A method for preventing infection of a tomato plant by a pathogenic fungus after continuous cropping of the tomato plant using calcium propionate in combination with trichoderma harzianum

Abstract

The present application relates to the field of agricultural cultivation technology, and specifically provides a method for preventing and treating pathogenic fungi infection after continuous cropping of tomatoes by using calcium propionate and Trichoderma harzianum, which can enhance the disease resistance of tomatoes after continuous cropping, improve the quality of tomatoes, and promote the growth of tomatoes. It is found for the first time that the combination of calcium propionate and Trichoderma harzianum can comprehensively prevent and treat common mixed fungi infection after continuous cropping of tomatoes. In particular, the present application has a comprehensive prevention and treatment effect on mixed fungi infection of Colletotrichum higginsianum, Colletotrichum gloeosporioides, Fusarium graminearum and Septoria australis.

Description

Technical Field

[0001] This invention relates to the field of agricultural cultivation technology, specifically to a method for controlling pathogenic fungal infection in tomatoes after continuous cropping using calcium propionate in combination with Trichoderma harzianum. Background Technology

[0002] In my country, continuous cropping of tomatoes leads to severe pathogenic fungal infections, particularly the frequent occurrence of co-infection by multiple pathogens. This is challenging to control, as it's difficult to completely eradicate several pathogens simultaneously. Combined pesticide application is also difficult, requiring a wide range of choices and complex dosage selection. Slight misjudgment can inhibit tomato growth, solving the infection problem while hindering crop development. Therefore, there is an urgent need to develop a method to control mixed infections of multiple pathogens in tomatoes after continuous cropping, while simultaneously promoting crop growth. Summary of the Invention

[0003] The purpose of this invention is to provide a method for controlling pathogenic fungal infection in tomatoes after continuous cropping using calcium propionate in combination with Trichoderma harzianum, thereby enhancing the disease resistance of tomatoes, improving tomato quality, and promoting their growth. To achieve the above objectives, this invention provides the following technical solution.

[0004] A method for controlling pathogenic fungal infection in tomatoes after continuous cropping using calcium propionate in combination with Trichoderma harzianum includes the following steps:

[0005] (1) Preparation of culture medium: Wash, peel and cut fresh potatoes into pieces, weigh 200g, add appropriate amount of distilled water and boil for about 15 minutes until the potatoes are soft. Filter with multiple layers of gauze, dissolve 20g glucose and 2.5g calcium propionate powder in the filtrate, and make up the volume of the solution to 1L. Dispense into conical flasks with 1.5% agar powder, seal and sterilize at 121℃ for 20 minutes.

[0006] (2) Preparation of Trichoderma harzianum conidium: Trichoderma harzianum strain was inoculated into the above-mentioned culture medium and placed under white fluorescent light irradiation, specifically at a light intensity of 100-200 μM / m. 2 / s, wavelength 400-700nm, incubated with shaking at 25℃ until the viable count of Trichoderma harzianum conidia per 1g is 10. 9 ~10 10 CFU was stopped, and activated Trichoderma harzianum conidia were obtained;

[0007] (3) When pathogenic fungal infection occurs after tomato transplanting, apply the Trichoderma harzianum conidia solution from step (2) along with irrigation water. Apply once every 3 days for a total of 3 times. The amount applied each time is 400-1600 kg per mu.

[0008] (4) On the day after irrigation in step (3) and on the fifth day, spray the tomato leaves with a mixture of equal volumes of gibberellin at a concentration of 20-50 mg / L and strigolactone at a concentration of 3-5 μM / L, once in the morning and once in the evening.

[0009] Furthermore, the tomato leaves are uniformly sprayed with a mixed solution of equal volumes of gibberellin at a concentration of 20 mg / L and strigolactone at a concentration of 5 μM / L.

[0010] Furthermore, the viable count of *Trichoderma harzianum* is 10 per 1g of *Trichoderma harzianum* conidia. 9 ~10 10 CFU.

[0011] This invention also provides a method for preparing an agent to prevent infection by pathogenic fungi after continuous cropping of tomatoes, comprising the following steps:

[0012] (1) Preparation of culture medium: Wash, peel and cut fresh potatoes into pieces, weigh 200g, add appropriate amount of distilled water and boil for about 15 minutes until the potatoes are soft. Filter with multiple layers of gauze, dissolve 20g glucose and 2.5g calcium propionate powder in the filtrate, and make up the volume of the solution to 1L. Dispense into conical flasks with 1.5% agar powder, seal and sterilize at 121℃ for 20 minutes.

