Applications of cladosporium sp. Strain and synthetic flora thereof in plant growth promotion and bacterial wilt prevention and control

Abstract

The invention relates to the technical field of microorganisms, in particular to application of a cladosporium spp. Strain and a synthetic flora thereof in plant growth promotion and bacterial wilt prevention and treatment, the cladosporium spp. HX2 is a new strain, has a good symbiotic relationship with tomatoes, has a good prevention effect on tomato bacterial wilt, and has a good application prospect in plant growth promotion and bacterial wilt prevention and treatment. After the bacterial strain is mixed with congeneric strains LJ1, MS2 and LC3 to prepare a cladosporium bacterial agent, the growth-promoting and disease-resisting capabilities of the bacterial agent are enhanced, and experiments show that the synthetic flora has a good growth-promoting function on tomatoes and fresh ginger, the yield of the fresh ginger is remarkably increased, and the growth vigor of tomato plants is improved; meanwhile, the synthetic flora also has a good prevention and treatment effect on bacterial wilt of tomatoes, fresh ginger and tobaccos, and can improve the bacterial wilt resistance of plants, so that the strain HX2 and the synthetic flora prepared from the strain HX2 can be used as an anti-microbial agent or a raw material for preparing a bio-organic fertilizer in the later period.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to application of a Cladosporium strain and a synthetic bacterial flora thereof in promoting plant growth and preventing and controlling bacterial wilt. Background Art

[0002] Bacterial wilt is a common soil-borne disease of plants. Its pathogen Ralstonia solanacarum can cause diseases in many plants. In diseased areas, the pathogen Ralstonia solanacarum often remains in the soil. If the sterilization is not thorough, it is easy to cause cross-infection between plants. The pathogen often infects plants from the roots, causing plants to wilt and causing serious economic losses. At present, there are three main countermeasures for bacterial wilt caused by Ralstonia solanacarum: physical control, chemical control and biological control. Physical and chemical control cannot be effectively implemented due to time and space limitations, pesticide residues, environmental pollution and other problems. Biological control uses biocontrol bacteria as the main body, which not only ensures the prevention effect but also meets the requirements of green environmental protection. It is regarded as an important means of sustainable agricultural development. Therefore, screening biocontrol bacteria for the prevention and control of crop bacterial wilt is of great significance to the green development of agriculture.

[0003] At present, the biocontrol agents on the market are mainly Bacillus and Trichoderma products, with relatively single strains, and most of the strains used in production are single strains, which makes it difficult to exert an ideal antagonistic effect on crop bacterial wilt, which has serious and complex differentiation of pathogenic bacteria. In addition, affected by the complex soil environment in the field, most biocontrol strains have poor field colonization ability and cannot create a microecological environment that is conducive to the healthy growth of plants but not conducive to the occurrence of diseases, resulting in unstable control effects of biocontrol bacteria in field applications. Therefore, it is still urgent to discover new biocontrol microorganisms and conduct compound evaluation of strains in production.

[0004] Dark septate endophyte (DSE) refers to a type of small soil fungi that colonizes plants, forms dark septate hyphae and microsclerotia in intercellular spaces and cells, and is non-pathogenic to the host. They live in mutualistic symbiosis with the host, can improve the resistance of plants to biotic and abiotic stresses and promote plant growth. DSE has shown obvious advantages in the field of biocontrol because it can establish a relatively stable symbiotic relationship with the host plant. Studies have found that DSE biocontrol bacteria can enter the body of new plants through plant seed germination and seedling treatment, preferentially occupy ecological niches, and can more stably exert their biocontrol functions. Cladosporium is a dominant genus of DSE distributed in various habitats around the world. It was previously found that strains of this genus have a good control effect on tomato bacterial wilt. Since the disease control mechanisms of different strains are different, mixed use will have an effect of increasing or decreasing the effect. Therefore, it is necessary to compound different strains to find a synthetic bacterial community suitable for broad-spectrum disease resistance and growth promotion. Summary of the invention

[0005] In view of the above, it is necessary to compound different strains to find out synthetic bacterial flora with broad-spectrum disease resistance and growth-promoting effects.

[0006] To achieve the above-mentioned purpose, the new strain of the present application is: Cladosporium phial fungus strain CladophiaphoraguangxienseHX2, the preservation number of the Cladosporium phial fungus strain Cladophiaphoraguangxiense HX2 is CGMCC NO.41498, and the strain classification name is: Cladophiaphora guangxiense is preserved in the General Microbiology Center of China Microorganism Culture Collection Administration, address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, and the preservation date is September 5, 2024.

[0007] The present invention also includes a Cladosporium synthetic bacterial community comprising the Cladosporium strain Cladophiaphora guangxiense HX2, comprising: Cladosporium strain Cladophiaphora sp.LJ1, Cladosporium strain Cladophiaphora sp.MS2 and Cladosporium strain Cladophiaphora immunda LC3;

[0008] The deposit number of the Cladosporium sp. strain Cladophiaphora sp. LJ1 is CGMCC NO.18140; the strain is classified and named as: Cladophiaphora sp. and is deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit date is July 4, 2019. The strain has been disclosed in the patent application document: 201910884589.9.

[0009] The deposit number of the Cladosporium strain Cladophiaphora sp.MS2 is CGMCC NO.20238; the strain is classified and named as: Cladophiaphora sp. and is deposited in the General Microbiology Center of China Culture Collection Administration, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit date is August 10, 2020. The strain has been disclosed in the patent application document: 202011239027.8.

