A synthetic bacterial community for preventing and treating macadamia decline disease 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 for preventing and treating macadamia nut decay disease, and a construction method and application thereof. An application of a synthetic bacterial community in preventing and treating macadamia nut decay disease, wherein the synthetic bacterial community is formed by mixing equal volumes of Bacillus subtilis X17, Pseudomonas fluorescens X62 and Trichoderma harzianum Z27; the pathogen of the macadamia nut decay disease is Pseudomonas J1 and / or Cladosporium J2. The present invention isolates and screens strains from healthy rhizosphere soil, and conducts a pairwise confrontation test on the well-growing strains to screen out three strains that have no antagonistic effect between the two and have an inhibitory effect on the pathogen of macadamia nut decay disease. Molecular biological identification determines that the three strains are: 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 effectively prevent and treat macadamia nut decay disease, 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 in particular relates to a synthetic bacterial community for preventing and treating macadamia nut decline disease, and a construction method and application thereof. Background Art

[0002] Macadamia nuts are favored by the mass market for their rich nutritional value and medicinal value, and are one of the most popular nuts in the world. With the increasing demand for macadamia nuts in the market, China's macadamia nut industry has flourished, and the planting area has increased year by year. As of 2020, China's macadamia nut planting area reached 268,700 hectares, accounting for more than 70.4% of the world's planting area, making it the country with the largest planting area and the fastest growth rate in the world. However, with the continuous expansion of macadamia nut planting, the accompanying fruit tree diseases have also continued to occur, seriously affecting the healthy development of the macadamia nut industry. There are many types of macadamia nut diseases, and the diseases and pathogens in different regions are different. Among them, macadamia decline is one of the most serious soil-borne diseases of macadamia nuts. Macadamia decline is caused by a variety of pathogens, and is mainly manifested in root rot, leaf browning, branch and seedling wilting and other symptoms, posing a huge threat to the industrial development of macadamia nuts.

[0003] Biological control has become the most promising method for the control of soil-borne plant diseases. For example, a variety of biological control agents such as Bacillus, Pseudomonas, Trichoderma, Streptomyces, Flavobacteria, Enterobacter, Actinomycetes, Serratia, Alcaligenes and Klebsiella have been widely used to control various pathogens 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 long-term colonization and biocontrol functions in rhizosphere soils. These synthetic microbial communities work by providing antibiotics, secondary metabolites, enzymes and other compounds that have inhibitory effects on pathogens. Although the research and development of biocontrol agents for plant pathogens has achieved a lot of application results, there are few studies on the biological control of woody diseases, especially macadamia decline disease. Therefore, it is urgent to develop effective synthetic bacterial flora to prevent and control macadamia decline disease.

[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 preventing and treating macadamia nut decline disease, and a construction method and application thereof.

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

[0007] A use of a synthetic bacterial community in preventing and treating macadamia nut decline disease, wherein the synthetic bacterial community is formed by mixing equal volumes of Bacillus subtilis X17, Pseudomonas fluorescens X62 and Trichoderma harzianum Z27; the pathogenic bacteria of the macadamia nut decline disease are Polytrichomonas J1 and / or Cladosporium J2.

[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] 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.

[0012] The Pestalotiopsis microspore J1, whose taxonomic name is Pestalotiopsis microspore J1, was deposited in Guangdong Microbiological Culture Collection Center on August 7, 2024, with the deposit number of GDMCC No: 64984;

[0013] The Cladosporium sp. J2, with the taxonomic name Cladosporium sp. J2, was deposited in the Guangdong Provincial Microbiological Culture Collection Center on August 7, 2024, with the deposit number GDMCC No: 64985.

[0014] The second object of the present invention is to provide a method for constructing a synthetic bacterial flora for preventing and controlling macadamia nut decline disease, comprising the following steps:

[0015] S1. Collect rhizosphere soil infected with macadamia nut decline disease, prepare diseased rhizosphere soil suspension, dilute with sterile water for bacterial and fungal culture, and isolate, purify and identify the pathogen of macadamia nut decline disease;

[0016] S2. Collect healthy rhizosphere soil, prepare healthy rhizosphere soil suspension, dilute it with sterile water, culture bacteria and fungi, separate, purify and screen cultivable fungi and bacteria; then screen fungi and bacteria that have inhibitory effects on macadamia decline disease pathogens, conduct pairwise plate confrontation tests on the screened fungi and bacteria, select bacteria and fungi that have antagonistic effects on pathogens and are not antagonistic to each other, and then conduct molecular biological identification to construct a synthetic bacterial community;

[0017] S3. Verify the antibacterial effect of the synthetic bacterial community through pot plant experiments.

