Streptomyces sporeans

Abstract

The present invention discloses a Desmospora profundinema GS1 strain. In a plate confrontation test, it was found that the GS1 strain has the activity of inhibiting the growth of multiple plant pathogenic bacteria, and the maximum diameter of the inhibition zone can reach 26 mm; in a potted inoculation test, it was found that the GS1 strain can inhibit the infection of citrus canker bacteria. The GS1 strain of the present invention also has a high efficiency inhibitory effect on pathogens such as peach root cancer, tobacco bacterial wilt, rice white leaf blight, apricot fire blight, melon bacterial fruit spot, and Chinese cabbage soft rot. The Desmospora profundinema of the present invention has a high efficiency antibacterial activity against plant pathogenic bacteria, and therefore, the Desmospora profundinema GS1 can be used as a biocontrol bacterium in the prevention and treatment of plant bacterial diseases.

Description

Technical Field

[0001] The invention belongs to the technical field of biological control, and specifically relates to a deep-sea spore-forming streptozotocin and an application thereof in preventing and controlling plant diseases. Background Art

[0002] Plant bacterial diseases refer to a series of physiological changes in cells and tissues that occur after plants are infected with pathogenic bacteria. Plant bacterial diseases are extremely harmful and difficult to prevent and control. Once infected, the planting area may become a lifelong epidemic area; because of its characteristics of outbreak, epidemic and destructiveness, it usually causes serious reduction in plant yield. The more serious bacterial diseases that occur in production include citrus canker, green orange disease of Solanaceae plants, rice white leaf blight, soft rot of cruciferous vegetables, bacterial fruit spot of melons, and fire blight of pears.

[0003] In production, antibiotics and copper-based chemical drugs such as kasugamycin, sinomenine, copper oxychloride, and thiophanate-methyl are often used in large quantities to prevent and control bacterial diseases. On the one hand, this has caused the pathogens to gradually increase their drug resistance and decrease their prevention efficacy, forcing them to increase the dosage of drugs, forming a vicious circle. On the other hand, pesticide residues pose great risks to the food safety of agricultural products and the ecological environment, and there is an urgent need to develop more green and environmentally friendly prevention and control technologies.

[0004] The control technology based on biocontrol microorganisms has the advantages of low cost, high efficiency and no pollution. It plays an important role in reducing the use of chemical pesticides and improving the quality of agricultural products, and has received more and more attention in agricultural production. Antagonistic microorganisms against plant bacterial diseases are widely present in nature, such as Bacillus, Pseudomonas, Actinomycetes, Streptomyces, etc. Antagonistic microorganisms kill plant pathogens by competing or secreting a variety of antimicrobial active substances such as antibiotics, antimicrobial proteins and peptides, reducing the damage of pathogens to plants. Antagonistic microorganisms that are easy to reproduce and have strong resistance to adverse environments are very suitable for the development, storage and application of biological agents. Among them, deep-sea spore-forming streptobacterium has shown great development value in the prevention and control of different types of bacterial and fungal plant diseases, and the application and research of this type of strain in the prevention and control of bacterial diseases is still in its infancy. Summary of the invention

[0005] The purpose of the present invention is to provide a deep-sea spore-forming streptozotocin and its application in preventing and controlling plant diseases.

[0006] A strain of Desmospora profundinema GS1, whose deposit number is CGMCCNO.23983.

[0007] The application of Desmospora profundinema in preventing and treating citrus canker.

[0008] Furthermore, Desmospora profundinema was added to the mixture at a concentration of 1.0×10 7 ~1.5×10 9 cfu / mL fermented bacterial solution spray or root irrigation treatment.

[0009] The application of Desmospora profundinema in inhibiting or killing pathogenic bacteria of plant diseases.

[0010] Preferably, the plant disease pathogens include citrus canker (Xanthomonas citri), Ralstonia solanacearum (Ralstonia solanacearum), peach root cancer (Agrobacterium tumefaciens), melon bacterial fruit spot (Acidovorax citrulli), amylovora (Erwinia amylovora), rice bacterial leaf blight (Xanthomonas oryzae pv.oryzae), and Chinese cabbage soft rot (Pectobacterium versatile).

[0011] A biological preparation comprising the Desmospora profundinema or its metabolite.

[0012] Furthermore, the dosage form of the biological agent is an aqueous suspension, a powder, a wettable powder, an aqueous solution, a microemulsion, a granule or a water-dispersible granule.

[0013] The deep-sea spore-forming bacteria (Desmospora profundinema) GS1 of the present invention is classified and named as deep-sea spore-forming bacteria Desmospora profundinema, which was deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration on November 26, 2021, and has a survival certificate. The deposit address is: No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, and the strain deposit number is CGMCC NO.23983.

