Delftia palustris and application thereof in prevention and treatment of plant diseases

Abstract

The invention discloses Delftia tsuhuensis and an application of the Delftia tsuhuensis in prevention and treatment of plant diseases. The Delftia tsuhuensis CDJ2 is preserved in the Guangdong Microbial Culture Collection Center on October 18, 2024, and the preservation number is GDMCC (China General Microbiological Culture Collection Center) NO: 65290. The strain has a good prevention and treatment effect on citrus canker, and the prevention and treatment effect can reach 84%. Meanwhile, the strain can also effectively prevent and treat soft rot of taro, bacterial basal rot of rice, bacterial wilt of crops, kiwifruit canker, angular leaf spot of mango, black rot of brassicaceous vegetables, tomato scab, gummosis of sugarcane, bacterial wilt of walnuts and bacterial leaf spot of plums. Wide application prospects are realized in the aspects of preventing and treating diseases of crops and commercial crops and promoting healthy growth of plants.

Description

Technical Field

[0001] The present invention relates to the technical field of plant disease control, and specifically, to a Delftia tsuruhatensis and its application in controlling plant diseases. Background Art

[0002] Plant diseases are the limiting factors for the yields of major crops and affect the product quality of important cash crops. Solving the problem of plant diseases is one of the prerequisites for agricultural development. Microorganisms such as Fusarium oxysporum and Rhizoctonia solani are one of the main pathogens that cause significant losses in crop yields, and the control of these plant pathogens mainly relies on chemical pesticides. However, the extensive use of chemical pesticides has gradually led to the development of drug resistance in plant pathogens, resulting in a gradual increase in the application rate of pesticides, increasing the control cost. In addition, the excessive use of chemical pesticides will also damage the ecological environment and increase the risk of phytotoxicity in plants. In recent years, biological control technology has shown its unique advantages in controlling plant diseases due to its advantages such as low toxicity, low residue, and pollution-free.

[0003] Currently, more than 1,000 species of biocontrol microorganisms have been discovered, mainly including biocontrol bacteria, biocontrol actinomycetes, and biocontrol fungi. The genus Delftia is a new genus established in 1999, belonging to the class β-Proteobacteria, Comamonadaceae, and has certain biocontrol potential. Chinese invention patent CN116445369A discloses that Delftia HW1 has the function of controlling citrus canker, kiwifruit soft rot, banana wilt, ponkan blue mold, mango anthracnose, and root rot. However, there are a wide variety of plant diseases, and it is still necessary to find a biocontrol bacterium that can control more disease types, so as to improve plant disease resistance and promote healthy plant growth. Summary of the Invention

[0004] To overcome the above-mentioned defects and deficiencies in the prior art, the present invention provides a Delftia tsuruhatensis and its application in controlling plant diseases.

[0005] The first object of the present invention is to provide a Delftia tsuruhatensis CDJ2.

[0006] The second object of the present invention is to provide the application of the above-mentioned Delftia tsuruhatensis CDJ2 in treating and / or preventing plant diseases.

[0007] The third object of the present invention is to provide the use of the above-mentioned Delftia tsuruhatensis CDJ2 in the preparation of a biological agent for treating and / or preventing plant diseases.

[0008] The fourth object of the present invention is to provide the use of the above-mentioned Delftia tsuruhatensis CDJ2 in the treatment and / or prevention of plant diseases caused by Xanthomonas.

[0009] The fifth object of the present invention is to provide the use of the above-mentioned Delftia tsuruhatensis CDJ2 in the preparation of a biological agent for treating and / or preventing plant diseases caused by Xanthomonas.

[0010] The sixth object of the present invention is to provide the use of the above-mentioned Delftia tsuruhatensis CDJ2 in the preparation of a fungicide.

[0011] The seventh object of the present invention is to provide a biocontrol agent.

[0012] The eighth object of the present invention is to provide a method for treating and / or preventing plant diseases.

[0013] The present invention claims the following:

[0014] A Delftia tsuruhatensis CDJ2, which was deposited at the Guangdong Provincial Microbial Culture Collection Center on October 18, 2024, with the deposit number GDMCC NO: 65290.

