We thank David Meints, University of Arkansas for his assistance in developing and analyzing the transgenic maize during the early generations. This work received the financial support of USDA-ARS CRIS project 6054-42000-025-00D. Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the US Department of Agriculture. USDA-ARS&#8217; Equal Employment Opportunity (EEO) Policy mandates equal opportunity for all persons and prohibits discrimination in all aspects of the Agency&#8217;s personnel policies, practices, and operations.

1. Plasmid constructs and maize transformation
<ol>
	<li>PCR amplify Lablab AILP insert using the primers 5&#8217;-TATCTAGAACTAGTGATTACCATGGCTCC-3&#8217; and 5&#39;-ATACTGCAGGATTGCATGCAGAGTAGTACTG-3&#39;. The PCR conditions include an initial denaturation step at 98 &#176;C for 30 s (step 1), followed by denaturation at 98 &#176;C for 10 s (step 2), annealing at 55 &#176;C for 30 s (step 3), elongation at 72 &#176;C for 20 s (step 4), 31 cycles of step 2 to step 4, and a final elongation step at 72 &#176;C for 5 min. Clone the PCR product into a modified pCAMBIA 1,300 vector using XbaI and PstI restriction sites. Sequence the final plant destination vector to confirm the orientation and sequence of the cloned AILP gene in the vector.</li>
	<li>Transform Agrobacterium strain EHA101 with the final vector construct (Figure 1) as earlier described 16.</li>
	<li>Use transformed Agrobacterium containing the final plant destination vector to transform immature maize (Zea mays L. Hi-II) embryos (performed at the Plant Transformation Facility of Iowa State University) 16.</li>
	<li>Grow T0 plants in the greenhouse (26&#8211;29 &#176;C; 16/8 h photoperiod supplemented with High Pressure Na-lamps) and repeatedly self-pollinate to obtain T6 generation to achieve homozygosity for the transgenic trait.</li>
</ol>
2. Spore germination assay
<ol>
	<li>Harvest leaf samples and store at -80 &#176;C. Grind leaf samples with a mortar and pestle using liquid nitrogen. Weigh out 0.5 g in 2 mL microcentrifuge tubes, add 17 &#181;L of protease inhibitor, and place tubes on ice.</li>
	<li>Centrifuge tubes for 10 min at 16,000 x g. Transfer 225 &#181;L of plant extract to 0.5 mL microcentrifuge tubes and place on ice.</li>
	<li>In a 15 mL centrifuge tube, prepare a 5 mL spore suspension of Aspergillus flavus 70 &#8211; GFP in 1% potato dextrose broth (w/v). Vortex and adjust spore concentration to 105 spores/mL with a haemocytometer. 
	CAUTION: A. flavus produces aflatoxins, all work with this fungus should be conducted in a biological safety cabinet.</li>
	<li>Incubate in PDB at 28 &#176;C until culture achieves 50% initiation of spore germination as indicated by bulging of spores.</li>
	<li>Vortex and aliquot 25 &#181;L spore solution to the 225 &#181;L plant extract. Incubate samples for 20 hours at 28 &#176;C with intermittent shaking.</li>
	<li>Aliquot 25 &#181;L of spore solution on a microscope slide and measure the length of germ tubes spores using the digital camera software. Use a minimum of 20 replicate measurements for each line. 
	NOTE: At this stage, place all cultures at 4 &#176;C to stop colony growth until ready to count.</li>
</ol>
3. Kernel Screening Assay (KSA)
<ol>
	<li>Construct KSA caps by gluing 4 snap caps (22 mm) in a 60 x 15 mm petri dish. Allow glue to dry for 48 H before using caps. Each KSA cap constitutes one rep. (Figure 2).</li>
	<li>In a sterile biological safety cabinet, spray a square bioassay tray (24 cm x 24 cm) with 70% ethanol:H2O (v/v) and let air dry. Add sterile chromatography paper to the tray. Spray 9 KSA caps with 70% ethanol, let air dry, and place in the bioassay tray.</li>
	<li>For each transgenic maize line being tested, select 20 undamaged kernels and place in a 50 mL centrifuge tube. Add 70% ethanol and let sit for 4 minutes. Gently shake tubes during sterilization. Pour off the ethanol and rinse kernels three times in sterile deionized H2O.</li>
	<li>Prepare a spore suspension of Aspergillus flavus 70 &#8211; GFP17 from a 6 day old culture grown on V8 medium (5% V8 Juice, 2% agar, pH 5.2).
	<ol>
		<li>Add 20 mL sterile 0.02% Triton X-100/deionized H2O (v/v) and scrap off spores with a sterile loop. Pipet off the inoculum and place in a 300 mL sterile beaker.</li>
		<li>Prepare a 5-fold dilution with 0.02% Triton X-100, then perform a spore count with a haemocytometer. Dilute again if necessary to obtain 100 mL with a concentration of 4 x 106 spores/mL.</li>
	</ol>
	</li>
	<li>Place kernels in a sterile 300 mL beaker with a stir bar. Add inoculum to the beaker. 
	CAUTION: Adding kernels to inoculum will cause solution to splash. Place beaker on a stir plate for 3 minutes. After 3 minutes, pour off inoculum into an empty beaker.</li>
	<li>Using a forceps, place kernels in the bioassay dish (1 kernel/cap). Add 30 mL deionized water to the bottom of each bioassay tray and incubate at 31 &#176;C in the dark for 7 days.</li>
	<li>Prepare kernels for photography.
	<ol>
		<li>Set aside 4 kernels from each line for microscopic analysis and photography. 
		NOTE: Kernels can be stored at 4 &#176;C for no longer than 5 days before completing photography.</li>
		<li>Take pictures of kernels after seven days of inoculation with A. flavus (Figure 3).</li>
		<li>Clean exterior of kernels with a soft tissue and deionized water. Perform longitudinal sections of kernels and immediately take photographs under the fluorescent microscope.</li>
	</ol>
	</li>
	<li>Prepare kernels for analysis.
	<ol>
		<li>Clean exterior of remaining kernels with a soft tissue and deionized water.</li>
		<li>Place 4 kernels, constituting 1 rep, in a 15 mL screw cap polycarbonate vial containing 2 stainless steel balls. Immediately freeze vials in liquid nitrogen and store at -80 &#176;C until further processing and analysis.</li>
		<li>Remove vials from -80 &#176;C freezer and grind kernels in a homogenizer at 1,500 rpm for 3 minutes. 
		NOTE: Keep kernels frozen with liquid nitrogen.</li>
	</ol>
	</li>
</ol>
4. PCR screening of transgenic maize kernels
<ol>