[0010] The deposit number of the Cladosporium strain Cladophiaphora immunda LC3 is CGMCCNO.41540; the strain is classified and named as: Cladophiaphora immunda and is deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit date is October 12, 2024.

[0011] The present invention also includes the use of the Cladosporium strain Cladophiaphora guangxiense HX2 or the synthetic bacterial community of Cladosporium in resisting plant bacterial wilt.

[0012] Furthermore, the plant is: tomato, ginger and / or tobacco.

[0013] The present invention also includes the use of the Cladosporium strain Cladophiaphora guangxiense HX2 or the Cladosporium synthetic flora in promoting plant growth.

[0014] Furthermore, the plant is: tomato and / or ginger.

[0015] The present invention also includes a method for preparing the synthetic flora of Cladosporium phialum, which comprises: inoculating the strains into PDA plates for activation and then inoculating into PDB liquid culture medium, collecting mycelium by filtering with sterile gauze, washing the mycelium with sterile water and squeezing out the water, mixing the four kinds of mycelium in equal mass ratios and preparing them into a concentration of 5×10 5 CFU / mL of bacterial solution.

[0016] The present invention also includes a method for cultivating plants using the Cladosporium phialina strain Cladophiaphora guangxiense HX2 or the Cladosporium synthetic flora or the synthetic flora prepared by the method, the method comprising: inoculating a bacterial solution containing the Cladosporium synthetic flora into the plants, and then cultivating the plants.

[0017] Furthermore, the inoculation method is: irrigating the plant roots with the bacterial liquid of the synthetic flora of Cladosporium; or soaking the plant roots in the bacterial liquid of the synthetic flora of Cladosporium; or soaking the plant seeds in the bacterial liquid of the synthetic flora of Cladosporium.

[0018] The present invention has the following beneficial effects:

[0019] 1. The Cladosporium phial strain Cladophiaphora guangxiense HX2 of the present invention belongs to a new species of Cladosporium. It has been verified that the strain has a good symbiotic effect with tomatoes and can effectively prevent the occurrence of tomato bacterial wilt. The strain is mixed with strains of the same genus LJ1, MS2 and LC3 to obtain a synthetic bacterial community of the genus Cladosporium. We found that the synthetic bacterial community can symbiotically coexist with more plants and enhance the disease resistance of plants. Experiments show that the synthetic bacterial community has an obvious growth-promoting function on tomatoes and ginger compared with the single strain HX2, significantly increases the ginger yield, and improves the growth of tomato plants; at the same time, the synthetic bacterial community also has a good prevention and control effect on bacterial wilt of plants such as tomatoes, ginger and tobacco, and can more significantly improve the plant's resistance to bacterial wilt, indicating that the new strain HX2 and the synthetic bacterial community prepared by the strain can be used as raw materials for the later development of antimicrobial agents or the preparation of biological organic fertilizers. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the morphological identification result of strain LC3.

[0021] Figure 2 This is the phylogenetic tree of strain LC3 constructed based on the ITS sequence.

[0022] Figure 3 This is the morphological identification result of strain HX2.

[0023] Figure 4 This is the phylogenetic tree of strain HX2 constructed based on ITS sequence.

[0024] Figure 5 The tomato-strain HX2 symbiont and its colonization in the roots of tomato seedlings; where a is the co-cultivation result of strain HX2 and tomato seedlings; b is the dry weight of tomato seedlings; cd is the intracellular colonization (black arrow) and intercellular colonization (red arrow) of HX2 hyphae.

[0025] Figure 6 This is a graph showing the potted plant protection effect of strain HX2 against tomato bacterial wilt.

[0026] Figure 7 These are the results of the ginger potted plant experiment with the synthetic bacterial community of Cladosporium; Figure A is the plant height experiment result, Figure B is the bacterial wilt disease index, Figure C is the bacterial wilt prevention effect result, and Figure D is the bacterial wilt incidence result.

[0027] Figure 8 These are the growth results of the ginger potted plant experiment with the synthetic bacterial community of Cladosporium. The upper picture shows the CK potted plant without the synthetic bacterial community, and the lower picture shows the potted plant of the experimental group inoculated with the synthetic bacterial community. The arrows in the pictures point to the new seedlings that have grown again.

[0028] Fig. 9 This is a graph showing the growth results of ginger field experiments inoculated with the synthetic bacterial community of Cladosporium; among them, CK is the control group not inoculated with the synthetic bacterial community, and T4 is the experimental group inoculated with the synthetic bacterial community.

[0029] Fig.10 This is a graph of the ginger yield results of the field experiment with the synthetic flora of Cladosporium. From left to right, they are: tiller number experiment results, plant width experiment results, underground root tuber height experiment results, biological yield experiment results and economic yield experiment results.

[0030] Fig.11 This is a graph showing the growth characteristics of field ginger at the harvest stage of a field experiment with a synthetic flora of Cladosporium.

[0031] Fig.12 These are the results of a potted experiment on tomatoes using the synthetic bacterial community of Cladosporium. Figure a shows the experimental results for the number of tomato leaves, and figure b shows the experimental results for the tomato plant height.

[0032] Fig.13 This is a graph showing the growth results of a tomato pot experiment with the synthetic bacterial community of Cladosporium. CK is the control group not inoculated with the synthetic bacterial community, and T4 is the experimental group inoculated with the synthetic bacterial community.

[0033] Fig.14 These are the experimental results of the synthetic bacterial community of Cladosporium on tomato bacterial wilt; among them, Figure a is the incidence of bacterial wilt, Figure b is the disease index of bacterial wilt, and Figure c is the prevention effect of bacterial wilt.

