tolerance in aggressive and non-aggressive isolates of Ceratocystis ulmi

In selection experiments, tolerance to 0–5 ppm methyl benzimidazole-2-ylcarbamate (MBC) occurred at a frequency of c. 1 in 1–3 ×10⁸ conidia in both aggressive and non-aggressive isolates of Ceratocystis ulmi. The tolerant strains were inhibited by 5 ppm MBC, however, and attempts to select strains tolerant to 10 ppm were unsuccessful. In each of three isolates examined, tolerance remained stable after fifteen successive transfers on fungicide-free medium. Genetic control was nuclear and probably conditioned by a single gene. It is thought unlikely that the appearance of tolerant strains in nature will jeopardize the use of MBC for the control of Dutch elm disease.     

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.     

The phytopathogenic fungi Leptosphaeria maculans and Leptosphaeria biglobosa: chemotaxonomical characterization of isolates and metabolite production in different culture media

Previous molecular chemotaxonomic analyses of isolates of the plant pathogenic fungus Leptosphaeria maculans (Desm.) Ces. et de Not. (asexual stage Phoma lingam (Tode ex Fr.) Desm.) in a chemically defined medium suggested that this species complex was composed of at least three distinct groups. Subsequently, a group within L. maculans was classified as Leptosphaeria biglobosa, on the basis of morphologic characteristics and the lack of sexual crossing. To obtain clarification regarding the metabolite profiles of the various groups or species of blackleg fungi, the objectives of this work were (i) to determine the chemical structures of metabolites produced by Canadian V isolates and Polish-type isolates in potato dextrose broth (PDB) and (ii) to determine the chemotaxonomic relationship among French isolates of L. biglobosa and among Canadian W isolates and Thlaspi isolates of L. maculans. Here, we report for the first time that Canadian V isolates grown in PDB produced 2,4-dihydroxy-3,6-dimethylbenzaldehyde, a metabolite never reported from L. maculans, but none of the usual phytotoxins (sirodesmins). In addition, we report a new metabolite, 2-[2-(5-hydroxybenzofuranyl)]-3-(4-hydroxyphenyl)propanenitrile, from Polish-type isolates of L. maculans grown in PDB and the metabolite profiles of 16 Thlaspi isolates. The metabolite profiles of Thlaspi isolates indicate that these are part of two distinct groups, the Polish W group and the Canadian W group, i.e., L. biglobosa. Finally, we demonstrate that the metabolite profiles of the French isolates classified as L. biglobosa are similar to those of Canadian W isolates.     

Identification of rickettsial isolates at the species level using multi-spacer typing

Background  

In order to estimate whether multi-spacer typing (MST), based on the sequencing of variable intergenic spacers, could serve for the identification of Rickettsia at the species level, we applied it to 108 rickettsial isolates or arthropod amplicons that include representatives of 23 valid Rickettsia species.     

Detoxification of the phytoalexin brassinin by isolates of Leptosphaeria maculans pathogenic on brown mustard involves an inducible hydrolase

Brassinin is a phytoalexin produced by plants from the family Brassicaceae that displays antifungal activity against a number of pathogens of Brassica species, including Leptosphaeria maculans (Desm.) Ces. et de Not. [asexual stage Phoma lingam (Tode ex Fr.) Desm.] and L. biglobosa. The interaction of a group of isolates of L. maculans virulent on brown mustard (Brassica juncea) with brassinin was investigated. The metabolic pathway for degradation of brassinin, the substrate selectivity of the putative detoxifying hydrolase, as well as the antifungal activity of metabolites and analogs of brassinin are reported. Brassinin hydrolase activity was detectable only in cell-free homogenates resulting from cultures induced with brassinin, N'-methylbrassinin, or camalexin. The phytoalexin camalexin was a substantially stronger inhibitor of these isolates than brassinin, causing complete growth inhibition at 0.5mM.     

