CN112266884B - Application of bacillus subtilis N-42-2 in preventing and controlling mango anthracnose - Google Patents
Application of bacillus subtilis N-42-2 in preventing and controlling mango anthracnose Download PDFInfo
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Abstract
The invention relates to application of bacillus subtilis N-42-2 in preventing and controlling mango anthracnose. The invention screens out a bacillus subtilis N-42-2 from mango peel, and the preservation number is CGMCC No.20670. The fermentation liquor of the bacillus subtilis N-42-2 can inhibit the growth of mango anthracnose hyphae, and the generated volatile gas can inhibit the growth of the mango anthracnose hyphae, and has a good control effect in indoor control of anthracnose after mango harvest.
Description
Technical Field
The invention relates to the prevention and treatment of plant diseases by antagonistic microorganisms, in particular to application of bacillus subtilis N-42-2 in preventing and treating mango anthracnose.
Background
Mango is an important cash crop in subtropical and tropical areas in China, and is popular among people because of strong fragrance, tender juice, sweet and sour taste and high nutritive value, and is called as tropical fruit king. The fructus Mangifera Indicae is rich in vitamin C and carotene, and also contains higher sugar, protein, etc., and has high nutritive value [2] . Besides higher nutritive value, mango also has high utilization value. In China, mango is mainly planted in Hainan,Yunnan, sichuan, guangdong and Fujian, guizhou provinces.
The mango production brings great economic benefit to fruit industry in China, but mango diseases also cause serious threat to mango industry, especially postharvest diseases. Mango anthracnose is the first major disease of mangoes, commonly occurs in mango main production areas at home and abroad, and can cause 30% -50% of mangoes to rot during storage. Anthracnose can sometimes cause more than 60% of disease fruit rate, so that the quality of mango is reduced, the annual economic loss is more than 8000 ten thousand yuan, and the development of mango industry is greatly influenced. The mango anthracnose in China is mainly caused by colletotrichum gloeosporioides (Colletotrichum gloeosporioides).
At present, chemical prevention and control are mostly adopted in terms of germ inhibition and fresh-keeping after mango harvest, but the cost of chemical prevention and control is high, and the long-term use of the chemical agents can cause pathogenic bacteria to generate drug resistance. Therefore, the search for a green, environment-friendly and efficient control method is needed. Therefore, the control of postharvest diseases of mangoes by biological control means is particularly important. The control effect of the antagonistic bacteria on mango anthracnose is determined by screening antagonistic bacteria with inhibition effect on mango anthracnose, and a theoretical basis is provided for biological control of mango anthracnose after picking in future.
Disclosure of Invention
The invention aims to provide the bacillus subtilis N-42-2 for preventing and controlling mango anthracnose, which not only can prevent and control the mango anthracnose by using the bacillus subtilis N-42-2 fermentation liquor, but also can prevent and control the mango anthracnose by using volatile gas, and has better non-contact prevention and control effect and is more beneficial to preventing and controlling mango postharvest diseases.
A bacillus subtilis N-42-2 has a preservation number of CGMCC No.20670.
The application of the bacillus subtilis N-42-2 in preventing and controlling plant anthracnose.
The plant anthracnose is mango anthracnose.
The control is to spray the fermentation liquor of bacillus subtilis N-42-2 on the surface of mango to realize the inhibition effect on the mango anthracnose hyphae on the fruit trees and the indoor control on the picked mango anthracnose.
The viable count of the fermentation liquor is 1 multiplied by 10 8 CFU/ml。
The preparation method of the fermentation liquor comprises the following steps:
(1) Strain activation: inoculating bacillus subtilis N-42-2 on NA solid culture medium, and culturing at 27 ℃ for 1-2 days;
(2) Preparation of strain fermentation liquor: inoculating the activated strain into NB liquid culture medium, and shake culturing at 27deg.C for 12-24 hr to obtain fermentation broth of Bacillus subtilis N-42-2.
The NA solid medium formula in the step (1) is as follows: 3g of beef extract, 5g of peptone powder, 18g of agar powder, 1000mL of distilled water and pH 7.0-7.2;
the NB liquid medium formula in the step (2) is as follows: 3g of beef extract, 5g of peptone powder, 1000mL of distilled water and 7.0-7.2.
The control is that the surviving bacillus subtilis N-42-2 is placed in a storage space of mango fruits, and the bacillus subtilis N-42-2 strain is not in direct contact with the mango fruits, so that volatile gas generated by the bacillus subtilis N-42-2 has an inhibition effect on mango anthrax hyphae on fruit trees and indoor control of mango anthracnose after picking.
