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Black nightshade (Solanum nigrum, S. nigrum L.) and red nightshade ( Solanum villosum, S. villosum Mill.) are medicinal plants from the Solanaceae family that synthesize glycoalkaloids and other secondary metabolites. To recognize the potential insecticide activity of these compounds, leaf extracts (containing glycoalkaloid and methanol fractions) were tested for enzyme inhibition, antifeedant activity and toxicity. For in-vitro glutathione S-transferase (GST) inhibition activity, we used insecticide-resistant Colorado potato beetle, Leptinotarsa decemlineata ( L. decemlineata; Say) midgut and fat-body homogenate. In-vivo toxicity and the antifeedant activity were performed using larval bioassays. The methanol extracts had greater GST inhibitory activity compared to the glycoalkaloids, as well as greater 2nd instar larvae mortality and antifeedant activity. Furthermore, the green leaf volatile compound, cis-hex-3-enyl acetate, at the concentration of 5 ppm, caused 50% mortality of 2nd instar larvae. Our findings suggest the potential usefulness of S. nigrum and S. villosum extracts to control L. decemlineata., (© 2019 Wiley Periodicals, Inc.)

The expression of cold-induced genes is critical for plants to survive under freezing stress. However, the underlying mechanisms for the decision of when, where, and which genes to express are unclear when a plant meets a sudden temperature drop. Previous studies have demonstrated epigenetics to play a central role in the regulation of gene expression in plant responses to environmental stress. DNA methylation and histone deacetylation are the two most important epigenetic modifications. This study was conducted to investigate the effects of inhibiting DNA methylation and histone deacetylation on gene expression, and to explore the potential role of epigenetics in plant responses to cold stress. The results revealed that histone deacetylase inhibitors (trichostatin A) and DNA methylation inhibitors (5-Aza-2'-deoxycytosine) treatment enhanced cold tolerance. DNA microarray analysis and the gene ontology method revealed 76 cold-induced differently expressed genes in Arabidopsis thaliana seedlings that were treated to 0°C for 24 h following Trichostatin A and 5-Aza-2'-Deoxycytidine. Furthermore, analyses of metabolic pathways and transcription factors of 3305 differentially expressed genes were performed. Each four metabolic pathways were significantly affected (p < 0.01) by Trichostatin A and 5-Aza-2'-Deoxycytidine. Finally, 10 genes were randomly selected and verified via qPCR analysis. Our study indicated that Trichostatin A and 5-Aza-2'-Deoxycytidine can improve the plant cold resistance and influence the expression of the cold-induced gene in A. thaliana. This result will advance our understanding of plant freezing responses and may provide a helpful strategy for cold tolerance improvement in crops.

A recently characterized calmodulin-like protein is an endogenous RNA silencing suppressor that suppresses sense-RNA induced post-transcriptional gene silencing (S-PTGS) and enhances virus infection, but the mechanism underlying calmodulin-like protein-mediated S-PTGS suppression is obscure. Here, we show that a calmodulin-like protein from Nicotiana benthamiana (NbCaM) interacts with Suppressor of Gene Silencing 3 (NbSGS3). Deletion analyses showed that domains essential for the interaction between NbSGS3 and NbCaM are also required for the subcellular localization of NbSGS3 and NbCaM suppressor activity. Overexpression of NbCaM reduced the number of NbSGS3-associated granules by degrading NbSGS3 protein accumulation in the cytoplasm. This NbCaM-mediated NbSGS3 degradation was sensitive to the autophagy inhibitors 3-methyladenine and E64d, and was compromised when key autophagy genes of the phosphatidylinositol 3-kinase (PI3K) complex were knocked down. Meanwhile, silencing of key autophagy genes within the PI3K complex inhibited geminivirus infection. Taken together these data suggest that NbCaM acts as a suppressor of RNA silencing by degrading NbSGS3 through the autophagy pathway.

At the time of this research, there were only two insecticides registered for control of cabbage maggot, Delia radicum L., in rutabaga in Canada, one of which (diazinon) will be deregistered by 2017, and resistance having been reported in some areas for the other (chlorpyrifos). To screen for chemistries to replace these organophosphates, and obtain efficacy data comparable between key vegetable brassica production areas in Canada, four small plot field studies were conducted concurrently in British Columbia, Saskatchewan, Ontario, and Quebec in 2009. These studies followed standardized protocols for seeding, application of insecticide drenches, sampling and damage assessment, and generally tested the same products. Of the insecticides evaluated, none provided maggot control comparable with the industry standard, chlorpyrifos. However, cyantraniliprole (Cyazypyr 200SC; registered in 2015 as Verimark) applied at 3 g AI (15.0 ml product)/100 m row of seeded rutabagas consistently provided the next highest reduction in % culls, suggesting the efficacy of this chemical may be improved if used at higher rates. The results of these studies are discussed in the context of current literature on D. radicum management in rutabaga. Future management strategies are also discussed, including a transplant plug treatment approach for increasing the dosage per plant and efficacy of chemistries such as Cyazypyr 200SC in the field., (© Crown copyright 2016.)

Drosophila suzukii is an emerging global pest of soft fruit; although it likely overwinters as an adult, larval cold tolerance is important both for determining performance during spring and autumn, and for the development of temperature-based control methods aimed at larvae. We examined the low temperature biology of third instar feeding and wandering larvae in and out of food. We induced phenotypic plasticity of thermal biology by rearing under short days and fluctuating temperatures (5.5-19°C). Rearing under fluctuating temperatures led to much slower development (42.1days egg-adult) compared to control conditions (constant 21.5°C; 15.7days), and yielded larger adults of both sexes. D. suzukii larvae were chill-susceptible, being killed by low temperatures not associated with freezing, and freezing survival was not improved when ice formation was inoculated externally via food or silver iodide. Feeding larvae were more cold tolerant than wandering larvae, especially after rearing under fluctuating temperatures, and rearing under fluctuating temperatures improved survival of prolonged cold (0°C) to beyond 72h in both larval stages. There was no evidence that acute cold tolerance could be improved by rapid cold-hardening. We conclude that D. suzukii has the capacity to develop at low temperatures under fluctuating temperatures, but that they have limited cold tolerance. However, phenotypic plasticity of prolonged cold tolerance must be taken into account when developing low temperature treatments for sanitation of this species., (Crown Copyright © 2016. Published by Elsevier Ltd. All rights reserved.)