	<li>Use DNA isolation kit to isolate genomic DNA (gDNA) from pulverized maize kernels infected with A. flavus according to the manufacturer&#8217;s instructions. Briefly, add 280 &#181;L buffer F, 20 &#181;L protease, and 3 &#181;L dithiothreitol (DTT) to 10-15 mg of pulverized maize kernels. Incubate and shake in a thermomixer (56 &#176;C, 1,200 rpm, 30 min). Centrifuge at 10,000 x g for 1 min and transfer the clear supernatant to equilibrated column. Incubate at room temperature for 3 min and centrifuge at 700 x g for 1 min to elute gDNA.</li>
	<li>Take 0.8 &#181;L of extracted gDNA to set up a 20 &#181;L PCR reaction for each sample using a PCR kit. Follow thermocycling conditions as recommended in the manufacturer&#8217;s protocol. The PCR conditions include an initial denaturation step at 98 &#176;C for 5 min (step 1) followed by denaturation at 98 &#176;C for 5 s (step 2), annealing at 55 &#176;C for 5 s (step 3), elongation at 72 &#176;C for 20 s (step 4), 40 cycles of step 2 to step 4, and a final elongation step at 72 &#176;C for 1 min (step 5).</li>
	<li>Use forward primer 5&#8217;-TATACCACCCCCATCCGTGT-3&#8217; and reverse primer 5&#8217;-AGCTCGGAAGCAAAAGACCA-3&#8217; to confirm the presence of the Lablab AILP gene in the transgenic maize kernels.</li>
</ol>
5. RNA isolation, cDNA synthesis, and semi-quantitative RT-PCR
<ol>
	<li>Take homogenized A. flavus infected maize kernels previously stored at -80&#176;C for RNA extraction. Extract RNA using a total RNA isolation kit according to the manufacturer&#8217;s protocol but with slight modification18. After adding 50 mg of homogenized maize kernel powder in the extraction buffer, mix it well (without vortexing) using a 1 mL pipet tip and leave it on ice for 5-6 minutes before proceeding to subsequent steps as described in the manufacturer&#8217;s protocol.</li>
	<li>Prepare cDNA using a cDNA synthesis kit according to the manufacturer&#8217;s protocol 18.</li>
	<li>Use 0.5 &#181;L of undiluted cDNA per PCR reaction for semi-quantitative RT-PCR as earlier described18. Use forward and reverse primers qAILP-F 5&#8217;-TCCCAACAAGGCAACTACTG-3&#8217; and qAILP-R 5&#8217;- CGGTGTCGAAGAGACCTAGATA-3&#8217; respectively to detect Lablab AILP gene expression, and forward and reverse primers qRib-F 5&#697;-GGCTTGGCTTAAAGGAAGGT-3&#697; and qRib-R 5&#697;-TCAGTCCAACTTCCAGAATGG-3&#697; respectively for expression of maize ribosomal structural gene (Rib), GRMZM2G02483819 as a house-keeping gene.</li>
</ol>
6. GFP quantitation
<ol>
	<li>Prepare 50 mL each of 0.2 M monobasic sodium phosphate (NaH2PO4) and 0.2 M dibasic sodium phosphate (Na2HPO4&#183;7H2O) in deionized H2O.</li>
	<li>Make up 100 mL of pH 7.0 Sorenson&#8217;s phosphate buffer. For pH 7.0, add 19.5 mL NaH2PO4 stock, 30.5 mL NaHPO4&#183;7H2O stock, and 50 mL deionized H2O.</li>
	<li>Weigh out 25 mg ground kernel material (fresh weight, FW) and place in a 1.0 mL microcentrifuge tube. Add 500 &#181;L phosphate buffer to the centrifuge tube and vortex for 30 s.</li>
	<li>Centrifuge samples for 15 minutes at 16,000 x g. Pipet 100 &#181;L supernatant into a black 96 well plate. Read the samples with a fluorometer (excitation 485 nm, emission 528 nm).</li>
</ol>
7. Total aflatoxin analysis
<ol>
	<li>Sample processing and aflatoxin extraction
	<ol>
		<li>Dry kernel samples in a forced air oven for 2 days at 60 &#176;C. Weigh samples and place in a 50 mL Erlenmeyer flask with a glass stopper.</li>
		<li>In a fume hood, add 25 mL methylene chloride to each flask. 
		CAUTION: Methylene chloride is highly volatile and is considered hazardous (irritant, carcinogen).&#160;Neither latex nor nitrile gloves are safe for working with Methylene Chloride. Use IMPROVED organic solvent resistant PolyVinylAlcohol gloves.</li>
		<li>Shake samples for 30 min on a wrist action shaker. After 30 min, slowly pour the extract through a fluted filter paper circle into an 80 mL beaker. Let the beakers dry overnight in a fume hood.</li>
		<li>The next day, squirt methylene chloride (approximately 5 mL) around the inside rim of the dry beakers, swirl around, and pour into 2 dram glass vials. Leave vials open to dry in a fume hood overnight.</li>
	</ol>
	</li>
	<li>Aflatoxin analysis
	<ol>
		<li>Add 4.0 mL 80% methanol:deionized H2O (v/v) to vials.</li>
		<li>Use an aflatoxin extraction kit to filter purify methanol solution with the provided column. Analyze total aflatoxin levels with a fluorometer, according to the manufacturer&#8217;s instructions.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Ismaiel, A., Papenbrock, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mycotoxins:+Producing+fungi+and+mechanisms+of+phytotoxicity.">Mycotoxins: Producing fungi and mechanisms of phytotoxicity.</a> Agriculture. 5 (3), 492-537 (2015).</li><li>Mitchell, N., Bowers, E., Hurburgh, C., Wu, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potential+economic+losses+to+the+USA+corn+industry+from+aflatoxin+contamination.">Potential economic losses to the USA corn industry from aflatoxin contamination.</a> Food Additives &amp; Contaminants: Part A. 33 (3), 540-550 (2016).</li><li>Umesha, S., Manukumar, H. M., Chandrasekhar, B., Shivakumara, P., Shiva Kumar, J., Raghava, S., Avinash, P., Shirin, M., Bharathi, T. R., Rajini, S. B., Nandhini, M., Vinaya Rani, G., Shobha, M., Prakash, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aflatoxins+and+food+pathogens:+Impact+of+biologically+active+aflatoxins+and+their+control+strategies.">Aflatoxins and food pathogens: Impact of biologically active aflatoxins and their control strategies.</a> Journal of the Science of Food and Agriculture. , (2016).</li><li>Brown, R. L., Menkir, A., Chen, Z. Y., Bhatnagar, D., Yu, J., Yao, H., Cleveland, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breeding+aflatoxin-resistant+maize+lines+using+recent+advances+in+technologies+-+a+review.">Breeding aflatoxin-resistant maize lines using recent advances in technologies - a review.</a> Food Additives &amp; Contaminants - Part A Chemistry, Analysis, Control, Exposure &amp; Risk Assessment. 30 (8), 1382-1391 (2013).</li><li>Abbas, H., Accinelli, C., Shier, W. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+control+of+aflatoxin+contamination+in+U.S.+crops+and+the+use+of+bioplastic+formulations+of+Aspergillus+flavus+biocontrol+strains+to+optimize+application+strategies.">Biological control of aflatoxin contamination in U.S. crops and the use of bioplastic formulations of Aspergillus flavus biocontrol strains to optimize application strategies.