Mapping the adaptive landscape of a major agricultural pathogen reveals evolutionary constraints across heterogeneous environments

The adaptive potential of pathogens in novel or heterogeneous environments underpins the risk of disease epidemics. Antagonistic pleiotropy or differential resource allocation among life-history traits can constrain pathogen adaptation. However, we lack understanding of how the genetic architecture of individual traits can generate trade-offs. Here, we report a large-scale study based on 145 global strains of the fungal wheat pathogen Zymoseptoria tritici from four continents. We measured 50 life-history traits, including virulence and reproduction on 12 different wheat hosts and growth responses to several abiotic stressors. To elucidate the genetic basis of adaptation, we used genome-wide association mapping coupled with genetic correlation analyses. We show that most traits are governed by polygenic architectures and are highly heritable suggesting that adaptation proceeds mainly through allele frequency shifts at many loci. We identified negative genetic correlations among traits related to host colonization and survival in stressful environments. Such genetic constraints indicate that pleiotropic effects could limit the pathogen’s ability to cause host damage. In contrast, adaptation to abiotic stress factors was likely facilitated by synergistic pleiotropy. Our study illustrates how comprehensive mapping of life-history trait architectures across diverse environments allows to predict evolutionary trajectories of pathogens confronted with environmental perturbations.Subject terms: Population genetics, Plant sciences, Molecular evolution, Fungi     

Functional genetics of human gut commensal Bacteroides thetaiotaomicron reveals metabolic requirements for growth across environments

1. Download : Download high-res image (203KB)
2. Download : Download full-size image

     

Identification of potential vaccine target antigens by immunoproteomic analysis of a virulent and a non-virulent strain of the fish pathogen Flavobacterium psychrophilum

Flavobacterium psychrophilum is the etiological agent of bacterial coldwater disease (CWD) and rainbow trout fry syndrome (RTFS). To identify antigens associated with virulence or host immunity, we compared total and immunogenic proteins of cellular and extracellular products (ECP) between a virulent (CSF-259-93) and non-virulent (ATCC 49418) strain of F. psychrophilum. One-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis of total cellular proteins revealed only minor differences between the strains; however, separation of ECP showed that proteins were differentially expressed. Western blot analysis using rainbow trout (Oncorhynchus mykiss) anti-CSF-259-93 sera showed greater reactivity to proteins of the virulent strain, including many > 50 kDa. Further analysis by 2-dimensional electrophoresis (2DE) identified numerous differences between the strains. Western blot analysis combined with 2DE identified several immunogenic proteins that reacted with the antisera and were shared between the 2 strains. However, at least 15 immunogenic proteins appeared to be unique to the virulent strain, while 4 such proteins were identified in the non-virulent strain; 8 proteins unique to the virulent strain and 6 shared proteins were further analyzed for identification by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) analysis. Of these, 3 immunogenic proteins (heat shock proteins HSP 60 and HSP 70) and 2 other proteins (ATP synthase and thermolysin) were conclusively identified. The 2 highly immunogenic heat shock proteins were shown to share extensive homology with heat shock proteins of related bacteria. This approach for antigen identification may provide a basis for targeted vaccine development against CWD and RTFS.     

Identifying microorganisms which fill a niche similar to that of the pathogen: a new investigative approach for discovering biological control organisms

Understanding the mechanisms of suppressive soils should lead to the development of new strategies to manage pests and diseases. For suppressive soils that have a biological nature, one of the first steps in understanding them is to identify the organisms contributing to this phenomenon. Here we present a new approach for identifying microorganisms involved in soil suppressiveness. This strategy identifies microorganisms that fill a niche similar to that of the pathogen by utilizing substrate utilization assays in soil. To demonstrate this approach, we examined an avocado grove where a Phytophthora cinnamomi epidemic created soils in which the pathogen could not be detected with baiting techniques, a characteristic common to many soils with suppressiveness against P. cinnamomi. Substrate utilization assays were used to identify rRNA genes (rDNA) from bacteria that rapidly grew in response to amino acids known to attract P. cinnamomi zoospores. Six bacterial rDNA intergenic sequences were prevalent in the epidemic soils but uncommon in the non-epidemic soils. These sequences belonged to bacteria related to Bacillus mycoides, Renibacterium salmoninarum, and Streptococcus pneumoniae. We hypothesize that bacteria such as these, which respond to the same environmental cues that trigger root infection by the pathogen, will occupy a niche similar to that of the pathogen and contribute to suppressiveness through mechanisms such as nutrient competition and antibiosis.     

