Application of Streptomyces sigangensis strain SGX33 in microbial fertilizer

Abstract

The present invention belongs to the technical field of the application of soil microorganisms and the prevention and control of crop diseases. The present invention provides the application of Streptomyces morookaense strain SGX33 in the preparation of a microbial fertilizer for preventing and controlling plant diseases, wherein the plant diseases are wheat root rot or banana wilt. The fertilizer containing Streptomyces morookaense strain SGX33 of the present invention increases the plant height of wheat seeds in soil containing wheat root rot pathogens, and can basically reach the growth level of wheat seeds in soil without pathogens. At the same time, for fields with a banana wilt incidence rate of more than 50% in the previous year, when bananas are replanted, the prevention and control of banana wilt reaches more than 75%, and the yield increases by more than 80%.

Description

Technical Field

[0001] The present invention belongs to the technical fields of the application of soil microorganisms and the prevention and control of crop diseases. Background Art

[0002] Fungal diseases are the main reasons for reducing the yield and quality of crops. The frequent use of chemical pesticides has led to a sharp increase in the drug resistance of some pathogenic fungi, causing environmental and agricultural product pollution, and disrupting the microbial balance inside and outside plants, thus exacerbating the outbreak and prevalence of diseases. Biological control is an important way to achieve the green control of fungal diseases, and related research has always been a hot spot and a difficult point. Over the years, many biocontrol bacteria antagonistic to fungi have been screened, such as Bacillus spp. and Pseudomonas spp. for the biological control of various fungal diseases, Trichoderma for the biological control of various diseases including sheath blight and root rot, and many Streptomyces spp. for the control of fungal diseases, such as Streptomyces griseofuscus for the control of rice blast, Streptomyces griseoviridis for the control of cotton wilt, Streptomyces hydrogenans and Streptomyces aureus verticalis for the control of various soil-borne fungal diseases, etc. Although these biocontrol strains all have strong antifungal activities, many strains have poor acid and salt tolerance and are not suitable for colonization in acidic soils, saline-alkali and salt-sodic soils, which limits their application scope and the exertion of biocontrol efficacy. There are great limitations in the actual application of agricultural production.

[0003] Soil acidification and salinization can lead to a decrease in soil biological properties and an imbalance in the soil microbial ecosystem. Especially, the occurrence of fungal diseases in acidic soils is aggravated, seriously reducing the production performance of the soil. There is an urgent need in production for beneficial microorganisms that can colonize in acidic soils, saline-alkali soils and salt-sodic soils and can exert biocontrol functions for the biological control of fungal diseases. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide the application of Streptomyces morookaense strain SGX33 in the preparation of fertilizers for preventing and controlling plant diseases, and the plant diseases are wheat root rot or banana wilt.

[0005] The fertilizer containing Streptomyces morookaense strain SGX33 of the present invention increases the plant height of wheat seeds in the soil containing wheat root rot pathogens, and can basically reach the growth level of wheat seeds in the soil without pathogenic bacteria. At the same time, for the fields where the incidence of banana wilt was more than 50% in the previous year, bananas were replanted, and the prevention and control of banana wilt reached more than 75%, with an increase in production of more than 80%. Description of the Drawings

[0006] Figure 1 It is the growth morphology of Streptomyces morookaense SGX33 on MS medium and the observation diagram by scanning electron microscope.

[0007] Figure 2 It is a diagram showing the antagonistic effect of Streptomyces shigangensis SGX33 against various plant pathogens. Detailed implementation methods

[0008] The following examples facilitate a better understanding of the present invention, but do not limit the present invention. The experimental methods in the following examples are all conventional methods unless otherwise specified. The test materials used in the following examples are all obtained from regular biochemical reagent stores unless otherwise specified.

[0009] In the following implementation regulations, the following media are used:

[0010] Gause's No. 1 medium: Soluble starch 20 g, KNO3 1 g, K2HPO4 0.5 g, MgSO4·7H2O 0.5 g, NaCl 0.5 g, FeSO4·7H2O 0.01 g, NaCl 0.5 g, agar 20 g, made up to 1000 mL with tap water, pH 7.2 - 7.4.

[0011] PDA medium: Potato dextrose agar medium, 200 g of potatoes are boiled for 30 minutes and then filtered to obtain the filtrate, 20 g of glucose, 15 - 20 g of agar, made up to 1000 ml with tap water, natural pH; sterilized at 115 °C.

[0012] MS medium: 20 g of soybean powder is boiled in advance with water until it becomes a paste, 20 g of mannitol, 15 - 20 g of agar, made up to 1000 mL with tap water, natural pH.

[0013] 2×YT liquid medium: 16 g of peptone, 10 g of yeast extract, 5 g of NaCl, made up to 1000 mL with tap water, natural pH.

