Antagonistic bacterial strain Bacillus subtilis subsp. deserticola and its application

Abstract

The invention discloses an antagonistic bacterial strain Bacillus subtilis deserticola and application thereof, belonging to the technical field of plant disease prevention and control. Bacillus subtilis subsp. Inaquosorum )DHJ1189, deposited in: Guangdong Microbial Culture Collection Center, deposit date: February 15, 2023, deposit number: GDMCC NO.63171. Bacillus subtilis subsp. deserticola is used in the prevention and treatment of plant diseases, including diseases caused by Fusarium oxysporum, Botrytis cinerea, Alternaria alternata, Fusarium spp., Phytophthora sojae and tobacco root rot. The present invention can effectively prevent and treat 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 Bacillus subtilis deserticola and an 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, Yunnan polygonatum root rot, as a devastating disease, is called "plant cancer". The field incidence of Yunnan polygonatum root rot is 5%-15%, and in severe cases it can reach more than 30%. Root rot is a global disease. For perennial medicinal plants such as polygonatum, Panax notoginseng, and ginseng, this disease has become one of the main obstacles threatening their healthy and sustainable development. The main pathogen of Yunnan polygonatum root rot is Fusarium oxysporum ( Fusarium oxysporum ), this pathogen exists mainly 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 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 address 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 Bacillus subtilis deserticola, 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 Bacillus subtilis deserticola, wherein the Bacillus subtilis deserticola ( Bacillus subtilis subsp. Inaquosorum)DHJ1189, deposit unit: Guangdong Provincial Microbiological Culture Collection Center, deposit date: February 15, 2023, deposit number: GDMCC NO.63171.

[0007] The present invention also provides an application of an antagonistic bacterial strain Bacillus subtilis deserticola, wherein the application of the Bacillus subtilis deserticola is in the prevention and treatment of plant diseases, wherein the plant diseases include diseases caused by Fusarium oxysporum, Botrytis cinerea, Alternaria alternata, Neofusella cylindrica, 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, Bacillus subtilis desert subspecies can effectively prevent and control plant diseases, among which the prevention and control effect of Yunnan polygonatum root rot reached 46.15%. It can be seen that the fermentation liquid of antagonistic bacteria of Bacillus subtilis desert subspecies can effectively prevent and control Yunnan polygonatum root rot. At the same time, inoculation of Bacillus subtilis desert subspecies fermentation liquid significantly reduced the incidence of Yunnan polygonatum and had a certain promoting effect on the root growth of Yunnan polygonatum.

[0017] Secondly, Bacillus subtilis desert subspecies can significantly increase the activity of superoxide dismutase (SOD), peroxidase (POD), phenylalanine ammonia lyase (PAL) and polyphenol oxidase (PPO) in the roots of Polygonatum cyrtonema. It can be seen that inoculation with Bacillus subtilis desert subspecies will increase the resistance of the defense enzymes in the roots of Polygonatum cyrtonema, thereby reducing the invasion of pathogens. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is a potted plant test effect diagram of the present invention;

[0019] Figure 2 The effect of Bacillus subtilis deserticola subsp. deserticola on SOD (A) and POD (B) in the roots of Polygonatum cyrtonema;

[0020] Figure 3 The effect of Bacillus subtilis deserticola subsp. deserticola on PPO (A) and PAL (B) in the roots of Polygonatum cyrtonema;

[0021] Figure 4 The effect of Bacillus subtilis subsp. deserticola on soluble sugar (A) and malondialdehyde (B) in the roots of Polygonatum cyrtonema;

[0022] Figure 5 The effect of Bacillus subtilis deserticola on chlorophyll in the leaves of Polygonatum cyrtonema;

[0023] Figure 6 It is the effect of Bacillus subtilis subsp. deserticola on the germination of sclerotia of the pathogen F. oxysporum;

[0024] Figure 7 It is the effect of Bacillus subtilis deserticola subsp. on the hyphae of pathogenic fungus F. oxysporum;

[0025] Figure 8 The effect of Bacillus subtilis subsp. deserticola treated with different pH on the antibacterial effect of F. oxysporum;

[0026] Fig. 9 is the effect of different inoculation amounts of Bacillus subtilis subsp. deserticola on the antibacterial effect of F. oxysporum;

[0027] Fig.10 The effect of different temperatures of Bacillus subtilis deserticola treatment 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] An antagonistic bacterial strain of Bacillus subtilis subsp. deserticola, whose plant disease is Fusarium oxysporum ( Fusarium oxysporum ) caused by root rot of Polygonatum cyrtonema.

