We thank Dr. Emir Islamovic for laboratory and greenhouse assistance. We also thank Dr. Sherry Flint-Garcia for providing the maize x teosinte introgression lines.

1. Growth of Plant Material
<ol>
	<li>Select plant lines for inoculation and screening. Two maize lines, five teosinte lines, and forty maize x teosinte lines with uncharacterized resistance to U. maydis were used for this work (Table 1).</li>
	<li>Plant seeds for experimental (U. maydis injection) and control (water injection) needle injection inoculation experiments. Do this for each plant line.</li>
	<li>Plant four seeds (replicates) for each plant line in small flats by pushing the seeds about &#189; inch into the soil with finger and covering with soil lightly (Figures 1A&#160;and 1B). Do not pack the soil over the seed. Planting the seed deeper or packing the soil over the seed may cause problems with seedling emergence.</li>
	<li>Water the seeds into the soil. Ensure that the soil is soaked and the seeds remain under the soil after watering.</li>
	<li>After watering, place plants in a growth chamber with day and night environments of 28/20 &#176;C temperature and 14/10 hr of photoperiod, respectively and approximately&#160;500&#160;&#956;mol/m2&#160;sec&#160;photosynthetically active radiations at the top of the canopy. Maintain the relative humidity during the day and night at approximately 70% and 90%, respectively.</li>
	<li>Keep all plants in the same growth chamber to maintain a growth environment that is congruent across the experiment.</li>
	<li>After 10 days, remove the plants from the growth chamber and inoculate the plants with the U. maydis cell suspension culture using a needle injection inoculation method. Note: Maize plants can be inoculated 7 days after planting8-10. However, the teosinte plants are too small after 7 days. Therefore, inoculate both maize and teosinte plants 10 days after planting for consistency within the experiment (see step 2.12).</li>
</ol>
2. Needle Injection Inoculation
<ol>
	<li>Do all work in a laminar flow hood. Remove U. maydis glycerol stocks from freezer storage. Use a sterile loop and streak glycerol stocks of U. maydis wild-type strains &#189; (mating type a1b1) and 2/9 (mating type a2b2, near isogenic to &#189;) on to potato dextrose agar (PDA) plates. Maintain strains separately.</li>
	<li>Place PDA plates streaked with U. maydis in a 30 &#176;C incubator for two days. If using a different biotrophic pathogen use the appropriate strain, media and growth conditions. Monitor the growth of the pathogen over the two day period to ensure that the U. maydis strain is growing well.</li>
	<li>Remove the PDA plates from the incubator after two days. The plates should have good pathogen growth and contain single colonies (Figure 2A). It is important to obtain single colonies. If single colonies are not present restreak the plates at a lower concentration.</li>
	<li>Do all of the work in a laminar flow hood. Use a sterile toothpick to select a single colony for each strain from the PDA plates. Place the toothpick containing a single colony into a 3 ml&#160;potato dextrose broth (PDB). It is advised to have 2-3 cultures.</li>
	<li>Place the 3 ml PDB cultures into a 30 &#176;C incubator/shaker for two days at 200 rpm. Monitor the growth of the culture over the two day period to ensure growth of the culture. The culture should appear very cloudy.</li>
	<li>Remove the liquid cultures from the incubator/shaker and measure the concentration at OD600 to ensure that the cells were grown to an OD of 1.0 (~1 x 107 cells/ml)17.</li>
	<li>Bring the U. maydis cell suspension cultures to a final concentration of 1 x 106 cells/ml, using water in a final 30 ml culture volume. This concentration consistently results in good infection of the plants with the pathogen cell suspension culture.17 </li>
</ol>
Note: Various cell suspension concentrations should be tested when using different pathogen strains to determine the appropriate cell titer needed for inoculation18,19.&#160;The given final concentration for the cell suspension culture can be used as a starting point for tittering. The appropriate concentration of the pathogen cell suspension culture should be verified by visualizing the plant phenotypes with good infection (Figures 3A-E).
<ol start="8">
	<li>Mix equal volumes of the two U. maydis strains prior to inoculation. If using one pathogen strain proceed to step 2.9. Prepare fresh U. maydis cell suspension cultures for each inoculation experiment and discard cell suspension cultures after two days.</li>
	<li>For the experimental needle injection inoculation, fill a 3 ml syringe with the U. maydis cell suspension culture by drawing the cell suspension culture into the syringe.</li>
	<li>For the control needle injection inoculation, fill a 3 ml syringe with water17. Use the same procedure for the experimental needle injection inoculation.</li>
	<li>Attach a 0.457 mm x 1.3 cm hypodermic needle to the end of each 3 ml syringe. The selected needle size will deliver the cell suspension culture in between the plant leaves with minimal damage to the plant tissue.</li>
	<li>Remove the experimental and control plants from the growth chamber 10 days after planting in preparation for needle injection inoculations (Figure 2B) (see step 1.7).</li>
	<li>Carefully insert the hypodermic needle containing the U. maydis cell suspension culture into the stem of an experimental plant at a 90&#176; angle just above the soil line. Insert the needle until it is in the middle of the stem. Do not push the needle through the stem (Figure 2C).</li>
	<li>Inject the experimental plant with about 100 &#956;l of the U. maydis cell suspension culture18,19. This will vary slightly depending on the height of the seedling. The cell suspension culture will push through the stem and move into the whirl of the plant. The cell suspension culture will be visible in the whirl of the plant. Continue injecting 100 &#956;l of the cell suspension culture into each individual plant until the 3 ml syringe is empty.</li>
	<li>After the injection, carefully remove the needle from the plant stem. Remove the needle from the now empty 3 ml syringe and fill with water. Attach the needle back to the syringe and push the water through the needle to remove any plant tissue that may be caught in the needle tip.</li>
	<li>Repeat steps 2.9-2.15 for each experimental plant. Follow the same protocol for the control plants by injecting water.</li>
	<li>Place the inoculated experimental and control plants back into the growth chamber. Water the plants daily by wetting the soil not the plant tissue.</li>
	<li>Check the plants daily to detect pathogen development and plant resistance reactions.</li>
</ol>
3. Resistance Reaction Screening
<ol>
	<li>Score and record the resistance reactions for each plant 7, 10, 14, and 21 days post inoculation (dpi) using a 1 to 5 resistance reaction rating scale. Disease severity increases as the numerical values on the rating scale increases (Table 2). A 1C (Leaf chlorosis), 1A (Leaf anthocyanin production), or 2 (small leaf galls) resistance reaction indicates resistance. A 3 (stem galls), 4 (basal gall), or 5 (plant death) resistance reaction indicates susceptibility (Figures 3A-E&#160;and Table 2)18,19.</li>
	<li>Score both experimental and control plants and record resistance reaction ratings.</li>
	<li>Compare the resistance reactions of the experimental and control plants. Select experimental plants with a 1C, 1A, or 2 resistance reaction rating. These plants are considered to be resistant to U. maydis18,19.</li>
	<li>Repeat the entire experiment to verify the plant phenotypes.</li>
</ol>

