An antagonistic bacterial strain Streptomyces cinnamomi and its application

Abstract

The present invention discloses an antagonistic bacterial strain Streptomyces cinnamonensis and its application, which belongs to the technical field of plant disease prevention and control, Streptomyces cinnamonensis DHJ986, deposited by Guangdong Microbial Culture Collection Center, deposited on February 15, 2023, and deposited on GDMCC NO.63170. Streptomyces cinnamonensis is used in plant disease prevention and control, and plant diseases include diseases caused by Fusarium oxysporum, Botrytis cinerea, Alternaria alternata, Fusarium cynaroides, Phytophthora sojae and tobacco root rot. The present invention can effectively prevent and control plant diseases, significantly reduce the incidence of plants, and has a certain promoting effect on the root growth of plants.

Description

Technical Field

[0001] The invention relates to the technical field of plant disease prevention and control, in particular to an antagonistic bacterial strain Streptomyces cinnamomi and application thereof. Background Art

[0002] Plant diseases are common problems in agricultural production, which have a great impact on plant growth and yield. In the past few decades, scientists have conducted a lot of research and exploration on the prevention and control of plant diseases, and have made some important progress, but there are also some problems and challenges. First of all, there are many types of plant diseases, and each disease has different pathogens and symptoms, so different prevention and control measures need to be taken for different diseases.

[0003] For example, the root rot of Polygonatum sibiricum, which is a devastating disease and is known as "plant cancer". The field incidence of root rot of Polygonatum sibiricum in Yunnan is 5%-15%, and can reach more than 30% in severe cases. Root rot is a global disease. For perennial medicinal plants such as Polygonatum sibiricum, Panax notoginseng, and ginseng, this disease has become one of the main obstacles threatening their healthy and sustainable development. The main pathogen of root rot of Polygonatum sibiricum is Fusarium oxysporum, which mainly exists in the rhizosphere soil in the form of mycelium. The mycelium of this fungus is a slender structure composed of multiple cells that can penetrate the soil pores and grow near the plant rhizosphere. Fusarium oxysporum can also invade from the apical cells of the plant root hairs and reproduce in the parenchyma, causing secondary xylem necrosis, and the cork layer continues to extend inward until the entire root is completely rotten.

[0004] Traditional agricultural control measures have many limiting factors, high costs and difficult implementation. The use of chemical pesticides to control plant diseases is prone to problems such as pesticide residues, resistance, ecological environment, safety and sustainability. The use of chemical pesticides not only has adverse effects on human health and the environment, but also easily leads to pathogens developing resistance and destroying the ecological balance. Improper operation may cause harm to users. Therefore, it is necessary to seek more environmentally friendly and sustainable control methods to cope with the many problems and limitations faced by traditional agricultural control of root rot. Summary of the invention

[0005] In view of the above problems, the present invention provides an antagonistic bacterial strain Streptomyces cinnamomi and its application, which has efficient, safe and sustainable prevention and control effects and is of great significance to agricultural production and environmental protection.

[0006] A technical purpose of the present invention is to provide an antagonistic bacterial strain Streptomyces cinnamonensis, wherein the Streptomyces cinnamonensis (Streptomyces cinnamonensis) DHJ986, deposited in: Guangdong Provincial Microbiological Culture Collection Center, deposited on February 15, 2023, and deposited on: GDMCC NO.63170.

[0007] The present invention also provides an application of an antagonistic bacterial strain Streptomyces cinnamomi, and the application of the Streptomyces cinnamomi in the prevention and treatment of plant diseases includes diseases caused by Fusarium oxysporum, Botrytis cinerea, Alternaria alternata, Neofusella cymoides, Phytophthora sojae and Fusarium solani.

[0008] Further preferably, the disease caused by Fusarium oxysporum is Polygonatum cyrtonema root rot.

[0009] Further preferably, the disease caused by Fusarium oxysporum is pitaya wilt.

[0010] Further preferably, the disease caused by Botrytis cinerea is tomato gray mold.

[0011] Further preferably, the disease caused by Alternaria alternata is citrus brown spot.

[0012] Further preferably, the disease caused by the Neofusobacteria sp. is kiwifruit rot.

[0013] Further preferably, the disease caused by Phytophthora sojae is Phytophthora sojae blight.

[0014] Further preferably, the disease caused by Fusarium solani is tobacco root rot.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] First, Streptomyces cinnamomi can effectively prevent and control plant diseases, among which the prevention and control effect of Yunnan polygonatum root rot reached 53.85%. It can be seen that the fermentation liquid of Streptomyces cinnamomi antagonistic bacteria can effectively prevent and control Yunnan polygonatum root rot. At the same time, inoculation of Streptomyces cinnamomi fermentation liquid significantly reduced the incidence of Yunnan polygonatum, and had a certain promoting effect on the root growth of Yunnan polygonatum.

