Biocontrol pandoraea KBA33 inhibiting botryosphaeria dothidea and application thereof

Abstract

The application discloses a biocontrol pantoea agglomerans KBA33 for inhibiting botryosphaeria dothidea and application thereof. The strain KBA33 disclosed by the application can effectively inhibit botryosphaeria dothidea at 16 DEG C and 28 DEG C, and can inhibit botryosphaeria dothidea through cell-cell contact.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of agricultural biotechnology, and particularly relates to a biocontrol Pantoea vagans KBA33 for inhibiting Pseudomonas syringae pv. actinidiae and application thereof. BACKGROUND

[0002] Pseudomonas syringae pv. actinidiae is a kind of bacterial disease caused by Pseudomonas syringae pv. actinidiae, and its symptoms include flower rot, yellow halo leaf spot, brown wood of branches and stems, and white or brown bacterial pus flowing out of the wound. Therefore, it is necessary to find more effective prevention and control means.

[0003] Temperature has a significant impact on the disease, and the disease is serious when the average temperature in spring is 12-18℃. The biocontrol bacteria for Pseudomonas syringae pv. actinidiae are mainly Bacillus and Pseudomonas, but these existing biocontrol bacteria for Pseudomonas syringae pv. actinidiae are not targeted for the characteristics of Pseudomonas syringae pv. actinidiae at low temperature, that is, they have certain prevention and control effect at normal temperature, but poor prevention and control effect at the early and outbreak stages of the disease. At the same time, these strains also have the problem of poor prevention and control effect of living biocontrol bacteria. SUMMARY

[0004] The present application aims to provide a biocontrol Pantoea vagans KBA33 for inhibiting Pseudomonas syringae pv. actinidiae and application thereof according to the characteristics of Pseudomonas syringae pv. actinidiae.

[0005] The present application provides a Pantoea vagans strain KBA33, which was preserved in the China General Microbiological Culture Collection Center on September 6, 2023, and the address is No. 1, Yihuangyuan, Beichen West Road, Chaoyang District, Beijing, China, and the Institute of Microbiology, Chinese Academy of Sciences, and the strain preservation number is CGMCC NO. 28366.

[0006] The present application also provides a bacterial liquid containing the strain KBA33.

[0007] The bacterial liquid can be obtained by strain culture, and the bacterial concentration of the commonly used bacterial liquid is 1*10 7 cfu / mL-1*10 9 cfu / mL.

[0008] The present application also provides the use of the Pantoea vagans strain KBA33 or the bacterial liquid in inhibiting Pseudomonas syringae pv. actinidiae.

[0009] The strain KBA33 of the present application can inhibit Pseudomonas syringae pv. actinidiae through direct cell-to-cell contact. In specific use, the bacterial liquid containing the strain KBA33 can be directly sprayed on the leaves, flowers and branches with Pseudomonas syringae pv. actinidiae.

[0010] The application also provides the application of the Pantoea strain KBA33 or the bacterial liquid in preventing and treating the disease caused by Pseudomonas syringae pv. actinidiae.

[0011] In some embodiments, the disease caused by Pseudomonas syringae pv. actinidiae is kiwifruit canker.

[0012] Compared with the prior art, the application has the following beneficial effects:

[0013] (1) The strain KBA33 can effectively inhibit Pseudomonas syringae pv. actinidiae at both 16℃ and 28℃, which can effectively ensure the control effect in the early and outbreak stages of the disease.

[0014] (2) The biocontrol strain KBA33 can inhibit Pseudomonas syringae pv. actinidiae through cell-cell contact, which makes up for the deficiency of most common biocontrol bacteria which rely on secreted antibacterial substances to kill bacteria, and the expression of the antibacterial substance synthesis gene cluster is easily disturbed by temperature and other environmental factors, resulting in extremely low antibacterial substance yield and poor control effect. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 . is a result graph of the strain KBA33 inhibiting the growth of Pseudomonas syringae pv. actinidiae through direct cell-cell contact;

[0016] Figure 2 . is a result graph of the strain KBA33 failing to inhibit Pseudomonas syringae pv. actinidiae after being separated by a filter membrane;

[0017] Figure 3 . is a control effect graph of the strain KBA33 on kiwifruit branches;

[0018] Figure 4 . is a phylogenetic tree of the strain KBA33 constructed by multiple sites in series. DETAILED DESCRIPTION

[0019] The application will be further described below in combination with specific examples, and the application examples are not limited thereto, and should not be understood as limiting the scope of the application. It should be pointed out that any modification, equivalent replacement and improvement made within the spirit and principle of the application should be included in the protection scope of the application.

[0020] Unless otherwise specified, the Pseudomonas syringae pv. actinidiae strain C48 used in the following examples is provided by the team of Zhong Caihong of the Kiwifruit Institute of the Wuhan Botanical Garden of the Chinese Academy of Sciences, and is now preserved in the present research group, and the applicant promises to permanently open to the public.

