METHOD FOR INDOORS AND RAPIDLY IDENTIFYING THE RESISTANCE OF WHEAT TO BLACK POINT DISEASE CAUSED BY BIPOLARIS SOROKINIANA

Abstract

The present application discloses a method for rapidly identifying the resistance of wheat to black point disease caused by Bipolaris sorokiniana, where testing takes places in an indoor, controlled environment. The test method includes surface sterilization of wheat seeds, and cultivating wheat seedlings from the sterilized wheat seeds; preparing conidial suspension of Bipolaris sorokiniana, and spraying the conidial suspension on the seedlings at one-leaf-one-shoot stage; recording the percentage of the diseased leaf area in total leaf area of the first leaf of the wheat seedling on the 10.sup.th day of inoculation; calculating the black point incidence of the wheat according to an equation, and then evaluating the resistance of wheat to black point disease caused by Bipolaris sorokiniana through the black point incidence. Compared with existing field identification methods, the method of the present disclosure shortens identification time, simplifies identification procedure, greatly improves identification efficiency, and improves the accuracy and reliability of identification results.

Claims

1. A method for rapid identification of resistance of wheat to black point disease caused by Bipolaris sorokiniana in a controlled, indoor environment, comprising the steps of: a) a step of surface sterilization of wheat seeds, comprising: selecting healthy and full wheat seeds and immersing the wheat seeds in alcohol, and rinsing the wheat seeds with sterilized distilled water following the immersion to generate sterilized wheat seeds; b) a step of cultivating wheat seedlings from sterilized wheat seeds, comprising: placing sterilized wheat seeds from step a) in Petri dishes, placing said Petri dishes in an incubator, and then cultivating said sterilized wheat seeds for 10 days; c) a step of preparing a conidial suspension of Bipolaris sorokiniana, comprising: cutting Bipolaris sorokiniana that have been stored in a refrigerator at 4° C. into 0.3 cm.sup.2 pieces and inoculating in a potato dextrose agar medium, culturing at 25° C. in the dark to generate a cultured colony, gently scraping conidia from the cultured colony with a glass slide, rinsing the potato dextrose agar mediums with distilled water to obtain a fungal suspension, and filtering the fungal suspension with gauze to remove mycelial fragments and obtain a conidia suspension; d) a step of inoculating and identifying, comprising: shaking the conidia suspension of Bipolaris sorokiniana obtained in step c) and spraying the conidia suspension on leaves of a wheat seedling which has grown to a one-leaf-one-shoot stage in step b), covering the wheat seedling with a transparent plastic cover, and placing the wheat seedling in an incubator and culturing for 10 days at 25° C.; e) a step of recording identification results, comprising: recording a percentage of diseased leaf area within a total leaf area of a leaf of the wheat seedling cultivated in step d) on the 10.sup.th day of inoculation to obtain a percentage of diseased leaf area; f) a step of calculating black point incidence in wheat, comprising: calculating a percentage of black point grains (black point incidence) from the percentage of diseased leaf area by using the equation y=−1.0037x+66.1360; wherein in the equation, y and x are the black point incidence and the percentage of diseased leaf area, respectively, comprising; g) a step of evaluating resistance to black point disease, comprising: evaluating resistance of wheat to black point disease caused by Bipolaris sorokiniana based on the black point incidence calculated in step f).

2. The method according to claim 1, wherein in step a), the volume percentage of alcohol is 70% by volume, duration of time for immersion in alcohol is 2 minutes; and wheat seeds are rinsed with sterilized distilled water 3 times.

3. The method according to claim 1, wherein in step a), rinsing wheat seeds with sterilized distilled water comprises adding distilled water and shaking gently for 5 seconds so as to dissolve the alcohol on the surface of the seeds in the distilled water, thereby preventing alcohol from inhibiting germination of the wheat seeds.

4. The method according to claim 1, wherein in step b), the step of cultivating the young wheat seedlings comprises steps of: i). placing wheat seeds in Petri dishes with a diameter of 9 cm, in which two layers of sterilized filter paper and 4 mL of sterilized distilled water have been added in advance, and placing 5 wheat cultivars or lines in each Petri dish with 15 grains for each cultivar or line; ii). placing the Petri dishes with the wheat seeds in a 25° C. incubator and culturing the wheat seeds for 3 days in the dark, removing wheat grains with inconsistent germinability, adding 10 mL of sterilized distilled water to each Petri dish, and removing covers of the Petri dishes; iii). after 3 days, alternating a temperature and light cycle in the incubator between a first cycle comprising a temperature of 25° C. and a light duration of 11 hours and a second cycle comprising a temperature of 21° C. and a dark duration of 13 hours and continuing to incubate the wheat seeds for 7 another days.

5. The method according to claim 1, wherein, in step c), after incubating Bipolaris sorokiniana at 25° C. in darkness for 7 days, observing the state of conidia production, wherein a gray color of colonies indicates a low yield of conidia production and a dark indicates a high yield of conidia, and continuing the culture process while removing Petri dishes when the colonies become dark color.

6. The method according to claim 1, wherein in step c), the step of rinsing the Petri dishes with distilled water and filtering with a gauze comprises washing the Petri dishes with distilled water for at least two times, filtering the fungal suspension with four layers of gauze to obtain the conidial suspension of B. sorokiniana, determining concentration of spores in the conidia suspension by using a hemocytometer, and adjusting spore concentration to 1×10.sup.5/mL.