Usefulness of a TaqMan-based polymerase chain reaction assay for the detection of the fish pathogen Flavobacterium psychrophilum

AIMS: This study developed a new diagnostic method for the bacterium Flavobacterium psychrophilum based on a TaqMan polymerase chain reaction (PCR) assay. METHODS AND RESULTS: Based on reported and newly designed PCR probes, a rapid procedure, that requires no post-PCR processing, was developed for the detection of F. psychrophilum by measuring the fluorescence produced during PCR amplification. Primers were designed to amplify a 971-bp fragment of the 16S rRNA as the target. When different F. psychrophilum strains and other bacterial species, that are taxonomically and ecologically related, were assayed the fluorogenic test was 100% specific in identifying all of the F. psychrophilum strains. The sensitivity of the assay was found to be 1.1 pg DNA and the assay was linear over a range of 0.1 pg-11.2 ng. With pure cultures of F. psychrophilum, the assay was linear over the range 0.4-4.7 x 104 cfu and was able to detect 4.7 cfu per reaction. The analysis was reproducible using either extracted DNA or pure culture. Results using artificially infected fish and diseased fry from natural fish farm outbreaks showed that the assay was useful for diagnosis. CONCLUSIONS: The data showed that the assay was as specific, sensitive, reproducible and rapid but less toxic than the PCR assays described and so very useful for the diagnosis of these micro-organisms. SIGNIFICANCE AND IMPACT OF THE STUDY: This new approach permits a rapid, easy and safe routine laboratory diagnosis of F. psychrophilum.     

Intra- and interlaboratory performance of antibiotic disk-diffusion-susceptibility testing of bacterial control strains of relevance for monitoring aquaculture environments

In the course of an international research project on hazard analysis of antimicrobial resistance in SE Asian aquaculture environments, 2 European Union and 3 SE Asian laboratories attempted to harmonize a procedure for antimicrobial agent susceptibility testing based on disk diffusion (DD). For this purpose, a selected panel of 10 bacterial control strains of relevance for monitoring warm-water aquaculture environments was sent by the central laboratory to the other participating laboratories. In each laboratory, 10 independently replicated DD determinations of each control strain to 6 antibiotics were performed using Iso-Sensitest Agar (ISA) according to a standard operating procedure (SOP); in total, this study thus yielded 300 data sets for all 5 laboratories. At the end of the study, strain authenticity of subcultures of the control strains used by the respective participating laboratories was verified by the central laboratory. Based on the arithmetic mean of 10 inhibition-zone diameter measurements and standard deviation (SD), intralaboratory SD variations ranged from 0 to 2 mm when 79% of the recorded data sets were considered. In 8% of the data sets, the SD value exceeded 4 mm, which in most cases could be attributed to the fact that the data points for a given strain-disk combination were not normally distributed in one of the laboratories. At the interlaboratory level, 81% of the SD values based on global averaging of 50 data points per strain-disk combination were situated in the 0 to 5 mm range. Comparison with a minimal data set from literature of DD testing performed with Mueller-Hinton (MH) medium indicated that the use of either ISA or MH medium in DD testing has a limited impact on the method's precision among different laboratories. In conclusion, the current study has provided a validated SOP to promote the coordination and harmonization of DD-susceptibility methodologies for aquaculture-associated organisms at an international level. As one of the main action items for the future, new interpretive breakpoints should be specifically designed and validated for aquaculture drugs and organisms.     

