Name: screening of tobacco genotypes for phytophthora nicotianae resistance
Uploaded: 2022-04-15T12:30:00.000+00:00
Duration: 10 min 10 s
Description: Watch this Scientific Journal Video about Screening of Tobacco Genotypes for Phytophthora nicotianae Resistance at JoVE.com

This research was funded by the National Natural Science Foundation of China (31571738) and the Agricultural Science and Technology Innovation Program of China (ASTIP-TRIC01).

1. Materials
<ol>
	<li>Obtain tobacco varieties. 
	NOTE: For this experiment &#34;Beinhart1000-1&#34; (a selection of Beinhart 1000) (BH) and &#34;Xiaohuangjin1025&#34; (XHJ) were obtained from the National Medium-term Genbank of the Tobacco Germplasm Resource of China. BH is resistant, whereas XHJ is susceptible to P. nicotianae infection16. A field isolate of P. nicotianae race 0, which was preserved in the Tobacco Research Institute of the Chinese Academy of Agricultural Sciences, was used for all inoculations throughout the study.</li>
</ol>
2. Planting tobacco genotypes for P. nicotianae resistance evaluation
<ol>
	<li>Mix tobacco seeds with vermiculite and broadcast the seeds gently on the sterilized potting soil. Place the pots in the growth chamber. Maintain a constant temperature of 25 &#176;C, under a 16 h light/8 h dark photoperiod.</li>
	<li>Prepare hydroponic devices with trays and foam sheets. After the seeds germinate, prick seedlings out from the potting soil, wash the roots gently with sterile deionized water, and transplant them into the hydroponic devices.</li>
	<li>Place the devices in climate chambers at 25 &#176;C under a 16 h light/8 h dark photoperiod for 24 h.</li>
	<li>Prepare Hoagland nutrient solution beforehand (Table 1). Transplant the seedlings to hydroponic devices with Hoagland nutrient solution (approximately 125 mL per plant).</li>
	<li>Place the devices in a climate chamber at 25 &#176;C under a 16 h light/8 h dark photoperiod for 2 weeks.</li>
</ol>
3. Preparation of P. nicotianae zoospore suspension
<ol>
	<li>Prepare oatmeal agar medium.
	<ol>
		<li>Weigh 33 g of oatmeal and transfer to glassware and add 1,000 mL of sterile water. Boil on an electromagnetic stovetop oven.</li>
		<li>After the oatmeal becomes sticky, strain the liquid solution through a piece of sterile gauze.</li>
		<li>Pour the liquid solution into a 1,000 mL graduated cylinder and adjust the volume to 1,000 mL with sterile water.</li>
		<li>Pour the liquid solution into a glass reagent bottle and add 18 g of agar. Shake well and autoclave the mixture (oatmeal agar medium) at 121 &#176;C for 15 min. Leave it at room temperature for 30 min.</li>
		<li>Pour around 20 mL of the sterilized oatmeal agar medium into each Petri dish. Leave the Petri dishes at room temperature to cool thoroughly before mycelial cultivation.</li>
	</ol>
	</li>
	<li>Carry out mycelial cultivation.
	<ol>
		<li>Prepare 1 cm diameter punches and toothpicks beforehand by autoclaving them at 121 &#176;Cfor 15 min.</li>
		<li>Punch holes in the P. nicotianae mycelial agar cultures to make round mycelial mats.</li>
		<li>Pick out the mycelial mats, place the mycelial side down on the oatmeal agar medium, and incubate the mycelium at 25 &#176;C in the dark for 14 days.</li>
	</ol>
	</li>
	<li>Prepare P. nicotianae zoospore suspension.
	<ol>
		<li>Add 0.1% KNO3 solution to each mycelial cultivation (15 mL/dish), followed by culturing at 4 &#176;C for 20 min to induce sporangium.</li>
		<li>Keep the dishes at 25 &#176;C for 25 min to release zoospores.</li>
		<li>Collect the zoospore suspension in a beaker and measure the zoospore concentration using a microscope andhemocytometer.</li>
		<li>Adjust the zoospore concentration to 1 x 104 zoospores/mL with sterile water.</li>
	</ol>
	</li>
</ol>
4. Identification of disease-resistant tobacco varieties
<ol>
	<li>Take the seedlings from the Hoagland nutrient solution and inoculate them by immersing the roots in 20 mL of P. nicotianae zoospore suspension in a Petri dish (90 mm) at 25 &#176;C for 3 h in the dark.</li>
	<li>After inoculation, put the tobacco seedlings into new Petri dishes with 10 mL of sterile water immersing the roots. Keep the roots moist by covering with two pieces of filter paper. Keep the dishes at 25 &#176;C with a 16 h light/8 h dark photoperiod.</li>
	<li>After 2-3 days, observe the disease severity.
	<ol>
		<li>For the control treatment, put the tobacco seedlings directly in the Petri dishes with 10 mL sterile water immersing the roots, and cover the roots with two pieces of filter paper. 
		NOTE: Each treatment had three replications, with 8 plants per experiment.</li>
	</ol>
	</li>
</ol>
5. Evaluation of P. nicotianae infection
<ol>
	<li>Evaluate the disease severity 4-5 days after inoculation. Based on the Chinese national standard18, score individual plant disease severity on a scale from 0 to 9 (Table 2, Figure 1).</li>
	<li>Calculate the disease index using the following formula18: 
	Disease index = <img alt="Equation 1" src="/files/ftp_upload/63054/63054eq01.jpg" style="margin: auto;" /> 
	where a, b, c, d, e, and f are the number of plants in each disease severity grade. 
	NOTE: Disease severity was divided into 6 grades18 (Table 3).</li>
</ol>

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The authors have nothing to disclose.