[0031] Example 2

[0032] An antagonistic bacterial strain of Bacillus subtilis subsp. deserticola, whose plant disease is Fusarium oxysporum ( Fusarium oxysporum) caused by dragon fruit wilt.

[0033] Example 3

[0034] An antagonistic bacterial strain of Bacillus subtilis subsp. deserticola, whose plant disease is Botrytis cinerea ( Botrytis cinerea ) caused by tomato gray mold.

[0035] Example 4

[0036] An antagonistic bacterial strain of Bacillus subtilis subsp. deserticola, whose plant disease is Alternaria alternata ( Alternaria alternata ) caused by citrus brown spot disease.

[0037] Example 5

[0038] An antagonistic bacterial strain of Bacillus subtilis subsp. deserticola, whose plant disease is Fusarium nematodes ( Neofusicoccum parvum ) caused by kiwifruit rot.

[0039] Example 6

[0040] An antagonistic bacterial strain of Bacillus subtilis subsp. deserticola, whose plant disease is Phytophthora sojae ( Phytophthora sojae ) is caused by soybean blight.

[0041] Example 7

[0042] An antagonistic bacterial strain of Bacillus subtilis subsp. deserticola, whose plant disease is Fusarium solani ( Fusarium solani ) caused by tobacco root rot.

[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] Under sterile conditions, plant roots, stems and leaves were ground into a homogenate with liquid nitrogen. First, 2 g of the crushed plant roots, stems and leaves and rhizosphere soil were weighed and transferred to a container containing glass slag and 18 mL of saline. Then, they were shaken and incubated at 37°C for 20 min to mix, and immediately ultrasonicated for 40 s to make 10 -1 Use 10-fold gradient dilution method to dilute step by step to make 10 -2 , 10 -4 and 10 -5 Dilution test solution (10-2 , 10 -4 and 10 -5 The plant sap / rhizosphere soil sample is diluted in a gradient, first by adding 2 g of sample to 18 ml of diluent, equivalent to a 10 -1 On this basis, dilute to 10 -2 , 10 -4 and 10 -5 ), take 0.2 mL of the test solution with different dilutions, inoculate it on 6 kinds of separation culture media by spreading method, and then culture it in a 28°C incubator for more than one week. Finally, 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 1.5 mL centrifuge tubes for DNA extraction.

[0046] Results and Analysis

[0047] (1) In this study, 224 antagonistic bacteria were isolated from three different varieties of Polygonatum. After excluding duplicates, a total of 121 antagonistic bacteria belonging to 43 genera were obtained. In both the endophytic and rhizosphere soils of Polygonatum, 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 abundance of Bacillus and Streptomyces was the highest in the roots of Polygonatum cyrtonema and Polygonatum cyrtonema, and the largest number was isolated from 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 Storage of Bacillus subtilis subsp. deserticola

[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] Preliminary screening of antagonistic bacteria by plate confrontation method

[0055] 121 antagonistic bacteria isolated and purified in the early stage were used as test strains. The plate confrontation method was used to determine that there were five strains including 1189 with an inhibition width >10 mm (+++) among the 121 antagonistic bacteria. 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 filtrate of 1189. The results (Table 1) showed that the antibacterial rate of 1189 reached 79.06%. Combining the results of the primary screening and the secondary screening, the strain 1189 was used as the test strain for subsequent studies.

[0058]

[0059] Identification of antagonistic bacteria 1189

[0060] Morphological observation

[0061] On ISP2 solid medium, 1189 was dark gray, opaque, serrated, wrinkled in the middle, and warm in texture. Gram staining showed that 1189 was blue-purple, so 1189 was a Gram-positive bacterium.

