Ribosomal Intergenic Spacer Analysis Research Articles

Profiling of rhizobacteria to alleviate drought stress in oil palm using ribosomal intergenic spacer analysis

During the drought stress period, oil palms experienced increasing ACC substance synthesis converted to ethylene. It decreases root growth and plant tolerance to drought stress. Ethylene production can be controlled by transforming ACC into α-ketobutyrate and ammonia via ACC deaminase (ACCd). This enzyme is produced by bacteria in the plant rooting system when the plant experiences drought stress. This study aimed to characterize ACCd bacteria diversity from selected oil palm progenies with difference responses to drought stress using ribosomal intergenic spacer analysis (RISA). The method used was by isolating bacteria from oil palm root surface using the Dworkin-Foster media enriched with ACC. Bacteria were then isolated, identified molecularly based on 16S rRNA genes, and profiling their population from selected oil palm progenies. The study results show that nine isolates managed to be isolated and characterized based on their morphology. Molecular identification based on their gene bank and phylogenetic analysis revealed that the ACCd bacteria community were divided into three major groups, i.e., Firmicutes, Actinobacteria, and Proteobacteria. Genus Pantoea, Acinetobacter, Pseudomonas, Burkholderia, Kocuria, and Bacillus were identified and could be utilized as bioagents to overcome the drought stress on oil palm crops. Based on the composition of the PCR-RISA fragments, showed that the oil palm rhizosphere of progeny P8 had a higher functional genetic diversity than progeny P1 and P13. Analysis of the similarity pattern of the ACC deaminase producing bacterial community divided 2 large clusters with a similar pattern of up to 69 %.

Exploring the impact of fungicide exposure and nutritional stress on the microbiota and immune response of the Cape honey bee (Apis mellifera capensis)

Honey bees (Apis mellifera) harbour a stable core microbial community within their gut, that is suggested to play a role in metabolic functioning, immune regulation, and host homeostasis. This microbiota presents a unique opportunity to observe the effects of stressors on honey bee health. We examined the effects of two common honey bee stressors: indirect fungicide contamination and nutrient limitation. These effects were observed through changes in their hind- and midgut microbiota using Automated Ribosomal Intergenic Spacer Analysis (ARISA), alongside high-throughput amplicon sequencing. Expression of the honey bees’ immune response was examined through the expression of three immune-related genes, namely, immune deficiency (imd), proPhenolOxidase (proPO), and spaetzle (spz). Additionally, longevity of the honey bees was monitored through observation of the expression levels of Vitellogenin (Vg). Both treatment groups were compared to a negative control, and a diseased positive control. There was no effect on the hindgut microbiota due to the stressors, while significant changes in the midgut was observed. This was also observed in the expression of the immune-related genes within the treatment groups. The Imd pathway was substantially downregulated, with upregulation in the prophenoloxidase pathway. However, no significant effect was observed in the expression of spz, and only the pollen treatment group showed reduced longevity through a downregulation of Vg. Overall, the effect of these two common stressors indicate a compromise in honey bee immunity, and potential vulnerabilities within the immune defence mechanisms.

Microbial Diversity of Traditional Livno Cheese from Bosnia and Herzegovina

Traditional dairy products, especially cheeses, represent part of the cultural food heritage of many countries. In addition, these cheeses constitute microbiological “reservoirs”, of which many have been lost due to the introduction of the pasteurization of milk in the dairy industry. Increased awareness of the importance of microorganisms that make up the biodiversity of traditional cheeses, as well as the development of molecular methods in recent decades, have enabled efforts to identify and preserve them. Traditional Livno cheese is a full-fat hard cheese, considered one of the most famous traditional cheeses of Bosnia and Herzegovina and is seasonally produced from a mixture of raw sheep’s milk supplemented with cow’s milk. Often, Livno cheese has variable quality, due to microbial contamination and poor milk quality. In this study, traditional Livno cheese was studied during the ripening of cheeses produced by different producers during two seasons. Culture-dependent analyses were made during ripening using microbiological plating on suitable media. Likewise, culture-independent methods Denaturing Gradient Gel Electrophoresis (DGGE) and Automated Ribosomal Intergenic Spacer Analysis (ARISA) were used to elucidate the cheese microbiota. Results of analysis showed Lactococcus spp., Streptococcus spp., Lactobacillus spp., Pediococcus spp. and Leuconostoc spp. to be dominant species in traditional Livno cheese. However, when comparing the use of culture-dependent and culture-independent methods in the evaluation of Livno cheese microbiota, Enterococcus was not detected by culture-independent DGGE methods. The microbial population of both the milk and the environment determines the fermentation processes during cheese production and ripening, and thereby defines the quality of this cheese. The numbers of bacteria in the cheese were shown to be dependent on the manufacturer, the degree of ripening and the production season.

