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The sequencing platform and workflow strongly influence microbial community analyses through potential errors at each step. Effective diagnostics and experimental controls are needed to validate data and improve reproducibility. This cross-laboratory study evaluates sources of variability and error at three main steps of a standardized amplicon sequencing workflow (DNA extraction, polymerase chain reaction [PCR], and sequencing) using Oxford Nanopore MinION to analyze agricultural soils and a simple mock community. Variability in sequence results occurs at each step in the workflow with PCR errors and differences in library size greatly influencing diversity estimates. Common bioinformatic diagnostics and the mock community are ineffective at detecting PCR abnormalities. This work outlines several diagnostic checks and techniques to account for sequencing depth and ensure accuracy and reproducibility in soil community analyses. These diagnostics and the inclusion of a reference soil can help ensure data validity and facilitate the comparison of multiple sequencing runs within and between laboratories., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Cover crop biomass is helpful for weed and pest control, soil erosion control, nutrient recycling, and overall soil health and crop productivity improvement. These benefits may vary based on cover crop species and their biomass. There is growing interest in the agricultural sector of using remotely sensed imagery to estimate cover crop biomass. Four small plot study sites located at the United States Department of Agriculture Agricultural Research Service, Crop Production Systems Research Unit farm, Stoneville, MS with different cereals, legumes, and their mixture as fall-seeded cover crops were selected for this analysis. A randomized complete block design with four replications was used at all four study sites. Cover crop biomass and canopy-level hyperspectral data were collected at the end of April, just before cover crop termination. High-resolution (3 m) PlanetScope imagery (Dove satellite constellation with PS2.SD and PSB.SD sensors) was collected throughout the cover crop season from November to April in the 2021 and 2022 study cycles. Results showed that mixed cover crop increased biomass production up to 24% higher compared to single species rye. Reflectance bands (blue, green, red and near infrared) and vegetation indices derived from imagery collected during March were more strongly correlated with biomass ( r = 0-0.74) compared to imagery from November ( r = 0.01-0.41) and April ( r = 0.03-0.57), suggesting that the timing of imagery acquisition is important for biomass estimation. The highest correlation was observed with the near-infrared band ( r = 0.74) during March. The R 2 for biomass prediction with the random forest model improved from 0.25 to 0.61 when cover crop species/mix information was added along with Planet imagery bands and vegetation indices as biomass predictors. More study with multiple timepoint biomass, hyperspectral, and imagery collection is needed to choose appropriate bands and estimate the biomass of mix cover crop species.

Research supports the theory that the microbiome of plants and mushrooms produce potent activators of pathogen recognition receptors which are principal contributors to the stimulation of macrophages. We have previously reported that the in vitro macrophage stimulatory activity of water-soluble extracts from 13 different types of edible mushrooms is predominantly due to bacterial components originating from the naturally occurring bacterial communities within these materials. The purpose of the current study was to further investigate the bacterial-dependent activity of the water-soluble extracts and assess whether these 13 types of mushrooms contain water-insoluble beta glucans that activate the dectin-1b signaling pathway. Activity of the water-soluble extracts was predominantly due to Toll-like receptor 2 (TLR2) and TLR4 agonists. For dectin-1b-dependent activity (indicative of water-insoluble beta glucans), culinary mushrooms (Agaricus bisporus varieties) were essentially inactive, whereas most of the medicinal mushrooms (Lentinula edodes, Grifola frondosa, Hypsizygus marmoreus varieties, Flammulina velutipes) exhibited potent activation. A. bisporus samples with no detectable dectin-1b-dependent activity had yeast colony forming units that were 687 times lower than L. edodes exhibiting high activity, indicating that the active insoluble beta glucans are derived from colonizing yeast. In addition, co-stimulation of macrophages with the TLR agonists and insoluble beta glucan was found to result in a synergistic enhancement of in vitro cytokine production. Taken together, these findings indicate that the in vitro macrophage activating potential of edible mushrooms is due to the collaborative interaction of water-soluble TLR agonists (derived from colonizing bacteria) and water-insoluble beta glucans (derived from colonizing yeast).