[0017] Second, Streptomyces cinnamomi can significantly increase the activity of SOD, POD, PAL and PPO in the roots of Polygonatum cyrtonema. It can be seen that inoculation with Streptomyces cinnamomi can improve the defense enzyme resistance of the roots of Polygonatum cyrtonema, thereby reducing the invasion of pathogens. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a potted plant test effect diagram of the present invention (a: culture medium + pathogenic bacteria, b: Streptomyces cinnamomi + pathogenic bacteria);

[0019] Figure 2 The effect of Streptomyces cinnamomi on SOD (a) and POD (b) in the roots of Polygonatum cyrtonema;

[0020] Figure 3 The effect of Streptomyces cinnamomi on PPO (a) and PAL (b) in the roots of Polygonatum cyrtonema;

[0021] Figure 4 The effect of Streptomyces cinnamomi on soluble sugar (a) and malondialdehyde (b) in the roots of Polygonatum cyrtonema;

[0022] Figure 5 The effect of Streptomyces cinnamomi on the chlorophyll in the leaves of Polygonatum cyrtonema;

[0023] Figure 6 The effect of Streptomyces cinnamomi on the germination of sclerotia of the pathogen F.oxysporum (a: CK; b: Streptomyces cinnamomi);

[0024] Figure 7 The effect of Streptomyces cinnamomi on the hyphae of pathogenic fungus F.oxysporum (a: CK; b: Streptomyces cinnamomi);

[0025] Figure 8 The effect of different pH treatments on the antibacterial effect of Streptomyces cinnamomi on F.oxysporum;

[0026] Fig. 9 The effect of different inoculation amounts of Streptomyces cinnamomi on the antibacterial effect of F.oxysporum;

[0027] Fig.10 The effect of Streptomyces cinnamomi treatment at different temperatures on the antibacterial effect of F.oxysporum. DETAILED DESCRIPTION

[0028] In order to make those skilled in the art better understand the technical solution of the present invention, the present invention is further described in detail below in conjunction with embodiments. It will be understood by those skilled in the art that the following embodiments are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention.

[0029] Example 1

[0030] The invention discloses an antagonistic bacterial strain Streptomyces cinnamomi and application thereof, wherein the plant disease is root rot of Polygonatum cyrtonema caused by Fusarium oxysporum.

[0031] Example 2

[0032] The invention discloses an antagonistic bacterial strain Streptomyces cinnamomi and application thereof, wherein the plant disease is pitaya wilt disease caused by Fusarium oxysporum.

[0033] Example 3

[0034] The invention discloses an antagonistic bacterial strain Streptomyces cinnamomi and application thereof, wherein the plant disease is tomato gray mold caused by Botrytis cinerea.

[0035] Example 4

[0036] The invention discloses an antagonistic bacterial strain Streptomyces cinnamomi and application thereof, wherein the plant disease is citrus brown spot caused by Alternaria alternata.

[0037] Example 5

[0038] The invention discloses an antagonistic bacterial strain Streptomyces cinnamomi and application thereof, wherein the plant disease is kiwifruit rot caused by Neofusicoccum parvum.

[0039] Example 6

[0040] The invention discloses an antagonistic bacterial strain Streptomyces cinnamomi and application thereof, wherein the plant disease caused by Phytophthora sojae is soybean phytophthora.

[0041] Example 7

[0042] The invention discloses an antagonistic bacterial strain Streptomyces cinnamomi and application thereof, wherein the plant disease is tobacco root rot caused by Fusarium solani.

[0043] Screening and identification of antagonistic bacteria against root rot of Polygonatum cyrtonema

[0044] The surfaces of three kinds of polygonatum samples, namely, polygonatum cyrtonema, polygonatum yunnanensis and polygonatum cyrtonema, were disinfected. After disinfection, the sterile water used to clean the samples was spread on 6 different culture medium plates to observe whether colonies would grow. If not, it means that the samples were thoroughly disinfected, which means that the isolated strains were all endophytic bacteria.

[0045] Then, the samples were ground into a homogenate with liquid nitrogen under sterile conditions. First, the crushed plant roots, stems and leaves were transferred to a container containing glass slag and 18 mL of saline, and the rhizosphere soil was directly transferred to a container containing glass slag and 18 mL of saline. Then, they were shaken and incubated at 37°C for 20 minutes to mix, and then immediately treated with ultrasound for 40 seconds. Finally, the mixed plant juice / rhizosphere soil was diluted with saline to 10 -2 , 10 -4 and 10-5 Then, 0.2 mL was applied to 6 kinds of separation culture media, and then cultured in a 28°C incubator for more than one week. Observe the morphology and size of the antagonistic bacteria, pick out single colonies with different morphologies and inoculate them again on ISP2 solid culture medium for zoning and streaking culture. The purified strains were stored in a -80°C refrigerator with 30% glycerol, and the rest were picked into a 1.5 mL centrifuge tube for DNA extraction.

[0046] Results and Analysis

[0047] (1) In this study, 224 antagonistic bacteria were isolated from three different varieties of Polygonatum cyrtonema. After excluding duplicates, a total of 121 antagonistic bacteria belonging to 43 genera were obtained. In both the endophytic and rhizosphere soils of Polygonatum cyrtonema, Streptomyces accounted for the largest proportion, followed by Bacillus, accounting for 35.54% and 6.61% of the total number of isolated strains, respectively.