The goal of this study was to monitor Shiga toxin-producing Escherichia coli (STEC) serogroups and virulence genes in cattle (n = 30) originating from a closed herd. Fecal samples were collected (1) at weaning, (2) upon arrival to a feedlot, (3) after 30 days on feed (DOF), and (4) after 135 DOF. DNA was extracted from feces for detection of virulence and serogroup genes by polymerase chain reaction (PCR) and immunomagnetic separation and pulsed-field gel electrophoresis (PFGE) were performed to collect and subtype STEC isolates. The prevalence of each serogroup measured by PCR from weaning to 135 DOF was 23.3-80.0% for O26, 33.3-46.7% for O45, 70.0-73.3% for O103, 36.7-86.7% for O111, 56.7-6.7% for O121, 26.7-66.7% for O145, and 66.7-90.0% for O157. Total fecal samples positive for virulence genes were 87.5% for ehxA, 85.8% for stx 1 , 60.0% for stx 2 , 52.5% for eae, and 44.2% for the autoagglutinating adhesion gene, saa. The prevalence of each serogroup and virulence gene tended to increase by 135 DOF, with the exception of O121, stx 2 , and saa. The frequency of detection of some virulence genes was largely affected over time, most notably with saa and stx 2 decreasing, and eae increasing when cattle were transitioned to concentrate-based diets. PFGE analysis of O157 and O103 fecal isolates revealed dominant pulsotypes, but the presence of identical O103 isolates, which differed in virulence profiles. Overall, this study showed that fecal shedding of E. coli serogroups and virulence-associated genes are highly variable over time as cattle move from ranch to feedlot. To mitigate STEC, it is important to understand the factors affecting both prevalence of individual serogroups and the presence of virulence factors.

The inositol requiring enzyme (IRE1) is an endoplasmic reticulum (ER) stress sensor. When activated, it splices the bZIP60 mRNA, producing a truncated transcription factor that upregulates genes involved in the unfolded protein response. Bax inhibitor 1 (BI-1) is another ER stress sensor that regulates cell death in response to environmental assaults. The potyvirus 6K2 and potexvirus TGB3 proteins are known to reside in the ER, serving, respectively, as anchors for the viral replicase and movement protein complex. This study used green fluorescent protein (GFP)-tagged Turnip mosaic virus (TuMV), Plantago asiatica mosaic virus (PlAMV), Potato virus Y (PVY), and Potato virus X (PVX) to determine that the IRE1/bZIP60 pathway and BI-1 machinery are induced early in virus infection in Arabidopsis thaliana, Nicotiana benthamiana, and Solanum tuberosum. Agrodelivery of only the potyvirus 6K2 or TGB3 genes into plant cells activated bZIP60 and BI-1 expression in Arabidopsis thaliana, N. benthamiana, and S. tuberosum. Homozygous ire1a-2, ire1b-4, and ire1a-2/ire1b-4 mutant Arabidopsis plants were inoculated with TuMV-GFP or PlAMV-GFP. PlAMV accumulates to a higher level in ire1a-2 or ire1a-2/ire1b-4 mutant plants than in ire1b-4 or wild-type plants. TuMV-GFP accumulates to a higher level in ire1a-2, ire1b-4, or ire1a-2/ire1b-4 compared with wild-type plants, suggesting that both isoforms contribute to TuMV-GFP infection. Gene silencing was used to knock down bZIP60 and BI-1 expression in N. benthamiana. PVX-GFP and PVY-GFP accumulation was significantly elevated in these silenced plants compared with control plants. This study demonstrates that two ER stress pathways, namely IRE1/bZIP60 and the BI-1 pathway, limit systemic accumulation of potyvirus and potexvirus infection. Silencing BI-1 expression also resulted in systemic necrosis. These data suggest that ER stress-activated pathways, led by IRE1 and BI-1, respond to invading potyvirus and potexviruses to restrict virus infection and enable physiological changes enabling plants to tolerate virus assault.

Antimicrobial resistance is one of the most significant challenges facing the global medical community and can be attributed to the use and misuse of antibiotics. This includes use as growth promoters or for prophylaxis and treatment of bacterial infection in intensively farmed livestock from where antibiotics can enter the environment as residues in manure. We characterised the impact of the long-term application of a mixture of veterinary antibiotics alone (tylosin, sulfamethazine and chlortetracycline) on class 1 integron prevalence and soil microbiota composition. Class 1 integron prevalence increased significantly (P < 0.005) from 0.006% in control samples to 0.064% in the treated plots. Soil microbiota was analysed using 16S rRNA gene sequencing and revealed significant alterations in composition. Of the 19 significantly different (P < 0.05) OTUs identified, 16 were of the Class Proteobacteria and these decreased in abundance relative to the control plots. Only one OTU, of the Class Cyanobacteria, was shown to increase in abundance significantly; a curiosity given the established sensitivity of this class to antibiotics. We hypothesise that the overrepresentation of Proteobacteria as OTUs that decreased significantly in relative abundance, coupled with the observations of an increase in integron prevalence, may represent a strong selective pressure on these taxa., (© FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Agricultural operations generate large quantities of manure which must be eliminated in a manner that is consistent with public health guidelines. Meanwhile, construction and demolition waste makes up about 25% of total solid municipal waste. Co-composting of manure with construction and demolition waste offers a potential means to make manure safe for soil amendment and also divert construction and demolition waste from municipal landfills. Therefore, the archaeal, bacterial, and fungal microbiota of two different types of composted cattle manure and one co-composted with construction and demolition waste, were assessed over a 99-day composting period. The microbiota of the three compost mixtures did not differ, but significant changes over time and by sampling depth were observed. Bacillus and Halocella, however, were more relatively abundant in composted manure from cattle fed dried distillers' grains and solubles. Proteobacteria and Bacteroidetes were enriched at day 0 and Firmicutes at day 99. The fungal genus Kernia was the most relatively abundant overall and was enriched at day 0. The concentration of 12 antimicrobial resistance determinants in the compost mixtures was also determined, and 10 of these determinants decreased significantly from days 0 to 99. The addition of construction and demolition waste did not affect the persistence of antimicrobial resistance genes or community structure of the compost microbiota and therefore co-composting construction and demolition waste with cattle manure offers a safe, viable way to divert this waste from landfills.

