A biological control bacterium for Solanaceae plants and its cultivation method

Abstract

The present invention belongs to the field of biological control, and particularly relates to a biocontrol fungus capable of infecting and controlling solanaceous plants and an optimization method for its fermentation and culture conditions. The present invention discloses a method for preventing bacteria in solanaceous plants and optimizing its fermentation and culture conditions. Among them, the biocontrol bacteria are Alternaria alternata, Epichloë sorghi, Fusarium equiseti, Curvularia hawaiiensis and / or Nigrospora oryzae. The present invention obtains pathogenic fungi with strong pathogenicity to solanaceous plants by collecting naturally diseased plants, isolating, culturing and purifying them indoors, and verifying the pathogenic bacteria by Koch's postulates.

Description

Technical Field

[0001] The present invention belongs to the field of biological control, and particularly relates to a biocontrol fungus capable of infecting and controlling solanaceous plants and a cultivation method thereof. Background Art

[0002] The invasion of alien organisms threatens the ecological safety of farmland environment, agricultural production and human health, and thus has received extensive attention from researchers in various countries. Solanum rostratum, also known as Solanum aculeatissimum and yellow-flowered Solanum aculeatissimum, is a malignant plant with serious harm in the genus Solanum of the Solanaceae family. It is listed as a quarantine plant with great invasion risk in China. Solanum rostratum has a taproot system, with a plant height of more than 80 cm. The whole plant is densely covered with yellow thorns of different lengths, with the thorn length of 0.3 - 1.0 cm, and there are stellate hairs with stalks on the thorns; the leaves are ovate or elliptical, 6 - 18 cm long and 4 - 9 cm wide, with irregular pinnate deep lobes and some lobes being pinnately semi-lobed, the lobes are elliptical or nearly circular, alternate single leaves, with thorns on both the front and back of the leaves, and the petiole is 0.5 - 5.0 cm long; racemose inflorescence, the corolla is yellow, radially symmetric. Solanum rostratum has high reproductive ability and strong adaptability, is tolerant to barrenness and drought, often grows in wastelands, grasslands, river beaches and overgrazed pastures, and can also invade farmlands and orchards. Since it was first discovered in Chaoyang County, Liaoning Province, China in 1981, it has been successively discovered in many places in China in the past 40 years, and the spreading trend is still continuously expanding. It has strong competitiveness and can severely inhibit the growth of other plants, often forming a large area of single communities and destroying local biodiversity. The whole plant is densely covered with stinging hairs, which can harm livestock and affect grazing and wool production. The plant is poisonous. After ingestion, it can cause severe enteritis and bleeding. The fruit contains the neurotoxin solanine, which can cause the death of livestock. At the same time, this species is also a wild host of the Colorado potato beetle and the potato leafroll virus, causing serious economic losses and ecological environment damage.

[0003] Currently, the control technologies for Solanum rostratum mainly include three types: manual and mechanical removal, chemical agent control, and alternative plant planting. Mechanical removal is carried out in the early growth stage, especially in the seedling stage before 4 true leaves. Physical control is simple and easy to implement and has no pollution to the environment, but it consumes a large amount of manpower and material resources. Moreover, Solanum rostratum grows extremely fast. If the plants are allowed to mature, due to the thorns on the whole plant, it will bring certain difficulties to the removal work. Chemical control involves spraying chemical pesticides, which has a good effect on removing Solanum rostratum and a fast effect, but it is prone to problems such as pollution, residue and drug resistance, and is not suitable for long-term application; alternative control can effectively inhibit the growth of Solanum rostratum but cannot completely remove this weed.

[0004] To achieve the green control of such weeds, it is an inevitable choice to develop microbial herbicides using pathogenic fungi. However, the infection effect of pathogenic fungi is affected by the environment, so the fermentation conditions are particularly important. At present, there is a lack of research on the fermentation medium and fermentation conditions of the biocontrol bacteria of Solanum rostratum, making it impossible to further develop and produce microbial herbicides. Therefore, this study focuses on important control parameters during the fermentation process, such as carbon and nitrogen sources, culture temperature, pH value, and culture time, in order to clarify the relationship between various factors and the fermentation production of the strain, thereby providing a theoretical basis for the large-scale production of the microbial agent. Summary of the Invention

[0005] First, the present invention provides a herbicide for inhibiting the growth of Solanaceae plants, which contains biocontrol bacteria of Solanaceae plants and / or their fermentation broth. The biocontrol bacteria of Solanaceae plants include Alternaria spp., Epichloë sorghi, Fusarium equiseti, Curvularia hawaiiensis, and / or Nigrospora oryzae.

[0006] Furthermore, the Solanaceae plants include, but are not limited to, one or more of Solanum aculeatissimum, Solanum nivalomontanum, Solanum souliei, Solanum rostratum, Solanum nigrum, and Solanum violaceum.

[0007] Furthermore, the Solanaceae plant is selected from Solanum rostratum.

[0008] Second, the present invention provides a culture medium for the fermentation culture of biocontrol bacteria of Solanaceae plants, which mainly contains carbon sources, nitrogen sources, as well as other inorganic salts and water.

[0009] Furthermore, the carbon source is selected from one or more of maltose, glucose, soluble starch, sucrose, lactose, and glycerol.

[0010] Furthermore, the carbon source is selected from lactose and glycerol.

[0011] Furthermore, the nitrogen source is selected from one or more of peptone, beef extract, yeast extract, ammonium sulfate, ammonium chloride, and urea.

[0012] Furthermore, the nitrogen source is selected from yeast and ammonium sulfate.