[0014] Example 1

[0015] Isolation, identification and biological characteristics of the strain

[0016] I. Isolation and purification of the strain

[0017] In September 2018, several strains of actinomycetes were isolated from the fields in Nanning, Guangxi. The process is as follows: 1 g of the sample was added to a 300 ml triangular flask containing sterilized small glass beads and 99 ml of sterile physiological saline, and cultured on a shaker at 28 °C for 1 h. Take 1 ml of the suspension and dilute it to 10 -4 , take the original solution, 10 -2 dilution and 10 -4 dilution and spread them on Gause's No. 1 medium with a final concentration of 50 μg / ml of potassium dichromate. Pick single colonies on the 5th day, 7th day and 10th day respectively, and then use the plate streaking method to purify and isolate them on PDA medium, named strain SGX33.

[0018] The obtained purified strain was preserved at -80 °C using 20% glycerol.

[0019] II. Identification of the strain

[0020] 1. Morphological characteristics

[0021] For the morphological characteristics of the strain SGX33 isolated from the fields in Guangxi, after inoculating and culturing on the medium using the streak plate method, morphological observation was carried out. The colony characteristics of the strain SGX33 on various media are shown in Table 1.

[0022] Table 1. Colony characteristics of strain SGX33

[0023]

[0024] The colony characteristics of the strain SGX33 on various media are shown in Table 1. Among them, the growth rate is relatively fast on MS and PDA media, and relatively slow on Gause's No. 1 medium. On MS medium, the aerial mycelium is slightly white ( Figure 1 A). Observed by scanning electron microscopy, the hyphae are straight and have few branches ( Figure 1 B).

[0025] 2. Molecular identification

[0026] For PCR amplification of 16S rDNA, universal primers were used: 27F: 5'-AGAGTTTGATCCTGGCTCAG-3'; 1492R: 5'-GGTTACCTTGTTACGACTT-3. The size of the amplified fragment is about 1473 bp, and the annealing temperature is 55 °C.

[0027] Primers for gyrB amplification: gyrBF: 5'-TTATCTACGACCTTAGACG-3'; gyrBR: 5'-TAAATTGAAGTCTTCTCCG-3'. The size of the amplified fragment is about 1045 bp, and the annealing temperature is 55 °C.

[0028] The PCR reaction program was: 95 °C for 5 min; 95 °C for 30 s, 56 °C for 90 s, 72 °C for 60 s (33 cycles); 72 °C for 10 min; 4 °C.

[0029] After purification and sequencing of the amplified sequences, homology comparison was carried out through the Blastn program of NCBI. According to the sequence alignment results of the three gene fragments and combined with the morphological characteristics of the strain, the strain SGX33 was identified as Streptomyces morookaense. Among them, the 16S rDNA sequence is as shown in SEQ ID NO.1 in the sequence listing, and the gyrB sequence is as shown in SEQ ID NO.2 in the sequence listing.

[0030] III. Biological Characteristics of the Strain

[0031] The growth temperature, pH, and salt tolerance concentration range of Streptomyces morookaense strain SGX33 isolated from the fields in Guangxi were determined, and the specific results are shown in Table 2 below.

[0032] The growth temperature range of Streptomyces morookaense SGX33 is 15 - 45 °C, the growth pH range is pH 4 - 10, and it can tolerate 2% NaCl by mass concentration. This indicates that Streptomyces morookaense SGX33 is an acid- and salt-tolerant streptomycete.

[0033] Table 2. Growth Temperature, pH, and Salt Tolerance Concentration Range of Streptomyces morookaense SGX33

[0034] Growth temperature Tolerance Growth pH Tolerance Tolerant salt concentration Tolerance 4℃ - pH 3 - 1% NaCl + 15℃ + pH 4 + 2% NaCl + 25℃ +++ pH 5 + 5% NaCl - 37℃ +++ pH 6 + 7% NaCl - 45℃ + pH 7 + 10% NaCl - 55℃ - pH 8 + 12% NaCl - - pH 9 + 15% NaCl - - pH 10 +

[0035] Note: + indicates tolerance, - indicates intolerance

[0036] IV. Preservation of the Strain

[0037] Streptomyces morookaense strain SGX33 isolated from the fields in Guangxi was deposited at the China General Microbiological Culture Collection Center on July 20, 2022. The center is abbreviated as CGMCC, and its address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The deposit number of strain SGX33 is CGMCC No. 25350.

[0038] Example 2

[0039] Antifungal activity test of Streptomyces morookaense SGX33 isolated from the fields in Guangxi.

[0040] The antifungal activity of Streptomyces morookaense SGX33 was determined by the plate confrontation method:

[0041] (1) Streptomycete culture and collection: Streptomyces morookaense SGX33 was inoculated on an MS plate by the streaking method and cultured at 28 °C for 7 days for standby.

