Advancing the automation of plant nucleic acid extraction for rapid diagnosis of plant diseases in space

Haveman, Natasha J.; Schuerger, Andrew C.; Yu, Pei-Ling; Brown, Mark J.; Doebler, Robert; Paul, Anna‐Lisa; Ferl, Robert J.

doi:10.3389/fpls.2023.1194753

Cited by 3 publications

(1 citation statement)

References 62 publications

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“…Specifically, an OmniLyse micro-homogenizer was used to lyse the petioles and midveins of citrus to produce a crude lysate, followed by one of two strategies, serial dilution and paper-disk-assisted extraction, to enable downstream use in qPCR-based assays (Figure 1b). To the best of our knowledge, the OmniLyse micro-homogenizer has not been used for the quick lysis of plant tissues, except for one report in 2023 [45]. Thus, this current study expands the application of the device successfully to plant tissues that are mechanically robust and difficult to lyse.…”

Section: Introductionmentioning

confidence: 87%

Quick Plant Sample Preparation Methods Using a Micro-Homogenizer for the Detection of Multiple Citrus Pathogens

Liu,

Bodaghi,

Vidalakis

et al. 2024

Chemosensors

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Effective pathogen detection is essential for plant disease control. However, plant sample preparation for downstream assays, such as quantitative polymerase chain reaction (qPCR), is challenging to perform outside of a laboratory. This paper reports two sample preparation methods featuring chemical and mechanical lysis and nucleic acid extraction using a micro-homogenizer, followed by serial dilution or nucleic acid purification with a paper disk before assay. Five minutes of lysis and extraction resulted in DNA and RNA yields of up to 76.5% and 63.3%, respectively, compared to mortar and pestle controls. Crude lysates were unsuitable for direct use in qPCR assays; however, serial dilution or quick wash using chromatography paper rendered samples ready for such assays. Additionally, the nucleic acids stored on paper disks under various storage conditions remained stable for one month. These methods can facilitate the in-field preparation of citrus samples and allow for both onsite and mail-in diagnostics for growers.

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Section: Introductionmentioning

confidence: 87%

Quick Plant Sample Preparation Methods Using a Micro-Homogenizer for the Detection of Multiple Citrus Pathogens

Liu,

Bodaghi,

Vidalakis

et al. 2024

Chemosensors

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Effectiveness of bimetallic ZnO-B2O3 nanoparticles produced by Streptomyces gancidicus as prospective antifungal agents and therapeutic nutrients to enhance pea plant immunity against damping off-causing Pythium irregulare: in vivo and in vitro investigations

Elkhodary,

Attia,

El-Sayyad

et al. 2023

Biomass Conv. Bioref.

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Pythium irregulare (P. irregulare) is one of the soil-borne pathogens that is the primary cause of damage to several plants each year. The novelty and originality of this work were the ability of Streptomyces gancidicus (S. gancidicus OR229936) to synthesize bimetallic zinc oxide-boron oxide nanoparticles (ZnO-B2O3 NPs) for reducing P. irregulare growth and safeguarding pea plant from damping off disease. The produced bimetallic ZnO-B2O3 NPs’ XRD results highlighted the ZnO diffraction peaks at 2Ɵ = 27.50°, 31.15°, 45.15°, 56.89°, 67.98°, and 75.25°, which are complemented by the standard card JCPDS number 361451 and correspond to (002), (101), (102), (110), (103), and (201) Bragg’s reflections. Along with the standard card JCPDS number 300019, they additionally include the B2O3 NP diffraction peaks at 2Ɵ = 15.25°, 28.69°, 31.99°, and 41.28°. Bimetallic ZnO-B2O3 NPs were tested against P. irregular for their antifungal activities. The findings indicated that ZnO-B2O3 NPs exhibited potential anti P. irregulare activity, with an inhibition zone of 33 mm at a concentration of 1000 µg/mL and a promising MIC of 0.01 µg/mL. Bimetallic ZnO-B2O3 NPs (0.01 ppm) application appeared to significantly lessen the severity of the pea post-emergence damaging off disease by 10% and to provide significant protection by 88%. In comparison to fungicide (difenoconazole 25%) treatments, all metabolic resistance indicators significantly enhanced after the usage of bimetallic ZnO-B2O3 NPs, ZnO NPs, and B2O3 NPs with ethyl acetate extract of S. gancidicus. The beneficial impacts of the bimetallic ZnO-B2O3 NPs, ZnO NPs, and B2O3 NPs have been broadened to increase the enzyme activities of peroxidase (POD) and polyphenol oxidase (PPO) in both healthy and infected pea plant in comparison to control. Reduction of Malondialdehyde content (MDA) in response to S. gancidius filtrate, bimetallic ZnO-B2O3 NPs, ZnO NPs, B2O3 NPs, and difenoconazole by 41.68%, 36.51%, 26.15, 26.15, and 15.25%, respectively. Also, contents of H2O2 in infected pea plant were diminished by 50%, 45%, 40%, 37.5%, and 22.5% at bimetallic ZnO-B2O3 NPs, S. gancidicus filtrate, ZnO NPs, difenoconazole, and B2O3 NPs comparing to P. irregular-infected pea plant is strong evidence to induce disease recovery. The application of bimetallic ZnO-B2O3 NPs seems to be a significant approach to relieve the toxic influences of P. irregulare on infected pea plant as green and alternative therapeutic nutrients of chemical fungicides.

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Rapid colorimetric detection ofcitrus tristeza viruscombining portable sample preparation and reverse transcription-loop mediated isothermal amplification

Liu,

Bodaghi,

Keremane

et al. 2024

Preprint

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A sensing platform combining semi-automated sample preparation protocol and one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) is reported for rapid colorimetric detection of Citrus tristeza virus (CTV) in a greenhouse. An OmniLyse micro-homogenizer and cellulose paper disks were integrated for quick sample preparation of total nucleic acids (<15 min). RT-LAMP assays were optimized in terms of primers' concentrations and minimization of false positives for both CTV and cytochrome oxidase (COX) detections. Specifically, the optimal reaction time for lab-based RT-LAMP assays was determined as 40 minutes with the detection limits of CTV and COX as 43 copies/μL (equivalent to 86 copies/mg of tissue) and 5 copies/μL (equivalent to 10 copies/mg of tissue), respectively. Additionally, an in-greenhouse colorimetric RT-LAMP assay with lyophilized reaction mix for endpoint CTV detection was successfully conducted in 35 minutes without a false response in either colorimetric or fluorometric assays. Overall, this quick sample preparation protocol integrated with the lyophilized RT-LAMP assays showed high efficiency and reliability in plant pathogen detection in a greenhouse. This strategy holds great potential to be integrated into a portable, autonomous system and be universally adopted for in-field diagnosis of different pathogens.

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Advancing the automation of plant nucleic acid extraction for rapid diagnosis of plant diseases in space

Cited by 3 publications

References 62 publications

Quick Plant Sample Preparation Methods Using a Micro-Homogenizer for the Detection of Multiple Citrus Pathogens

Quick Plant Sample Preparation Methods Using a Micro-Homogenizer for the Detection of Multiple Citrus Pathogens

Effectiveness of bimetallic ZnO-B2O3 nanoparticles produced by Streptomyces gancidicus as prospective antifungal agents and therapeutic nutrients to enhance pea plant immunity against damping off-causing Pythium irregulare: in vivo and in vitro investigations

Rapid colorimetric detection ofcitrus tristeza viruscombining portable sample preparation and reverse transcription-loop mediated isothermal amplification

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