The Diversity of Shallot Rhizomicrobiome and Twisted Disease Suppression with The Applicationof Bacillus spp. and Trichoderma asperellum

Twisted disease (Fusarium spp.) is an endemic disease that reduces shallot production in the coastal land area of Samas, Bantul, Yogyakarta. The application of Bacillus spp. can suppress the twisted disease by secreting secondary metabolites and enhancing soil suppressiveness. This study aimed to determine the effectiveness of adding spraying Bacillus spp. on the disease incidence, production of shallots, and their effect on the diversity of rhizomicrobiome by culture microbe approaches. Bacillus spp. with a density 108 cfu mL-1, Trichoderma asperellum 106 cfu mL-1 was applied by spraying to the shallot. Fungicide chlorothalonil, propiconazole, and prochloraz were used to control the disease. The diversity of rhizobacteria and fungi was analyzed using the ribosomal intergenic spacer analysis (RISA) method. Based on the analysis result, the addition of spraying B. velezensis B-27, combination B. velezensis B-27 and B. cereus RC76, and T. asperellum was unable to enhance the suppression of twisted disease, but it was able to enhance the production of shallot bulbs reaching 7.10, 7.80, and 8.43 ton ha-1. Furthermore, the result revealed the diversity of the rhizomicrobiome, spraying Bacillus sp. showed 39% differences in bacterial diversity with control while T. asperellum caused 43% difference in the diversity. Spraying Bacillus spp. has not been able to suppress the incidence of twisted diseases compared to control. However, the similar disease incidence on a spraying Bacillus spp. and control showed a higher production until 70% compared to control. This result showed that the addition of spraying Bacillus spp. able to increase the tolerance of shallot plants toward twisted disease.

Spatiotemporal transitions in Pseudo-nitzschia species assemblages and domoic acid along the Alaska coast.

The toxic diatom genus Pseudo-nitzschia is distributed from equatorial to polar regions and is comprised of >57 species, some capable of producing the neurotoxin domoic acid (DA). In the Pacific Arctic Region spanning the Bering, Chukchi, and Beaufort seas, DA is recognized as an emerging human and ecosystem health threat, yet little is known about the composition and distribution of Pseudo-nitzschia species in these waters. This investigation characterized Pseudo-nitzschia assemblages in samples collected in 2018 during summer (August) and fall (October-November) surveys as part of the Distributed Biological Observatory and Arctic Observing Network, encompassing a broad geographic range (57.8° to 73.0°N, -138.9° to -169.9°W) and spanning temperature (-1.79 to 11.7°C) and salinity (22.9 to 32.9) gradients associated with distinct water masses. Species were identified using a genus-specific Automated Ribosomal Intergenic Spacer Analysis (ARISA). Seventeen amplicons were observed; seven corresponded to temperate, sub-polar, or polar Pseudo-nitzschia species based on parallel sequencing efforts (P. arctica, P. delicatissima, P. granii, P. obtusa, P. pungens, and two genotypes of P. seriata), and one represented Fragilariopsis oceanica. During summer, particulate DA (pDA; 4.0 to 130.0 ng L-1) was observed in the Bering Strait and Chukchi Sea where P. obtusa was prevalent. In fall, pDA (3.3 to 111.8 ng L-1) occurred along the Beaufort Sea shelf coincident with one P. seriata genotype, and south of the Bering Strait in association with the other P. seriata genotype. Taxa were correlated with latitude, longitude, temperature, salinity, pDA, and/or chlorophyll a, and each had a distinct distribution pattern. The observation of DA in association with different species, seasons, geographic regions, and water masses underscores the significant risk of Amnesic Shellfish Poisoning (ASP) and DA-poisoning in Alaska waters.

