A fungicide for preventing and controlling bacterial fruit spot of melons and its application

Abstract

The invention discloses a fungicide for preventing and treating bacterial fruit spot of melons and its application, belonging to the technical field of pesticides. The invention discloses a new use of albendazole in preventing and treating bacterial fruit spot of melons. The melon seeds are disinfected with albendazole to kill the melon fruit spot pathogen carried on the surface of the melon seeds, effectively blocking the occurrence and spread of the disease from the source, and the prevention and control effect reaches 100%. The seed disinfection technology provided by the invention can significantly improve the seed germination rate and emergence rate.

Description

Technical Field

[0001] The invention relates to the technical field of pesticides, and in particular to a fungicide containing an effective ingredient, albendazole, and application thereof in preventing and controlling bacterial fruit spot of melons. Background Art

[0002] Bacterial fruit spot of melons is a bacterial disease caused by the acidophilic bacteria Acidovorax citrulli. It harms cucurbitaceous crops such as watermelon, melon, cantaloupe, bitter melon, pumpkin, zucchini, cucumber, loofah, gourd, etc., especially watermelon and melon. The disease mainly harms leaves and fruits. The lesions are water-soaked in the early stage. When the humidity is high, pus overflows and is further spread by rain and insects. In the later stage, dark brown necrotic lesions are formed on the leaves. The lesions are slightly yellow around the lesions. Multiple lesions can fuse, and in severe cases, the whole plant will die. In the later stage, cracked lesions are formed on the peel, and the flesh is rotten in a water-soaked state or necrotic in a cotton-like state, causing the fruit to lose its economic value.

[0003] Bacterial fruit spot of melons is a seed-borne disease that spreads over long distances through seeds and seedlings. The seeds harvested from diseased melons carry pathogens. If the diseased seeds are not properly handled, they will become the source of infection at the beginning of the next year, infecting the plants again and causing diseases, resulting in significant economic losses. The relevant agricultural management departments require seed production companies to conduct origin monitoring and inspection. All melon seeds of seed marketing units must have inspection and quarantine certificates issued by professional institutions. On this basis, the plant inspection and quarantine department or the agricultural law enforcement department will sample melon seeds and have them tested by professional institutions. If the seeds are found to carry melon fruit spot bacteria, the seeds of the same batch will be required to be sealed and destroyed, causing significant losses to the seed production company. Since origin monitoring and inspection and seed testing require strong professionalism, and the technical levels of professionals in various places are uneven, it is inevitable that the infected seeds will flow into the hands of the majority of growers, causing the plants to become sick in the field, resulting in reduced production or even total crop failure.

[0004] Therefore, correct seed treatment is an important method to effectively prevent and control bacterial fruit spot of melons and reduce or avoid economic losses.

[0005] At present, there are physical and chemical methods for seed treatment. Commonly used physical methods include dry heat treatment and warm water soaking. Studies have shown that the amount of live bacteria in dry heat treated seeds decreases with increasing treatment temperature or time, but seed vigor and germination rate also decrease (Zhang Yongping, Zhang Wenxian, et al. Effect of dry heat treatment on melon seed vigor and bacterial fruit spot inhibition ability [J]. Journal of Northwest Botany, 2017, 37(7): 1364-1371.); warm water soaking cannot completely kill bacteria, causing some pathogens to enter a viable but non-culturable state (VBNC) (Wang Mengyu, Jiang Na, et al. Study on induction of watermelon acidophilic bacteria into the VBNC state under inappropriate temperature conditions [J]. Journal of Plant Pathology, 2021, 51(1): 104-114.). Therefore, it is a problem that technicians in this field need to solve to develop a seed treatment method that is effective in sterilization and does not affect seed vigor.

[0006] Using chemical agents to soak seeds is the main means of preventing and controlling bacterial fruit spot disease. Patent document CN 106063486A discloses that using peracetic acid and hydrochloric acid to treat Hami melon seeds can effectively prevent and control the disease in seedlings and has no effect on the germination rate, but it does not mention whether it has an effect on the emergence rate.

[0007] Albendazole, chemical formula is C 12 H 15 N3O2S is a broad-spectrum anthelmintic drug of the imidazole derivative class. Patent document CN 113662002 A discloses that albendazole has inhibitory activity against rice blast fungus. However, there is no relevant research on the use of albendazole in the prevention of bacterial fruit spot disease of melons. Summary of the invention

[0008] The object of the present invention is to provide a fungicide for preventing and controlling bacterial fruit blotch of melons, so as to solve the problem that bacterial fruit blotch of melons is spread through infected seeds and causes the occurrence of the disease.

