CN105861397A - Chili antagonistic endophyte P5 strain - Google Patents

Abstract

An antagonistic endophyte Bacillus subtilis (B.subtilis) P5 strain was preserved in the China Center for Type Culture Collection on March 18, 2016, and the preservation number is CCTCC NO: M2016131. The fermentation broth of this strain P5 has broad-spectrum antibacterial activity, and is effective against plant pathogens such as capsicum anthracnose, citrus penicillium, cabbage black spot, rape sclerotinia, capsicum phytophthora, capsicum spp., tomato early blight The fungi have obvious inhibitory effect, among which the antagonism ability to the mycelial growth of S. capsici is the strongest, and the inhibition rate reaches 48.57%. The development of new biological plant fungicides provides a basis.

Description

A Capsicum Antagonistic Endophyte P5 Strain

technical field

The present invention relates to an antagonistic endophytic bacterium, in particular to an endophytic bacterium capable of antagonizing various plant pathogenic fungi such as pepper anthracnose (C.gloeosporioides), in particular to a kind of Bacillus subtilis (B.subtilis) P5 strain.

technical field

At present, the main measures to control postharvest diseases of fruits and vegetables are still chemical fungicides. However, the long-term use of chemical fungicides leads to the resistance of bacteria and greatly reduces its disease prevention effect. At the same time, the frequent use of high-concentration chemical fungicides in production increases their residues in fruit and vegetable pulp and threatens human health. Therefore, seeking safe and effective post-harvest disease prevention technologies for fruits and vegetables has become a current research hotspot.

Biological control has the advantages of no pollution to the environment, no pesticide residues, no drug resistance, and low treatment costs. Plant endophytes parasitize in plant tissues and form a mutually beneficial symbiotic relationship with their hosts during the long-term evolution process. Plant endophytes protect host plants from disease by producing a large number of biologically active substances. At present, there have been related reports on the use of plant endophytes for postharvest sterilization of fruits and vegetables. For example, the fermentation broth of endophyte EJS has a significant inhibitory effect on nectarine postharvest blue mold.

Bacillus can produce a variety of lipopeptide antibiotics, such as iturin, surfactin, fenmgycin and so on. These compounds have strong antibacterial activity and can effectively control the occurrence of postharvest diseases of fruits and vegetables. As a species of Bacillus, Bacillus subtilis widely exists in nature and is a common endophytic bacterium in plants. It is non-toxic and harmless to the human body and does not pollute the environment. It can produce two types of antibacterial substances in the process of growth and metabolism, one is lipopeptide antibiotics synthesized by non-ribosomes, such as surfactin, iturin, plipastatin, etc., which usually have a significant inhibitory effect on plant pathogenic fungi; Classes are bacteriocins synthesized by ribosomes, such as subtilosin, sublancin, subtilin, which can effectively kill related bacteria. At present, there have been many domestic research reports on the biological control of plant pathogenic bacteria by Bacillus subtilis. For example, He Hong et al. found that the extracellular secretions of Bacillus subtilis BS-2 strain had a strong inhibitory effect on the pathogenic bacteria of pepper red anthracnose; inhibition.

Therefore, screening antagonistic strains from plant endophytic flora, and using the bacteriostatic substances produced by them to make new biological fungicides, so as to replace chemical fungicides or reduce the use of chemical fungicides has great application prospects.

Contents of the invention

The technical problem to be solved by the present invention is to provide an antagonistic endophyte Bacillus subtilis (Bacillus subtilis) P5 strain, which has obvious antagonistic effect on plant pathogenic bacteria such as capsicum anthracnose.

The Bacillus subtilis P5 (Bacillus subtilis) P5 strain described in the present invention has been preserved in the China Center for Type Culture Collection on March 18, 2016. The preservation address is Wuhan University, Wuhan, China, and the preservation number is CCTCC NO: M2016131.

Morphological characteristics of bacterial strain P5 of the present invention:

Bacterial strain P5 of the present invention was cultivated in beef extract peptone solid medium at 28°C. Under a high-power microscope, the bacterium was rod-shaped, and Gram staining was positive (Figure 1). After 24 hours of cultivation at 28°C, a single colony was round. , milky white, opaque, with neat edges, and the colony size is about 2-8mm, see Figure 2.

Physiological and biochemical characteristics of the strain P5 of the present invention: aerobic property+, V.P experiment+, contact enzyme reaction+, starch hydrolysis experiment+, hydrogen sulfide experiment+, glucose oxidation fermentation+, gelatin liquefaction+, resistance to 7% NaC+, resistance to 50°C temperature +. Among them, "+" indicates positive or can grow and utilize; "-" indicates negative or cannot grow and utilize.

The total DNA of the bacterial strain P5 of the present invention was extracted by the CTAB method, PCR amplification of the 16S rDNA was carried out using universal primers 16S-27F and 16S-1492R, and the measured 16S rDNA sequences were compared by BLAST homology on NCBI, and it was found that the The sequence has 98% homology with the 16S rDNA sequences of 30 bacterial strains of Bacillus subtilis (B.subtilis), wherein the accession number is the sequence size of the 16S rDNA of the Bacillus subtilis bacterial strain FHGXJ1 of KF527194.1 and the sequence size of the bacterial strain P5 of the present invention They are basically the same, and the two belong to the same branch on the evolutionary tree, and the evolutionary distance is the closest. Combined with the morphological and physiological and biochemical characteristics of the strain P5 of the present invention, it is further shown that the strain P5 of the present invention is Bacillus subtilis (B. subtilis).