[0034] Fig.15 These are the experimental results of the synthetic bacterial community of Cladosporium on tobacco bacterial wilt; among them, Figure a shows the incidence of bacterial wilt, and Figure b shows the prevention effect of bacterial wilt.

[0035] Fig.16These are the results of a tobacco pot experiment inoculated with bacterial wilt pathogens for 13 days; CK is the control group not inoculated with synthetic flora, and T4 is the experimental group inoculated with synthetic flora.

[0036] Fig.17 These are the results of a tobacco pot experiment inoculated with bacterial wilt pathogens for 23 days; CK is the control group not inoculated with synthetic flora, and T4 is the experimental group inoculated with synthetic flora.

[0037] Biomaterial Deposit Information

[0038] Four strains were used in the present application examples, and their deposit information is as follows:

[0039] ① Cladophiaphora sp. strain LJ1, whose classification name is: Cladophiaphora sp., Chinese classification name is: Cladophiaphora sp., and the preservation number is CGMCC NO.18140; the strain is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, the preservation date is July 4, 2019, and the strain has been disclosed in the patent application document: 201910884589.9.

[0040] ② Cladophiaphora sp. strain MS2, whose classification name is: Cladophiaphora sp., Chinese classification name is: Cladophiaphora sp., and the preservation number is CGMCC NO.20238; the strain is deposited in the General Microbiology Center of China Culture Collection Administration, address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, the preservation date is August 10, 2020, and the strain has been disclosed in the patent application document: 202011239027.8.

[0041] ③ Cladophiaphora immunda strain LC3, its classification name is: Cladophiaphoraimmunda, its Chinese classification name is: Cladosporium immunda, and its preservation number is CGMCC NO.41540; the strain is deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, and the preservation date is October 12, 2024.

[0042] ④ Cladophiaphora guangxiense strain HX2, its classification name is: Cladophiaphora guangxiense, its Chinese classification name is: Cladophiaphora guangxiense, and its preservation number is CGMCC NO.41498; the strain is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, and the preservation date is September 5, 2024. DETAILED DESCRIPTION

[0043] All features disclosed in this specification, or steps in all methods or processes disclosed, except mutually exclusive features and / or steps, can be combined in any manner.

[0044] Any feature disclosed in this specification (including any appended claims and abstract), unless otherwise stated, is merely an example of a series of equivalent or similar features.

[0045] The culture medium and formula used in the examples of this application are as follows:

[0046] Potato dextrose agar medium (PDA): 200 g potato, 20 g glucose, 15 g agar, 1000 mL distilled water, sterilized by high pressure steam at 121°C for 30 min.

[0047] Potato dextrose liquid medium (PDB): 200 g potato, 20 g glucose, 1000 mL distilled water, sterilized at 121°C for 30 min.

[0048] LA agar medium (Luria-Agar): Tryptone 10 g, yeast extract 5 g, sodium chloride (NaCl) 10 g, agar 15 g, distilled water 1000 mL, pH 7.0, high-pressure steam sterilization at 121°C for 30 min.

[0049] LB liquid medium (Luria-Bertani): Tryptone 10 g, yeast extract 5 g, sodium chloride (NaCl) 10 g, distilled water 1000 mL, pH 7.0, high-pressure steam sterilization at 121°C for 30 min.

[0050] Malt extract medium (CMMY): 10.0 g malt extract, 2.0 g yeast extract, 8.5 g corn flour agar, 7.5 g agar, 1000 mL distilled water, sterilized at 121°C with high pressure steam for 30 min. Used to observe the colony morphology of Cladosporium sporeans.

[0051] Oatmeal medium (OMA): MgSO4 7H20 1.0g, KH2PO4 15g, NaNO3 1.0g, oatmeal powder 10g, agar powder 11g, distilled water 1000mL, 121℃ high pressure steam sterilization for 30min. Used to observe the spore structure of Cladosporium strains.

[0052] 1.5% agar medium (WA): 15 g agar, 1000 mL distilled water, sterilized by high pressure steam at 121°C for 30 min. OMA medium for carrying small pieces.

[0053] Example 1

[0054] This example is the separation and identification of strain LC3 and strain HX2.

[0055] 1. Isolation and identification of Cladosporium LC3.

[0056] 1. Cladosporium LC3 was isolated from sugarcane rhizosphere soil in Guangxi.

[0057] 2. Identification of Cladosporium LC3:

[0058] ① Morphological identification: The strain was inoculated on CMMY medium and the colony morphology was observed after 14 days of culture. At the same time, a sterilized coverslip was placed on WA medium, and a 0.5 cm × 0.5 cm OMA medium was placed in the center of the coverslip. At the same time, the LC3 strain was inoculated on OMA medium and covered with another sterilized coverslip. After culturing at 28°C for 2 weeks, it was placed in a 4°C refrigerator to stimulate spore production. After one week, the coverslip was removed and the morphology of hyphae, spore production structure and conidia was observed under an Olympus BX53 microscope.

[0059] like Figure 1 As shown, the colony grows at a moderate rate on CMMY medium. After 2 weeks of culture at 28°C, the colony is 28 mm in diameter, gray, tapered, round, and black on the back. The hyphae are transparent to light brown, septate, and 2.3-4.1 μm in diameter. Conidiophores are not significantly different from hyphae, slightly thicker than hyphae, 2.6-5.5 μm in diameter, light brown, 1-8 septa, upright, branched or unbranched. The conidial cells are drop-shaped, 1.5-4.2 μm in diameter, often branched, and the conidia on them will leave obvious tooth-like spore scars after falling off. Conidia are single-celled, terminal, often chain-arranged or head-shaped, translucent to light brown, spherical, elliptical to lemon-shaped, spindle-shaped or teardrop-shaped, smooth, and unseptate. Some spores have a hilum at both ends or one end. Non-spherical spores are 3.2-6.4×2.1-3.6μm in size; spherical spores are 2.2-4.2μm in diameter.

