CN110129238B - Bacillus amyloliquefaciens with various disease prevention effects, application thereof and biocontrol microbial inoculum - Google Patents
Bacillus amyloliquefaciens with various disease prevention effects, application thereof and biocontrol microbial inoculum Download PDFInfo
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- CN110129238B CN110129238B CN201910471155.6A CN201910471155A CN110129238B CN 110129238 B CN110129238 B CN 110129238B CN 201910471155 A CN201910471155 A CN 201910471155A CN 110129238 B CN110129238 B CN 110129238B
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- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C12R2001/07—Bacillus
Abstract
The invention relates to bacillus amyloliquefaciens with various disease prevention functions, application thereof and a biocontrol microbial inoculum, belonging to the technical field of plant disease prevention and control. In the invention, 1 strain of bacillus amyloliquefaciens MS021 with antibacterial activity is separated and screened from healthy branches of Malus sieversii; the antibacterial peptide has obvious antagonistic action on staphylococcus, colletotrichum gloeosporioides, phomopsis maltica, rhizoctonia cerealis, wheat take-all pathogen, botrytis cinerea, fusarium graminearum, kaffir basicola, nandina anthracnose and Erwinia amylovora, and antibacterial objects have broad spectrum; provides a new microbial resource for the biological control of plant diseases such as apple trees and the like.
Description
Technical Field
The invention relates to bacillus amyloliquefaciens with various disease prevention functions, application thereof and a biocontrol microbial inoculum, belonging to the technical field of plant disease prevention and control.
Background
At present, the most applied biocontrol bacteria mainly comprise Bacillus (Bacillus spp.), Pseudomonas (Pseudomonas spp.), Streptomyces spp) and other beneficial bacteria (Chengliang, Youchun, Xiaoaiping, research progress of antagonistic bacteria, university of Jiangxi agriculture, 2003,25(5): 732-737), wherein the application of the Bacillus is the most widely applied. Bacillus is widely present in air, water, soil and other environments, is widely applied to biological control of plant diseases, and plays a role in biological control by producing bacteriostatic proteins or antibacterial peptides and the like (step T. Bacillus subtilis antibodies: structures, synthesis and specific functions. molecular Microbiology,2010,56(4):845 + 857.). For example, Bacillus subtilis F3 is capable of producing an antimicrobial protein (ZHao XY, ZHao XM, Wei YM, Shang QX, Liu ZP.isolation and identification of a novel antibacterial protein from a rhizobacterium Bacillus subtilis strain F3.journal of Phytopathology,2012,161(1):43-48.) that inhibits Monilinia fructicola; bacillus cereus (Bacillus cereus) has strong bacteriostatic activity on alternaria pyricularis (Altrnaria alternata) and citrus green mold (Penicillium digitatum) (Yehia HM, Salmen SH, Alhargi SA, Khiyami MA, Wainwright M, Alhargi NS, Chinnathambi A. antibacterial protein 35kDa produced by Bacillus cereus inhibitus the growth of microorganisms and properties. journal of Pure and real Microbiology,2013,7: 401. 406.); zhangsheng and the like utilize Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) to control rice stripe disease, the control effect is over 60 percent, surfactin substances generated by the Bacillus amyloliquefaciens LX-11 have stronger inhibition effect on stripe disease bacteria and are one of the main substances for inhibiting stripe disease bacteria at present (Zhangsheng, Chengxia, Wangxiang, Luchuping, Liuyongfeng, Chenshiyi, Zhangsheng and the like, 2013).
Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) is a species of Bacillus and can secrete a plurality of antibacterial active substances to the outside of cells during the growth process. The bacillus amyloliquefaciens can prevent and control various plant diseases and promote plant growth. Cao Jing et al found that Bacillus amyloliquefaciens has good prevention and treatment effects on wheat yellow mosaic disease, and the prevention effect is obviously superior to the treatment effect (Cao Jing. biological prevention and treatment research on wheat yellow mosaic disease by Bacillus amyloliquefaciens [ D ]. Yangling: northwest university of agriculture and forestry, 2015.); the Yangheng, etc. found that the root irrigation of the bacillus amyloliquefaciens CC09 strain on the wheat has the prevention and control rate of wheat seedling gibberellic disease caused by the fusarium graminearum infection as high as 90.7% (Yangheng, Endaoyang, Xue Yarong, etc.. the electron microscope observation and the prevention effect of the endogenetic bacillus amyloliquefaciens CC09 in the wheat root colonization [ J ]. Chinese biological prevention and control academy, 2014,30(6): 839-844.). Shenli et al found that the inhibition rate of Bacillus amyloliquefaciens Ba33 strain on the number of withered spots of tobacco mosaic virus on tobacco leaves is 67.8% (Shenli, Wang Fenglong, Qianyume, etc.. Bacillus amyloliquefaciens Ba33 has growth promoting and TMV resisting effects on tobacco [ J ] Proc. of Jilin university of agriculture, 2010,32(4): 383-. Zhaojiang tao et al (2014) found that bacillus amyloliquefaciens BGP14 strain has strong antagonistic activity on carrot soft rot Erwinia (Zhao, Zhao Yangsheng, Huangkaihong, et al. research on antagonistic bacteria of soft rot of taro [ J ] food science, 2014,35(7):155 + 159.).
However, there are few reports that bacillus amyloliquefaciens has an antagonistic effect on various diseases of fruit trees and the like at the same time.
Disclosure of Invention
The invention aims to provide bacillus amyloliquefaciens with various disease prevention effects, and the bacillus amyloliquefaciens can resist 10 plant pathogenic bacteria.
The invention also provides application of the bacillus amyloliquefaciens, and provides a new way for preventing and treating plant diseases.
The invention also provides a bio-control microbial inoculum with various disease prevention effects, which can inhibit the occurrence of plant diseases.
In order to achieve the purpose, the invention adopts the technical scheme that:
the Bacillus amyloliquefaciens strain with various disease prevention functions is Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) MS021, and the preservation number is CCTCC NO: M2019268.
In the invention, a strain of endogenous Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) MS021 with bacteriostatic activity is obtained by separation; it has specific antagonistic action on various plant pathogenic bacteria, and provides a new microbial resource for biological control of plant diseases of apple trees and the like.
The bacillus amyloliquefaciens with various disease prevention functions is applied to antagonism of plant pathogenic bacteria. Specifically, the plant pathogenic bacteria are staphylococcus, colletotrichum gloeosporioides and/or phomopsis maltica.
Experiments show that the bacteriostatic rate of the bacillus amyloliquefaciens MS021 on the staphylococcus lumen is 78.87 +/-0.00%, the bacteriostatic rate on colletotrichum gloeosporioides is 73.45 +/-1.96%, the bacteriostatic rate on phomopsis maltica reaches 78.26 +/-2.90%, and the growth of 3 kinds of pathogenic hypha of apple branch and stem diseases can be well inhibited.
Specifically, the plant pathogenic bacteria are rhizoctonia cerealis, wheat take-all pathogen, botrytis cinerea, gibberella zeae, kaffir lily rhizoctonia solani and/or nandina anthracnose pathogen.
Experiments show that the bacteriostatic rate of the bacillus amyloliquefaciens MS021 on Nandina domestica anthracnose pathogen is 87.98%, the bacteriostatic rate on tomato botrytis cinerea is 84.46%, the bacteriostatic rates on Rhizoctonia cerealis, wheat take-all pathogen, kaffir lily rhizoctonia solani and wheat gibberellic disease pathogen are respectively 80.23%, 78.34%, 69.91% and 65.25%, and the bacillus amyloliquefaciens has an inhibiting effect on the growth of 6 plant pathogenic bacteria.
