Evaluating Options to Increase the Efficacy of Biocontrol Agents for the Management of Pantoea spp. Under Field Conditions.

Center rot of onion is caused by a complex of plant pathogenic Pantoea species, which can lead to significant yield losses in the field and during storage. Conventional growers use foliar protectants such as a mixture of copper bactericides and an ethylene-bis-dithiocarbamate (EBDC) fungicide to manage the disease; however, organic growers have limited management options besides copper-protectants. Biocontrol agents (BCAs) provide an alternative; however, their efficacy could be compromised due in part to their inability to colonize the foliage. We hypothesized that pretreatment with peroxide (OxiDate 2.0: a.i., hydrogen peroxide and peroxyacetic acid) enhances the colonizing ability of the subsequently applied BCAs, leading to effective center rot management. Field trials were conducted in 2020 and 2021 to assess the efficacy of peroxide, BCAs (Serenade ASO: Bacillus subtilis and BlightBan: Pseudomonas fluorescens), and an insecticide program (tank mix of spinosad and neem oil) to manage center rot. We observed no significant difference in foliar area under the disease progress curve (AUDPC) between the peroxide pretreated P. fluorescens plots and only P. fluorescens-treated plots in 2020 and 2021. Peroxide pretreatment before B. subtilis application significantly reduced the foliar AUDPC as compared with the stand-alone B. subtilis treatment in 2020; however, no such difference was observed in 2021. Similarly, peroxide pretreatment before either of the BCAs did not seem to reduce the incidence of bulb rot as compared with the stand-alone BCA treatment in any of the trials (2020 and 2021). Additionally, our foliar microbiome study showed comparatively higher P. fluorescens retention on peroxide pretreated onion foliage; however, at the end of the growing season, P. fluorescens was drastically reduced and was virtually nonexistent (<0.002% of the total reads). Overall, the pretreatment with peroxide had a limited effect in improving the foliar colonizing ability of BCAs and consequently a limited effect in managing center rot.

Pseudomonas capsici sp. nov., a plant-pathogenic bacterium isolated from pepper leaf in Georgia, USA.

Three phytopathogenic bacterial strains (Pc19-1T, Pc19-2 and Pc19-3) were isolated from seedlings displaying water-soaked, dark brown-to-black, necrotic lesions on pepper (Capsicum annuum) leaves in Georgia, USA. Upon isolation on King's medium B, light cream-coloured colonies were observed and a diffusible fluorescent pigment was visible under ultraviolet light. Analysis of their 16S rRNA gene sequences showed that they belonged to the genus Pseudomonas, with the highest similarity to Pseudomonas cichorii ATCC 10857T (99.7 %). The fatty acid analysis revealed that the majority of the fatty acids were summed feature 3 (C16  :  1 ω7c/C16  :  1 ω6c), C16  :  0 and summed feature 8 (C18  :  1 ω7c/C18  :  1 ω6c). Phylogenomic analyses based on whole genome sequences demonstrated that the pepper strains belonged to the Pseudomonas syringae complex with P. cichorii as their closest neighbour, and formed a separate monophyletic clade from other species. Between the pepper strains and P. cichorii, the average nucleotide identity values were 91.3 %. Furthermore, the digital DNA-DNA hybridization values of the pepper strains when compared to their closest relatives, including P. cichorii, were 45.2 % or less. In addition, biochemical and physiological features were examined in this study and the results indicate that the pepper strains represent a novel Pseudomonas species. Therefore, we propose a new species Pseudomonas capsici sp. nov., with Pc19-1T (=CFBP 8884T=LMG 32209T) as the type strain. The DNA G+C content of the strain Pc19-1T is 58.4 mol%.

Identification of Two Novel Pathovars of Pantoea stewartii subsp. indologenes Affecting Allium sp. and Millets.

