We gratefully acknowledge Agriculture and Agri-Food Canada for the funding under which this research was made possible (projects J-001843 and J-002021). We thank Brett van Heyningen for filming the video content for this protocol. We would also like to thank former graduate students (Jennifer Vacon and Mark Nabuurs) for their insights into the methods described in this manuscript.

The details of the reagents and the equipment used in this study are listed in the Table of Materials.
1. Selecting the microbial biocontrol candidates
<ol>
	<li>Using competition plate assays, select microbial isolates that display antagonism against the pathogen of interest. 
	NOTE: Nine Bacillus isolates that exhibited antagonism against S. sclerotiorum were selected, including Bacillus atrophaeus, mojavensis and subtilis species, to demonstrate this protocol.</li>
</ol>
2. Media preparation
<ol>
	<li>Prepare potato dextrose agar (PDA) by dissolving 39 g of medium per liter of water.</li>
	<li>Prepare Pseudomonas F agar by adding 45 g of media per liter of water.</li>
	<li>Prepare potato dextrose broth (PDB) with 1% agar by adding 24 g of medium, and 0.24 g agar per liter of water.</li>
	<li>Prepare yeast-glucose-manganese (YGM) broth. Create a buffer solution consisting of 2.5 g of KH2PO4 and 2.5 g K2HPO4 in 100 mL of water, and a salt solution consisting of 0.58 g of MnSO4.H20, 0.5 g of NaCl and 0.05 g of FeSO4.7H20 in 100 mL of water.
	<ol>
		<li>Next, add 1 g of yeast extract, 1.25 g of dextrose, 400 mL of water, 5 mL of the buffer solution, 5 mL of the salt solution and 90 mL of water to ensure that the metal salts do not precipitate in solution.</li>
	</ol>
	</li>
	<li>Autoclave-sterilize the media, pour the agar into 100 x 15 mm Petri plates, and let all cool to room temperature before use.</li>
</ol>
3. Pathogen preparation
<ol>
	<li>Culture five plates of Sclerotinia sclerotiorum on PDA (prepared in step 2.1) and incubate at 25 &#176;C for 3 days.</li>
</ol>
4. Natural product extract preparation
<ol>
	<li>Culture each bacterial isolate on Pseudomonas F Agar (prepared in step 2.2) and grow until individual colonies are observed.</li>
	<li>Subculture single bacterial colonies into 25 mL of YGM media in centrifuge or culture tubes and incubate with shaking for 3 days.</li>
	<li>Transfer 5 mL of aliquots of each extract into 1 L of YGM and incubate in the same conditions for another 3 days.</li>
	<li>Centrifuge each culture at 3000 x g for 15 min at 25 &#176;C to pellet the cells.</li>
	<li>Decant and wash the supernatant three times with ethyl acetate to extract metabolites from the culture medium.</li>
	<li>Combine and dry the ethyl acetate layer of each using rotary evaporation.</li>
	<li>Re-dissolve the extract in minimal methanol, transfer it to a small scintillation vial, and dry it again by rotary evaporation. 
	NOTE: If the extract dries to an oily or resinous material, lyophilize the material to obtain a dry powder that can be accurately weighed.</li>
</ol>
5. TLC plate preparation
<ol>
	<li>Prepare the 20 cm x 20 cm TLC plate by drawing a line 2 cm from the bottom and 2 cm from the top of the plate. On the bottom line, place nine dots 2 cm apart. Above the top line, place four dots 4 cm apart.</li>
	<li>Dissolve 5 mg of each extract in 15 &#181;L of methanol and apply 5 &#181;L of each extract on the nine dots marked on the bottom line using a pipette. Let each extract spot dry on the TLC plate and apply another 5 &#181;L aliquot to the same spot. Repeat until all of the material is loaded onto the TLC plate.</li>
	<li>Develop the TLC plate in a developing tank using a 1:2 solution of dichloromethane and methanol until the solvent reaches the line marked 2 cm from the top of the plate (approximately 45 min).</li>
	<li>Once developed, remove the TLC plate and apply a series of positive controls on the four dots marked above the solvent line. Four concentrations of the same control are used. For S. scl., 0.5 &#181;g/mL, 5 &#181;g/mL, 50 &#181;g/mL, and 500 &#181;g/mL of Hygromycin B are positive controls.</li>
	<li>Before all solvent evaporates from the plate, place it in an ethanol-sterilized TLC plate box.</li>
</ol>
6. Direct bioautography assay
<ol>
	<li>Autoclave the materials to be used in the assay (glass components of the chromatography sprayer, metal spatula, four sheets of paper towel, cellulose or filter paper, water, and media) in a large beaker covered with tinfoil. 
	NOTE: Paper materials (paper towel, cellulose paper) should be wrapped in tin foil to avoid becoming wet in the autoclave.</li>
	<li>Connect an air source, pressure gauge, and chromatography sprayer using ethanol-sterilized PTFE tubing. Connect a HEPA filter between the chromatography sprayer and the pressure gauge.</li>
	<li>Collect the mycelial mat of the five pathogen plates using the sterile metal spatula into a 50 mL centrifuge tube using a sterile spatula. 
	NOTE: Use a small volume of liquid broth to help dislodge the mycelia if needed, and transfer with a sterile pipette.</li>
	<li>Add 25 mL of PDB amended with agar and sterile glass beads to the 50 mL centrifuge tube and vortex for 5 min to break up the mycelial mat.</li>
	<li>Transfer the mycelial suspension to a chromatography sprayer using a sterile syringe with a needle tip to ensure large pieces of mycelia will not clog the chromatography sprayer.</li>
	<li>Place the previously developed TLC plates on sterile paper towels in a laminar flow hood to reduce hand contact with the plate while applying the media suspension.</li>
	<li>Increase the air pressure to approximately 4 bars and apply three coats of the suspension to the TLC plate, allowing the plate to dry completely between applications. 
	NOTE: Three layers were found to be the optimal amount. Less than this, and the pathogen does not have enough media to grow on. More than this, the media is too thick, which may not allow for pathogen contact with active metabolites.</li>
	<li>Add 500 mL of sterile water into a sterilized plastic plate box and place sterilized folded cellulose sheets or filter paper on each side to hold moisture.</li>
	<li>Place the completed assay plate onto four empty Petri dishes in the box.</li>
	<li>Incubate the assay for 3 days to 1 week or until an even layer of mycelia grows evenly across the TLC plate except for around the positive controls and zone of inhibition (ZOIs).</li>
</ol>
7. Liquid chromatography mass spectrometry analysis
<ol>
	<li>Image the completed assay using photography.</li>
	<li>Calculate the retention factor for each zone of inhibition by dividing the distance from the bottom line to the ZOI by the distance from the bottom line to the top line.</li>
	<li>Scrape the silica off the inhibition zones and place it into microcentrifuge tubes.</li>
	<li>Add 500 &#181;L of methanol to each tube and vortex to extract the metabolites from the silica.</li>
	<li>Centrifuge at 8,500 x g at 20 &#176;C for 10 min, and transfer the supernatant to a small vial.</li>
	<li>Evaporate to dryness by rotary evaporation or under a stream of nitrogen.</li>
	<li>Re-suspend the extract in 50 &#181;L of methanol in an LC vial.</li>
	<li>Analyze the metabolites in the zone of inhibition by LC-MS and compare them to the metabolites in the crude extract to determine which metabolites in the crude extract are responsible for the inhibition of the pathogen16.</li>
	<li>Using the genus and species of the source microorganism and the masses identified in the ZOI, search the literature and relevant databases such as Antibase and the Dictionary of Natural Products to identify the metabolites.</li>
</ol>