</a> Journal of Agricultural and Food Chemistry. 65, 7081-7087 (2017).</li><li>Udomkun, P., Wiredu, A. N., Nagle, M., M&#252;ller, J., Vanlauwe, B., Bandyopadhyay, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innovative+technologies+to+manage+aflatoxins+in+foods+and+feeds+and+the+profitability+of+application+&#8211;+A+review.">Innovative technologies to manage aflatoxins in foods and feeds and the profitability of application &#8211; A review.</a> Food Control. 76, 127-138 (2017).</li><li>Dohroo, N. P., Bhardwaj, S. S., Shyram, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amylase+and+invertase+activity+as+influenced+by+Pythium+pleroticum+causing+rhizome+rot+of+ginger.">Amylase and invertase activity as influenced by Pythium pleroticum causing rhizome rot of ginger.</a> Plant Disease Research. 2, 106-107 (1987).</li><li>Singh, R., Saxena, V. S., Singh, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pectinolytic,+cellulolytic,+amylase+and+protease+production+by+three+isolates+of+Fusarium+solani+variable+in+their+virulence.">Pectinolytic, cellulolytic, amylase and protease production by three isolates of Fusarium solani variable in their virulence.</a> Indian Journal of Mycology and Plant Pathology. 19, 22-29 (1989).</li><li>Fakhoury, A. M., Woloshuk, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amy1,+the+&#945;-amylase+gene+of+Aspergillus+flavus:+Involvement+in+aflatoxin+biosynthesis+in+maize+kernels.">Amy1, the &#945;-amylase gene of Aspergillus flavus: Involvement in aflatoxin biosynthesis in maize kernels.</a> Phytopathology. 89 (10), 908-914 (1999).</li><li>Bluhm, B. H., Woloshuk, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amylopectin+induces+Fumonisin+B1+production+by+Fusarium+verticillioides+during+colonization+of+maize+kernels.">Amylopectin induces Fumonisin B1 production by Fusarium verticillioides during colonization of maize kernels.</a> Molecular Plant-Microbe Interactions. 18 (12), 1333-1339 (2005).</li><li>Gilbert, M. K., Majumdar, R., Rajasekaran, K., Chen, Z. Y., Wei, Q., Sickler, C. M., Lebar, M. D., Cary, J. W., Frame, B. R., Wang, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+interference-based+silencing+of+the+a-amylase+(amy1)+gene+in+Aspergillus+flavus+decreases+fungal+growth+and+aflatoxin+production+in+maize+kernels.">RNA interference-based silencing of the a-amylase (amy1) gene in Aspergillus flavus decreases fungal growth and aflatoxin production in maize kernels.</a> Planta. 247 (6), 1465-1473 (2018).</li><li>Chen, Z. Y., Brown, R. L., Russin, J. S., Lax, A. R., Cleveland, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+corn+trypsin+inhibitor+with+antifungal+activity+inhibits+Aspergillus+flavus+&#945;-amylase.">A corn trypsin inhibitor with antifungal activity inhibits Aspergillus flavus &#945;-amylase.</a> Phytopathology. 89 (18944733), 902-907 (1999).</li><li>Fakhoury, A. M., Woloshuk, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+growth+of+Aspergillus+flavus+and+fungal+&#945;-amylases+by+a+lectin-like+protein+from+Lablab+purpureus.">Inhibition of growth of Aspergillus flavus and fungal &#945;-amylases by a lectin-like protein from Lablab purpureus.</a> Molecular Plant-Microbe Interactions. 14 (8), 955-961 (2001).</li><li>Mirkov, T. E., Wahlstrom, J. M., Hagiwara, K., Finardi-Filho, F., Kjemtrup, S., Chrispeels, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolutionary+relationships+among+proteins+in+the+phytohemagglutinin-arcelin-a-amylase+inhibitor+family+of+the+common+bean+and+its+relatives.">Evolutionary relationships among proteins in the phytohemagglutinin-arcelin-a-amylase inhibitor family of the common bean and its relatives.</a> Plant Molecular Biology. 26 (4), 1103-1113 (1994).</li><li>Kim, Y. H., Woloshuk, C. P., Cho, E. H., Bae, J. M., Song, Y. S., Huh, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning+and+functional+expression+of+the+gene+encoding+an+inhibitor+against+Aspergillus+flavus+a-amylase,+a+novel+seed+lectin+from+Lablab+purpureus+(Dolichos+lablab).">Cloning and functional expression of the gene encoding an inhibitor against Aspergillus flavus a-amylase, a novel seed lectin from Lablab purpureus (Dolichos lablab).</a> Plant Cell Reports. 26 (4), 395-405 (2007).</li><li>Frame, B., Main, M., Schick, R., Wang, K., Thorpe, T. A., Yeung, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ch.+22.">Ch. 22.</a> Plant Embryo Culture. 710, 327-341 (2011).</li><li>Rajasekaran, K., Sickler, C. M., Brown, R. L., Cary, J. W., Bhatnagar, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+resistance+to+aflatoxin+contamination+in+kernels+of+maize+genotypes+using+a+GFP-expressing+Aspergillus+flavus+strain.">Evaluation of resistance to aflatoxin contamination in kernels of maize genotypes using a GFP-expressing Aspergillus flavus strain.</a> World Mycotoxin Journal. 6 (2), 151-158 (2013).</li><li>Rajasekaran, K., Sayler, R. J., Sickler, C. M., Majumdar, R., Jaynes, J. M., Cary, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+Aspergillus+flavus+growth+and+aflatoxin+production+in+transgenic+maize+kernels+expressing+a+tachyplesin-derived+synthetic+peptide,+AGM182.">Control of Aspergillus flavus growth and aflatoxin production in transgenic maize kernels expressing a tachyplesin-derived synthetic peptide, AGM182.</a> Plant Science. , 150-156 (2018).</li><li>Shu, X., Livingston, D. P., Franks, R. G., Boston, R. S., Woloshuk, C. P., Payne, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-specific+gene+expression+in+maize+seeds+during+colonization+by+Aspergillus+flavus+and+Fusarium+verticillioides.">Tissue-specific gene expression in maize seeds during colonization by Aspergillus flavus and Fusarium verticillioides.</a> Molecular Plant Pathology. 16 (4), 662-674 (2015).</li><li>Savary, S., Ficke, A., Aubertot, J. -. N., Hollier, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crop+losses+due+to+diseases+and+their+implications+for+global+food+production+losses+and+food+security.">Crop losses due to diseases and their implications for global food production losses and food security.</a> Food Security. 4, 519-537 (2012).</li><li>Damalas, C. A., Eleftherohorinos, I. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pesticide+exposure,+safety+issues,+and+risk+assessment+indicators.">Pesticide exposure, safety issues, and risk assessment indicators.</a> International Journal of Environmental Research and Public Health. 8 (5), 1402-1419 (2011).</li><li>Kowalska, A., Walkiewicz, K., Kozie&#322;, P., Muc-Wierzgo&#324;, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aflatoxins:+Characterisitcs+and+impact+on+human+health.">Aflatoxins: Characterisitcs and impact on human health.</a> Post&#281;py Higieny i Medycyny Do&#347;wiadczalnej (Online). 71, 315-327 (2017).</li><li>Rajasekaran, K., Cary, J. W., Cotty, P. J., Cleveland, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+GFP-expressing+Aspergillus+flavus+strain+to+study+fungal+invasion,+colonization,+and+resistance+in+cottonseed.">Development of a GFP-expressing Aspergillus flavus strain to study fungal invasion, colonization, and resistance in cottonseed.</a> Mycopathologia. 165 (2), 89-97 (2008).</li><li>Punt, P., Dingemanse, M. A., Kuyvenhoven, A., Soede, R. D., Pouwels, P. H., van den Hondel, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+elements+in+the+promoter+region+of+the+Aspergillus+nidulans+gpdA+gene+encoding+glyceraldehyde-3-phosphate+dehydrogenase.">Functional elements in the promoter region of the Aspergillus nidulans gpdA gene encoding glyceraldehyde-3-phosphate dehydrogenase.</a> Gene. 93 (1), 101-109 (1990).</li><li>Lee, L. W., Chiou, C. H., Klomparens, K. L., Cary, J. W., Linz, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subcellular+localization+of+aflatoxin+biosynthetic+enzymes+Nor-1,+Ver-1,+and+OmtA+in+time-dependent+fractionated+colonies+of+Aspergillus+parasiticus.">Subcellular localization of aflatoxin biosynthetic enzymes Nor-1, Ver-1, and OmtA in time-dependent fractionated colonies of Aspergillus parasiticus.</a> Archives of Microbiology. 181 (3), 204-214 (2004).</li><li>Bhatnagar, D., Cary, J. W., Ehrlich, K., Yu, J., Cleveland, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+the+genetics+of+regulation+of+aflatoxin+production+and+Aspergillus+flavus+development.">Understanding the genetics of regulation of aflatoxin production and Aspergillus flavus development.</a> Mycopathologia. 162, 155-166 (2006).</li><li>Williams, W. P., Krakowsky, M. D., Scully, B. T., Brown, R. L., Menkir, A., Warburton, M. L., Windham, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identifying+and+developing+maize+germplasm+with+resistance+to+accumulation+of+aflatoxins.">Identifying and developing maize germplasm with resistance to accumulation of aflatoxins.</a> World Mycotoxin Journal. 8 (2), 193-209 (2015).</li><li>Broekaert, W. F., van Parijs, J., Leyns, F., Joos, H., Peumans, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+chitin-binding+lectin+from+stinging+nettle+rhizomes+with+antifungal+properties.">A chitin-binding lectin from stinging nettle rhizomes with antifungal properties.</a> Science. 245 (4922), 1100-1102 (1989).</li><li>Vanparijs, J., Broekaert, W. F., Goldstein, I. J., Peumans, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hevein-an+antifungal+protein+from+rubber-tree+(Hevea+brasiliensis)+latex.">Hevein-an antifungal protein from rubber-tree (Hevea brasiliensis) latex.</a> Planta. 183, 258-264 (1991).</li><li>Gozia, O., Ciopraga, J., Bentia, T., Lungu, M., Zamfirescu, I., Tudor, R., Roseanu, A., Nitu, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antifungal+properties+of+lectin+and+new+chitinases+from+potato+tubers.">Antifungal properties of lectin and new chitinases from potato tubers.</a> Comptes Rendus de l'Academie des Sciences - Series III. 316 (8), 788-792 (1993).</li><li>Wisessing, A., Choowongkomon, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amylase+inhibitors+in+plants:+Structures,+Functions+and+Applications.">Amylase inhibitors in plants: Structures, Functions and Applications.</a> Functional Plant Science and Biotechnology. 6 (1), 31-41 (2012).</li><li>Tyagi, B., Trivedi, N., Dubey, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=a-amylase+inhibitor:+A+compelling+plant+defense+mechanism+against+insect/pests.">a-amylase inhibitor: A compelling plant defense mechanism against insect/pests.</a> Environment &amp; Ecology. 32 (3), 995-999 (2014).</li><li>Powers, J. R., Culbertson, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+effect+of+bean+amylase+inhibitor+on+insect+amylases.">In vitro effect of bean amylase inhibitor on insect amylases.</a> Journal of Food Protection. 45, 655-657 (1982).</li><li>Gatehouse, A. M. R., Fenton, K. A., Jepson, I., Pavey, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+a-amylase+inhibitors+on+insect+storage+pests:+Inhibition+of+a-amylase+in+vitro+and+effects+on+development+in+vivo.">The effects of a-amylase inhibitors on insect storage pests: Inhibition of a-amylase in vitro and effects on development in vivo.</a> Journal of the Science of Food and Agriculture. 37, 727-734 (1986).</li><li>Blanco-Labra, A., Chagolla-Lopez, A., Martinez-Gallardo, N., Valdes-Rodriguez, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Further+characterization+of+the+12-kDa+protease+a-amylase+inhibitor+present+in+maize+seeds.">Further characterization of the 12-kDa protease a-amylase inhibitor present in maize seeds.</a> Journal of Food Biochemistry. 19, 27-41 (1995).</li><li>Abdollahi, A., Buchanan, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+aflatoxin+biosynthesis:+Induction+of+aflatoxin+production+by+various+carbohydrates.">Regulation of aflatoxin biosynthesis: Induction of aflatoxin production by various carbohydrates.</a> Journal of Food Science. 46, 633-635 (1981).</li><li>Liu, J., Sun, L., Zhang, N., Zhang, J., Guo, J., Li, C., Rajput, S. A., Qi, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+nutrients+in+substrates+of+different+grains+on+aflatoxin+B1+production+by+Aspergillus+flavus.">Effects of nutrients in substrates of different grains on aflatoxin B1 production by Aspergillus flavus.</a> BioMed Research International. 2016, (2016).</li><li>Uppala, S. S., Bowen, K. L., Woods, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pre-harvest+aflatoxin+contamination+and+soluble+sugars+of+peanut.">Pre-harvest aflatoxin contamination and soluble sugars of peanut.</a> Peanut Science. 40 (1), 40-51 (2013).</li></ol>

