Acinetobacter pittii and its application in prevention and treatment of tomato bacterial wilt

Abstract

This invention discloses a strain of Acinetobacter piterum and its application in the control of bacterial wilt in tomatoes. The Acinetobacter piterum provided by this invention (… Acinetobacter pittii Acinetobacter pituitaria BB307, with accession number GDMCC NO. 67769, exhibits broad-spectrum antibacterial activity against eight sequence variants of Ralstonia solanaceae, making it suitable for controlling bacterial wilt in tomatoes. Furthermore, it possesses nitrogen-fixing, phosphorus-solubilizing, siderophore-producing, and indoleacetic acid-promoting abilities, which can promote plant growth in tomatoes. The Acinetobacter pituitaria BB307 provided by this invention demonstrates synergistic potential for biocontrol and growth promotion, offering a highly efficient microbial resource for green production of solanaceous crops and providing a more economical, efficient, and environmentally friendly solution for the prevention and control of bacterial wilt.

Description

Technical Field

[0001] This invention belongs to the field of bacterial wilt control technology, specifically involving a strain of Acinetobacter pietrogenis and its application in the control of bacterial wilt in tomatoes. Background Technology

[0002] Bacterial wilt is caused by the complex of Rhesperidium spp. (Solanaceae) Ralstonia solanacearum This is a devastating global disease caused by the Species Complex (RSSC). The pathogen can infect more than 450 species of plants from over 50 families, with particularly severe damage to Solanaceae crops such as tomatoes, potatoes, eggplants, and tobacco, often leading to yield losses exceeding 50% or even total crop failure, resulting in incalculable economic losses. Currently, in my country, except for Tibet and Macau where there are no reported cases, the disease has been recorded in all 30 provinces and regions, with Guangdong and Guangxi in southern China being high-incidence and severely affected areas.

[0003] In Guangdong Province, the dominant pathogen of bacterial wilt in tomatoes is RSSC evolution type I – *Corynebacterium pseudosolaniae* (RSSC evolution type I). Ralstonia pseudosolanacearum Currently, multiple sequence variants, including 13, 14, 15, 17, 18, 34, 44, and 57, have been identified. It is noteworthy that in recent years, the pathogenicity of the pathogen population has continued to evolve, with new highly pathogenic variants constantly emerging, leading to the degradation of crop resistance. Existing control agents have limited effectiveness, and field control techniques are severely inadequate. In production, outbreaks of bacterial wilt have frequently resulted in yield reductions and even crop failures, causing significant economic losses to growers.

[0004] Current pest control relies excessively on chemical pesticides, but highly effective agents are scarce. Farmers often use multiple fungicides in rotation, sometimes even applying them weekly during peak disease season. This not only significantly increases pesticide costs and environmental pressure but also raises the risk of pathogen resistance. Therefore, developing biological control technologies has become a key breakthrough in addressing the bacterial wilt problem. Unfortunately, the resource bank of biocontrol microorganisms suitable for tomato bacterial wilt remains weak and urgently needs in-depth exploration and innovative application. Summary of the Invention

[0005] This invention addresses the shortcomings of the existing technology by providing a strain of Acinetobacter pitera (…). Acinetobacter pittii The BB307 strain (accession number: GDMCC NO. 67769, accession date: January 29, 2026, depository institution: GDMCC-Guangdong Provincial Microbial Culture Collection Center) and its application in the prevention and control of bacterial wilt in tomatoes provide a more economical, efficient and environmentally friendly solution for the prevention and control of bacterial wilt in tomatoes.

[0006] The first objective of this invention is to provide a strain of Acinetobacter pitera (… Acinetobacter pittii BB307, with accession number GDMCC NO. 67769.

[0007] A second objective of this invention is to provide an agent for the prevention and control of bacterial wilt in plants, which contains a culture of Acinetobacter pituitaria BB307 as an active ingredient.

[0008] Preferably, the culture of Acinetobacter pituitaria BB307 is obtained by inoculating Acinetobacter pituitaria BB307 into LB medium and culturing it.

[0009] Preferably, the concentration of Acinetobacter pituitaria BB307 culture in the formulation is 1×10⁻⁶. 8 cfu / mL or higher.