Using TLC-Direct Bioautography Assay to Identify Natural Products for Biocontrol

<ol>
	<li>Fisher, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+fungal+threats+to+animal,+plant+and+ecosystem+health.">Emerging fungal threats to animal, plant and ecosystem health.</a> Nature. 484 (7393), 186-194 (2012).</li><li>Savary, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+global+burden+of+pathogens+and+pests+on+major+food+crops.">The global burden of pathogens and pests on major food crops.</a> Nat Ecol Evol. 3 (3), 430-439 (2019).</li><li>Lin, B. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resilience+in+agriculture+through+crop+diversification:+Adaptive+management+for+environmental+change.">Resilience in agriculture through crop diversification: Adaptive management for environmental change.</a> BioSci. 61 (3), 183-193 (2011).</li><li>Piquerez, S. J., Harvey, S. E., Beynon, J. L., Ntoukakis, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+crop+disease+resistance:+Lessons+from+research+on+arabidopsis+and+tomato.">Improving crop disease resistance: Lessons from research on arabidopsis and tomato.</a> Front Plant Sci. 5, 671 (2014).</li><li>Damalas, C., Koutroubas, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+status+and+recent+developments+in+biopesticide+use.">Current status and recent developments in biopesticide use.</a> Agriculture. 8 (1), 13 (2018).</li><li>Syed Ab Rahman, S. F., Singh, E., Pieterse, C. M. J., Schenk, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+microbial+biocontrol+strategies+for+plant+pathogens.">Emerging microbial biocontrol strategies for plant pathogens.</a> Plant Sci. 267, 102-111 (2018).</li><li>Tudi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exposure+routes+and+health+risks+associated+with+pesticide+application.">Exposure routes and health risks associated with pesticide application.</a> Toxics. 10 (6), 335 (2022).</li><li>Santos, E. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacillus+thuringiensis:+From+biopesticides+to+anticancer+agents.">Bacillus thuringiensis: From biopesticides to anticancer agents.</a> Biochimie. 192, 83-90 (2022).</li><li>Kumar, J., Ramlal, A., Mallick, D., Mishra, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+overview+of+some+biopesticides+and+their+importance+in+plant+protection+for+commercial+acceptance.">An overview of some biopesticides and their importance in plant protection for commercial acceptance.</a> Plants. 10 (6), 1185 (2021).</li><li>Marrone, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pesticidal+natural+products+-+status+and+future+potential.">Pesticidal natural products - status and future potential.</a> Pest Manag Sci. 75 (9), 2325-2340 (2019).</li><li>Ayilara, M. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biopesticides+as+a+promising+alternative+to+synthetic+pesticides:+A+case+for+microbial+pesticides,+phytopesticides,+and+nanobiopesticides.">Biopesticides as a promising alternative to synthetic pesticides: A case for microbial pesticides, phytopesticides, and nanobiopesticides.</a> Front. Microbiol. 14, 1040901 (2023).</li><li>Abdelaziz, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biocontrol+of+soil+borne+diseases+by+plant+growth+promoting+rhizobacteria.">Biocontrol of soil borne diseases by plant growth promoting rhizobacteria.</a> Trop. Plant Pathol. 48 (2), 105-127 (2023).</li><li>Zhao, Z., Liu, D., Ruan, L., Wang, T., Liang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antifungal+mechanism+of+Bacillus+amyloliquefaciens+SC-B15+and+its+application+in+cereal+mildewproof+and+grape+preservation.">Antifungal mechanism of Bacillus amyloliquefaciens SC-B15 and its application in cereal mildewproof and grape preservation.</a> Food Biosci. 56, 103287 (2023).</li><li>Jamshidi-Aidji, M., Dimkic, I., Ristivojevic, P., Stankovic, S., Morlock, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect-directed+screening+of+bacillus+lipopeptide+extracts+via+hyphenated+high-performance+thin-layer+chromatography.">Effect-directed screening of bacillus lipopeptide extracts via hyphenated high-performance thin-layer chromatography.</a> J Chromatogr A. 1605, 460366 (2019).</li><li>Prichystal, J., Schug, K. A., Lemr, K., Novak, J., Havlicek, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+analysis+of+natural+products.">Structural analysis of natural products.</a> Anal Chem. 88 (21), 10338-10346 (2016).</li><li>De Souza, C. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+quantification+of+lipopeptide+isoforms+by+UPLC-MS+in+the+fermentation+broth+from+Bacillus+subtilis+CNPMS22.">Simultaneous quantification of lipopeptide isoforms by UPLC-MS in the fermentation broth from Bacillus subtilis CNPMS22.</a> Anal Bioanal Chem. 410 (26), 6827-6836 (2018).</li><li>Dewanjee, S., Gangopadhyay, M., Bhattacharya, N., Khanra, R., Dua, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioautography+and+its+scope+in+the+field+of+natural+product+chemistry.">Bioautography and its scope in the field of natural product chemistry.</a> J Pharm Anal. 5 (2), 75-84 (2015).</li><li>Choma, I., Jesionek, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TLC-direct+bioautography+as+a+high+throughput+method+for+detection+of+antimicrobials+in+plants.">TLC-direct bioautography as a high throughput method for detection of antimicrobials in plants.</a> Chromatography. 2 (2), 225-238 (2015).</li><li>Attia, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thin-layer+chromatography-bioautographic+method+for+the+detection+of+arginase+inhibitors.">Thin-layer chromatography-bioautographic method for the detection of arginase inhibitors.</a> J Sep Sci. 43 (12), 2477-2486 (2020).</li><li>Legerska, B., Chmelova, D., Ondrejovic, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TLC-bioautography+as+a+fast+and+cheap+screening+method+for+the+detection+of+alpha-chymotrypsin+inhibitors+in+crude+plant+extracts.">TLC-bioautography as a fast and cheap screening method for the detection of alpha-chymotrypsin inhibitors in crude plant extracts.</a> J Biotechnol. 313, 11-17 (2020).</li><li>Agatonovic-Kustrin, S., Doyle, E., Gegechkori, V., Morton, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-performance+thin-layer+chromatography+linked+with+(bio)assays+and+FTIR-ATR+spectroscopy+as+a+method+for+discovery+and+quantification+of+bioactive+components+in+native+Australian+plants.">High-performance thin-layer chromatography linked with (bio)assays and FTIR-ATR spectroscopy as a method for discovery and quantification of bioactive components in native Australian plants.</a> J Pharm Biomed Anal. 184, 113208 (2020).</li><li>Hilaire, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+method+for+screening+anti-leishmania+compounds+in+plants+extracts+by+HPTLC-bioautography.">New method for screening anti-leishmania compounds in plants extracts by HPTLC-bioautography.</a> J Chromatogr B. 1188, 123061 (2022).</li><li>Stankovic, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HPTLC-direct+bioautography-guided+isolation+of+isogeranic+acid+as+the+main+antibacterial+constituent+of+Artemisia+santonicum+essential+oil.">HPTLC-direct bioautography-guided isolation of isogeranic acid as the main antibacterial constituent of Artemisia santonicum essential oil.</a> J Serb Chem Soc. 84 (12), 1355-1365 (2019).</li><li>Su, Y., Qi, Y., Cai, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+sporulation+in+plant+pathogenic+fungi.">Induction of sporulation in plant pathogenic fungi.</a> Mycology. 3, 195-200 (2012).</li><li>Wedge, E., Nagle, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+2D-TLC+bioautography+method+for+the+discovery+of+novel+antifungal+agents+to+control+plant+pathogens.">A new 2D-TLC bioautography method for the discovery of novel antifungal agents to control plant pathogens.</a> J Nat Prod. 63, 1050-1054 (2000).</li></ol>

