Section 1 update: DNA and RNA extraction from soil

Griffiths, Robert I.; Manefield, Mike; Whiteley, Andrew S.; Bailey, Mark

doi:10.1007/978-1-4020-2177-0_111

Cited by 13 publications

(14 citation statements)

References 28 publications

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“…DNA was isolated from all bacterial strains using the DNeasy UltraClean microbial Kit (Qiagen) while DNA was isolated from soil using the protocol from Griffiths et al . (). The qPCR reaction was performed in a final volume of 20 μ l and consisted of: 10 μ l of 10X iTaq Mix (Bio‐Rad Laboratories, Mississauga, Canada), 0·8 μ l of 5 μ mol l −1 of probe, 0·8 μ l of 5 μ mol l −1 of each primer, 3·0 μ l of DNA and 4·6 μ l of DEPC‐treated H 2 O.…”

Section: Methodsmentioning

confidence: 97%

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Persistence ofPseudomonas fluorescensLBUM677 in the rhizosphere of corn gromwell (Buglossoides arvensis) under field conditions and its impact on seed oil and stearidonic acid bioaccumulation

Novinscak

Filion

2019

J Appl Microbiol

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Aims The aim of this study was to evaluate the persistence of Pseudomonas fluorescens LBUM677 in the rhizosphere of Buglossoides arvensis under agricultural field conditions and determine if B. arvensis intraspecific genetic variations affect the capacity of LBUM677 to colonize its rhizosphere and increase seed oil and stearidonic acid (SDA) accumulation. Methods and Results Two field experiments were performed to: (i) study the persistence of various concentrations of LBUM677 inoculated in the rhizosphere of B. arvensis and determine a minimum inoculation threshold required to maximize biological activity; and (ii) study the impact of B. arvensis intraspecific genetic variations on LBUM677 rhizosphere colonization and seed oil and SDA accumulation. In order to track LBUM677 populations in soil over time, a specific quantitative polymerase chain reaction assay was developed. Inoculation with a minimum of 109 LBUM677 bacterial cells per plant was determined as a threshold to promote maximum B. arvensis rhizosphere colonization and seed oil and SDA accumulation. Buglossoides arvensis intraspecific genetic variations had an impact on rhizosphere colonization, B. arvensis seed oil and SDA accumulation, where two cultivars benefited more than others from LBUM677 inoculation. Conclusions LBUM677 can colonize the rhizosphere and increase seed oil and SDA yields in B. arvensis plants in a cultivar‐dependant manner. Significance and Impact of the Study LBUM677 shows potential to be used as a biofertilizer to specifically increase seed oil and SDA yields in B. arvensis. This will in turn promote the development of an economically viable agricultural‐based approach as an alternative for producing high‐quality polyunsaturated fatty acids.

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Section: Methodsmentioning

confidence: 97%

“…Total DNA was extracted from each composite soil sample using the previously mentioned protocol developed by Griffiths et al . (). Following DNA extraction, all samples were diluted 1 : 10 with sterile distilled water before being used in qPCR.…”

Section: Methodsmentioning

confidence: 97%

Persistence ofPseudomonas fluorescensLBUM677 in the rhizosphere of corn gromwell (Buglossoides arvensis) under field conditions and its impact on seed oil and stearidonic acid bioaccumulation

Novinscak

Filion

2019

J Appl Microbiol

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“…The quantity and purity of DNA samples were verified by spectrophotometry (NanoDrop Inc., Wilmington, DE). DNA was also isolated directly from eight soil samples collected in this study using the DNA extraction protocol developed by Griffiths et al (31). The quantity and purity of the DNA samples were verified by spectrophotometry (NanoDrop Inc.).…”

Section: Methodsmentioning

confidence: 99%

Novel P450nor Gene Detection Assay Used To Characterize the Prevalence and Diversity of Soil Fungal Denitrifiers

