Potamogeton malabaricus phyllosphere attachment bacterium ML5 with algae-lysing activity and application thereof

Abstract

The invention discloses potamogeton malabaricus phyllosphere attachment bacteria ML5 with algae-lysing activity and application of the potamogeton malabaricus phyllosphere attachment bacteria ML5, and belongs to the technical field of ecological microorganisms. The bacterium ML5 is named as Priesta aryabhattai ML5, the strain is preserved in Guangdong Province Microbial Culture Collection Center located at No.100, Xianlie Middle Road, Guangzhou City, Guangdong Province, the preservation number is GDMCC No: 65778, the preservation date is January 13, 2025, and the strain can be separated from potamogeton malabaricus phyllosphere. The invention further provides a microbial algicidal preparation containing the potamogeton malabaricus phyllosphere attached bacterium ML5. The method is used for biological prevention and control of freshwater cyanobacterial bloom, especially invasion cyanobacteria cuscuta larakii, and has a wide application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of ecological microbiology, and particularly relates to a Potamogeton malaianus leaf-attached bacterium ML5 with algicidal activity and its application. Background Art

[0002] In the past few decades, many methods have been adopted to remove the invasive cyanobacterium Raphidiopsis raciborskii. For example, physical methods such as mechanical salvage, physical aeration, and ultrasonic treatment are usually costly and time-consuming; chemical methods such as chemical oxidants are efficient and low-cost methods for controlling Raphidiopsis raciborskii in a short time, but chemical substances are likely to cause secondary pollution and pose a potential threat to the aquatic ecosystem. In contrast, biological approaches such as allelopathy of plants, aquatic animals, and algicidal microorganisms are considered an economical and environmentally friendly way to inhibit or biodegrade cyanobacteria. The microbial method is considered a promising method for controlling cyanobacterial blooms due to its high efficiency, low cost, and environmental friendliness. Therefore, screening for microorganisms with high algicidal properties has been widely studied.

[0003] Raphidiopsis raciborskii shows an invasive and expanding trend in deep lakes and reservoirs worldwide and has developed into one of the main bloom cyanobacteria threatening the health of the aquatic ecosystem after Microcystis. In the southeastern region of China, Raphidiopsis raciborskii producing cylindrospermopsin has been isolated, and this toxin is also the second most common cyanotoxin globally. At present, the bloom of toxigenic Raphidiopsis raciborskii and its induced ecological effects have become the focus of close attention of algologists, ecologists, and management departments. Scholars in this field are committed to screening for algicidal bacteria that have a significant inhibitory effect on the growth of the invasive cyanobacterium Raphidiopsis raciborskii, studying secondary metabolites with algicidal activity, developing microbial algicidal preparations, and applying them to the safe and efficient solution of cyanobacterial bloom problems, which is of great value for the safe, economical, and efficient control and treatment of water blooms. Summary of the Invention

[0004] In view of this, the main object of the present invention is to provide a Potamogeton malaianus leaf-attached bacterium ML5 with algicidal activity, which is a strain Priestia aryabhattai with algicidal activity.

[0005] Another object of the present invention is to provide the application of the Potamogeton malaianus leaf-attached bacterium ML5 in the biological control of the invasive cyanobacterium Raphidiopsis raciborskii.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] The invention provides a Priestia aryabhattai leaf-attached bacterium ML5, named as Priestia aryabhattaiML5, the strain is preserved in Guangdong Microbiological Culture Collection Center located at No. 100, Xianlie Middle Road, Guangzhou City, Guangdong Province, with a preservation number of GDMCC No: 65778 and a preservation date of January 13, 2025.

[0008] Preferably, the Potamogeton phyllosphere-attached bacteria ML5 are isolated from the Potamogeton phyllosphere.

[0009] Preferably, the Potamogeton phyllosphere-attached bacteria ML5 has algicidal activity and belongs to the genus Priestia aryabhattai.

[0010] The present invention also provides a microbial algae-dissolving preparation, which comprises the Potamogeton phyllosphere-attached bacteria ML5.

[0011] The present invention also provides the use of the leaf-attached bacteria ML5 of Potamogeton oleifera or the microbial algae-lysing preparation in biological control of the invasive cyanobacteria Acrocephalus lagerstroemia.

[0012] Compared with the prior art, the beneficial effects of the present invention are as follows: the present invention proposes a leaf-attached bacterium ML5 of Potamogeton raphis with algae-lytic properties, the fermentation broth of the strain has a strong inhibitory effect on the growth of the invasive cyanobacteria Raphidiopsis raciborskii, and the desalination culture of the strain ML5 does not affect its algae-lytic effect. The novel aquatic ecological algae inhibitor is developed by the present invention for the biological control of freshwater cyanobacteria blooms, especially the invasive cyanobacteria Raphidiopsis raciborskii, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 This is a preliminary screening of the inhibitory effects of 5 active attachment strains screened from the leaves of Potamogeton rapa on the photosynthetic activity and chlorophyll content of the invasive cyanobacteria Acrocephalus lamarckii at 0, 48, and 120 h in the examples; (a) Preliminary screening of the inhibitory effects of bacteria attached to the leaves of Potamogeton rapa on the actual photosynthetic efficiency of Acrocephalus lamarckii; (b) Changes in chlorophyll content of Acrocephalus lamarckii.