7. The method according to claim 1, wherein in step d) the step of culturing the seedlings after inoculation in an incubator comprises: incubating the seedlings in the dark for 24 hours, setting alternating temperature and light cycle of the incubator at a third cycle of a temperature of 25° C. and light for 11 hours and a fourth cycle of a temperature of 25° C. and dark for 13 hours; spraying 3 mL of sterilized distilled water evenly onto cultured seedlings twice a day during the culturing process so as to maintain a high humidity required for infection of Bipolaris sorokiniana and for development of leaf blight.

8. The method according to claim 1, wherein step of evaluating the resistance of wheat to black point disease in step g) comprises: recording a value of “I” if there are no diseased grains, wherein “I” is considered to be immune, recording a value of “HR” if the black point incidence is from 0.1% to 1.9%, wherein “HR” is considered highly resistant, recording a value of “R” if the black point rate is from 2.0% to 4.9%, wherein “R” is considered to be resistant, recording a value of “SS” if the black point incidence is from 5.0% to 14.9%, wherein “SS” is considered to be slightly susceptible, recording a value of “MS” if the black point incidence is from 15.0% to 30.0%, wherein “MS” is considered to be moderately susceptible, and recording a value of “HS” if the black point incidence is greater than 30%, wherein “HS” is considered to be highly susceptible.

Description

DESCRIPTION OF THE DRAWINGS

[0051] FIG. 1 shows the results for regression analysis of the black point incidence and percentage of diseased leaf area for 54 wheat cultivars/lines inoculated with B. sorokiniana in the present disclosure.

[0052] In FIG. 1, the percentage of black point incidence and the percentage of leaf blights shows significantly negative correlation, the correlation coefficient (r) is −0.95, the regression equation of the two is y=−1.0037x+66.1360, and the coefficient of determination (r.sup.2) is 0.90, y and x in the equation are the percentage of black point incidence and the diseased area, respectively.

[0053] FIG. 2 shows the results for the regression analysis of the black point incidence and the black point incidence by the field identification methods for 54 wheat cultivars or lines infected by B. sorokiniana in the present disclosure.

[0054] In FIG. 2, the black point incidence calculated in the present disclosure has a significantly positive correlation with the black point incidence identified in the field condition, and the correlation coefficient (r) is 0.96.

[0055] FIG. 3 shows the typical wheat lines screened by the technical scheme of the present disclosure, which are resistant or susceptible to black point disease caused by B. sorokiniana.

[0056] In FIG. 3, the upper part shows the status of the spots of the leaf blight after inoculation with B. sorokiniana; the lower part shows the status of black point disease after inoculation with B. sorokiniana. A is the wheat line of Zhumai 6097, which is susceptible to black point disease; B is the wheat line of Shannong 530070, which is resistant to black point disease; CK is a control in which distilled water is sprayed, Bs represents the lines that is sprayed with conidia suspension of B. sorokiniana.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0057] The present disclosure will be further described below in conjunction with the embodiments, but these embodiments are not intended to limit the protection scope of the present disclosure.

Example 1

[0058] In the winter of 2016, the method of the present disclosure for rapid indoor identification of the resistance of wheat to black point disease caused by Bipolaris sorokiniana was used to identify the resistance of 54 wheat cultivars or lines harvested in Xingyang to black point disease. The detailed steps are described as follows.

[0059] Surface sterilization of wheat seeds: healthy and full wheat seeds from 54 wheat cultivars or lines were selected and soaked in 70% (volume ratio) alcohol for 2 minutes, then the seeds were rinsed with sterilized distilled water for 3 times, and shaken gently for 5 seconds each time after adding distilled water, so that the alcohol remaining on the surface of the seeds was fully dissolved in the water to prevent the alcohol from inhibiting the germination of wheat seeds;

[0060] Wheat seedling cultivation: the wheat seeds that had been surface-sterilized in step a) were placed in Petri dishes with a diameter of 9 cm. To each Petri dish were added 2 layers of sterilized filter paper and 4 mL of sterilized distilled water in advance. Five wheat cultivars or lines were placed in each Petri dish, 15 seeds per cultivar/line. Then the Petri dishes were placed in a 25° C. incubator for 3 days in the dark. After 3 days, the wheat seeds with inconsistent germination potential were picked out, 10 mL of sterilized distilled water was supplemented to each Petri dish, and the cover of the Petri dishes were removed. The temperature and light cycle in the incubator was alternated between “25° C., light, 11 hours” and “21° C., dark, 13 hours”, and then 7-day culturing was continued.

[0061] Preparation of suspensions of B. sorokiniana conidia: the B. sorokiniana stored in a 4° C. refrigerator ready for use was cut into 0.3 cm.sup.2 pieces and inoculated into potato dextrose agar (PDA) medium, and the B. sorokiniana were cultured in the dark at 25° C. for 7 days. The conidia production was observed. If the color of the colony was gray, it indicated that the number of conidia was too small. The sealing film of the Petri dishes needed to be removed for further culture. The Petri dishes were taken out when the color of the colonies became deep black and a large number of conidia were produced. The conidia were gently scraped off the Petri dishes with a sterilized glass slide. The Petri dishes were rinsed 3 times with sterilized distilled water, and mycelial fragment were filtered with a four-layer gauze to obtain the suspension of B. sorokiniana conidia, and the concentration of conidia suspension was measured by means of a hemocytometer and adjusted to a spore concentration of 1×10.sup.5/mL;