Genome assisted molecular typing and pathotyping of rice blast pathogen,Magnaporthe oryzae,reveals a genetically homogenous population with high virulence diversity

Genome sequence-driven molecular typing tools have the potential to uncover the population biology and genetic diversity of rapidly evolving plant pathogens like Magnaporthe oryzae. Here, we report a new molecular typing technique -a digitally portable tool for population genetic analysis of M. oryzae to decipher the genetic diversity. Our genotyping tool exploiting allelic variations in housekeeping and virulence genes coupled with pathotyping revealed a prevalence of genetically homogenous populations within a single-field and plant niches such as leaf and panicle. The M. oryzae inciting leaf-blast and panicle-blast were confirmed to be genetically identical with no or minor nucleotide polymorphism in 17 genomic loci analyzed. Genetic loci such as Mlc1, Mpg1, Mps1, Slp1, Cal, Ef-Tu, Pfk, and Pgk were highly polymorphic as indicated by the haplotype-diversity, the number of polymorphic sites, and the number of mutations. The genetically homogenous single field population showed high virulence variability or diversity on monogenic rice differentials. The study indicated that the genetic similarity displayed by the isolates collected from a particular geographical location had no consequence on their virulence pattern on rice differentials carrying single/multiple resistance genes. The data on virulence diversity showed by the identical Sequence Types (STs) is indicative of no congruence between polymorphic virulence genes-based pathotyping and conserved housekeeping genes-based genotyping.     

Rapid detection of columnaris disease in channel catfish (Ictalurus punctatus) with a new species-specific 16-S rRNA gene-based PCR primer for Flavobacterium columnare

A 16-S rRNA gene from the chromosomal DNA of the fish-pathogenic bacterium Flavobacterium columnare (formerly Flexibacter columnaris), strain ARS-I, was cloned, sequenced and used to design a polymerase chain reaction (PCR) primer set. The primer set amplified a specific 1193-bp DNA fragment from F. columnare strains but not from related bacteria, F. psychrophilum, F. aquatile, F. branchiophilum, or other bacterial pathogens of fish, Flexibacter maritimus, Cytophaga johnsonae, Edwardsiella ictaluri, E. tarda, Aeromonas hydrophila, and Streptococcus iniae or from the non-fish pathogen Escherichia coli. The PCR reaction conditions were optimized to permit detection of the organism from agar plates, broth culture, frozen samples, dead fish tissue, and live fish in less than 5 h (8 h, if the more sensitive nested PCR is used). DNA was extracted by a boiled-extraction method or by commercial column purification. The PCR product was detected at DNA concentrations below 0.1 ng and from as few as 100 bacterial cells. Nested PCR using universal eubacterial primers increased the sensitivity five-fold, allowing detection of F. columnare strains at DNA concentrations below 0.05 ng and from as few as 10 bacterial cells in apparently healthy, asymptomatic fish. The efficiency of this primer set was compared to the 16-S rRNA gene primer sets of Toyama et al. [Fish Pathol. 29 (1994) 271.] and that of Bader and Shotts [J. Aquat. Anim. Health 10 (1998) 311.]. The new primer set is as good or better than the previously published primer sets for detecting F. columnare in all samples and under all conditions tested.     

The crystal structure of a plant 3-ketoacyl-CoA thiolase reveals the potential for redox control of peroxisomal fatty acid beta-oxidation

Crystal structures of peroxisomal Arabidopsis thaliana 3-ketoacyl-CoA thiolase (AtKAT), an enzyme of fatty acid beta-oxidation, are reported. The subunit, a typical thiolase, is a combination of two similar alpha/beta domains capped with a loop domain. The comparison of AtKAT with the Saccharomyces cerevisiae homologue (ScKAT) structure reveals a different placement of subunits within the functional dimers and that a polypeptide segment forming an extended loop around the open catalytic pocket of ScKAT converts to alpha-helix in AtKAT, and occludes the active site. A disulfide is formed between Cys192, on this helix, and Cys138, a catalytic residue. Access to Cys138 is determined by the structure of this polypeptide segment. AtKAT represents an oxidized, previously unknown inactive form, whilst ScKAT is the reduced and active enzyme. A high level of sequence conservation is observed, including Cys192, in eukaryotic peroxisomal, but not mitochondrial or prokaryotic KAT sequences, for this labile loop/helix segment. This indicates that KAT activity in peroxisomes is influenced by a disulfide/dithiol change linking fatty acid beta-oxidation with redox regulation.     

