CN105754926B - Method for rapidly inducing spore production of stemphylium stolonifera - Google Patents
Method for rapidly inducing spore production of stemphylium stolonifera Download PDFInfo
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Abstract
The invention relates to a method for rapidly inducing botrytis cinerea to produce spores. The method is used for quickly and easily inducing the spore production of the stemphylium stolonifera, greatly shortens the spore production time, shortens the identification period, effectively saves a large amount of precious time of manpower, material resources and scientific research workers, and has great application potential and higher economic value.
Description
[ technical field ] A method for producing a semiconductor device
The invention belongs to the technical field of induced spore production of plant pathogenic bacteria. More particularly, the present invention relates to a method for rapidly inducing sporulation of stemphylium stolonifera.
[ background of the invention ]
The fungus Stemphylium (stemphyllium) is capable of infecting a wide variety of crops worldwide, possibly causing a variety of vegetable diseases, including for example, primarily tomatoes, lettuce, peppers, cabbage, spinach, garlic, with tomatoes being particularly serious. Tomato leaf spot disease caused by stemphylium nodosum has been reported in the united states, israel, new zealand, etc., causing significant economic loss to local tomato production. In recent years, 50 ten thousand mu of tomato gray leaf spot disease occurs in northeast, east and south China, and the like, particularly serious, wherein the disease is the most serious in Shandong shou guang, Hainan Haikou and Guangxi Tianyang, the total disease area exceeds 10 ten thousand mu, and the loss exceeds thousand yuan.
The tomato is a very important vegetable in solanaceous vegetables and is one of the main cultivated crops in the vegetable planting industry in China. At present, tomato planting, processing and export in China are in a continuously increasing situation, and after more than 20 years of development, China has become the most important tomato product producing country and export country in the world, and is the third production region and the first export country following the United states and European Union. Since 2002, the leaf spot of the stemphylium stolonifera of the tomato is continuously generated in main tomato production areas in China, secondary diseases gradually rise to be main diseases, and the development of the tomato industry in China is seriously influenced.
In recent years, researches on classification and identification of the stemphylium botrytis, pathogenicity and pathogenic mechanism, resistance gene excavation and the like are gradually concerned, wherein induction of spore production is always a key link in classification and identification researches, and how to quickly and easily obtain a large number of spores determines research progress and success and failure to a great extent. The main method for inducing spore production by pure culture of the fungi is to induce spore production by utilizing sunlight or near ultraviolet irradiation, low temperature, mechanical damage or low nutrient culture and other abnormal growth environments. At present, some reports are provided for a culture medium for producing spores of the stemona flagellata, and the culture medium mainly comprises a PCA (principal component analysis), a V8 culture medium, a PDA (personal digital assistant) culture medium, an MEA (membrane electrode assembly) culture medium, a PSA (pressure swing adsorption) culture medium, an oat culture medium, a TWA-W (two-way rolling) culture medium and the like, but tests show that the culture medium has poor stability and special requirements on strains, and cannot ensure that each strain produces spores; moreover, even if spore production can be realized, a large amount of time is consumed, the average spore production time is more than 20 days, and the precious time of manpower, energy and scientific research workers is greatly wasted.
Therefore, the present inventors have completed the present invention through a large number of experimental studies and analyses based on the summary of the prior art for overcoming the technical defects of the prior art.
[ summary of the invention ]
[ problem to be solved ]
The invention aims to provide a method for rapidly inducing the spore production of the stemphylium stolonifera.
[ solution ]
The invention is realized by the following technical scheme.
The invention relates to a method for rapidly inducing botrytis cinerea to produce spores.
The method for rapidly inducing the gene comprises the following steps:
A. plate culture
Inoculating 1-3 environmental-friendly stemphylium stolonifera stored on the inclined plane of the PDA solid culture medium on the PDA plate culture medium, and culturing for 5 days at 25 ℃ in a constant-temperature culture box according to a dark light period of 12 hours illumination and 12 hours, so that hyphae grow over a culture dish;
B. tomato seedling cultivation
Wrapping tomato seeds with clean gauze, soaking the tomato seeds in water at the temperature of 55 ℃ at a constant temperature, transferring the tomato seeds into normal-temperature water, continuously soaking the tomato seeds for 24 hours, accelerating germination in a germination accelerating box at the temperature of 28 ℃ for 48 hours, sowing the accelerated tomato seeds into an 8 x 8 seedling pot, and allowing the tomato seeds to germinate and grow leaves until 4 tomato seedlings with true leaves spread are obtained;
C. inducing spore production
C, spraying clear water on the leaf surfaces of the tomato seedlings obtained in the step C;
and B, taking a stemphylium piece at the hypha growth edge of the step A, sticking the hypha surface of the stemphylium piece on the tomato leaves sprayed with clear water, putting the tomato seedlings in an artificial climate box, culturing for 3 days under the conditions of 25 ℃ of temperature, 60-80% of relative humidity and 12 hours of illumination for 12 hours of dark light period, and picking a large amount of stemphylium spores on the surface of the stemphylium piece.
