Fungicide composition for preventing and treating peanut southern blight as well as preparation and application thereof

Abstract

The invention relates to a fungicide composition for preventing and treating peanut southern blight as well as preparation and application of the fungicide composition. The invention aims to solve the technical problems of unstable field control effect, high chemical pesticide residue and high drug resistance risk of biological control in peanut southern blight control. Bacillus velezensis YJ02 with good biocompatibility is compounded with thifluzamide (a biological agent is used for generating antibacterial substances and inducing plant defensive enzyme, and a chemical bactericide is combined for rapidly inhibiting respiration of pathogenic bacteria) to prevent and treat the peanut southern blight; the technical effects that the efficient synergistic control effect is as high as 94.87%, the chemical pesticide application amount is reduced (the thifluzamide application amount is only 2.08% of the conventional thifluzamide application amount), the lasting period is prolonged (20-30 days), the morbidity and disease index are reduced, and environment friendliness is achieved are achieved.

Description

Technical Field

[0001] This invention relates to the field of plant disease control technology, and in particular to a fungicide composition for controlling peanut white mold disease, its preparation and application. Background Technology

[0002] Peanut white mold disease is caused by Sclerotium sclerotiorum (Sclerotium sclerotiorum). Sclerotium rolfsii This disease, caused by [unspecified pathogen], primarily affects the base of the peanut stem, pedicel, pods, and roots, and is one of the major diseases affecting peanut yield. Peanut white mold can infect peanuts throughout their entire growth cycle, mainly affecting the base of the stem, pedicel, pods, and roots. In the early stages of the disease, the plant... The main stem and lateral branches of the plant turn yellow and wilt, with white, silky mycelium growing near the soil at the base of the stem. In the later stages of the disease, the tissue at the base of the stem rots and becomes fibrous, the leaves turn yellow and scorch, and the plant withers and dies. At the same time, the white mycelium aggregates to produce a large number of rapeseed-shaped sclerotia, which gradually change from white to yellowish-brown and eventually to dark brown. In recent years, affected by factors such as climate warming, high-density planting, and straw returning to the field, peanut white mold disease has become widespread in most peanut-producing areas of my country, and the incidence rate has been increasing year by year, seriously affecting the yield and quality of peanuts.

[0003] Based on an analysis of existing technologies, there are currently certain limitations in the control of peanut white mold: ① While chemical control methods offer advantages such as rapid effectiveness and ease of operation, they lead to increased pesticide residues in peanuts. This not only poses a safety hazard of poisoning to humans and animals but also results in stronger drug resistance in the disease and may even trigger a resurgence, thus threatening sustainable agricultural development. ② Biological control methods, such as the use of plant immune activators like chitosan and alginate, enhance disease resistance by inducing the plant to produce disease-resistant factors. This method is environmentally friendly, pollution-free, and does not induce drug resistance in the disease. However, its disadvantages include a relatively slow onset of action and susceptibility to environmental factors.

[0004] In summary, there is still a lack of effective measures to prevent and control peanut white mold; therefore, finding economical and efficient methods to prevent and cure peanut white mold has become a key measure to improve peanut yield and quality.

[0005] The information disclosed in this background section is intended only to enhance the understanding of the background technology of this disclosure and should not be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art. Summary of the Invention

[0006] The inventors have found that Bacillus velezensis YJ02 and thifluzamide have good biocompatibility, and the combination of the two can significantly inhibit Sclerotium rolfsii and has excellent synergistic effect, which can solve the problem of unstable field control effect of biocontrol bacteria, reduce the application amount of chemical pesticides, and reduce the incidence and disease index of peanut white thread blight, and significantly improve the disease resistance of peanuts, thereby providing a new way for the prevention and control of peanut white thread blight.

[0007] According to one aspect of the present disclosure, a bacterium-pesticide composition is provided, which contains effective components of thifluzamide 0.05-5 μg / mL, Bacillus velezensis (Bacillus velezensis) YJ02 with preservation number CGMCC NO. 23110 0.5×10 Bacillus velezensis CFU / mL. 2 ~0.5×10 8 CFU / mL.

[0008] In some embodiments of the present disclosure, the bacterium-pesticide composition contains thifluzamide 0.5-5 μg / mL, Bacillus velezensis (Bacillus velezensis) YJ02 with preservation number CGMCC NO. 23110 0.5×10 Bacillus velezensis ~0.5×10 5 ~0.5×10 8 CFU / mL.

