CN116083308B - Biocontrol strain Pse147 for preventing and treating sugarcane white streak and application thereof - Google Patents

Biocontrol strain Pse147 for preventing and treating sugarcane white streak and application thereof Download PDF

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CN116083308B
CN116083308B CN202310038524.9A CN202310038524A CN116083308B CN 116083308 B CN116083308 B CN 116083308B CN 202310038524 A CN202310038524 A CN 202310038524A CN 116083308 B CN116083308 B CN 116083308B
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张木清
李美霖
暴怡雪
姚伟
陈保善
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Abstract

The invention discloses a biocontrol strain Pse147 for preventing and treating sugarcane white streak and application thereof, and relates to the technical field of plant protection. The biocontrol strain Pse147 is Pseudomonas aeruginosa (Pseudomonas asmosselii) which is preserved in China center for type culture Collection with the preservation address of China, university of Wuhan and the preservation number of CCTCCNO: m20221133. According to the invention, the biocontrol strain Pse147 is obtained by separating and screening sugarcane leaves, and the biocontrol strain Pse147 is pseudomonas aeruginosa through identification, and is proved by a flat plate counter culture experiment and a greenhouse barrel planting experiment, the biocontrol strain Pse147 has a good effect of preventing and treating sugarcane white streak, and the prevention effect can reach more than 66%.