[0060] The morphological characteristics of strain LC3 were basically consistent with the description of Cladophialophora immunda by Badali et al. (2008), so the strain was identified as Cladophialophora immunda.

[0061] ② Molecular identification: After the LC3 strain was activated in PDA medium, the mycelium was scraped and the genomic DNA of the strain was extracted using a fungal DNA kit. The extracted DNA was used as a template and the ITS rRNA sequence was amplified by PCR using the universal fungal primers ITS1 and ITS4. The amplification procedure was as follows: after pre-denaturation at 94℃ for 5min; 35 cycles were performed in the order of denaturation at 94℃ for 40s, annealing at 56℃ for 40s, and extension at 72℃ for 1min60s; and then extended at 72℃ for 10min. The PCR amplification product was detected by 0.8% agarose gel electrophoresis and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The obtained sequence was uploaded to NCBI, and the approximate genus-level classification status of the strain LC3 was confirmed by BLAST comparison. Then the ITS sequences of the known species model strains of the genus and the representative strains of its closely related species were downloaded from NCBI. The phylogenetic evolutionary tree was constructed by the neighbor-joining method using MEGA 11.0 to determine the taxonomic status of the target strain. The ITS similarity between strains was calculated by NCBI's BLAST comparison.

[0062] The comparison results in NCBI are as follows Figure 2 As shown, the strain with the highest ITS sequence similarity to strain LC3 is Cladophialophora immunda FMR20179, with a similarity of 99.7% (578 / 580). In the phylogenetic tree constructed based on the ITS sequences of the type strains of known species of the genus and the representative strains of the closely related species of strain LC3, strain LC3 was clustered in a monophyletic evolutionary branch with 13 representative strains of Cladophialophora immunda, including the type strain, with a support rate of 54%. Among them, strain LC3 and FMR20179 from marine sediments (Guerra-Mateo et al., 2024) and CBS126725 from healthy palm stems (Vicente et al., 2014) formed a small evolutionary branch, while the type strain CBS 834.96 was clustered in another small evolutionary branch with two strains related to environmental pollution with a support rate of 100%, and the remaining strains related to plant pathogens or animals were clustered in a third small evolutionary branch with a support rate of 96%. The ITS similarity between strain LC3 and the Cladophialophora immunda model strain CBS 834.96 was 98.1%, which further proved the reliability of strain LC3 identification as Cladophialophora immunda.

[0063] 2. Isolation and identification of Cladosporium HX2.

[0064] 1. Cladosporium phalla HX2 was isolated from the sugarcane rhizosphere soil of Liujia Village, Baili Town, Hengzhou City, Nanning City, Guangxi (109°27′37″N, 22°41′23″E, 60.74 m above sea level), and the sampling time was January 2016.

[0065] 2. Identification of Cladosporium HX2:

[0066] ① Morphological identification: The strain was inoculated on CMMY medium and the colony morphology was observed after 14 days of culture. At the same time, a sterilized coverslip was placed on WA medium, and a 0.5 cm × 0.5 cm OMA medium was placed in the center of the coverslip. At the same time, the HX2 strain was inoculated on the OMA medium and covered with another sterilized coverslip. After culturing at 28°C for 2 weeks, it was placed in a 4°C refrigerator to stimulate spore production. After one week, the coverslip was removed and the morphology of hyphae, spore production structure and conidia was observed under an Olympus BX53 microscope.

[0067] The morphological identification results are as follows Figure 3 As shown: Strain HX2 grew slowly on CMMY, and after 14 days of cultivation at 28°C, the colony diameter reached 18-20 mm. The colonies were roughly round, ranging in color from gray to dark gray, and showed black borders. The colonies had a slightly hard texture and a fluffy surface ( Figure 3 a). HX2 hyphae on OMA are septate, olive to yellow-brown, and 1.3-4.4 μm in diameter (average 3.0±0.7 μm, n=30). Meristematic cells are terminal or lateral, olive to light yellow-brown, rectangular, cylindrical to slightly fusiform, (6.0-17.8)×(3.3-6.4) μm [average (10.7±2.6)×(4.7±0.8) μm, n=44]( Figure 3 bh). Conidia are unicellular, unbranched, occasionally branched, forming short chains containing 2-17 conidia, elliptical, cylindrical to sub-cylindrical, or curled sausage-shaped in chains, olive to yellow-brown, with a diameter of (4.9-10.5) × (4.0-6.4) μm [average (6.7 ± 1.2) × (5.1 ± 0.6) μm, n = 37]. Bottle-shaped bud cells ( Figure 3 i), chlamydospores and sexual states were not observed.

[0068] The morphological characteristics of strain HX2 were similar to those of the genus Cladophialophora. However, strain HX2 exhibited a unique species form characterized by the production of unseptate and dark cylindrical and fusiform conidia, curled and arranged into continuous and unbranched sausage-shaped conidia strings, which was different from any known species of the genus Cladophialophora. Therefore, strain HX2 was described as a new species of the genus Cladophialophora and named after Guangxi, where the strain was located. Therefore, the strain was named Cladophialophora guangxiense.