[0010] A third objective of this invention is to provide the application of the aforementioned Acinetobacter pituitaria BB307 or the aforementioned preparation in the control of bacterial wilt in plants.

[0011] Preferably, the pathogen causing bacterial wilt is one or more sequence variants of Ralstonia solanaceae, namely 13, 14, 15, 17, 18, 34, 44 or 57.

[0012] Preferably, sequence variant 13 is Ralstonia solanacearum RS180, sequence variant 14 is Ralstonia solanacearum BY-2 and / or SSF-4, sequence variant 15 is Ralstonia solanacearum RS58 and / or RS208, sequence variant 17 is Ralstonia solanacearum RS397, sequence variant 18 is Ralstonia solanacearum GMI1000 and / or TS-2, sequence variant 34 is Ralstonia solanacearum SM-GZZC-127, RS196, RS210, RS404 and / or RS550, sequence variant 44 is Ralstonia solanacearum RS129 and / or SG-2, and sequence variant 57 is Ralstonia solanacearum GY-2.

[0013] Preferably, the plant is a tomato.

[0014] Preferably, the method includes the step of applying Acinetobacter pituitariae BB307 of claim 1 or the preparation of claim 2 to tomato plants.

[0015] Preferably, the application to the tomato plant is applied to the roots of the tomato plant.

[0016] The beneficial effects of this invention are: This invention discovered a strain of Acinetobacter pitera (… Acinetobacter pittii The BB307 strain possesses multiple functions, including broad-spectrum antibacterial activity, growth promotion, and control of bacterial wilt in tomatoes. (1) Acinetobacter piezoides BB307 has broad-spectrum antibacterial activity against Solanacria cocos. Specifically, eight sequence variants (sequence variants 13, 14, 15, 17, 18, 34, 44, and 57 in RSSC evolution type I) were tested and all showed antibacterial activity, with inhibition zone diameters ranging from 0.88 to 2.01 cm.

[0017] (2) Acinetobacter piezoides BB307 has the potential to combine nitrogen fixation, phosphorus solubilization, iron production and indoleacetic acid biocontrol and growth promotion. After root irrigation treatment, tomato plants showed a significant increase in root weight by 57.14%, fresh weight by 31.72% and plant height by 15.43%.

[0018] (3) After pretreating tomatoes with Acinetobacter pituitaria BB307 by root irrigation, they were inoculated with Solanacorhizium anisopliae, which causes bacterial wilt of tomatoes. It was found that Acinetobacter pituitaria BB307 could effectively control bacterial wilt of tomatoes, with a control efficacy of 58%.

[0019] Preservation Instructions Acinetobacter piezophyllum BB307 of the present invention Acinetobacter pittii BB307 was deposited on January 29, 2026 at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), with accession number GDMCC NO. 67769. The depository address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Academy of Sciences, Institute of Microbiology. Attached Figure Description

[0020] Figure 1 This is the colony morphology of Acinetobacter pituitaria BB307.

[0021] Figure 2 This is a circulated genome diagram of Acinetobacter pituitaria BB307.

[0022] Figure 3 It is an evolutionary tree constructed based on the core genes of Acinetobacter pituitaria BB307.

[0023] Figure 4 These are the results of biological function assays for Acinetobacter pituitaria BB307.

[0024] Figure 5 The growth-promoting effect of Acinetobacter pituitaria BB307 on tomato plants; where A represents the phenotype of tomato plants without and with Acinetobacter pituitaria BB307, and B represents the physiological indicators of tomato plants without and with Acinetobacter pituitaria BB307; different letters indicate significant differences between different treatments for the same indicator (p<0.05).

[0025] Figure 6 This is a phenotypic diagram showing the control effect of Acinetobacter pituitaria BB307 bacterial suspension on bacterial wilt of tomato. Detailed Implementation

[0026] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0027] The culture medium and its formulation used in the examples are as follows: LB solid medium: 10 g tryptone, 5 g yeast extract, 10 g sodium chloride, 15 g agar powder, bring to a final volume of 1000 mL with deionized water, pH 7.0-7.2, sterilize at 121℃ for 20 min, then cool to room temperature before use.