[0048] (2) There are obvious differences in community structure among different varieties. At the genus level, 20, 19, and 23 genera were detected in different samples of Polygonatum cyrtonema, Polygonatum cyrtonema, and Polygonatum cyrtonema, respectively, and the number of unique genus species was 6, 10, and 13, respectively, accounting for 13.95%, 23.26%, and 30.23% of the total genus types. A total of five common antagonistic bacterial genera were found in the three varieties of Polygonatum, namely Stenotrophomonas, Microbacterium, Curtobacterium, Rhodococcus, and Streptomyces, among which Streptomyces was the most dominant genus in all three varieties. It can be seen that there are obvious differences in the antagonistic bacterial community structure among different Polygonatum varieties.

[0049] (3) The most strains of Bacillus and Streptomyces were isolated from Polygonatum cyrtonema, with 89 strains isolated. Polygonatum cyrtonema was the second most abundant, with 52 strains isolated. Polygonatum cyrtonema had the least number of strains isolated, with only 14 strains isolated. Among the different varieties, the highest abundance of Bacillus and Streptomyces was found in the roots of Polygonatum cyrtonema and Polygonatum cyrtonema, and the highest abundance was found in the stems and leaves of Polygonatum cyrtonema. It can be seen that the diversity of antagonistic bacteria in Polygonatum cyrtonema is richer, and the abundance of Bacillus and Streptomyces is the highest.

[0050] Isolation, Purification and Preservation of Streptomyces cinnamomi

[0051] Experimental design and process

[0052] Antagonistic bacteria isolated from endophytic and rhizosphere soils of Polygonatum cyrtonema, Polygonatum cyrtonema and Polygonatum cyrtonema by dilution coating method were used as test strains, and Fusarium oxysporum, the pathogen of root rot of Polygonatum cyrtonema, was used as target strain, aiming to screen strains with high antagonism. At the same time, the biocontrol potential of active strains was further verified by measuring the antibacterial spectrum of highly active strains and observing their effects on the mycelial growth of pathogens by scanning electron microscopy. In addition, morphological observation, 16SrDNA sequencing, physiological and biochemical identification and phylogenetic tree construction of highly active strains were carried out to clarify the taxonomic status of biocontrol bacteria.

[0053] Results and Analysis

[0054] Initial screening of antagonistic bacteria by plate confrontation method

[0055] The 121 antagonistic bacteria separated and purified in the early stage were used as test strains. The plate confrontation method was used to determine that among the 121 antagonistic bacteria, the following strains had an inhibition width >10mm(+++): 807, 810, 849, 986 and 1189. The five biocontrol antagonistic bacteria were rescreened using the growth rate method.

[0056] Rescreening of Antagonistic Bacteria by Growth Rate Method

[0057] In this study, the mycelium growth rate method was used to determine the antibacterial activity of the fermentation filtrates of strains 807, 810, 849, 986 and 1189. The results (Table 1) showed that 1189 had the highest antibacterial rate, followed by 986. The antibacterial rates of these two strains were significantly higher than those of other plants, reaching 79.06% and 31.66% respectively. Combined with the results of the initial screening and rescreening, strains 986 and 1189 were selected as test strains for subsequent studies.

[0058] Table 1 The effect of biocontrol strains on Fusarium oxysporum

[0059]

[0060]

[0061] Note: Different letters after the values ​​in the same column indicate significant differences (P<0.05)

[0062] Identification of antagonistic bacteria 986 and 1189

[0063] Morphological observation

[0064] On ISP2 solid medium, strain 986 was light purple, and the colonies formed were nearly round, with ridges, central wrinkles, and a diameter of 5-6 mm. 1189 was dark gray, opaque, and had a serrated shape, central wrinkles, and a warm texture. Gram staining showed that both 986 and 1189 were blue-purple, so both 986 and 1189 were Gram-positive bacteria.

[0065] Physiological and biochemical identification

[0066] The results of physiological and biochemical identification of 986 and 1189 are shown in Table 2. The results show that both 986 and 1189 have the ability to produce D-xylose, oxidase, starch hydrolysis, methyl red and VP test. In addition, 1189 can also produce D-mannitol, catalase and gelatin liquefaction.

[0067] In addition, it can be seen from Table 3 that the effects of different pH values, different temperatures and different NaCl concentrations on the growth of strains 986 and 1189 were determined. The results show that both 986 and 1189 grow better when the pH is 5-7; both 986 and 1189 can grow normally at 22℃-37℃, and 986 grows better at 28℃-37℃. 1189 grows better at 37℃. In addition, 986 can grow normally in a culture medium with a salt concentration of 1%. When the salt concentration reaches 3%, the colony no longer grows at this salt concentration. The salt tolerance of strain 1189 gradually decreases when the salt concentration increases. When the salt concentration reaches 7%, the strain concentration decreases, but it can still grow normally. However, when the salt concentration reaches 10%, the strain no longer grows normally.