The bacterial strain P5 of the present invention is inserted into the beef extract peptone liquid medium for fermentation and culture, and the fermentation conditions are: inoculum size 3%, rotation speed 150-180r/min, temperature 28-30°C, time 46-52h, and the bacterial strain P5 of the present invention can be obtained of fermentation broth. The fermentation broth has broad-spectrum antibacterial activity, and has obvious antibacterial activity against plant pathogenic fungi such as capsicum anthracnose, citrus penicillium, cabbage black spot, rape sclerotinia, capsicum phytophthora, capsicum spp., tomato early blight Among them, the antagonism to the mycelial growth of S. capsici is the strongest, and the inhibition rate reaches 48.57%. Plant fungicides provide the basis.

Description of drawings

Fig. 1 is a graph of Gram staining (×100) of strain P5 of the present invention.

Fig. 2 is a single colony morphological diagram of the strain P5 of the present invention.

detailed description

Example 1

Fresh peppers were taken from the vegetable garden of Changsha City, Hunan Province, washed with tap water, soaked in 75% mass concentration of ethanol for 3 minutes, wiped with 5% mass concentration of NaClO for 5 minutes, rinsed repeatedly with sterile water for 6 times, and peeled with sterile water. Cut into small pieces with a bacterial knife and grind into a slurry in a sterile mortar. Dilute 5ml of the grinding solution 10 times with sterile water, spread 0.1ml of the diluted solution on NA solid medium, incubate in the dark at 28°C for 72 hours, pick a single colony and separate it by streaking on the corresponding plate, repeat this operation until The purified strain was obtained, namely the strain P5 of the present invention.

Embodiment 2 The preparation of bacterial strain P5 fermentation filtrate of the present invention

Pick a ring of the activated bacterial strain P5 of the present invention and insert it into the beef extract peptone liquid medium, cultivate it at 180r/min and 28°C for 48h to obtain the fermentation stock solution, shake it up, centrifuge at 10000r/min for 10min, and take the supernatant in Under sterile conditions, the fermentation filtrate was obtained by passing through a 0.22 μm sterile filter membrane.

Embodiment 3 antibacterial activity assay

Antagonism experiments were carried out using the plate confrontation method. Pour about 5ml of PDA medium into a 90mm-diameter petri dish, add 2ml of the above-mentioned fermentation filtrate after solidification, shake well, and then add 15ml of PDA medium. In the PDA medium of the control group, 2 ml of sterile water was added to replace the fermentation filtrate, and then a piece of pathogenic mycelium with a diameter of 3 mm was inserted on the plate, and cultured in the dark at 28°C for 72 hours, the diameter of the colony was measured, and the inhibition rate was calculated. Inhibition rate = (D _CK - D _experiment ) / D _CK × 100%, where D _experiment is the diameter of the bacterial block in the experimental group, and _{DC CK} is the diameter of the bacterial block in the control group. The results are shown in Table 1 below.

The confrontation test results show that the fermentation filtrate of bacterial strain P5 of the present invention has obvious inhibitory effect on seven kinds of phytopathogenic fungi tested, among which the antagonism to the mycelia growth of S. capsici is the strongest, and the inhibition rate is 48.57%. The antagonism ability of mycelial growth of P. early blight was the worst, but the inhibition rate still reached 21.74%.

The antagonism of table 1 bacterial strain P5 fermentation filtrate of the present invention to seven kinds of common plant pathogenic fungi

Claims (10)

1. An antagonistic endophyte Bacillus subtilis P5 strain, which was deposited in China Center for Type Culture Collection on March 18, 2016, with a preservation number of CCTCC NO: M2016131. 2. a method for preparing an antagonistic endophyte fermented liquid by a fermentation method, is characterized in that, the method is that the bacterial strain described in claim 1 is inserted in the beef extract peptone liquid medium for fermentation and cultivation, and the fermentation condition is: inoculum size 3% , Speed 150-180r/min, temperature 28-30℃, time 46-52h. 3. the fermented liquid that the described method of claim 2 makes. 4. the strain described in claim 1 or the application of fermented liquid described in claim 3 in suppressing capsicum anthracnose. 5. the strain described in claim 1 or the application of fermented liquid described in claim 3 in suppressing citrus penicillium. 6. the strain described in claim 1 or the application of fermented liquid described in claim 3 in suppressing Chinese cabbage black spot. 7. the strain described in claim 1 or the application of fermented liquid described in claim 3 in suppressing Sclerotinia sclerotiorum. 8. the strain described in claim 1 or the application of fermented liquid described in claim 3 in suppressing capsicum phytophthora. 9. the bacterial strain described in claim 1 or the application of fermented liquid described in claim 3 in suppressing capsicum candidum. 10. the strain described in claim 1 or the application of fermented liquid described in claim 3 in suppressing early blight of tomato.