Evaluation of the mechanism of action of Bacillus spp. to manage Meloidogyne incognita with split root assay, RT-qPCR and qPCR

The goal of this research is to determine the mechanism of action of two Bacillus spp. that can manage Meloidogyne incognita population density in cotton. The overall objectives are 1) determine the efficacy and direct antagonistic capabilities of the Bacillus spp. and 2) determine the systemic capabilities of the Bacillus spp. The greenhouse in planta assay indicated B. amyloliquefaciens QST713 and B. firmus I-1582 could manage M. incognita similarly to the chemical standard fluopyram. An in vitro assay determined that B. firmus I-1582 and its extracted metabolites were able to directly manage M. incognita second stage juveniles by increasing mortality rate above 75%. A split root assay, used to determine systemic capabilities of the bacteria, indicated B. amyloliquefaciens QST713 and B. firmus I-1582 could indirectly decrease the nematode population density. Another species, B. mojavensis strain 2, also demonstrated systemic capabilities but was not a successful biological control agent because it supported a high population density in greenhouse in planta assay and in the split root assay. A RT-qPCR assay was used to confirm any systemic activity observed in the split root assay. At 24 hours both B. amyloliquefaciens QST713 and B. firmus I-1582 upregulated one gene involved in the initial stages of JA synthesis pathway but not another gene involved in the later stages of JA synthesis. These results point to a JA intermediate molecule, most likely OPDA, stimulated by the bacteria rather than JA in a short-term systemic response. After 1 week, the Bacillus spp. stimulated a SA-responsive defense related gene. The long-term systemic response to the Bacillus spp. indicates salicylic acid also plays a role in defense conferred by these bacteria. The final assay was a qPCR to determine the concentration of the bacteria on the cotton roots after 24 days. Bacillus amyloliquefaciens QST713 and B. firmus I-43 1582 were able to colonize the root successfully, with the concentration after 24 days not significantly differing from the concentration at inoculation. This study identifies two bacteria that work via systemic resistance and will help aid in implementing these species in an integrated management system

\u3ci\u3ePlectus\u3c/i\u3e of the Prairie: A Case Study of Taxonomic Resolution from a Nematode Biodiversity Survey

Taxonomic resolution is a critical component of biodiversity assessments. In this case study, we examined a single taxon within a larger study of nematode diversity to evaluate the taxonomic resolution of different diversity assessment methods. The selected taxon was the microbial-feeding genus Plectus, a group considered to include multiple cosmopolitan species. The methods included a morphological evaluation by light microscopy, Sanger sequencing of PCR amplicons of COI and 18S gene regions, and 18S metabarcoding sequencing. The study sites were 15 remnant tallgrass prairie plots in eastern Nebraska. In the morphological analysis, we observed two basic morphotypes, a short-tailed form with a small amphid and a long-tailed form with a large amphid. Sanger sequencing of COI sorted Plectus diversity into six distinct clades. The largest two of these six clades keyed to P. parietinus and P. rhizophilus based on morphology. BLAST analysis with COI revealed no close matches in GenBank. Sanger sequencing of the 18S region did not differentiate the six clades. These results illustrate that the method of diversity assessment strongly influences estimates of biodiversity. An additional 95 Plectus specimens, from outside the remnant sites, added taxonomic breadth to the COI phylogenetic tree. There were no geographically widespread COI haplotypes and no evidence of cosmopolitan Plectus species

18S-NemaBase: Curated 18S rRNA Database of Nematode Sequences

Nematodes are the most abundant and diverse animals on the planet but lack representation in biodiversity research. This presents a problem for studying nematode diversity, particularly when molecular tools (i.e., barcoding and metabarcoding) rely on well-populated and curated reference databases, which are absent for nematodes. To improve molecular identification and the assessment of nematode diversity, we created and curated an 18S rRNA database specific to nematodes (18S-NemaBase) using sequences sourced from the most recent publicly available 18S rRNA SILVA v138 database. As part of the curation process, taxonomic strings were standardized to contain a fixed number of taxonomic ranks relevant to nematology and updated for the most recent accepted nematode classifications. In addition, apparent erroneous sequences were removed. To test the efficacy and accuracy of 18S-NemaBase, we compared it to an older but also curated SILVA v111 and the newest SILVA v138 by assigning taxonomies and analyzing the diversity of a nematode dataset from the Western Nebraska Sandhills. We showed that 18S-NemaBase provided more accurate taxonomic assignments and diversity assessments than either version of SILVA, with a much easier workflow and no need for manual corrections. Additionally, observed diversity further improved when 18S-NemaBase was supplemented with reference sequences from nematodes present in the study site. Although the 18S-NemaBase is a step in the right direction, a concerted effort to increase the number of high-quality, accessible, full-length nematode reference sequences is more important now than ever

