<i>Albugo candida</i> race diversity, ploidy and host-associated microbes revealed using DNA sequence capture on diseased plants in the field

• Physiological races of the oomycete Albugo candida are biotrophic pathogens of diverse plant species, primarily the Brassicaceae, and cause infections that suppress host immunity to other pathogens. However, A. candida race diversity and the consequences of host immunosuppression are poorly understood in the field. • We report a method that enables sequencing of DNA of plant pathogens and plant-associated microbes directly from field samples (Pathogen Enrichment Sequencing: PenSeq). We apply this method to explore race diversity in A. candida and to detect A. candida-associated microbes in the field (91 A. candida-infected plants).• We show with unprecedented resolution that each host plant species supports colonization by one of 17 distinct phylogenetic lineages, each with an unique repertoire of effector candidate alleles. These data reveal the crucial role of sexual and asexual reproduction, polyploidy and host domestication in A. candida specialization on distinct plant species. Our bait design also enabled phylogenetic assignment of DNA sequences from bacteria and fungi from plants in the field.• This paper shows that targeted sequencing has a great potential for the study of pathogen populations while they are colonizing their hosts. This method could be applied to other microbes, especially to those that cannot be cultured

Development of high-throughput methods to screen disease caused by Rhizoctonia solani AG 2-1 in oilseed rape

Background: Rhizoctonia solani (Kühn) is a soil-borne, necrotrophic fungus causing damping off, root rot and stem canker in many cultivated plants worldwide. Oilseed rape (OSR, Brassica napus) is the primary host for anastomosis group (AG) 2-1 of R. solani causing pre- and post-emergence damping-off resulting in death of seedlings and impaired crop establishment. Presently, there are no known resistant OSR genotypes and the main methods for disease control are fungicide seed treatments and cultural practices. The identification of sources of resistance for crop breeding is essential for sustainable management of the disease. However, a high-throughput, reliable screening method for resistance traits is required. The aim of this work was to develop a low cost, rapid screening method for disease phenotyping and identification of resistance traits. Results: Four growth systems were developed and tested: (1) nutrient media plates, (2) compost trays, (3) light expanded clay aggregate (LECA) trays, and (4) a hydroponic pouch and wick system. Seedlings were inoculated with virulent AG 2-1 to cause damping-off disease and grown for a period of 4–10 days. Visual disease assessments were carried out or disease was estimated through image analysis using ImageJ. Conclusion: Inoculation of LECA was the most suitable method for phenotyping disease caused by R. solani AG 2-1 as it enabled the detection of differences in disease severity among OSR genotypes within a short time period whilst allowing measurements to be conducted on whole plants. This system is expected to facilitate identification of resistant germplasm

Altered Neurocircuitry in the Dopamine Transporter Knockout Mouse Brain

The plasma membrane transporters for the monoamine neurotransmitters dopamine, serotonin, and norepinephrine modulate the dynamics of these monoamine neurotransmitters. Thus, activity of these transporters has significant consequences for monoamine activity throughout the brain and for a number of neurological and psychiatric disorders. Gene knockout (KO) mice that reduce or eliminate expression of each of these monoamine transporters have provided a wealth of new information about the function of these proteins at molecular, physiological and behavioral levels. In the present work we use the unique properties of magnetic resonance imaging (MRI) to probe the effects of altered dopaminergic dynamics on meso-scale neuronal circuitry and overall brain morphology, since changes at these levels of organization might help to account for some of the extensive pharmacological and behavioral differences observed in dopamine transporter (DAT) KO mice. Despite the smaller size of these animals, voxel-wise statistical comparison of high resolution structural MR images indicated little morphological change as a consequence of DAT KO. Likewise, proton magnetic resonance spectra recorded in the striatum indicated no significant changes in detectable metabolite concentrations between DAT KO and wild-type (WT) mice. In contrast, alterations in the circuitry from the prefrontal cortex to the mesocortical limbic system, an important brain component intimately tied to function of mesolimbic/mesocortical dopamine reward pathways, were revealed by manganese-enhanced MRI (MEMRI). Analysis of co-registered MEMRI images taken over the 26 hours after introduction of Mn^(2+) into the prefrontal cortex indicated that DAT KO mice have a truncated Mn^(2+) distribution within this circuitry with little accumulation beyond the thalamus or contralateral to the injection site. By contrast, WT littermates exhibit Mn^(2+) transport into more posterior midbrain nuclei and contralateral mesolimbic structures at 26 hr post-injection. Thus, DAT KO mice appear, at this level of anatomic resolution, to have preserved cortico-striatal-thalamic connectivity but diminished robustness of reward-modulating circuitry distal to the thalamus. This is in contradistinction to the state of this circuitry in serotonin transporter KO mice where we observed more robust connectivity in more posterior brain regions using methods identical to those employed here

Development of a biosensor for urea assay based on amidase inhibition, using an ion-selective electrode