Dr. Susan Boyetchko passed away prior to the submission of this work (Feb 8, 2023). All other authors have declared to have no conflicts of interest.

TLC-DB is a valuable and well-established NP research tool and a simple and inexpensive alternative to microplate bioassay-guided isolation methods17. It requires minimal time and material resources compared to microplate assays, which require metabolite separation using liquid chromatography techniques. It is a highly versatile assay that can be used to detect antibacterial, antifungal, anti-parasitic, and antioxidant NPs in addition to enzyme inhibitors18,19,20,21,22,23. Though most commonly used for detecting and identifying bioactive phytochemicals, the same method can be applied for bacterial NPs as explored in this protocol18. Additionally, the protocol can be optimized for use with a variety of NP sources and target pathogens to aid in discovering and assessing novel bioactive NPs.
The media used for bacterial growth and solvent used for extraction can greatly impact natural product discovery results. The media used in this protocol is optimized for bacteria that produce cyclic lipopeptides, including Pseudomonas and Bacillus spp. but other media and nutrient sources should be considered if exploring other genera. One may choose to complete TLC-DB using the same microorganism grown in a range of media to evaluate the full breadth of metabolite diversity from one isolate or use identical growth conditions for a wider range of isolates. The choice of extraction solvent also impacts the natural products detected. It is generally understood that most bioactive NPs have low to moderate polarity, making ethyl acetate a suitable choice due to its low boiling point, which makes it easy to remove. However, if one also wishes to examine the polar and non-polar fractions, multiple extractions with other solvents can be carried out. Alternatively, the cell-free extract can be lyophilized and used in the assay to see all metabolites released into the media. However, more material will often need to be loaded onto the TLC plate to account for the dried media components in the lyophilized material. Similarly, if this method is used with a different pathogen, such as inoculum, the media used and incubation conditions must be optimized to obtain the 5 agar plates of mycelium used for the TLC-DB assay.
TLC-DB is advantageous compared to contact and immersion bioautography as it uses the thinnest layer of agar and inoculum, minimizing the reliance on the diffusion of metabolites into the agar layer, which can allow smaller amounts of natural products to induce pathogen inhibition17. Previously published findings using TLC bioautographic methods have used a spore suspension of the pathogen to complete the assay17. Although this does allow for precise control over the suspension concentration, it can be exceedingly difficult and time-consuming to induce the sporulation of certain fungi24. This modification greatly simplifies the assay and allows for the completion of the assay using fungal pathogens that are difficult to sporulate and may have previously been avoided for this method.
The mass of bacterial extract used in the assay can impact results. If too little extract is applied to the TLC plate, it is possible that the minimum inhibitory concentration of an active compound will not be surpassed, and bioactivity will not be detected. As a result, in some cases, and as seen in Figure 1 and Figure 2, it is worthwhile to overload the TLC plate, compromising separation for the ability to easily detect activity. Similarly, the pathogen load sprayed onto the plate must not be too low, as there will not be enough media applied to support pathogen growth. This method can be easily tuned to accommodate a variety of bacterial extracts and pathogens, and the quantities of microbial extract and inoculum outlined in the protocol have ensured bioactivity can be detected for multiple pathogens and microbial extracts. If no zones of inhibition are observed upon the completion of the assay, it may indicate one of the following. First, active metabolites may not be present in the extract applied due to incompatible media being used for bacterial growth or due to the activity of the bacteria not being a result of the NP production. A disc diffusion assay with the extract can be completed to confirm or deny the existence of active NPs in the extract. If the disk diffusion assay shows no pathogen suppression, other media can be tested to determine if other conditions produce bioactive NPs. If the disk diffusion assay does indicate that the extract suppresses the pathogen, then a larger mass of the bacterial extract may need to be applied to the TLC plate, in which case another assay can be attempted.
Comparing metabolites in the ZOI to the crude extract is essential for identifying active NPs. In the assay, metabolites from the crude extract can be metabolized or modified by the pathogen, which can be observed via LC-MS. Thus, only metabolites that occur in both the crude extract and the ZOI can be considered as NPs produced by the bacteria under study. If one cannot correlate the metabolites extracted from the ZOI to metabolites in the crude extract, a TLC plate can be prepared, as described in step 5. Without completing the bioautography assay, extract the metabolites from the TLC plate at the same retention time observed in the completed assay. This should allow for an easier correlation between the metabolites in the ZOIs and crude extract, causing pathogen suppression.
One drawback of TLC-DB is that the resolution of TLC is considerably less than that achieved when using traditional microwell screening techniques, which require liquid chromatography for separation. Thus, it is common for multiple metabolites to exist in the zone of inhibition where some metabolites may not be contributing to the bioactivity. This issue can be further caused by the practice of overloading the TLC plate to observe bioactivity more clearly. Recent work has been published using high-performance TLC (HP-TLC), which greatly improves resolution and can allow for the automation of TLC development that is otherwise impossible when using conventional TLC14,21,22,23. Also, plates can be developed in a second dimension (2D-TLC) to further separate metabolites with similar retention times. That being said, one should evaluate whether the increased time and material cost is a worthwhile compromise for increased resolution obtained from HP- and 2D-TLC25.