18S-NemaBase: Curated 18S rRNA Database of Nematode Sequences

Nematodes are the most abundant and diverse animals on the planet but lack representation in biodiversity research. This presents a problem for studying nematode diversity, particularly when molecular tools (i.e., barcoding and metabarcoding) rely on well-populated and curated reference databases, which are absent for nematodes. To improve molecular identification and the assessment of nematode diversity, we created and curated an 18S rRNA database specific to nematodes (18S-NemaBase) using sequences sourced from the most recent publicly available 18S rRNA SILVA v138 database. As part of the curation process, taxonomic strings were standardized to contain a fixed number of taxonomic ranks relevant to nematology and updated for the most recent accepted nematode classifications. In addition, apparent erroneous sequences were removed. To test the efficacy and accuracy of 18S-NemaBase, we compared it to an older but also curated SILVA v111 and the newest SILVA v138 by assigning taxonomies and analyzing the diversity of a nematode dataset from the Western Nebraska Sandhills. We showed that 18S-NemaBase provided more accurate taxonomic assignments and diversity assessments than either version of SILVA, with a much easier workflow and no need for manual corrections. Additionally, observed diversity further improved when 18S-NemaBase was supplemented with reference sequences from nematodes present in the study site. Although the 18S-NemaBase is a step in the right direction, a concerted effort to increase the number of high-quality, accessible, full-length nematode reference sequences is more important now than ever

Effects of drought-induced stress on nematode communities in aquatic and terrestrial habitats of the Nebraska Sandhills

IntroductionGlobal change events (e.g., worsening drought) are increasing environmental stress in a variety of terrestrial and aquatic habitats. The degree to which communities in soils and sediments are driven by temporal environmental changes across multiple habitat types from the same region is not clear.MethodsWe used nematodes, a common bioindicator of soil and sediment health, to determine how community diversity and composition are altered by rising alkalinity across lakes, shorelines, and prairies in the western Nebraska Sandhills. We sampled these three habitats from five lake basins spanning an alkalinity gradient (pH 7–11) across three years (2019, 2020, 2021). During our sampling, the Sandhills experienced a range of drought intensities, with 2019 being a wet year, followed by severe drought in 2020, and abnormally dry/moderate drought in 2021. To determine if diversity and composition of nematodes responded to increased alkalinity and drought-induced stress we used different modelling approaches, including Random Forest and pairwise comparisons.Results and discussionOverall, nematode diversity in lakes was most affected by increasing alkalinity over time, whereas in shorelines and prairies diversity was most reliant on bacterial diversity and potential nematode-nematode interactions. In comparison to shorelines and prairies, community composition in lakes was the least variable and consistently driven by pH and lake water levels. In contrast, compositions in the shorelines and prairies were more variable and explained at varying degrees by pH, year, lake basin, and climate-associated variables. In addition, relative abundance and compositional nature of select copious taxa were highly unpredictable, indicating potential instability in these habitats. Future research is necessary to address the ecologic stability of the Sandhills and determine where conservation efforts are most needed

Host identity is the dominant factor in the assembly of nematode and tardigrade gut microbiomes in Antarctic Dry Valley streams

Abstract Recent work examining nematode and tardigrade gut microbiomes has identified species-specific relationships between host and gut community composition. However, only a handful of species from either phylum have been examined. How microbiomes differ among species and what factors contribute to their assembly remains unexplored. Cyanobacterial mats within Antarctic Dry Valley streams host a simple and tractable natural ecosystem of identifiable microinvertebrates to address these questions. We sampled 2 types of coexisting mats (i.e., black and orange) across four spatially isolated streams, hand-picked single individuals of two nematode species (i.e., Eudorylaimus antarcticus and Plectus murrayi) and tardigrades, to examine their gut microbiomes using 16S and 18S rRNA metabarcoding. All gut microbiomes (bacterial and eukaryotic) were significantly less diverse than the mats they were isolated from. In contrast to mats, microinvertebrates’ guts were depleted of Cyanobacteria and differentially enriched in taxa of Bacteroidetes, Proteobacteria, and Fungi. Among factors investigated, gut microbiome composition was most influenced by host identity while environmental factors (e.g., mats and streams) were less important. The importance of host identity in predicting gut microbiome composition suggests functional value to the host, similar to other organisms with strong host selected microbiomes

18S-NemaBase: Curated 18S rRNA Database of Nematode Sequences

Nematodes are the most abundant and diverse animals on the planet but lack representation in biodiversity research. This presents a problem for studying nematode diversity, particularly when molecular tools (i.e., barcoding and metabarcoding) rely on well-populated and curated reference databases, which are absent for nematodes. To improve molecular identification and the assessment of nematode diversity, we created and curated an 18S rRNA database specific to nematodes (18S-NemaBase) using sequences sourced from the most recent publicly available 18S rRNA SILVA v138 database. As part of the curation process, taxonomic strings were standardized to contain a fixed number of taxonomic ranks relevant to nematology and updated for the most recent accepted nematode classifications. In addition, apparent erroneous sequences were removed. To test the efficacy and accuracy of 18S-NemaBase, we compared it to an older but also curated SILVA v111 and the newest SILVA v138 by assigning taxonomies and analyzing the diversity of a nematode dataset from the Western Nebraska Sandhills. We showed that 18S-NemaBase provided more accurate taxonomic assignments and diversity assessments than either version of SILVA, with a much easier workflow and no need for manual corrections. Additionally, observed diversity further improved when 18S-NemaBase was supplemented with reference sequences from nematodes present in the study site. Although the 18S-NemaBase is a step in the right direction, a concerted effort to increase the number of high-quality, accessible, full-length nematode reference sequences is more important now than ever