[0015] The use of the above-mentioned Delftia tsuruhatensis CDJ2 in the treatment and / or prevention of plant diseases, where the plant diseases are one or more of soft rot of taro, bacterial foot rot of rice, bacterial wilt of crops, citrus canker, kiwifruit canker, mango angular leaf spot, black rot of cruciferous vegetables, tomato scab, gummosis of sugarcane, bacterial wilt of walnut, and bacterial leaf spot of plum.

[0016] The use of the above-mentioned Delftia tsuruhatensis CDJ2 in the preparation of a biological agent for treating and / or preventing plant diseases, where the plant diseases are one or more of soft rot of taro, bacterial foot rot of rice, bacterial wilt of crops, citrus canker, kiwifruit canker, mango angular leaf spot, black rot of cruciferous vegetables, tomato scab, gummosis of sugarcane, bacterial wilt of walnut, and bacterial leaf spot of plum.

[0017] Preferably, the soft rot of taro is caused by Pectobacterium colocasium and / or Dickeya fangzhongdai;

[0018] The bacterial foot rot of rice is caused by Dickeya oryzae;

[0019] The bacterial wilt of crops is caused by Ralstonia solanacearum;

[0020] The citrus canker is caused by Xanthomonas citri pv. citri;

[0021] The kiwifruit canker is caused by Pseudomonas syringae pv. actinidiae;

[0022] The mango angular leaf spot is caused by Xanthomonas citri pv. mangiferaeindicae;

[0023] The black rot of cruciferous vegetables is caused by Xanthomonas campestris pv. campestris;

[0024] The tomato scab is caused by Xanthomonas perforans;

[0025] The gummosis of sugarcane is caused by Xanthomonas axonopodis;

[0026] The bacterial blight of walnut is caused by Xanthomonas euroxanthea;

[0027] The bacterial leaf spot of plum is caused by Xanthomonas arboricola pv. pruni.

[0028] Use of the above-mentioned Delftia tsuruhatensis CDJ2 in the treatment and / or prevention of plant diseases caused by Xanthomonas.

[0029] Use of the above-mentioned Delftia tsuruhatensis CDJ2 in the preparation of a biological agent for the treatment and / or prevention of plant diseases caused by Xanthomonas.

[0030] Preferably, the Xanthomonas includes one or more of Xanthomonas citri pv. citri, Xanthomonas citri pv. mangiferaeindicae, Xanthomonas campestris pv. campestris, Xanthomonas perforans, Xanthomonas axonopodis, Xanthomonas euroxanthea, and Xanthomonas arboricola pv. pruni.

[0031] Use of the above-mentioned Delftia tsuruhatensis CDJ2 in the preparation of a fungicide, the fungicide being a fungicide for killing one or more of Pectobacterium colocasium, Dickeya fangzhongdai, Dickeya oryzae, Ralstonia solanacearum, Pseudomonas syringae pv. actinidiae, Xanthomonas citri pv. citri, Xanthomonas citri pv. mangiferaeindicae, Xanthomonas campestris pv. campestris, Xanthomonas perforans, Xanthomonas axonopodis, Xanthomonas euroxanthea, and Xanthomonas arboricola pv. pruni.

[0032] A biocontrol agent, comprising the above-mentioned Delftia tsuruhatensis CDJ2.

[0033] A method for treating and / or preventing plant diseases, by applying the above-mentioned Delftia tsuruhatensis CDJ2 and / or the biocontrol agent to plants.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] The present invention discloses a Delftia tsuruhatensis and its application in preventing and controlling plant diseases. The Delftia tsuruhatensis CDJ2 was deposited at the Guangdong Provincial Culture Collection of Microorganisms on October 18, 2024, with the deposit number GDMCC NO: 65290 and the deposit address being the 5th floor of Building 59, 100th Yard, Xianlie Middle Road, Guangzhou. This strain has a good control effect on citrus canker, and the control effect can reach 84%. At the same time, this strain can also effectively control soft rot of taro, bacterial foot rot of rice, bacterial wilt of crops, kiwifruit canker, mango angular leaf spot, black rot of cruciferous vegetables, tomato scab, gummosis of sugarcane, bacterial wilt of walnut, and bacterial leaf spot of plum. The microbial inoculum prepared from the Delftia tsuruhatensis CDJ2 of the present invention is environmentally friendly and will not cause plants to develop drug resistance, and has broad application prospects in the prevention and control of diseases of agricultural and cash crops and promoting the healthy growth of plants. Description of the Drawings

[0036] Figure 1 It is the colony morphology of the CDJ2 strain on the LB medium.