According to a preferred embodiment of the invention, said Stemphylium species are Stemphylium botrytis (Stemphylium lycopersici) and Stemphylium solani (Stemphylium solani).
According to another preferred embodiment of the present invention, the PDA solid medium is composed of 20 parts by weight of potato, 2 parts by weight of glucose and 100 parts by weight of deionized water.
According to another preferred embodiment of the invention, the tomato seeds are red hetero 10 tomato seeds or medium hetero 105 tomato seeds.
According to another preferred embodiment of the invention, the tomato seeds are soaked in water for 15-25 min at constant temperature.
According to another preferred embodiment of the present invention, the leaf surface of the tomato seedling is uniformly sprayed with clear water in a spray manner.
According to another preferred embodiment of the present invention, the stolonifera pieces are taken using a 5mm punch.
According to another preferred embodiment of the invention, each tomato leaf is stuck with a stemphylium plate.
The present invention will be described in more detail below.
The invention relates to a method for rapidly inducing botrytis cinerea to produce spores.
By adopting the induced spore production method and the induced spore production method of 7 common fungi, the induced spore production is carried out on the stemphylium stolonifera (including the stemphylium stolonifera and the stemphylium solani) from different areas, and the practicability and the reliability of the method are verified based on the conidium quantity index generated by each method; the method for rapidly inducing the botrytis cinerea to produce spores promotes the observation of spore morphology of the botrytis cinerea and the related research of classification to a great extent, and has important theoretical and academic significance and higher economic value.
The strain of Podospora stolonifera used in the method of the present invention is described in detail below.
Collecting typical diseased leaves of tomato gray leaf spot from the field, rinsing the leaves with sterile water for three times in a laboratory under an aseptic condition, putting the diseased leaves into NaClO with the concentration of 0.6 percent by weight, soaking for 2-3 min, rinsing the leaves with sterile water for three times, drying the leaves, airing the leaves according to 5 blocks per plate, inoculating the aired leaves onto a PDA (personal digital assistant) plate, repeatedly setting 3 dishes, inverting the leaves in a constant-temperature incubator at the temperature of 25 ℃ for light-proof culture, observing at any time, and picking the staphylium stolonifera filaments by using an inoculating needle for transfer purification. After 5 days, the plate producing a large amount of hyphae is purified by adopting a mode of picking the stemphylium hyphae with an inoculating needle and transferring the stemphylium hyphae to a new culture medium, and then is continuously cultured for 10 days under the same condition;
selecting 5mm bacterial plaque, placing the bacterial plaque into 150mL PD culture medium, performing shake culture on a constant-temperature shaking table with the rotation speed of 80rpm and the temperature of 25 ℃ for 6-7 days to generate a large amount of hyphae, filtering the mycelium by using sterile gauze, absorbing excessive moisture by using filter paper, then placing the mycelium into tinfoil paper, then placing the tinfoil paper into a refrigerator with the temperature of-20 ℃ for freezing for 24h, then using a vacuum extraction dryer for drying for 24-48 h, and placing the obtained freeze-dried mycelium into the refrigerator with the temperature of-20 ℃ for storage and standby.
Grinding the freeze-dried mycelium in liquid nitrogen, mixing the freeze-dried mycelium with an equivalent amount of CTAB (cetyl trimethyl ammonium bromide) extract to form paste, extracting the paste in a water bath kettle at the temperature of 65 ℃ for 30min, shaking the paste once every 10min, centrifuging the paste in a centrifuge at the temperature of 4 ℃ and 12000rpm for 10min, then extracting supernatant, adding a chloroform-isopropanol mixture (volume ratio is 24: 1) with the same volume as the supernatant, slowly turning the mixture up and down for 6 to 10 times, centrifuging the mixture in the centrifuge at the temperature of 4 ℃ for 10min, and repeating the steps in the same manner until a protein layer appears in the centrifuge tube. The last supernatant was removed and pre-frozen isopropanol was added thereto in a volume of 2/3 volumes of the supernatant. And when flocculent precipitates appear in the centrifugal tube, carrying out centrifugal separation, discarding supernatant, washing the obtained precipitates for 2 times by using 75% ethanol water solution in volume, carrying out air drying under the aseptic condition, and dissolving in 50-80 mu L of secondary distilled water to obtain a solution containing the DNA of the pathogenic bacteria.