[0013] Furthermore, the ratio of the carbon source to the nitrogen source is 1 - 3:1 - 3.

[0014] In an embodiment of the present application, the biocontrol bacteria of the present application are selected from Alternaria spp. In the liquid fermentation medium of Alternaria spp., the ratio of glucose, lactose, yeast, and ammonium sulfate is 3:3:1:3; the fermentation culture conditions are: the amount of mother liquor is 5%, the filling amount in the shake flask is 15%, the temperature is 30°C, the initial pH value is 6, and the culture time is 6 days.

[0015] In one embodiment of the present application, the biocontrol bacteria of the present application are selected from Epichloë sorghi. The ratio of maltose, sucrose, yeast, and ammonium sulfate in the liquid fermentation medium of Epichloë sorghi is 2:1:3:3; the culture conditions for fermentation are: the mother liquor volume is 25%, the shaking flask loading is 25%, the temperature is 20°C, the initial pH value is 7, and the culture is carried out for 8 days.

[0016] In one embodiment of the present application, the biocontrol bacteria of the present application are selected from Fusarium equiseti. The ratio of maltose, sucrose, yeast, and ammonium sulfate in the liquid fermentation medium of Fusarium equiseti is 1:3:3:2; the culture conditions for fermentation are: the mother liquor volume is 15%, the shaking flask loading is 15%, the temperature is 20°C, the initial pH value is 5, and the culture is carried out for 4 days.

[0017] In one embodiment of the present application, the biocontrol bacteria of the present application are selected from C. hawaiiensis. The ratio of soluble starch, sucrose, beef extract, and peptone in the liquid fermentation medium of C. hawaiiensis is 2:2:1:2; the culture conditions for fermentation are: the mother liquor volume is 25%, the shaking flask loading is 15%, the temperature is 20°C, the initial pH value is 5, and the culture is carried out for 4 days.

[0018] In one embodiment of the present application, the biocontrol bacteria of the present application are selected from Nigrospora oryzae. The ratio of lactose, glycerol, yeast, and ammonium sulfate in the liquid fermentation medium of Nigrospora oryzae is 2:2:1:2; the culture conditions for fermentation are: the mother liquor volume is 25%, the shaking flask loading is 25%, the temperature is 20°C, the initial pH value is 5, and the culture is carried out for 4 days.

[0019] Thirdly, the present invention provides a screening method for a biocontrol bacteria medium of solanaceous plants, and the method includes the following steps:

[0020] S1. Preparation of liquid mother species: Punch a pure cultured fungal cake and inoculate it into a PDA liquid medium, and carry out shaking flask culture under certain conditions. The mycelia in the obtained liquid mother species are tightly entangled with each other to form particles and are evenly suspended in the culture solution;

[0021] S2. Single-factor screening of carbon sources: Add 6 different carbon sources to the basic medium I, and then inoculate the liquid mother species into different media respectively, compare the effects of the 6 carbon sources on the biomass, and determine the carbon source with the highest mycelial biomass;

[0022] S3. Single-factor screening of nitrogen sources: Add 6 different nitrogen sources to the basic medium II, and then inoculate the liquid mother species into different media respectively, compare the effects of the 6 nitrogen sources on the biomass, and determine the nitrogen source with the highest mycelial biomass;

[0023] S4. Selection of carbon-nitrogen ratio: Taking two carbon sources and two nitrogen sources with the highest obtained biomass as factors, without adding any other components, an orthogonal experiment is designed according to four factors and three levels to compare the effects of different C / N ratios on the growth of fungi, and the optimal carbon-nitrogen ratio is screened out.

[0024] Further, in step S1, the shake flask culture is carried out under the culture conditions of a medium loading of 20%, 25 °C, 120 rpm, and shake flask culture for 72 h.

[0025] Further, in step S2, the basic medium I is based on 2% peptone, 0.05% MgSO4·H2O, and 0.1% KH2PO4.

[0026] Further, the carbon source is selected from one or more of maltose, glucose, soluble starch, sucrose, lactose, and glycerol.

[0027] Further, in step S3, the basic medium II is based on 2% sucrose, 0.05% MgSO4·H2O, and 0.1% KH2PO4.

[0028] Further, in steps S2 and S3, the inoculation amount of the liquid mother culture is 5%.

[0029] Further, the nitrogen source is selected from one or more of peptone, beef extract, yeast extract, ammonium sulfate, ammonium chloride, and urea.

[0030] Further, the ratio of the carbon source to the nitrogen source is: 1-3:1-3.

[0031] Fourthly, the present invention provides an application of a biocontrol agent in the preparation of a herbicide for inhibiting the growth of solanaceous plants. The solanaceous plant biocontrol bacteria include Alternaria alternata, Epichloë sorghi, Fusarium equiseti, Curvularia hawaiiensis, and / or Nigrospora oryzae.

[0032] Further, the solanaceous plants include but are not limited to one or more of Solanum aculeatissimum, Solanum nivalomontanum, Solanum luridum, Solanum rostratum, Solanum nigrum, and Solanum violaceum.