[0009] To achieve the above object, the present invention adopts the following technical solution:

[0010] The invention provides the use of albendazole in preparing a fungicide for preventing and treating bacterial fruit spot disease of melons. The structural formula of the albendazole is shown in formula (I).

[0011]

[0012] Furthermore, the melon is a Cucurbitaceae crop, and the disease is caused by the watermelon acidophilic bacteria Acidovorax citrulli.

[0013] The present invention shows that albendazole has a significant inhibitory effect on the pathogen of melon bacterial fruit spot. After being applied to the disinfection of melon seeds, the occurrence of seed-transmitted melon bacterial fruit spot can be effectively controlled, and the germination rate and emergence rate of seeds can be significantly improved.

[0014] Furthermore, the fungicide is used for seed treatment. The effective concentration of albendazole in the fungicide for preventing and treating bacterial fruit spot of melons is greater than 0.03 mg / L.

[0015] The fungicide uses albendazole as an active ingredient, and can also be added with auxiliary ingredients required by the pesticide formulation.

[0016] The present invention also provides a seed treatment agent for preventing and treating bacterial fruit spot of melons. The seed treatment agent comprises albendazole and auxiliary ingredients. The auxiliary ingredients comprise wetting and dispersing agents sodium methylene bis naphthalene sulfonate and agricultural milk 700#, thickening agents xanthan gum and white carbon black, antifreeze agent ethylene glycol and preservative sodium benzoate.

[0017] Preferably, the components of the seed treatment agent include, by mass percentage: 5-20% albendazole, 1-5% sodium methylene bisnaphthalene sulfonate, 1-5% Nongru 700#, 0.1-1% xanthan gum, 0.1-1% white carbon black, 1-10% ethylene glycol, 0.1-0.5% sodium benzoate, and the balance is water.

[0018] More preferably, the components of the seed treatment agent include, by mass percentage, 10% albendazole, 2% sodium methylene bisnaphthalene sulfonate, 3% agricultural milk 700#, 0.3% xanthan gum, 0.5% white carbon black, 5% ethylene glycol, 0.2% sodium benzoate, and the balance is water.

[0019] The present invention also provides a seed treatment method for preventing and controlling bacterial fruit spot of melons, the seed treatment method comprising: diluting the seed treatment agent with water by 100-600 times to prepare a drug dilution solution, then soaking the seeds in the drug dilution solution for 2-24 hours, then taking them out, washing them with clean water and air-drying them.

[0020] Preferably, the seed treatment agent is diluted 350 times with water to obtain a diluent, and then the melon seeds are soaked in the diluent and stirred continuously for 12-24 hours before being fished out. In the above method, the seeds are soaked in the agent and stirred continuously to fully contact the agent.

[0021] Furthermore, the seeds are seeds of Cucurbitaceae crops, and Cucurbitaceae crops include but are not limited to: watermelon, melon, bitter melon, pumpkin, zucchini, cucumber, loofah, and bottle gourd.

[0022] The present invention has the following beneficial effects:

[0023] (1) The present invention discloses a new use of albendazole in preventing and controlling bacterial fruit spot disease of melons. The melon seeds are disinfected with albendazole to kill the fruit spot pathogens carried by the melon seeds, effectively blocking the occurrence and spread of the disease from the source, with a prevention and control effect of 100%. The invention can effectively avoid risks for seed production companies, seed and seedling management units and melon farmers, and reduce the economic losses caused by fruit spot disease.

[0024] (2) The seed disinfection technology provided by the present invention can significantly improve the seed germination rate and emergence rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a single colony morphology of the fruit spot pathogen A. citrulli QT0801 on LB medium.

[0026] Figure 2 This is a symptom diagram of the pathogenicity test of strain A. citrulli QT0801 on gourd leaves.

[0027] Figure 3 Are the PCR amplification results of strain-specific fragments, where (A) is the amplification result of primers SEQID4 / SEQID5, (B) is the amplification result of primers BX-L1 / BX-S-R2, lane M is Marker, lane 1 is the negative control, lanes 2-4 are the identified strains of cucurbit fruit blotch A.citrullixj112, A.citrullipslb116 and A.citrullitw21, and lanes 5-9 are QT0801, QT0806, QT0808, QT0812 and QT0815, respectively.

[0028] Figure 4 This is the MALDI-TOF mass spectrometry identification report.

[0029] Figure 5 This is the regression curve of the toxicity of albendazole against melon fruit spot pathogen A. citrulli QT0801.