[0014] Beneficial effects of the present invention: The present invention isolates and identifies a strain of Desmospora profundinema GS1, and the research results show that strain GS1 can effectively inhibit the growth of a variety of plant pathogenic bacteria, and has significant antagonistic activity against a variety of plant pathogenic bacteria. Strain GS1 can successfully and rapidly reproduce, has stable antagonistic activity against pathogens, and has great application potential. The promotion and application of GS1 will provide more basis for reducing chemical residues in agricultural products and achieving green and safe production. The biocontrol agent of the present invention has the characteristics of persistence, stability, pollution-free, safety for humans and animals, no residue, strong specificity, etc. in the biological control of plant diseases, and has the advantages of protecting the environment and maintaining ecological balance. The deep-sea spore-forming streptococcus GS1 disclosed in the present invention can be used to prevent the occurrence of bacterial diseases of plants, effectively reduce their incidence, and has important application potential in the preparation of biocontrol microbial agents. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 These are the colony morphological characteristics of deep-sea spore-forming bacteria GS1.

[0016] Figure 2 It is the antagonistic activity of deep-sea spore-forming bacteria GS1 against a variety of pathogenic bacteria. DETAILED DESCRIPTION

[0017] In order to facilitate the understanding of the present invention, the present invention will be described more fully below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0018] Example 1 Isolation of strains

[0019] Take 1g of seabed sediment, add it to 10ml of sterile water, and culture it at 180rpm for 30min. Then dilute it with water, and the dilution gradient is 10×, 100× and 1000×. Take 100μl of each gradient dilution liquid and spread it on LB medium. Culture it at 28℃ for 1d, pick a single colony and streak it on LB medium for purification. After purification, transfer 62 single colonies to new LB medium for storage.

[0020] Example 2 Screening of antagonistic strains

[0021] The plate confrontation method was used to determine the inhibitory activity of the 62 isolated bacteria against citrus canker bacteria. The specific operation was as follows: the citrus canker bacteria were cultured at 28°C and 180rpm until the OD600 was about 1, then spread on LB solid culture medium, and the isolated bacteria were picked onto the plate and cultured overnight at 28°C. Strain GS1 had significant antagonistic activity against citrus canker bacteria, and the diameter of the inhibition zone reached more than 20 mm.

[0022] Example 3 Identification of deep sea spore-forming Streptozotocin GS1

[0023] Morphological identification: The colonies are white, round, with irregular edges, protrusions, translucent, and have a rough membrane coating on the surface in the early stage, and wrinkles on the surface in the later stage (such as Figure 1 shown).

[0024] Molecular biological identification as 16S rRNA gene analysis: The total DNA of strain GS1 was extracted using a bacterial DNA extraction kit. The bacterial universal primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-TACGACTTAACCCCAATCGC-3′) were used to amplify the 16S rRNA gene of the strain by PCR. The amplification program was: 94℃ pre-denaturation for 5min; 36 cycles (94℃ denaturation for 45s, 57℃ annealing for 30s, 72℃ extension for 120s); 72℃ extension for 10min. The PCR product was detected by 1.7% (w / v) agarose gel electrophoresis, and then sequenced by Sangon Biotech (Shanghai) Co., Ltd. The results of molecular biological identification showed that the sequence of strain GS1 was 99% consistent with the 16S rRNA sequence data of deep-sea spore-forming bacteria strain SCSIO 11154 (GenBank: KM368342). Therefore, GS1 was identified as deep-sea spore-forming bacteria.

[0025] Example 4 Physiological and biochemical characteristics of deep-sea spore-forming Streptozoa GS1

[0026] Starch hydrolysis: negative; cellulose decomposition: negative; methyl red (MR): positive; acetylmethylcarbinol (VP): positive; catalase: negative; gelatin liquefaction: positive; hydrogen sulfide production: negative; nitrate reduction: positive; indole: negative.

[0027] Example 5 Antagonistic effect of Streptozotocin GS1 on bacterial plant disease pathogens

[0028] The strain GS1 was cultured against bacterial pathogens such as citrus canker, including Ralstonia solanacearum, Agrobacterium tumefaciens, Acidovorax citrulli, Erwinia amylovora, Xanthomonas oryzae pv.oryzae and Pectobacterium versatile, and its antibacterial effect was observed after culturing overnight at 28°C. The test results (Table 1) showed that the strain GS1 had antagonistic effects on these seven pathogens, with the diameter of the inhibition zone ranging from 10-26mm ( Figure 2 ).