[0013] (2) Preparation of Trichoderma harzianum conidium: Trichoderma harzianum strain was inoculated into the above-mentioned culture medium and placed under white fluorescent light irradiation, specifically at a light intensity of 100-200 μM / m. 2 / s, wavelength 400-700nm, incubated with shaking at 25℃ until the viable count of Trichoderma harzianum conidia per 1g is 10. 9 ~10 10 CFU was stopped, and activated Trichoderma harzianum conidia were obtained.

[0014] The above method yields a formulation for preventing fungal infection of tomatoes after continuous cropping.

[0015] The above-mentioned methods for preventing fungal infection of tomatoes after continuous cropping can be applied in any of the following ways:

[0016] (1) Application in the prevention and control of one or more fungal infections, such as Fusarium graminearum, Anthrax hygroscopicus, Anthrax oleifera, and Alternaria esculenta.

[0017] (2) Application in the prevention and control of tomato wilt caused by Fusarium oxysporum f.sp. Lycopersici, Fol after continuous cropping of tomatoes;

[0018] (3) Its application in promoting the growth and development of tomatoes;

[0019] (4) Application in improving the salt tolerance of tomatoes.

[0020] This application also provides a method for preventing pathogenic fungal infection in tomatoes after continuous cropping, comprising the following steps:

[0021] (1) Preparation of culture medium: Wash, peel and cut fresh potatoes into pieces, weigh 200g, add appropriate amount of distilled water and boil for about 15 minutes until the potatoes are soft. Filter with multiple layers of gauze, dissolve 20g glucose and 2.5g calcium propionate powder in the filtrate, and make up the volume of the solution to 1L. Dispense into conical flasks with 1.5% agar powder, seal and sterilize at 121℃ for 20 minutes.

[0022] (2) Preparation of Trichoderma harzianum conidium: Trichoderma harzianum strain was inoculated into the above culture medium and placed under white fluorescent light irradiation, specifically at a light intensity of 100 μM / m. 2 / s, wavelength 400nm, shake culture at 25℃ until the viable count of Trichoderma harzianum conidia per 1g is 10 9 ~10 10 CFU was stopped, and activated Trichoderma harzianum conidia were obtained;

[0023] (3) Apply Trichoderma harzianum when transplanting tomatoes. Use the hole application method. The dosage is 5g of Trichoderma harzianum conidia solution from step (2) per hole. Sprinkle it into the hole.

[0024] The beneficial effects of this application are:

[0025] 1. This invention is the first to discover that the combined use of calcium propionate and Trichoderma harzianum can provide comprehensive control against common fungi such as Higgins anthracnose, Camellia oleifera anthracnose, Fusarium graminearum, and Eimeria spp. In particular, it addresses the problem of mixed infection by multiple fungi after continuous cropping of tomatoes.

[0026] 2. This invention is the first to study the pathogenic fungi of continuous cropping in tomatoes, specifically a solution for when these pathogenic fungi appear in large areas and in multiple combinations. Comparisons were made with the control group (CK), the experimental group using calcium propionate alone, the experimental group using *Trichoderma harzianum* alone, and the combined group of calcium propionate and *Trichoderma harzianum*. The results showed that the combination of *Trichoderma harzianum* and 0.25% CP had good control effects against several mixed fungal infections: *Fusarium graminearum*, *Anthracis higginense*, *Anthracis oleifera*, and *Alternaria emoreca*. For the control of mixed fungal infections of *Anthracis higginense*, *Anthracis oleifera*, and *Alternaria emoreca*, either *Trichoderma harzianum* alone or in combination with 0.25% CP can achieve good technical results.

[0027] 3. This invention is the first to propose a method for controlling tomato wilt caused by *Fusarium oxysporum* f. sp. *Lycopersici* (Fol), one of the most serious fungal diseases in tomato production, causing huge economic losses to tomato agriculture worldwide. The combined use of calcium propionate and *Trichoderma harzianum* can effectively control *Fol* fungal infection.

[0028] 4. Adding calcium propionate and *Trichoderma harzianum* to irrigation water can significantly prevent the spread of various fungal infections in the soil after continuous cropping. However, this also leads to soil salinization. Applying gibberellin and strigolactone can promote plant growth and enhance the plant's salt tolerance. A foliar spray experiment using an equal volume mixture of 20 mg / L gibberellin and 5 μM / L strigolactone, combined with an application of this solution, showed that it had the best effect on promoting tomato plant growth. Higher concentrations of both gibberellin and strigolactone inhibited leaf growth and chlorophyll synthesis, thus hindering tomato growth.