Inheritance of pathogenicity and cultural characters in Ceratocystis ulmi: hybridization of aggressive and non-aggressive strains

When British isolates of Ceratocystis ulmi were surveyed for compatibility type, both A- and B-types were found in the non-aggressive strain, but only the B-type in the aggressive strain. Single ascospore progeny from crosses between compatible aggressive and non-aggressive isolates showed a near-normal growth rate distribution, with a mean lying between the parents. Many grew either faster than the aggressive or slower than the non-aggressive parent. The progeny were highly variable in culture morphology and could not be classified in terms of the parental types. When inoculated into English elm they showed a marked skewness towards low pathogenicity. None approached the aggressive strain in pathogenicity. It is concluded that the above characters are under polygenic control, and that the aggressive strain could not arise from the non-aggressive by a simple mutation. The results suggest that the two strains may be reproductively isolated.     

Seedling and adult plant peaetions of Brassica napus-B. juncea recombinant lines towards A- and B-group isolates of Leptosphaeria maculans

The Brassica napus-B. juncea recombinant lines MX and MXS carrying a B. juncea major gene (JLml) in the genetic background of a spring- or a winter type B. napus cultivar, respectively, were tested for their resistance level to Leptosphaeria maculans under controlled conditions. Inoculation with three A-and four B-group individual isolates and with different mixtures of isolates realised within or between these groups was performed on cotyledons, leaves and stems. Cotyledons and leaves of the two recombinant lines were more resistant to A-group isolates than those of B. napus cultivars, except for one isolate recovered from the MX line. The recombinant lines were susceptible at cotyledon stage and resistant on leaves to B-group isolates, as were B. napus cultivars. On stems, severe cortical damage was usually produced on B. napus cultivars by some A-group isolates, whereas B-group isolates induced pith blackening on all genotypes. Stems of the MX line and the resistant donor species (B. juncea cv. Picra) were more resistant than those of the susceptible B. napus (cv. Westar) to the individual A-group isolates. Cultivar Picra was the most susceptible genotype to pith infection caused by the B-group isolates. The consequence of the host pathogen differential interactions on the durability of the monogenic resistance to L. maculans introduced from B. juncea into B. napus is discussed.     

Genome structure impacts molecular evolution at the AvrLm1 avirulence locus of the plant pathogen Leptosphaeria maculans

Leptosphaeria maculans, a dothideomycete fungus causing stem canker on oilseed rape, develops gene-for-gene interactions with its host plants. It has the ability to rapidly adapt to selection pressure exerted by cultivars harbouring novel resistance genes as exemplified recently by the 3-year evolution towards virulence at the AvrLm1 locus in French populations. The AvrLm1 avirulence gene was recently cloned and shown to be a solo gene within a 269 kb non-coding, heterochromatin-like region. Here we describe the sequencing of the AvrLm1 genomic region in one avirulent and two virulent isolates to investigate the molecular basis of evolution towards virulence at the AvrLm1 locus. For these virulent isolates, the gain of virulence was linked to a 260 kb deletion of a chromosomal segment spanning AvrLm1 and deletion breakpoints were identical or similar. Among the 460 isolates analysed from France, Australia and Mexico, a similar large deletion was apparent in > 90% of the virulent isolates. Deletion breakpoints were also strongly conserved in most of the virulent isolates, which led to the hypothesis that a unique deletion event leading to the avrLm1 virulence has diffused in pathogen populations. These data finally suggest that retrotransposons are key drivers in genome evolution and adaptation to novel selection pressure in L. maculans.     

