Molecular Assay Development to Monitor the Kinetics of Viable Populations of Two Biocontrol Agents, Bacillus subtilis QST 713 and Gliocladium catenulatum J1446, in the Phyllosphere of Lettuce Leaves.

Optimising the use of biocontrol agents (BCAs) requires the temporal tracking of viable populations in the crop phyllosphere to ensure that effective control can be achieved. No sensitive systems for quantifying viable populations of commercially available BCAs, such as Bacillus subtilis and Gliocladium catenulatum, in the phyllosphere of crop plants are available. The objective of this study was to develop a method to quantify viable populations of these two BCAs in the crop phyllosphere. A molecular tool based on propidium monoazide (PMA) (PMAxx™-qPCR) capable of quantifying viable populations of these two BCAs was developed. Samples were treated with PMAxx™ (12.5-100 µM), followed by 15 min incubation, exposure to a 800 W halogen light for 30 min, DNA extraction, and quantification using qPCR. This provided a platform for using the PMAxx™-qPCR technique for both BCAs to differentiate viable from dead cells. The maximum number of dead cells blocked, based on the DNA, was 3.44 log10 for B. subtilis and 5.75 log10 for G. catenulatum. Validation studies showed that this allowed accurate quantification of viable cells. This method provided effective quantification of the temporal changes in viable populations of the BCAs in commercial formulations on lettuce leaves in polytunnel and glasshouse production systems.

Reducing Drosophila suzukii emergence through inter-species competition.

BACKGROUND: Drosophila suzukii has dispersed widely from its native Asian range since 2008. Its arrival in the UK is resulting in economic losses in soft- and stone-fruit crops caused by larvae feeding on the flesh of ripening fruit. Although a large amount of research has been directed at controlling this pest, it is presently unknown how this invasive species interacts with native Drosophila species. RESULTS: In the work reported here, D. suzukii or Drosophila melanogaster adults were introduced to substrates pre-inoculated with the eggs of the same or the other species in a laboratory choice assay. Drosophila melanogaster adult emergence was not affected by pre-inoculation with D. suzukii. The rate of emergence of D. suzukii was significantly lower from medium pre-inoculated by D. melanogaster than from blank medium. In a subsequent experiment, significantly more D. suzukii eggs were laid in blank medium than in D. melanogaster pre-inoculated medium. CONCLUSION: The presence of D. melanogaster in a substrate significantly reduced D. suzukii emergence and egg laying. This study raises research questions about how this reduction mechanism is driven and how it could be exploited as part of future integrated pest management practices. © 2017 Society of Chemical Industry.

A new three-locus model for rootstock-induced dwarfing in apple revealed by genetic mapping of root bark percentage.

Rootstock-induced dwarfing of apple scions revolutionized global apple production during the twentieth century, leading to the development of modern intensive orchards. A high root bark percentage (the percentage of the whole root area constituted by root cortex) has previously been associated with rootstock-induced dwarfing in apple. In this study, the root bark percentage was measured in a full-sib family of ungrafted apple rootstocks and found to be under the control of three loci. Two quantitative trait loci (QTLs) for root bark percentage were found to co-localize to the same genomic regions on chromosome 5 and chromosome 11 previously identified as controlling dwarfing, Dw1 and Dw2, respectively. A third QTL was identified on chromosome 13 in a region that has not been previously associated with dwarfing. The development of closely linked sequence-tagged site markers improved the resolution of allelic classes, thereby allowing the detection of dominance and epistatic interactions between loci, with high root bark percentage only occurring in specific allelic combinations. In addition, we report a significant negative correlation between root bark percentage and stem diameter (an indicator of tree vigour), measured on a clonally propagated grafted subset of the mapping population. The demonstrated link between root bark percentage and rootstock-induced dwarfing of the scion leads us to propose a three-locus model that is able to explain levels of dwarfing from the dwarf 'M.27' to the semi-invigorating rootstock 'M.116'. Moreover, we suggest that the QTL on chromosome 13 (Rb3) might be analogous to a third dwarfing QTL, Dw3, which has not previously been identified.