Authentication scheme for routine verification of genetically similar laboratory colonies: a trial with Anopheles gambiae

Background  

When rearing morphologically indistinguishable laboratory strains concurrently, the threat of unintentional genetic contamination is constant. Avoidance of accidental mixing of strains is difficult due to the use of common equipment, technician error, or the possibility of self relocation by adult mosquitoes ("free fliers"). In many cases, laboratory strains are difficult to distinguish because of morphological and genetic similarity, especially when laboratory colonies are isolates of certain traits from the same parental strain, such as eye color mutants, individuals with certain chromosomal arrangements or high levels of insecticide resistance. Thus, proving genetic integrity could seem incredibly time-consuming or impossible. On the other hand, lacking proof of genetically isolated laboratory strains could question the validity of research results.     

Interaction of fishes with pathogenic micro-organisms and application of phages for their control: a review

The present condition of aquacultural industry is influenced by the economical losses due to various aquacultural diseases causing fish mortality. The present review provides benchmark information related to application of bacteriophages in aquacultural industries over available traditional treatment procedures like antibiotics and chemotherapy. The traditional methods are mostly less advantageous due to development of resistance, non-specific targeting of bacteria including intestinal microflora, etc. In short here we discuss the interaction between fish, bacteria and their phages in order to have an alternate treatment method for the pathogens responsible for aquacultural diseases.     

Comparative mapping of QTLs for agronomic traits of rice across environments using a doubled haploid population

We report here the RFLP mapping of quantitative triat loci (QTLs) that affect some important agronomic traits in cultivated rice. An anther culturederived doubled haploid (DH) population was established from a cross between an indica and a japonica rice variety. On the basis of this population a molecular linkage map comprising 137 markers was constructed that covered the rice genome at intervals of 14.8cM on average. Interval mapping of the linkage map was used to locate QTLs for such important agronomic traits as heading date, plant height, number of spikelets per panicle, number of grains Per panicle, 1000-grain weight and percentage of seed set. Evidence of genotype-byenvironment interaction was found by comparing QTL maps of the same population grown in three diverse environments. A total of 22 QTLs for six agronomic traits were detected that were significant in at least one environment, but only 7 were significant in all three environments, 7 were significant in two environments and 8 could only be detected in a single environment. However, QTL-by-environment interaction was traitdependent. QTLs for spikelets and grains per panicle were common across environments, while traits like heading date and plant height were more sensitive to environment.     

Comparative mapping of QTLs for agronomic traits of rice across environments by using a doubled-haploid population

We report here the RFLP mapping of quantitative trait loci (QTLs) which affect some important agronomic traits in cultivated rice. An anther culture-derived doubled-haploid (DH) population was established from a cross between indica and japonica rice varieties. A molecular linkage map comprising 137 markers was constructed based on this population which covered the rice genome at intervals of 14.8 cM on average. The linkage map was used to locate QTLs for such important agronomic traits as heading date, plant height, number of spikelets per panicle, number of grains per panicle, 1 000-grain weight and the percentage of seed set, by interval mapping. Evidence of genotype-by-environment interaction was found by comparing QTL maps of the same population grown in three diverse environments. A total of 22 QTLs for six agronomic traits was detected which were significant in at least one environment, but only seven were significant in all three environments; seven were significant in two environments and eight could only be detected in a single environment. However, QTLs-by-environment interaction was trait dependent. QTLs for spikelets and grains per panicle were common across environments while traits like heading date and plant height were more sensitive to environment. Received: 22 February 1996 / Accepted: 10 May 1996     

Combined use of eBeam irradiation and the potential probiotic Lactobacillus rhamnosus Vahe for control of foodborne pathogen Klebsiella pneumoniae