Aims: The purpose of this study was to assess impacts of long-term reduced tillage and no till management on bacterial communities in agricultural field soils., Methods and Results: Samples from surface soils were collected from field plots maintained under reduced tillage and no till conditions for 14 years. No till soils had significantly higher microbial biomass, as well as β-glucosidase activity, which is linked to organic matter breakdown. Sequencing of the 16S rRNA gene revealed most variability in bacterial community composition was observed in low abundance community members. Diversity estimates (Chao 1, ACE, and Shannon indices) were lower in no till soils, and several bacterial taxa linked to organic matter breakdown were significantly higher in no till compared to tilled soils., Conclusions: Long-term no till management can significantly enhance the size of soil microbial communities while negatively impacting bacterial diversity, through the lack of soil disturbance and breakdown of crop residues left on soil surfaces., Significance and Impact of the Study: This study provides insights into how no till management can influence the microbial community's contribution to soil health and suggests that long-term no till field plots may benefit from occasional tillage., (Published 2019. This article is a U.S. Government work and is in the public domain in the USA.)

We previously demonstrated that extracts from Echinacea purpurea material varied substantially in their ability to activate macrophages in vitro and that this variation was due to differences in their content of bacterial components. The purpose of the current study was to identify soil conditions (organic matter, nitrogen, and moisture content) that alter the macrophage activation potential of E. purpurea and determine whether these changes in activity correspond to shifts in the plant-associated microbiome. Increased levels of soil organic matter significantly enhanced macrophage activation exhibited by the root extracts of E. purpurea (p < 0.0001). A change in soil organic matter content from 5.6% to 67.4% led to a 4.2-fold increase in the macrophage activation potential of extracts from E. purpurea. Bacterial communities also differed significantly between root materials cultivated in soils with different levels of organic matter (p < 0.001). These results indicate that the level of soil organic matter is an agricultural factor that can alter the bacterial microbiome, and thereby the activity, of E. purpurea roots. Since ingestion of bacterial preparation (e.g., probiotics) is reported to impact human health, it is likely that the medicinal value of Echinacea is influenced by cultivation conditions that alter its associated bacterial community.

Recent studies have indicated that a major contributor to the innate immune enhancing properties of some medicinal plants is derived from the cell wall components of bacteria colonizing these plants. The purpose of the current study was to assess if the bacteria present within edible and medicinal mushrooms substantially contribute to the innate immune stimulating potential of these mushrooms. Whole mushrooms from thirteen types of edible fungi and individual parts from Agaricus bisporus were analyzed for in vitro macrophage activation as well as bacterial lipopolysaccharides (LPS) content, cell load, and community composition. Substantial variation between samples was observed in macrophage activation (over 500-fold), total bacterial load (over 200-fold), and LPS content (over 10 million-fold). Both LPS content (ρ = 0.832, p < 0.0001) and total bacterial load (ρ = 0.701, p < 0.0001) correlated significantly with macrophage activation in the whole mushroom extracts. Extract activity was negated by treatment with NaOH, conditions that inactivate LPS and other bacterial components. Significant correlations between macrophage activation and total bacterial load (ρ = 0.723, p = 0.0001) and LPS content (ρ = 0.951, p < 0.0001) were also observed between different tissues of Agaricus bisporus. Pseudomonas and Flavobacterium were the most prevalent genera identified in the different tissue parts and these taxa were significantly correlated with in vitro macrophage activation (ρ = 0.697, p < 0.0001 and ρ = 0.659, p = 0.0001, respectively). These results indicate that components derived from mushroom associated bacteria contribute substantially to the innate immune enhancing activity exhibited by mushrooms and may result in similar therapeutic actions as reported for ingestion of bacterial preparations such as probiotics.