<ol>
	<li>Smith, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crop+fungal+resistance+developed+using+genetic+engineering+and+antifungal+proteins+from+viruses.">Crop fungal resistance developed using genetic engineering and antifungal proteins from viruses.</a> , (2011).</li><li>Sher, A. F., MacNab, A. 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> Vegetable diseases and their control. , 223-226 (1986).</li><li>Crepet, W. L., Feldman, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+earliest+remains+of+grasses+in+the+fossil+record.">The earliest remains of grasses in the fossil record.</a> Am. J. Bot. 78, 1010-1014 (1991).</li><li>Iltis, H. H., Scoderstrom, T. R., Hilu, K. W., Campbell, C. S., Barkworth, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maize+evolution+and+agricultural+origins.">Maize evolution and agricultural origins.</a> Grass systematic and evolution. , 195-213 (1997).</li><li>Mangelsdorf, P. C., Reeves, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+origin+of+corn.+III.+Modern+races,+the+product+of+tesonite.">The origin of corn. III. Modern races, the product of tesonite.</a> Bot. Mus. Leafl.. 18, 389-411 (1957).</li><li>Agrios, G. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Plant Pathology. , (1997).</li><li>Dean, 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=The+top+10+fungal+pathogens+in+molecular+plant+pathology.">The top 10 fungal pathogens in molecular plant pathology.</a> Mol. Plant. Pathol. 13, 414-430 (2012).</li><li>Estrada, A. E., Jonkers, W., Kistler, H. C., May, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+beteen+Fusarium+verticillioides,+Ustilago+maydis,+and+Zea+mays:+An+endophyte,+a+pathogen,+and+their+shared+plant+host.">Interactions beteen Fusarium verticillioides, Ustilago maydis, and Zea mays: An endophyte, a pathogen, and their shared plant host.</a> Fung. Genet. Biol. 49, 578-587 (2012).</li><li>Freeman, S., Rodriguez, R. 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+rapid+technique+for+assessing+pathogenicity+of+Fusarium+oxysporum+f.+sp+niveum+and+F.+o.+melonis+on+cucrbits.">A rapid technique for assessing pathogenicity of Fusarium oxysporum f. sp niveum and F. o. melonis on cucrbits.</a> Plant Dis. 77, 1198-1201 (1993).</li><li>Gottwald, T. R., Graham, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+device+for+precise+and+nondisruptive+stomatal+inoculation+of+leaf+tissue+with+bacterial+pathogens.">A device for precise and nondisruptive stomatal inoculation of leaf tissue with bacterial pathogens.</a> Phytopathol. 82, 930-935 (1992).</li><li>Posada, F., Aime, M. C., Peterson, S. W., Rehner, S. A., Vega, F. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inoculation+of+coffee+plants+with+the+fungal+entomopathogen+Beauveria+bassiana+(Asomycota:+Hypocreales).">Inoculation of coffee plants with the fungal entomopathogen Beauveria bassiana (Asomycota: Hypocreales).</a> Mycolog. Res. 111, 748-757 (2007).</li><li>Bolker, M., Bohnert, H. U., Braun, K. H., Gorl, J., Kahmann, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tagging+pathogenicity+genes+in+Ustilago+maydis+by+restriction+enzyme-mediated+intergratior+(REMI).">Tagging pathogenicity genes in Ustilago maydis by restriction enzyme-mediated intergratior (REMI).</a> Mol. Gen. Genet. 6, 274-283 (1991).</li><li>Brachmann, A., Weinzierl, G., Kamper, J., Kahmann, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+genes+in+the+bW/bE+regulatory+cascade+in+Ustilago+maydis.">Identification of genes in the bW/bE regulatory cascade in Ustilago maydis.</a> Mol. Microbiol. 42, 1047-1063 (2001).</li><li>Christensen, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corn+smut+caused+by+Ustilago+maydis.