The authors have no conflict of interest.

Yield losses in agricultural crops due to pathogens and pests is a global problem20. Currently, application of synthetic fungicides and pesticides is the predominant means for controlling plant pathogens and pests, but residual toxicity of these biochemicals in food and feed can pose serious threat to human and animal health21. Considering the economic importance of maize as a food and feed crop, reduction in or elimination of aflatoxin contamination is of utmost importance...

Mycotoxin contamination by the fungal genera, Aspergillus, Fusarium, Penicillium, and Alternaria is a major problem of food and feed crops grown worldwide1,2,3. Among these phytopathogenic fungi, Aspergillus has the highest adverse impact on crop value and human and animal health. Aspergillus flavus (A. flavus) is an opportunistic plant pathogen that infects oil rich crops such as maize, cottonseed and peanut and produces the potent carcinogens, aflatoxins, as well as numerous toxic secondary metabolites (SMs). Maize is an important food and feed crop grown worldwide and is highly susceptible to contamination by A. flavus. The economic impact of aflatoxin contamination on loses and reduced value in maize can be as much as $686.6 million/year in the U.S.2 With predicted changes in global climate, the impact of aflatoxins could result in greater economic losses in maize with estimates as high as $1.68 billion/year in the near future2. Given the adverse economic and health effects of aflatoxins in humans and livestock, pre-harvest aflatoxin control in maize might be the most efficient way to prevent aflatoxin contamination in food and feed products.
The major pre-harvest control approach for aflatoxin resistance in maize that has been used extensively in the last few decades is primarily through breeding, which requires a significant amount of time4. Recently, biocontrol has had some success in aflatoxin reduction in large scale field applications5,6. Besides biocontrol, application of cutting-edge molecular tools such as &#8216;Host Induced Gene Silencing&#8217; (HIGS) through RNAi and transgenic expression of resistance-associated proteins has had some success in reduction of A. flavus growth and aflatoxin production in small scale laboratory and field studies. These approaches are currently being optimized in addition to identifying new potential A. flavus gene targets for future manipulation.
Besides genes that are directly involved in mycotoxin production as potential targets of transgenic control strategies, fungal amylases have been shown to play a critical role in maintaining successful pathogenesis and mycotoxin production during early stages of host plant infection. A few examples include Pythium pleroticum (causal agent of ginger rhizome rot), Fusarium solani (causal agent of cauliflower wilt), where positive correlations between pathogenicity and &#945;-amylase expression and activity were observed7,8. Inhibition of &#945;-amylase activity either through gene knockout or knockdown approaches negatively affects fungal growth and toxin production. An &#945;-amylase knockout mutant of A. flavus was unable to produce aflatoxins when grown on starch substrate or degermed maize kernels9. Similarly, in Fusarium verticillioides an &#945;-amylase knockout strain failed to produce fumonisin B1 (mycotoxin) during infection of maize kernels10. In a more recent study, Gilbert et al. (2018) demonstrated that an RNAi-based knock down of A. flavus &#945;-amylase expression through HIGS significantly reduced A. flavus growth and aflatoxin production during maize kernel infection11.
Specific inhibitors of &#945;-amylase activity have also produced similar results as obtained from down-regulation of &#945;-amylase expression. The first report on the role of an &#945;-amylase inhibitor in fungal resistance came from the isolation and characterization of a 14-kDa trypsin-&#945;-amylase inhibitor from maize lines resistant to A. flavus12. Further screening of several hundreds of plant species by Fakhoury and Woloshuk led to the identification of a 36-kDa &#945;-amylase inhibitor-like protein (AILP) from the seeds of hyacinth beans, Lablab purpureus L.13. The peptide sequence of AILP resembled lectins belonging to the lectin&#8211;arcelin&#8211;&#945;-amylase inhibitor family reported in common bean14,15. Purified AILP does not exhibit any inhibitory activity towards mammalian trypsin and further in vitro characterization showed significant inhibition of A. flavus growth and conidial germination13. The reports presented here clearly shows &#945;-amylase can serve as a target in control approaches to restrict pathogens or pests that depend on starch mobilization (through &#945;-amylase activity) and acquisition of soluble sugars as an energy source during their pathogenic interaction with host plants.
Alpha-amylase is known to be critical in A. flavus pathogenicity9,10,11, and given the importance of AILP as a potent anti-A. flavus agent (&#945;-amylase inhibition/antigrowth)13, we generated transgenic maize plants expressing Lablab AILP gene under the constitutive CaMV 35S promoter. The goal was to investigate if heterologous expression of this &#945;-amylase inhibitor in maize is effective against A. flavus pathogenesis and aflatoxin production during maize kernel infection. Our results demonstrate that transgenic maize kernels expressing AILP significantly reduced A. flavus growth and aflatoxin production during kernel infection.

<table>
	<tbody>
		<tr>
			<td>Agar</td>
			<td>Caisson</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Amazing Marine Goop</td>
			<td>Eclectic Products</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>C1000 Touch CFX96 Real-Time System</td>
			<td>Bio-Rad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Corning&#160;Falcon Tissue Culture Dishes, 60 mm</td>
			<td>Fisher Scientific</td>
			<td>08-772F</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf&#160;5424 Microcentrifuge</td>
			<td>Fisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Erlenmeyer flask with stopper, 50 mL</td>
			<td>Ace Glass</td>
			<td>6999-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FluoroQuant Afla</td>
			<td>Romer Labs</td>
			<td>COKFA1010</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluted Qualitative Filter Paper Circles, 15 cm</td>
			<td>Fisher Scientific</td>
			<td>09-790-14E</td>
			<td></td>
		</tr>
		<tr>
			<td>Force Air Oven</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FQ-Reader</td>
			<td>Romer Labs</td>
			<td>EQFFM3010</td>
			<td></td>
		</tr>
		<tr>
			<td>Geno/Grinder 2010</td>
			<td>OPS Diagnostics</td>
			<td>SP 2010-115</td>
			<td></td>
		</tr>
		<tr>
			<td>Innova 44 Incubator Shaker</td>
			<td>Brunswick Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>iScript cDNA Synthesis Kit</td>
			<td>Bio-Rad</td>
			<td>1708890</td>
			<td></td>
		</tr>
		<tr>
			<td>liquid Nitrogen</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Low Form Griffin Beakers, 100 mL</td>
			<td>DKW Life Sciences</td>
			<td>14000-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Methylene Chloride</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nexttec 1-step DNA Isolation Kit for Plants</td>
			<td>Nexttec</td>
			<td>47N</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon Eclipse E600 microscope with Nikon DS-Qi1 camera</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon SMZ25 stereomicroscope with C-HGFI Episcopic Illuminator and Andor Zyla 4.2 sCMOS camera</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nunc Square BioAssay Dishes</td>
			<td>ThermoFisher Scientific</td>
			<td>240835</td>
			<td></td>
		</tr>
		<tr>
			<td>Phire Plant Direct PCR Kit</td>
			<td>ThermoFisher Scientific</td>
			<td>F130WH</td>
			<td></td>
		</tr>
		<tr>
			<td>Polycarbonate Vials, 15 ml</td>
			<td>OPS Diagnostics</td>
			<td>PCRV 15-100-23</td>
			<td></td>
		</tr>
		<tr>
			<td>Potato Dextrose Broth</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Snap Cap, 22 mm</td>
			<td>DKW Life Sciences</td>
			<td>242612</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Phosphate dibasic heptahydrate</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Phosphate monobasic</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrum Plant Total RNA Kit</td>
			<td>Sigma-Aldrich</td>
			<td>STRN50</td>
			<td></td>
		</tr>
		<tr>
			<td>Stainless Steel Grinding Balls, 3/8&#39;&#39;</td>
			<td>OPS Diagnostics</td>
			<td>GBSS 375-1000-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Stir Plate</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Synergy 4 Fluorometer</td>
			<td>Biotek</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>T100 Thermal Cycler</td>
			<td>Bio-Rad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>T-9284</td>
			<td></td>
		</tr>
		<tr>
			<td>V8 juice</td>
			<td>Campbell&#39;s</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Whatman Qualitative Grade Plain Sheets, Grade 3</td>
			<td>Fisher Scientific</td>
			<td>09-820P</td>
			<td></td>
		</tr>
		<tr>
			<td>Wrist-Action Shaker</td>
			<td>Burrell Scientific</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

inhibition of aspergillus flavus growth and aflatoxin production in transgenic maize expressing the &#945;-amylase inhibitor from lablab purpureus l.

Aflatoxin contamination in food and feed crops is a major challenge worldwide. Aflatoxins, produced by the fungus Aspergillus flavus (A. flavus) are potent carcinogens that substantially reduce crop value in maize and other oil rich crops like peanut besides posing serious threat to human and animal health. Different approaches, including traditional breeding, transgenic expression of resistance associated proteins, and RNA interference (RNAi)-based host-induced gene silencing of critical A. flavus gene targets, are being evaluated to increase aflatoxin resistance in susceptible crops. Past studies have shown an important role of &#945;-amylase in A. flavus pathogenesis and aflatoxin production, suggesting this gene/enzyme is a potential target to reduce both A. flavus growth and aflatoxin production. In this regard, the current study was undertaken to evaluate heterologous expression (under control of the constitutive CaMV 35S promoter) of a Lablab purpureus L. &#945;-amylase inhibitor-like protein (AILP) in maize against A. flavus. AILP is a 36-kDa protein, which is a competitive inhibitor of A. flavus &#945;-amylase enzyme and belongs to the lectin&#8211;arcelin&#8211;&#945;-amylase inhibitor protein family in common bean. In vitro studies prior to the current work had demonstrated the role of AILP in inhibition of A. flavus &#945;-amylase activity and fungal growth. Fungal growth and aflatoxin production in mature kernels were monitored in real time using a GFP-expressing A. flavus strain. This kernel screening assay (KSA) is very simple to set up and provides reliable and reproducible data on infection and the extent of spread that could be quantified for evaluation of germplasm and transgenic lines. The fluorescence from the GFP strain is closely correlated to fungal growth and, by extension, it is well-correlated to aflatoxin values.&#160; The goal of the current work was to implement this previous knowledge in a commercially important crop like maize to increase aflatoxin resistance. Our results show a 35%&#8211;72% reduction in A. flavus growth in AILP-expressing transgenic maize kernels which, in turn, translated into a 62%&#8211;88% reduction in aflatoxin levels.

