CN114891702B - Bacillus amyloliquefaciens L19 with biocontrol effect, microbial inoculum and application thereof - Google Patents
Bacillus amyloliquefaciens L19 with biocontrol effect, microbial inoculum and application thereof Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
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Abstract
The invention belongs to the technical field of microbial agent preparation, and in particular relates to bacillus amyloliquefaciens L19 with a biocontrol effect, a microbial agent and application thereof, wherein the bacillus amyloliquefaciens L19 has antagonism on plant fusarium wilt bacteria and can effectively control plant diseases; meanwhile, the bacillus amyloliquefaciens L19 can improve photosynthesis of plants, increase plant height, root length, overground fresh weight and underground fresh weight of the plants, and effectively promote plant growth.
Description
Technical Field
The invention belongs to the technical field of microbial agent preparation, and particularly relates to bacillus amyloliquefaciens L19 with a biocontrol effect, a microbial agent and application thereof.
Background
Potato is the fourth main grain crop after wheat, corn and rice in China, and can provide essential proteins, crude fibers, multiple vitamins and the like for human bodies. With the implementation of potato staple food strategy, the potato cultivation area in China is continuously enlarged, so that the problem of continuous cropping obstacle is remarkable, and soil-borne diseases are aggravated. Wherein, the potato wilt is taken as a main soil-borne disease, and occurs in various planting areas, so that the yield of the potato is seriously influenced, the yield reduction rate is up to 30% -50%, and the potato is one of main diseases which restrict the development of the potato industry.
At present, the prevention and treatment of potato wilt mainly depend on chemical agents, but the problems of pathogen resistance, pesticide residues, environmental pollution and the like are increasingly prominent due to the annual increase of the dosage and the variety of the chemical agents. In recent years, biological control has been receiving a great deal of attention because of its advantages of good environmental compatibility, safety to humans and animals and natural enemies, and the like. Among them, bacillus has the advantages of easy colonization, broad-spectrum antifungal activity and good growth promoting effect, so that Bacillus thallus and its secondary metabolite become hot spots for preventing and treating plant diseases in present agricultural production. Bacillus can produce various secondary metabolites such as lipopeptid antibiotics, antibacterial proteins and the like, and inhibit the growth of hyphae and spore germination of pathogenic bacteria by dissolving the cell wall or cell membrane of the pathogen and interfering the effects of protein synthesis, energy metabolism, cell division and the like. At present, related reports on the research of preventing and treating fusarium wilt by bacillus are presented, and the bacillus atrophaeus MQ19ST15 can obviously inhibit the hypha growth of cabbage fusarium wilt, so that the hypha has distortion such as expansion, branching increase and the like, and the bacteriostasis rate reaches 54.95%. The antibacterial radius of the bacillus amyloliquefaciens SFJ11 to the watermelon fusarium wilt is 8mm, and the field control effect of the strain fermentation liquor can reach 78 percent at most. Bacillus bailii Blz02 can effectively prevent and treat banana vascular wilt, and the prevention effect can reach 63.33%.
The quality of the growth promoting ability is an important index for evaluating bacillus. Bacillus may promote plant growth directly or indirectly by secreting phytohormones, phytase, and ferrite. As disclosed in the prior art, the bacillus amyloliquefaciens PHODB35 has obvious growth promoting effect on tomato seedlings, and the plant height, the overground fresh weight, the underground fresh weight, the organic matters and the effective phosphorus content are respectively increased by 28.21%, 22.59%, 113.06%, 45.08% and 16.24% after the strain fermentation liquor is applied. The bacillus amyloliquefaciens T-6 strain fermentation liquor can improve the dry weight, root total length and chlorophyll of tomato seedlings by 31.81%, 50.79% and 35.11% respectively compared with a control component, and has obvious growth promoting effect. The bacillus cymbidium 1Y4 fermentation inoculant is used for treating cucumber and capsicum, the plant heights of the cucumber and the capsicum are respectively increased by 153.06% and 117.33%, and the effect of promoting effect is obvious. However, bacillus which antagonizes potato fusarium wilt and has a growth promoting effect on potato is not reported.
Disclosure of Invention
The invention aims to provide bacillus amyloliquefaciens L19 with a biocontrol effect, a microbial inoculum and application thereof, wherein the bacillus amyloliquefaciens L19 can effectively prevent and treat plant diseases and promote plant growth.
The invention provides bacillus amyloliquefaciens (Bacillus amyloliquefaciens) L19 with a biocontrol effect, wherein the preservation number of the bacillus amyloliquefaciens L19 is CGMCC No.24596.
The invention provides a disease-resistant growth-promoting microbial agent, and the effective components of the disease-resistant growth-promoting microbial agent comprise bacillus amyloliquefaciens L19, fermentation liquor of bacillus amyloliquefaciens L19 and one or more of metabolites of bacillus amyloliquefaciens L19.