[0029] 5. We found that when Trichoderma harzianum conidia with calcium propionate were used for the combined control of various continuous cropping pathogens, the best control effects were observed when applied with irrigation water after tomato transplanting, specifically on the second day and the fifth day after irrigation. Excessive calcium propionate significantly inhibited tomato biomass accumulation and plant development. With increasing calcium propionate content, the fresh weight of the above-ground parts decreased, chlorophyll content also decreased significantly, and the disease index increased. This indicates that excessive calcium propionate inhibited tomato plant growth, while low calcium propionate content was ineffective in controlling fungal infection, and the disease index also increased accordingly.

[0030] 6. This application represents a significant amount of creative research, yielding the result that the activated Trichoderma harzianum conidial solution exhibits the highest antibacterial activity, specifically at a light intensity of 100 μM / m². 2 / s, wavelength 400nm, shake culture at 25℃ until the viable count of Trichoderma harzianum conidia per 1g is 10 9 ~10 10 When CFU was stopped, the activated Trichoderma harzianum conidial solution showed the highest antibacterial activity. Increased light intensity and wavelength both led to a significant increase in its disease index. Attached Figure Description

[0031] Figure 1 Different fungal colony morphologies under PDA medium (A);

[0032] Figure 2 PDA medium with different fungal colony diameters (B) for 7 days;

[0033] Figure 3Different fungal colony growth rates (C) in PDA medium;

[0034] Figure 4 Different fungal colony morphologies (A) on PDA medium (+0.25% CP);

[0035] Figure 5 PDA medium (+0.25% CP) 7-day colony diameter (B) of different fungi;

[0036] Figure 6 Growth rates (C) of different fungal colonies in PDA medium (+0.25% CP);

[0037] Figure 7 Different fungal colonies and Trichoderma harzianum growth morphology in PDA medium (A);

[0038] Figure 8 Diameter of different fungal colonies confronting Trichoderma harzianum for 7 days (B);

[0039] Figure 9 The growth rate (C) of different fungal colonies in PDA medium in confrontation with Trichoderma harzianum;

[0040] Figure 10 Different fungal colonies and Trichoderma harzianum growth morphology in PDA medium (+0.25% CP) in confrontation (A);

[0041] Figure 11 Diameter of different fungal colonies confronting Trichoderma harzianum for 7 days (B);

[0042] Figure 12 Growth rate (C) of different fungal colonies confronting Trichoderma harzianum in PDA medium (+0.25% CP);

[0043] Figure 13 A comprehensive comparison of the growth diameter of different bacterial groups over 7 days under four experimental conditions;

[0044] Figure 14 A comprehensive comparison of the growth rates of different bacterial populations over 7 days under four experimental conditions.

[0045] Figure 15 ITS amplification bands isolated from soil. Detailed Implementation

[0046] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0047] Example 1

[0048] A method for controlling pathogenic fungal infection in tomatoes after continuous cropping using calcium propionate in combination with Trichoderma harzianum includes the following steps:

[0049] (1) Preparation of culture medium: Wash, peel and cut fresh potatoes into pieces, weigh 200g, add appropriate amount of distilled water and boil for about 15 minutes until the potatoes are soft. Filter with multiple layers of gauze, dissolve 20g glucose and 2.5g calcium propionate powder in the filtrate, and make up the volume of the solution to 1L. Dispense into conical flasks with 1.5% agar powder, seal and sterilize at 121℃ for 20 minutes.

[0050] (2) Preparation of Trichoderma harzianum conidium: Trichoderma harzianum strain was inoculated into the above culture medium and placed under white fluorescent light irradiation, specifically at a light intensity of 100 μM / m. 2 / s, wavelength 400nm, shake culture at 25℃ until the viable count of Trichoderma harzianum conidia per 1g is 10 9 ~10 10 CFU was stopped, and activated Trichoderma harzianum conidia were obtained;

[0051] (3) When pathogenic fungal infection occurs after tomato transplanting, apply the Trichoderma harzianum conidium solution from step (2) along with irrigation water. Apply once every 3 days for a total of 3 times. Each application amount is 400 kg of Trichoderma harzianum conidium solution from step (2) per mu.