[0062] Physiological and biochemical identification

[0063] The results of physiological and biochemical identification of 1189 are shown in Table 2. The results show that 1189 has the ability to produce D-xylose, oxidase, starch hydrolysis, methyl red and VP test. In addition, 1189 also has the ability to produce D-mannitol, catalase and gelatin liquefaction.

[0064] In addition, as shown in Table 3, the effects of different pH values, different temperatures and different NaCl concentrations on the growth of strain 1189 were determined. The results showed that 1189 grew better when the pH was 5-7; 1189 could grow normally at 22℃-37℃, and 1189 grew better at 37℃. When the salt concentration increased, the salt tolerance of strain 1189 gradually decreased. When the salt concentration reached 7%, the concentration of the strain decreased, but it could still grow normally. However, when the salt concentration reached 10%, the strain no longer grew normally.

[0065]

[0066] Molecular Biology Identification

[0067] Phylogenetic tree analysis of 16S rDNA sequence of strain 1189

[0068] The 16S rDNA sequence cloned product of strain 1189 was sent to Kunming Qingke Biotechnology Co., Ltd. for sequencing, and a 1377 bp DNA fragment was obtained. The sequence was then submitted to the GenBank database for registration. The similarity between 1189 and B. cabrialesii was high, reaching 99.93%. At the same time, the morphological characteristics and physiological and biochemical characteristics of strain 1189 were compared with the "Handbook of Identification of Bergey's Antagonistic Bacteria" and the "Handbook of Identification of Common Antagonistic Bacteria Systems". 1189 met the requirements of Bacillus cabrialesii ( B. cabrialesii ) based on the 16S rDNA homology, 24 model strains with high homology were downloaded from the NCBI website, and the Neighbor-Joining phylogenetic tree was constructed using MEGA 5.1 software. B. cabrialesii In summary, 1189 was identified as Bacillus subtilis deserticola ( Bacillus inaqusorum ).

[0069] Finally, 1198 strains were sent to Guangdong Microbial Culture Collection Center for preservation. Their taxonomic names are: Bacillus subtilis subsp. Inaquosorum ,Deposit date: February 15, 2023, Deposit number: GDMCC NO.63171, translated name is Bacillus subtilis desert subsp.

[0070] Study on the control effect of Bacillus subtilis deserticola on root rot of Polygonatum cyrtonema

[0071] Antagonistic bacteria tested: Bacillus subtilis subsp. deserticola ( Bacillus subtilis subsp. Inaquosorum ).

[0072] Pathogenic fungi tested: Fusarium oxysporum, the pathogen of Polygonatum cyrtonema root rot Fusarium oxysporum ) was provided by Yunnan University of Chinese Medicine.

[0073] Test materials and cultivation medium

[0074] Yunnan Polygonatum sibiricum are all one-year-old tubers, provided by the Muqi Black Polygonatum sibiricum planting base in Bozhu Town, Yunnan Province.

[0075] Cultivation medium: The planting soil is mainly red soil, humus and perlite, which are mixed in a ratio of 3:3:1. After mixing, the pH is 7.23, the organic matter is 91.11 g / kg, and the effective phosphorus, effective potassium and effective nitrogen contents are 72.18 mg / kg, 312.14 mg / kg and 484 mg / kg respectively.

[0076] 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.

[0077] The main reagents used in the experiment include formalin, trichloroacetic acid, EDTA-Na2, riboflavin, guaiacol, sulfur salicylic acid, toluene, Coomassie brilliant blue, etc.

[0078] Experimental design and process

[0079] In December 2021, a potted inoculation experiment was conducted to explore the biocontrol effect of Bacillus subtilis desert subspecies 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 Bacillus subtilis desert subspecies fermentation liquid; 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 Bacillus subtilis desert subspecies fermentation liquid in the early stage, and then inoculation of pathogens (Bacillus subtilis desert subspecies + B) in the later stage. A total of 5 treatments were set up (Table 4), with 10 replicates for each treatment.

