A disease-resistant and growth-promoting strawberry biological bacteria and a method for preparing a biological organic fertilizer

Abstract

The disease-resistant and growth-promoting strawberry biological bacteria of the present invention has a deposit number of CGMCC No. 19859, and the disease-resistant and growth-promoting strawberry biological bacteria is used in fertilizer. The preparation method of the disease-resistant and growth-promoting strawberry biological agent includes preparing a strain biological agent, preparing a disease-resistant and growth-promoting mixed agent, and preparing a bio-organic fertilizer.

Description

Technical Field

[0001] The present invention relates to the field of agricultural microorganisms, in particular to a microorganism with nutrient activation, growth promotion and antagonism functions and a method for preparing bio-organic fertilizer from the microorganism. Background Art

[0002] Strawberry is a plant of the genus Fragaria in the Rosaceae family. It has high nutritional and economic value and a large market demand because of its bright color, sweet taste, and rich content of vitamins, carotene, tannins and other nutrients. It is one of the most widely grown economic crops in the world.

[0003] The rapid development of the strawberry industry and the long-term, large-scale continuous cropping have exacerbated soil ecological damage, exacerbated the problem of continuous cropping disorder, and increased pests and diseases, hindering the sustainable development of strawberry production. This is particularly true in greenhouse cultivation, where strawberry cultivation primarily relies on a lack of rainfall, high temperatures and humidity, and secondary soil salinization, acidification, and the accumulation of pathogens are particularly prominent. This deteriorates the soil microbiome and increases the prevalence of diseases. It has been reported that continuous cropping for four years can reduce strawberry yields by over 40%. Currently, the generally accepted mechanisms for continuous cropping disorder include soil quality degradation, accumulation of pathogens, imbalance of soil nutrients, allelopathy, and imbalance of the soil microbiome. Researchers generally agree that an imbalance of the soil microbiome is the root cause of continuous cropping disorder.

[0004] Existing research has found significant differences in the dominant fungi in soils after three years of continuous strawberry cropping compared to soils from newly cropped crops. The abundance of dominant fungi in newly cropped soils decreased significantly, while fungi with increased abundance in continuously cropped soils are mostly associated with plant diseases. For example, the dominant fungi in continuously cropped soils, such as Rhizoctonia, Verticillium, and Fusarium, increase significantly after continuous cropping. These pathogens can cause diseases such as black root, damping-off, leaf rot, stem rot, and wilt in a variety of plants (including strawberry), exacerbating continuous cropping problems.

[0005] Therefore, the effective solution to strawberry-growing soil problems is to utilize modern biotechnology and beneficial microorganisms to regulate the soil microbial flora, improve soil physical and chemical properties, inhibit the growth of pathogenic microorganisms, and thus effectively prevent and control soil-borne diseases. However, there are currently no effective microorganisms or fertilizers that can effectively control these diseases. Summary of the Invention

[0006] The present invention aims to provide a disease-resistant and growth-promoting strawberry biological bacterium and a method for preparing a biological organic fertilizer.

[0007] The invention aims to solve the problem of continuous cropping obstacles in existing strawberry continuous cropping soil.

[0008] The present invention is achieved through the following technical solutions:

[0009] The disease-resistant and growth-promoting strawberry biological bacteria described in the present invention is Bacillus amyloliquefaciens, which was deposited in the General Microbiology Center of the China Culture Collection Administration (CGMCC) on May 21, 2020, with the deposit number CGMCC No. 19859.

[0010] The disease-resistant and growth-promoting strawberry biological bacteria is applied to fertilizer, and the fertilizer is a microbial fertilizer.

[0011] The isolation and screening method of a disease-resistant and growth-promoting strawberry biological bacteria comprises at least the following steps:

[0012] Screening medium for rhizosphere functional microbial strains LB medium was used for screening rhizosphere functional microbial strains.

