CN109022304B - Bacillus belgii, broad-spectrum antibacterial action thereof and application thereof in preventing and treating banana diseases - Google Patents
Bacillus belgii, broad-spectrum antibacterial action thereof and application thereof in preventing and treating banana diseases Download PDFInfo
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Abstract
The invention discloses a Bacillus belgii strainBacillus velezensis)3-10 and application thereof in preventing and treating banana diseases. The strain is preserved in Guangdong province microorganism culture collection center in 2018, 4 and 30 days, and the preservation number is GDMCC NO. 60366. The invention finds that 3-10 strains have broad-spectrum antibacterial effects on banana bacterial soft rot, ralstonia solanacearum, xanthomonas bacterium, peach rot, yellow skin rot, pepper colletotrichum, colletotrichum gloeosporioides, banana fusarium oxysporum, rice sheath blight, rice leaf blight, phytophthora litchi, rice blast, smut, crucifer black rot and the like. In addition, the invention discovers that the strains 3-10 can obviously reduce the occurrence of banana bacterial soft rot and banana wilt, and the effect is better in a pot experiment. The strain can be used for developing broad-spectrum biological pesticides to prevent and control important crop bacterial and fungal diseases, particularly banana diseases.
Description
Technical Field
The present invention belongs to the field of biological plant disease preventing and controlling technology. More particularly, relates to a Bacillus belgii strainBacillus velezensis)3-10 and its broad-spectrum bacteriostasis and application in preventing and treating banana diseases.
Background
Bananas are one of four fruits in the world, China banana producing areas mainly focus on Yunnan, Guangdong, Guangxi, Fujian, Hainan and Taiwan, the total yield is 1207.5238 ten thousand tons, and the banana producing area is listed in the second world. The bananas are often infected by diseases in the production process, and the common scab, black spot, leaf spot, bunchy top, rot, blight and the like seriously affect the yield and the quality of the bananas.
Such as bacterial soft rot of banana (Dickeya zeae) In 2009, it was first discovered in Guangdong that more than 80% of the fields examined were infected with the disease, the incidence rate was 20-40% unequal, and only 2010-2012 caused bananas of about 6000 hectares in Guangdong province to be affected; at present, the disease has been expanded to Fujian Zhangzhou, eastern Hainan county, Ledong Li autonomous county, Lingao county, Chengmei county, Yunnan Xishuangbanna, Guangxi Nanning and the like. The disease has the characteristics of high diffusion speed and high harmfulness, and once outbreak occurs, serious economic loss is caused to the local banana industry. Banana wilt was first discovered in Hawaii of the United states in 1904, and is now reported to occur in many countries and regions and cause serious harm, and is called cancer on bananas. In 12 months 1995, the number 4 physiological races were first found in the area of Guangzhou city Muyu wine ((C.))Fusarium oxysporumf.sp.cubensisFOC 4) infestation caused banana wilt. At present, the disease widely occurs in multiple banana production areas such as Yangtze delta, Yuexi and the like, the rate of banana plants in the banana orchard is generally 10% -40%, and seriously exceeds 90%, so that the banana orchard is destroyed, and the banana orchard is abandoned.
At present, the prevention and control of banana diseases are mainly chemical prevention and control and biological prevention and control. The use of a large amount of chemical pesticides not only causes serious environmental pollution, but also causes pathogenic bacteria to generate stress resistance; the existing biocontrol bacteria resource library is not perfect.
Disclosure of Invention
The invention aims to solve the technical problem of overcoming the defects and shortcomings of the existing banana disease control technology and provide a biocontrol bacterium with better inhibition effect on various banana disease bacteria. The strain has broad-spectrum antibacterial activity and no pathogenicity, provides a new development resource for using microorganisms to replace chemical synthesis bactericides and utilizing a biological control method to prevent and treat banana diseases, and can be developed and utilized as a biological pesticide.
The invention aims to provide a Bacillus beiLeisi 3-10 strain.
Another purpose of the invention is to provide the bacteriostasis range of the Bacillus beilaisi 3-10 to pathogenic bacteria and pathogenic fungi of various important crops.
The invention also aims to provide application of the Bacillus belgii 3-10 in preventing and treating various banana diseases.
The above purpose of the invention is realized by the following technical scheme:
the invention obtains a strain of Bacillus belgii through screening and identificationBacillus sp.)3-10, and is preserved in Guangdong province microorganism culture collection (GDMCC) in 2018, 4 and 30 months, the number of the strain is GDMCC number 60366, and the preservation address is No. 59, No. 5 storied building of Michelia Tokyo 100, Guangzhou city.