Absence of isolation by distance patterns at the regional scale in the fungal plant pathogen Leptosphaeria maculans

Outcomes of host-pathogen coevolution are influenced by migration rates of the interacting species. Reduced gene flow with increasing spatial distance between populations leads to spatial genetic structure, as predicted by the isolation by distance (IBD) model. In wind-dispersed plant-pathogenic fungi, a significant spatial genetic structure is theoretically expected if local spore dispersal is more frequent than long-distance dispersal, but this remains to be documented by empirical data. For 29 populations of the oilseed rape fungus Leptosphaeria maculans sampled from two French regions, genetic structure was determined using eight minisatellite markers. Gene diversity (H = 0.62-0.70) and haplotypic richness (R = 0.96-1) were high in all populations. No linkage disequilibrium was detected between loci, suggesting the prevalence of panmictic sexual reproduction. Analysis of molecular variance showed that > 97% of genetic diversity was observed within populations. Genetic differentiation was low among populations (F(st) < 0.05). Although direct methods previously revealed short-distance dispersal for L. maculans, our findings of no correlation between genetic and geographic distances among populations illustrate that the IBD model does not account for dispersal of the fungus at the spatial scale we examined. These results indicate high gene flow among French populations of L. maculans, suggesting high dispersal rates and/or large effective population sizes, two characteristics giving the pathogen high evolutionary potential against the deployment of resistant oilseed rape cultivars.     

Evolution of linked avirulence effectors in Leptosphaeria maculans is affected by genomic environment and exposure to resistance genes in host plants

Brassica napus (canola) cultivars and isolates of the blackleg fungus, Leptosphaeria maculans interact in a 'gene for gene' manner whereby plant resistance (R) genes are complementary to pathogen avirulence (Avr) genes. Avirulence genes encode proteins that belong to a class of pathogen molecules known as effectors, which includes small secreted proteins that play a role in disease. In Australia in 2003 canola cultivars with the Rlm1 resistance gene suffered a breakdown of disease resistance, resulting in severe yield losses. This was associated with a large increase in the frequency of virulence alleles of the complementary avirulence gene, AvrLm1, in fungal populations. Surprisingly, the frequency of virulence alleles of AvrLm6 (complementary to Rlm6) also increased dramatically, even though the cultivars did not contain Rlm6. In the L. maculans genome, AvrLm1 and AvrLm6 are linked along with five other genes in a region interspersed with transposable elements that have been degenerated by Repeat-Induced Point (RIP) mutations. Analyses of 295 Australian isolates showed deletions, RIP mutations and/or non-RIP derived amino acid substitutions in the predicted proteins encoded by these seven genes. The degree of RIP mutations within single copy sequences in this region was proportional to their proximity to the degenerated transposable elements. The RIP alleles were monophyletic and were present only in isolates collected after resistance conferred by Rlm1 broke down, whereas deletion alleles belonged to several polyphyletic lineages and were present before and after the resistance breakdown. Thus, genomic environment and exposure to resistance genes in B. napus has affected the evolution of these linked avirulence genes in L. maculans.     

Differential Reactions Between the Genus Brassica and Aggressive Single Spore Isolates of Leptosphaeria maculans

Based on sirodesmin production and pathogenicity tests with Brassica cotyledons, strains of Leptosphaeria maculans were classified as aggressive (pathotype group A), or non-aggressive (pathotype group NA). NA strains caused no differential reactions. However, the pathotype group A could be divided into 5 sub-groups. AO isolates caused non-sporulating lesions with dark margins while Al isolates sporulated on cotyledons of most Brassica hosts tested. Only the cv. Erfurter Zwerg (B. oleracea var. botrytis) reacted resistant against AO and Al strains. A2 isolates caused resistance reactions on cotyledons of the cvs. Quinta (B. napus var. oleifera) and Runde (B. rapa var. rapa). A3 and A4 isolates were not detectable in our material. Isolates of these pathotype groups, supplied by Dr. P. H. Williams, Madison, USA, caused differential reactions on the oilseed rape cvs. Glacier, Quinta and Jet Neuf. In glasshouse and field experiments strains of pathotype groups Al, A2 and NA were tested on true leaves and hypocotyls of different oilseed rape cultivars. The low aggressiveness of NA isolates was evident under all experimental conditions. A2 strains caused resistance reactions not only on cotyledons but also on true leaves and hypocotyls of Quinta. Moreover, compared with Al, pathotype group A2 was more aggressive on hypocotyls of Jet Neuf. The resistance of this cultivar against Al isolates was clearly visible on hypocotyls and true leaves but not on cotyledons.     