A biosensor for urea has been developed based on the observation that urea is a powerful active-site inhibitor of amidase, which catalyzes the hydrolysis of amides such as acetamide to produce ammonia and the corresponding organic acid. Cell-free extract from Pseudomonas aeruginosa was the source of amidase (acylamide hydrolase, EC 3.5.1.4) which was immobilized on a polyethersulfone membrane in the presence of glutaraldehyde; anion-selective electrode for ammonium ions was used for biosensor development. Analysis of variance was used for optimization of the biosensorresponse and showed that 30 mu L of cell-free extract containing 7.47 mg protein mL(-1), 2 mu L of glutaraldehyde (5%, v/v) and 10 mu L of gelatin (15%, w/v) exhibited the highest response. Optimization of other parameters showed that pH 7.2 and 30 min incubation time were optimum for incubation ofmembranes in urea. The biosensor exhibited a linear response in the range of 4.0-10.0 mu M urea, a detection limit of 2.0 mu M for urea, a response timeof 20 s, a sensitivity of 58.245 % per mu M urea and a storage stability of over 4 months. It was successfully used for quantification of urea in samples such as wine and milk; recovery experiments were carried out which revealed an average substrate recovery of 94.9%. The urea analogs hydroxyurea, methylurea and thiourea inhibited amidase activity by about 90%, 10% and 0%, respectively, compared with urea inhibition

A bacterial glycan core linked to surface (S)-layer proteins modulates host immunity through Th17 suppression

Tannerella forsythia is a pathogen implicated in periodontitis, an inflammatory disease of the tooth-supporting tissues often leading to tooth loss. This key periodontal pathogen is decorated with a unique glycan core O-glycosidically linked to the bacterium's proteinaceous surface (S)-layer lattice and other glycoproteins. Herein, we show that the terminal motif of this glycan core acts to modulate dendritic cell effector functions to suppress T-helper (Th)17 responses. In contrast to the wild-type bacterial strain, infection with a mutant strain lacking the complete S-layer glycan core induced robust Th17 and reduced periodontal bone loss in mice. Our findings demonstrate that surface glycosylation of this pathogen may act to ensure its persistence in the host likely through suppression of Th17 responses. In addition, our data suggest that the bacterium then induces the Toll-like receptor 2–Th2 inflammatory axis that has previously been shown to cause bone destruction. Our study provides a biological basis for pathogenesis and opens opportunities in exploiting bacterial glycans as therapeutic targets against periodontitis and a range of other infectious diseases

Attenuation of Helicobacter pylori-induced gastric inflammation by prior cag− strain (AM1) infection in C57BL/6 mice

Helicobacter pylori, colonize in stomach of ~50% of the world population. cag pathogenicity Island of H. pylori is one of the important virulent factors that attributed to gastric inflammation. Coinfection with H. pylori strain with different genetic makeup alters the degree of pathogenicity and susceptibility towards antibiotics. The present study investigates host immunomodulatory effects of H. pylori infection by both cag+ strain (SS1) and cag− strain (AM1). C57BL/6 mice were infected with AM1 or SS1 strain as well as AM1 followed by SS1 (AM1/SS1) and vice versa. Results: Mice infected with AM1/SS1 strain exhibited less gastric inflammation and reduced proMMP9 and proMMP3 activities in gastric tissues as compared to SS1/SS1 and SS1/AM1 infected groups. The expression of both MMP9 and MMP3 followed similar trend like activity in infected tissues. Both Th1 and Th17 responses were induced by SS1 strain more profoundly than AM1 strain infection which induced solely Th1 response in spleen and gastric tissues. Moreover, IFN-γ, TNF-α, IL-1β and IL-12 were significantly downregulated in mice spleen and gastric tissues infected by AM1/SS1 compared to SS1/SS1 but not with SS1/AM1 coinfection. Surprisingly, IL-17 level was dampened significantly in AM1/ SS1 compared to SS1/AM1 coinfected groups. Furthermore, number of Foxp3+ T-regulatory (Treg) cells and immunosuppressive cytokines like IL-10 and TGF-β were reduced in AM1/SS1 compared to SS1/SS1 and SS1/AM1 coinfected mice gastric tissues. Conclusions: These data suggested that prior H. pylori cag− strain infection attenuated the severity of gastric pathology induced by subsequent cag+ strain in C57BL/6 mice. Prior AM1 infection induced Th1 cytokine IFN-γ, which reduced the Th17 response induced by subsequent SS1 infection. The reduced gastritis in AM1/SS1-infected mice might also be due to enrichment of AM1- primed Treg cells in the gastric compartment which inhibit Th1 and Th17 responses to subsequent SS1 infection. In summary, prior infection by non-virulent H. pylori strain (AM1) causes reduction of subsequent virulent strain (SS1) infection by regulation of inflammatory cytokines and MMPs expressio