[0009] According to another aspect of the present disclosure, a preparation method of a bacterium-pesticide composition is provided, which comprises the following steps: (1) Bacillus velezensis YJ02 with preservation number CGMCC NO. 23110 is activated and inoculated in NA culture solution, and then cultured in a 28°C shaking incubator at 160 r / min for 18-30 h to obtain a bacterium agent raw material; (2) The bacterium agent raw material, thifluzamide technical material, and corresponding adjuvants are uniformly mixed in proportion, so that the content of thifluzamide reaches 0.05-5 μg / mL, and the number of Bacillus velezensis YJ02 bacteria reaches 0.5×10 2 ~0.5×10 8 CFU / mL.

[0010] According to a third aspect of the present disclosure, the bacterium-pesticide composition is applied to inhibit Sclerotium rolfsii.

[0011] According to a fourth aspect of the present disclosure, the bacterium-pesticide composition is applied to improve the disease resistance of peanuts.

[0012] According to a fifth aspect of the present disclosure, the bacterium-pesticide composition is applied to prevent and control peanut white thread blight.

[0013] In some embodiments of the present disclosure, the root drenching is repeated 2-4 times at intervals of 5-8 days before or after the occurrence of the white thread disease, and each time 5-15 mL of the solution is used for each plant.

[0014] The one or more technical solutions provided in the embodiments of the present application have at least any of the following technical effects or advantages: 1. Synergistic effect, improve the prevention effect: Bacillus velezensis YJ02 produces protease, cellulase, amylase and siderophore and other antibacterial substances to inhibit the growth of pathogenic bacteria (Sclerotium rolfsii) and destroy its mycelium structure and sclerotium formation. At the same time, it can induce peanut plants to produce defense enzymes (such as phenylalanine ammonia lyase, polyphenol oxidase) to enhance the disease resistance of the plants; thifluzamide as a chemical fungicide can quickly kill pathogenic bacteria by inhibiting the succinate dehydrogenase of fungi and blocking their respiration. The combination of the two achieves the combination of the durability of biological control and the rapidity of chemical control, realizing the whole cycle control of "prevention-treatment-persistence", and the data of the embodiments show that the prevention effect of the optimal combination is as high as 94.87%, which is significantly better than using biological agents or chemical agents alone.

[0015] 2. Prolong the persistence period and reduce the frequency of use: Bacillus velezensis YJ02 can survive in the soil for a long time and continuously inhibit the reproduction of pathogenic bacteria, with a persistence period of 20-30 days. After being combined with thifluzamide, the rapid fungicidal effect of the chemical agent can quickly control the spread of the disease, and the sustained effect of the biological agent reduces the risk of recurrence of pathogenic bacteria in the later period, thereby reducing the overall frequency of use.

[0016] 3. Environmentally friendly, reduce the risk of drug resistance: The biological control mechanism of Bacillus velezensis YJ02 is not prone to cause drug resistance of pathogenic bacteria, and the combination with thifluzamide can delay the development of drug resistance of chemical agents, prolong the service life of the agents, and the use of biological agents reduces the amount of chemical agents, with the amount of thifluzamide being only 2.08% of the conventional amount in the field, greatly reducing the pollution risk of soil and water, and meeting the requirements of green agriculture and sustainable development.

[0017] 4. Innovative disease control strategy: Bacillus velezensis YJ02 with good biocompatibility is first selected to be combined with thifluzamide to control peanut white thread disease, breaking through the limitations of single active ingredient, and providing an efficient and environmentally friendly new solution for the control of peanut white thread disease. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 In an embodiment of the present application, YJ02 0.5x10 2 The synergistic inhibition effect of the combination of cfu / mL + thifluzamide 0.05 μg / mL on Sclerotium rolfsii is shown in the result graph, wherein A is single dose of Bacillus velezensis YJ02 1x10 2Plate inhibition effect of cfu / mL on Sclerotium rolfsii; B is the plate inhibition effect of 0.1 μg / mL thifluzamide on Sclerotium rolfsii; C is the plate inhibition effect of 0.5×10 2 Plate inhibition effect of cfu / mL+thifluzamide 0.05 μg / mL on Sclerotium rolfsii; D is the blank control of Sclerotium rolfsii. DETAILED DESCRIPTION

[0019] In order to better understand the technical scheme of the present application, the above technical scheme will be described in detail below in combination with the drawings of the specification and specific embodiments.