Evidence supports the theory that bacterial communities colonizing Echinacea purpurea contribute to the innate immune enhancing activity of this botanical. Previously, we reported that only about half of the variation in in vitro monocyte stimulating activity exhibited by E. purpurea extracts could be accounted for by total bacterial load within the plant material. In the current study, we test the hypothesis that the type of bacteria, in addition to bacterial load, is necessary to fully account for extract activity. Bacterial community composition within commercial and freshly harvested (wild and cultivated) E. purpurea aerial samples was determined using high-throughput 16S rRNA gene pyrosequencing. Bacterial isolates representing 38 different taxa identified to be present within E. purpurea were acquired, and the activity exhibited by the extracts of these isolates varied by over 8000-fold. Members of the Proteobacteria exhibited the highest potency for in vitro macrophage activation and were the most predominant taxa. Furthermore, the mean activity exhibited by the Echinacea extracts could be solely accounted for by the activities and prevalence of Proteobacteria members comprising the plant-associated bacterial community. The efficacy of E. purpurea material for use against respiratory infections may be determined by the Proteobacterial community composition of this plant, since ingestion of bacteria (probiotics) is reported to have a protective effect against this health condition., (Georg Thieme Verlag KG Stuttgart · New York.)

Background: Plants harbor a diverse bacterial community, both as epiphytes on the plant surface and as endophytes within plant tissue. While some plant-associated bacteria act as plant pathogens or promote plant growth, others may be human pathogens. The aim of the current study was to determine the bacterial community composition of organic and conventionally grown leafy salad vegetables at the point of consumption using both culture-dependent and culture-independent methods., Results: Total culturable bacteria on salad vegetables ranged from 8.0 × 10(3) to 5.5 × 10(8) CFU g(-1). The number of culturable endophytic bacteria from surface sterilized plants was significantly lower, ranging from 2.2 × 10(3) to 5.8 × 10(5) CFU g(-1). Cultured isolates belonged to six major bacterial phyla, and included representatives of Pseudomonas, Pantoea, Chryseobacterium, and Flavobacterium. Eleven different phyla and subphyla were identified by culture-independent pyrosequencing, with Gammaproteobacteria, Betaproteobacteria, and Bacteroidetes being the most dominant lineages. Other bacterial lineages identified (e.g. Firmicutes, Alphaproteobacteria, Acidobacteria, and Actinobacteria) typically represented less than 1% of sequences obtained. At the genus level, sequences classified as Pseudomonas were identified in all samples and this was often the most prevalent genus. Ralstonia sequences made up a greater portion of the community in surface sterilized than non-surface sterilized samples, indicating that it was largely endophytic, while Acinetobacter sequences appeared to be primarily associated with the leaf surface. Analysis of molecular variance indicated there were no significant differences in bacterial community composition between organic versus conventionally grown, or surface-sterilized versus non-sterilized leaf vegetables. While culture-independent pyrosequencing identified significantly more bacterial taxa, the dominant taxa from pyrosequence data were also detected by traditional culture-dependent methods., Conclusions: The use of pyrosequencing allowed for the identification of low abundance bacteria in leaf salad vegetables not detected by culture-dependent methods. The presence of a range of bacterial populations as endophytes presents an interesting phenomenon as these microorganisms cannot be removed by washing and are thus ingested during salad consumption.

Much of the nutrient cycling and carbon processing in natural environments occurs through the activity of extracellular enzymes released by microorganisms. Thus, measurement of the activity of these extracellular enzymes can give insights into the rates of ecosystem level processes, such as organic matter decomposition or nitrogen and phosphorus mineralization. Assays of extracellular enzyme activity in environmental samples typically involve exposing the samples to artificial colorimetric or fluorometric substrates and tracking the rate of substrate hydrolysis. Here we describe microplate based methods for these procedures that allow the analysis of large numbers of samples within a short time frame. Samples are allowed to react with artificial substrates within 96-well microplates or deep well microplate blocks, and enzyme activity is subsequently determined by absorption or fluorescence of the resulting end product using a typical microplate reader or fluorometer. Such high throughput procedures not only facilitate comparisons between spatially separate sites or ecosystems, but also substantially reduce the cost of such assays by reducing overall reagent volumes needed per sample.