[0018] More specifically, in step S1, the pathogens of macadamia decline disease were identified as Pseudomonas J1 and Cladosporium J2, wherein:

[0019] The Pestalotiopsis microspore J1, whose taxonomic name is Pestalotiopsis microspore J1, was deposited in Guangdong Microbiological Culture Collection Center on August 7, 2024, with the deposit number of GDMCC No: 64984;

[0020] The Cladosporium sp. J2, with the taxonomic name Cladosporium sp. J2, was deposited in the Guangdong Provincial Microbiological Culture Collection Center on August 7, 2024, with the deposit number GDMCC No: 64985.

[0021] More specifically, in step S2, the strains screened for inhibitory effects on the pathogen of macadamia nut decline disease are Bacillus subtilis X17, Pseudomonas fluorescens X62 and Trichoderma harzianum Z27, wherein the Bacillus subtilis X17, with a taxonomic name of Bacillus subtilis X17, was deposited in Guangdong Provincial Microbiological Culture Collection Center on August 7, 2024, with a deposit number of GDMCC No: 64986;

[0022] 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;

[0023] 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.

[0024] 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.

[0025] 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.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] The present invention isolates and screens strains from healthy rhizosphere soil, and conducts a pairwise confrontation test on the well-growing strains, and screens out three strains that have no antagonistic effect between each other and have an inhibitory effect on the pathogenic bacteria of macadamia nut decline disease. Molecular biological identification determined that the three strains were: 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 effectively prevent and treat macadamia nut decline disease, and can provide support for the cultivation and economic development of macadamia nuts.

[0028] Collection Information

[0029] Pestalotiopsis microspore J1, with the taxonomic name Pestalotiopsis microspore J1, was deposited in Guangdong Microbial 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: 64984.

[0030] Cladosporium sp. J2, with the taxonomic name Cladosporium sp. J2, was deposited in the Guangdong Microbial 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 GDMCCNo: 64985.

[0031] 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.

[0032] Pseudomonas fluorescens X62, with the 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.

[0033] 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

[0034] Figure 1 The culture and microscopic morphological characteristics of the pathogens J1 and J2 of the present invention;

[0035] Figure 2 is a systematic development tree of the pathogen J1 of the present invention;

[0036] Figure 3 is the phylogenetic tree of the pathogen J2 of the present invention;

[0037] Figure 4 The inhibitory effect of the antagonistic bacteria X17 and X62 of the present invention on the pathogen J1;

[0038] Figure 5 The inhibitory effect of the antagonistic fungus X27 of the present invention on pathogens J1 and J2;

[0039] Figure 6 Shown are the morphological characteristics and phylogenetic tree of the three strains of the present invention. DETAILED DESCRIPTION

[0040] 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.

[0041] 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.

[0042] 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.

[0043] 1. Isolation, purification and identification of the pathogen causing macadamia decline disease

[0044] 1.1 Isolation and purification of pathogens

[0045] The rhizosphere soil of macadamia decline disease was collected, 5 g was weighed into a sterile conical flask, 45 mL of sterile water was added to make a suspension, and the suspension was shaken at 160 r / min for 1 h. Then 10 mL of the suspension was drawn into a centrifuge tube and gradiently diluted to 10 -7 Take 0.1mL10 -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 2-3 days. Two pathogenic fungi were isolated and recorded as J1 and J2, which were purified and cultured until they covered the entire PDA medium.

[0046] 1.2 Morphological characteristics of pathogens

[0047] The microbial morphology of J1 and J2 was observed respectively. The culture and microscopic morphological characteristics of J1 and J2 are shown in Figure 1 .

[0048] It was observed that the mycelium of J1 was white, with feathery or coarse velvety surface and light yellow or milky white on the reverse side. After 7 days of culture on LB medium, it covered the entire plate, with a diameter of more than 90 mm. It was white, coarse velvety or cotton-like, with small black clay particles visible on the surface, which were conidiophores and conidia, and the reverse side was soybean yellow.

