This work was supported by the Special Scientific Research Project of the Beijing Agriculture University (YQ201603) and the Scientific Project of the Beijing Educational Committee (KM201610020005).

1. Spray Inoculation with a Suspension of M. grisea Conidia
<ol>
	<li>Fungal culture for M.grisea
	<ol>
		<li>Prepare the oatmeal tomato agar (OTA) culture medium for fungal strains.</li>
		<li>Weigh 30 - 50 g of oatmeal, add this to 800 mL of distilled/deionized water (ddH2O) and boil the mixture for 30 min in the electric pot.</li>
		<li>Filter the boiled oatmeal juice into the beaker through a piece of gauze.</li>
		<li>Add 150 mL of tomato juice and 20 g of agar to the filtrate in the beaker and add ddH2O up to 1,000 mL.</li>
	</ol>
	</li>
	<li>Preparation of experimental materials
	<ol>
		<li>Soak about 50 seeds of rice (Oryza sativa) cultivar Lijiangxintuan-heigu (LTH) in ddH2O for 3 d or soak about 50 seeds of barley (Hordeum vulgare cv Golden Promise) in ddH2O for about 1 d.</li>
		<li>Wrap the seeds of rice or barley in moist gauze and germinate them on moist filter paper in about 30 Petri dishes with a diameter of 10 cm x 10 cm at 28 &#176;C. The rice seed germination time is about 2 - 3 d, the barley seed germination time is about 2 d. The relative humidity in the greenhouse is approximately 70%.</li>
		<li>Plant the seedlings of the rice or barley in pots using autoclaved potting soil and watering and then cover them with a layer of vermiculite.</li>
		<li>Place the plants in a suitable glasshouse or growth cabinet at 25 &#176;C for about 1 ~ 2 weeks.</li>
		<li>Cut 3 layers of filter paper in circles with sterile surgical scissors (each circle should have an 8 cm diameter) and place them onto 100 mm sterile plastic plates.</li>
		<li>Add ddH2O to each dish to soak the filter paper.</li>
		<li>Make sure the filter paper is completely wet but add no extra water.</li>
		<li>Remove any excess water with a vacuum pump.</li>
		<li>Place 2 sterile toothpicks into the culture dish to support the rice/barley leaves; space the toothpicks ~2 - 3 cm apart.</li>
		<li>Collect the leaves of the rice 2 weeks after sowing the seeds or take the leaves of the barley 7 d after sowing the seeds.</li>
		<li>Using 4- to 6-leaf seedlings of rice/barley, cut the lower part of the stem at ~5 cm from the top and collect the leaves.</li>
	</ol>
	</li>
	<li>Spray inoculation protocol
	<ol>
		<li>Culture the fungal strain on OTA plates in a thermostatic incubator (25 &#176;C) for ~4 d.</li>
		<li>Add ~2 mL of ddH2O using a 0.5 - 5 mL pipette to each 4-day-old plate.</li>
		<li>With an inoculation loop, scrape the mycelia of the M. grisea wild-type strain and the mutant strain into mycelia debris.</li>
		<li>Collect the mycelia debris and transfer it to a new OTA plate. Take the mycelia debris and blow dry in the clean bench.</li>
		<li>Cover the plate with 3 layers of gauze to ensure the humidity required for the growth of the conidia in a greenhouse at 25 &#176;C in the day (14 h) and 23 &#176;C in the night (10 h) for 24 - 48 h.</li>
		<li>Add 2 mL of ddH2O to each dish and scrape the conidia gently with sterile cotton swabs followed by a filtration through 2 layers of lens paper. Be careful not to scratch the surface of the culture medium.</li>
		<li>Transfer the conidia suspension into a new 50 mL tube with a 100 - 1,000 &#181;L pipette.</li>
		<li>Centrifuge for 5 min at a minimum of 5,000 x g at 25 &#176;C.</li>
		<li>Remove the supernatant and resuspend the pellet to give 2 x 104 conidia per mL in a 0.025% (v/v) Tween-20 solution. The Tween-20 solution is usually about 10 - 20 mL.</li>
		<li>Pour the spore suspension into a hand-held sprayer.</li>
		<li>Spay about 10 mL of the conidial suspension onto the rice leaves of 2-week-old rice seedlings or barley leaves of 7-day-old barley seedlings and incubate them at 25 &#176;C in a dark, humid chamber for ~24 h. Spray the control plants with the 0.025% (v/v) Tween-20 solution.</li>
		<li>Transfer the leaves into another moist chamber under fluorescent light at 25 &#176;C for a photoperiod of 12 h.</li>
		<li>Record the disease symptoms at 5 d after the inoculation. Examine the diseased rice/barley blades of ~6 cm in length. The evaluation standard is according to the scoring system for blast resistance of the International Rice Research Institute (IRRI). The details of the scoring system for blast resistance are shown in Table 1.</li>
		<li>Photograph the leaves to evaluate the infection of the tested strains. The infection was assessed by the number of lesions per 3.6 cm2.</li>
	</ol>
	</li>
</ol>
2. Wounding Inoculation with Mycelium Cubes or Droplets of Conidial Suspension of M. grisea
<ol>
	<li>Using an anatomical needle, scrape three 2 - 3 cm long wounds in the main veins of detached rice/barley leaves; take care not to penetrate the leaves.</li>
	<li>Put the scraped leaves on toothpicks and spray a 0.02% (v/v) Tween-20 solution on the leaves to form a layer of droplets.</li>
	<li>Cut a 0.5 cm x 0.5 cm mycelial plug for each M. grisea strain (wild-type, mutant, complement strains, or other test strains) from an OTA plate.</li>
	<li>Put the mycelial plugs or 25 &#181;L droplets of conidial suspension onto the wounded leaves and incubate the leaves at 25 &#176;C in a humid chamber for 3 - 8 d.</li>
	<li>Examine the lesions at 5 - 7 d post-inoculation. The method of examination is the same as it was for the spray inoculation method (see step 1.3.13).</li>
	<li>Examine the diseased rice/barley blades of ~6 cm in length and photograph them to evaluate the infection of the tested strains. The infection was assessed by the number of lesions per 3.6 cm2.</li>
</ol>

