CN116103186B - Enterobacter cloacae and application thereof in preventing and treating litchi frost epidemic diseases - Google Patents
Enterobacter cloacae and application thereof in preventing and treating litchi frost epidemic diseases Download PDFInfo
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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Abstract
The invention discloses enterobacter cloacae and application thereof in preventing and treating litchi frost epidemic diseases, and belongs to the technical field of plant disease biocontrol. The Enterobacter cloacae is named as Enterobacter cloacae (Enterobacter cloacae) YG-7 which is deposited in Guangdong province microorganism strain collection of No. 59 building of Guangdong province, guangzhou city, xiuxiu martyr at day 14 of 2022, with the deposition number of GDMCC No:62546. The enterobacter cloacae is derived from rhizosphere soil of healthy litchi fruit trees, and is mutually symbiotic with litchi for a long time, so that the possibility of damaging the stability of the microecological environment on the plant surface is avoided; the enterobacter cloacae and the biological preparation based on the enterobacter cloacae have strong inhibition effect on the phytophthora litchi and have good development and application prospects.
Description
Technical Field
The invention belongs to the technical field of plant disease biocontrol, and particularly relates to enterobacter cloacae YG-7 and application thereof in controlling litchi frost epidemic diseases.
Background
Litchi (LITCHI CHINENSIS Sonn.) is originally produced in southern China, and is a subtropical evergreen arbor, which is commonly called as 'four big fruits in south China' together with bananas, longans and pineapples. The fruit juice has good taste and juice, has extremely high nutritive value and very high medicinal value, can be used as medicine for fruits, fruit cores, fruit shells, even spica and root peels, and has the effects of relieving cough, promoting spirit, strengthening qi, benefiting human colors and the like. The litchi is easily affected by diseases and insect pests in the field planting and post-harvest storage processes, the quality and yield of the litchi are seriously reduced, and the litchi is easily browned and rotten due to the limitation of the storage period, which is one of important factors for severely limiting the litchi production. The rot and deterioration of fruits can be caused by the plague of litchi frost, anthracnose, acid rot and the like, and the harm of the plague caused by the phytophthora litchi is the most serious.
When the litchi frost epidemic disease is developed, brown disease spots are generated when the fruit stalks and fruiting branches of litchi develop. The fruits are mostly onset at the pedicel, brown irregular lesions are generated on the surface of the fruit peel, the whole fruit is rapidly expanded, the fruit peel is dark brown to black, the pulp is rotten to give off wine flavor or sour flavor, and brown juice is discharged. White downy mildew grows on the surface of the affected part when wet. Phytophthora litchi (Peronophythora LITCHII CHEN ex Ko et al) which causes frosty blight can infect not only nearly mature fruits, but also flowers, young fruits, branches, stalks, even tender leaves and the like of litchi to cause flower and fruit dropping. In the picking, storage and transportation processes of litchi fruits, a few fruits carrying litchi frost epidemic disease fruits can cause the diseases of large-area adjacent fruits, the fruits of the whole trees or the whole baskets are decomposed and deteriorated, the storage and the export of fresh fruits are seriously influenced, and huge economic losses are caused.
At present, chemical control is mainly used for controlling the litchi frost epidemic disease in production. This not only leaves a potential risk for the food safety of litchi fruits, but also promotes the pathogenic bacteria to develop drug resistance. For this reason, many researches have been focused on developing a safe and efficient biocontrol method. Antagonistic microorganisms are screened from litchi rhizosphere soil, so that the stability of the microecological environment of the plant rhizosphere soil can be effectively maintained, and the method has important practical significance.
Enterobacter cloacae (Enterobacter cloacae) of the genus Enterobacter (Enterobacter) has no spore or no capsule, forms large and moist mucinous colony, is facultative anaerobic gram-negative Brevibacterium, can be detected in human and animal feces, water, soil and plants, and is widely found in nature. Research shows that the saline-alkali tolerant enterobacter cloacae can be used for promoting the growth of the capsicum and the biological control of phytophthora capsici, in particular to the biological control of cultivating the capsicum epidemic disease in saline-alkali soil (Sun Zhongtao and the like, 2020). Peng Yinan (2022) found that the addition of enterobacter cloacae to the composite microbial preparation can significantly improve the agronomic traits and yield of corn, and effectively exert the effect of promoting growth after corn application. At present, no related report research is carried out on the biological control of the downy mildew of litchis, so that the successful application of the downy mildew of litchis in the aspect of biological control is capable of effectively controlling the storage of the disease after litchi picking, has an important role in the biological control of plant diseases, is an important natural resource, is more concerned about the inhibition effect of the downy mildew of litchis, and has the potential of developing biological pesticides.
