A kind of black Aspergillus and its fermentation method and application

Abstract

The invention belongs to the field of microbial technology, relates to the screening, identification and application of functional bacteria, and discloses a black Aspergillus and its fermentation method and application. The black Aspergillus provided by the invention is Aspergillus niger YXlfs22.3, and its deposit number is CGMCC No.40261. The black Aspergillus Aspergillus niger YXlfs22.3 bacterial body and / or fermentation liquid provided by the invention can effectively prevent and control aphids: after the fermentation liquid stock solution is applied to the leaves of melon plants for 5 days, the insect population reduction rate is 96.19%, and the corrected control effect is 95.88%; after the fermentation liquid diluted 10 times is applied to the leaves of watermelon plants for 48 hours, the insect population reduction rate is 90.01%, and the corrected control effect is 86.89%. The invention can replace traditional pesticides and be used for preventing and controlling plant aphid pests, and has the advantages of low preparation cost, no pollution to the environment, high cost performance, etc.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, relates to the separation, identification and application of functional bacteria, and specifically relates to black Aspergillus and a fermentation method and application thereof. Background Art

[0002] Aphids are the main pests in plant cultivation and crops. A total of 10 families and about 4,400 species have been found. Due to the characteristics of aphids being small, damaging many crops, wide range, fast reproduction, and difficult to eradicate, pesticides have become the top priority in solving the problem. At present, chemical pesticides play a leading role in the market. With the increase of aphid resistance, the cost of prevention and control continues to rise, the agricultural ecological environment deteriorates, and the environmental pollution is serious, forming a vicious cycle of more pesticides, more rampant, and more rampant, more pesticides. Therefore, the development of biological pesticides is particularly important in the sustainable development of agriculture.

[0003] Aspergillus niger can secrete a variety of enzymes with strong activity. The enzymes produced by Aspergillus niger are the most among the enzyme production strains for food industry permitted by the U.S. FDA. In food processing, the enzymes produced by Aspergillus niger can be used to produce excellent functional food additives and health food raw materials. Aspergillus niger can also produce a large amount of organic acids, so it is often used in organic acid fermentation in industry. In addition, Aspergillus niger is also a cheap raw material for extracting chitin and chitosan, and the use of its secondary metabolites can reduce the root knot index and root knot nematode number of plants and improve disease resistance. The Chinese patent with publication number CN104365679A discloses that Aspergillus niger is used for soybean aphid control, which can significantly enhance the anti-aphid effect. Summary of the invention

[0004] In summary, developing more safe and efficient microbial agents as pesticide substitutes is an urgent task and a research hotspot in the field of pesticide and formulation development technology. Based on the good application prospects of Aspergillus niger and its metabolites in plant disease and insect pest control, the inventors isolated an endophytic Aspergillus niger from Ginkgo biloba leaves. The deposit information of the endophytic Aspergillus niger is as follows:

[0005] Species name: Aspergillus niger

[0006] Latin name: Aspergillus niger

[0007] Strain ID: YXlfs22.3

[0008] Depository: China National Microbiological Culture Collection Administration General Microbiology Center

[0009] Abbreviation of depository institution: CGMCC

[0010] Address of the depository institution: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing

[0011] Deposit date: August 1, 2022

[0012] Deposit number: CGMCC No.40261

[0013] Furthermore, the ITS rDNA sequence of Aspergillus niger YXlfs22.3 is shown in SEQ ID NO.1.

[0014] Furthermore, the fungus body and / or fermentation liquid of Aspergillus niger YXlfs22.3 has the function of preventing and controlling plant aphids.

[0015] In addition, the present invention also provides a fermentation method of Aspergillus niger YXlfs22.3, comprising:

[0016] Activating the Aspergillus niger YXlfs22.3, wherein the culture medium selected for activation is PDA culture medium, and the activation conditions are: 28° C. to 33° C., and culturing for 3 to 4 days;

[0017] Prepare seed solution, fermentation conditions: 28℃~33℃, 150r / min shaking culture for 2~4d;

[0018] The seed liquid is inoculated and fermented, and the seed liquid is inoculated into the fermentation medium at an inoculation rate of 5% by volume, and the fermentation conditions are: 28°C to 33°C, 150r / min shaking culture for 2 to 4 days;