+Monograph+number+2.">Corn smut caused by Ustilago maydis. Monograph number 2.</a> , (1963).</li><li>Skibbe, D. S., Doehlemann, G., Fernandes, J., Walbot, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maize+tumors+caused+by+Ustilago+maydis+require+organ-specific+genes+in+host+and+pathogen.">Maize tumors caused by Ustilago maydis require organ-specific genes in host and pathogen.</a> Sci.. 328, 89-92 (2010).</li><li>Kamper, 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=Insights+from+the+genome+of+the+biotrophic+fungal+plant+pathogen+Ustilago+maydis.">Insights from the genome of the biotrophic fungal plant pathogen Ustilago maydis.</a> Nature. 444, 97-101 (2006).</li><li>Allen, A., Kaur, J., Gold, S., Shah, D., Smith, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+maize+plants+expressing+the+Totivirus+antifungal+protein,+KP4,+are+highly+resistant+to+corn+smut.">Transgenic maize plants expressing the Totivirus antifungal protein, KP4, are highly resistant to corn smut.</a> Plant Biotechnol. J. 8, 857-864 (2011).</li><li>Gold, S. E., Brogdon, S. M., Mayorga, M. E., Kronstad, 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=The+Ustilago+maydis+regulatory+subunit+of+a+cAMP-Dependent+protein+kinase+is+required+for+gall+formation+in+maize.">The Ustilago maydis regulatory subunit of a cAMP-Dependent protein kinase is required for gall formation in maize.</a> , (1997).</li><li>Gold, S. E., Kronstad, 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=Disruption+of+two+chitin+syn-+thase+genes+in+the+phytopathogenic+fungus+Ustilago+maydis.">Disruption of two chitin syn- thase genes in the phytopathogenic fungus Ustilago maydis.</a> Mol. Microbiol. 11, 897-902 (1994).</li><li>Brefort, T., Doehlemann, G., Mendoza-Mendoza, A., Reissmann, S., Djamei, A., Kahmann, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ustilago+maydis+as+a+Pathogen.">Ustilago maydis as a Pathogen.</a> Annu. Rev. Phytopathol. 47, 423-445 (2005).</li><li>Doehlemann, G., Wahl, R., Vranes, M., de Vries, R., K&#228;mper, J., Kahmann, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+compatibility+in+the+Ustilago+maydis/maize+pathosystems.">Establishment of compatibility in the Ustilago maydis/maize pathosystems.</a> J. Plant Physiol. 165, 29-40 (2008).</li><li>Reineke, G., Heinze, B., Schirawski, J., Buettner, H., Kahmann, R., Base, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Indole-3-acetic+acid+(IAA)+biosynthesis+in+the+smut+fungus+Ustilago+maydis+and+its+relevance+for+increased+IAA+levels+in+infected+tissue+and+host+tumor+formation.">Indole-3-acetic acid (IAA) biosynthesis in the smut fungus Ustilago maydis and its relevance for increased IAA levels in infected tissue and host tumor formation.</a> Mol. Plant Pathol. 9, 339-355 (2008).</li><li>Mart&#237;nez-Espinoza, A., Garc&#237;a-Pedrajas, M. D., Gold, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Ustilaginales+as+Plant+Pests+and+Model+Systems.">The Ustilaginales as Plant Pests and Model Systems.</a> Fungal Genet. Biol. 35, 1-20 (2002).</li><li>Banuett, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetics+of+Ustilago+maydis,+a+fungal+pathogen+that+induces+tumors+in+maize.">Genetics of Ustilago maydis, a fungal pathogen that induces tumors in maize.</a> Annu. Rev. Genet. 29, 179-208 (1995).</li><li>Keen, N. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+century+of+plant+pathology:+a+retrospective+view+on+understanding+host-parasite+interactions.">A century of plant pathology: a retrospective view on understanding host-parasite interactions.</a> Annu. Rev. Phytopathol. 38, 31-48 (2000).</li></ol>