Description

Biocontrol strain Pse147 for preventing and treating sugarcane white streak and application thereof
Technical Field
The invention relates to the technical field of plant protection, in particular to a biocontrol strain Pse147 for preventing and treating sugarcane white streak and application thereof.
Background
Sugarcane white streak (Sugarcane leafscald) is a bacterial disease caused by xanthomonas Saccharum Xanthomonas albilineans (Xal). Sugarcane white streaks are classified into acute and chronic. The acute symptoms can lead to reddening of the cane stems, cause rapid death of the cane, and are particularly obvious after the cane is overqi in rainy days. The chronic disease firstly grows a pencil-shaped stripe parallel to the vein on the leaf, and the leaf becomes white in a large area along with the aggravation of the disease, and finally the leaf becomes yellow and dies. The typical symptom of sugarcane white streak is that a pencil-shaped white streak parallel to veins is generated on leaves, and the sugarcane white streak is characterized in that the sugarcane white streak is in a state of latency in a period of months or even years when pathogenic bacteria infest plants, and does not show symptoms.
Research shows that the occurrence of sugarcane white streak can cause huge loss of sugarcane yield, and the serious damage can reach 10% -34% of loss per hectare.
The pathogenic bacteria causing sugarcane white streak is sugarcane xanthomonas, the pathogenic bacteria is gram-negative bacteria, the capsule is in a rod shape, the diameter is (0.6-1.0) × (0.2-0.3) mu m, the optimal culture temperature is 25-28 ℃, and the propagation mode is through leaves and leaves, roots and roots, even through air. Although the study on sugarcane white streaks was earlier reported since 1994, there has been no stable and effective measure for disease control. The traditional control method such as improvement of sugarcane seed disinfection, cultivation measures, selection of disease-resistant varieties, chemical control and the like is difficult to find resistance genes and overcome the problems of disease resistance, quality property and the like; if chemical control is adopted, pathogenic bacteria are easy to generate drug resistance, the environment is polluted, and life safety of human beings and organisms is threatened, so that development of new green pollution-free biological control and antagonistic bacteria is also an important subject in the next agricultural production.
Disclosure of Invention
The invention aims to provide a biocontrol strain Pse147 for preventing and treating sugarcane white streak and application thereof, so as to solve the problems in the prior art.
In order to achieve the above object, the present invention provides the following solutions:
the invention provides a biocontrol strain for preventing and treating sugarcane white streak, which is pseudomonas aeruginosa (Pseudomonas mosselii) Pse147, and is preserved in Wuhan university in China at the year of 2022 and 7 months and 18 days, and the preservation address is the university of Wuhan and the preservation number is CCTCC No. M20221133.
The invention also provides application of the biocontrol strain in preparation of biocontrol bactericides for preventing and treating sugarcane white streak.
The invention also provides a biocontrol microbial inoculum for preventing and treating sugarcane white streak, which comprises the biocontrol strain.
The invention also provides application of the biocontrol strain or biocontrol microbial inoculum in preventing and treating sugarcane white streak.
Further, the pathogenic bacteria of sugarcane white streak disease is xanthomonas sugarcane (Xanthomonas albilineans).
The invention also provides a preparation method of the biocontrol microbial inoculum, which comprises the following steps: fermenting and culturing the biocontrol strain to obtain fermentation liquor, and diluting the fermentation liquor to obtain the biocontrol microbial agent.
Further, the conditions of the fermentation culture include: the temperature is 28 ℃, the rotating speed is 180-210rpm, and the culture time is 12-16h.
Further, the diluted OD of the fermentation broth 600 2.
The invention also provides application of the biocontrol strain in preparing a bacteria inhibitor of xanthomonas.
The invention also provides a bacteria inhibitor of Xanthomonas, which comprises the biocontrol strain.
The invention discloses the following technical effects:
the biocontrol strain Pse147 is obtained by separating and screening sugarcane leaves, and the biocontrol strain Pse147 is pseudomonas aeruginosa through identification, and the biocontrol strain Pse147 has good effect of preventing and treating sugarcane white streak and has the prevention effect of more than 66 percent through a flat plate counter culture experiment and a greenhouse barrel planting experiment.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a colony morphology of Pse147 strain on LB solid medium;
FIG. 2 is a phylogenetic tree constructed in example 1;
FIG. 3 shows the results of a first set of plate counter experiments in example 3;
FIG. 4 shows the results of a second set of plate counter experiments in example 3, wherein the Pse147 bacterial solutions of A-D had respective OD concentrations 600 ＝0.2、OD 600 ＝0.5、OD 600 =1 and OD 600 ＝2。
Detailed Description
Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples of the present invention are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.
The preparation method of the medium used in the following examples was as follows:
MW solid Medium: 5g of peptone, 0.5g of monopotassium phosphate, 10g of sucrose, 0.25g of magnesium sulfate heptahydrate, 0.05g of sodium sulfite and 10g of agar, adding deionized water to 1000mL, and sterilizing at 115 ℃ for 21 minutes.
MW liquid Medium: 5g of peptone, 0.5g of monopotassium phosphate, 10g of sucrose, 0.25g of magnesium sulfate heptahydrate and 0.05g of sodium sulfite, adding deionized water to 1000mL, and sterilizing at 115 ℃ for 21 minutes.
LB solid medium: tryptone (Tryptone) 10g/L, yeast extract (Yeast extract) 5g/L, sodium chloride (NaCl) 10g/L, agar 10g/L, pH=7.4, deionized water to 1000mL, and sterilizing at 121deg.C for 15 min.
Xanthomonas Saccharum Xal JG43 used in the examples below is disclosed in the literature "Li, M., et al, comparative genome analysis unravelspathogenicity ofXanthomonas albilineans causing sugarcane leaf scaleddisease.BMC Genomics,2022.23 (1): p.671".
Example 1
1. Isolation and screening of strains
In 7 months 2021, a strain of bacteria obtained by separation and screening in sugarcane leaves is named as Pse147, and the separation and screening method is as follows:
(1) Putting sugarcane leaves into a sterilized plastic bag, rapidly taking the sugarcane leaves back to a laboratory, taking 5g of sugarcane leaves, sterilizing, shearing the sugarcane leaves, putting the sugarcane leaves into a culture dish with 90mm, and soaking the sugarcane leaves in alcohol under a sterile environment.
Rinsing with sterile water, cutting, loading into 1.5mL ep tube, adding sterile water 1mL, and cooling to-4deg.CAnd (5) preserving in a refrigerator. The following day is taken out and configured to be 10 -2 ～10 -4 The sample was diluted and the dilution was spread on LB solid medium, which was placed at 28℃for 3d.