[0069] ② Molecular identification: After the strain HX2 was activated in PDA medium, the mycelium was scraped and the genomic DNA of the strain was extracted using a fungal DNA kit. Using the extracted DNA as a template, the universal primers ITS1 (5-'TCCGTAGGTGAACCTGCGG-3') and LR5 (5-'TCCTGAGGGAAACTTCG-3') were used to amplify the ITS1-5.8S-ITS4-28S sequence of rDNA; NS1 (5-'GTAGTCATATGCTTGTCTC-3') and NS4 (5-'CTTCCGTCAATTCCTTTAAG-3') were used to amplify the 18S rRNA sequence of rDNA. The amplification procedure was as follows: pre-denaturation at 94℃ for 5min; 35 cycles were performed in the order of denaturation at 94℃ for 40s, annealing at 56℃ for 40s, and extension at 72℃ for 1min 60s; and then extension at 72℃ for 10min. The PCR amplification products were detected by 0.8% agarose gel electrophoresis and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The obtained sequences were uploaded to NCBI, and the approximate genus-level classification status of strain HX2 was confirmed by BLAST comparison. Then the ITS sequences of the known species model strains of the genus and the representative strains of closely related species were downloaded from NCBI. The phylogenetic evolutionary tree was constructed by the neighbor-joining method using MEGA 11.0 to determine the taxonomic status of the target strain. The ITS similarity between strains was calculated by NCBI BLAST comparison, and the results are shown in the figure. Figure 4 As shown: The ITS, LSU and SSU regions of the HX2 strain were BLAST analyzed using the NCBI database (http: / / www.ncbi.nlm.nih.gov / accessed on August 3, 2023). Phylogenetic analysis showed that strains HX2 and Cladophialophora inabaensis (EUCL1) formed a monophyletic group with a support rate of 87%. The similarities of the ITS and LSU sequences of HX2 and Cladophialophora inabaensis (EUCL1) were 92.07% and 98.91%, respectively ( Figure 4). This further proves that strain HX2 is a new species of the genus Cladophialophora.

[0070] Combined with morphological identification, strain HX2 was classified and named Cladophialophora guangxiense.

[0071] Example 2

[0072] This example is a study on the performance of a new strain of Cladosporium HX2, specifically as follows:

[0073] 1. Determination of symbiosis between strain HX2 and tomato:

[0074] In order to verify the symbiosis between strain HX2 and tomato, HX2 was co-cultured with tomato seedlings under sterile conditions for 14 days, and no disease symptoms were shown. Compared with the uninoculated CK, the plant height was higher, the leaves were more stretched, and the growth was better ( Figure 5 a), there was no significant difference in the average dry weight of seedlings between the HX2-treated and control groups ( Figure 5 b). Through sectioning and lactophenol cotton blue staining, it was observed under a microscope that HX2 can colonize the epidermis and cortical cells of tomato roots ( Figure 5 c, d). The re-isolated strain was completely consistent with the original inoculated strain HX2 by ITS sequence alignment. These results indicate that the hyphae of the HX2 strain can penetrate the tomato root cells and establish colonization, confirming the symbiotic relationship between tomato and HX2.

[0075] 2. Effect of strain HX2 on control of bacterial wilt of potted tomatoes:

[0076] It was carried out in the greenhouse of the Institute of Plant Protection of Guangxi Academy of Agricultural Sciences. Germination period of tomato seeds: soak in 60℃ warm water for 2h, remove and place in a culture dish (covered with a wet towel on the top and bottom), and germinate in a light incubator (temperature 28℃, humidity 80℃, no light) for 72h. Seedling period: select plump seeds with consistent bud length, soak them in HX2 bacterial suspension for 30min, soak them in the same amount of sterilized water as the control (CK), sow them in the seedling tray for the first root irrigation with HX2 bacterial suspension, inoculate 10mL of root irrigation for each seedling as root water, and place them on a light culture rack for cultivation (lighting conditions: 16h of light, light intensity 30000lx, open at 6:00 am and close at 22:00 pm); 10d after sowing, the second root irrigation with HX2 bacterial suspension was carried out, and 10mL of root irrigation was carried out for each seedling. Growth period: After 25 days of seedling cultivation, transplant to a pot (20.6cm×17.5cm×15cm) filled with 2 / 3 diseased soil, and irrigate the roots with HX2 suspension for the third time, 10mL per seedling, and irrigate the roots with HX2 suspension for the fourth time 10 days after transplanting, 10mL per seedling, and place in a greenhouse (temperature 30±5℃, relative humidity 80±5%, natural light). Each replicate has 12 tomato seedlings, and each treatment is repeated 3 times. Investigate the disease situation on the 45th day after transplanting, and calculate the incidence rate, disease index and control effect.

[0077] The grading standard of tomato bacterial wilt disease index is as follows: Grade 0, no wilting symptoms; Grade 1, 1%-25% leaf wilting; Grade 2, 26%-50% leaf wilting; Grade 3, 51%-75% leaf wilting; Grade 4, 76%-100% leaf wilting. Incidence rate (%) = (number of diseased plants / total number of plants) × 100; Disease index = 100 × ∑ (number of plants at each disease level × representative value of each level) / (total number of plants surveyed × highest representative value); Control effect (%) = 100 × (control group disease index - treatment group disease index) / control group disease index. The results are shown in Table 1 and Figure 6 As shown:

[0078] Table 1 Control effect of strain HX2 on tomato bacterial wilt

[0079]

[0080] "*" in the table indicates significant difference.