Authors have nothing to disclose.

In this study the needle injection inoculation method used to deliver a strain of U. maydis into the stem of 700 maize and teosinte plants was successful. Additionally, a revised disease resistance rating scale was used to screen the plants and detect pathogen development. As a result of using both methods, plant lines that are resistant to U. maydis were identified among 700 maize and teosinte plants that can now be combined and tested in breeding programs for improved disease resistance.
<p class=...

Fungal diseases of plants represent one of the most eminent threats to agriculture. The need to develop crops with improved disease resistance is increasing due to the food needs of a growing world population. Plant pathogens naturally infect crop plants in the field causing diseases that negatively impact crop yield6. It has been shown that identifying and utilizing resistant plants can improve resistance and decrease yield loss. Resistant cultivars have been identified in many plant species including maize, wheat, rice, and sorghum by inoculating the plants with a plant pathogen and selecting for resistant lines7. Therefore, development and use of an efficient inoculation method would allow many plants to be inoculated and screened for resistance. Various inoculation methods have been used including dip inoculation, pipetting the pathogen cell suspension culture into the whirl of the plant, and needle injection inoculation8-11.&#160;With each method, the pathogen must reliably be introduced in between the plant leaves where the pathogen enters the plant through the formation of appresoria to ensure pathogen development and plant infection12,13.
The dip inoculation method involves submerging a plant seedling into a pathogen cell suspension culture, while the pipetting method requires placing the pathogen cell suspension culture into the whirl of the plant seedling. However, there are issues with both methods. First, both methods depend on the natural movement of the pathogen from the leaf surface into the plant tissue which is highly variable. Most pathogens naturally enter the plant through stomatal openings or wounds on the plant leaf surface. However, there is significant variability in the pathogens ability to penetrate the plant leaf surface through the stomata and/or wounds on the leaf surface. Therefore, pathogen penetration cannot be controlled with either inoculation method potentially resulting in inconsistent data. Second, when screening a large number of plants, submerging the seedlings into a pathogen cell suspension culture can be time consuming and may limit the number of plants that can be screened. Conversely, the needle injection inoculation protocol described herein delivers the pathogen cell suspension culture in between the plant leaves facilitating the formation of appressoria14. The pathogen then utilizes the newly developed appressoria to enter the plant eliminating the pathogen penetration issue. Additionally, the needle injection inoculation protocol provides a range of phenotypes for maize and teosinte plants that have been inoculated with U. maydis and demonstrate good infection. The phenotypes can be used as a marker to determine the best concentration for the pathogen cell suspension culture resulting in consistent plant phenotypes within and between different experiments.
Following plant inoculation with a pathogen cell suspension culture, plants are typically screened to detect a resistant or susceptible phenotype8-11,15. While disease rating scales have being used extensively to screen and classify plant phenotypes, rating scales differ depending on the pathogen being analyzed. Therefore, a disease rating scale protocol establishment for U. maydis and maize interactions can be utilized for similar fungal pathogens16.
The present series of protocols details needle injection inoculation with a U. maydis cell suspension culture and disease resistance reaction screening of maize, teosinte, and maize x teosinte introgression lines. The present protocols are not limited to needle injection inoculation of U. maydis into maize plants but can be utilized for relatively any fungal pathogen and plant species. Therefore, including the details of both methods in the same protocol will enable researchers to directly utilize the protocols for inoculation and screening or to manipulate the original protocols to better fit the pathogen and plant species of interest.

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Seed for plants</td>
			<td></td>
			<td></td>
			<td>Collected from original crosses</td>
		</tr>
		<tr>
			<td>Growth chamber</td>
			<td>Conviron</td>
			<td>PGR14 REACH-IN</td>
			<td></td>
		</tr>
		<tr>
			<td>Planting flats</td>
			<td>Hummert International</td>
			<td>14-3385-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Soil (3 parts pine bark; 1 part peat moss with perlite)</td>
			<td>Hummert International</td>
			<td>10-1059-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminar flow hood</td>
			<td>Lab Conoco</td>
			<td>70875372</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol stock of pathogen (U. maydis) or fungal pathogen of interest</td>
			<td></td>
			<td></td>
			<td>Stocks were grown from original culture</td>
		</tr>
		<tr>
			<td>Sterile loop</td>
			<td>Fisher Scientific</td>
			<td>S17356A</td>
			<td></td>
		</tr>
		<tr>
			<td>Potato dextrose agar (PDA) plates</td>
			<td>Fisher Scientific</td>
			<td>R454311</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator set to 30 &#176;C</td>
			<td>Fisher Scientific</td>
			<td>11-690-650F</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile toothpicks</td>
			<td>Walmart</td>
			<td></td>
			<td>Purchased from Walmart and sterilized by autoclave</td>
		</tr>
		<tr>
			<td>Potato dextrose broth (PDB)</td>
			<td>Fisher Scientific</td>
			<td>ICN1008617</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator-shaker set to 30 &#176;C</td>
			<td>New Brunswick</td>
			<td>14-278-179</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrophotometer</td>
			<td>Fisher Scientific</td>
			<td>4001000</td>
			<td></td>
		</tr>
		<tr>
			<td>U. maydis&#160;cell suspension culture (1 x 106 cells/ml)</td>
			<td></td>
			<td></td>
			<td>Grown from glycerol stock as described in the methods</td>
		</tr>
		<tr>
			<td>3 ml Syringes</td>
			<td>Becton Dickinson</td>
			<td>309606</td>
			<td></td>
		</tr>
		<tr>
			<td>.457 mm x 1.3 cm Hypodermic needles</td>
			<td>Kendall Brands</td>
			<td>8881250321</td>
			<td></td>
		</tr>
	</tbody>
</table>