[0020] The main test raw materials and reagents involved in the following examples include: Test strain: Sclerotium rolfsii (Sclerotium rolfsii) Sclerotium rolfsii ), Bacillus velezensis (Bacillus velezensis) Bacillus velezensis YJ02, preservation number CGMCC NO. 23110, preservation date August 4, 2021; see Chinese patent document CN113817642A.

[0021] PDA culture medium: potato 200.0 g, glucose 20.0 g, agar 17.0 g, distilled water 1000 mL.

[0022] NA culture medium: yeast extract powder 3.0 g, peptone 5.0 g, glucose 10.0 g, agar 17.0 g, distilled water 1000 mL, NA culture solution does not add agar, and the rest of the ingredients are the same.

[0023] Tested peanut variety: Yuhua 22, purchased from the market.

[0024] The tested fungicides are shown in Table 1.

[0025] Table 1 Test fungicides .

[0026] The Bacillus velezensis agent described in the present application is prepared by the following method: After the NA plate YJ02, a single colony is inoculated in the NA culture solution and placed in a 28℃ shaking incubator at 160 r / min for 18-30 h, and then obtained.

[0027] Example 1, Bacillus velezensis YJ02 and common chemical pesticide virulence determination 1. Test method The inhibition rates of Bacillus velezensis YJ02 and 9 fungicides on S. arachidicola were determined by using toxic medium method. The PDA medium was cooled to about 50 ℃, and then different concentrations of prepared fungicide solution (Table 2) were added and mixed evenly. The control group was prepared with sterile water. The 5.0 mm diameter S. arachidicola mycelium disc was inoculated in the prepared PDA medium, and each concentration was repeated 3 times. After 5 days of culture at 25 ℃ in the dark, the colony diameter of S. arachidicola was measured, and the inhibition rate was calculated.

[0028] Inhibition rate (%) = (control colony diameter - treatment colony diameter) / (control colony diameter - mycelium disc diameter) x 100.

[0029] Table 2 Concentration gradient of Bacillus velezensis YJ02 and 9 fungicides Note: The concentration in the table is the final concentration in the medium.

[0030] 2. Test results The indoor toxicity of Bacillus velezensis YJ02 and 9 fungicides is shown in Table 3. The EC 50 value of Bacillus velezensis YJ02 on S. arachidicola was 1.04 x 10 2 cfu / mL. Among the 9 chemical fungicides, thifluzamide, pyraclostrobin, fludioxonil and tebuconazole had higher fungicidal activity, with EC 50 values of 0.10, 0.46, 0.47 and 0.66 μg / mL, respectively. Therefore, thifluzamide, pyraclostrobin, fludioxonil and tebuconazole were selected for bio-compatibility determination with Bacillus velezensis YJ02. To facilitate the determination of concentration, the EC 50 of Bacillus velezensis YJ02 on S. arachidicola was rounded to 1.0 x 10 2 cfu / mL in subsequent tests.

[0031] Table 3 Results of toxicity determination of Bacillus velezensis YJ02 and 9 fungicides on S. arachidicola .

[0032] Example 2, determination of compatibility of Bacillus velezensis YJ02 with fungicides 1. Test method The compatibility of the selected fungicides with higher toxicity to S. arachidicola and Bacillus velezensis YJ02 was determined by plate counting method. The chemical fungicide was added to the NA plate to make the final concentration 50, 100, 150, 200 and 250 μg / mL; 1 mL of 1.0 x 10 8The Bacillus velezensis YJ02 liquid with CFU / mL was diluted by 10 times in series for 5 times, 100 μL of the diluted bacterial suspension was uniformly coated on the NA plate, sterile water was used as control, and the plate was cultured at 28 ℃ in dark condition, and the number of colonies of the Bacillus velezensis YJ02 was counted after 48 h, the number of colonies per milliliter of the YJ02 liquid was calculated, and each group was repeated for 3 times. The DPS software was used for statistical analysis of the biocompatibility.

