Candidatus liberibacter asiaticum and method for detecting same

Abstract

The present application relates to a strain of Caulobacter korsakowii and application thereof. The present application finds that the strain Ya-17 of Caulobacter korsakowii has salt tolerance, and has the effects of promoting germination and growth on crops, such as wheat, in a salt environment, and thus can be applied to farmland with high salt content to reduce the damage of the salt environment to crops. Shouchella clausii The present application relates to a strain of Caulobacter korsakowii and application thereof. The present application finds that the strain Ya-17 of Caulobacter korsakowii has salt tolerance, and has the effects of promoting germination and growth on crops, such as wheat, in a salt environment, and thus can be applied to farmland with high salt content to reduce the damage of the salt environment to crops.

Description

Technical Field

[0001] This invention relates to the field of microbiology, and particularly to a salt-tolerant microorganism. Background Technology

[0002] Soil salinization is a significant global issue affecting agricultural production and the ecological environment. Its formation is influenced by a combination of natural and anthropogenic factors, including drought, high evaporation rates, high groundwater mineralization, improper irrigation, and inappropriate fertilization. Globally, the area of ​​saline-alkali land exceeds 950 million hectares and continues to expand, seriously threatening food security and ecological stability. In China, saline-alkali land is widely distributed, mainly concentrated in the Songnen Plain in Northeast China, the inland basins of Northwest China, the coastal areas of North China, and the Yellow River Delta, covering a total area of ​​over 100 million hectares. Of this, approximately 30 million hectares are exploitable. Salt-alkali stress causes multiple damages to plants, including osmotic stress, ion toxicity, and oxidative stress, severely impacting crop growth and development. Summary of the Invention

[0003] One of the inventions provides *Schönleinii krais* ( Shouchella clausii The strain Ya-17 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 50183.

[0004] The second invention provides a composition comprising the *Schönleinii krais* strain Ya-17 as described in the first invention.

[0005] In one specific embodiment, the dosage form of the composition is one of a suspension, powder, and granules.

[0006] In one specific embodiment, the dosage form of the composition is an oil suspension or a wettable powder.

[0007] The third invention provides the use of the *Schönleinii Klaus* strain Ya-17 as described in the first invention or the composition as described in any one of the second inventions for salt tolerance.

[0008] In one specific embodiment, the application is the use of the *Schönleinii Klaus* strain Ya-17 or the composition thereof in promoting the germination or growth of crops in a saline environment.

[0009] In one specific embodiment, the crop is at least one of the Poaceae family of crops.

[0010] In one specific embodiment, the crop is wheat ( Triticum aestivum ),barley( Hordeum vulgare L), oats ( Avena sativaL.), corn ( Zea mays L.), rice ( Oryza sativa L.) and sorghum ( Sorghum bicolor At least one of (L.) Moench.

[0011] Beneficial effects of the present invention: The present invention discovered *Schönleinii krais* (… Shouchella clausii Strain Ya-17 has salt tolerance and can promote germination and growth of crops, such as wheat, in saline environments. Therefore, it can be applied to farmland with high salt content to reduce the damage of saline environment to crops.

[0012] Strain Preservation: The strain screened in this invention was named Ya-17. This strain is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 50183, deposited on August 31, 2026, at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Its systematic classification is *Schönleinii* clade. Shouchella clausii . Attached Figure Description

[0013] Figure 1 The image shows a typical plate from which colonies grew after a soil sample was plated on a medium containing 10% NaCl.

[0014] Figure 2 The results show the growth of wheat plants after treatment with strain Ya-17 in a saline environment. In the figures, a represents the water control, b represents the salt treatment control, and c represents the plants treated with strain Ya-17 in a saline environment.

[0015] Figure 3 The growth of strain Ya-17 on LB solid medium with different salt concentrations is shown.

[0016] Figure 4 The growth curves of strain Ya-17 in LB liquid medium with different salt concentrations are shown.

[0017] Figure 5 A phylogenetic tree of strain Ya-17, constructed based on the gyrB gene, is shown. Detailed Implementation

[0018] The present invention will be further described in detail below through preferred embodiments, but these embodiments do not constitute a limitation thereof.

[0019] Unless otherwise specified, the reagents and other materials used in the embodiments of this invention can be purchased commercially.

[0020] LB liquid medium: 10.0 g peptone, 5.0 g yeast extract, 10.0 g sodium chloride, diluted to 1.0 L with distilled water, incubated at 121°C for 20 min.

[0021] LB liquid medium containing 10% NaCl: 10.0 g peptone, 5.0 g yeast extract, 100.0 g sodium chloride, diluted to 1.0 L with distilled water, incubated at 121°C for 20 min.

[0022] LB liquid medium containing 15% NaCl: 10.0 g peptone, 5.0 g yeast extract, 150.0 g sodium chloride, diluted to 1.0 L with distilled water, incubated at 121°C for 20 min.

[0023] LB liquid medium containing 20% ​​NaCl: 10.0 g peptone, 5.0 g yeast extract, 200.0 g sodium chloride, diluted to 1.0 L with distilled water, incubated at 121°C for 20 min.

