Marsinia sp., microbial inoculum prepared from marsinia sp. And application of microbial inoculum

Abstract

The invention discloses massidium sp., a microbial inoculum prepared from the massidium sp. And application of the massidium sp. The strain RC3207 of the invention is preserved in the China General Microbiological Culture Collection Center (CGMCC); the address of the preservation unit is No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing; the preservation time is April 8, 2025; the preservation number is CGMCC (China General Microbiological Culture Collection Center) According to the present invention, with the application of the Marsinia sp. Strain, the growth of the pine needle red spot pathogenic bacteria (Dothistoma septospora) can be effectively inhibited, and the good inhibition effect on the truffle pink rot pathogenic bacteria can be provided; field control tests show that when the microbial inoculum is applied to the early stage of the pine needle red spot disease, the obvious control effect can be achieved, the average control effect reaches up to 73.4%, and the microbial inoculum is suitable for application and popularization in biocontrol practice of pine red spot disease and truffle pink rot.

Description

Technical Field

[0001] This application relates to the field of biological control, and more specifically, to a Massilia sp., bacterial agents prepared therefrom, and their potential applications. Background Technology

[0002] Pine trees refer to species in the genus *Pinus*, which includes many important economic and commercial forest species, such as Korean pine (*P. koraiensis*), Scots pine (*P. sylvestris var. mongolica*), Chinese pine (*P. armandii*), and oil pine (*P. tabuliformis*). However, in commercial pine forest production, pine needle red spot disease is widespread and severely damaging, leading to massive needle shedding, slowed growth, reduced timber yield, and even death, causing significant economic losses. The pathogens of pine needle red spot disease are considered to be two species in the genus *Dothistroma*: *Dothistroma pini* and *Dothistroma septospora*. Previous reports have suggested that *Dothistroma pini* is the pathogen of pine needle red spot disease occurring in my country, but since pure cultures were not obtained in previous studies, the identification may be inaccurate. Our team's research has found that pine needle red spot disease in Northeast my country and Inner Mongolia is caused by infection with *D. septospora*. This pathogen can infect over 80 species of pine trees worldwide and is listed as a quarantine pathogen in my country and the European Union. The disease can be distributed in regions between 35° and 51° north latitude, and the pathogen has strong adaptability. Under natural conditions, the disease's spread distance is limited, mainly through the transportation of seedlings and the presence of diseased leaves in seeds. Currently, the main control measures remain strengthening quarantine, silvicultural practices, and chemical control. Studies have shown that fungicides such as chlorothalonil, thiram, zineb, carbendazim, and Bordeaux mixture are effective against pine needle red spot disease. Early symptoms of pine needle red spot disease are often subtle and difficult to detect, and conventional chemical control techniques can cause environmental pollution; therefore, there is an urgent need to develop a green control technology system for this harmful organism.

[0003] Truffles are a type of ascomycete fungus, also known as pig truffles, and commercially as truffles. They are ectomycorrhizal fungi that often form symbiotic relationships with the roots of angiosperms or gymnosperms such as pines, willows, betulaceae, Fagaceae, and elms. Truffles have unique habitats, rich flavors, and delicious tastes. They are rich in inorganic salts, amino acids, and other nutrients, making them one of the most prized large edible fungi. The black truffle (T. melanosporum) and the Italian white truffle (T. magnatum), both from Europe and abroad, are highly sought after by consumers both domestically and internationally. my country's Indian truffle (T. indicum), pseudohimalayense (T. pseudohimalayense), and Chinese truffle (T. sinense) are produced in large quantities, widely distributed, and concentrated in major producing areas, and are also highly praised. Currently, my country's annual export of fresh truffles is estimated to reach 200 tons. A common disease affecting truffle products is pink rot (caused by *Trichothecium roseum*), which primarily infects *Truffles indicus* and *Truffles thaliana*. It can cause losses exceeding 10% during the storage and transportation of fresh truffles. Truffles growing in soil generally do not exhibit disease or show very little disease; in most cases, disease only occurs after emergence. Currently, there is a lack of green control measures for diseases during the storage and transportation of truffles.

[0004] Massilia is a genus of Gram-negative bacteria belonging to the family Oxalicobacteriaceae. This genus contains 43 viable species. The bacteria are short rod-shaped, with apical flagella, and are non-spore-forming. They are heterotrophic aerobic microorganisms, mainly distributed in soil, ice cores, plants, water bodies, and air. Some strains possess phosphorus-solubilizing, phenanthrene-degrading, and heavy metal-tolerant properties, making them valuable for soil remediation and the development of microbial inoculants. Summary of the Invention

[0005] This invention provides a Massilia sp. strain named Massilia sp. RC3207, which is deposited at the China General Microbiological Culture Collection Center (CGMCC); address of the depository: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; deposit date: April 8, 2025; accession number: CGMCC No. 34129.

[0006] This invention also provides a *Bacillus simonii* inoculant prepared from *Bacillus simonii* strain RC3207. Specifically, the inoculant contains ≥10 live *RC3207* bacteria. 8 CFU / mL.