[0033] Further, the solanaceous plant is selected from Solanum rostratum. Description of the Drawings

[0034] Figure 1Morphological characteristics of the colonies and conidia of the pathogens of Solanum rostratum Dunal (A-1, A-2: Colony morphology of Alternaria alternata; A-3: Morphology of conidia of Alternaria alternata; B-1, B-2: Colony morphology of Epicoccum sorghinum; B-3: Morphology of conidia of Epicoccum sorghinum; C-1, C-2: Colony morphology of Fusarium equiseti; C-3: Morphology of conidia of Fusarium equiseti; D-1, D-2: Colony morphology of Curvularia sp.; D-3: Morphology of conidia of Curvularia sp.; E-1, E-2: Colony morphology of Nigrospora oryzae; E-3: Morphology of conidia of Nigrospora oryzae);

[0035] Figure 2 Inhibitory effects of five pathogens on the radicle germination of Solanum rostratum Dunal seeds;

[0036] Figure 3 Disease incidence of leaves inoculated with pathogen discs (A-1, A-2, A-3: Disease incidence of leaves 3, 7, and 11 days after inoculation with Alternaria alternata; B-1, B-2, B-3: Disease incidence of leaves 3, 7, and 11 days after inoculation with Epicoccum sorghinum; C-1, C-2, C-3: Disease incidence of leaves 3, 7, and 11 days after inoculation with Fusarium equiseti; D-1, D-2, D-3: Disease incidence of leaves 3, 7, and 11 days after inoculation with Curvularia sp.; E-1, E-2, E-3: Disease incidence of leaves 3, 7, and 11 days after inoculation with Nigrospora oryzae); Figure 4 Disease incidence of leaves inoculated with pathogen spore suspensions (A-1, A-2, A-3: Disease incidence of leaves 3, 7, and 11 days after inoculation with Alternaria alternata; B-1, B-2, B-3: Disease incidence of leaves 3, 7, and 11 days after inoculation with Epicoccum sorghinum; C-1, C-2, C-3: Disease incidence of leaves 3, 7, and 11 days after inoculation with Fusarium equiseti; D-1, D-2, D-3: Disease incidence of leaves 3, 7, and 11 days after inoculation with Curvul aria hawaiiensis; E-1, E-2, E-3: Disease incidence of leaves 3, 7, and 11 days after inoculation with Nigrospora oryzae);

[0037] Figure 5 Effects of carbon and nitrogen sources in the fermentation broth of Alternaria alternata on mycelial biomass (A: Effects of carbon sources on mycelial biomass; B: Effects of nitrogen sources on mycelial biomass);

[0038] Figure 6 Effects of carbon and nitrogen sources in the fermentation broth of Epicoccum sorghinum on mycelial biomass (A: Effects of carbon sources on mycelial biomass; B: Effects of nitrogen sources on mycelial biomass);

[0039] Figure 7 Effects of carbon and nitrogen sources in the fermentation broth of Fusarium equiseti on mycelial biomass (A: Effects of carbon sources on mycelial biomass; B: Effects of nitrogen sources on mycelial biomass);

[0040] Figure 8 Effect of carbon and nitrogen sources in the fermentation broth of Curvularia hawaiiensis on mycelial biomass (A: Effect of carbon source on mycelial biomass; B: Effect of nitrogen source on mycelial biomass);

[0041] Figure 9 Effect of carbon and nitrogen sources in the fermentation broth of Nigrospora oryzae on mycelial biomass (A: Effect of carbon source on mycelial biomass; B: Effect of nitrogen source on mycelial biomass);

[0042] Figure 10 Effect of inoculum amount of mother liquor on mycelial biomass of pathogenic bacteria;

[0043] Figure 11 Effect of shaking flask volume on mycelial biomass of pathogenic bacteria;

[0044] Figure 12 Effect of culture temperature on mycelial biomass of pathogenic bacteria;

[0045] Figure 13 Effect of initial pH value on mycelial biomass of pathogenic bacteria;

[0046] Figure 14 Effect of culture time on mycelial biomass of pathogenic bacteria. Specific implementation manners

[0047] The present invention will be described in detail below in conjunction with specific embodiments, but the protection scope of the present invention is not limited to the following embodiments. Unless otherwise specified, the experimental methods adopted in the present invention are all conventional methods, and the experimental equipment, materials, reagents, etc. used can be obtained from commercial channels.

[0048] Example 1 Isolation and identification of biocontrol bacteria of Solanum rostratum

[0049] 1. Test materials

[0050] Seeds of Solanum rostratum plants, diseased leaves of Solanum rostratum, 75% alcohol, 12% sodium hypochlorite, potato dextrose medium, culture dishes (d = 90 mm), artificial climate chamber (ZRG - 1500A - L), fungal genomic DNA extraction kit (Sangon Biotech Co., Ltd.).

[0051] 2. Isolation and culture of pathogenic microorganisms on leaves

[0052] Select diseased plants and isolate the plant pathogenic bacteria in the samples by tissue isolation method. Inoculate on PDA medium and culture at 28 °C for 7 d. Classify all the growing colonies, use a puncher (d = 5 mm) to punch the medium at the edge of each fungus and transplant it onto a new PDA medium, and culture at 28 °C for 7 d to obtain pure culture strains. Preserve the successfully isolated and purified strains on PDA slants and store them in a 4 °C refrigerator.

[0053] 3. Identification of pathogenic microorganisms in leaves

[0054] Observe the colony characteristics and record their morphological, color and other characteristics. Use an inoculation needle to pick up a small amount of hyphae and place them on a glass slide, add a few drops of sterile water, cover with a coverslip to make a temporary mount, and observe the hyphal morphology; after sporulation, collect the spores, add sterile water to prepare a spore suspension, use a pipette to aspirate 5 μL of the spore suspension onto a washed and dried glass slide to make a temporary slide, observe the morphological characteristics such as conidia and conidiophores under an optical microscope and take pictures. Refer to the "Fungal Identification Manual" for morphological identification of the pathogen.

