Helminthosporium yamadaensis TL05 and its application

Abstract

The present invention discloses a Yamada flat navel helminth TL05 and its application. The present invention separates a Yamada flat navel helminth TL05 from a black spot diseased plant of long spiny tribulus terrestris Bipolaris yamadae ) TL05, whose deposit number is CGMCC No.41290. Yamada Hiranchusporium TL05 and its fungal agent can control weeds such as long-spined thorn grass and can effectively inhibit the growth of weeds such as long-spined thorn grass; at the same time, it is highly safe for pastures and crops such as corn, eggplant, tomato, pepper, cabbage, Chinese cabbage, wheat, oats, cucumber, spinach, Elymus dahliae, sheep grass, and alfalfa. It can be safely and effectively used to control weeds such as long-spined thorn grass in such pastures and farmlands, reduce the use of chemical herbicides, and is environmentally friendly.

Description

Technical Field

[0001] The invention belongs to the technical field of biological control, and specifically relates to Yamada Hiraumbilicus TL05 and applications thereof. Background Art

[0002] Tribulus terrestris Cenchrus longispinus ) is one of the invasive alien species in the "National Key Management Alien Invasive Plant List (First Batch)" announced in 2013. Due to its high stress resistance, high reproductive capacity, and easy spread and diffusion, long-spined tribulus has erupted in large areas in the agricultural and pastoral transition zone in northern my country in recent years. The southern part of Jilin Province, the northwest of Liaoning Province, and the eastern part of Inner Mongolia are the main distribution areas of long-spined tribulus in my country; among them, Tongliao City, Inner Mongolia is the hardest hit area of ​​long-spined tribulus, and the invasion area has reached 1.1389 million hm 2 .

[0003] Physical measures such as manual pulling and eradication can completely kill Tribulus terrestris plants, with a control effect of more than 85%. However, this measure alone cannot completely control Tribulus terrestris, because the grassland soil seed bank in areas where Tribulus terrestris is seriously invaded can be as high as 12,000 seeds / m -2 The above, and the number of germinated soil seeds accounts for a very low proportion of the soil seed bank. Even if all the germinated surface plants are removed every year, it will take many years to exhaust the soil seed bank. Moreover, more than 10% of the plants can bear fruit every year. Therefore, the number of soil seed banks will show an increasing trend. Chemical control and weed control has a good effect, but its control effect is best in the seedling stage of long-spined thorn grass. The control effect will decrease after entering the flowering period. If the remaining plants bear fruit, the number of seeds in the soil will still increase. Therefore, a second application of pesticides must be carried out to completely control the long-spined thorn grass that germinates in the season, thereby increasing the input of manpower and material resources, and the large-scale application of chemical agents increases environmental hazards, seriously affecting the sustainability of agricultural ecological development; and some special production areas such as grasslands and tidal flats require restrictions on the use of chemical herbicides. Summary of the invention

[0004] The purpose of the present invention is to provide a strain of Helicoverpa yamadaensis TL05, a bacterial agent and an application thereof.

[0005] The present invention isolated a strain TL05 with strong pathogenicity from naturally occurring long-spined Tribulus terrestris, which was identified as Helicoverpa yamadaensis ( Bipolaris yamadae ), has an inhibitory effect on the growth of weeds such as long-spined tribulus terrestris, and can safely and effectively prevent and control weeds such as long-spined tribulus terrestris.

[0006] The purpose of the present invention is implemented by the following technical solutions.

[0007] The present invention provides a strain of Helicoverpa yamadaensis ( Bipolaris yamadae)TL05, currently deposited in the China General Microbiological Culture Collection Center (CGMCC), located at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. Its deposit number is CGMCC No.41290 and the deposit date is April 19, 2024.

[0008] The present invention also provides Yamada Hiraumuan Helminthosporium ( Bipolaris yamadae ) TL05 bacterial agent, which includes Helicoverpa yamadaensis ( Bipolaris yamadae ) TL05 and / or its fermentation broth.

[0009] The present invention also provides Yamada Hiraumuan Helminthosporium ( Bipolaris yamadae ) A method for preparing a fermentation broth, comprising the following steps: 1) inoculation: Inoculating Helicoverpa yamadaensis ( Bipolaris yamadae 1) TL05 was inoculated on mung bean solid culture medium and cultured at 25°C for 7 days to obtain activated strains; 2) Fermentation: The activated strains were inoculated into PDB liquid culture medium and cultured at 25°C; 3) Filtration: The bacterial solution after 14 days of culture was filtered to obtain a crude metabolite stock solution, which was the fermentation broth.