[0007] This invention also provides the application of *M. muscarinatum* strain RC3207 or an agent prepared from *M. muscarinatum* strain RC3207 in the control of pine needle red spot disease or truffle pink rot. Specifically, the pathogen of pine needle red spot disease is *D. septospora*, and the pathogen of truffle pink rot is *T. roseum*.

[0008] Preferably, when controlling pine needle red spot disease, the number of active bacteria of RC3207 used is ≥10. 8 CFU / mL; the application method is spraying, and the application period is before or at the early stage of pine needle erythema.

[0009] The beneficial effects of this invention include:

[0010] This invention isolated over 1000 bacterial strains from truffle inoculum and surrounding soil, and screened out the *Morchella esculenta* strain RC3207. The inoculant prepared using this *Morchella esculenta* strain RC3207 effectively inhibited the growth of *D. septospora*, the pathogen causing pine needle red spot disease, and showed good control efficacy against pine needle red spot. Field control trials revealed that applying this inoculant at the early stage of pine needle red spot disease development resulted in significant control, with an average efficacy of up to 73.4%, making it suitable for widespread application in the biocontrol of pine needle red spot disease. Furthermore, confrontation culture revealed that this *Morchella esculenta* strain RC3207 also inhibited the growth of *T. roseum*, the pathogen causing truffle pink rot, and can also be used as an inoculant for controlling truffle pink rot. Attached Figure Description

[0011] Figure 1 The Masseuse strain RC3207 (concentration of 10) in Example 1 of this invention. 8 An antibacterial experiment was conducted on the pine needle red spot pathogen (D. septospora) using CFU / mL. In this experiment, A was a pure culture of the pine needle red spot pathogen as a control, and B was the inhibition of the growth of the pine needle red spot pathogen by the Masseuse strain RC3207 spread on a petri dish.

[0012] Figure 2 This is an antibacterial test of *M. muscarinii* RC3207 against *T. roseum*, the pathogen of pink rot of truffles, in Example 1. A is a control culture of the pink rot pathogen; B is the inhibition of the growth of the pink rot pathogen by *M. muscarinii* strain RC3207 inoculated into a petri dish.

[0013] Figure 3 The image shows the control efficacy of pine needle red spot disease observed in September during the field control trial in Example 2. In the image, A is the negative control without the application of biocontrol agent; B is the control with the application of biocontrol agent RC3207.

[0014] Biological Preservation Information

[0015] Masseille strain RC3207 is deposited at the China General Microbiological Culture Collection Center (CGMCC); address of the depository: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing; deposit date: April 8, 2025; accession number: CGMCC No. 34129. Detailed Implementation

[0016] The present invention will be further described and illustrated below with reference to embodiments. However, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the present invention and the embodiments, all other inventions and embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0017] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0018] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0019] Example 1: Screening and Identification of Biocontrol Functional Strains RC3207

[0020] (1) Isolation of bacteria

[0021] Soil from the truffle incubation pond and surrounding area was collected from Panzhihua. 1.0g of soil was weighed and poured into a test tube containing 9ml of sterile water. The tube was then capped and shaken at 220rpm for 5 minutes. The tube was then removed and allowed to stand at room temperature for 5 minutes. The supernatant was then diluted 10... 1 -10 3 After dilution, the bacteria were spread on NA plates and incubated upside down at 30°C for 2 days. Each dilution gradient was replicated in 3 steps. Then, single colonies were picked and streaked to purify the target bacteria. The pure culture strains were stored at -70°C for later use. In this study, more than 1,000 bacterial strains from the truffle pond and surrounding soil were isolated.

[0022] (2) Screening of antagonistic strains

[0023] Screening of antagonistic bacteria against pine needle red spot pathogen

[0024] Screening of biocontrol strains using plate confrontation culture method: First, prepare 100 μL of cell suspension of the strain to be screened (10 8Tissue blocks of *D. septospora* (the pathogen causing pine needle red spot disease, as identified) were inoculated into the center of PDA plates. The bacteria to be screened were then spread around the periphery of the *D. septospora* tissue blocks. Three replicates were performed. The culture dishes were incubated upside down at 26°C for 4 weeks, and the inhibition zone was observed and measured. This experiment successfully screened a strain with antagonistic activity against *D. septospora*, obtaining the functional strain RC3207 of this invention. Figure 1 As shown, this strain completely inhibited the growth of pine needle red spot pathogen in the confrontation experiment.

[0025] Experiment on the antagonism of RC3207 strain against pink rot fungus

[0026] Biocontrol strains were screened using a plate confrontation culture method: First, a tissue block of *T. roseum* (identified as *T. roseum*) was inoculated into the center of a PDA plate. Then, the bacteria to be screened were spotted around the periphery of the *T. roseum* tissue block. The plates were incubated upside down at 26°C for 7 days, after which the inhibition zone was observed and measured. Figure 2 As shown, the confrontation experiment revealed that strain RC3207 had a strong antagonistic effect on the pine needle red spot pathogen, forming a clear inhibition zone. The inhibition rate (inhibition rate = (colon radius of control group *Trichoderma pinkis* - colony radius of treatment group *Trichoderma pinkis*) / colony radius of control group *Trichoderma pinkis* × 100%) ranged from 67% to 74.4%, with an average inhibition rate of 70.2%.