Preferably, the concentration of the bacillus amyloliquefaciens L19 in the disease-resistant growth-promoting microbial agent is 5 multiplied by 10 3 ～5×10 7 CFU/mL。
Preferably, the concentration of the bacillus amyloliquefaciens L19 in the disease-resistant growth-promoting microbial agent is 5 multiplied by 10 5 CFU/mL。
The invention also provides application of the bacillus amyloliquefaciens L19 or the disease-resistant growth-promoting microbial inoculum in the technical scheme in preventing and treating plant diseases and/or promoting plant growth.
Preferably, the promoting plant growth comprises promoting photosynthesis of the plant.
Preferably, the promotion of plant growth includes increasing plant height, root circumference, fresh weight of the aerial part and fresh weight of the subsurface part of the plant.
Preferably, the plant disease includes plant blight.
Preferably, the plant disease comprises potato blight.
The invention also providesA method of promoting plant growth is provided, the method comprising the steps of: the bacillus amyloliquefaciens L19 or the disease-resistant growth promoting microbial agent disclosed in the technical scheme is used for root irrigation treatment of potato seedlings; the concentration of the bacillus amyloliquefaciens L19 in the disease-resistant growth-promoting microbial agent is 5 multiplied by 10 3 ～5×10 7 CFU/mL。
The beneficial effects are that:
the invention provides bacillus amyloliquefaciens (Bacillus amyloliquefaciens) L19 with a biocontrol effect, and the biological preservation is completed. The bacillus amyloliquefaciens L19 has antagonism to plant fusarium wilt bacteria and can effectively prevent and treat plant diseases; meanwhile, the bacillus amyloliquefaciens L19 can improve photosynthesis of plants, increase plant height, root length, overground fresh weight and underground fresh weight of the plants, and effectively promote plant growth.
Biological preservation information
Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) L19 is preserved in China general microbiological culture Collection center (CGMCC) for 25 months in 2022, and the preservation address is North Chen West Lu No.1 and No.3 in the Korean area of Beijing, and the preservation number is CGMCC No.24596.
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 required to be used in the embodiments will be briefly described below.
FIG. 1 shows the inhibition of potato wilt pathogens by strain L19;
FIG. 2 is an identification chart of strain L19, wherein A is colony morphology of strain L19, B is gram staining result, and C is molecular biology identification phylogenetic tree;
FIG. 3 is a diagram showing the morphology of the opposite hyphae of Bacillus amyloliquefaciens L19, wherein the diagram A is a control group, and the diagram B is a diagram showing the morphology of the opposite hyphae after the treatment of the fermentation broth of Bacillus amyloliquefaciens L19;
FIG. 4 is a measurement of metabolic substances produced by Bacillus amyloliquefaciens L19, wherein FIG. A is Congo red medium containing cellulose, FIG. B is casein medium, and FIG. C is pectin medium;
FIG. 5 shows the effect of Bacillus amyloliquefaciens L19 volatile gas on the colonies of fusarium wilt pathogens, wherein A is a control group and B is a treatment group;
FIG. 6 shows the teratogenic effect of Bacillus amyloliquefaciens L19 volatile gas on the hyphae of fusarium wilt, wherein A is control group (20×), B is treatment group (20×), C is treatment group (20×), D is control group (40×), E is treatment group (40×), F is treatment group (40×); wherein B and C are hyphae results in different fields of treatment group (magnification of 20X), E and F are hyphae results in different fields of treatment group (magnification of 40X);
FIG. 7 shows the effect of Bacillus amyloliquefaciens L19 volatile gas on the hypha permeability of fusarium wilt pathogens;
FIG. 8 shows the effect of Bacillus amyloliquefaciens L19 fermentation broth at various concentrations on potato root circumference, wherein graph A is CK group and graph B has a concentration of 5×10 3 CFU/mL, C concentration 5X 10 5 CFU/mL, D plot concentration 5X 10 7 CFU/mL；
FIG. 9 is an OJIP curve of potato root-irrigation treated leaves with Bacillus amyloliquefaciens L19 fermentation broth of different concentrations;
FIG. 10 shows the effect of different concentrations of Bacillus amyloliquefaciens L19 fermentation broth on chlorophyll fluorescence parameters of potato leaf blades, wherein A is ABS/RC, B is TRo/RC, C is DIo/CSo, and D is TRo/CSo.
Detailed Description
The invention provides bacillus amyloliquefaciens (Bacillus amyloliquefaciens) L19 with a biocontrol effect, wherein the preservation number of the bacillus amyloliquefaciens L19 is CGMCC No.24596.
