Appropriate citrobacter and application thereof

Abstract

The invention relates to a suitable citrobacter strain and application thereof. The invention relates to citrobacter suitabilis strain J35, which is preserved in the China General Microbiological Culture Collection Center (CGMCC), the preservation number of the citrobacter suitabilis strain J35 is CGMCC No.36708, and the citrobacter suitabilis strain J35 has insecticidal activity on Sitobion avenae, Rhopalosiphum padi, Amyzus persicae, Lygocoris lucorum, Bemisia tabaci, Plutella xylostella and Spodoptera litura. The citrobacter suitabilis strain J35 has the advantages that the strain J35 is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number of the citrobacter suitabilis strain J35 is CGMCC

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microorganisms, in particular to the field of biocontrol microorganisms. BACKGROUND

[0002] Plant pests and diseases can cause serious losses to agricultural production, and chemical control has been the most important control method since the founding of our country, which has significant control effect, high popularity and easy operation. However, due to non-standard use of drugs, ecological environment is damaged, soil is polluted, and residue problem is increasingly prominent, which greatly limits the sustainable development of China's agriculture. Therefore, developing biological control and reducing the dependence on chemical pesticides can effectively slow down the resistance of pests and diseases to chemical pesticides, and at the same time meet the new requirements of people for green, environmentally friendly and healthy life. SUMMARY

[0003] One of the present application provides a suitable Citrobacter strain J35, Citrobacter koseri which is preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC No. 36708.

[0004] The second of the present application provides a composition comprising the suitable Citrobacter strain J35 according to the first of the present application, and an acceptable carrier.

[0005] The third of the present application provides the use of the suitable Citrobacter strain J35 according to the first of the present application or the composition according to the second of the present application in the control of at least one of aphidoidea, miridae, aleyrodidae, plutellidae and noctuidae.

[0006] In a specific embodiment, the aphidoidea is at least one of Sitobion avenae, Sitobion avenae Schizaphis graminum and Acyrthosiphon pisum. Rhopalosiphum padi Acyrthosiphon pisum In a specific embodiment, the miridae is Lyus lineolaris.

[0007] In a specific embodiment, the aleyrodidae is Bemisia tabaci. Lygocoris lucorum

[0008] In a specific embodiment, the plutellidae is Plutella xylostella. Bemisia tabaci

[0009] In a specific embodiment, the noctuidae is Spodoptera litura. Plutella xylostella

[0010] In a specific embodiment, the noctuidae is Spodoptera litura. Spodoptera litura

[0011] ​​​​​In one specific embodiment, the aphids, the plant bugs, the whiteflies, the diamondback moths and the armyworms are in respective larval instars.

[0012] Advantages of the present application: The present application finds that the suitable Citrobacter sp. Citrobacter koseri ) J35 has insecticidal activity against the Sitobion avenae ( Sitobion avenae ), the Schizaphis graminum ( Rhopalosiphum padi ), the Acyrthosiphon pisum ( Acyrthosiphon pisum ), the Lygus lineolaris ( Lygocoris lucorum ), the Bemisia tabaci ( Bemisia tabaci ), the Plutella xylostella ( Plutella xylostella ) and the Spodoptera litura ( Spodoptera litura ). This not only broadens the use of the suitable Citrobacter sp., but also broadens the means of controlling the above-mentioned pests.

[0013] Strain preservation: The suitable Citrobacter sp. Citrobacter koseri screened by the present application is named J35, and the strain is preserved in the China General Microbiological Culture Collection Center, with a preservation number of CGMCC No. 36708, a preservation date of December 4, 2025, and a preservation address of No. 3, Yikuangli, Beichenxi Road, Beijing, China Institute of Microbiology, Chinese Academy of Sciences. Its systematic classification is Citrobacter sp. Citrobacter koseri. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 The figure shows the colony of the J35 strain.

[0015] Figure 2 The figure shows the phylogenetic tree of 16S of the J35 strain. DETAILED DESCRIPTION

[0016] The above content of the present application is further described in detail in the form of preferred implementation cases, but it does not constitute a limitation on the present application.

[0017] Unless otherwise specified, the strains, reagents, etc. in the embodiments of the present application can be purchased through commercial channels.

[0018] Straw medium preparation: dry the wheat straw naturally to a water content of less than 3wt%, then cut the long sections into small sections, crush them with a crusher, and pass them through a 100-mesh sieve to obtain wheat straw powder. Soak 100g of wheat straw powder with 1000ml of distilled water for 30min, heat to 100 degrees Celsius, continue heating for 10min, filter with gauze to obtain the extraction liquid; add 400ml of distilled water and 8g of agar to 100ml of the extraction liquid, sterilize at 121 degrees Celsius for 20 minutes.