Maize transformation and molecular screening of transgenic plants
Immature embryos of maize Hi-II lines were transformed using Agrobacterium tumefaciens EHA101 strain containing the final plant destination vector expressing the Lablab purpureus AILP gene under the control of CaMV 35S promoter. Five independently transformed maize lines were advanced to the T6 generation for su...

Watch this Scientific Journal Video about Inhibition of Aspergillus flavus Growth and Aflatoxin Production in Transgenic Maize Expressing the Î±-amylase Inhibitor from Lablab purpureus L. at JoVE.co

Inhibition of Aspergillus flavus Growth and Aflatoxin Production in Transgenic Maize Expressing the &#945;-amylase Inhibitor from Lablab purpureus L.

Here we present a protocol to analyze Aspergillus flavus growth and aflatoxin production in maize kernels expressing an antifungal protein.&#160; Using a GFP-expressing A. flavus strain we monitored the infection and spread of the fungus in mature kernels in real time. The assay is rapid, reliable, and reproducible.

Inhibition of Aspergillus flavus Growth and Aflatoxin Production in Transgenic Maize Expressing the &#945;-amylase Inhibitor from Lablab purpureus L.

Aflatoxin contamination in food and feed crops is a major challenge worldwide. Aflatoxins, produced by the fungus Aspergillus flavus (A. flavus) are ...

inhibition-aspergillus-flavus-growth-aflatoxin-production-transgenic

Research

JoVE Journal

Environment

10.4K Views.  USDA-ARS, Food and Feed Safety Research Unit. Here we present a protocol to analyze Aspergillus flavus growth and aflatoxin production in maize kernels expressing an antifungal protein.  Using a GFP-expressing A. flavus strain we monitored the infection and spread of the fungus in mature kernels in real time. The assay is rapid, reliable, and reproducible.

Video: Inhibition of Aspergillus flavus Growth and Aflatoxin Production in Transgenic Maize Expressing the α-amylase Inhibitor from Lablab purpureus L.

Mass Production of Genetically Modified Aedes aegypti for Field Releases in Brazil

New techniques and methods are being sought to try to win the battle against mosquitoes. Recent advances in molecular techniques have led to the development of new and innovative methods of mosquito control based around the Sterile Insect Technique (SIT)1-3. A control method known as RIDL&#160;(Release of Insects carrying a Dominant Lethal)4, is based around SIT, but uses genetic methods to remove the need for radiation-sterilization5-8. A RIDL&#160;strain of Ae. aegypti was successfully tested in the field in Grand Cayman9,10; further field use is planned or in progress in other countries around the world.
Mass rearing of insects has been established in several insect species and to levels of billions a week. However, in mosquitoes, rearing has generally been performed on a much smaller scale, with most large scale rearing being performed in the 1970s and 80s. For a RIDL&#160;program it is desirable to release as few females as possible as they bite and transmit disease. In a mass rearing program&#160;there are several stages to produce the males to be released: egg production, rearing eggs until pupation, and then sorting males from females before release. These males are then used for a RIDL&#160;control program,&#160;released as either pupae or adults11,12.
To suppress a mosquito population using RIDL&#160;a large number of high quality male adults need to be reared13,14. The following describes the methods for the mass rearing of OX513A, a RIDL&#160;strain of Ae. aegypti 8, for release and covers the techniques required for the production of eggs and mass rearing RIDL&#160;males for a control program.

Mass Production of Genetically Modified Aedes aegypti for Field Releases in Brazil

To achieve population suppression of Aedes aegypti using the RIDL&#174; (Release of Insects carrying a Dominant Lethal) system, large numbers of male mosquitoes need to be released. This requires the use of mass rearing techniques and technology to provide reliable systems to obtain the maximum number of high quality male mosquitoes.

New techniques and methods are being sought to try to win the battle against mosquitoes. Recent advances in molecular techniques have led to the ...

Determination of Microbial Extracellular Enzyme Activity in Waters, Soils, and Sediments using High Throughput Microplate Assays

Much of the nutrient cycling and carbon processing in natural environments occurs through the activity of extracellular enzymes released by microorganisms. Thus, measurement of the activity of these extracellular enzymes can give insights into the rates of ecosystem level processes, such as organic matter decomposition or nitrogen and phosphorus mineralization. Assays of extracellular enzyme activity in environmental samples typically involve exposing the samples to artificial colorimetric or fluorometric substrates and tracking the rate of substrate hydrolysis. Here we describe microplate based methods for these procedures that allow the analysis of large numbers of samples within a short time frame. Samples are allowed to react with artificial substrates within 96-well microplates or deep well microplate blocks, and enzyme activity is subsequently determined by absorption or fluorescence of the resulting end product using a typical microplate reader or fluorometer. Such high throughput procedures not only facilitate comparisons between spatially separate sites or ecosystems, but also substantially reduce the cost of such assays by reducing overall reagent volumes needed per sample.

Microplate based procedures are described for the colorimetric or fluorometric analysis of extracellular enzyme activity. These procedures allow for the rapid assay of such activity in large numbers of environmental samples within a manageable time frame.

Much of the nutrient cycling and carbon  processing in natural environments occurs through the activity of extracellular  enzymes released by ...

A Rapid and Efficient Method for Assessing Pathogenicity of Ustilago maydis on Maize and Teosinte Lines

Maize is a major cereal crop worldwide. However, susceptibility to biotrophic pathogens is the primary constraint to increasing productivity. U. maydis is a biotrophic fungal pathogen and the causal agent of corn smut on maize. This disease is responsible for significant yield losses of approximately $1.0 billion annually in the U.S.1 Several methods including crop rotation, fungicide application and seed treatments are currently used to control corn smut2. However, host resistance is the only practical method for managing corn smut. Identification of crop plants including maize, wheat, and rice that are resistant to various biotrophic pathogens has significantly decreased yield losses annually3-5. Therefore, the use of a pathogen inoculation method that efficiently and reproducibly delivers the pathogen in between the plant leaves, would facilitate the rapid identification of maize lines that are resistant to U. maydis. As, a first step toward indentifying maize lines that are resistant to U. maydis, a needle injection inoculation method and a resistance reaction screening method was utilized to inoculate maize, teosinte, and maize x teosinte introgression lines with a U. maydis strain and to select resistant plants.
Maize, teosinte and maize x teosinte introgression lines, consisting of about 700 plants, were planted, inoculated with a strain of U. maydis, and screened for resistance. The inoculation and screening methods successfully identified three teosinte lines resistant to U. maydis. Here a detailed needle injection inoculation and resistance reaction screening protocol for maize, teosinte, and maize x teosinte introgression lines is presented. This study demonstrates that needle injection inoculation is an invaluable tool in agriculture that can efficiently deliver U. maydis in between the plant leaves and has provided plant lines that are resistant to U. maydis that can now be combined and tested in breeding programs for improved disease resistance.

