CN116694505A - A kind of Streptomyces purpurecane that can prevent and treat clubroot and its application - Google Patents

Abstract

The invention relates to Streptomyces crimson CC2-6 capable of preventing and treating clubroot, which is preserved in China general microbiological culture Collection center (CGMCC) No.26458 in 2023 and 1-12 days, and also relates to application of the Streptomyces crimson CC2-6 in clubroot. The Streptomyces roseoformis CC2-6 has strong inhibition effect on various plant pathogenic bacteria on a flat plate, and the fermentation liquor metabolite has good prevention and control effects on clubroot. The germination inhibition rate of the dormant spores of the plasmodiophora radicis is 51.01%, the root and root hair infection inhibition rate of the Chinese cabbage is 77.41%, the pot control effect of the Chinese cabbage treated by the CC2-6 fermentation liquor is 27.46%, and the pot control effect of the Chinese cabbage is 53.20% when the Chinese cabbage is treated in 7d in advance. In addition, the strain has natural harmony and compatibility with natural ecology, and has no toxic or side effect and no residue on soil ecology compared with chemical pesticides. Therefore, the method has potential commercial development and application value in biological control practice of diseases.

Description

A kind of Streptomyces purpurecane that can prevent and treat clubroot and its application

technical field

The invention relates to the field of crop disease prevention and control, and more particularly relates to a Streptomyces fuchsinosa which can prevent and treat clubroot and its application.

Background technique

Clubroot is a soil-borne disease that mainly infects some economic crops in the cruciferous family, such as rapeseed, radish, mustard and cabbage. Clubroot is widely distributed in more than 50 countries and regions in the world, including Africa, South America, North America, Europe, Asia and Oceania, and the annual crop yield loss due to clubroot around the world is as high as 10-15 %, so clubroot is regarded as one of the important diseases of cruciferous crops. According to statistics, clubroot can occur in an area of 48-60 million mu in my country in a year with less damage, and in an outbreak year, the damage area is as high as 135 million mu.

Clubroot occurs and harms the roots of plants, and the plants can be infected by clubroot throughout the growth period, but the seedling stage is the most important period of infection. After the underground root is infected by Plasmodium, it causes changes in plant hormones, accelerates plant cell division, and the main root and lateral root swell to form tumors. In the early stage of swollen root formation, the surface of the root is smooth, and later the root rots, the color becomes dark brown, and emits an odor, the tumor expands, and the root hairs are reduced or even absent, which leads to the inhibition of the plant's absorption and conduction of water and nutrients, so the aboveground parts will appear. Symptoms such as wilting and yellowing of leaves due to lack of water, and in severe cases, the whole plant will die.

At present, the prevention and treatment of clubroot is still dominated by chemical agents, which are relatively simple and quick to operate, low in cost, and quick in effect. However, the use of chemical fungicides is likely to lead to drug resistance of pathogenic bacteria, pollution to the environment, and residual toxicity. In the end, there will be problems such as poor control effect, endangering ecological safety and human health.

With the further development of scientific research, it is found that biocontrol microorganisms have unique advantages in overcoming drug resistance of plant diseases, reducing environmental pollution by chemical fungicides, ecological damage, and chemical pesticide residues in agricultural and sideline products. Biopesticides prepared from antibacterial secondary metabolites have the characteristics of no pollution, no residue, no resistance to harmful organisms, high compatibility with the environment, and safety for humans and animals; and the use of metabolites produced by microorganisms to treat plant pathogenic bacteria , to achieve biological control of disease research is a hot spot. The biocontrol potential of secondary metabolites produced by actinomycetes is mainly reflected in the inhibition of pathogen mycelium growth, spore germination and infection, and more and more actinomycetes originate from endogenous plants or different extreme environments. When bacteria are discovered, the types of metabolites produced by actinomycetes will become more and more abundant. Therefore, vigorously carrying out biological control research is an important way for the sustainable development of agriculture, and has important practical significance in production.