Rapid, automated detection of stem canker symptoms in woody perennials using artificial neural network analysis.

BACKGROUND: Pseudomonas syringae can cause stem necrosis and canker in a wide range of woody species including cherry, plum, peach, horse chestnut and ash. The detection and quantification of lesion progression over time in woody tissues is a key trait for breeders to select upon for resistance. RESULTS: In this study a general, rapid and reliable approach to lesion quantification using image recognition and an artificial neural network model was developed. This was applied to screen both the virulence of a range of P. syringae pathovars and the resistance of a set of cherry and plum accessions to bacterial canker. The method developed was more objective than scoring by eye and allowed the detection of putatively resistant plant material for further study. CONCLUSIONS: Automated image analysis will facilitate rapid screening of material for resistance to bacterial and other phytopathogens, allowing more efficient selection and quantification of resistance responses.

Inoculation of drought-stressed strawberry with a mixed inoculum of two arbuscular mycorrhizal fungi: effects on population dynamics of fungal species in roots and consequential plant tolerance to water deficiency.

The effect of inoculation with two arbuscular mycorrhizal fungi (AMF) on growth and drought tolerance of cultivated strawberry (Fragaria × ananassa) was studied. Three treatments (a single treatment either of Funneliformis mosseae BEG25, Funneliformis geosporus BEG11 or a 50:50 mixed inoculation treatment of both species) were compared to uninoculated plants. Species-specific primers for qPCR quantification of F. geosporus and F. mosseae DNA were developed to quantify the relative abundance of each fungus in roots of strawberry under different conditions of water stress. Co-occupation of the same root by both species was shown to commonly occur, but their relative abundance varied with water stress (reduced irrigation of up to 40%). Greater root colonisation was observed microscopically under water stress, but this increased colonisation was often accompanied with decreased amounts of fungal DNA in the root. F. mosseae tended to become more abundant under water stress relative to F. geosporus. There was significant correlation in the fungal colonisation measurements from the microscopic and qPCR methods under some conditions, but the nature of this relationship varied greatly with AMF inoculum and abiotic conditions. Single-species inoculation treatments gave similar benefits to the host to the mixed inoculation treatment regardless of irrigation regime; here, amount of colonisation was of greater importance than functional diversity. The addition of AMF inocula to plants subjected to reduced irrigation restored plant growth to the same or higher values as the non-mycorrhizal, fully-watered plants. The water use efficiency of plants was greater under the regulated deficit irrigation (RDI) regime and in AMF-inoculated plants, but there were no significant differences between plants inoculated with the single or combined inoculum. This study demonstrated that the increase in plant growth was directly influenced by an increase in root colonisation by AMF when individual plants were examined.

Concurrent infection with Cryptococcus neoformans/gattii species complex and Mycobacterium avium affecting the subcutis and bone of a pelvic limb in a cat.

This paper describes a cat with severe localised infections with Cryptococcus neoformans/gattii species complex and Mycobacterium avium affecting the subcutis and underlying fascia and bone of the right pelvic limb. The simultaneous isolation of both pathogens in this patient was unexpected and posed unique issues concerning both diagnosis and clinical management. The aetiopathogenesis of this infection is discussed in relation to aspects of diagnosis and therapy.

Small molecules targeting hepatitis C virus-encoded NS5A cause subcellular redistribution of their target: insights into compound modes of action.