The pathogen DNA was PCR amplified at 25 μ L using ribosomal DNA (rdna) primers ITS1(5 '-TCCGTAGGTGAACCTGCGG-3') and ITS4(5 '-TCCTCCGCTTATTGATATGC-3') by a reaction of 2.5 μ L Buffer, 0.5 μ L Taq enzyme and 2 μ L dNTP, each at 1 μ L, with double distilled water to make up the remaining volume.
The PCR amplification procedure was as follows: 5min at 94 ℃; 30 cycles of 94 ℃ for 30s, 60 ℃ for 30s, and 72 ℃ for 90 s; 10min at 72 ℃. The amplified product is detected by 1 percent agarose gel electrophoresis, the PCR product is directly sequenced by a bidirectional sequencing method, the sequencing work is completed by Beijing Nuosace genome research center, Inc., the sequencing result is determined by Blast sequence homology comparison through GenBank, and the separated strain is a botrytis cinerea strain.
Carrying out pathogenicity determination on a strain which is identified as the phoma stolonifera in molecular biology, directly spraying broken hypha onto tomato leaves by adopting a hypha spraying method, and selecting the strain with the same disease symptoms as those in the field for storage.
Three staphylotrichum strains, namely FQ14092805 (the Guangxi resource county, the Tantao village of the seedling family of the car field, the county, the Countryptian), FQ140618115 (the Shandong Shouguang field, Liujia village of Liujia, the town of Sichuan village) and FQ14091004 (the Liaoning Dianjin county, the Tanopectum hybridum) are obtained through molecular biological identification and pathogenicity determination, and are stored in a vegetable pathogen storage warehouse of vegetable floriculture institute of Chinese agricultural academy by adopting a mode of injecting paraffin.
The method for rapidly inducing the gene comprises the following steps:
A. plate culture
Inoculating 1-3 environmental-friendly stemphylium stolonifera stored on the inclined plane of the PDA solid culture medium on the PDA plate culture medium, and culturing for 5 days at 25 ℃ in a constant-temperature culture box according to a dark light period of 12 hours illumination and 12 hours, so that hyphae grow over a culture dish; see in particular fig. 1.
The method induces 3 stemphylium strains separated from different areas to produce spores, observes through an optical microscope, and identifies the stemphylium strains by using morphology.
The PDA solid culture medium is composed of 20 parts by weight of potatoes, 2 parts by weight of glucose and 100 parts by weight of deionized water.
In the present invention, the light irradiation and the dark irradiation are alternately performed to promote the growth of hyphae. The illumination intensity in illumination is generally 20000 LX;
in the present invention, dark is generally understood to mean culturing under light-shielding conditions.
The constant temperature light incubator used in the present invention is a product currently sold in the market, for example, a product sold under the trade name "Ningbo apparatus" by Ningbo south of the Yangtze, Instrument works, and the model is a "GXZ light incubator".
B. Tomato seedling cultivation
Wrapping tomato seeds with clean gauze, soaking the tomato seeds in water at the temperature of 55 ℃ at a constant temperature, transferring the tomato seeds into normal-temperature water, continuously soaking the tomato seeds for 24 hours, accelerating germination in a germination accelerating box at the temperature of 28 ℃ for 48 hours, sowing the accelerated tomato seeds into an 8 x 8 seedling pot, and allowing the tomato seeds to germinate and grow leaves until 4 tomato seedlings with true leaves spread are obtained;
according to the invention, the tomato seeds are red weed 10 tomato seeds or medium weed 105 tomato seeds, and the tomato seeds are from medium vegetable science and technology (Beijing) Co.
According to the invention, the tomato seeds are soaked in water for 15-25 min at a constant temperature. If the seed soaking time of the tomato seeds is less than 15min, the tomato seeds are irregular in sprouting; if the tomato seeds are soaked for more than 25min, the germination rate is reduced; accordingly, it is appropriate that the tomato seeds are soaked for 15-25 min, preferably 18-22 min.
