A new strain of Xanthomonas aeruginosa and its application in the prevention and treatment of citrus canker

Abstract

The present invention discloses a novel yellow bacterium and its application in preventing and treating citrus canker. The strain is a potential new species of the genus Luteibacter, named Luteibacter sp. F1009. The strain has been deposited in the Guangdong Provincial Microbial Culture Collection Center on May 24, 2023, with a deposit number of GDMCC No: 63499. The novel yellow bacterium F1009 of the present invention can effectively inhibit the growth of citrus canker pathogens, and can also produce siderophores and indoleacetic acid. Therefore, the novel yellow bacterium F1009 of the present invention has great application potential in preventing and treating citrus canker and promoting plant iron absorption and growth.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, in particular to a novel yellow bacterium and application thereof in preventing and treating citrus canker. Background Art

[0002] Citrus canker disease (Citrus canker disease), a bacterial disease caused by Xanthomonas citri subsp. citri (Xcc), has been designated a priority quarantine disease in over 30 countries and regions. Citrus canker damages citrus leaves, branches, and fruit, typically forming crater-like lesions on their surfaces. In mild cases, this affects the appearance of the tree and fruit, while in severe cases, it can cause massive leaf and fruit drop, even branch dieback, severely impacting citrus quality and yield. Citrus canker disease is widely transmitted, spreads rapidly, and is difficult to control, causing significant economic losses to the citrus industry, particularly oranges and grapefruits.

[0003] Currently, the prevention and control strategy for citrus canker is primarily chemical, supplemented by agricultural control. Compared to agricultural control, chemical control is the most widely used method in citrus production. Copper preparations, with their significant bactericidal efficacy and long-lasting effectiveness, are the preferred agents for the prevention and control of citrus canker. However, these copper-containing pesticides pose significant environmental risks. Long-term use can lead to copper accumulation in the soil, causing heavy metal pollution. They can also increase the emergence of copper-resistant strains of Xcc, leading to a vicious cycle of frequent spraying without effective control. More importantly, excessive use of copper preparations can render citrus fruit toxic, and copper can threaten human health and safety through multiple pathways. Therefore, the search for greener, more natural, and more effective agents for the prevention and control of citrus canker is an urgent and highly significant task.

[0004] Compared to chemical control, which can easily pollute the environment and disrupt the ecological balance, the use of microorganisms to control citrus canker offers advantages such as high efficiency, environmental friendliness, and resistance resistance. Currently, researchers have isolated a large number of biocontrol microorganisms with highly effective antagonistic activity against Xcc from citrus leaves, fruit, rhizosphere, and surrounding growth environments. Citrus canker biocontrol microorganisms can be classified into biocontrol bacteria, biocontrol fungi, and biocontrol phages based on taxonomic differences. Among these biocontrol bacteria, the most extensively studied, classified, and widely used groups are Bacillus, Pseudomonas, and Actinomycetes. These biocontrol bacteria control citrus canker through a variety of mechanisms, including the production of active substances, induction and activation of the citrus immune system, and competition with Xcc for nutrients and ecological niches. However, there are currently no reports on the use of Luteibacter bacteria for the control of citrus canker. Summary of the Invention

[0005] The first objective of the present invention is to overcome the shortcomings and deficiencies of the prior art and provide a potential new species of the genus Luteibacter, Luteibacter sp. F1009. This strain has been deposited with the Guangdong Provincial Microbiological Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100, Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province, 510070, China. The deposit number is GDMCC No. 63499, and the deposit date is May 24, 2023.

[0006] The morphological characteristics of the yellow bacillus F1009 are as follows: Gram staining is negative, and single colonies are round, milky yellow, smooth, opaque, and have regular edges.

[0007] The second object of the present invention is to provide the use of the yellow bacterium F1009 in preventing and treating citrus canker.

[0008] The third object of the present invention is to provide the use of the yellow bacterium F1009 in promoting plant iron absorption.

[0009] The fourth object of the present invention is to provide the use of the yellow bacterium F1009 in promoting plant growth.

[0010] The fifth object of the present invention is to provide the use of the said Flavobacterium F1009 in the preparation of biological preparations for preventing and treating citrus canker and / or promoting plant iron absorption and growth.