Bacterial morphology observation shows that bacterial colonies conforming to the morphology characteristics of pseudomonas aeruginosa are selected and separated by a plate streaking method. And separating, purifying and preserving strains according to the size and the morphology of the bacterial colonies on the culture medium. Each test was repeated 3 times.
2. Authentication
(1) Morphological and physiological biochemical identification
The colony morphology of the Pse147 strain on LB solid medium is shown in FIG. 1. The biological properties of the identified Pse147 strain were: aerobic respiration, gram-negative staining, rod-shaped thallus, optimal growth temperature of 28-37 ℃ and optimal growth pH of 6.0-7.0.
(2) Molecular biological identification
Through comparison of the evolutionary trees, the relation between the Pse147 and other pseudomonas aeruginosa is found: phylogenetic trees were constructed using neighbor-joining (NJ) and Bayesian Inference (BI), see FIG. 2. The Pse147 strain was identified to be of Pseudomonas aeruginosa (Pseudomonas mosselii).
3. Preserving
At day 7 and 18 of 2022, the strain Pse147 is preserved in China center for type culture Collection with a preservation address of China university of Wuhan and a preservation number of CCTCC NO: m20221133.
Example 2
Inoculating Pse147 strain into LB liquid medium, shake culturing at 28deg.C and 180rpm (180-210 rpm can achieve the same effect) for 16 hr (12-16 hr can achieve the same effect), and diluting with sterilized water to obtain OD 600 ＝0.2、OD 600 ＝0.5、OD 600 ＝1、OD 600 Bacterial liquid of=2, ready for use.
Example 3
The first panel counter experiment of Pse147 against sugarcane white streak pathogen (i.e., xanthomonas saccharum) was performed as follows:
(1) Inoculating Xanthomonas Saccharum Xal JG43 in MW liquid culture medium, shaking at 28deg.COD after overnight incubation 600 1, regulating the bacterial liquid to OD 600 =0.5 and evenly spread into MW solid medium.
(2) An oxford cup was inserted in the center of the MW solid medium and 150. Mu.L of the OD prepared in example 2 was injected into the center of the oxford cup with a pipette 600 Bacterial liquid=0.5. The inoculated MW plate is placed in an incubator at 28 ℃ for culture.
(3) After 7d, judging whether the strain has antagonism or not by judging whether a bacteriostasis ring and the size of the bacteriostasis ring appear on the flat plate, photographing to record the diameter of the bacteriostasis ring regularly, and calculating the bacteriostasis rate, wherein the results are shown in table 1, figure 3 and figure 4.
Bacteriostatic ratio = length of zone diameter/plate diameter x 100%.
(4) A second set of plate counter experiments (experimental methods were performed with the first set of plates) was performed by density gradient dilution, finding the optimum antagonistic concentration of Pse147 and periodically recording the diameter of the zone of inhibition.
TABLE 1
Pse147 bacterial liquid OD 600 Antibacterial diameter (cm) Bacteriostasis rate (%)
0.2 3.6 41.4％
0.5 5.7 65.5％
1 6.5 74.7％
2 7.9 90.8％
The results in Table 1 show that the Pse147 strain has stronger antagonism to the pathogenic bacteria of sugarcane white streak, wherein the optimal antagonism concentration is OD 600 ＝2.0
The criteria used to evaluate the inhibition of E.coli, staphylococcus aureus by Bacillus coagulans with Yao Xiaogong were based on: "high sensitivity" (DIZ)>20 mm), "middle sensitivity" (10 mm)<DIZ<20 mm) and "hypo-sensitivity" (DIZ)<10 mm) is used as a judgment standard. As is known from the first group of plate counter experiments, when the bacterial liquid concentration is OD 600 When the bacterial strain Pse147 is=0.5, the bacterial strain Pse147 has an inhibition effect on pathogenic bacteria of sugarcane white streak, and the size of a bacteriostasis zone is shown in fig. 2; through density gradient dilution, the plate facing experiment is repeatedly developed, and from the second group of plate facing experiments, it can be seen that the plate facing experiment is performed on the sugarcane white streak pathogenic bacteria by selecting Pse147 bacterial liquids with different concentrations under the premise of being fully coated with the sugarcane white streak pathogenic bacteria Xal JG43 under the influence of Pse 147. The concentrations are OD 600 ＝0.2、OD 600 ＝0.5、OD 600 ＝1、OD 600 =2. As can be seen from Table 1, the concentration (OD 600 ) The inhibition ring also gradually increases, at OD 600 When=2, the inhibition ring is larger; the maximum restraint loop reaches 7.9CM, and the length of the flat plate is 8.7CM. This example illustrates that the strain Pse147 has a strong antagonism against sugarcane white streak.
Example 4
The greenhouse barrel planting experiment detects the control effect of the Pse147 on the sugarcane white streak, and the specific method is as follows:
(1) Selecting a susceptible variety of sugarcane material cinnamyl sugar 46 (GT 46), and taking the sugarcane material cinnamyl sugar 46 as an inoculation experimental material for a bacterial strain truncated inoculation experiment when the sugarcane material cinnamyl sugar 46 grows to 5 knots;
(2) After cutting off sugarcane sheaths above the plant growth points inoculated with the experimental materials, the following treatments are respectively carried out:
treatment group one: absorbent cotton is used for dipping Xanthomonas Saccharum Xanthomonas Xal JG43 bacterial liquid (OD 600 =0.5) and covered on the wound, and then the cotton-covered wound was wrapped with a tinfoil.
Treatment group two: the same treatment group I is different in that the Xanthomonas Saccharum Xanthomonas Xal JG43 bacterial solution is replaced by a mixed bacterial solution, and the mixed bacterial solution is OD prepared in example 2 600 Bacterial liquid and od=0.5 600 Xanthomonas saccharalis Xanthomonas Xal JG43 bacterial liquid with the volume ratio of 1:1 is mixed to obtain the microbial inoculum.
Control group: the same treatment group I is different only in that the Xanthomonas Saccharum Xanthomonas Xal JG43 bacterial liquid is replaced by sterile water.
(3) Each group of experimental materials is treated for 15 strains respectively, the treatment is repeated for 3 times, the disease condition of sugarcane white streak is counted for three months continuously, and whether pencil-shaped white streak appears on the leaves or not; the morbidity and the control efficiency were calculated from the formulas (1) and (2), and the results are shown in table 2.
Incidence = total number of lesions/total number of investigation x 100% (1)
Biocontrol effect= (control group incidence-treatment group incidence)/control group incidence x 100% (2)
TABLE 2 biocontrol Effect of Pse147 on sugarcane white streaks in greenhouses
Treatment of Incidence of disease Biocontrol effect
Treatment group I 54.44％ ——
Treatment group two 18.09％ 66.77％
Control group —— ——
The table shows that the prevention and treatment effect of the Pse147 biocontrol microbial inoculum on the sugarcane white streak reaches 66.77%, which shows that the Pse147 strain has good prevention and treatment effect on the sugarcane white streak.
The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.