Researches show that the strains 3-10 can form an obvious inhibition zone on a flat plate containing bacterial liquid of banana bacterial soft rot fungi MS2 and MS3, the growth of the banana seedlings is not influenced in a greenhouse potting test, and the infection of MS2 on the banana seedlings can be effectively inhibited. In addition, 3-10 has obvious growth inhibition effect on a plurality of plant pathogenic bacteria and fungi and obvious prevention and control effect on banana vascular wilt in a pot experiment, and the 3-10 strain can be used for developing biological pesticides aiming at banana bacterial and fungal diseases. The discovery of the strain is beneficial to reducing the abuse problem of chemical agents, and provides resources for preventing and treating banana diseases by using a biological control method.
Therefore, the application of the Bacillus belgii 3-10 in the aspect of controlling crop bacterial and fungal diseases also falls within the protection scope of the invention.
Preferably, the banana bacterial diseases include, but are not limited to, bananaDickeya zeaeOrErwiniaCausing bacterial soft rot. The banana fungal diseases include, but are not limited to, fungal diseases caused byFusarium oxysporumf.sp.cubensisFOC4 caused banana wilt.
Preferably, in view of the determination result of the 3-10 bacterial inhibition spectrum, the action range of the strains 3-10 also comprises solanaceae ralstonia solanacearum, cruciferae nivale solani, rice leaf blight, peach rot, yellow skin rot, anthrax, banana wilt, rice sheath blight, litchi downy mildew, rice blast, sugarcane smut and the like.
More specifically, the bacterial disease comprises bacterial soft rotDickeya zeaeMS2 or MS3Bacterial wilt of solanaceaeRalstonia solanacearumEP1, Leptosphaeria maculans of BrassicaceaeXanthomonas campetrispv. campetrisXcc, rice leaf blight germXanthomonasoryzae pv.oryzaeXoo and peach rot pathogenPantoea ananatisPP1, yellow skin rot pathogenPantoea anthophilaDisease caused by C L1.
The fungal diseases comprise diseases caused by colletotrichum capsici, colletotrichum gloeosporioides, fusarium oxysporum FOC4, rhizoctonia solani, peronospora litchii, pyricularia oryzae and/or sugarcane smut.
In addition, the banana disease biocontrol agent containing the Bacillus belgii 3-10 also falls within the scope of the present invention.
Preferably, the concentration of the Bacillus belgii 3-10 in the biocontrol preparation for preventing and controlling banana bacterial diseases is OD600= 1.0-1.5, concentration of OD for preventing and treating banana fungal diseases600= 0.1。
As an alternative embodiment, the invention also provides a method for controlling crop diseases, which comprises inoculating the biocontrol agent into plant materials or soil.
Preferably, the inoculation can be by injection inoculation.
Preferably, the inoculation can adopt a soil root irrigation inoculation method.
The invention has the following beneficial effects:
the invention provides a Bacillus belgii strain 3-10, which has obvious inhibiting effect on various crop pathogenic bacteria and fungi, in particular to bacterial soft rot, ralstonia solanacearum, xanthomonas and Pantoea (R) ((R))Pantoeaspp), anthrax bacteria, banana wilt bacteria, rice sheath blight bacteria, peronophythora litchii, rice blast bacteria, sugarcane smut bacteria and the like have good inhibition effects, reduce the occurrence of banana diseases, and have good control effect in pot experiments.
The research of the invention shows that the bacterial strains 3-10 can form an antibacterial zone on a flat plate containing bacterial liquids of MS2, MS3, EP1, Xcc, Xoo, PP1 and C L1, an obvious antibacterial zone can be formed on a hypha flat plate containing anthracnose bacteria, banana fusarium wilt bacteria, rice sheath blight bacteria, peronophythora litchi and rice blast bacteria, the bacterial strains have an inhibition effect on the growth of single cells of sugarcane smut bacteria to influence the sexual coordination of the sugarcane smut bacteria, and dinuclear hypha with the infection capacity can not be formed, and the bacterial strains 3-10 have no pathogenicity on radish, carrot, Chinese cabbage, potato and banana in an inoculation test, but can well inhibit the infection of pathogenic bacteria on radish, carrot, Chinese cabbage, potato and banana seedlings, reduce the incidence rate of bacterial soft rot and obviously reduce the occurrence of blight symptoms.
Moreover, the strains 3-10 have no pathogenicity, can be used for developing biopesticides aiming at banana fungus and bacterial diseases, and provides a new idea for biologically preventing and treating banana diseases.