[0037] Figure 2 It is the Neighbor-Joining phylogenetic tree constructed by the CDJ2 strain based on the 16S rRNA gene.

[0038] Figure 3 It is the determination result of the antibacterial spectrum of Delftia tsuruhatensis CDJ2 against plant pathogens.

[0039] Figure 4 It is the control effect diagram of Delftia tsuruhatensis CDJ2 on citrus canker in detached leaves; a: control group, b: prevention group, c: treatment group. Detailed Embodiments

[0040] The following specific embodiments are used to further illustrate the present invention, but the embodiments do not impose any form of limitation on the present invention. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field.

[0041] Unless otherwise specified, the reagents and materials used in the following embodiments are all commercially available.

[0042] LB liquid medium: 10 g of peptone, 5 g of yeast extract, 10 g of sodium chloride, 1000 mL of water, pH = 7.0; sterilized at 121 °C for 20 min.

[0043] LB solid medium: 10 g of peptone, 5 g of yeast extract, 10 g of sodium chloride, 1000 mL of water, 15 g of agar, pH = 7.0; sterilized at 121 °C for 20 min.

[0044] Screening of biocontrol strains in Example 1

[0045] 1. Leaf source

[0046] Citrus leaves collected from Zengcheng District, Guangzhou City, Guangdong Province on January 9, 2023.

[0047] 2. Sample treatment

[0048] (1) Leaf collection

[0049] Collect leaves in the citrus orchard, 5 samples from each tree. Collect 3 trees from each plot, and record the sampling time, location and type. The collected samples are stored at room temperature for bacterial isolation.

[0050] (2) Isolation of bacteria

[0051] Plate dilution method: Weigh 3 g of leaves into a 50 mL centrifuge tube, surface disinfect with 1% sodium hypochlorite solution (v / v) and 75% ethanol solution (v / v), then add 27 mL of sterile water, and then shake in a shaker at 200 rpm and 28 °C. After 20 h, take it out and let it stand at room temperature for 10 min to prepare the original leaf bacterial suspension. Gradient dilute this original solution to obtain 6 gradient dilutions of 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 respectively. Take 100 μL of each leaf bacterial suspension dilution and spread it evenly on LB and R2A plates, with 3 replicates for each gradient. Place the plates in an incubator at 28 °C and incubate for 48 h, then observe.

[0052] 3. Bacterial purification

[0053] Observe and pick out single colonies, streak purify on LB and R2A plates, incubate in an inverted position in an incubator at 28 °C, pick out single colonies after 12 h, and number them in sequence.

[0054] 4. Bacterial preservation

[0055] Inoculate the single colonies of the strains into LB and R2A liquid media, culture in a shaker at 28 °C and 180 rpm for 12 h, then pipette 1 mL of the bacterial liquid and mix it with 0.5 mL of 60% sterile glycerol solution (v / v), gently shake and mix well, and store at -80 °C for long-term preservation.

[0056] 5. Strain screening

[0057] The plate bacteriostatic circle method was used to screen biocontrol bacteria, and the specific operation is as follows:

[0058] Xanthomonas citri pv. citri and candidate biocontrol strains were activated on LB plates, and then inoculated into liquid LB medium and cultured until the OD 600 value reached 1.0. LB medium was used as the screening medium. 49 mL of well-dissolved LB medium was added to a 50 mL centrifuge tube. When the temperature of the medium dropped to about 50 °C, the Xanthomonas citri pv. citri bacterial solution was fully mixed with the LB medium at a volume ratio of 1:49 and poured into a square dish (13 cm × 13 cm) and flattened. After cooling and solidifying, holes were punched in the medium using a 5 mm puncher and the agar blocks were picked out with a sterile toothpick. 10 μL of the candidate bacterial solution was added to the holes, and after the bacterial solution was dried, the plate was sealed and cultured upside down in an incubator at 28 °C. After culturing for 24 h, observe whether there is a transparent bacteriostatic circle, and take pictures and record the results.

