Biotechnological and Medical Aspects of Lactic Acid Bacteria Used for Plant Protection: A Comprehensive Review.

The use of chemical pesticides in agriculture goes hand in hand with some crucial problems. These problems include environmental deterioration and human health complications. To eliminate the problems accompanying chemical pesticides, biological alternatives should be considered. These developments spark interest in many environmental fields, including agriculture. In this review, antifungal compounds produced by lactic acid bacteria (LABs) are considered. It summarizes the worldwide distribution of pesticides and the effect of pesticides on human health and goes into detail about LAB species, their growth, fermentation, and their antifungal compounds. Additionally, interactions between LABs with mycotoxins and plants are discussed.

Respiratory syncytial, parainfluenza and influenza virus infection in young children with acute lower respiratory infection in rural Gambia.

Respiratory viral infections contribute significantly to morbidity and mortality worldwide, but representative data from sub-Saharan Africa are needed to inform vaccination strategies. We conducted population-based surveillance in rural Gambia using standardized criteria to identify and investigate children with acute lower respiratory infection (ALRI). Naso- and oropharyngeal swabs were collected. Each month from February through December 2015, specimens from 50 children aged 2-23 months were randomly selected to test for respiratory syncytial (RSV), parainfluenza (PIV) and influenza viruses. The expected number of viral-associated ALRI cases in the population was estimated using statistical simulation that accounted for the sampling design. RSV G and F proteins and influenza hemagglutinin genes were sequenced. 2385 children with ALRI were enrolled, 519 were randomly selected for viral testing. One or more viruses were detected in 303/519 children (58.4%). RSV-A was detected in 237 and RSV-B in seven. The expected incidence of ALRI associated with RSV, PIV or influenza was 140 cases (95% CI, 131-149) per 1000 person-years; RSV incidence was 112 cases (95% CI, 102-122) per 1000 person-years. Multiple strains of RSV and influenza circulated during the year. RSV circulated throughout most of the year and was associated with eight times the number of ALRI cases compared to PIV or IV. Gambian RSV viruses were closely related to viruses detected in other continents. An effective RSV vaccination strategy could have a major impact on the burden of ALRI in this setting.

Biological activities associated with the volatile compound 2,5-bis(1-methylethyl)-pyrazine.

Pyrazines are 1,4-diazabenzene-based volatile organic compounds and known for their broad-spectrum antimicrobial activity. In the present study, we assessed the antimicrobial activity of 2,5-bis(1-methylethyl)-pyrazine, produced by Paenibacillus sp. AD87 during co-culture with Burkholderia sp. AD24. In addition, we were using transcriptional reporter assays in E. coli and mammalian cells to decipher the possible mode of action. Bacterial and mammalian luciferase reporter strains were deployed to elucidate antimicrobial and toxicological effects of 2,5-bis(1-methylethyl)-pyrazine. At high levels of exposure, 2,5-bis(1-methylethyl)-pyrazine exerted strong DNA damage response. At lower concentrations, cell-wall damage response was observed. The activity was corroborated by a general toxicity reporter assay in E. coli ΔampD, defective in peptidoglycan turnover. The maximum E. coli cell-wall stress activity was measured at a concentration close to the onset of the mammalian cytotoxicity, while other adverse outcome pathways, such as the activation of aryl hydrocarbon and estrogenic receptor, the p53 tumour suppressor and the oxidative stress-related Nrf2 transcription factor, were induced at elevated concentrations compared to the response of mammalian cells. Because of its broad-spectrum antimicrobial activity at lower concentrations and the relatively low mammalian toxicity, 2,5-bis(1-methylethyl)-pyrazine is a potential bio-based fumigant with possible applications in food industry, agriculture or logistics.

Draft Genome Sequence of Bacillus toyonensis VU-DES13, Isolated from Folsomia candida (Collembola: Entomobryidae).

We present here the draft genome of Bacillus toyonensis VU-DES13, which was isolated from the midgut of the soil-living springtail Folsomia candida Previous research revealed the presence of gene clusters for the biosynthesis of various secondary metabolites, including ß-lactam antibiotics, in the host's genome. The genome data are discussed in the light of the antimicrobial properties against fungi and oomycetes and a high level of ß-lactam resistance of the isolate.

Exploring bacterial interspecific interactions for discovery of novel antimicrobial compounds.