[0052] (4) On the day after irrigation in step (3) and on the fifth day, spray an equal volume mixture of gibberellin at a concentration of 20 mg / L and strigolactone at a concentration of 5 μM / L on the tomato leaves, once in the morning and once in the evening.

[0053] Example 2

[0054] A method for preventing pathogenic fungal infection in tomatoes after continuous cropping includes the following steps:

[0055] (1) Preparation of culture medium: Wash, peel and cut fresh potatoes into pieces, weigh 200g, add appropriate amount of distilled water and boil for about 15 minutes until the potatoes are soft. Filter with multiple layers of gauze, dissolve 20g glucose and 2.5g calcium propionate powder in the filtrate, and make up the volume of the solution to 1L. Dispense into conical flasks with 1.5% agar powder, seal and sterilize at 121℃ for 20 minutes.

[0056] (2) Preparation of Trichoderma harzianum conidium: Trichoderma harzianum strain was inoculated into the above-mentioned culture medium and cultured under white fluorescent light at 25°C with shaking, specifically at a light intensity of 100 μM / m² / s and a wavelength of 400 nm, until the viable count of Trichoderma harzianum conidium was 10 per 1 g. 9 ~10 10 CFU was stopped, and activated Trichoderma harzianum conidia were obtained;

[0057] (3) Apply Trichoderma harzianum when transplanting tomatoes. Use the hole application method. The dosage is 5g of Trichoderma harzianum conidia solution from step (2) per hole. Sprinkle it into the hole.

[0058] Example 3

[0059] A method for preparing an agent to control pathogenic fungal infection in tomatoes after continuous cropping includes the following steps:

[0060] (1) Preparation of culture medium: Wash, peel and cut fresh potatoes into pieces, weigh 200g, add appropriate amount of distilled water and boil for about 15 minutes until the potatoes are soft. Filter with multiple layers of gauze, dissolve 20g glucose and 2.5g calcium propionate powder in the filtrate, and make up the volume of the solution to 1L. Dispense into conical flasks with 1.5% agar powder, seal and sterilize at 121℃ for 20 minutes.

[0061] (2) Preparation of Trichoderma harzianum conidium: Trichoderma harzianum strain was inoculated into the above culture medium and placed under white fluorescent light irradiation, specifically at a light intensity of 100 μM / m. 2 / s, wavelength 400nm, shake culture at 25℃ until the viable count of Trichoderma harzianum conidia per 1g is 10 9 ~10 10 CFU was stopped, and activated Trichoderma harzianum conidia were obtained.

[0062] Experimental methods:

[0063] The strains used in the following experiments—Trichoderma harzianum, Fusarium graminearum, Colletotrichum higginsianum, Colletotrichum phomoides, Didymella americana, Colletotrichum boninense, and Fusarium oxysporum f.sp. Lycopersici, Fol—are all known strains and commercially available. The materials used in this application were purchased from Mingzhou Biotechnology and Gray Algae Biotechnology.

[0064] Culture medium: All fungi were cultured on PDA medium (potato dextrose agar). Fresh potatoes were washed, peeled, and cut into chunks. 200g of each potato was weighed and boiled in an appropriate amount of distilled water for about 15 minutes until the potatoes softened. The mixture was filtered through multiple layers of gauze. 20g of glucose was dissolved in the filtrate (if treated with 0.25% calcium propionate, 2.5g of calcium propionate powder was added). The solution volume was brought to 1L and dispensed into Erlenmeyer flasks containing 1.5% agar powder. The flasks were sealed and sterilized at 121°C for 20 minutes. The resulting flasks were then unpacked and ready for use.

[0065] Culture conditions: All strains were cultured under white fluorescent light (light intensity up to 100 μM / m²). 2 / s; wavelength 400nm), and cultured at 25℃.

[0066] Experiment 1: Measurement of growth rate and colony diameter of different fungi

[0067] Experimental Methods: Fusarium graminearum, Anthracnose spp., Anthracnose spp., Alternaria oleifera, and Trichoderma harzianum were grown on PDA medium for 3 days. Young mycelia along the colony edge were collected in a fungal dish and inoculated into the center of PDA medium without 0.25% calcium propionate. Markings were made on days 1, 2, and 7 of growth. The distance between two marks represented the fungal growth rate. After 7 days of growth, colony morphology (A), colony diameter (B), and colony growth rate (C) were directly measured, with three replicates. Different letters represent significant differences (P < 0.01), with an error range of ±SDs (n = 3).