Specifically, the plant pathogenic bacteria is Erwinia amylovora. Experiments show that the bacillus amyloliquefaciens MS021 has an obvious inhibiting effect on the erwinia amyloliquefaciens, so that the bacillus amyloliquefaciens can be used for antagonizing the erwinia amyloliquefaciens.
A biocontrol microbial inoculum with various disease prevention effects comprises biocontrol bacteria and auxiliary materials, wherein the biocontrol bacteria is Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) MS021 with a preservation number of CCTCC NO: M2019268.
The bacillus amyloliquefaciens MS021 can antagonize plant pathogenic bacteria, so that other auxiliary materials can be added to be used as a biocontrol microbial inoculum.
Drawings
FIG. 1 is a graph showing the result of the confronting of Bacillus amyloliquefaciens MS021 on a flat plate of a. viticola in test example 2 of the present invention;
FIG. 2 is a graph showing the results of plate confrontation of Bacillus amyloliquefaciens MS021 against colletotrichum gloeosporioides in test example 2 of the present invention;
FIG. 3 is a graph showing the result of the confronting of Bacillus amyloliquefaciens MS021 on a Phomopsis Malloti plate in test example 2 of the present invention;
FIG. 4 is a graph showing the plate confronting result of Bacillus amyloliquefaciens MS021 on Nandina domestica anthracnose pathogen in test example 4 of the present invention;
FIG. 5 is a graph showing the results of the plate confronting of Bacillus amyloliquefaciens MS021 on Botrytis cinerea in test example 4 of the present invention;
FIG. 6 is a graph showing the plate confronting result of Bacillus amyloliquefaciens MS021 on Rhizoctonia cerealis in test example 4 of the present invention;
FIG. 7 is a graph showing the plate-confrontation result of Bacillus amyloliquefaciens MS021 against wheat take-all pathogen in test example 4 of the present invention;
FIG. 8 is a graph showing the results of the confronting of Bacillus amyloliquefaciens MS021 on a Clivia stem basal rot fungi plate in test example 4 of the present invention;
FIG. 9 is a graph showing the plate confronting result of Bacillus amyloliquefaciens MS021 on Gibberella zeae in test example 4 of the present invention;
FIG. 10 is a graph showing the plate inhibition of Bacillus amyloliquefaciens MS021 against Erwinia amyloliquefaciens in test example 5 of the present invention.
Detailed Description
The present invention will be described in further detail with reference to specific examples. The equipment and reagents used in the examples and the experimental examples were commercially available except as specifically indicated. Examples of pathogenic bacteria to be tested used in the following test examples include Staphylococus Botrytis (Botryosphaeria dothidea), Colletotrichum gloeosporioides (Colletotrichum gloeosporioides), Phomopsis malli (Phomopsis mali), Rhizoctonia cerealis (Rhizoctonia cerealis), Rhizoctonia cerealis (Gaeumannomyces graminis), Botrytis cinerea (Botrytis cinerea), Gibberella graminis (Fusarium graminearum), Rhizopus kawachii (Fusarium oxysporum), Rhizoctonia solani (Colletotrichum karstii), and Erwinia amylovora (Erwinia amylovora).
Wherein, the botrytis cinerea, colletotrichum gloeosporioides, phomopsis maltica, rhizoctonia cerealis, phomopsis tritici, botrytis cinerea, fusarium graminearum, kaffir lily stalk rot, and anthracnose of Nandina domestica are collected by the group of the invention to the field disease plants in Luoyang city, are verified by Koch's law after purification and separation, are observed in microscopic morphology and identified in molecular biology, and are stored in forestry biotechnology laboratories of the institute of forestry, university of Henan science and technology. The Erwinia amyloliquefaciens (Erwinia amylovora) strain is provided by the technical center of the entry and exit inspection and quarantine office in Luoyang.
