CN114149935A - Lysobacter enzymogenes and application thereof - Google Patents
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Abstract
The invention belongs to the technical field of biological control, and discloses lysobacter enzymogenes and application thereof, wherein the lysobacter enzymogenes is a lysobacter enzymogenes BN3-15 strain, the strain is separated from rhizosphere soil of fresh corn Dai nationality of autonomous State of Xishuangbanna, Yunnan province and is classified and named as lysobacter enzymogenes, the lysobacter enzymogenes BN3-15 strain is preserved in the common microorganism center of China Committee for culture preservation and management of microorganisms, and the preservation number is as follows: CGMCC No. 21259. The lysobacter enzymogenes BN3-15 provided by the invention is safe, has no residue, has the effects of preventing diseases and promoting growth, can be prepared into water aqua, wettable powder or seed coating agent, and is used for preventing and treating diseases such as corn leaf blight, pepper anthracnose, Chinese cabbage clubroot, pseudo-ginseng root rot and the like in fields. The biological agent provided by the invention has better comprehensive properties, has obvious prevention effect on fungal diseases, is beneficial to reducing the use amount and residual quantity of chemical agents in agricultural products, is safer to use, and is easy for industrial production.
Description
Technical Field
The invention belongs to the technical field of biological control, and particularly relates to lysobacter enzymogenes and application thereof.
Background
At present, corn (Zea mays Linn.) Gramineae annual herbaceous plants are important grain-feed-industrial raw material crops in China, are one of the crops with the widest planting area and the largest land utilization rate in China, and are crops with large planting areas in Yunnan province. Yunnan province can be divided into four corn planting areas: a cold and cool corn area in the northwest of Yunnan, a warm corn area in the Yunnan, a warm and hot corn area in the Yunnan and a warm and cool corn area in the northeast of Yunnan. In recent years, the planting area of fresh corn in our province is gradually enlarged, and the fresh corn is a supporting industry which is the key development of plateau characteristics. The main disease types of the corn with kernel in Yunnan province are corn rust disease, corn gray leaf spot, corn big leaf spot, corn stalk rot and the like, and the fresh corn is mainly seriously damaged by the corn big leaf spot and the corn stalk rot. The northern leaf blight of corn is a leaf spot disease caused by Deuteromycotina major (Exserohilum turcicum), and in recent years, the harm of the northern leaf blight of corn is obviously aggravated due to the problems of rapid physiological race variation, serious environmental pollution caused by chemical control and the like of the northern leaf blight of corn. Corn stalk rot and ear rot are also diseases which are seriously popular in recent years in corn producing areas in China, and most researches report that Fusarium graminearum (Fusarium moniliforme) and Fusarium moniliforme (Fusarium moniliforme) are main pathogenic fungi causing stalk rot/ear rot in China. The disease not only causes the rotting of the ear of grain in the corn yield to reduce the yield, but also causes the pathogenic fungi to generate a large amount of mycotoxin to reduce the quality of the corn. In addition, the toxins have strong pathogenic, teratogenic and carcinogenic properties, cause diseases of human beings and livestock, and pose a serious threat to the life health of human beings and livestock.
The pepper is one of the most important vegetables and seasonings in China, mainly plays a role in appetizing and adjusting taste, and is an important economic crop in China. With the spread of spicy food flavor and the development of functions of pepper extract in medical treatment, health care, beauty treatment and the like in recent years, the market demand of pepper is expanding. The pepper anthracnose is one of three diseases in the production process of pepper, is wide in distribution and serious in harm, generally reduces the yield by 20-50%, and seriously affects the yield and quality of pepper. Colletotrichum oxysporum (Colletotrichum acutatum) is a major fungus causing anthracnose of capsicum.