[0019] LB liquid medium: 10 g of proteose peptone, 5 g of yeast powder, 10 g of NaCl, 1000 mL of distilled water, 121 degrees Celsius sterilization for 20 minutes. Example 1: Isolation of strains and preliminary screening of insecticidal activity

[0020] Straw medium was used to isolate strains from the collected beetle sand of Anomala daimiana.

[0021] Collection of beetle sand: 15 three-year-old larvae were taken, washed once with pure water, washed twice with 75% alcohol, washed once with pure water, and finally wiped the surface of the larvae with water-absorbing paper. The larvae were placed in a clean plastic box, covered with a lid and placed in a clean bench to wait for the larvae to defecate.

[0022] Strain isolation: 0.5 g of collected beetle sand was added to a 10 mL centrifuge tube, 5 mL of sterilized water containing one thousandth Tween-80 was added, vortexed to mix, and sequentially diluted to a concentration of 10 -2 to 10 -4 . 100 microliters of each concentration was coated on a straw medium plate and incubated in a 30 degree Celsius constant temperature incubator for 17 hours. Single colonies of different morphologies were picked and purified using LB solid medium streaking. This method was used for purification and culture for three times to obtain purified isolated strains, which were numbered.

[0023] Insecticidal strain screening: aphids were used as target organisms, and each isolated strain was cultured in LB liquid medium. The culture was incubated in a 30 degree Celsius, 200 rpm shaking bed for 36 hours. The bacterial content in the culture was adjusted to 1.25 x 10 16 CFU / mL using LB liquid medium as a negative control. Leaf immersion method was used to detect the touch-killing activity of the strains on aphids. Specifically, each strain was repeated three times, with 20 heads per repeat. Healthy and well-conditioned four-year-old wheat long-tube aphid alates colonized on wheat seedlings were selected and immersed in bacterial suspension with wheat leaves for 10 seconds, dried, and placed in a 10 cm x 10 cm culture dish. Observation began after 72 hours, and the number of dead insects was recorded. Insect bodies that did not react when touched with a soft brush were determined to be dead. The observation was continued until the end of the experiment at 1 week (144 hours). The number of dead insects was recorded, and strains with better insecticidal effects were selected. Example 2: Strain identification

[0024] The strains with better insecticidal effects selected were identified by morphology and molecular biology.

[0025] Strain J35 was cultured in LB medium at 30 degrees Celsius for 15 hours. The colonies of strain J35 were observed to be round, grayish white, translucent, smooth and moist on the surface, and Figure 1The characteristics were compared with the strain morphology described in the Systematic Manual of Common Bacteria, and it was preliminarily speculated to be a strain of Bacillus or Citrobacter.

[0026] DNA of the strain J35 was extracted using a TSINGKE extraction kit, and 16S rDNA sequences of the strain J35 were amplified using bacterial 16S rDNA universal primers 27F (as shown in SEQ ID No. 1) and 1492R (as shown in SEQ ID No. 2) with the DNA of the strain J35 as a template, and a 1402 bp fragment was obtained. The sequence obtained is shown as SEQ ID No. 3. The sequence was submitted to the NCBI website for homology comparison, and a phylogenetic tree was constructed using MEGA11, as shown in Figure 2 According to the phylogenetic tree of Figure 2 , it is known that it is relatively close to the suitable Citrobacter strain of Citrobacter, and therefore the systematic classification of the strain J35 is suitable Citrobacter Citrobacter koseri .

[0027] The J35 strain was preserved in the General Microbial Center of the Chinese Microbial Strain Preservation Management Committee, with a preservation number of CGMCC No. 36708, a preservation date of December 4, 2025, and a preservation address of No. 3, Yikuangli, Beichen West Road, Beijing, China Institute of Microbiology, Chinese Academy of Sciences. Its systematic classification is suitable Citrobacter Citrobacter koseri . Example 3: Insecticidal activity of suitable Citrobacter J35

[0028] The J35 strain was inoculated into a test tube containing 5 mL of LB liquid medium, activated by culturing at 30 degrees Celsius for 12 hours, and then the activated bacterial liquid was inoculated into 12 mL of LB liquid medium at 1%, and cultured at 30 degrees Celsius with 200 rpm shaking for 36 hours. The bacterial content in the culture liquid was adjusted to 1.25 x 10 16 CFU / mL by LB liquid medium by plate counting method, to obtain a J35 suspension.

[0029] Healthy and well-conditioned 4th instar winged nymphs of S. graminum colonized on wheat seedlings were immersed in the J35 suspension together with the wheat leaves for 10 s, air-dried, and placed in 10 cm x 10 cm culture dishes, and reared under the conditions of temperature 22 ± 2 degrees Celsius, humidity 65 ± 5%, and photoperiod L:D = 16:8. The wheat leaves were replaced every day during the rearing period. There were 3 replicates, with 20 individuals in each replicate. LB liquid medium was used as a negative control. The number of dead insects was counted and the mortality and corrected mortality were calculated at 7 d.

