Patent Publication Number: US-2023136991-A1

Title: Iturin family lipopeptides in pest control

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application claims priority to Chinese Patent Application No. 202110445504.4, filed on Apr. 25, 2021, the contents of which are hereby incorporated by reference as if fully rewritten herein. 
     TECHNICAL FIELD 
     The application relates generally to the technical field of pest control, and in particular to an application of iturin family lipopeptides in pest control. 
     BACKGROUND 
     Iturin family compounds were separated from  Bacillus subtilis  by Delcambe and Devignat in 1957. So far, many lipopeptides belonging to iturin family have been found, such as Iturin A, B, C, D, E, bacillomycin D, F, L and mycosporin. Recently, it has been found that  Paenibacillus elgii, Bacillus amyloliquefaciens  and  Bacillus pumilus  also produce iturin family lipopeptides. Iturin family compounds are linked by cyclo-heptapeptide and β-amino fatty acid chain with 14 to 17 carbons in length. Although the iturin family lipopeptides also have strong hemolysis, their biological activities are different from surfactants. Iturin family lipopeptides have strong in vitro resistance to a variety of yeasts and fungi, but have weak activity against bacteria and no antiviral activity. Comparing the different structures and functions of various lipopeptides of iturin family, it is found that the position and length of free carboxyl groups have an effect on their biological activity, and the antibacterial activity of the active substance becomes stronger with the increase of carbon atom number of the β-amino fatty acid chain. 
     Pest control is a very important part of agricultural production. With the change of agricultural production mode and the progress of science and technology, pest control strategies have successively gone through agricultural control, chemical control, comprehensive control and green control. With the continuous development of various pest control technologies, such as monitoring and early warning technology, precise pesticide application technology, biological control, physical control and transgenic technology, an integrated control technology system, which takes single crop production process and major disaster induced insects as the object, has been constructed successively, and played a key role in ensuring China&#39;s grain output and food safety and promoting ecological control of agricultural pests. In recent years, influenced by many factors, such as global climate change, change of farming mode, adjustment of industrial structure and increasing frequency of biological invasion brought by international trade exchange, the occurrence species and damage rules of agricultural pests in China have also changed obviously, and the agricultural pest control is facing new challenges. 
     Chemical control is widely adopted at present, but the problems of pesticide residues and biological resistance caused by long-term use of chemical pesticides have become increasingly prominent. In recent years, with people&#39;s growing concern for environmental ecology and food safety, it has been an inevitable trend to develop eco-friendly new pesticides. Iturin family lipopeptides are suitable for developing as new pest control drugs due to their broad antifungal spectrum, low toxicity, easy biodegradation, no allergic reaction and many other advantages. 
     SUMMARY 
     The objective of the present application is to provide an application of iturin family lipopeptides in pest control, so as to solve the problems existing in the prior art mentioned above. 
     To achieve the above objective, the present application provides theapplication of iturin family lipopeptides as active ingredients in pest control. 
     Optionally, the iturin family lipopeptides are one of or a mixture of two or more of iturin A, iturin B, iturin C, iturin D, iturin E,bacillomycin D, bacillomycin F, bacillomycin L and mycosubtilin. 
     Optionally, the pests are Homoptera insects or Coleoptera insects. 
     Optionally, the HOMOPTERA insects are one or more of  Aphis craccivora  Koch,  Aphis gossypii, Brevicoryne brassicae  and  Macrosiphum avenae.    
     Optionally, the Coleoptera insects are one or more of  Protaetia brevitarsis, Holotrichia parallela, Anomala corpulenta  and  Polyphylla gracilicornis.    
     Optionally, the application is to prepare the iturin family lipopeptides into liquid and then treat plants or soil to control pests. 
     The present application also provides a method for protecting seeds and later formed plant organs from pests, including treating seeds with the iturin family lipopeptides. 
     Optionally, the mass concentration of the iturin family lipopeptides is 0.01-100 μg/mL 
     Optionally, the seeds are coated with a coating agent containing the iturin family lipopeptides. 
     Optionally, the coating agent further includes a film forming agent or an adhesive. 
     The present application discloses a new application of iturin in pest control. Experiments have proven that iturin has obvious insecticidal activity against Homoptera insects and Coleoptera insects, and its insecticidal effect may reach more than 90% in 24 hours, so iturin may be applied to the control of agricultural pests. 
    
