Koreanibrio strain having a preventive and therapeutic effect on tomato bacterial wilt and use thereof

Abstract

The present invention discloses a strain of Korean Priesteria Priestia koreensis ) strain, with the deposit number CGMCC No.35027, named SY6-81. In a potted biocontrol test, the strain achieved a 39.8% control effect against tomato bacterial wilt. In a potted growth promotion test, Korean Priesteria koreaensis ( P. koreensis ) The aboveground plant height, fresh weight, root length and underground fresh weight of the SY6-81 treatment group increased by 56.3%, 42.6%, 27.7% and 43.4% respectively compared with the water control, which has the effect of promoting tomato growth.

Description

Technical Field

[0001] The invention belongs to the technical field of agricultural microorganisms and biological control, and particularly relates to a Korean Priesteria strain capable of preventing and treating tomato bacterial wilt and an application thereof. Background Art

[0002] tomato( Solanum lycopersicum Tomato, also known as tomato, sweet tomato or tomato, is an annual herbaceous crop of the genus Solanum in the Solanaceae family. Tomatoes occupy an extremely important position in global agricultural cultivation, with their cultivated area accounting for approximately 20% of the global vegetable cultivated area, highlighting their important value in the agricultural economy. As a key player in the global tomato industry, China not only ranks among the top in the world in tomato planting area and output, but also occupies an important position in tomato processing and export (Song Xuanyu et al., 2024). In recent years, China has further consolidated its dominant position in the global tomato industry by optimizing planting techniques, improving variety quality, and strengthening agricultural infrastructure construction. Tomatoes generally have high commercial traits and are deeply loved by consumers. However, they face the threat of many diseases and pests during their growth process, among which soil-borne tomato diseases are particularly concerning (PANNO et al., 2021). These diseases not only affect the yield and quality of tomatoes, but can also cause serious economic losses.

[0003] Tomato bacterial wilt is caused by the Gram-negative bacterium Ralstonia solanacearum ( Ralstonia solanacearum A typical soil-borne bacterial disease caused by Solanaceae (Solanum spp.) causes severe economic losses to Solanaceae crop-producing regions worldwide through a specific infection mechanism involving root vascular bundles. It can persist in the soil for several years and spread through irrigation water, forming a regional reservoir of pathogens (Kwak et al., 2018). Symptoms are staged: initial leaf wilting, browning and necrosis of the vascular bundles in the middle stage, and systemic plant death in the final stage. Under high temperature and high humidity conditions, the disease can reach 80% in continuously cropped fields, resulting in a sharp drop of over 50% in marketable fruit yield and, in severe cases, crop failure. Prevention and control are challenging, and once an outbreak occurs, it is difficult to eradicate in the short term (ATTIA et al., 2022).

[0004] Soilborne tomato diseases can be quickly controlled through the application of fungicides, disinfectants, and fumigants (LEE et al., 2012). Fungicides include carbendazim, thiophanate-methyl, bethiam-methyl, and thiophanate-methyl, which work by inhibiting the growth of pathogens or directly killing them (PANG et al., 2015). Disinfectants such as peracetic acid and hydrogen peroxide can be used to treat seeds or soil to reduce pathogen populations (LI Xiao, 2019). Although chemical control is effective quickly, long-term use may lead to the development of antibiotic resistance in pathogens and pose a threat to the environment and food safety (SRINIVAS et al., 2019).

[0005] Biological control uses microorganisms, plant compounds, or natural enemies to achieve environmentally friendly prevention and control, and is also a commonly used method for preventing and controlling tomato soil-borne diseases (KUMARI et al., 2020). Antagonistic microorganisms such as Bacillus, Pseudomonas, Trichoderma, Streptomyces, etc. are widely used to inhibit various plant pathogens (ZHANG et al., 2016). In addition, SynComs can also be synthesized in production, combining antagonistic bacteria, growth-promoting bacteria, and signal molecules to reshape the rhizosphere microbial network (such as the combination of Trichoderma and Bacillus to reduce the incidence of root rot by 60%). In addition, there are plant-derived compounds, such as azadirachtin, which interferes with the molting and feeding behavior of root-knot nematodes, reducing the infection rate by 50%-80%, and allicin inhibits the quorum sensing (QS) system of Ralstonia solanacearum, blocking the expression of its pathogenic genes (AYAZ et al., 2023). Biological control provides a safe and sustainable solution for tomato diseases through the ecological mode of "controlling bacteria with bacteria and insects with insects".