The surviving bacillus subtilis N-42-2 is bacillus subtilis N-42-2 inoculated in the NA solid culture medium.
The applicant screens out a bacillus subtilis N-42-2 from mango epidermis, and the preservation number is CGMCC No.20670.
According to experimental study, the inhibition rate of volatile gas of fermentation broth of bacillus subtilis N-42-2 on mango anthracnose pathogen hypha reaches 87.47% under laboratory conditions; the concentration is 1 multiplied by 10 8 The inhibiting effect of CFU/ml bacillus subtilis N-42-2 fermentation liquor on isolated mango anthracnose hyphae reaches 67.21%, and the inhibiting effect of the inhibiting effect on indoor control of picked mango anthracnose is good. The bacillus subtilis N-42-2 does not need to be in direct contact with mango, and can achieve the control effect of anthracnose after mango harvest only by storing the active strain and the mango in the same space, namely the bacillus subtilisThe bacillus N-42-2 carries out natural fumigation treatment on the picked mango anthracnose, can effectively inhibit the occurrence of the mango anthracnose, and has a control effect reaching 67.57 percent.
The beneficial effects of the invention are as follows: the bacillus subtilis N-42-2 fermentation liquor can prevent and treat mango anthracnose, volatile gas of the bacillus subtilis N-42-2 can also prevent and treat mango anthracnose indoors, and the effect is better, the volatile gas is prevented and treated by a different prevention and treatment method, such as fumigation, from the bacillus subtilis N-42-2 fermentation liquor, the strain does not need to be in direct contact with mango fruits, and secondary pollution of the bacillus subtilis N-42-2 fermentation liquor or the bacillus subtilis N-42-2 to mango is reduced. Compared with the existing agent control, the microorganism has the characteristics of green, no pollution and no residue in the application of controlling anthracnose after mango picking, has better effect and prospect, and can be used as a resource for development of biological pesticides.
Bacillus subtilis N-42-2 Strain preservation information:
classification and naming of strains: bacillus subtilis Bacillus subtilis.
Strain deposit number: CGMCC No.20670.
Preservation address: china general microbiological culture Collection center of China Committee for culture Collection of microorganisms
Preservation date: 2020, 9 and 18 days
Drawings
FIG. 1 shows colony morphology of Bacillus subtilis N-42-2 on NA plates;
FIG. 2 shows the morphology of Bacillus subtilis N-42-2 cells observed after gram staining;
FIG. 3 shows the inhibition of B.subtilis N-42-2 volatile gas on growth of mango anthrax hyphae in a plate butt buckling method;
wherein: a is the normal mycelium form of the anthrax, B is the inhibition condition of the volatile gas of bacillus subtilis N-42-2 on the mango anthrax mycelium;
FIG. 4 shows the control effect of the Bacillus subtilis N-42-2 fermentation broth on anthracnose of in vitro mango fruits;
wherein: a is the disease condition of in-vitro mango fruits treated by sterile water; b is the morbidity of the in-vitro mango fruits treated by the bacillus subtilis N-42-2 fermentation broth;
FIG. 5 shows the control effect of bacillus subtilis N-42-2 volatile gas on anthracnose of in-vitro mango fruits;
wherein: a is the disease condition of the isolated fruits treated by sterile water; b is the morbidity of the isolated fruits treated by the volatile gas of the bacillus subtilis N-42-2.
Detailed Description
Preferred embodiments of the present invention will be described in detail below with reference to examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention may be made by those skilled in the art without departing from the spirit and scope of this invention.
The experimental methods used in the following examples are conventional methods unless otherwise specified.
EXAMPLE 1 isolation, screening and characterization of Bacillus subtilis N-42-2
1.1 isolation of bacteria
Taking healthy mango skin, sterilizing for 10s with 75% alcohol in an ultra-clean workbench, sterilizing for 1min with NaClO with concentration of 2%, rinsing with sterile water for 3 times, shearing into tissue blocks with the thickness of about 3X 3mm, and airing. Placing 3-4 tissue blocks on PDA culture medium, and culturing in incubator at 25deg.C for 3-5d. When the tissue block grows out of colonies, bacteria with different colony forms are picked up, purified and cultured on a PDA culture medium and marked.
