Bacillus siamensis PL55 and application thereof in prevention and treatment of Korla pear brown rot

Abstract

The invention discloses bacillus siamensis PL55 and application of the bacillus siamensis PL55 in prevention and treatment of brown rot of Korla pear, and belongs to the technical field of microbial preparations. The strain is preserved in the China Center for Type Culture Collection on May 19, 2025, and the preservation number of the strain is CCTCC NO: M 20251089. The biocontrol bacterium PL55 disclosed by the invention has broad-spectrum antibacterial activity, has a good antagonistic effect on pear brown rot pathogen, pear valsa ceratosperma, apple tree valsa ceratosperma, walnut tree valsa ceratosperma and cotton fusarium wilt pathogen, and is a potential biological agent for preventing and treating various plant diseases. In addition, the bacillus siamensis also has good capabilities of producing protease, synthesizing iron carriers, dissolving inorganic phosphorus, dissolving organic phosphorus and fixing nitrogen. The biocontrol bacterium bacillus siamensis PL55 has a wide development space, and a new candidate strain is provided for biological control of the five diseases.

Description

Technical Field

[0001] This invention relates to the field of microbial preparation technology, and in particular to a strain of Bacillus simonii PL55 and its application in the control of brown rot disease in Korla fragrant pear. Background Art

[0002] Korla fragrant pear is an ancient and superior variety from Xinjiang, with a cultivation history of 1400 years. It has thin skin, crisp flesh, is juicy and sweet, has few stone cells, a rich aroma, strong resistance to adverse conditions, is rich in nutrients, and is extremely resistant to storage. Korla fragrant pears are highly regional, mainly distributed in Bayingolin Mongol Autonomous Prefecture and Aksu Prefecture of Xinjiang, exhibiting distinct regional characteristics.

[0003] Brown rot in Korla fragrant pears is a significant disease affecting pear production. In recent years, with climate change, brown rot has occurred and spread rapidly in all Korla pear-growing areas, severely impacting the quality and yield of the pears and significantly affecting fruit growers' income. Currently, effective control methods for pear brown rot include chemical pesticides, agricultural control, and biological control. While traditional chemical control methods and techniques can suppress plant diseases to some extent, they also cause environmental pollution, which is detrimental to ecological protection. Furthermore, food residues can affect public health. Agricultural control requires substantial human and material resources. Therefore, developing environmentally friendly and sustainable biological control measures is receiving increasing attention.

[0004] Biological control utilizes beneficial microorganisms found in nature to suppress and control diseases, which is not only environmentally friendly but also aligns with the concept of "green plant protection." Current research both domestically and internationally has shown that Bacillus subtilis has a strong inhibitory effect on postharvest pear brown rot, and this inhibitory effect increases with increasing concentration. Therefore, screening and obtaining antagonistic microbial strains that can effectively inhibit pear brown rot is of paramount importance for promoting green control strategies for this disease, reducing reliance on chemical pesticides, and fostering sustainable development. Summary of the Invention

[0005] The purpose of this invention is to provide a strain of Bacillus sicca PL55 and its application in the control of brown rot in Korla fragrant pear, so as to solve the problems existing in the prior art.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] One of the technical solutions of this invention provides a strain of Bacillus siamensis PL55, which was deposited at the China Center for Type Culture Collection on May 19, 2025, at Wuhan University, Wuhan, China, with accession number CCTCC NO: M 20251089.

[0008] The second technical solution of the present invention provides a microbial inoculant, including the Bacillus sicca PL55.

[0009] The third technical solution of the present invention provides the application of the Bacillus tataricus PL55 or the microbial agent in the prevention and / or treatment of brown rot disease of Korla fragrant pear caused by M. yunnanensis.

[0010] The fourth technical solution of the present invention provides the application of the Bacillus sicca PL55 or the microbial agent in the prevention and / or treatment of plant diseases caused by V. pyri (the pathogen causing canker of Korla fragrant pear), F. oxysporum (the pathogen causing wilt of cotton), V. mali (the pathogen causing canker of apple trees), and / or V. nivea (the pathogen causing canker of walnut trees).

[0011] The fifth technical solution of the present invention is a method for preventing and controlling plant diseases, which involves spraying plants with the Bacillus simonii PL55 or the microbial agent to prevent and control plant diseases.

[0012] The plant diseases mentioned include those caused by *Brassica juncea*, *Brassica juncea*, *Brassica fusca*, *Brassica juncea*, *Brassica juncea*, and / or *Brassica juncea*.