Pantoea stewartii subsp. indologenes is a causative agent of leafspot of foxtail millet and pearl millet; however, novel strains were recently identified that are pathogenic on onion. We phenotypically and genotypically characterized 17 P. stewartii subsp. indologenes strains from onion and other sources (pearl millet, foxtail millet, guar pulse, verbena, and corn). Based on the host range evaluation, we propose two pathovars: P. stewartii subsp. indologenes pv. cepacicola pv. nov. and P. stewartii subsp. indologenes pv. setariae pv. nov. P. stewartii subsp. indologenes pv. cepacicola pv. nov. causes symptoms on Allium spp. (leek, onion, chive, and Japanese bunching onion) and on foxtail millet, pearl millet, and oat. However, P. stewartii subsp. indologenes pv. setariae pv. nov. can only infect the members of Poaceae family (foxtail millet, pearl millet, and oat). We also propose that the type strain of P. stewartii subsp. indologenes (LMG 2632T) should be designated as a pathotype strain of P. stewartii subsp. indologenes pv. setariae and recommend that the strain PNA 14-12 be designated as the pathotype strain of P. stewartii subsp. indologenes pv. cepacicola. The digital DNA-DNA hybridization (dDDH), average nucleotide identity (ANI), and multilocus sequence analysis study showed that the two pathovars are genotypically closely related. Our study also showed that P. stewartii subsp. indologenes pathovars and P. stewartii subsp. stewartii share high genotypic relatedness and cannot be differentiated by dDDH and ANI values. Although the newly proposed pathovars are not clearly distinguishable by their fatty acid and methyl esterase profiles and substrate use patterns, a fatty acid (unknown with retention time: 10.9525) and a few metabolites (3-methyl glucose, Na butyrate, and fusidic acid) can be potentially used to distinguish them. We also report the distribution of previously known pathogenicity (HiVir, hrcC) and virulence (alt) factors of Pantoea spp. in the new pathovars. The impact of these new pathovars in the center rot pathosystem of onion is yet to be determined.

Two Tandem Mechanisms Control Bimodal Expression of the Flagellar Genes in Salmonella enterica.

Flagellar gene expression is bimodal in Salmonella enterica Under certain growth conditions, some cells express the flagellar genes whereas others do not. This results in mixed populations of motile and nonmotile cells. In the present study, we found that two independent mechanisms control bimodal expression of the flagellar genes. One was previously found to result from a double negative-feedback loop involving the flagellar regulators RflP and FliZ. This feedback loop governs bimodal expression of class 2 genes. In this work, a second mechanism was found to govern bimodal expression of class 3 genes. In particular, class 3 gene expression is still bimodal, even when class 2 gene expression is not. Using a combination of experimental and modeling approaches, we found that class 3 bimodality results from the σ28-FlgM developmental checkpoint.IMPORTANCE Many bacterial use flagella to swim in liquids and swarm over surface. In Salmonella enterica, over 50 genes are required to assemble flagella. The expression of these genes is tightly regulated. Previous studies have found that flagellar gene expression is bimodal in S. enterica, which means that only a fraction of cells express flagellar genes and are motile. In the present study, we found that two separate mechanisms induce this bimodal response. One mechanism, which was previously identified, tunes the fraction of motile cells in response to nutrients. The other results from a developmental checkpoint that couples flagellar gene expression to flagellar assembly. Collectively, these results further our understanding of how flagellar gene expression is regulated in S. enterica.

Reciprocal Regulation of l-Arabinose and d-Xylose Metabolism in Escherichia coli.

UNLABELLED: Glucose is known to inhibit the transport and metabolism of many sugars in Escherichia coli. This mechanism leads to its preferential consumption. Far less is known about the preferential utilization of nonglucose sugars in E. coli. Two exceptions are l-arabinose and d-xylose. Previous studies have shown that l-arabinose inhibits d-xylose metabolism in Escherichia coli. This repression results from l-arabinose-bound AraC binding to the promoter of the d-xylose metabolic genes and inhibiting their expression. This mechanism, however, has not been explored in single cells. Both the l-arabinose and d-xylose utilization systems are known to exhibit a bimodal induction response to their cognate sugar, where mixed populations of cells either expressing the metabolic genes or not are observed at intermediate sugar concentrations. This suggests that l-arabinose can only inhibit d-xylose metabolism in l-arabinose-induced cells. To understand how cross talk between these systems affects their response, we investigated E. coli during growth on mixtures of l-arabinose and d-xylose at single-cell resolution. Our results showed that mixed, multimodal populations of l-arabinose- and d-xylose-induced cells occurred at intermediate sugar concentrations. We also found that d-xylose inhibited the expression of the l-arabinose metabolic genes and that this repression was due to XylR. These results demonstrate that a strict hierarchy does not exist between l-arabinose and d-xylose as previously thought. The results may also aid in the design of E. coli strains capable of simultaneous sugar consumption. IMPORTANCE: Glucose, d-xylose, and l-arabinose are the most abundant sugars in plant biomass. Developing efficient fermentation processes that convert these sugars into chemicals and fuels will require strains capable of coutilizing these sugars. Glucose has long been known to repress the expression of the l-arabinose and d-xylose metabolic genes in Escherichia coli. Recent studies found that l-arabinose also represses the expression of the d-xylose metabolic genes. In the present study, we found that d-xylose also represses the expression of the l-arabinose metabolic genes, leading to mixed populations of cells capable of utilizing l-arabinose and d-xylose. These results further our understanding of mixed-sugar utilization and may aid in strain design.