[0013] (1) Collection of soil samples and isolation of rhizosphere functional microorganisms

[0014] Healthy plants were collected from the diseased area of the greenhouse in Mazhan Town, Cangnan County, Wenzhou. The plants were gently pulled out with soil and brought back to the laboratory for isolation and screening of disease-resistant and growth-promoting strains. The collection time was 2019. During isolation and screening, the loose soil attached to the roots of the plants was gently shaken off, the plants were cut off from the rhizomes, 10g of plant roots with rhizosphere soil were weighed and placed in a conical flask containing sterilized glass beads and 90mL of sterile water, and the mixture was incubated at 28℃ and 170r·min. -1 Oscillate for 30 min to obtain a suspension, dilute it according to the gradient concentration, and spread it on LB medium (select 10 －4 , 10 －5 , 10 －6 Three gradients were plated, and each gradient was repeated three times), and the plate was inverted and cultured in a constant temperature incubator at 28°C for 18 hours. Single colonies with rapid growth and different morphologies were picked and placed on new plates. They were streaked and purified 3-4 times, numbered respectively, and colonies were picked and added with 15% sterile glycerol by volume and stored at -80°C until use.

[0015] (2) Screening of antagonistic microorganisms

[0016] The plate confrontation method was used, with Fusarium oxysporum as the target pathogen strain. The pathogen was inoculated into the PDA plate, and the isolated rhizosphere microorganisms were inoculated around the plate. By comparing the size of the inhibition zone, functional microbial strains with high-efficiency disease resistance were screened.

[0017] (3) Identification of functional microorganisms

[0018] The functional microorganisms were identified using 16S rDNA sequencing. PCR amplification of the 16S rDNA fragment used a pair of universal primers: forward primer BSF8 / 20: 5'-AGAGT TTGAT CCTGG CTCAG-3'; reverse primer BSR1541 / 20: 5'-AAGGA GGTGA TCCAG CCGCA-3'. The PCR reaction system (50 μl) consisted of: 5.0 μl of 10× PCR buffer, 3.0 μl of 25 mM MgCl2, 4.0 μl of dNTPs, 1.0 μl each of primers BSF8 / 20 and BSR1541 / 20, 1.0 μl of template DNA, 0.25 μl of Taq enzyme (10,000 U / mL), and 36.0 μl of distilled water. The PCR procedure was as follows: (1) 94°C for 5 min; (2) 94°C for 1 min, 55°C for 1 min, 72°C for 1.5 min, and 35 cycles in step 2; (3) 72°C for 10 min; and (4) 4°C for 10 min. The 16S rDNA sequence amplified by PCR was sequenced by Sangon Biotech (Shanghai) Co., Ltd. and then compared to the NCBI website to obtain the taxonomic information of the strain.

[0019] (4) Isolation of antagonistic functional microorganisms

[0020] To screen for functional microorganisms with highly effective antagonistic effects, rhizosphere microorganisms isolated from healthy plant roots were inoculated onto slant cultures. A total of 13 strains with diverse morphologies were obtained. These strains were then inoculated onto PDA plates seeded with Fusarium oxysporum in the center for a head-on test. After incubation in an incubator, the size of the inhibition zone was observed, and strains with larger inhibition zones were selected for further validation and purification. The results showed that among the isolated strains, KS12 exhibited a significant inhibition zone, indicating strong antibacterial activity. It was selected for further identification and functional validation. In accordance with strain collection requirements, this isolate, KS12, was designated WSW1.

[0021] (5) Identification of strain WSW1

[0022] Identification of the strain: Universal primers were used to amplify the 16S rDNA sequence of strain WSW1. Sequence alignment was performed on the RDP website, and the sequence showed the highest similarity to Bacillus amyloliquefaciens. The strain was named Bacillus amyloliquefaciens WSW1.

[0023] Described bio-organic fertilizer preparation method is as follows:

[0024] (1) Preparation of Bacillus amyloliquefaciens strain (WSW1) biological agent

[0025] Preparation of antagonistic bacterial agent: The Bacillus amyloliquefaciens strain (WSW1) stored in a low-temperature refrigerator was inoculated onto an LB slant for activation. The activated slant strain was inoculated into LB liquid culture medium and cultured in a shaker at 30°C and 150 rpm for 3 days to form a bacterial agent with a large number of spores, thereby obtaining the antagonistic bacterial agent.