​Identification of Lactic Acid Bactria in Dairy Products using Culture-independent Methods: A Review

The microbiota of dairy products is composed of starter and non-starter lactic acid bacteria, as well as other bacteria, yeasts and filamentous fungi that form secondary microbiota and play an important role in cheese ripening. The use of combined LAB strains based on compatibility and/or coexistence tests plays an important role in food production and preservation, while combinations of strains that exhibit mutually inhibitory properties must be avoided. For centuries, researchers have focused on the exploration of autochthonous LAB biodiversity from various ecological dairy niches in order to address its role in texture and flavour formation and to select strains for use as starter cultures. Recent advanced technological tools like next-generation sequencing machines have played a significant role in the fast and accurate characterization and isolation of microbial communities from dairy products. However, the characterization and isolation of LAB using culture-independent methods is also a reliable and cost-effective method for studying a bacterial community without prior cultivation. These methods include single-strand conformation polymorphism (SSCP-PCR), denaturing gradient gel electrophoresis (DGGE), thermal gradient gel electrophoresis (TGGE), real-time PCR (qPCR), terminal restriction fragment length polymorphism (T-RFLP), length heterogeneity PCR (LH-PCR), automated ribosomal intergenic spacer analysis (ARISA), and fluorescence in situ hybridization (FISH). Therefore the goal of this review is to compare and describe the most recent culture-independent molecular-based methods for the characterization and isolation of LAB in dairy products.

Comparison of Three DNA Extraction Kits for Assessment of Bacterial Diversity in Activated Sludge, Biofilm, and Anaerobic Digestate

Direct DNA analysis is the most widely used approach for microorganism characterization in natural and built environments; therefore, reliable and effective methods of nucleic acid extraction for samples from particular types of environments are needed. In this study, we compared three commercial kits for metagenomic DNA extraction from three types of biomass: activated sludge, biofilm, and anaerobic digestate. The yield, purity, and quality of DNA were measured, and the effect that the DNA kit had on the subsequent microbial community analysis was assessed with amplified ribosomal intergenic spacer analysis (ARISA). Amplicons were analyzed automatically utilizing capillary electrophoresis. For the activated sludge and digestate, the suggested kit is FastDNA™ Spin Kit for Soil (MP Biomedicals). This kit allowed the highest DNA yield to be obtained and provided the highest biodiversity. For biofilm with a high content of extracellular polymeric substances, the FavorPrep™ Soil DNA Isolation Mini Kit (FAVORGEN) is recommended. This kit allowed to obtain the highest biodiversity and provided the most reliable results of genetic distance assessment in this type of biomass.

Characterization of Two Zymomonas mobilis Wild Strains and Analysis of Populations Dynamics during Their Leavening of Bread-like Doughs.

Two Zymomonas mobilis wild strains (UMB478 and 479) isolated from water kefir were characterized for their biomass production levels and leavening performance when used as the inoculum of a real bread-like dough formulation. The obtained baked product would be consumable by people with adverse responses to Saccharomyces cerevisiae. In liquid cultures, the two strains reached similar biomass concentration (0.7 g CDW/L). UMB479 showed an interesting resistance to NaCl (MBC 30 g/L), that may be useful in the bakery sector. When inoculated in doughs, UMB479 produced the maximum dough volume (650 mL) after 5 h, glucose was almost consumed and 1 g/100 g of ethanol produced, +200% respective to UMB478. Using S. cerevisiae for comparison purposes, the dough doubled its volume fast, in only 2 h, but reached a final level of 575 mL, lower than that achieved by Z. mobilis. The analysis of bacterial and fungal population dynamics during dough leavening was performed through the Automated Ribosomal Intergenic Spacer Analysis (ARISA); doughs leavened by UMB479 showed an interesting decrease in fungal richness after leavening. S. cerevisiae, instead, created a more complex fungal community, similar before and after leavening. Results will pave the way for the use of Z. mobilis UMB479 in commercial yeast-free leavened products.