[0028] TZC solid medium: 10 g peptone, 1 g casein lysate, 5 g glucose, 15 g agar powder, bring the volume to 1000 mL with deionized water, pH 7.0, sterilize at 121℃ for 20 min, cool to about 40℃, add 0.005% 2,3,5-triphenyltetrazolium chloride, and cool to room temperature before use.

[0029] Phosphate-solubilizing medium: 5 g tricalcium diphosphate, 0.5 g ammonium sulfate, 0.3 g sodium chloride, 0.3 g potassium chloride, 0.3 g magnesium sulfate heptahydrate, 0.003 g ferric sulfate heptahydrate, 0.5 g yeast extract, 10 g glucose, 15 g agar powder, pH 7-7.5, diluted to 1000 mL with deionized water, sterilized at 121℃ for 20 min, and then cooled to room temperature before use.

[0030] Ashbee's nitrogen-fixing medium: 0.2 g dipotassium hydrogen phosphate, 0.2 g sodium chloride, 2 g calcium carbonate, 10 g mannitol, 0.1 g calcium sulfate, 0.2 g magnesium sulfate heptahydrate, 15 g agar powder, pH 7-7.5, diluted to 1000 mL with deionized water, sterilized at 121℃ for 20 min, and then cooled to room temperature before use.

[0031] CAS detection medium: 1 mL of 20% glucose, 3 mL of 10% chromium (casein amino acids), 100 μL of 1 mmol / L calcium chloride, 2 mL of 1 mmol / L magnesium sulfate heptahydrate, 2.3 g of agar powder, bring the volume to 100 mL with deionized water, sterilize at 121℃ for 20 min, cool to 60℃ and slowly add 5 mL each of phosphate buffer and CAS staining solution, cool to room temperature and use.

[0032] King medium: 100 mL of LB liquid medium was sterilized at 121°C for 20 min, cooled to 40°C, and then 0.01 g of filtered and sterilized tryptophan was added.

[0033] Protease medium: 8 g skim milk powder, 15 g agar powder, deionized water to a final volume of 300 mL, pH 7.0, sterilized at 121℃ for 20 min, then cooled to room temperature before use.

[0034] Cellulase medium: 10 g peptone, 10 g yeast extract, 10 g sodium carboxyformate, 5 g sodium chloride, 1 g potassium dihydrogen phosphate, 18 g agar, bring the volume to 1000 mL with deionized water, pH 7.0, sterilize at 121℃ for 20 min, and then cool to room temperature before use.

[0035] β-1,3-glucanase medium: 10 g sodium carboxycarboxylate, 10 g tryptone, 10 g sodium chloride, 5 g yeast extract, 15 g agar powder, diluted to 1000 mL with deionized water, pH 7.0-7.2, sterilized at 121℃ for 20 min, then cooled to room temperature before use.

[0036] All of the above culture media are solid culture media. If no agar powder is added, a liquid culture medium can be prepared.

[0037] Example 1: Identification and preservation of strain BB307 I. Isolation and Identification of Strain BB307 The tested bacterial strains were isolated from soil in a forest area in Dapu County, Meizhou City. The isolation method employed was a dilution isolation method combined with the standard operating procedure for soil microbial isolation. Briefly, 1 g of soil was serially diluted with 1 mL of sterile water, and then 10 μL of each diluted sample was taken. - ³、10 -4 10 -5 Three concentration gradients of soil dilution (g / mL, 100 μL each) were added and spread onto LB agar plates. After standing for 10 min to allow for full bacterial adsorption, the plates were inverted and incubated at 30℃ for 1 day to obtain uniformly dispersed single colonies. The plate inhibition zone method was used for preliminary screening of strains antagonistic to Ralstonia solanacearum strain RS550, resulting in strain BB307. The colony phenotype of strain BB307 is shown below. Figure 1 As shown.