Multiple resistance sources have been used to improve P. nicotianae resistance in cultivated tobacco. Single dominant R genes, Php and Phl, have been introgressed from Nicotiana plumbaginifolia and Nicotiana longiflora, respectively10. The cigar tobacco variety Beinhart 1000 has the highest reported level of quantitative resistance to P. nicotianae13. Multiple interval mapping experiments have suggested that at least six quantitative trait loci (QTLs) may contribute to the resistancein this line13,22. In addition to the resistant sources mentioned above, another alien gene, Wz, has also been found to confer a high level of resistance to race 0 and race 123,24. Considering the multiple resistant sources and the complicated genetic background of resistant varieties, a precise and reliable method for resistance evaluation is required for 1) the identification of P. nicotianae-resistant&#160;tobacco genotypes, 2) breeding resistant varieties, and 3) molecular mechanism studies of plant-pathogen interactions. The previous methods used to assess P. nicotianae infection severities were time-consuming or the inoculation intensities were difficult to control. Hence, it is urgent to set a new method for P. nicotianae resistance evaluation.
Imitating natural infection processes is the key point in our new method. Firstly, during the typical life cycle of P. nicotianae, zoospores are the major infective agents that initiate plant diseases. Once the zoospores reach the plant surface, they become immobile cysts, subsequently germinate, and form germ tubes. Then, appressoria are produced at the tip of the germ tubes25. In our protocol, zoospore suspension was used as inoculum, which imitates the natural infection situation. Secondly, on Nicotiana benthamiana leaves, the cysts germinated and colonized epidermal cells at 3 h26. In Arabidopsis roots, all the encysted zoospores successfully penetrated the roots at 6 h post inoculation27. In our approach, the inoculation process involved immersing roots in a P. nicotianae zoospore suspension for 3 h in the dark, which imitates the natural environment and promotes the colonization of the pathogen, hence leading to stable and effective infection.
The most frequently used approach to assess P. nicotianae resistance in tobacco, the oat grains method, is efficient and easy for inoculating a large number of plants7,11,12,13,14,15,16. However, it has some drawbacks. The main disadvantage is the uncontrolled inoculate intensity, which limits its application in precision breeding. Compared to the oat grains method and the other two existing approaches, in this new approach, zoospore suspension is used as inoculum, so the inoculate intensity is controllable and adjustable. As the plants are cultured in a hydroponic environment, the interference of soil is eliminated, which increases the evaluation accuracy. However, one limitation exists for this method, which is that it cannot be usedon adult plants. The oat grain method, on the other hand, is applicable for large-scale resistance screening of adult plants7,13. Therefore, this method and the oat grain method can complement each other for different growth periods of the tobacco plant and in different situations.
The protocol described here is an efficient and reliable method for evaluating the resistance of tobacco to infection by P. nicotianae at the seedling stage. This protocol can be used for breeding and for molecular mechanism research.

P. nicotianae is destructive to many solanaceous crops. It can cause tobacco &#34;black shank&#34;1, potato foliar and tuber rot2, tomato and sweet pepper crown and root rot3, and Goji collar and root rot4. P. nicotianae can attack all parts of tobacco plants, including the roots, stems, and leaves at any growing stage5. The most common symptom of the disease is the black base of the stalk. The roots are initially visible as water-soaked and then become necrotic, and the leaves show large circular lesions5. This disease can be devastating to a tobacco plant in the greenhouse, as well as in the field6. The most practical and economical method for controlling P. nicotianae is the use of resistant varieties7. However, an effective screening protocol is required for the identification of P. nicotianae-resistant accessions from tobacco germplasm collections.
Various identification methods have been described to assess P. nicotianae resistance in tobacco7,8,9,10,11,12,13,14,15,16. In general, three major approaches have been used for the identification of P. nicotianae-resistant tobacco genotypes. The first includes mixing mycelia with agar medium on Petri plates containing P. nicotianae. The mycelia are then cultivated in the dark at room temperature for 2 weeks. 1 L of deionized water is added to the mycelia and homogenized for 30 s. The inoculum is kept on ice until needed. Two holes (1 cm in diameter and 4-5 cm deep) are made on each side of the plant, and 10 mL of the inoculum is poured into each hole. The holes are then filled with the surrounding soil, and disease development is monitored daily for 2 weeks8,10.
In the second method, the plants are inoculated with pathogen-infested toothpicks. For this approach, the plants should be used approximately 6 weeks after transplanting and should have a minimum height of 30 cm. Autoclaved toothpicks are placed on the surface of cultures containing P. nicotianae mycelia. The culture dishes are then stored under the light at room temperature for 7 days. Then, colonized toothpicks are used to inoculate the plants. Toothpicks are inserted into the tobacco stems between the fourth and fifth nodes. The plants are monitored daily for 5 days9,15. This method is not applicable for small seedlings. As the inoculum is pathogen-infested toothpicks, the inoculation intensity cannot be precisely controlled.
The most frequently used approach involves oat grains for inoculation. In this case, oat grains are prepared by autoclaving 500 mL of oats and 300 mL of deionized water at 121 &#176;C for 1 h once per day for 3 days. Then, oat grains are added to the pathogen-colonized culture medium. The dishes are sealed with paraffin film and incubated at 25 &#176;C in light for 7-12 days. Four separate 5 cm deep holes are madeon the potting soil, 4 cm from each plant, and one pathogen-infested oat grain is placed into each hole. The incubation period is determined based on when the first aboveground symptom occurrs7,11,12,13,14,15,16. This method is efficient and applicable for large-scale resistance screening. However, one limitation of this approach is that the inoculum is pathogen-infested oat grains, hence the inoculation intensity cannot be precisely controlled.
However, presented here is a more accurate method that is applicable to growth chamber resistance evaluation. Compared to the other approaches, the inoculum is zoospore suspension, hence the inoculation intensity is controllable and adjustable. As the tobacco plants in this study are cultivated without soil, the results are easier to observe. Moreover, sampling plant roots from soil always causes damage to the roots, which induces a series of physiological responses17. In this method, as plants are cultivated without soil, the interference in root damage can be eliminated. In conclusion, this method is more practical for molecular mechanism research and precision breeding. Using this protocol, data are typically obtained within 5 days, with more than 200 plants evaluated in a single experiment.