ITS rDNA sequences of gluconobacter vinifera, colletotrichum gloeosporioides, phomopsis malorum, kaffir lily physalis and southern bamboo colletotrichum are cloned and sequenced and are submitted to a Genbank database, and the corresponding sequence numbers are respectively: MH250045, MH156052, MH250047, MF471668 and KX 987162. The ITS rDNA sequences of Rhizoctonia cerealis, Ustilago cerealis, Botrytis cinerea and Gibberella cerealis are cloned and sequenced, and are not submitted to a Genbank database temporarily, and the corresponding sequences are respectively shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO. 4.
Example 1 of Bacillus amyloliquefaciens having various disease preventing effects
In the embodiment, the Bacillus amyloliquefaciens with various disease prevention functions is Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) MS021, and the preservation number is CCTCC NO: M2019268; the preservation date is as follows: 17/04/2019, depository: china center for type culture Collection, collection address: wuhan university in Wuhan, Hubei (preservation center of Wuchang Lojia mountain Wuhan university, Wuhan city, Hubei province).
Example 1 application of Bacillus amyloliquefaciens having various disease-preventing effects to antagonism of plant pathogenic bacteria
The bacterial strain used in the example is Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) MS021, and the preservation number is CCTCC NO: M2019268. The plant pathogenic bacteria are Acinetobacter, colletotrichum gloeosporioides and/or Phomopsis malorum.
Example 2 application of Bacillus amyloliquefaciens having various disease-preventing effects to antagonism of plant pathogenic bacteria
The bacterial strain used in the example is Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) MS021, and the preservation number is CCTCC NO: M2019268. The plant pathogenic bacteria are Rhizoctonia cerealis, Ustilago graminis, Botrytis cinerea, Gibberella cerealis, Clivia fujielii Stem rot bacteria, and/or Colletotrichum nandinense.
Example 3 application of Bacillus amyloliquefaciens having various disease-preventing effects to antagonism of plant pathogenic bacteria
The bacterial strain used in the example is Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) MS021, and the preservation number is CCTCC NO: M2019268. The plant pathogenic bacteria is Erwinia amylovora.
Example 1 of biocontrol microbial inoculum having various disease-preventing effects
The bio-control bacterium agent with various disease prevention effects in the embodiment comprises bio-control bacteria and auxiliary materials, wherein the bio-control bacteria is Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) MS021, and the preservation number is CCTCC NO: M2019268. The adjuvants may be those commonly used in the art.
Test example 1
The invention separates and screens 1 strain of apple disease inhibiting bacteria from healthy branches of Malus sieversii in Xinjiang: the bacillus amyloliquefaciens MS021 provides a new microbial resource for biological control of plant diseases such as apple trees and the like.
1. Morphological characteristics of Bacillus amyloliquefaciens MS021
The bacillus amyloliquefaciens MS021 is cultured on NA culture medium and PDA culture medium at the constant temperature of 28 ℃ for 2d, and the colony morphology is observed and recorded (shown in Table 1). The thallus is rod-shaped, has spores and is provided with periphytic flagellum.
TABLE 1 characteristics of Bacillus amyloliquefaciens MS021 colonies
2. Physiological and biochemical characteristics of bacillus amyloliquefaciens MS021
The physiological and biochemical characteristics of Bacillus amyloliquefaciens MS021 are shown in Table 2.
TABLE 2 physiological and biochemical characteristics of Bacillus amyloliquefaciens MS021
Note: + indicates positive; one indicates negative.