[0011] Preferably, the biological agent is a microbial agent, a microbial fertilizer, a microbial pesticide or a microbial additive.

[0012] A sixth object of the present invention is to provide a biological preparation for preventing and treating citrus canker and / or promoting plant iron absorption and growth, comprising Flavobacterium xanthomonas F1009 or its fermentation liquid as an active ingredient.

[0013] The seventh object of the present invention is to provide a method for preventing and treating citrus canker, which comprises spraying the fermentation liquid of Flavobacterium flavum F1009 to the area to be prevented and treated.

[0014] An eighth object of the present invention is to provide a method for promoting plant iron absorption and growth, wherein the method comprises applying Flavobacterium flavum F1009 to cultivated plants, specifically, watering the plants with the fermentation liquid of Flavobacterium flavum F1009 to promote plant iron absorption and growth.

[0015] The present invention has the following advantages and effects compared to the prior art:

[0016] This study reports for the first time a potential new species in the genus Xanthomonas that exhibits excellent efficacy against citrus canker. The strain also produces siderophores and indoleacetic acid, promoting plant iron absorption and growth. The discovery and identification of this strain enriches my country's resources for biocontrol and growth-promoting microorganisms. Its stable, highly effective, and environmentally friendly antibacterial effects hold great promise for applications in preventing and controlling citrus canker and promoting plant iron absorption and growth.

[0017] Luteibacter sp. F1009 was deposited in the Guangdong Provincial Microbial Culture Collection Center (GDMCC) on May 24, 2023, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province, Postal Code: 510070, and the deposit number is: GDMCC No: 63499. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the colony morphology of the new yellow bacterium F1009 on NA medium.

[0019] Figure 2 Phylogenetic tree of the novel Flavobacterium F1009 based on the 16S rRNA gene.

[0020] Figure 3 This is the phylogenetic tree of the novel yellow bacterium F1009 based on gene sequences.

[0021] Figure 4 This study tested the inhibitory effect of the novel yellow bacterium F1009 on the citrus canker pathogen Xcc.

[0022] Figure 5 Test for the production of siderophores by the novel Flavobacterium sp. F1009.

[0023] Figure 6 Test for the production of indoleacetic acid by the novel Flavobacterium sp. F1009. DETAILED DESCRIPTION

[0024] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0025] Example 1: Isolation and identification of novel Xanthomonas sp. F1009

[0026] Healthy citrus trees were selected from the orchard of Fuzhu Group, Xiaying Village, Huangkeng Town, Renhua County, Shaoguan City, Guangdong Province (N 25°4′17″, E 113°46′50″), and fresh branches and leaves were cut with sterile scissors and placed in sterile sampling bags. Subsequently, the samples were stored in a 4°C sampling box and brought back to the laboratory. A total of 10 g of leaves were cut on the clean bench, added to a 500 mL triangular flask containing 190 mL of sterile PBS buffer (0.2 mol / L, pH = 7.0), shaken well, and placed in a 28°C constant temperature shaker, shaking at 200 rpm for 30 minutes. Subsequently, the triangular flask was placed in an ultrasonic oscillator, 40 KHz, working for 8 seconds, resting for 2 seconds, and ultrasonic for 10 minutes. Take 1 mL of the eluate and add it to a test tube containing 9 mL of PBS buffer, and dilute to 10 -1 , 10 -2 , 10 -3 and 10 -4 , draw the original solution of eluent, 10 -1 , 10 -2 , 10 -3 and 10 -4 100 μL of each diluted sample was spread onto R2A medium (Qingdao Haibo, Catalog No. HB0167) and incubated in a 28°C biochemical incubator for 7 days. Based on characteristics such as colony size, color, dryness, smoothness, and the presence of halos, individual colonies were selected from the culture medium and transferred to fresh R2A medium. Purification was performed by multiple streaking until a pure culture was obtained. The purified strains were numbered, mixed with 25% glycerol (v / v), and stored in an ultra-low temperature freezer, resulting in strain F1009.

[0027] The strain F1009 was Gram-negative. The colony morphology of the strain after culturing on R2A medium for 4 days was as follows: Figure 1 As shown, the single colony is round, 2 to 4 mm in diameter, milky yellow, with a smooth, opaque surface and neat edges.