Overwintering insects must survive the multiple-stress environment of winter, which includes low temperatures, reduced food and water availability, and cold-active pathogens. Many insects overwinter in diapause, a developmental arrest associated with high stress tolerance. Drosophila suzukii (Diptera: Drosophilidae), spotted wing drosophila, is an invasive agricultural pest worldwide. Its ability to overwinter and therefore establish in temperate regions could have severe implications for fruit crop industries. We demonstrate here that laboratory populations of Canadian D. suzukii larvae reared under short-day, low temperature, conditions develop into dark 'winter morph' adults similar to those reported globally from field captures, and observed by us in southern Ontario, Canada. These winter-acclimated adults have delayed reproductive maturity, enhanced cold tolerance, and can remain active at low temperatures, although they do not have the increased desiccation tolerance or survival of fungal pathogen challenges that might be expected from a more heavily melanised cuticle. Winter-acclimated female D. suzukii have underdeveloped ovaries and altered transcript levels of several genes associated with reproduction and stress. While superficially indicative of reproductive diapause, the delayed reproductive maturity of winter-acclimated D. suzukii appears to be temperature-dependent, not regulated by photoperiod, and is thus unlikely to be 'true' diapause. The traits of this 'winter morph', however, likely facilitate overwintering in southern Canada, and have probably contributed to the global success of this fly as an invasive species., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

As an organelle only found in plant cells and some protists, the chloroplast is not only the main metabolic energy originator, but also the abiotic/biotic stress sensor and defense signal generator. For a long time, chloroplasts have been recognized as a common target by many plant viruses. Viruses may directly modify chloroplast membranes to assemble their replication complex for viral genome replication. Viruses may downregulate chloroplast-related and photosynthesis-related genes via an as yet unknown mechanism to support their infection. Viruses may also interrupt functionality of the photosynthetic machinery through protein-protein interactions. This review briefly summarizes current knowledge about modifications of the photosynthetic machinery by plant viruses, highlights the important role of chloroplasts in the infection process and discusses chloroplast-associated pathogenesis., (Crown Copyright © 2015. Published by Elsevier B.V. All rights reserved.)

The histone deacetylases play important roles in the regulation of gene expression and the subsequent control of a number of important biological processes, including those involved in the response to environmental stress. A specific group of histone deacetylase genes, HD2, is present in plants. In Arabidopsis, HD2s include HD2A, HD2B, HD2C, and HD2D. Previous research showed that HD2A, HD2B, and HD2C are more related in terms of expression and function, but not HD2D. In this report, we studied different aspects of AtHD2D in Arabidopsis with respect to plant response to drought and other abiotic stresses. Bioinformatics analysis indicates that HD2D is distantly related to other HD2 genes. Transient expression in Nicotiana benthamiana and stable expression in Arabidopsis of AtHD2D fused with gfp showed that AtHD2D was expressed in the nucleus. Overexpression of AtHD2D resulted in developmental changes including fewer main roots, more lateral roots, and a higher root:shoot ratio. Seed germination and plant flowering time were delayed in transgenic plants expressing AtHD2D, but these plants exhibited higher degrees of tolerance to abiotic stresses, including drought, salt, and cold stresses. Physiological studies indicated that the malondialdehyde (MDA) content was high in wild-type plants but in plants overexpressing HD2D the MDA level increased slowly in response to stress conditions of drought, cold, and salt stress. Furthermore, electrolyte leakage in leaf cells of wild type plants increased but remained stable in transgenic plants. Our results indicate that AtHD2D is unique among HD2 genes and it plays a role in plant growth and development regulation and these changes can modulate plant stress responses.

Leguminous plants selectively initiate primary responses to rhizobial nodulation factors (NF) that ultimately lead to symbiotic root nodule formation. Functioning downstream, cytokinin has emerged as the key endogenous plant signal for nodule differentiation, but its role in mediating rhizobial entry into the root remains obscure. Nonetheless, such a role is suggested by aberrant infection phenotypes of plant mutants with defects in cytokinin signaling. We postulate that cytokinin participates in orchestrating signaling events that promote rhizobial colonization of the root cortex and limit the extent of subsequent infection at the root epidermis, thus maintaining homeostasis of the symbiotic interaction. We further argue that cytokinin signaling must have been crucial during the evolution of plant cell predisposition for rhizobial colonization., (Crown Copyright © 2015. Published by Elsevier Ltd. All rights reserved.)

Detoxification by glutathione S-transferases (GSTs) and esterases are important mechanisms associated with insecticide resistance. Discovery of novel GST and esterase inhibitors from phytochemicals could provide potential new insecticide synergists. Conifer tree species contain flavonoids, such as taxifolin, that inhibit in vitro GST activity. The objectives were to test the relative effectiveness of taxifolin as an enzyme inhibitor and as an insecticide synergist in combination with the organophosphorous insecticide, Guthion (50% azinphos-methyl), and the botanical insecticide, pyrethrum, using an insecticide-resistant Colorado potato beetle (CPB) Leptinotarsa decemlineata (Say) strain. Both taxifolin and its isomer, quercetin, increased the mortality of 1(st) instar CPB larvae after 48h when combined with Guthion, but not pyrethrum. Taxifolin had greater in vitro esterase inhibition compared with the commonly used esterase inhibitor, S, S, S-tributyl phosphorotrithioate (DEF). An in vivo esterase and GST inhibition effect after ingestion of taxifolin was measured, however DEF caused a greater suppression of esterase activity. This study demonstrated that flavonoid compounds have both in vitro and in vivo esterase inhibition, which is likely responsible for the insecticide synergism observed in insecticide-resistant CPB., (Crown Copyright © 2015. Published by Elsevier Inc. All rights reserved.)