[0055] Take the fungal hyphae and grind them into powder in liquid nitrogen, and extract DNA using the UNIQ-10 Column Fungal Genomic DNA Extraction Kit from Sangon Biotech (Shanghai) Co., Ltd. Use the universal primers ITS4 (5’-TCCTCCGCTTATTGATATGC-3’) and ITS5 (5’-GGAAGTAAAAGTCGTAACAAGG-3’) as PCR amplification primers. The 25 μL PCR reaction system includes: 12.5 μL of Taq PCR MasterMix, 1 μL each of the two forward and reverse primers ITS4 and ITS5, 2 μL of genomic template DNA, and 8.5 μL of ddH2O. The amplification conditions include: pre-denaturation at 94 °C for 3 min, denaturation at 94 °C for 30 s, annealing at 51 °C for 30 s, extension at 72 °C for 45 s, and extension at 72 °C for 10 min for a total of 30 cycles. The PCR products are sequenced by Sangon Biotech (Shanghai) Co., Ltd., and the sequences are analyzed using Chromas software to obtain the complete amplified sequence of the target fragment. The results are submitted to the NCBI database and compared with the known sequences in the database by Blast to determine the species of the pathogen.

[0056] 4. Results of isolation and identification of pathogens of Solanum rostratum Dunal

[0057] Isolate the pathogens from 120 diseased leaves at different investigation sites. After culturing and observation, their culture characteristics and colony morphologies on PDA plates are divided into 5 types (see Figure 1) The descriptions of colony and spore morphology are shown in Table 1. Morphological identification was carried out based on colony morphological characteristics and spore morphological characteristics. The five pathogenic bacteria were preliminarily identified as Alternaria sp., Epicoccum sp., Fusarium sp., Curvularia sp., and Nigrospora sp. Further, through molecular biological identification to the species level, the five pathogenic bacteria were identified as Alternaria alternata, Epicoccum sorghinum, Fusarium equiseti, Curvularia hawaiiensis, and Nigrospora oryzae respectively. The ITS sequence lengths of the five strains were 574bp, 541bp, 544bp, 533bp, and 499bp respectively. Blast homology comparison was performed with the known sequences in the NCBI database, and the highest homologies were found with Alternaria alternata, Epicoccum sorghinum, Fusarium equiseti, Curvularia hawaiiensis, and Nigrospora oryzae, which were 99%, 100%, 100%, 100%, and 99% respectively.

[0058] Table 1 Identification characteristics and morphological identification results of Solanum rostratum Dunal pathogenic bacteria

[0059]

[0060]

[0061] Example 2 Determination of the pathogenicity of five pathogenic bacteria to Solanum rostratum Dunal

[0062] 1. Determination of the germination inhibition rate of five pathogenic bacteria on Solanum rostratum Dunal seeds

[0063] In a sterile laminar flow hood, place two layers of sterilized filter paper into a sterile petri dish with a diameter of 90 mm. Pour the prepared pathogenic bacteria spore suspension into the petri dish respectively and mark the numbers. Set 3 replicates for each type. The control group is replaced with sterile water. The pathogenic bacteria spore suspension poured into the petri dish should just moisten the sterile filter paper. Place Solanum rostratum Dunal seeds on the two layers of filter paper, put 20 Solanum rostratum Dunal seeds in each petri dish, and place them in an incubator at 28 °C under dark conditions with a humidity of 70% in the incubator. When the radicle appears, it is considered to have germinated. During the experiment, add the suspension in a timely manner to keep it moist. Observe the seed germination situation every day. After the seeds germinate, measure the radicle germination length every day.

[0064] Results: After treatment with the five pathogenic bacteria, all had an inhibitory effect on the radicle germination length of the seeds. Among them, Nigrospora oryzae had the strongest inhibitory effect (see Figure 2) After being treated with *Nigrospora oryzae* for 24 h, the average germination length of the radicles of the seeds was 1.00 mm, which was significantly lower than that of the control group (F = 4.35, df = 5,114, P < 0.05). There were no significant differences after treatment with other pathogenic fungi. After being treated with sterile water for 72 h, the average germination length of the radicles was 2.55 mm. After being treated with the five pathogenic fungi for 72 h, the average germination lengths of the radicles were 1.79 mm, 1.62 mm, 1.63 mm, 1.68 mm, and 1.54 mm, respectively, all of which were significantly lower than that of the control group (F = 8.69, df = 5,114, P < 0.05). Moreover, *Nigrospora oryzae* had the highest inhibition rate on the germination length of the radicles, which was 47.76% (Table 2).

[0065] Table 2 Radicle germination length and inhibition rate of *Solanum rostratum* seeds after treatment with five pathogenic fungi

[0066]

[0067] 2. Pathogenicity determination of five pathogenic fungi on detached leaves of *Solanum rostratum*

[0068] Select *Solanum rostratum* plants with uniform growth vigor, and take leaves of the same size from each plant and place them in petri dishes. First, lay a layer of sterile filter paper at the bottom of the petri dish, and moisten the filter paper with sterile water. After rinsing the detached leaves with clean water, blot the surface moisture with filter paper, disinfect them with 75% alcohol for 30 s, disinfect them with 3% NaClO solution for 1 min, and rinse them 3 times with sterile water. Use a sterilized insect pin to make 6 horizontal scratches on each leaf at a distance of 2 mm from the main vein, and each wound is 2 mm. The standard is to pierce the lower epidermis while keeping the upper epidermis intact. Punch out a fungal cake (5 mm) cultured on a PDA plate for 7 d, and attach the culture medium with mycelium to the wound position close to the leaf, using the PDA medium without inoculation as a control, and culture it under the conditions of a temperature of 25 °C and a humidity of 90%.