[0014] Figure 2 The figure shows the effect of the leaf-attached bacterium ML5 of Potamogeton malayi selected from 5 strains of bacteria in the examples on the photosynthetic activity of the invasive cyanobacterium Acrocephalum ramospermum.

[0015] Figure 3 The figure shows the effect of the leaf-attached bacteria ML5 of Potamogeton malayi screened out from 5 strains of bacteria in the example on the chlorophyll content of the invasive cyanobacterium Acrocephalum ramospermum.

[0016] Figure 4The figure shows the effect of the leaf-attached bacterium ML5 of Potamogeton malayi selected from 5 strains in the examples on the photosynthetic biomass of the invasive cyanobacterium Acrocephalum ramospermum.

[0017] Figure 5 The figures show the inhibitory effect of the fermentation broth of the leaf-attached bacteria ML5 of Potamogeton malayi cultured in diluted BPM medium on the growth of the invasive cyanobacterium Acutum lamarckii in the examples; (a) The inhibitory effect of the leaf-attached bacteria ML5 of Potamogeton malayi cultured in 1 / 5 BPM medium on the actual photosynthetic efficiency of Acutum lamarckii; (b) The change in the actual chlorophyll content of Acutum lamarckii. DETAILED DESCRIPTION

[0018] In order to more fully understand and demonstrate the technical solutions, purposes and advantages of the present invention, the technical effects produced by the present invention are further described in detail and completely in conjunction with the accompanying drawings and specific embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, not all. It should be pointed out that for ordinary technicians in this field, other embodiments obtained without departing from the concept of the present invention all belong to the protection scope of the present invention.

[0019] In the following examples, a Priestia aryabhattai leaf-attached bacterium ML5 was screened and identified, named Priestia aryabhattai ML5, which was deposited in the Guangdong Provincial Microbiological Culture Collection Center located at No. 100, Xianlie Middle Road, Guangzhou City, Guangdong Province, with a deposit number of GDMCC No: 65778 and a deposit date of January 13, 2025.

[0020] In some embodiments, the phyllosphere-attached bacteria ML5 of Potamogeton malayi were isolated from the phyllosphere of Potamogeton malayi, and the strain ML5 was cultured in a diluted BPM medium and was found to have a highly efficient algicidal activity.

[0021] In some embodiments, the method for screening the leaf-attached bacteria ML5 of Potamogeton malayi comprises the following steps:

[0022] (1) Weigh the submerged plant Potamogeton malayi and place it in a sterile tube. Add an appropriate amount of sterile water, wash with ultrasound for 1 min, and vortex for 10 s. Repeat this step twice.

[0023] (2) Mix the obtained water sample with the bacterial culture medium and culture with shaking at 37°C for 24-48 hours until the color of the culture medium changes. Take a certain amount of the culture solution and use fresh bacterial culture medium to make up the volume in proportion. Repeat the operation at least 3 times using the same culture conditions as above to obtain an enriched bacterial solution.

[0024] (3) Dilute the enriched bacterial solution, spread the diluted bacterial solution on a plate, and further culture it for 2-3 days. Pick different single colonies and streak them on the plate multiple times to obtain a purified strain.

[0025] (4) Detect the algicidal effect of the algal solution of *Raphidiopsis raciborskii* on each purified and isolated single colony, and screen out the Potamogeton malaianus leaf-attached bacterium ML5 with high algicidal activity.

[0026] In some embodiments, the dilution culture method of strain ML5 is as follows:

[0027] ① Ferment and culture strain ML5;

[0028] ② Inoculate the fermentation broth into the diluted BPM medium for secondary fermentation;

[0029] ③ Measure the growth curve of ML5 with BPM media at different concentrations.

[0030] Among them, the initial medium of strain ML5 is the BPM medium prepared with first-grade water and sterilized. The fermentation conditions are as follows: Strain ML5 is inoculated into the sterilized BPM medium with pH 7.0 and fermented at 37 °C and 180 rpm for 2 days to obtain the fermentation broth of strain ML5, where the BPM medium is prepared with first-grade water.

[0031] Among them, the gradients of the diluted BPM medium are 1 / 2, 1 / 5, and 1 / 10 volume ratios (diluted with BG11 medium). The results show that strain ML5 can grow normally and has algicidal activity in the diluted BPM medium.