<table><tbody><tr><td>(NH&lt;sub&gt;4&lt;/sub&gt;)&lt;sub&gt;2&lt;/sub&gt;SO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sinopharm</td><td>10002917</td><td>Analytical Reagent</td></tr><tr><td>(NH&lt;sub&gt;4&lt;/sub&gt;)&lt;sub&gt;6&lt;/sub&gt; Mo&lt;sub&gt;7&lt;/sub&gt;O&lt;sub&gt;24&lt;/sub&gt;&amp;bull;2 H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sinopharm</td><td>XW131067681</td><td>Analytical Reagent</td></tr><tr><td>1.5 ml Safe-lock Microcentrifuge Tubes</td><td>Eppendorf</td><td>30120086</td><td>Used for Sample Extarction</td></tr><tr><td>2 ml Safe-lock Microcentrifuge Tubes</td><td>Eppendorf</td><td>30120094</td><td>Used for Sample Extarction</td></tr><tr><td>Agar</td><td>MDBio, Inc</td><td>9002-18-0</td><td>Materials of Culture Medium</td></tr><tr><td>Analytical Balance</td><td>AOHAOSI</td><td>AX2202ZH</td><td>Equipment</td></tr><tr><td>Autoclave</td><td>Yamatuo</td><td>SQ510C</td><td>Equipment</td></tr><tr><td>Autoclave</td><td>YAMATUO</td><td>SQ510C</td><td>Equipment</td></tr><tr><td>Beaker</td><td>Bio Best</td><td>DHSB-2L</td><td>Materials of Culture Medium</td></tr><tr><td>Biological Incubator</td><td>JINGHONG</td><td>SHP-250</td><td>Equipment</td></tr><tr><td>Ca(NO&lt;sub&gt;3&lt;/sub&gt;)&lt;sub&gt;2&lt;/sub&gt;&amp;bull;4 H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sinopharm</td><td>80029062</td><td>Analytical Reagent</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sinopharm</td><td>10005817</td><td>Analytical Reagent</td></tr><tr><td>CuSO&lt;sub&gt;4&lt;/sub&gt;&amp;bull;5 H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sinopharm</td><td>10008218</td><td>Analytical Reagent</td></tr><tr><td>Electromagnetic Oven</td><td>Bio Best</td><td>DHDCL</td><td>Equipment</td></tr><tr><td>FeSO&lt;sub&gt;4&lt;/sub&gt;&amp;bull;7 H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sinopharm</td><td>10002918</td><td>Analytical Reagent</td></tr><tr><td>Filter Paper</td><td>Bio Best</td><td>DHLZ-9CM</td><td>Material</td></tr><tr><td>Fluorescence Ration PCR Instrument</td><td>Roche</td><td>LightCycler96</td><td>Equipment</td></tr><tr><td>Gauze</td><td>Bio Best</td><td>17071202</td><td>Materials of Culture Medium</td></tr><tr><td>H&lt;sub&gt;3&lt;/sub&gt;BO&lt;sub&gt;3&lt;/sub&gt;</td><td>Phytotechnology</td><td>B210-500G</td><td>Analytical Reagent</td></tr><tr><td>Hemocytometer</td><td>Solarbio</td><td>17072801</td><td>Material for disease-resistant&amp;nbsp; identification</td></tr><tr><td>K&lt;sub&gt;2&lt;/sub&gt;SO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sinopharm</td><td>10017918</td><td>Analytical Reagent</td></tr><tr><td>KNO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sinopharm</td><td>10017218</td><td>Analytical Reagent</td></tr><tr><td>KT Foam Sheet</td><td>Bio Best</td><td>DHKTB</td><td>Material for Seedling</td></tr><tr><td>Low Constant Incubator</td><td>Jinghong</td><td>SHP-250</td><td>Equipment</td></tr><tr><td>Measuring Cylinder</td><td>Bio Best</td><td>DHBLLT-1000ML</td><td>Materials of Culture Medium</td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;&amp;bull;7 H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sinopharm</td><td>10013080</td><td>Analytical Reagent</td></tr><tr><td>Microscope</td><td>ECHO</td><td>RVL-100-G</td><td>Equipment</td></tr><tr><td>MnCl&lt;sub&gt;2&lt;/sub&gt;&amp;bull;4 H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sinopharm</td><td>G5468154</td><td>Analytical Reagent</td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;-EDTA</td><td>Sinopharm</td><td>G21410-250</td><td>Analytical Reagent</td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;&amp;bull;2 H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sinopharm</td><td>20040717</td><td>Analytical Reagent</td></tr><tr><td>NH&lt;sub&gt;4&lt;/sub&gt;NO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sinopharm</td><td>B64586-100g</td><td>Analytical Reagent</td></tr><tr><td>Oatmeal</td><td>Bio Best</td><td>DHYMP-1.5KG</td><td>Materials of Culture Medium</td></tr><tr><td>Petri Dish</td><td>Bio Best</td><td>DHPYM-9CM</td><td>Material for disease-resistant&amp;nbsp; identification</td></tr><tr><td>Pipettor</td><td>THERMO</td><td>S1</td><td>Equipment</td></tr><tr><td>Potting</td><td>Bio Best</td><td>DHYCXHP-12CM</td><td>Material for Seedling</td></tr><tr><td>Potting Soil</td><td>Bio Best</td><td>DHYMJZ-50L</td><td>Seedling Material</td></tr><tr><td>Punch</td><td>Bio Best</td><td>DHDKW</td><td>Material</td></tr><tr><td>qRT-PCR Plate</td><td>Monad</td><td>MQ50401S</td><td>qRT-PCR Plate</td></tr><tr><td>SYBR&amp;nbsp;Green Premix&amp;nbsp;Pro Taq&amp;nbsp;HS qPCR Kit</td><td>Accurate Biology</td><td>AG11718</td><td>PCR Reagent</td></tr><tr><td>Toothpick</td><td>Bio Best</td><td>DHYQ-900</td><td>Material</td></tr><tr><td>Total RNA Kit II</td><td>Omega</td><td>R6934-01</td><td>PCR Reagent</td></tr><tr><td>TransScript&amp;reg;&amp;nbsp;II One-Step gDNA Removal and cDNA Synthesis SuperMix</td><td>Transgen</td><td>AH311-02</td><td>PCR Reagent</td></tr><tr><td>Trays</td><td>Bio Best</td><td>DHYMTP-90G</td><td>Material for Seedling</td></tr><tr><td>Vermiculite</td><td>Bio Best</td><td>DHZS</td><td>Seedling Material</td></tr><tr><td>Water Purification System</td><td>HEAL FORCE</td><td>HSE68-2</td><td>Equipment</td></tr><tr><td>ZnSO&lt;sub&gt;4&lt;/sub&gt;&amp;bull;7 H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sinopharm</td><td>10024018</td><td>Analytical Reagent</td></tr></tbody></table>

screening of tobacco genotypes for phytophthora nicotianae resistance

Black shank, caused by the oomycetes Phytophthora nicotianae, is destructive to tobacco, and this pathogen is highly pathogenic to many solanaceous crops. P. nicotianae is well adapted to high temperatures; therefore, research on this pathogen is gaining importance in agriculture worldwide because of global warming. P. nicotianae-resistant varieties of tobacco plants are commonly screened by inoculation with oat grains colonized by P. nicotianae and monitoring for the disease symptoms. However, it is difficult to quantify the inoculation intensity since accurate inoculation is crucial in this case. This study aimed to develop an efficient and reliable method for evaluating the resistance of tobacco to infection with P. nicotianae. This method has been successfully used to identify resistant varieties, and the inoculation efficiency was confirmed by real-time PCR. The resistance evaluation method presented in this study is efficient and practical for precision breeding, as well as molecular mechanism research.

