A non-saccharolytic Pseudochrobactrum asaccharolyticum Nan-Y, bacterial suspension and its preparation method and application

Abstract

The present invention provides a strain of Palleronella apachucensis Nan-Y that does not decompose sugar and its application, which relates to the technical field of microorganisms. The Palleronella apachucensis Nan-Y disclosed in the present invention has a preservation number of GDMCC NO: 63377. The present invention isolates the Palleronella apachucensis Nan-Y strain from the dead bodies of Euproctis pseudoconspersa, and this strain has strong pathogenicity to the larvae of Euproctis pseudoconspersa and can be applied to the biological control of Euproctis pseudoconspersa.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbiology, and particularly relates to a Pseudochrobactrum asaccharolyticum Nan-Y and its application. Background Art

[0002] Plants of the genus Camellia L. are mostly famous beverage, oil, and ornamental tree species, with high utilization value. Among them, species especially favored by the Euproctis pseudoconspersa, such as Camellia oleifera Abel and Camellia sinensis (L.) O. Kuntze, are all important economic tree species. The low-instar larvae of the Euproctis pseudoconspersa gather on the back of the leaves to feed, causing the leaves to wither and turn into a semi-transparent film. After the 3rd instar, they disperse and migrate, feeding on the tender shoots, and then gnawing on the leaves, tender branch bark, and fruit peels, affecting the growth of tea trees and the yield of tea seeds. When the damage is severe, the tender shoots, old leaves, bud leaves, and flowers of the tea trees are all eaten up, leaving only bare branches. The entire tea forest looks like it has been burned by fire, the fruits fall prematurely, and the yield drops sharply. If damaged continuously for several years, the tea trees can die, causing serious economic losses. Traditional chemical control methods are prone to cause the 3R problems, namely Resistance, Resurgence, and Residue. Therefore, biological control has begun to be taken seriously. At present, there are the following problems in the biological control of the Euproctis pseudoconspersa: (1) The research on the biological control of this pest mainly focuses on predatory natural enemies, and there is less research on pathogenic microorganisms, especially pathogenic bacteria; (2) There have been no reports on the isolation and identification of pathogenic bacteria of the Euproctis pseudoconspersa, the determination of pathogenicity, the pathogenic mechanism, and the epidemic induction mechanism.

[0003] Insect pathogenic bacteria are an important part of pest biological control. The larvae of the Euproctis pseudoconspersa mainly gather and damage on the back of the leaves of Camellia oleifera. After being infected with the pathogen, the persistence of the insect corpses is beneficial to the spread, reinfection, and induction of epidemics of the pathogen. Therefore, it is of great significance to isolate pathogenic bacterial strains with pathogenicity to the Euproctis pseudoconspersa. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a Pseudochrobactrum asaccharolyticum Nan-Y, which has strong pathogenicity to the larvae of the Euproctis pseudoconspersa and can be applied to the biological control of the Euproctis pseudoconspersa.

[0005] In order to achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0006] The present invention provides a Pseudochrobactrum asaccharolyticum Nan-Y, and the Pseudochrobactrum asaccharolyticum Nan-Y was deposited with the Guangdong Provincial Culture Collection of Microorganisms on April 21, 2023, and the deposit number is GDMCC NO: 63377.

[0007] Preferably, the Pseudogracilibacter asaccharolyticus Nan-Y is derived from the dead bodies of Euproctis pseudoconspersa larvae.

[0008] The present invention provides a bacterial suspension containing the Pseudogracilibacter asaccharolyticus Nan-Y.

[0009] The present invention also provides a method for preparing the bacterial suspension. The Pseudogracilibacter asaccharolyticus Nan-Y is inoculated onto an NA plate medium. After activation for 36 to 72 hours, it is cultured with shaking for 8 to 15 minutes to obtain the bacterial suspension of Pseudogracilibacter asaccharolyticus Nan-Y.

[0010] Preferably, the NA plate medium comprises the following raw materials: 8 to 15 g of peptone, 2 to 8 g of NaCl, 2 to 5 g of beef extract, and 15 to 25 g of agar.

[0011] The present invention also provides an insecticide for controlling Euproctis pseudoconspersa larvae, which contains the Pseudogracilibacter asaccharolyticus Nan-Y or the bacterial suspension of the Pseudogracilibacter asaccharolyticus Nan-Y.

[0012] Preferably, the effective content of the Pseudogracilibacter asaccharolyticus Nan-Y is (1 ± 0.5) × 10 10 ~(1 ± 0.5) × 10 14 cfu·mL -1 .