The bacillus amyloliquefaciens L19 is separated from potato root layer soil. The invention adopts gyrB-F:5'-TTGRCGGHRGYGGHTATAAAGT-3' (SEQ ID NO. 1) with gyrB-R:5'-TCCDCCSTAGARTCWCCCTC-3' (SEQ ID NO. 2) after PCR amplification and sequencing of the gyrB gene in the isolated strain DNA genome, a phylogenetic tree (FIG. 1) is constructed using the amplified gyrB gene sequence SEQ ID NO.3, which is specifically 5'-AATGGTTTGCATGATATGCGTGTATGCCCGGCTGCATCTATCGAGTGTCATAGGGGCGTCTGTCGTAAACGCCTTGTCGACCACTCTTGACGTTACGGTTCATCGTGACGGAAAAATCCACTATCAGGCGTACGAGCGCGGTGTACCTGTGGCCGATCTTGAAGTGATCGGTGATACTGATAAGACCGGAACGATTACGCACTTCGTTCCGGATCCGGAAATTTTCAAAGAAACAACCGTATACGACTATGATCTGCTTTCAAACCGTGTCCGGGAATTGGCCTTCCTGACAAAAGGCGTAAACATCACGATTGAAGACAAACGTGAAGGACAAGAACGGAAAAACGAGTACCACTACGAAGGCGGAATCAAAAGCTATGTTGAGTACTTAAACCGTTCCAAAGAAGTCGTTCATGAAGAGCCGATTTATATCGAAGGCGAGAAAGACGGCATAACGGTTGAAGTTGCATTGCAATACAACGACAGCTATACAAGCAATATTTATTCTTTCACGAATAATATCAACACATACGAAGGCGGCACGCACGAGGCCGGATTTAAAACCGGTCTGACCCGTGTCATAAACGACTATGCAAGAAGAAAAGGGATTTTCAAAGAAAATGATCCGAATTTAAGCGGGGATGATGTGAGAGAAGGGCTGACTGCCATTATTTCAATTAAGCACCCTGATCCGCAATTCGAAGGTCAGACGAAAACGAAGCTCGGCAACTCCGAAGCGAGAACGATCACTGATACGCTGTTTTCTTCTGCGCTGGAAACATTCCTTCTTGAAAATCCGGACTCAGCCCGCAAAATCGTTGAAAAAGGTTTAATGGCCGCAAGAGCGCGGATGGCAGCGAAAAAAGCGCGGGAATTGACCCGCCGCAAAAGTGCGCTTGAGATTTCCAATCTGCCGGGCAAACTGGCGGACTGTTCTTCTAAAGATCCGAGCATTTCCGAGCTGTATATCGTAGAGGGTGACTCTGCGGGCGGATCAGCGAAACAGGGACGGGACCGTCATTTCCAAGCCATTCTGCCGCTGCGCGGTAAGATTCTGAACGTTGAGAAAGCCAGACTTGATAAGATTCTCTCAAACAATGAGGTCAGATCAATGATCACGGCCCTCGGAACAGGAATCGGAGAAGATTTTAATCTTGAAAAAGCGCGTATCCGAGACAGTGTACTCCCGTGGTGCGTCAG-3'. The bacillus amyloliquefaciens L19 has 86% similarity with the bacillus amyloliquefaciens HUA8 and the bacillus amyloliquefaciens BJ-6, which shows that the strain has high similarity with the existing bacillus amyloliquefaciens.
The bacterial colony of the bacillus amyloliquefaciens L19 is milky irregular and nearly circular, the surface of the bacterial colony is provided with concave-convex folds, and the edges of the bacterial colony are uneven; the purple short rod shape is positive in gram color.
The bacillus amyloliquefaciens L19 has antagonism on various fungal pathogens including fusarium wilt, for example, the bacillus amyloliquefaciens L19 can damage cell walls of the fungal pathogens to cause hyphae deformity; meanwhile, the bacillus amyloliquefaciens L19 has the capability of secreting cellulase, can degrade cell walls of fungi, deform hyphae and lose infection capability. In the embodiment of the invention, the diameter of the inhibition zone of the bacillus amyloliquefaciens L19 on potato fusarium wilt is 26.0mm.
The bacillus amyloliquefaciens L19 can improve the photosynthetic performance of plants, increase the energy transferred and captured in unit area by allocating energy distribution and reducing the pigment content of an antenna, improve the electron transfer yield, further improve the light energy utilization rate of the plants and achieve the purpose of promoting growth.
The invention provides a disease-resistant growth-promoting microbial agent, and the effective components of the disease-resistant growth-promoting microbial agent comprise bacillus amyloliquefaciens L19, fermentation liquor of bacillus amyloliquefaciens L19 and one or more of metabolites of bacillus amyloliquefaciens L19, preferably the fermentation liquor of bacillus amyloliquefaciens L19 or bacillus amyloliquefaciens L19. The concentration of the bacillus amyloliquefaciens L19 in the disease-resistant growth-promoting microbial agent is preferably 5 multiplied by 10 3 ～5×10 7 CFU/mL, more preferably 5X 10 5 CFU/mL。
The invention also provides a preparation method of the disease-resistant growth-promoting microbial agent, which comprises the following steps: activating the bacillus amyloliquefaciens L19 strain, inoculating the activated bacillus amyloliquefaciens L19 bacterial liquid into an LB culture medium, and culturing overnight at 37 ℃ to obtain a single bacterial colony of the bacillus amyloliquefaciens L19 strain; the single colony of the bacillus amyloliquefaciens L19 strain is selected and cultured in an LB liquid medium for the first time to obtain bacillus amyloliquefaciens L19 strain seed liquid; mixing the bacillus amyloliquefaciens L19 strain with an LB liquid culture medium, and performing secondary culture to obtain a bacillus amyloliquefaciens L19 strain fermentation broth, namely the disease-resistant growth-promoting microbial agent.