<ol><li>Li, W. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+natural+allele+of+a+transcription+factor+in+rice+confers+broad-spectrum+blast+resistance%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">A natural allele of a transcription factor in rice confers broad-spectrum blast resistance.</a> Cell. 170 (1), 114-126 (2017).</li>
<li>Chi, M. H., Park, S. Y., Kim, S., Lee, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+pathogenicity+gene+is+required+in+the+rice+blast+fungus+to+suppress+the+basal+defenses+of+the+host%5BTitle%5D)+AND+%22PLoS+Pathogens%22%5BJournal%5D)">A novel pathogenicity gene is required in the rice blast fungus to suppress the basal defenses of the host.</a> PLoS Pathogens. 5 (4), 1000401(2009).</li>
<li>Jia, Y., Valent, B., Lee, F. N. <a target="_blank" href="https://apsjournals.apsnet.org/doi/10.1094/PDIS.2003.87.2.129">Determination of host responses to Magnaporthe grisea.on detached rice leaves using a spot inoculation method.</a> Plant Disease. 87 (2), 129-133 (2003).</li>
<li>Ebbole, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Magnaporthe+as+a+model+for+understanding+host-pathogen+interactions%5BTitle%5D)+AND+%22Annual+Review+of+Phytopathology%22%5BJournal%5D)">Magnaporthe as a model for understanding host-pathogen interactions.</a> Annual Review of Phytopathology. 45, 437-456 (2007).</li>
<li>Hamer, J. E., Talbot, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Infection-related+development+in+the+rice+blast+fungus+Magnaporthe+grisea%5BTitle%5D)+AND+%22Current+Opinion+in+Microbiology%22%5BJournal%5D)">Infection-related development in the rice blast fungus Magnaporthe grisea.</a> Current Opinion in Microbiology. 1 (6), 693-697 (1998).</li>
<li>Howard, R. J., Valent, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Breaking+and+entering%3A+host+penetration+by+the+fungal+rice+blast+pathogen+Magnaporthe+grisea%5BTitle%5D)+AND+%22Annual+Review+of+Microbiology%22%5BJournal%5D)">Breaking and entering: host penetration by the fungal rice blast pathogen Magnaporthe grisea.</a> Annual Review of Microbiology. 50, 491-512 (1996).</li>
<li>Chen, X. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((N-Glycosylation+of+Effector+Proteins+by+an+%CE%B1-1%2C3-+Mannosyltransferase+Is+Required+for+the+Rice+Blast+Fungus+to+Evade+Host+Innate+Immunity%5BTitle%5D)+AND+%22The+Plant+Cell%22%5BJournal%5D)">N-Glycosylation of Effector Proteins by an α-1,3- Mannosyltransferase Is Required for the Rice Blast Fungus to Evade Host Innate Immunity.</a> The Plant Cell. 26 (3), 1360-1376 (2014).</li>
<li>Zhang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((M.ARG1%2C+MoARG5%2C6+and+MoARG7+involved+in+arginine+biosynthesis+are+essential+for+growth%2C+conidiogenesis%2C+sexual+reproduction%2C+and+pathogenicity+in+Magnaporthe+oryzae%5BTitle%5D)+AND+%22Microbiological+Research%22%5BJournal%5D)">M.ARG1, MoARG5,6 and MoARG7 involved in arginine biosynthesis are essential for growth, conidiogenesis, sexual reproduction, and pathogenicity in Magnaporthe oryzae.</a> Microbiological Research. 180, 11-22 (2015).</li>
<li>Du, Y. X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+serine%2Fthreonine-protein+phosphatase+PP2A+catalytic+subunit+is+essential+for+asexual+development+and+plant+infection+in+Magnaporthe+oryzae%5BTitle%5D)+AND+%22Current+Genetics%22%5BJournal%5D)">A serine/threonine-protein phosphatase PP2A catalytic subunit is essential for asexual development and plant infection in Magnaporthe oryzae.</a> Current Genetics. 59 (1-2), 33-41 (2013).</li>