Fungal agricultural pathogens cause significant losses in crop quality and yields worldwide, contributing to the economic and supply challenges of a stable global food production system1,2. Pathogen damage can be prevented by breeding cultivars resistant to infection and using integrated crop management systems, including crop rotations and land management practices to suppress pathogen proliferation3,4. Though these methods reduce damage to crops, chemical pesticides are generally used in tandem to actively kill fungal reproductive structures in the field and further prevent damage and yield reductions. Although effective, chemical pesticide usage has many drawbacks, including damage to surrounding ecosystems, a decline in soil fertility, associated human health risks, and the development of pathogen resistance, the latter causing higher doses of pesticides to be required each year5,6,7.
Microbial-based pest and pathogen management products have long been considered potential alternatives or complementary to synthetic pesticides. Since the early 1900s, Bacillus thuringiensis has been widely used to control agricultural pests and pathogens as a seed treatment, as a foliar spray, and in direct soil treatment8. Such products have been named biopesticides and are characterized as naturally occurring microorganisms or biochemicals that can kill, suppress, or reduce the vigor of a target pest or pathogen. Biopesticides can control the growth of a pathogen through various mechanisms but most commonly do so through the secretion of secondary metabolites9. Secondary metabolites, often referred to as natural products, are not involved in primary metabolism but are produced as an evolutionary advantage to outcompete other microorganisms10.
Biopesticides offer many advantages over their synthetic counterparts. They pose a low toxicity risk to the environment, fauna, and humans compared to synthetic pest management products9,10. Since most biopesticides have existed naturally in the environment for millennia, biodegradation pathways for microbial metabolites exist in the environment, limiting the possibility of soil or ecosystem contamination and reducing residence times that contribute to synthetic pesticides being so environmentally destructive11. Additionally, many biopesticides used for mitigating pathogen infection also exhibit plant growth-promoting properties, which can increase nutrient bioavailability and induce plant systemic resistance12.
Most commonly, biopesticides are applied in the form of microbial inoculum, and NPs are secreted by live microorganisms in situ12,13. In such a case, identifying the source of the activity of a biopesticide is of great value. Doing so provides insight into the mechanism of action of the biopesticide, aids with building a case for the protection of a microorganism with a patent, and can have a significant scientific impact if their structures are novel. Most importantly, however, identification of the bioactive source informs formulation possibilities for a downstream biopesticide product. If the NP itself is active, one can use the microorganism as a biomolecule factory for large-scale biopesticide production. Additionally, many NPs that have been explored for biocontrol also have potential applications in human medicine, making them even more economically valuable8.
Thin layer chromatography-direct bioautography (TLC-DB) assays are an inexpensive and straightforward method for the bioactivity-guided isolation and identification of biopesticidal metabolites. Although the technique is commonly used for separating bioactivity testing of phytochemicals from crude plant extracts, it also has great potential for the analysis of microbial extracts14. TLC provides fast and inexpensive separation of NPs in a crude microbial extract, and after coating with a media pathogen suspension, zones that contain active metabolites are easily visualized. Those zones can be scraped from the plate and extracted for chemical analysis by ultra-high-performance liquid chromatography coupled with mass spectrometry (UPLC-MS) to identify known metabolites. Metabolites that do not match previously reported compounds can be isolated in larger quantities via liquid chromatography to undergo structure elucidation studies using techniques such as nuclear magnetic resonance spectroscopy and X-ray crystallography15.
This article describes a model system for discovering and identifying biopesticide NPs using TLC-DB with Bacillus spp. and the agricultural pathogen Sclerotinia sclerotiorum. Figure 1 provides a schematic overview of the TLC-DB procedure.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66967/66967fig01.jpg" /> 
Figure 1: Schematic overview of steps 4-7 of the procedure of TLC-DB. <a href="https://www.jove.com/files/ftp_upload/66967/66967fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table><tbody><tr><td>0.5-5 mL single channel Pipette</td><td>VWR</td><td>CA11020-004</td><td></td></tr><tr><td>10 mL Thin Layer Chromatography Sprayer</td><td>VWR</td><td>KT422530-0010</td><td></td></tr><tr><td>100 x 15 mm Petri plates</td><td>VWR</td><td>89038-970</td><td></td></tr><tr><td>100-1000 &amp;micro;L pipette tips</td><td>VWR</td><td>76322-164</td><td></td></tr><tr><td>100-1000 &amp;micro;L single channel pipette</td><td>VWR</td><td>76169-240</td><td></td></tr><tr><td>15 mL sterile centrifuge tubes</td><td>VWR</td><td>CA21008-918</td><td></td></tr><tr><td>1 L glass bottle</td><td>Millipore Sigma</td><td>CLS13951L</td><td>Must be autoclave safe</td></tr><tr><td>1 mL sterile syringe with needle</td><td>Thomas Scientific</td><td>8935L75</td><td>Detachable needle is recommended</td></tr><tr><td>2 mL Microcentrifuge tube</td><td>VWR</td><td>87003-298</td><td></td></tr><tr><td>50 mL sterile centrifuge tubes</td><td>VWR</td><td>CA21008-940</td><td></td></tr><tr><td>5 mL pipette tips</td><td>VWR</td><td>CA11020-008</td><td></td></tr><tr><td>7 mL scintilation vials</td><td>VWR</td><td>76538-962</td><td></td></tr><tr><td>95% ethanol</td><td>Thermo Fisher Scientific</td><td>A412-500</td><td></td></tr><tr><td>Autoclave</td><td>Cole-Parmer</td><td>UZ-01850-34</td><td>8 L, 115 VAC</td></tr><tr><td>Bacteriological agar</td><td>VWR</td><td>97064-336</td><td></td></tr><tr><td>bin</td><td>Thomas Scientific</td><td>1216H91</td><td></td></tr><tr><td>D-Glucose</td><td>VWR</td><td>BDH9230-500G</td><td></td></tr><tr><td>Dichloromethane &amp;ge;99.8% ACS</td><td>VWR</td><td>BDH1113-4LG</td><td></td></tr><tr><td>Ethyl Acetate &amp;ge;99.8% ACS</td><td>VWR</td><td>BDH1123-4LG</td><td></td></tr><tr><td>Filter paper</td><td>VWR</td><td>CA28297-846</td><td></td></tr><tr><td>Grinding Beads</td><td>VWR</td><td>12621-148</td><td></td></tr><tr><td>Hygromycin B</td><td>VWR</td><td>CA80501-074</td><td></td></tr><tr><td>Iron Sulfate Heptahydrate</td><td>VWR</td><td>97061-542</td><td></td></tr><tr><td>Laminar flow hood</td><td>CleanTech</td><td>1000-6-A</td><td></td></tr><tr><td>LC-MS</td><td>Waters</td><td>LITR10064178</td><td>UPLC/MS/MS TQD system</td></tr><tr><td>Lyophilizer</td><td>Labconco</td><td>700201000</td><td>Temperature collector&amp;nbsp;-50 &amp;deg;C&amp;nbsp;</td></tr><tr><td>Manganese Sulfate Hydrate</td><td>VWR</td><td>CAAA10807-14</td><td></td></tr><tr><td>Methanol &amp;ge;99.8% ACS</td><td>VWR</td><td>BDH2018-5GLP</td><td></td></tr><tr><td>Paper towel</td><td>VWR</td><td>89402-824</td><td></td></tr><tr><td>Potato Dextrose Agar</td><td>VWR</td><td>CA90000-758</td><td></td></tr><tr><td>Potato Dextrose Broth</td><td>VWR</td><td>CA90003-494</td><td></td></tr><tr><td>Potsasium Phosphate Dibasic</td><td>VWR</td><td>470302-246</td><td></td></tr><tr><td>Potsasium Phosphate Monobasic</td><td>VWR</td><td>470302-252</td><td></td></tr><tr><td>Pressure Gauge 6mm Union Straight 0-10 bar (0-145 psi)</td><td>Tameson</td><td>F25U6</td><td></td></tr><tr><td>Pseudomonas F Agar</td><td>VWR</td><td>90003-352</td><td>Also known as Flo Agar</td></tr><tr><td>PTFE Tubing</td><td>Sigma Aldrich</td><td>58697-U</td><td>1/16 inch inner diameter&amp;nbsp;</td></tr><tr><td>Sodium Chloride</td><td>VWR</td><td>BDH9286-500G</td><td></td></tr><tr><td>Spatula</td><td>VWR</td><td>82027-490</td><td></td></tr><tr><td>Sterile Inoculation loops with needle</td><td>VWR</td><td>76534-512</td><td></td></tr><tr><td>Tinfoil</td><td>Thomas Scientific</td><td>1086F24</td><td>Can be purchased from supermarket</td></tr><tr><td>TLC Silica Gel 60 RP-18 F254S 25 Glass Plates 20 X 20 cm</td><td>Thomas Scientific</td><td>1205Q12</td><td></td></tr><tr><td>Vacuum Pump</td><td>Labconco</td><td>1472100</td><td>98 L/min</td></tr><tr><td>Vortex</td><td>VWR</td><td>76549-928</td><td>Must accomadate 15 mL and 50 mL centrifuge tubes</td></tr><tr><td>Whatman in-line HEPA-VENT</td><td>Millipore Sigma</td><td>WHA67235000</td><td>10 filters, 1/4 to 3/8 inch inlet/outlet</td></tr><tr><td></td><td>VWR</td><td>97063-370</td><td></td></tr></tbody></table>