[0028] Example 2: 16S rRNA gene sequence analysis of the novel Xanthomonas sp. F1009

[0029] The genomic DNA of strain F1009 was extracted and purified using the HiPure bacterial DNA extraction kit (Guangzhou Meiji Biotechnology, catalog number D3146-03). Using the purified genomic DNA as a template, the bacterial 16S rRNA gene amplification universal primers 27F / 1492R, i.e. 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3') were used to amplify the 16S rRNA gene sequence of strain F1009. After amplification, an appropriate amount of PCR product was taken for gel electrophoresis detection. When the presence of the target band was confirmed, the remaining PCR product was sent to Suzhou Jinweizhi Biotechnology Co., Ltd. for sequencing. The sequence is shown in SEQ ID NO.1, with a length of 1408 bp. The 16S rRNA gene sequence of strain F1009 was submitted to the EzBioCloud database (www.ezbiocloud.net) for sequence homology comparison. The comparison results showed that strain F1009 and the model strain Luteibacter pinisoli MAH-14 T The 16SrRNA gene sequence of the Luteibacter anthropic CCUG 25036 (accession number CP041046) has the highest similarity of 99.2%. T The 16S rRNA gene sequence of Luteibacter rhizovicinus LJ96 (accession number FM212561) has the second highest similarity, at 98.9%. T (Accession No. CP017480) has a similarity of 98.7%; the similarity of the 16S rRNA gene with other type strains of Luteibacter is less than 98.6%. Figure 2 As shown, 16S rRNA gene phylogenetic analysis showed that strain F1009 had the closest phylogenetic relationship with Luteibacter pinisoli. However, the exact taxonomic status of strain F1009 cannot be determined solely through 16S rRNA gene sequencing analysis.

[0030] The 16S rRNA gene sequence of strain F1009 is shown in SEQ ID NO. 1, specifically:

[0031] ATGCAGTCGAACGGCAGCACAGCAGAGCTTGCTCTGTGGGTGGCGAGTGGCGGACGGGTGAGTAAT

[0032] GCATCGGGACCTACCCAGACGTGGGGGATAACGTAGGGAAACTTACGCTAATACCGCATACGTCCT

[0033] ACGGGAGAAAGCGGGGGATCGCAAGACCTCGCGCGGTTGGATGGACCGATGTGCGATTAGCTAGTT

[0034] GGTGAGGTAACGGCTCACCAAGGCGACGATCGCTAGCTGGTCTGAGAGGATGATCAGCCACACTGG

[0035] GACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGCAAGC

[0036] CTGATCCAGCAATGCCGCGTGTGTGAAGAAGGCCCTCGGGTTGTAAAGCACTTTTATCAGGAGCGAA

[0037] ATCTGCAAGGTTAATACCTTTGCAGTCTGACGGTACCTGAGGAATAAGCACCGGCTAACTCCGTGCC

[0038] AGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGTGCGTAGG

[0039] CGGTTCGTTAAGTCTGTTGTGAAAGCCCCGGGCTCAACCTGGGAATGGCAATGGATACTGGCGAGCT

[0040] AGAGTGTGTCAGAGGATGGTGGAATTCCCGGTGTAGCGGTGAAATGCGTAGAGATCGGGAGGAACA

[0041] TCAGTGGCGAAGGCGGCCATCTGGGACAACACTGACGCTGAGGCACGAAAGCGTGGGGAGCAAAC

[0042] AGGATTAGATACCCTGGTAGTCCACGCCCTAAACGATGCGAACTGGATGTTGGTCTCAACTCGGAGA

[0043] TCAGTGTCGAAGCTAACGCGTTAAGTTCGCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAA

[0044] GGAATTGACGGGGGCCCGCACAAGCGGTGGAGTATGTGGTTTAATTCGATGCAACGCGAAGAACCT

[0045] TACCTGGCCTTGACATGTCCGGAATCCAGCAGAGATGCAGGAGTGCCTTCGGGAATCGGAACACAG

[0046] GTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCC

[0047] TTGTCCTTAGTTGCCAGCGAGTAATGTCGGGAACTCTAAGGAGACTGCCGGTGACAAACCGGAGGA

[0048] AGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGGCCAGGGCTACACACGTACTACAATGGTCG