[0027] (3) Molecular identification of bacterial strains

[0028] Bacterial identification: DNA was extracted from strain RC3207 using the CTAB method. PCR amplification was performed using the universal primers 27F / 1492R for bacteria, and the PCR products were sent to a sequencing company for gel sequencing. After obtaining the sequence, the bacterial strain RC3207 of this invention was identified using an online identification tool (https: / / eztaxon-e.ezbiocloud.net / identify). Its 16S rRNA gene sequence information was entered into a database for comparison. Strain RC3207 of this invention showed the highest homology (98.17%) with the type strain *Massilia violacea* CAVIO(T). This strain can be considered a different species within the same genus as the type strain (bacterial 16S rRNA gene homology greater than 95% is considered to be in the same genus; 16S rRNA gene homology greater than 98.5% is considered to be in the same species). The homology between RC3207 and other known species in the genus *Massilia* was also less than 98.5%. Therefore, strain RC3207 is considered a new species in the genus *Massilia*.

[0029] Based on molecular identification results, the strain RC3207 of this invention was identified as Massilia sp., which is deposited at the China General Microbiological Culture Collection Center (CGMCC); address of the depository: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing; deposit date: April 8, 2025; accession number: CGMCCNo. 34129.

[0030] Example 2: Field control experiment

[0031] (1) Preparation of inoculum of Bacillus simae strain RC3207

[0032] The preserved strain RC3207 was streaked and cultured for three generations via single-colony transfer. Single colonies were then transferred to TSB liquid medium for propagation and cultured at 30°C with shaking at 220 rpm for 36 hours. The cultured strain was then centrifuged at 10,000 rpm for 5 minutes to precipitate the culture. The precipitate was diluted with sterile water to a viable count of 10⁻⁶ RC3207 cells. 8 Prepare a bacterial agent with approximately CFU / mL.

[0033] (2) Field efficacy trials

[0034] The field efficacy experiment was conducted in a pine forest within the Chinese Academy of Forestry. In May 2024, the fungal agent prepared in step (1) of this embodiment was sprayed twice consecutively on the pine needles of diseased pine trees at the early stage of the disease (identified as *D. septospora*). The spraying continued until droplets dripped from the pine needles, and a second spray was applied two weeks later. The control effect was observed and recorded in September, with the number of diseased pine needles being the primary focus. Ten pine branches with similar disease conditions and a comparable number of pine needles were selected as experimental subjects. Five branches were used for the control experiment, and the other five branches were sprayed with an equal amount of water as a control. Because the number of pine needles was large and the lesions were small, making recording and measurement difficult, the control effect of this experiment was calculated based on the number of diseased pine needles and the incidence rate.

[0035]

[0036] A1 represents the number of diseased pine needles on pine branches in the control group after treatment; A2 represents the number of diseased pine needles on pine branches in the control group before treatment; B1 represents the number of diseased pine needles on pine branches in the treatment group before treatment; B2 represents the number of diseased pine needles on pine branches in the treatment group after treatment; C represents the total number of pine needles in each treatment.

[0037] The incidence of disease recorded in the experimental observation of the prevention and control effect is as follows: Figure 3 As shown in Table 1, the application of fungicide RC3207 resulted in fewer diseased pine branches, with a control effect ranging from 69% to 76%, and an average control efficacy of 73.4%. Figure 3It can be seen that the number of pine needles was reduced after applying the bacterial agent RC3207, and even the diseased lesions were smaller than those of the control. In summary, the biocontrol strain RC3207 of this invention has a good control effect in the early stages of pine needle red spot disease.

[0038] Table 1 Prevention and control effects

[0039]

Claims

1. A Massilia sp., characterized in that, The *Massezia* strain was named *Massezia* strain RC3207 and is deposited at the China General Microbiological Culture Collection Center (CGMCC); address of the depository: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; deposit date: April 8, 2025; accession number: CGMCC No. 34129.

2. A *Bacillus simonii* inoculant, characterized in that, The bacterial agent is prepared from the Masseilles strain RC3207 as described in claim 1.

3. The *Morseella asiatica* inoculant according to claim 2, characterized in that, The number of active RC3207 bacteria in the bacterial agent is ≥10. 8 CFU / mL.

4. The application of the *Mosas* strain according to claim 1 or the fungal agent according to any one of claims 2-3 in the prevention and control of pine needle red spot disease or truffle pink rot, wherein the pathogen of pine needle red spot disease is *Dothistromaseptosporum*, and the pathogen of truffle pink rot is *Trichothecium roseum*.

5. The application according to claim 4, characterized in that, When controlling pine needle red spot disease, the number of active bacteria of RC3207 should be ≥10. 8 CFU / mL.

6. The application according to claim 5, characterized in that, The application method is spraying.

7. The application according to claim 6, characterized in that, The application period is before or at the early stage of pine needle erythroplasia.

Images