    
     DETAILED DESCRIPTION OF THE EMBODIMENTS 
     The technical solutions in the embodiments of the present application will be clearly and completely described below. Obviously, the described embodiments are only part of the embodiments of the present application, but not all of them. Based on the embodiments of the present application, all other embodiments obtained by ordinary technicians in the field without creative labor are within the scope of the present application. 
     In order to make the above objects, features and advantages of the present application more obvious and understandable, the present application will be further explained in detail with reference to the specific embodiments. 
     Unless otherwise specified, materials used in the embodiments may be obtained from commercial sources; The experimental methods used in the embodiments are conventional experimental methods in the field unless otherwise specified. 
     Embodiment 1 Preparation of Iturins 
     Preparation of iturin family popeptides from  Bacillus amyloliquefaciens    
     S1, carrying out amplification culture on the  Bacillus amyloliquefaciens  WS-8 (preservation number: CGMCC No.11787) to obtain fermentation liquor; 
     S2, centrifuging the fermentation liquor to remove thallus and then obtain sterile supernatant; 
     S3, adding amberlity XAD-7HP macroporous resin to the sterile supernatant for adsorption, and shaking at 4° C. for 12 hours; 
     S4, filtrating and collecting the resin, washing the resin with distilled water for three times, then with 60% ethanol (volume/volume), and concentrating the eluent to obtain a crude extract; 
     S5, using high performance liquid chromatography for further purification, including, loading mobile phase A, which is acetonitrile containing 0.1% (volume ratio) trifluoroacetic acid, and mobile phase B, which is ultrapure water containing 0.1% (volume ratio) trifluoroacetic acid, into WondaSil C18(4.6 mm×150 mm) chromatographic column with a particle size of 5 μm, using 10%-90% acetonitrile for linear gradient elution with the flow rate 1 mL/min, detecting the wavelength as 214 nm and the column temperature as 30° C., collecting the components corresponding to 30-50 min, and using the mass spectrometry (Table 1) to confirm that iturins are obtained. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Mass spectrometry detection results of purified products 
               
            
           
           
               
               
               
               
               
            
               
                   
                 Compound 
                   
                   
                   
               
               
                   
                 serial 
                 m/z 
                 m/z 
                 Determination  
               
               
                   
                 number 
                 [M + H] +   
                 [M + Na] +   
                 result 
               
               
                   
                   
               
               
                   
                 1 
                 1043.5474 
                 1065.5284 
                 C 14 Iturin A 
               
               
                   
                 2 
                 1044.5328 
                 1066.5145 
                 C 14 Iturin B 
               
               
                   
                 3 
                 1044.5334 
                 1066.5149 
                 C 14 Iturin B 
               
               
                   
                 4 
                 1057.5643 
                 1079.5454 
                 C 15 Iturin A 
               
               
                   
                 5 
                 1057.5636 
                 1079.5449 
                 C 15 Iturin A 
               
               
                   
                   
               
            
           
         
       
     
     Embodiment 2 
     Insecticidal Effect of the Iturins on HOMOPTERA Insects 
     Dissolving the iturins prepared in embodiment 1 in methanol to prepare 200 μg/mL mother liquor, and then diluting the mother liquor with water to 5 μg/mL, 10 μg/mL and 20 μg/mL liquid medicine, and using clean water treatment as a control; soaking young broad bean stems in insecticidal experiment of  Aphis craccivora  Koch, young cotton stems in insecticidal experiment of  Aphis gossypii , young cabbage leaves in insecticidal experiment of  Brevicoryne brassicae,  young stems of wheat in insecticidal experiment of  Macrosiphum avenae,  soaking the young broad bean stems, the young cotton stems, the young cabbage leaves and the young stems of wheat in the above-mentioned liquid medicine for 10 minutes, then absorbing water with filter paper, taking them out, putting them in a plate with filter paper, adding 2 mL of deionized water to keep moisture, then covering the plate with fresh-keeping film, puncturing holes, and adding 1 mL of water every 12 hours; testing 50 aphids in each concentration respectively, repeating for 3 times, using a brush head to pick 50 aphids of each species with similar age and size, putting them on the plate to starve for 4 hours, then adding the young broad bean stems, the young cotton stems, the young cabbage leaves and the young wheat stems treated with the above-mentioned liquid medicine onto the plate respectively, changing every 12 hours, culturing overnight in an incubator at 25° C., recording the number of aphids killed, and calculating the mortality rates of aphids at different dilution ratios. 
     
       
         
           
             
               The 
               ⁢ 
                   
               mortality 
               ⁢ 
                   
               rate 
             
             = 
             
               
                 
                   the 
                   ⁢ 
                       
                   number 
                   ⁢ 
                       
                   of 
                   ⁢ 
                       
                   aphids 
                   ⁢ 
                       
                   killed 
                 
                 
                   the 
                   ⁢ 
                       
                   number 
                   ⁢ 
                       
                   of 
                   ⁢ 
                       
                   aphids 
                   ⁢ 
                       
                   for 
                   ⁢ 
                       
                   testing 
                 
               
               × 
               100 
               ⁢ 
               % 
             
           
         
       
     
     Measuring the mortality rates of the aphids: After the aphids are treated overnight, touching the aphids with a brush head, and if the aphid does not move, it is regarded as dead. Count and record the number of dead aphids. It can be seen from the implementation data results in Table 2 that the iturins have a good control effect on Homoptera insects. 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Control effect of the iturins on Homoptera insects 
               