[0026] (2) Preparation of disease-resistant and growth-promoting mixed bacterial agents

[0027] In order to improve the disease resistance and growth promotion effect, the bacterial agent prepared by the antagonistic functional microorganism Bacillus amyloliquefaciens strain (WSW1) and the bacterial agent prepared by Bacillus arnoldii were mixed in a volume ratio of 1:1 to obtain a disease resistance and growth promotion mixed bacterial agent.

[0028] (3) Preparation of bio-organic fertilizer

[0029] The prepared Bacillus amyloliquefaciens inoculant is added to fully decomposed pig manure organic fertilizer at a ratio of 1%, and after sufficient stirring, a bio-organic fertilizer is prepared. The pig manure organic fertilizer used for the preparation of the bio-organic fertilizer is prepared by high-temperature fermentation of pig manure and wood chips or straw at a ratio of 4:1. The high-temperature fermentation time (composting temperature>55°C) should be no less than 30 days. After the fermentation is completed and before inoculation, the pH of the compost should be measured, and the pH should be between 5.5-7.5.

[0030] The beneficial effects of the present invention are as follows: The present invention uses highly efficient antagonistic microorganisms isolated from the soil to prepare a microbial fertilizer, which solves the continuous cropping problem in strawberry continuous cropping soil. The microbial fertilizer prepared by the present invention is applied to the soil with continuous cropping problems to proliferate and form a colony advantage, decompose the nitrogen, phosphorus, and potassium elements fixed in the soil, and fix the free nitrogen in the air for plant absorption and utilization, thereby increasing soil fertility and improving soil physical and chemical properties. At the same time, the antagonistic microorganisms can effectively inhibit the proliferation of pathogens, thereby forming a disease-suppressing soil, reducing the occurrence of soil-borne diseases, and effectively preventing and controlling the occurrence of continuous cropping problems. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The following are photos comparing the growth of strawberries under different treatments.

[0032] Figure 2 A comparison chart of strawberry stem lengths under different treatments.

[0033] Figure 3 A comparison chart of strawberry root lengths under different treatments.

[0034] Figure 4 This is a comparison chart of strawberry root weights under different treatments.

[0035] Figure 5 The figure is a comparison of the survival rates of strawberries in each treatment.

[0036] Figure 6 This is a comparison chart of strawberry yields in each treatment.

[0037] Figure 7 A comparison chart of soil pH values in each treatment.

[0038] Figure 8 Comparison chart of soil EC values in each treatment.

[0039] Figure 9 Comparison of soil organic matter content in each treatment.

[0040] Figure 10 Comparison of total nitrogen content in soil under different treatments.

[0041] Figure 11 Comparison of soil alkaline nitrogen in each treatment.

[0042] Figure 12 A comparison chart of available phosphorus in each treatment.

[0043] Figure 13 This is a comparison chart of available potassium in soils of different treatments. DETAILED DESCRIPTION

[0044] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0045] The disease-resistant and growth-promoting strawberry biological bacteria described in the present invention is Bacillus amyloliquefaciens, which was deposited in the General Microbiology Center of the China Culture Collection Administration (CGMCC) on May 21, 2020, with the deposit number CGMCC No. 19859.

[0046] The disease-resistant and growth-promoting strawberry biological bacteria is applied to fertilizer, and the fertilizer is a microbial fertilizer.

[0047] The method for isolating and screening disease-resistant and growth-promoting strawberry bacteria comprises at least the following steps:

[0048] Rhizosphere functional microorganism strain screening culture medium: LB culture medium is used for screening rhizosphere functional microorganism strains.

[0049] (1) Collection of soil samples and isolation of rhizosphere functional microorganisms

[0050] Healthy plants were collected from the diseased area of the greenhouse in Mazhan Town, Cangnan County, Wenzhou. The plants were gently pulled out with soil and brought back to the laboratory for isolation and screening of disease-resistant and growth-promoting strains. The collection time was 2019. During isolation and screening, the loose soil attached to the roots of the plants was gently shaken off, the plants were cut off from the rhizomes, 10g of plant roots with rhizosphere soil were weighed and placed in a conical flask containing sterilized glass beads and 90mL of sterile water, and shaken at 28℃ and 170r·min-1 for 30min to obtain a suspension, which was diluted according to gradient concentration and spread on LB medium (select 10 －4 , 10 －5 , 10 －6 Three gradients were plated, and each gradient was repeated three times), and the plate was inverted and cultured in a constant temperature incubator at 28°C for 18 hours. Single colonies with rapid growth and different morphologies were picked and placed on new plates. They were streaked and purified 3-4 times, numbered respectively, and colonies were picked and added with 15% sterile glycerol by volume and stored at -80°C until use.