The Brassicaceae crops include rape, Chinese cabbage, cauliflower, stem mustard, leaf mustard, radish, Chinese cabbage, leaf mustard (hot pickled mustard tuber), and special crops such as radix Isatidis and horseradish. Clubroot of cruciferous crops is a soil-borne disease caused by infection with Plasmodiophora brassicae, which mainly damages the roots of cruciferous crops. Because the pathogenic bacteria in the soil have long survival time, strong infectivity, high transmission speed and various transmission ways, the clubroot disease is more and more serious. The clubroot of cruciferae seriously affects the yield of vegetables, and the yield is reduced by 70-80% when the clubroot is extremely serious. With the increasing demand for cruciferous vegetables at home and abroad, the cultivation area of the cruciferous vegetables is continuously enlarged, so that the continuous cropping planting phenomenon of the cruciferous vegetables is more and more common, the quantity of clubroot pathogens in soil is accumulated year by year, the clubroot is developed in a large area, and the land suitable for planting the cruciferous vegetables is less and less.
At present, agricultural cultivation measures, disease-resistant varieties, chemical agent control measures and the like are mainly adopted for controlling the diseases, but the problems of quick variation of physiological races, serious environmental pollution caused by chemical control and the like cause unsatisfactory control effect of fungal diseases of crops and have certain limitations. Therefore, the development of biological agents for corn leaf blight, corn ear rot, pepper anthracnose and crucifer clubroot has important practical significance.
Lysobacterpenes belong to the group of gram-negative bacteria of the genera lysobacteroides, the order xanthomonas, the family xanthomonas, the genus lysobacter, and occupy an important position in the field of biocontrol microorganisms. From 1978, lysobacter species were established, named 4 species, and were: lysobacter enzymogenes (l.enzymogenes), lysobacter antibioticus (l.antibioticus), lysobacter palmatum (l.brunescens) and lysobacter colloquii (l.gummosus). A large number of domestic and foreign researches find that the metabolic products of the lysobacter contain a large number of small molecular compounds which have good antagonistic action on crop pathogenic fungi, bacteria and nematodes. For example, the reported lysobacter enzymogenes can produce antifungal cyclic peptide active substances HSAF, which shows that the lysobacter enzymogenes has great development potential in the aspect of biological control; according to the reports of domestic and foreign documents, few reports of lysobacter enzymogenes as a biocontrol microbial inoculum are reported, and few biological microbial inocula prepared for preventing and controlling crop diseases in fields are available. In conclusion, the development of the lysobacter enzymogenes strain with safe, efficient and broad-spectrum biocontrol potential has important significance for the construction of a biocontrol lysobacter resource library, the protection of the ecological environment and the reduction of the use of chemical pesticides, and can promote the development of ecological agriculture and green agriculture.
Through the above analysis, the problems and defects of the prior art are as follows:
(1) the harm of the northern leaf blight of corn presents a remarkable aggravating trend due to the problems of quick physiological race variation, serious environmental pollution caused by chemical control and the like of the northern leaf blight of corn and vegetable clubroot.
(2) Along with the problems of quick variation of physiological race, serious environmental pollution caused by chemical control and the like, the control effect of fungal diseases of crops is not ideal and has certain limitation.
(3) Lysobacter enzymogenes is mostly reported from the aspects of bacterial inhibition spectrum and disease prevention mechanism, but is not reported from the rhizosphere of fresh corn, and few biological agents prepared from the lysobacter enzymogenes are used for preventing and treating diseases in fields.
The difficulty in solving the above problems and defects is: the zymogenic lysobacter is cultured and fermented to prepare a water agent or wettable powder and a seed coating agent, or is matched with an organic fertilizer for use, so that the operation and the production are easy. The biological agent is used as spraying and root irrigation, is also a common agricultural use method, is simple to operate and is convenient to use.
The significance of solving the problems and the defects is as follows: the prepared zymogen bacillus biological agent can prevent and control main diseases of crops, achieve the aims of reducing weight, reducing drug, improving quality and improving efficiency, and promote the green development of industries such as vegetables, traditional Chinese medicinal materials and the like. Meanwhile, a green prevention and control technology system for crop diseases is constructed, and a new product and a new technology support are provided as green product production.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides lysobacter enzymogenes and application thereof.
