Evaluation and optimization of lysis method for microbial DNA extraction from epiphytic phyllosphere samples

Yang, Seongmin; Lee, Jaejin; Mahmood, Maheen; Deng, Min; Im, Jeongdae

doi:10.1016/j.mimet.2023.106677

Cited by 4 publications

(2 citation statements)

References 31 publications

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“…Microbial nucleic acid extraction from plant material, particularly from the epiphytic phyllosphere, poses challenges due to plant-derived biomolecules such as proteins and nucleic acids (58, 59). In this study, we used a microbial DNA extraction method optimized for silage samples, as detailed in our previous study (60). Briefly, for DNA extraction, five grams of the sample was suspended in 45 mL of sterile 0.85% NaCl solution, shaken on a rotary shaker at 120 rpm for 2 h at room temperature, filtered through two layers of gauze cloth to remove large plant debris, and centrifuged at 12,000 g for 15 min at 4°C (61).…”

Section: Methodsmentioning

confidence: 99%

Forage conservation is a neglected nitrous oxide source

Yang,

Mahmood,

Baral

et al. 2024

Preprint

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Agricultural activities are the major source of nitrous oxide (N2O), an important greenhouse gas and ozone [ndash]depleting substance. However, the role of forage conservation as a potential source of N2O has hardly been studied. We investigated N2O production from the simulated silage of the three major crops—maize, alfalfa, and sorghum—used for silage in the US, which comprises over 90% of the total silage production. We demonstrated a substantial N2O generation, reaching up to 169.7 g [ndash]CO2 eq. per kgDM–forage, which places forage conservation as the third largest N2O source in the agricultural sector. Notably, the addition of chlorate significantly reduced N2O production, but neither acetylene nor intermittent exposure to oxygen showed any impact, highlighting that denitrifiers are responsible for N2O production from silage—a hypothesis confirmed by molecular analyses. In addition, we calculated the social cost of the N2O emission from forage conservation and proposed market incentives for mitigation. Our study reveals a previously unexplored source of N2O and provides a crucial mechanistic understanding for effective mitigation strategies.

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Section: Methodsmentioning

confidence: 99%

Forage conservation is a neglected nitrous oxide source

Yang,

Mahmood,

Baral

et al. 2024

Preprint

Self Cite

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“…In addition, enzymatic lysis (and chemical lysis) is better suited for large sample numbers than most machine-dependent physical disruption methods. However, the DNA quality may be lower (Yang et al, 2023). Therefore, the quantity and quality requirements should be considered when deciding the use of an enzymatic disgest approach.…”

Section: Enzymatic Digestmentioning

confidence: 99%

A guide to DNA extraction protocols for ecologists, conservation managers, and environmental DNA researchers

Rieder,

Jemmi,

Adrian-Kalchhauser

2023

Preprint

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Ecologists, conservation biologists, and environmental managers are increasingly dependent on environmental DNA (eDNA) data for research and decision making. The inherent complexity of eDNA samples, coupled with different choices involved during the DNA extraction process, can introduce biases into the final eDNA dataset. Therefore, optimization and troubleshooting of DNA extraction protocols are pivotal for the successful execution of eDNA projects. Knowledge of the basic steps and principles of DNA extraction is essential for eDNA analysis. However, traditional education in ecology, conservation, and environmental management typically does not include in-depth training in molecular methods. While DNA extraction box kits are typically user-friendly, they may fail to deliver the desired results with eDNA samples, necessitating protocol adaptations or educated selection of alternative approaches. The primary objective of this paper is to enable scientists with an ecological background who employ DNA extraction protocols to understand the four key steps of DNA extraction, and to use this expertise to their advantage. Furthermore, we describe the purpose of commonly used reagents and chemicals, point out alternatives for each key step, explain the impact of certain choices on DNA integrity and purity, and advocate for an adaptable “mix and match” protocol when applicable. We anticipate that the paper will enable field ecologists to develop a deeper understanding of the mechanisms and chemistry underlying DNA extraction, thus allowing them to make informed decisions regarding the best DNA extraction method for their research. Our intention is not to provide comprehensive, step-by-step protocols, but to offer guiding principles while highlighting alternative solutions. Finally, we hope that this paper will act as a useful resource to support knowledge transfer and teaching.

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Forage conservation is a neglected nitrous oxide source

Yang,

Mahmood,

Baral

et al. 2024

PNAS Nexus

View full text Add to dashboard Cite

Agricultural activities are the major anthropogenic source of nitrous oxide (N2O), an important greenhouse gas and ozone-depleting substance. However, the role of forage conservation as a potential source of N2O has rarely been studied. We investigated N2O production from the simulated silage of the three major crops—maize, alfalfa, and sorghum—used for silage in the United States, which comprises over 90% of the total silage production. Our findings revealed that a substantial N2O could be generated, potentially placing forage conservation as the third largest N2O source in the agricultural sector. Notably, the application of chlorate as an additive significantly reduced N2O production, but neither acetylene nor intermittent exposure to oxygen showed any impact. Overall, the results highlight that denitrifiers, rather than nitrifiers, are responsible for N2O production from silage, which was confirmed by molecular analyses. Our study reveals a previously unexplored source of N2O and provides a crucial mechanistic understanding for effective mitigation strategies.

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Evaluation and optimization of lysis method for microbial DNA extraction from epiphytic phyllosphere samples

Cited by 4 publications

References 31 publications

Forage conservation is a neglected nitrous oxide source

Forage conservation is a neglected nitrous oxide source

A guide to DNA extraction protocols for ecologists, conservation managers, and environmental DNA researchers

Forage conservation is a neglected nitrous oxide source

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