[0051] (2) Screening of antagonistic microorganisms

[0052] The plate confrontation method was used, with Fusarium oxysporum as the target pathogen strain. The pathogen was inoculated into the PDA plate, and the isolated rhizosphere microorganisms were inoculated around the plate. By comparing the size of the inhibition zone, functional microbial strains with high-efficiency disease resistance were screened.

[0053] (3) Identification of functional microorganisms

[0054] The functional microorganisms were identified using 16S rDNA sequencing. PCR amplification of the 16S rDNA fragment used a pair of universal primers: forward primer BSF8 / 20: 5'-AGAGT TTGAT CCTGG CTCAG-3'; reverse primer BSR1541 / 20: 5'-AAGGA GGTGA TCCAG CCGCA-3'. The PCR reaction system (50 μl) consisted of: 5.0 μl of 10× PCR buffer, 3.0 μl of 25 mM MgCl2, 4.0 μl of dNTPs, 1.0 μl each of primers BSF8 / 20 and BSR1541 / 20, 1.0 μl of template DNA, 0.25 μl of Taq enzyme (10,000 U / mL), and 36.0 μl of distilled water. The PCR procedure was as follows: (1) 94°C for 5 min; (2) 94°C for 1 min, 55°C for 1 min, 72°C for 1.5 min, and 35 cycles in step 2; (3) 72°C for 10 min; and (4) 4°C for 10 min. The 16S rDNA sequence amplified by PCR was sequenced by Sangon Biotech (Shanghai) Co., Ltd. and then compared to the NCBI website to obtain the taxonomic information of the strain.

[0055] (4) Isolation of antagonistic functional microorganisms

[0056] To screen for functional microorganisms with highly effective antagonistic effects, rhizosphere microorganisms isolated from healthy plant roots were inoculated onto slant cultures. A total of 13 strains with diverse morphologies were obtained. These strains were then inoculated onto PDA plates seeded with Fusarium oxysporum in the center for a head-on test. After incubation in an incubator, the size of the inhibition zone was observed, and strains with larger inhibition zones were selected for further validation and purification. The results showed that among the isolated strains, KS12 exhibited a significant inhibition zone, indicating strong antibacterial activity. It was selected for further identification and functional validation. In accordance with strain collection requirements, this isolate, KS12, was designated WSW1.

[0057] (4) Identification of strain WSW1

[0058] Identification of the strain: Universal primers were used to amplify the 16S rDNA sequence of strain WSW1. Sequence alignment was performed on the RDP website, and the sequence showed the highest similarity to Bacillus amyloliquefaciens. The strain was named Bacillus amyloliquefaciens WSW1.

[0059] Described bio-organic fertilizer preparation method is as follows:

[0060] (1) Preparation of Bacillus amyloliquefaciens strain (WSW1) biological agent

[0061] Preparation of antagonistic bacterial agent: The Bacillus amyloliquefaciens strain (WSW1) stored in a low-temperature refrigerator was inoculated onto an LB slant for activation. The activated slant strain was inoculated into LB liquid culture medium and cultured in a shaker at 30°C and 150 rpm for 3 days to form a bacterial agent with a large number of spores, thereby obtaining the antagonistic bacterial agent.

[0062] (2) Preparation of disease-resistant and growth-promoting mixed bacterial agents

[0063] In order to improve the disease resistance and growth promotion effect, the bacterial agent prepared by the antagonistic functional microorganism Bacillus amyloliquefaciens strain (WSW1) and the bacterial agent prepared by Bacillus arnoldii were mixed in a volume ratio of 1:1 to obtain a disease resistance and growth promotion mixed bacterial agent.