[0019] Furthermore, in the above fermentation method, the components of the seed solution are: 35 g / L soluble starch, 15 g / L sucrose, 12.5 g / L yeast extract powder, 7.5 g / L soybean cake powder, 1.0 g / L KH2PO4, 1.1 g / L anhydrous MgSO4, 1.0 g / L NaCl, and 1 L distilled water;

[0020] Further, in the above fermentation method, the components of the fermentation medium are: soluble starch 70g / L, sucrose 30g / L, yeast extract powder 25g / L, soybean cake powder 15g / L, KH2PO4 2.0g / L, anhydrous MgSO4 2.2g / L, NaCl 2.0g / L, distilled water 1L;

[0021] The Aspergillus niger YXlfs22.3 of the present invention can utilize a variety of carbon sources and form bacterial pellets in a sucrose-containing medium within 48 hours. 2+The number of mycelial balls was small and the size was extremely uneven, while Mg 2+ 、Na + It can not only promote the increase of the number of mycelial balls, but also generate mycelial balls of uniform size and smooth surface, have adsorption capacity for melanin, and can promote the spore polymerization process. Therefore, the culture medium provided by the present invention adopts a more efficient, time-saving, economical and environmentally friendly formula.

[0022] The invention also provides application of the Aspergillus niger YXlfs22.3 in controlling plant aphids.

[0023] Furthermore, in the application provided by the present invention, the fungus body of Aspergillus niger YXlfs22.3 and / or its fermentation liquid has a control effect on plant aphids.

[0024] The present invention also provides a bacterial agent for controlling aphids, wherein the active ingredient of the bacterial agent comprises the bacterial body and / or fermentation liquid of the above-mentioned Aspergillus niger YXlfs22.3. In order to expand the application of Aspergillus niger YXlfs22.3, the present invention also requests protection for the application of the above-mentioned Aspergillus niger YXlfs22.3 in the preparation of a bacterial agent for controlling plant aphids.

[0025] Compared with the prior art, the present invention "A black Aspergillus and its fermentation method and application" has the following beneficial effects:

[0026] Based on the good application prospect of Aspergillus niger, the present invention isolates an endophytic Aspergillus niger from ginkgo leaves, which is Aspergillus niger YXlfs22.3. The Aspergillus niger YXlfs22.3 bacterial body and / or fermentation liquid provided by the present invention can effectively prevent and control the growth of aphids: the insect population reduction rate of the fermentation liquid stock solution on the leaves of melon plants after 5 days of application is 96.19%, and the corrected control effect is 95.88%; the insect population reduction rate of the fermentation liquid diluted 10 times on the leaves of watermelon plants after 48 hours of application is 90.01%, and the corrected control effect is 86.89%, and the effect is remarkable. The Aspergillus niger provided by the present invention can be used as a raw material for developing a new type of biological microbial agent, has the advantages of safety, high efficiency, low cost, no pollution to the environment, etc., and will play an important role in the prevention and control of plant diseases and insect pests. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the phylogenetic tree of Aspergillus niger YXlfs22.3.

[0028] Figure 2 This is the colony growth morphology of Aspergillus niger YXlfs22.3 after being cultured on PDA medium for 4 days.

[0029] Figure 3 This is the morphology of the fungus ball of Aspergillus niger YXlfs22.3 after 3 days of fermentation.

[0030] Figure 4 This is a picture of the spore morphology of Aspergillus niger YXlfs22.3 under an optical microscope magnified 1000 times.

[0031] Figure 5 This is a morphological picture of the sporangium of Aspergillus niger YXlfs22.3 under an optical microscope at a magnification of 1000 times.

[0032] Figure 6 This is a morphological image of the sporangium of Aspergillus niger YXlfs22.3 under a scanning electron microscope at a magnification of 550 times.

[0033] Figure 7 This is a 1000-fold magnification of the mycelium of Aspergillus niger YXlfs22.3 attached to the epidermis of aphids under a scanning electron microscope.

[0034] Figure 8 This is a morphological picture of aphids infected by mycelium of Aspergillus niger YXlfs22.3 at a magnification of 1400 times under a scanning electron microscope. DETAILED DESCRIPTION

[0035] The present invention is explained below in conjunction with the embodiments, and the technical solutions in the embodiments of the present invention are clearly and completely described. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0036] Example 1

[0037] This example provides the separation, purification process and identification results of Aspergillus niger YXlfs22.3.