Reduced representation sequencing accurately quantifies relative abundance and reveals population-level variation in Pseudo-nitzschia spp.

Certain species within the genus Pseudo-nitzschia are able to produce the neurotoxin domoic acid (DA), which can cause illness in humans, mass-mortality of marine animals, and closure of commercial and recreational shellfisheries during toxic events. Understanding and forecasting blooms of these harmful species is a primary management goal. However, accurately predicting the onset and severity of bloom events remains difficult, in part because the underlying drivers of bloom formation have not been fully resolved. Furthermore, Pseudo-nitzschia species often co-occur, and recent work suggests that the genetic composition of a Pseudo-nitzschia bloom may be a better predictor of toxicity than prevailing environmental conditions. We developed a novel next-generation sequencing assay using restriction site-associated DNA (2b-RAD) genotyping and applied it to mock Pseudo-nitzschia communities generated by mixing cultures of different species in known abundances. On average, 94% of the variance in observed species abundance was explained by the expected abundance. In addition, the false positive rate was low (0.45% on average) and unrelated to read depth, and false negatives were never observed. Application of this method to environmental DNA samples collected during natural Pseudo-nitzschia spp. bloom events in Southern California revealed that increases in DA were associated with increases in the relative abundance of P. australis. Although the absolute correlation across time-points was weak, an independent species fingerprinting assay (Automated Ribosomal Intergenic Spacer Analysis) supported this and identified other potentially toxic species. Finally, we assessed population-level genomic variation by mining SNPs from the environmental 2bRAD dataset. Consistent shifts in allele frequencies in P. pungens and P. subpacifica were detected between high and low DA years, suggesting that different intraspecific variants may be associated with prevailing environmental conditions or the presence of DA. Taken together, this method presents a potentially cost-effective and high-throughput approach for studies aiming to evaluate both population and species dynamics in mixed samples.

Comparison of Bacterial and Archaeal Microbiome in Two Bioreactors Fed with Cattle Sewage and Corn Biomass

The bacterial and archaeal communities of two full-scale biogas producing plants (P1 and P2), associated with a 999 kW cogeneration unit, both located in North Italy, were analyzed at start up and fully operating phases, by means of various molecular approaches: (i) Automated Ribosomal Intergenic Spacer Analysis; (ii) cloning and sequencing of PCR amplicons of archaeal genes 16Srrna and mcrA; (iii) 16S rDNA high throughput next generation sequencing. P1 and P2 use the same technology and both were fed with cattle manure and corn silage. During the study of P1 also the post digester (fed with pig manure) was analyzed. The aim of this research was to characterize the bacterial and archaeal communities in two very similar plants to profile the core microbiota. The results of this analysis highlighted that the two plants (producing comparable quantities of volatile fatty acids, biogas, and energy) differed in anerobic microbiota (Bacteria and Archaea). Notably the methanogenic community of P1 was dominated by the strict acetoclastic Methanosaeta (Methanothrix) (up to 23.05%) and the unculturable Candidatus Methanofastidiosum (up to 32.70%), while P2 was dominated by the acetoclastic, but more substrate-versatile, Methanosarcina archaeal genus (49.19%). The data demonstrated that the performances of plants with identical design, in similar operating conditions, yielding comparable amount of biogas (average of 7237 m3 day−1 and 7916 m3 day−1 respectively for P1 and P2), VFA (1643 mg L−1 and 1634 mg L−1) and energy recovery (23.90–24 MWh day−1), depend on the stabilization of effective and functionally optimized methanogenic communities, but these communities are taxonomically different in the two biodigesters.

