Quick in-vivo screening method of antagonistic trichoderma

[0055] Example 1

[0056] This embodiment relates to a rapid screening method for antagonistic Trichoderma in vivo, which includes the following steps:

[0057] 1. Cultivate the cucumber anthracnose pathogen on PDA plates for 7 days (a gift from researcher Li Baoju, Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences). Take the bacterial cake from the edge of the colony, transfer it to PD liquid medium, and culture it on a constant temperature shaker at 28°C and 180r/min for 5 days. The cultured fermentation broth was transferred to a centrifuge tube, and centrifuged at 10000r for 10 minutes in a centrifuge to obtain the bacterial fermentation supernatant.

[0058] 2. In a 5ml centrifuge tube, mix the supernatant of the fermentation broth of the pathogen and MS medium 1:4 for a total volume of 4ml to obtain the pathogen inoculum.

[0059] 3. Place cucumber seeds (Xiafeng No. 2) in a petri dish covered with moist filter paper, and germinate for 2 days in a 28°C constant temperature incubator under dark conditions. Put the seeds after germination at a concentration of 10 6 After being soaked in the Trichoderma spore suspension per ml for 12 hours, the Trichoderma spore suspension attached to the surface of the seed was absorbed by filter paper.

[0060] 4. The cucumber seed radicles after the treatment in step 3 are placed down in the pathogen inoculum and cultivated as a treatment group. Cucumber seeds were cultured in a culture medium without pathogenic bacteria and Trichoderma as the control group (CK). Placed in a constant temperature incubator (light 16h/dark 8h) at 28°C, 4000lx light intensity and 75% relative humidity for 5 days, the seedling emerges ( figure 1 ).

[0061] 5. Cut off the cotyledons of the test tube seedlings of the treatment group after cultivation in step 4, and scan the cotyledon area S1 with a colony area scanner ( figure 2 ), the larger the cotyledon area S1, the stronger the corresponding Trichoderma antagonistic. At the same time, scan the cotyledon leaf area S2 of the test tube seedlings of the control group to compare the values ​​of S1/S2. The antagonistic threshold was set according to the ratio of the cotyledon area of ​​the treatment group to that of the control healthy cotyledon, and the antagonism of Trichoderma was divided into three levels:

[0062] S1/S2<1/2 is the first level;

[0063] 1/2 <3/4 is the second level;

[0064] S1/S2>3/4 are three levels.

[0065] 6. The 12 strains of Trichoderma were tested for potted control to test the correlation between the results of rapid screening in vivo and the results of potted trials.

[0066] 7. Potted screening method: activate the tested Trichoderma strain and the pathogen (the pathogen of cucumber anthracnose) on the PDA. Rinse the pathogenic spores and Trichoderma spores on the culture medium with sterile water, filter the bacteria liquid with sterilized filter paper to remove the hyphae, and dilute the pathogenic bacteria filtrate and the Trichoderma filtrate with the appropriate concentration of 0.5% Tween 60 solution. Spore concentration is 10 6 Pieces/mL. The cucumber seedlings of Xiafeng No. 2 with 3-5 leaves were sprayed on the cucumber seedlings with the prepared Trichoderma spore solution uniformly by the method of stem and leaf spray. Then, the prepared cucumber anthracnose pathogen spore suspension was sprayed and inoculated with 3 replicates for each treatment group. After inoculation, move to a constant temperature room humidity box to shade and moisturize (humidity 100%) to induce. After 24h, remove the melon seedlings out of the moisturizing box, and move them to a shelf with light exposure (4000 Lux), and investigate the disease 14 days later ( image 3 ), compare with the blank control to calculate the control effect.

[0067] Disease index==[100×∑(the number of diseased plants at each level×the disease classification value)]/(total number of surveys×9)

[0068] Control effect=[(control disease index-candidate strain disease index)/control disease index]×100%

[0069] The anthracnose grading standard (that is, the disease classification value) is: 0 grade, no lesions; 1 grade, the lesion area accounts for less than 5% of the entire leaf area; 3 grades, the lesion area accounts for 6% to 10% of the entire leaf area ; Grade 5, diseased spot area accounts for 11%-25% of the entire leaf area; Grade 7 diseased spot area accounts for 26%-50% of the entire leaf area; Grade 9 diseased spot area accounts for more than 50% of the entire leaf area.

[0070] 8. Compare the results of the rapid live screening method adopted in steps 1-6 with the results of the potted plant screening method adopted in step 7, as shown in Table 2. Among the first 6 strains that performed well in the rapid screening in vivo, 4 strains were still ranked in the top 6 in the pot screening. It shows that the screening accuracy of this method is about 70%. And the control effect obtained by the two screening methods has a significant correlation P<0.05, see Table 1.

[0071] Control effect of rapid screening in vivo (%) = area of ​​cotyledons in the treatment group/area of ​​healthy cotyledons in the control group×100%

[0072] Table 1. Rapid screening in vivo and control effect of pot experiment

[0073]

[0074] Note: P<0.05

[0075] Table 2. Sorting results of different screening methods (ranking antagonism from low to high)

[0076]

[0077]

[0078] Note: a: CTCCSJ-W-SBW10260; b: CTCCSJ-W-AW10287; c: CTCCSJ-W-RW10420

[0079]; D: CTCCSJ-W-RW10569; e: CTCCSJ-F-KY10032; f: CTCCSJ-F-KZ10037; g: CTCCSJ-F-KY10039; h: CTCCSJ-F-KY10043; i: CTCCSJ-A-YM12048; j : CTCCSJ-A-YM12057; k: CTCCSJ-A-YM12082; l: CTCCSJ-A-YM12091.