[0068] Table 2986 and 1189 physiological and biochemical identification results

[0069]

[0070]

[0071] Table 3 Effects of different pH values, temperatures and NaCl concentrations on the growth of strains 986 and 1189

[0072]

[0073] Note: Growth status: “+” can grow, but the growth is poor; “++” grows well; “+++” grows vigorously; “-” cannot grow

[0074] Molecular Biology Identification

[0075] Phylogenetic tree analysis of strains 986 and 118916S rDNA sequences

[0076] The 16S rDNA sequence cloned products of strains 986 and 1189 were sent to Kunming Qingke Biotechnology Co., Ltd. for sequencing, and DNA fragments of 1369bp and 1377bp were obtained, respectively. The sequences were then submitted to the GenBank database for registration. 986 had a high similarity with Streptomyces virginiae and Streptomyces cinnamonensis, reaching 100%; Komakai et al. reclassified S. cinnamonensis as S. virginiae in 2021. 1189 had a high similarity with B. cabrialesii, reaching 99.93%. At the same time, the morphological characteristics and physiological and biochemical characteristics of strains 986 and 1189 were compared with the Bergey Antagonistic Bacteria Identification Manual and the Common Antagonistic Bacteria System Identification Manual. Strain 986 conforms to the physiological, biochemical and morphological characteristics of Streptomyces virginiae (S. virginiae), and 1189 conforms to the physiological and biochemical characteristics of Bacillus cabrialesii (B. cabrialesii). Based on 16S rDNA homology, 24 model strains with high homology were downloaded from the NCBI website, and the Neighbor-Joining phylogenetic tree was constructed using MEGA5.1 software. Strain 986 and S. virginiae were in the same evolutionary branch, and 1189 and B. cabrialesii were in the same evolutionary branch. In summary, 986 was identified as Streptomyces cinnamonensis, and 1189 was identified as Bacillus inaqusorum.

[0077] Finally, strain 986 was sent to the Guangdong Provincial Microbial Culture Collection Center for preservation. Its taxonomic name is Streptomyces cinnamonensis, preservation date is February 15, 2023, and preservation number is GDMCC NO.63170, which is translated as Streptomyces cinnamonensis.

[0078] Study on the control effect of Streptomyces cinnamomi on root rot of Polygonatum cyrtonema

[0079] Test bacteria: Streptomyces cinnamonensis.

[0080] Test pathogenic fungi: Fusarium oxysporum, the pathogen of Polygonatum cyrtonema root rot, was provided by Yunnan University of Traditional Chinese Medicine.

[0081] Test materials and cultivation medium

[0082] Yunnan Polygonatum sibiricum are all one-year-old tubers, provided by the Muqi Black Polygonatum sibiricum planting base in Bozhu Town, Yunnan Province.

[0083] Cultivation medium: The planting soil is mainly red soil, humus and perlite, mixed in a ratio of 3:3:1. After mixing, the pH is 7.23, the organic matter is 91.11g / kg, and the effective phosphorus, effective potassium and effective nitrogen contents are 72.18mg / kg, 312.14mg / kg and 484mg / kg respectively.

[0084] The main instruments used in the experiment include: UV spectrophotometer (Shanghai Jinghua Science Park Instrument Co., Ltd.), electric constant temperature shaker (HYG-A), constant temperature incubator (DPR-9052), desktop centrifuge (TD4K-Z), digital constant temperature water bath (HH-2), vortex shaker (QL-866), gel imaging system (Syngene lnereniasc), PCR instrument (A200), pH meter (KL-009), etc.

[0085] The main reagents used in the experiment include formalin, trichloroacetic acid, EDTA-Na2, riboflavin, guaiacol, sulfur salicylic acid, toluene, Coomassie brilliant blue, etc.

[0086] Experimental design and process

[0087] In December 2021, a potted inoculation experiment was conducted to explore the biocontrol effect of Streptomyces cinnamomi on root rot of Polygonatum cyrtonema. A total of 5 treatments were designed for the potted inoculation experiment of antagonistic bacteria, A: continuous root irrigation liquid culture medium (CK); B: continuous inoculation of antagonistic bacteria cinnamon Streptomyces fermentation liquid (cinnamon Streptomyces); C: root irrigation with liquid culture medium in the early stage, and then inoculation of pathogens in the later stage (P+B); D: inoculation of pathogens only (B); E: root irrigation with cinnamon Streptomyces fermentation liquid in the early stage, and then inoculation of pathogens in the later stage (cinnamon Streptomyces+B), a total of 5 treatments were set, as shown in Table 4, with 10 replicates for each treatment.

[0088] The plastic flower pots used in the experiment (sterilized by soaking in 0.1% potassium permanganate solution for 24 hours before use) were 15 cm high and 16.50 cm in diameter. Each pot was filled with 1.5 kg of sterilized soil, and one-year-old tubers of Polygonatum cyrtonema were transplanted. After Polygonatum cyrtonema had grown for about 180 days, potted Polygonatum cyrtonema seedlings with consistent growth were selected for the experiment. For treatments A, B, and C, 10 mL of Streptomyces cinnamomi and liquid culture medium were added to the roots for root irrigation, and the roots were irrigated once every 7 days, for a total of 3 times. For treatments D and F, 10 mL of Streptomyces cinnamomi and liquid culture medium were used for root irrigation in the early stage. After 12 days, the pathogen Fusarium oxysporum was inoculated using the root injury root irrigation method (10 7 CFU / mL, 5mL). Treatment D was inoculated with only the pathogen Fusarium oxysporum (10 7CFU / mL, 5mL). After 21 days, the disease situation was observed, the incidence rate and disease index were calculated, and the enzyme activity indexes of the root system of Polygonatum cyrtonema were measured (SOD, POD, PAL and PPO, etc.), and the physical and chemical properties of the rhizosphere soil and the microbial community structure were measured.

