A synthetic bacterial community for promoting the growth of macadamia nuts and its construction method and application

Abstract

The present invention belongs to the field of microbial technology, and specifically relates to a synthetic bacterial community that promotes the growth of macadamia nuts, and a construction method and application thereof. A synthetic bacterial community, the synthetic bacterial community is formed by mixing equal volumes of Bacillus subtilis X17, Pseudomonas fluorescens X62 and Trichoderma harzianum Z27. The present invention separates and screens strains from healthy rhizosphere soil, and conducts a pairwise confrontation test on the well-growing strains to screen out strains that have no antagonistic effect between the two. Through molecular biological identification, three strains with growth-promoting effects are screened, namely: Bacillus subtilis X17, Pseudomonas fluorescens X62 and Trichoderma harzianum Z27, and then the three strains are constructed into a synthetic bacterial community. It is verified by potted plant experiments that the synthetic bacterial community constructed by the present invention can promote the growth of macadamia nut plants and the accumulation of biomass, and can also activate the soil, improve the soil nutrient content, enzyme activity and microbial biomass carbon, nitrogen and phosphorus content, and can provide support for the planting and economic development of macadamia nuts.

Description

Technical Field

[0001] The present invention belongs to the field of microbial technology, and specifically relates to a synthetic bacterial community for promoting the growth of macadamia nuts, and a construction method and application thereof. Background Art

[0002] Macadamia nuts are favored by the mass market due to their rich nutritional value and medicinal value. They are one of the most popular nuts in the world. With the increasing demand for macadamia nuts, China's macadamia nut industry is booming and the planting area is increasing year by year.

[0003] Biological agents have become the most promising method for promoting plant growth. For example, a variety of biological agents such as Bacillus, Pseudomonas, Trichoderma, Streptomyces, Flavobacteria, Enterobacter, Actinomycetes, Serratia, Alcaligenes and Klebsiella have been widely used to promote the increase of yield and volume of various crops and are widely used in commercial production and applications. In addition, synthetic microbial communities (SynCom) have been shown to be more effective than single strains in promoting crop growth. These synthetic microbial communities provide antibiotics, secondary metabolites, enzymes and other inhibitory effects on pathogens, and can promote plant root growth. Although the research on plant growth-promoting bacteria has achieved a lot of application results, there is little research on the growth-promoting effect on woody plants, especially macadamia nuts. Therefore, it is urgent to develop effective synthetic microbial communities to promote the growth of macadamia nuts.

[0004] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention and should not be regarded as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art. Summary of the invention

[0005] The object of the present invention is to provide a synthetic bacterial community for promoting the growth of macadamia nuts and a construction method and application thereof.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] The first object of the present invention is to provide a synthetic bacterial community, which is formed by mixing equal volumes of Bacillus subtilis X17, Pseudomonas fluorescens X62 and Trichoderma harzianum Z27.

[0008] More specifically, the concentrations of Bacillus subtilis X17, Pseudomonas fluorescens X62, and Trichoderma harzianum Z27 were all 1.0×10 8 cfu / mL.

[0009] More specifically, the Bacillus subtilis X17, whose taxonomic name is Bacillus subtilis X17, was deposited in Guangdong Microbiological Culture Collection Center on August 7, 2024, with the deposit number of GDMCC No: 64986;

[0010] The Pseudomonas fluorescens X62, whose taxonomic name is Pseudomonasfluorescens X62, was deposited in Guangdong Microbiological Culture Collection Center on August 7, 2024, with the deposit number of GDMCC No: 64987;

[0011] Trichoderma harzianum Z27, with the taxonomic name Trichoderma harzianum Z27, was deposited in Guangdong Provincial Microbiological Culture Collection Center on August 7, 2024, with the deposit number GDMCC No: 64988.

[0012] The second object of the present invention is to provide the use of the synthetic bacterial flora in promoting the growth of macadamia nuts.