The invention is realized in such a way that the lysobacter enzymogenes BN3-15 new strain is a new strain separated from rhizosphere soil of fresh-eating corn of Dai nationality of the Xishuangbanna, Yunnan province, and is classified and named as lysobacter enzymogenes, the new strain of the lysobacter enzymogenes BN3-15 is preserved in the common microorganism center of the China Committee for culture preservation and management of microorganisms, and the preservation numbers are as follows: CGMCC No. 21259.
Further, the lysobacter enzymogenes BN3-15 is highly viscous, creamy in color, capable of sliding, free of flagellum, rod-shaped in thallus, round in colony shape and smooth in edge.
The invention also aims to provide application of the lysobacter enzymogenes BN3-15 in field disease control, wherein the diseases comprise corn northern leaf blight, pepper anthracnose, cruciferous crop clubroot and panax notoginseng root rot.
Further, the lysobacter enzymogenes BN3-15 can be prepared into any one of a water agent, a wettable powder and a coating agent for use.
Further, the preparation method of the lysobacter enzymogenes BN3-15 aqueous solution comprises the following steps:
(1) strain culture
Rejuvenation culture: sterilizing an NA culture medium at 121 ℃ for 30min, adding the NA culture medium into a test tube, making an inclined plane, inoculating a strain, and culturing for 24-48 h at 26 ℃ in a constant-temperature incubator;
expansion culture of strains by using a shaking table: inoculating the slant seeds on a slant NA culture medium of an eggplant bottle, and culturing for 24-48 h at 26 ℃ to obtain eggplant bottle seeds;
(2) fermentation of
Preparation of seed suspension: washing the lawn on the slope of the eggplant bottle with sterilized water to obtain a seed suspension;
inoculation: the seed suspension is inoculated to a fermentation tank culture medium by a pressure difference through an inoculation port;
③ fermenting liquid: after inoculation, culturing the fermentation tank culture medium for 36-48 h; the tank for the culture medium of the fermentation tankThe temperature is 25-32 ℃, and the pot pressure is 0.5kg/cm2The stirring speed in the tank is 200r/min, and the tank pressure is not more than 1.5kg/cm in the process of inoculation pressure increase2。
Further, the formula and the processing method of the NA culture medium are as follows: 0g parts of glucose l, 5 parts of peptone, 3 parts of beef extract, 1 part of yeast extract and 17 parts of agar powder, wherein the volume of the yeast extract is 1000mL, and the pH value is adjusted to 7.0.
Further, the formula and the processing method of the fermentation tank culture medium are as follows: the fermentation medium contained 5% peptone, 10% corn flour, 20% soybean flour, 2% yeast extract, 20% sucrose, 3% dipotassium hydrogen phosphate, and 10% glycerol, the pH was adjusted to 7.0 before sterilization, and sterilization was carried out at 121 ℃ for 30 min.
Further, the using method of the lysobacter enzymogenes strain BN3-15 aqueous solution comprises spraying, root irrigation and seed soaking.
Further, the preparation method of the lysobacter enzymogenes strain BN3-15 coating agent comprises the following steps:
(1) inoculating lysobacter enzymogenes into KB liquid culture solution, performing shake-flask fermentation culture at 28 deg.C and 160r/min for 48h to obtain fermentation solution with initial lysobacter enzymogenes content of not less than 1 × 109cfu/mL；
(2) Mixing polyvinyl alcohol with a chitosan mixture, OP-10, sodium lignosulfonate, xanthan gum, glycerol and eosin, uniformly stirring, then putting into a grinding machine for grinding for 4-8 hours until the average particle size of the materials is less than 5 microns, and preparing into an auxiliary agent;
(3) mixing the zymogenic lysobacter zymogenes obtained in the step (1) and the auxiliary agent obtained in the step (2) according to the volume ratio of 1: 99 to prepare the biological seed coating agent.