[0033] 2. Test results The compatibility test results of the chemical fungicides and the Bacillus velezensis YJ02 showed that the YJ02 could not grow on the pyraclostrobin and fludioxonil plates with a concentration of 50 μg / mL; the number of colonies of the YJ02 on the plate with a concentration of 50 μg / mL of the tebuconazole was only 0.20×10 8 CFU / mL, which was significantly lower than that of the control group (P<0.05); but on the plates with a concentration of 50, 100, 150 μg / mL of the thifluzamide, the number of colonies of the YJ02 was 1.08×10 8 , 1.05×10 8 , 1.02×10 8 CFU / mL, which had no significant difference with the control group (Table 4), indicating that the Bacillus velezensis YJ02 had good biocompatibility with the thifluzamide with a concentration lower than 150 μg / mL, and could be used for mixing.

[0034] Table 4 Effects of four fungicides on the growth of the Bacillus velezensis YJ02 .

[0035] Example 3, screening of the Bacillus velezensis YJ02 and different chemical mixing ratios 1. Test method According to the indoor toxicity determination results of the 9 fungicides and the compatibility test results between the Bacillus velezensis YJ02 and the fungicides in the foregoing examples, the thifluzamide with the highest toxicity to the Sclerotium rolfsii and the least effect on the growth of the Bacillus velezensis YJ02 was selected to be compounded with the Bacillus velezensis YJ02, and the interactive determination method was used for screening the best mixing ratio. Based on the EC 50 of the two single agents, 11 mixed treatment groups with different mixing ratios were set, and the toxicity determination was carried out. Among them, the two single agents were respectively set as 100% of the EC 50 , in the mixed combination, the amount of the A group was respectively 100% of the EC 50 .10 / 10, 9 / 10, 8 / 10, 7 / 10, 6 / 10, 5 / 10, 4 / 10, 3 / 10, 2 / 10, 1 / 10 and 0, while the amount of B component is 0 / 10, 1 / 10, 2 / 10, 3 / 10, 4 / 10, 5 / 10, 6 / 10, 7 / 10, 8 / 10, 9 / 10, 10 / 10 of its EC50, respectively, to prepare 11 different mixed combinations of mixed agents with different mixing ratios, and perform toxicity determination respectively. The toxicity determination results of each mixed agent treatment are calculated using the following method.

[0036] Toxicity ratio = test inhibition rate / theoretical inhibition rate Theoretical inhibition rate = test inhibition rate of agent A at its EC 50 Dose x proportion of agent A + test inhibition rate of agent B at its EC 50 Dose x proportion of agent B; In the formula: A is 1 x 10 2 cfu / mL Bacillus velezensis YJ02, and B is 0.1 μg / mL thifluzamide.

[0037] Toxicity ratio < 0.75 shows antagonism; toxicity ratio around 1 shows additive effect; and toxicity ratio > 1.25 shows synergistic effect.

[0038] 2. Test results The inhibition effect of different proportions of Bacillus velezensis YJ02 and thifluzamide on Sclerotium rolfsii is shown in Table 5 and Figure 1 The results show that the toxicity ratio of all combinations of Bacillus velezensis YJ02 and thifluzamide to Sclerotium rolfsii is greater than 1, among which the toxicity ratio of the 0.5:0.5 ratio is the highest, up to 1.95, and the synergistic effect is the most significant, indicating that the synergistic effect of thifluzamide and Bacillus velezensis YJ02 is the strongest at this ratio, and the control effect is the best.

[0039] Table 5 Inhibition rate and toxicity ratio of Bacillus velezensis YJ02 and thifluzamide to Sclerotium rolfsii .

[0040] Example Four, verification of control effect test 1. Test method The peanut Sclerotinia sclerotiorum cake was inoculated in sterilized wheat grains for expansion culture, and after 7-10 days of culture at 25°C in the dark, it was ready for use. The peanut seeds were sterilized with 75% alcohol for 1 min, rinsed with sterile water, and then sown in 19 cm x 14 cm x 10 cm square flowerpots, with 0.2 kg of sterilized soil in each pot. All the pots were placed in a greenhouse at 22-28°C, 12 h light, and a relative humidity of about 70%, and were watered regularly. When the peanut seedlings grew to 4-5 leaf stage, the control effect test was conducted.