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 successful needle injection inoculation can be determined by visualizing the phenotype of the plants inoculated with U. maydis (experimental). The majority of the experimental plants were susceptible to U. maydis infection. The susceptible plants showed very severe disease development demonstrated by stem and basal gall formation with black teliospores (Figures 3D&#160;and&#160;3E,&#160;Table 2). Several plants were dead after inoculation due to the s...

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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.

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 ...

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JoVE Journal

Environment

8.4K Views.  University of Georgia. يوصف استخدام طريقة حقن الإبر لتطعيم الذرة ونباتات التيوسينت مع مسببات الأمراض النيروبية الحيوية أوستيلاغو مايديس. تسهل طريقة تلقيح حقن الإبر التسليم المراقب لمسببات الأمراض الفطرية بين أوراق النبات حيث يدخل الممرض النبات من خلال تشكيل الأبريسوريا. هذه ا...

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

Use of Chironomidae (Diptera) Surface-Floating Pupal Exuviae as a Rapid Bioassessment Protocol for Water Bodies

Rapid bioassessment protocols using benthic macroinvertebrate assemblages have been successfully used to assess human impacts on water quality. Unfortunately, traditional benthic larval sampling methods, such as the dip-net, can be time-consuming and expensive. An alternative protocol involves collection of Chironomidae surface-floating pupal exuviae (SFPE). Chironomidae is a species-rich family of flies (Diptera) whose immature stages typically occur in aquatic habitats. Adult chironomids emerge from the water, leaving their pupal skins, or exuviae, floating on the water&#8217;s surface. Exuviae often accumulate along banks or behind obstructions by action of the wind or water current, where they can be collected to assess chironomid diversity and richness. Chironomids can be used as important biological indicators, since some species are more tolerant to pollution than others. Therefore, the relative abundance and species composition of collected SFPE reflect changes in water quality. Here, methods associated with field collection, laboratory processing, slide mounting, and identification of chironomid SFPE are described in detail. Advantages of the SFPE method include minimal disturbance at a sampling area, efficient and economical sample collection and laboratory processing, ease of identification, applicability in nearly all aquatic environments, and a potentially more sensitive measure of ecosystem stress. Limitations include the inability to determine larval microhabitat use and inability to identify pupal exuviae to species if they have not been associated with adult males.

Use of Chironomidae Diptera Surface-Floating Pupal Exuviae as a Rapid Bioassessment Protocol for Water Bodies

Rapid bioassessment protocols using benthic macroinvertebrates are often used to monitor and assess water quality. An efficient protocol involves collections of Chironomidae surface-floating pupal exuviae (SFPE). Here, techniques for field collection, laboratory processing, slide mounting, and identification of Chironomidae SFPE are described.

Rapid bioassessment protocols using benthic macroinvertebrate assemblages have been successfully used to assess human impacts on water quality. ...

The Use of an Automated System (GreenFeed) to Monitor Enteric Methane and Carbon Dioxide Emissions from Ruminant Animals

Ruminant animals (domesticated or wild) emit methane (CH4) through enteric fermentation in their digestive tract and from decomposition of manure during storage. These processes are the major sources of greenhouse gas (GHG) emissions from animal production systems. Techniques for measuring enteric CH4 vary from direct measurements (respiration chambers, which are highly accurate, but with limited applicability) to various indirect methods (sniffers, laser technology, which are practical, but with variable accuracy). The sulfur hexafluoride (SF6)&#160;tracer gas method is commonly used to measure enteric CH4 production by animal scientists and more recently, application of an Automated Head-Chamber System (AHCS) (GreenFeed, C-Lock, Inc., Rapid City, SD), which is the focus of this experiment, has been growing. AHCS is an automated system to monitor CH4 and carbon dioxide (CO2) mass fluxes from the breath of ruminant animals. In a typical AHCS operation, small quantities of baiting feed are dispensed to individual animals to lure them to AHCS multiple times daily. As the animal visits AHCS, a fan system pulls air past the animal&#8217;s muzzle into an intake manifold, and through an air collection pipe where continuous airflow rates are measured. A sub-sample of air is pumped out of the pipe into non-dispersive infra-red sensors for continuous measurement of CH4 and CO2 concentrations. Field comparisons of AHCS to respiration chambers or SF6 have demonstrated that AHCS produces repeatable and accurate CH4 emission results, provided that animal visits to AHCS are sufficient so emission estimates are representative of the diurnal rhythm of rumen gas production. Here, we demonstrate the use of AHCS to measure CO2 and CH4 fluxes from dairy cows given a control diet or a diet supplemented with technical-grade cashew nut shell liquid.