[0049] The colony of J2 on PDA medium was flocculent, with a black center surrounded by a layer of yellow hyphae, yellow-white edges, and a bulge in the center.

[0050] 1.3 Molecular Biological Identification

[0051] DNA was extracted using the PowerSoil DNA kit (Mobio Laboratories, Carlsbad, CA, USA), and the amplified PCR products were subjected to agarose gel electrophoresis (2 μL sample + 6 μL bromophenol blue) at 300 V for 12 min to obtain the identification gel map. ITS rDNA amplicon sequencing was used, and the spliced ​​sequences were aligned in the NCBI database (blast.ncbi.nlm.nih.gov) to construct a phylogenetic tree (see Figure 2 and Figure 3 ) to determine the taxonomic status of the pathogen.

[0052] Results: Molecular biological identification showed that J1 and J2 were Pestalotiopsis microspore and Cladosporium sp., respectively. Their base sequences are shown in SEQ ID No.1 and SEQ ID No.2.

[0053] 2. Isolation, purification and identification of pathogenic bacteria antagonistic strains

[0054] 2.1 Isolation and purification of pathogen antagonistic strains

[0055] 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.

[0056] 2.2 Screening of antagonistic fungi and bacteria

[0057] The purified bacteria and fungi were subjected to plate confrontation tests with pathogens J1 and J2, respectively, the colony diameters of the pathogens were measured and the inhibition rates were calculated, and then antagonistic strains with high efficiency in inhibiting the pathogens were screened out.

[0058] Inhibition rate = (control pathogen colony diameter - test pathogen colony diameter) / control pathogen colony diameter × 100%.

[0059] The results showed that 22 and 27 bacteria with antagonistic effects on pathogens J1 and J2 were screened out respectively. However, due to the slow growth of J2, it was impossible to screen bacteria with obvious antagonistic effects on J2. Then rescreening was carried out, and the results are shown in Table 1.

[0060] In addition, the results showed that 56 and 47 fungi were antagonistic to pathogens J1 and J2, respectively, and then rescreened. The results are shown in Table 2.

[0061] Table 1 Growth inhibition rate of antagonistic bacteria on pathogens

[0062]

[0063] As shown in Table 1, 10 bacterial strains with strong antagonistic effects on pathogen J1 were screened out, and the inhibition rates of these 10 antagonistic bacteria on pathogen J1 were all over 55%.

[0064] Table 2 Growth inhibition rate of antagonistic fungi on pathogens

[0065]

[0066] As shown in Table 2, 14 and 13 fungi with strong antagonistic effects on pathogens J1 and J2 were screened out respectively. The inhibition rates of the antagonistic fungi on pathogen J1 were more than 55%, and the inhibition rates on pathogen J2 were more than 45%.

[0067] 2.3 Tablet confrontation

[0068] Antagonistic fungi and bacteria were tested in pairs. The results of the confrontation test are shown in Figure 4 and Figure 5 .

[0069] After pairwise confrontation tests, two antagonistic bacteria and one antagonistic fungus were screened out. The antagonistic bacteria were numbered X17 and X62, and the antagonistic fungus was numbered Z27.

[0070] 2.4 Identification of antagonistic fungi and bacteria

[0071] DNA was extracted using the PowerSoil DNA kit (Mobio Laboratories, Carlsbad, CA, USA), and 16S / ITS rDNA amplicon sequencing was performed to clarify the taxonomic status of the antagonistic strains.

[0072] Through molecular biological identification: X17 is Bacillus subtilis, X62 is Pseudomonasfluorescens, and Z27 is Trichoderma harzianum. The morphological characteristics and phylogenetic tree of the three strains are shown in Figure 6 , and its base sequences are shown in SEQ ID No.3, SEQ ID No.4 and SEQ IDNo5.

[0073] 3. Construction of synthetic bacterial communities

[0074] 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.

[0075] 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.

[0076] 4. Pot Plant Test

[0077] A mixture of equal volumes of three strains was selected as a synthetic bacterial flora, and macadamia seedlings were used as the research objects to study the control effect of the synthetic bacterial flora on macadamia decline disease.