[0064] (3) Preparation of bio-organic fertilizer

[0065] The prepared Bacillus amyloliquefaciens strain (WSW1) inoculum is added to fully decomposed pig manure organic fertilizer at a ratio of 1%, and after sufficient stirring, a bio-organic fertilizer is prepared. The pig manure organic fertilizer used for the preparation of the bio-organic fertilizer is prepared by high-temperature fermentation of pig manure and wood chips or straw at a ratio of 4:1. The high-temperature fermentation time (composting temperature>55°C) should be no less than 30 days. After the fermentation is completed and before inoculation, the pH of the compost should be measured, and the pH should be between 5.5-7.5.

[0066] Application of biological organic fertilizer (disease-resistant and growth-promoting bacteria) in strawberry cultivation:

[0067] The main antibacterial agent is Bacillus amyloliquefaciens WSW1.

[0068] NKY1 disease-resistant and growth-promoting bacterial agent is composed of Bacillus amyloliquefaciens WSW1 and Bacillus aeruginosa, which are mixed in a volume ratio of 1:1.

[0069] The N-P2O5-K2O composition of compound fertilizer is 17-17-17. Application

[0070] The experiment included one control and two biological inoculant treatments. Conventional fertilizer was applied to both the control and treatments: 1000 kg of rabbit manure, 300 kg of vegetable cake, and 15 kg of compound fertilizer were applied per mu (approximately 1 acre) as base fertilizer, followed by irrigation and suffocation. Strawberry seedlings were then established, and both treatments were topdressed with four applications of fruit-enhancing fertilizer, 2 kg each time, for a total of 8 kg. The control received no inoculant. Treatment 1 applied WSW1, an anti-disease agent, diluted 100-fold and applied to the roots during strawberry establishment, followed by irrigation every 30 days. Treatment 2 applied NKY1, diluted 100-fold and applied to the roots during strawberry establishment, followed by irrigation every 30 days.

[0071] Strawberry colonization: Strawberries were planted at a rate of 5,000 plants per acre. Strawberry management: Strawberry management followed normal practices. Records: Regularly record strawberry growth and measure tiller number and branch length. Sample testing: After strawberry cultivation, soil and plant samples were collected to test the physical and chemical properties of the soil. Test results:

[0072] Effects of microbial agents on strawberry growth

[0073] After the strawberry yield calculation is completed, strawberry plant samples are collected to measure the plant traits. Figure 1 and Figure 2 The average plant height of the control was 15.29 cm, that of T1 was 21.67 cm, and that of T2 was 20.55 cm. ANOVA showed that there were significant differences among the treatments (P<0.01).

[0074] Determine the root length of strawberries. Figure 3 The average root length of the control was 13.1 cm, that of T1 was 14.44 cm, and that of T2 was 15.15 cm. Analysis of variance showed that there were significant differences among the treatments (P<0.01), with the root lengths of T1 and T2 being significantly longer than that of the control.

[0075] Measure the root weight. Figure 4 As shown, the average root weight of CK was 19.13 g, T1 was 22.51 g, and T2 was 33.00 g, with T2 being significantly higher than both CK and T1. Analysis of variance showed that root weights differed significantly between treatments (P < 0.01), with T2 significantly higher than both CK and T1. Therefore, the application of microbial inoculants can promote strawberry growth.

[0076] Strawberry survival rate, Figure 5 The average survival rate of CK was 64.26%, T1 was 82.25%, and T2 was 89.45%. T2 was higher than CK and T1, and T1 was higher than CK. ANOVA showed that the survival rates of the treatments were significantly different (P<0.05), and T2 was significantly higher than CK.

[0077] Strawberry production, Figure 6 The average yield of CK was 1681.9 kg / mu, T1 was 2189.11 kg / mu, and T2 was 2384.78 kg / mu. T2 was higher than both CK and T1, and T1 was higher than the control. ANOVA showed significant differences in strawberry yield among treatments (P < 0.05), with T1 and T2 significantly higher than CK.

[0078] Effects of microbial agents on soil physical and chemical properties

[0079] (1) Changes in soil pH under different treatments

[0080] Soil pH under different treatments, such as Figure 7 As shown in Figure 2, the pH of the control and both treatments was above 6.0, with the pH of the control at 6.55, T1 at 6.59, and T2 at 6.32. T2 was lower than both the CK and T1. Analysis of variance revealed significant differences in soil pH between treatments (p < 0.01), with T2 pH significantly lower than both the control and T1.