[0059] Through the above steps, primary screening and re-screening were carried out to obtain a strain with the best bacteriostatic effect, named CDJ2. The colony morphology of strain CDJ2 is as Figure 1 shown. By observing the strain morphology, it was found that strain CDJ2 grew faster on LB medium, and obvious colonies could be seen in about 24 h in an incubator at 28 °C. Strain CDJ2 was white and opaque on LB medium, approximately circular, with a rough and dry surface, irregular and blurred edges, and had an obvious peculiar smell after 24 h of fermentation culture of the bacterial solution.

[0060] Example 2 Identification of the 16S rRNA gene of the biocontrol strain

[0061] I. Experimental method

[0062] A small amount of fresh colonies were picked with a pipette tip as a template, and PCR amplification was carried out using the bacterial 16S rRNA gene primers 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO: 2) and 1492R: 5'-TACGGCTACCTTGTTACGACTT-3' (SEQ ID NO: 3) to obtain the target fragment. The PCR reaction system was: 2×Taq PCR Mix 12.5 μL, upstream primer (10 μM) 0.5 μL, downstream primer (10 μM) 0.5 μL, sterile water 9.5 μL, and fresh colonies.

[0063] The PCR reaction conditions were as follows: 95°C pre-denaturation for 3 min; 95°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 60 s, 34 cycles; 72°C pre-extension for 10 min, and storage at 16°C. After the reaction, the PCR products were tested by 1% agarose gel electrophoresis. The PCR stock solution was sent to Bio-Tech for sequencing. The sequencing results were compared by BLAST on the NCBI website, and a phylogenetic tree was constructed using MEGA 11 software to determine the species of closely related bacterial strains.

[0064] 2. Experimental Results

[0065] The nucleotide sequence of the 16S rRNA gene of the CDJ2 strain is shown in SEQ ID NO: 1. The phylogenetic tree was constructed by neighbor-joining analysis. The results showed that the 16S rRNA gene of the CDJ2 strain had a similarity of 99.86% with that of Delftia tsuruhatensis and was in the same evolutionary tree branch as Delftia tsuruhatensis ( Figure 2 ), so the CDJ2 strain belongs to Delftia tsuruhada.

[0066] The CDJ2 strain was deposited in the Guangdong Provincial Microbiological Culture Collection Center with the deposit number GDMCC NO: 65290. The deposit address is 5th Floor, Building 59, 100 Courtyard, Xianlie Middle Road, Guangzhou. The preservation date is October 18, 2024. The classification name is Delftia tsuruhatensis.

[0067] Example 3 Determination of the antibacterial spectrum of Delftia tsuruhada CDJ2

[0068] 1. Experimental Methods

[0069] Single colonies of plant pathogens (Table 1) were picked up in LB medium, and single colonies of Delftia tsuruhada CDJ2 (GDMCC NO: 65290) of Example 2 were picked up in LB medium, and cultured in a shaking incubator at 28°C and 180 rpm for 12 h. The concentration of the plant pathogen solution was adjusted to OD 600 = 1, the concentration of Delftia tsuruhada CDJ2 bacterial solution is OD 600 = 1. Pipette 1 mL of plant pathogenic bacteria solution and about 49 mL of LB solid medium and mix thoroughly, then pour the solution into a plate, and then punch a hole with a diameter of 8 mm in the center of the LB plate, and inoculate 5 μL of Delftia tsuruhada CDJ2 solution into the hole. Repeat three times and place in a 28°C incubator. After culturing for 48 hours, observe the presence of an inhibition zone, record the size of the inhibition zone, and take photos.

[0070] Table 1 Plant pathogens

[0071]

[0072] Note: (1) The citrus canker pathogen was collected by our laboratory and identified as Xanthomonas citri pv. citri.