[0062] Inoculation and identification: the suspension of B. sorokiniana conidia prepared in step c) was shaken and sprayed on the wheat leaves growing to one-leaf-one-shoot stage in step b), and the wheat leaves were covered with a transparent plastic cover and then placed in an incubator, cultured in the dark at 25° C. for 24 hours. After 24 hours, the temperature and light cycle in the incubator was alternated between “25° C., light, 11 hours” and “25° C., dark, 13 hours”, and cultured for another 9 days. 3 mL of sterilized distilled water was sprayed evenly into the cover by using a hand sprayer, twice a day in the morning and in the evening to maintain a high humidity required for infection of B. sorokiniana and for development of leaf blight;

[0063] Recording of identification results: the percentage of the diseased leaf area in the total leaf area of the first leaf of the wheat seedling in step d) was recorded on the 10.sup.th day of inoculation, and the percentage of diseased leaf area was obtained;

[0064] Calculation of Black Point Incidence of the Wheat:

[0065] Calculate the percentage of black point grain (namely the black point incidence) from the identified diseased leaf area percentage by using the equation y=−1.0037x+66.1360. In the equation, y and x are the black point incidence and the percentage of diseased leaf area, respectively.

[0066] g) Evaluation of resistance to black point disease:

[0067] The resistance of wheat to black point disease caused by B. sorokiniana was evaluated based on the black point incidence calculated in step f) (the method for evaluating the resistance of wheat to black point disease comprises recording as I if there are no diseased grains, which is considered to be immune; recording as HR if the black point incidence is 0.1% to 1.9%, which is considered to be highly resistance; recording as R if the black point incidence is from 2.0% to 4.9%, which is considered to be resistant; recording as SS if the black point incidence is from 5.0% to 14.9%, which is considered to be of slightly susceptible; recording as MS if the black point incidence is from 15.0% to 30.0%, which is considered to be moderately susceptible; and recording as HS if the black point incidence is greater than 30%, which is considered to be highly susceptible).

[0068] The identification results for 54 wheat cultivars or lines are shown in Table 2 and FIG. 1 in detail. It can be seen from FIG. 1 that the larger the diseased leaf area of leaf blight caused by B. sorokiniana at the seedling stage of wheat, the lower the black point incidence caused by B. sorokiniana at the field filling stage is. That is, there is a significantly negative correlation between the area of the diseased leaf area of the leaf blight at the seedling stage and the black point incidence, the correlation coefficient reached −0.95. Through regression analysis, the coefficient of determination was 0.90. The comparison of the resistance evaluation of 54 wheat strains using the method of the present disclosure to the field identification results for inoculation showed that 50 in 54 of wheat cultivars or lines presented consistent identification results, and resistance identification results for only 4 lines had inconsistent results (see Table 2). The black point incidence calculated based on the diseased leaf area of leaf blight was significantly correlated with the actual black point incidence identified in the field, with a correlation coefficient being 0.96. The above results indicate that the technical scheme of the present disclosure can better evaluate the resistance of wheat to black point disease caused by B. sorokiniana.

TABLE-US-00002 TABLE 2 Comparison of results for field inoculation identification to results for resistance identification of the present disclosure for 54 wheat cultivars/lines harvested in 2016 Black point Black point Resistance incidence Resistance incidence by evaluation by calculated by evaluation by field field method of the method of the Wheat identification identification present present No. cultivar/lines method (%) method disclosure (%) disclosure 1 Guomai 2 0.9 HR 0.1 HR 2 Wenmai 10 2.0 R 0.2 HR 3 Shannong 2.4 R 0.0 I 530070 4 SN4143 3.3 R 1.6 HR 5 11-253 4.2 R 4.1 R 6 L661 (LPG) 4.2 R 5.8 SS 7 Xinong 4.4 R 13.7 SS 9871-1 8 Wenmai 8 4.9 R 0.1 HR 9 481/274 5.1 SS 0.8 HR 10 Huixianhong 5.6 SS 10.8 SS 11 04ZP16 5.6 SS 15.5 MS 12 11-696 5.7 SS 0.0 I 13 11-269 6.2 SS 10.3 SS 14 11-285 7.5 SS 17.4 MS 15 Bima 1 7.6 SS 17.0 MS 16 Shi 98-7136 7.6 SS 19.1 MS 17 SP1777-6-8 8.3 SS 20.9 MS 18 Wangshuibai 9.4 SS 9.8 SS 19 Sumai 3 9.6 SS 18.2 MS 20 92R137 9.6 SS 20.7 MS 21 15YD55 12.0 SS 13.9 SS 22 GM15 15.0 MS 20.5 MS 23 15YD77 16.0 MS 24.0 MS 24 15YD14 17.0 MS 12.2 SS 25 15YD56 19.0 MS 18.0 MS 26 PH691 22.0 MS 27.8 MS 27 15YQ24 22.0 MS 23.4 MS 28 Xuke 316 24.0 MS 29.9 MS 29 15YQ39 27.0 MS 24.2 MS 30 15YQ41 29.0 MS 26.1 MS 31 11-239 33.2 HS 29.7 MS 32 15YQ33 35.0 HS 33.0 HS 33 Xumai 9169 37.3 HS 46.3 HS 34 15YQ29 38.0 HS 34.4 HS 35 Yumai 47 41.2 HS 45.1 HS 36 11YC173 42.4 HS 45.3 HS 37 Zheng 2062 42.5 HS 55.7 HS 38 Wanyuanbai 1 42.9 HS 47.2 HS 39 Jinan 31 43.0 HS 40.7 HS 40 Xinaizao 818 44.2 HS 40.3 HS 41 Danti (NIU) 44.3 HS 45.3 HS 42 Aifeng 66 44.4 HS 45.9 HS 43 YN177 46.7 HS 48.3 HS 44 Helandazi 47.8 HS 45.8 HS 45 Chinese 50.3 HS 48.8 HS Spring 46 Tunmai 127 50.4 HS 46.5 HS 47 He 0927 54.4 HS 45.7 HS 48 Zhumai 6097 58.1 HS 53.2 HS 49 PZSCL6 60.0 HS 50.4 HS 50 Bainong 107 60.0 HS 53.6 HS 51 10M24 60.7 HS 55.9 HS 52 11-229 64.7 HS 54.7 HS 53 Jicheng 2 64.7 HS 56.5 HS 54 Yuaniia 69 64.7 HS 55.9 HS Note: The diseased leaf area of leaf blight is the identification result for the indoor inoculation with B. sorokiniana in the winter of 2016 in the present disclosure. The black point incidence is the identification result for the field inoculation with B. sorokiniana during the grain filling stage in 2016. The method for field identification of resistance to black point disease referred to the Chinese patent (ZL 2015105396457). In the black point resistance evaluation: I represents immune (black point incidence is 0.0%), HR represents highly resistant (0.1-1.9%), R represents resistant (2.0-4.9%), SS represents slightly susceptible (5.0-14.9%), MS represents moderately susceptible (15.0-30.0%), HS represents highly susceptible (black point incidence >30%). The identification result is that the immune, highly resistant and resistant are considered resistant cultivars/lines, while the slightly susceptible, the moderately susceptible strains and highly susceptible are considered susceptible cultivars/lines.

