Name: screening of tobacco genotypes for phytophthora nicotianae resistance
Uploaded: 2022-04-15T12:30:00
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 Acade...

<ol>
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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...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>(NH4)2SO4</td>
			<td>Sinopharm</td>
			<td>10002917</td>
			<td>Analytical Reagent</td>
		</tr>
		<tr>
			<td>(NH4)6 Mo7O24&#8226;2 H2O</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(NO3)2&#8226;4 H2O</td>
			<td>Sinopharm</td>
			<td>80029062</td>
			<td>Analytical Reagent</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sinopharm</td>
			<td>10005817</td>
			<td>Analytical Reagent</td>
		</tr>
		<tr>
			<td>CuSO4&#8226;5 H2O</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>FeSO4&#8226;7 H2O</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>H3BO3</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&#160; identification</td>
		</tr>
		<tr>
			<td>K2SO4</td>
			<td>Sinopharm</td>
			<td>10017918</td>
			<td>Analytical Reagent</td>
		</tr>
		<tr>
			<td>KNO3</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>MgSO4&#8226;7 H2O</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>MnCl2&#8226;4 H2O</td>
			<td>Sinopharm</td>
			<td>G5468154</td>
			<td>Analytical Reagent</td>
		</tr>
		<tr>
			<td>Na2-EDTA</td>
			<td>Sinopharm</td>
			<td>G21410-250</td>
			<td>Analytical Reagent</td>
		</tr>
		<tr>
			<td>NaH2PO4&#8226;2 H2O</td>
			<td>Sinopharm</td>
			<td>20040717</td>
			<td>Analytical Reagent</td>
		</tr>
		<tr>
			<td>NH4NO3</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&#160; 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>
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			<td>Punch</td>
			<td>Bio Best</td>
			<td>DHDKW</td>
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			<td>qRT-PCR Plate</td>
			<td>Monad</td>
			<td>MQ50401S</td>
			<td>qRT-PCR Plate</td>
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			<td>SYBR&#160;Green Premix&#160;Pro Taq&#160;HS qPCR Kit</td>
			<td>Accurate Biology</td>
			<td>AG11718</td>
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			<td>Toothpick</td>
			<td>Bio Best</td>
			<td>DHYQ-900</td>
			<td>Material</td>
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			<td>Total RNA Kit II</td>
			<td>Omega</td>
			<td>R6934-01</td>
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			<td>TransScript&#174;&#160;II One-Step gDNA Removal and cDNA Synthesis SuperMix</td>
			<td>Transgen</td>
			<td>AH311-02</td>
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			<td>Trays</td>
			<td>Bio Best</td>
			<td>DHYMTP-90G</td>
			<td>Material for Seedling</td>
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			<td>Vermiculite</td>
			<td>Bio Best</td>
			<td>DHZS</td>
			<td>Seedling Material</td>
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			<td>Water Purification System</td>
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			<td>HSE68-2</td>
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			<td>ZnSO4&#8226;7 H2O</td>
			<td>Sinopharm</td>
			<td>10024018</td>
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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 ...

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. ...