[0073] (2) Dickeya oryzeae EC1 was disclosed in PMID: 30336787.

[0074] (3) Dickeya fangzhongdai CL3 was disclosed in PMID: 35273588.

[0075] (4) Ralstonia solanacearum EP1 was disclosed in PMID: 27833603.

[0076] II. Experimental Results

[0077] The results of the antibacterial spectrum determination of Delftia tsuruhatensis CDJ2 are shown in Figure 3 Table 1 and Table 2. The results show that Delftia tsuruhatensis CDJ2 has significant antagonistic effects against the pathogens of taro soft rot, rice bacterial foot rot, crop bacterial wilt, citrus canker, kiwifruit canker, mango angular leaf spot, cruciferous vegetable black rot, tomato scab, sugarcane gummosis, walnut bacterial wilt, and plum bacterial leaf spot.

[0078] Table 2 The diameter of the antibacterial circle of Delftia tsuruhatensis CDJ2 against plant pathogens

[0079] Strain Host disease Diameter of inhibition zone (cm) Pectobacterium colocasium Soft rot of taro 1.3±0.3 Dickeya fangzhongdai CL3 Soft rot of taro 1.8±0.2 Dickeya fangzhongdai ZXC1 Soft rot of taro 1.2±0.1 Dickeya oryzae EC1 Bacterial foot rot of rice 1.5±0.1 Ralstonia solanacearum Bacterial wilt of crops 1.0±0.1 Xanthomonas citri pv.citri Citrus canker 1.4±0.1 Pseudomonas syringae pv.actinidiae Kiwi canker 3.2±0.1 Xanthomonas citri pv.mangiferaeindicae Mango angular leaf spot 1.3±0.1 Xanthomonas campestris pv.campestris XC1 Black rot of cruciferous vegetables 1.67±0.17 Xanthomonas perforans TC2-1 Tomato scab 1.16±0.06 Xanthomonas axonopodis Gummosis of sugarcane 1.6±0.2 Xanthomonas euroxanthea Bacterial blight of walnut 1.27±0.07 Xanthomonas arboricola pv.pruni Xap Bacterial leaf spot of plum 1.93±0.13

[0080] Example 4 Determination of the control effect of Delftia tsuruhatensis CDJ2 on citrus canker in detached leaves

[0081] I. Experimental Method

[0082] Pick a single colony of the citrus canker pathogen Xanthomonas citri pv. citri into LB medium, and at the same time pick a single colony of Delftia tsuruhatensis CDJ2 (GDMCC NO: 65290) into LB medium. After culturing in a shaker at 28 °C and 180 rpm for 24 h, adjust the concentration of the citrus canker pathogen Xanthomonas citri pv. citri bacterial solution to OD 600 = 1, and the concentration of the Delftia tsuruhatensis CDJ2 bacterial solution to OD 600 = 1.

[0083] Control group: Select the leaves of living plants with consistent growth status, prick wounds with a needle, inoculate the pathogen of citrus canker, Xanthomonas citri pv. citri, by spraying, with 3 replicates, bag the plants, and observe the disease incidence of the leaves (canker spots, yellow halos) after growing for 14 days, take pictures and record.

[0084] Prevention group: Select the leaves of living plants with consistent growth status, prick wounds with a needle, inoculate Delftia tsuruhatensis CDJ2 by spraying, with 3 replicates, bag the plants overnight, and 24 hours later, inoculate the pathogen of citrus canker, Xanthomonas citri pv. citri, by spraying. Observe the disease incidence of the leaves (canker spots, yellow halos) after growing for 14 days, take pictures and record.

[0085] Treatment group: Select the leaves of living plants with consistent growth status, prick wounds with a needle, inoculate the pathogen of citrus canker, Xanthomonas citri pv. citri, by spraying, with 3 replicates, bag the plants overnight, and 24 hours later, inoculate Delftia tsuruhatensis CDJ2 by spraying. Observe the disease incidence of the leaves (canker spots, yellow halos) after growing for 13 days, take pictures and record.