<li>Yang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+protein+com1+is+required+for+normal+conidium+morphology+and+full+virulence+in+Magnaporthe+oryzae%5BTitle%5D)+AND+%22Molecular+Plant-Microbe+Interactions%22%5BJournal%5D)">A novel protein com1 is required for normal conidium morphology and full virulence in Magnaporthe oryzae.</a> Molecular Plant-Microbe Interactions. 23 (1), 112-123 (2010).</li>
<li>Cao, Z. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+ash1-like+protein+MoKMT2H+null+mutant+is+delayed+for+conidium+germination+and+pathogenesis+in+Magnaporthe+oryzae%5BTitle%5D)+AND+%22BioMed+Research+International%22%5BJournal%5D)">An ash1-like protein MoKMT2H null mutant is delayed for conidium germination and pathogenesis in Magnaporthe oryzae.</a> BioMed Research International. 2016, 1575430(2016).</li>
<li>Bryan, G. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+single+amino+acid+difference+distinguishes+resistant+and+susceptible+alleles+of+the+rice+blast+resistance+gene+Pi-ta%5BTitle%5D)+AND+%22The+Plant+Cell%22%5BJournal%5D)">A single amino acid difference distinguishes resistant and susceptible alleles of the rice blast resistance gene Pi-ta.</a> The Plant Cell. 12 (11), 2033-2045 (2000).</li>
<li>Zhou, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Plant+pathology%3A+a+life+and+death+struggle+in+rice+blast+disease%5BTitle%5D)+AND+%22Current+Biology%22%5BJournal%5D)">Plant pathology: a life and death struggle in rice blast disease.</a> Current Biology. 26 (18), 843-845 (2016).</li>
<li>Guo, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((MoGrr1%2C+a+novel+F-box+protein%2C+is+involved+in+conidiogenesis+and+cell+wall+integrity+and+is+critical+for+the+full+virulence+of+Magnaporthe+oryzae%5BTitle%5D)+AND+%22Applied+Microbiology+and+Biotechnology%22%5BJournal%5D)">MoGrr1, a novel F-box protein, is involved in conidiogenesis and cell wall integrity and is critical for the full virulence of Magnaporthe oryzae.</a> Applied Microbiology and Biotechnology. 99 (19), 8075-8088 (2015).</li>
<li>Talbot, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((On+the+trail+of+a+cereal+killer%3A+Exploring+the+biology+of+Magnaporthe+grisea%5BTitle%5D)+AND+%22Annual+Review+of+Microbiology%22%5BJournal%5D)">On the trail of a cereal killer: Exploring the biology of Magnaporthe grisea.</a> Annual Review of Microbiology. 57, 177-202 (2009).</li>
<li>Wilson, R. A., Talbot, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Under+pressure%3A+investigating+the+biology+of+plant+infection+by+Magnaporthe+oryzae%5BTitle%5D)+AND+%22Nature+Reviews+Microbiology%22%5BJournal%5D)">Under pressure: investigating the biology of plant infection by Magnaporthe oryzae.</a> Nature Reviews Microbiology. 7, 185-195 (2009).</li>
<li>Jia, Y. L., Lee, F. N., McClung, A. <a target="_blank" href="https://apsjournals.apsnet.org/doi/10.1094/PDIS-93-6-0639">Determination of Resistance Spectra of the Pi-ta and Pi-k Genes to U.S. Races of Magnaporthe oryzae Causing Rice Blast in a Recombinant Inbred Line Population.</a> Plant Disease. 93, 639-644 (2009).</li>
<li>Peng, Y. L., Shishiyama, J. <a target="_blank" href="http://www.nrcresearchpress.com/doi/abs/10.1139/b88-107?journalCode=cjb1#.WvnHgogvzIU">Temporal sequence of cytological events in rice leaves infected with Pyricularia oryzae.</a> Canadian Journal of Botany. 66 (4), 730-735 (1988).</li>
</ol>