The current standard of care for hepatitis C virus (HCV)-infected patients consists of lengthy treatment with interferon and ribavirin. To increase the effectiveness of HCV therapy, future regimens will incorporate multiple direct-acting antiviral (DAA) drugs. Recently, the HCV-encoded NS5A protein has emerged as a promising DAA target. Compounds targeting NS5A exhibit remarkable potency in vitro and demonstrate early clinical promise, suggesting that NS5A inhibitors could feature in future DAA combination therapies. Since the mechanisms through which these molecules operate are unknown, we have used NS5A inhibitors as tools to investigate their modes of action. Analysis of replicon-containing cells revealed dramatic phenotypic alterations in NS5A localization following treatment with NS5A inhibitors; NS5A was redistributed from the endoplasmic reticulum to lipid droplets. The NS5A relocalization did not occur in cells treated with other classes of HCV inhibitors, and NS5A-targeting molecules did not cause similar alterations in the localization of other HCV-encoded proteins. Time course analysis of the redistribution of NS5A revealed that the transfer of protein to lipid droplets was concomitant with the onset of inhibition, as judged by the kinetic profiles for these compounds. Furthermore, analysis of the kinetic profile of inhibition for a panel of test molecules permitted the separation of compounds into different kinetic classes based on their modes of action. Results from this approach suggested that NS5A inhibitors perturbed the function of new replication complexes, rather than acting on preformed complexes. Taken together, our data reveal novel biological consequences of NS5A inhibition, which may help enable the development of future assay platforms for the identification of new and/or different NS5A inhibitors.

Feline cholecystitis and acute neutrophilic cholangitis: clinical findings, bacterial isolates and response to treatment in six cases.

Clinicopathological findings from six cats with confirmed cholecystitis or acute neutrophilic cholangitis are presented. Historical findings included lethargy and anorexia or inappetence of up to five days duration. On physical examination all cats were pyrexic and four out of six were jaundiced and had cranial abdominal pain. Bile samples were obtained by cholecystocentesis at exploratory coeliotomy (two cases) or by percutaneous, ultrasound-guided cholecystocentesis (four cases). Gall bladder rupture and bile peritonitis occurred subsequent to ultrasound-guided cholecystocentesis in one case. The most common bacterial isolate was Escherichia coli (four cases); E coli was isolated alone in two cases, in combination with a Streptococcus species (one case) and in combination with a Clostridium species (one case). Streptococcus species alone was isolated from one case, as was Salmonella enterica serovar Typhimurium. The latter is the first reported case of Salmonella-associated cholecystitis in a cat. Concurrent pancreatic or intestinal disease was detected histologically in three cases. All cases were treated with antimicrobials based on in vitro susceptibility results. Treatment was successful in five cases. One cat with concurrent diffuse epitheliotropic intestinal lymphoma was euthanased. Percutaneous ultrasound-guided cholecystocentesis is an effective, minimally-invasive technique enabling identification of bacterial isolates in cats with inflammatory hepatobiliary disease.

Herbicide risk assessment for non-target aquatic plants: sulfosulfuron--a case study.

Herbicides entering the aquatic environment by spray drift, run-off and leaching to field drains may cause adverse effects on non-target aquatic vegetation. The potential for such effects has typically been evaluated from tests with floating, monocotyledonous Lemna sp. However, concern has been expressed as to whether this species could be used to indicate potential effects on other vegetation types, particularly rooted, submerged, emergent or dicotyledonous species. In 1997, the Centre for Aquatic Plant Management undertook development of new tests based on the additional species, Glyceria maxima (Hartm) Holmb, Lagarosiphon major (Ridl) Moss and Myriophyllum spicatum L. The resulting methodology was used to assess the effects of the sulfonylurea herbicide, sulfosulfuron on these species. Data presented here demonstrate that exposure to initial sulfosulfuron concentrations of 3.33 microg litre(-1) for up to 21 days was tolerated by these species and that adverse effects were observed only when plants were exposed to initial concentrations of 3.33 and 10 microg litre(-1) for 70 days. As the occurrence of such high initial concentrations for long periods is unlikely in the aquatic environment, sulfosulfuron is not expected to have adverse effects on the growth of these species. This study has also demonstrated that G maxima, L major and M spicatum grown in small outdoor tanks can be used successfully to assess the effects of crop-protection products on non-target aquatic flora.