[0029] Table 1 Antagonistic effect of Streptozotocin GS1 on bacterial plant disease pathogens

[0030]

[0031] Example 6 The control effect of deep sea spore-forming bacteria GS1 on citrus canker

[0032] The bacterial solution was evenly sprayed on the leaves of potted citrus by spraying the positive and negative leaves. Sterile water was used as a non-inoculated control. The disease situation of citrus was observed and recorded every day. This experiment set up 4 treatments, each with 5 replicates. Treatment T1 was first sprayed with a concentration of 10 8 cfu / mL of biocontrol bacteria GS1, wait for 24 hours to dry, and then spray an equal volume of 10 8 cfu / mL pathogen; Treatment T2 was first sprayed with a concentration adjusted to 10 8 cfu / mL of pathogen (GJK3), and spray with an equal volume concentration of 10 8 cfu / mL of biocontrol bacteria GS1; treatment T3 was first sprayed with a concentration of 10 8 cfu / mL of biocontrol bacteria GS1, wait for 24 hours to dry and then spray with an equal volume of sterile water; treatment CK was first sprayed with a concentration of 10 8 cfu / mL of pathogens, and spray with an equal amount of sterile water after drying for 24 hours. The potted seedlings were placed in an artificial climate box and kept moist at 28℃. The disease was observed after 15 days, and the disease statistics under different treatments were conducted to analyze the control effect of citrus canker.

[0033] The grading standard for citrus canker on citrus plants is as follows: Grade 0, no lesions; Grade 1, the lesion area is less than 5% of the entire leaf; Grade 3, the lesion area is 5% to 25% of the entire leaf; Grade 5, the lesion area is 26% to 50% of the entire leaf; Grade 7, the lesion area is 51% to 75% of the entire leaf; Grade 9, the lesion area is 76% to 100% of the entire leaf.

[0034] Different degrees of disease occurred on the leaves of citrus in different treatments of spraying and inoculating pathogens, but there were large differences in the disease index between different treatments. Needle-sized yellow-green oily spots appeared on the surface of the diseased leaves, and the spots were gray-white ridges in the shape of craters, and there were nearly circular brown spots at the ridges, which were completely consistent with the symptoms of citrus canker, while the leaves inoculated with sterile water did not get sick. Among them, the number and area of ​​diseased spots on the leaves of citrus in the two treatments (T1 and T2) inoculated with biocontrol bacteria GS1 were significantly smaller than those of the control treatment CK without biocontrol bacteria GS1. The control effect of treatment T1, which was first sprayed with biocontrol bacteria GS1 and then inoculated with pathogens, on citrus canker was as high as 83.09%, which was significantly higher than that of treatment T2, which was first sprayed with pathogens and then inoculated with biocontrol bacteria GS1 (the control effect was only 27.69%). This shows that deep-sea spore-forming Streptozotocin GS1 has an important preventive effect on the occurrence of citrus canker. Pre-spraying and inoculating the culture solution of this strain can significantly reduce the occurrence of citrus canker and protect citrus plants to the greatest extent. When citrus is infected with citrus canker and then inoculated with GS1, its prevention and control effect is greatly reduced, far lower than the treatment method of pre-spraying and inoculation. Therefore, it is recommended to reduce the occurrence of the disease and protect the healthy growth of plants by pre-spraying and inoculating deep-sea spore-forming Streptozotocin GS1 during the production season when citrus canker is prone to occur (Table 2).

[0035] Table 2 The control effect of deep sea spore Streptozotocin GS1 on citrus canker under different treatments

[0036]

[0037]

[0038] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A strain of deep-sea spore-forming bacteria ( Desmospora profundinema ) GS1, its deposit number is CGMCC NO.23983.

2. The deep sea spore-forming bacteria of claim 1 ( Desmospora profundinema ) in the prevention and treatment of citrus canker.

3. According to claim 2, the deep sea spore streptozotocin ( Desmospora profundinema ) for preventing and treating citrus canker, characterized in that: Streptozotocin Desmospora profundinema ) at a concentration of 1.0×10 7 ~1.5×10 9 cfu / mL fermented bacterial solution spray or root irrigation treatment.

4. The deep sea spore-forming bacteria of claim 1 ( Desmospora profundinema ) for inhibiting or killing plant disease pathogens, characterized in that: The plant disease pathogens include citrus canker bacteria ( Xanthomonas citri )、Ralstonia solanacearum( Ralstonia solanacearum )、Peach root cancer bacteria( Agrobacterium tumefaciens )、Bacterial fruit spot of cucurbits( Acidovorax citrulli )、Ernstria amylovora( Erwinia amylovora ), Xanthomonas oryzae ( Xanthomonas oryzae pv. oryzae ) and Chinese cabbage soft rot pathogen ( Pectobacterium versatile ).

5. A biological agent, characterized in that: Containing the deep sea spore streptozotocin as claimed in claim 1 ( Desmospora profundinema ).

6. The biological agent according to claim 5, characterized in that: The dosage form of the biological preparation is water suspension, powder, aqueous solution, microemulsion, and granule.

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