[0038] The Aspergillus niger YXlfs22.3 provided by the present invention is isolated from ginkgo leaves, which are collected from ginkgo trees in the north campus of Northwest Agriculture and Forestry University and are endophytic fungi. The specific separation and purification process is as follows:

[0039] Clean the ginkgo leaves, cut them into small pieces of 2cm×2cm, rinse them with sterile water and place them on a clean bench; soak them in 75% alcohol for 30s and then rinse them with sterile water for 4 times; soak them in 1% sodium hypochlorite solution for 10min after rinsing, and then rinse them with sterile water for 4 times to obtain surface-sterilized ginkgo leaves; put them into a sterilized mortar and crush them to extract the juice; dilute them with sterile water for 10 minutes respectively. 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 times; 100 μL of each gradient was inoculated on PDA medium by coating method, and three culture dishes were inoculated for each gradient, and cultured in a constant temperature incubator at 28°C for 3 to 5 days until colonies grew; repeated streaking purification was performed until a single colony strain was obtained, and the strain was numbered YXlfs22.3.

[0040] Morphological characteristics of colonies on PDA medium: The strain grows rapidly, has a dense texture, and the colonies are flat. In the early stage, they are white short velvety hyphae that grows divergently in all directions. The edges of the colonies are regular. In the later stage, the center of the colonies turns black and grows a large number of black spores. Figure 2 .

[0041] Morphological characteristics of the strain in the fermentation medium: The strain grows rapidly. In the early stage, the spores aggregate and swell to grow hyphae, which aggregate and entangle to form a bacterial ball. In the later stage, the culture medium gradually becomes clear, the bacterial ball is clearly visible, and the color of the center of the bacterial ball gradually turns black. Figure 3 .

[0042] Microscopic morphology of the bacteria: A spherical capsule is formed on the top of the bacteria, which is fully covered with a layer of peduncles and a layer of peduncles, and the peduncles have strings of brown-black spheres; the conidia are brown-black radial, of varying lengths, with a spherical capsule and double-layered peduncles. The microscopic morphology of the bacteria and spores can be seen in Figures 4 to 6 . Figure 4 This is a picture of the spore morphology of Aspergillus niger YXlfs22.3 under an optical microscope magnified 1000 times. Figure 5 This is a morphological picture of the sporangium of Aspergillus niger YXlfs22.3 under an optical microscope at a magnification of 1000 times. Figure 6This is a morphological image of the sporangium of Aspergillus niger YXlfs22.3 under a scanning electron microscope at a magnification of 550 times.

[0043] The single colony obtained above was subjected to bacterial species identification: DNA of the single colony was extracted and the ITS rDNA sequence was amplified. The primers used in the PCR amplification of the ITS rDNA gene were universal primers, which were commissioned to be synthesized by the Xi'an Branch of Beijing Qingke Biotechnology Co., Ltd. The primer sequences were:

[0044] ITSI: 5'-TCCGTAGGTGAACCTGCGG-3'

[0045] ITS4: 5'-TCCTCCGCTTATTGATATGC-3'

[0046] The amplified product was subjected to Sanger bidirectional sequencing, and the ITS rDNA sequence of the strain was obtained by sequencing as shown in SEQ ID NO.1. The sequencing results were compared and analyzed on the NCBI website, and the classification status of the strain was determined by the bacterial species with homology greater than 97% and the highest homology. The similarity between the gene sequence of the Aspergillus niger strain and the closest strain Aspergillus niger (whose GenBank accession number is MK886749) is 99.83%, and the accession number of the Aspergillus niger strain on GenBank is: OP080740.

[0047] Combined with the strain morphology observation and ITS rDNA sequence comparison results, the above colony was finally identified as Aspergillus niger YXlfs22.3, and its phylogenetic tree is shown in Figure 1 .

[0048] Example 2

[0049] This example provides an activation and fermentation method for Aspergillus niger YXlfs22.3.

[0050] The medium used for activation is PDA medium, and the activation conditions are: 33°C, 3 to 4 days;

[0051] The black Aspergillus seed liquid is prepared, and the components of the seed liquid are: 35g / L soluble starch, 15g / L sucrose, 12.5g / L yeast extract powder, 7.5g / L soybean cake powder, 1.0g / L KH2PO4, 1.1g / L anhydrous MgSO4, 1.0g / L NaCl, and 1L distilled water. The fermentation conditions are: 28°C to 33°C, 150r / min shaking culture for 2 to 4d, and the seed liquid is inoculated into the fermentation medium with an inoculation amount of 5% volume ratio. The composition of the fermentation medium is: 70g / L soluble starch, 30g / L sucrose, 25g / L yeast extract powder, 15g / L soybean cake powder, 2.0g / L KH2PO4, 2.2g / L anhydrous MgSO4, 2.0g / L NaCL, and 1L distilled water. The fermentation conditions are: 28°C-33°C, 150r / min shaking culture for 2-4 days, and Aspergillus niger YXlfs22.3 bacteria and fermentation liquid are obtained after the fermentation.