Black and white with some shades of grey: the diverse responses of inducible metabolic pathways in Escherichia coli.

The metabolic pathways for many sugars are inducible. This process has been extensively studied in the case of Escherichia coli lactose metabolism. It has long been known that gratuitous induction of the lac operon with non-metabolizable lactose analogues generates an all-or-nothing response, where some cells express the lac genes at a maximal rate and others not at all. However, the response to lactose itself is graded, where all cells express the lac genes in proportion to lactose concentrations. The mechanisms generating these distinct behaviours in lactose metabolism have been a topic of many studies. Despite this large body of work, little is known about how other pathways respond to their cognate sugars. An article of Molecular Microbiology investigated the response of eight metabolic pathways in E. coli to their cognate sugars at single-cell resolution. The authors demonstrate that these pathways exhibit diverse responses, ranging from graded to all-or-nothing responses and combinations thereof. Remarkably, they were able to interpret these responses using a simple mathematical model and identify the mechanisms likely giving rise to each.

Pattern of Deaths among Women of Reproductive Age in Major Autopsy Centres of Capital Cities of the Seven Provinces of Nepal.

BACKGROUND: The females in the reproductive age group are vulnerable to injuries and diseases. There is no reliable evidence of the pattern of deaths in reproductive age group females from Nepal. This study was conducted to explore the patterns of deaths of females of the reproductive age group in Nepal. METHODS: A multicentric quantitative cross-sectional study was conducted among the 611 deceased females of age 15 to 49 years who had undergone medico-legal autopsy in major autopsy centers of the capital cities of seven provinces of Nepal in a duration of one year. The demographic, clinical, and causes of death data were retrieved from the records and analyzed. The intentional and unintentional causes were compared with the marital status, age groups, ethnicity, and deceased of rural and urban communities. RESULTS: Female reproductive age group deaths constituted 611 (20·64%) of the total autopsies. Suicide was the most common manner of death (413, 67·59%) followed by accident (121, 19·80%). The most common cause of suicide was hanging (258, 62·47%) followed by poisoning (149, 36·08%) whereas road traffic accident (72, 59·5%) was the commonest cause of accidental death. Intentional deaths were associated with higher risk to the females of younger age groups (p<0·001), ethnicity of the hilly region (p<0·001), and unmarried women (p=0·001). CONCLUSIONS: Suicide was the commonest manner of death among the autopsies of females of the reproductive age group in Nepal. Appropriate preventive strategies need to be developed to uplift the overall health, socioeconomic status, and general wellbeing of the females.

Unnatural Deaths among Autopsy Cases Brought at Tertiary Care Hospital of Western Nepal during COVID-19 Pandemic Period: A Descriptive Cross-sectional Study.

INTRODUCTION: The outbreak of COVID-19  as changed patterns of mortality in different setups. The rate of suicide has increased in some countries during the pandemic while the overall death rates  have decreased. The study was conducted with objective to find out the prevalence of unnatural deaths among the autopsy cases brought at tertiary care hospital during COVID-19 pandemic period. METHODS: This is a descriptive cross-sectional study using the records of the medico legal autopsies conducted from 24th March 2020 to 23rd August 2020 during the COVID-19 pandemic in Pokhara Academy of Health Sciences. Ethical approval was taken from Institutional Review Committee of Pokhara Academy of Health Sciences (Reference number 28.2077/78). Whole sampling method was used. Records which were available were included in the study whereas those cases whose complete records were not available were excluded. Point estimate at 95% confidence interval was calculated along with frequency and proportion for binary data. RESULTS: Out of 188 deaths studied at the autopsy during the COVID-19 pandemic period, the prevalence of unnatural deaths was 147 (78.19%) (71.04-85.33 at 95% Confidence Interval). Among these deaths, 109 (74.14%) were males and 38 (25.85%) were females. Suicide was the most common manner attributing to 78 (53.06%) of the unnatural deaths. CONCLUSIONS: The prevalence of suicide was more than those demonstrated by earlier observations in similar settings before the pandemic period. Suicidal deaths were more common during the COVID 19 pandemic. This is an indicator of frustration of the people and necessary steps have to be taken to decrease such deaths in similar conditions to come.