Example 2

[0069] Ten (10) disease-resistant wheat cultivars/lines were identified for 3 consecutive years by using the method for quickly identifying wheat resistance to black point disease caused by B. sorokiniana in the present disclosure, and the results were compared with the field identification results.

[0070] The detailed steps are as follows

[0071] Surface sterilization of wheat seeds: healthy and full wheat seeds were selected and soaked in 70% (volume ratio) alcohol for 2 minutes, then the seeds were rinsed with sterilized distilled water for 3 times, and shaken gently for 5 seconds each time after adding distilled water, so that the alcohol remaining on the surface of the seeds was fully dissolved in the water to prevent the alcohol from inhibiting the germination of wheat seeds;

[0072] Wheat seedling cultivation: the wheat seeds that had been surface-sterilized in step a) were placed in Petri dishes with a diameter of 9 cm. Each Petri dish was added 2 layers of sterilized filter paper and 4 mL of sterilized distilled water in advance. Five wheat cultivars or lines were placed in the each Petri dish, 15 grains per cultivar or line. Then the Petri dishes were placed in a 25° C. incubator for 3 days in the dark. After 3 days, the wheat seeds with inconsistent germination potential were picked out, 10 mL of sterilized distilled water was supplemented to each Petri dish, and the cover of the Petri dishes were removed. The temperature and light cycle in the incubator was alternated between “25° C., light, 11 hours” and “21° C., dark, 13 hours”, and then 7-day culturing was continued.

[0073] c) Preparation of suspension of B. sorokiniana conidia: the B. sorokiniana stored in a 4° C. refrigerator ready for use was cut into 0.3 cm.sup.2 pieces and inoculated into potato dextrose agar (PDA) medium, and the B. sorokiniana conidia were cultured in the dark at 25° C. for 7 days. The conidia production was observed. If the color of the colony was gray, it indicated that the number of conidia was too small. The sealing film over the Petri dishes needed to be removed for further culture. The Petri dishes were taken out when the color of the colonies became deep black and a large number of conidia were produced. The conidia were gently scraped off the Petri dishes with a sterilized glass slide. The Petri dishes were rinsed 3 times with sterilized distilled water, and mycelial fragment were filtered with a four-layer gauze to obtain the suspension of B. sorokiniana conidia, and the concentration of conidia suspension was measured by means of a hemocytometer and adjusted to a conidia concentration of 1×10.sup.5/mL;

[0074] d) Inoculation and identification: the suspension of B. sorokiniana conidia prepared in step c) was shaken and sprayed on the wheat leaves growing to one-leaf-one-shoot stage in step b), and the wheat leaves were covered with a transparent plastic cover and then placed in an incubator, cultured in the dark at 25° C. for 24 hours. After 24 hours, the temperature and light cycle in the incubator was alternated between “25° C., light, 11 hours” and “25° C., dark, 13 hours”, and cultured for another for 9 days. 3 mL of sterilized distilled water was sprayed evenly into the cover by using a hand sprayer, twice a day in the morning and in the evening to maintain a high humidity required for infection of B. sorokiniana and for development of leaf blight;

[0075] e) Recording of identification results: the percentage of the diseased leaf area in the total leaf area of the first leaf of the wheat seedling in step d) was recorded on the 10.sup.th day of inoculation, and the percentage of diseased leaf area was obtained;

[0076] f) Calculation of black point incidence of the wheat:

[0077] Calculate the percentage of black point grain (namely the black point incidence) from the identified percentage of diseased leaf area by using the equation y=−1.0037x+66.1360. In the equation, y and x are the black point incidence and the percentage of diseased leaf area, respectively.