Here we report the complete genomic sequence of the bacterium Arthrobacter sp. strain LS16, consisting of a single circular chromosome of 3.85 Mb with no identified plasmid. Data contained within will facilitate future genetic modification and engineering of the Arthrobacter sp. LS16 metabolic network to enhance traits relevant to bioremediation and bioproducts., (Copyright © 2016 Hassan et al.)

The receptor binding domain of the tailspike protein Gp9 from the P22 bacteriophage was recently shown to reduce Salmonella colonization in the chicken gut. In this study, we transiently expressed the receptor binding domain of the Gp9 tailspike protein in Nicotiana benthamiana, and targeted it to the endoplasmic reticulum (ER) or to the chloroplasts. Gp9 was also fused to either an elastin-like polypeptide (ELP) or hydrophobin I tag, which were previously described to improve accumulation levels of recombinant proteins. The highest levels of recombinant protein accumulation occurred when unfused Gp9 was targeted to the ER. Lower levels of chloroplast-targeted Gp9 were also detected. ELP-fused Gp9 was purified and demonstrated to bind to Salmonella enterica serovar Typhimurium in vitro. Upon oral administration of lyophilized leaves expressing Gp9-ELP to newly hatched chickens, we found that this tailspike protein has the potential to be used as a therapeutic to control Salmonella contamination in chickens.

It is envisioned that a more precise study of the association between the traits and biomarkers will dramatically decrease the time and costs required to bring new improved disease resistance lines to market. The field of omics has an enormous potential to assess diseases more precise, including the identification and understanding of pathogenic mechanisms in legume crops, and have been exemplified by a relatively large number of studies. Recently, molecular genetic studies have accumulated a huge amount of genotypic data, through a more affordable next generation sequencing (NGS) technology, causing the omics approaches to fall behind. In this paper I provide an overview of genomics and proteomics and their use in legume crops, including the use of comparative genomics to identify homologous markers within legume crops.

Rationale: Fumonisins and AAL-toxins are structurally similar mycotoxins that contaminate agricultural crops and foodstuffs. Traditional analytical screening methods are designed to target the known compounds for which standards are available but there is clear evidence that many other derivatives exist and could be toxic. A fast, semi-targeted method for the detection of all known fumonisins, AAL-toxins and related emerging toxins is required., Methods: Strains of Fusarium verticillioides, Alternaria arborescens and Aspergillus welwitschiae were grown on their associated crops (maize, tomatoes, and grapes, respectively). Extracts were first analyzed in negative mode using product ion filtering to detect the tricarballylic ester product ion that is common to fumonisins and AAL-toxins (m/z 157.0142). During the same liquid chromatography (LC) run, rapid polarity switching was then used to collect positive mode tandem mass spectrometric (MS(2) ) data for characterization of the detected compounds., Results: Fumonisin B1 , B2 , B3 and B4 were detected on Fusarium contaminated maize, AAL-toxins TA, TB, TD, TE were detected on Alternaria inoculated tomatoes and fumonisin B2 , B4 and B6 on Aspergillus contaminated grapes. Additionally, over 100 structurally related compounds possessing a tricarballylic ester were detected from the mould inoculated plant material. These included a hydroxyl-FB1 from F. verticillioides inoculated maize, keto derivatives of AAL-toxins from A. arborescens inoculated tomatoes, and two previously unreported classes of non-aminated fumonisins from Asp. welwitschiae contaminated grapes., Conclusions: A semi-targeted method for the detection of all fumonisins and AAL-toxins in foodstuffs was developed. The use of the distinctive tricarballylic ester product anion for detection combined with rapid polarity switching and positive mode MS(2) is an effective strategy for differentiating between known isomers such as FB1 and FB6 . This analytical tool is also effective for the identification of new compounds as evident from the discoveries of the previously unreported hydroxyl-FB1 , keto-AAL-toxins, and the two new families of non-aminated fumonisins., (© 2015 Her Majesty the Queen in Right of Canada Rapid Communications in Mass Spectrometry © 2015 John Wiley & Sons Ltd. Reproduced with the permission of the Ministers of Agriculture and Agri-Food Canada.)

14-3-3s are a class of conserved regulatory proteins ubiquitously found in eukaryotes, which play important roles in a variety of cellular processes including response to diverse stresses. Although much has been learned about 14-3-3s in several plant species, it remains unknown in common bean. In this study, 9 common bean 14-3-3s (PvGF14s) were identified by exhaustive data mining against the publicly available common bean genomic database. A phylogenetic analysis revealed that each predicted PvGF14 was clustered with two GmSGF14 paralogs from soybean. Both epsilon-like and non-epsilon classes of PvGF14s were found in common bean, and the PvGF14s belonging to each class exhibited similar gene structure. Among 9 PvGF14s, only 8 are transcribed in common bean. Expression patterns of PvGF14s varied depending on tissue type, developmental stage and exposure of plants to stress. A protein-protein interaction study revealed that PvGF14a forms dimer with itself and with other PvGF14 isoforms. This study provides a first comprehensive look at common bean 14-3-3 proteins, a family of proteins with diverse functions in many cellular processes, especially in response to stresses.