[0089] Table 4 Marking numbers for different treatments

[0090]

[0091] Preparation of antagonistic bacteria fermentation broth: The isolated strain was inoculated into ISP2 medium and cultured at 28°C for 5 days. Then the antagonistic bacteria were inoculated into liquid culture medium and cultured at 28°C and 180r / min for 3 days. ISP2 liquid culture medium was used for dilution to obtain 10 8 CFU / mL of fermentation broth.

[0092] Preparation of pathogen spore suspension: Inoculate the pathogen onto a fresh PDA plate, incubate at 28°C for 8-10 days, brush off the spores with a brush, mix using a hemocytometer, and dilute with sterile water to obtain 10 7 The pathogen spore suspension with CFU / mL is ready for use.

[0093] Effect of biocontrol bacteria on root rot of Polygonatum cyrtonema

[0094] The root rot of Polygonatum cyrtonema was graded and statistically analyzed, and the incidence rate, disease index and disease prevention effect were calculated.

[0095] Grading method:

[0096] Level 0: No disease occurs;

[0097] Level 1: The taproot remains unchanged, and the tips of the fibrous roots turn slightly brown, which does not affect plant growth;

[0098] Level 3: The taproot turns slightly black, and the tips of the fibrous roots turn seriously black, which does not affect the growth of the plant;

[0099] Level 5: The taproot turns darker, and the fibrous roots decrease or disappear, seriously affecting the growth of the plant;

[0100] Level 7: Roots rot and the plant cannot grow.

[0101] Calculation formula:

[0102]

[0103]

[0104] Determination of Growth Physiological Indexes of Polygonatum cyrtonema

[0105] The plant height, fresh weight of underground and aboveground parts of Polygonatum yunnanensis were determined by conventional methods. The activity of superoxide dismutase (SOD) was determined by nitroblue tetrazolium photoreduction method; the activity of peroxidase (POD) was determined by guaiacol colorimetry; the activity of polyphenol oxidase (PPO) was determined by catechol method; the activity of phenylalanine ammonia lyase (PAL) was determined by mercaptoethanol method; the content of malondialdehyde (MDA) was determined by thiobarbituric acid colorimetry; the content of soluble sugar was determined by phenol method; and the chlorophyll was determined by ethanol acetone-ethanol mixture (1:1) extraction method.

[0106] Determination of Physical and Chemical Properties of Rhizosphere Soil of Polygonatum cyrtonema

[0107] The soil pH value was determined using a pH-25 meter; the soil organic matter content (SOM) was determined using the potassium dichromate external heating method; the total phosphorus (TP) content was determined using the HClO4-H2SO4 digestion method; the total potassium (TK) content was determined using the acid-atomic absorption method using a Thermo Scientific S series atomic absorption spectrometer; the alkaline nitrogen (AN) content was determined using the diffusion dish method; the available phosphorus (AP) content was determined using the sodium bicarbonate extraction-molybdenum antimony colorimetry method, and the available potassium (AK) content was determined using the ammonium acetate extraction-atomic absorption method.

[0108] Results and Analysis

[0109] Effects of Streptomyces cinnamomi on the incidence and disease index of Polygonatum cyrtonema root rot

[0110] Five different treatments were set up for potted plant experiments. The results are shown in Table 5 and Figure 1 As shown, Figure 1 In the treatment group (a: medium + B, b: Streptomyces cinnamomi + B), the incidence rate of the pathogen alone reached 86.67%, which was significantly higher than that of other treatment groups; the incidence rate of the pathogen alone (Streptomyces cinnamomi) was less than 20% when the pathogen was not inoculated; the incidence rate of the pathogen was 42.22% when the antagonistic bacteria fermentation liquid of Streptomyces cinnamomi was inoculated in the early stage, and the control effect reached 53.85%. It can be seen that the antagonistic bacteria fermentation liquid of Streptomyces cinnamomi can effectively prevent and control the root rot of Polygonatum cyrtonema.

[0111] Table 5 Effect of strain Streptomyces cinnamomi on root rot of Polygonatum cyrtonema in potted plants

[0112]

[0113] Note: Different letters after the values ​​in the same column indicate significant differences (P<0.05), the same below

[0114] Effects of Streptomyces cinnamomi on the growth parameters of Polygonatum cyrtonema

[0115] As shown in Table 6, whether inoculated with pathogens or not, inoculation with the fermentation liquid of Streptomyces cinnamomi will promote the growth of the roots of Polygonatum yunnanensis. In the case of single antagonistic bacteria treatment, the weight of the root system reached 11.55g, which was 9.48% higher than CK. The root length reached 16.50cm, which was 22.95% higher than the treatment. In the case of inoculation with pathogens, the weight of the root system reached 9.79g, which was 22.99% higher than the P+B treatment. The root length reached 15.38cm, which was 61.89% higher than P+B.