bioassay-guided identification of natural products for biocontrol by thin layer chromatography-direct bioautography

Thin layer chromatography-direct bioautography (TLC-DB) is a well-established bioassay used to separate and identify natural products (NPs) that are antagonistic against a target pathogen. It is a rapid, inexpensive, and simple option for the bioassay-guided isolation and identification of NPs that hinges on separation by TLC coupled with the direct application of a target pathogen to examine bioactivity. It is typically used for the analysis of bioactive plant extracts, detecting inhibitory activity against bacteria, fungi, and enzymes. That being said, it has great potential in bacterial NP discovery, particularly for evaluating bacterial NPs against pertinent agricultural pathogens, which is valuable for discovering and developing novel biopesticides for the agriculture industry. Furthermore, it is a tunable protocol that could be applied to other target pathogens or sources of NPs in research programs concerning the discovery and identification of bioactive compounds. Herein, we describe a model system for discovering and identifying biopesticide NPs using TLC-DB with Bacillus spp. and the agricultural pathogen Sclerotinia sclerotiorum.

Upon the separation of microbial extracts by TLC, metabolites should be dispersed along the TLC plate vertically. Under visible light, it can be difficult to view metabolites that do not absorb in the visible light range. Thus, imaging under UV light can help view the separation of metabolites, as seen in Figure 2A,B. After the incubation period, the pathogen should appear to grow evenly across the entire plate except over the positive controls and the inhibition zones where active metabolites reside, pictured in Figure 2C.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66967/66967fig02v2.jpg" /> 
Figure 2: Separation of microbial extracts by TLC. (A) Developed TLC plate containing nine Bacillus extracts imaged under visible light and (B) under 320 nm UV light prior to the application of fungal inoculum. (C) Completed bioautography assay with pathogen growth observed across the plate except for where growth is inhibited around the positive controls, and ZOI of each extract. <a href="https://www.jove.com/files/ftp_upload/66967/66967fig02largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Metabolites extracted from the zones of inhibition are analyzed by LC-MS and compared to the crude extract to determine the NPs responsible for the antagonism against the pathogen. Matching metabolites should have the same retention time and molecular ion species to be considered a match. Once the active metabolites are identified, the bacterial culture can be grown in bulk to isolate active metabolites of interest for structural chemistry or biological study.