Drawings
FIG. 1 is a cluster map of strains 3-10 based on the 16S rDNA gene sequence.
FIG. 2 shows that strains 3 to 10 are based ongyrAAndgyrBjoint cluster map of gene sequences.
FIG. 3 is the determination of the bacteriostasis spectrum of biocontrol bacteria 3-10 on pathogenic bacteria of important crops.
FIG. 4 is the determination of the bacteriostasis spectrum of biocontrol bacteria 3-10 on the pathogenic fungi of important crops.
FIG. 5 shows the inhibitory effect of biocontrol bacteria 3-10 on sexual coordination of sugarcane smut bacteria.
FIG. 6 shows that the strains 3-10 can remarkably relieve the occurrence of bacterial soft rot of dicotyledonous host plants.
FIG. 7 strains 3-10 significantly reduced the occurrence of symptoms of bacterial soft rot in bananas.
FIG. 8 strains 3-10 significantly reduced bacterial strainsFusarium oxysporumf.sp.cubensisFOC4 caused banana wilt disease symptoms.
Detailed Description
The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
Example 1 isolation and selection of Bacillus 3-10
1. Strain isolation
Collecting samples: in vegetable fields in the flower city district of Guangzhou, Guangdong province, soil samples with soil surface less than 5cm are dug out and bagged for storage, and the soil samples are taken back to a laboratory for strain separation.
Separating strains, namely separating strains from a soil sample by adopting a dilution coating plate method, specifically, weighing 4.0 g of the collected soil sample after fully and uniformly mixing the soil sample, soaking and shaking the soil sample by 20m L sterile water, then culturing the soil sample for 20min by shaking a shaking table at 28 ℃ and 180 rpm, carrying out gradient dilution, and taking the dilution multiple as 105-109The diluted solution 100 mu L is coated on a L B flat plate, the flat plate is sealed after being dried by blowing at room temperature, the flat plate is inverted and cultured in an incubator at 28 ℃ for 24 h, a single colony growing on the L B flat plate is picked, the single colony is marked, the single colony is placed in a 2 m L centrifugal tube containing 500 mu L liquid L B, and shaking culture is carried out on a shaker at 28 ℃ and 180 rpm overnight for later use.
2. Strain screening, namely activating banana bacterial soft rot MS2 on a L B flat plate, picking a single colony in a 50 m L centrifugal tube containing 15 m L L0B culture solution, shaking and culturing at 28 ℃ and 180 rpm overnight in a shaking table for later use, pouring a melted 15 m L solid L B culture medium in a square culture dish of 10 × 10 cm, drying, cooling agarose with the concentration of 15 m L being 1% to 50-60 ℃, adding 150 mu L MS2 bacterial solution, pouring the agarose in the square culture dish paved with L B, drying, punching a hole with a hole puncher with the diameter of 5 mm, after the culture medium is picked out by a sterilized toothpick, injecting the bacterial solution to be tested into the hole for overnight culture of 20 mu L, drying, sealing, putting the hole in a 28 ℃ culture box, culturing for 16-18 h, observing whether a transparent antibacterial ring exists or not, and obtaining a biocontrol strain with excellent antibacterial effect, namely 3-10.
EXAMPLE 2 identification of Bacillus 3-10
1. Morphological identification
The strain 3-10 is gram positive; can reduce nitrate, liquefy gelatin, produce acid, peptone, and catalase, and can utilize fructose, trehalose, glucose and sucrose.
2. Molecular identification
To clarify the taxonomic status of the strains 3-10 obtained in example 1, we used a method of Multilocus sequence analysis (M L SA) in combination with Phylogenetic tree analysis (Phylogenetic analysis) to identify the strains 3-10.
The 16S rDNA sequence analysis is a molecular biological identification means for identifying bacteria, and the invention combines the conserved housekeeping gene by analyzing the 16S rDNA sequencegyrAAndgyrBthe sequence, the MEGA 5.0 software is used for constructing a phylogenetic tree by adopting a Neighbor-join method, the evolutionary relationship of 3-10 is comprehensively analyzed, and researches show that the 16S rDNA sequence of 3-10 and the sequenceBacillus velezensisThe homologies of GH1-13, GY L4, CGMCC 11640 and PG12 strains are respectively as high as 99.73%, 99.67% and 99.54%, and the strains have homology withB. subtilisThe homology of P52 is up to 99.73%,B. siamensisIHB 14741 has homology as high as 99.60%, and B. amyloliquefaciensALB69L-H15, L-S60 have homologies of up to 99.67%, 99.34%, 99.47%, withB. nakamuraiNRRL B-41091、B. methylotrophicusThe 16S rDNA sequence homology of strains such as CBMB205 and the like is up to 99 percent, a phylogenetic tree of the 16S rDNA is constructed by utilizing MEGA6.0, and the results show that the 16S rDNA evolution coefficient of 3-10 strains and a plurality of strainsB. velezensisBacterial strain,B. siamensisSCSIO 05746、 B. amyloliquefaciens L-S60、 B. nakamuraiNRR L B-41091 andB. methylotrophicusCBMB205 is most closely located in one branch (FIG. 1), and thus, it is difficult to determine the classification status of 3-10 specific species simply by the 16S rDNA sequence.