[0024] LB liquid medium containing 25% NaCl: 10.0 g peptone, 5.0 g yeast extract, 250.0 g sodium chloride, diluted to 1.0 L with distilled water, incubated at 121°C for 20 min.

[0025] LB liquid medium containing 30% NaCl: 10.0 g peptone, 5.0 g yeast extract, 300.0 g sodium chloride, diluted to 1.0 L with distilled water, incubated at 121°C for 20 min.

[0026] LB solid medium and LB solid medium containing 10%, 15%, 20%, 25% and 30% NaCl: Add 15 g / L agar powder to LB liquid medium and LB liquid medium containing 10%, 15%, 20%, 25% and 30% NaCl respectively, and incubate at 121 degrees Celsius for 20 min.

[0027] wheat( Triticum aestivum The seeds are Shijiazhuang No. 9. Example 1: Isolation of strains

[0028] Soil samples were collected in Cangzhou, Hebei Province in August 2024. A certain amount of soil sample was weighed, added to sterile distilled water, and mixed thoroughly to prepare a soil suspension. This suspension was then serially diluted, and 200 μL of each concentration of the soil suspension was evenly spread onto LB agar plates containing 10% NaCl. The plates were incubated upside down at 30°C for 2 to 3 days. Some colonies showed good growth, exhibiting round shapes with neat or irregular edges, and colors including yellow, white, and red. Some plates with colonies showed growth as shown in the image. Figure 1As shown. Colonies of different morphologies were selected and streaked onto LB agar plates for purification. The purified single colonies were stored in a 25% glycerol-80°C freezer, resulting in a total of 84 strains, which were then numbered. Example 2: Screening of salt-tolerant bacterial strains that promote wheat seed germination under salt stress

[0029] Each isolated strain was inoculated into LB liquid medium, shaken, and centrifuged. The bacterial cells were collected, and bacterial suspensions of each strain were prepared using 150 mmol / L sterile NaCl aqueous solution. The concentration of each bacterial suspension was adjusted to OD0.05. 600 =0.8. Wheat seeds were disinfected with a 10% NaClO aqueous solution and then soaked in sterile water overnight. Ten seeds were then evenly distributed in each petri dish, with 10 mL of bacterial suspension of each isolated strain added to each dish. The treatment with 10 mL of sterile water was used as the water control (CK0), and the treatment with 10 mL of 150 mmol / L NaCl aqueous solution was used as the salt control (CK1). Each petri dish was a replicate, and each treatment was replicated in triplicate. Germination of wheat seeds was investigated on days 4 and 8, and root and shoot lengths were measured. Germination potential, germination rate, germination index, vigor index, seedling length, salt tolerance index, and relative salt damage rate were calculated using the following formulas. The results are shown in Table 1.

[0030] Germination potential (%) = Number of seeds germinated on day 4 × 100 / Number of seeds tested.

[0031] Germination rate (%) = Number of seeds germinated on day 8 × 100 / Number of seeds tested.

[0032] Germination index GI = ∑(Gt / Dt), where: Gt is the number of germinated days t; Dt is the number of germination days.

[0033] Seedling length = root length + bud length.

[0034] Vitality Index VI = GI × Seedling Length.

[0035] Salt tolerance index = Vi salt × 100 / Vi water, where: Vi salt is the seed viability index under salt stress; Vi water is the seed viability index under sterile water treatment.

[0036] Relative salt damage rate (%) = (germination rate of clean water control - germination rate of treatment) × 100 / germination rate of clean water control.

[0037] The screening results showed that most isolated strains did not promote germination of wheat seeds under salt treatment. However, one strain, Ya-17, exhibited excellent germination and growth-promoting effects on wheat seeds under salt treatment. Its data, compared with the water control and the salt treatment control, are shown in Table 1. (Photos are available in the table below.) Figure 2 .

[0038] Table 1

[0039] Note: Data in the table are mean ± standard error. Different letters after the data in the same row indicate significant differences at the P<0.05 level as determined by Duncan's new multiple range test.

[0040] According to Table 1 and Figure 2 The results showed that strain Ya-17 significantly promoted the germination and growth of wheat seeds under saline conditions. Compared with the salt-treated CK1, the germination potential, germination rate, germination index, vigor index, seedling length, and salt tolerance index of wheat seeds treated with Ya-17 were all improved, with improvement rates of 13.0%, 16.8%, 23.3%, 37.1%, 12.3%, and 38.6%, respectively. In particular, the germination potential, germination rate, germination index, and salt tolerance index of seeds treated with Ya-17 were significantly higher than those of the salt-treated CK1, and the relative salt damage rate was reduced by 16.1% compared with the salt-treated CK1, indicating that the Ya-17 treatment significantly reduced the salt damage to seeds and seedlings. Example 3: Salt tolerance determination of strain Ya-17 1. Growth of strain Ya-17 in solid culture media with different salt concentrations

[0041] The Ya-17 strain was streaked onto LB solid medium containing 10%, 15%, 20%, 25%, and 30% NaCl, respectively, and incubated at 37°C for 48 h. Photos were taken, and one set of replicate photos is shown below. Figure 3 The growth of salt-tolerant strains on culture media with different salt concentrations was observed, as shown in Table 2.