Contents of the invention

In order to solve the above problems, the present invention provides a Streptomyces purpurea that can prevent and treat clubroot, which was preserved in the General Microorganism Center of China Microbiological Culture Collection Management Committee on January 12, 2023, and the preservation number is CGMCC No.26458 .

The present invention also provides the application of the above-mentioned Streptomyces fumaratus in preventing and treating clubroot.

The present invention also provides a method for preventing and treating clubroot, comprising the step of applying the above-mentioned cultured product of Streptomyces fumarusus to the soil where crops are planted.

In a specific embodiment, the method comprises the steps of:

S1: Cultivate the Streptomyces ruberophorus to prepare Streptomyces ruborophorus fermentation broth;

S2: Applying the fermented liquid of Streptomyces fumarusus to the soil where the crops are planted.

In a specific embodiment, in S1, the Streptomyces fumarusus is cultured in Gao's No. 1 medium.

In a specific embodiment, S1 comprises the following steps:

S11: inoculate the Streptomyces purpureus into Gao's No. 1 liquid culture medium;

S12: Cultivate under the conditions of 28°C and 180rpm for 7 days to obtain the fermentation broth of Streptomyces ruthenicus.

In a specific embodiment, in S2, the fermented liquid of Streptomyces fumarusus is used for regular irrigation of the crops.

In a specific embodiment, the time interval between every two root irrigation treatments is 7 days.

The Streptomyces purpureus CC2-6 strain of the present invention has a strong inhibitory effect on various plant pathogenic bacteria on a plate, and its fermentation broth metabolites have a good control effect on clubroot. The germination inhibition rate of dormant spores of Plasmodium was 51.01%. The water seedling test showed that after treatment with CC2-6 fermentation broth for 9 days, the inhibition rate of root hair infection of cabbage roots was 77.41%. After 50 days of simultaneous inoculation with nematodes fermentation liquid, the control effect of potted cabbage treated with CC2-6 fermentation liquid was 27.46%, and when treated 7 days in advance, the control effect of potted cabbage was 53.20%. In addition, the strain is isolated from the soil and has a natural harmony with the natural ecology. Compared with chemical pesticides, it has no toxic side effects and no residues on the soil ecology. Therefore, it has potential commercial development and application value in the practice of biological control of diseases.

biological material deposit

Purified Streptomyces crimsonophore CC2-6 of the present invention was preserved on January 12, 2023 in the General Microorganism Center of China Microorganism Culture Preservation Management Committee ( CGMCC), the deposit number is CGMCC No.26458.

Description of drawings

Fig. 1 is the plate morphology photo of Streptomyces rufosa CC2-6;

Fig. 2 is the single colony morphological photo of Streptomyces rufosa CC2-6;

Fig. 3 is the micrograph of spore chain and aerial mycelia of Streptomyces rufosa CC2-6;

Fig. 4 shows that Streptomyces fumarusus CC2-6 produces multiple hydrolytic enzyme identifications;

Fig. 5 shows the inhibitory effect of Streptomyces fusarium fusarium CC2-6 on cotton Fusarium wilt pathogen;

Figure 6 shows the disease control effect of Streptomyces fumarusus CC2-6 clubroot.

Detailed ways

The principles and features of the present invention are described below in conjunction with the accompanying drawings, and the examples given are only used to explain the present invention, and are not intended to limit the scope of the present invention.

1. Identification and preservation of Streptomyces rufosa CC2-6

The team collected mainly the rhizosphere soil of non-farmland ecosystems such as lawns, bamboo forests, and street trees from Beibei, Shapingba, Banan, and Nan'an in Chongqing City. The isolated strains were screened and re-screened. A strain CC2-6 was obtained, and it was confirmed that the strain has a good control effect on clubroot through the inoculation test of living plants in indoor pots.

The bacterial strain CC2-6 was cultured on Gaoshi No. 1 culture plate at 28°C for 5-7 days, and the photo of the plate morphology was shown in Figure 1, and the photo of a single colony was shown in Figure 2.