[0006] Peruvian tomato ( S. peruvianum ) is native to the western coast of the Andes Mountains at the border between Peru and Chile, and its ecological distribution range covers hilly and mountainous areas. As a wild relative of cultivated tomato ( S.lycopersicum ), Peruvian tomato has shown significant genetic improvement potential in improving the disease resistance, environmental adaptability, and fruit quality of cultivated varieties (TAPIA et al., 2021). Plant rhizosphere microorganisms refer to the complex microbial community formed around plant roots, which directly affects plant health, nutrient uptake, and disease resistance (NAKAYASU et al., 2022). Among them, plant disease resistance genes (such as NBS-LRR type disease resistance genes) not only directly resist pathogen invasion, but also recruit specific beneficial microorganisms by regulating root exudates (such as phenolic acids and flavonoids) (PEREIRA et al., 2023). There is no report on the screening of beneficial microorganisms from the rhizosphere of Peruvian tomato. SUMMARY

[0007] The purpose of the present application is to provide a Korean Pseudomonas strain for preventing and controlling tomato bacterial wilt and its application.

[0008] A strain of Korean Pseudomonas ( Priestia koreensis ) with the accession number CGMCC No.35027, named SY6-81.

[0009] Application of the Korean Pseudomonas ( Priestia koreensis ) in preventing and controlling tomato bacterial wilt.

[0010] A microbial pesticide for preventing and controlling tomato bacterial wilt, comprising the Korean Pseudomonas ( Priestiakoreensis ).

[0011] A Korean Priesterol ( Priestia koreensis ) fermentation broth, which is prepared from the Korean Plastidiomyces pulmonis ( Priestia koreensis ) obtained by fermentation.

[0012] A microbial fungicide for preventing and treating tomato bacterial wilt, the fungicide containing the Korean Plastidia koreaensis ( Priestia koreensis ) and / or their fermentation products.

[0013] The method for using the microbial fungicide is root irrigation.

[0014] The classification name of the Korean Priesterol SY6-81 of the present invention is: Korean Priesterol Priestia koreensis The strain was deposited at the General Microbiology Center of the China Culture Collection Administration on June 27, 2025, with a certificate of survival. The deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing. The culture deposit number is CGMCC No. 35027.

[0015] Beneficial effects of the present invention: The present invention discovered a strain of Korean Priesterol Priestia koreensis In the potted plant biocontrol test, the control effect of the fungus was 39.8%. In the potted plant growth promotion test, Korean Priesteria P.koreensis ) The aboveground plant height, fresh weight, root length and underground fresh weight of the SY6-81 treatment group increased by 56.3%, 42.6%, 27.7% and 43.4% respectively compared with the water control, which has the effect of promoting tomato growth. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a diagram showing the different levels of inhibition of Bacillus against tomato bacterial wilt pathogens.

[0017] Figure 2 Korean Priesterol ( Priestia koreensis ) Inhibitory effect of SY6-81 on Ralstonia solanacearum plate.

[0018] Figure 3 Korean Priesterol ( Priestia koreensis ) Colony morphology.

[0019] Figure 4 Korean Priesterol ( Priestia koreensis ) Phylogenetic tree of SY6-81.

[0020] Figure 5 Korean Priesterol ( Priestia koreensis ) The control effect of SY6-81 on bacterial wilt pathogen in tomatoes.

[0021] Figure 6 K. koreensis SY6-81 on tomato against R. solanacearum. Priestia koreensis Table 1. Control effect (disease index) of K. koreensis SY6-81 on tomato against R. solanacearum.

[0022] Figure 7 K. koreensis SY6-81 on tomato growth. P. koreensis Table 2. Effect of K. koreensis SY6-81 on tomato growth. DETAILED DESCRIPTION

[0023] For the purpose of facilitating the understanding of the present application, a more comprehensive description will be given below. However, the present application can be realized in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present application more thorough and comprehensive.