Then we further analyzegyrAAndgyrBgene sequence discovery, 3-10gyrAGene sequences andB. velezensisthe homology of GFP-2, GH1-13 and PG12 is as high as 99.51%, 99.51% and 99.02%, and the homology with the GH1-13 and PG12 is as high as 99.51%, 99.51% and 99.02% respectivelyB. AmyloliquefaciensHomologies of Y14, 9192, L-H15 and L-S60 are respectively as high as 99.41%, 99.12%, 99.02%, and 3-10%gyrBGene sequences andB. velezensishx05, J01, Z L918,B. methylotrophicusB25、B. amyloliquefaciensThe homology of Y14 is as high as 98.95%, andB. velezensisPG12 has 98.95% homologyB. amyloliquefaciensL-H15 and L-S60 have homology as high as 98.79 percentgyrAAndgyrBcombining phylogenetic trees to find sequences from 3 to 10B. velezensisThe homology of the strain PG12 is as high as 98.97 percent, and the strain PG12 has high homology with the strain PG12B. AmyloliquefaciensL-S60, L-H15 have homology as high as 97.82% (see FIG. 2).
In summary, the biocontrol bacteria were identified as being of the genus Bacillus in combination with phylogenetic analysis of three housekeeping genesBacillus velezensisDesignated Bacillus belgii (B.), (Bacillus sp.)3-10. And the strain is preserved in Guangdong province microbial strain preservation center in 2018, 4 and 30 days, wherein the preservation number is GDMCC number 60366, and the preservation address is No. 59, No. 5, of the Michelia Tokyo No. 100 college in Guangzhou city.
EXAMPLE 3 measurement of bacteriostatic spectra of biocontrol bacteria 3-10
1. In order to better study the biocontrol potential of the strain 3-10 of the invention, the bacterial inhibition spectrum of the strain is studied. The specific operation is as follows:
(1) 3-10 ability to inhibit growth of other pathogenic bacteria Using the same method as that used in the screening of the strains of example 1, the pathogenic bacteria MS2 was replaced with the same amount of basal rot fungi of rice EC1, bacterial soft rot fungi of banana MS3, Ralstonia solanacearum EP1 (the first layer L B of 15 m L was replaced with TTC of 15 m L), Hierosporus sphaericus of Cruciferae Xcc, Hierosporus oryzae Xoo, Hierosporus of Prunus persica, all of which were cultured under the same conditions and methodsPantoea ananatisPP1, yellow skin rot pathogenPantoea anthophilaC L1, and the rest steps are unchanged.
(2) The method comprises the steps of probing whether 3-10 fungi have the capability of inhibiting the growth of pathogenic fungi, wherein the fungi to be tested comprise ustilago, peronospora parasitica, fusarium oxysporum, pyricularia oryzae, rhizoctonia solani, colletotrichum gloeosporum and colletotrichum gloeosporioides, a plate confronting method is adopted for the ustilago, 20m L PDA is poured on a square culture dish of 13 × 13, the plate is dried and then cut into rectangular strips with the width of 0.5cm and the length of 6 cm, 3-10 bacterial solutions obtained by overnight culture of 1 mu L are taken every 0.5 cm. and are respectively poured at one end of the PDA strip, 3 bacterial solutions are in one group, and ustilago '+' basidiospore and a mixed solution obtained by equivalently mixing the basidiospore and the two basidiospore spores, the mixture is dried and then placed in a 28 ℃ culture box to be inversely cultured for 1-2 days, the growth condition of the triticale is observed, white villous hyphae is formed on the control plate, and the test result is recorded.
Punching holes with a 0.5cm puncher after the rest fungi are recovered, inversely placing the fungus cakes in the center of a PDA plate, dripping 2 mu L of 3-10 bacteria liquid cultured overnight and escherichia coli DH5 α bacteria liquid at the same distance from the fungus cakes, sealing after the bacteria liquid is dried, just placing the bacteria liquid in an incubator at 28 ℃ for culturing for 3-4 days, observing the growth condition of hyphae and recording the experimental result.