[0049] GTACAGAGGGTTGCGATACCGCGAGGTGGAGCTAATCCCAGAAAGCCGATCCCAGTCCGGATTGGA [[ID=​​​​​​​​​The novel yellow bacterium F1009 genome extracted in Example 2 was sent to Shanghai Meiji Biopharmaceutical Technology Co., Ltd. for genome sequencing. An Illumina PE library was constructed and sequenced using the Illumina Novaseq platform. After quality inspection of the original reads, the high-quality data obtained was spliced ​​using SPAdes v3.15.3 for genome sequence assembly. According to the parameters of the coverage of the spliced ​​contigs ≥ 100 and the length ≥ 500bp, low-quality contig sequences were filtered and removed. The strain F1009 genome was quality inspected using the software CheckM v1.1.3. The results showed that the integrity of the strain F1009 genome was 99.57% and the contamination was 0.43%. Generally, if the genome integrity is ≥ 95% and the contamination is ≤ 5%, the genome quality is considered to be high. Therefore, the strain F1009 genome is a high-quality genome that can ensure the accuracy of subsequent analysis. The strain F1009 genome was analyzed using the software QUAST v5.0.2. The results showed that the genome sequence of strain F1009 contained 32 contigs, with a total genome length of 4471016bp, an N50 length of 282690bp, and a genomic DNA G+C content of 66.28%.

[0054] A digital DNA-DNA hybridization (dDDH) value of 70% is the gold standard for prokaryotic species identification. The online tool Genome-to-Genome Distance Calculator 3.0 (https: / / ggdc.dsmz.de / ggdc.php#) was used to calculate the genome sequence of strain F1009 and the closely related species model strain Luteibacter pinisoli MAH-14. T (Genome accession number CP041046), Luteibacter anthropic CCUG25036 T (genome accession number JAARLZ000000000) and Luteibacter rhizovicinus LJ96 T The dDDH values ​​between the genome sequences of strain F1009 and its closest relatives (genome accession number CP017480) were 23.0, 23.2, and 23.9%, respectively. These three dDDH values ​​are all below the 70% species classification standard, indicating that strain F1009 is different from the three closest related species and is a potential new species. The phylogenetic tree of strain F1009 and its closest reference strains was constructed using the software UBCG v3.0. Figure 3As shown, strain F1009 formed an independent phylogenetic branch with a support bootstrap value of 100, indicating independent taxonomic status. Based on the results of dDDH and phylogenetic analyses, strain F1009 is a potential new species in the genus Luteibacter and is therefore named Luteibacter sp. F1009. This strain has been deposited with the Guangdong Provincial Microbial Culture Collection (GDMCC), 5th Floor, Building 59, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province, China, under the GDMCC accession number 63499, and the date of deposit is May 24, 2023.

[0055] Example 4: Identification of the inhibitory effect of the novel Flavobacterium F1009 on the pathogen of citrus canker

[0056] The double-layer agar diffusion method was used to determine the inhibitory effect of the new yellow bacterium F1009 on the pathogen of citrus canker. The citrus canker pathogen Xcc and the new yellow bacterium F1009 were inoculated into NB liquid culture medium for activation, and the fermentation broth was collected after 24 hours of culture. Prepare the bottom NA culture medium (agar powder content 1.5%), and the culture medium thickness is moderate. Place Oxford cups on the bottom NA culture medium, 3 on each plate. Take 1mL of Xcc fermentation broth and add 300mL of NA culture medium (agar powder content 1%), and the final concentration of Xcc is 10 6 ~10 7 cfu / mL. After solidification, remove the Oxford cup, which is the upper culture medium. Add 30 μL of the fermentation broth of the novel yellow bacterium F1009 to the wells and incubate at 28°C for 36 hours. Use a vernier caliper to measure the diameter of the inhibition zone.

[0057] like Figure 4 As shown in the figure, after adding the new Flavobacterium F1009, the citrus canker pathogen Xcc formed a clear transparent ring around the wells, with diameters of 20.37, 20.77, and 20.76 mm, respectively, and an average of 20.63 mm. The results show that the new Flavobacterium F1009 has a significant inhibitory effect on the growth of the citrus canker pathogen Xcc.