Recent studies indicated that the production of secondary metabolites by soil bacteria can be triggered by interspecific interactions. However, little is known to date about interspecific interactions between Gram-positive and Gram-negative bacteria. In this study, we aimed to understand how the interspecific interaction between the Gram-positive Paenibacillus sp. AD87 and the Gram-negative Burkholderia sp. AD24 affects the fitness, gene expression and the production of soluble and volatile secondary metabolites of both bacteria. To obtain better insight into this interaction, transcriptome and metabolome analyses were performed. Our results revealed that the interaction between the two bacteria affected their fitness, gene expression and the production of secondary metabolites. During interaction, the growth of Paenibacillus was not affected, whereas the growth of Burkholderia was inhibited at 48 and 72 h. Transcriptome analysis revealed that the interaction between Burkholderia and Paenibacillus caused significant transcriptional changes in both bacteria as compared to the monocultures. The metabolomic analysis revealed that the interaction increased the production of specific volatile and soluble antimicrobial compounds such as 2,5-bis(1-methylethyl)-pyrazine and an unknown Pederin-like compound. The pyrazine volatile compound produced by Paenibacillus was subjected to bioassays and showed strong inhibitory activity against Burkholderia and a range of plant and human pathogens. Moreover, strong additive antimicrobial effects were observed when soluble extracts from the interacting bacteria were combined with the pure 2,5-bis(1-methylethyl)-pyrazine. The results obtained in this study highlight the importance to explore bacterial interspecific interactions to discover novel secondary metabolites and to perform simultaneously metabolomics of both, soluble and volatile compounds.

Fungus-associated bacteriome in charge of their host behavior.

Bacterial-fungal interactions are widespread in nature and there is a growing number of studies reporting distinct fungus-associated bacteria. However, little is known so far about how shifts in the fungus-associated bacteriome will affect the fungal host's lifestyle. In the present study, we describe for the first time the bacterial community associated with the saprotrophic fungus Mucor hiemalis, commonly found in soil and rhizosphere. Two broad-spectrum antibiotics that strongly altered the bacterial community associated with the fungus were applied. Our results revealed that the antibiotic treatment did not significantly reduce the amount of bacteria associated to the fungus but rather changed the community composition by shifting from initially dominating Alpha-Proteobacteria to dominance of Gamma-Proteobacteria. A novel approach was applied for the isolation of fungal-associated bacteria which also revealed differences between bacterial isolates obtained from the original and the antibiotic-treated M. hiemalis. The shift in the composition of the fungal-associated bacterial community led to significantly reduced fungal growth, changes in fungal morphology, behavior and secondary-metabolites production. Furthermore, our results showed that the antibiotic-treated isolate was more attractive and susceptible to mycophagous bacteria as compared to the original isolate. Overall, our study highlights the importance of the fungus-associated bacteriome for the host's lifestyle and interactions and indicate that isolation with antibacterials is not sufficient to eradicate the associated bacteria.

Soil Microbiome Is More Heterogeneous in Organic Than in Conventional Farming System.

Organic farming system and sustainable management of soil pathogens aim at reducing the use of agricultural chemicals in order to improve ecosystem health. Despite the essential role of microbial communities in agro-ecosystems, we still have limited understanding of the complex response of microbial diversity and composition to organic and conventional farming systems and to alternative methods for controlling plant pathogens. In this study we assessed the microbial community structure, diversity and richness using 16S rRNA gene next generation sequences and report that conventional and organic farming systems had major influence on soil microbial diversity and community composition while the effects of the soil health treatments (sustainable alternatives for chemical control) in both farming systems were of smaller magnitude. Organically managed system increased taxonomic and phylogenetic richness, diversity and heterogeneity of the soil microbiota when compared with conventional farming system. The composition of microbial communities, but not the diversity nor heterogeneity, were altered by soil health treatments. Soil health treatments exhibited an overrepresentation of specific microbial taxa which are known to be involved in soil suppressiveness to pathogens (plant-parasitic nematodes and soil-borne fungi). Our results provide a comprehensive survey on the response of microbial communities to different agricultural systems and to soil treatments for controlling plant pathogens and give novel insights to improve the sustainability of agro-ecosystems by means of beneficial microorganisms.

Combined Transcriptomics Analysis for Classification of Adverse Effects As a Potential End Point in Effect Based Screening.