The authors have nothing to disclose.

Plant disease resistance genes play an essential role in preventing infections by pathogens, including fungal pathogens1,12. Rice blast has been used as a model to understand the nature of pathogen population structures and to identify plant resistance genes4. Therefore, it is necessary to examine the disease resistance genotype and avirulence genotypes of the main varieties of agricultural plants on a large scale to identify disease-resistant plants that can be cultivated continuously. Furthermore, the advantages of avirulence genotypes of field pathogens necessitate the rational distribution of the different resistant genotypes of host varieties12,13. However, current inoculation methods commonly interfere with the screening for resistance and pathogen identification14,15,16,17.
Here, we present a rapid and accurate method to test for plant infection. This inoculation method is suitable for the identification of resistant plants during breeding trials and of the phenotype of a progeny population during the cloning of resistance genes. Here, we established a method for inoculating wounded plant leaves with M. grisea. Because the host resistance plays a major role in preventing the spreading of a pathogen, this in vitro method, which produced wounds on the tested rice leaves and inoculated the rice blast on the wound, is convenient for creating an infection directly into the leaves without having to penetrate the leaf epidermis and the epidermal cell wall. Furthermore, this inoculation method is suitable for different leaf ages. The inoculation results are stable and accurate. The method of wounding inoculation can be used for inoculation with mycelia to identify the pathogenicity of fungal strains which produce only low amounts of conidia.
This protocol will further contribute to our understanding of the pathogenic mechanisms that are conserved in fungi, as well as the pathogen-specific factors that permit a fungus to resist and suppress the innate immunity of its host13. However, the wound inoculation is not applicable when trying to determine the rate of a conidial invasion and mycelia expansion. But in the natural state, the spray inoculation method can better reflect the pathogenicity of a pathogen. The spray inoculation method is easy to operate and helps to improve work efficiency. Taken together, these results indicating an accurate and stable inoculation method are necessary for screening plant resistance genes and determining pathogenicity.

Rice blast, caused by M. grisea, is one of the most serious diseases for rice varieties worldwide1,2. The process by which M. grisea infects host plants includes a conidia production and surface attachment, a conidia germination and appressorium formation, a formation of the penetration peg and infectious hypha differentiation, and a disease spread3. All these stages are common in many other plant pathogenic fungi, and, indeed, a blockade of any single stage prevents the infection of host plants. Owing to its economic importance and genetic tractability, M. grisea has emerged as a model organism for studying the mechanisms of plant-fungal pathogen interactions1,4. Therefore, studying the molecular basis of these developmental stages in M. grisea will help to elucidate the molecular mechanisms underlying fungal pathogenicity and the identification of candidate target genes for screening and designing novel fungicides5.
Recent reports concerning M. grisea infection have focused on the molecular mechanisms of the pre-penetration stages, especially the conidiation, the appressorium formation, the penetration pegs, and the infectious growth3,6. Therefore, it is essential to develop a detailed protocol to test M. grisea infection. Herein, we present a detailed method for an infection test that utilizes spray-mediated infection assays with a conidial suspension and the inoculation of wounds with mycelial plugs of M. grisea. In this report, the protocol focuses on the culture of strains, the preparation of the conidiation solution for spraying, and the mycelial plug-mediated inoculation of plants with M. grisea. These steps are described in detail below, and a schematic view showing the entire workflow of the method and a typical lesion are shown in Figures 1 and 2, respectively.

<table><tbody><tr><td>Filter paper</td><td>GE Healthcare brand(Sweden)&amp;nbsp;&amp;nbsp;</td><td>10311387</td><td></td></tr><tr><td>50-mL tube</td><td>CORNING(Amercia)</td><td>430290</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf(Amercia)</td><td>5804R</td><td></td></tr><tr><td>Culture dish</td><td>Thermofisher(Amercia)</td><td>150326</td><td></td></tr><tr><td>0.5-5 mL pipette</td><td>Eppendorf</td><td>&amp;nbsp;4920000105</td><td></td></tr><tr><td>100-1000uL pipette</td><td>Eppendorf</td><td>4920000083</td><td></td></tr><tr><td>Vacuum pump</td><td>Leybold</td><td>D25B</td><td></td></tr><tr><td>Dissection needle</td><td>FST</td><td>26000-35</td><td></td></tr><tr><td>Incubator</td><td>MEMMERT</td><td>PYX313</td><td></td></tr><tr><td>Inoculation ring</td><td>Greiner Bio One</td><td>731175</td><td></td></tr></tbody></table>

the plant infection test: spray and wound-mediated inoculation with the plant pathogen magnaporthe grisea

Plants possess a powerful system to defend themselves against potential threats by pathogenic fungi. For agriculturally important plants, however, current measures to combat such pathogens have proved too conservative and, thus, not sufficiently effective, and they can potentially pose environmental risks. Therefore, it is extremely necessary to identify host-resistance factors to assist in controlling plant diseases naturally through the identification of resistant germplasm, the isolation and characterization of resistance genes, and the molecular breeding of resistant cultivars. In this regard, there is need to establish an accurate, rapid, and large-scale inoculation method to breed and develop plant resistance genes. The rice blast fungal pathogen Magnaporthe grisea causes severe disease symptoms and yield losses. Recently, M. grisea has emerged as a model organism for studying the mechanisms of plant-fungal pathogen interactions. Hence, we report the development of a plant virulence test method that is specific for M. grisea. This method provides for both spray inoculation with a conidial suspension and wounding inoculation with mycelium cubes or droplets of conidial suspension. The key step of the wounding inoculation method for detached rice leaves is to make wounds on plant leaves, which avoids any interference caused by host penetration resistance. This spray/wounding protocol contributes to the rapid, accurate, and large-scale screening of the pathotypes of M. grisea isolates. This integrated and systematic plant infection method will serve as an excellent starting point for gaining a broad perspective of issues in plant pathology.