1.2 preliminary screening of bacteria
And (3) primary screening: the bacterial strain obtained by separating and purifying 1.1 is screened on a PDA plate by adopting a plate facing method and taking a strain of mango anthracnose as an indicator bacterium, and the bacterial strain with antagonism is preserved for standby.
And (3) re-screening: and screening bacteria with good effect in the primary screening by adopting a flat plate buckling method, and preserving the bacterial strain with antagonism for later use.
Wherein, the strain N-42-2 has better inhibition effect on mango anthrax hyphae, so that the strain N-42-2 can be subjected to a next control effect test.
1.3 identification of bacteria
The strain N-42-2 is identified by morphological, physiological and biochemical identification and molecular biological analysis, and the results show that the strain N-42-2 is milky and thick in quality, and single colony is round or oval and irregular in edge on NA culture medium. Gram staining showed positive and the cells were observed under a microscope to be rod-shaped. The physiological and biochemical measurement results show that the strain is positive in a methyl red test and positive in a V-P test, can be subjected to oxidative fermentation in a glucose culture medium, can utilize starch and hydrolyze the starch, has the characteristic that gelatin is obviously positive in liquefaction, cannot generate hydrogen sulfide, and can utilize various carbon sources such as fructose, glucose, mannitol, galactose, sucrose and the like. Based on morphological observation, physiological biochemical assay, molecular phylogenetic analysis was performed in combination with 16S rDNA and gryA genes, and strain N-42-2 was identified as Bacillus subtilis Bacillus subtilis. See fig. 1. Preservation number: CGMCC No.20670.
1.4 preparation of Bacillus subtilis N-42-2 fermentation broth
Culturing bacillus subtilis N-42-2 on NA solid culture medium by streaking, and culturing at 27 ℃ for 1-2 days; selecting N-42-2 single colony, inoculating into NB liquid culture medium, shake culturing at 27deg.C for 12-24 hr to obtain Bacillus subtilis fermentation broth, and adjusting concentration to 10 8 CFU/mL was ready for use.
Example 2 Effect of volatile gases produced by Bacillus subtilis N-42-2 on mango anthrax hyphae
Placing sterile filter paper sheets with the diameter of 5mm in the center of an NA flat plate by adopting a buckling method, and dripping 50 mu L of N-42-2 fermentation liquor; the bacterial cake was picked up by a punch with a diameter of 5mm at the edge of the colony of mango anthrax Colletotrichum fructicola cultured for 5-7d, and inoculated in the center of the PDA plate. The PDA plate inoculated with mango anthrax was snapped with the NA plate inoculated with bacterial broth and sealed. As a control, NA plates without bacterial fermentation broth were incubated at 27℃in 3 replicates per treatment. When the control colony grows on the flat plate, the diameter of the pathogenic bacteria colony is measured by a crisscross method, and the average value and the bacteriostasis rate are calculated. Antibacterial ratio= (1-treatment group colony diameter/control group colony diameter) ×100%. The result shows that the volatile gas generated by the bacillus subtilis N-42-2 in the flat plate has an inhibition effect on mango anthracnose, and the inhibition rate is 87.47%. See fig. 3.
Example 3 control experiment of antagonistic bacterial fermentation liquor on mango anthracnose
The mango fruits with intact surfaces, physiological maturity and size as uniform as possible are selected. Surface-sterilizing with 75% ethanol for 10s,1% NaClO for 1min, washing with sterile water for 3 times, and air drying. Pricking the surface of the fruit, inoculating 20 μl of N-42-2 fermentation broth (10 8 CFU/mL), and air-dried for 10-30 minutes. Then inoculating 20. Mu.L of the strain with a concentration of 10 6 individual/mL of anthrax (Colletotrichum fructicola) fungal spore suspension. In a sterile fresh-keeping box, culturing at 25deg.C for 7 days, observing disease condition, and measuring the diameter of disease spots, each treatment consisting of 5 fruits, and each treatment being repeated three times.
The prevention and treatment effects of the bacterial fermentation liquid on mango fruits are studied, and analysis is carried out from the morbidity, wherein the total morbidity of the CK group is 100%; warp concentration 10 8 The incidence of mango treated with CFU/mL bacterial broth was only 33.33%. In the control effect of the bacterial fermentation liquor on mango fruits, the bacillus subtilis N-42-2 is 10 8 The control effect in CFU/mL concentration treatment reaches 67.21 percent. See fig. 4.