[0013] The third object of the present invention is to provide a method for constructing a synthetic bacterial community that promotes the growth of macadamia nuts, comprising the following steps:

[0014] S1. Collect healthy rhizosphere soil, prepare healthy rhizosphere soil suspension, dilute it with sterile water for bacterial and fungal culture, and separate and purify the well-growing fungi and bacteria for culture;

[0015] S2. Conduct plate confrontation test on the isolated and purified fungi and bacteria with good growth, select bacteria and fungi with no antagonistic effect between them for molecular biological identification, and select strains reported in the literature to promote crop growth to construct synthetic bacterial flora;

[0016] S3. Verify the growth-promoting effect of synthetic bacteria on crops through pot experiments.

[0017] More specifically, in step S2, the strains screened for promoting crop growth are Bacillus subtilis X17, Pseudomonas fluorescens X62 and Trichoderma harzianum Z27, wherein:

[0018] The Bacillus subtilis X17, whose taxonomic name is Bacillus subtilis X17, was deposited in Guangdong Microbiological Culture Collection Center on August 7, 2024, with the deposit number of GDMCC No: 64986;

[0019] The Pseudomonas fluorescens X62, whose taxonomic name is Pseudomonasfluorescens X62, was deposited in Guangdong Microbiological Culture Collection Center on August 7, 2024, with the deposit number of GDMCC No: 64987;

[0020] The Trichoderma harzianum Z27, whose taxonomic name is Trichoderma harzianum Z27, was deposited in the Guangdong Provincial Microbiological Culture Collection Center on August 7, 2024, with the deposit number GDMCC No: 64988.

[0021] More specifically, in step 2, the constructed synthetic bacterial community is formed by mixing equal volumes of Bacillus subtilis X17, Pseudomonas fluorescens X62 and Trichoderma harzianum Z27.

[0022] More specifically, in step 2, the concentrations of Bacillus subtilis X17, Pseudomonas fluorescens X62 and Trichoderma harzianum Z27 are all 1.0×10 8 cfu / mL.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The present invention separates and screens fungi and bacteria from healthy rhizosphere soil, and conducts a plate confrontation test on the well-growing strains, and screens out strains with no antagonism between each other for molecular biological identification. Three strains reported in the literature to promote crop growth were selected, namely: Bacillus subtilis X17, Pseudomonas fluorescens X62 and Trichoderma harzianum Z27, and then the three strains were constructed into a synthetic bacterial community. It was verified through potted plant experiments that the synthetic bacterial community constructed by the present invention can promote the growth of macadamia plants and the accumulation of biomass, and at the same time, it can also increase the content of soluble organic nitrogen, nitrate nitrogen and ammonium nitrogen in the soil, increase the enzyme activity of β-glucosidase, β-acetylglucosaminidase and acid phosphatase, and increase the soil microbial biomass carbon, nitrogen and phosphorus, which can provide support for the planting and economic development of macadamia nuts.

[0025] Preservation Information

[0026] Bacillus subtilis X17, with the taxonomic name Bacillus subtilis X17, was deposited in Guangdong Microbiological Culture Collection Center on August 7, 2024. The deposit address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the deposit number is GDMCC No: 64986.

[0027] Pseudomonas fluorescens X62, taxonomic name Pseudomonasfluorescens X62, was deposited in Guangdong Microbiological Culture Collection Center on August 7, 2024. The deposit address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, and the deposit number is GDMCC No: 64987.

[0028] Trichoderma harzianum Z27, with the taxonomic name Trichoderma harzianum Z27, was deposited in the Guangdong Provincial Microbiological Culture Collection Center on August 7, 2024. The collection address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, and the collection number is GDMCC No: 64988. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The morphological characteristics and phylogenetic tree of the three strains of the present invention are shown in FIG. DETAILED DESCRIPTION

[0030] The following is a clear and complete description of the technical solution of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by technicians in this field without creative work are within the scope of protection of the present invention.

[0031] In the following examples, the PDA medium formula is: 200 g potato, 20 g glucose, 15 g agar, 1 L sterile water, pH 7.0.

[0032] The LB medium formula is: 10 g tryptone, 5 g yeast extract, 10 g sodium chloride, 15 g agar powder, 1 L sterile water, pH 7.0.