Novinscak

Zebarth

Burton

et al. 2016

Appl Environ Microbiol

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Denitrifying fungi produce nitrous oxide (N 2 O), a potent greenhouse gas, as they generally lack the ability to convert N 2 O to dinitrogen. Contrary to the case for bacterial denitrifiers, the prevalence and diversity of denitrifying fungi found in the environment are not well characterized. In this study, denitrifying fungi were isolated from various soil ecosystems, and novel PCR primers targeting the P450nor gene, encoding the enzyme responsible for the conversion of nitric oxide to N 2 O, were developed, validated, and used to study the diversity of cultivable fungal denitrifiers. This PCR assay was also used to detect P450nor genes directly from environmental soil samples. Fungal denitrification capabilities were further validated using an N 2 O gas detection assay and a PCR assay targeting the nirK gene. A collection of 492 facultative anaerobic fungi was isolated from 15 soil ecosystems and taxonomically identified by sequencing the internal transcribed spacer sequence. Twenty-seven fungal denitrifiers belonging to 10 genera had the P450nor and the nirK genes and produced N 2 O from nitrite. N 2 O production is reported in strains not commonly known as denitrifiers, such as Byssochlamys nivea, Volutella ciliata, Chloridium spp., and Trichocladium spp. The prevalence of fungal denitrifiers did not follow a soil ecosystem distribution; however, a higher diversity was observed in compost and agricultural soils. The phylogenetic trees constructed using partial P450nor and nirK gene sequences revealed that both genes clustered taxonomically closely related strains together. IMPORTANCE A PCR assay targeting the P450nor gene involved in fungal denitrification was developed and validated. The newly developedP450nor primers were used on fungal DNA extracted from a collection of fungi isolated from various soil environments and on DNA directly extracted from soil. The results indicated that approximatively 25% of all isolated fungi possessed this gene and were able to convert nitrite to N 2 O. All soil samples from which denitrifying fungi were isolated also tested positive for the presence of P450nor. The P450nor gene detection assay was reliable in detecting a large diversity of fungal denitrifiers. Due to the lack of homology existing between P450nor and bacterial denitrification genes, it is expected that this assay will become a tool of choice for studying fungal denitrifiers.

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“…Soil DNA extraction and qPCR quantification of P. fluorescens LBUM677 DNA was extracted from 0Á25 g of rhizosphere soil samples using the method described in (Griffiths et al 2008). The DNA quantity and quality was evaluated using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE).…”

Section: Plant Harvest and Soil Samplingmentioning

confidence: 99%

Pseudomonas fluorescens LBUM677 differentially increases plant biomass, total oil content and lipid composition in three oilseed crops

2019

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Aims Pseudomonas spp. have been widely studied for their plant growth–promoting effects. However, their capacity to promote lipid accumulation in oilseed crops is not well characterized. In this study, we evaluated the effect of Pseudomonas fluorescens LBUM677 on lipid accumulation in three oilseed crops: soybean (Glycine max), canola (Brassica napus) and corn gromwell (Buglossoides arvensis), a plant of high nutraceutical interest for its accumulation of the omega‐3 stearidonic acid. Methods and Results Pot experiments were conducted under controlled conditions where seeds were inoculated or not with LBUM677 and plants were harvested at 4, 8 and 12 weeks. A qPCR assay specifically targeting LBUM677 was used in parallel to correlate LBUM677 soil rhizosphere competency to growth promotion and seed lipid accumulation. Total oil seed content and fatty acid composition were analysed at seed maturity. Results showed that LBUM677 was able to establish itself in the rhizosphere of the three plant species at similar levels, but it differentially increased plant biomass, total oil content and lipid composition in a plant‐specific manner. Conclusions Despite some species‐specific differences observed in P. fluorescens LBUM677's effect on different crops, the strain appears to be a generalist plant growth–promoting rhizobacteria of oilseed crops. Significance and Impact of the Study LBUM677 shows great potential to be used as an inoculum to promote oil yield and fatty acid accumulation in oilseed crops.

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Section 1 update: DNA and RNA extraction from soil

Cited by 13 publications

References 28 publications

Persistence ofPseudomonas fluorescensLBUM677 in the rhizosphere of corn gromwell (Buglossoides arvensis) under field conditions and its impact on seed oil and stearidonic acid bioaccumulation

Persistence ofPseudomonas fluorescensLBUM677 in the rhizosphere of corn gromwell (Buglossoides arvensis) under field conditions and its impact on seed oil and stearidonic acid bioaccumulation

Novel P450nor Gene Detection Assay Used To Characterize the Prevalence and Diversity of Soil Fungal Denitrifiers

Pseudomonas fluorescens LBUM677 differentially increases plant biomass, total oil content and lipid composition in three oilseed crops

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