[0068] Experimental results: Figure 1 and Figure 4 In comparison, it can be clearly seen that the inhibition zones of *Hicks anthrax* and *Camellia oleifera* were significantly smaller in the experimental group with PDA medium (+0.25% calcium propionate) compared to the control group without calcium propionate. Figure 2 It can be seen that after 7 days of colony growth, there are significant differences in the diameter of different fungi, with Trichoderma harzianum having the largest diameter, followed by Camellia anthracnose. Figure 3 Based on the statistical results of the growth rate of different fungal colonies, it can be seen that Trichoderma harzianum has the fastest growth rate, followed by Anthracnosea oleifera, and the results are consistent with the trend of fungal colony diameter (B).

[0069] Experiment 2: Effects of adding 0.25% CP to the culture medium on the growth rate and colony diameter of different fungi.

[0070] Experimental Methods: Fusarium graminearum, Anthracnose spp., Anthracnose spp., Anthracnose spp., Alternaria spp., and Trichoderma harzianum, grown on PDA medium for 3 days, were inoculated into the center of PDA medium supplemented with 0.25% calcium propionate (CP) after being collected along the edge of the colony by casting a fungal disc. Markings were made on days 1, 2, and 7 of growth. The distance between two marks represented the fungal growth rate. The experiment was repeated three times. After 7 days of growth, colony morphology (A), colony diameter (B), and colony growth rate (C) were directly measured. Statistical results were presented. Different letters represent significant differences (P < 0.01), with an error range of ±SDs (n = 3).

[0071] Experimental results: Figure 4 Based on the colony morphology results of different fungi on PDA medium (with 0.25% CP), the growth of Fusarium graminearum, Anthracnose spp., Anthracnose spp., and Alternaria spp. was significantly inhibited, while Trichoderma harzianum was unaffected.

[0072] Figure 5 Based on the measurement results of different fungal colony diameters on PDA medium, consistent with the colony morphology observations, the diameters of *Fusarium graminearum*, *Anthracis higgins*, *Anthracis oleifera*, and *Alternaria emarginatus* were significantly lower than those obtained in Experiment 1 without the addition of 0.25% CP. However, *Trichoderma harzianum* differed from other fungi; the addition of 0.25% CP promoted its growth.

[0073] Figure 6 Based on the statistical results of the growth rate of different fungal colonies, it can be seen that the trend is consistent with the results of the fungal colony diameter (B), that is, Trichoderma harzianum has the fastest growth rate, while the growth rate of other fungi is inhibited by 0.25% CP.

[0074] Experiment 3: Colony morphology of different plant pathogenic fungi and Trichoderma harzianum in confrontational growth

[0075] Experimental Methods: *Fusarium graminearum*, *Anthracis higginense*, *Anthracis oleifera*, and *Alternaria emarginatus* were used to confront *Trichoderma harzianum* on PDA plates without the addition of 0.25% calcium propionate. Colony morphology (A), colony diameter (B), and colony growth rate (C) were recorded after 7 days. Results were statistically analyzed, with three replicates. Different letters represent significant differences (P < 0.01), with an error margin of ± SDs (n = 3).

[0076] Experimental results: Combining Figure 7 and Figure 8 The results of the growth morphology (A) and diameter (B) of different fungal colonies in confrontation with *Trichoderma harzianum* show that *Trichoderma harzianum* has the greatest inhibitory effect on *Alternaria alternata*, followed by *Anthracis higgins*. *Trichoderma harzianum* has the least inhibitory effect on *Fusarium graminearum*. Figure 9 Based on the statistical results of the growth rate of different fungal colonies confronting Trichoderma harzianum, it can be seen that the trend is consistent with the results of the fungal colony confrontation diameter (B), that is, Trichoderma harzianum alone has the greatest inhibitory effect on Eimeria spp.

[0077] Experiment 4: Colony morphology of different plant pathogenic fungi confronting Trichoderma harzianum on plates supplemented with 0.25% CP for 7 days.

[0078] Experimental Methods: *Fusarium graminearum*, *Anthracis higgins*, *Anthracis oleifera*, and *Alternaria emarginatus* were placed on PDA plates supplemented with 0.25% CP and confronted with *Trichoderma harzianum* for 7 days. Colony morphology (A), colony diameter (B), and colony growth rate (C) were recorded. The results were statistically analyzed, with three replicates. Different letters represent significant differences (P < 0.01), with an error range of ± SDs (n = 3).