Disclosure of Invention
In order to overcome the defects and the shortcomings of the prior art in preventing and treating litchi frost epidemic diseases and develop application of enterobacter cloacae, the primary aim of the invention is to provide the enterobacter cloacae. The strain is obtained by taking litchi fruit tree rhizosphere microorganisms as screening objects and has a good inhibition effect on phytophthora litchi. The strain provides new resources and directions for enterobacter cloacae for biological control instead of chemical control, and can be developed and utilized as biological pesticide in future.
The invention also aims to provide a biological agent prepared based on the enterobacter cloacae.
Another object of the present invention is to provide the use of the above Enterobacter cloacae.
In order to achieve the above object, the present invention adopts the following technical scheme:
A strain of Enterobacter cloacae, designated Enterobacter cloacae (Enterobacter cloacae) YG-7, was deposited at No. 59 building of Guangdong province microorganism strain collection (GDMCC) in Guangzhou, kyowa, martyr on day 14 of 2022, with a deposit number of GDMCC No:62546.
A biological preparation contains thallus and/or culture solution of Enterobacter cloacae YG-7.
Further, the culture medium may include the culture medium itself, a concentrate thereof, or a lyophilized product thereof obtained by culturing the strain, or may include a culture medium supernatant, a concentrate thereof, or a lyophilized product obtained by removing the strain from the culture medium.
Further, the cultured strain preferably comprises the steps of: inoculating enterobacter cloacae YG-7 into LB liquid culture medium for culture to obtain culture solution.
The pH value of the LB liquid medium is 7.0.
The preparation of each liter of LB liquid medium is as follows: 10.0g of tryptone, 5.0g of yeast extract and 10.0g of sodium chloride are weighed, 1000mL of water is added, the mixture is stirred and mixed uniformly, 15.0g of agar is added, and after the mixture is fully heated and dissolved, the mixture is subjected to wet heat sterilization at 121 ℃ for 20min for later use.
The culture is carried out for 24 hours under the conditions that the temperature is 28-30 ℃ and the shaking speed is 180-220 rpm.
The application of the enterobacter cloacae or the biological agent is one or a combination of the following applications:
(1) Application in inhibiting growth of Phytophthora litchii;
(2) The application in preparing the litchi downy mildew inhibitor;
(3) Application in orthodontics litchi downy mildew colony
(4) The application in preparing the downy mildew morphology regulator;
(5) The application in preventing and treating litchi frost epidemic diseases.
Compared with the prior art, the invention has the following advantages and effects:
1. the enterobacter cloacae obtained by the invention is derived from rhizosphere soil of healthy litchi fruit trees, and is mutually symbiotic with litchi for a long time, so that the possibility of damaging the stability of the microecological environment on the plant surface is avoided;
2. The biological agent has strong inhibition effect on the phytophthora litchi, and the biological agent has environmental protection and no toxicity, and has little influence on ecological environment;
3. the enterobacter cloacae obtained by the invention has low requirements on culture conditions and has good development and application prospects.
Drawings
FIG. 1 is a single colony chart of Enterobacter cloacae YG-7 on LB medium;
FIG. 2 is a morphology of normal growing colonies of Phytophthora litchii;
FIG. 3 is a graph showing the effect of Enterobacter cloacae YG-7 on inhibiting Phytophthora litchi;
FIG. 4 is a graph showing the effect of the biological preparation of Enterobacter cloacae YG-7 on inhibiting Phytophthora litchii.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but embodiments of the present invention are not limited thereto. The reagents involved in the examples are all available from commercial sources.
Example 1: isolation, purification and preservation of Enterobacter cloacae YG-7
(1) Preparation of LB medium: 10g of tryptone, 5g of yeast extract and 10g of sodium chloride are weighed, water is added to 1000mL, stirring and mixing are carried out, 15g of agar is added, the mixture is fully heated and dissolved, and then the mixture is packaged into 250mL triangular flasks, sterilized by heat and humidity at 121 ℃ for 20min, and preserved for standby.
(2) Isolation and purification of enterobacter cloacae: soil samples for separating antagonistic bacteria are collected in healthy fruit tree rhizosphere soil in litchi orchards at agricultural university of south China. Weighing 10g of soil sample, adding the soil sample into a triangular flask containing 90mL of sterile physiological saline (0.9%) under aseptic condition, placing the triangular flask on a 180rpm shaking table at 28 ℃ for shaking for 30min to prepare a soil diluent with dilution multiple of 10 -1, and standing at room temperature for 10min to separate solid impurities such as sand, stone, soil and the like. 1mL of the soil dilution with the dilution factor of 10 -1 is taken and added into a test tube with 9mL of physiological saline, and the mixture is fully and uniformly mixed to prepare the soil dilution with the dilution factor of 10 -2. And so on, the soil liquid is gradually diluted to 10 -6. 100. Mu.L of dilutions (dilution factors: 10 -4、10-5 and 10 -6, respectively) were applied to the LB solid plate prepared in step (1), 3 replicates were set for each dilution factor, and incubated at 30℃for 3d to isolate various bacteria in the soil. And (3) selecting bacteria with obvious characteristic differences of size, shape, color, texture, surface structure and the like, and purifying by a plate streaking method to obtain single colonies for later use.