[0013] The sixth technical solution of the present invention is the application of Bacillus sicca PL55 or the microbial agent in the production of proteases, iron carriers, and the decomposition of inorganic and organic phosphorus.

[0014] Based on the above technical solution, the present invention has the following technical effects:

[0015] The biocontrol agent PL55 for brown rot of Korla fragrant pear of this invention has broad-spectrum antibacterial activity, exhibiting good antagonistic effects against various pathogens including those causing brown rot of Korla fragrant pear, canker of Korla fragrant pear, wilt of cotton, canker of apple, and canker of walnut. It is a potential biological agent for the control of these diseases. Furthermore, PL55 also possesses good abilities in protease production, iron carrier synthesis, inorganic phosphorus solubilization, organic phosphorus solubilization, and nitrogen fixation. PL55 has broad development potential and provides a candidate resource for the control of brown rot of Korla fragrant pear and other plant diseases. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 The strain PL55 exhibits antagonistic activity against *Pterocarya stenoptera*, the causal agent of brown rot in Korla fragrant pear. In the diagram, A represents normally growing *Pterocarya stenoptera* colonies; B represents *Pterocarya stenoptera* colonies after antagonism by strain PL55.

[0018] Figure 2 The culture morphology of strain PL55 on LB medium.

[0019] Figure 3 Phylogenetic tree of strain PL55 based on 16S rDNA.

[0020] Figure 4 The effect of PL55 aseptic fermentation filtrate on colony diameter and inhibition rate of brown rot pathogens in Korla fragrant pear.

[0021] Figure 5 This study investigated the protective and therapeutic effects of sterile fermentation filtrate of strain PL55 against brown rot disease in Korla fragrant pear. Specifically, A represents a control group inoculated only with *Pyrrosia lingua* without the addition of sterile fermentation filtrate of strain PL55; B represents the therapeutic effect of inoculation with *Pyrrosia lingua* followed by sterile fermentation filtrate of strain PL55; C represents the protective effect of inoculation with sterile fermentation filtrate of strain PL55 followed by inoculation with *Pyrrosia lingua*; and D represents a blank control group without inoculation with either *Pyrrosia lingua* or sterile fermentation filtrate of strain PL55.

[0022] Figure 6 The protective and therapeutic effects of strain PL55 on brown rot disease of Korla fragrant pear fruit were studied, including the length of lesions.

[0023] Figure 7 The study investigated the inhibitory effects of strain PL55 on five important pathogenic fungi in Xinjiang.

[0024] Figure 8 The growth-promoting characteristics of strain PL55 are shown. A represents CAS agar medium, B represents Assumption medium, C represents casein medium, D represents inorganic phosphorus medium, and E represents organic phosphorus medium. Detailed Implementation

[0025] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0026] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any intermediate value within a stated value or stated range and any other stated value or intermediate value within the stated range is also encompassed by the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0027] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0028] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This application specification and embodiments are merely exemplary.

[0029] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0030] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field, and the reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.

[0031] This invention provides a strain of Bacillus siamensis PL55, which was deposited on May 19, 2025, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M 20251089.

[0032] This invention also provides a microbial agent, including the Bacillus sicca PL55.

[0033] This invention also provides the application of the Bacillus tataricus PL55 or the microbial agent in the prevention and / or treatment of brown rot disease of Korla fragrant pear caused by M. yunnanensis.

[0034] This invention also provides the application of the Bacillus sicca PL55 or the microbial agent in the prevention and / or treatment of plant diseases caused by Bacillus thuringiensis var. chinensis, Bacillus wiltus, Bacillus thuringiensis var. chinensis, and / or Bacillus thuringiensis var. chinensis.

[0035] This invention also provides a method for preventing and controlling plant diseases, which involves spraying plants with the Bacillus sicca PL55 or the microbial agent to prevent and control plant diseases;

[0036] The plant diseases mentioned include those caused by *Brassica juncea*, *Brassica juncea*, *Brassica fusca*, *Brassica juncea*, *Brassica juncea*, and / or *Brassica juncea*.

[0037] This invention also provides the application of the Bacillus simonii PL55 or the microbial agent in the production of proteases, siderophores, and the decomposition of inorganic and organic phosphorus.