Background: MicroRNAs (miRNAs) have functions in diverse biological processes such as growth, signal transduction, disease resistance, and stress responses in plants. Thermotherapy is an effective approach for elimination of viruses from fruit trees. However, the role of miRNAs in this process remains elusive. Previously, we showed that high temperature treatment reduces the titers of Apple stem grooving virus (ASGV) from the tips of in vitro-grown Pyrus pyrifolia plants. In this study, we identified high temperature-altered pear miRNAs using the next generation sequencing technology, and futher molecularly characterized miRNA-mediated regulaton of target gene expression in the meristem tip and base tissues of in vitro-grown, ASGV-infected pear shoots under different temperatures., Results: Using in vitro-grown P. pyrifolia shoot meristem tips infected with ASGV, a total of 22,592,997 and 20,411,254 clean reads were obtained from Illumina high-throughput sequencing of small RNA libraries at 24 °C and 37 °C, respectively. We identified 149 conserved and 141 novel miRNAs. Seven conserved miRNAs and 77 novel miRNAs were differentially expressed at different temperatures. Target genes for differentially expressed known and novel miRNAs were predicted and functionally annotated. Gene Ontology (GO) analysis showed that high-ranking miRNA target genes were involved in metabolic processes, responses to stress, and signaling, indicating that these high temperature-responsive miRNAs have functions in diverse gene regulatory networks. Spatial expression patterns of the miRNAs and their target genes were found to be expressed in shoot tip and base tissues by qRT-PCR. In addition, high temperature reduced viral titers in the shoot meristem tip, while negatively regulated miRNA-mediated target genes related to resistance disease defense and hormone signal transduction pathway were up-regulated in the P. pyrifolia shoot tip in response to high temperature. These results suggested that miRNAs may have important functions in the high temperature-dependent decrease of ASGV titer in in vitro-grown pear shoots., Conclusions: This is the first report of miRNAs differentially expressed at 24 °C and 37 °C in the meristem tip of pear shoots infected with ASGV. The results of this study provide valuable information for further exploration of the function of high temperature-altered miRNAs in suppressing viral infections in pear and other fruit trees.

Double-stranded RNA (dsRNA) is an important type of RNA that plays essential roles in diverse cellular processes in eukaryotic organisms and a hallmark in infections by positive-sense RNA viruses. Currently, no in vivo technology has been developed for visualizing dsRNA in living cells. Here, we report a dsRNA binding-dependent fluorescence complementation (dRBFC) assay that can be used to efficiently monitor dsRNA distribution and dynamics in vivo. The system consists of two dsRNA-binding proteins, which are fused to the N- and C-terminal halves of the yellow fluorescent protein (YFP). Binding of the two fusion proteins to a common dsRNA brings the split YFP halves in close proximity, leading to the reconstitution of the fluorescence-competent structure and restoration of fluorescence. Using this technique, we were able to visualize the distribution and trafficking of the replicative RNA intermediates of positive-sense RNA viruses in living cells., (Crown Copyright © 2015. Published by Elsevier Inc. All rights reserved.)

Rationale: Many species of Alternaria damage important agricultural crops, including small grains and tomatoes. These fungi can produce a variety of secondary metabolites, some of which are toxic to humans and animals. Interest in screening for conjugated or 'modified' mycotoxins has increased because of their tendency to evade traditional analytical screening methods. Two sulfoconjugated Alternaria toxins have been reported and the potential exists for many more., Methods: One hundred and forty-eight Canadian strains of Alternaria spp., about half of them isolated from grain, were grown on Potato Dextrose Agar in Petri dishes for 7 days. Plugs of each strain were removed, extracted and screened by a rapid liquid chromatography (LC)/data-dependent tandem mass spectrometry (MS(2)) method in negative electrospray ionization mode. Data generated on an Orbitrap Q-Exactive mass spectrometer was processed by post-acquisition neutral loss filtering (NLF). Seven isolates that produced sulfoconjugates of known Alternaria toxins were selected for growth on three additional types of fermentation media., Results: Collision-induced dissociation of sulfoconjugated ions displayed a distinctive neutral loss of SO3 (79.957 Da) that was detected in the MS(2) datasets using post-acquisition NLF. A total of 108 of the 148 isolates screened produced sulfoconjugated metabolites on agar plates. Analysis of the seven isolates grown in liquid culture, on rice and Cheerios, led to the discovery of six new, two previously reported and 30 unidentified sulfoconjugated compounds., Conclusions: NLF of HRMS(2) data from an Orbitrap Q-Exactive is a powerful tool for the rapid discovery of sulfoconjugated fungal metabolites. This technique could also be applied to the detection of other important conjugated mycotoxins such as glucosides. The majority of the Canadian isolates of Alternaria spp. studied produced sulfoconjugated metabolites, some of which had no known 'free' Alternaria precursor metabolite, indicating that they are possibly new metabolites. The advantage of sulfoconjugation to Alternaria spp. is unknown, and warrants further study into the mechanisms behind the sulfur assimilatory pathways., (Copyright © 2015 John Wiley & Sons, Ltd.)

Utilization of timed virus acquisition access probes in studies of plum pox virus (PPV) transmission by aphids demonstrated that endemic species transmitted the virus readily from plum, Prunus domestica (L.) Batsch; peach, P. persica (L.); or dwarf flowering almond, P. glandulosa Thunberg., to peach seedlings. The green peach aphid, Myzus persicae (Sulzer), was shown to be the most efficient vector. Acquisition of virus by green peach aphids from infected peach leaves resulted in 18-28% infected peach seedlings, while aphids previously fed on infected leaves of plum transferred virus to 36% of peach seedlings. Although the spirea aphid, Aphis spiraecola (Patch), was a less efficient vector than M. persicae it is perhaps more important for the spread of PPV due to its greater abundance and occurrence earlier in the season when peach trees are thought to be more susceptible to infection. Virus transmission rates varied depending on the virus source and healthy test plant species. In contrast to many previous studies, aphid inoculation of the experimental host Nicotiana benthamiana Domin occurred at a low rate, never exceeding 4%. Acquisition of PPV by M. persicae from infected peach fruit was greatly reduced compared with acquisition from leaves. The results of this research indicate that the Ontario isolate of PPV-D is readily transmissible by aphids to peach and natural spread of the virus needs to be considered in future management or eradication programs., (© Her Majesty in Right of Canada, as represented by the Minister of Agriculture and Agri-Food Canada. Published by Oxford University Press on behalf of Entomological Society of America.)