4-week-old plants of the resistant variety BH and susceptible variety XHJ were challenged with P. nicotianae using the method presented in this article. The experiment was designed with three replicates, each with 8 plants per group. P. nicotianae infection of the two tobacco varieties, BH and XHJ, is presented in Figure 2. At 3 days post inoculation, for XHJ, stem lesions covered approximately one-half of the stem girth, and one-half of the leaves were slightly wilted; in the resistant variety BH, no symptoms were observed. At 4 days post inoculation, leaf wilting and severe stem lesions occurred in XHJ, whereas these symptoms did not appear in BH.
At 5 days post inoculation, individual plant disease severity was recorded and calculated based on the Chinese national standard for the &#34;Grade and Investigation Method of Tobacco Diseases and Insect Pests&#34;18 (Table 4). The mean disease index of BH was 6.48, which was considered as resistant (R) as per the standard, and the mean disease index of XHJ was 76.85, which was considered susceptible (S) as per the standard.
To confirm the inoculation efficiency, relative pathogen biomass was quantified by real-time PCR&#160;(Figure 3). Plant and pathogen genomic DNA were isolated from infected BH and XHJ and collected at 24 h, 72 h, and 168 h post infection using the CTAB protocol19. Relative pathogen biomass was quantified using the primer pairs Pn-Fw (5&#39;-CTCCAGAACGTGTACATCCG-3&#39;) / Pn-Rev (5&#39;-TAGCGCCCTTCTCCTCAG-3&#39;), amplifying the 40S ribosomal protein S3A (WS21) of P. nicotianae20,&#160;and Nt-Fw (5&#39;-CAAGGAAATCACCGCTTTGG-3&#39;) / Nt-Rev (5&#39;-AAGGGATGCGAGGATGGA-3&#39;), amplifying the tobacco reference gene 26S rRNA gene21. Expression fold changes between pathogen and tobacco gDNA were calculated using the 2-&#916;&#916;CT Ct method. The results of the real-time PCR confirmed the phenotypic observations.
Five progenies from the BC4F2 population of a cross between BH and XHJ and two intermediate resistance varieties, K326 and Yunyan87, were evaluated using the zoospore suspension inoculation method and the oat grains inoculation method (Table 5). The zoospore suspension method was performed on small seedlings, and the oat grains inoculation method was performed on adult plants. For the five progenies from the BC4F2 population, the disease index ranged from 16.49 to 77.60 for the zoospore suspension method and from 10.33 to 83.08 for the oat grains method. The resistance classifications between the two infection methods were mostly consistent with each other. With the two intermediate resistance varieties, K326 and Yunyan87, the evaluation showed &#34;resistant&#34; in both methods. These data illustrate the correlation between the two inoculation methods, despite them being performed on tobacco plants in different growth periods.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Original liquor</td>
			<td>Proportion</td>
			<td>Original liquor volume (mL)</td>
			<td>Nutrient elements</td>
			<td>Content (g)</td>
		</tr>
		<tr>
			<td>OL 1</td>
			<td>1000&#215;</td>
			<td>500</td>
			<td>MgSO4&#8226;7 H2O</td>
			<td>30.81</td>
		</tr>
		<tr>
			<td>OL 2</td>
			<td>1000&#215;</td>
			<td>500</td>
			<td>Ca(NO3)2&#8226;4 H2O</td>
			<td>221.39</td>
		</tr>
		<tr>
			<td>OL 3</td>
			<td>1000&#215;</td>
			<td>500</td>
			<td>NaH2PO4&#8226;2 H2O</td>
			<td>19.5</td>
		</tr>
		<tr>
			<td>OL 4</td>
			<td>1000&#215;</td>
			<td>500</td>
			<td>NH4NO3</td>
			<td>75.04</td>
		</tr>
		<tr>
			<td>OL 5</td>
			<td>1000&#215;</td>
			<td>500</td>
			<td>(NH4)2SO4</td>
			<td>123.75</td>
		</tr>
		<tr>
			<td>OL 6</td>
			<td>1000&#215;</td>
			<td>500</td>
			<td>CaCl2</td>
			<td>104.06</td>
		</tr>
		<tr>
			<td rowspan="2">OL 7</td>
			<td rowspan="2">1000&#215;</td>
			<td rowspan="2">500</td>
			<td>FeSO4&#8226;7 H2O</td>
			<td>2.78</td>
		</tr>
		<tr>
			<td>Na2-EDTA</td>
			<td>3.73</td>
		</tr>
		<tr>
			<td>OL 8</td>
			<td>1000&#215;</td>
			<td>500</td>
			<td>K2SO4</td>
			<td>87.1</td>
		</tr>
		<tr>
			<td rowspan="5">OL 9</td>
			<td rowspan="5">4000&#215;</td>
			<td rowspan="5">1000</td>
			<td>MnCl2&#8226;4 H2O</td>
			<td>7.24</td>
		</tr>
		<tr>
			<td>H3BO3</td>
			<td>11.44</td>
		</tr>
		<tr>
			<td>ZnSO4&#8226;7 H2O</td>
			<td>0.88</td>
		</tr>
		<tr>
			<td>CuSO4&#8226;5 H2O</td>
			<td>0.32</td>
		</tr>
		<tr>
			<td>(NH4)6 Mo7O24&#8226;2 H2O</td>
			<td>0.36</td>
		</tr>
	</tbody>
</table>
Table 1: Hoagland nutrient solution.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Grade</td>
			<td>Appearance of phenotype</td>
		</tr>
		<tr>
			<td>Grade 0</td>
			<td>No symptoms on the whole plant</td>
		</tr>
		<tr>
			<td>Grade 1</td>
			<td>Stem lesions &#60; 1/3 of stem girth or &#60;1/3 of leaves wilting</td>
		</tr>
		<tr>
			<td>Grade 3</td>
			<td>Stem lesions between 1/3 and 1/2 of stem girth or between 1/3 and 1/2 of leaves slightly wilting</td>
		</tr>
		<tr>
			<td>Grade 5</td>
			<td>Stem lesions &#62;1/2 of stem girth, but not completely around the girth, or between 1/2 and 2/3 of leaves wilting</td>
		</tr>
		<tr>
			<td>Grade 7</td>
			<td>Stem lesions around whole stem girth or &#62;2/3 of leaves wilting</td>
		</tr>
		<tr>
			<td>Grade 9</td>
			<td>Plants look dead</td>
		</tr>
	</tbody>
</table>
Table 2: Black shank disease severity ranking. Based on the Chinese national standard for the &#34;Grade and Investigation Method of Tobacco Diseases and Insect Pests&#34; (GB/T 23222-2008), individual plant disease severity was scored on a scale from 0 to 9.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Disease index</td>
			<td>Evaluation of resistance</td>
		</tr>
		<tr>
			<td>0</td>
			<td>Highly resistant or immune (I)</td>
		</tr>
		<tr>
			<td>0.1 to 20</td>
			<td>Resistant (R)</td>
		</tr>
		<tr>
			<td>20.1 to 40</td>
			<td>Moderately resistant (MR)</td>
		</tr>
		<tr>
			<td>40.1 to 60</td>
			<td>Moderately susceptible (MS)</td>
		</tr>
		<tr>
			<td>60.1 to 80</td>
			<td>Susceptible (S)</td>
		</tr>
		<tr>
			<td>80.1 to 100</td>
			<td>Highly susceptible (HS)</td>
		</tr>
	</tbody>
</table>
Table 3: Evaluation of resistance according to disease index. Disease severity was divided into 6 grades according to the disease index. 0 means highly resistant or immune (I), 0.1 to 20 means resistant (R), 20.1 to 40 means moderately resistant (MR), 40.1 to 60 means moderately susceptible (MS), 60.1 to 80 means susceptible (S), 80.1 to 100 means highly susceptible (HS).
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td style="min-width: 70px;">Cultivar</td>
			<td style="min-width: 70px;">Repeat</td>
			<td style="min-width: 70px;">1</td>
			<td style="min-width: 70px;">2</td>
			<td style="min-width: 70px;">3</td>
			<td style="min-width: 70px;">4</td>
			<td style="min-width: 70px;">5</td>
			<td style="min-width: 70px;">6</td>
			<td style="min-width: 70px;">7</td>
			<td style="min-width: 70px;">8</td>
			<td style="min-width: 70px;">Disease index</td>
			<td style="min-width: 70px;">Mean</td>
			<td colspan="2" style="min-width: 70px;">Evaluation of resistance</td>
		</tr>
		<tr>
			<td rowspan="3" style="min-width: 70px;">BH</td>
			<td style="min-width: 70px;">1</td>
			<td style="min-width: 70px;">0</td>
			<td style="min-width: 70px;">1</td>
			<td style="min-width: 70px;">0</td>
			<td style="min-width: 70px;">0</td>
			<td style="min-width: 70px;">1</td>