Author Spotlight: Discovering New Biopesticides from Bioactive Soil Microbe-Derived Natural Products

We describe the use of thin-layer chromatography direct bioautography assay and liquid chromatography-mass spectrometry to identify microbial natural products that display antagonism against fungal pathogens using the pathogen Sclerotinia sclerotiorum and biopesticidal Bacillus isolates as a model system.

Bioassay-Guided Identification of Natural Products for Biocontrol by Thin Layer Chromatography-Direct Bioautography

Thin layer chromatography-direct bioautography (TLC-DB) is a well-established bioassay used to separate and identify natural products (NPs) that are ...

bioassay-guided-identification-natural-products-for-biocontrol-thin

Research

JoVE Journal

Biochemistry

1.4K Views.  Agriculture and Agri-Food Canada. In de landbouwsector is het onderzoek naar de ontdekking van alternatieven voor synthetische pesticiden de afgelopen twee decennia aanzienlijk toegenomen. Door de natuurlijke producten te onderzoeken die bioactieve bodemmicroben produceren, stelt ons team zich voor om nieuwe biopesticiden te ontdekken die al in het milieu voorkomen. De laagchromatografie directe biotagrafie of TLCDB is voornamelijk gebruikt om de bioactiviteit van plantenextracten tegen patho...

Video: Auteur in de schijnwerpers: nieuwe biopesticiden ontdekken uit bioactieve bodemmicrobe-afgeleide natuurlijke producten

Thin-layer Chromatographic (TLC) Separations and Bioassays of Plant Extracts to Identify Antimicrobial Compounds

A common screen for plant antimicrobial compounds consists of separating plant extracts by paper or thin-layer chromatography (PC or TLC), exposing the chromatograms to microbial suspensions (e.g. fungi or bacteria in broth or agar), allowing time for the microbes to grow in a humid environment, and visualizing zones with no microbial growth.&#160;The effectiveness of this screening method, known as bioautography, depends on both the quality of the chromatographic separation and the care taken with microbial culture conditions.&#160;This paper describes standard protocols for TLC and contact bioautography&#160;with a novel application to amino acid-fermenting bacteria. The extract is separated on flexible (aluminum-backed) silica TLC plates, and bands are visualized under ultraviolet (UV) light.&#160;Zones are cut out and incubated face down onto agar inoculated with the test microorganism.&#160;Inhibitory bands are visualized by staining the agar plates with tetrazolium red.&#160;The method is applied to the separation of red clover (Trifolium pratense cv. Kenland) phenolic compounds and their screening for activity against Clostridium sticklandii, a hyper ammonia-producing bacterium (HAB) that is native to the bovine rumen.&#160;The TLC methods apply to many types of plant extracts&#160;and other bacterial species (aerobic or anaerobic), as well as fungi, can be used as test organisms if culture conditions are modified to fit the growth requirements of the species.

Thin-layer Chromatographic TLC Separations and Bioassays of Plant Extracts to Identify Antimicrobial Compounds

Methods are described for thin-layer chromatographic (TLC) separation of plant extracts and contact bioautography to identify antibacterial metabolites. The methods are applied to the screening of red clover phenolic compounds inhibiting hyper&#160;ammonia-producing bacteria (HAB) native to the bovine rumen.

A common screen for plant antimicrobial compounds consists of separating plant extracts by paper or thin-layer chromatography (PC or TLC), exposing the ...

Chemistry

Rapid High-throughput Species Identification of Botanical Material Using Direct Analysis in Real Time High Resolution Mass Spectrometry

We demonstrate that direct analysis in real time-high resolution mass spectrometry can be used to produce mass spectral profiles of botanical material, and that these chemical fingerprints can be used for plant species identification. The mass spectral data can be acquired rapidly and in a high throughput manner without the need for sample extraction, derivatization or pH adjustment steps. The use of this technique bypasses challenges presented by more conventional techniques including lengthy chromatography analysis times and resource intensive methods. The high throughput capabilities of the direct analysis in real time-high resolution mass spectrometry protocol, coupled with multivariate statistical analysis processing of the data, provide not only class characterization of plants, but also yield species and varietal information. Here, the technique is demonstrated with two psychoactive plant products, Mitragyna speciosa (Kratom) and Datura (Jimsonweed), which were subjected to direct analysis in real time-high resolution mass spectrometry followed by statistical analysis processing of the mass spectral data. The application of these tools in tandem enabled the plant materials to be rapidly identified at the level of variety and species.

A method for species identification of botanical material by direct analysis in real time-high resolution mass spectrometry and multivariate statistical analysis is presented.

We demonstrate that direct analysis in real time-high resolution mass spectrometry can be used to produce mass spectral profiles of botanical material, ...

A Loop-mediated Isothermal Amplification (LAMP) Assay for Rapid Identification of Bemisia tabaci

The whitefly Bemisia tabaci (Gennadius) is an invasive pest of considerable importance, affecting the production of vegetable and ornamental crops in many countries around the world. Severe yield losses are caused by direct feeding, and even more importantly, also by the transmission of more than 100 harmful plant pathogenic viruses. As for other invasive pests, increased international trade facilitates the dispersal of B. tabaci to areas beyond its native range. Inspections of plant import products at points of entry such as seaports and airports are, therefore, seen as an important prevention measure. However, this last line of defense against pest invasions is only effective if rapid identification methods for suspicious insect specimens are readily available. Because the morphological differentiation between the regulated B. tabaci and close relatives without quarantine status is difficult for non-taxonomists, a rapid molecular identification assay based on the loop-mediated isothermal amplification (LAMP) technology has been developed. This publication reports the detailed protocol of the novel assay describing rapid DNA extraction, set-up of the LAMP reaction, as well as interpretation of its read-out, which allows identifying B. tabaci specimens within one hour. Compared to existing protocols for the detection of specific B. tabaci biotypes, the developed method targets the whole B. tabaci species complex in one assay. Moreover the assay is designed to be applied on-site by plant health inspectors with minimal laboratory training directly at points of entry. Thorough validation performed under laboratory and on-site conditions demonstrates that the reported LAMP assay is a rapid and reliable identification tool, improving the management of B. tabaci.

A Loop-mediated Isothermal Amplification LAMP Assay for Rapid Identification of Bemisia tabaci

This paper reports the protocol for a rapid identification assay for Bemisia tabaci based on loop-mediated isothermal amplification (LAMP) technology. The protocol requires minimal laboratory training and can, therefore, be implemented on-site at points of entry for plant imports such as seaports and airports.