[0013] The present invention also provides an application of the Pseudogracilibacter asaccharolyticus Nan-Y or the bacterial suspension of the Pseudogracilibacter asaccharolyticus Nan-Y in controlling Euproctis pseudoconspersa.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] In the present invention, an entomopathogenic bacterium Pseudogracilibacter asaccharolyticus Nan-Y is isolated from the dead bodies of Euproctis pseudoconspersa. Fresh tea tree leaves are soaked in different concentrations of the Nan-Y bacterial suspension and then fed to Euproctis pseudoconspersa. After 2 days of treatment, the cumulative mortality rate of the larvae increases significantly. After 6 days, the cumulative lethality rate of Euproctis pseudoconspersa is 61.00% at the lowest concentration and 100.00% at the highest concentration. The results show that the obtained Pseudogracilibacter asaccharolyticus Nan-Y strain has strong pathogenicity to Euproctis pseudoconspersa larvae, provides an application basis and strain resources for the biological control of Euproctis pseudoconspersa on oil tea and tea trees, and has great development and utilization prospects.

[0016] Biological preservation information

[0017] Nan-Y, Latin name: Pseudochrobactrum asaccharolyticum, is deposited in Guangdong Provincial Microbiological Culture Collection Center, with the deposit number GDMCCNO:63377, the deposit date is April 21, 2023, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 is the 16S rDNA phylogenetic tree of Pseudochryobacterium insaccharolyticum Nan-Y strain;

[0019] Figure 2 This is the microscopic morphology of the Nan-Y strain of Pseudochaobacillum insaccharolyticum;

[0020] Figure 3 The death dynamics of tea tussock moth larvae. DETAILED DESCRIPTION

[0021] The present invention provides a Pseudochrobactrum asaccharolyticum Nan-Y, wherein the Pseudochrobactrum asaccharolyticum Nan-Y was deposited in Guangdong Provincial Microbiological Culture Collection Center on April 21, 2023, with a deposit number of GDMCC NO: 63377.

[0022] In the present invention, Pseudomonas aglycones Nan-Y is a Gram-negative rod without spores.

[0023] In the present invention, the pseudo-orange bacillus insaccharolyticus Nan-Y is derived from the diseased and dead larvae of the tea tussock moth.

[0024] The invention provides a bacterial suspension containing the pseudochlamydial bacterium Nan-Y.

[0025] The present invention also provides a method for preparing the bacterial suspension. Preferably, the non-saccharolytic Pseudomonas aeruginosa Nan-Y is inoculated into a NA plate culture medium, activated for 36 to 72 hours, and then shaken and cultured for 8 to 15 minutes to obtain the non-saccharolytic Pseudomonas aeruginosa Nan-Y bacterial suspension; more preferably, the non-saccharolytic Pseudomonas aeruginosa Nan-Y is inoculated into a NA plate culture medium, activated for 48 hours, and then shaken and cultured for 10 minutes to obtain the non-saccharolytic Pseudomonas aeruginosa Nan-Y bacterial suspension.

[0026] In the present invention, the NA plate culture medium preferably includes the following raw materials: 8-15 g peptone, 2-8 g NaCl, 2-5 g beef extract, and 15-25 g agar; more preferably includes the following raw materials: 10 g peptone, 5 g NaCl, 3 g beef extract, and 20 g agar.

[0027] The present invention also provides an insecticide for controlling the larvae of Euproctis pseudoconspersa, which comprises the Paenibacillus apapillosus Nan-Y that does not decompose sugar or the bacterial suspension of the Paenibacillus apapillosus Nan-Y that does not decompose sugar.

[0028] In the present invention, the preferred effective content of the Paenibacillus apapillosus Nan-Y is (1±0.5)×10 10 ~(1±0.5)×10 14 cfu·mL -1 ; the more preferred effective content is (1±0.5)×10 11 ~(1±0.5)×10 13 cfu·mL -1 ; the most preferred effective content is (1±0.5)×10 12 cfu·mL -1 .

[0029] The present invention also provides an application of the above-mentioned Paenibacillus apapillosus Nan-Y or the bacterial suspension of the above-mentioned Paenibacillus apapillosus Nan-Y in controlling Euproctis pseudoconspersa.

[0030] Next, the technical solutions in the present invention will be clearly and completely described in conjunction with the embodiments in the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

[0031] Example 1 Isolation and Identification of Paenibacillus apapillosus Nan-Y Strain

[0032] Under aseptic operation, the body fluid of the diseased and pest Euproctis pseudoconspersa was scraped with a sterile inoculation loop, appropriately diluted in sterile water, and spread on an NA plate medium (10 g of peptone, 5 g of NaCl, 3 g of beef extract, 20 g of agar, added water to 1 L, pH natural), and cultured in an inverted manner in a constant temperature incubator at 30°C. After 2 d, the four-zone streaking method was used for three streaking separations until a single strain was observed under microscopic examination and named Nan-Y.