[0078] 4.1 Experimental setup

[0079] Treatment 1: sterile PDA liquid medium (PDA liquid medium formula: 300 g potato, 20 g glucose, 1 L water);

[0080] Treatment 2: X17, bacterial concentration 1.0×10 8 cfu / mL;

[0081] Treatment 3: X62, bacterial concentration 1.0×10 8 cfu / mL;

[0082] Treatment 4: Z27, bacterial concentration 1.0×10 8 cfu / mL;

[0083] Treatment 5: X17+X62, mixed in equal volumes, concentration of each bacterium 1.0×10 8 cfu / mL;

[0084] Treatment 6: X17+Z27, mixed in equal volumes, concentration of each bacterium 1.0×10 8 cfu / mL;

[0085] Treatment 7: X62+Z27, mixed in equal volumes, concentration of each bacterium 1.0×10 8 cfu / mL;

[0086] Treatment 8: X17+X62+Z27, mixed in equal volumes, concentration of each bacterium 1.0×10 8 cfu / mL.

[0087] The six-month-old macadamia seedlings with the same growth were planted in pots, with one seedling in each 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, with 30 seedlings in each group. Among them, the organic fertilizer was purchased from Guangdong Jinfanwan Organic Agriculture Development Co., Ltd. The organic matter was ≥40.0%, the implementation standard was: NY884-2012, and the registration certificate number was: Microbial Fertilizer (2023) Standard (1111).

[0088] One week after the seedlings were colonized, 8 groups of seedlings were irrigated with 50 mL of spore suspension of pathogens J1 and J2, respectively. The concentration of the spore suspension was 1.0×10 6 cfu / mL. After 5 days, one group of seedlings were treated with treatments 1 to 8 respectively, and each pot was irrigated with 150 mL. The pathogen and treatments 1 to 8 were irrigated once every 2 months, for a total of 8 times. During the period, the soil was kept moist.

[0089] 4.2 Statistics of incidence

[0090] The disease incidence of potted seedlings was statistically analyzed every three months to analyze the disease trend over time and reveal the disease dynamics of macadamia decline disease among different treatment groups.

[0091] The diseased plants were graded according to the disease grading standard of Chen et al. (2013): Grade 0: healthy plant growth, no diseased leaves; Grade 1: 1-25% of leaves withered; Grade 2: 26-50% of leaves withered; Grade 3: 51-75% of leaves withered; Grade 4: 76-100% of leaves withered.

[0092] Disease index = [Σ(number of cases at each level × corresponding level) / (total number of plants surveyed × highest level)] × 100

[0093] The disease index at 18 months of age is shown in Table 3.

[0094] Table 3 The control effect of the synthetic bacterial flora of the present invention on macadamia seedling decline disease

[0095] Different treatment Disease index (%) Process 1 41.0 Process 2 31.2 Process 3 36.4 Process 4 33.9 Process 5 28.4 Process 6 19.3 Process 7 30.5 Process 8 7.5

[0096] As shown in Table 3, the disease index of macadamia seedling decline disease in treatment 8 was 7.5%, which was significantly lower than that in treatments 1 to 7. This indicates that the synthetic bacterial flora constructed by the present invention can effectively prevent and control the occurrence of macadamia decline disease.

[0097] In summary, the synthetic bacterial community constructed by the present invention can effectively prevent and control the occurrence of macadamia decline disease, and can provide support for the cultivation and economic development of macadamia.

[0098] 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. An application of a synthetic bacterial flora in preventing and treating macadamia nut decline disease, characterized in that: The synthetic bacterial community is composed of equal volumes of Bacillus subtilis X17, Pseudomonas fluorescens X62 and Trichoderma harzianum Z27; the pathogen of macadamia nut decline disease is Polytrichomoniasis J1 and / or Cladosporium J2; wherein, The concentrations of Bacillus subtilis X17, Pseudomonas fluorescens X62 and Trichoderma harzianum Z27 were 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 Microbiological Culture Collection Center on August 7, 2024, with the deposit number GDMCC No: 64988; The described polytrichomonas fungus J1 is taxonomically named Pestalotiopsis microspore J1 ， It was deposited in Guangdong Microbiological Culture Collection Center on August 7, 2024, with the deposit number GDMCC No: 64984; The Cladosporium species J2 is taxonomically named Cladosporium sp.J2 ， It was deposited in Guangdong Provincial Microbiological Culture Collection Center on August 7, 2024, with the deposit number GDMCC No: 64985.

Images