[0010] The present invention also provides Yamada Hiraumuan Helminthosporium ( Bipolaris yamadae )TL05 or Yamada Hiraumbiloba ( Bipolaris yamadae ) Application of TL05 microbial agent in controlling long-spined tribulus terrestris.

[0011] The present invention also provides Yamada Hiraumuan Helminthosporium ( Bipolaris yamadae )TL05 or Yamada Hiraumbiloba ( Bipolaris yamadae ) Application of TL05 microbial agent in the preparation of products for controlling long-spined tribulus terrestris.

[0012] The present invention also provides Yamada Hiraumuan Helminthosporium ( Bipolaris yamadae )TL05 or Yamada Hiraumbiloba ( Bipolaris yamadae ) Application of TL05 microbial agent in inhibiting weed growth.

[0013] Preferably, the weeds include at least one of barnyard grass, chloris, Amaranthus retroflexus, and Chenopodium album.

[0014] Beneficial effects: The present invention isolates and obtains the Yamada Hiraumphalis spores ( Bipolaris yamadae) TL05. Yamada flat navel worm spore TL05 has a strong inhibitory effect on the growth of stems and leaves of weeds such as long-spined thorn grass. At the same time, it is highly safe for pastures and crops such as corn, eggplant, tomato, pepper, cabbage, Chinese cabbage, wheat, oats, cucumber, spinach, Elymus dahliae, sheep grass, and alfalfa. Yamada flat navel worm spore TL05 can be used as the main component of biological herbicides to safely and effectively control weeds such as long-spined thorn grass in such pastures or farmlands, reduce the use of chemical herbicides, and be environmentally friendly. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0016] Figure 1 This is the colony morphology of Yamada Hiraumbilical Helminthosporium TL05 in Example 2; the left picture is the front morphology of Yamada Hiraumbilical Helminthosporium TL05 colony, and the right picture is the back morphology of Yamada Hiraumbilical Helminthosporium TL05 colony.

[0017] Figure 2 This is the structural feature of the Yamada Hirachusporium TL05 under a microscope in Example 2.

[0018] Figure 3 The results of the pathogenicity test of Helminthosporium yamadaensis TL05 to Tribulus terrestris in Example 6; the left figure shows the treatment group inoculated with TL05 bacterial cake, and the right figure shows the control group inoculated with mung bean solid culture medium. DETAILED DESCRIPTION

[0019] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0020] The formula of the culture medium used in the examples of the present invention is as follows.

[0021] PDA solid culture medium: potato 300 g / L, glucose 20 g / L, agar 15 g / L, chloramphenicol 0.1 g / L.

[0022] PDB liquid medium: potato 300 g / L, glucose 20 g / L.

[0023] Mung bean solid culture medium: mung bean powder 40 g / L, agar 20 g / L.

[0024] Example 1: Isolation and screening of strain TL05.

[0025] 1. Sample collection: In October 2021, typical black spot diseased plants were collected from the long-spined Tribulus terrestris occurrence area in Dongfeng Village, Dongfeng Town, Kailu County, Tongliao City, Inner Mongolia (43.658N, 121.577E), stored in paper bags and numbered, and brought back to the laboratory for immediate pathogen isolation.

[0026] 2. Sample processing: Use sterile scissors to cut the diseased tissue sample (0.5 cm × 0.5 cm) at the junction of the diseased and healthy parts of the diseased plants, treat it with 30% sodium hypochlorite for 3 min, rinse it with sterile water 3 to 5 times, place it on PDA solid culture medium containing cephalosporin, and culture it in a constant temperature incubator at 28°C for 3 days.

[0027] 3. Isolation and purification of bacterial strains: After hyphae grow around the diseased tissue block, use an inoculation needle to pick up the hyphae at the edge of the colony, place it diagonally on PDA solid culture medium for purification and culture, and repeat the purification until the colony morphology is consistent; the purified colony is named and numbered TL05, and placed in a 4°C refrigerator to preserve the strain for future use.

[0028] Example 2: Identification of strain TL05.

[0029] 1. Morphological identification.

[0030] The fermentation liquid of the isolated and purified TL05 strain was inoculated on PDA solid medium and cultured at 25°C and 12 h photoperiod for 7 days. The colony morphology and color were observed, the growth diameter of the colony was measured, and its structural characteristics were observed under a 40x microscope. Figure 1 , Figure 2 As shown, the colony grew rapidly. After 7 days of cultivation, the colony had a diameter of 84 mm and a flat morphology. The entire border was composed of felt-gray aerial mycelium, and spore formation was rare.

