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Abstract 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.

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 R2 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.

Crop varieties resistant to multiple herbicides have been developed to provide better control of weed populations in row-crop fields where glyphosate resistance has become common. These new varieties include lines of maize (Zea mays) resistant to both glyphosate and 2,4-dichlorophenoxyacetic acid (2,4-D). As these herbicides have the potential to impact microbial communities in soil, there is concern that their co-application may have a greater effect on activities linked to soil nutrient cycling than if they were applied individually. To investigate this possibility, a field study was conducted on 2,4-D+glyphosate-resistant maize to determine the impact of 2,4-D alone and 2,4-D+glyphosate on extracellular enzyme activity in both bulk and rhizosphere soil. Maize was treated at the V2 and V8 developmental stages. Changes in soil activities were small in magnitude and inconsistent between timepoints. 2,4-D+glyphosate-treated plots had higher beta-glucosidase, cellobiohydrolase, and phosphatase activities, but only after the V2 application in bulk soil in the first year of the study, while no significant effects were observed in the rhizosphere. Enzyme activities were more impacted by soil organic matter than herbicide treatments. These results suggest that, when applied at label rates, 2,4-D+glyphosate application will not adversely affect soil microbial enzyme activities.

Conservation management practices can improve soil health while minimizing deleterious effects of agriculture on the environment. However, adoption of these practices, particularly cover crops, is not widespread, as they often reduce crop yields compared to traditional management practices. The purpose of the current study was to determine if a two-species cover crop treatment of rye (Secale cereale L.) and crimson clover (Trifolium incarnatum L.) could increase soil health parameters and maximize soybean (Glycine max L.) yield greater than rye only in tilled and no-till Mississippi field soils. Enhanced microbial biomass and organic matter input from cover crops increased the activities of β-glucosidase, cellobiohydrolase, fluorescein diacetate hydrolysis, N-acetylglucosaminidase, and phosphatase in surface soils. Rye plus clover tended to elicit higher activities than rye only in no-till plots. Both cover crop treatments inhibited soybean yield in tilled plots by 11–25%. These results indicate that tillage exacerbates yield inhibition by cover crops in soybean and that double-species cover crop treatments were more consistent in increasing activities linked to nutrient cycling. Further study examining different combinations of cover crops in no-till systems is necessary to gain a better understanding of how they can be implemented to enhance soil health while maximizing crop yield.

Plants harbor a diverse microbiome existing as bacterial populations on the leaf surface (the phyllosphere) and within plant tissues (endophytes). The composition of this microbiome has been largely unexplored in fresh produce vegetables, where studies have tended to focus on pathogen detection and survival. However, the application of next-generation 16S rRNA gene sequencing approaches is beginning to reveal the diversity of this produce-associated bacterial community. In this article we review what is known about the composition of the microbiome of fresh produce vegetables, placing it in the context of general phyllosphere research. We also demonstrate how next-generation sequencing can be used to assess the bacterial assemblages present on fresh produce, using fresh herbs as an example. That data shows how the use of such culture-independent approaches can detect groups of taxa (anaerobes, psychrophiles) that may be missed by traditional culture-based techniques. Other issues discussed include questions as to whether to determine the microbiome during plant growth or at point of purchase or consumption, and the potential role of the natural bacterial community in mitigating pathogen survival.

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.