Field strains of tomato bacterial spot pathogen (Xanthomonas euvesicatoria, X. vesicatoria, X. perforans, and X. gardneri) were characterized for sensitivity to copper and species composition. A total of 98 strains were isolated from symptomatic leaf and fruit samples collected from 18 tomato fields in Ontario. In greenhouse pathogenicity tests, most of the field strains caused severe (37 strains) to highly severe (23 strains) symptoms on 'Bonny Best' tomato plants, whereas 38 strains caused moderate symptoms. In MGY agar plates amended with various concentrations of copper sulfate, 11 strains were completely sensitive (no growth) and 87 strains were resistant (grew on 1.0 mmol/L or higher copper concentration). PCR analysis of the hrp gene cluster followed by restriction digestion with HaeIII and sequencing identified X. gardneri (35 strains) and X. perforans (26 strains) as predominant species and X. euvesicatoria and X. vesicatoria as less common species in Ontario tomato fields. Separation of field strains into various species was also confirmed with starch hydrolysis activity on agar medium. Moreover, 72 field strains produced shiny greenish-yellow colonies surrounded by a milky zone on xanthomonad differential (Xan-D) medium, and the colonies of 26 strains did not produce a milky zone. Thirty-four strains could not be clustered into any species and 25 of those strains were negative for the hrp gene PCR and also did not produce a milky zone around colonies on Xan-D medium. Our results suggest a widespread existence of copper-resistant strains and an increase in X. perforans strains of bacterial spot pathogen in Ontario. This information on copper resistance and species composition within bacterial spot pathogens in Ontario will be helpful for developing effective disease management strategies, making cultivar selection, and breeding new tomato cultivars.

In soybean seed, a correlation has been observed between the concentration of free asparagine at mid-maturation and protein concentration at maturity. In this study, a Phaseolus vulgaris K + -dependent asparaginase cDNA, PvAspG2, was expressed in transgenic soybean under the control of the embryo specific promoter of the β-subunit of β-conglycinin. Three lines were isolated having high expression of the transgene at the transcript, protein and enzyme activity levels at mid-maturation, with a 20- to 40-fold higher asparaginase activity in embryo than a control line expressing β-glucuronidase. Increased asparaginase activity was associated with a reduction in free asparagine levels as a percentage of total free amino acids, by 11-18%, and an increase in free aspartic acid levels, by 25-60%. Two of the lines had reduced nitrogen concentration in mature seed as determined by nitrogen analysis, by 9-13%. Their levels of extractible globulins were reduced by 11-30%. This was accompanied by an increase in oil concentration, by 5-8%. The lack of change in nitrogen concentration in the third transgenic line was correlated with an increase in free glutamic acid levels by approximately 40% at mid-maturation., (© 2015 Scandinavian Plant Physiology Society.)

Protein bodies (PBs) are endoplasmic reticulum (ER) derived organelles originally found in seeds whose function is to accumulate seed storage proteins. It has been shown that PB formation is not limited to seeds and green fluorescent protein (GFP) fused to either elastin-like polypeptide (ELP) or hydrophobin (HFBI) fusion tags induce the formation of PBs in leaves of N. benthamiana. In this study, we compared the ELP- and HFBI-induced PBs and showed that ELP-induced PBs are larger than HFBI-induced PBs. The size of ELP- and HFBI-induced PBs increased over time along with the accumulation levels of their fused protein. Our results show that PB formation is a concentration-dependent mechanism in which proteins accumulating at levels higher than 0.2% of total soluble protein are capable of inducing PBs in vivo. Our results show that the presence of fusion tags is not necessary for the formation of PBs, but affects the distribution pattern and size of PBs. This was confirmed by PBs induced by fluorescent proteins as well as fungal xylanases. We noticed that in the process of PB formation, secretory and ER-resident molecules are passively sequestered into the lumen of PBs. We propose to use this property of PBs as a tool to increase the accumulation levels of erythropoietin and human interleukin-10 by co-expression with PB-inducing proteins., (© 2015 Her Majesty the Queen in Right of Canada. Plant Biotechnology Journal © 2015 John Wiley & Sons Ltd and Society for Experimental Biology and The Association of Applied Biologists. Reproduced with the permission of the Minister of Agriculture and Agri-Food Canada.)

Bell pepper endornavirus (BPEV) is a double-stranded RNA virus infecting economically important crops, such as peppers. Next-generation sequencing of small RNAs extracted from the leaves of a pepper plant showing mild viral symptoms, along with subsequent analysis, identified BPEV. The complete genome of this isolate was cloned and sequenced., (Copyright © 2015 Chen et al.)

Drosophila suzukii (Matsumura) (Diptera: Drosophilidae) is a worldwide emerging pest of soft fruits, but its cold tolerance has not been thoroughly explored. We determined the cold tolerance strategy, low temperature thermal limits, and plasticity of cold tolerance in both male and female adult D. suzukii. We reared flies under common conditions (long days, 21°C; control) and induced plasticity by rapid cold-hardening (RCH, 1h at 0°C followed by 1h recovery), cold acclimation (CA, 5 days at 6°C) or acclimation under fluctuating temperatures (FA). D. suzukii had supercooling points (SCPs) between -16 and -23°C, and were chill-susceptible. 80% of control flies were killed after 1h at -7.2°C (males) or -7.5°C (females); CA and FA improved survival of this temperature in both sexes, but RCH did not. 80% of control flies were killed after 70 h (male) or 92 h (female) at 0°C, and FA shifted this to 112 h (males) and 165 h (females). FA flies entered chill coma (CTmin) at approximately -1.7°C, which was ca. 0.5°C colder than control flies; RCH and CA increased the CTmin compared to controls. Control and RCH flies exposed to 0°C for 8h took 30-40 min to recover movement, but this was reduced to <10 min in CA and FA. Flies placed outside in a field cage in London, Ontario, were all killed by a transient cold snap in December. We conclude that adult phenotypic plasticity is not sufficient to allow D. suzukii to overwinter in temperate habitats, and suggest that flies could overwinter in association with built structures, or that there may be additional cold tolerance imparted by developmental plasticity., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