[0031] 2. Molecular Biology Identification

[0032] The pathogen DNA was extracted by CTAB method and stored in a -20℃ refrigerator for future use. PCR amplification was performed using rDNA-ITS4 / 5 and gpdh sequence primers. The sequence primers are shown in Table 1, the PCR reaction system is shown in Table 2, and the PCR reaction procedure is shown in Table 3. After the PCR amplification reaction, the PCR amplification product was stored at -20℃.

[0033] Table 1 PCR amplification primer sequences

[0034]

[0035] Table 2 PCR amplification reaction system

[0036]

[0037] Table 3 PCR amplification reaction program

[0038]

[0039] After the PCR amplification product was tested with 1% agarose gel and qualified, it was sent to BGI for sequencing. The nucleotide sequence length of the rDNA-ITS4 / 5 of the strain was 547 bp using ITS1 / ITS4 as primers, and the sequencing result of the ITS gene is shown in SEQ ID No. 5; the nucleotide sequence length of the gpdh of the strain was 615 bp using GPD1 / GPD2 as primers, and the sequencing result of the gpdh gene is shown in SEQ ID No. 6.

[0040] The obtained sequence was compared with the known sequences in the NCBI database (http: / / www.ncbi.nlm.nih.gov / ) by BLAST and found that the strain TL05 was similar to Helicoverpa yamadaensis Bipolaris yamadae The highest similarity was found between the amplified sequences and Helicoverpa yamadaensis Bipolaris yamadae The homology of ITS amplified sequence is: Bipolaris yamadae The homology of GG3 is 99.81% (accession number MT514520.1), and the gpdh amplified sequence is similar to that of Helicoverpa yamadaensis Bipolaris yamadae The homology of CPC 28807 is 100% (accession number MF490835.1). Combined with the results of microscopic morphological identification, it can be known that the pathogen TL05 of black spot disease of Tribulus terrestris is Helminthosporium yamadaensis Bipolaris yamadae .

[0041] Helminthosporium yamadaensis Bipolaris yamadae )TL05, currently deposited in the China General Microbiological Culture Collection Center (CGMCC), located at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. Its deposit number is CGMCC No.41290 and the deposit date is April 19, 2024.

[0042] Example 3: A Yamada Hiraumbifolia ( Bipolaris yamadae )TL05 bacterial agent.

[0043] Provided is a Yamada Hiraumuan Helminthosporium ( Bipolaris yamadae ) TL05 bacterial agent, which includes Helicoverpa yamadaensis ( Bipolaris yamadae)TL05 and its fermentation broth.

[0044] Example 4: A Yamada Hiraumbifolia ( Bipolaris yamadae )TL05 bacterial agent.

[0045] Provided is a Yamada Hiraumuan Helminthosporium ( Bipolaris yamadae ) TL05 bacterial agent, which includes Helicoverpa yamadaensis ( Bipolaris yamadae )TL05 fermentation broth.

[0046] Example 5: Yamada Hiraumbifolia ( Bipolaris yamadae )TL05 fermentation broth.

[0047] Helminthosporium yamadaensis Bipolaris yamadae ) TL05 fermentation broth (hereinafter referred to as fermentation broth), the preparation method of which includes the following steps.

[0048] 1) Inoculation: Inoculate Helminthosporium yamadaensis ( Bipolaris yamadae ) TL05 was inoculated on mung bean solid culture medium and cultured at 25℃ for 7 days to obtain activated strains.

[0049] 2) Fermentation: Inoculate the activated strain into PDB liquid culture medium and culture in a shaker at 25°C and 180 rpm.

[0050] 3) Filtration: After 14 days of shaking culture, the bacterial liquid was filtered through 4 layers of gauze to remove the mycelium to obtain the filtrate, which is the crude metabolite stock solution, i.e., the fermentation liquid.

[0051] Example 6: Yamada Hiraumbifolia ( Bipolaris yamadae ) Pathogenicity determination of TL05 to Tribulus terrestris.