[0069] Measure the lesion area, and determine the pathogenicity of the inoculated strain according to the area size (grading according to the percentage of the diseased leaf area in the total leaf area: grade 0 = no disease on the leaf; grade 1 = 1% - 5% of the leaf is diseased; grade 2 = 5% - 25% of the leaf is diseased; grade 3 = 25% - 50% of the leaf is diseased; grade 4 = 50% - 75% of the leaf is diseased; grade 5 = 75% - 100% of the leaf is diseased (including dead plants)).

[0070] Disease index = ∑(number of inoculation points at the disease level × corresponding disease level) / (total number of inoculation points × highest disease level) × 100%

[0071] Pathogenicity situation: When the disease index is 0, it is non-pathogenic; when the disease index is 0 - 45, it has weak pathogenicity; when the disease index is 45 - 75, it has moderate pathogenicity; when the disease index is 75 - 100, it has strong pathogenicity.

[0072] Results: After inoculating the mycelial discs of five pathogens on the excised leaves of Solanum rostratum for 3 days, obvious lesions began to appear on the leaves, and the tissues near the inoculation sites turned gray. The lesions started to spread along the inoculation sites, and after 7 days, the lesion area expanded, the leaves at the inoculation sites were damaged, and the lesion color changed from gray to grayish black. After 11 days, the whole leaves turned yellow, and the leaves inoculated with Alternaria alternata, Epichloë sorghi, and Nigrospora oryzae gradually withered (see Figure 3 ). After re-isolating and identifying the pathogens from the diseased leaves, they were exactly the same as the pathogens used during inoculation, verifying Koch's postulates.

[0073] There were differences in the pathogenicity of different strains. Among them, the incidence rates of the leaves inoculated with Alternaria alternata, Fusarium equiseti, and Nigrospora oryzae were all 100%. Followed by Epichloë sorghi and C. hawaiiensis, with incidence rates of 92.46% and 88.10% respectively. After inoculating with Nigrospora oryzae for 3 days and 7 days, the proportion of the diseased leaf area was significantly larger than that of the other four pathogens (F = 26.23, df = 5,12, P < 0.05; F = 8.21, df = 5,12, P < 0.05). After 11 days, the disease index of the leaves was the highest, at 60%, followed by Alternaria alternata, with a disease index of 46.67%, both having moderate pathogenicity (Table 3).

[0074] Table 3 Analysis of the pathogenicity of pathogen mycelia on the excised leaves of Solanum rostratum

[0075]

[0076]

[0077] Note: Pathogenicity situation: The disease index is 0, non-pathogenic; the disease index is 0 - 45, weak pathogenicity; the disease index is 45 - 75, moderate pathogenicity; the disease index is 75 - 100, strong pathogenicity.

[0078] 3. Determination of the pathogenicity of five pathogens on potted plants of Solanum rostratum

[0079] Eight horizontal scratches, each 2 mm long, were made on each leaf with a sterilized needle 1 mm away from the main vein. The conidial suspension was used to spray and inoculate the healthy leaves of Solanum rostratum, and sterile water was used to spray and inoculate the healthy leaves as a control. After inoculation, the plants were covered with a fresh-keeping bag to keep them moist for 2 days and cultured at a temperature of 25°C and a humidity of 90%. The measurement method was the same as that in "Determination of the pathogenicity of five pathogens on the excised leaves of Solanum rostratum".

[0080] Results: After inoculating the spore suspension of five pathogens on the potted plants of Solanum rostratum, typical lesions appeared on the leaves of Solanum rostratum (see Figure 4) After 3 days of inoculation, small lesions appeared on the leaves. After 7 days, the lesion area expanded, and the tissue around the inoculated leaves became necrotic. After 11 days, the leaves inoculated with Epichloë sorghi and Nigrospora oryzae were damaged and curled, with yellowish-brown edges. After re-isolating and identifying the pathogenic bacteria from the diseased leaves, they were exactly the same as the pathogenic bacteria used during inoculation, verifying Koch's postulates.

[0081] After inoculation with five pathogenic bacteria, the incidence rates of the other four pathogenic bacteria except C. hawaiiensis were all 100%. After 11 days of inoculation with Epichloë sorghi and Nigrospora oryzae, the proportion of the diseased leaf area was significantly larger than that of the other three pathogenic bacteria (F = 8.22, df = 4,55, P < 0.05). There were significant differences in the pathogenicity of different pathogenic bacteria to potted Solanum rostratum plants. Among them, Epichloë sorghi had the strongest pathogenicity, and the disease index of the diseased plants was the highest, reaching 78.30%. Followed by Nigrospora oryzae, the disease index of the diseased plants was 70.00%, showing medium pathogenicity (Table 4).

[0082] Table 4 Analysis of the pathogenicity of the spore suspension of pathogenic bacteria to potted Solanum rostratum plants

[0083]

[0084] Isolation and identification of pathogenic bacteria from naturally diseased Solanum rostratum preliminarily identified that its pathogenic microorganisms were Alternaria sp., Epichloë sorghi, Fusarium equiseti, Curvularia sp. C. hawaiiensis, and Nigrospora oryzae. Pathogenicity tests were conducted on the seeds and leaves of this weed. The results showed that Nigrospora oryzae had the strongest inhibitory effect on the germination length of the radicle of the seeds after treatment. After inoculating the leaves with Nigrospora oryzae and Epichloë sorghi, the diseased area was significantly larger than that of other pathogenic bacteria.