The implementation of electron beam radiation coupled with the use of probiotics is one of the newest food processing technologies that may be used to ensure food safety and improve shelf life of food products. The purpose of this study was to evaluate the effect of 50–150-Gy electron beam irradiation on the antimicrobial activity of the putative probiotic strain Lactobacillus rhamnosus Vahe. Low-dose electron beam irradiation of lactobacilli cells was performed using the Advanced Research Electron Accelerator Laboratory’s electron accelerator, and the agar well diffusion method and Verhulst logistic function were used to evaluate the effect of radiation on anti–Klebsiella pneumoniae activity of the cell free supernatant of L. rhamnosus Vahe cells in vitro. Our results suggest that 50–150-Gy electron beam irradiation decreases the viability of the investigated lactobacilli, but does not significantly change the probiotic’s activity against K. pneumoniae. Results indicate that the combined use of irradiation and L. rhamnosus Vahe might be suggested for non-thermal food sterilizing technologies.     

High-yield production of a chitinase from Aeromonas veronii B565 as a potential feed supplement for warm-water aquaculture

Chitin, present in crustacean shells, insects, and fungi, is the second most plentiful natural organic fiber after wood. To effectively use chitin in a cost-saving and environmentally friendly way in aquaculture, crustacean shells (e.g., shrimp-shell meal) are supplemented into aquafeed after degradation by chemical methods. Herein, we describe a chitinase from Aeromonas veronii B565, designated ChiB565, which potently degrades shrimp-shell chitin and resists proteolysis. We isolated recombinant ChiB565 of the expected molecular mass in large yield from Pichia pastoris. ChiB565 is optimally active at pH 5.0 and 50 °C and stable between pH 4.5 and 9.0 at 50 °C and below. Compared with the commercial chitinase C-6137, which cannot degrade shrimp-shell chitin, ChiB565 hydrolyzes shrimp-shell chitin in addition to colloidal chitin, powdered chitin, and β-1,3-1,4-glucan. The optimal enzyme concentration and reaction time for in vitro degradation of 0.1 g of powdered shrimp shell are 30 U of ChiB565 and 3 h, respectively. A synergistic protein-release effect occurred when ChiB565 and trypsin were incubated in vitro with shrimp shells. Tilapia were fed an experimental diet containing 5 % (w/w) shrimp bran and 16.2 U/kg ChiB565, which significantly improved growth and feed conversion compared with a control diet lacking ChiB565. Dietary ChiB565 enhanced nitrogen digestibility and downregulated intestinal IL-1β expression. The immunologically relevant protective effects of dietary ChiB565 were also observed for 2 to 3 days following exposure to pathogenic Aeromonas hydrophila.     

Characterization of a novel gene for strain typing reveals substructuring of Aspergillus fumigatus across North America

Fifty-five epidemiologically linked Aspergillus fumigatus isolates obtained from six nosocomial outbreaks of invasive aspergillosis were subtyped by sequencing the polymorphic region of the gene encoding a putative cell surface protein, Afu3g08990 (denoted as CSP). Comparative sequence analysis showed that genetic diversity was generated in the coding region of this gene by both tandem repeats and point mutations. Each unique sequence in an outbreak cluster was assigned an arbitrary number or CSP sequence type. The CSP typing method was able to identify "clonal" and genotypically distinct A. fumigatus isolates, and the results of this method were concordant with those of another discriminatory genotyping technique, the Afut1 restriction fragment length polymorphism typing method. The novel single-locus sequence typing (CSP typing) strategy appears to be a simple, rapid, discriminatory tool that can be readily shared across laboratories. In addition, we found that A. fumigatus isolates substructured into multiple clades; interestingly, one clade consisted of isolates predominantly representing invasive clinical isolates recovered from cardiac transplant patients from two different outbreak situations. We also found that the A. fumigatus isolate Af293, whose genome has been sequenced, possesses a CSP gene structure that is substantially different from those of the other A. fumigatus strains studied here, highlighting the need for further taxonomic study.