[0081] As shown in Table 1, under potted conditions, the incidence of bacterial wilt in the tomato seedlings transplanted into the soil with bacterial wilt for 45 days after inoculation with HX2 was significantly lower than that in CK, and the disease index was significantly lower than that in CK, with a control efficiency of 72.48%, showing excellent control effect and potential for bacterial wilt resistance. Figure 6 As shown: the growth of tomato plants inoculated with HX2 strain was significantly better than that of the control group not inoculated with HX2 strain, and the tomato plants in the control group not inoculated with HX2 strain showed obvious wilting phenomenon.

[0082] Example 3

[0083] This example is a method for preparing a synthetic bacterial community of Cladosporium using strain HX2, strain LC3 of Example 1, strain LJ1 disclosed in Patent 201910884589.9, and strain MS2 disclosed in Patent 202011239027.8, as follows:

[0084] Cladosporium synthetic flora (hereinafter referred to as T4): strains LJ1, MS2, LC3 and HX2 were inoculated into PDA plates for activation, cultured in a 28°C mold incubator for 10 days, and then the colonies were cut into small blocks with a sterilized scalpel and inoculated into PDB liquid culture medium. After culturing on a shaker at 28°C and a speed of 120 r / min for 10 days, the mycelium was collected by filtration with double-layer sterilized gauze, washed with sterile water three times and squeezed dry, and the four myceliums were mixed at a mass ratio of 1:1:1:1 and prepared with a homogenizer to a concentration of 5×10 5 CFU / mL of bacterial solution.

[0085] This embodiment also includes a method for preparing a pathogenic bacteria liquid: the pathogen (Ralstonia solanacearum) is inoculated onto an LA plate by streaking to activate, a single colony is picked out and inoculated into an LB medium after 2 days, and the culture is carried out on a shaker at 30°C and a speed of 180 r / min for 2 days, and the ginger solanacearum is prepared to an OD of 600 =1 concentration for standby use, and the tobacco solanacearum was prepared to OD 600 =0.2 concentration for standby use.

[0086] Example 4

[0087] This example is the biocontrol effect of the synthetic bacterial consortium of Cladosporium (T4) prepared in Example 3 on ginger bacterial wilt, as follows:

[0088] (1) Potted experiment: After the ginger tissue culture seedlings were hardened from bottle seedlings, they were first treated with a concentration of 5×10 5CFU / mL T4 bacterial solution was soaked for 30 minutes, and the same amount of sterilized water was used as the control (CK). Healthy and uniform ginger tissue culture seedlings were selected, and one plant was transplanted per pot. Each treatment was repeated 3 times, and 12 pots of ginger seedlings were placed in a greenhouse (temperature 30±5℃, relative humidity 90±5%, natural light). The plant height was measured 80 days after transplanting, and then each pot of ginger seedlings was inoculated with 50mL T4 bacterial solution, and the same amount of sterilized water was used as the CK. After the ginger roots were irrigated with T4 bacterial solution for 14 days, the pathogen (Ralstonia solanacarum) was inoculated. After the stem base of the ginger was pierced with a pin with a diameter of 0.4mm, each plant was inoculated with 100mL Gg24 bacterial solution. The disease situation was investigated 10d, 50d and 100d after the pathogen was inoculated, and the incidence rate, disease index and prevention effect were calculated.

[0089] After 80 days of transplanting, the average plant height of ginger seedlings treated with T4 was significantly increased by 58.5% compared with the control, reaching an extremely significant level ( Figure 7 A). Seven days after the pathogen was inoculated, ginger treated with CK showed typical symptoms of bacterial wilt, with dull leaves, gradually withering and turning yellow, curling edges, and finally dying. The aboveground stems were dark purple, and the internal tissues turned brown and rotted, while T4 had no obvious symptoms. Ten days after the pathogen was inoculated, both treatments had become diseased. The survey results at different times showed that the disease index of T4 treatment was lower than that of CK, showing a trend of first increasing and then decreasing, while CK was always on an upward trend, and the difference changed from significant to extremely significant compared with CK ( Figure 7 B). The control effect of T4 showed a trend of decreasing first and then increasing, and finally reached 51.5% (100 days after inoculation of pathogens) Figure 7 C), indicating that it has a certain therapeutic effect on ginger bacterial wilt. The incidence of T4 treatment also showed a trend of first increasing and then decreasing, and finally decreased by 22.2% compared with CK at 100 days after inoculation of pathogens, while the incidence of CK reached 100% 50 days after inoculation of pathogens and remained high. ( Figure 7 D). In the late stage of disease, 44% of the plants in the T4 treatment died, and 50% of them grew new seedlings again; while 55.6% of the plants in the CK treatment died, and none of them grew new seedlings again ( Figure 8 ), Figure 8 In the figure, the arrows point to the new seedlings that have grown again.

[0090] (2) Field experiment: Each plot has an area of ​​75m 2 66 ginger seedlings were planted, and each treatment was repeated 3 times, with random plots arranged in the field. Two treatments were set: (1) conventional management as control (CK); (2) Cladosporium synthetic bacterial community T4 (T4): 10 times 5×10 5CFU / mL T4 bacterial solution was sprayed on the whole plant and irrigated the roots once, for a total of 1 application. Each treatment was sprayed with a backpack electric sprayer, and the application amount per plot was 30L. The occurrence of bacterial wilt was natural in the field. At the harvest period, growth indicators were investigated, and the incidence rate, disease index and control effect were calculated.