Example 4 test for controlling the efficacy of antagonizing bacterial volatile substances against mango anthracnose
Selecting fresh picked healthy mango with approximate size and maturity, and carrying out surface disinfection treatment: treating with 75% ethanol for 10s, sterilizing with 1% NaClO for 1min, washing with sterile water for 3 times, and air drying. Each water surface was needled with 6 small holes to form a ring of about 5mm diameter, and 20. Mu.L of mango anthrax Colletotrichum fructicola conidiophore suspension (10 6 and/mL). 5 inoculated fruits were placed in 6.5L containers, one for each 5, one for each 10 antagonistic pathogenic bacteria, and three replicates were set. Advance 20mL of N-42-2 fermentation broth (1X 10) 8 CFU/mL) was inoculated on NA medium, cultured at 27 ℃ for 24h, and 6 cultures were placed in a container without direct contact with mango. The control group was incubated with the same volume of sterile water instead of the antagonist. After 7 days of incubation at 25 ℃, the spot diameter was measured by the crisscross method, and the indoor control effect was compared, and the control effect=100× (spot diameter of mango in the control group-spot diameter of the treatment group)/(spot diameter of the control group).
In the test of bacteria producing volatile substances on in-vitro mango anthracnose, the average spot diameter of the mango anthracnose treated by the strain N-42-2 is only 0.60cm, and the control effect is 67.57%. See fig. 5.
Claims (10)
1. A bacillus subtilis N-42-2 has a preservation number of CGMCC No.20670.
2. Use of bacillus subtilis N-42-2 according to claim 1 for controlling anthracnose in plants.
3. The use according to claim 2, wherein the plant anthracnose is mango anthracnose.
4. The application of claim 3, wherein the control is to spray the fermentation liquor of the bacillus subtilis N-42-2 in claim 1 on the surface of mango to realize the inhibition effect on the mango anthracnose hyphae on fruit trees and the indoor control on the mango anthracnose after harvest.
5. The use according to claim 4, wherein the viable count of the fermentation broth is 1X 10 8 CFU/ml。
6. The use according to claim 5, wherein the fermentation broth is prepared by the steps of:
(1) Strain activation: inoculating the bacillus subtilis N-42-2 according to claim 1 on NA solid medium, and culturing at 27 ℃ for 1-2 days;
(2) Preparation of strain fermentation liquor: inoculating the activated strain in the step (1) into NB liquid culture medium, and culturing for 12-24 hours at 27 ℃ in a shaking way to obtain fermentation liquor of bacillus subtilis N-42-2.
7. The use of claim 6, wherein the NA solid medium formulation in step (1) is: beef extract 3g, peptone powder 5g, agar powder 18g, distilled water 1000mL, pH 7.0-7.2.
8. The use of claim 2, wherein the NB liquid media formulation in step (2) is: 3g of beef extract, 5g of peptone powder, 1000mL of distilled water and 7.0-7.2.
9. The use according to claim 3, wherein the control is to place the surviving bacillus subtilis N-42-2 in the storage space of mango fruits and the bacillus subtilis N-42-2 strain is not in direct contact with the mango fruits, so that the volatile gas generated by the bacillus subtilis N-42-2 has an inhibiting effect on mango anthrax hyphae on fruit trees and indoor control of mango anthracnose after harvest.
10. The use according to claim 9, wherein the surviving bacillus subtilis N-42-2 is bacillus subtilis N-42-2 inoculated in NA solid medium.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101092599A (en) * | 2007-05-30 | 2007-12-26 | 江苏省农业科学院 | Preventing Bacillus subtilis grown inside diseased plant |
| CN102071162A (en) * | 2009-11-26 | 2011-05-25 | 中国农业大学 | Bacillus subtilis LFB112 as well as bacteriocin produced by same and application thereof |
| CN103114055A (en) * | 2013-01-21 | 2013-05-22 | 广西壮族自治区农业科学院植物保护研究所 | Bacillus subtilis 9A and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101092599A (en) * | 2007-05-30 | 2007-12-26 | 江苏省农业科学院 | Preventing Bacillus subtilis grown inside diseased plant |
| CN102071162A (en) * | 2009-11-26 | 2011-05-25 | 中国农业大学 | Bacillus subtilis LFB112 as well as bacteriocin produced by same and application thereof |
| CN103114055A (en) * | 2013-01-21 | 2013-05-22 | 广西壮族自治区农业科学院植物保护研究所 | Bacillus subtilis 9A and application thereof |
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