[0078] g) Evaluation of resistance to disease:

[0079] The resistance of wheat to black point disease caused by B. sorokiniana was evaluated based on the black point incidence calculated in step f) (the method for evaluating the resistance of wheat to black point disease comprises recording as I if there are no diseased grains, which is considered to be immune; recording as HR if the black point incidence is from 0.1% to 1.9%, which is considered to be highly resistant; recording as R if the black point incidence is from 2.0% to 4.9%, which is considered to be resistant; recording as SS if the black point incidence is from 5.0% to 14.9%, which is considered to be of slightly susceptible; recording as MS if the black point incidence is from 15.0% to 30.0%, which is considered to be moderately susceptible; and recording as HS if the black point incidence is greater than 30%, which is considered to be highly susceptible).

[0080] In the present disclosure, 10 wheat cultivars/lines were identified for the resistance to black point disease caused by B. sorokiniana in three consecutive years and the results were compared with the results for the field identification, the comparison results are shown in Table 3 and FIG. 3. The results in Table 3 show that during the three years, there is consistency among the results for resistance to the black point disease caused by B. sorokiniana for 10 cultivars/lines identified by the method of the present disclosure. However, under field conditions, due to the effect of the meteorological factors such as temperature, humidity, sunshine length etc. the resistance evaluation of 9 in 10 cultivars or lines are inconsistent from year to year. For example, the black point incidence for Guomai 2 varies from 0.9% to 9.1% in three years, and the resistance evaluation results are R, HR, and SS, respectively. This result will lead to deviation in the resistance evaluation for the same wheat cultivar/line. In contrast, the use of the identification method of the present disclosure gives consistency between the identification results for three years, which ensures the consistency of the resistance evaluation.

TABLE-US-00003 TABLE 3 Comparison of the results for identification of wheat resistance to black point disease caused by B. sorokiniana by method of the present disclosure with the results for field identification for three consecutive years Black point incidence by method Identification result by method Wheat of the present disclosure (%) of the present disclosure cultivars/lines 2016 2017 2018 2016 2017 2018 Wenmai 10 0.1 0.1 1.1 HR HR HR 481/274 1.6 0.2 0.4 HR HR HR 11-253 4.0 0.0 3.5 R I R 11-696 4.6 0.0 3.0 R I R SN4143 8.3 5.8 5.0 SS SS SS Shannong 4.9 0.0 3.0 R I R 530070 L661 (LPG) 10.3 13.7 5.3 SS SS SS Wenmai 8 1.5 0.1 0.0 HR HR HR Xinong 2.5 0.8 0.0 R HR HR 9871-1 Guomai 2 4.1 0.0 1.9 R I HR Black point incidence Field identification Wheat by Field identification results cultivars/lines 2016 2017 2018 2016 2017 2018 Wenmai 10 1.9 2.0 2.5 HR R R 481/274 4.4 5.1 4.8 R SS R 11-253 4.4 4.2 5.4 R R SS 11-696 3.5 5.7 6.1 R SS SS SN4143 6.8 3.3 7.4 SS R SS Shannong 1.5 2.4 7.6 HR R SS 530070 L661 (LPG) 9.1 4.2 8.7 SS R SS Wenmai 8 5.0 4.9 8.8 SS R SS Xinong 4.4 4.4 8.9 R R SS 9871-1 Guomai 2 4.5 0.9 9.1 R HR SS Note: The three-year field resistance identification test was completed in Xingyang Breeding Field (Yulong Town, Xingyang City, Henan Province, China), and the method for field identification of resistance to black point disease referred to the Chinese invention patent (ZL 2015105396457). In the resistance evaluation, I, HR, R, and SS represent immune, highly resistant, resistant, and slightly susceptible, respectively.

[0081] When identified using the method of the present disclosure, there is consistent evaluation of resistance of 10 strains to black point disease caused by B. sorokiniana during 3 years. However, under field conditions, 9 of 10 strains have inconsistent results for resistance evaluation between two years.

Example 3

[0082] By using the method for rapid indoor identification of the resistance of wheat to the black point disease caused by B. sorokiniana, two of ten advanced lines from the F.sub.4 and F.sub.5 were identified to be resistant lines in March 2018. Crossing was conducted in April 2018 in the field, and breeding efficiency is improved compared with field identification method. Cross breeding may be carried out one year earlier. The detailed steps are described as follows.

[0083] Surface sterilization of wheat seeds: healthy and full wheat seeds from 10 advanced F.sub.4 and F.sub.5 wheat lines were selected and soaked in 70% (volume ratio) alcohol for 2 minutes, then the seeds were rinsed with sterilized distilled water for 3 times, and shaken gently for 5 seconds each time after adding distilled water, so that the alcohol remaining on the surface of the seeds was fully dissolved in the water to prevent the alcohol from inhibiting the germination of wheat seeds;

[0084] Wheat seedling cultivation: the wheat seeds that had been surface-sterilized in step a) were placed in Petri dishes with a diameter of 9 cm. Each Petri dish was added 2 layers of sterilized filter paper and 4 mL of sterilized distilled water in advance. Five wheat lines were placed in each Petri dish, 15 grains per lines. Then the Petri dishes were placed in a 25° C. incubator for 3 days in the dark. After 3 days, the wheat seeds with inconsistent germination potential were picked out, 10 mL of sterilized distilled water was supplemented to each Petri dish, and the cover of the Petri dishes were removed. The temperature and light cycle in the incubator was alternated between “25° C., light, 11 hours” and “21° C., dark, 13 hours”, and then 7-day culturing was continued.