[0091] (3) Investigation of ginger bacterial wilt: The disease index grading standard is as follows: 0, the whole plant is disease-free; 1, less than 10% of the leaves turn slightly yellow, and the fleshy stems have no obvious symptoms; 3, 11%-30% of the leaves turn yellow, the leaf margins curl slightly, and the fleshy stems have water-soaked spots; 5, 31%-50% of the leaves turn yellow, the leaf margins curl, the plant dwarfs, and the fleshy stems partially rot; 7, more than 51% of the leaves turn yellow, the plant wilts, and most of the fleshy stems rot; 9, the plant dies, the fleshy stems rot or only the filamentous vascular bundles remain. Incidence rate (%) = (number of diseased plants / total number of plants) × 100; Disease index = 100 × ∑ (number of plants at each disease level × representative value of each level) / (total number of plants surveyed × highest level representative value); Prevention effect (%) = [(control disease index - treatment disease index) / control disease index] × 100.

[0092] The survey results are shown in Table 2 and Figure 9-11 As shown:

[0093] Table 2 Control effect of Cladosporium synthetic bacterial consortium T4 on ginger bacterial wilt

[0094]

[0095] Note: Data are expressed as mean ± standard deviation. Capital letters after data in the same column indicate extremely significant differences (P < 0.01), and different lowercase letters indicate significant differences (P < 0.05).

[0096] As shown in Table 2, the incidence rate and disease index of T4 treatment were the lowest, 73.30% and 16.05 respectively, which were 25.0% and 68.6% lower than those of CK, reaching a highly significant difference, and the control efficiency was 68.42%. From the field disease situation, CK treatment had a large area of ​​disease, with yellow stems and leaves, while T4 treatment had a mild disease, with better overall growth, green stems and leaves, and strong rhizomes ( Fig. 9 ).

[0097] Six months after the ginger seedlings were grown in T4 treatment, the tiller number, plant radius, rhizome height, biological yield and economic yield of ginger were the highest, increasing by 50.4%, 12.8%, 36.4%, 50% and 55.6% respectively compared with those in CK treatment ( Fig.10 ), reaching a very significant level of difference. In terms of ginger growth, compared with the CK treatment, the T4 treatment had more branches and roots, more luxuriant leaves, larger ginger pieces, and a significantly lower disease loss rate ( Fig.11 ).

[0098] Example 5

[0099] This example is the biocontrol effect of the synthetic bacterial consortium of Cladosporium (T4) prepared in Example 3 on tomato bacterial wilt, as follows:

[0100] Soak in 60℃ warm water for 2h, and germinate in a light incubator (temperature 28℃, humidity 80℃, no light) for 72h. Select seeds with full buds and consistent bud length, soak them in bacterial T4 solution for 30min, soak them in the same amount of sterilized water as the control (CK), sow them in seedling trays, and inoculate 10mL T4 solution for each seedling. After 7d of seedling cultivation, water the bacteria for the second time, 10mL / plant. After 25d of seedling cultivation, transplant them into a pot (20.6cm×17.5cm×15cm) filled with 2 / 3 tomato bacterial wilt disease soil (collected from the Lijian Base of Guangxi Academy of Agricultural Sciences), water the bacteria for the third time, 50mL / plant, and place them in a greenhouse (temperature 30±5℃, relative humidity 80±5%, natural light). Each treatment was repeated 3 times, with 12 tomato seedlings in each repeat. The number of leaves of tomato seedlings was investigated on the 10th and 18th day after sowing in the seedling tray, the plant height of tomato seedlings was measured on the 25th day after transplanting to the diseased soil, the disease situation was investigated on the 15th, 25th and 35th day, and the incidence rate, disease index and control effect were calculated.

[0101] The grading standard for the disease index of tomato bacterial wilt is as follows (Wu Renmin, 2020): Level 0, no wilting symptoms; Level 1, 1%-25% leaf wilting; Level 2, 26%-50% leaf wilting; Level 3, 51%-75% leaf wilting; Level 4, 76%-100% leaf wilting. Incidence rate (%) = (number of diseased plants / total number of plants) × 100; disease index = 100 × ∑ (number of plants at each disease level × representative value of each level) / (total number of plants surveyed × highest representative value); prevention and control effect (%) = 100 × (disease index of control group - disease index of treatment group) / disease index of control group. The results obtained are as follows Figure 12-14 As shown:

[0102] After 10 days of tomato seedling cultivation, the number of leaves in the T4 treatment was significantly greater than that in the CK treatment. As the seedling cultivation time increased, the difference in the number of leaves between the T4 treatment and the CK treatment became increasingly significant at 18 days of seedling cultivation. The number of leaves in the T4 treatment increased by 57.7% ( Fig.12 a). 25 days after the tomato seedlings were transplanted into the diseased soil, the plant heights of the tomatoes in the T4 treatment were significantly higher than those in the CK treatment, increasing by 128.8% ( Fig.12 b, Fig.13 ).

[0103] From the 15th to the 35th day after transplanting the tomato seedlings into the diseased soil, the incidence and disease index of the T4 treatment remained unchanged, both significantly lower than those of the CK treatment, and reached a significant difference level at the 35th day ( Fig.14 a and Fig.14 b). The control efficiency of the three surveys was above 60%, among which the increase was relatively large in the first 15-25 days after transplanting the diseased soil, and remained unchanged at 67.78% in the later 25-35 days ( Fig.14 c).

[0104] Example 6

[0105] This example is the biocontrol effect of the synthetic bacterial consortium of Cladosporium (T4) prepared in Example 3 on tobacco bacterial wilt, as follows:

[0106] After 45 days of cultivation, the tobacco seedlings with consistent growth were selected and transplanted into 32-hole seedling trays. Each seedling was inoculated with 10 mL of bacterial group T4 solution. After 10 days, the healthy tobacco seedlings were wounded with an inoculation knife and inoculated with 30 mL of Ralstonia solanacarum solution. The blank control group was treated with an equal amount of water after the wounded roots. Thirty-two tobacco seedlings were placed in a greenhouse at 28±5℃ for daily observation. The disease index was calculated after the disease of the tobacco seedlings stabilized.