Automated Ribosomal Intergenic Spacer Analysis to Detect Pathogenic Fungi in Dark-Colored Spots on Apple Fruits

Dark-colored spots (DCSs) found on apples at harvest time worldwide significantly reduce the commercial value of the fruits. In this study, we conducted an automated ribosomal intergenic spacer analysis (ARISA) to reliably detect fungal pathogens in these lesions. The fragment sizes of peaks for eight species and two species complexes that might cause DCSs on apples in northern Honshu Island, Japan, were determined on the basis of the ARISA. The fragment sizes for six species ( Botryosphaeria kuwatsukai, Diplocarpon mali, Mycosphaerella pomi, Neonectria galligena, Phlyctema vagabunda, and Venturia inaequalis) were unique, enabling their detection in a single ARISA trial. However, two other species and two species complexes had similar fragment sizes. To identify these fungi, PCR amplifications using species- or genus-specific primers were needed. Using this method to examine the fresh tissue of DCSs (i.e., after removing the epidermis) on commercially produced apples resulted in the detection of six fungal species and two Colletotrichum species complexes. Because the pathogenic fungi in the tissue underlying lesions are most likely the causal agents, these fungi could be responsible for the DCSs on apple fruits. An automated analysis of the PCR products amplified for the ARISA enables the rapid examination of many samples. These results indicate that the ARISA-based technique developed in the present study is useful for the accurate, reliable, and comprehensive detection of the pathogens causing DCSs on apple fruits. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY 4.0 International license .

The Root Mycobiota of Betula aetnensis Raf., an Endemic Tree Species Colonizing the Lavas of Mt. Etna (Italy)

Betula aetnensis is an endemic tree of high conservation value, which thrives on the nutrient-poor volcanic soils of Mount Etna. Since plant–microbe interactions could play a crucial role in plant growth, resource uptake, and resistance to abiotic stresses, we aimed to characterize the root and rhizosphere microbial communities. Individuals from natural habitat (NAT) and forest nursery (NURS) were surveyed through microscopy observations and molecular tools: bacterial and fungal automated ribosomal intergenic spacer analysis (ARISA), fungal denaturing gradient gel electrophoresis (DGGE). B. aetnensis was found to be simultaneously colonized by arbuscular (AM), ectomycorrhizal (ECM), ericoid (ERM) fungi, and dark septate endophytes (DSE). A high diversity of the bacterial community was observed whilst the root fungal assemblage of NAT plants was richer than that of NURS. Root and rhizosphere fungal communities from NAT plants were characterized by Illumina MiSeq sequencing. Most of the identified sequences were affiliated to Helotiales, Pezizales, and Malasseziales. Ascomycota and Basidiomycota dominated roots and rhizosphere but differed in community structure and composition. ECM in the roots mainly belonged to Tylospora and Leccinum, while Rhizopogon was abundant in the rhizosphere. The Helotiales, including ERM (mostly Oidiodendron) and DSE (mostly Phialocephala), appeared the dominant component of the fungal community. B. aetnensis harbors an extraordinarily wide array of root-associated soil microorganisms, which are likely to be involved in the adaptation and resistance mechanisms to the extreme environmental conditions in volcano Etna. We argue that nursery-produced seedlings could lack the necessary microbiota for growth and development in natural conditions.

The microbiology of rebuilding soils with water treatment residual co-amendments: Risks and benefits.