The bacillus amyloliquefaciens L19 strain is activated, activated bacillus amyloliquefaciens L19 bacterial liquid is inoculated into an LB culture medium, and the bacillus amyloliquefaciens L19 strain is cultured overnight at 37 ℃ to obtain single bacterial colony of the bacillus amyloliquefaciens L19 strain; the Bacillus amyloliquefaciens L19 strain is preferably activated in an incubator, and the activation temperature is preferably 37 ℃. The invention preferably adopts a streak inoculation mode to inoculate the activated bacillus amyloliquefaciens L19 bacterial liquid into an LB culture medium.
After obtaining the single colony of the bacillus amyloliquefaciens L19 strain, the single colony of the bacillus amyloliquefaciens L19 strain is selected to be subjected to first culture in an LB liquid culture medium, so as to obtain the bacillus amyloliquefaciens L19 strain seed liquid. The first culture according to the present invention is preferably shaking culture, and the rotation speed of the shaking culture is preferably 180 to 220r/min, more preferably 200r/min. The temperature of the first culture according to the present invention is preferably 30 to 37 ℃, more preferably 37 ℃; the time of the first culture is preferably 8 to 12 hours, more preferably 12 hours.
After seed liquid of the bacillus amyloliquefaciens L19 strain is obtained, the bacillus amyloliquefaciens L19 strain is mixed with an LB liquid culture medium, and is subjected to secondary culture to obtain fermentation liquid of the bacillus amyloliquefaciens L19 strain, namely the disease-resistant growth-promoting microbial agent. The preferred values of the bacillus amyloliquefaciens L19 strain and the LB liquid medium are 100:1. the second culture according to the present invention is preferably shaking culture, and the rotation speed of the shaking culture is preferably 180 to 220r/min, more preferably 200r/min. The temperature of the second culture according to the present invention is preferably 30 to 37 ℃, more preferably 37 ℃; the time of the second culture is preferably 6 to 8 hours, more preferably 6 hours. The initial effective viable count of the bacillus amyloliquefaciens L19 in the bacillus amyloliquefaciens L19 strain fermentation liquid obtained by the invention is preferably 5 multiplied by 10 8 CFU/mL, followed by gradient dilution.
The invention also provides application of the bacillus amyloliquefaciens L19 or the disease-resistant growth-promoting microbial inoculum in the technical scheme in preventing and treating plant diseases and/or promoting plant growth.
The promotion of plant growth according to the present invention preferably includes promotion of photosynthesis of plants, more preferably includes increasing plant height, root circumference, fresh weight of aerial parts and fresh weight of subsurface parts of plants. The plant diseases according to the invention preferably comprise plant blight, more preferably potato blight.
The invention also provides a method for promoting plant growth, comprising the following steps: the bacillus amyloliquefaciens L19 or the disease-resistant growth promoting microbial agent disclosed in the technical scheme is used for root irrigation treatment of potato seedlings; the concentration of the bacillus amyloliquefaciens L19 in the disease-resistant growth-promoting microbial agent is 5 multiplied by 10 3 ～5×10 7 CFU/mL。
The inventionThe dosage of the root irrigation treatment is preferably 20 mL/plant, the specific process of the root irrigation treatment is not particularly limited, and the root irrigation treatment is carried out by adopting the conventional root irrigation treatment process in the field. The potato seedlings of the present invention are preferably 6-8 seedling clusters. The concentration of the Bacillus amyloliquefaciens L19 in the disease-resistant growth-promoting microbial agent is preferably 5 multiplied by 10 5 CFU/mL。
The technical solutions provided by the present invention are described in detail below with reference to the drawings and examples for further illustrating the present invention, but they should not be construed as limiting the scope of the present invention.
The culture medium and the formula thereof are as follows:
PDA medium: 200g/L of potato, 20g/L of agar powder and 20g/L of glucose;
LB solid medium: 10g/L of peptone, 10g/L of NaCl, 5g/L of yeast extract powder, 20g/L of agar powder and pH 7.0;
LB liquid medium: peptone 10g/L, naCl 10g/L, yeast extract 5g/L, pH 7.0;
cellulose medium (congo red medium containing cellulose): k (K) 2 HPO 4 1.0 g/L, ammonium sulfate 2.0g/L, mgSO 4 ·7H 2 0.5g/L of O, 0.5g/L of NaCl, 2.0g/L of microcrystalline cellulose, 0.4g/L of Congo red and 20.0g/L of agar;
casein medium: glucose 10g/L, yeast extract 4g/L, casein 10g/L, KH 2 PO 4 1 g/L，MgSO 4 ·7H 2 O0.2 g/L, agar 20g/L, pH 7.0;
pectin medium: 20g/L of peptone, 5g/L of yeast extract, 5g/L of beef extract, 5g/L of NaCl, 2g/L of pectin, 20g/L of agar, pH 7.0 and 15min of saturated copper acetate solution.
Example 1
Separation, screening and identification of bacillus amyloliquefaciens L19:
(1) Test soil sample: and (5) collecting soil samples of 0-20cm root layers in a potato continuous cropping 5 years by using a five-point sampling method, and preserving the soil samples in a refrigerator at 4 ℃ for later use.