[0038] 1. Whole genome sequencing and identification The whole genome of strain BB307 was sequenced using a combination of second-generation Illumina and third-generation PacBio sequencing platforms. Flye and pilon sequencing were used for genome assembly and correction, and CheckM2 analysis confirmed 100% genome integrity. The genome consists of one circular chromosome and four circular plasmids, with sizes of 3,837,403 bp, 94,386 bp, 50,599, 11,158, and 8,469 bp, respectively, and G+C contents of 38.90%, 37.49%, 40.20%, 35.11%, and 39.08%, respectively. It encodes 3725 genes, including 74 tRNAs, 18 rRNAs, and 37 ncRNAs. The BB307 genome also contains four prephage sequences and 20 genomic island sequences. Figure 2 Based on average nucleotide identity analysis and core gene phylogenetic results, strain BB307 is related to... Acinetobacter pittii ATCC19004 has an ANI value of 97.67% and clusters into one branch on the phylogenetic tree. Figure 3 Therefore, BB307 was preliminarily identified as Acinetobacter piezophyllum (Piezophyllum). Acinetobacter pittii Its 16S rRNA nucleotide sequence is shown in SEQ ID NO.1.

[0039] II. Preservation of strain BB307 Strain BB307 was deposited at the Guangdong Provincial Microbial Culture Collection Center on January 29, 2026, and was indeed identified as Acinetobacter piezoides ( ). Acinetobacter pittii The accession number is GDMCC 67769, therefore strain BB307 is named Acinetobacter petrificus BB307. Acinetobacter pittii BB307).

[0040] Example 2: Antibacterial effect of Acinetobacter piezoides BB307 against Ralstonia solanacearum Activated Acinetobacter pituitaria BB307 and 16 different sequence variants of Ralstonia solanacearum strains (published in Ding S., Li P., Tang Y., He Z., She X., 2025. Identification and genomic insights into Bacillus siamensis strains with host colonization potential and activity against tomato bacterial wilt. Pest Management Science 81, 1547-1561.) were cultured at 30°C for 2 days and the biocontrol bacteria for 1 day. Single strains of Acinetobacter pituitaria BB307 were inoculated into 5 mL of LB liquid medium, and single strains of Ralstonia solanacearum were inoculated into TZC liquid medium. The cultures were incubated overnight at 30°C with shaking at 160 r / min to obtain bacterial suspensions for use. Use a pipette to draw 500 μL of overnight Ralstonia solanacearum culture and add it to 150 mL of TZC solid medium that has been cooled to about 30°C but has not yet solidified. Shake to mix the medium with the bacteria, then pour the medium into a 9 mm Petri dish. After the medium solidifies, prepare TZC solid medium plates containing Ralstonia solanacearum. Place 3 filter paper discs in each plate, and slowly add 5 μL of Acinetobacter piezoides BB307 culture to the filter paper discs in the 9 cm plates. Repeat 3 times. After drying, incubate upside down in a 30°C incubator for 48 h. Measure and record the diameter of the inhibition zone using the cross-cross method.

[0041] Table 1. Results of antibacterial activity assay of strain BB307 against different sequence variants of Ralstonia solanacearum. From Table 1 and Figure 4 It was found that Acinetobacter pitera BB307 exhibited certain antibacterial activity against different sequence variants of Ralstonia solanacearum. The antibacterial effects of Acinetobacter pitera BB307 against 16 Ralstonia solanacearum strains with 8 sequence variants varied, with inhibition zone diameters ranging from 0.88 cm to 2.01 cm.

[0042] Example 3: Biological functions of Acinetobacter piezophyllum BB307 Indoleacetic acid (IAA) production capacity assay: Add 5 mL of King liquid culture medium and 100 μL of OD to each test tube. 600A 1-unit (=1) culture of Acinetobacter pylori BB307 was prepared, with 100 μL of sterile water added to King's Liquid Medium as a blank control. The culture was incubated on a shaker at 30°C and 180 r / min for 2 days. After centrifugation, 200 μL of supernatant and 200 μL of IAA colorimetric solution were placed in 1.5 mL centrifuge tubes. 100 μL of sterile water added to King's Liquid Medium served as a control. After incubation at room temperature for 20 min, the color change was observed. A reddish color in the test tube indicated the production of IAA.