Test example 2 determination of bacteriostatic activity of Bacillus amyloliquefaciens MS021 against 3 apple pathogens
Inoculating MS021 into PDA liquid culture medium, 28 deg.C, 180r.min-1Shaking and culturing for 24h for later use. A plate confronting method is adopted, 3 pathogenic bacteria of staphylococcus, colletotrichum gloeosporioides and phomopsis maltica are taken as test bacteria, 7mm bacterial cakes are respectively punched by a puncher and inoculated in the center of a PDA plate, 4 points are equidistantly taken at a position 25mm away from the bacterial cakes and are placed with sterile filter paper discs with the diameter of 4mm, and 3 mu L of MS012 bacterial liquid is added into each filter paper disc. As a control, 3. mu.L of sterile water was added. And (4) when the hypha of the control pathogenic bacteria grows over the culture dish at 28 ℃, recording the diameters of the pathogenic bacteria in the antibacterial confronting treatment and taking a picture. The bacteriostasis rate is [ (control colony radius-radius of treated colony)/(control colony radius-radius of cake)]×100％。
The plate confrontation method is used for detecting the bacteriostasis of the bacillus amyloliquefaciens MS021 on 3 apple pathogens (in figures 1-3, the left side picture is a control pathogen, and the right side picture is the inhibition effect of the MS012 on the pathogen). The bacteriostatic rate of the strain MS012 on the staphylococcus lumen bacteria is 78.87 +/-0.00 percent (shown in figure 1); the bacteriostasis rate to colletotrichum gloeosporioides is 73.45 +/-1.96% (see figure 2); the inhibition rate of the phomopsis malloti is 78.26 +/-2.90% (see figure 3).
Test example 3 Bacillus amyloliquefaciens MS021 is not pathogenic to apple shoots
Selecting healthy red Fuji apple two-year-old branches, cutting into branch sections with the length of 40cm, performing surface sterilization by using 75% alcohol, washing with sterile water for 3 times, naturally drying, and sealing the top end with paraffin. Scalding 3 parts of each branch, and screening 10 μ L (10 μ L) of endophytic bacteria strain8cfu/mL) was dropped on the scald area. After the bacterial liquid is naturally dried, attaching sterile and moist absorbent cotton to the wound, wrapping the wound with a preservative film, inserting the lower end of the wound into tap water, and culturing in an incubator at 28 ℃ with the humidity not lower than 80%. 10 mu L of sterile water is dripped into the wound to be used as a negative control, and the phomopsis maltica cake is placed into the wound to be used as a positive control. Observing the disease condition of the inoculation partAfter 25 days, the number of the wounds of the branches is counted, and the incidence rate is calculated, wherein the incidence rate is (the number of the wounds with the disease/the total number of the wounds) multiplied by 100%. And (3) performing pathogenicity analysis on the screened bacillus amyloliquefaciens MS021, wherein the morbidity of a positive control is 100%, and the morbidity of a negative control group and the bacterial strain MS021 is 0, and the result proves that the bacillus amyloliquefaciens MS021 has no pathogenicity on apple branches.
Test example 4 Bacillus amyloliquefaciens MS021 has broad bacteriostatic spectrum
Inoculating MS021 into PDA liquid culture medium, 28 deg.C, 180r.min-1Shaking and culturing for 24h for later use. A plate confronting method is adopted, 6 pathogenic bacteria of rhizoctonia cerealis, wheat take-all pathogen, botrytis cinerea, fusarium graminearum, kaffir lily rhizoctonia solani and nandina anthracnose pathogen are taken as indicator bacteria, 7mm fungus cakes are respectively punched by a puncher and inoculated in the center of a PDA plate, 4 points are taken at equal distance of 25mm from the fungus cakes, aseptic filter paper discs with the diameter of 4mm are placed, and 3 mu L of MS012 bacterium liquid is added into each filter paper disc. As a control, 3. mu.L of sterile water was added. And (4) when the hypha of the control pathogenic bacteria grows over the culture dish at 28 ℃, recording the diameters of the pathogenic bacteria in the antibacterial confronting treatment and taking a picture.
The results are shown in FIGS. 4-9, the left photographs showing the control pathogen, and the right photographs showing the pathogen inhibitory effect of MS 021; the results show that the bacillus amyloliquefaciens MS021 has a wide antibacterial spectrum, the antibacterial rate to the southern bamboo anthracnose pathogen is 87.98% (see figure 4), the antibacterial rate to the tomato botrytis cinerea is 84.46% (see figure 5), and the antibacterial rates to the wheat sheath blight pathogen, the wheat take all, the kaffir lily stem rot pathogen and the wheat gibberellic disease are respectively 80.23% (see figure 6), 78.34% (see figure 7), 69.91% (see figure 8) and 65.25% (see figure 9). MS021 has good bacteriostatic activity on various plant pathogenic bacteria, and the bacterial strain has broad bacteriostatic activity.