[0042] According to Table 2 and Figure 3 The results show that Ya-17 has strong salt tolerance. It can still maintain vigorous growth at salt concentrations of 10% and 15%, and although its growth is somewhat inhibited at salt concentrations of 20% to 30%, it can still grow well.

[0043] Table 2

[0044] Note: "++" indicates that the strain grows normally and vigorously on a salt plate; "+" indicates that the strain can grow on a salt plate but its growth is inhibited; "-" indicates that the strain does not grow much on a salt plate. 2. Growth of strain Ya-17 in liquid culture media with different salt concentrations

[0045] Strain Ya-17 was activated in LB liquid medium, and the activated bacterial culture was adjusted to OD. 600=0.8, to obtain the inoculum solution. The inoculum solution was inoculated at a volume ratio of 1% into LB liquid medium containing 10%, 15%, 20%, 25%, and 30% NaCl, respectively, and cultured at 37°C with shaking at 170 rpm. OD values ​​were measured at 0 h, 4 h, 8 h, 12 h, 24 h, 36 h, and 48 h. 600 Standard LB liquid medium was used as a control. Three replicates were set up. Growth curves were plotted, see [link to diagram]. Figure 4 .

[0046] according to Figure 4 The growth curves show that at a 10% salt concentration, Ya-17 showed almost no growth from 0 to 8 hours, began to grow at 8 hours, and entered a rapid growth phase after 12 hours, with a rapid increase in bacterial count. At 24 hours, the bacterial count was close to that of the control. An independent samples t-test using SPSS software (P=0.081) showed no significant difference in bacterial count between LB liquid medium containing 10% NaCl and ordinary LB liquid medium after 24 hours, with the count reaching a stationary phase after 24 hours. This indicates that a 10% salt concentration had some inhibitory effect on bacterial growth in the early stages, but the inhibitory effect weakened in the later stages, allowing the strain to gradually adapt to the salt concentration and begin to grow well, adapting to the salt environment more quickly. At a 15% salt concentration, the bacterial count entered a rapid growth phase after 24 hours, reaching a level close to that of the control colonies at 48 hours (P=0.415), indicating that Y-17 could still grow at a 15% salt concentration, but the adaptation period to this salt concentration was relatively long. At salt concentrations of 20% and above, the bacterium could hardly grow. Example 4: Classification and identification of Ya-17 strain

[0047] Genomic DNA was extracted from strain Ya-17 using a bacterial genomic DNA extraction kit (Beijing Solarbio Science & Technology Co., Ltd.). Using gyrB-F (as shown in SEQ ID No. 1) and gyrB-R (as shown in SEQ ID No. 2) as primers and the extracted genomic DNA as a template, PCR amplification of the gyrB gene was performed. The amplified products were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing, and the sequencing results are shown in SEQ ID No. 3. The sequence shown in SEQ ID No. 3 was compared with the NCBI database using BLAST, and sequences with high similarity were selected. The results showed that the gyrB gene of Ya-17 is similar to that of *Schönleinii* (*Schönleinii*). Shouchella clausii The closest sequence was found to be gyrB. Multiple sequence comparison analysis of nucleic acid sequences was performed using MEGA11 software. A phylogenetic tree of Ya-17 was constructed based on the gyrB gene using the Neighbor-joining algorithm. (See [link to documentation]). Figure 5 .from Figure 5 It can be seen that strain Ya-17 is related to *Schönleinii krais* (…). Shouchella clausii They all gathered together.

[0048] In summary, strain Ya-17 is systematically classified as *Showerella clausti* (…). Shouchella clausii ).

[0049] The Ya-17 strain was deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 50183, on August 31, 2026. The deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Its systematic classification is *Schuchera clade*. Shouchella clausii .

Claims

1. *Schönleinii* var. *c.* Shouchella clausii The strain Ya-17 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 50183.

2. A composition comprising the *Schönleinii krais* strain Ya-17 as described in claim 1.

3. The composition according to claim 2, characterized in that, The composition is in the form of a suspension, powder, or granules.

4. The composition according to claim 2, characterized in that, The composition is in the form of an oil suspension or a wettable powder.

5. The use of the *Schönleinii* strain Ya-17 as described in claim 1 or the composition as described in any one of claims 2 to 4 for salt tolerance.

6. The application according to claim 5, characterized in that, The application refers to the use of the *Schönleinii Klaus* strain Ya-17 or the composition thereof in promoting the germination or growth of crops in a saline environment.

7. The application according to claim 6, characterized in that, The crop is at least one of the Poaceae family of crops.

8. The application according to claim 6, characterized in that, The crop mentioned is wheat ( Triticum aestivum ),barley( Hordeum vulgare L), oats ( Avena sativa L.), corn ( Zea mays L.), rice ( Oryza sativa L.) and sorghum ( Sorghum bicolor At least one of (L.) Moench.

Images