[0081] (2) Changes in soil EC under different treatments

[0082] Soil EC of different treatments, such as Figure 8 As shown in Figure 2 , the EC values for the control and both treatments were all above 500 μs / cm, indicating a mild salinization trend: 647 μs / cm for the control, 700 μs / cm for T1, and 795.33 μs / cm for T2. T2 was significantly higher than both the CK and T1. Analysis of variance revealed highly significant differences in soil EC among the treatments (p < 0.01), with T2 being significantly higher than both the control and T1.

[0083] (3) Changes in soil organic matter content under different treatments

[0084] Changes in soil organic matter content under different treatments, such as Figure 9 The soil organic matter content in the control was 25.11 g / kg, in T1 it was 23.92 g / kg, and in T2 it was 25.6 g / kg. T2 was higher than both the CK and T1. ANOVA analysis showed no significant differences in soil organic matter content among the treatments.

[0085] (4) Changes in total nitrogen content in soil under different treatments

[0086] Changes in total nitrogen content in soil under different treatments, such as Figure 10 The total nitrogen content in the soil of the control was 1.61 g / kg, that of T1 was 1.57 g / kg, and that of T2 was 1.65 g / kg. T2 was higher than that of both CK and T1. Analysis of variance showed no significant differences in soil total nitrogen content among the treatments.

[0087] (5) Changes in soil alkaline nitrogen content under different treatments

[0088] Changes in soil alkaline nitrogen content under different treatments, such as Figure 11 As shown. The alkaline nitrogen content in the control was 146.44 mg / kg, in T1 it was 127.55 g / kg, and in T2 it was 137.25 g / kg. The CK soil had higher alkaline nitrogen content than both T1 and T2. Analysis of variance showed significant differences in soil alkaline nitrogen content among treatments (P < 0.01). The CK soil had significantly higher alkaline nitrogen content than the T1 and T2 soils, which may be related to the lower disease incidence and greater growth in the T1 and T2 treatments.

[0089] (6) Changes in available phosphorus content in soils under different treatments

[0090] Changes in available phosphorus content in soils under different treatments, such as Figure 12 As shown. The available phosphorus content of the control was 129.25 mg / kg, that of T1 was 127.41 mg / kg, and that of T2 was 152.90 mg / kg. T2 was higher than that of CK and T1. Variance analysis showed that the available phosphorus content in the soil was significantly different among the treatments (P<0.05). The alkaline nitrogen content in the soil of T2 was significantly higher than that of CK and T1. This may be because the growth-promoting bacteria were added to T2, which had a phosphorus-solubilizing effect and increased the available phosphorus content in the soil. (7) Changes in the available potassium content in the soil under different treatments

[0091] Changes in soil available potassium content under different treatments, such as Figure 13The available potassium content in the control was 148.84 mg / kg, in T1 it was 173.06 mg / kg, and in T2 it was 231.11 mg / kg, with T2 being higher than in both CK and T1. Analysis of variance showed significant differences in soil available phosphorus content among treatments (P < 0.01). The available potassium content in T2 soil was significantly higher than in CK and T1. This may be due to the addition of growth-promoting bacteria to T2, which has a potassium-releasing effect and increases the available potassium content in the soil.

[0092] The 16S rDNA sequence of the strain is shown in the sequence table. Sequence comparison results show that the strain belongs to Bacillus amyloliquefaciens.

Claims

1. A disease-resistant and growth-promoting strawberry bio-bacterium, characterized in that The disease-resistant and growth-promoting strawberry biological bacteria is Bacillus amyloliquefaciens, which was deposited in the General Microbiology Center of the China Culture Collection Administration (CGMCC) on May 21, 2020, with the deposit number CGMCC No. 19859.

2. The application of a disease-resistant and growth-promoting strawberry biological bacteria according to claim 1, characterized in that The disease-resistant and growth-promoting strawberry biological bacteria are applied to fertilizers, and the fertilizers are microbial fertilizers.

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