[0033] Cultivate the strain to be tested in liquid medium for 12 h, extract bacterial DNA, and use PCR technology (the universal primers for bacterial 16S rDNA are the upstream primer 27F (SEQ ID NO.1) and the downstream primer 1492R (SEQ ID NO.2). PCR reaction system: 10×Ex Taq buffer 2.0 μL, 5 U Ex Taq 0.2 μL, 2.5 mM dNTP Mix 1.6 μL, 27F / ITS1 1.0 μL, 1492R / ITS4 1.0 μL, DNA 0.5 μL, ddH2O 13.7 μL. PCR reaction program: pre-denaturation at 95 °C for 5 min; denaturation at 95 °C for 30 s, annealing at 56 °C for 30 s, extension at 72 °C for 30 s, 30 cycles; additional extension at 72 °C for 10 min.) to obtain its DNA sequence information. Conduct a homologous alignment on the NCBI website, and use MEGA 7.0 software to perform sequence analysis on the molecular identification results and construct a molecular phylogenetic tree ( Figure 1 ). Through Blast homology alignment on the NCBI website, it was found that the 16S rDNA sequence of the Nan-Y strain had a 99.71% identity with the corresponding sequence of the Pseudochrobactrum asacchrolyticum strain CCUG 46016 (accession number NR 042474.1). Use MEGA 7.0 software to perform sequence matching alignment and construct a phylogenetic tree to determine that the Nan-Y strain is Pseudochrobactrum asacchrolyticum.

[0034] Example 2 Morphological identification of Pseudochrobactrum asacchrolyticum strain Nan-Y

[0035] After inoculating the Nan-Y strain obtained in Example 1 on NA medium and culturing for 24 h, stain the bacterial cells by Gram staining method and observe under a Nikon E100 optical microscope. It was found that the bacterial cells were rod-shaped and without spores ( Figure 2 ).

[0036] Example 3 Determination of the pathogenicity of Pseudochrobactrum asacchrolyticum strain Nan-Y to the larvae of Euproctis pseudoconspersa

[0037] Inoculate the Nan-Y strain onto NA plate medium. After activation for 48 h, wash it with sterile water into a sterile Erlenmeyer flask (100 mL), and shake it on a SHA-C oscillator for 10 min (150 r·min -1 ), and prepare it with sterile water into (1±0.5)×10 10 , (1±0.5)×10 11 , (1±0.5)×10 12 , (1±0.5)×10 13 and (1±0.5)×10 14 cfu·mL-1 Bacterial suspensions of 5 concentrations. After soaking fresh young tea leaves in the bacterial solution for 20 s, they were placed in a culture dish (D = 15 cm). Four-instar healthy Euproctis pseudoconspersa larvae with basically the same size were selected and placed in the culture dish for feeding. Absorbent cotton soaked in sterile water was used for moisturizing, and they were raised in an artificial climate chamber at a temperature of (25 ± 1) °C and a relative humidity of RH 80%. Each concentration was one treatment, with 5 replicates for each treatment and 20 larvae for each replicate. Feeding with young tea leaves soaked in sterile water was used as the control.

[0038] According to Figure 3 It can be seen that after treating with Nan-Y bacterial suspensions of different concentrations for 2 d, the cumulative mortality rate of the larvae increased significantly. After 6 d, the cumulative lethality rate was 61.00% at the lowest concentration, and the cumulative mortality rate reached 100.00% at the highest concentration.

[0039] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. An Achromobacter xylosoxidans subsp. denitrificans Nan-Y, characterized in that, The Pseudochrobactrum asaccharolyticum Nan-Y was deposited at the Guangdong Microbial Culture Collection Center on April 21, 2023, with the deposit number GDMCC NO: 63377.

2. The Pseudogulbenkiania asaccharolytica Nan-Y according to claim 1, characterized in that, The Pseudochrobactrum asaccharolyticum Nan-Y is derived from the dead bodies of Euproctis pseudoconspersa larvae.

3. A bacterial suspension containing the Pseudochrobactrum asaccharolyticum Nan-Y described in claim 1.

4. A method for preparing the bacterial suspension according to claim 3, characterized in that, The Pseudochrobactrum asaccharolyticum Nan-Y is inoculated onto an NA plate medium. After activation for 36 - 72 h, it is cultured with shaking for 8 - 15 min to obtain a bacterial suspension of Pseudochrobactrum asaccharolyticum Nan-Y.

5. The preparation method according to claim 4, characterized in that, The NA plate medium comprises the following raw materials: 8 - 15 g of peptone, 2 - 8 g of NaCl, 2 - 5 g of beef extract, and 15 - 25 g of agar.

6. An insecticide for controlling the larvae of Euproctis pseudoconspersa, characterized in that, A bacterial suspension containing the Pseudochrobactrum asaccharolyticum Nan-Y described in claim 1 or 2 or the bacterial suspension of Pseudochrobactrum asaccharolyticum Nan-Y described in claim 3.

7. The pesticide according to claim 6, characterized in that, The effective content of the non-saccharolytic Pseudoclavibacter nan-yensis is (1±0.5)×10 10 ~(1±0.5)×10 14 cfu·mL -1 .

8. Use of the bacterial suspension of the Pseudochrobactrum asaccharolyticum Nan-Y described in claim 1 or 2 or the bacterial suspension of Pseudochrobactrum asaccharolyticum Nan-Y described in claim 3 in controlling Euproctis pseudoconspersa.

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