			<td style="min-width: 70px;">0</td>
			<td style="min-width: 70px;">1</td>
			<td style="min-width: 70px;">0</td>
			<td style="min-width: 70px;">4.17</td>
			<td rowspan="3" style="min-width: 70px;">6.48</td>
			<td colspan="2" style="min-width: 70px;"></td>
		</tr>
		<tr>
			<td style="min-width: 70px;">2</td>
			<td style="min-width: 70px;">1</td>
			<td style="min-width: 70px;">0</td>
			<td style="min-width: 70px;">0</td>
			<td style="min-width: 70px;">0</td>
			<td style="min-width: 70px;">3</td>
			<td style="min-width: 70px;">1</td>
			<td style="min-width: 70px;">0</td>
			<td style="min-width: 70px;">1</td>
			<td style="min-width: 70px;">8.33</td>
			<td colspan="2" style="min-width: 70px;">R</td>
		</tr>
		<tr>
			<td style="min-width: 70px;">3</td>
			<td style="min-width: 70px;">1</td>
			<td style="min-width: 70px;">0</td>
			<td style="min-width: 70px;">0</td>
			<td style="min-width: 70px;">0</td>
			<td style="min-width: 70px;">0</td>
			<td style="min-width: 70px;">0</td>
			<td style="min-width: 70px;">3</td>
			<td style="min-width: 70px;">1</td>
			<td style="min-width: 70px;">6.94</td>
			<td colspan="2" style="min-width: 70px;"></td>
		</tr>
		<tr>
			<td rowspan="3" style="min-width: 70px;">XHJ</td>
			<td style="min-width: 70px;">1</td>
			<td style="min-width: 70px;">7</td>
			<td style="min-width: 70px;">5</td>
			<td style="min-width: 70px;">7</td>
			<td style="min-width: 70px;">7</td>
			<td style="min-width: 70px;">9</td>
			<td style="min-width: 70px;">7</td>
			<td style="min-width: 70px;">7</td>
			<td style="min-width: 70px;">7</td>
			<td style="min-width: 70px;">77.78</td>
			<td rowspan="3" style="min-width: 70px;">76.85</td>
			<td colspan="2" style="min-width: 70px;"></td>
		</tr>
		<tr>
			<td style="min-width: 70px;">2</td>
			<td style="min-width: 70px;">9</td>
			<td style="min-width: 70px;">7</td>
			<td style="min-width: 70px;">9</td>
			<td style="min-width: 70px;">7</td>
			<td style="min-width: 70px;">5</td>
			<td style="min-width: 70px;">9</td>
			<td style="min-width: 70px;">7</td>
			<td style="min-width: 70px;">5</td>
			<td style="min-width: 70px;">80.56</td>
			<td colspan="2" style="min-width: 70px;">S</td>
		</tr>
		<tr>
			<td style="min-width: 70px;">3</td>
			<td style="min-width: 70px;">7</td>
			<td style="min-width: 70px;">5</td>
			<td style="min-width: 70px;">7</td>
			<td style="min-width: 70px;">9</td>
			<td style="min-width: 70px;">5</td>
			<td style="min-width: 70px;">9</td>
			<td style="min-width: 70px;">5</td>
			<td style="min-width: 70px;">5</td>
			<td style="min-width: 70px;">72.22</td>
			<td colspan="2" style="min-width: 70px;"></td>
		</tr>
	</tbody>
</table>
Table 4: Disease index and resistance in BH and XHJ. The mean disease index of BH was 6.48, which shows resistance (R) according to the standard, and the mean disease index of XHJ was 76.85, which shows susceptibility (S) according to the standard.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td rowspan="2">Genotype</td>
			<td colspan="4">Zoospore suspension</td>
			<td colspan="4">Oat grains method</td>
		</tr>
		<tr>
			<td>Experiment 1</td>
			<td>Experiment 2</td>
			<td>Mean</td>
			<td>Classification</td>
			<td>Experiment 1</td>
			<td>Experiment 2</td>
			<td>Mean</td>
			<td>Classification</td>
		</tr>
		<tr>
			<td>BH</td>
			<td>4.17</td>
			<td>8.33</td>
			<td>6.94</td>
			<td>R</td>
			<td>0</td>
			<td>0</td>
			<td>0</td>
			<td>I</td>
		</tr>
		<tr>
			<td>XHJ</td>
			<td>77.78</td>
			<td>80.56</td>
			<td>72.22</td>
			<td>S</td>
			<td>84.89</td>
			<td>90</td>
			<td>87.45</td>
			<td>HS</td>
		</tr>
		<tr>
			<td>K326</td>
			<td>12.5</td>
			<td>12.5</td>
			<td>12.5</td>
			<td>R</td>
			<td>5.39</td>
			<td>15.67</td>
			<td>10.53</td>
			<td>R</td>
		</tr>
		<tr>
			<td>Yunyan87</td>
			<td>19.45</td>
			<td>8.33</td>
			<td>13.89</td>
			<td>R</td>
			<td>4.7</td>
			<td>28.89</td>
			<td>16.8</td>
			<td>R</td>
		</tr>
		<tr>
			<td>C42-4</td>
			<td>21.28</td>
			<td>21.89</td>
			<td>21.59</td>
			<td>MR</td>
			<td>7.56</td>
			<td>13.11</td>
			<td>10.33</td>
			<td>R</td>
		</tr>
		<tr>
			<td>C13-4</td>
			<td>18.16</td>
			<td>14.81</td>
			<td>16.49</td>
			<td>R</td>
			<td>38.89</td>
			<td>19.66</td>
			<td>29.27</td>
			<td>MR</td>
		</tr>
		<tr>
			<td>C9-5</td>
			<td>77.41</td>
			<td>77.78</td>
			<td>77.6</td>
			<td>S</td>
			<td>79.83</td>
			<td>82.86</td>
			<td>81.34</td>
			<td>HS</td>
		</tr>
		<tr>
			<td>C46-8</td>
			<td>55.56</td>
			<td>51.11</td>
			<td>53.34</td>
			<td>MS</td>
			<td>62.91</td>
			<td>72.65</td>
			<td>67.78</td>
			<td>S</td>
		</tr>
		<tr>
			<td>C66-9</td>
			<td>79.88</td>
			<td>74.07</td>
			<td>76.98</td>
			<td>S</td>
			<td>93.94</td>
			<td>72.22</td>
			<td>83.08</td>
			<td>HS</td>
		</tr>
	</tbody>
</table>
Table 5: P. nicotianae infection response in different genotypes with the zoospore suspension inoculation method and the oat grains inoculation method. The zoospore suspension inoculation method was performed on small seedlings, and the oat grains inoculation method was performed on adult plants. These data illustrate the correlation between the zoospore suspension inoculation method and the oat grains inoculation method. K326 and Yunyan87 have intermediate levels of resistance, and C42-4, C13-4, C9-5, C46-8, C66-9 are progenies from the BC4F2 population of a cross between BH and XHJ. The plants were classified as immune, resistant, moderately resistant, moderately susceptible, susceptible, or high susceptible.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63054/63054fig01.jpg" /> 
Figure 1: Symptom of each grade. (A) Grade 0, whole plant symptom free. (B) Grade 1, stem lesion &#60;1/3 of stem girth or &#60;1/3 leaves wilting. (C) Grade 3, stem lesions between 1/3 and 1/2 of stem girth or between 1/3 and 1/2 of leaves slightly wilting. (D) Grade 5, stem lesions &#62;1/2 of stem girth, but not completely around the girth, or between 1/2 and 2/3 of leaves wilting. (E) Grade 7, stem lesions around whole stem girth or &#62;2/3 of leaves wilting. (F) Grade 9, plants look dead. Red arrows indicate the stem lesions. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63054/63054fig01large.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/63054/63054fig02.jpg" /> 
Figure 2: Variation observed in two tobacco genotypes. (A) Whole plant symptom free in resistant variety BH 3 days post inoculation. (B) Stem lesions are around 1/2 of stem girth and 1/2 of leaves slightly wilting in XHJ 3 days post inoculation. (C) Whole plant symptom free in resistant variety BH 4 days post inoculation. (D) Stem lesions &#62;1/2 of stem girth, but not completely around the girth, and between 1/2 and 2/3 of leaves wilting in susceptible variety XHJ 4 days post inoculation. Red arrows indicate the stem lesions. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63054/63054fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63054/63054fig03.jpg" /> 
Figure 3: Relative P. nicotianae biomass quantification in infected BH and XHJ at 24 h, 72 h, and 168 h post inoculation. This is calculated as the ratio of P. nicotianae WS21 gene amplification in comparison with the tobacco 26S rRNA gene. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63054/63054fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Screening of Tobacco Genotypes for Phytophthora nicotianae Resistance at JoVE.com