[0058] Example 5: Identification of the siderophore-producing novel Xanthomonas sp. F1009

[0059] The activated novel Flavobacterium F1009 was inoculated into R2A medium and cultured at 28°C for 48 h. Subsequently, CAS assay medium (Qingdao Haibo, product number HB9132) was poured onto the R2A medium and cultured at 28°C for 48 h. The diameter of the siderophore secretion zone (orange) was measured using a vernier caliper.

[0060] like Figure 5As shown in the CAS test medium, a clearly visible orange-yellow siderophore secretion ring formed around the novel Flavobacterium F1009, with diameters of 31.0, 32.0, and 32.0 mm, respectively, and an average of 31.7 mm. These test results indicate that the siderophore secreted by the novel Flavobacterium F1009 can efficiently bind to iron in the surrounding environment, forming a stable chelate with it, thereby effectively absorbing low-soluble iron in the environment to promote plant growth.

[0061] Example 6: Identification of IAA production by the novel Xanthomonas sp. F1009

[0062] The Salkowski colorimetric test was used to determine the production of indoleacetic acid (IAA) by the novel yellow bacterium F1009. The absorbance value OD 530 The standard curve was prepared by taking the concentration of IAA as the vertical axis and the concentration of IAA as the horizontal axis. Figure 6 The fermentation liquid of the new yellow bacterium F1009 was centrifuged at 4°C and 5000 rpm for 10 min, and the supernatant was mixed with Salkowski's color reagent at a ratio of 1:1 (V:V). The mixture was reacted in the dark for 20 min and the color of the mixture was observed. At the same time, the OD 530 The absorbance value of the new type of yellow bacterium F1009 was calculated using the standard curve, and the uninoculated IAA detection medium was used as a control.

[0063] like Figure 6 As shown, the supernatant obtained from the fermentation broth of the novel Flavobacterium F1009 was reacted with a colorimetric reagent in the dark for 20 minutes, turning the solution pink, indicating its ability to produce IAA. Based on the standard curve, the amount of IAA produced by the novel Flavobacterium F1009 was calculated to be 47.07 mg / L. These test results demonstrate that the novel Flavobacterium F1009 can produce high concentrations of indoleacetic acid, thereby promoting plant growth.

[0064] These results demonstrate that the novel Luteibacter sp. F1009 is a potential new species of the genus Luteibacterium, with significant inhibition of the growth of Xcc, the pathogen of citrus canker. Furthermore, it is a highly effective biocontrol strain that can be used for the prevention and treatment of citrus canker through direct spraying, as well as in the production of microbial fertilizers and microbial agents, which can be used to control the aforementioned plant diseases. Luteibacter sp. F1009 can produce siderophores and indoleacetic acid, thus showing great potential for promoting iron absorption and growth in crops.

[0065] The above are merely preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be construed as limiting the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims. Persons skilled in the art will appreciate that improvements and modifications may be made without departing from the spirit and scope of the present invention, and such improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Yellow bacillus ( Luteibacter sp.) F1009, deposit number: GDMCC No: 63499.

2. Use of the Flavobacterium F1009 according to claim 1 in preventing and treating citrus canker.

3. Use of the Flavobacterium F1009 according to claim 1 in promoting plant iron absorption.

4. Use of the Flavobacterium F1009 according to claim 1 in promoting plant growth.

5. Use of the Flavobacterium F1009 according to claim 1 in the preparation of a biological preparation for preventing and treating citrus canker and / or promoting plant iron absorption and growth.

6. The use according to claim 5, characterized in that The biological agent is a microbial agent, a microbial fertilizer, a microbial pesticide or a microbial additive.

7. A biological agent for preventing and treating citrus canker and / or promoting plant iron absorption and growth, characterized in that: Contains the Flavobacterium sp. F1009 according to claim 1 or a fermentation liquid containing Flavobacterium sp. F1009 as an active ingredient.

8. A method for preventing and treating citrus canker, characterized in that: The method comprises spraying the fermentation liquid of the yellow bacterium F1009 according to claim 1 to the area to be controlled.

9. A method for promoting plant iron absorption and growth, characterized in that: The method comprises applying the flavobacterium F1009 according to claim 1 to plant cultivation.

10. The method according to claim 9, characterized in that The plant is irrigated with the fermentation liquid of the yellow bacterium F1009 according to claim 1.

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