[0038] Example 1

[0039] 1. Experimental Methods

[0040] 1.1 Isolation and Screening of Biocontrol Bacteria

[0041] Test samples: Branches, bark, and leaves of Korla fragrant pear were used for the isolation of biocontrol bacteria. Collection information is shown in Table 1. Korla fragrant pear fruits were collected from the fragrant pear orchard of Tarim University (81°17′23″, 40°32′53″) for the efficacy test of biocontrol bacteria against brown rot fungus of Korla fragrant pear.

[0042] Table 1

[0043] Collection time Collection location and coordinates Number of specimens collected 2024.05 80°39′51″, 40°37′20″, Tamen Town, Alar City, First Division 11 2024.05 First Division, Alar City, Tarim University, 81°17′22″, 40°32′53″ 15 2024.05 81°37′26″, 40°40′40″, Xinjingzi Town, Alar City, First Division 13 2024.08 Korla City, Bayingolin Mongol Autonomous Prefecture, 86°0′31″, 41°36′50″ 16 2024.08 Korla City, Bayingolin Mongol Autonomous Prefecture, 86°5′39″, 41°40′45″ 11 2024.08 The Third Division of Tumushuke City: 79°8′24″, 39°49′34″ 18

[0044] The dilution separation method was used. The collected samples were washed with sterile water, air-dried, and then cut to 0.5 cm in a clean bench. 2 After thorough disinfection with 75% alcohol for 1 minute and 1% sodium hypochlorite for 3 minutes, the tissue was rinsed three times with sterile water. The final rinse was then applied to a plate to check for complete disinfection. The disinfected healthy Korla pear tree tissue was ground into a paste using a sterile mortar and pestle, diluted with sterile water to obtain a tissue homogenate. The homogenate was then diluted 10 times... -1 10 -2 10 -3 10 -4 Then, using a sterile spreader, the culture was evenly spread onto LB agar plates, with four replicates for each concentration. The culture medium was incubated at 28°C for 1 day. Purification was performed by streak plating based on colony color and morphology. The purified single colonies were then stored in glycerol tubes at -20°C.

[0045] The biocontrol bacteria were screened using the plate confrontation method. 5 mm diameter *M. yunnanensis* (from the Green Air Defense Laboratory of Tarim University) were inoculated in the center of PDA plates, and purified biocontrol bacteria were inoculated 2.5 cm from the edge of the mycelial cake. Four biocontrol mycelial cakes were inoculated on each plate. Plates inoculated only with *M. yunnanensis* served as a control. The plates were incubated in the dark at 25°C for 8 days. Each treatment was repeated four times. After the control colonies had completely covered the plate, the colony diameter was measured using the cross-crossing method, and the inhibition rate was calculated.

[0046] Inhibition rate = [(control colony diameter - treated colony diameter) / control colony diameter] × 100%.

[0047] In this embodiment, 55 bacterial strains were isolated from 129 pear tree tissue samples using the dilution separation method. After plate confrontation screening, 26 strains were found to have antagonistic effects against the rot pathogen of Korla fragrant pear, among which one strain showed the best antagonistic effect against M. yunnanensis. Figure 1 It achieved an inhibition rate of 88.18% and was named PL55.

[0048] 1.2 Identification of biocontrol strain PL55

[0049] Morphological identification: Biocontrol strain PL55 was streaked on LB medium and incubated at 28℃ for 3 days. The morphological characteristics of the colonies on LB plates were observed according to Berger's Manual of Bacterial Identification (Buchanan Berger, 1984) and the Manual of Systematic Identification of Common Bacteria (Cai Miaoying and Dong Xiuzhu, 2001).

[0050] DNA was extracted from the biocontrol strain PL55 using a bacterial DNA extraction kit. PCR amplification was performed using universal bacterial primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3'). The PCR reaction system was as follows: 95℃ pre-denaturation for 3 min, 95℃ denaturation for 45 s, 55℃ annealing for 45 s, 72℃ extension for 90 s, 30 cycles of 72℃ extension for 5 min. The amplified products were detected by 1% agarose gel electrophoresis, then excised and sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. The sequencing results were subsequently compared for homology in GenBank and NCBI databases, and a phylogenetic tree was constructed using MEGA11 software.

[0051] Colony morphology characteristics of strain PL55: After culturing on LB medium for 1 day, the colonies of strain PL55 are milky white, opaque, with regular or slightly wavy edges, and the surface is dry or slightly wrinkled. Figure 2 ).