Screening of Tobacco Genotypes for Phytophthora nicotianae Resistance

Here, a protocol is presented for the efficient and accurate screening of tobacco genotypes for Phytophthora nicotianae resistance in seedlings. This is a practical approach for precision breeding, as well as molecular mechanism research.

Screening of Tobacco Genotypes for Phytophthora nicotianae Resistance

Black shank, caused by the oomycetes Phytophthora nicotianae, is destructive to tobacco, and this pathogen is highly pathogenic to many solanaceous crops. ...

screening-of-tobacco-genotypes-for-phytophthora-nicotianae-resistance

Nanyang Technological University

Research

JoVE Journal

Biology

3.3K Views.  Chinese Academy of Agricultural Sciences. Hier wordt een protocol gepresenteerd voor de efficiënte en nauwkeurige screening van tabaksgenotypes op Phytophthora nicotianae-resistentie bij zaailingen. Het is praktisch voor precisieveredeling, evenals onderzoek naar moleculaire mechanismen. Materiaal. Verkrijg tabaksvariëteiten Beinhart1000-1, een selectie van Beinhart1000 BH, en Xiaohuangjin1025 XHJ van de National Medium-term Geneback of the Tobacco Germplasm Resource of China.BH is resistent, terwijl XHJ gevoelig is voor Phy...

Video: Screening of Tobacco Genotypes for Phytophthora nicotianae Resistance

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 ...

Environment

Protocols for Robust Herbicide Resistance Testing in Different Weed Species

Robust protocols to test putative herbicide resistant weed populations at whole plant level are essential to confirm the resistance status. The presented protocols, based on whole-plant bioassays performed in a greenhouse, can be readily adapted to a wide range of weed species and herbicides through appropriate variants. Seed samples from plants that survived a field herbicide treatment are collected and stored dry at low temperature until used. Germination methods differ according to weed species and seed dormancy type. Seedlings at similar growth stage are transplanted and maintained in the greenhouse under appropriate conditions until plants have reached the right growth stage for herbicide treatment. Accuracy is required to prepare the herbicide solution to avoid unverifiable mistakes. Other critical steps such as the application volume and spray speed are also evaluated. The advantages of this protocol, compared to others based on whole plant bioassays using one herbicide dose, are related to the higher reliability and the possibility of inferring the resistance level. Quicker and less expensive in vivo or in vitro diagnostic screening tests have been proposed (Petri dish bioassays, spectrophotometric tests), but they provide only qualitative information and their widespread use is hindered by the laborious set-up that some species may require. For routine resistance testing, the proposed whole plant bioassay can be applied at only one herbicide dose, so reducing the costs.

A robust and flexible approach to confirm herbicide resistance in weed populations is presented. This protocol allows the herbicide resistance levels to be inferred and applied to a wide range of weed species and herbicides with minor adaptations.

Robust protocols to test putative herbicide resistant weed populations at whole plant level are essential to confirm the resistance status. The presented ...

High and Low Throughput Screens with Root-knot Nematodes Meloidogyne spp.