The pregermination chamber used in the present invention is a product currently marketed, for example, by Shanghai Yixi instruments and meters, Inc. under the trade name "Yixi".
The nursery pot used in the present invention is a product currently sold in the market, for example, a product sold under the trade name "Shengda" by Hangzhou Anhua plastics Co.
C. Inducing spore production
C, spraying clear water on the leaf surfaces of the tomato seedlings obtained in the step C;
according to the invention, clean water is uniformly sprayed on the leaf surfaces of the tomato seedlings in a spraying manner. And B, taking a stemphylium plate at the hypha growth edge of the step A, sticking the hypha surface of the stemphylium plate on the tomato leaves sprayed with clear water (see attached figure 2), putting the tomato seedlings in an artificial climate box, culturing for 3 days under the conditions of 25 ℃ of temperature, 60% -80% of relative humidity and 12 hours of illumination for a dark and dark period, and picking a large amount of stemphylium spores growing on the surface of the stemphylium plate, see attached figures 3 and 4. The conidiogenesis numbers were observed and counted under a microscope in a conventional manner.
Specifically, a drop of clear water is dropped on a glass slide, a mold layer on the surface of a fungus piece is picked by an inoculating needle and placed in the center of the clear water, a cover glass is used for covering the surface of the water drop lightly, excessive water is absorbed by using water absorption paper, then the water drop is placed under an optical microscope for observation, and the species of the stemphylium stolonifera is identified by morphology. Morphological observation the strain FQ14092805 was morphologically identified as a strain of Pholiota lycopersici (Stemphylium lycopersici), and the strains FQ140618115 and FQ14091004 were of Pholiota solani (Stemphylium solani), and the results are shown in Table 1.
Table 1: comparison table for microscopical morphological differences of stemphylium
In the present invention, a piece of stemphylium was taken using a 5mm punch.
Each tomato leaf is stuck with a stemphylium stolonifera tablet.
The Stemphylium (stemphilium) is Stemphylium Lycopersicum and Stemphylium Solani.
The climatic chamber used in the present invention is a product currently marketed, for example, by Ningbo south of the Yangtze, Instrument works under the trade name "Ningbo instruments", model "GXZ light incubator".
[ advantageous effects ]
The invention has the beneficial effects that:
(1) a large number of conidia are generated in 3 days by inducing the mycelia of the stemphylium botryoides through the live tomato seedlings, and the number of the conidia generated on each bacterial sheet can basically reach more than 500.
(2) By utilizing the induction mode of living tomato seedlings, pure culture hypha spore production can be simply induced to be used for morphological identification of the stemphylium stolonifera, and an important technical platform is provided for identification and classification of the stemphylium stolonifera;
(3) the induced spore production identification method has simple steps, easy operation and short spore production time, greatly improves the spore production efficiency of the stemphylium stolonifera, shortens the identification period, effectively saves a large amount of precious time of manpower and material resources and scientific research workers, and has great application potential.
[ description of the drawings ]
FIG. 1 is the normal colony morphology of Stemphylium strain on PDA medium;
FIG. 2 shows the mushroom pieces just stuck to the tomato leaves;
FIG. 3 shows the growth state of the fungus pieces after inducing sporulation on tomato leaves;
FIG. 4 shows conidiophores and conidia generated by the Stemphylium strain after induction of sporulation on tomato leaves.
[ detailed description ] embodiments
The invention will be better understood from the following examples.
Example 1: quickly inducing botrytis
The implementation steps of this example are as follows:
A. plate culture
Inoculating 3 environmentally-friendly tomato stemphylium stolonifera FQ14092805 stored on an inclined plane of a PDA solid culture medium consisting of 20 parts by weight of potatoes and 2 parts by weight of glucose on a PDA plate culture medium, culturing for 5 days at 25 ℃ according to a 12-hour illumination 12-hour dark light period in a constant-temperature illumination incubator sold by Ningbo south China instrument factory under the trade name 'Ningbo instrument' and the model 'GXZ illumination incubator', and allowing hypha to grow over the culture dish; the stemphylium is stemphylium botryoides and stemphylium solani.