The entire workflow for the technique is shown in Figure 1. The plant infection assays were performed on 14-day-old susceptible rice seedlings (O. sativa cv CO-39) or susceptible 7-day-old barley leaves (H. vulgare cv Golden Promise)7,8,9. To test for an infection on the rice leaves, a conidial suspension (1.0 x 105 spores/mL) of the M. grisea wild-type strain P131 and the Com1 deletion mutant strain were prepared and then sprayed onto the leaf sheaths of the 14-day-old susceptible CO-39 seedlings, which were then kept in a moist chamber for 5 d10. The P131-inoculated CO-39 leaves displayed the typical robust lesions of rice blast, but the Com1-inoculated leaves showed obvious infection defects and could not elicit a full infection (Figure 2A). The wild-type strain P131 is a strain obtained by You-Liang Peng in 198818. The MoKMT2H null mutant (&#916;MoKMT2H) was obtained by Cao et al. in our lab using a target gene replacement strategy11. The Com1 mutant was isolated by Yang et al. in You-Liang Peng&#39;s lab10.
To check whether the M. grisea &#916;MoKMT2H could infect the host cells through wounds, abraded leaves of wild-type rice plants were inoculated with mycelial plugs of &#916;MoKMT2H via the spray method or wounding method11. The leaves that were spray-inoculated with &#916;MoKMT2H revealed no obvious defects in the &#916;MoKMT2H infection compared with the P131/wild-type strain; however, obvious defects were observed for leaves inoculated with &#916;MoKMT2H&#160;via wounds (Figure 2A). To further test the plant infection with barley leaves, healthy or wounded leaves (cv Golden Promise) were inoculated with conidial droplets or mycelial plugs, respectively, of Com1, &#916;MoKMT2H, or P131. At 5 d post-inoculation, typical rice blast lesions had fully developed on the leaves inoculated with either the &#916;MoKMT2H or P131 strain, whereas fewer and smaller lesions were found on the leaves inoculated with the Com1 mutant (Figure 2B).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57675/57675fig1.jpg" /> 
Figure 1: A scheme illustrating plant infection. Plant seeds were germinated in soil in plastic pots (50 mm2 x 50 mm deep, with a drainage hole), two seeds per pot, and the seedlings were grown in a greenhouse. The culture and inoculations of the blast fungus M. grisea were grown on OTA plates. For the conidia spraying inoculation method, the conidia suspension was suspended in a 0.02% (v/v) Tween-20 solution and sprayed onto the rice/barley plants. For the scratched inoculation method, the scraped rice/barley plant leaves were put on plastic plates and then inoculated by a mycelial plug or the conidia suspension. And then, all the treated plant leaves were dark-cultured for 24 h and light-cultured for 12 h. Finally, the number of colonies within units of 3.6 cm2 was recorded. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57675/57675fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57675/57675fig2.jpg" /> 
Figure 2: Com1 and MoKMT2H are required for a conidium formation and pathogenicity on rice leaves. (A) This panel shows the inoculation of rice leaves via conidia spray (left) or mycelial plug (right) with conidia from the M. grisea wild-type P131, mutant Com1, or &#916;MoKMT2H. Typical leaves were observed 7 d after the mycelial plug-mediated inoculation, which was conducted on abraded rice leaves. Typical lesions were observed 5 d after the mycelial plug-mediated inoculation. (B) Barley leaves were inoculated via conidia spray (left) or mycelial plug (right). As a mock treatment control, the same volume of a 0.025% (v/v) Tween-20 solution was sprayed. For the wound inoculation, the figure of P131 has been modified from Cao et al.11. The MoKMT2H in the ascomycete fungi is a functional homolog of Ash1, which is implicated in H3K4 and H3K36 methylation11. The bar = 1 cm. The &#916;com1 mutants were significantly reduced in virulence on the rice and barley seedlings10. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57675/57675fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Scale</td>
			<td>Description</td>
		</tr>
		<tr>
			<td>1</td>
			<td>Small brown specks of pin-point size</td>
		</tr>
		<tr>
			<td>2</td>
			<td>Small roundish to slightly elongated, necrotic gray spots, about 1-2 mm in diameter ,with a distinct brown margin .Lesions are mostly found on the upper leaves</td>
		</tr>
		<tr>
			<td>3</td>
			<td>Lesion type is the same as in 2, but significant number of lesion are on theupper leaves</td>
		</tr>
		<tr>
			<td>4</td>
			<td>Typical susceptible blast lesions, 3 mm or longer, infecting less than 4% of theleaf area</td>
		</tr>
		<tr>
			<td>5</td>
			<td>Typical susceptible blast lesions, 3 mm or longer, infecting less than 4-10% ofthe leaf area</td>
		</tr>
		<tr>
			<td>6</td>
			<td>Typical susceptible blast lesions, 3 mm or longer, infecting less than 11-25% ofthe leaf area</td>
		</tr>
		<tr>
			<td>7</td>
			<td>Typical susceptible blast lesions, 3 mm or longer, infecting less than 26-50% ofthe leaf area</td>
		</tr>
		<tr>
			<td>8</td>
			<td>Typical susceptible blast lesions, 3 mm or longer, infecting less than 51-75% ofthe leaf area, many leaves dead</td>
		</tr>
		<tr>
			<td>9</td>
			<td>Typical susceptible blast lesions, 3 mm or longer, infecting more than 75% ofthe leaf area</td>
		</tr>
	</tbody>
</table>
Table 1: A scoring system for blast resistance. The table is cited from the International Rice Research Institute (IRRI).