The Use of an Automated System GreenFeed to Monitor Enteric Methane and Carbon Dioxide Emissions from Ruminant Animals

Accuracy and precision of the techniques used to measure methane emissions from ruminant animals are critically important for the success of greenhouse gas mitigation efforts. This manuscript describes the principles and operation of an automated system to monitor methane and carbon dioxide mass fluxes from the breath of ruminant animals.

Ruminant animals (domesticated or wild) emit methane (CH4) through enteric fermentation in their digestive tract and from decomposition of manure during ...

Administering and Detecting Protein Marks on Arthropods for Dispersal Research

Monitoring arthropod movement is often required to better understand associated population dynamics, dispersal patterns, host plant preferences, and other ecological interactions. Arthropods are usually tracked in nature by tagging them with a unique mark and then re-collecting them over time and space to determine their dispersal capabilities. In addition to actual physical tags, such as colored dust or paint, various types of proteins have proven very effective for marking arthropods for ecological research. Proteins can be administered internally and/or externally. The proteins can then be detected on recaptured arthropods with a protein-specific enzyme-linked immunosorbent assay (ELISA). Here we describe protocols for externally and internally tagging arthropods with protein. Two simple experimental examples are demonstrated: (1) an internal protein mark introduced to an insect by providing a protein-enriched diet and (2) an external protein mark topically applied to an insect using a medical nebulizer. We then relate a step-by-step guide of the sandwich and indirect ELISA methods used to detect protein marks on the insects. In this demonstration, various aspects of the acquisition and detection of protein markers on arthropods for mark-release-recapture, mark-capture, and self-mark-capture types of research are discussed, along with the various ways that the immunomarking procedure has been adapted to suit a wide variety of research objectives.

Proteins are often used to mark arthropods for dispersal research. Methods for administering internal and external protein marks on arthropods are demonstrated. Protein mark detection techniques, either through indirect or sandwich enzyme-linked immunosorbent assays (ELISAs), are also illustrated.

Monitoring arthropod movement is often required to better understand associated population dynamics, dispersal patterns, host plant preferences, and other ...

Reliable Method for Assessing Seed Germination, Dormancy, and Mortality under Field Conditions

We describe techniques for approximating seed bank dynamics over time using Helianthus annuus as an example study species. Strips of permeable polyester fabric and glue can be folded and glued to construct a strip of compartments that house seeds and identifying information, while allowing contact with soil leachate, water, microorganisms, and ambient temperature. Strips may be constructed with a wide range of compartment numbers and sizes and allow the researcher to house a variety of genotypes within a single species, different species, or seeds that have experienced different treatments. As opposed to individual seed packets, strips are more easily retrieved as a unit. While replicate packets can be included within a strip, different strips can act as blocks or can be retrieved at different times for observation of seed behavior over time. We used a high temperature glue gun to delineate compartments and sealed the strips once the seed and tags identifying block and removal times were inserted. The seed strips were then buried in the field at the desired depth, with the location marked for later removal. Burrowing animal predators were effectively excluded by use of a covering of metal mesh hardware cloth on the soil surface. After the selected time interval for burial, strips were dug up and seeds were assessed for germination, dormancy and mortality. While clearly dead seeds can often be distinguished from ungerminated living ones by eye, dormant seeds were conclusively identified using a standard Tetrazolium chloride colorimetric test for seed viability.

Here we present a protocol for assessing seed survivorship, germination and dormancy under field conditions using buried, labeled seed strips and tetrazolium chloride (TZ) viability testing.

We describe techniques for approximating seed bank dynamics over time using Helianthus annuus as an example study species. Strips of permeable polyester ...

Protocol for Assessing the Relative Effects of Environment and Genetics on Antler and Body Growth for a Long-lived Cervid

Cervid phenotype can be placed into one of two categories: efficiency, which promotes survival over extravagant morphometric growth, and luxury, which promotes growth of large weaponry and body size. Populations of the same species display each phenotype depending on environmental conditions. Although antler and body size of male white-tailed deer (Odocoileus virginianus) varies by physiographic region in Mississippi, USA and is strongly correlated with regional variation in nutritional quality, the effects of population-level genetics from native stocks and previous re-stocking efforts cannot be disregarded. This protocol describes how we designed a controlled study, where other factors that influence phenotype, such as age and nutrition, are controlled. We brought wild-caught pregnant females and six-month-old fawns from three distinct physiographic regions in Mississippi, USA to the Mississippi State University Rusty Dawkins Memorial Deer Unit. Deer from the same region were bred to produce a second generation of offspring, allowing us to assess generational responses and maternal effects. All deer ate the same high-quality (20% crude protein deer pellet) diet ad libitum. We uniquely marked each neonate and recorded body mass, hind foot, and total body length. Each subsequent fall, we sedated individuals via remote injection and sampled the same morphometrics plus antlers of adults. We found that all morphometrics increased in size from first to second generation, with full compensation of antler size (regional variation no longer present) and partial compensation of body mass (some evidence of regional variation) evident in the second generation. Second generation males that originated from our poorest quality soil region displayed about a 40% increase in antler size and about a 25% increase in body mass when compared to their wild harvested counterparts. Our results suggest phenotypic variation of wild male white-tailed deer in Mississippi are more related to differences in nutritional quality than population-level genetics.