The invention also aims to provide application of the lysobacter enzymogenes BN3-15 as a growth promoter in promoting growth of cruciferous vegetables, corns and pseudo-ginseng.
By combining all the technical schemes, the invention has the advantages and positive effects that: the lysobacter enzymogenes BN3-15 provided by the invention is safe, has no residue, and has the effects of preventing diseases and promoting growth, and the strain is separated from rhizosphere soil of corn in the Dai autonomous state of the Xishuangbanna, Yunnan province; the lysobacter enzymogenes strain BN3-15 is prepared into water aqua, wettable powder or seed coating agent, and can be used for preventing and treating diseases such as corn leaf blight, pepper anthracnose, Chinese cabbage clubroot, and radix Notoginseng root rot in fields on corn, cruciferous crops, radix Notoginseng, etc.
The invention provides a biocontrol microbial inoculum which is efficient, nontoxic and residue-free, is convenient to use and can prevent and treat soil-borne diseases such as root rot, clubroot of cruciferous vegetables and the like, the effective active ingredients of the biocontrol microbial inoculum are lysobacter enzymogenes BN3-15 and secondary metabolites thereof, antibacterial active substances produced by the biocontrol microbial inoculum can degrade the cell wall structure of pathogenic bacteria, and the biocontrol microbial inoculum has strong inhibiting effect on pathogenic fungi causing diseases such as corn northern leaf blight, corn ear rot, pepper anthracnose, pseudo-ginseng root rot and the like. Therefore, the compound has the development and utilization prospect as a biological control preparation.
The invention provides a biological preparation which takes Lysobacter enzymogenes (Lysobacter enzymogenes) BN3-15 strain and metabolites thereof as main active effective components, the biocontrol strain has better inhibition effect on various pathogenic fungi, and the inhibition effect on pathogenic fungi such as corn northern leaf blight, corn ear rot, pepper anthracnose, panax notoginseng rust spot, panax notoginseng root rot and the like can reach 46.15-68.60%. Lysobacter enzymogenes (Lysobacter enzymogenes) BN3-15 can degrade the cell wall of pathogenic bacteria and inhibit the spore germination and the extension of the germ tube of the pathogenic bacteria by generating secondary metabolites, such as bacteriostatic active substances of glucanase, protease, chitinase, cellulase, heat-stable antifungal factor (HSAF) and the like, thereby influencing the metabolic pathway of the pathogenic bacteria and further realizing high-efficiency biocontrol effect. The biological preparation prepared by the strain has the advantages of wide antibacterial spectrum, outstanding antibacterial ability, strong colonization ability and the like, has the characteristics of high efficiency, no toxicity, safety, no residue, better disease prevention and growth promotion effects and easy industrial production, and can be applied in various modes such as root irrigation, spraying, coating and the like. The experiments of preventing and promoting the growth of the ground greenhouse and the field of Wen-greenhouse and field of Hunan Dian, Denghua and Delhong, etc. of Yunnan agricultural university show that the biological agent has obvious preventing effect and stable effect, can prevent the clubroot of cruciferous vegetables by 50.1-77.99 percent, and can prevent and control the corn northern leaf blight by 40.42 percent. And has good growth promoting effect on cruciferous vegetables, corn and pseudo-ginseng.
The biological agent created by the invention has been produced by cooperating with a plurality of microorganism bacterium agent production enterprises in Yunnan province, is used for jointly researching and developing new products such as seed coating agents, biological organic fertilizers and the like, is widely used for preventing and controlling fungal diseases of crops in fields, reduces chemical agents and the using amount and residual quantity of chemical fertilizers in the fields, and obtains remarkable ecological and economic benefits.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments of the present invention will be briefly described below, and it is obvious that the drawings described below are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a flow chart of a preparation method of an aqueous solution of lysobacter enzymogenes BN3-15 provided by the embodiment of the invention.