Agricultural pesticide use is necessary to help meet the increased demand for a safe and secure food supply for the United States, as well as the global community. Even with proper application and careful management, the possibility of pesticide leaching and detachment in runoff still exists following certain storm events. Several different management practices have been designed to reduce the impacts of pesticides on aquatic receiving systems. Many such practices focus on the use of vegetation to slow runoff and allow for sorption of the various contaminants. Three common drainage ditch macrophytes, Leersia oryzoides (cutgrass), Typha latifolia (cattail), and Sparganium americanum (bur-reed) were assessed for their ability to reduce effluent loads of atrazine, diazinon, and permethrin in simulated agricultural runoff water in 379L individual mesocosms. Of the three macrophytes examined, L. oryzoides was the most effective at mitigating atrazine, and permethrin. L. oryzoides and T. latifolia significantly reduced overall atrazine loads (45±7%, p=0.0073 and 35±8%, p=0.0421, respectively) when compared to unvegetated controls (13±20%). No significant differences in overall diazinon load retention were noted between plant species. Each plant species significantly decreased the initial load (after 6h) of trans-permethrin, while both L. oryzoides and T. latifolia significantly reduced the overall trans-permethrin loads (88±5%, p=0.0022 and 88±5%, p=0.0020, respectively) when compared to unvegetated controls (68±8%). Reversible adsorption of atrazine and diazinon to plants, noted during the flushing events, was greater than that observed in either cis- or trans-permethrin. These results demonstrate the ability of native ditch vegetation to mitigate pesticides associated with agricultural runoff. Likewise, they provide farmers and action agencies with supportive data for selection of vegetation in drainage ditches used as management practices., (Published by Elsevier Ltd.)

Passage of agricultural runoff through vegetated drainage ditches has been shown to reduce the amount of pesticides, such as atrazine, exiting out of agricultural watersheds. Previous studies have found that microbial communities in soil from fields treated with atrazine display enhanced rates of atrazine degradation. However, no studies have examined the potential for atrazine degradation in ditches used to drain these lands. The purpose of the current study was to determine the potential of the drainage ditch soil microbial community for atrazine degradation. Soil samples were collected from fields and adjacent drainage ditches and from nonagricultural land with no previous exposure to atrazine. Polymerase chain reaction analysis indicated widespread presence of atrazine degradation genes in fields and ditches. Potential for degradation was determined by following the decrease of atrazine in spiked soil samples over a 28-d incubation period. Greater than 95% of atrazine was degraded in field and ditch soils, whereas only 68.5 ± 1.3% was degraded in the nonagricultural control. Comparison with autoclaved soil samples indicated the primary mechanism of atrazine degradation in agricultural soils was microbially mediated, whereas its breakdown in nonagricultural soil appeared to be the byproduct of abiotic processes. Therefore, microbial communities in drainage ditch sediments have the potential to play a role in atrazine removal from agricultural runoff by breaking down atrazine deposited in sediments and limiting the amount of this herbicide carried into downstream ecosystems., (Copyright © by the American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America, Inc.)

Gluconacetobacter diazotrophicus PAl 5 is of agricultural significance due to its ability to provide fixed nitrogen to plants. Consequently, its genome sequence has been eagerly anticipated to enhance understanding of endophytic nitrogen fixation. Two groups have sequenced the PAl 5 genome from the same source (ATCC 49037), though the resulting sequences contain a surprisingly high number of differences. Therefore, an optical map of PAl 5 was constructed in order to determine which genome assembly more closely resembles the chromosomal DNA by aligning each sequence against a physical map of the genome. While one sequence aligned very well, over 98% of the second sequence contained numerous rearrangements. The many differences observed between these two genome sequences could be owing to either assembly errors or rapid evolutionary divergence. The extent of the differences derived from sequence assembly errors could be assessed if the raw sequencing reads were provided by both genome centers at the time of genome sequence submission. Hence, a new genome sequence standard is proposed whereby the investigator supplies the raw reads along with the closed sequence so that the community can make more accurate judgments on whether differences observed in a single stain may be of biological origin or are simply caused by differences in genome assembly procedures.