Morphological feature analysis: culturing in LB medium at 28deg.C for 24 hr, and along with the extension of culture time, single colony is round, milky white, protruding, smooth, and neat in edge, and opaque (see FIG. 1).
16S sequence analysis: the purified strain is subjected to 16S sequence analysis, and the 16S sequence (shown as SEQ ID NO. 1) has 99.58 percent of homology with Enterobacter cloacae with the accession number of MN062622.1 in GenBank.
The classification of Enterobacter cloacae (Enterobacter cloacae) was confirmed by combining the above morphological characteristics and 16S sequence results. Further, the strain was designated as Enterobacter cloacae (Enterobacter cloacae) YG-7 and deposited at No. 59 building of Guangdong province in Xiuzhou Kogyu martyr, guangzhou City, guangdong in 2022, at 6 months and accession number GDMCC No:62546.
16S sequence fragment (SEQ ID NO. 1):
CATTGGGGGCAGCTACACATGCAAGTCGAACGGTAGCACAGAGAGCTTGCTCTCGGGTGACGAGTGGCGGACGGGTGAGTAATGTCTGGGAAACTGCCTGATGGAGGGGGATAACTACTGGAAACGGTAGCTAATACCGCATAATGTCGCAAGACCAAAGAGGGGGACCTTCGGGCCTCTTGCCATCAGATGTGCCCAGATGGGATTAGCTAGTAGGTGGGGTAACGGCTCACCTAGGCGACGATCCCTAGCTGGTCTGAGAGGATGACCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCCATGCCGCGTGTATGAAGAAGGCCTTCGGGTTGTAAAGTACTTTCAGCGGGGAGGAAGGTGTTGTGGTTAATAACCMCAGCAATTGACGTTACCCGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCACGCAGGCGGTCTGTCAAGTCGGATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATTCGAAACTGGCAGGCTGGAGTCTTGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACAAAGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCGATTTGGAGGTTGTGCCCTTGAGGCGTGGCTTCCGGAGCTAACGCGTTAAATCGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTGGTCTTGACATCCACAGAACTTTCCAGAGATGGATTGGTGCCTTCGGGAACTGTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATCCTTTGTTGCCAGCGGTTAGGCCGGGAACTCAAAGGAGACTGCCAGTGATAAACTGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGACCAGGGCTACACACGTGCTACAATGGCGCATACAAAGAGAAGCGACCTCGCGAGAGCAAGCGGACCTCATAAAGTGCGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGTAGATCAGAATGCTACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCAAAAGAAGTAGGTAGCTTAACCTTCGGGAGGGCGCTACCACTTTGAATCAGGG.
Example 2: activity of Enterobacter cloacae YG-7 by counter culture
(1) The preparation of LB medium was the same as in example 1.
(2) Preparing carrot culture medium: weighing 200g of carrot, fully squeezing with a juicer, filtering with 16 layers of gauze, supplementing water to 1000mL, adding 15g of agar, fully heating for dissolving, packaging in a conical flask, sealing, sterilizing at 121deg.C for 20min, and preserving for later use.
(3) Preparation of enterobacter cloacae single colony: and (3) streaking and inoculating enterobacter cloacae onto an LB culture medium plate for activation culture for 24 hours, and then picking single bacterial colony for streak preservation.
(4) Culturing the lychee phytophthora: inoculating Phytophthora litchi to the carrot plate, culturing for 7d, and taking out bacterial cake at the edge of bacterial colony by using a puncher with the diameter of 7 mm.
(5) Activity measurement: inoculating enterobacter cloacae obtained in the step (3) on the edge of a carrot culture medium flat plate in a streaking way, inoculating the phytophthora litchi cake obtained in the step (4) at a position 3cm away from the streaking area, taking enterobacter cloacae which is not inoculated as a control in an experiment, repeating the steps for 3 times, culturing at a constant temperature of 25 ℃ for 5 days, observing the colony morphology of the antibacterial zone edge and the control phytophthora litchi, and the results are shown in figures 2 and 3. As can be seen from the comparison of the two, the enterobacter cloacae YG-7 has obvious inhibition effect on the growth of the downy mildew, and causes the malformation of hyphae.