Water treatment residual (WTR) is composed of sludges from the potable water treatment process, currently largely destined for landfill. This waste can be diverted to rebuild degraded soils, aligning with the UN's Sustainable Development Goals 12 (Consumption and Production) and 15 (Terrestrial Ecosystems). Biosolids are tested against stringent pathogen guidelines, yet few studies have explored the microbial risk of WTR land application, despite anthropogenic impacts on water treatment. We explored the microbial risks and benefits of amending nutrient-poor sandy soil with WTRs. Our results showed that the culturable pathogen load of wet and dry WTRs did not warrant pre-processing before land application, according to South African national quality guidelines, with fecal coliforms not exceeding 104 colony forming units per gram dry weight in wet sludges sampled from four South African and Zimbabwean water treatment plants and decreasing upon drying and processing. There was no culturable pathogenic (fecal coliforms, enterococci, Salmonella, and Shigella) regrowth in soil incubations amended with dry WTR. However, the competition (microbial load and diversity) introduced by a WTR co-amendment did not limit pathogen survival in soils amended with biosolids. Application of WTR to nutrient-poor sandy soils for wheat (Triticum aestivum L.) growth improved the prokaryotic and eukaryotic culturable cell concentrations, similar to compost. However, the compost microbiome more significantly affected the bacterial beta diversity of the receiving soil than WTR when analyzed with automated ribosomal intergenic spacer analysis. Thus, although there was a low pathogen risk for WTR amendment in receiving soils and total soil microbial loads were increased, microbial diversity was more significantly enhanced by compost than WTR.

Characterisation of Environmental Biofilms Colonising Wall Paintings of the Fornelle Cave in the Archaeological Site of Cales.

Caves present unique habitats for the development of microbial communities due to their peculiar environmental conditions. In caves decorated with frescoes, the characterization of microbial biofilm is important to better preserve and safeguard such artworks. This study aims to investigate the microbial communities present in the Fornelle Cave (Calvi Risorta, Caserta, Italy) and their correlation with environmental parameters. The cave walls and the wall paintings have been altered by environmental conditions and microbial activity. We first used light microscopy and scanning electron microscopy (SEM) and X-ray diffraction to characterise the biofilm structure and the mineral composition of substrata, respectively. Then, using both culture-dependent (Sanger sequencing) and culture-independent (automated ribosomal intergenic spacer analysis, ARISA) molecular methods, we demonstrated that the taxonomic composition of biofilms was different across the three substrata analysed and, in some cases, positively correlated with some environmental parameters. We identified 47 taxa in the biofilm samples, specifically 8 bacterial, 18 cyanobacterial, 14 algal and 7 fungal taxa. Fungi showed the highest number of ARISA types on the tuff rock, while autotrophic organisms (cyanobacteria and algae) on the frescoes exposed to light. This study confirms that caves constitute a biodiversity-rich environment for microbial taxa and that, in the presence of wall paintings, taxonomic characterization is particularly important for conservation and restoration purposes.

Inoculation of grape musts with single strains of Saccharomyces cerevisiae yeast reduces the diversity of chemical profiles of wines.

Anecdotal evidence suggests that spontaneous alcoholic fermentation of grape juice is becoming a more popular option in global wine production. Wines produced from the same grape juice by inoculation or spontaneous fermentation usually present distinct chemical and sensorial profiles. Inoculation has been associated with more similar end-products, a loss of typicity, and lower aroma complexity, and it has been suggested that this may be linked to suppression of the local or regional wine microbial ecosystems responsible for spontaneous fermentations. However, whether inoculated fermentations of different juices from different regions really end up with a narrower, less diverse chemical profile than those of spontaneously fermented juices has never been properly investigated. To address this question, we used grape juice from three different varieties, Grüner Veltliner (white), Zweigelt (red), and Pinot noir (red), originating from different regions in Austria to compare spontaneous and single active dry yeast strains inoculated fermentations of the same grape samples. The chemical analysis covered primary metabolites such as glycerol, ethanol and organic acids, and volatile secondary metabolites, including more than 40 major and minor esters, as well as higher alcohols and volatile fatty acids, allowing an in depth statistical evaluation of differences between fermentation strategies. The fungal (mainly yeast) communities throughout fermentations were monitored using automated ribosomal intergenic spacer analysis. The data provide evidence that inoculation with single active dry yeast strains limits the diversity of the chemical fingerprints. The fungal community profiles clearly show that inoculation had an effect on fermentation dynamics and resulted in chemically less diverse wines.

Effects of in situ Remediation With Nanoscale Zero Valence Iron on the Physicochemical Conditions and Bacterial Communities of Groundwater Contaminated With Arsenic.

