Novel primers for quantification of Priestia megaterium populations in soil using qPCR

Kaminsky, Laura M.; Bell, Terrence H.

doi:10.1016/j.apsoil.2022.104628

Cited by 7 publications

(6 citation statements)

References 41 publications

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“…DNA extracted from soil usually contains high quantities of PCR inhibitors, so that DNA has to be diluted before processing [15]. Lower detection limits in soil can be achieved using DNA extraction kits that are more efficient to remove soil inhibitors or do not require high dilution levels before qPCR processing [16]. Nonetheless, the quantification limits measured in copies per ng of DNA or copies per reaction is in accordance with those commonly found in the literature for qPCR primers [17].…”

Section: Resultsmentioning

confidence: 99%

Generic workflow for a rapid and easy design of strain-specific PCR and qPCR primers, applied to the assessment of bacterial strains survival in soil

Polrot,

Béguet,

Devers-Lamrani

et al. 2024

Preprint

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Background: Detecting bacteria at the strain level is crucial in microbiology. Although qPCR is widely used, designing strain-specific primers remains a challenge due to nucleotide sequence similarities among related strains. Methods and Results: This paper introduces a simplified, web-based workflow for designing strain-specific primers using publicly available microbial genomes. The method does not require advanced bioinformatics skills and can be applied using a basic computer. Primers designed using this workflow are applied to assess the survival of two close Bacillus strains in soil microcosms. Conclusion: The workflow offers an accessible solution for accurate bacterial strain detection, and fills a gap for researchers without specialized training in bioinformatics.

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Section: Resultsmentioning

confidence: 99%

Generic workflow for a rapid and easy design of strain-specific PCR and qPCR primers, applied to the assessment of bacterial strains survival in soil

Polrot,

Béguet,

Devers-Lamrani

et al. 2024

Preprint

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“…In conclusion, the in vitro characterization comprising biofertilization, plant growth promotion, and rhizocompetence traits together with in planta assays and in silico genomic analysis of strains of Bacillus sensu lato, points at ILBB592 and ILBB95 -both ascribed to Priestia megateriumas candidates to be used as PGPR with an impact on N and P nutrition of soybean plants. This species has already been associated with a positive effect on plant growth and also P solubilization (Kaminsky and Bell, 2022). Their development as biofertilizers with PGPR activity, alone or in a combined formulation, is envisaged.…”

Section: Discussionmentioning

confidence: 99%

Phenotypic and genomic characterization ofBacillus sensu latofor their biofertilization effect and plant growth promotion features in soybean plants

Torres,

Altier,

Beyhaut

et al. 2023

Preprint

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Bacillus sensu lato were screened for their capacity to mineralize organic phosphorus (P) and promote plant growth, improving nitrogen (N) and phosphorus nutrition of soybean plants. Isolates were first identified based on their genomic sequences through TYGS and ANII. ILBB95, ILBB510 and ILBB592 were identified as Priestia megaterium, ILBB139 as Bacillus wiedmannii, ILBB44 as a member of a sister clade of B. pumilus (together with a human pathogenic strain), ILBB15 as Peribacillus butanolivorans and ILBB64 as Lysinibacillus sp. These strains were evaluated for their capacity to mineralize organic P as sodium phytate and solubilize inorganic P forms in liquid medium. These in vitro assays allowed the strains to be ranked according to their P mobilization potential, with ILBB15 and ILBB64 showing the highest orthophosphate production from phytate, ILBB592 the lowest and ILBB510 nil. In addition, features related to their rhizocompetence and plant growth promotion were evaluated in vitro and in silico. Finally, plant bioassays were deployed to assess the effect of the co-inoculation of Bacillus s.l. strains and rhizobial inoculant on nodulation, plant growth and nutrition. In planta bioassays showed that B. pumilus ILBB44 and P. megaterium ILBB95 increased P absorbed in plants grown on a poor substrate of sand and vermiculite and also on the richer mix of sand, vermiculite and peat. Priestia megaterium ILBB592 increased rhizobial nodulation and N content in plants grown on sand, vermiculite and peat mixture only. ILBB15 reduced plant growth and nutrition on both substrates. Genomes of ILBB95 and ILBB592 were characterized by genes related with plant growth and biofertilization whereas ILBB15 was differentiated by genes related to bioremediation. Priestia megaterium ILBB592 can be described as nodule-enhancing rhizobacteria (NER) and together with ILBB95, can be envisaged as prospective PGPR with the capacity to exert a positive effect on N and P nutrition of soybean plants.