Slides sterilized in advance were inserted obliquely into the plate at 30°C, cultured upside down in a 28°C incubator for 7 days, then the coverslips were taken out, a little distilled water was dropped on the slides, and the aerial hyphae of the strains were observed using an optical microscope , The shape of the morphological characteristics of the spore chain, as shown in Figure 3.

The genomic DNA of the bacterial strain was extracted with bacterial universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3', SEQ ID NO:1,) and 1492R (5'-TACGGCTACC TTGACGAC-3', SEQ ID NO:2). Using the genomic DNA of strain CC2-6 as a template, after PCR amplification, electrophoresis detection and sequencing, the 16S rDNA sequence was obtained as shown in SEQ ID NO:3. The obtained sequence results were compared by Blast in NCBI, and the 16S rDNA sequences of related strains were selected, and the phylogenetic tree was constructed using the Neighbor-Joining method of MEGA 7.0 software, and combined with morphological characteristics, the strain was identified as belonging to the magenta chain Mold (Streptomyces purpeofuscus).

The above strains were deposited on January 12, 2023 at the General Microbiology Center (CGMCC) of the Chinese Academy of Sciences, Institute of Microbiology, Chinese Academy of Sciences, No. 1, Beichen West Road, Chaoyang District, Beijing, and the preservation number: CGMCCNo.26458 .

2. Preparation of fermentation broth of Streptomyces purpureus CC2-6 and analysis of metabolites

The bacteria cake of the antagonistic actinomycete XDS3-6 was placed in the sterile Gaoshi Synthetic No. 1 liquid medium, and the culture was shaken and fermented at 28°C and 180rpm for 7 days. Take the fermentation broth at 7500rpm, centrifuge for 10min, discard the precipitate and take the supernatant. Freeze at -80°C, and freeze-dry in a freeze dryer for 12 hours.

3. Prevention and treatment of pathogenic bacteria by Streptomyces purpureus CC2-6

3.1 Bacteriostatic activity of Streptomyces ruberunus CC2-6 on pathogenic bacteria

Antagonistic actinomycetes were spotted in some hydrolase selection media. Place the plate upside down in an incubator at 28°C, and observe the growth of the strain after 7 days and record the radius of the transparent circle. After testing, Streptomyces fumarusus CC2-6 can produce a variety of hydrolytic enzymes (Figure 4). It is speculated that it has good antibacterial effect on some pathogenic bacteria.

Further, the influence of the antagonistic bacteria on the mycelial growth of the pathogenic fungus (Fusarium wilt of cotton) was determined by using the plate confrontation streaking method. Inoculate pathogenic fungus 8 mm bacteria cake in the center of the plate, inoculate antagonistic actinomycetes at both sides of the bacteria cake 25 mm, draw two 30 mm straight lines, actinomycetes inoculate 3 days earlier than pathogenic fungi, control group inoculate pathogenic fungi in the center of the plate, no Actinomycetes were inoculated, and each treatment was replicated three times, and cultured in a constant temperature incubator at 28°C. When the pathogenic fungi in the control group covered the plate, it was observed whether the treatment group produced a bacteriostatic zone, and the diameter of the pathogenic fungal colony was measured to calculate the bacteriostatic rate. To determine the inhibitory effect of CC2-6 strain on pathogenic fungi. The results are shown in Figure 5, the CC2-6 strain has a significant antibacterial effect on the bacteria.

3.2 Prevention and control of clubroot by Streptomyces ruberunus CC2-6

Using the root exudates of Chinese cabbage as the culture medium, add 5mL root exudates, 0.5mL Plasmodium dormant spore suspension and 0.5mL fermentation broth of strain CC2-6 into a sterilized Erlenmeyer flask, and place in the dark at 24°C Under culture, sterilized liquid medium was used as the control, and each treatment was repeated in 3 groups. On the 5th day, the germination of dormant spores was observed under a microscope, stained with 1% lichenin (dissolved in 45% acetic acid) for 10-15s, and the spore germination rate and germination inhibition rate were calculated. The results showed that the germination inhibition rate of dormant spores of Plasmodium root was 51.01%.