[0079] Experimental results: Figure 10 Figure 11 The results of the contrast growth morphology (A) and diameter (B) of different fungal colonies with Trichoderma harzianum show that the addition of 0.25% CP significantly increased the inhibitory effect of Trichoderma harzianum on different fungal colonies, with the greatest inhibitory effect on Anthracnose of Higgins, followed by Fusarium graminearum. Figure 12 Based on the statistical results of the growth rate of different fungal colonies confronting Trichoderma harzianum, it can be seen that the trend is consistent with the results of the fungal colony confrontation diameter (B), that is, Trichoderma harzianum has the greatest inhibitory effect on Higgins anthracnose, followed by Fusarium graminearum.

[0080] Figure 13 Figure 14A comprehensive review of the control group (CK), the experimental group treated with calcium propionate alone, the experimental group treated with *Trichoderma harzianum* alone, and the combined treatment group of calcium propionate and *Trichoderma harzianum* showed that the treatment of *Trichoderma harzianum* combined with 0.25% CP (0.25% CP + *Trichoderma harzianum*) had a significant inhibitory effect on all bacterial groups, especially *Fusarium graminearum*, where the inhibitory effect was particularly significant. The treatment with 0.25% CP alone was not effective against the four bacterial groups, especially *Fusarium graminearum* and *Eimeria tenella*. The treatment with *Trichoderma harzianum* alone also showed good inhibitory effects against *Anthracis higgins*, *Anthracis oleifera*, and *Eimeria tenella*, but the effect on *Fusarium graminearum* was not significant. Therefore, for the control of Fusarium graminearum, the method of using Trichoderma harzianum in combination with 0.25% CP should be adopted; while for the control of Anthracnose of Higgins, Anthracnose of Camellia oleifera and Alternaria spp., the confrontation method of Trichoderma harzianum can be adopted, or the method of using Trichoderma harzianum in combination with 0.25% CP can be used to achieve better results.

[0081] Experiment 5: Effects of gibberellin + strigolactone on salt tolerance and growth of tomatoes

[0082] Experimental methods: Figure 15 Three plant pathogenic fungal bands isolated from soil samples obtained after five years of continuous tomato cropping were amplified using fungal ITS sequences. The ITS sequence alignment results of the isolated plant pathogenic fungi from the soil were as follows: *Anthracis oleifera*, *Fusarium oxysporum* (tomato-specific strain Fol), and *Alternaria emoreca*, indicating that these pathogens are common species in soil samples obtained after continuous tomato cropping. Using experimental fields with five years of continuous cropping and tomato seedlings as research materials, *Fusarium graminearum* (*Fusarium oxysporum* (tomato-specific strain Fol), *Anthracis higgins*, *Anthracis oleifera*, *Colletotrichum gloeosporioides*, and *Alternaria emoreca* were added to the irrigation water at the beginning of the experiment. The samples were completely randomized to three replicates per treatment, with five plants per replicate. Specifically, the tomato crops were treated using the method described in Example 1. Gibberellin and strigolactone were sprayed on the leaves of transplanted tomato seedlings twice a day, morning and evening. The spraying concentrations are shown in Table 1. For Experiments 1-5, Controls 1-2, and the blank group, equal volumes of a mixed solution of gibberellin and strigolactone were sprayed evenly on the tomato leaves on the day after irrigation and the 5th day, once in the morning and once in the evening. Experiment 6 was sprayed on the day of irrigation and the 5th day. Experiment 7 was sprayed on the day after irrigation and the 3rd day. Controls 3-4 only changed the calcium propionate concentration in Example 1 to 0.2% and 0.3%, respectively. Control 5 only increased the light intensity in Example 1 to 200 μM / m². 2 / s, wavelength 400nm; Comparative Example 6 only uses light intensity up to 100μM / m in Example 1. 2 / s, wavelength 700nm; 20 days after the above-mentioned treatment with the growth regulator, the aboveground parts of the whole plant were collected to determine the fresh weight, disease index, total chlorophyll content, and MDA content.