(2) Separation and screening of biocontrol bacteria: separating the strain from the soil sample in the step (1) by adopting a soil dilution method, and screening the strain by combining a plate counter method. The method comprises the following steps: a filter paper sheet with the diameter of 5mm is inoculated at the center of a PDA culture medium, 5 mu L of the separated candidate bacterial suspension is inoculated, 5mm potato Fusarium wilt bacteria cakes (Fusarium oxysporum f.sp.turbosi) are placed at the positions of 2.5cm around each, the potato Fusarium wilt bacteria cakes are preserved by a potato disease research center of the university of Hebei agriculture plant protection college, sterile water is used as a control, and after the control grows full of the flat plate, the diameter of a bacteriostasis ring is measured.
The antagonistic bacteria 25 strains having an inhibitory effect on fusarium wilt were obtained by co-screening in the above steps, as shown in table 1:
TABLE 1 antibacterial zone width of bacillus antagonizing potato blight
(3) Further screening 25 antagonistic bacteria obtained by screening:
selecting single bacterial colony of the screened strain, inoculating the single bacterial colony into a liquid LB (LB) culture medium, culturing for 12 hours at 37 ℃ at 200r/min to prepare strain seed liquid, inoculating 100 mu L of the strain seed liquid into 100mL of LB liquid culture medium by using a pipette, culturing for 6 hours at 37 ℃ at 200r/min for standby, placing potato fusarium wilt cakes with the diameter of 5mm in the center of a PDA (personal digital assistant) culture medium flat plate, respectively placing sterile filter paper sheets with the diameter of 5mm for inoculating the 25 bacillus strain antagonistic bacteria at 25mm away from the cakes in a crisscross manner, culturing at the constant temperature of 25 ℃ for 4 days, and measuring the width of a bacteria inhibition zone. Each group was repeated 4 times with the spotted LB liquid medium as a blank, and the results are shown in fig. 1: wherein the graph A is a control group, the graph B is the inhibition effect of the strain L19 on potato fusarium wilt pathogens in a counter culture medium, and the diameter of a inhibition zone of the strain L19 on the potato fusarium wilt pathogens reaches 26.0mm.
(4) Colony morphology characterization: the bacterial strain L19 bacterial liquid obtained by screening in the step (3) is filteredDiluted to a concentration of 1X 10 8 CFU/mL, then spread on LB plates, cultured at 37℃for 24h. As a result of streaking, the colony of the strain L19 was milky white, irregular and nearly circular, and had uneven wrinkles on the surface of the colony and uneven edges (FIG. 2A). Strain L19 was in the form of a purple short rod and gram-positive (panel B in fig. 2).
(5) Molecular biology identification: strain L19 genomic DNA was extracted using the whole gold bacteria genome extraction kit. gyrB-F was used: 5'-TTGRCGGHRGYGGHTATAAAGT-3' with gyrB-R:5'-TCCDCCSTAGARTCWCCCTC-3' amplification of the gyrB gene. PCR amplification system: template DNA 2. Mu. L, mix 12.5. Mu. L, gyrB-F (1. Mu.M) 1.5. Mu. L, gyrB-R (1. Mu.M) 1.5. Mu. L, ddH 2 O7.5 μl; amplification conditions: 94 ℃ for 5min;94℃30s,55℃45s,72℃1min,35 cycles; and at 72℃for 10min. After the amplification result is detected by electrophoresis, sequencing is performed by Shanghai workers. BLAST analysis is carried out on the sequencing result through logging in Gen Bank, a phylogenetic tree is constructed, the result is shown as a C diagram in FIG. 2, the strain L19 has 86% similarity with the bacillus amyloliquefaciens HUA8 and the bacillus amyloliquefaciens BJ-6, the strain has high similarity with the existing bacillus amyloliquefaciens, the strain is identified as the bacillus amyloliquefaciens, and the strain is named as the bacillus amyloliquefaciens L19.
Example 2
Antagonism of bacillus amyloliquefaciens L19 on potato fusarium wilt bacteria
(1) Antagonizing bacillus amyloliquefaciens L19 to fusarium wilt pathogen hypha morphology observation: mycelia on the edges of colonies of Fusarium oxysporum (fusarium oxysporum) were picked up on the counter plates in example 1, and the mycelia morphology of Fusarium oxysporum (fusarium oxysporum) was observed under an optical microscope.
The results are shown in FIG. 3: wherein, A is control hypha (blank control hypha without inoculating bacillus fermentation broth), and B is fusarium wilt pathogenic hypha on the opposing plate with inoculating bacillus fermentation broth. The result shows that the fusarium wilt hyphae in the control group grow normally and have uniform morphology; after the bacillus amyloliquefaciens L19 fermentation liquid is treated, the growth of fusarium wilt hyphae is uneven, the shape is curved at the top end, cavitation is increased and the phenomenon of end digestion is eliminated, so that the metabolic substances generated by the bacillus amyloliquefaciens L19 can damage the cell wall of fusarium oxysporum, and the hyphae are distorted.