Put the cabbage seeds in a petri dish lined with wet filter paper, and place them under light and humid conditions at 24°C to accelerate germination. After 3 days, the seedlings were transplanted into 10 mL centrifuge tubes filled with Hoagland nutrient solution for hydroculture, and after 5 days, they were inoculated with dormant spores of Plasmodium. And CC2-6 strain fermentation broth, the final concentration of Plasmodium dormant spores was 10 ⁷ /mL, the treatment group was added to the fermentation broth at a ratio of 1:30, and the control group was added to Hoagland nutrient solution. After 9 days of hydroponics, the roots of the seedlings were washed with clean water, dyed in fluorescent pink dyeing solution for 30 minutes, and moved under a microscope to observe the infection of the root hairs of the seedlings. The results showed that after treatment with CC2-6 fermentation broth for 9 days, the inhibition rate of root hair infection of cabbage root was 77.41%.

Cabbage was inoculated by root irrigation method. Take out the swollen root from the refrigerator and put it in a dark environment at 25°C to rot for 5 days, mix the swollen root and water at a ratio of 1:5, stir and filter the juicer intermittently, prepare and adjust the concentration of the dormant spore suspension of Plasmodium root 2.5×10 ⁸ cells/mL. The antagonistic bacteria cake was inoculated into Gao's No. 1 liquid medium, and cultured at 28°C and 180r·min ^-1 for 7 days to obtain an actinomycete fermentation broth with a concentration of 10 ⁷ CFU/mL. Each cabbage plant was treated with 30mL dormant spore suspension of Plasmodium spores, so that the final bacterial content per gram of soil was 3×10 ⁷ . Treatment A: Inoculate antagonistic bacteria and Plasmodium at the same time, pour 20 mL of actinomycete fermentation broth every 7 days, and water five times in total. Treatment B: Inoculate antagonistic bacteria 7 days in advance, water 20 mL of actinomycete fermentation liquid every 7 days, and water five times. Inoculate the dormant spore suspension of Plasmodium spp. and pour Gaoshi No. 1 liquid medium as the disease control, repeat 20 cabbage plants for each treatment, investigate the disease 50 days after the inoculation, and make statistical records. The results showed that after Plasmodium and CC2-6 fermented liquid were inoculated simultaneously for 50 days, the control effect of potted cabbage treated with CC2-6 fermented liquid was 27.46%, and when CC2-6 fermented liquid was treated 7 days in advance, the control effect of potted cabbage was 53.20% (Figure 6).

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the protection of the present invention. within range.

Claims (8)

1. A Streptomyces purpurea that can prevent and treat clubroot, characterized in that it was preserved in the General Microorganism Center of China Committee for the Collection of Microorganisms on January 12, 2023, and the preservation number is CGMCCNo.26458. 2. the application of the Streptomyces fumaratus described in claim 1 in the prevention and treatment of clubroot. 3. A method for preventing and treating clubroot, characterized in that it comprises the step of applying the cultured product of Streptomyces fumaratus according to claim 1 to the soil where crops are planted. 4. The method according to claim 3, characterized in that, comprising the steps of: S1: Cultivate the Streptomyces ruberophorus to prepare Streptomyces ruborophorus fermentation broth; S2: Applying the fermented liquid of Streptomyces fumarusus to the soil where the crops are planted. 5. The method according to claim 4, characterized in that, in S1, the Streptomyces fumarusus is cultivated in Gao's No. 1 medium. 6. The method according to claim 5, characterized in that S1 comprises the following steps: S11: inoculate the Streptomyces purpureus into Gao's No. 1 liquid culture medium; S12: Cultivate under the conditions of 28°C and 180rpm for 7 days to obtain the fermentation broth of Streptomyces ruthenicus. 7. The method according to claim 4, characterized in that, in S2, the Streptomyces fumarus fermented liquid is used to carry out regular root irrigation treatment to the crops. 8. The method according to claim 7, characterized in that, the time interval between every two root irrigation treatments is 7 days.