[0042] (2) Cultivation of pathogenic bacteria: Inoculate Fusarium oxysporum f.sp. cubense (the Cuban special form of Fusarium oxysporum), Fusarium oxysporum f.sp. lycopersici (the tomato special form of Fusarium oxysporum), Bipolaris sorokiniana (the causal agent of wheat root rot), Rhizoctonia solani (the causal agent of cucumber damping-off), Cladosporium cladsporioides (the causal agent of tomato leaf spot), Colletotrichum gloeosporioides (the causal agent of citrus anthracnose), and Botrytis cinerea (the causal agent of vegetable gray mold) on PDA plates and use them after culturing at 28 °C for one week.

[0043] (3) Plate confrontation experiment: Inoculate the pathogen culture blocks in the center of PDA plates, and inoculate the culture blocks of Streptomyces shigangensis SGX33 about 2.0 cm away from the pathogen. Incubate at 28 °C for 3 - 5 days and observe the growth of the pathogen.

[0044] (4) Results: The plate confrontation experiment showed that after culturing for 3 - 5 days, the colonies on the plates inoculated only with plant pathogens were circular and covered the plates. However, after culturing for 3 - 5 days with the simultaneous inoculation of Streptomyces shigangensis SGX33 and plant pathogens, the growth of Fusarium oxysporum f.sp. cubense, Fusarium oxysporum f.sp. lycopersici, Bipolaris sorokiniana, Rhizoctonia solani, Cladosporium cladsporioides, Colletotrichum gloeosporioides, and Botrytis cinerea was significantly inhibited ( Figure 2 ), indicating that Streptomyces shigangensis SGX33 has obvious antagonistic effects against a variety of pathogenic fungi.

[0045] Example 3

[0046] Microbial inoculant with Streptomyces shigangensis SGX33 as the material:

[0047] (1) Preparation of liquid seeds

[0048] Pick the colonies of Streptomyces shigangensis SGX33 that have grown on MS plates for 5 - 7 days and inoculate them into 2×YT liquid medium. Incubate at 28 - 30 °C with shaking at 180 rpm for 2 - 3 days and use it as the seed liquid.

[0049] (2) Preparation of solid microbial inoculant

[0050] Using agricultural waste such as livestock and poultry manure, edible mushroom residue, bran, or soybean meal as basic raw materials, a certain proportion of MnSO₄ and (NH₂SO₄) is added. Liquid fermentation seeds are inoculated at a mass ratio of 10%, and the material-to-water ratio is adjusted to approximately 1:1. The mixture is incubated at 28-30°C for 5-7 days. After fermentation, it is air-dried and pulverized into a fine powder. The resulting Streptomyces fermentation powder can be used directly as a solid microbial inoculant.

[0051] (3) Preparation of liquid microbial agents

[0052] Liquid seeds are added into a liquid fermentation tank at a mass ratio of 5%, and fermented at 28-30° C. for 4-6 days. The obtained bacterial fermentation liquid or the filtered bacterial fermentation liquid can be used as a liquid microbial agent.

[0053] Example 4

[0054] Preparation of microbial fertilizer using Streptomyces SGX33 as material:

[0055] (1) Preparation of liquid seeds

[0056] A colony of Streptomyces SGX33 grown on an MS plate for 5-7 days was picked and inoculated into 2×YT liquid culture medium. The culture was shaken at 28-30° C. and 180 rpm for 2-3 days and then used as seed liquid.

[0057] (2) Preparation of microbial fertilizer

[0058] Using agricultural waste such as livestock and poultry manure, edible mushroom residue, bran, or soybean meal as basic raw materials, a certain proportion of MnSO4 and (NH)2SO4 is added. Liquid fermentation seeds are inoculated at a mass ratio of 10%, and the material-water ratio is adjusted to approximately 1:1. The culture is incubated at 28-30°C for 5-7 days. After fermentation, it is air-dried and pulverized into a fine powder. The resulting Streptomyces fermentation powder can be used directly as a solid microbial fertilizer. Alternatively, the resulting Streptomyces fermentation powder can be mixed with organic fertilizer in a certain ratio to produce a bio-organic fertilizer.

[0059] Example 5

[0060] Application of biological agents or microbial fertilizers of Streptomyces SGX33 isolated from the fields of Guangxi to prevent and control plant fungal diseases:

[0061] 1. Prevention and control of wheat root rot with the microbial agent Streptomyces SGX33

[0062] (1) Experimental location and environmental conditions: Three potted plant experiments were conducted in the outdoor natural environment of the Institute of Microbiology, Chinese Academy of Sciences.