[0107] Among them, the classification survey of tobacco bacterial wilt disease refers to the standard of "Industry Standard of the People's Republic of China YC / T39-1996", as shown in Table 3:

[0108] Table 3 Grading of tobacco bacterial wilt

[0109]

[0110]

[0111] In addition, the calculation formulas for disease index and prevention and treatment effect are as follows:

[0112] Disease index = 100 × ∑ (number of plants at each disease level × representative value of each level) / (total number of plants surveyed × highest representative value);

[0113] The control effect (%) = 100 × (control group disease index - treatment group disease index) / control group disease index. Figure 15-17 As shown:

[0114] After 13, 18 and 23 days of inoculation with pathogens, the incidence rate of T4 treatment was significantly lower than that of CK, and it was 55.36% lower than that of CK at 23 days of inoculation with pathogens ( Fig.15 a). The protective effects of T4 treatment were 91.70%, 87.06% and 77.86% at 13d, 18d and 23d after inoculation of pathogens, respectively. Fig.15 b).

[0115] From the perspective of plant growth status, CK treatment had obvious disease 13 days after inoculation of pathogens, with leaves at the base of the plants turning yellow and dead, and the base of the stems turning black, with an incidence rate of 47.50%; while T4 treatment had no obvious disease, with an incidence rate of only 4.88% ( Fig.16 ); 23 days after inoculation with the pathogen, almost all the plants in the CK treatment died, with an incidence rate of 77.50%, while only a small number of plants in the T4 treatment became sick, with an incidence rate of 22.14%. ( Fig.17 ).

[0116] The statistical analysis method of the data in Examples 2 to 5 of the present application was performed using Microsoft Excel 2016 and DPS7.05 system. Duncan's new multiple range method in one-way analysis of variance was used for significant difference test (P < 0.05, P < 0.01), and GraphPad Prism 8 was used for plotting. All results are expressed as mean ± standard deviation (SD).

[0117] In summary, the Cladosporium strain HX2 of the present invention is identified as a new species of the genus Cladosporium, has good symbiotic ability with tomatoes, and has the effect of resisting tomato bacterial wilt. After it is mixed with strains LJ1, MS2 and LC3 of the same genus to prepare a Cladosporium synthetic flora, the synthetic flora has better prevention and treatment ability for bacterial wilt. After experiments, in addition to the growth-promoting and disease-resistant effects on tomatoes, the synthetic flora also has good growth-promoting and disease-resistant functions on ginger and tobacco. It is a good synthetic flora that can provide rich germplasm resources for the later development of antimicrobial agents or the preparation of biological organic fertilizers.

[0118] The above-described embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. It should be pointed out that, for a person of ordinary skill in the art, several modifications and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the attached claims.

Claims

1. Cladosporium phial fungus strain Cladophiaphora guangxiense HX2, characterized in that The deposit number of the Cladosporium phialium strain Cladophiaphora guangxiense HX2 is CGMCC NO.41498.

2. A Cladosporium synthetic flora comprising the Cladosporium strain Cladophiaphoraguangxiense HX2 according to claim 1, characterized in that: The Cladosporium synthetic flora also includes: Cladosporium strain Cladophiaphora sp.LJ1, Cladosporium strain Cladophiaphora sp.MS2 and Cladosporium strain Cladophiaphoraimmunda LC3; The deposit number of the Cladosporium sp. LJ1 is CGMCC NO.18140; The deposit number of the Cladosporium sp. strain Cladophiaphora sp. MS2 is CGMCC NO.20238; The deposit number of the Cladosporium strain Cladophiaphora immunda LC3 is CGMCC NO.41540.

3. Use of the Cladosporium strain Cladophiaphora guangxiense HX2 according to claim 1 or the synthetic bacterial consortium of Cladophiaphora according to claim 2 in resisting plant bacterial wilt.

4. The use according to claim 3, characterized in that: The plants are: tomato, ginger and / or tobacco.

5. Use of the Cladosporium strain Cladophiaphora guangxiense HX2 according to claim 1 or the Cladosporium synthetic flora according to claim 2 in promoting plant growth.

6. The use according to claim 5, characterized in that: The plants are: tomato and / or ginger.

7. A method for preparing the synthetic flora of Cladosporium as claimed in claim 2, characterized in that: The method comprises: inoculating the strains into a PDA plate for activation and then inoculating into a PDB liquid culture medium, collecting mycelia by filtering with sterile gauze, washing the mycelia with sterile water and squeezing out the water, mixing the four kinds of mycelia in equal mass ratios and preparing them into a concentration of 5×10 5 CFU / mL of bacterial solution.

8. A method for plant cultivation using the Cladosporium phialaria strain Cladophiaphoraguangxiense HX2 according to claim 1 or the Cladosporium phialaria synthetic flora according to claim 2 or the synthetic flora prepared by the method according to claim 7, characterized in that: The method comprises the following steps: inoculating a bacterial solution containing the synthetic bacterial flora of Cladosporium into plants, and then carrying out cultivation.

9. The method according to claim 8, characterized in that The inoculation method comprises: irrigating the plant roots with the bacterial liquid of the synthetic bacterial flora of Cladosporium; or soaking the plant roots in the bacterial liquid of the synthetic bacterial flora of Cladosporium; or soaking the plant seeds in the bacterial liquid of the synthetic bacterial flora of Cladosporium.

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