[0030] Figure 6 This is the regression curve of the toxicity of zinc thiazole to melon fruit spot pathogen A. citrulli QT0801.

[0031] Figure 7 The control effect of seed treatment agents on watermelon fruit spot disease: A, positive control, B, negative control, C, fungicide No. 1, D, 5% albendazole, E, 10% albendazole, F, 20% albendazole.

[0032] Figure 8Effects of soaking seeds with seed treatment agents for 12 hours on seed germination rate: A, positive control, B, negative control, C, fungicide No. 1, D, 5% albendazole, E, 10% albendazole, F, 20% albendazole. DETAILED DESCRIPTION

[0033] The present invention will be further described below in conjunction with specific embodiments. The following embodiments are only used to illustrate the present invention and are not intended to limit the scope of application of the present invention. Without departing from the spirit and essence of the present invention, modifications or replacements made to the inventive method, steps or conditions all belong to the scope of the present invention.

[0034] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0035] Albendazole technical, CAS: 54965-21-8; sodium methylene bisnaphthalene sulfonate, CAS: 36290-04-7; agricultural milk 700#, Shandong Tiandao Bioengineering Co., Ltd.; xanthan gum, CAS: 11138-66-2; white carbon black, CAS: 10279-57-9; ethylene glycol, CAS: 2219-51-4; sodium benzoate, CAS: 532-32-1.

[0036] Test strain: Acidophilus citrulli QT0801, isolated and identified by Professor Lou Binggan of the Institute of Biotechnology, Zhejiang University, and stored at ultra-low temperature after pathogenicity test. The specific isolation, identification and pathogenicity test are as follows:

[0037] 1. Isolation, culture and identification

[0038] Samples with typical symptoms of melon fruit spot were collected and prepared into sample suspensions. The colonies were streaked and separated on LB medium plates. The colonies were white, raised, and had smooth and neat edges. The colonies were cultured at 28°C for 48 hours, and suspicious single colonies were picked for purification. After three transfers, five single colonies were picked and recorded as strains QT0801, QT0806, QT0808, QT0812, and QT0815. The single colony morphology of strain QT0801 on LB medium was as follows: Figure 1 As indicated, pathogenicity assays were then performed.

[0039] 2. Pathogenicity determination

[0040] The very early-growing gourd seeds were disinfected with 1% sodium hypochlorite and then sown to germinate until the 4-6 leaf stage. The isolated and preserved strains were prepared into 1×10 8CFU / mL bacterial suspension. The gourd leaves were inoculated by spraying, and then placed in a 25℃ plant growth room, covered with bags to keep moist for 48 hours. The disease was investigated after 72 hours. All five strains showed typical symptoms of melon fruit spot disease. The fruit spot symptoms of strain QT0801 after infection were as follows: Figure 2 shown.

[0041] 3. Specific primer PCR identification

[0042] 3.1 PCR primer sequences are shown in Table 1.

[0043] Table 1

[0044]

[0045] 3.2 PCR reaction system and amplification conditions

[0046] PCR reaction system (20 μL): Green Taq Mix 10 μL, dd H2O 8 μL, upstream and downstream primers 0.5 μL each (10 μmol / L), bacterial liquid template 1 μL.

[0047] The PCR reaction program of primers SEQID4 / SEQID5 was as follows: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 53°C for 30 s, extension at 72°C for 30 s, 35 cycles; extension at 72°C for 5 min.

[0048] The PCR reaction program of primers BX-L1 / BX-S-R2 was as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 68.1°C for 30 s, extension at 72°C for 30 s, 35 cycles; extension at 72°C for 8 min.

[0049] 3.3 Agarose gel electrophoresis results

[0050] The PCR product was electrophoresed with 1.5% agarose gel. After the electrophoresis, the gel imaging system was used to observe and take pictures. The size of the amplified product fragment: Primer SEQID4 / SEQID5 is 246 bp ( Figure 3 A) Primer BX-L1 / BX-S-R2 is 279 bp ( Figure 3 B); consistent with the identified strains of melon fruit blotch A. citrullixj112, A. citrullipslb116 and A. citrullitw21.

[0051] 4. MALDI-TOF mass spectrometry identification report such as Figure 4 As shown, the strain QT0801 was identified as A. citrulli.

[0052] Example 1: Preparation of 10% albendazole suspension as a seed disinfectant

[0053] 10.2 g of 98% albendazole technical, 2 g of sodium methylene bisnaphthalene sulfonate, 3 g of Nongru 700#, 0.3 g of xanthan gum, 0.5 g of white carbon black, 5 g of ethylene glycol, 0.2 g of sodium benzoate and 75.8 g of deionized water were fully mixed, put into a high-speed shear emulsifier for coarse grinding and homogenization, and then transferred to a sand mill for sand grinding until the particle size was less than 5 μm, and then filtered to obtain a 10% albendazole suspension.