[0063] (2) Test agents: blank control, fermentation suspension of Streptomyces SGX33 (liquid microbial agent)

[0064] (3) Test method:

[0065] Seed treatment: Take a number of wheat seeds, treat them with 10% 84 disinfectant solution for 5 minutes, rinse twice with sterile water, and then soak the seeds in sterile water for 4 hours;

[0066] Use the Streptomyces shigangensis SGX33 bacterial agent for seed soaking and root irrigation: Take the soaked seeds in a petri dish, soak the seeds with 10 ml of SGX33 fermentation suspension for 20 minutes, and the SGX33 fermentation suspension is used for root irrigation.

[0067] (4) Treatment arrangement: Plant 10 seeds in each flower pot, make four parallels for each treatment, arrange them randomly, and cultivate them under outdoor natural environmental conditions for 2 - 3 weeks to observe the growth situation. This experiment is repeated three times.

[0068] (5) Statistical data: Record the height of the plants, count the plant heights of the control group, the group inoculated with Bipolaris sorokiniana, and the group inoculated with Bipolaris sorokiniana and applied with the Streptomyces shigangensis SGX33 bacterial agent at the same time, calculate the average value, and conduct a significant difference test.

[0069] (6) Result analysis: In the pot experiment for controlling wheat root rot, by using the methods of seed soaking and root irrigation, the application of the Streptomyces shigangensis SGX33 bacterial agent increased the emergence rate of wheat seeds in the soil containing Bipolaris sorokiniana. The plant heights were all higher than those of the diseased group control, and basically reached the growth level of wheat seeds in the soil without pathogenic bacteria (Table 3).

[0070] Table 3. Prevention and control of wheat root rot by Streptomyces shigangensis SGX33 (plant height cm)

[0071]

[0072] II. Field experiment on the microbial bacterial agent / microbial fertilizer with Streptomyces shigangensis SGX33 as the raw material against banana wilt.

[0073] (1) Test site and environmental conditions: This experiment was set up in Jinling Town, Nanning, Guangxi. The test field had been planted with bananas for many years, and the incidence of banana wilt was over 50%, accumulating a large number of banana wilt pathogens.

[0074] (2) Test design and treatment: The land was plowed before the experiment. Banana seedlings were transplanted in January, and the banana seedling variety was a disease - susceptible banana variety (Williams). When transplanting, a control (without applying the bacterial agent) and treatment with the Streptomyces shigangensis SGX33 microbial bacterial agent / microbial fertilizer were set. The samples treated with the bacterial agent were applied by hole - application. For each hole, the excavated part of the soil was mixed with 30 g of the microbial bacterial agent / microbial fertilizer, and then backfilled for planting the seedlings. Six months later, the microbial bacterial agent / microbial fertilizer (30 g per plant) was supplemented by hole - application around the banana roots.

[0075] (3) Data statistics: An incidence survey was conducted in November. The number of banana plants per mu planted in the control group and the treatment group applying Streptomyces shigangensis SGX33 agent was counted respectively, as well as the number of banana plants infected with Fusarium wilt. Incidence rate = (number of infected plants / total number of plants) × 100%, control efficacy = (incidence rate of the control group - incidence rate of the treatment group) / incidence rate of the control group × 100%. At the same time, the total yield per mu of the control group and the treatment group was counted, and the yield increase rate = (yield of the control group - yield of the treatment group) / yield of the control group × 100%.

[0076] (4) Test results: Through hole application treatment, the use of Streptomyces shigangensis SGX33 microbial agent / microbial fertilizer can significantly overcome the continuous cropping obstacle of crops caused by Fusarium wilt: When replanting bananas in fields where the incidence rate of banana Fusarium wilt was over 50% in the previous year, the incidence rate of banana Fusarium wilt in the control group was 51.5%, and that in the group applying Streptomyces shigangensis SGX33 agent was 12.8%. The prevention and control of banana Fusarium wilt in the group applying biological pesticide preparation reached over 75%, and the yield increased by over 80% (Table 4).

[0077] Table 4 Investigation results of field control test on banana Fusarium wilt

[0078]

[0079] The fertilizer containing Streptomyces shigangensis strain SGX33 of the present invention improves the emergence rate and plant height of wheat seeds in soil containing wheat root rot pathogen, and can basically reach the growth level of wheat seeds in soil without pathogen. At the same time, when replanting bananas in fields where the incidence rate of banana Fusarium wilt was over 50% in the previous year, the prevention and control of banana Fusarium wilt reached over 75%, and the yield increased by over 80%.

Claims

1. Use of Streptomyces morookaense strain SGX33 in preparing a fertilizer for preventing and controlling plant diseases, characterized in that, The plant disease is wheat root rot; the preservation number of the Streptomyces shigangensis strain SGX33 is CGMCC No. 25350.

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