[0052] Example 3

[0053] This example provides a field test on the efficacy of Aspergillus niger YXlfs22.3 against aphids on leaves of melon plants.

[0054] The field test was applied on June 18, 2023. The test site was located in a melon greenhouse in a farmer's house in Yangling, Shaanxi. The test plant was melon. Aphids were seriously infested, and the effect of chemical pesticide spraying was not obvious. The test set 0.1% Tween 20 as the negative control, and the fermentation liquid of Aspergillus nigerYXlfs22.3, the fermentation liquid diluted 10 times, and the fermentation liquid diluted 50 times as the test group. Three batches were sprayed, with three treatments in each batch, and three parallel tests were performed for each treatment. One leaf was selected for each parallel test. Use a manual sprayer to spray the medicine once, so that the front and back of the leaves are evenly covered with the liquid, and at the same time, the leaves near the leaf are sprayed with the liquid. Before spraying, the number of live insects before the medicine was investigated, and the number of live aphids on each leaf was investigated 1d, 3d, and 5d after spraying. The insect population reduction rate and the corrected control effect were calculated. The calculation formulas for the insect population reduction rate and the corrected control effect are as follows:

[0055]

[0056]

[0057] SPSS26.0 was used for data analysis. The field control test results of each group on aphids on melon plant leaves are shown in Table 1.

[0058] Table 1. Field test results of aphid control on melon plant leaves

[0059]

[0060]

[0061] Note: No. 1: original fermentation solution; No. 2: 10-fold dilution of fermentation solution; No. 3: 50-fold dilution of fermentation solution; No. 4: negative control. The data in the table are mean ± standard deviation. Different lowercase letters after the data in the same column indicate significant differences between the reagent treatments (P<0.05).

[0062] As shown in Table 1, the corrected control effect of the fermentation liquid was 91.91% after 3 days of application, and 95.88% after 5 days of application. The corrected control effect of the fermentation liquid diluted 10 times after 3 days of application was 89.15%, and the corrected control effect after 5 days of application was 92.82%. The corrected control effect of the YXlfs22.3 fermentation liquid diluted 50 times after 3 days of application was 86.23%, and the corrected control effect after 5 days of application was 89.76%. It can be seen that the fermentation liquid of Aspergillus nigerYXlfs22.3 has the effect of killing aphids, and the corrected control effect of the fermentation liquid original liquid test group is gradually improved, the efficacy lasts for more than 5 days, and the corrected control effect after 5 days of application reaches 95.88%.

[0063] In order to further verify that the fermentation liquid of Aspergillus nigerYXlfs22.3 has killing activity against aphids, scanning electron microscopy was used for verification. Figure 7 This is a 1000-fold magnification of the mycelium of Aspergillus niger YXlfs22.3 attached to the epidermis of aphids under a scanning electron microscope. Figure 7 In the experiment, a few conidia of Aspergillus niger YXlfs22.3 distributed on the abdomen of aphids have begun to germinate hyphae and attach to the epidermis of aphids in search of invasion points. Figure 8 This is a morphological picture of aphids infected by mycelium of Aspergillus niger YXlfs22.3 at a magnification of 1400 times under a scanning electron microscope. Figure 8 In the process, the strain and its fermentation products secrete extracellular enzymes and other active substances to decompose and rupture the aphid's body wall, while the mycelium invades the aphid's body.

[0064] Example 4

[0065] This example provides a field test on the efficacy of fermentation liquid of Aspergillus niger YXlfs22.3 against aphids on leaves of watermelon plants.