[0041] Since the actual field drug concentration is usually much higher than the plate inhibition concentration, the two mixed components were respectively set as EC 50 for low concentration, and the medium and high concentrations were added in proportion. Bacillus velezensis YJ02 was set as 1000 times gradient (1 x 10 2 CFU / mL, 1 x 10 5 CFU / mL, 1 x 10 8 CFU / mL), and thifluzamide was set as 10 times gradient (0.1 μg / mL, 1 μg / mL, 10 μg / mL). The mixed combination of low, medium and high gradients was mixed with the corresponding gradients of the two components at a ratio of 0.5:0.5. The preventive treatment group and the treatment group were set up to explore the action mode of Bacillus velezensis YJ02 and thifluzamide complex on peanut Sclerotinia sclerotiorum.

[0042] Preventive treatment settings: T1: Bacillus velezensis YJ02 1 x 10 2 CFU / mL (single dose); T2: Thifluzamide 0.1 μg / mL; T3: Bacillus velezensis YJ02 0.5 x 10 2 CFU / mL + thifluzamide 0.05 μg / mL (complex combination); T4: Bacillus velezensis YJ02 1 x 10 5 CFU / mL (single dose); T5: Thifluzamide 1 μg / mL; T6: Complex combination of Bacillus velezensis YJ02 0.5 x 10 5 CFU / mL + thifluzamide 0.5 μg / mL (complex combination); T7: Bacillus velezensis YJ02 1 x 10 8 CFU / mL (single dose); T8: Thifluzamide 10 μg / mL; T9: Bacillus velezensis YJ02 0.5 x 10 8CFU / mL + thifluzamide 5 pg / mL (complex combination) T10: 240 pg / mL thifluzamide (control drug); T11: blank control.

[0043] The application method was root irrigation, and the amount of irrigation per peanut plant was 10 mL. All treatments were inoculated uniformly 48 h after the first application. Wheat grains infected with S. sphaerodes were placed at the base of each peanut seedling stem, with 5 grains per plant. Root irrigation was repeated 7 d after the first application, for a total of 3 times. The treatment group was treated with irrigation 48 h after inoculation, and the treatment settings were the same as the prevention group. The blank control group was treated with water. There were 20 peanuts per treatment, and a total of 3 replicates were set. After the blank control group was fully diseased, the disease conditions of each treatment were investigated and the control effect was calculated.

[0044] The disease classification referred to the standard of Yan Liying et al., and the details were as follows: 0 level: no symptoms on the plant; 1 level: only symptoms at the stem base; 2 level: necking symptoms at the stem base, and less than 1 / 3 of the whole plant showing systemic symptoms (wilting, withering, death, etc.); 3 level: less than 2 / 3 of the whole plant showing systemic symptoms; 4 level: more than 2 / 3 of the whole plant showing systemic symptoms.

[0045] Disease index =∑(disease level x number of plants at that disease level) / (maximum disease level x total number of plants) x 100 Control effect (%) = (disease index of the blank control group - disease index of the treatment group) / disease index of the blank control group x 100 The control effect was statistically analyzed by DPS software.