Example 3: oxford cup method for measuring activity of enterobacter cloacae YG-7 biological preparation
(1) Preparation of LB medium was the same as in example 1; the preparation of LB liquid medium is not added with agar, the other ingredients are the same as the formula of LB solid medium, the weighed medicament is fully dissolved and then is split into conical flasks (100 mL of culture solution per flask), the conical flasks are plugged and wrapped, sterilization is carried out for 20min at 121 ℃, and the conical flasks are cooled and stored for standby.
(2) Carrot culture media were prepared as in example 2.
(3) Preparation of enterobacter cloacae biological preparation: and (3) taking a single colony of the enterobacter cloacae prepared in the step (3) in the example 2, putting the single colony into the conical flask of the LB culture solution prepared in the step (1), and culturing for 24 hours at 28 ℃ at a shaking table speed of 180rpm to obtain the biocontrol preparation of the enterobacter cloacae.
(4) The culture of Phytophthora litchii was the same as in example 2.
(5) Activity measurement: placing an oxford cup at the edge of a carrot culture medium flat plate, sucking 200 mu L of the enterobacter cloacae biological agent prepared in the step (3) into the oxford cup, inoculating the downy mildew cake prepared in the step (4) at a position 3cm away from the oxford cup, taking the uninoculated enterobacter cloacae as a control in experiments, culturing for 5 days at the constant temperature of 25 ℃ repeatedly for 3 times, and observing the colony morphology of the downy mildew on the edge of a bacteriostatic zone and the control, wherein the result is shown in figure 4. As can be seen from comparison of FIG. 2, the biological preparation of Enterobacter cloacae YG-7 of the present invention has an obvious inhibitory effect on the growth of Phytophthora litchii and causes malformation of colonies.
The embodiments described above are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the embodiments described above, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principles of the present invention should be made in the equivalent manner, and are included in the scope of the present invention.
Claims (10)
1. The enterobacter cloacae is characterized in that: the strain is named as Enterobacter cloacae (Enterobacter cloacae) YG-7 which is deposited at No. 59 building of Guangdong province in Xiuzhou Kouzuo district martyr of Guangdong province on day 14 of 2022, and the deposited number is GDMCC No: 62546.
2. A biologic, characterized in that: a bacterial cell and/or a culture solution containing the Enterobacter cloacae YG-7 of claim 1.
3. The biologic of claim 2, wherein:
the culture solution is a culture solution itself obtained by culturing a strain, a concentrate thereof or a freeze-dried product thereof.
4. The biologic of claim 2, wherein:
the culture solution is a culture solution supernatant obtained by removing a strain from a culture solution obtained by culturing the strain, a concentrate thereof or a freeze-dried product.
5. The biologic of claim 3 or 4, wherein:
The cultured strain comprises the following steps: inoculating enterobacter cloacae YG-7 into LB liquid culture medium for culture to obtain culture solution.
6. The biologic of claim 5, wherein:
The pH value of the LB liquid medium is 7.0.
7. The biologic of claim 5, wherein:
The preparation of each liter of LB liquid medium is as follows: 10.0g of tryptone, 5.0g of yeast extract and 10.0g of sodium chloride are weighed, 1000mL of water is added, the mixture is stirred and mixed uniformly, 15.0g of agar is added, and after the mixture is fully heated and dissolved, the mixture is subjected to wet heat sterilization at 121 ℃ for 20min for later use.
8. The biologic of claim 5, wherein:
the culture is carried out for 24 hours under the conditions that the temperature is 28-30 ℃ and the shaking speed is 180-220 rpm.
9. The biologic of claim 5, wherein:
the culture is carried out at 28 ℃ for 24 hours under the condition of shaking speed of 180 rpm.
10. Use of enterobacter cloacae according to claim 1 or a biological agent according to any one of claims 2 to 3, characterised in that: is one or a combination of the following applications:
(1) Application in inhibiting growth of Phytophthora litchii;
(2) The application in preparing the litchi downy mildew inhibitor;
(3) The application in the colony of the orthodontics downy mildew of litchi.
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| CN110468075A (en) * | 2019-08-21 | 2019-11-19 | 华南农业大学 | One plant height mountain bacillus and its application in prevention and treatment lichee frost epidemic disease |
| CN112204038A (en) * | 2019-08-17 | 2021-01-08 | 浙江新农化工股份有限公司 | Agricultural bactericide and application and preparation method thereof |
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| CN112204038A (en) * | 2019-08-17 | 2021-01-08 | 浙江新农化工股份有限公司 | Agricultural bactericide and application and preparation method thereof |
| CN110468075A (en) * | 2019-08-21 | 2019-11-19 | 华南农业大学 | One plant height mountain bacillus and its application in prevention and treatment lichee frost epidemic disease |
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