Formulation and evaluation of mucoadhesive buccal tablets of aceclofenac.

This project was aimed to formulate and characterize mucoadhesive buccal tablets of aceclofenac, utilizing different proportions of three polymers carbopol 934, hydroxypropyl methylcellulose, and sodium carboxymethylcellulose. Twelve batches of buccoadhesive aceclofenac were prepared by the direct compression method. The compressed tablets were then evaluated for physicochemical parameters such as hardness, thickness, weight variation, drug content, friability, swelling index, surface pH, and ex vivo mucoadhesion. In vitro dissolution test was conducted for 12 h according to Indian Pharmacopeia 2018, using the rotating paddle method in phosphate buffer of pH 7.4. Physiochemical parameters like weight variation (231.25-268.75 mg), hardness (8.32-11.56 kg), friability (0.04-0.2%), diameter (9.00 mm), thickness (3.8-4.05 mm), and drug content ((97.67-102.25%) were within the acceptable limit as per Indian Pharmacopeia 2018. The swelling index was reported to be in the range of 112.93-450.19%, at 8 h. The surface pHs of all the batches were in between 6.72 to 6.96. The mucoadhesive strengths (40.5-50 g) varied with the change in polymer concentrations especially of carbopol 934. The dissolution profile of all the batches varied greatly, with a maximum release of 109.41% (in batch 12 at 6 h) to a minimum release of 44.82% (in batch 3 at 12 h). Among them, only batch 1 ensured sustained and effective drug release (88.34% at 12 h) with appropriate swelling index (112.93%) and mucoadhesive strength (40 g). Fourier Transform Infrared Spectroscopy analysis showed no evidence of drug excipients interaction. Hence, the results concluded that buccal mucoadhesive aceclofenac tablets can be formulated. Furthermore, the property of the tablet not only depends on the concentration but also the behavior of the polymers used.

The Distribution of Onion Virulence Gene Clusters Among Pantoea spp.

Pantoea ananatis is a gram-negative bacterium and the primary causal agent of center rot of onions in Georgia. Previous genomic studies identified two virulence gene clusters, HiVir and alt, associated with center rot. The HiVir gene cluster is required to induce necrosis on onion tissues via synthesis of pantaphos, (2-hydroxy[phosphono-methyl)maleate), a phosphonate phytotoxin. The alt gene cluster aids in tolerance to thiosulfinates generated during onion tissue damage. Whole genome sequencing of other Pantoea species suggests that these gene clusters are present outside of P. ananatis. To assess the distribution of these gene clusters, two PCR primer sets were designed to detect the presence of HiVir and alt. Two hundred fifty-two strains of Pantoea spp. were phenotyped using the red onion scale necrosis (RSN) assay and were genotyped using PCR for the presence of these virulence genes. A diverse panel of strains from three distinct culture collections comprised of 24 Pantoea species, 41 isolation sources, and 23 countries, collected from 1946-2019, was tested. There is a significant association between the alt PCR assay and Pantoea strains recovered from symptomatic onion (P < 0.001). There is also a significant association of a positive HiVir PCR and RSN assay among P. ananatis strains but not among Pantoea spp., congeners. This may indicate a divergent HiVir cluster or different pathogenicity and virulence mechanisms. Last, we describe natural alt positive [RSN+/HiVir+/alt +] P. ananatis strains, which cause extensive bulb necrosis in a neck-to-bulb infection assay compared to alt negative [RSN+/HiVir+/alt -] P. ananatis strains. A combination of assays that include PCR of virulence genes [HiVir and alt] and an RSN assay can potentially aid in identification of onion-bulb-rotting pathogenic P. ananatis strains.

FliZ induces a kinetic switch in flagellar gene expression.

FliZ is an activator of class 2 flagellar gene expression in Salmonella enterica. To understand its role in flagellar assembly, we investigated how FliZ affects gene expression dynamics. We demonstrate that FliZ participates in a positive-feedback loop that induces a kinetic switch in class 2 gene expression.

Dynamic Measures of Flagellar Gene Expression.