[0021] The LB medium components are as follows: 10 g of tryptone, 5 g of yeast extract, 10 g of sodium chloride, 15 g of agar powder, and pure water to 1 L. The solid medium is added with 15 g / L of agar powder.

[0022] Example 1

[0023] The strain KBA33 is isolated from the xylem of a healthy branch of the wild kiwi long spindle early maturing superior line 3-0-2-1. A wound is caused by cutting from the surface of the branch to the inside, exposing the xylem of the branch. A sterilized LB medium agar block is placed on the wound of the kiwi branch, and the cutting site is wrapped with plastic wrap and placed in a 16°C constant temperature incubator for enrichment for 7-15 days. Then the agar block is taken out, ground and crushed in sterile water, and then 10-fold gradient dilution is performed and plated on LB medium plates, and cultured at 16°C for 16-24 hours. Different single colony strains are picked and purified. After picking single colonies and purifying by streaking for three times in succession, they are stored for use.

[0024] The single colonies of the purified strain are picked from the LB medium plates, inoculated into 3 ml of liquid LB medium, and cultured at 220 rpm and 28°C for 12 hours to obtain a seed solution. The seed solution is inoculated into 20 ml of fresh liquid LB medium at a ratio of 1:100 (v / v) and cultured for 5 hours. The absorbance value at 600 nm is detected by a spectrophotometer, and the OD 600 = 1.5. The biocontrol strain KBA33 is subjected to phylogenetic analysis. According to the 16S rDNA sequence alignment results, primers are designed for five housekeeping genes, fusA (SEQ ID NO. 2), leuS (SEQ ID NO. 3), pyrG (SEQ ID NO. 4), rlpB (SEQ ID NO. 5), and rpoB (SEQ ID NO. 6), for multi-gene concatenation phylogenetic tree construction. The KBA33 strain genomic DNA is used as a template, and the fragments amplified by the corresponding primers are used.

[0025] Table 1. Primers used in this patent.

[0026]

[0027] The amplification system of 16S rDNA (SEQ ID NO. 1) of strain KBA33 and the sequences of the remaining 5 housekeeping genes is as follows: 2x Phnanta Flash Master Mix (Dye Plus) 25 μl, forward primer (10 μM) 2 μl, reverse primer (10 μM) μl, DNA template 1 μl, double distilled water 20 μl. The PCR program is: 98℃, 30s; [95℃, 10s; 55℃, 5s; 72℃, 5s]x30 cycles; 72℃, 1s. After the PCR product is verified by agarose gel, it is sent to a sequencing company for sequencing.

[0028] The corresponding reference sequences are obtained from NCBI. The sequences of the 5 housekeeping genes are concatenated, and a phylogenetic tree is constructed using MEGA software and the neighbor-joining method. According to the constructed phylogenetic tree, it is determined that strain KBA33 belongs to Pantoea vagans (Pantoea vagans). Figure 4 On September 6, 2023, it was preserved in the China General Microbiological Culture Collection Center, located at No. 3, Beichen West Road, Haidian District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with the strain preservation number CGMCC NO. 28366.

[0029] The red fluorescent protein (RFP) labeled biocontrol bacteria KBA33 were activated overnight in a 28℃ constant temperature incubator on LB agar medium plates, and single colonies were picked and cultured in liquid LB medium in a 28℃ constant temperature shaker overnight. The next day, the concentration of the overnight culture was adjusted to OD 600 = 1.0 with sterile water after washing away the culture medium. This is the biocontrol bacteria KBA33 liquid.

[0030] Example 2

[0031] The green fluorescent protein (GFP) labeled kiwifruit canker bacteria were used as an indicator. The green fluorescent protein labeled kiwifruit canker bacteria C48 strain were activated on LB agar medium plates, and single colonies were picked and cultured in liquid LB medium in a 28℃ constant temperature shaker overnight. The next day, the concentration of the overnight culture was adjusted to OD 600= 1.0. The biocontrol bacteria KBA33 prepared in Example 1 was mixed with the green fluorescent labeled Psa in equal volume, and the biocontrol bacteria or the pathogenic bacteria mixed with sterile water in equal volume was used as a control. The mixture was dropped on the surface of LB solid medium (10 g of tryptone, 5 g of yeast extract, 10 g of sodium chloride, 15 g of agar powder, and pure water to 1 L, sterilized at 121℃) to provide cell-to-cell contact conditions for the biocontrol bacteria and Psa, and was placed at 16℃ and 28℃ for co-culture. After 24-48 hours, whether the green fluorescent signal existed was observed under a fluorescence stereomicroscope to determine whether the pathogenic bacteria survived.

[0032] After 24 and 48 hours, the fluorescent signals of the co-cultured colonies at 28℃ and 16℃ were observed, and the results are shown in Table 1. Figure 1 As shown in Table 1, only the red fluorescent signal carried by the biocontrol bacteria KBA33 remained, and the green fluorescent signal carried by Psa did not exist, indicating that the biocontrol bacteria KBA33 killed Psa.