(2) Detection of degradation of hypha cell wall substances by bacillus amyloliquefaciens L19:
after streaking the stored bacillus amyloliquefaciens L19 fermentation liquor, picking a single colony, and carrying out shake culture for 24 hours at 37 ℃ to prepare strain seed liquor; inoculating 1% of the strain into an LB liquid culture medium, and carrying out shaking culture at 37 ℃ for 48 hours to obtain activated bacillus amyloliquefaciens L19 fermentation liquor;
respectively taking 1 mu L of activated bacillus amyloliquefaciens L19 fermentation liquor, respectively inoculating to a Congo red culture medium containing cellulose, a casein culture medium and a pectin culture medium, culturing at 37 ℃ for 48 hours, observing whether transparent rings are generated or not, repeating for 3 times, and recording the result by photographing, wherein the result is shown in a figure 4, wherein a figure is the Congo red culture medium containing cellulose, a figure B is the casein culture medium, and a figure C is the pectin culture medium;
as can be seen from FIG. 4, after staining with iodine solution, bacillus amyloliquefaciens L19 was cultured on a cellulose Congo red medium for 48 hours to produce an obvious transparent ring, which indicates that the Bacillus amyloliquefaciens L19 strain has the ability to secrete cellulase, and can degrade the cell wall of fungi, deform hyphae and lose infectivity. The bacillus amyloliquefaciens L19 did not change in the casein and pectin media, indicating that the bacillus amyloliquefaciens L19 did not produce protease and pectase.
(3) Influence of Bacillus amyloliquefaciens L19 volatile gas on bacterial colony and hypha of fusarium wilt pathogen
200 mu L of the stored bacillus amyloliquefaciens L19 fermentation liquor is absorbed by a double-dish buckling method and uniformly coated on an LB plate, and the other half of the PDA plate is inoculated with fusarium wilt pathogenic bacteria and sealed by a sealing film (treatment group). The control group was not added with the strain L19 broth, 200. Mu.L of sterile water was pipetted onto LB plates and incubated in the dark at 25℃for 3 replicates. When the colony grows to 2/3 of the control colony, the colony and the mycelium form are observed by photographing, as shown in figures 5-6.
As can be seen from fig. 5: after co-culturing bacillus amyloliquefaciens L19 with fusarium wilt pathogenic bacteria, the colony size of the fusarium wilt pathogenic bacteria in the treatment group is obviously smaller than that of the control group, and the colony color is changed into yellow white.
From fig. 6, it can be derived that: the mycelium of the wilt pathogen of the treatment group showed a small amount of blisters (B and E in fig. 6) and a large amount of deformities, sticking (C and F in fig. 6).
The above results demonstrate that the volatile gas of Bacillus amyloliquefaciens L19 has the effect of inhibiting the growth of bacterial colonies and hyphae of fusarium wilt, and that Bacillus amyloliquefaciens L19 has antagonism to fusarium wilt pathogenic bacteria.
(4) Influence of Bacillus amyloliquefaciens L19 volatile gas on the penetration of hyphae of fusarium wilt pathogen
200 mu L of stored bacillus amyloliquefaciens L19 fermentation liquor is absorbed by a double-dish buckling method and uniformly coated on an LB plate, a layer of cellophane is uniformly paved on the other half of PDA plate, the center is inoculated with fusarium wilt pathogen, and a sealing film is sealed (treatment group). Taking LB plate without adding strain L19 fermentation broth as control, removing glasspaper after dark culture at 25deg.C for 5d, continuously culturing for 3d, and photographing and recording as shown in FIG. 7:
from fig. 7, it can be derived that: after the glass paper is removed, the hyphae of the wilt pathogenic bacteria of the control group can normally grow, which indicates that the hyphae can penetrate the glass paper; the culture dish of the treatment group has no colony growth, which indicates that hyphae are malformed after gas fumigation and cannot penetrate through cellophane, and indicates that the volatile gas of the bacillus amyloliquefaciens L19 can reduce the penetrability of the hyphae of pathogenic bacteria of the fusarium wilt.
Example 3
(1) The preparation method of the bacillus amyloliquefaciens L19 disease-resistant growth-promoting microbial agent comprises the following steps:
taking out the frozen bacterial liquid in a refrigerator at the temperature of minus 80 ℃, activating an incubator at the temperature of 37 ℃, then streaking the bacterial liquid on an LB plate by using an inoculating loop, and culturing single bacterial colonies at the temperature of 37 ℃ overnight. The bacillus amyloliquefaciens L19 strain single colony is selected and inoculated in a liquid LB culture medium, and is cultured for 12 hours at 37 ℃ at 200r/min to prepare bacillus amyloliquefaciens L19 strain seed liquid. Mixing the shaken seed liquid with LB liquid culture medium 1:100, culturing at 37deg.C for 6-8 hr/min to give colony concentration of 5×10 8 CFU/mL, performing gradient dilution.
(2) Seedling growth promotion pot test of potatoes:
the sterilized vermiculite, soil and nutrient soil are mixed according to the mass ratio of 1:2:2, uniformly mixing, putting into a sterilized flowerpot, cutting and sowing the germinated Holland fifteen kinds of potatoes, treating 10 pots in each group, putting the treated 10 pots in a sunlight greenhouse at 16 ℃ for 7d, and selecting potato seedlings with consistent growth vigor for later use.