Root-knot nematodes (genus Meloidogyne) are obligate plant parasites. They are extremely polyphagous and considered one of the most economically important plant parasitic nematodes. The microscopic second-stage juvenile (J2), molted once in the egg, is the infective stage. The J2s hatch from the eggs, move freely in the soil within a film of water, and locate root tips of suitable plant species. After penetrating the plant root, they migrate towards the vascular cylinder where they establish a feeding site and initiate feeding using their stylets. The multicellular feeding site is comprised of several enlarged multinuclear cells called 'giant cells' which are formed from cells that underwent karyokinesis (repeated mitosis) without cytokinesis. Neighboring pericycle cells divide and enlarge in size giving rise to a typical gall or root knot, the characteristic symptom of root-knot nematode infection. Once feeding is initiated, J2s become sedentary and undergo three additional molts to become adults. The adult female lays 150-250 eggs in a gelatinous matrix on or below the surface of the root. From the eggs new infective J2s hatch and start a new cycle. The root-knot nematode life cycle is completed in 4-6 weeks at 26-28&deg;C.
Here we present the traditional protocol to infect plants, grown in pots, with root-knot nematodes and two methods for high-throughput assays. The first high-throughput method is used for plants with small seeds such as tomato while the second is for plants with large seeds such as cowpea and common bean. Large seeds support extended seedling growth with minimal nutrient supplement. The first high throughput assay utilizes seedlings grown in sand in trays while in the second assay plants are grown in pouches in the absence of soil. The seedling growth pouch is made of a 15.5 x 12.5cm paper wick, folded at the top to form a 2-cm-deep trough in which the seed or seedling is placed. The paper wick is contained inside a transparent plastic pouch. These growth pouches allow direct observation of nematode infection symptoms, galling of roots and egg mass production, under the surface of a transparent pouch. Both methods allow the use of the screened plants, after phenotyping, for crossing or seed production. An additional advantage of the use of growth pouches is the small space requirement because pouches are stored in plastic hanging folders arranged in racks.

High and Low Throughput Screens with Root-knot Nematodes Meloidogyne spp.

Two distinct methods to screen plants with root-knot nematodes are described. The described approaches include high-throughput screens with nematodes in a nondestructive manner facilitating the use of these plants in breeding programs.

Root-knot nematodes (genus Meloidogyne) are obligate plant parasites. They are extremely polyphagous and considered one of the most economically important ...

Immunology and Infection

Screening Cotton Genotypes for Reniform Nematode Resistance

A rapid non-destructive reniform nematode (Rotylenchulus reniformis) screening protocol is needed for the development of resistant cotton (Gossypium hirsutum) varieties to improve nematode management. Most protocols involve extracting vermiform nematodes or eggs from the cotton root system or potting soil to determine population density or reproduction rate. These approaches are generally time-consuming with a small number of genotypes evaluated. An alternative approach is described here in which the root system is visually examined for nematode infection. The protocol involves inoculating cotton seedling 7 days after planting with vermiform nematodes and determining the number of females attached to the root system 28 days after inoculation. Data are expressed as the number of females per gram of fresh root weight to adjust for variation in root growth. The protocol provides an excellent method for evaluating host-plant resistance associated with the ability of the nematode to establish an infection site; however, resistance that hinders nematode reproduction is not assessed. As with other screening protocols, variation is commonly observed in nematode infection among individual genotypes within and between experiments. Data are presented to illustrate the range of variation observed using the protocol. To adjust for this variation, control genotypes are included in experiments. Nonetheless, the protocol provides a simple and rapid method to evaluate host-plant resistance. The protocol has been successfully used to identify resistant accessions from the G. arboreum germplasm collection and evaluate segregating populations of more than 300 individuals to determine the genetics of resistance. A vegetative propagation method for recovering plants for resistance breeding was also developed. After removal of the root system for nematode evaluation, the vegetative shoot is replanted to allow the development of a new root system. More than 95% of the shoots typically develop a new root system with plants reaching maturity.

Here, a protocol is presented for the rapid non-destructive screening of cotton genotypes for reniform nematode resistance. The protocol involves visually examining the roots of nematode-infected cotton seedlings to determine infection response. The vegetative shoot from each plant is then propagated to recover plants for seed production.

A rapid non-destructive reniform nematode (Rotylenchulus reniformis) screening protocol is needed for the development of resistant cotton (Gossypium ...

Genetics

VIGS-Mediated Forward Genetics Screening for Identification of Genes Involved in Nonhost Resistance

Nonhost disease resistance of plants against bacterial pathogens is controlled by complex defense pathways. Understanding this mechanism is important for developing durable disease-resistant plants against wide range of pathogens. Virus-induced gene silencing (VIGS)-based forward genetics screening is a useful approach for identification of plant defense genes imparting nonhost resistance. Tobacco rattle virus (TRV)-based VIGS vector is the most efficient VIGS vector to date and has been efficiently used to silence endogenous target genes in Nicotiana benthamiana.

	In this manuscript, we demonstrate a forward genetics screening approach for silencing of individual clones from a cDNA library in N. benthamiana and assessing the response of gene silenced plants for compromised nonhost resistance against nonhost pathogens, Pseudomonas syringae pv. tomato T1, P. syringae pv. glycinea, and X. campestris pv. vesicatoria. These bacterial pathogens are engineered to express GFPuv protein and their green fluorescing colonies can be seen by naked eye under UV light in the nonhost pathogen inoculated plants if the silenced target gene is involved in imparting nonhost resistance. This facilitates reliable and faster identification of gene silenced plants susceptible to nonhost pathogens. Further, promising candidate gene information can be known by sequencing the plant gene insert in TRV vector. Here we demonstrate the high throughput capability of VIGS-mediated forward genetics to identify genes involved in nonhost resistance. Approximately, 100 cDNAs can be individually silenced in about two to three weeks and their relevance in nonhost resistance against several nonhost bacterial pathogens can be studied in a week thereafter. In this manuscript, we enumerate the detailed steps involved in this screening. VIGS-mediated forward genetics screening approach can be extended not only to identifying genes involved in nonhost resistance but also to studying genes imparting several biotic and abiotic stress tolerances in various plant species.

Virus-induced gene silencing is an useful tool for identifying genes involved in nonhost resistance of plants. We demonstrate the use of bacterial pathogens expressing GFPuv in identifying gene silenced plants susceptible to nonhost pathogens. This approach is easy, fast and facilitates large scale screening and similar protocol can be applied to studying various other plant-microbe interactions.


	Nonhost disease resistance of plants against bacterial pathogens is controlled by complex defense pathways. Understanding this mechanism is important ...

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 ...

High-Throughput Identification of Resistance to Pseudomonas syringae pv. Tomato in Tomato using Seedling Flood Assay

Tomato is an agronomically important crop that can be infected by Pseudomonas syringae, a Gram-negative bacterium, resulting in bacterial speck disease. The tomato-P. syringae pv. tomato pathosystem is widely used to dissect the genetic basis of plant innate responses and disease resistance. While disease was successfully managed for many decades through the introduction of the Pto/Prf gene cluster from Solanum pimpinellifolium into cultivated tomato, race 1 strains of P. syringae have evolved to overcome resistance conferred by the Pto/Prf gene cluster and occur worldwide.
Wild tomato species are important reservoirs of natural diversity in pathogen recognition, because they evolved in diverse environments with different pathogen pressures. In typical screens for disease resistance in wild tomato, adult plants are used, which can limit the number of plants that can be screened due to their extended growth time and greater growth space requirements. We developed a method to screen 10-day-old tomato seedlings for resistance, which minimizes plant growth time and growth chamber space, allows a rapid turnover of plants, and allows large sample sizes to be tested. Seedling outcomes of survival or death can be treated as discrete phenotypes or on a resistance scale defined by amount of new growth in surviving seedlings after flooding. This method has been optimized to screen 10-day-old tomato seedlings for resistance to two P. syringae strains and can easily be adapted to other P. syringae strains.