FIG. 2 is a flow chart of a preparation method of a coating agent of lysobacter enzymogenes strain BN3-15 provided in the example of the invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Aiming at the problems in the prior art, the invention provides lysobacter enzymogenes and application thereof, and the invention is described in detail with reference to the accompanying drawings.
The lysobacter enzymogenes provided by the embodiment of the invention is a lysobacter enzymogenes BN3-15 strain, the strain is separated from rhizosphere soil of fresh corn in Dai nationality of the Xishuangbanna, Yunnan province, and the strain is classified and named as the lysobacter enzymogenes, the lysobacter enzymogenes BN3-15 is preserved in the common microorganism center of the culture preservation management committee of Chinese microorganisms, and the preservation numbers are as follows: CGMCC No. 21259.
The lysobacter enzymogenes BN3-15 provided by the embodiment of the invention is highly viscous, creamy in color, capable of sliding, free of flagella, rod-shaped in thallus, round in colony and smooth in edge.
As shown in fig. 1, the preparation method of the lysobacter enzymogenes BN3-15 aqueous solution provided by the embodiment of the present invention includes:
s101, rejuvenation culture: sterilizing an NA culture medium at 121 ℃ for 30min, adding the NA culture medium into a test tube, making an inclined plane, inoculating a strain, and culturing for 24-48 h at 26 ℃ in a constant-temperature incubator;
s102, strain shake cultivation: inoculating the slant seeds on a slant NA culture medium of an eggplant bottle, and culturing for 24-48 h at 26 ℃ to obtain eggplant bottle seeds;
s103, preparing a seed suspension: washing the lawn on the slope of the eggplant bottle with sterilized water to obtain a seed suspension;
s104, inoculation: the seed suspension is inoculated to a fermentation tank culture medium by a pressure difference through an inoculation port;
s105, liquid fermentation: after inoculation, culturing the fermentation tank culture medium for 36-48 h; the tank temperature of the culture medium of the fermentation tank is 25-32 ℃, and the tank pressure is 0.5kg/cm2The stirring speed in the tank is 200r/min, and the tank pressure is not more than 1.5kg/cm in the process of inoculation pressure increase2。
As shown in FIG. 2, the preparation method of the lysobacter enzymogenes strain BN3-15 coating agent provided by the embodiment of the invention comprises the following steps:
s201, inoculating lysobacter enzymogenes to KB liquid culture solution, performing shake-flask fermentation culture at 28 ℃ and 160r/min for 48h to prepare fermentation solution, wherein the content of the lysobacter enzymogenes initially is not less than 1 × 109cfu/mL；
S202, mixing the mixture of polyvinyl alcohol and chitosan, OP-10, sodium lignosulfonate, xanthan gum, glycerol and eosin, uniformly stirring, then putting the mixture into a grinding machine for grinding for 4-8 hours until the average particle size of the materials is less than 5 microns, and preparing into an auxiliary agent;
s203, mixing the zymogen lysobacter zymogenes obtained in the step S201 and the auxiliary agent obtained in the step S202 according to the volume ratio of 1: 99 to prepare the biological seed coating agent.
The technical solution of the present invention will be further described with reference to the following examples.
Example 1: preparation of Lysobacter enzymogenes (Lysobacter enzymogenes) BN3-15 aqueous solution
(1) The strain source is as follows: lysobacter enzymogenes (BN 3-15) is separated from rhizosphere soil of corn in Dai autonomous State of Western Banna, Yunnan province.
(2) Strain culture
a. Culturing test tube species (the following are all in weight percent):
placing the NA culture medium into a test tube, making a slant, inoculating lysobacter enzymogenes BN3-15 strain, and culturing at 26 ℃ for 24 h. The preparation and sterilization method of the NA culture medium comprises the following steps: 5g of peptone, 0g g of sucrose, lg of yeast powder, 3g of beef extract and 17-20g of agar, supplementing the mixture to 1000mL by using distilled water, adjusting the pH value to 7.0, and sterilizing at 121 ℃ for 30 min.
b. And (3) expanding culture of strains by using a shaking table:
inoculating the seeds obtained in the above steps to eggplant bottle slant culture medium (the culture medium is NA culture medium, and the formula is the same as that of the test tube seed culture), and culturing at 26 deg.C for 48h to obtain eggplant bottle seeds.