[0052] Plant the seeds of Tribulus terrestris in a plastic pot containing nutrient soil and vermiculite at a ratio of 1:2. Use a sterile needle to pierce 3 to 4 wounds on the leaves and stem base of healthy Tribulus terrestris. Bipolaris yamadae TL05 was inoculated on mung bean solid medium and cultured at 25℃ for 7 days. A 5 mm diameter bacterial cake was taken from the edge of the colony and inoculated on the wound as the treatment group; and mung bean solid medium was inoculated on the wound as the control group. Each treatment was repeated 3 times. The culture was placed in a growth room with 18 h light and 6 h dark alternating for cultivation, and the disease situation was observed and recorded. According to Koch's postulates, the strains were isolated and purified from the diseased part, and the isolated strains were identified to see if they were consistent with the inoculum.

[0053] The results showed that in the live grafting, the healthy leaves of the treated group began to become diseased after 3 days. At first, the area around the pinholes turned black and wrinkled, and then the lesions expanded to a maximum of 1 cm, and the leaves turned yellow, while the leaves of the control group remained normal. Figure 3The symptoms of the plants inoculated with the pathogen were consistent with those in the field, and the pathogen identical to the inoculum could be reisolated from the lesion tissue. According to Koch's postulates, Bipolaris yamadae TL05 was identified as the pathogen.

[0054] Example 7: Yamada Hiraumbifolia ( Bipolaris yamadae ) Determination of the herbicidal activity of TL05 against Tribulus terrestris.

[0055] The long-spined Tribulus terrestris was sown in a small plastic pot, and the small pot was placed in a tray filled with water, and then placed in an incubator with 18 h light and 6 h dark light to grow naturally. When the long-spined Tribulus terrestris grew to the 3-5 leaf stage, the treatment group sprayed the fermentation liquid prepared in Example 5 evenly on the leaves of the long-spined Tribulus terrestris until they were covered with dewdrops, and then covered with a black plastic bag in the dark for 24 h, and the bacterial liquid was sprayed continuously for 3 days. The control group sprayed with sterile water. Each treatment was repeated 3 times, and the growth was observed every two days and the data was recorded.

[0056] Crop disease susceptibility evaluation criteria: plants have no lesions, grow normally, and are asymptomatic, which is indicated by NS; plants have sporadic lesions on their leaves, and their growth and development are slightly restricted, indicating a slight impact, which is indicated by LS; 1 / 5-1 / 4 of the plant's leaf area is diseased and its growth is inhibited, which is indicated by MS; a large number of plants die and their growth and development are severely restricted, which is indicated by SS.

[0057] The results are shown in Table 4. The pathogenicity level of the fermentation liquid to Tribulus terrestris was MS, while that of the control group was NS, indicating that the fermentation liquid had a serious inhibitory effect on the growth of stems and leaves of Tribulus terrestris and had strong herbicidal activity. The fermentation liquid contained metabolites produced by Helicoverpa yamadaensis TL05 during the fermentation process, which could enhance the herbicidal effect and better adapt to different environmental conditions to maintain its stable state, simplifying the operation process of biological weed control and improving the application efficiency; the preparation method of the fermentation liquid was simple, and the large-scale production cost was relatively low, which made biological weed control more economical.

[0058] Table 4 Effects of fermentation liquid on the growth of Tribulus terrestris

[0059]

[0060] Example 8: Yamada Hiraumbifolia ( Bipolaris yamadae ) Pathogenicity of TL05 to common farmland weeds.

[0061] The seeds of six kinds of weeds, including barnyard grass, foxtail grass, tiger tail grass, crabgrass, Amaranthus retroflexus, and quinoa, were sown in a small plastic pot, and the small pot was placed in a tray filled with water, and then placed in an incubator with 18 h light and 6 h dark light for natural growth. When the above six kinds of weeds grew to the 3-5 leaf stage, the fermentation liquid prepared in Example 5 was evenly sprayed on the stems and leaves of the weeds to be tested until they were covered with dewdrops, and then covered with a black plastic bag and treated in the dark for 24 h, and the bacterial liquid was sprayed continuously for 3 d. The control group was set to spray sterile water, and each treatment was repeated 3 times. The growth was observed every two days and the data was recorded. The criteria for judging crop disease susceptibility are the same as those in Example 5.

[0062] The results are shown in Table 5. The fermentation liquid of the strain is most pathogenic to barnyard grass, chloris, Amaranthus retroflexus, and Chenopodium album. It is not obviously pathogenic to Setaria viridis and Digitaria sanguinea, and needs to be used in combination with other herbicides for joint prevention and control.

[0063] Table 5 Effects of fermentation liquid on the growth of common weeds

[0064]

[0065] Example 9: Yamada Hiraumbifolia ( Bipolaris yamadae ) Biosafety determination of TL05.