[0083] Table 1. Proportions of different concentrations of plant growth regulators

[0084]

[0085]

[0086] Table 2. Effects of different treatments on salt tolerance and growth of tomatoes

[0087]

[0088] Experimental results:

[0089] Biomass is the most direct data representation of seedling growth. Compared with controls 1-2, which were treated with gibberellin or strigolactone alone, and the blank group, the foliar spraying of an equal volume mixture of 20 mg / L gibberellin and 5 μM / L strigolactone, applied twice daily (morning and evening), significantly improved tomato leaf growth (fresh weight of above-ground parts). Adding calcium propionate and Trichoderma harzianum to irrigation water significantly prevented various fungal infections caused by continuous cropping. Simultaneously, it could lead to soil salinization. Applying gibberellin and strigolactone promoted plant growth and enhanced salt tolerance.

[0090] Furthermore, we compared several salt tolerance regulators commonly used in tomatoes and other crops, such as gibberellin and strigolactone, along with salicylic acid, melatonin, and brassinolide. We found that gibberellin and strigolactone showed the best effect, significantly improving the stress caused by calcium propionate spraying after pathogenic fungal infection. In addition, the total chlorophyll content also showed that gibberellin + strigolactone had a synergistic effect. With the addition of calcium propionate and Trichoderma harzianum for control, Experiment Example 1 of this application also effectively increased the total chlorophyll content of tomatoes. In the treatments with gibberellin and strigolactone, the accumulation of MDA in tomato cells was reduced in both treatment groups, alleviating and mitigating the damage of salt to tomato seedlings.

[0091] The application of this invention, involving the uniform foliar spraying of a mixture of equal volumes of gibberellin at a concentration of 20 mg / L and strigolactone at a concentration of 5 μM / L, in conjunction with a control experiment against multiple fungal infections following continuous cropping of tomatoes, showed that this mixture had the best growth-promoting effect on tomato plants. In contrast, in comparative experiments 2-5, increasing the concentrations of both gibberellin and strigolactone hindered leaf growth and chlorophyll synthesis, thereby impeding tomato growth.

[0092] In addition, we found that when Trichoderma harzianum conidia with calcium propionate were used for the combined control of various continuous cropping pathogens, the best control effect was achieved when applied with irrigation water after tomato transplanting, on the second day and the fifth day after irrigation. In comparative experiments 6-7, the disease index of spraying on the day of irrigation and the fifth day, as well as spraying on the second day and the third day after irrigation, was higher than that of spraying on the second day and the fifth day after irrigation in experiment 1.

[0093] We also found that excessive calcium propionate significantly inhibited tomato biomass accumulation and plant development. With increasing calcium propionate levels, the fresh weight of the above-ground parts decreased, chlorophyll content also decreased significantly, and the disease index increased. This indicates that excessive calcium propionate inhibits tomato plant growth, while low levels of calcium propionate are ineffective in preventing fungal infection, and the disease index also increases accordingly.

[0094] Light intensity 100 μM / m 2 / s, wavelength 400nm, shake culture at 25℃ until the viable count of Trichoderma harzianum conidia per 1g is 10 9 ~10 10 When CFU was stopped, the activated Trichoderma harzianum conidial solution exhibited the highest antibacterial activity. Increased light intensity and wavelength were detrimental to the comprehensive prevention and control effect of the Trichoderma harzianum conidial solution combined with calcium propionate, leading to a significant increase in the disease index.

[0095] This experiment utilizes materials proposed for the control of tomato wilt caused by *Fusarium oxysporum* var. *tomato* (Fol) and *Colletotrichum candida*. Fol is one of the most serious fungal diseases in tomato production, causing huge economic losses to tomato agriculture worldwide. The experiment shows that the method of using calcium propionate combined with *Trichoderma harzianum* is effective in controlling *Fol* and *Colletotrichum candida* fungal infections. Furthermore, this method can also effectively control co-infection by *Fusarium graminearum* (*Fusarium oxysporum* var. *tomato* Fol), *Anthracnose glomeratus*, *Anthracnose oleifera*, *Colletotrichum candida*, and *Eimeria emarginatus* after continuous cropping of tomatoes.