[0066] The field test was applied on July 4, 2022. The test materials were collected from the watermelon greenhouse of a farmer in Yangling, Shaanxi. The test plant was watermelon. Aphids were seriously infested, which seriously affected the growth of watermelon. The test set 0.1% Tween 20 as the negative control, and the fermentation liquid of Aspergillus nigerYXlfs22.3 was diluted 10 times, the fermentation liquid was diluted 50 times, and the fermentation filtrate was used as the test group. Three batches were sprayed, with three treatments in each batch, and three parallel tests were performed for each treatment. One leaf was selected for each parallel test. A manual sprayer was used to spray the medicine once, so that the front and back of the leaves were evenly covered with the liquid. At the same time, a moistened filter paper was placed in the culture dish, and the leaves were placed on the filter paper. The rhizomes of the leaves were tied with cotton and dripped with clean water. A layer of plastic wrap was covered on the culture dish, and a certain number of small holes were pierced to ensure oxygen flow. Before spraying, the insect population was investigated before application, and the insect population on each leaf was investigated 16h, 24h, and 48h after spraying, and the insect population reduction rate and the corrected control effect were calculated. The calculation method of the insect population reduction rate and the corrected control effect was the same as that in Example 3.

[0067] SPSS26.0 was used for data analysis. The results of the field control efficacy test of each group on aphids on the leaves of watermelon plants are shown in Table 2.

[0068] Table 2. Field test results of aphid control on watermelon plant leaves

[0069]

[0070] Note: No. 1: Aspergillus niger YXlfs22.3 fermentation broth diluted 10 times; No. 2: Fermentation broth diluted 50 times; No. 3: Fermentation filtrate; No. 4: Negative control. The data in the table are mean ± standard deviation. Different lowercase letters after the data in the same column indicate significant differences between the reagent treatments (P < 0.05).

[0071] As shown in Table 2, the corrected control effect of Aspergillus nigerYXlfs22.3 fermentation liquid diluted 10 times was 86.89% 48 hours after application; the corrected control effect of the fermentation liquid diluted 50 times was 84.50% 48 hours after application. The corrected control effect of the fermentation filtrate was 83.74% 48 hours after application. It can be seen that the fermentation liquid of Aspergillus nigerYXlfs22.3 has the effect of killing aphids, while the control effect of the fermentation filtrate is slightly weaker. This result can provide a basis for further research.

[0072] The embodiments described above are only some embodiments of the present invention, not all embodiments. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention claimed for protection, but only represents selected embodiments of the present invention. All other embodiments obtained without creative work and related deductions and substitutions made by ordinary technicians in the field under the conditions of the concept of the present invention belong to the scope of protection of the present invention.

Claims

1. A strain of Aspergillus niger ( Aspergillus niger )YXlfs22.3, deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number CGMCC No.40261.

2. The black Aspergillus niger of claim 1 ( Aspergillus niger )YXlfs22.3 fermentation method, characterized in that, In the fermentation method, the components of the seed liquid are: 35g / L soluble starch, 15g / L sucrose, 12.5g / L yeast extract powder, 7.5g / L soybean cake powder, 1.0g / L KH2PO4, 1.1g / L anhydrous MgSO4, and 1.0g / L NaCl; the components of the fermentation medium are: 70g / L soluble starch, 30g / L sucrose, 25g / L yeast extract powder, 15g / L soybean cake powder, 2.0g / L KH2PO4, 2.2g / L anhydrous MgSO4, 2.0g / L NaCl, and 1L distilled water.

3. The fermentation method according to claim 2, characterized in that Activate the Aspergillus niger ( Aspergillus niger ) The culture medium used for YXlfs22.3 is PDA culture medium, and the activation conditions are: 28℃~33℃, culture for 3~4 days; Prepare seed solution, fermentation conditions: 28℃~33℃, 150r / min shaking culture for 2~4d; The seed liquid is inoculated and fermented. The seed liquid is inoculated into the fermentation medium at an inoculation rate of 5% by volume. The fermentation conditions are: 28° C. to 33° C., 150 r / min shaking culture for 2 to 4 days.

4. Aspergillus niger described in claim 1 ( Aspergillus niger )Application of YXlfs22.3 in plant aphid control.

5. The use according to claim 4, characterized in that: The Aspergillus niger ( Aspergillus niger ) The bacteria of YXlfs22.3 and / or its fermented liquid have a control effect on plant aphids.

6. A bacterial agent for controlling aphids, characterized in that: The active ingredient of the bacterial agent comprises the Aspergillus niger according to claim 1 ( Aspergillus niger ) YXlfs22.3 bacteria and / or its fermentation broth.

7. The black Aspergillus niger of claim 1 ( Aspergillus niger ) Application of YXlfs22.3 in the preparation of bacterial agents for controlling plant aphids.

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