[0080] The plastic flower pots used in the experiment (sterilized by soaking in 0.1% potassium permanganate solution for 24 h before use) were 15 cm high and 16.50 cm in diameter. Each pot was filled with 1.5 kg of sterilized soil, and the one-year-old tubers of Yunnan Polygonatum were transplanted. After Yunnan Polygonatum grew for about 180 days, Yunnan Polygonatum potted seedlings with consistent growth were selected for the experiment. For treatments A and B, 10 mL of Bacillus subtilis desert subsp. 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 Bacillus subtilis desert subsp. and liquid culture medium were used for root irrigation in the early stage. After 12 days, the pathogen Fusarium oxysporum was inoculated by root irrigation method (10 7 CFU / mL, 5 mL). Treatment D was inoculated with only the pathogen Fusarium oxysporum (10 7 CFU / 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 roots of Polygonatum cyrtonema were measured, including superoxide dismutase (SOD), peroxidase (POD), phenylalanine ammonia lyase (PAL) and polyphenol oxidase (PPO), and the physical and chemical properties of the rhizosphere soil and the microbial community structure were measured.

[0081]

[0082] 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 180 r / min for 3 days. ISP2 liquid culture medium was used for dilution to obtain 10 8 CFU / mL of fermentation broth.

[0083] 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.

[0084] Effect of biocontrol bacteria on root rot of Polygonatum cyrtonema

[0085] The root rot of Polygonatum cyrtonema was graded and statistically analyzed, and the incidence rate, disease index and disease prevention effect were calculated.

[0086] Grading method:

[0087] Level 0: No disease occurs;

[0088] Level 1: The taproot remains unchanged, and the tips of the fibrous roots turn slightly brown, which does not affect plant growth;

[0089] 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;

[0090] Level 5: The taproot turns darker, and the fibrous roots decrease or disappear, seriously affecting the growth of the plant;

[0091] Level 7: Root rot and plant cannot grow.

[0092] Calculation formula:

[0093]

[0094] Determination of Growth Physiological Indexes of Polygonatum cyrtonema

[0095] 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.

[0096] Determination of Physical and Chemical Properties of Rhizosphere Soil of Polygonatum cyrtonema

[0097] Soil pH was determined using a pH-25 meter; soil organic matter content (SOM) was determined using potassium dichromate external heating method; total phosphorus (TP) content was determined using HClO4-H2SO4 digestion method; total potassium (TK) content was determined using the acid-atomic absorption method using a Thermo Scientific S series atomic absorption spectrometer; alkaline nitrogen (AN) content was determined using the diffusion dish method; available phosphorus (AP) content was determined using sodium bicarbonate extraction-molybdenum antimony colorimetry, and available potassium (AK) content was determined using ammonium acetate extraction-atomic absorption method.

[0098] Results and Analysis

[0099] Effects of Bacillus subtilis subsp. deserticola on the incidence and disease index of Polygonatum cyrtonema root rot

[0100] 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: Bacillus subtilis desert subspecies + B; B: P + 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-free group and the single antagonistic bacteria (Bacillus subtilis desert subspecies) treatment was less than 20%; the incidence rate of the pathogen-inoculated group was 44.45% after the inoculation of Bacillus subtilis desert subspecies in the early stage, and the control effect reached 46.15%. It can be seen that Bacillus subtilis desert subspecies can effectively prevent and control the root rot of Polygonatum cyrtonema.

[0101]

[0102] Effects of Bacillus subtilis subsp. deserticola on the growth parameters of Polygonatum cyrtonema

[0103] As shown in Table 6, whether inoculated with pathogens or not, inoculation with Bacillus subtilis subsp. deserticola promoted the growth of the roots of Polygonatum yunnanensis. In the case of single antagonistic bacteria treatment, the weight of the root system reached 11.84 g, which was 12.23% higher than CK. The root length reached 16.73 cm, which was 24.67% higher than the treatment. In the case of pathogen inoculation, the weight of the root system reached 10.59 g, which was 33.04% higher than the P+B treatment. The root length reached 15.37 cm, which was 61.79% higher than P+B.