Many genes are required to assemble flagella. These genes encode not only the structural elements of the flagellum but also a number of regulators that control how the flagellar genes are temporally expressed during the assembly process. These regulators also specify the likelihood that a given cell will express the flagellar genes. In particular, not all cells express the flagellar genes, resulting in mixed populations of motile and non-motile cells. Nutrients provide one signal that specifies the motile fraction. In this chapter, we describe two methods for measuring flagellar gene expression dynamics using fluorescent proteins in Salmonella enterica. Both the methods can be used to investigate the mechanisms governing flagellar gene expression dynamics.

Growth rate control of flagellar assembly in Escherichia coli strain RP437.

The flagellum is a rotary motor that enables bacteria to swim in liquids and swarm over surfaces. Numerous global regulators control flagellar assembly in response to cellular and environmental factors. Previous studies have also shown that flagellar assembly is affected by the growth-rate of the cell. However, a systematic study has not yet been described under controlled growth conditions. Here, we investigated the effect of growth rate on flagellar assembly in Escherichia coli using steady-state chemostat cultures where we could precisely control the cell growth-rate. Our results demonstrate that flagellar abundance correlates with growth rate, where faster growing cells produce more flagella. They also demonstrate that this growth-rate dependent control occurs through the expression of the flagellar master regulator, FlhD4C2. Collectively, our results demonstrate that motility is intimately coupled to the growth-rate of the cell.

Escherichia coli swimming is robust against variations in flagellar number.

Bacterial chemotaxis is a paradigm for how environmental signals modulate cellular behavior. Although the network underlying this process has been studied extensively, we do not yet have an end-to-end understanding of chemotaxis. Specifically, how the rotational states of a cell's flagella cooperatively determine whether the cell 'runs' or 'tumbles' remains poorly characterized. Here, we measure the swimming behavior of individual E. coli cells while simultaneously detecting the rotational states of each flagellum. We find that a simple mathematical expression relates the cell's run/tumble bias to the number and average rotational state of its flagella. However, due to inter-flagellar correlations, an 'effective number' of flagella-smaller than the actual number-enters into this relation. Data from a chemotaxis mutant and stochastic modeling suggest that fluctuations of the regulator CheY-P are the source of flagellar correlations. A consequence of inter-flagellar correlations is that run/tumble behavior is only weakly dependent on number of flagella. DOI: http://dx.doi.org/10.7554/eLife.01916.001.

A nutrient-tunable bistable switch controls motility in Salmonella enterica serovar Typhimurium.

UNLABELLED: Many bacteria are motile only when nutrients are scarce. In contrast, Salmonella enterica serovar Typhimurium is motile only when nutrients are plentiful, suggesting that this bacterium uses motility for purposes other than foraging, most likely for host colonization. In this study, we investigated how nutrients affect motility in S. enterica and found that they tune the fraction of motile cells. In particular, we observed coexisting populations of motile and nonmotile cells, with the distribution being determined by the concentration of nutrients in the growth medium. Interestingly, S. enterica responds not to a single nutrient but apparently to a complex mixture of them. Using a combination of experimentation and mathematical modeling, we investigated the mechanism governing this behavior and found that it results from two antagonizing regulatory proteins, FliZ and YdiV. We also found that a positive feedback loop involving the alternate sigma factor FliA is required, although its role appears solely to amplify FliZ expression. We further demonstrate that the response is bistable: that is, genetically identical cells can exhibit different phenotypes under identical growth conditions. Together, these results uncover a new facet of the regulation of the flagellar genes in S. enterica and further demonstrate how bacteria employ phenotypic diversity as a general mechanism for adapting to change in their environment. IMPORTANCE: Many bacteria employ flagella for motility. These bacteria are often not constitutively motile but become so only in response to specific environmental cues. The most common is nutrient starvation. Interestingly, in Salmonella enterica serovar Typhimurium, nutrients enhance the expression of flagella, suggesting that motility is used for purposes other than foraging. In this work, we investigated how nutrients affect motility in S. enterica and found that nutrients tune the fraction of motile cells within a population. Using both experimental and mathematical analysis, we determined the mechanism governing this tunable response. We further demonstrated that the response is bistable: that is, genetically identical cells can exhibit different phenotypes under identical growth conditions. These results reveal a new facet of motility in S. enterica and demonstrate that nutrients determine not only where these bacteria swim but also the fraction of them that do so.