[0116] Table 6 Effects of the strain Streptomyces cinnamomi on the growth indexes of Polygonatum cyrtonema

[0117]

[0118] Effects of Streptomyces cinnamomi on the physicochemical properties of rhizosphere soil of Polygonatum cyrtonema

[0119] As can be seen from Table 7, the pH of the rhizosphere soil of Polygonatum cyrtonema under different treatments was neutral, ranging from 7.23 to 7.40. Under the condition of single antagonistic bacteria treatment, compared with CK, the organic matter content in the rhizosphere soil was reduced by 16.79% after treatment with the fermentation liquid of Streptomyces cinnamomi; the contents of available phosphorus and available potassium were reduced, and the Streptomyces cinnamomi treatment group decreased by 2.70% and 1.91% respectively. Compared with CK, the total phosphorus content of the Streptomyces cinnamomi treatment group increased (6.29%), and the change in total potassium did not reach a significant level.

[0120] In the treatment of pathogen inoculation, there was no significant change in organic matter content compared with the P+B treatment group. The treatment with the fermentation liquid of Streptomyces cinnamomi significantly increased the content of alkaline nitrogen and available phosphorus, with alkaline nitrogen being 153.61% higher than that of the P+B treatment; the available phosphorus being 18.46% higher than that of the P+B treatment; there was no significant difference in total phosphorus content among different treatments. In terms of potassium content, the effective potassium content of the Streptomyces cinnamomi+B treatment group was the highest at 293.52 mg / kg.

[0121] Table 7 Effects of the strain Streptomyces cinnamomi on the physical and chemical properties of rhizosphere soil of Polygonatum cyrtonema

[0122]

[0123] Effects of Streptomyces cinnamomi on POD and SOD of Polygonatum cyrtonema

[0124] Depend on Figure 2It can be seen that the fermentation liquid of Streptomyces cinnamomi has a significant effect on the root enzyme activity of Polygonatum cyrtonema. From the results of root enzyme activity, under the condition of single antagonistic bacteria treatment, the fermentation liquid of Streptomyces cinnamomi can significantly increase the activity of POD and SOD of Polygonatum cyrtonema, with POD increased by 3.83% and SOD increased by 398.57%, respectively. Under the condition of inoculation of pathogens, Streptomyces cinnamomi can also increase the POD and SOD of Polygonatum cyrtonema roots, with POD increased by 28.17% and SOD increased by 237.03%. It can be seen that the fermentation filtrate of Streptomyces cinnamomi can increase the POD and SOD enzyme activities of Polygonatum cyrtonema roots.

[0125] Effects of Streptomyces cinnamomi on the activities of PPO and PAL in Polygonatum cyrtonema

[0126] Effects of inoculation with the fermentation broth of Streptomyces cinnamomi on the activities of PPO and PAL in the roots of Polygonatum cyrtonema Figure 3 After the roots of Polygonatum cyrtonema were irrigated with the fermentation liquid of Streptomyces cinnamomi, the root PPO and PAL activities showed a trend of Streptomyces cinnamomi > CK. It can be seen that Streptomyces cinnamomi can significantly increase the PPO and PAL activities of Polygonatum cyrtonema roots.

[0127] Effects of Streptomyces cinnamomi on soluble sugar and malondialdehyde in Polygonatum cyrtonema

[0128] Effects of inoculation with Streptomyces cinnamomi on soluble sugar and malondialdehyde activity in roots of Polygonatum cyrtonema Figure 4 , under the condition of single inoculation of antagonistic bacteria, compared with CK, the soluble sugar of Streptomyces cinnamomi increased significantly, 49% higher than CK, while the activity of malondialdehyde was not significantly different from CK. Under the condition of inoculation of pathogens, compared with CK, Streptomyces cinnamomi significantly reduced the soluble sugar and malondialdehyde contents of Polygonatum yunnanensis by 27.58% and 48.50%, respectively. It can be seen that under the condition of inoculation of pathogens, the activity of soluble sugar and malondialdehyde in plant roots will be reduced, and under the condition of single inoculation of antagonistic bacteria, the soluble sugar content of plants will be increased, and the content of malondialdehyde will not be significantly affected.

[0129] Effects of Streptomyces cinnamomi on chlorophyll in Polygonatum cyrtonema

[0130] After inoculation with Streptomyces cinnamomi, the effect on the chlorophyll content of plants can be seen Figure 5 The results showed that the chlorophyll content was significantly increased under the treatment of pathogen inoculation and single antagonistic bacteria, among which the chlorophyll content under the conditions of pathogen inoculation and single antagonistic bacteria treatment was 22.46% and 106.18% higher than that of CK, respectively.

[0131] The conclusions drawn from the above experiments are:

[0132] (1) This experiment adopted the root injury perfusion method to test the control effect of cinnamon Streptomyces fermentation liquid on the root rot of Polygonatum cyrtonema. The experimental results showed that after inoculation with cinnamon Streptomyces fermentation liquid, the incidence of root rot of Polygonatum cyrtonema was 42.22%, and the control effect reached 53.85%. It can be seen that the antagonistic bacteria fermentation liquid of cinnamon Streptomyces can effectively prevent and control the root rot of Polygonatum cyrtonema. At the same time, the weight and length of the root system of Polygonatum cyrtonema treated with cinnamon Streptomyces fermentation liquid were significantly higher than those of other treatment groups. It can be seen that inoculation with cinnamon Streptomyces fermentation liquid significantly reduced the incidence of Polygonatum cyrtonema and had a certain promoting effect on the root growth of Polygonatum cyrtonema.