[0024] Example 1 Screening and activity identification of biocontrol bacillus strains against tomato bacterial wilt

[0025] (1) Strain screening

[0026] In this embodiment, bacillus strains were isolated from the rhizosphere soil of Peruvian tomato LA3858 by the following method:

[0027] 5 g of the rhizosphere soil sample of Peruvian tomato LA3858 was taken into a sterile triangular conical flask, 50 mL of physiological saline solution (0.85% NaCl) was added, and the mixture was placed in a shaking bed for 20 min of vibration culture at 28°C and 250 r / min, followed by 10 min of room temperature standing, to prepare a rhizosphere soil suspension; the above suspension was water-bathed at 80°C for 30 min, and gradient diluted with physiological saline to 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 times. 100 uL of each of the above 10 -4 , 10 -5 , 10 -6 times gradient diluted bacterial suspension was uniformly coated on a sterile LB culture medium, and the plate was inverted and cultured in a 30°C constant temperature incubator for 3 days; the colony size was checked every day, and the number of colonies was counted when independent colonies grew on the plate, 100 single colonies were selected by systematic sampling method, covering typical morphological characteristics, and after each target colony was purified and cultured on an LB plate, subsequent work was carried out;

[0028] R. solanacearum stored in -80℃ cryotubes was streaked on TTC plates and incubated for 48h. Pathogenic strong colonies (colony center pink and white edge wide, like flowing) were picked and incubated on TTC plates for 42h. The single colonies on the TTC plates were inoculated into FY liquid medium and incubated at 28℃, 200r / min for 48h.

[0029] LB plates were used to activate the LA3858 rhizosphere Bacillus stored in -80℃ cryotubes. Single colonies were picked and incubated in LB liquid medium at 28℃, 200r / min for 48h to obtain the bacterial suspension.

[0030] The OD value of the pathogenic bacteria was measured. The OD600 of the bacterial suspension was adjusted to 0.8. 200μL of the bacterial suspension was added to 15mL of FY solid medium. The solid medium was cooled to 50℃ before adding the bacterial suspension to prepare the target pathogenic bacteria plate.

[0031] After the plate was completely solidified, the test bacterial suspension was inoculated and dried. The plate was inverted and incubated in a 28℃ incubator. The blank control was inoculated with the test bacterial suspension. The diameter of the inhibition zone was measured after 48h. Each group had three replicates. The diameter of the inhibition zone was calculated and the antagonistic effect was statistically analyzed.

[0032] Evaluation criteria: A-level antagonism was characterized by complete restriction of pathogen growth, forming a large and clear inhibition zone. B-level antagonism was characterized by significant inhibition of pathogen growth, forming a clear inhibition zone. C-level antagonism was characterized by low-level inhibition of pathogen growth, with a blurred inhibition zone. Figure 1 .

[0033] The LA3858 rhizosphere recruited Bacillus was tested for its ability to inhibit R. solanacearum. Forty-two strains were found to have inhibitory activity against R. solanacearum. Among them, B. velezensis ( B. velezensis ), B. amyloliquefaciens ( B. amyloliquefaciens ), B. cereus ( B. cereus ), B. thuringiensis ( B. thuringiensis ), and P. koreensis ( Priestia koreensis ) had strong inhibitory effects, forming a large and clear inhibition zone. Among them, P. koreensis ( Priestia koreensis ) was a new species that had not been reported before. It was named SY6-81. Its inhibitory effect on R. solanacearum plates is shown in Figure 2 .

[0034] (2) Strain identification

[0035] The colony morphology on LB solid medium is shown in FIG. 1, and the obtained strain colony is light yellow and irregular round in shape with neat edges. Figure 3

[0036] Subsequently, the sequence of the strain was determined by 16S sequence fragment (amplification primer and sequencing primer were 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' and 1492R: 5'-GGTTACCTTGTTACGACTT-3'), and the determination result is shown in the sequence table; sequence 16S rRNA sequence homology analysis, phylogenetic analysis ( Figure 4 ), through BLAST homology comparison, we determined that the closest species of the strain is Korean Pseudomonas ( Priestia koreensis ). The sequence determination result is shown in:

[0037] ​

[0038] Example 2 Korean Priesterol ( Priestia koreensis ) Potted control experiment on tomato bacterial wilt

[0039] Culture of the fermentation broth: Korean Priesterol ( Priestia koreensis ) SY6-81 strain was activated using LB plates and cultured in an incubator at 28°C for 24 hours. A single colony was picked and inoculated into LB liquid medium and shaken at 28°C, 180 r / min.

[0040] After 24 h of culture, 1 mL of seed solution was inoculated into 100 mL of culture medium and cultured at 28 °C, 180 r / min, with shaking for 3 days. The concentration was 1×10 8 cfu / mL, and the fermentation broth of active strains was obtained.