[0085] Preparation of suspension of B. sorokiniana conidia: the B. sorokiniana stored in a 4° C. refrigerator ready for use was cut into 0.3 cm.sup.2 pieces and inoculated into potato dextrose agar (PDA) medium, and B. sorokiniana were cultured in the dark at 25° C. for 7 days. The conidia production was observed. If the color of the colony was gray, it indicated that the number of conidia was too small. The sealing film over the Petri dishes needed to be removed for further culture. The Petri dishes were taken out when the color of the colonies became deep black and a large number of conidia were produced. The conidia were gently scraped off the Petri dishes with a sterilized glass slide, the Petri dishes was rinsed 3 times with sterilized distilled water, and mycelial fragment were filtered with a four-layer gauze to obtain the suspension of B. sorokiniana conidia, and the concentration of conidia suspension was measured by means of a hemocytometer and adjusted to a conidia concentration of 1×10.sup.5/mL;

[0086] Inoculation and identification: the suspension of B. sorokiniana conidia prepared in step c) was shaken and Tween 20 in amount of 0.02% by volume with respect to the suspension of B. sorokiniana was added, and sprayed with a hand sprayer on the wheat leaves growing to one-leaf-one-shoot stage in step b), and the wheat leaves were covered with a transparent plastic cover and then placed in an incubator, cultured in the dark at 25° C. for 24 hours. After 24 hours, the temperature and light cycle in the incubator was alternated between “25° C., light, 11 hours” and “25° C., dark, 13 hours”, and cultured for another 9 days. 3 mL of sterilized distilled water was sprayed evenly into the cover by using a watering can, twice a day in the morning and in the evening to maintain a high humidity required for B. sorokiniana infection and development of leaf blight;

[0087] Recording of identification results: the percentage of the diseased leaf area in the total leaf area of the first leaf of the wheat seedling in step d) was recorded on the 10.sup.th day of inoculation, and the percentage of diseased leaf area was obtained;

[0088] Calculation of Black Point Incidence of the Wheat:

[0089] Calculate the percentage of black point grains (namely the black point incidence), from the identified percentage of diseased leaf area by using the equation y=−1.0037x+66.1360. In the equation, y and x are the black point incidence and the percentage of diseased leaf area, respectively;

[0090] g) Evaluation of resistance to disease:

[0091] The resistance of wheat to black point disease caused by B. sorokiniana was evaluated based on the black point incidence calculated in step f) (the method for evaluating the resistance of wheat to black point disease comprises recording as I if there are no diseased grains, which is considered to be immune; recording as HR if the black point incidence is from 0.1% to 1.9%, which is considered highly resistant; recording as R if the black point incidence is from 2.0% to 4.9%, which is considered to be resistant; recording as SS if the black point rate is from 5.0% to 14.9%, which is considered to be of slightly susceptible; recording as MS if the black point incidence is from 15.0% to 30.0%, which is considered to be moderately susceptible; and recording as HS if the black point incidence is greater than 30%, which is considered to be highly susceptible).

[0092] In March 2018, ten advanced lines were identified in an indoor, controlled environment for black point disease, and the results are shown in Table 4. According to the results of the indoor identification, two lines resistant to black point disease were screened among the 10 advanced lines (17YH24 and 17YQ13). These two lines were crossed with high-yielding wheat cultivars in April 2018. If identified in the field, the identification had to be conducted at the end of April and the crossing could be done till the year of 2019. Therefore, by adopting the method of the present disclosure, the breeding process of wheat resistant to black point disease can be preceded by one year.

TABLE-US-00004 TABLE 4 Identification results for the resistance of ten advanced F.sub.4 and F.sub.5 wheat lines to black point disease caused by B. sorokiniana by using the method of the present disclosure Identification result by the Field Identification method of the Crossing identification No. Combination Generation time present disclosure time time 17YH22 Zhoumai F.sub.4 March 2018 SS April-June 22.sup.2/Zhengfeng 2018 99745 17YH23 Zhoumai F.sub.4 March 2018 SS April-June 22.sup.2/Zhengfeng 2018 99745 17YH24 Shengnong F.sub.4 March 2018 R April 2018 April-June 2.sup.2/Zhengfeng 2018 99745 17YH25 Shengnong F.sub.4 March 2018 SS April-June 2.sup.2/Zhengfeng 2018 99745 17YH26 Shengnong F.sub.4 March 2018 HS April-June 2.sup.2/Zhengfeng 2018 99745 17YH27 Shengnong F.sub.4 March 2018 HS April-June 2.sup.2/Zhengfeng 2018 99745 17YQ13 Shengnong F.sub.5 March 2018 R April 2018 April-June 2/Shannong 4143 2018 17YQ14 Shengnong F.sub.5 March 2018 MS April-June 2/Shannong 4143 2018 17YQ15 Zhoumai F.sub.5 March 2018 MS April-June 22/Shannong 2018 4143 17YQ16 Zhoumai F.sub.5 March 2018 SS April-June 22/Shannong 2018 4143 Note: In the resistance evaluation, I, HR, R, SS, MS, HS represent immune, highly resistant, resistant, slightly susceptible, moderately susceptible, and highly susceptible. The texts in bold (row 17YH24 and row 17YQ13) represent two resistant advanced lines screened for crossing. The susceptible lines are the eliminated breeding materials which were no longer used for cross-breeding.