[0104]

[0105] Effects of Bacillus subtilis subsp. deserticola on the physicochemical properties of rhizosphere soil of Polygonatum cyrtonema

[0106] 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 8.30% after treatment with Bacillus subtilis desert subspecies fermentation liquid; the available phosphorus and available potassium contents were reduced, and the Bacillus subtilis desert subspecies treatment group decreased by 48.81% and 26.29%, respectively. Compared with CK, the total phosphorus content of the Bacillus subtilis desert subspecies treatment group decreased (15.55%), and from the perspective of total potassium, its change did not reach a significant level.

[0107] In the treatment of pathogen inoculation, there was no significant change in organic matter content compared with the P+B treatment group. The fermentation liquid of Bacillus subtilis subsp. deserticola significantly increased the content of alkaline nitrogen and available phosphorus, with alkaline nitrogen being 144.58% higher than that of the P+B treatment; available phosphorus being 35.95% higher than that of the P+B treatment; total phosphorus content was not significantly different among different treatments. In terms of potassium content, the total potassium content of the Bacillus subtilis subsp. deserticola + B treatment group was higher than that of the other two treatment groups, reaching 1100.82 mg / kg.

[0108]

[0109] Effects of Bacillus subtilis subsp. deserticola on POD and SOD of Polygonatum cyrtonema

[0110] Depend on Figure 2It can be seen that the fermentation liquid of Bacillus subtilis subsp. deserticola 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 Bacillus subtilis subsp. deserticola can significantly increase the activity of POD and SOD of Polygonatum cyrtonema, with POD increased by 8.83% and SOD increased by 343.54%, respectively. Under the condition of inoculation of pathogens, Bacillus subtilis subsp. deserticola can also increase the POD and SOD of Polygonatum cyrtonema roots, with POD increased by 34.92% and SOD increased by 101.47%. It can be seen that the fermentation filtrate of Bacillus subtilis subsp. deserticola can increase the POD and SOD enzyme activities of Polygonatum cyrtonema roots.

[0111] Effects of Bacillus subtilis subsp. deserticola on the activities of PPO and PAL in Polygonatum cyrtonema

[0112] Effects of inoculation with Bacillus subtilis subsp. deserticola fermentation broth on the activities of PPO and PAL in the roots of Polygonatum cyrtonema Figure 3 After the roots of Polygonatum cyrtonema were irrigated with fermented liquid of Bacillus subtilis deserticola, the root PPO and PAL activities showed a trend of Bacillus subtilis deserticola > CK. It can be seen that Bacillus subtilis deserticola can significantly increase the PPO and PAL activities of Polygonatum cyrtonema roots.

[0113] Effects of Bacillus subtilis subsp. deserticola on soluble sugar and malondialdehyde (MDA) in Polygonatum cyrtonema

[0114] Effects of inoculation with Bacillus subtilis subsp. deserticola 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 Bacillus subtilis desert subsp. desert was significantly increased, 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, Bacillus subtilis desert subsp. desert significantly reduced the soluble sugar and malondialdehyde contents of Polygonatum cyrtonema by 10.95% and 17.54%, 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 have little effect.

[0115] Effects of Bacillus subtilis subsp. deserticola on chlorophyll in Polygonatum cyrtonema

[0116] After inoculation with Bacillus subtilis deserticola, the effect on the chlorophyll content of plants can be seen Figure 5 The results showed that the chlorophyll content was significantly increased when inoculated with pathogens or treated with single antagonistic bacteria. The chlorophyll content of the fermentation liquid of Bacillus subtilis subsp. deserticola was 15.99% and 47.39% higher than that of CK under the conditions of inoculation with pathogens and single antagonistic bacteria, respectively.

[0117] The conclusions drawn from the above experiments are:

[0118] (1) This experiment used the root injury infusion method to test the control effect of Bacillus subtilis subsp. deserticola fermentation liquid on the root rot of Polygonatum cyrtonema. The experimental results showed that after inoculation with the fermentation liquid of Bacillus subtilis subsp. deserticola, the incidence of root rot of Polygonatum cyrtonema was 44.45%, and the control effect reached 46.15%. It can be seen that the fermentation liquid of antagonistic bacteria of Bacillus subtilis subsp. deserticola 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 the fermentation liquid of Bacillus subtilis subsp. deserticola were significantly higher than those of other treatment groups. It can be seen that inoculation with the fermentation liquid of Bacillus subtilis subsp. deserticola significantly reduced the incidence of Polygonatum cyrtonema and had a certain promoting effect on the root growth of Polygonatum cyrtonema.