[0086] The calculation formula for the control effect is as follows: (the number of diseased wounds in the control group - the number of diseased wounds in the treatment group) / the number of diseased wounds in the control group × 100%.

[0087] II. Experimental results

[0088] The schematic diagram of the control effect of Delftia tsuruhatensis CDJ2 on citrus canker is as Figure 4 shown. The results show that Delftia tsuruhatensis CDJ2 has a good control effect on citrus canker, and the control effect reaches 84%.

[0089] The above embodiments are the preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A Delftia tsuruhatensis CDJ2, characterized in that It was deposited in Guangdong Provincial Microbiological Culture Collection Center on October 18, 2024, with the deposit number GDMCC NO: 65290.

2. The use of Delftia tsuruhatensis CDJ2 according to claim 1 in treating and / or preventing plant diseases, characterized in that: The plant diseases are one or more of taro soft rot, rice bacterial basal rot, crop bacterial wilt, citrus canker, kiwi canker, mango angular spot, cruciferous vegetable black rot, tomato scab, sugarcane gummosis, walnut bacterial wilt and plum bacterial spot.

3. The use of Delftia tsuruhatensis CDJ2 according to claim 1 in the preparation of a biological agent for treating and / or preventing plant diseases, characterized in that: The plant diseases are one or more of taro soft rot, rice bacterial basal rot, crop bacterial wilt, citrus canker, kiwi canker, mango angular spot, cruciferous vegetable black rot, tomato scab, sugarcane gummosis, walnut bacterial wilt and plum bacterial spot.

4. The use according to claim 2 or 3, characterized in that: The taro soft rot is caused by Pectobacterium colocasium and / or Dickeya fangzhongdai; The rice bacterial basal rot is caused by Dickeya oryzae; The crop bacterial wilt is caused by Ralstonia solanacearum; The citrus canker is caused by Xanthomonas citri pv. citri; The kiwifruit canker is caused by Pseudomonas syringaepv.actinidiae; The mango angular spot disease is caused by Xanthomonas citripv. mangiferaeindicae; The black rot of cruciferous vegetables is caused by cruciferous black rot fungus (Xanthomonas campestrispv. campestris); The tomato scab disease is caused by Xanthomonas perforans; The sugarcane gummosis is caused by Xanthomonas axonopodis; The walnut bacterial wilt is caused by Xanthomonas euroxanthea; The plum bacterial spot disease is caused by Xanthomonas arborifolia pruni (Xanthomonas arborifolia pruni).

5. Use of the Delftia tsuruhatensis CDJ2 according to claim 1 in treating and / or preventing plant diseases caused by Xanthomonas.

6. Use of the Delftia tsuruhatensis CDJ2 according to claim 1 in the preparation of biological preparations for treating and / or preventing plant diseases caused by Xanthomonas.

7. The use according to claim 5 or 6, characterized in that: The xanthomonas include one or more of Xanthomonas citri pv. citri, Xanthomonas citripv. mangiferaeindicae, Xanthomonas campestris pv. campestris, Xanthomonas perforans, Xanthomonas axonopodis, Xanthomonas euroxanthea and Xanthomonas arboricolapv. pruni.

8. The use of Delftia tsuruhatensis CDJ2 according to claim 1 in the preparation of a bactericide, characterized in that: The fungicide is used to kill Pectobacterium colocasium, Dickeya fangzhongdai, Dickeya oryzae, Ralstonia solanacearum, Pseudomonas syringae pv. actinidiae, Xanthomonas citri pv. citri, Xanthomonas citri pv. mangiferae indicae, Xanthomonas campestris pv. campestris, Xanthomonas perforans, Xanthomonas axonopodis, Xanthomonas euroxanthea and Xanthomonas arboricola pv.pruni) one or more fungicides.

9. A biocontrol agent, characterized in that: The method comprises the Delftia tsuruhatensis CDJ2 according to claim 1.

10. A method for treating and / or preventing plant diseases, characterized in that: The Delftia tsuruhatensis CDJ2 of claim 1 and / or the biocontrol agent of claim 9 are applied to plants.

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