Nanoscale Zero-Valent Iron (nZVI) is a cost-effective nanomaterial that is widely used to remove a broad range of metal(loid)s and organic contaminants from soil and groundwater. In some cases, this material alters the taxonomic and functional composition of the bacterial communities present in these matrices; however, there is no conclusive data that can be generalized to all scenarios. Here we studied the effect of nZVI application in situ on groundwater from the site of an abandoned fertilizer factory in Asturias, Spain, mainly polluted with arsenic (As). The geochemical characteristics of the water correspond to a microaerophilic and oligotrophic environment. Physico-chemical and microbiological (cultured and total bacterial diversity) parameters were monitored before and after nZVI application over six months. nZVI treatment led to a marked increase in Fe(II) concentration and a notable fall in the oxidation-reduction potential during the first month of treatment. A substantial decrease in the concentration of As during the first days of treatment was observed, although strong fluctuations were subsequently detected in most of the wells throughout the six-month experiment. The possible toxic effects of nZVI on groundwater bacteria could not be clearly determined from direct observation of those bacteria after staining with viability dyes. The number of cultured bacteria increased during the first two weeks of the treatment, although this was followed by a continuous decrease for the following two weeks, reaching levels moderately below the initial number at the end of sampling, and by changes in their taxonomic composition. Most bacteria were tolerant to high As(V) concentrations and showed the presence of diverse As resistance genes. A more complete study of the structure and diversity of the bacterial community in the groundwater using automated ribosomal intergenic spacer analysis (ARISA) and sequencing of the 16S rRNA amplicons by Illumina confirmed significant alterations in its composition, with a reduction in richness and diversity (the latter evidenced by Illumina data) after treatment with nZVI. The anaerobic conditions stimulated by treatment favored the development of sulfate-reducing bacteria, thereby opening up the possibility to achieve more efficient removal of As.

Impact of cigarette butts on bacterial community structure in soil.

Cigarette butts contribute significantly to global pollution present on the planet. The filters found in cigarette butts contain a microplastic, cellulose acetate, as well as toxic metals and metalloids which are responsible for pollution in the environment. Although cigarette butt litter is prevalent in many soils, research on the effects of these cigarette butts is limited. In this study, we used Automated Ribosomal Intergenic Spacer Analysis (ARISA) to generate DNA fingerprints of bacterial communities in soil before and after the addition of cigarette butt leachate treatments. An ICP-MS analysis of the biodegradable and non-biodegradable cigarette butts revealed the presence of various elements: Al, As, B, Ba, Cd, Co, Cr, Cu, Fe, Hg, Mn, Mo, Ni, Pb, Sb, Se, Sn, Sr, V, and Zn. The analysis also specified which metals were present at the highest concentrations in the biodegradable and non-biodegradable cigarette butts, and these were, respectively, Al (1,31 g/kg and 2,35 g/kg), Fe (2,03 g/kg and 1,11 g/kg), and Zn (3,18 mg/kg and 15,70 mg/kg). Our results show that biodegradable cigarette butts had a significant effect on bacterial community composition (beta diversity), unlike the non-biodegradable butts. This effect can be attributed to higher concentrations of certain metals and metalloids in the leachate of biodegradable cigarette butts compared to the non-biodegradable ones. Our findings suggest that biodegradable and non-biodegradable cigarette butts can significantly affect bacterial communities in soil as a result of the leaching of significant quantities of certain elements into the surrounding soils.