B. Tomato seedling cultivation
Wrapping the tomato seeds of No. 10 Hongza with clean gauze, placing the wrapped tomato seeds into water with the temperature of 55 ℃ for soaking for 15min at constant temperature, transferring the wrapped tomato seeds into normal-temperature water for further soaking for 24h, then accelerating germination for 48h at the temperature of 28 ℃ in a germination accelerating box sold under the trade name of Yinxi by Shanghai Yinxi Instrument and meters Co., Ltd, sowing the accelerated germination tomato seeds into an 8 x 8 seedling pot sold under the trade name of Shengda by Hangzhou Anhua plastic industry Co., Ltd, and allowing the tomato seeds to germinate and grow leaves until 4 tomato seedlings with true leaves unfolded are obtained;
C. inducing spore production
C, spraying clear water on the leaf surfaces of the tomato seedlings obtained in the step C;
a stolonifera mold sheet is taken at the hypha growth edge of the step A by using a 5mm puncher, the hypha surface of the stolonifera mold sheet is attached to tomato leaves uniformly sprayed with clear water in a spraying mode, one stolonifera mold sheet is attached to each tomato leaf, the tomato seedlings are placed in an artificial climate box sold by Ningbo Jiangnan instrument factory under the trade name 'Ningbo instrument', the model is 'GXZ illumination incubator', the artificial climate box is cultured for 3 days under the conditions of 25 ℃ of temperature, 80% of relative humidity and 12 hours of illumination in a dark light period of 12 hours, a large number of stolonifera mold spores are picked up to grow on the surface of the stolonifera mold sheet, the formation number of the stolonifera spores is observed and calculated under a microscope, and the results are listed in Table 2.
Example 2: quickly inducing botrytis
The implementation of this example was carried out as in example 1, except that 1 the strain of botrytis cinerea FQ140618115 environmentally protected on the slant of the PDA solid culture medium was taken; soaking seeds of the Zhongza No. 105 tomato for 20min at constant temperature; the tomato leaves to which the stemphylium pieces were attached were cultured under conditions of a temperature of 25 deg.c, a relative humidity of 70% and a 12-hour light irradiation for a dark period of 12 hours, and the number of formed conidia was observed and calculated under a microscope, and the results thereof are shown in table 2.
Example 3: quickly inducing botrytis
The implementation of the example is carried out according to the implementation of the example 1, except that 2 environmental protection stolonifera FQ14091004 stored on the inclined plane of the PDA solid culture medium is taken; soaking the red-impurity No. 10 tomato seeds for 25min at constant temperature; the tomato leaves to which the stemphylium pieces were attached were cultured under conditions of a temperature of 25 deg.c, a relative humidity of 60% and a 12-hour light irradiation for a dark period of 12 hours, and the number of formed conidia was observed and calculated under a microscope, and the results thereof are shown in table 2.
Comparative example 1:
the comparative example was carried out as follows: the 3 strains used in examples 1 to 3 were activated and transferred to PCA plates, and each strain was transferred to 6 PCA plates, incubated at 25 ℃ for 20 days under a 12-hour light irradiation for 12 hours in a dark cycle, observed under a microscope and counted for the number of conidia formed, and the results are shown in Table 2. The PCA medium consisted of: 200g of potato, 200g of carrot, 15g of agar and 1L of distilled water.
Comparative example 2:
the comparative example was carried out as follows: the 3 strains used in examples 1 to 3 were activated and transferred to V8 plates, and each strain was transferred to 6V 8 plates, incubated at 25 ℃ for 20 days under a 12-hour light-dark cycle for 12 hours, observed under a microscope and counted for the number of conidia, and the results are shown in Table 2. The composition of V8 medium was as follows: 200g V8 vegetable juice, 3g CaCO315g of agar, 1L of distilled water.
Comparative example 3:
the comparative example was carried out as follows: the 3 strains used in examples 1 to 3 were activated and transferred to PDA plates, and each strain was transferred to 6 PDA plates, incubated at 25 ℃ for 20 days under a 12-hour light-exposure period in dark for 12 hours, observed under a microscope and counted for the number of conidia, and the results are shown in Table 2. The PDA medium consisted of: 200g of potato, 200g of carrot, 15g of agar and 1L of distilled water.
Comparative example 4:
the comparative example was carried out as follows: the 3 strains used in examples 1 to 3 were activated and transferred to MEA plates, each of the strains was transferred to 6 MEA plates, incubated at 25 ℃ for 20 days under a 12-hour light irradiation for 12-hour dark period, observed under a microscope and counted for the number of conidia, and the results are shown in Table 2. The MEA medium consisted of: 30g of malt extract, 3g of soybean peptone, 15g of agar and 1L of distilled water.