[0054] Example 2: Preparation of 5% albendazole suspension as a seed disinfectant

[0055] Fully mix 5.1 g of 98% albendazole technical, 1 g of sodium methylene bisnaphthalene sulfonate, 5 g of Nongru 700#, 0.1 g of xanthan gum, 1 g of white carbon black, 1 g of ethylene glycol, 0.1 g of sodium benzoate and 91.6 g of deionized water, put them into a high-speed shear emulsifier for coarse grinding and homogenization, then transfer them to a sand mill for sand grinding until the particle size is less than 5 μm, and then filter to obtain a 5% albendazole suspension.

[0056] Example 3: Preparation of 20% albendazole suspension as a seed disinfectant

[0057] 20.4 g of 98% albendazole technical, 5 g of sodium methylene bisnaphthalene sulfonate, 1 g of Nongru 700#, 1 g of xanthan gum, 0.1 g of white carbon black, 10 g of ethylene glycol, 0.5 g of sodium benzoate and 62 g of deionized water were fully mixed, put into a high-speed shear emulsifier for coarse grinding and homogenization, and then transferred to a sand mill for sand grinding until the particle size was less than 5 μm, and then filtered to obtain a 20% albendazole suspension.

[0058] Test Example 1: Determination of the antibacterial effect of albendazole on melon fruit spot pathogen

[0059] 1. Test strain: A. citrulli QT0801.

[0060] 2. Test agent: 98% albendazole original drug.

[0061] 3. Comparative agent: 95% zinc thiazole technical drug.

[0062] 4. Test methods:

[0063] Dissolve 98% albendazole stock drug in glacial acetic acid and add it to sterile LB medium to dilute it to the following concentration gradient: 0.03, 0.04, 0.05, 0.07, 0.09 mg / L; dissolve 95% thiazole zinc stock drug in dimethyl sulfoxide and add it to sterile LB medium to dilute it to the following concentration gradient: 16.00, 22.40, 31.36, 43.90, 61.48 mg / L. Repeat 3 bottles for each concentration, 50 mL per bottle.

[0064] Add 100 μL of seed bacterial solution, use 50 mL of LB medium plus 100 μL of seed bacterial solution as the positive control, and use 50 mL of LB medium plus 100 μL of sterile water as the negative control. Cultivate at 28°C and 200 rpm until the positive control enters the logarithmic growth phase, and then measure the absorbance of the culture solution at 600 nm. Calculate the inhibition rate of each concentration of fungicide, and use the Probit regression analysis method of SPSS21.0 to calculate the inhibition concentration EC 50 .

[0065]

[0066] 5. Results

[0067] Test results Table 2 and Figure 5-6 The results show that the inhibitory concentration of 98% albendazole on melon fruit spot pathogen EC 50 It is 0.03mg / L, showing strong toxicity.

[0068] Table 2. Indoor toxicity test of 98% albendazole original drug against melon fruit spot pathogen

[0069]

[0070] Test Example 2: Effect of seed treatment agents on the prevention and treatment of melon fruit spot

[0071] 1. Preparation of seed treatment agent

[0072] Experimental group: The seed treatment agent of Examples 1 to 3 was diluted 350 times with clean water to prepare a seed treatment agent;

[0073] Drug control group: Fungicide No. 1 (Institute of Plant Protection, Chinese Academy of Agricultural Sciences) diluted 200 times as the control drug treatment;

[0074] Positive control (CK1): The infected seeds were treated with clean water instead of fungicide as positive control;

[0075] Negative control (CK2): Healthy seeds treated with clean water were used as negative control.

[0076] 2. Treatment method:

[0077] Immerse the infected seeds in the seed treatment agent prepared in step 1 and stir continuously to make the seeds fully contact the solution. After soaking for 2h, 12h, and 24h, remove the seeds, rinse them with clean water 3 times, and place them on absorbent paper to air dry. Place the air-dried seeds in a sterile culture dish covered with moist absorbent paper and place them in a constant temperature incubator at 28℃ in the dark for germination for 3 days. Each treatment was repeated 3 times, with a total of 100 watermelon seeds in each treatment.

[0078] The germinated seeds were sown into seedling pots. After 21 days, the number of diseased plants was investigated, and the disease index and control effect were calculated.