Test example 5 Bacillus amyloliquefaciens MS021 has strong bacteriostatic action on Erwinia amyloliquefaciens
Erwinia amyloliquefaciens in NA medium at 28 deg.C and 180r.min-1Shaking and culturing to OD value of 1.0, and spreading 100. mu.L of bacterial liquid on NA solid culture medium. Each solid medium plate surface placed 34 mm diameter sterile filter paper sheet, set up 3 dish repeat. Bacillus amyloliquefaciens MS021 is cultured in NA culture medium at 28 deg.c for 180r.min-1Shaking culture to OD 2.0, and adding 3. mu.L of bacterial liquid to each filter paper sheet. A blank control was prepared by adding 3. mu.L of sterile water to each filter paper slide. After the plate is placed in an incubator at 28 ℃ for 24 hours, an obvious inhibition zone appears around the filter paper sheet (see figure 10, the left side picture is blank control, and the right side picture is an obvious inhibition zone generated by MS 021), the diameter of the inhibition zone is measured by a cross method, and the average diameter of the inhibition zone is statistically 21 mm. The result shows that the bacillus amyloliquefaciens MS021 has strong bacteriostatic action on the Erwinia amyloliquefaciens.
<110> university of Henan science and technology
<120> bacillus amyloliquefaciens with various disease prevention effects, application thereof and biocontrol microbial inoculum
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Claims (3)
1. Bacillus amyloliquefaciens with various disease prevention functions is characterized in that: the strain is bacillus amyloliquefaciens (Bacillus amyloliquefaciens) MS021 with preservation number of CCTCC NO of M2019268.
2. The use of bacillus amyloliquefaciens with multiple disease prevention effects according to claim 1 for antagonizing plant pathogenic bacteria that are plasmopara viticola, colletotrichum gloeosporioides, and/or phomopsis malloti; or the plant pathogenic bacteria are rhizoctonia cerealis, totrichum cerealis, kaffir lily rhizoctonia solani and/or nandina anthracnose.
3. A biological control agent with various disease prevention effects is characterized in that: comprises biocontrol bacteria and auxiliary materials, wherein the biocontrol bacteria is bacillus amyloliquefaciens (A), (B) and (C)Bacillus amyloliquefaciens) MS021 with preservation number of CCTCC NO of M2019268.
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| CN107058146A (en) * | 2016-08-05 | 2017-08-18 | 河南科技大学 | A kind of bacillus amyloliquefaciens RH9, screening technique and purposes |
| CN107820392A (en) * | 2014-12-29 | 2018-03-20 | Fmc有限公司 | Bacillus amyloliquefaciens RTI472 compositions and for beneficial to plant growth and treatment plant disease method |
| CN104762223B (en) * | 2014-11-06 | 2018-05-08 | 河北省科学院生物研究所 | A kind of bacillus amyloliquefaciens BA-KA3 and its application |
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| CN107820392A (en) * | 2014-12-29 | 2018-03-20 | Fmc有限公司 | Bacillus amyloliquefaciens RTI472 compositions and for beneficial to plant growth and treatment plant disease method |
| CN107058146A (en) * | 2016-08-05 | 2017-08-18 | 河南科技大学 | A kind of bacillus amyloliquefaciens RH9, screening technique and purposes |
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| Isolation of anteiso-C17, iso-C17, iso-C16, and iso-C15 Bacillomycin D from Bacillus amyloliquefaciens SD-32 and Their Antifungal Activities against Plant Pathogens;Keijitsu Tanaka等;《Journal of Agricultural and Food Chemistry》;20140121;第62卷(第7期);第1469-1476页 * |
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