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The Plant Infection Test: Spray and Wound-Mediated Inoculation with the Plant Pathogen Magnaporthe Grisea

Here, we present a protocol to test plant virulence with the plant pathogen Magnaporthe grisea. This report will contribute to the large-scale screening of the pathotypes of fungi isolates and serve as an excellent starting point for understanding the resistant mechanisms of plants during molecular breeding.

The Plant Infection Test: Spray and Wound-Mediated Inoculation with the Plant Pathogen Magnaporthe Grisea

Plants possess a powerful system to defend themselves against potential threats by pathogenic fungi. For agriculturally important plants, however, current ...

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12.6K Views.  Beijing University of Agriculture. Hier presenteren we een protocol om te testen van de virulentie van de plant met de plant pathogen Magnaporthe grisea. Dit verslag zal bijdragen tot de grootschalige screening van de biotypen van schimmels isolaten en dienen als een uitstekend uitgangspunt voor het begrijpen van de resistente mechanismen van planten tijdens de moleculaire veredeling.

Video: The Plant Infection Test: Spray and Wound-Mediated Inoculation with the Plant Pathogen Magnaporthe Grisea

Assay for Pathogen-Associated Molecular Pattern (PAMP)-Triggered Immunity (PTI) in Plants

To perceive potential pathogens in their environment, plants use pattern recognition receptors (PRRs) present on their plasma membranes. PRRs recognize conserved microbial features called pathogen-associated molecular patterns (PAMPs) and this detection leads to PAMP-triggered immunity (PTI), which effectively prevents colonization of plant tissues by non-pathogens1,2. The most well studied system in PTI is the FLS2-dependent pathway3. FLS2 recognizes the PAMP flg22 that is a component of bacterial flagellin. 
Successful pathogens possess virulence factors or effectors that can suppress PTI and allow the pathogen to cause disease1. Some plants in turn possess resistance genes that detect effectors or their activity, which leads to effector-triggered immunity (ETI)2.
We describe a cell death-based assay for PTI modified from Oh and Collmer4. The assay was standardized in N. benthamiana, which is being used increasingly as a model system for the study of plant-pathogen interactions5. PTI is induced by infiltration of a non-pathogenic bacterial strain into leaves. Seven hours later, a bacterial strain that either causes disease or which activates ETI is infiltrated into an area overlapping the original infiltration zone. PTI induced by the first infiltration is able to delay or prevent the appearance of cell death due to the second challenge infiltration. Conversely, the appearance of cell death in the overlapping area of inoculation indicates a breakdown of PTI. 
Four different combinations of inducers of PTI and challenge inoculations were standardized (Table 1). The assay was tested on non-silenced N. benthamiana plants that served as the control and plants silenced for FLS2 that were predicted to be compromised in their ability to develop PTI.

Assay for Pathogen-Associated Molecular Pattern PAMP-Triggered Immunity PTI in Plants

A cell death-based assay for PTI in Nicotiana benthamiana plants is described.

To perceive potential pathogens in their environment, plants use pattern recognition receptors (PRRs) present on their plasma membranes. PRRs recognize ...

Immunology and Infection

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 ...

Establishing Fungal Entomopathogens as Endophytes: Towards Endophytic Biological Control

Beauveria bassiana is a fungal entomopathogen with the ability to colonize plants endophytically. As an endophyte, B. bassiana may play a role in protecting plants from herbivory and disease. This protocol demonstrates two inoculation methods to establish B. bassiana endophytically in the common bean (Phaseolus vulgaris), in preparation for subsequent evaluations of endophytic biological control. Plants are grown from surface-sterilized seeds for two weeks before receiving a B. bassiana treatment of 108 conidia/ml (or water) applied either as a foliar spray or a soil drench. Two weeks later, the plants are harvested and their leaves, stems and roots are sampled to evaluate endophytic fungal colonization. For this, samples are individually surface sterilized, cut into multiple sections, and incubated in potato dextrose agar media for 20 days. The media is inspected every 2-3 days to observe fungal growth associated with plant sections and record the occurrence of B. bassiana to estimate the extent of its endophytic colonization. Analyses of inoculation success compare the occurrence of B. bassiana within a given plant part (i.e. leaves, stems or roots) across treatments and controls. In addition to the inoculation method, the specific outcome of the experiment may depend on the target crop species or variety, the fungal entomopathogen species strain or isolate used, and the plant's growing conditions.

This protocol demonstrates two inoculation methods to introduce the fungal entomopathogen Beauveria bassiana as an endophyte in the common bean (Phaseolus vulgaris), in preparation for subsequent evaluations of endophytic biological control.