[0041] Fermentation culture of pathogenic bacteria: Ralstonia solanacearum stored in a glycerol tube at -80°C was inoculated on a TTC plate to form a single colony. A red and slightly mobile colony was picked and inoculated into FY liquid medium. The culture was shaken at 28°C and 180 rpm for 24 h to prepare a seed solution. 1 mL of the seed solution was inoculated into 100 mL of culture medium and shaken at 28°C and 180 rpm for 3 days to a concentration of 1 × 10 8 cfu / mL, and obtain the pathogen fermentation culture fluid.

[0042] Potted biocontrol effect test: A total of three treatments were set up, treatment 1 (clear water), treatment 2 (Zhongshengmycin), and treatment 3 (SY6-81 bacteria, marked as SY6-81). Tomato seeds were sown in seedling pots. When the seedlings grew to two leaves and one heart, 30 seedlings were selected for each treatment and divided into 3 replicates, with 10 seedlings in each replicate. They were transplanted into disposable nutrient pots and irrigated with 50 mL of clear water, sterile LB liquid medium, 1000 times Zhongshengmycin, and bacterial fermentation liquid, respectively. After 5 days, the fermentation liquid of the bacterial wilt pathogen was inoculated using the root wound irrigation method. The disease development was recorded on the 21st day after the inoculation of the bacterial wilt pathogen and the control effect was calculated. The formulas are shown in (1), (2), and (3):

[0043]

[0044] Growth promotion experiment: Three treatments were set up: Treatment 1 (clear water), Treatment 2 (LB medium), and Treatment 3 (SY6-81). Tomato seeds were sown in seedling pots. When the seedlings grew to two leaves and one heart, 30 seedlings were selected from each treatment and divided into three replicates, with 10 seedlings per replicate, for a total of 30 seedlings. These seedlings were transplanted into disposable nutrient pots, and plant growth was recorded after 21 days.

[0045] The incidence of tomato bacterial wilt is graded according to the technical regulations for identification of tomato bacterial wilt resistance NY / T 1858.4-2010:

[0046] Level 0: No wilt symptoms;

[0047] Level 1: One leaf wilts;

[0048] Level 2: 2-3 leaves wilt;

[0049] Level 3: All leaves except the top 2-3 are wilted;

[0050] Level 4: The leaves of the entire plant wilt.

[0051] In the potted biocontrol test, the water group began to become ill 7 days after inoculation, with the leaves of the plants shrinking due to water loss and accompanied by plant wilting symptoms; after 14 days, the incidence of the water group reached 50%, and the disease index reached level 3; after 21 days, the water group was completely ill, with the disease index reaching level 4 and the incidence reaching 100%, with leaves shrinking and tomato stems turning yellow due to lack of water. In severe cases, the plants died. Figure 5 When the water control reached the susceptible level, the disease level of all treatments was investigated and the average disease index of all treatments was statistically analyzed. The disease index of the water control was 66.9%, the disease index of the positive control Zhongshengmycin was 16.7%, and the disease index of Korean Priesterol ( P. koreensis ) The disease index of SY6-81 treatment was 40.3%, and the results were as follows Figure 6 Zhongshengmycin, Korean Priesterol ( P.koreensis ) The control effects of SY6-81 were 75.1% and 39.8% respectively.

[0052] In the potted plant growth promotion experiment, four growth indicators, namely, plant height above ground, fresh weight above ground, root length and fresh weight below ground, were investigated and statistically analyzed. Figure 7 As shown. Korean Priesterol ( P.koreensis ) The aboveground plant height, fresh weight, root length and underground fresh weight of the SY6-81 treatment group increased by 56.3%, 42.6%, 27.7% and 43.4% respectively compared with the water control. P. koreensis )SY6-81 has a growth-promoting effect on tomato growth.

[0053] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A strain of Korean Priesterol ( Priestia koreensis ) strain, with the deposit number CGMCC No.35027, named SY6-81.

2. The Korean Priesteria bacteria according to claim 1 ( Priestia koreensis ) in the prevention and control of tomato bacterial wilt.

3. A microbial pesticide for preventing and controlling tomato bacterial wilt, characterized in that: Containing the Korean Priesterbacterium according to claim 1 ( Priestia koreensis ).

4. A Korean Priesterol ( Priestia koreensis ) fermentation broth, characterized in that The Korean Priesterol bacterium ( Priestia koreensis ) obtained by fermentation.

5. A microbial fungicide for preventing and treating tomato bacterial wilt, characterized in that: The bactericide contains the Korean Priesteria koreaensis ( Priestia koreensis ).

6. The microbial fungicide for preventing and controlling tomato bacterial wilt according to claim 5, characterized in that: The method for using the microbial fungicide is root irrigation.

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