[0033] 1. Isolation and purification of strains

[0034] Healthy rhizosphere soil was collected to prepare healthy rhizosphere soil suspension. The gradient dilution plate method was used to take 0.1 mL 10 -3 , 10 -4 , 10 -5 The dilutions were used to screen fungi on PDA medium, with 3 replicates for each gradient, and cultured in a 28°C constant temperature incubator for 3-10 days. At the same time, 0.1 mL of 10 -5 , 10 -6 , 10 -7 The dilutions were used to screen bacteria on LB medium, with 3 replicates for each gradient, and placed in a 37°C constant temperature incubator for 1-2 days. Fungi and bacteria that grew well were then isolated, purified, cultured, and numbered.

[0035] 2. Screening and identification of growth-promoting fungi and bacteria

[0036] The isolated and purified strains with good growth were subjected to plate confrontation test to screen out strains with no antagonistic effect between each other. DNA was extracted using PowerSoil DNA kit (Mobio Laboratories, Carlsbad, CA, USA), and the taxonomic status of the strains was clarified by 16S / ITS rDNA amplicon sequencing.

[0037] Two growth-promoting bacteria and one growth-promoting fungus were screened and identified by molecular biology: X17 was Bacillus subtilis, X62 was Pseudomonasfluorescens, and Z27 was Trichoderma harzianum. The morphological characteristics and phylogenetic tree of the three strains are shown in Figure 1 , base sequences such as SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3.

[0038] 3. Construction of synthetic bacterial communities

[0039] The antagonistic strain Z27 was inoculated into PDA medium and placed in a constant temperature incubator at 28°C for 3-10 days. After spore production, a spore suspension was prepared and the concentration of the spore suspension was calculated using a hemocytometer. Similarly, the antagonistic bacteria X17 and X62 were inoculated into LB liquid medium, placed in a shaker at 37°C and 180 rpm / min for 1-2 days, and the absorbance OD value of the bacterial solution was measured using a UV spectrophotometer, and the bacterial concentration was calculated using the OD value.

[0040] The bacterial concentrations of the three strains were adjusted to 1.0×10 8 cfu / mL, and then mixed in equal volumes to construct a synthetic bacterial colony.

[0041] 4. Pot Plant Test

[0042] A mixture of equal volumes of three strains was selected as a synthetic bacterial community, and macadamia seedlings were used as the research object to study the effect of the synthetic bacterial community on the growth of macadamia seedlings

[0043] 4.1 Experimental setup

[0044] Treatment 1: clean water;

[0045] Treatment 2: X17, bacterial concentration 1.0×10 8 cfu / mL;

[0046] Treatment 3: X62, bacterial concentration 1.0×10 8 cfu / mL;

[0047] Treatment 4: Z27, bacterial concentration 1.0×10 8 cfu / mL;

[0048] Treatment 5: X17+X62, mixed in equal volumes, concentration of each bacterium 1.0×10 8 cfu / mL;

[0049] Treatment 6: X17+Z27, mixed in equal volumes, concentration of each bacterium 1.0×10 8 cfu / mL;

[0050] Treatment 7: X62+Z27, mixed in equal volumes, concentration of each bacterium 1.0×10 8 cfu / mL;

[0051] Treatment 8: X17+X62+Z27, mixed in equal volumes, concentration of each bacterium 1.0×10 8 cfu / mL.

[0052] Six-month-old macadamia seedlings with consistent growth were planted in pots, with one seedling per pot, and each pot was filled with 6 kg of fresh soil containing 0.8% organic fertilizer. The planted seedlings were randomly divided into 8 groups, and the 8 groups of seedlings corresponded to treatments 1 to 8, with 30 seedlings in each group. After one week of planting, treatments 1 to 8 corresponded to one group of seedlings, and each pot was irrigated with 50 mL, once every 2 months, for a total of 8 times. Among them, the organic fertilizer was purchased from Guangdong Jinfanwan Organic Agriculture Development Co., Ltd.'s biological organic fertilizer, with an organic matter ≥40.0%, implementation standard: NY884-2012, registration certificate number: Microbial Fertilizer (2023) Standard No. (1111).

[0053] 4.2 Determination of potted seedling indicators

[0054] 4.2.1 The growth indicators of seedlings such as plant height, stem diameter and number of leaves were measured every two months. The results of the measurement when the seedlings grew to 18 months are shown in Table 1 (the data are average values). The measurement method is as follows:

[0055] Plant height: Use a steel measuring tape to measure the distance from the root collar to the top of the plant.

