Name: visualizing early infection sites of rice blast disease (magnaporthe oryzae) on barley (hordeum vulgare) using a basic microscope and a smartphone
Uploaded: 2023-03-17T15:00:00
Description: Watch this Scientific Journal Video about Visualizing Early Infection Sites of Rice Blast Disease Magnaporthe oryzae on Barley Hordeum vulgare Using a Basic Microscope and a Smartphone at JoVE.com

The authors acknowledge funding from the USDA-NIFA award 2016-67013-24816.

1. Preparation of experimental materials
<ol>
	<li>Prepare oatmeal agar (OMA) by blending oatmeal until it is a fine powder. Add 25 g of oatmeal powder and 15 g of agar to 500 mL of ddH2O, and autoclave on media cycle (alternatively bring to a boil for 20 min). Pour the media into sterile 60 mm Petri dishes. 
	NOTE: Other media types that induce sporulation, such as V8 agar, are acceptable for this protocol.</li>
	<li>Plate M. oryzae filter stocks directly onto the OMA plates u...

<ol>
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A., 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+genome+sequence+of+the+rice+blast+fungus+Magnaporthe+grisea.">The genome sequence of the rice blast fungus Magnaporthe grisea.</a> Nature. 434 (7036), 980-986 (2005).</li><li>Wilson, R. A., Talbot, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Under+pressure:+investigating+the+biology+of+plant+infection+by+Magnaporthe+oryzae.">Under pressure: investigating the biology of plant infection by Magnaporthe oryzae.</a> Nature Reviews. Microbiology. 7 (3), 185-195 (2009).</li><li>Giraldo, M. 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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emergence+of+wheat+blast+in+Bangladesh+was+caused+by+a+SouthAmerican+lineage+of+Magnaporthe+oryzae.">Emergence of wheat blast in Bangladesh was caused by a SouthAmerican lineage of Magnaporthe oryzae.</a> BMC Biology. 14 (1), 84 (2016).</li><li>Langridge, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Economic+and+Academic+Importance+of+Barley.">Economic and Academic Importance of Barley.</a> The Barley Genome. Compendium of Plant Genomes. , 1-10 (2018).</li><li>Heath, M. C., Valent, B., Howard, R. J., Chumley, F. 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+of+two+strains+of+Magnaporthe+grisea+with+rice,+goosegrass,+and+weeping+lovegrass.">Interactions of two strains of Magnaporthe grisea with rice, goosegrass, and weeping lovegrass.</a> Canadian Journal of Botany. 68 (8), 1627-1637 (1990).</li><li>Gowda, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+analysis+of+rice-blast+fungus+Magnaporthe+oryzae+field+isolates+from+southern+India.">Genome analysis of rice-blast fungus Magnaporthe oryzae field isolates from southern India.</a> Genomics Data. 5, 284-291 (2015).</li><li>Luginbuehl, L. H., El-Sharnouby, S., Wang, N., Hibberd, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+reporters+for+functional+analysis+in+rice+leaves.">Fluorescent reporters for functional analysis in rice leaves.</a> Plant Direct. 4 (2), 00188 (2020).</li><li>Fernandez, J., Wilson, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Why+no+feeding+frenzy?+Mechanisms+of+nutrient+acquisition+and+utilization+during+infection+by+the+rice+blast+fungus+Magnaporthe+oryzae.">Why no feeding frenzy? Mechanisms of nutrient acquisition and utilization during infection by the rice blast fungus Magnaporthe oryzae.</a> Molecular Plant-Microbe Interactions. 25 (10), 1286-1293 (2012).</li><li>Cooper, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identifying+Genetic+Control+of+Reactive+Oxygen+Species+in+Magnaporthe+oryzae+(the+Rice+Blast+Fungus)+through+Development,+Screening,+and+Characterization+of+a+Random+Insert+Mutant+Library.">Identifying Genetic Control of Reactive Oxygen Species in Magnaporthe oryzae (the Rice Blast Fungus) through Development, Screening, and Characterization of a Random Insert Mutant Library.</a> University of Delaware. , (2022).</li><li>Zhang, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=al.The+plant+infection+test:+Spray+and+wound-mediated+inoculation+with+the+plant+pathogen+Magnaporthe+grisea.">al.The plant infection test: Spray and wound-mediated inoculation with the plant pathogen Magnaporthe grisea.</a> Journal of Visualized Experiments. (138), e57675 (2018).</li><li>Koga, H., Dohi, K., Nakayachi, O., Mori, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+inoculation+method+of+Magnaporthe+grisea+for+cytological+observation+of+the+infection+process+using+intact+leaf+sheaths+of+rice+plants.">A novel inoculation method of Magnaporthe grisea for cytological observation of the infection process using intact leaf sheaths of rice plants.</a> Physiological and Molecular Plant Pathology. 64 (2), 67-72 (2004).</li><li>Hamer, J. E., Howard, R. J., Chumley, F. G., Valent, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mechanism+for+surface+attachment+in+spores+of+a+plant+pathogenic+fungus.">A mechanism for surface attachment in spores of a plant pathogenic fungus.</a> Science. 239 (4837), 288-290 (1988).</li><li>Khang, C. 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There are many commonly used assays available to test M. oryzae strains that provide a macroscopic-level visual of a compatible or incompatible infection response, such as spray or droplet inoculations, and the use of rating systems to quantifylesion sizes13,14. Another common assay for M. oryzae is to test the ability of the pathogen to form its specialized penetration structure, the apppressorium15. Described here is an...