Example 4

[0093] By using the method of the present disclosure for rapid indoor identification of wheat resistance to black point disease caused by B. sorokiniana, 105 wheat cultivars or lines that were planted on a large scale or under pilot production were identified in an indoor, controlled environment on March 2018, and 3 resistant lines were screened, saving more than 50% of manpower and material resources. The detailed steps are described as follows.

[0094] Surface sterilization of wheat seeds: healthy and full seeds from 105 wheat strains were selected and soaked in 70% (volume ratio) alcohol for 2 minutes, then the seeds were rinsed with sterilized distilled water for 3 times, and shaken gently for 5 seconds each time after adding distilled water, so that the alcohol remaining on the surface of the seeds was fully dissolved in the water to prevent the alcohol from inhibiting the germination of wheat seeds;

[0095] Wheat seedling cultivation: the wheat seeds that had been surface-sterilized in step a) were placed in Petri dishes with a diameter of 9 cm. Each Petri dish was added 2 layers of sterilized filter paper and 4 mL of sterilized distilled water in advance. Five wheat cultivars/lines were placed in each Petri dish, 15 grains per cultivar/line. Then the Petri dishes were placed in a 25° C. incubator for 3 days in the dark. After 3 days, the wheat seeds with inconsistent germination potential were picked out, 10 mL of sterilized distilled water was supplemented to each Petri dish, and the cover of the Petri dishes were removed. The temperature and light cycle in the incubator was alternated between “25° C., light, 11 hours” and “21° C., dark, 13 hours”, and then a 7-day culturing was continued.

[0096] Preparation of suspension of B. sorokiniana conidia: the B. sorokiniana stored in a 4° C. refrigerator ready for use was cut into 0.3 cm.sup.2 pieces and inoculated into potato dextrose agar (PDA) medium, and B. sorokiniana were cultured in the dark at 25° C. for 7 days. The conidia production was observed. If the color of the colony was gray, it indicated that the number of conidia was too small. The sealing film over the Petri dishes needed to be removed for further culture. The Petri dishes were taken out when the color of the colonies became deep black and a large number of conidia were produced. The conidia were gently scraped off the Petri dishes with a sterilized glass slide, the Petri dishes was rinsed 3 times with sterilized distilled water, and mycelial fragment were filtered with a four-layer gauze to obtain the suspension of B. sorokiniana conidia, and the concentration of conidia suspension was measured by means of a hemocytometer and adjusted to a spore concentration of 1×10.sup.5/mL;

[0097] Inoculation and identification: the suspension of B. sorokiniana conidia prepared in step c) was shaken and Tween 20 in amount of 0.02% by volume with respect to the suspension of B. sorokiniana was added, and sprayed with a hand sprayer on the wheat leaves growing to one-leaf-one-shoot stage in step b), and the wheat leaves were covered with a transparent plastic cover and then placed in an incubator, cultured in the dark at 25° C. for 24 hours. After 24 hours, the temperature and light cycle in the incubator was alternated between “25° C., light, 11 hours” and “25° C., dark, 13 hours”, and cultured for another 9 days. 3 mL of sterilized distilled water was sprayed evenly into the cover by using a hand sprayer twice a day in the morning and in the evening to maintain a high humidity required for B. sorokiniana infection and development of leaf blight;

[0098] Recording of identification results: the percentage of the diseased leaf area in the total leaf area of the first leaf of the wheat seedling in step d) was recorded on the 10.sup.th day of inoculation, and the percentage of diseased leaf area was obtained;

[0099] Calculation of Black Point Incidence of the Wheat:

[0100] Calculate the percentage of black point grain (namely the black point incidence), from the identified percentage of diseased leaf area by using the equation y=−1.0037x+66.1360. In the equation, y and x are the black point incidence and the percentage of diseased leaf area, respectively;

[0101] g) Evaluation of resistance to disease:

[0102] The resistance of wheat to black point disease caused by B. sorokiniana was evaluated based on the black point incidence calculated in step f) (the method for evaluating the resistance of wheat to black point disease comprises: recording as I if there are no diseased grains, which is considered to be immune; recording as HR if the black point incidence is from 0.1% to 1.9%, which is considered highly resistant; recording as R if the black point incidence is from 2.0% to 4.9%, which is considered to be resistant; recording as SS if the black point incidence is from 5.0% to 14.9%, which is considered to be of slightly susceptible; recording as MS if the black point incidence is from 15.0% to 30.0%, which is considered to be moderately susceptible; and recording as HS if the black point incidence is greater than 30%, which is considered to be highly susceptible).

[0103] The indoor identification results for 105 wheat cultivars/lines in 2018 are shown in Table 5. Four resistant cultivar/lines were selected from 105 wheat strains. In 2019, the identified resistant cultivar/lines were verified by field inoculation, and 3 wheat cultivar/lines resistant to black point disease were determined. Indoor identification of 105 wheat cultivar/lines took only 20 days, 2 incubators were used, and only one staff is sufficient to complete. If identified in the field, it will require planting at least 105 rows of wheat, a wheat growth season of 200 days, and 2-3 staff to cooperate to complete the field identification. Therefore, the method of the present disclosure for indoor identification of wheat resistance to black point disease caused by B. sorokiniana saves at least 50% of manpower and material resources.