[0085] Example 3 Fermentation medium optimization test

[0086] Preparation method of liquid mother culture: Punch a pure cultured fungal cake (d = 5 mm) and inoculate it into a PDA liquid medium. The medium filling volume is 20%, at 25 °C, 120 rpm, and shake flask culture for 72 h. The mycelia in the obtained liquid mother culture are tightly entangled with each other to form particles, evenly suspended in the culture solution, which is beneficial to the transfer of oxygen and the delivery of nutrients, and is beneficial to the growth of mycelia and the production of metabolites.

[0087] Single-factor screening of carbon sources: Using 2% peptone, 0.05% MgSO4·H2O, and 0.1% KH2PO4 as the basal medium, add 6 carbon sources: maltose, glucose, soluble starch, sucrose, lactose, and glycerol to the medium at a volume fraction of 2% respectively to prepare the medium. The medium filling volume is 20%. Then, inoculate the liquid mother culture into different media at an inoculation amount of 5% respectively, at 120 rpm, 26 °C, and shake flask culture for 6 days to compare the effects of 6 carbon sources on biomass.

[0088] Single-factor screening of nitrogen source: Using 2% sucrose, 0.05% MgSO4·H2O, and 0.1% KH2PO4 as the basal medium, six nitrogen sources, namely peptone, beef extract, yeast extract, ammonium sulfate, ammonium chloride, and urea, were added at a volume fraction of 2% respectively to prepare the medium. The medium filling volume was 20%. Then, the liquid mother culture was inoculated into different media at an inoculation amount of 5% respectively, and cultured in a shaking flask at 120 rpm and 26°C for 6 days to compare the effects of the six nitrogen sources on biomass.

[0089] Method for measuring biomass: 1 mL of spore suspension was inoculated into each medium treatment, with three replicates for each medium, and the liquid filling volume was V. The mycelium was poured onto a filter paper (filter paper mass, M1) that had been dried to a constant weight, vacuum filtered, and then dried to a constant weight in an oven at 80°C, and weighed with an analytical balance (total mass of filter paper and mycelium, M2): Biomass (g / L) = (M2 - M1) / V.

[0090] Selection of carbon-nitrogen ratio: According to the single-factor results obtained, two carbon sources and two nitrogen sources with the highest biomass were selected as factors respectively, without adding any other components, and an orthogonal experiment was designed according to the four-factor three-level method to compare the effects of different C / N ratios on the growth of fungi.

[0091] The orthogonal design is shown in Table 5.

[0092] Table 5 Carbon-nitrogen ratio L9(3 4 ) Orthogonal experimental design table

[0093]

[0094]

[0095] Results:

[0096] 1. Screening of carbon and nitrogen sources for Alternaria alternata fermentation broth

[0097] In the six carbon source media, the mycelium of Alternaria alternata could grow. Among them, in the medium with glucose as the carbon source, the mycelium biomass was the highest, reaching 1.06 g / L. Followed by the medium with lactose as the carbon source, with a biomass of 0.48 g / L( Figure 5 A). Therefore, glucose and lactose were selected as the carbon sources for the medium.

[0098] In the six nitrogen source media, the mycelium of Alternaria alternata could grow in the media with ammonium chloride, ammonium sulfate, yeast, and peptone. Among them, in the medium with yeast as the carbon source, the mycelium biomass was 14.09 g / L, which was significantly greater than the other five nitrogen sources (F = 4.87, df = 5,12, P < 0.05). Followed by the medium with ammonium sulfate as the carbon source, with a biomass of 2.06 g / L( Figure 5B). Therefore, yeast and ammonium sulfate were selected as the nitrogen sources for the medium.

[0099] 2. Screening of carbon and nitrogen sources for the fermentation broth of Epichloe sorghi

[0100] In the 6 carbon source media, the mycelia of Epichloe sorghi could grow. Among them, in the media with maltose and sucrose as carbon sources, the mycelial biomass was the highest, being 10.47 g / L and 10.68 g / L respectively, significantly higher than the other four carbon sources (F = 29.82, df = 5,12, P < 0.05)( Figure 6 A). Therefore, maltose and sucrose were selected as the carbon sources for the medium.

[0101] In the 6 nitrogen source media, the mycelia of Epichloe sorghi could grow in the media with ammonium chloride, ammonium sulfate, yeast, beef extract, and peptone as nitrogen sources. Among them, in the medium with yeast as the nitrogen source, the mycelial biomass was 20.87 g / L, significantly greater than the other 5 nitrogen sources (F = 154.85, df = 5,12, P < 0.05). Secondly, it was the medium with ammonium sulfate as the nitrogen source, and the biomass was 1.13 g / L( Figure 6 B). Therefore, yeast and ammonium sulfate were selected as the nitrogen sources for the medium.

[0102] 3. Screening of carbon and nitrogen sources for the fermentation broth of Fusarium equiseti

[0103] In the 6 carbon source media, the mycelia of Fusarium equiseti could grow. Among them, in the media with glucose and soluble starch as carbon sources, the mycelial biomass was the highest, being 11.95 g / L and 10.70 g / L respectively( Figure 7 A), significantly higher than the media with maltose, glycerol, lactose, and sucrose as carbon sources (F = 11.03, df = 5,12, P < 0.05). Therefore, glucose and soluble starch were selected as the carbon sources for the medium.

[0104] In the 6 nitrogen source media, the mycelia of Fusarium equiseti could grow in the media with ammonium chloride, ammonium sulfate, yeast, beef extract, and peptone as nitrogen sources. Among them, in the medium with yeast as the nitrogen source, the mycelial biomass was the highest, being 16.58 g / L( Figure 7 B), significantly higher than the other five media (F = 394.47, df = 5,12, P < 0.05). Secondly, it was the media with ammonium chloride and ammonium sulfate as carbon sources, and the biomass was 3.84 g / L and 2.98 g / L respectively, significantly higher than the other three media, and the mycelial biomass of ammonium sulfate was higher than that of ammonium chloride. Therefore, yeast and ammonium sulfate were selected as the nitrogen sources for the medium.