[0056] Stem diameter: Use a vernier caliper to measure the stem diameter 10 cm away from the root neck.

[0057] Number of leaves: The number of leaves per seedling in each treatment was recorded.

[0058] Root length: At the end of the potting period, roots of 5 plants from each treatment were collected and taproot length was measured using a steel measuring tape.

[0059] Table 1 Effects of the synthetic bacterial flora of the present invention on plant height, stem diameter, leaf number and root length of macadamia seedlings

[0060] Different treatment Plant height (cm) Number of leaves (pieces / plant) Stem diameter (mm) Taproot length (cm) Process 1 61.99 57 10.79 24.82 Process 2 62.32 58 10.93 25.03 Process 3 64.19 59 11.02 26.19 Process 4 62.67 61 10.88 25.83 Process 5 65.82 60 11.09 28.19 Process 6 66.39 62 11.13 29.01 Process 7 68.19 61 11.08 27.39 Process 8 82.68 64 11.23 30.31

[0061] As shown in Table 1, treatment 8 is superior to treatments 1 to 7 in terms of plant height, number of leaves, stem thickness and root length, indicating that the synthetic bacterial flora of the present invention can promote the growth of macadamia plants.

[0062] 4.2.2 When the plants grew to 18 months old, 5 plants from each treatment were collected and the fresh weight of the aboveground and underground biomass of the macadamia nut seedlings was weighed using an electronic balance. The plants were then dried at 80℃ to constant weight (24h) and the dry weight of the aboveground and underground biomass of the plants was weighed respectively. The results are shown in Table 2 (data are average values).

[0063] Table 2 Effect of the synthetic bacterial flora of the present invention on the biomass of macadamia seedlings

[0064]

[0065] As shown in Table 2, the aboveground biomass and underground biomass of treatment 8 were higher than those of treatments 1 to 7, indicating that the synthetic bacterial community of the present invention can promote the accumulation of macadamia nut biomass.

[0066] 4.2.3 When the seedlings grew to 18 months old, the physical, chemical and nutrient parameters of the soil of macadamia nut seedlings were measured using a continuous flow analyzer (AutoAnalyzer 3, SEALAnalytic, Germany). The results are shown in Table 3 (data are average values).

[0067] Table 3 Effects of the synthetic bacterial flora of the present invention on soil physicochemical properties and nutrients of macadamia seedlings

[0068]

[0069]

[0070] As shown in Table 3, the contents of soluble organic nitrogen, nitrate nitrogen and ammonium nitrogen in the soil of treatment 8 are all higher than those in treatments 1 to 7. This indicates that the synthetic bacterial flora of the present invention can activate the soil and increase the contents of soluble organic nitrogen, ammonium nitrogen and liquid nitrogen in the soil.

[0071] 4.2.4 When the seedlings grew to 18 months old, the soil enzyme activity of macadamia nut seedlings was measured using the MUB fluorescence spectrophotometer. The results are shown in Table 4 (data are average values).

[0072] Table 4 Effects of the synthetic bacterial flora of the present invention on soil enzyme activities of macadamia seedlings

[0073]

[0074] As shown in Table 4, the enzyme activities of β-glucosidase, β-acetylglucosamidosidase and acid phosphatase in the soil of treatment 8 are all higher than those in treatments 1 to 7. This indicates that the synthetic bacterial consortium of the present invention can activate the soil and increase the enzyme activities of β-glucosidase, β-acetylglucosamidosidase and acid phosphatase in the soil.

[0075] 4.2.5 When the macadamia seedlings were grown to 18 months old, the soil microbial biomass, carbon, nitrogen and phosphorus indicators were measured. The results are shown in Table 4 (data are average values). The measurement method is as follows:

[0076] Sampling fumigation method: The organic carbon in the fumigated and non-fumigated soil samples was extracted with 0.50 mol / L potassium sulfate solution, and the extract was determined using an organic carbon analyzer.

[0077] Table 5 Effects of the synthetic bacterial flora of the present invention on soil microbial biomass carbon, nitrogen and phosphorus of macadamia seedlings

[0078]

[0079] As shown in Table 5, the carbon, nitrogen and phosphorus contents of soil microbial biomass in treatment 8 were all higher than those in treatments 1 to 7. This indicates that the synthetic bacterial flora of the present invention can activate the soil and increase the carbon, nitrogen and phosphorus contents of soil microbial biomass.