[0133] (2) In this experiment, the enzyme activity of the root system of Polygonatum cyrtonema was measured. The results showed that inoculation with the fermentation liquid of Streptomyces cinnamomi significantly increased the activities of SOD, POD, PAL and PPO in the root system of Polygonatum cyrtonema. It can be seen that inoculation with Streptomyces cinnamomi can improve the defense enzyme resistance of the root system of Polygonatum cyrtonema, thereby reducing the invasion of pathogens.

[0134] Determination of the antibacterial spectrum of Streptomyces cinnamomi

[0135] Determination of the antibacterial spectrum of Streptomyces cinnamomi against six different pathogens

[0136] The broad-spectrum activity of the antagonistic strain was tested against tomato gray mold (B. cinerea), citrus brown spot (A. alternata), kiwi rot (N. parvum), soybean phytophthora (P. sojae), pitaya wilt (F. oxysporum) and tobacco root rot (F. solani). The plate confrontation and cross-inoculation method were used. The results are shown in Table 8. Streptomyces cinnamomi had a good inhibitory effect on the six plant pathogens, and the maximum inhibition rate of Streptomyces cinnamomi reached 38%.

[0137] Table 8 The effect of biocontrol strains on multiple plant pathogens

[0138]

[0139] Note: Different letters after the values ​​in the same column indicate significant differences (P<0.05)

[0140] Effects of Streptomyces cinnamomi on sclerotial germination of pathogenic bacteria

[0141] Pick a single colony of activated antagonistic bacteria and inoculate it in the liquid culture medium of ISP2. Cultivate it at 28℃ and 160r / min for 3 days to make a fermentation liquid. Dip the fermentation liquid with a sterilized pipette tip, draw a nine-square grid containing 9 small squares (1.5cm*1.5cm) on the PDA plate, inoculate the sclerotia of the pathogen obtained by 15 days of culture, and observe the germination of sclerotia. The control is streaked with ISP2 culture medium, and repeated 3 times.

[0142] The results are as follows Figure 6 (a: CK; b: Streptomyces cinnamomi). The results showed that the control group pathogens almost covered the entire plate after 3 days of growth, while the mycelium of sclerotia germinated in the nine-square grid drawn by Streptomyces cinnamomi had been inhibited from growing, and there was an obvious antibacterial zone in the nine-square grid drawn by Streptomyces cinnamomi. It can be seen that Streptomyces cinnamomi can effectively inhibit the germination of Fusarium oxysporum sclerotia.

[0143] Effects of Streptomyces cinnamomi on the hyphae of pathogenic bacteria

[0144] In order to further explore the effect of biocontrol bacteria on the internal structure of pathogenic hyphae, after 3 days of confrontation culture with the antagonistic bacteria of Streptomyces cinnamomi, the hyphae of the pathogen close to the antagonistic bacteria were picked up and the morphological changes of the pathogenic hyphae were observed under a scanning electron microscope. The hyphae of the pathogenic fungus that had not been treated with the antagonistic bacteria were used as a control.

[0145] The results are as follows Figure 7 (a: CK; b: Streptomyces cinnamomi). After the antagonistic strain and Fusarium oxysporum were cultured for 3 days, the hyphae close to the antagonistic strain were picked, and the hyphae of the pathogen without antagonistic strain were used as the control. The morphological changes of the hyphae were observed under a scanning electron microscope. The results showed that the antagonistic strains could cause the hyphae of the pathogen to be deformed and have more branches. The hyphae grew twisted, the hyphae branches increased, and even expanded.

[0146] Effect of different pH on antibacterial effect

[0147] The activated Streptomyces cinnamomi was inoculated into ISP2 liquid medium with pH values ​​of 1, 3, 5, 7, 9, and 11. The culture was shaken at 28°C and 160rpm for 3 days, and the obtained fermentation liquid was centrifuged at 4°C and 8000rpm for 30 minutes, and the supernatant was collected and filtered with a 0.22μm filter membrane. PDA solid medium containing the fermentation filtrate of Streptomyces cinnamomi was prepared at a ratio of 1:9. Finally, the bacterial cake was placed in the center of the culture medium, and the antibacterial rate was calculated by inverting and culturing at 28°C for 7-9 days.

[0148] The results are as follows Figure 8 As shown in the figure, the antibacterial effect of the fermentation filtrate of Streptomyces cinnamomi first increased and then decreased with the increase of initial pH. The fermentation filtrate of Streptomyces cinnamomi had the highest antibacterial rate when the initial pH was 5. The antibacterial rate of Streptomyces cinnamomi reached 81.56%. It can be seen that the antagonistic effect of Streptomyces cinnamomi antagonists is better in acidic and neutral conditions.