2. Experimental results show that the strains 3-10 have strong bacteriostatic effects on banana bacterial soft rot MS2 and MS3, solanaceae ralstonia solanacearum EP1, cruciferae black rot Xcc, rice bacterial leaf blight Xoo, peach rot PP1 and yellow skin rot C L1 (as shown in figure 3), and have obvious bacteriostatic effects on pepper colletotrichum, colletotrichum gloeosporioides, banana fusarium oxysporum, rice sheath blight, peronophythora litchii, rice blast and ustilago smut (as shown in figures 4 and 5), which indicates that 3-10 can be used for developing biocontrol agents for crop diseases.
Example 4 determination of Effect of biocontrol bacteria 3-10 on bacterial Soft rot
1. Activating banana bacterial soft rot MS2 and biocontrol bacteria 3-10 strains on an L B solid plate, selecting a single colony in a 50 m L centrifugal tube containing 10 m L L B, placing the single colony in a shaker at 28 ℃ and 180 rpm for overnight culture, and taking 1 m L OD600About 1.3 of 3-10 bacterial liquid is mixed with the same amount of MS2 bacterial liquid, MS2 bacterial liquid is added with 1 m L L B bacterial liquid to be mixed as positive control, L B culture medium is used as negative control.
And respectively inoculating potatoes, Chinese cabbages and dwarf banana seedlings in different treatments, repeating the treatment for 3 times, selecting 12 dwarf banana seedlings with similar growth vigors, respectively taking mixed liquid of L B, MS2+ L B, 3-10+ L B and 3-10+ MS2 of 200 mu L by using a 1 m L injector, injecting the mixed liquid into pseudostems of the dwarf bananas, repeating the treatment for 3 times, observing the growth condition of the dwarf banana seedlings every day, and recording.
2. Experimental results show that the strains 3-10 can obviously reduce the morbidity of bacterial soft rot on radish, carrot, Chinese cabbage, potato (shown in figure 6) and banana powder (shown in figure 7).
Example 5 determination of controlling Effect of biocontrol bacteria 3-10 on Banana wilt disease
1. Inoculation on PDA platesFusarium oxysporumf.sp.cubensisFOC4, culturing in 26 deg.C incubator until the mycelia are spread over the whole plate; FOC4 plates were rinsed with sterile water to rinse conidia off for use.
Carrying out appropriate root injury treatment on banana seedlings by using sterilizing scissors, and putting 6 untreated banana seedlings into a pot; another 6 plants are soaked in water with banana vascular wilt for 20min and then taken out to be planted in a basin filled with a certain amount of soil; using sterile water to make biocontrol bacteria 3-10 OD600Diluting the value to 0.1, respectively pouring biocontrol bacteria into 3 pots of plants which are not inoculated with FOC4 and are inoculated with FOC4 until the soil is wet, pouring sterile water into other pots (including those without treatment) until the soil is wet, and observing the growth condition of banana seedlings every day.
2. Experimental results show that the strains 3-10 can obviously reduce the incidence rate of banana vascular wilt (as shown in figure 8).
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (7)
1. 3-10 of a strain of Bacillus velezensis, which is preserved in Guangdong province collection center of microorganism strains in 2018, 4-30 days, and the preservation number is GDMCC NO. 60366.
2. Use of bacillus belgii 3-10 according to claim 1 for the control of bacterial and/or fungal diseases;
the bacterial diseases comprise diseases caused by bacterial soft rot, solanaceae ralstonia solanacearum, cruciferae black rot, rice white leaf blight, peach rot and yellow skin rot; the fungal diseases comprise diseases caused by colletotrichum capsici, colletotrichum gloeosporioides, fusarium oxysporum f.sp.cubense, phytophthora littoralis, rice blast and/or sugarcane smut.
3. A biological agent for controlling banana diseases, characterized by comprising the Bacillus belgii 3-10 of claim 1.
4. The biological agent according to claim 3, wherein the biological agent is prepared from a bacterial solution and/or a fermentation broth of Bacillus belgii strain 3-10.
5. A method for controlling banana diseases, characterized in that the method is a treatment with the Bacillus belgii 3-10 of claim 1 or the biological agent of claim 3.
6. The method for controlling according to claim 5, characterized in that the method for treatment is a vaccination treatment.
7. A method of controlling according to claim 6, characterised in that the method of inoculation is injection inoculation or root irrigation inoculation.
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