High-Throughput Identification of Resistance to Pseudomonas syringae pv. Tomato in Tomato using Seedling Flood Assay

The seedling flood assay facilitates rapid screening of wild tomato accessions for the resistance to the Pseudomonas syringae bacterium. This assay, used in conjunction with the seedling bacterial growth assay, can assist in further characterizing the underlying resistance to the bacterium, and can be used to screen mapping populations to determine the genetic basis of resistance.

Tomato is an agronomically important crop that can be infected by Pseudomonas syringae, a Gram-negative bacterium, resulting in bacterial speck disease. ...

Bioengineering

Agroinfiltration and PVX Agroinfection in Potato and Nicotiana benthamiana

Agroinfiltration and PVX agroinfection are two efficient transient expression assays for functional analysis of candidate genes in plants. The most commonly used agent for agroinfiltration is Agrobacterium tumefaciens, a pathogen of many dicot plant species. This implies that agroinfiltration can be applied to many plant species. Here, we present our protocols and expected results when applying these methods to the potato (Solanum tuberosum), its related wild tuber-bearing Solanum species (Solanum section Petota) and the model plant Nicotiana benthamiana. In addition to functional analysis of single genes, such as resistance (R) or avirulence (Avr) genes, the agroinfiltration assay is very suitable for recapitulating the R-AVR interactions associated with specific host pathogen interactions by simply delivering R and Avr transgenes into the same cell. However, some plant genotypes can raise nonspecific defense responses to Agrobacterium, as we observed for example for several potato genotypes. Compared to agroinfiltration, detection of AVR activity with PVX agroinfection is more sensitive, more high-throughput in functional screens and less sensitive to nonspecific defense responses to Agrobacterium. However, nonspecific defense to PVX can occur and there is a risk to miss responses due to virus-induced extreme resistance. Despite such limitations, in our experience, agroinfiltration and PVX agroinfection are both suitable and complementary assays that can be used simultaneously to confirm each other&#39;s results.

Agroinfiltration and PVX Agroinfection in Potato and Nicotiana benthamiana

Agroinfiltration and PVX agroinfection are routine functional assays for transient ectopic expression of genes in plants. These methods are efficient assays in effectoromics strategies (rapid resistance and avirulence gene discovery) and crucial to modern research in molecular plant pathology. They meet the demand for robust high-throughput functional analysis in plants.

Agroinfiltration and PVX agroinfection are two efficient transient expression assays for functional analysis of candidate genes in plants. The most ...

A Contrast of Three Inoculation Techniques used to Determine the Race of Unknown Fusarium oxysporum f.sp. niveum Isolates

Fusarium wilt of watermelon (Citrullus lanatus), caused by Fusarium oxysporum f. sp. niveum (Fon), has reemerged as a major production constraint in the southeastern USA, especially in Florida. Deployment of integrated pest management strategies, such as race-specific resistant cultivars, requires information on the diversity and population density of the pathogen in growers' fields. Despite some progress in developing molecular diagnostic tools to identify pathogen isolates, race determination often requires bioassay approaches.
Race typing was conducted by root-dip inoculation, infested kernel seeding method, and the modified tray-dip method with each of the four watermelon differentials (Black Diamond, Charleston Grey, Calhoun Grey, Plant Introduction 296341-FR). Isolates are assigned a race designation by calculation of disease incidence five weeks after inoculation. If less than 33% of the plants for a particular cultivar were symptomatic, they were categorized as resistant. Those cultivars with incidence greater than 33% were regarded as susceptible. This paper describes three different methods of inoculation to ascertain race, root-dip, infested kernel, and modified tray-dip inoculation, whose applications vary according to the experimental design.

A Contrast of Three Inoculation Techniques used to Determine the Race of Unknown Fusarium oxysporum f.sp. niveum Isolates

Managing Fusarium wilt of watermelon requires knowledge of the pathogen races present. Here, we describe the root-dip, infested kernel seeding, and modified tray-dip inoculation methods to demonstrate their efficacy in race-typing of the pathogenic fungus Fusarium oxysporum f. sp niveum (Fon).

Fusarium wilt of watermelon (Citrullus lanatus), caused by Fusarium oxysporum f. sp. niveum (Fon), has reemerged as a major production constraint in the ...

Rapid In Vivo Leaf Inoculation for Screening Poplar Stem Canker Resistance

In Vivo Leaf Inoculation: An Alternative Method to Assess the Disease Resistance of Hybrid Clones in Poplar Breeding of Stem Canker Disease

Stem canker diseases caused by the pathogen Cytospora chrysosperma (Pers.) Fr.) and Botryosphaeria dothidea (Moug. ex Fr.) Ces. &#38; de Not. are the two major forest diseases in the poplar plantations in China, sometimes which can destroy all the poplar seedlings or severely damage mature poplar forests. Hybrid breeding is the most direct and efficient method of controlling and managing tree diseases. However, assessing disease resistance or selecting disease-resistance clones based on In vitro stem inoculation is inefficient, time-consuming, and expensive, limiting the development of hybrid breeding of poplar stem canker disease. In this study, we proposed an alternative method to assess disease resistance to stem canker pathogens through in vivo leaf inoculation. The test materials used in this method can be on 1-year-old poplar saplings or the annual branches of perennial poplars in the greenhouse or the field. The critical step of this alternative method is the selection of inoculating leaves: the 5-7th newly matured leaves might be the most suitable. The second critical step of the leaf inoculation method is to make wounds on plant leaves through needle pierces, providing sufficient lesions to measure disease severity. For the adequate number of leaves produced in the early stage of poplar breeding, this in vivo leaf inoculation contributes to the rapid, accurate, and large-scale screening of the disease-resistance poplar clones to stem canker pathogens. Moreover, this leaf inoculation method will also serve as an efficient method for screening pathotypes of stem canker disease pathogen C. chrysosperma, B.dothidea, or other poplar stem canker pathogens.

Author Spotlight: High-Throughput In Vivo Leaf Inoculation for Accelerating Disease Resistance Screening in Poplar Hybrid Breeding

We provide a step-by-step protocol for assessing poplar resistance to stem canker pathogens using an in vivo leaf inoculation method. This method is especially suitable for large-scale assessment of Cytospora chrysosperma and Botryosphaeria dothidea canker disease resistance in poplar breeding progeny in China.

Stem canker diseases caused by the pathogen Cytospora chrysosperma (Pers.) Fr.) and Botryosphaeria dothidea (Moug. ex Fr.) Ces. &#38; de Not. are the two ...

Screening of Tobacco Genotypes for Phytophthora nicotianae Resistance

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