(3) Fermentation:
the specific fermentation production process is as follows:
a. preparation of seed suspension: washing the lawn on the slant of eggplant bottle with sterilized water to obtain seed suspension.
b. Inoculation: the seed suspension was inoculated through an inoculation port into a fermentation tank by using a pressure difference (fermentation medium: 5% peptone, 10% corn flour, 20% soybean flour, 2% yeast extract, 20% sucrose, 3% dipotassium hydrogenphosphate, 10% glycerol, pH was adjusted to 7.0 before sterilization, and sterilization was carried out at 121 ℃ for 30 min). The culture temperature of the fermentation tank is 25-32 deg.C, and the tank pressure is 0.5kg/cm2The stirring speed in the tank is 200r/min, and attention is paid to avoid pollution; the pot pressure can not exceed 1.5kg/cm during pressure increase2。
c. Liquid fermentation: after inoculation, the fermentation tank is cultured for 36h until the number of bacteria is not increased any more, the fermentation tank is immediately placed, and the bacteria content is determined by plate counting and reaches 180 hundred million CFU/mL.
Example 2: preparation of wettable powder of Lysobacter enzymogenes (Lysobacter enzymogenes) BN3-15
(1) Preparing a liquid biocontrol microbial inoculum: obtaining fermentation liquor in the same way as in example 1;
(2) adding a filling agent: pressing the fermentation liquid into a storage tank, adding diatomite (light calcium carbonate) as filler according to the following calculation formula, and stirring for 30 min;
the amount of the filler added (kg) is [ number of fermentation broth bacteria (hundred million/ml) × volume of tank (liter) × yield ]/number of finished product bacteria-the residue (kg) in the fermentation broth. Then, the wettable powder is prepared by plate-and-frame filter pressing, pulping, drying and crushing, and is used for field application.
(3) Plate-frame filtration: using 2kg/cm2The pressure of the filter cake is adjusted at any time, the bacteria content in the filtrate is not more than 0.2 hundred million/ml, and a filter cake and the filtrate are obtained;
(4) pulping: discharging the filter cake into a pulping tank, adding concentrated milk No. 100 according to 8% of the calcium carbonate, or adding SDS according to 3%, adding appropriate amount of filtrate, and stirring for 30 min;
(5) and (3) drying: placing the slurry in a drying room below 60 ℃ for ventilation drying until the water content is 5% -8%, and obtaining a semi-finished product;
(6) crushing: crushing the semi-finished product, wherein the discharging temperature is less than or equal to 60 ℃ during crushing;
(7) and (3) measuring the quality index of the finished product: the determination of the bacteria content, the water content, the suspension percentage and the fineness is carried out according to the national enterprise standard (Q/KWL02-2003), the bacteria content is 18 hundred million CFU/g, and other indexes all meet the standard.
Example 3: ex vivo bacteriostasis experiment of Lysobacter enzymogenes (Lysobacter enzymogenes) BN3-15 on pathogenic fungi of corn leaf blight, corn ear rot, pepper anthracnose and pseudo-ginseng root rot
1.1 test Medium
NA culture medium (peptone 5g, beef extract 3g, yeast extract 1g, sucrose 10g, agar 17g, water 1000ml, pH 7.0); PDA medium (potato 200g, glucose 15g, agar 17g, water 1000ml, pH 7.0).
1.2 test strains: lysobacter enzymogenes (Lysobacter enzymogenes) BN 3-15; test pathogenic fungi: northern leaf blight (Exserohilum turcicum) HXMY13, maize head rot (Fusarium moniliforme) SF, Colletotrichum gloeosporioides (Colletotrichum acratum) L2-18, Panax notoginseng root rot (Fusarium oxysporum) SLJD2, Panax notoginseng rust (Cylindrocarpon destructors) RS006, Panax notoginseng root rot (Fusarium solani) PN 21. Both provided by national engineering center for biological diversity and disease control of Yunnan agricultural university bacteria research laboratory.