[0079] Disease grade classification standards: Grade 0, no lesions on leaves; Grade 1, fewer lesions, with the lesion area accounting for less than 5% of the total leaf area; Grade 3, more lesions, with the lesion area accounting for 5% to 30% of the total leaf area; Grade 5, lesions begin to fuse, with the lesion area accounting for 30% to 50% of the total leaf area; Grade 7, many lesions or fused into large spots, with the lesion area accounting for more than 50% of the total leaf area, and the leaves are not scorched or dead; Grade 9, lesions are fused over a large area, with the lesion area accounting for more than 50% of the total area, and the leaves are often scorched or dead.

[0080]

[0081]

[0082] 3. Results

[0083] Test results Table 3 and Figure 7 It shows that the control effect of using the 10% and 20% albendazole suspension provided by the present invention to treat the infected watermelon seeds for 12h and 24h on bacterial fruit spot disease reaches 100%, which is significantly higher than the control effect of other treatment groups.

[0084] Table 3. Effect of fungicide soaking on bacterial fruit spot

[0085]

[0086] Note: The disease index values ​​are the mean ± standard error of 3 replicates. Different lowercase letters indicate significant differences in the same column at the P < 0.05 level tested by Duncan's new multiple range method.

[0087] Test Example 3: Effects of seed treatment agents on seed germination rate and emergence rate

[0088] 1. The test method is the same as that of test example 2. The germination rate of seeds is investigated 3 days after germination. The germination rate is investigated 10 days after the germinated seeds are sown in seedling pots.

[0089]

[0090]

[0091] 2. Results

[0092] 2.1 Effect of seed treatment agents on seed germination rate

[0093] The results of the experiment on the effect of fungicide treatment on seed germination rate (Table 4 and Figure 8) showed that the germination rate of watermelon seeds treated with the seed treatment agent provided by the present invention was significantly higher than that of the positive control. After soaking the seeds for 12-24 hours, the germination rate of the seeds treated with 10% albendazole suspension was the highest among the four treatment groups, close to the negative control.

[0094] Table 4. Effects of fungicide treatment on seed germination rate

[0095]

[0096] Note: The germination rate values ​​are the mean ± standard error of 3 replicates. Different lowercase letters indicate significant differences in the same column at the P < 0.05 level by Duncan's new multiple range test.

[0097] 2.2 Effect of seed treatment agents on seed germination rate

[0098] The test results of the effect of fungicide treatment on seed germination rate (Table 5) show that the germination rate of seeds treated with the seed treatment agent provided by the present invention is significantly higher than that of the positive control, among which the germination rate of seeds soaked in 10% albendazole suspension for 12h-24h is higher or significantly higher than that of the negative control.

[0099] Table 5. Effect of fungicide treatment on germination rate

[0100]

[0101] Note: The values ​​of germination rate are the mean ± standard error of 3 replicates. Different lowercase letters indicate significant differences in the same column at the P < 0.05 level by Duncan's new multiple range test.

Claims

1. A seed treatment agent for preventing and treating bacterial fruit spot of melons, the disease being caused by acidophilic bacteria Acidovorax citrulli of watermelon, characterized in that: The components of the seed treatment agent include, by mass percentage, 5-20% albendazole, 1-5% sodium methylene bisnaphthalene sulfonate, 1-5% agricultural milk 700#, 0.1-1% xanthan gum, 0.1-1% white carbon black, 1-10% ethylene glycol, 0.1-0.5% sodium benzoate, and the balance is water.

2. The seed treatment agent for preventing and treating melon bacterial fruit spot according to claim 1, characterized in that: The components of the seed treatment agent include, by mass percentage, 10% albendazole, 2% sodium methylene bisnaphthalene sulfonate, 3% agricultural milk 700#, 0.3% xanthan gum, 0.5% white carbon black, 5% ethylene glycol, 0.2% sodium benzoate, and the balance is water.

3. A seed treatment method for preventing and controlling bacterial fruit spot of melons, the disease being caused by acidophilic bacteria Acidovorax citrulli of watermelon, characterized in that: include: The seed treatment agent according to claim 1 or 2 is diluted 100-600 times with water to prepare a diluent, and then the seeds are soaked in the diluent for 2-24 hours, taken out, washed with clean water and air-dried.

4. The processing method according to claim 3, characterized in that: The seed treatment agent is diluted 350 times with water to prepare a diluent, and then the melon seeds are soaked in the diluent and continuously stirred for 12-24 hours before being fished out.

5. The processing method according to claim 3, characterized in that: The seeds are Cucurbitaceae crop seeds.

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