A study was undertaken to determine the prevalence and diversity of species of the genus Arcobacter in pig and dairy cattle manure, which led to the identification of strains AF1440T, AF1430 and AF1581. Initially identified as Arcobacter butzleri based on colony morphology and initial PCR-confirmation tests, analyses of 16S rRNA gene sequences of these strains confirmed that they belonged to the genus Arcobacter and were different from all known species of the genus. The isolates formed a distinct group within the genus Arcobacter based on their 16S rRNA, gyrB, rpoB, cpn60, gyrA and atpA gene sequences and fatty acid profiles. Their unique species status was further supported by physiological properties and DNA-DNA hybridization that allowed phenotypic and genotypic differentiation of the strains from other species of the genus Arcobacter. The isolates were found to be oxidase, catalase and esterase positive and urease negative; they grew well at 30 °C under microaerophilic conditions and produced nitrite and acetoin. Based on their common origin and various physiological properties, it is proposed that the isolates are classified as members of a novel species with the name Arcobacter lanthieri sp. nov. The type strain is AF1440T ( = LMG 28516T = CCUG 66485T); strains AF1430 ( = LMG 28515 = CCUG 66486) and AF1581 ( = LMG 28517 = CCUG 66487) are reference strains.

Background: Non-typhoidal Salmonella enterica serovars, associated with different foods including poultry products, are important causes of bacterial gastroenteritis worldwide. The colonization of the chicken gut by S. enterica could result in the contamination of the environment and food chain. The aim of this study was to compare the genomes of 25 S. enterica serovars isolated from broiler chicken farms to assess their intra- and inter-genetic variability, with a focus on virulence and antibiotic resistance characteristics., Methodology/principal Finding: The genomes of 25 S. enterica isolates covering five serovars (ten Typhimurium including three monophasic 4,[5],12:i:, four Enteritidis, three Hadar, four Heidelberg and four Kentucky) were sequenced. Most serovars were clustered in strongly supported phylogenetic clades, except for isolates of serovar Enteritidis that were scattered throughout the tree. Plasmids of varying sizes were detected in several isolates independently of serovars. Genes associated with the IncF plasmid and the IncI1 plasmid were identified in twelve and four isolates, respectively, while genes associated with the IncQ plasmid were found in one isolate. The presence of numerous genes associated with Salmonella pathogenicity islands (SPIs) was also confirmed. Components of the type III and IV secretion systems (T3SS and T4SS) varied in different isolates, which could explain in part, differences of their pathogenicity in humans and/or persistence in broilers. Conserved clusters of genes in the T3SS were detected that could be used in designing effective strategies (diagnostic, vaccination or treatments) to combat Salmonella. Antibiotic resistance genes (CMY, aadA, ampC, florR, sul1, sulI, tetAB, and srtA) and class I integrons were detected in resistant isolates while all isolates carried multidrug efflux pump systems regardless of their antibiotic susceptibility profile., Conclusions/significance: This study showed that the predominant Salmonella serovars in broiler chickens harbor genes encoding adhesins, flagellar proteins, T3SS, iron acquisition systems, and antibiotic and metal resistance genes that may explain their pathogenicity, colonization ability and persistence in chicken. The existence of mobile genetic elements indicates that isolates from a given serovar could acquire and transfer genetic material. Conserved genes in the T3SS and T4SS that we have identified are promising candidates for identification of diagnostic, antimicrobial or vaccine targets for the control of Salmonella in broiler chickens.

BRAHMA (BRM), a SWI/SNF chromatin remodeling ATPase, is essential for the transcriptional reprogramming associated with development and cell differentiation in Arabidopsis thaliana. In this study, we show that loss-of-function mutations in BRM led to defective maintenance of the root stem cell niche, decreased meristematic activity, and stunted root growth. Mutations of BRM affected auxin distribution by reducing local expression of several PIN-FORMED (PIN) genes in the stem cells and impaired the expression of the stem cell transcription factor genes PLETHORA (PLT1) and PLT2. Chromatin immunoprecipitation assays showed that BRM could directly target to the chromatin of PIN1, PIN2, PIN3, PIN4, and PIN7. In addition, genetic interaction assays indicate that PLTs acted downstream of BRM, and overexpression of PLT2 partially rescued the stem cell niche defect of brm mutants. Taken together, these results support the idea that BRM acts in the PLT pathway to maintain the root stem cell niche by altering the expression of PINs., (© 2015 American Society of Plant Biologists. All rights reserved.)

Background: A survey of insecticide resistance in over 150 Canadian populations of Colorado potato beetle was completed between 2008 and 2011. Three neonicotinoid and two anthranilic diamide insecticides were tested at a discriminating concentration (DC) with second-instar larvae in a leaf-disc bioassay., Results: The mean mortality for the imidacloprid (Admire) DC was 46-67% between 2008 and 2011 respectively. Over the 4 years, 10-46% and 26-40% of the populations were classified as resistant or showed reduced susceptibility to imidacloprid. The mean mortality for thiamethoxam (Actara) and clothianidin (Poncho/Titan) ranged from 56-76% in 2008 to 81-84% in 2010 for each insecticide respectively, indicating continuous susceptibility to clothianidin but reduced susceptibility to thiamethoxam. In 2008 and 2009, susceptibility to chlorantraniliprole (Coragen) was observed in 85% of populations. Similarly, cyantraniliprole (Cyazypyr) affected 93% of the 2009 and 74% of the 2010 populations. There was a significant (P < 0.05) and high positive correlation (R = 0.4-0.84) between the three neonicotinoids, indicating the potential for cross-resistance., Conclusions: The trend observed in decreasing susceptibility for thiamethoxam and clothianidin will continue unless resistance management practices are followed., (© 2014 Her Majesty the Queen in Right of Canada Pest Management Science © 2014 Society of Chemical Industry.)