Setting the concentration of the bacillus amyloliquefaciens L19 bacterial liquid to be 0 (clear water treatment) and 5 multiplied by 10 respectively 3 CFU/mL、5×10 5 CFU/mL and 5X 10 7 The CFU/mL 4 concentration gradients were root irrigated and repeated 6 times. After the root irrigation treatment grows for 7d, the plants in each pot are pulled up, washed and dried, and indexes such as the height of each treated plant, the root length, the fresh weight of the overground part and the underground part, chlorophyll content and the like are respectively measured, and the results are shown in Table 2:
TABLE 2 various physiological indexes of potato after 7 days of root-filling of Bacillus amyloliquefaciens L19 fermentation broth with different concentrations
Note that: data in the table are mean ± standard deviation. The different letters indicate significant differences at P <0.05 levels as tested by Duncan's new complex polar error method.
As can be seen from table 2: after 7d treatment by fermentation liquid of bacillus amyloliquefaciens L19 with different concentrations, the plant height, root circumference, fresh weight of overground parts and dry weight of underground parts of the potato seedlings are obviously higher than those of a control group. Wherein the root canal is most obviously increased, and the three concentration fermentation liquid is respectively increased by 52.3 percent, 64.29 percent and 78.57 percent compared with the control after the root canal is irrigated.
As can be seen from fig. 8: after the fermentation liquid is treated by the bacillus amyloliquefaciens L19, fibrous roots of the potatoes in the treated group are obviously increased, and root circumferences are obviously increased compared with those in the control group, so that the potato plants can absorb moisture and nutrients. Wherein 5×10 5 The plant height and the fresh weight of the overground part of the CFU/mL concentration treatment are greatly increased, the growth promotion effect is most obvious, and the growth rates are respectively29.56% and 33.38%. The bacillus amyloliquefaciens L19 has obvious growth promotion effect on potatoes.
(3) After the fermentation liquid with different concentrations of the bacillus amyloliquefaciens L19 treated in the step (2) is treated for 7d, the leaves of potato seedlings are fully dark adapted for 20min respectively, and a portable plant efficiency instrument, namely, hansat-ech, UK, is adopted to measure fluorescence induction kinetics curves (OJIP curves) of the leaves.
Effect of bacillus amyloliquefaciens L19 strain broth on leaf OJIP curve:
chlorophyll fluorescence-induced kinetics (OJIP) can reflect information on photochemical reactions at the PS ii reaction center, and the results are shown in fig. 9: after 7d treatment of the fermentation broth, the fluorescence intensities at each of the concentrations of the treated OJIP curve at O, J (2 ms), I (30 ms) and P points were significantly different from the control at 5X 10 7 The CFU/mL concentration treatment had the highest rise. The results show that the photosynthetic mechanism and the function of the plant leaf are changed after the plant leaf is treated by fermentation liquor.
Effect of fermentation broth of bacillus amyloliquefaciens L19 strain on fluorescence properties of potato leaf blades:
as can be seen from the a-graph in fig. 10: the light energy (ABS/RC) absorbed by the center of reaction of PS II units of potato leaf and the light energy (TRo/RC) captured by the center of reaction of PS II units of potato leaf are gradually increased along with the increase of the concentration of the bacillus L19 and are increased at 5 multiplied by 10 7 The maximum value is reached when CFU/mL is reached, wherein ABS/RC is 1.31 times of the control, and under the treatment of the other two concentrations, the ABS/RC is 1.18 times and 1.28 times of the control respectively, and the ABS/RC is obviously higher than the control treatment;
as can be seen from the B diagram in fig. 10: at each concentration, TRo/RC was significantly increased by 24.89%, 23.54% and 17.50% compared to CK, respectively.
From this, it can be derived that: the light energy (ABS/RC) absorbed by the unit reaction center and the energy (TRo/RC) captured by the unit reaction center of the potato leaf treated by the fermentation liquid with different concentrations of the bacillus amyloliquefaciens L19 are both higher, which means that the antenna pigment content is low, the absorbed light energy is more, and the energy for electron transfer by the PS II reaction center is more.
As can be seen from the graph C in fig. 10: solution at each concentrationThe heat dissipation per unit area (DIo/CSo) under Bacillus amyloliquefaciens L19 fermentation broth treatment was significantly higher than that of the control (P<0.05 At 5X 10) 7 CFU/mL reached a maximum of 59.40, and the three treatments increased 16.60%, 16.32% and 23.42% over the control, respectively.
As can be seen from the D diagram in fig. 10: the captured light energy (TRo/CSo) per unit area has a tendency of increasing and decreasing with the increase of the concentration of the fermentation liquor, and is 5 multiplied by 10 3 CFU/mL and 5X 10 5 CFU/mL treatment was significantly higher than control, but to 5X 10 7 The CFU/mL treatment concentration was somewhat decreased.