Environmental risk assessment relies on the use of bioassays to assess the environmental impact of chemicals. Gene expression is gaining acceptance as a valuable mechanistic end point in bioassays and effect-based screening. Data analysis and its results, however, are complex and often not directly applicable in risk assessment. Classifier analysis is a promising method to turn complex gene expression analysis results into answers suitable for risk assessment. We have assembled a large gene expression data set assembled from multiple studies and experiments in the springtail Folsomia candida, with the aim of selecting a set of genes that can be trained to classify general toxic stress. By performing differential expression analysis prior to classifier training, we were able to select a set of 135 genes which was enriched in stress related processes. Classifier models from this set were used to classify two test sets comprised of chemical spiked, polluted, and clean soils and compared to another, more traditional classifier feature selection. The gene set presented here outperformed the more traditionally selected gene set. This gene set has the potential to be used as a biomarker to test for adverse effects caused by chemicals in springtails to provide end points in environmental risk assessment.

Influence of adaptive evolution of cadmium tolerance on neutral and functional genetic variation in Orchesella cincta.

Adaptation to environmental toxicants, such as metals, can affect population genetic diversity, both at neutral and selectable loci. At the transcriptional level, evolution of metal tolerance is possible due to the existence of polymorphisms in the cis-regulatory sequences of stress-responsive genes such as the metallothionein gene (mt). This study investigated the influence of cadmium adaptation on genetic diversity of soil-living Orchesella cincta (Collembola) populations in neutral (microsatellites and AFLP) and in functional (mt promoter) markers. Also, the influence of cis- and trans-acting factors on increased tolerance was addressed. No reduced genetic diversity was observed in two tolerant populations compared to five sensitive populations, either in neutral or in selectable markers. Extensive migration along with a large population size may explain the high genetic diversity measured. The metal-tolerant phenotype seems to be mostly influenced by genetic factors acting in cis on mt gene expression. The results suggest that certain promoter genotypes, which are found mainly or exclusively in Cd-tolerant populations, contribute to higher constitutive mt gene expression in individuals from these populations. However, more studies are needed to clearly unravel the influence of cis/trans-regulatory evolution in tolerant populations.

Micro-evolution of toxicant tolerance: from single genes to the genome's tangled bank.

Two case-studies published 55 years ago became textbook examples of evolution in action: DDT resistance in houseflies (Busvine) and the rise of melanic forms of the peppered moth (Kettlewell). Now, many years later, molecular studies have elucidated in detail the mechanisms conferring resistance. In this paper we focus on the case of metal tolerance in a soil-living arthropod, Orchesella cincta, and provide new evidence on the transcriptional regulation of a gene involved in stress tolerance, metallothionein. Evolution of resistance is often ascribed to cis-regulatory change of such stress-combatting genes. For example, DDT resistance in the housefly is due to insertion of a mobile element into the promoter of Cyp6g1, and overexpression of this gene allows rapid metabolism of DDT. The discovery of these mechanisms has promoted the idea that resistance to environmental toxicants can be brought about by relatively simple genetic changes, involving up-regulation, duplication or structural alteration of a single-gene. Similarly, the work on O. cincta shows that populations from metal-polluted mining sites have a higher constitutive expression of the cadmium-induced metallothionein (Mt) gene. Moreover, its promoter appears to include a large degree of polymorphism; Mt promoter alleles conferring high expression in cell-based bioreporter assays were shown to occur at higher frequency in populations living at polluted sites. The case is consistent with classical examples of micro-evolution through altered cis-regulation of a key gene. However, new data on qPCR analysis of gene expression in homozygous genotypes with both reference and metal-tolerant genetic backgrounds, show that Mt expression of the same pMt homozygotes depends on the origin of the population. This suggests that trans-acting factors are also important in the regulation of Mt expression and its evolution. So the idea that metal tolerance in Orchesella can be viewed as a single-gene adaptation must be abandoned. These data, added to a genome-wide gene expression profiling study reported earlier shows that evolution of tolerance takes place in a complicated molecular network, not unlike an internal tangled bank.

Adaptive differences in gene expression associated with heavy metal tolerance in the soil arthropod Orchesella cincta.