Comparative example 5:
the comparative example was carried out as follows: the 3 strains used in examples 1 to 3 were activated and transferred to PSA plates, and each strain was transferred to 6 PSA plates, incubated at 25 ℃ for 20 days under 12-hour light for a dark cycle for 12 hours, observed under a microscope and counted for the number of conidia, and the results are shown in Table 2. The PSA medium consisted of: 30g of malt extract, 3g of soybean peptone, 15g of agar and 1L of distilled water.
Comparative example 6:
the comparative example was carried out as follows: the 3 strains used in examples 1 to 3 were activated and transferred to oat plates, and each strain was transferred to 6 oat plates, incubated at 25 ℃ for 20 days under a 12-hour light-exposure dark cycle for 12 hours, observed under a microscope and counted for the number of conidia, and the results are shown in Table 2. The oat culture medium comprises the following components: 30g of oat, 15g of agar and 1L of distilled water.
Comparative example 7:
the comparative example was carried out as follows: the 3 strains used in examples 1 to 3 were activated and transferred to TWA-W plates, and 6 TWA-W plates were transferred to each strain, incubated at 25 ℃ for 20 days under a 12-hour light-exposure and dark cycle for 12 hours, and observed under a microscope and the number of conidia formed was calculated, and the results are shown in Table 2. TWA-W medium consisted of: 20g of agar and 1000mL of tap water were melted and sterilized. Cutting wheat straw into small sections with the diameter smaller than that of the culture medium, and sterilizing. Sterile wheat straw is put into a sterilized culture dish, 3-4 drops of 25% lactic acid are added, and a melted agar culture medium is poured.
Table 2: effect of different cultivation methods on spore yield
-: no spore production +: sporadic sporulation
++: medium spore production +++: mass production of spores
It can be seen that the method of the present invention has simple operation steps and can stably produce a large number of conidia in a minimum time, compared to the other 7 spore production methods (comparative examples).
Claims (7)
1. A method for rapidly inducing the sporulation of the stemphylium stolonifera is characterized by comprising the following steps:
A. plate culture
Inoculating 1-3 environmental-friendly stemphylium stolonifera stored on the inclined plane of the PDA solid culture medium on the PDA plate culture medium, and culturing for 5 days at 25 ℃ in a constant-temperature incubator according to 12-hour illumination and 12-hour dark light cycle to allow hyphae to grow over a culture dish; the Stemphylium (Stemphylium) is Stemphylium Lycopersici and Stemphylium Solani solani;
B. tomato seedling cultivation
Wrapping tomato seeds with clean gauze, soaking the tomato seeds in water at the temperature of 55 ℃ at a constant temperature, transferring the tomato seeds into normal-temperature water, continuously soaking the tomato seeds for 24 hours, accelerating germination in a germination accelerating box at the temperature of 28 ℃ for 48 hours, sowing the accelerated tomato seeds into an 8 x 8 seedling pot, and allowing the tomato seeds to germinate and grow leaves until 4 tomato seedlings with true leaves spread are obtained;
C. inducing spore production
C, spraying clear water on the leaf surfaces of the tomato seedlings obtained in the step C;
and B, taking a stemphylium piece at the hypha growth edge of the step A, sticking the hypha surface of the stemphylium piece on the tomato leaves sprayed with clear water, putting the tomato seedlings in an artificial climate box, culturing for 3 days under the conditions of 25 ℃ of temperature, 60-80% of relative humidity and 12 hours of illumination for 12 hours of dark light period, and picking a large amount of stemphylium spores on the surface of the stemphylium piece.
2. The method as set forth in claim 1, wherein the PDA solid medium is composed of 20 parts by weight of potato, 2 parts by weight of glucose and 100 parts by weight of deionized water.
3. The method according to claim 1, wherein the tomato seed is a red-hybrid tomato seed No. 10 or a medium-hybrid tomato seed No. 105.
4. The method according to claim 1, wherein the tomato seeds are soaked in water at a constant temperature for 15-25 min.
5. The method according to claim 1, characterized in that the leaf surface of the tomato seedlings is uniformly sprayed with clear water in a spray pattern.
6. The method according to claim 1, wherein the stolonifera pieces are taken using a 5mm punch.
7. The method of claim 1, wherein each tomato leaf is coated with a sheet of stemphylium stolonifera.
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| CN109609589B (en) * | 2018-12-20 | 2022-01-28 | 东北农业大学 | Method for stably germinating conidia of tomato gray leaf spot and identifying seedling stage artificial inoculation |
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