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“…The slope of the resulting calibration curve of quantification cycle (Cq) values vs. log10 of amplicon molecules initially present in the PCR tube was used to calculate assay efficiency according to the following equation: PCR efficiency = 10 -1/slope – 1 (Bustin et al, 2009). To determine if there was qPCR inhibition caused by the extracted DNA sample, we followed a method similar to (Kaminsky and Bell 2022). Five concentrations of insect DNA were prepared from the 12 insect extractions obtained in the previous step: undiluted, 5-fold, 10-fold, 50-fold, and 100-fold dilutions in sterile nuclease-free water.…”

Section: Methodsmentioning

confidence: 99%

Insects visitFusarium xyrophilumpseudoflowers on the hostXyris surinamensis(Xyridaceae) and carry fungal DNA on their bodies

Torres-Cruz,

Cofer,

Kaminsky

et al. 2024

Preprint

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The fungusFusarium xyrophilumproduces flower-like structures (i.e., pseudoflowers) that were recently discovered on yellow-eyed grasses (Xyrisspp.) in Guyana. It is unknown whether these pseudoflowers, which are composed entirely of fungal tissue, are true mimics that attract insects as a means of fungal dispersal. We evaluated the potential ofF. xyrophilumto affect insect visitation patterns to flowers and pseudoflowers by 1) documenting insect visitation toX. surinamensisin Guyana, 2) measuring the presence ofF. xyrophilumDNA on insects, and 3) evaluating fluorescence and volatile production on flowers and pseudoflowers. We report for the first time Vespidae, Formicidae, Salticidae, Acrididae, and Tetrigidae visitingXyris. Diverse insects, including Conocephalini spp. (meadow katydids; Tettigoniidae),Camponotusspp. (carpenter ants; Formicidae), and a Geometridae sp. (geometer moths) were found to visit flowers and pseudoflowers.Fusarium xyrophilumDNA was detected on 3/12 (25%) of captured insect bodies using conventional and quantitative PCR. Volatiles produced in the field by pseudoflowers and flowers were similar, except for the presence of a sesquiterpene, putatively identified here as α-gurjunene, which was detected both inF. xyrophilumpure cultures and field-collected pseudoflower samples, but not from flowers. The production of this sesquiterpene byF. xyrophilumand the fluorescence ofX. surinamensispeduncles represent potential signals involved in insect attraction for this system. These observations, along with the overlap in insect visitors of flowers and pseudoflowers and the detection ofF. xyrophilumDNA on insect bodies, are consistent with insect visitors being vectors ofXyrispollen andF. xyrophilumpropagules between host plants.

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Novel primers for quantification of Priestia megaterium populations in soil using qPCR

Cited by 7 publications

References 41 publications

Generic workflow for a rapid and easy design of strain-specific PCR and qPCR primers, applied to the assessment of bacterial strains survival in soil

Generic workflow for a rapid and easy design of strain-specific PCR and qPCR primers, applied to the assessment of bacterial strains survival in soil

Phenotypic and genomic characterization ofBacillus sensu latofor their biofertilization effect and plant growth promotion features in soybean plants

Insects visitFusarium xyrophilumpseudoflowers on the hostXyris surinamensis(Xyridaceae) and carry fungal DNA on their bodies

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