 Beauveria bassiana is a fungal entomopathogen with the ability to colonize plants endophytically. As an endophyte, B. bassiana may play a role in ...

Bioengineering

An Effective Inoculation Method for Phytophthora capsici on Black Pepper Plants

Piper nigrum L. (black pepper) is a typical woody vine that is an economically important spice crop across the world. Black pepper production is significantly impacted by root rot disease caused by Phytophthora capsici, which has seriously influenced the industry development as a "choke point" problem. However, the molecular genetic mechanism of resistance in black pepper is unclear, leading to slow progress in the development of new black pepper varieties. An effective inoculation and precise sampling system for Phytophthora capsici on black pepper plants is essential for studying this plant-pathogen interaction. The main aim of this study is to demonstrate a detailed methodology where the basal head of black pepper is inoculated with Phytophthora capsici, while also providing a reference for the inoculation of woody vine plants. The basal head of the black pepper plant was pinpricked to damage it, and mycelial pellets covered the three holes to retain the moisture so the pathogen could infect the plant well. This method provides a better way of solving the instability that is caused by traditional inoculation methods including soil drench or root dipping. It also provides a promising means for studying the mode of action between plants and other soil-borne plant pathogens in agricultural precision breeding.

An Effective Inoculation Method for Phytophthora capsici on Black Pepper Plants

Pinpricking the basal head of the black pepper plant is a brief and time-saving method to damage it. Here, we provided detailed steps with a video for infecting black pepper plants.

Piper nigrum L. (black pepper) is a typical woody vine that is an economically important spice crop across the world. Black pepper production is ...

Genetics

Inoculation Strategies to Infect Plant Roots with Soil-Borne Microorganisms

The rhizosphere harbors a highly complex microbial community in which plant roots are constantly challenged. Roots are in close contact with a wide variety of microorganisms, but studies on soil-borne interactions are still behind those performed on aboveground organs. Although some inoculation strategies for infecting model plants with model root pathogens are described in the literature, it remains difficult to get a comprehensive methodological overview. To address this problem, three different root inoculation systems are precisely described that can be applied to gain insights into the biology of root-microbe interactions. For illustration, Verticillium species (namely, V. longisporum and V. dahliae) were employed as root invading model pathogens. However, the methods can be easily adapted to other root colonizing microbes - both pathogenic and beneficial. By colonizing the plant xylem, vascular soil-borne fungi such as Verticillium spp. exhibit a unique lifestyle. After root invasion, they spread via the xylem vessels acropetally, reach the shoot, and elicit disease symptoms. Three representative plant species were chosen as model hosts: Arabidopsis thaliana, economically important oilseed rape (Brassica napus), and tomato (Solanum lycopersicum). Step-by-step protocols are given. Representative results of pathogenicity assays, transcriptional analyses of marker genes, and independent confirmations by reporter constructs are shown. Furthermore, the advantages and disadvantages of each inoculation system are thoroughly discussed. These proven protocols can assist in providing approaches for research questions on root-microbe interactions. Knowing how plants cope with microbes in the soil is crucial for developing new strategies to improve agriculture.

This protocol presents a detailed summary of strategies to inoculate plant roots with soil-borne microbes. Exemplified for the fungi Verticillium longisporum and Verticillium dahliae, three different root infection systems are described. Potential applications and possible downstream analyses are highlighted, and advantages or disadvantages are discussed for each system.

The rhizosphere harbors a highly complex microbial community in which plant roots are constantly challenged. Roots are in close contact with a wide ...

Bacterial Leaf Infiltration Assay for Fine Characterization of Plant Defense Responses using the Arabidopsis thaliana-Pseudomonas syringae Pathosystem

In the absence of specialized mobile immune cells, plants utilize their localized programmed cell death and Systemic Acquired Resistance to defend themselves against pathogen attack. The contribution of a specific Arabidopsis gene to the overall plant immune response can be specifically and quantitatively assessed by assaying the pathogen growth within the infected tissue. For over three decades, the hemibiotrophic bacterium Pseudomonas syringae pv. maculicola ES4326 (Psm ES4326) has been widely applied as the model pathogen to investigate the molecular mechanisms underlying the Arabidopsis immune response. To deliver pathogens into the leaf tissue, multiple inoculation methods have been established, e.g., syringe infiltration, dip inoculation, spray, vacuum infiltration, and flood inoculation. The following protocol describes an optimized syringe infiltration method to deliver virulent Psm ES4326 into leaves of adult soil-grown Arabidopsis plants and accurately screen for enhanced disease susceptibility (EDS) towards this pathogen. In addition, this protocol can be supplemented with multiple pre-treatments to further dissect specific immune defects within different layers of plant defense, including Salicylic Acid (SA)-Triggered Immunity (STI) and MAMP-Triggered Immunity (MTI).

Bacterial Leaf Infiltration Assay for Fine Characterization of Plant Defense Responses using the Arabidopsis thaliana-Pseudomonas syringae Pathosystem

Quantification of pathogen growth is a powerful tool to characterize various Arabidopsis thaliana (hereafter: Arabidopsis) immune responses. The method described here presents an optimized syringe infiltration assay to quantify the Pseudomonas syringae pv. maculicola ES4326 growth in adult Arabidopsis leaves.