[0052] Molecular biological identification of strain PL55: Sequencing results showed that the 16S rDNA of strain PL55 was 1433 bp in length. Comparison with GenBank sequences revealed that strain PL55 shared the highest homology with *Bacillus sicca*, with a similarity exceeding 99%. Phylogenetic tree construction using MEGA11 showed that it clustered in one clade. Figure 3 Based on morphological and physiological-biochemical characteristics, strain PL55 was identified as Bacillus siamensis.

[0053] This invention discloses a strain of Bacillus siamensis PL55, which was deposited at the China Center for Type Culture Collection (CCTCC) on May 19, 2025, at Wuhan University, Wuhan, China, with accession number CCTCC NO: M 20251089.

[0054] 1.3 Preparation of aseptic fermentation filtrate of strain PL55

[0055] The biocontrol strain PL55 was purified by streaking in LB medium in a clean bench. A small amount of the purified biocontrol strain was transferred to 100 ml of liquid LB and cultured at 37°C and 120 rpm for 2 days to obtain the biocontrol strain fermentation broth. The fermentation broth was transferred to a centrifuge tube and centrifuged at 4°C and 13000 rpm for 10 min. The supernatant was then filtered through a 0.22 μm pore size microporous membrane to obtain the biocontrol strain PL55 fermentation filtrate.

[0056] 1.4 Inhibitory effect of sterile fermentation filtrate of strain PL55 on mycelial growth of *M. yunnanensis*, the causal agent of brown rot in Korla fragrant pear.

[0057] The aseptic fermentation filtrate of strain PL55 was poured into 100ml Erlenmeyer flasks at 55℃ at volume fractions of 2%, 4%, 6%, 8%, and 10%, respectively. After thorough mixing, the filtrate was poured onto plates, and a 5mm diameter *M. yunnanensis* mycelial cake was inoculated in the center of each plate. Plates containing only *M. yunnanensis* mycelial cake and without the addition of antibacterial aseptic fermentation filtrate served as controls. Each treatment was repeated five times. The plates were incubated at 25℃ for 8 days. After 8 days, the colony diameter was measured, and the inhibition rate was calculated.

[0058] Inhibition rate = (control colony diameter - treated colony diameter) / control colony diameter × 100%.

[0059] Using *M. yunnanensis*, the causal agent of brown rot in Korla fragrant pear, as the target bacterium, the effect of different concentrations of sterile fermentation filtrate from strain PL55 on the mycelial growth of *M. yunnanensis* was determined. The results are as follows: Figure 4As shown, the aseptic fermentation filtrate of strain PL55 inhibited the growth of M. yunnanensis mycelia. The inhibition effect was best when the volume fraction of the aseptic fermentation filtrate was 10%, reaching 97.65%. The inhibition effect decreased with decreasing volume fraction of the fermentation filtrate of biocontrol strain PL55, and the lowest inhibition effect (74.85%) was observed at a volume fraction of 2%.

[0060] 1.5 Determination of the control effect of aseptic fermentation filtrate of strain PL55 on Korla fragrant pear fruit

[0061] Korla fragrant pears of good growth and uniform size were picked from the fragrant pear orchard of Tarim University. After washing with clean water, they were soaked in 75% alcohol for 2 minutes, disinfected with 1% sodium hypochlorite for 90 seconds, rinsed with sterile water after surface disinfection, and then air-dried. A conidial suspension (1×10⁻⁶) of the cultured *M. yunnanensis* strain was prepared. 6 (units / mL).

[0062] Treatment 1 was a protective effect test: sterile fermentation filtrate was sprayed onto the fruit surface and incubated at 26℃ for 1 day. Then, M. yunnanensis conidia suspension was sprayed onto the fruit surface and incubated at 26℃ with humidity.

[0063] Treatment 2 was a therapeutic experiment: M. yunnanensis conidial suspension was sprayed onto the fruit surface and incubated at 26℃ for 1 day. Then, sterile fermentation filtrate was sprayed onto the fruit surface and incubated at 26℃ with humidity.

[0064] The treatment involving spraying only with a suspension of M. yunnanensis conidia served as a control, while the treatment involving spraying only with water served as a blank control. Each treatment was replicated three times. The length and size of lesions were observed on day 7, and the control efficacy was calculated.

[0065] Control efficacy = (Leg length of control group - lesion length of treatment group) / lesion length of control group × 100%.