A Rapid and Efficient Method for Assessing Pathogenicity of Ustilago maydis on Maize and Teosinte Lines

The use of a needle injection method to inoculate maize and teosinte plants with the biotrophic pathogen Ustilago maydis is described. The needle injection inoculation method facilitates the controlled delivery of the fungal pathogen in between the plant leaves where the pathogen enters the plant through the formation of appresoria. This method is highly efficient, enabling reproducible inoculations with U. maydis.

Maize is a major cereal crop worldwide. However, susceptibility to biotrophic pathogens is the primary constraint to increasing productivity. U. maydis is ...

Optimize Flue Gas Settings to Promote Microalgae Growth in Photobioreactors via Computer Simulations

Flue gas from power plants can promote algal cultivation and reduce greenhouse gas emissions1. Microalgae not only capture solar energy more efficiently than plants3, but also synthesize advanced biofuels2-4. Generally, atmospheric CO2 is not a sufficient source for supporting maximal algal growth5. On the other hand, the high concentrations of CO2 in industrial exhaust gases have adverse effects on algal physiology. Consequently, both cultivation conditions (such as nutrients and light) and the control of the flue gas flow into the photo-bioreactors are important to develop an efficient &ldquo;flue gas to algae&rdquo; system. Researchers have proposed different photobioreactor configurations4,6 and cultivation strategies7,8 with flue gas. Here, we present a protocol that demonstrates how to use models to predict the microalgal growth in response to flue gas settings. We perform both experimental illustration and model simulations to determine the favorable conditions for algal growth with flue gas. We develop a Monod-based model coupled with mass transfer and light intensity equations to simulate the microalgal growth in a homogenous photo-bioreactor. The model simulation compares algal growth and flue gas consumptions under different flue-gas settings. The model illustrates: 1) how algal growth is influenced by different volumetric mass transfer coefficients of CO2; 2) how we can find optimal CO2 concentration for algal growth via the dynamic optimization approach (DOA); 3) how we can design a rectangular on-off flue gas pulse to promote algal biomass growth and to reduce the usage of flue gas. On the experimental side, we present a protocol for growing Chlorella under the flue gas (generated by natural gas combustion). The experimental results qualitatively validate the model predictions that the high frequency flue gas pulses can significantly improve algal cultivation.

Flue gas from power plants is a cheap CO2 source for algal growth. We have built prototype &#34;flue gas to algal cultivation&#34; systems and described how to scale up the algal cultivation process. We have demonstrated the use of a mass-transfer bio-reaction model to simulate and to design the optimal operation of flue gas for the growth of Chlorella sp. in algal photo-bioreactors.

Flue gas from power plants can promote algal cultivation and reduce greenhouse gas emissions1. Microalgae not only capture solar energy more efficiently ...

Methods for Facilitating Microbial Growth on Pulp Mill Waste Streams and Characterization of the Biodegradation Potential of Cultured Microbes

The kraft process is applied to wood chips for separation of lignin from the polysaccharides within lignocellulose for pulp that will produce a high quality paper. Black liquor is a pulping waste generated by the kraft process that has potential for downstream bioconversion. However, the recalcitrant nature of the lignocellulose resources, its chemical derivatives that constitute the majority of available organic carbon within black liquor, and its basic pH present challenges to microbial biodegradation of this waste material. Methods for the collection and modification of black liquor for microbial growth are aimed at utilization of this pulp waste to convert the lignin, organic acids, and polysaccharide degradation byproducts into valuable chemicals. The lignocellulose extraction techniques presented provide a reproducible method for preparation of lignocellulose growth substrates for understanding metabolic capacities of cultured microorganisms. Use of gas chromatography-mass spectrometry enables the identification and quantification of the fermentation products resulting from the growth of microorganisms on pulping waste. These methods when used together can facilitate the determination of the metabolic activity of microorganisms with potential to produce fermentation products that would provide greater value to the pulping system and reduce effluent waste, thereby increasing potential paper milling profits and offering additional uses for black liquor.

Industrial wastes can be collected and modified to analyze microbial growth. Lignocellulose extraction techniques provide components to analyze specific biodegradation ability. Gas chromatography-mass spectrometry identifies fermentation products of microorganisms grown on pulping waste. These methods determine the metabolic capacity of microorganisms to degrade pulping waste.

The kraft process is applied to wood chips for separation of lignin from the polysaccharides within lignocellulose for pulp that will produce a high ...

RNAi-mediated Control of Aflatoxins in Peanut: Method to Analyze Mycotoxin Production and Transgene Expression in the Peanut/Aspergillus Pathosystem

The Food and Agriculture Organization of the United Nations estimates that 25% of the food crops in the world are contaminated with aflatoxins. That represents 100 million tons of food being destroyed or diverted to non-human consumption each year. Aflatoxins are powerful carcinogens normally accumulated by the fungi Aspergillus flavus and A. parasiticus in cereals, nuts, root crops and other agricultural products. Silencing of five aflatoxin-synthesis genes by RNA interference (RNAi) in peanut plants was used to control aflatoxin accumulation following inoculation with A. flavus. Previously, no method existed to analyze the effectiveness of RNAi in individual peanut transgenic events, as these usually produce few seeds, and traditional methods of large field experiments under aflatoxin-conducive conditions were not an option. In the field, the probability of finding naturally contaminated seeds is often 1/100 to 1/1,000. In addition, aflatoxin contamination is not uniformly distributed. Our method uses few seeds per transgenic event, with small pieces processed for real-time PCR (RT-PCR) or small RNA sequencing, and for analysis of aflatoxin accumulation by ultra-performance liquid chromatography (UPLC). RNAi-expressing peanut lines 288-72 and 288-74, showed up to 100% reduction (p&#8804;0.01) in aflatoxin B1 and B2 compared to the control that accumulated up to 14,000 ng.g-1 of aflatoxin B1 when inoculated with aflatoxigenic A. flavus. As reference, the maximum total of aflatoxins allowable for human consumption in the United States is 20 ng.g-1. This protocol describes the application of RNAi-mediated control of aflatoxins in transgenic peanut seeds and methods for its evaluation. We believe that its application in breeding of peanut and other crops will bring rapid advancement in this important area of science, medicine and human nutrition, and will significantly contribute to the international effort to control aflatoxins, and potentially other mycotoxins in major food crops.

RNAi-mediated Control of Aflatoxins in Peanut: Method to Analyze Mycotoxin Production and Transgene Expression in the Peanut/Aspergillus Pathosystem

We demonstrate a method for the analysis of aflatoxins and transgene expression in peanut seeds that contain RNA-interference signals for silencing aflatoxin-synthesis genes in the fungus Aspergillus flavus. RNAi-mediated control of mycotoxins in plants has not been reported previously.

The Food and Agriculture Organization of the United Nations estimates that 25% of the food crops in the world are contaminated with aflatoxins. That ...