Here, a protocol is presented for the efficient and accurate screening of tobacco genotypes for Phytophthora nicotianae resistance in seedlings. It is practical for precision breeding, as well as molecular mechanism research.Materials. Obtain tobacco varieties Beinhart1000-1, a selection of Beinhart1000 BH, and Xiaohuangjin1025 XHJ from the National Medium-term Geneback of the Tobacco Germplasm Resource of China.BH is resistant, whereas XHJ is susceptible to Phytophthora nicotianae infection. Planting tobacco genotypes for Phytophthora nicotianae resistance evaluation. 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 degrees Celsius under a 16-hour light/eight-hour dark photo period. Prepare hydroponic devices with trays and foam sheets.After seeds germinate, prick seedlings out from the potting soil. Wash the roots gently with sterile deionized water and transplant them into the hydroponic devices. Place the devices in climate chambers at 25 degrees Celsius under a six-hour light/eight-hour dark photo period for 24 hours.Prepare Hoagland nutrient solution beforehand. Transplant the seedlings to hydroponic devices with Hoagland nutrient solution. Place the devices in climate chamber at 25 degrees Celsius under 16-hour light/eight-hour dark photo period for two weeks.Preparation of Phytophthora nicotianae zoospore suspension. Preparation of oatmeal agar medium. Weigh 33 grams of oatmeal into a hollowware and add 1, 000 milliliters of sterile water.Boil on an electromagnetic oven. After the oatmeal becomes sticky, strain the liquid through a piece of sterile gauze. Pour the liquid into a 1, 000 milliliter graduated cylinder and adjust the volume to 1, 000 milliliter with sterile water.Pour the liquid into a glass reagent bottle and add 18 grams of agar. Shake well and autoclave the mixture oatmeal agar medium at 121 degrees Celsius for 15 minutes. Leave it at room temperature for 30 minutes.Pour around 20 milliliters of sterilized oatmeal agar medium to each Petri dish. Leave the Petri dishes at room temperature to cool thoroughly before mycelial cultivation. Mycelial cultivation.Prepare one centimeter diameter punches and toothpicks beforehand by autoclaving them in an autoclave at 121 degrees Celsius for 15 minutes. Punch holes in Phytophthora nicotianae mycelial agar cultures to make round mycelial mats. Pick out mycelial mats, place the mycelial side down on oatmeal agar medium and incubate the mycelium at 25 degrees Celsius in the dark for 14 days.Preparation of Phytophthora nicotianae zoospore suspension. Add 0.1%potassium nitrate solution to each mycelial cultivation 15 milliliters per dish, followed by culturing at four degrees Celsius for 20 minutes to induce sporangium. Keep the dishes at 25 degrees Celsius for 25 minutes to release zoospores.Collect the zoospore suspension to a beaker and measure the zoospore concentration by microscope and hemocytometer. Adjust the zoospore concentration to one times 10 to the fourth zoospore per milliliter with sterile water. Identification of disease-resistant tobacco varieties.Take seedlings from Hoagland nutrient solution and inoculate them by immersing the roots in 20 milliliters of Phytophthora nicotianae zoospore suspension in a Petri dish 90 millimeters at 25 degrees Celsius for three hours in the dark. After inoculation, put tobacco seedlings to new Petri dishes with 10 milliliters of sterile water, immersing the roots. Keep the roots moist by covering with two pieces of filter paper.Keep the dishes in 25 degrees Celsius 16-hour light/eight-hour dark photo period. After two to three days, observe the disease severity. For the control treatment, put the tobacco seedlings directly in the Petri dishes with 10 milliliter sterile water, immersing the roots and cover the roots by two pieces of filter paper.Evaluation of Phytophthora nicotianae infection. Evaluate disease severity four to five days after inoculation. Based on the Chinese national standard, score individual plant disease severity on a scale from zero to nine.Grade 0 means no symptoms on the whole plant. Grade 1 means stem lesions less than 1/3 of stem girth or 1/3 of leaves wilting. Grade 3 means stem lesions between 1/3 and 1/2 of stem girth or between 1/3 and 1/2 of leaves slightly wilting.Grade 5 means stem lesions greater than 1/2 of stem girth, but not completely around the girth or between 1/2 and 2/3 of leaves wilting. Grade 7 means stem lesions around whole stem girth or greater than 2/3 of leaves wilting. Grade 9 means plants look dead.Calculate the disease index using the following formula. Disease severity was divided into six grades. Representative results.Four-week-old plants of the resistant variety BH and susceptible variety XHJ were challenged with Phytophthora nicotianae using the method presented in this article. At three days post inoculation, in XHJ, stem lesions covered approximately 1/2 of the stem girth and 1/2 of the leaves were slightly wilted. In the resistant variety BH, no symptoms were observed.At four days post inoculation, leaf wilting and severe stem lesions occurred in XHJ whereas these symptoms did not appear in BH.At five days post inoculation, individual plant disease severity was recorded and calculated based on the Chinese national standard for the grade and investigation method of tobacco diseases and insect pests. 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. To confirm the inoculation efficiency, relative pathogen biomass was quantified by realtime PCR.The result of the realtime PCR confirms the phenotypic observations. Five progenies from the BC4 F2 population of a cross between BH and XHJ, two intermediate resistance varieties, K326 and Yunyan87, were evaluated by zoospore suspension inoculation method and oat grains inoculation method. Zoospore suspension method was performed on small seedlings and oat grains inoculation method was performed on adult plants.For the five progenies from the BC4 F2 population, the disease index ranged from 16.49 to 77.60 by zoospore suspension method and ranged from 10.33 to 83.08 by oat grains method. The resistance classifications between two infection methods are mostly consistent with each other. With the two intermediate resistance varieties K326 and Yunyan87, the evaluation showed resistant in both methods.These data illustrate the correlation between two inoculation method despite they were performed on tobacco in different growth period. The protocol described here is an efficient and reliable method to evaluate the resistance of tobacco to infection by P.nicotianae seedling stage. This protocol is practical for breeding as well as for molecular mechanism research as the oat grain method is applicable for large-scale resistance screening of adult plants.This two methods can use complementary. Thanks for watching.

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