Comparative in situ analyses of cell wall matrix polysaccharide dynamics in developing rice and wheat grain

Cell wall polysaccharides of wheat and rice endosperm are an important source of dietary fibre. Monoclonal antibodies specific to cell wall polysaccharides were used to determine polysaccharide dynamics during the development of both wheat and rice grain. Wheat and rice grain present near synchronous developmental processes and significantly different endosperm cell wall compositions, allowing the localisation of these polysaccharides to be related to developmental changes. Arabinoxylan (AX) and mixed-linkage glucan (MLG) have analogous cellular locations in both species, with deposition of AX and MLG coinciding with the start of grain filling. A glucuronoxylan (GUX) epitope was detected in rice, but not wheat endosperm cell walls. Callose has been reported to be associated with the formation of cell wall outgrowths during endosperm cellularisation and xyloglucan is here shown to be a component of these anticlinal extensions, occurring transiently in both species. Pectic homogalacturonan (HG) was abundant in cell walls of maternal tissues of wheat and rice grain, but only detected in endosperm cell walls of rice in an unesterified HG form. A rhamnogalacturonan-I (RG-I) backbone epitope was observed to be temporally regulated in both species, detected in endosperm cell walls from 12 DAA in rice and 20 DAA in wheat grain. Detection of the LM5 galactan epitope showed a clear distinction between wheat and rice, being detected at the earliest stages of development in rice endosperm cell walls, but not detected in wheat endosperm cell walls, only in maternal tissues. In contrast, the LM6 arabinan epitope was detected in both species around 8 DAA and was transient in wheat grain, but persisted in rice until maturity

Endophytes vs tree pathogens and pests: can they be used as biological control agents to improve tree health?

Like all other plants, trees are vulnerable to attack by a multitude of pests and pathogens. Current control measures for many of these diseases are limited and relatively ineffective. Several methods, including the use of conventional synthetic agro-chemicals, are employed to reduce the impact of pests and diseases. However, because of mounting concerns about adverse effects on the environment and a variety of economic reasons, this limited management of tree diseases by chemical methods is losing ground. The use of biological control, as a more environmentally friendly alternative, is becoming increasingly popular in plant protection. This can include the deployment of soil inoculants and foliar sprays, but the increased knowledge of microbial ecology in the phytosphere, in particular phylloplane microbes and endophytes, has stimulated new thinking for biocontrol approaches. Endophytes are microbes that live within plant tissues. As such, they hold potential as biocontrol agents against plant diseases because they are able to colonize the same ecological niche favoured by many invading pathogens. However, the development and exploitation of endophytes as biocontrol agents will have to overcome numerous challenges. The optimization and improvement of strategies employed in endophyte research can contribute towards discovering effective and competent biocontrol agents. The impact of environment and plant genotype on selecting potentially beneficial and exploitable endophytes for biocontrol is poorly understood. How endophytes synergise or antagonise one another is also an important factor. This review focusses on recent research addressing the biocontrol of plant diseases and pests using endophytic fungi and bacteria, alongside the challenges and limitations encountered and how these can be overcome. We frame this review in the context of tree pests and diseases, since trees are arguably the most difficult plant species to study, work on and manage, yet they represent one of the most important organisms on Earth

Endophytes vs tree pathogens and pests: can they be used as biological control agents to improve tree health?

Like all other plants, trees are vulnerable to attack by a multitude of pests and pathogens. Current control measures for many of these diseases are limited and relatively ineffective. Several methods, including the use of conventional synthetic agro-chemicals, are employed to reduce the impact of pests and diseases. However, because of mounting concerns about adverse effects on the environment and a variety of economic reasons, this limited management of tree diseases by chemical methods is losing ground. The use of biological control, as a more environmentally friendly alternative, is becoming increasingly popular in plant protection. This can include the deployment of soil inoculants and foliar sprays, but the increased knowledge of microbial ecology in the phytosphere, in particular phylloplane microbes and endophytes, has stimulated new thinking for biocontrol approaches. Endophytes are microbes that live within plant tissues. As such, they hold potential as biocontrol agents against plant diseases because they are able to colonize the same ecological niche favoured by many invading pathogens. However, the development and exploitation of endophytes as biocontrol agents will have to overcome numerous challenges. The optimization and improvement of strategies employed in endophyte research can contribute towards discovering effective and competent biocontrol agents. The impact of environment and plant genotype on selecting potentially beneficial and exploitable endophytes for biocontrol is poorly understood. How endophytes synergise or antagonise one another is also an important factor. This review focusses on recent research addressing the biocontrol of plant diseases and pests using endophytic fungi and bacteria, alongside the challenges and limitations encountered and how these can be overcome. We frame this review in the context of tree pests and diseases, since trees are arguably the most difficult plant species to study, work on and manage, yet they represent one of the most important organisms on Earth