The whitefly Bemisia tabaci (Gennadius) is an invasive pest of considerable importance, affecting the production of vegetable and ornamental crops in many ...

Environment

A Platform of Anti-biofilm Assays Suited to the Exploration of Natural Compound Libraries

Biofilms are regarded as one of the most challenging topics of modern biomedicine, and they are potentially responsible for over 80% of antibiotic-tolerant infections. Biofilms have displayed an exceptionally high tolerance for chemotherapy, which is thought to be multifactorial. For instance, the matrix provides a physical barrier that decreases the penetration of antibiotics into the biofilm. Also, cells within the biofilms are phenotypically diverse. Likely, biofilm resilience arises from a combination of these and other, yet unknown, mechanisms. All of the currently existing antibiotics have been developed against single-cells (planktonic) bacteria. Therefore, so far, a very limited repertoire of molecules exists that can selectively act on mature biofilms. This situation has driven a progressive paradigm shift in drug discovery, in which searching for anti-biofilms has been urged to occupy a more prominent place. An additional challenge is that there are a very limited number of standardized methods for biofilm research, especially those that can be used for large-throughput screening of chemical libraries. Here, an experimental anti-biofilm platform for chemical screening is presented. It uses three assays to measure biofilm viability (with resazurin staining), total biomass (with crystal violet staining), and biofilm matrix (using a wheat germ agglutinin, WGA-fluorescence-based staining of the poly-N-acetyl-glucosamine, PNAG, fraction). All the assays were developed using Staphylococcus aureus as the model bacteria. Examples of how the platform can be used for primary screening as well as for functional characterization of identified anti-biofilm hits are presented. This experimental sequence further allows for the classification of the hits based upon the measured end-points. It also provides information on their mode of action, especially on long-term versus short-term chemotherapeutic effects. Thus, it is very advantageous for the quick identification of high-quality hit compounds that can serve as starting points for various biomedical applications.

Biofilm infections show high tolerance towards chemotherapy. No single assay captures the complexity of biofilms. Instead, complementary assays are needed. We present a screening platform (developed for S. aureus) that combines assays for viability, biomass, and biofilm matrix. It allows anti-biofilm drug discovery, including the assessment of long-term chemotherapeutic effects.

Biofilms are regarded as one of the most challenging topics of modern biomedicine, and they are potentially responsible for over 80% of ...

Immunology and Infection

Classification of Tropane Alkaloids from Plant Metabolites Using LC-MS/MS

Applications of Liquid-Chromatography Tandem Mass Spectrometry in Natural Products Research: Tropane Alkaloids as a Case Study

Although many drugs utilized today are synthetic in origin, natural products still provide a rich source of novel chemical diversity and bioactivity, and can yield promising leads for resistant or emerging diseases. The challenge, however, is twofold: not only must researchers find natural products and elucidate their structures, but they must also identify what is worth isolating and assaying (and what is already known - a process known as dereplication). With the advent of modern analytical instrumentation, the pace of natural product discovery and dereplication has accelerated. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has become an especially valuable technique for identifying and classifying chemical structures. Tropane alkaloids (TAs) are plant-derived compounds of great medicinal and toxicological significance. In this study, we developed an LC-MS/MS-based screening workflow utilizing the multiple MS/MS configurations available on a triple-quadrupole (QQQ) mass spectrometer to annotate and classify TA structures based on their distinct fragmentation patterns. By using a combination of data-dependent (DD) product ion scans, precursor ion scans (PrIS), and neutral loss scans (NLS), we applied this method to TA-rich extracts of the nightshades Datura stramonium and Datura metel. This method is rapid, sensitive, and was successfully employed for both preliminary dereplication of complex TA-containing samples and for the discovery of a novel candidate for isolation, purification (and eventual bioassay).

Author Spotlight: Discovering New Alkaloids in Plants with Advanced Mass Spectrometry Techniques

We present a method for rapid mass spectrometry (MS)/mass spectrometry (MS)-based annotation and classification of tropane alkaloids, useful for both preliminary dereplication of tropane-containing samples and discovery of novel alkaloids for isolation.

Although many drugs utilized today are synthetic in origin, natural products still provide a rich source of novel chemical diversity and bioactivity, and ...

Electroantennographic Bioassay as a Screening Tool for Host Plant Volatiles

Plant volatiles play an important role in plant-insect interactions. Herbivorous insects use plant volatiles, known as kairomones, to locate their host plant.1,2 When a host plant is an important agronomic commodity feeding damage by insect pests can inflict serious economic losses to growers. Accordingly, kairomones can be used as attractants to lure or confuse these insects and, thus, offer an environmentally friendly alternative to pesticides for insect control.3 Unfortunately, plants can emit a vast number volatiles with varying compositions and ratios of emissions dependent upon the phenology of the commodity or the time of day. This makes identification of biologically active components or blends of volatile components an arduous process. To help identify the bioactive components of host plant volatile emissions we employ the laboratory-based screening bioassay electroantennography (EAG). EAG is an effective tool to evaluate and record electrophysiologically the olfactory responses of an insect via their antennal receptors. The EAG screening process can help reduce the number of volatiles tested to identify promising bioactive components. However, EAG bioassays only provide information about activation of receptors. It does not provide information about the type of insect behavior the compound elicits; which could be as an attractant, repellent or other type of behavioral response. Volatiles eliciting a significant response by EAG, relative to an appropriate positive control, are typically taken on to further testing of behavioral responses of the insect pest. The experimental design presented will detail the methodology employed to screen almond-based host plant volatiles4,5 by measurement of the electrophysiological antennal responses of an adult insect pest navel orangeworm (Amyelois transitella) to single components and simple blends of components via EAG bioassay. The method utilizes two excised antennae placed across a "fork" electrode holder. The protocol demonstrated here presents a rapid, high-throughput standardized method for screening volatiles. Each volatile is at a set, constant amount as to standardize the stimulus level and thus allow antennal responses to be indicative of the relative chemoreceptivity. The negative control helps eliminate the electrophysiological response to both residual solvent and mechanical force of the puff. The positive control (in this instance acetophenone) is a single compound that has elicited a consistent response from male and female navel orangeworm (NOW) moth. An additional semiochemical standard that provides consistent response and is used for bioassay studies with the male NOW moth is (Z,Z)-11,13-hexdecadienal, an aldehyde component from the female-produced sex pheromone.6-8

A method to rapidly screen host plant volatiles by measurement of the electrophysiological response of adult navel orangeworm (Amyelois transitella) antennae to single components and blends via electroantennographic analysis is demonstrated.