Experimental Protocol for Biodiesel Production with Isolation of Alkenones as Coproducts from Commercial Isochrysis Algal Biomass

The need to replace petroleum fuels with alternatives from renewable and more environmentally sustainable sources is of growing importance. Biomass-derived biofuels have gained considerable attention in this regard, however first generation biofuels from edible crops like corn ethanol or soybean biodiesel have generally fallen out of favor. There is thus great interest in the development of methods for the production of liquid fuels from domestic and superior non-edible sources. Here we describe a detailed procedure for the production of a purified biodiesel from the marine microalgae Isochrysis. Additionally, a unique suite of lipids known as polyunsaturated long-chain alkenones are isolated in parallel as potentially valuable coproducts to offset the cost of biodiesel production. Multi-kilogram quantities of Isochrysis are purchased from two commercial sources, one as a wet paste (80% water) that is first dried prior to processing, and the other a dry milled powder (95% dry). Lipids are extracted with hexanes in a Soxhlet apparatus to produce an algal oil (&#34;hexane algal oil&#34;) containing both traditional fats (i.e., triglycerides, 46-60% w/w) and alkenones (16-25% w/w). Saponification of the triglycerides in the algal oil allows for separation of the resulting free fatty acids (FFAs) from alkenone-containing neutral lipids. FFAs are then converted to biodiesel (i.e., fatty acid methyl esters, FAMEs) by acid-catalyzed esterification while alkenones are isolated and purified from the neutral lipids by crystallization. We demonstrate that biodiesel from both commercial Isochrysis biomasses have similar but not identical FAME profiles, characterized by elevated polyunsaturated fatty acid contents (approximately 40% w/w). Yields of biodiesel were consistently higher when starting from the Isochrysis wet paste (12% w/w vs. 7% w/w), which can be traced to lower amounts of hexane algal oil obtained from the powdered Isochrysis product.

Experimental Protocol for Biodiesel Production with Isolation of Alkenones as Coproducts from Commercial Isochrysis Algal Biomass

Detailed methods are presented for the production of biodiesel along with the co-isolation of alkenones as valuable coproducts from commercial Isochrysis microalgae.

The need to replace petroleum fuels with alternatives from renewable and more environmentally sustainable sources is of growing importance. ...

Extraction and Analysis of Microbial Phospholipid Fatty Acids in Soils

Phospholipid fatty acids (PLFAs) are key components of microbial cell membranes. The analysis of PLFAs extracted from soils can provide information about the overall structure of terrestrial microbial communities. PLFA profiling has been extensively used in a range of ecosystems as a biological index of overall soil quality, and as a quantitative indicator of soil response to land management and other environmental stressors.
The standard method presented here outlines four key steps: 1. lipid extraction from soil samples with a single-phase chloroform mixture, 2. fractionation using solid phase extraction columns to isolate phospholipids from other extracted lipids, 3. methanolysis of phospholipids to produce fatty acid methyl esters (FAMEs), and 4. FAME analysis by capillary gas chromatography using a flame ionization detector (GC-FID). Two standards are used, including 1,2-dinonadecanoyl-sn-glycero-3-phosphocholine (PC(19:0/19:0)) to assess the overall recovery of the extraction method, and methyl decanoate (MeC10:0) as an internal standard (ISTD) for the GC analysis.

Phospholipid fatty acids provide information about the structure of soil microbial communities. We present methods for extraction from soil samples with a single-phase chloroform mixture, fractionation of extracted lipids using solid phase extraction columns, and methanolysis to produce fatty acid methyl esters, which are analyzed by capillary gas chromatography.

Phospholipid fatty acids (PLFAs) are key components of microbial cell membranes. The analysis of PLFAs extracted from soils can provide information about ...

A Gusseted Thermogradient Table to Control Soil Temperatures for Evaluating Plant Growth and Monitoring Soil Processes

Thermogradient tables were first developed in the 1950s primarily to test seed germination over a range of temperatures simultaneously without using a series of incubators. A temperature gradient is passively established across the surface of the table between the heated and cooled ends and is lost quickly at distances above the surface. Since temperature is only controlled on the table surface, experiments are restricted to shallow containers, such as Petri dishes, placed on the table. Welding continuous aluminum vertical strips or gussets perpendicular to the surface of a table enables temperature control in depth via convective heat flow. Soil in the channels between gussets was maintained across a gradient of temperatures allowing a greater diversity of experimentation. The gusseted design was evaluated by germinating oat, lettuce, tomato, and melon seeds. Soil temperatures were monitored using individual, battery-powered dataloggers positioned across the table. LED lights installed in the lids or along the sides of the gradient table create a controlled temperature chamber where seedlings can be grown over a range of temperatures. The gusseted design enabled accurate determination of optimum temperatures for fastest germination rate and the highest percentage germination for each species. Germination information from gradient table experiments can help predict seed germination and seedling growth under the adverse soil conditions often encountered during field crop production. Temperature effects on seed germination, seedling growth, and soil ecology can be tested under controlled conditions in a laboratory using a gusseted thermogradient table.

Traditional thermogradient tables create a range of temperatures across the surface. Welding gussets perpendicular to the surface of a thermogradient table will control temperature in depth increasing possible research applications.

Thermogradient tables were first developed in the 1950s primarily to test seed germination over a range of temperatures simultaneously without using a ...

Production and Measurement of Organic Particulate Matter in a Flow Tube Reactor

Organic particulate matter (PM) is increasingly recognized as important to the Earth's climate system as well as public health in urban regions, and the production of synthetic PM for laboratory studies have become a widespread necessity. Herein, experimental protocols demonstrate approaches to produce aerosolized organic PM by &#945;-pinene ozonolysis in a flow tube reactor. Methods are described for measuring the size distributions and morphology of the aerosol particles. The video demonstrates basic operations of the flow tube reactor and related instrumentation. The first part of the video shows the procedure for preparing gas-phase reactants, ozonolysis, and production of organic PM. The second part of the video shows the procedures for determining the properties of the produced particle population. The particle number-diameter distributions show different stages of particle growth, namely condensation, coagulation, or a combination of both, depending on reaction conditions. The particle morphology is characterized by an aerosol particle mass analyzer (APM) and a scanning electron microscope (SEM). The results confirm the existence of non-spherical particles that have grown from coagulation for specific reaction conditions. The experimental results also indicate that the flow tube reactor can be used to study the physical and chemical properties of organic PM for relatively high concentrations and short time frames.

This paper describes the operation procedure for the flow tube reactor and related data collection. It shows the protocols for setting the experiments, recording data and generating the number-diameter distribution as well as the particle mass information, which gives useful information about chemical and physical properties of the organic aerosols.

Organic particulate matter (PM) is increasingly recognized as important to the Earth's climate system as well as public health in urban regions, and the ...

Inhibition of Aspergillus flavus Growth and Aflatoxin Production in Transgenic Maize Expressing the α-amylase Inhibitor from Lablab purpureus L.

Summary

Explore More Videos

Chapters in this video

Mass Production of Genetically Modified Aedes aegypti for Field Releases in Brazil

Determination of Microbial Extracellular Enzyme Activity in Waters, Soils, and Sediments using High Throughput Microplate Assays

A Rapid and Efficient Method for Assessing Pathogenicity of Ustilago maydis on Maize and Teosinte Lines

Optimize Flue Gas Settings to Promote Microalgae Growth in Photobioreactors via Computer Simulations

Methods for Facilitating Microbial Growth on Pulp Mill Waste Streams and Characterization of the Biodegradation Potential of Cultured Microbes

RNAi-mediated Control of Aflatoxins in Peanut: Method to Analyze Mycotoxin Production and Transgene Expression in the Peanut/Aspergillus Pathosystem

Experimental Protocol for Biodiesel Production with Isolation of Alkenones as Coproducts from Commercial Isochrysis Algal Biomass

Extraction and Analysis of Microbial Phospholipid Fatty Acids in Soils

A Gusseted Thermogradient Table to Control Soil Temperatures for Evaluating Plant Growth and Monitoring Soil Processes

Production and Measurement of Organic Particulate Matter in a Flow Tube Reactor