[0096] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for controlling the infestation of pathogenic fungi after the continuous cropping of tomatoes by calcium propionate in combination with Trichoderma harzianum, characterized in that, Comprising the following steps: (1) medium preparation, after washing and peeling fresh potatoes into pieces, 200g is weighed and added to appropriate amount of distilled water, boiled for 15min until the potatoes become soft, filtered with multiple layers of gauze, 20g of glucose and 2.5g of calcium propionate powder are dissolved in the filtrate, and the solution is made up to 1L and divided into conical flasks with 1.5% agar powder, sealed and sterilized at 121 ℃ for 20min, ready for use; (2) Preparation of Trichoderma harzianum conidium liquid: Trichoderma harzianum strain is inoculated into the above-mentioned culture medium, and is shaken and cultured at 25°C under the irradiation of white cold light fluorescent lamp with light intensity of 100-200 μM / m 2 2 and wavelength of 400-700 nm until the viable cell count of Trichoderma harzianum conidium liquid is 10 9 -10 10 CFU per 1 g of Trichoderma harzianum conidium liquid, and the activated Trichoderma harzianum conidium liquid is obtained. (3) when the pathogenic fungi infects after the tomato is transplanted, the conidial suspension of the Trichoderma harzianum in step (2) is applied with irrigation water, once every 3 days, for a total of 3 times, with an application amount of 400kg per mu each time; (4) the next day and the 5th day after irrigation in step (3), a mixed solution of gibberellin with a concentration of 20-50mg / L and strigolactone with a concentration of 3-5μM / L is uniformly sprayed on the tomato leaves, once in the morning and once in the evening; The tomato leaves are evenly sprayed with a mixed solution of gibberellin with a concentration of 20 mg / L and strigolactone with a concentration of 5 μM / L in equal volume; the light intensity is 100 μM / m 2 / s, and the wavelength is 400 nm.

2. A method for the preparation of a preparation for the control of pathogenic fungi after the cultivation of tomatoes, characterized in that, Comprising the following steps: (1) medium preparation, after washing and peeling fresh potatoes into pieces, 200g is weighed and added to appropriate amount of distilled water, boiled for 15min until the potatoes become soft, filtered with multiple layers of gauze, 20g of glucose and 2.5g of calcium propionate powder are dissolved in the filtrate, and the solution is made up to 1L and divided into conical flasks with 1.5% agar powder, sealed and sterilized at 121 ℃ for 20min, ready for use; (2) Preparation of Trichoderma harzianum conidium liquid: Trichoderma harzianum strain is inoculated into the above-mentioned culture medium, and is shaken and cultured at 25°C under the irradiation of white cold light fluorescent lamp with light intensity of 100-200 μM / m 2 2, wavelength of 400-700 nm, until the viable cell count of Trichoderma harzianum conidium liquid is 10 9 ~ 10 10 CFU per 1 g of Trichoderma harzianum conidium liquid, and the activated Trichoderma harzianum conidium liquid is obtained. The light intensity 100 μM / m 2 / s, wavelength 400 nm.

3. The preparation for preventing and treating pathogenic fungi infection after continuous cropping of tomatoes obtained by the method of claim 2.

4. The use of the preparation for preventing and treating pathogenic fungi infection after continuous cropping of tomatoes of claim 3 in any one of the following, (1) in the prevention and treatment of any one or several of Fusarium graminearum, Guignardia bidwellii, Guignardia exitiosa and Septoria artemisiae infection; (2) After continuous cropping of tomatoes, Fusarium oxysporum tomato-specific strain ( Fusarium oxysporumf.sp.Lycopersici Applications of Fol), Higgins anthrax, Camellia anthrax, Colletotrichum candida, and Alternaria esculenta in infections caused by one or more fungi.

5. A method for preventing infestation by pathogenic fungi after continuous cropping of tomatoes, characterized in that, Comprising the following steps: (1) medium preparation, after washing and peeling fresh potatoes into pieces, 200g is weighed and added to appropriate amount of distilled water, boiled for 15min until the potatoes become soft, filtered with multiple layers of gauze, 20g of glucose and 2.5g of calcium propionate powder are dissolved in the filtrate, and the solution is made up to 1L and divided into conical flasks with 1.5% agar powder, sealed and sterilized at 121 ℃ for 20min, ready for use; (2) Preparation of Trichoderma harzianum conidium liquid: Trichoderma harzianum strain is inoculated into the above-mentioned culture medium, and is shaken and cultured at 25°C under the irradiation of white cold light fluorescent lamp with light intensity of 100 μM / m 2 2, wavelength of 400 nm, until the viable cell count of Trichoderma harzianum conidium liquid is 10 9 ~ 10 10 CFU per 1 g of Trichoderma harzianum conidium liquid, and the activated Trichoderma harzianum conidium liquid is obtained. (3) Trichoderma harzianum is applied when the tomato is transplanted, using hole application method, with a dosage of 5g of the conidial suspension of Trichoderma harzianum in step (2) per hole, which is scattered into the hole.

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