[0066] A total of 16 plants, including 11 crops (corn, soybean, eggplant, tomato, pepper, cabbage, Chinese cabbage, wheat, oats, cucumber, and spinach) and 5 forage grasses (Elymus tinctorius, Leymus chinensis, alfalfa, Sweet Clover, and Polygonum multiflorum), were selected as test subjects for the effects of fermentation liquid on the growth of stems and leaves of forage grasses and crops.

[0067] 16 kinds of forage grasses and crops were sown in small plastic pots, and the small pots were placed in a tray filled with water, and then placed in an incubator with 18 hours of light and 6 hours of dark light for natural growth. When the forage grasses and crops grew to the 3-5 leaf stage, the treatment group sprayed the fermentation liquid prepared in Example 5 evenly on the leaves of the forage grasses and crops until they were covered with dewdrops, and then covered with black plastic bags for 24 hours in the dark, and sprayed with the bacterial liquid for 3 days continuously. The control group sprayed with sterile water, and each treatment was repeated 3 times. The growth was observed every two days and the data was recorded. The criteria for judging crop disease susceptibility were the same as those in Example 5.

[0068] The results are shown in Table 6. The fermentation liquid had no effect on the growth of three forage grasses, including Elymus dahliae, Leymus chinensis, and Alfalfa, and had a slight effect on Sweet Clover and Polygonum multiflorum, indicating that the TL105 fermentation liquid can be used to control Tribulus terrestris on these forage grasslands. The fermentation liquid had a slight effect on the growth of soybean stems and leaves, and was safe for crops such as corn, eggplant, tomato, pepper, cabbage, Chinese cabbage, wheat, oats, cucumber, and spinach, and can be used safely.

[0069] Table 6 Effects of fermentation liquid on the growth of forage and crop stems and leaves

[0070]

[0071] The selection of biological herbicides is mainly based on two aspects: effectiveness (good control effect on target weeds) and safety (safety for crops other than target weeds). The strain TL05 isolated from the leaves of the diseased long-spined Tribulus terrestris in the present invention was identified as Helicoverpa yamadaensis ( Bipolaris yamadae ), which has a mild inhibitory effect on the growth of soybean, sweet clover, and schizonepeta, and has no harm to other crops and forage, indicating that Helicoverpa yamadaensis ( Bipolaris yamadae ) TL05 can be used to control Tribulus terrestris in forage and crop fields except soybean, sweet osmanthus and schizonepeta; In summary, Yamada Hirachus umbilical cord worm spore ( Bipolaris yamadae ) TL05 is both effective and safe, showing that the bacterium has great potential to be developed into a commercial bioherbicide.

[0072] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. Yamada Hiraumbiloba Bipolaris yamadae ) TL05, characterized in that, Its deposit number is CGMCC No.41290.

2. Helminthosporium yamadaensis Bipolaris yamadae ) TL05 bacterial agent, characterized in that: It includes the Yamada Hiraumbiloba as claimed in claim 1 ( Bipolaris yamadae ) TL05 and / or its fermentation broth.

3. Yamada Hiraumbiloba Bipolaris yamadae ) A method for preparing TL05 fermentation broth, characterized in that: It includes the following steps: 1) Inoculation: Inoculate the Yamada Hiraumphalis spores described in claim 1 ( Bipolaris yamadae ) TL05 was inoculated on mung bean solid culture medium and cultured at 25°C for 7 days to obtain activated strains; 2) Fermentation: Inoculate the activated strain into PDB liquid culture medium and culture at 25°C; 3) Filtration: Filter the bacterial solution after 14 days of culture to obtain the crude metabolite stock solution, which is the fermentation broth.

4. The Yamada Hiraumbilicus according to claim 1 ( Bipolaris yamadae ) TL05 or the Yamada Hiraumbiloba described in claim 2 ( Bipolaris yamadae ) Application of TL05 microbial agent in controlling long-spined tribulus terrestris.

5. The Yamada Hiraumbilicus according to claim 1 ( Bipolaris yamadae ) TL05 or the Yamada Hiraumbiloba described in claim 2 ( Bipolaris yamadae ) Application of TL05 microbial agent in the preparation of products for controlling long-spined tribulus terrestris.

6. The Yamada Hiraumphalis spore according to claim 1 ( Bipolaris yamadae ) TL05 or the Yamada Hiraumbiloba described in claim 2 ( Bipolaris yamadae ) The application of TL05 microbial agent in inhibiting weed growth is characterized in that: The weeds are at least one of barnyard grass, chloris, amaranth and quinoa.

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