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.