In the absence of specialized mobile immune cells, plants utilize their localized programmed cell death and Systemic Acquired Resistance to defend ...

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.

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.

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

Developmental Biology

An Easy and Flexible Inoculation Method for Accurately Assessing Powdery Mildew-Infection Phenotypes of Arabidopsis and Other Plants

Reducing crop losses due to fungal diseases requires improved understanding of the mechanisms governing plant immunity and fungal pathogenesis, which in turn requires accurate determination of disease phenotypes of plants upon infection with a particular fungal pathogen. However, accurate disease phenotyping with unculturable biotrophic fungal pathogens such as powdery mildew is not easy to achieve and can be a rate-limiting step of a research project. Here, we have developed a safe, efficient, and easy-to-operate disease phenotyping system using the Arabidopsis-powdery mildew interaction as an example. This system mainly consists of three components: (i) a wooden inoculation box fitted with a removable lid mounted with a stainless steel or nylon mesh of ~50 &#181;m pores for inoculating a flat of plants with fungal spores, (ii) a transparent plastic chamber with a small front opening for minimizing spore escape while conducting inoculation inside, and (iii) a spore-dislodging and distribution method for even and effective inoculation. The protocols described here include the steps and parameters for making the inoculation box and the plastic chamber at a low cost, and a video demonstration of how to use the system to enable even inoculation with powdery mildew spores, thereby improving accuracy and reproducibility of disease phenotyping.

We present a protocol for constructing a simple spore-distribution system consisting of an inoculation box with a ~50 &#181;m mesh and a transparent plastic chamber. This can be used to evenly inoculate plants with powdery mildew spores, thereby enabling accurate and reproducible assessment of disease phenotypes of plants under study.

Reducing crop losses due to fungal diseases requires improved understanding of the mechanisms governing plant immunity and fungal pathogenesis, which in ...

Biology

Visualization of Cellular Interactions Between the Maize Sheaths and Fungi

Detached Maize Sheaths for Live-Cell Imaging of Infection by Fungal Foliar Maize Pathogens

We have optimized a protocol to inoculate maize leaf sheaths with hemibiotrophic and necrotrophic foliar pathogenic fungi. The method is modified from one originally applied to rice leaf sheaths and allows direct microscopic observation of fungal growth and development in living plant cells. Leaf sheaths collected from maize seedlings with two fully emerged leaf collars are inoculated with 20 &#181;L drops of 5 x 105 spores/mL fungal spore suspensions and incubated in humidity chambers at 23 &#176;C under continuous fluorescent light. After 24-72 h, excess tissue is removed with a razor blade to leave a single layer of epidermal cells, an optically clear sample that can be imaged directly without the necessity for chemical fixation or clearing. Plant and fungal cells remain alive for the duration of the experiment and interactions can be visualized in real-time. Sheaths can be stained or subjected to plasmolysis to study the developmental cytology and viability of host and pathogen cells during infection and colonization. Fungal strains transformed to express fluorescent proteins can be inoculated or co-inoculated on the sheaths for increased resolution and to facilitate the evaluation of competitive or synergistic interactions. Fungal strains expressing fluorescent fusion proteins can be used to track and quantify the production and targeting of these individual proteins in planta. Inoculated sheath tissues can be extracted to characterize nucleic acids, proteins, or metabolites. The use of these sheath assays has greatly advanced the detailed studies of the mechanisms of fungal pathogenicity in maize and also of fungal protein effectors and secondary metabolites contributing to pathogenicity.

Author Spotlight: Investigating Fungal Pathogenicity Mechanisms in Maize 

This manuscript details an optimized inoculation protocol that uses detached maize leaf sheaths for reproducible cytological, physiological, and molecular studies of maize interactions with fungal plant pathogens. The leaf sheaths facilitate real-time observation of cellular interactions between the living plant and fungus in unfixed tissues.

We have optimized a protocol to inoculate maize leaf sheaths with hemibiotrophic and necrotrophic foliar pathogenic fungi. The method is modified from one ...

A Spray Inoculation Technique to Establish Fungal Infection in Rice Plant Leaves

Source: Zhang, M., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/57675">The Plant Infection Test: Spray and Wound-Mediated Inoculation with the Plant Pathogen Magnaporthe Grisea.</a> J. Vis. Exp. (2018).
This video demonstrates a technique to establish the pathogenic fungus Magnaporthe grisea infection in rice plant leaves. The fungal conidia suspension is sprayed on sections of rice leaf, and the disease progression is assessed by scoring the formation of necrotic lesions.

1. Spray Inoculation with a Suspension of M. grisea Conidia

	Fungal culture for M.grisea
	
		Prepare the oatmeal tomato agar (OTA) culture medium for ...

The Plant Infection Test: Spray and Wound-Mediated Inoculation with the Plant Pathogen Magnaporthe Grisea

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