Plant volatiles play an important role in plant-insect interactions. Herbivorous insects use plant volatiles, known as kairomones, to locate their host ...

Biology

TurboID-Based Proximity Labeling for In Planta Identification of Protein-Protein Interaction Networks

Proximity labeling (PL) techniques using engineered ascorbate peroxidase (APEX) or Escherichia coli biotin ligase BirA (known as BioID) have been successfully used for identification of protein-protein interactions (PPIs) in mammalian cells. However, requirements of toxic hydrogen peroxide (H2O2) in APEX-based PL, longer incubation time with biotin (16&#8211;24 h), and higher incubation temperature (37 &#176;C) in BioID-based PL severely limit their applications in plants. The recently described TurboID-based PL addresses many limitations of BioID and APEX. TurboID allows rapid proximity labeling of proteins in just 10&#8201;min under room temperature (RT) conditions. Although the utility of TurboID has been demonstrated in animal models, we recently showed that TurboID-based PL performs better in plants compared to BioID for labeling of proteins that are proximal to a protein of interest. Provided here is a step-by-step protocol for the identification of protein interaction partners using the N-terminal Toll/interleukin-1 receptor (TIR) domain of the nucleotide-binding leucine-rich repeat (NLR) protein family as a model. The method describes vector construction, agroinfiltration of protein expression constructs, biotin treatment, protein extraction and desalting, quantification, and enrichment of the biotinylated proteins by affinity purification. The protocol described here can be easily adapted to study other proteins of interest in Nicotiana and other plant species.

Described here is a proximity labeling method for identification of interaction partners of the TIR domain of the NLR immune receptor in Nicotiana benthamiana leaf tissue. Also provided is a detailed protocol for the identification of interactions between other proteins of interest using this technique in Nicotiana and other plant species.

Proximity labeling (PL) techniques using engineered ascorbate peroxidase (APEX) or Escherichia coli biotin ligase BirA (known as BioID) have been ...

Field Identification of Matricaria chamomilla using a Portable qPCR System

Quality control in botanical products begins with the raw material supply. Traditionally, botanical identification is performed through morphological assessment and chemical analytical methods. However, the lack of availability of botanists, especially in recent years, coupled with the need to enhance quality control to combat the stresses on the supply chain brought by increasing consumer demand and climate change, necessitates alternative approaches. The goal of this protocol is to facilitate botanical species identification using a portable qPCR system on the field or in any setting, where access to laboratory equipment and expertise is limited. Target DNA is amplified using dye-based qPCR, with DNA extracted from botanical reference materials serving as a positive control. The target DNA is identified by its specific amplification and matching its melting peak against the positive control. A detailed description of the steps and parameters, from hands-on field sample collection, to DNA extraction, PCR amplification, followed by data interpretation, has been included to ensure that readers can replicate this protocol. The results produced align with traditional laboratory botanical identification methods. The protocol is easy to perform and cost-effective, enabling quality testing on raw materials as close to the point of origin of the supply chain as possible.

Field Identification of Matricaria chamomilla using a Portable qPCR System

Presented here is a protocol for field identification of Matricaria chamomilla using a portable qPCR system. This easy-to-perform protocol is ideal as a method to confirm the identity of a botanical species at locations where access to laboratory equipment and expertise is limited, such as farms and warehouses.

Quality control in botanical products begins with the raw material supply. Traditionally, botanical identification is performed through morphological ...

Systematic Approach to Identify Novel Antimicrobial and Antibiofilm Molecules from Plants' Extracts and Fractions to Prevent Dental Caries

Natural products provide structurally different substances, with a myriad of biological activities. However, the identification and isolation of active compounds from plants are challenging because of the complex plant matrix and time-consuming isolation and identification procedures. Therefore, a stepwise approach for screening natural compounds from plants, including the isolation and identification of potentially active molecules, is presented. It includes the collection of the plant material; preparation and fractionation of crude extracts; chromatography and spectrometry (UHPLC-DAD-HRMS and NMR) approaches for analysis and compounds identification; bioassays (antimicrobial and antibiofilm activities; bacterial &#34;adhesion strength&#34; to the salivary pellicle and initial glucan matrix treated with selected treatments); and data analysis. The model is simple, reproducible, and allows high-throughput screening of multiple compounds, concentrations, and treatment steps can be consistently controlled. The data obtained provide the foundation for future studies, including formulations with the most active extracts and/or fractions, isolation of molecules, modeling molecules to specific targets in microbial cells and biofilms. For example, one target to control cariogenic biofilm is to inhibit the activity of Streptococcus mutans glucosyltransferases that synthesize the extracellular matrix&#8217; glucans. The inhibition of those enzymes prevents the biofilm build-up, decreasing its virulence.

Natural products represent promising starting points for the development of new drugs and therapeutic agents. However, due to the high chemical diversity, finding new therapeutic compounds from plants is a challenging and time-consuming task. We describe a simplified approach to identify antimicrobial and antibiofilm molecules from plant extracts and fractions.

Natural products provide structurally different substances, with a myriad of biological activities. However, the identification and isolation of active ...

Performing 1D Thin Layer Chromatography

Source: Laboratory of Dr. Yuri Bolshan &#8212; University of Ontario Institute of Technology
Thin layer chromatography (TLC) is a chromatographic method used to separate mixtures of non-volatile compounds. A TLC plate consists of a thin layer of adsorbent material (the stationary phase) fixed to an appropriate solid support such as plastic, aluminum, or glass1. The sample(s) and reference compound(s) are dissolved in an appropriate solvent and applied near the bottom edge of the TLC plate in small spots. The TLC plate is developed by immersing the bottom edge in the developing solvent consisting of an appropriate mobile phase. Capillary action allows the mobile phase to move up the adsorbent layer.&#160;As the solvent moves up the TLC plate, it carries with it the components of each spot and separates them based on their physical interactions with the mobile and stationary phases.

Thin Layer Chromatography: Principle, Procedure, Applications

Source: Laboratory of Dr. Yuri Bolshan &#8212; University of Ontario Institute of Technology
Thin layer chromatography (TLC) is a chromatographic method ...

Bioassay-Guided Identification of Natural Products for Biocontrol by Thin Layer Chromatography-Direct Bioautography

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