[0033] The melted LB solid medium was introduced into a culture dish, and a sterile filter membrane with a pore size of 0.22 μm cut to an appropriate size was inserted into the medium. The filter membrane was fixed in the solid medium plate with sterilized tweezers. The biocontrol bacteria KBA33 prepared in Example 1 and the green fluorescent labeled Psa were dropped on both sides of the filter membrane, and 5 μl of the bacteria liquid was used as a control. After the liquid evaporated, the culture dish was placed in a 28℃ incubator for 24 hours. The luminescence was observed and recorded by taking pictures under a fluorescence stereomicroscope. If the green fluorescent signal carried by Psa existed, it indicated that the biocontrol bacteria KBA33 failed to kill Psa, and the results are shown in Table 2. Figure 2 As shown in Table 2, Psa was killed in the mixed culture liquid, but Psa placed on both sides of the filter membrane was not killed. The contact-dependent killing experiment of the filter membrane isolation proved that KBA33 killed Psa strains through cell-to-cell direct contact, rather than through the production of diffusible antibacterial substances.

[0034] Compared with the secretion of antibacterial substances, the KBA33 strain of the present application can exert stable prevention effect through cell-to-cell direct contact killing under the low-temperature pathogenic environment of Psa. Moreover, studies have shown that the common contact killing system (such as type IV and type VI secretion systems) of biocontrol bacteria functions stably under various environmental conditions. Therefore, the KBA33 strain of the present application makes up for the deficiency of most common biocontrol bacteria that rely on the secretion of antibacterial substances for killing, which is prone to be disturbed by environmental factors such as temperature, resulting in extremely low yield of antibacterial substances and poor prevention effect.

[0035] Example 3

[0036] The current year's healthy branches of kiwifruit were collected, disinfected with 4% NaClO3 for 20 min, washed with sterile water for 3 times, and dried. The branches were cut into 15 cm short branches, and two cuts were made respectively. 5 μl of the biocontrol bacteria KBA33 liquid prepared in Example 1 was inoculated into the cuts, and sterile water was used as a negative control. The treated kiwifruit branches were placed in an artificial climate incubator (16 h light, 8 h darkness, 16℃), after 7 days, the same concentration of P. ribis was inoculated into the cuts containing biocontrol bacteria and water control, and one group of negative controls were not inoculated with biocontrol bacteria and pathogenic bacteria. Based on the above artificial climate incubator, the same conditions were continued for 42 days, the branch bark was cut open to expose the xylem lesions, the results were recorded by taking pictures, and the lesion length was measured.

[0037] The results are shown in Figure 3 and Table 2, the biocontrol bacteria KBA33 co-inoculated with P. ribis treatment, compared with only inoculated with pathogenic bacteria treatment, the lesion length on the kiwifruit branches was significantly shortened, the average control effect was 59.61%, it can be seen that the biocontrol bacteria KBA33 described in the present patent has good control effect on the kiwifruit canker caused by P. ribis.

[0038] The specific calculation formula of the control effect is as follows:

[0039] Control effect % = [(control lesion length - treatment lesion length) / control lesion length] x 100

[0040] Table 2. Data statistics of control effect results.

[0041]

Claims

1. A pantothecin strain, KBA33, is... Pantoea vagans The accession number is CGMCC NO.28366.

2. The pantothecin strain KBA33 according to claim 1 inhibits the growth of *Actinidia cuspidatum*, the pathogen causing kiwifruit canker. Pseudomonas syringae pv. actinidiae Applications in [the context of the text].

3. The pantothecin strain KBA33 according to claim 1 in controlling kiwifruit canker pathogens. Pseudomonas syringae pv. actinidiae Applications in the treatment of diseases caused by it.

4. The application according to claim 3, characterized in that, The disease caused by the kiwifruit canker pathogen is kiwifruit canker.

5. A bacterial suspension containing the pantothecin strain KBA33 as described in claim 1.

6. The bacterial solution according to claim 5, characterized in that, The bacterial cell concentration of the bacterial solution is 1*10 7 cfu / mL ~ 1*10 9 cfu / mL.

7. The bacterial solution according to claim 5 or 6 in inhibiting the pathogenic bacteria of kiwifruit canker. Pseudomonas syringae pv. actinidiae Applications in [the context of the text].

8. The bacterial solution according to claim 5 or 6 in the prevention and control of kiwifruit canker pathogens. Pseudomonas syringae pv. actinidiae Applications in the treatment of diseases caused by it.

9. The application according to claim 8, characterized in that, The aforementioned kiwifruit ulcer pathogen Pseudomonas syringae pv. actinidiae The disease caused is kiwifruit canker.

10. The application according to any one of claims 7 to 9, characterized in that, Specifically, the bacterial solution described in claim 5 or 6 is sprayed onto areas infected with kiwifruit canker pathogens. Pseudomonas syringae pv. actinidiae The part.

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