[0030] The 4th instar aphid was replaced by the 4th instar aphid nymph of Sitobion avenae, and the other operations were the same as those of the aphid. The number of dead insects was counted and the mortality and corrected mortality were calculated on the 7th day.

[0031] The 4th instar aphid was replaced by the 4th instar aphid nymph of Sitobion avenae, and the other operations were the same as those of the aphid. The number of dead insects was counted and the mortality and corrected mortality were calculated on the 7th day.

[0032] The 4th instar aphid was replaced by the 4th instar aphid nymph of Sitobion avenae, and the other operations were the same as those of the aphid. The number of dead insects was counted and the mortality and corrected mortality were calculated on the 7th day.

[0033] The 4th instar aphid was replaced by the 4th instar aphid nymph of Sitobion avenae, and the other operations were the same as those of the aphid. The number of dead insects was counted and the mortality and corrected mortality were calculated on the 7th day.

[0034] The 4th instar aphid was replaced by the 4th instar aphid nymph of Sitobion avenae, and the other operations were the same as those of the aphid. The number of dead insects was counted and the mortality and corrected mortality were calculated on the 7th day.

[0035] The 4th instar aphid was replaced by the 4th instar aphid nymph of Sitobion avenae, and the other operations were the same as those of the aphid. The number of dead insects was counted and the mortality and corrected mortality were calculated on the 7th day.

[0036] The 4th instar aphid was replaced by the 4th instar aphid nymph of Sitobion avenae, and the other operations were the same as those of the aphid. The number of dead insects was counted and the mortality and corrected mortality were calculated on the 7th day.

[0037] The aphids were replaced with ladybugs 2nd instar larvae, which were directly immersed in J35 suspension or LB liquid medium for 10 s, dried, and fed with B. tabaci. During the feeding period, B. tabaci were supplemented daily. The feeding temperature was 22±2 degrees Celsius, the humidity was 65±5%, and the light cycle was L:D = 16:8. Each repetition had 20 individuals. The rest of the bioassay operations were the same as those for the aphids. The number of dead insects was counted and the mortality and corrected mortality were calculated at 7 days.

[0038] The results are shown in Table 1.

[0039] Table 1

Claims

1. Suitable Citrobacter ( Citrobacter koseri Strain J35 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 36708.

2. A composition comprising the Citrobacter diversus strain J35 according to claim 1 and an acceptable carrier.

3. Use of the Citrobacter diversus strain J35 according to claim 1 or the composition according to claim 2 for controlling at least one of aphidoidea, miridae, aleyrodidae, plutellidae and noctuidae.

4. Use according to claim 3, characterized in that, The aphids are at least one of Sitobion avenae (cereal aphid) Sitobion avenae ), Schizaphis graminum (English grain aphid) Rhopalosiphum padi ), and Acyrthosiphon pisum (pea aphid) Acyrthosiphon pisum ).

5. Use according to claim 3, characterized in that, The plant bug is Lyus lineolaris (Palisot de Beauvois) Lygocoris lucorum ).

6. Use according to claim 3, characterized in that, The whitefly is Bemisia tabaci (Gennadius) Bemisia tabaci ).

7. Use according to claim 3, characterized in that, The said moth is Plutella xylostella (L.) (Linnaeus) (Lepidoptera: Plutellidae) Plutella xylostella ).

8. Use according to claim 3, characterized in that, The noctuid is Spodoptera exigua (Hubner) (Lepidoptera: Noctuidae) Spodoptera litura ).

9. Use according to claim 3, characterized in that, in their respective larval instar stages.

4. The use according to claim 3, wherein the aphidoidea are Acyrthosiphon pisum, Rhopalosiphum padi, Sitobion avenae, Myzus persicae, Aphis fabae, Aphis craccivora, Aphis gossypii, Brachycorynella asclepiab, Hyalopterus pruni, Macrosiphum euphorbiae, Myzus cerasi, Myzus varians, Myzus nicotianae, Nasonovia ribis, and the miridae are Apolygus lucorum, Cyrtopimpla luteoventris, Distantiella theobroma, Eysarcoris parvus, Leptocorisa varivestis, Leptocorisa oratoria, Lygus lineolaris, Lygus pratensis, Lygus hesperus, and the aleyrodidae are Bemisia tabaci, Trialeurodes vaporariorum, and the plutellidae are Plutella xylostella, and the noctuidae are Spodoptera litura, Spodoptera exigua, Spodoptera eridania, Spodoptera cosmioides, Spodoptera littoralis, Spodoptera mauritia, Spodoptera nympha, Spodoptera ornithogalli, Spodoptera pelipleura, Spodoptera praevulata, Spodoptera signifera

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