Phenotypic differences among cervid populations may be related to population-level genetics or nutrition; discerning which is difficult in the wild. This protocol describes how we designed a controlled study where nutritional variation was eliminated. We found that phenotypic variation of male white-tailed deer was more limited by nutrition than genetics.

Cervid phenotype can be placed into one of two categories: efficiency, which promotes survival over extravagant morphometric growth, and luxury, which ...

Preparation of DMMTAV and DMDTAV Using DMAV for Environmental Applications: Synthesis, Purification, and Confirmation

Dimethylated thioarsenicals such as dimethylmonothioarsinic acid (DMMTAV) and dimethyldithioarsinic acid (DMDTAV), which are produced by the metabolic pathway of dimethylarsinic acid (DMAV) thiolation, have been recently found in the environment as well as human organs. DMMTAV and DMDTAV can be quantified to determine the ecological effects of dimethylated thioarsenicals and their stability in environmental media. The synthesis method for these compounds is unstandardized, making replicating previous studies challenging. Furthermore, there is a lack of information about storage techniques, including storage of compounds without species transformation. Moreover, because only limited information about synthesis methods is available, there may be experimental difficulties in synthesizing standard chemicals and performing quantitative analysis. The protocol presented herein provides a practically modified synthesis method for the dimethylated thioarsenicals, DMMTAV and DMDTAV, and will help in the quantification of species separation analysis using high performance liquid chromatography in conjunction with inductively coupled plasma mass spectrometry (HPLC-ICP-MS). The experimental steps of this procedure were modified by focusing on the preparation of chemical reagents, filtration methods, and storage.

Preparation of DMMTAV and DMDTAV Using DMAV for Environmental Applications: Synthesis, Purification, and Confirmation

This article presents modified experimental protocols for dimethylmonothioarsinic acid (DMMTAV) and dimethyldithioarsinic acid (DMDTAV) synthesis, inducing dimethylarsinic acid (DMAV) thiolation through mixing of DMAV, Na2S, and H2SO4. The modified protocol provides an experimental guideline, thereby overcoming limitations of the synthesis steps that could have caused experimental failures in quantitative analysis.

Dimethylated thioarsenicals such as dimethylmonothioarsinic acid (DMMTAV) and dimethyldithioarsinic acid (DMDTAV), which are produced by the metabolic ...

The Hawaii Protocol for Scientific Monitoring of Coffee Berry Borer: a Model for Coffee Agroecosystems Worldwide

Coffee berry borer (CBB) is the most devastating insect pest for coffee crops worldwide. We developed a scientific monitoring protocol that is aimed at capturing and quantifying the dynamics and impact of this invasive insect pest as well as the development of its host plant across a heterogeneous landscape. The cornerstone of this comprehensive monitoring system is timely georeferenced data collection on CBB movement, coffee berry infestation, mortality by the fungus Beauveria bassiana, and coffee plant phenology via a mobile electronic data recording application. This electronic data collection system allows field records to be georeferenced through built-in global positioning systems, and is backed by a network of weather stations and records of farm management practices. Comprehensive monitoring of CBB and host plant dynamics is an essential part of an area-wide project in Hawaii to aggregate landscape-level data for research to improve management practices. Coffee agroecosystems in other parts of the world that experience highly variable environmental and socioeconomic factors will also benefit from implementing this protocol, in that it will drive the development of customized integrated pest management (IPM) to manage CBB populations.

Comprehensive monitoring of coffee berry borer and host plant dynamics is essential for aggregating landscape-level data to improve management of this invasive pest. Here, we present a protocol for scientific monitoring of coffee berry borer movement, infestation, mortality, coffee plant phenology, weather, and farm management via a mobile electronic data recording application.

Coffee berry borer (CBB) is the most devastating insect pest for coffee crops worldwide. We developed a scientific monitoring protocol that is aimed at ...