            
           
           
               
               
            
               
                   
                 Mortality rate (%) 
               
            
           
           
               
               
               
               
            
               
                   
                 Aphis  
                   
                   
               
            
           
           
               
               
               
               
               
            
               
                 Processing 
                 craccivora 
                 Aphis  
                 Brevicoryne 
                 Macrosiphum 
               
               
                 group 
                 Koch 
                 gossypii 
                 brassicae 
                 avenae 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 CK 
                 2.67 
                 2.00 
                 1.33 
                 1.33 
               
               
                  5 μg/mL 
                 76.00 
                 72.00 
                 69.33 
                 69.33 
               
               
                 10 μg/mL 
                 85.33 
                 88.00 
                 81.33 
                 84.00 
               
               
                 20 μg/mL 
                 93.33 
                 93.33 
                 90.00 
                 90.67 
               
               
                   
               
            
           
         
       
     
     Embodiment 3 
     Insecticidal Effects of the Iturins on Coleoptera Insects 
     Dissolving the iturins in methanol to prepare 200 μg/mL mother liquor, and then diluting the mother liquor with water to prepare 5 μg/mL, 10 μg/mL and 20 μg/mL liquid medicine, using the liquid medicine to prepare medicine soil, with the mass ratio of the liquid medicine to soil 1:5, and using medicine soil prepared with clean water treatment as a control; using chopped peanuts as food,using plastic buckets with a capacity of 4 L as containers, and placing 2 L of evenly mixed medicinal soil in each bucket, and adding a proper amount of equal food to mix well; adopting a medicinal soil method, selecting the grubs of the third instar larvae of  Protaetia brevitarsis, Holotrichia parallela, Anomala corpulenta  and  Polyphylla gracilicornis  with the same size, and keeping them in the environment with relative humidity of 50% - 70% and temperature of 25° C.-28° C. for 24 hours; treating 15 third instar larvaes with the different medicine soil prepared above respectively, repeating for 3 times; after inoculating the larvaes, culturing them indoors at normal atmospheric temperature, and counting the mortality rate after 56 hours, as shown in Table 3. 
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Killing effects of the iturins on different Coleoptera larvaes 
               
            
           
           
               
               
            
               
                   
                 Mortality rate (%) 
               
            
           
           
               
               
               
               
               
            
               
                 Processing  
                 Protaetia 
                 Holotrichia 
                 Anomala 
                 Polyphylla 
               
               
                 group 
                 brevitarsis 
                 parallela 
                 corpulenta 
                 gracilicornis 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 CK 
                 0.00 
                 0.00 
                 0.00 
                 0.00 
               
               
                  5 μg/mL 
                 62.22 
                 55.56 
                 60.00 
                 60.00 
               
               
                 10 μg/mL 
                 82.22 
                 80.00 
                 82.22 
                 80.00 
               
               
                 20 μg/mL 
                 88.89 
                 91.11 
                 88.89 
                 86.67 
               
               
                   
               
            
           
         
       
     
     Embodiment 4 
     Dissolving the iturins in methanol to prepare 200 μg/mL mother liquor, and then diluting the mother liquor with water into 0.1 μg/mL, 0.5 μg/mL, 1 μg/mL, 5 μg/mL, 10 μg/mL, 20 μg/mL, 50 μg/mL and 100 μg/mL liquid medicine to soak peanuts, and using peanuts that soaking in clean water as a control; using plastic buckets with a capacity of 4 L as containers, and placing 2 L of evenly mixed soil in each bucket, and adding 50 treated peanuts to mix well. Selecting the grubs of the third instar larvae of  Holotrichia parallela  with the same size, and keeping them in the environment with relative humidity of 50%-70% and temperature of 25° C.-28° C. for 24 hours, repeating the treatment for 3 times. The number of each test insect in each treatment is 15, repeating the treatment for 3 times. After inoculating the larvaes, culturing them indoors at normal atmospheric temperature, and counting the mortality rate of the larvaes and intactness rate of the peanuts after 56 hours, as shown in Table 4. 
                     TABLE 4                  Insecticidal and protective effects of iturins on seeds                         Processing   Mortality rate   Peanut intactness       group   (%)   rate (%)                                 CK   0.00   8        0.1 μg/mL   44.44   46.00        0.5 μg/mL   55.56   50.67        1 μg/mL   57.78   54.67        5 μg/mL   62.22   60.00        10 μg/mL   82.22   71.33        20 μg/mL   91.11   74.00        50 μg/mL   97.78   76.67       100 μg/mL   100.00   78.67                    
The above are only preferred embodiments of the present application, and are not intended to limit the present application. On the premise of not departing from the design spirit of the present application, all kinds of modifications and improvements made by ordinary technicians in the field to the technical schemes of the present application should fall in the scope of protection defined by the claims of the present application.