[0080] In summary, the synthetic bacterial community constructed by the present invention can promote the growth of macadamia plants and the accumulation of biomass, and at the same time increase the content of soluble organic nitrogen, ammonium nitrogen and liquid nitrogen in the soil, increase the enzyme activity of β-glucosidase, β-acetylglucosaminidase and acid phosphatase, and increase the soil microbial biomass carbon, nitrogen and phosphorus, which can provide support for the cultivation and economic development of macadamia nuts.

[0081] The foregoing description of specific exemplary embodiments of the present invention is for the purpose of illustration and demonstration. These descriptions are not intended to limit the present invention to the precise form disclosed, and it is clear that many changes and variations can be made based on the above teachings. The purpose of selecting and describing the exemplary embodiments is to explain the specific principles of the present invention and its practical application, so that those skilled in the art can realize and utilize various different exemplary embodiments of the present invention and various different selections and changes. The scope of the present invention is intended to be limited by the claims and their equivalents.

Claims

1. A synthetic bacterial community, characterized in that: The synthetic bacterial community is formed by mixing equal volumes of Bacillus subtilis X17, Pseudomonas fluorescens X62 and Trichoderma harzianum Z27; wherein the concentrations of Bacillus subtilis X17, Pseudomonas fluorescens X62 and Trichoderma harzianum Z27 are all 1.0×10 8 cfu / mL; The Bacillus subtilis X17, taxonomically known as Bacillus subtilis X17 ， It was deposited in Guangdong Microbiological Culture Collection Center on August 7, 2024, with the deposit number GDMCC No: 64986; The Pseudomonas fluorescens X62 is taxonomically named Pseudomonas fluorescens X62 ， It was deposited in Guangdong Microbiological Culture Collection Center on August 7, 2024, with the deposit number GDMCC No: 64987; The Trichoderma harzianum Z27 is taxonomically named Trichoderma harzianum Z27 ， It was deposited in Guangdong Provincial Microbiological Culture Collection Center on August 7, 2024, with the deposit number GDMCC No: 64988.

2. Use of the synthetic bacterial flora according to claim 1 in promoting the growth of macadamia nuts.

3. A method for constructing a synthetic bacterial community that promotes the growth of macadamia nuts, characterized in that: The following steps are involved: S1. Collect healthy rhizosphere soil, prepare healthy rhizosphere soil suspension, dilute it with sterile water for bacterial and fungal culture, and separate and purify the well-growing fungi and bacteria for culture; S2. Conduct plate confrontation tests on isolated and purified fungi and bacteria with good growth, select bacteria and fungi with no antagonistic effects for molecular biological identification, and select strains reported in the literature to promote crop growth to construct synthetic bacterial communities; S3. Verify the growth-promoting effect of synthetic flora on crops through pot experiments; Wherein, in step S2, the strains screened for promoting crop growth are Bacillus subtilis X17, Pseudomonas fluorescens X62 and Trichoderma harzianum Z27, wherein, The Bacillus subtilis X17, taxonomically known as Bacillus subtilis X17 ， It was deposited in Guangdong Microbiological Culture Collection Center on August 7, 2024, with the deposit number GDMCC No: 64986; The Pseudomonas fluorescens X62 is taxonomically named Pseudomonas fluorescens X62 ， It was deposited in Guangdong Microbiological Culture Collection Center on August 7, 2024, with the deposit number GDMCC No: 64987; The Trichoderma harzianum Z27 is taxonomically named Trichoderma harzianum Z27 ， It was deposited in Guangdong Microbiological Culture Collection Center on August 7, 2024, with the deposit number GDMCC No: 64988; In step 2, the constructed synthetic bacterial community is composed of equal volumes of Bacillus subtilis X17, Pseudomonas fluorescens X62 and Trichoderma harzianum Z27; the concentrations of Bacillus subtilis X17, Pseudomonas fluorescens X62 and Trichoderma harzianum Z27 are all 1.0×10 8 cfu / mL.

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