[0046] 2. Test results: potting data (Table 6) showed that the control effects of the three complex agents in the prevention treatment group and the treatment group were significantly higher than those of the single agents, indicating that Bacillus velezensis YJ02 and thifluzamide 0.5:0.5 had a significant synergistic effect after complexing. The control effect of each treatment in the prevention treatment group was higher than that of the corresponding treatment in the treatment group, indicating that the complex agent and each single agent and thifluzamide at the conventional field use amount had a better preventive effect than a therapeutic effect on peanut Sclerotium blight, and the complex agent was more suitable for use before the occurrence of Sclerotium blight. Among the three concentrations of complex agents, the control effect of Bacillus velezensis YJ02 1x10 8The control effect of 0.5:0.5 complex combination of 10 μg / mL thifluzamide and 0.5 CFU / mL Bacillus velezensis YJ021 was the highest, reaching 94.87%, which was equivalent to the control effect of thifluzamide at the conventional field use amount, and the content of thifluzamide (5 μg / mL) was only 2.08% of the conventional field use amount (240 μg / mL). The control effect of 0.5:0.5 complex combination of 1 μg / mL thifluzamide and 0.5 CFU / mL Bacillus velezensis YJ021 was 87.62%, which was significantly higher than the control effect of thifluzamide at the conventional field use amount. 5 The control effect of 0.5:0.5 complex combination of 1 μg / mL thifluzamide and 0.5 CFU / mL Bacillus velezensis YJ021 was 87.62%, which was significantly higher than the control effect of thifluzamide at the conventional field use amount. 2 The control effect of 0.5:0.5 complex combination of 0.1 μg / mL thifluzamide and 0.5 CFU / mL Bacillus velezensis YJ021 was 71.37% and 25.21% respectively, which were significantly higher than the control effect of thifluzamide at the conventional field use amount, but the control effect of 0.5:0.5 complex combination of 0.1 μg / mL thifluzamide and 0.5 CFU / mL Bacillus velezensis YJ021 was significantly lower than the control effect of Bacillus velezensis YJ021 at the conventional field use amount. 2 The control effect of 0.5:0.5 complex combination of 0.1 μg / mL thifluzamide and 0.5 CFU / mL Bacillus velezensis YJ021 was 71.37% and 25.21% respectively, which were significantly higher than the control effect of thifluzamide at the conventional field use amount, but the control effect of 0.5:0.5 complex combination of 0.1 μg / mL thifluzamide and 0.5 CFU / mL Bacillus velezensis YJ021 was significantly lower than the control effect of Bacillus velezensis YJ021 at the conventional field use amount. 5 The control effect of 0.5:0.5 complex combination of 1 μg / mL thifluzamide and 0.5 CFU / mL Bacillus velezensis YJ021 was 87.62%, which was significantly higher than the control effect of thifluzamide at the conventional field use amount. 8 The control effect of 0.5:0.5 complex combination of 10 μg / mL thifluzamide and 0.5 CFU / mL Bacillus velezensis YJ021 was the highest, reaching 94.87%, which was equivalent to the control effect of thifluzamide at the conventional field use amount, and the content of thifluzamide (5 μg / mL) was only 2.08% of the conventional field use amount (240 μg / mL).

[0047] Table 6 Pot-culture control effect of complex combination on peanut Sclerotium blight .

[0048] Although some preferred embodiments of the present application have been described, those skilled in the art can make additional changes and modifications to these embodiments once they know the basic inventive concept. Therefore, the appended claims are intended to include all preferred embodiments and all changes and modifications falling within the scope of the present application.

[0049] Obviously, those skilled in the art can make various modifications and variations to the present application without departing from the spirit and scope of the present application. Thus, if these modifications and variations of the present application fall within the scope of the claims and their equivalents, the present application is also intended to include these modifications and variations.

Claims

1. A fungal drug composition, characterized in that, Bacillus belyssus, containing the active ingredient thifluzamide at a concentration of 0.05–5 μg / mL and with the preservation number CGMCC NO. 23110, is a strain of Bacillus belyssus. Bacillus velezensis YJ02 0.5×10 2 ~0.5×10 8 CFU / mL.

2. The fungal drug composition according to claim 1, characterized in that, The *Bacillus belyssus* YJ02, with a thifluzamide content of 0.5–5 μg / mL and preservation number CGMCC NO. 23110, contained 0.5 × 10⁻⁶ μg / mL thifluzamide. 5 ~0.5×10 8 CFU / mL.

3. A method for preparing the fungal drug composition according to claim 1, characterized in that, Includes the following steps: (1) Take Bacillus belye YJ02 with preservation number CGMCC NO. 23110, activate it and inoculate it into NA culture medium. Incubate it in a shaking incubator at 28℃ and 160 r / min for 18-30 h to obtain the bacterial agent raw material; (2) The bacterial agent raw material is mixed evenly with thifluzamide technical and corresponding adjuvants in a certain proportion, so that the thifluzamide content reaches 0.05~5 μg / mL and the Bacillus belyssus YJ02 colony count reaches 0.5×10⁻⁶. 2 ~0.5×10 8 CFU / mL.

4. The use of the fungal composition according to claim 1 or 2 in at least one of the following (1) to (3) (1) Inhibits *Sclerotium sclerotiorum*; (2) Improve peanut disease resistance; (3) Control peanut white mold disease.

5. The application according to claim 4, characterized in that, Before or after the occurrence of white mold disease, repeat the root drenching 2-4 times at 5-8 day intervals, with each application of 5-15 mL per plant.

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