[0043] Siderophore assay: Acinetobacter piezoides BB307 was inoculated onto CAS test medium using the single-point method and incubated at 30°C for 3 days. The presence of a yellow halo around the colony was then observed. If such a halo was present, the bacterium was capable of producing siderophores.

[0044] Nitrogen fixation and phosphorus solubilization capacity testing: Acinetobacter pituitaria BB307 was inoculated onto Assumption nitrogen-fixing medium using the streak method and incubated at 30°C for 2 days. The presence of a clear zone was observed. The Acinetobacter pituitaria BB307 culture was then inoculated onto phosphorus-solubilizing medium using the three-point method, and the presence of a clear zone was also observed. The presence of a clear zone indicated nitrogen fixation or phosphorus solubilization capacity; the absence of a clear zone indicated the absence of either nitrogen fixation or phosphorus solubilization capacity.

[0045] Enzyme production capacity assay: The hydrolytic activities of Acinetobacter pituitaria BB307 against cellulase, β-1,3-glucan hydrolase and protease were determined using cellulase medium, β-1,3-glucan hydrolase medium and protease medium, respectively.

[0046] The test results showed that *Acinetobacter pylori* BB307 possesses protease hydrolytic activity, exhibiting phosphorus solubilization, nitrogen fixation, and IAA production capabilities. It lacks cellulase and β-1,3-glucanase hydrolytic activity but has the ability to produce siderophores. Figure 4 ).

[0047] Example 4: Acinetobacter piezoides BB307 has the ability to promote the growth of tomato plants. 1. Preparation of Acinetobacter piezophyllum BB307 bacterial suspension A single colony of activated Acinetobacter pituitaria BB307 was picked from an LB solid medium plate and inoculated into a test tube containing 5 mL of LB liquid medium. The culture was then incubated at 30°C with shaking at 180 rpm for 24 h. For further culture, the resulting Acinetobacter pituitaria BB307 seed culture was inoculated at a volume fraction of 1% into an Erlenmeyer flask containing 150 mL of LB liquid medium, and cultured under the same conditions for another 24 h.

[0048] 2. Vaccination and Survey When the tomato plants have grown to two leaves and a central bud, select tomato seedlings with uniform growth for growth promotion capacity testing. Each seedling is irrigated with 40 mL of water at a concentration of 1×10⁻⁶. 8 CFU / mL Acinetobacter piezoides inoculum (diluted with sterile water) was used as a control, with 40 mL of LB liquid medium diluted according to the inoculum dilution ratio. Each treatment consisted of 15 seedlings, with 3 replicates. The treatments were randomly arranged. After 15 days, tomato plant height, root length, stem diameter, fresh weight, and root weight were measured, photographed, and the growth promotion effect (GPE%) was calculated.

[0049] Depend on Figure 5 As shown in Figure A, applying Acinetobacter pituitaria BB307 bacterial solution significantly reduced the growth of tomato plants. After treating tomato plants with Acinetobacter pituitaria BB307 bacterial solution for 15 days, plant height, root length, stem diameter, fresh weight, and root weight were measured. The results showed that compared with the control treatment, Acinetobacter pituitaria BB307 significantly promoted plant height, fresh weight, and root weight, with a promotion effect of 15.43% on plant height, 31.72% on fresh weight, and 57.14% on root weight. However, the promotion effect of Acinetobacter pituitaria BB307 on root length and stem diameter was not significantly different from the control treatment. Figure 5 (B in the middle).

[0050] Example 5: The control efficacy of Acinetobacter piezoides BB307 against bacterial wilt in tomato 1. Preparation of Ralstonia solanacearum fermentation broth Preparation of Ralstonia solanacearum strain RS550 suspension: Activated single colonies from TZC medium were picked and revitalized on TZC slant medium and incubated at 30℃ for 24 h. White colonies were then picked and inoculated into test tubes containing 5 mL of TZC liquid medium and incubated at 30℃ with shaking at 180 r / min for 24 h to obtain RS550 Ralstonia solanacearum suspension.