1.3 Experimental methods
Activation of biocontrol bacteria and pathogenic fungi: and (3) taking 20ul of the obtained lysobacter enzymogenes out of the preservation tube, adding the lysobacter enzymogenes into the NA culture medium, streaking by using an inoculating needle plate after the lysobacter enzymogenes grows to 2-3d, and purifying for later use. Putting a small block of pathogenic fungi into a PDA culture medium, picking fresh pollution-free hyphae after hyphae grow out, and inoculating the hyphae on a new culture medium for later use.
The determination method comprises the following steps: determination of the bacteriostatic action of pathogenic fungi: inoculating a pathogenic fungus plate (diameter of 5mm) at the center of the PDA plate, inoculating BN3-15 strain at 2.5cm positions on two sides, placing in an incubator at 28 ℃, and observing the bacteriostasis condition after 7 days.
The inhibition growth rate (%) (control colony radius-treated colony radius)/control colony radius 100.
1.4 test results and analysis
The test results are shown in Table 1
TABLE 1 bacteriostatic effect of lysobacter enzymogenes BN3-15 strain on different pathogenic fungi
Note: the average colony diameter was observed at 7d after inoculation.
As can be seen from Table 1, lysobacter enzymogenes BN3-15 has a good antagonistic effect against 6 pathogenic fungi, and has a growth inhibition rate of over 45% for the pathogenic fungi of northern leaf blight and ear rot, anthracnose of capsicum, root rot of panax notoginseng and rust of panax notoginseng (Exserohilum turcum) HXMY13, Fusarium graminearum (Fusarium moniliforme) SF, Fusarium oxysporum (Colletotrichum acutum) L2-18, Fusarium oxysporum (Fusarium oxysporum) SLJD2, Fusarium solani PN21 and Cylindrocarpon destructor (Cylindrocarpans) RS006, with a growth inhibition rate of over 68.60% for northern leaf blight.
Example 4: disease control and growth promotion experiment of Lysobacter enzymogenes (Lysobacter enzymogenes) BN3-15 on cruciferous vegetable clubroot disease
1. Experimental Material
1.1 test microbial inoculum: lysobacter enzymogenes BN3-15 biocontrol microbial inoculum;
1.2 test medium: KB liquid medium: peptone 20g, glycerol 10ml, dipotassium hydrogen phosphate 1.5g, magnesium sulfate heptahydrate 1.5g, distilled water 1000ml, pH 7.0, 121 ℃ sterilization 15min for standby.
1.3 test article: the cabbage variety 83-1 (Luchunbai No. 1) is infected with clubroot of cruciferous vegetables;
1.4 control subjects: clubroot of cruciferous vegetables;
1.5 test sites: greenhouse of plant protection institute of Yunnan agricultural university (Chinese cabbage variety 83-1 (Luchunbai No. 1) is planted in a seedling raising pot of 30cm × 15cm, 10 plants are planted in each pot, sterile culture soil for seeding is used, the volume ratio is prepared according to the common soil, turf, vermiculite and perlite, namely 1: 2: 1: 1, (90 × 210mm) culture pots are adopted, 5 holes are formed in each pot, 3 seeds are formed in each hole, experimental treatment is carried out, namely, zymogen bacillus subtilis BN3-15 biocontrol microbial inoculum, chemical pesticide cyazofamid and clear water inoculation treatment are used as positive and negative controls, and 3 repetitions are set in each treatment)
2. Experimental methods
2.1 preparation of a suspension of pathogenic spores
Crushing the clubroot tissue of Chinese cabbage, filtering with 8 layers of gauze, centrifuging for 5min at 2000rpm, discarding the supernatant, suspending with distilled water, centrifuging for 10min at 4000rpm, and repeating for 2 times. The supernatant was discarded, the precipitate was suspended in distilled water, and the spore concentration of the suspension was adjusted to 4X 107Pieces/g, stored at 4 ℃ for later use.