Field-selected tolerance to heavy metals has been reported for Orchesella cincta (Arthropoda: Collembola) populations occurring at metal-contaminated mining sites. This tolerance correlated with heritable increase in metal excretion efficiency, less pronounced cadmium (Cd)-induced growth reduction and overexpression of the metallothionein gene. We applied transcriptomics to determine differential gene expression caused by this abiotic stress in reference and Cd-tolerant populations. Many cDNAs responded to Cd exposure in the reference population. Significantly fewer clones were Cd responsive in tolerant animals. Analysis of variance revealed transcripts that interact between Cd exposure and population. Hierarchical cluster analysis of these clones identified two major groups. The first one contained cDNAs that were up-regulated by Cd in the reference culture but non-responsive or down-regulated in tolerant animals. This cluster was also characterized by elevated constitutive expression in the tolerant population. Gene ontology analysis revealed that these cDNAs were involved in structural integrity of the cuticle, anti-microbial defence, calcium channel-blocking, sulphur assimilation and chromatin remodelling. The second group consisted of cDNAs down-regulated in reference animals but not responding or slightly up-regulated in tolerant animals. Their functions involved carbohydrate metabolic processes, Ca(2+)-dependent stress signalling, redox state, proteolysis and digestion. The reference population showed a strong signature of stress-induced genome-wide perturbation of gene expression, whereas the tolerant animals maintained normal gene expression upon Cd exposure. We confirmed the micro-evolutionary processes occurring in soil arthropod populations and suggest a major contribution of gene regulation to the evolution of a stress-adapted phenotype.

Comparative population analysis of metallothionein promoter alleles suggests stress-induced microevolution in the field.

We investigate a model system for microevolution of transcriptional regulation: metallothionein expression in springtails. A previous survey of the metallothionein promoter in Orchesella cincta (Collembola) revealed nine alleles with differential basal activities and responses to cadmium and oxidative stress. In this study, 23 woodlands, with a divergent degree of pollution, were sampled, and heavy metals were measured. When grouped to their contamination degree, they were discriminated best on the pmtD2 metallothionein promoter allele frequency, which was higher in populations from heavily polluted sites. Taken together with previous work showing high inducibility of the pmtD2 promoter allele by Cd in a reporter assay, this suggests a fitness advantage of the pmtD2 allele in polluted sites. Redundancy analysis revealed associations between allele frequencies and specific metals in the environment, resulting in a subdivision between pollution associated alleles and others. A positive relationship between the pmtD2 allele frequency and the Cd content of the soil as well as between pmtE and Ni in the litter emerged. An increase of genetic diversity was observed with increasing Pb in the soil, reached through substitution of the pmtA1 allele, suggesting balancing selection. Our results illustrate that environmental factors can exert selection on promoter polymorphisms and cause adaptation through altered transcriptional regulation.

Recombinational micro-evolution of functionally different metallothionein promoter alleles from Orchesella cincta.

BACKGROUND: Metallothionein (mt) transcription is elevated in heavy metal tolerant field populations of Orchesella cincta (Collembola). This suggests that natural selection acts on transcriptional regulation of mt in springtails at sites where cadmium (Cd) levels in soil reach toxic values This study investigates the nature and the evolutionary origin of polymorphisms in the metallothionein promoter (pmt) and their functional significance for mt expression. RESULTS: We sequenced approximately 1600 bp upstream the mt coding region by genome walking. Nine pmt alleles were discovered in NW-European populations. They differ in the number of some indels, consensus transcription factor binding sites and core promoter elements. Extensive recombination events between some of the alleles can be inferred from the alignment. A deviation from neutral expectations was detected in a cadmium tolerant population, pointing towards balancing selection on some promoter stretches. Luciferase constructs were made from the most abundant alleles, and responses to Cd, paraquat (oxidative stress inducer) and moulting hormone were studied in cell lines. By using paraquat we were able to dissect the effect of oxidative stress from the Cd specific effect, and extensive differences in mt induction levels between these two stressors were observed. CONCLUSION: The pmt alleles evolved by a number of recombination events, and exhibited differential inducibilities by Cd, paraquat and molting hormone. In a tolerant population from a metal contaminated site, promoter allele frequencies differed significantly from a reference site and nucleotide polymorphisms in some promoter stretches deviated from neutral expectations, revealing a signature of balancing selection. Our results suggest that the structural differences in the Orchesella cincta metallothionein promoter alleles contribute to the metallothionein -over-expresser phenotype in cadmium tolerant populations.