ASSESSMENT OF CULTURABLE BACTERIA ASSOCIATED WITH FINE PARTICULATE MATTER COLLECTED IN ANTIOQUIA COLOMBIA- SOUTH AMERICA

In recent years, air pollution in urban and rural environments has been increasing and is one of the factors of greatest public health concern. PM 2.5 particulate matter is one of the main pollutants that deteriorate air quality. This particulate matter can carry bioaerosols as bacteria and fungi. Due to its size or aerodynamic diameter of less than 2.5 μm, it can be inhaled to the pulmonary alveoli, which is strongly associated with a rise in mortality and morbidity in exposed populations. In this research, the presence of bacteria associated with the PM 2.5 fraction of particulate matter was evaluated in two urban areas (Urban-C and Urban-NW) and one rural area (Rural-N) from the Aburra Valley (Antioquia, Colombia South America). In recent years, this region has been presenting high concentrations of PM 2.5 , exceeding several times the daily permissible limits (50 μg/m3) for this air pollutant. The isolation, characterization and identification of bacteria associated with PM 2.5 was performed by culture-dependent techniques, molecular characterization by ribosomal intergenic spacer analysis (RISA) and 16S rDNA sequencing, respectively. The dominant phylogenetic affiliations of the bacteria were grouped into three phyla: Proteobacteria, Actinobacteria and Firmicutes. The phylum Firmicutes dominated in all sampling points with several genera such as Bacillus, Staphylococcus, Paenibacillus, Lysinibacillus, Exiguobacterium and Macrococcus . Some species of these genera have been linked to pathogenicity in plants, animals and humans. Additionally, a greater presence of possible pathogenic microorganisms in urban areas was estimated, probably influenced by the concentration of PM 2.5 and environmental conditions. These results provide important information to understand the distribution and ecology of airborne bacteria and demonstrate that the atmosphere in Colombia (Aburra Valley) harbors bacteria that are clearly an important, but understudied, component of air quality that needs to be better integrated to the public health perspective.

Cover Crop Impact on Soil Organic Carbon, Nitrogen Dynamics and Microbial Diversity in a Mediterranean Semiarid Vineyard

Cover crop (CC) management in vineyards increases sustainability by improving soil chemical and biological fertility, but knowledge on its effects in semiarid soils is lacking. This study evaluated the effect of leguminous CC management on soil organic carbon (SOC) sequestration, soil nitrate content and microbial diversity in a semiarid vineyard, in comparison to conventional tillage (CT). SOC and nitrate were monitored during vine-growing season; soil respiration, determined by incubation experiments, microbial biomass and diversity was analyzed after CC burial. The microbial diversity was evaluated by bacterial and fungal automated ribosomal intergenic spacer analysis (ARISA) and high-throughput sequencing of 16SrDNA. CC increased nitrate content and, although it had no relevant effect on SOC, almost doubled its active microbial component, which contributes to SOC stabilization. An unexpected stability of the microbial communities under different soil managements was assessed, fungal diversity being slightly enhanced under CT while bacterial diversity increased under CC. The complete nitrifying genus Nitrospira and plant growth-promoting genera were increased under CC, while desiccation-tolerant genera were abundant in CT. Findings showed that temporary CC applied in semiarid vineyards does not optimize the provided ecosystem services, hence a proper management protocol for dry environments should be set up.

Botanical microbiomes on the cheap: Inexpensive molecular fingerprinting methods to study plant-associated communities of bacteria and fungi.

High‐throughput sequencing technologies have revolutionized the study of plant‐associated microbial populations, but they are relatively expensive. Molecular fingerprinting techniques are more affordable, yet yield considerably less information about the microbial community. Does this mean they are no longer useful for plant microbiome research? In this paper, we review the past 10 years of studies on plant‐associated microbiomes using molecular fingerprinting methodologies, including single‐strand conformation polymorphism (SSCP), denaturing gradient gel electrophoresis (DGGE), amplicon length heterogeneity PCR (LH‐PCR), ribosomal intergenic spacer analysis (RISA) and automated ribosomal intergenic spacer analysis (ARISA), and terminal restriction fragment length polymorphism (TRFLP). We also present data juxtaposing results from TRFLP methods with those generated using Illumina sequencing in the comparison of rhizobacterial populations of Brazilian maize and fungal surveys in Canadian tomato roots. In both cases, the TRFLP approach yielded the desired results at a level of resolution comparable to that of the MiSeq method, but at a fraction of the cost. Community fingerprinting methods (especially TRFLP) remain relevant for the identification of dominant microbes in a population, the observation of shifts in plant microbiome community diversity, and for screening samples before their use in more sensitive and expensive approaches.