A Flexible Low Cost Hydroponic System for Assessing Plant Responses to Small Molecules in Sterile Conditions

A wide range of studies in plant biology are performed using hydroponic cultures. In this work, an in vitro hydroponic growth system designed for assessing plant responses to chemicals and other substances of interest is presented. This system is highly efficient in obtaining homogeneous and healthy seedlings of the C3 and C4 model species Arabidopsis thaliana and Setaria viridis, respectively. The sterile cultivation avoids algae and microorganism contamination, which are known limiting factors for plant normal growth and development in hydroponics. In addition, this system is scalable, enabling the harvest of plant material on a large scale with minor mechanical damage, as well as the harvest of individual parts of a plant if desired. A detailed protocol demonstrating that this system has an easy and low-cost assembly, as it uses pipette racks as the main platform for growing plants, is provided. The feasibility of this system was validated using Arabidopsis seedlings to assess the effect of the drug AZD-8055, a chemical inhibitor of the target of rapamycin (TOR) kinase. TOR inhibition was efficiently detected as early as 30 min after an AZD-8055 treatment in roots and shoots. Furthermore, AZD-8055-treated plants displayed the expected starch-excess phenotype. We proposed this hydroponic system as an ideal method for plant researchers aiming to monitor the action of plant inducers or inhibitors, as well as to assess metabolic fluxes using isotope-labeling compounds which, in general, requires the use of expensive reagents.

A simple, versatile, and low-cost in vitro hydroponic system was successfully optimized, enabling large-scale experiments under sterile conditions. This system facilitates the application of chemicals in a solution and their efficient absorption by roots for molecular, biochemical, and physiological studies.

A wide range of studies in plant biology are performed using hydroponic cultures. In this work, an in vitro hydroponic growth system designed for ...

High-throughput, Microscale Protocol for the Analysis of Processing Parameters and Nutritional Qualities in Maize (Zea mays L.)

Maize is an important grain crop in the United States and worldwide. However, maize grain must be processed prior to human consumption. Furthermore, whole grain composition and processing characteristics vary among maize hybrids and can impact the quality of the final processed product. Therefore, in order to produce healthier processed food products from maize, it is necessary to know how to optimize processing parameters for particular sets of germplasm to account for these differences in grain composition and processing characteristics. This includes a better understanding of how current processing techniques impact the nutritional quality of the final processed food product. Here, we describe a microscale protocol that both simulates the processing pipeline to produce cornflakes from large flaking grits and allows for the processing of multiple grain samples simultaneously. The flaking grits, the intermediate processed products, or final processed product, as well as the corn grain itself, can be analyzed for nutritional content as part of a high-throughput analytical pipeline. This procedure was developed specifically for incorporation into a maize breeding research program, and it can be modified for other grain crops. We provide an example of the analysis of insoluble-bound ferulic acid and p-coumaric acid content in maize. Samples were taken at five different processing stages. We demonstrate that sampling can take place at multiple stages during microscale processing, that the processing technique can be utilized in the context of a specialized maize breeding program, and that, in our example, most of the nutritional content was lost during food product processing.

High-throughput, Microscale Protocol for the Analysis of Processing Parameters and Nutritional Qualities in Maize Zea mays L.

Here, we present a microscale protocol for processing grain samples and for incorporating this microscale approach into a high-throughput analytical pipeline. This is a higher throughput adaptation of currently available protocols.

Maize is an important grain crop in the United States and worldwide. However, maize grain must be processed prior to human consumption. Furthermore, whole ...

Assessing the Particulate Matter Removal Abilities of Tree Leaves

Based on the conventional cleaning methods (water cleaning (WC) + brush cleaning (BC)), this study evaluated the influence of ultrasonic cleaning (UC) on collecting various sized particulate matter (PM) retained on leaf surfaces. We further characterized the retention efficiency of leaves to various sized PM, which will help to assess the abilities of urban trees to remove PM from ambient air quantitatively. 
Taking three broadleaf tree species (Ginkgo biloba, Sophora japonica, and Salix babylonica) and two needleleaf tree species (Pinus tabuliformis and Sabina chinensis) as the research objects, leaf samples were collected 4 days (short PM retention period) and 14 days (long PM retention period) after the latest rainfall. PM retained on the leaf surfaces was collected by means of WC, BC, and UC in sequence. Then, retention efficiencies of leaves (AEleaf) to three types of the various sized PM, including easily removable PM (ERP), difficult-to-remove PM (DRP), and totally removable PM (TRP), were calculated. Only around 23%-45% of the total PM retained on leaves could be cleaned off and collected by WC. When the leaves were cleaned through WC+BC, the underestimation of the PM retention capacity of different tree species was in the range of 29%-46% for various sized PM. Almost all PM retained on leaves could be removed if UC was supplemented to WC+BC. 
In conclusion, if the UC was complemented after the conventional cleaning methods, more PM on leaf surfaces could be eluted and collected. The procedure developed in this study can be used for assessing the PM removal abilities of different tree species.

The ultrasonic cleaning method was applied to elute the particulate matter (PM) retained on leaf surfaces after PM was eluted by the conventional cleaning methods (water cleaning only or water cleaning plus brush cleaning). The methodology can help to improve the estimation accuracy for PM retention capacity of leaves.

Based on the conventional cleaning methods (water cleaning (WC) + brush cleaning (BC)), this study evaluated the influence of ultrasonic cleaning (UC) on ...