[0032] Example 1 Preliminary screening of active strains

[0033] A total of 5 culturable attached bacteria were isolated and purified from the leaf surface of *Potamogeton malaianus*, and then they were activated and cultured. The bacteria solution after secondary activation was added to the cultured *Raphidiopsis raciborskii* at a volume ratio of 10%. The co-cultured bacteria-algae system was placed in a full-automatic light incubator for culture. The culture conditions were as follows: temperature was 25 °C, light intensity was 1500 Lux, and the light-dark ratio was 12:12. Samples were taken regularly every day to measure the photosynthetic activity and chlorophyll content of *Raphidiopsis raciborskii*. It was found that the algicidal activities of 2 strains were not significant, but 3 strains of bacteria, including ML1, ML3, and ML5, had significant inhibitory effects on the growth of *Raphidiopsis raciborskii*. The results are as Figure 1 shown. When the addition amounts of the 3 attached bacteria (ML1, ML3, and ML5) were at a volume ratio of 10%, the inhibition rates on *Raphidiopsis raciborskii* within 48 h were 83.21%, 86.79%, and 96.28% respectively (the inhibition rate was indicated by the change rate of chlorophyll content); among them, strain ML5 had the highest inhibition rate (96.28%) and the best activity.

[0034] Example 2 Re-screening of the algicidal effect of the fermentation broth of strain ML5 on the invasive cyanobacterium *Raphidiopsis raciborskii*

[0035] Raphidiopsis raciborskii was cultured in sterile BG11 medium and placed in an illumination incubator. The temperature was 25 °C, the light-dark cycle was 12 h∶12 h, and the light intensity was 1500 Lux. The strain ML5 was inoculated into beef extract peptone liquid medium and incubated at 37 °C for 12 h. A rescreening experiment was carried out in a conical flask according to a 1:10 volume ratio of bacteria to algae. The photosynthetic activity, chlorophyll content and changes in photosynthetic biomass of Raphidiopsis raciborskii were measured at 0, 24, 48, 72, and 120 h. The results are as Figures 2 - 4 shown.

[0036] Example 3 Growth of strain ML5 under BPM conditions at different dilution ratios

[0037] The prepared BG11 medium and BPM medium were diluted into 1 / 2, 1 / 5, and 1 / 10 BPM media according to a certain volume ratio. The shaking incubator was set at: 37 °C, 180 rpm. After culturing in BPM medium for 12 h, the diluted BPM medium was injected according to a 5% volume ratio. The absorbance OD 600 was measured at 0, 2, 4, 6, 8, 10, 12, 24, 36, and 48 h. Three parallels were set for each group, and the growth curve of bacteria was recorded. Among them, the bacterial liquid of 1 / 5 BPM grew well and had no obvious inhibitory effect on Raphidiopsis raciborskii.

[0038] Example 4 Inhibitory effect of strain ML5 cultured in 1 / 5 BPM on the growth of invasive cyanobacterium Raphidiopsis raciborskii

[0039] The strain ML5 was inoculated into the prepared 1 / 5 BPM medium and fermented for 2 days. The fermentation conditions were: culturing in a shaking incubator at 37 °C and 180 rpm. The fermented bacterial liquid was inoculated into the invasive cyanobacterium Raphidiopsis raciborskii in the logarithmic growth phase at a 10% volume ratio. The sterile 1 / 5 BPM medium was added to the algal liquid at the same inoculation rate as a control. It was cultured in an illumination incubator for 4 days. Samples were taken every day to measure the biomass of Raphidiopsis raciborskii in the control group and the experimental group, and the algicidal efficiency was calculated. Three parallel samples were set for both the control group and the experimental group, and the results were expressed in the form of mean ± standard deviation.

[0040] As Figure 5 shown, the 4-day inhibition rate of the fermentation broth of strain ML5 cultured in 1 / 5 BPM on the invasive cyanobacterium Raphidiopsis raciborskii was 56.48%. The actual photosynthetic efficiency of Raphidiopsis raciborskii was already 0 at 48 h, indicating that strain ML5 could still maintain its algicidal activity after being cultured in the diluted BPM medium.

[0041] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A leaf-attached bacterium ML5 of Priestia aryabhattai, named Priestia aryabhattai ML5, which is deposited in Guangdong Microbiological Culture Collection Center, No. 100, Xianlie Middle Road, Guangzhou City, Guangdong Province, with a deposit number of GDMCC No: 65778 and a deposit date of January 13, 2025.

2. The leaf-adhering bacteria ML5 of Potamogeton oleifera according to claim 1, characterized in that The pectinata leaf-adhering bacteria ML5 are isolated from the pectinata leaf-adhering bacteria.

3. The leaf-adhering bacteria ML5 of Potamogeton oleifera according to claim 1, characterized in that The leaf-adherent bacteria ML5 of Potamogeton oleifera have algae-lytic activity.

4. A microbial algae-dissolving preparation, characterized in that: It comprises the leaf-adherent bacteria ML5 of Potamogeton oleifera according to any one of claims 1 to 3.

5. Use of the leaf-attached bacteria ML5 of Potamogeton oleraceus according to any one of claims 1 to 3 or the microbial algae-lytic preparation according to claim 4 in biological control of the invasive cyanobacteria Acanthopanax raschni.

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