[0119] (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 Bacillus subtilis subsp. deserticola significantly increased the activities of SOD, POD, PAL and PPO in the root system of Polygonatum cyrtonema. It can be seen that inoculation with Bacillus subtilis subsp. deserticola will increase the resistance of the defense enzymes in the root system of Polygonatum cyrtonema, thereby reducing the invasion of pathogens.

[0120] Determination of the antibacterial spectrum of Bacillus subtilis subsp. deserticola

[0121] Determination of the antibacterial spectrum of Bacillus subtilis subsp. deserticola against six different pathogens

[0122] Broad-spectrum activity assay against tomato gray mold ( B. cinerea )、Citrus brown spot( A. alternata )、Kiwifruit rot( N. parvum )、Phytophthora sojae( P. sojae )、Pitaya wilt disease( F. oxysporum ) and tobacco root rot ( F. solani The plate was inoculated in a cross-cross manner. The results are shown in Table 8. Bacillus subtilis deserticola had a good inhibitory effect on the six plant pathogens, and the maximum inhibition rate of Bacillus subtilis deserticola reached 34%.

[0123]

[0124] Note: Different letters after the values ​​in the same column indicate significant differences ( P <0.05)

[0125] Effects of Bacillus subtilis subsp. deserticola on sclerotial germination of pathogenic bacteria

[0126] Pick a single colony of activated antagonistic bacteria and inoculate it in ISP2 liquid culture medium. Cultivate it at 28℃ and 160 r / min for 3 days to make a fermentation liquid. Dip the fermentation liquid with a sterilized pipette tip, and draw a nine-square grid containing 9 small squares (1.5 cm*1.5 cm) on the PDA plate. Inoculate the pathogen sclerotia obtained after 15 days of cultivation into it and observe the germination of sclerotia. The control was streaked with ISP2 culture medium, and repeated 3 times.

[0127] The results are as follows Figure 6 , and further determination of sclerotial germination showed that the pathogen in the control group almost covered the entire plate after 3 days of growth, while the mycelium of sclerotial germination placed in the nine-square grid drawn by Bacillus subtilis desert subsp. had been inhibited from growing, and there was an obvious antibacterial zone in the nine-square grid drawn by Bacillus subtilis desert subsp. It can be seen that Bacillus subtilis desert subsp. can effectively inhibit the germination of Fusarium oxysporum sclerotia.

[0128] Effects of Bacillus subtilis subsp. deserticola on the hyphae of pathogenic bacteria

[0129] In order to further explore the effect of biocontrol bacteria on the internal structure of pathogenic hyphae, after culturing with the antagonistic bacteria of Bacillus subtilis subsp. deserticola for 3 days, 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 the control.

[0130] The results are as follows Figure 7 , (A:CK; B: Bacillus subtilis deserticola), after the antagonistic strain and Fusarium oxysporum were cultured for 3 days, the hyphae close to the antagonistic bacteria were picked, and the hyphae of the pathogen without antagonistic bacteria 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.

[0131] Effect of different pH on antibacterial effect

[0132] The activated Bacillus subtilis deserticola 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 160 rpm for 3 days, and the fermentation broth was centrifuged at 4°C and 8000 rpm for 30 min. The supernatant was collected and filtered with a 0.22 μm filter membrane. A PDA solid medium containing the fermentation filtrate of Bacillus subtilis deserticola was prepared at a ratio of 1:9. Finally, the bacterial cake was placed in the center of the culture medium and inverted at 28°C for 7-9 days to calculate the antibacterial rate.

[0133] The results are as follows Figure 8As shown in the figure, the antibacterial effect of the fermentation filtrate of Bacillus subtilis subsp. deserticola showed a trend of first increasing and then decreasing with the increase of initial pH. The antibacterial rate of the fermentation filtrate of Bacillus subtilis subsp. deserticola was the highest when the initial pH was 7. The antibacterial rate of Bacillus subtilis subsp. deserticola reached 38.32%. It can be seen that the antagonistic bacteria of Bacillus subtilis subsp. deserticola showed a good antagonistic effect under neutral conditions.