[0149] Effect of different fermentation broth inoculation amounts on antibacterial effects

[0150] The activated Streptomyces cinnamomi was inoculated into ISP2 liquid medium. The culture was shaken at 28℃ and 160rpm for 3 days. The fermentation liquid was centrifuged at 4℃ and 8000rpm for 30min, and the supernatant was collected and filtered with a 0.22μm filter membrane. PDA solid medium containing the fermentation filtrate of Streptomyces cinnamomi was prepared at a ratio of 1%, 5%, 10% and 15%. Finally, the bacterial cake was placed in the center of the culture medium and inverted at 28℃ for 7-9 days to calculate the antibacterial rate.

[0151] The results are as follows Fig. 9 As shown in the figure, the growth rate method was used to determine the effect of different concentrations of the fermentation filtrate of Streptomyces cinnamomi on the inhibition of pathogens. The results showed that with the increase of the concentration of the fermentation filtrate, the inhibition rate of the antagonistic bacteria gradually increased. When the proportion of the fermentation filtrate in the culture medium was greater than 10%, the inhibition rate tended to be stable, and the inhibition rate of the antagonistic bacteria of Streptomyces cinnamomi on pathogens exceeded 80%.

[0152] Effect of different temperatures on antibacterial effect

[0153] The activated Streptomyces cinnamomi was inoculated into ISP2 liquid medium. The culture was shaken at 28℃ and 160rpm for 3 days, and the collected fermentation liquid was centrifuged at 4℃ and 8000rpm for 30min to remove the bacteria and collect the supernatant. The supernatant was repeatedly filtered with a filter with a pore size of 0.22μm. The fermentation filtrate was treated at 25℃, 40℃, 60℃, 70℃ and 90℃ for 30 minutes. The PDA solid medium of the fermentation filtrate of Streptomyces cinnamomi was used. Finally, the bacterial cake was placed in the center of the culture medium and inverted at 28℃ for 7-9d to calculate the antibacterial rate.

[0154] The results are as follows Fig.10 As can be seen from the figure, the antibacterial rate of fermentation filtrate treated at 25℃ and 40℃ is significantly higher than that of other temperature treatments, and it can be seen that the antibacterial rate will be greatly affected when the temperature exceeds 40℃. As the temperature increases, the antibacterial rate gradually decreases, and the differences are all significant. It can be seen that high temperature treatment will affect the antibacterial rate of Streptomyces cinnamomi.

[0155] The conclusions drawn from the above experiments are:

[0156] (1) The inhibition spectrum determination and electron microscopic observation of the effects of antagonistic bacteria on the mycelium of pathogens further confirmed that Streptomyces cinnamomi has the potential to be a biocontrol bacterium and can be used for the prevention and control of plant diseases.

[0157] (2) The effects of different culture conditions of Streptomyces cinnamomi on the antibacterial effect were further studied. The results showed that the antibacterial rate of Streptomyces cinnamomi was the highest at pH = 7, and the antibacterial rate was not significantly affected when the fermentation filtrate was treated at 25°C and 40°C. In addition, the antibacterial rate gradually increased with the increase of inoculation amount.

[0158] The above embodiments are merely explanations of the present invention and are not limitations of the present invention. After reading this specification, those skilled in the art may make modifications to the embodiments without any creative contribution as needed. However, such modifications are protected by the patent law as long as they are within the scope of the claims of the present invention.

Claims

1. An antagonistic bacterial strain Streptomyces cinnamomi, characterized in that: The antagonistic bacterial strain Streptomyces cinnamomi is Streptomyces cinnamomi (Streptomyces cinnamonensis) DHJ986, deposit unit: Guangdong Provincial Microbiological Culture Collection Center, deposit date: February 15, 2023, deposit number: GDMCC NO.63170.

2. An application of the antagonistic bacterial strain Streptomyces cinnamomi according to claim 1, characterized in that: The application of Streptomyces cinnamomi in the prevention and treatment of plant diseases is a disease caused by any one of Fusarium oxysporum, Botrytis cinerea, Alternaria alternata, Neofusella cylindrica, Phytophthora sojae or Fusarium solani.

3. The use of an antagonistic bacterial strain Streptomyces cinnamomi according to claim 2, characterized in that: The disease caused by Fusarium oxysporum is Polygonatum cyrtonema root rot.

4. The use of an antagonistic bacterial strain Streptomyces cinnamomi according to claim 2, characterized in that: The disease caused by Fusarium oxysporum is pitaya wilt.

5. The use of an antagonistic bacterial strain Streptomyces cinnamomi according to claim 2, characterized in that: The disease caused by Botrytis cinerea is tomato gray mold.

6. The use of an antagonistic bacterial strain Streptomyces cinnamomi according to claim 2, characterized in that: The disease caused by Alternaria alternata is citrus brown spot disease.

7. The use of an antagonistic bacterial strain Streptomyces cinnamomi according to claim 2, characterized in that: The disease caused by the network fungus Fusarium is kiwifruit rot.

8. The use of an antagonistic bacterial strain Streptomyces cinnamomi according to claim 2, characterized in that: The disease caused by soybean phytophthora is soybean phytophthora disease.

9. The use of an antagonistic bacterial strain Streptomyces cinnamomi according to claim 2, characterized in that: The disease caused by Fusarium solani is tobacco root rot.

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