[0105] 4. Screening of carbon and nitrogen sources for the fermentation broth of Curvularia hawaiiensis

[0106] Among the six carbon source media, the mycelia of C. hawaiiensis could grow. Among them, in the media with soluble starch and sucrose as carbon sources, the mycelial biomass was the highest, reaching 11.36 g / L and 9.60 g / L respectively, which was significantly higher than those of the other four carbon sources (F = 42.65, df = 5,12, P < 0.05)( Figure 8 A). Therefore, soluble starch and sucrose were selected as the carbon sources for the medium.

[0107] Among the six nitrogen source media, the mycelia of C. hawaiiensis could grow in the media with ammonium chloride, ammonium sulfate, yeast, beef extract, and peptone as nitrogen sources. Among them, in the medium with beef extract as the nitrogen source, the mycelial biomass was 10.33 g / L( Figure 8 B), which was significantly higher than those of the other five media (F = 93.41, df = 5,12, P < 0.05). Secondly, the medium with peptone as the carbon source had a biomass of 8.09 g / L, which was significantly higher than those of the other four media. Therefore, beef extract and peptone were selected as the nitrogen sources for the medium.

[0108] 5. Screening of Carbon and Nitrogen Sources in the Fermentation Broth of Nigrospora oryzae

[0109] Among the six carbon source media, the mycelia of Nigrospora oryzae could grow. Among them, in the medium with glycerol as the carbon source, the mycelial biomass was the highest, reaching 0.78 g / L, which was significantly higher than those of the media with maltose, glucose, soluble starch, and sucrose as carbon sources (F = 11.03, df = 5,12, P < 0.05). Secondly, the medium with lactose as the carbon source had a biomass of 0.58 g / L( Figure 9 A). Therefore, glycerol and lactose were selected as the carbon sources for the medium.

[0110] Among the six nitrogen source media, the mycelia of Nigrospora oryzae could grow in the media with ammonium chloride, ammonium sulfate, yeast, beef extract, and peptone as nitrogen sources. Among them, in the medium with ammonium sulfate as the nitrogen source, the mycelial biomass was 4.58 g / L( Figure 9 B), which was significantly higher than those of the media with urea, beef extract, and peptone as nitrogen sources (F = 6.78, df = 5,12, P < 0.05), and the mycelial biomass of ammonium sulfate and yeast was the highest. Therefore, ammonium sulfate and yeast were selected as the nitrogen sources for the medium.

[0111] 6. Results of Carbon-Nitrogen Ratio in the Fermentation Broth

[0112] Table 6 Optimal Carbon-Nitrogen Ratios of Five Pathogenic Fungi

[0113]

[0114]

[0115] The results are shown in Table 6. The fermentation medium for the pathogen of Solanum rostratum Dunal. For Nigrospora oryzae, the carbon sources are lactose and glycerol, and the nitrogen sources are yeast and ammonium sulfate. The mycelial biomass of Treatment 2 (1% yeast, 2% ammonium sulfate, 2% lactose, 2% glycerol) is the highest, reaching 1.08 g / L. The optimal ratio of lactose, glycerol, yeast, and ammonium sulfate in the fermentation medium is 2:2:1:2.

[0116] Screening of liquid fermentation conditions in Example 4

[0117] Effect of inoculum size on biomass: The liquid stock culture was inoculated into the fermentation medium at amounts of 5%, 10%, 15%, 20%, and 25%, and cultured in a shaking flask at 120 rpm and 25 °C for 6 days to measure the biomass.

[0118] Effect of shaking flask volume on biomass: The liquid stock culture was inoculated into the liquid fermentation medium, and different amounts of the medium, 5%, 10%, 15%, 20%, and 25%, were respectively filled into Erlenmeyer flasks. After shaking flask fermentation for 6 days, the biomass was measured.

[0119] Effect of culture temperature on biomass: The liquid stock culture was inoculated into the liquid fermentation medium and fermented in a shaking flask at temperatures of 15 °C, 20 °C, 25 °C, 30 °C, and 35 °C for 6 days to measure the biomass.

[0120] Effect of initial pH value of culture components on biomass: The fermentation medium was adjusted to pH values of 5.0, 6.0, 7.0, 8.0, and 9.0 with 1 mL of NaOH or HCl respectively, and the liquid stock culture was inoculated and fermented in a shaking flask for 6 days to measure the biomass.

[0121] Effect of culture time on biomass: The liquid stock culture was inoculated into the liquid fermentation medium and fermented for 2, 4, 6, 8, and 10 days respectively to measure the biomass.

[0122] Optimization of fermentation conditions by orthogonal experiment: On the basis of the above single-factor experiments, orthogonal design was adopted. With the inoculum size, shaking flask volume, culture temperature, initial pH value of the medium, and culture time as the main research factors, an orthogonal design experiment of 5 factors was carried out using the orthogonal table of L25(55). Each factor took 5 levels, and each group of experiments was repeated 3 times. The biomass was used as the detection index for the growth of the strain. The orthogonal design is shown in Tables 7 and 8.