[0134] Effect of different fermentation broth inoculation amounts on antibacterial effects

[0135] The activated Bacillus subtilis deserticola was inoculated into ISP2 liquid medium. The culture was shaken at 28°C and 160 rpm for 3 days, and the obtained fermentation broth was centrifuged at 4°C and 8000 rpm for 30 min. The supernatant was collected and filtered with a 0.22 μm filter membrane. PDA solid medium containing fermentation filtrate of Bacillus subtilis deserticola 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°C for 7-9 days to calculate the antibacterial rate.

[0136] 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 fermentation filtrate of Bacillus subtilis subsp. deserticola on the inhibitory effect of pathogens. The results showed that with the increase of the concentration of fermentation filtrate, the inhibition rate of antagonistic bacteria gradually increased. When the proportion of fermentation filtrate in the culture medium was greater than 10%, the inhibition rate tended to be stable, and the inhibition rate of antagonistic bacteria of Bacillus subtilis subsp. deserticola on pathogens exceeded 50%.

[0137] Effect of different temperatures on antibacterial effect

[0138] The activated Bacillus subtilis desert subspecies was inoculated into ISP2 liquid medium. The culture was shaken at 28℃ and 160 rpm for 3 days, and the collected fermentation broth was centrifuged at 4℃ and 8000 rpm for 30 min to remove the bacteria and collect the supernatant. The supernatant was repeatedly filtered with a 0.22 μm pore filter. The fermentation filtrate was treated at 25℃, 40℃, 60℃, 70℃ and 90℃ for 30 minutes. The PDA solid medium of the fermentation filtrate of Bacillus subtilis desert subspecies was used. 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.

[0139] The results are as follows Fig.10 For Bacillus subtilis deserticola, the antibacterial rate of fermentation filtrate treated at 25℃ and 40℃ was significantly higher than that of other temperature treatments, which shows that the antibacterial rate will be greatly affected when the temperature exceeds 40℃. It can be seen that high temperature treatment will affect the antibacterial rate of Bacillus subtilis deserticola.

[0140] The conclusions drawn from the above experiments are:

[0141] (1) The inhibition spectrum determination and electron microscopic observation of the effects of antagonistic bacteria on pathogenic hyphae further confirmed that Bacillus subtilis deserticola has the potential to be a biocontrol bacterium and can be used for the prevention and control of plant diseases.

[0142] (2) The effects of different culture conditions of Bacillus subtilis subsp. deserticola on the antibacterial effect were further studied. The results showed that the antibacterial rate of Bacillus subtilis subsp. deserticola 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.

[0143] 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 Bacillus subtilis subsp. deserticola, characterized in that: The Bacillus subtilis subsp. deserticola ( Bacillus subtilis subsp. Inaquosorum )DHJ1189, deposit unit: Guangdong Provincial Microbiological Culture Collection Center, deposit date: February 15, 2023, deposit number: GDMCC NO.63171.

2. A use of the antagonistic bacterial strain Bacillus subtilis subsp. deserticola according to claim 1, characterized in that: The application of the Bacillus subtilis deserticola subspecies in the prevention and treatment of plant diseases includes diseases caused by Fusarium oxysporum, Botrytis cinerea, Alternaria alternata, Neofusella cylindrica, Phytophthora sojae and Fusarium solani.

3. The use of an antagonistic bacterial strain Bacillus subtilis subsp. deserticola 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 Bacillus subtilis subsp. deserticola according to claim 2, characterized in that: The disease caused by Fusarium oxysporum is pitaya wilt.

5. The use of an antagonistic bacterial strain Bacillus subtilis subsp. deserticola 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 Bacillus subtilis subsp. deserticola 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 Bacillus subtilis subsp. deserticola 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 Bacillus subtilis subsp. deserticola 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 Bacillus subtilis subsp. deserticola according to claim 2, characterized in that: The disease caused by Fusarium solani is tobacco root rot.

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