Multilocus sequence typing supports the hypothesis that cow- and human-associated Salmonella isolates represent distinct and overlapping populations

A collection of 179 human and 156 bovine clinical Salmonella isolates obtained from across New York state over the course of 1 year was characterized using serotyping and a multilocus sequence typing (MLST) scheme based on the sequencing of three genes (fimA, manB, and mdh). The 335 isolates were differentiated into 52 serotypes and 72 sequence types (STs). Analyses of bovine isolates collected on different farms over time indicated that specific subtypes can persist over time on a given farm; in particular, a number of farms showed evidence for the persistence of a specific Salmonella enterica serotype Newport sequence type. Serotypes and STs were not randomly distributed among human and bovine isolates, and selected serotypes and STs were associated exclusively with either human or bovine sources. A number of common STs were geographically widespread. For example, ST6, which includes isolates representing serotype Typhimurium as well as the emerging serotype 4,5,12:i:-, was found among human and bovine isolates in a number of counties in New York state. Phylogenetic analyses supported the possibility that serotype 4,5,12:i:- is closely related to Salmonella serotype Typhimurium. Salmonella serotype Newport was found to represent two distinct evolutionary lineages that differ in their frequencies among human and bovine isolates. A number of Salmonella isolates carried two copies of manB (33 isolates) or showed small deletion events in fimA (nine isolates); these duplication and deletion events may provide mechanisms for the rapid diversification of Salmonella surface molecules. We conclude that the combined use of an economical three-gene MLST scheme and serotyping can provide considerable new insights into the evolution and transmission of Salmonella.     

Structure and biological activity of maculansin A, a phytotoxin from the phytopathogenic fungus Leptosphaeria maculans

During a search for elicitors and phytotoxins produced by virulent isolates of the phytopathogenic fungus Leptosphaeria maculans (Desm.) Ces. et de Not. [asexual stage Phomalingam (Tode ex Fr.) Desm.], the selective phytotoxin maculansin A was isolated and its structure determined by analysis of spectroscopic data and chemical degradation. Maculansin A, a unique derivative of mannitol containing the unusual chromophore 2-isocyano-3-methyl-2-butenoyl, was isolated from potato dextrose cultures of L. maculans virulent on canola (Brassica napus L. cv. Westar). Surprisingly, maculansin A was more toxic to resistant plants (B. juncea L. cv. Cutlass, brown mustard) than to susceptible plants (canola). Maculansin A, however, did not elicit the production of phytoalexins either in resistant or susceptible plants. In addition, other maculansin type structures and the metabolite 2,4-dihydroxy-3,6-dimethylbenzaldehyde were isolated and the latter was found to be a strong inhibitor of root growth of both brown mustard and canola. Considering that L. maculans seems to be expanding its host range to infect brown mustard as well, maculansins could assist in chemotaxonomic studies to group the diverse isolates.     

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.     

Chemical suppression of the sexual stage of Leptosphaeria maculans on oil-seed rape and turnip seed crop straw

A selection of fungicides, herbicides and surfactants and urea were tested for their effect on the production of pseudothecia and ascospore release of Leptosphaeria maculans present on oil-seed rape straw and turnip seed crop straw. The fungicides ethyl mercury phosphate, triarimol, fenarimol, carbendazim, tridemorph and benomyl, each at 1 g/litre, the herbicides dinoseb and diquat, each at 10 g/litre the surfactants Bradasol, Cetrimide, Deciquam 222, Hyamine 1622 and Maxonol N, each at 50 g product/litre, and urea at 150 g/litre, applied to straw before pseudothecia had formed were more than 90% effective in preventing further development. These chemicals were also effective in preventing further ascospore production when applied to straw bearing mature pseudothecia but only dinoseb and urea prevented the release of mature ascospores. The results suggest that it may be possible to break the life cycle of L. maculans by chemical treatment and thereby obviate the need for subsequent control measures.