[0123] Table 7 Orthogonal factor level table for liquid fermentation / volume fraction

[0124]

[0125]

[0126] Table 8 Orthogonal experiment design table for liquid fermentation

[0127]

[0128] Result:

[0129] 1. Effect of inoculum size of mother liquor on the fermentation of five pathogenic fungi

[0130] For Alternaria alternata, the mycelial biomass was the highest at an inoculum size of 5% of the mother liquor, which was 22.31 g / L. For Fusarium equiseti, the mycelial biomass was the highest at an inoculum size of 15% of the mother liquor, which was 9.72 g / L. For Epicoccum sorghinum, C. hawaiiensis, and Nigrospora oryzae, the mycelial biomass was significantly higher than that of other inoculum sizes at an inoculum size of 25% of the mother liquor (F = 42.91, df = 4,10, P < 0.05; F = 6853.15, df = 4,10, P < 0.05; F = 138.87, df = 4,10, P < 0.05), which were 2.07 g / L, 5.81 g / L, and 1.89 g / L respectively. (See Figure 10 )

[0131] 2. Effect of shaking flask volume on the fermentation of five pathogenic fungi

[0132] For Alternaria alternata and Fusarium equiseti, the mycelial biomass was the highest at a shaking flask volume of 15%, which were 6.46 g / L and 5.71 g / L respectively. For Epicoccum sorghinum, C. hawaiiensis, and Nigrospora oryzae, the mycelial biomass was the highest at a shaking flask volume of 25%, which were 6.8 g / L, 0.17 g / L, and 0.20 g / L respectively. (See Figure 11 )

[0133] 3. Effect of culture temperature on the fermentation of five pathogenic fungi

[0134] For Epicoccum sorghinum, Fusarium equiseti, C. hawaiiensis, and Nigrospora oryzae, the mycelial biomass was significantly higher than that of other temperatures at a culture temperature of 20°C (F = 669.29, df = 4,10, P < 0.05; F = 794.08, df = 4,10, P < 0.05; F = 3011.96, df = 4,10, P < 0.05; F = 118.23, df = 4,10, P < 0.05), which were 7.57 g / L, 9.28 g / L, 8.15 g / L, and 5.80 g / L respectively. For Alternaria alternata, the mycelial biomass was significantly higher than that of other temperatures at a culture temperature of 30°C (F = 5208.47, df = 4,10, P < 0.05), which was 22.31 g / L. (See Figure 12 )

[0135] 4. Effect of initial pH value on the fermentation of five pathogenic fungi

[0136] The mycelial biomass of Fusarium equiseti, C. hawaiiensis, and Nigrospora oryzae was the highest at an initial pH of 5, being 9.78 g / L, 30.61 g / L, and 14.92 g / L, respectively. The mycelial biomass of Alternaria alternata was the highest at an initial pH of 6, being 9.78 g / L. The mycelial biomass of Epichloe sorghi was significantly higher than that of the other four groups at an initial pH of 7 (F = 250.07, df = 4,10, P < 0.05), being 8.86 g / L. (See Figure 13 )

[0137] 4.2.5 Effect of culture time on the fermentation of five pathogenic fungi

[0138] The mycelial biomass of Fusarium equiseti, C. hawaiiensis, and Nigrospora oryzae was the highest at a culture time of 4 d, being 5.05 g / L, 4.25 g / L, and 1.27 g / L, respectively. The mycelial biomass of Alternaria alternata was the highest at a culture time of 6 d, being 22.31 g / L. The mycelial biomass of Epichloe sorghi was significantly higher than that of the other four groups at a culture time of 8 d (F = 370.00, df = 4,10, P < 0.05), being 10.14 g / L. (See Figure 14 )

[0139] In summary, the experimental results show that:

[0140] For the liquid fermentation medium of Alternaria alternata, the ratio of glucose, lactose, yeast, and ammonium sulfate is 3:3:1:3; the fermentation culture conditions are: the mother liquor volume is 5%, the shaking flask loading is 15%, the temperature is 30 °C, the initial pH is 6, and the culture time is 6 d.

[0141] For the liquid fermentation medium of Epichloe sorghi, the ratio of maltose, sucrose, yeast, and ammonium sulfate is 2:1:3:3; the fermentation culture conditions are: the mother liquor volume is 25%, the shaking flask loading is 25%, the temperature is 20 °C, the initial pH is 7, and the culture time is 8 d.

[0142] For the liquid fermentation medium of Fusarium equiseti, the ratio of maltose, sucrose, yeast, and ammonium sulfate is 1:3:3:2; the fermentation culture conditions are: the mother liquor volume is 15%, the shaking flask loading is 15%, the temperature is 20 °C, the initial pH is 5, and the culture time is 4 d.

[0143] For the liquid fermentation medium of C. hawaiiensis, the ratio of soluble starch, sucrose, beef extract, and peptone is 2:2:1:2; the fermentation culture conditions are: the mother liquor volume is 25%, the shaking flask loading is 15%, the temperature is 20 °C, the initial pH is 5, and the culture time is 4 d.

[0144] The ratio of lactose, glycerol, yeast, and ammonium sulfate in the liquid fermentation medium of Nigrospora oryzae is 2:2:1:2; the fermentation culture conditions are: the mother liquor volume is 25%, the shaking flask loading is 25%, the temperature is 20 °C, the initial pH value is 5, and the culture time is 4 days.

Claims

1. Use of Nigrospora oryzae and / or its fermentation broth in the preparation of an inhibitor for inhibiting the germination length of the radicle of Solanum rostratum Dunal seeds.

2. The use according to claim 1, wherein glycerol or lactose is used as a carbon source in the fermentation medium of Nigrospora oryzae.

3. The use according to claim 2, wherein glycerol is used as a carbon source in the fermentation medium of Nigrospora oryzae.

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