TABLE-US-00005 TABLE 5 Results for identification of resistance to black point disease caused by B. sorokiniana for 105 wheat cultivars or lines by using the method of the present disclosure Identification Results results for the for field Wheat method of the identification No. cultivars/lines present disclosure method 1 Emai 526 HS HS 2 Guinong 22 R R 3 Nongda 2011 MS — 4 Zhengmai 1860 MS — 5 Zhenmai 3 MS — 6 Taihemai 2 SS — 7 Saidemai 1 MS — 8 Womai 66 HS HS 9 Zhoumai 36 MS — 10 Zhongmai 170 MS — 11 Zhengmai 132 HS MS 12 Fengdecunmai 16 MS — 13 Luomai 956 HS MS 14 Zhumai 305 HS HS 15 Junmai 0821 HS HS 16 Zhongyu 1526 MS — 17 Wenmai 168 HS MS 18 Taixue 518 MS — 19 Chenming 1 SS — 20 Hemai 0666 SS — 21 Dengmai 298 SS — 22 Xiangmai 182 SS — 23 Kemai 1609 SS — 24 Zhengmai 20 MS — 25 Xindifeng 058 SS — 26 Jinmai 23 HS HS 27 Zhongmai 255 SS — 28 Gaoyou 5218 R R 29 Zhengmai 158 SS — 30 Gaoyou 5766 SS — 31 Huaichuan 709 SS — 32 Zhengyuanmai 5 SS — 33 Xinong 238 SS — 34 Weimai 68 SS — 35 Changyimai 3 SS — 36 Fumai 368 SS — 37 Jinyou 18 SS — 38 Shannong 981 HS HS 39 Bainongyunluo 33 HS HS 40 Luomai 31 SS — 41 Zhengmai 379 SS — 42 Zhoumai 22 MS — 43 Zhoumai 18 MS — 44 Bainong 207 SS — 45 Xun 5366 MS — 46 Yunong 516 MS — 47 Shangdumai 137 HS HS 48 Jiyanmai 10 HS HS 49 Saidemai 8 HS MS 50 Xinong 511 SS — 51 Xinong 585 SS — 52 Ruiquanmai168 MS — 53 Yikemai 5 MS — 54 Ruihua 1426 SS — 55 Yangmai 158 HS MS 56 Zhoumai 32 HS MS 57 Ruihua 055 SS — 58 Xinong 528 HS MS 59 Ruihua 1426 SS — 60 Xinong 239 SS — 61 Nannong 0686 SS — 62 Junmai 802 HS HS 63 Junmai 830 MS — 64 Zhengmai 1867 HS HS 65 Ningmai 13 SS — 66 Ningmai 20 SS — 67 Ningmai 24 SS — 68 Yubao 5018 MS — 69 Yunong 517 SS — 70 Ningmaizi 126 SS — 71 Ningmaizi14213 R R 72 Zhenmai 10 SS — 73 Yangfumai 2166 SS — 74 Ningmaizi 67 R SS 75 Haomai 1 SS — 76 Longmai 28 SS — 77 Yangmai 14 MS — 78 Ningmaizi 119 SS — 79 Xiaoyan 22 SS — 80 Xunong 0029 SS — 81 Huaimai 28 HS HS 82 Annong 1124 SS — 83 Luokang 2 SS — 84 Yumai 18 SS — 85 Yangmai 15 SS — 86 Shannong 20 SS — 87 Gaochanxianfeng SS — 88 Zimai 19 HS MS 89 Yannong 19 SS — 90 Womai 8 SS — 91 Womai 182 SS — 92 Huaimai 28 SS — 93 Huaimai 29 MS — 94 Huaima 35 MS — 95 Weilai 0818 SS — 96 Annong 0711 SS — 97 Annong 1589 SS — 98 Shengxuan No. 5 SS — 99 Shengxuan 6 SS — 100 Tainong 9862 SS — 101 Huaimai 302 SS — 102 Huamai 0772 SS — 103 Wuhan 1 SS — 104 Fengdecunmai 5 MS — 105 Quanmai 29 MS — Note: The purpose of this example is to provide typical resistant and susceptible materials for disease resistance breeding and mechanism research on resistance to black point disease. Since field inoculation and identification are limited by the growth period, in order to reduce the workload, the wheat cultivars or lines used in indoor identification are not resistant or highly susceptible cultivars or lines, that is, materials that are not typical resistant or susceptible will no longer undergo field identification; In the resistance evaluation, R, SS, MS, and HS represent resistant, slightly susceptible, moderately susceptible, and highly susceptible, respectively; Texts in bold (rows 2, 28 and 71) represent the three lines identified resistant to black point disease.

[0104] The formulation and method for preparation of the potato dextrose agar (PDA) medium used in the above examples to cultivate the pathogen B. sorokiniana are as follows.

[0105] Formula of PDA medium: potato 200 g, glucose 20 g, agar powder 20 g and tap water 1000 mL, having a natural pH.

[0106] Preparation steps: Peeling and washing the potatoes, slicing the potatoes into small chips with a thickness of about 0.2 mm, placing the chips in boiling water and cooking the potato chips to an extent that the chips can be poked through with a glass rod. Then filtering the potato chips with four-layer gauze, and removing the potato chips. Adding glucose and agar powder, continuing to heat and stir them to mix, and then making up to 1000 mL with water after cooling the mixture. Dispensing the mixture into 150 mL flasks (90 mL/flask), sterilizing the mixture at 121° C. for 20 minutes. Cooling mixture to approximately 50° C. and pouring into Petri dishes with a diameter of 9 cm (10 mL/dish), the resulting PDA medium is used to culture B. sorokiniana after cooling.