The citrus disease Huanglongbing (HLB) is highly destructive in many citrus-growing regions of the world. The putative causal agent of this disease, 'Candidatus Liberibacter asiaticus', is difficult to culture, and Koch's postulates have not yet been fulfilled. As a result, efforts have focused on obtaining the genome sequence of 'Ca. L. asiaticus' in order to give insight on the physiology of this organism. In this work, three next-generation high-throughput sequencing platforms, 454, Solexa, and SOLiD, were used to obtain metagenomic DNA sequences from phloem tissue of Florida citrus trees infected with HLB. A culture-independent, polymerase chain reaction (PCR)-independent analysis of 16S ribosomal RNA sequences showed that the only bacterium present within the phloem metagenome was 'Ca L. asiaticus'. No viral or viroid sequences were identified within the metagenome. By reference assembly, the phloem metagenome contained sequences that provided 26-fold coverage of the 'Ca. L. asiaticus' contigs in GenBank. By the same approach, phloem metagenomic data yielded less than 0.2-fold coverage of five other alphaproteobacterial genomes. Thus, phloem metagenomic DNA provided a PCR-independent means of verifying the presence of 'Ca L. asiaticus' in infected tissue and strongly suggests that no other disease agent was present in phloem. Analysis of these metagenomic data suggest that this approach has a detection limit of one 'Ca. Liberibacter' cell for every 52 phloem cells. The phloem sample sequenced here is estimated to have contained 1.7 'Ca. Liberibacter' cells per phloem cell.

We report here the sequencing and analysis of the genome of the nitrogen-fixing endophyte, Klebsiella pneumoniae 342. Although K. pneumoniae 342 is a member of the enteric bacteria, it serves as a model for studies of endophytic, plant-bacterial associations due to its efficient colonization of plant tissues (including maize and wheat, two of the most important crops in the world), while maintaining a mutualistic relationship that encompasses supplying organic nitrogen to the host plant. Genomic analysis examined K. pneumoniae 342 for the presence of previously identified genes from other bacteria involved in colonization of, or growth in, plants. From this set, approximately one-third were identified in K. pneumoniae 342, suggesting additional factors most likely contribute to its endophytic lifestyle. Comparative genome analyses were used to provide new insights into this question. Results included the identification of metabolic pathways and other features devoted to processing plant-derived cellulosic and aromatic compounds, and a robust complement of transport genes (15.4%), one of the highest percentages in bacterial genomes sequenced. Although virulence and antibiotic resistance genes were predicted, experiments conducted using mouse models showed pathogenicity to be attenuated in this strain. Comparative genomic analyses with the presumed human pathogen K. pneumoniae MGH78578 revealed that MGH78578 apparently cannot fix nitrogen, and the distribution of genes essential to surface attachment, secretion, transport, and regulation and signaling varied between each genome, which may indicate critical divergences between the strains that influence their preferred host ranges and lifestyles (endophytic plant associations for K. pneumoniae 342 and presumably human pathogenesis for MGH78578). Little genome information is available concerning endophytic bacteria. The K. pneumoniae 342 genome will drive new research into this less-understood, but important category of bacterial-plant host relationships, which could ultimately enhance growth and nutrition of important agricultural crops and development of plant-derived products and biofuels., Competing Interests: The authors have declared that no competing interests exist.

Non-plant pathogenic endophytic bacteria can promote plant growth, improve nitrogen nutrition, and, in some cases, are human pathogens. Recent work in several laboratories has shown that enteric bacteria are common inhabitants of the interior of plants. These observations led to the experiments that showed the entry into plants of enteric human pathogens such as Salmonella and E. coli O157:H7. The extent of endophytic colonization by strains is regulated by plant defenses and several genetic determinants necessary for this interior colonization in endophytic bacteria have been identified. The genomes of four endophytic bacteria now available should promote discovery of other genes that contribute to this phenotype. Common virulence factors in plant and animal pathogens have also been described in bacteria that can infect both plant and animal models. Future directions in all of these areas are proposed.