This can be explained by: the energy transferred and captured in the unit area without fermentation liquid treatment is relatively reduced, the electron transfer yield is reduced, the energy transferred to the tail end of an electron chain is also reduced, the light energy conversion efficiency is reduced, the normal operation of photosynthesis is finally limited, the light energy utilization rate can be increased to a greater extent by the treatment of the fermentation liquid of the bacillus amyloliquefaciens L19, and the photosynthetic efficiency is improved.
From the above examples, the bacillus amyloliquefaciens L19 and the disease-resistant growth-promoting microbial inoculum prepared by the bacillus amyloliquefaciens L19 have higher growth-promoting effect on plant blight, and can improve photosynthesis of plants and promote plant growth.
Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.
Sequence listing
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gactgccatt atttcaatta agcaccctga tccgcaattc gaaggtcaga cgaaaacgaa 720
gctcggcaac tccgaagcga gaacgatcac tgatacgctg ttttcttctg cgctggaaac 780
attccttctt gaaaatccgg actcagcccg caaaatcgtt gaaaaaggtt taatggccgc 840
aagagcgcgg atggcagcga aaaaagcgcg ggaattgacc cgccgcaaaa gtgcgcttga 900
gatttccaat ctgccgggca aactggcgga ctgttcttct aaagatccga gcatttccga 960
gctgtatatc gtagagggtg actctgcggg cggatcagcg aaacagggac gggaccgtca 1020
tttccaagcc attctgccgc tgcgcggtaa gattctgaac gttgagaaag ccagacttga 1080
taagattctc tcaaacaatg aggtcagatc aatgatcacg gccctcggaa caggaatcgg 1140
agaagatttt aatcttgaaa aagcgcgtat ccgagacagt gtactcccgt ggtgcgtcag 1200
Claims (10)
1. The bacillus amyloliquefaciens (Bacillus amyloliquefaciens) L19 with the biocontrol effect is characterized in that the preservation number of the bacillus amyloliquefaciens L19 is CGMCC No.24596.
2. An anti-disease growth-promoting microbial agent, wherein the active ingredients of the anti-disease growth-promoting microbial agent comprise one or more of bacillus amyloliquefaciens L19, bacillus amyloliquefaciens L19 fermentation liquor and bacillus amyloliquefaciens L19 metabolites according to claim 1.
3. The disease-resistant growth-promoting bacterium agent according to claim 2, wherein the concentration of bacillus amyloliquefaciens L19 in the disease-resistant growth-promoting bacterium agent is 5×10 3 ～5×10 7 CFU/mL。
4. The disease-resistant growth-promoting bacterium agent according to claim 2 or 3, wherein the concentration of Bacillus amyloliquefaciens L19 in the disease-resistant growth-promoting bacterium agent is 5X 10 5 CFU/mL。
5. Use of the bacillus amyloliquefaciens L19 of claim 1 or the disease-resistant growth-promoting microbial agent of any one of claims 2 to 4 for controlling plant diseases and/or promoting plant growth.
6. The use of claim 5, wherein said promoting plant growth comprises promoting photosynthesis of a plant.
7. The use of claim 5, wherein said promoting plant growth comprises increasing plant height, root circumference, above-ground fresh weight, and below-ground fresh weight.
8. The use according to claim 5, wherein the plant disease comprises plant blight.
9. The use according to claim 7, wherein the plant disease comprises potato blight.
10. A method of promoting plant growth, the method comprising the steps of: root irrigation treatment is carried out on potato seedlings by using the bacillus amyloliquefaciens L19 according to claim 1 or the disease-resistant growth promoting microbial agent according to any one of claims 2 to 4;
the concentration of the bacillus amyloliquefaciens L19 in the disease-resistant growth-promoting microbial agent is 5 multiplied by 10 3 ～5×10 7 CFU/mL。
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20120094647A (en) * | 2011-02-17 | 2012-08-27 | 고려바이오주식회사 | Bacillus amyloliquefaciens kb-mjk 601 with antifungal activity against plant pathogenic fungi and microbial agent for preventing plant phthogenic fungi |
| CN105018385A (en) * | 2015-07-31 | 2015-11-04 | 长江大学 | Bacillus amyloliquefaciens and application thereof |
| CN107736379A (en) * | 2017-11-01 | 2018-02-27 | 广州市林业和园林科学研究院 | Application of the bacillus amyloliquefaciens in fungal diseases of plants is prevented and treated |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20120094647A (en) * | 2011-02-17 | 2012-08-27 | 고려바이오주식회사 | Bacillus amyloliquefaciens kb-mjk 601 with antifungal activity against plant pathogenic fungi and microbial agent for preventing plant phthogenic fungi |
| CN105018385A (en) * | 2015-07-31 | 2015-11-04 | 长江大学 | Bacillus amyloliquefaciens and application thereof |
| CN107736379A (en) * | 2017-11-01 | 2018-02-27 | 广州市林业和园林科学研究院 | Application of the bacillus amyloliquefaciens in fungal diseases of plants is prevented and treated |
Non-Patent Citations (1)
| Title |
|---|
| 解淀粉芽孢杆菌JF-1对黄瓜的防病促生作用;张德珍;李婷婷;代惠洁;迟文娟;乔宁;;北方园艺(10);22-27 * |
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