Magnaporthe oryzae, the rice blast fungus, infects an assortment of grain crops, including barley, wheat, and rice1. This pathogen causes devastating diseases and poses a worldwide threat to these valuable crops, causing complete crop loss if not controlled. Many labs around the world focus on rice blast disease because of its global threat and its attributes as an excellent model for plant-fungal interactions2. It has been fully sequenced, and the genetics of its infective cycle, particularly the early events, have been established3,4. The life cycle begins with a spore germinating on a leaf surface, forming the specialized penetration structure called the appressorium. The appressorium penetrates the leaf tissue, and infection continues with the development of lesions which start the process of sporulation and spread disease4. Preventing any of these early events would drastically inhibit this devastating disease. Consequently, most current research on blast disease has been focused on the early infection steps, from the germinated conidia forming an appressorium to the development of the invasive hyphae and the biotrophic interfacial complex (BIC)5.
The vast amount of research on blast disease has been conducted in rice, even though M. oryzae is a significant pathogen for a variety of crops, and newly evolved strains are emerging as a global threat to wheat6. While rice is one of the top three staple crops used to feed the population, along with wheat and corn, barley is the fourth cereal grain in terms of livestock feed and beer production7. As the craft beer industry grows, so does the economic value of barley. There are distinct advantages of using M. oryzae and barley as a pathosystem to study blast disease. First, there are strains of M. oryzae that infect only barley, as well as strains that can infect multiple grass species. For example, 4091-5-8 infects primarily only barley, while Guy11 and 70-15 can infect both barley and rice8. These strains are genetically similar, and the infection process is comparable9. Second, under standard laboratory and greenhouse conditions, barley is easier to grow, as it doesn&#39;t have the complicated requirements of rice (concise temperature control, high humidity, specific light spectra). There are also imaging challenges with rice due to the hydrophobicity of the leaf surface, which barley does not exhibit10.
This protocol presents a simple method for isolating and effectively utilizing barley leaf sheaths for microscopic analysis of multiple infection stages, using common laboratory supplies and a smartphone for data collection. This method for the barley leaf sheath assay is adaptable for labs across the world as it requires minimal supplies, and yet provides a clear picture of the microscopic interaction between the pathogen and the first few cells it infects. Whereas pathogenicity assays, such as a spray or droplet inoculation, can provide a macro view of the pathogen&#39;s ability to form lesions, this assay allows the researcher to visualize specific steps of early infection, from pre-penetration events to colonization of epidermal cells. Further, researchers can easily compare infection with the wild-type fungus to infection with a mutant reduced in virulence.