BREVIPEDICELLUS (BP or KNAT1), a class-I KNOTTED1-like homeobox (KNOX) transcription factor in Arabidopsis thaliana, contributes to shaping the normal inflorescence architecture through negatively regulating other two class-I KNOX genes, KNAT2 and KNAT6. However, the molecular mechanism of BP-mediated transcription regulation remains unclear. In this study, we showed that BP directly interacts with the SWI2/SNF2 chromatin remodeling ATPase BRAHMA (BRM) both in vitro and in vivo. Loss-of-function BRM mutants displayed inflorescence architecture defects, with clustered inflorescences, horizontally orientated pedicels, and short pedicels and internodes, a phenotype similar to the bp mutants. Furthermore, the transcript levels of KNAT2 and KNAT6 were elevated in brm-3, bp-9 and brm-3 bp-9 double mutants. Increased histone H3 lysine 4 tri-methylation (H3K4me3) levels were detected in brm-3, bp-9 and brm-3 bp-9 double mutants. Moreover, BRM and BP co-target to KNAT2 and KNAT6 genes, and BP is required for the binding of BRM to KNAT2 and KNAT6. Taken together, our results indicate that BP interacts with the chromatin remodeling factor BRM to regulate the expression of KNAT2 and KNAT6 in control of inflorescence architecture.

Amphetamine analogues are produced by plants in the genus Ephedra and by khat (Catha edulis), and include the widely used decongestants and appetite suppressants (1S,2S)-pseudoephedrine and (1R,2S)-ephedrine. The production of these metabolites, which derive from L-phenylalanine, involves a multi-step pathway partially mapped out at the biochemical level using knowledge of benzoic acid metabolism established in other plants, and direct evidence using khat and Ephedra species as model systems. Despite the commercial importance of amphetamine-type alkaloids, only a single step in their biosynthesis has been elucidated at the molecular level. We have employed Illumina next-generation sequencing technology, paired with Trinity and Velvet-Oases assembly platforms, to establish data-mining frameworks for Ephedra sinica and khat plants. Sequence libraries representing a combined 200,000 unigenes were subjected to an annotation pipeline involving direct searches against public databases. Annotations included the assignment of Gene Ontology (GO) terms used to allocate unigenes to functional categories. As part of our functional genomics program aimed at novel gene discovery, the databases were mined for enzyme candidates putatively involved in alkaloid biosynthesis. Queries used for mining included enzymes with established roles in benzoic acid metabolism, as well as enzymes catalyzing reactions similar to those predicted for amphetamine alkaloid metabolism. Gene candidates were evaluated based on phylogenetic relationships, FPKM-based expression data, and mechanistic considerations. Establishment of expansive sequence resources is a critical step toward pathway characterization, a goal with both academic and industrial implications.

It has been hypothesized that the relatively low concentration of sulfur amino acids in legume seeds might be an ecological adaptation to nutrient poor, marginal soils. SARC1 and SMARC1N-PN1 are genetically related lines of common bean (dry bean, Phaseolus vulgaris) differing in seed storage protein composition. In SMARC1N-PN1, the lack of phaseolin and major lectins is compensated by increased levels of sulfur-rich proteins, resulting in an enhanced concentration of cysteine and methionine, mostly at the expense of the abundant non-protein amino acid, S-methylcysteine. To identify potential effects associated with an increased concentration of sulfur amino acids in the protein pool, the response of the two genotypes to low and high sulfur nutrition was evaluated under controlled conditions. Seed yield was increased by the high sulfate treatment in SMARC1N-PN1. The seed concentrations of sulfur, sulfate, and S-methylcysteine were altered by the sulfur treatment in both genotypes. The concentration of total cysteine and extractible globulins was increased specifically in SMARC1N-PN1. Proteomic analysis identified arcelin-like protein 4, lipoxygenase-3, albumin-2, and alpha amylase inhibitor beta chain as having increased levels under high sulfur conditions. Lipoxygenase-3 accumulation was sensitive to sulfur nutrition only in SMARC1N-PN1. Under field conditions, both SARC1 and SMARC1N-PN1 exhibited a slight increase in yield in response to sulfur treatment, typical for common bean.

Profiling small RNAs in soybean Williams 82 (rsv), susceptible to Soybean mosaic virus (SMV, the genus Potyvirus, family Potyviridae) strains G2 and G7, and soybean PI96983 (Rsv1), resistant to G2 but susceptible to G7, identified the microRNA miR168 that was highly overexpressed only in G7-infected PI96983 showing a lethal systemic hypersensitive response (LSHR). Overexpression of miR168 was in parallel with the high-level expression of AGO1 mRNA, high-level accumulation of miR168-mediated AGO1 mRNA cleavage products but with severely repressed AGO1 protein. In contrast, AGO1 mRNA, degradation products and protein remained without significant changes in G2- and G7-infected Williams 82. Moreover, knock-down of SGS3, an essential component in RNA silencing, suppressed AGO1 siRNA, partially recovered repressed AGO1 protein, and alleviated LSHR severity in G7-infected Rsv1 soybean. These results suggest that both miRNA and siRNA pathways are involved in G7 infection of Rsv1 soybean, and LSHR is associated with breakdown of AGO1 homeostasis., (Crown Copyright © 2015. Published by Elsevier Inc. All rights reserved.)

In order to develop approaches for reducing the carbon footprint of the swine and dairy industries, it is important first to identify the methanogenic communities that drive methane emissions from stored manure. In this study, the metabolically active methanogens in substrate-starved manure samples taken from two dairy and one swine manure storage tanks were identified using [(13)C]-acetate and DNA stable-isotope probing (DNA-SIP). Molecular analysis of recovered genomic [(13)C]-DNA revealed that two distinct clusters of unclassified methanogen populations affiliated with the Methanoculleus genus, and the populations affiliated with Methanoculleus chikugoensis assimilated acetate-derived carbon (acetate-C) in swine and dairy starved manure samples, respectively. Furthermore, carbon flow calculations indicated that these populations were the primary contributors to methane emissions during these anoxic SIP incubations. Comparative analysis of mcrA gene abundance (coding for a key enzyme of methanogenesis) for Methanoculleus spp. in fresh feces and a wider range of stored dairy or swine manure samples, by real-time quantitative PCR using newly designed specific primers, demonstrated that the abundance of this genus significantly increased during storage. The findings supported the involvement of these particular methanogen populations as methane emitters from swine and dairy manure storage tanks. The study revealed that the ability to assimilate acetate-C for growth in manure differed within the Methanoculleus genus., (Crown Copyright © 2014. Published by Elsevier GmbH. All rights reserved.)