[0051] 2. Vaccination and Survey Sow tomatoes in a sterilized mixture of nutrient soil and garden soil in a 1:1 ratio. Transplant the seedlings when they have four leaves and a central bud. One week after transplanting, drench each seedling with 50 mL of Acinetobacter pylori BB307 bacterial solution (final concentration 1×10⁻⁶). 8 The concentration of *Ralstonia solanacearum* was 100 cfu / mL, with a control group receiving 50 mL of LB liquid medium. After 5 days, each treatment was administered 50 mL of RS550 *Ralstonia solanacearum* suspension (final concentration 1×10⁻⁶ cfu / mL). 8 (cfu / mL). Each treatment consisted of 15 tomato seedlings, with 3 replicates. Treatments were randomly arranged and cultured at 25-30℃ and 60%-80% humidity. After 6 days, the disease severity was assessed and the control effect was statistically analyzed.

[0052] The classification of bacterial wilt in tomatoes is as follows: Grade 0: The plant is healthy and there is no wilting. Grade 1: 1-25% of the leaves are wilting. Grade 2: 26-50% of the leaves are wilting. Grade 3: 51-75% of the leaves are wilting. Grade 4: 76-100% of the leaves are wilting.

[0053] The disease index and biocontrol efficiency of tomato bacterial wilt were calculated using the following formula: Disease index = ∑ (number of infected plants × disease severity level) / (total number of plants surveyed × highest severity level) × 100; Biological control rate (%) = (Control disease index - Treatment disease index) / Control disease index × 100%.

[0054] Table 2. Control efficacy of Acinetobacter piezoides BB307 against bacterial wilt in tomato. Note: Disease index data are mean ± standard error, with 3 biological replicates for each treatment.

[0055] The results showed that 6 days after treatment with RS550 Ralstonia solanacearum suspension, the average disease incidence rate of tomato plants treated with RS550 Ralstonia solanacearum suspension alone was 46.67%, with a disease index of 45.00; while the average disease incidence rate of tomato plants pretreated with Acinetobacter pylori BB307 suspension was 24.44%, with a disease index of 18.90. Acinetobacter pylori BB307 showed a significant control effect on tomato bacterial wilt, with a control efficacy of 58% (Table 2). Figure 6 ).

Claims

1. A strain of Acinetobacter piterum ( Acinetobacter pittii BB307, characterized in that, Its accession number is GDMCC NO3.67769.

2. A preparation for controlling bacterial wilt of plants, characterized in that, A culture containing Acinetobacter piezophyllum BB307 as described in claim 1 is used as the active ingredient.

3. The formulation according to claim 2, characterized in that, The culture of Acinetobacter pituitaria BB307 was obtained by inoculating Acinetobacter pituitaria BB307 into LB medium and culturing it.

4. The formulation according to claim 2, characterized in that, The concentration of Acinetobacter pituitaria BB307 culture in the formulation is 1×10⁻⁶. 8 cfu / mL or higher.

5. The use of Acinetobacter pituitariae BB307 as described in claim 1 or the preparation as described in claim 2 in the prevention and control of bacterial wilt in plants.

6. The application according to claim 5, characterized in that, The pathogen causing bacterial wilt is one or more of sequence variants 13, 14, 15, 17, 18, 34, 44 or 57 of Ralstonia solanaceae.

7. The application according to claim 6, characterized in that, The sequence variant 13 is Ralstonia solanacearum RS180, the sequence variant 14 is Ralstonia solanacearum BY-2 and / or SSF-4, the sequence variant 15 is Ralstonia solanacearum RS58 and / or RS208, the sequence variant 17 is Ralstonia solanacearum RS397, the sequence variant 18 is Ralstonia solanacearum GMI1000 and / or TS-2, the sequence variant 34 is Ralstonia solanacearum SM-GZZC-127, RS196, RS210, RS404 and / or RS550, the sequence variant 44 is Ralstonia solanacearum RS129 and / or SG-2, and the sequence variant 57 is Ralstonia solanacearum GY-2.

8. The application according to claim 5, characterized in that, The plant in question is a tomato.

9. The application according to claim 8, characterized in that, The method includes the step of applying Acinetobacter pituitariae BB307 as described in claim 1 or the preparation as described in claim 2 to tomato plants.

10. The application according to claim 9, characterized in that, The application to the tomato plant refers to the application to the roots of the tomato plant.

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