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	<tbody>
		<tr>
			<td>Acetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>A6283</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell phone&#160;</td>
			<td>Google&#160;</td>
			<td>Pixel 4A</td>
			<td>Any smartphone with a rear facing camera that can be mounted in an a holder will suffice.&#160;</td>
		</tr>
		<tr>
			<td>Cell phone Microscope adapter</td>
			<td>Vankey</td>
			<td>B01788LT3S</td>
			<td>https://www.amazon.com/Vankey-Cellphone-Telescope-Binocular-Microscope/dp/B01788LT3S/ref=sr_1_2_sspa?keywords=vankey+cellphone+telescope+adapter+mount&#38;qid=1662568182&#38;sprefix= 
			vankey+%2Caps%2C63&#38;sr=8-2 
			-spons&#38;psc=1&#38;spLa=ZW5jcnlwd 
			GVkUXVhbGlmaWVyPUFKNklBR 
			jlCREJaMEcmZW5jcnlwdGVkSWQ 
			9QTA2MDMxNjhBRFYxQTMzNk9E 
			M0YmZW5jcnlwdGVkQWRJZD1BM 
			DQxMzAzOTMxNzI1TzE3M1ZGTEI 
			md2lkZ2V0TmFtZT1zcF9hdGYmY 
			WN0aW9uPWNsaWNrUmVkaXJlY3 
			QmZG9Ob3RMb2dDbGljaz10cnVl</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G5516</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>AmScope</td>
			<td>FM690TC</td>
			<td>40x&#8211;2500x Trinocular upright epi-fluorescence microscope</td>
		</tr>
		<tr>
			<td>Oatmeal old fashioned rolled oats</td>
			<td>Quaker</td>
			<td>N/A</td>
			<td>https://www.amazon.com/Quaker-Oats-Old-Fashioned-Pack/dp/B00IIVBNK4/ref=asc_df_B00IIVBNK4/?tag=hyprod-20&#38;linkCode=df0 
			&#38;hvadid=312253390021&#38;hvpos= 
			&#38;hvnetw=g&#38;hvrand=98212627704 
			6839544&#38;hvpone=&#38;hvptwo=&#38;hvq 
			mt=&#38;hvdev=c&#38;hvdvcmdl=&#38;hvlocint 
			=&#38;hvlocphy=9007494&#38;hvtargid 
			=pla-568492637928&#38;psc=1</td>
		</tr>
		<tr>
			<td>ProMix BX</td>
			<td>ProMix</td>
			<td>1038500RG</td>
			<td></td>
		</tr>
		<tr>
			<td>Rectangular coverglass</td>
			<td>Corning</td>
			<td>CLS2975245</td>
			<td></td>
		</tr>
		<tr>
			<td>Slides, microscope</td>
			<td>Sigma-Aldrich</td>
			<td>S8902</td>
			<td></td>
		</tr>
		<tr>
			<td>Stage micrometer&#160;</td>
			<td>OMAX</td>
			<td>A36CALM7</td>
			<td>0.1 mm and 0.01 mm Microscope calibration slide</td>
		</tr>
		<tr>
			<td>Trypan blue</td>
			<td>Sigma-Aldrich</td>
			<td>T6146</td>
			<td></td>
		</tr>
	</tbody>
</table>

visualizing early infection sites of rice blast disease (magnaporthe oryzae) on barley (hordeum vulgare) using a basic microscope and a smartphone

Understanding how plants and pathogens interact, and whether that interaction culminates in defense or disease, is required to develop stronger and more sustainable strategies for plant health. Advances in methods that more effectively image plant-pathogen samples during infection and colonization have yielded tools such as the rice leaf sheath assay, which has been useful in monitoring infection and early colonization events between rice and the fungal pathogen, Magnaporthe oryzae. This hemi-biotrophic pathogen causes severe disease loss in rice and related monocots, including millet, rye, barley, and more recently, wheat. The leaf sheath assay, when performed correctly, yields an optically clear plant section, several layers thick, which allows researchers to perform live-cell imaging during pathogen attack or generate fixed samples stained for specific features. Detailed cellular investigations into the barley-M. oryzae interaction have lagged behind those of the rice host, in spite of the growing importance of this grain as a food source for animals and humans and as fermented beverages. Reported here is the development of a barley leaf sheath assay for intricate studies of M. oryzae interactions during the first 48 h post-inoculation. The leaf sheath assay, regardless of which species is being studied, is delicate; provided is a protocol that covers everything, from barley growth conditions and obtaining a leaf sheath, to inoculation, incubation, and imaging of the pathogen on plant leaves. This protocol can be optimized for high-throughput screening using something as simple as a smartphone for imaging purposes.

A depiction of the initial workflow for this technique is displayed in Figure 1. The sheaths were harvested from 14-day-old susceptible &#34;Lacey&#34; barley plants (H. vulgare). The conidia were harvested from 10-day-old sporulating M. oryzae OMA plates, with a conidial suspension prepared using sterile ddH2O for a final concentration of 5 x 104 spores per mL. The inoculum suspension was directly applied to the leaf sheaths, which were secured to ste...

Watch this Scientific Journal Video about Visualizing Early Infection Sites of Rice Blast Disease Magnaporthe oryzae on Barley Hordeum vulgare Using a Basic Microscope and a Smartphone at JoVE.com

Visualizing Early Infection Sites of Rice Blast Disease Magnaporthe oryzae on Barley Hordeum vulgare Using a Basic Microscope and a Smartphone

This is a straightforward protocol of a barley leaf sheath assay using minimal reagents and common laboratory equipment (including a basic smartphone). The purpose is to visualize the early infection process of blast disease in labs without access to advanced microscopy equipment.

Visualizing Early Infection Sites of Rice Blast Disease (Magnaporthe oryzae) on Barley (Hordeum vulgare) Using a Basic Microscope and a Smartphone

Understanding how plants and pathogens interact, and whether that interaction culminates in defense or disease, is required to develop stronger and more ...

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