Claims (9)

1. A biocontrol strain for preventing and treating sugarcane white streak is characterized by comprising pseudomonas morganiiPseudomonas mosselii) Pse147 was preserved in China center for type culture Collection with a preservation address of China university of Wuhan, and a preservation number of CCTCC NO: m20221133.
2. Use of the biocontrol strain of claim 1 in the preparation of a biocontrol microbial agent for controlling sugarcane white streak.
3. A biocontrol microbial agent for controlling sugarcane white streaks, comprising the biocontrol strain of claim 1.
4. A biocontrol strain or rights as claimed in claim 1The use of the biocontrol microbial inoculum according to claim 3 for preventing and treating sugarcane white streak, characterized in that the pathogenic bacteria of sugarcane white streak areXanthomonas albilineans。
5. A method for preparing the biocontrol microbial agent as claimed in claim 3, comprising the steps of: fermenting and culturing the biocontrol strain of claim 1 to obtain a fermentation liquid, and diluting the fermentation liquid to obtain the biocontrol microbial agent.
6. The method according to claim 5, wherein the conditions of the fermentation culture include: the temperature is 28 ℃, the rotating speed is 180-210rpm, and the culture time is 12-16h.
7. The method according to claim 5, wherein the diluted fermentation broth has an OD 600 2.
8. A preparation method of the biocontrol strain of claim 1Xanthomonas albilineansIs applied to the bacteriostat.
9. The method comprises the following steps ofXanthomonas albilineansComprising the biocontrol strain of claim 1.
CN202310038524.9A 2023-01-12 2023-01-12 Biocontrol strain Pse147 for preventing and treating sugarcane white streak and application thereof Active CN116083308B (en)

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Citations (3)

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Publication number Priority date Publication date Assignee Title
CN104254611A (en) * 2012-02-28 2014-12-31 马罗内生物创新公司 Control of phytopathogenic microorganisms with pseudomonas sp. and substances and compositions derived therefrom
CN108998389A (en) * 2018-07-26 2018-12-14 上海交通大学 There is pseudomonad and the application of antagonism to rice Xanthomonas campestris and Pyricularia oryzae
CN109234218A (en) * 2018-09-19 2019-01-18 中国水稻研究所 A kind of Mo Shi pseudomonad and its application

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104254611A (en) * 2012-02-28 2014-12-31 马罗内生物创新公司 Control of phytopathogenic microorganisms with pseudomonas sp. and substances and compositions derived therefrom
CN108998389A (en) * 2018-07-26 2018-12-14 上海交通大学 There is pseudomonad and the application of antagonism to rice Xanthomonas campestris and Pyricularia oryzae
CN109234218A (en) * 2018-09-19 2019-01-18 中国水稻研究所 A kind of Mo Shi pseudomonad and its application

Non-Patent Citations (1)

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Title
甘蔗白条病及其致病菌Xanthomonas albilineans研究进展;孟建玉;张慧丽;林岭虹;黄宏阳;高三基;;植物保护学报;20190415(第02期);全文 *

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