[0066] The results are as follows Figure 5-6 As shown, the aseptic fermentation filtrate of strain PL55 effectively inhibited the infection and lesion expansion of brown rot in Korla fragrant pears. Furthermore, the protective effect of inoculating with the aseptic fermentation filtrate of strain PL55 followed by *M. yunnanensis* was 69.11%. However, the curative effect of inoculating with *M. yunnanensis* followed by the aseptic fermentation filtrate of strain PL55 was only 19.59%. This indicates that the aseptic fermentation filtrate of strain PL55 has both protective and curative effects against brown rot in Korla fragrant pears, with the protective effect being significantly superior to the curative effect.

[0067] 1.6 Determination of the antibacterial spectrum of strain PL55

[0068] The inhibitory effect of strain PL55 on five plant pathogens—*Pterocarya stenoptera* (brown rot of Korla fragrant pear), *Pterocarya stenoptera* (tree rot of Korla fragrant pear), *Pterocarya stenoptera* (tree rot of apple), *Pterocarya stenoptera* (tree rot of walnut), and *Pterocarya stenoptera* (cotton wilt)—was determined using the plate confrontation method. Pathogens were inoculated in the center of PDA plates, and PL55 fungal cakes were inoculated 2.5 cm from the edge of the cake. Plates inoculated only with pathogens served as controls. The plates were incubated in the dark at 25°C. Each treatment was repeated in quadruplicates. The inhibition rate was calculated after the control group colonies had fully colonized the plates.

[0069] Inhibition rate (%) = (Coronary diameter of control group - Colony diameter of treatment group) / Colony diameter of control group × 100%.

[0070] The inhibition rate of mycelial growth of biocontrol strain PL55 against five tested pathogens was determined by the plate confrontation method. The results are as follows: Figure 7 As shown, strain PL55 exhibited varying degrees of antagonistic effects on the mycelial growth of the five tested pathogens. It showed the best inhibitory effect on the mycelial growth of *Pterocarya stenoptera*, the causal agent of brown rot in Korla pear, reaching 91.23%, while its inhibitory effect on the mycelial growth of *Fusarium wilt*, the causal agent of cotton wilt, was relatively poor, at 66.82%. These results indicate that strain PL55 has good biocontrol effects against the main diseases of Korla pear fruit and also possesses a certain degree of broad-spectrum activity.

[0071] 1.7 Determination of nitrogen fixation, phosphorus solubilization, potassium solubilization, cellulase production, and protease production capabilities of strain PL55

[0072] Strain PL55 was transferred to casein agar, Assumption medium, CAS agar, organic phosphorus bacteria medium, inorganic phosphorus bacteria medium, and sodium carboxymethyl cellulose medium. After incubation at 28°C for 7 days, it was determined whether a clear zone was formed around the inoculation site or whether the colonies could grow normally.

[0073] The results are as follows Figure 8 As shown, strain PL55 can produce clear zones on CAS agar and casein medium, and can grow normally on Assumption medium, inorganic phosphorus medium and organic phosphorus medium, indicating that strain PL55 has the ability to secrete proteases, synthesize siderophores, decompose inorganic phosphorus, decompose organic phosphorus and fix nitrogen.

[0074] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A strain of Bacillus siamensis PL55, characterized in that, This strain was deposited on May 19, 2025 at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCCNO: M 20251089.

2. A microbial inoculant, characterized in that, Includes the Bacillus sicca PL55 as described in claim 1.

3. The use of Bacillus sicca PL55 as described in claim 1 or the microbial agent as described in claim 2 in the prevention and / or treatment of brown rot of Korla fragrant pear caused by Monilia yunnanensis.

4. The application of Bacillus sicca PL55 as described in claim 1 or the microbial agent as described in claim 2 in antagonizing plant diseases caused by Bacillus thuringiensis of Korla fragrant pear, Bacillus wilt of cotton, Bacillus thuringiensis of apple and / or Bacillus thuringiensis of walnut.

5. A method for preventing and controlling plant diseases, characterized in that, Plants are sprayed with the Bacillus simonii PL55 of claim 1 or the microbial agent of claim 2 to prevent and control plant diseases; The plant diseases mentioned include those caused by *M. yunnanensis* (brown rot pathogen of Korla fragrant pear), *V. pyri* (tree rot pathogen of Korla fragrant pear), *F. oxysporum* (cotton wilt pathogen), *V. mali* (apple rot pathogen), and / or *V. nivea* (walnut rot pathogen).

6. The use of Bacillus simonii PL55 as described in claim 1 or the microbial agent as described in claim 2 in the preparation of protease-producing, iron-carrier-synthesizing, inorganic phosphorus-solubilizing, organic phosphorus-solubilizing, and nitrogen-fixing agents.

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