2.2 preparation of the biocontrol microbial inoculum
Activating strain BN3-15 on NA culture medium, performing inverted culture in 28 deg.C bacteria incubator for 48 hr, inoculating in liquid KB culture medium, and shaking at 160rpmCulturing for 3d, adjusting OD to 0.5 (concentration of 10) at 600nm with spectrophotometer7-108CFU/mL) for use.
2.3 inoculation method
Selecting Chinese cabbage with consistent growth after seedling emergence, and mixing the prepared clubroot spore suspension (concentration of 4 × 10)7Per gram) root irrigation is carried out on the white cabbage, the using amount of the plasmodiophora brassicae spore suspension inoculated in each pot is 10mL, and the biocontrol microbial inoculum (the concentration is 3 multiplied by 10) of lysobacter enzymogenes BN3-15 is used after the pathogenic bacteria are inoculated for 24 hours8CFU/mL), the dosage of the biocontrol microbial inoculum in each basin is 20mL, and the biocontrol microbial inoculum is applied for 2-3 times at the 7 th and 14 th days after the biocontrol microbial inoculum is used for root irrigation for the first time. Investigation is carried out 40 days after sowing, the disease condition of clubroot and the physiological indexes of plants are recorded, and the result is calculated and analyzed.
2.4 data investigation
And measuring the character indexes of the Chinese cabbage such as the plant height, the leaf number, the leaf area and the like 40 days after sowing, recording the occurrence condition of clubroot of the Chinese cabbage, and calculating the disease index and the relative prevention effect.
The grading standard of the clubroot disease of the Chinese cabbage is as follows: 0: the root has no tumor, and the root system develops normally; level 1: small tumors were found in lateral roots; and 3, level: root enlargement, the diameter of which is less than 2 times of the base of the stem; and 5, stage: the main root is swollen, and the diameter of the main root is 2 to 3 times of the base of the stem; and 7, stage: the main root is swollen, and the diameter of the main root is 3 to 4 times of the base of the stem; and 9, stage: the main root is swollen, and the diameter of the root is more than 4 times of the base of the stem or the swollen root is blackened.
The disease index [ ∑ (number of disease-onset-level representative values × number of disease-onset-level plants)/(number of survey total plants × highest-level disease-onset representative value) ] × 100%
Relative control (%) (control disease index-treatment disease index)/control disease index) x 100%.
2.5 Experimental results and analysis
TABLE 2 Lysobacter enzymogenes (Lysobacter enzymogenes) BN3-15 Effect on controlling clubroot disease of cabbage
From the results in table 2, it can be seen that: the bacillus alcaligenes BN3-15 fungicide has better prevention and treatment effect on the clubroot of the Chinese cabbage, the disease indexes of the clubroot treated by the bacillus alcaligenes BN3-15 fungicide are lower than those of chemical pesticide cyazofamid treatment and sterile water treatment, and the prevention and treatment effect on the clubroot is as high as 74.99 percent and higher than that of cyazofamid treatment (66.67 percent).
TABLE 3 growth promoting effect of Lysobacter enzymogenes (Lysobacter enzymogenes) BN3-15 on Chinese cabbage
Table 3 shows that the growth promoting effect of BN3-15 on Chinese cabbage is obvious. The plant height, the leaf number and the leaf area of the treated root irrigated by the BN3-15 are all higher than those of chemical pesticide cyazofamid treatment and sterile water treatment, which shows that the BN3-15 can well promote the growth of the Chinese cabbage and increase the plant height, the leaf number and the leaf area of the Chinese cabbage.
Example 5: lysobacter enzymogenes (Lysobacter enzymogenes) BN3-15 fresh corn seed coating field disease control and growth promotion experiment
1. Experimental setup
1.1 test article: fresh corn (two-color star);
1.2 control subjects: northern leaf blight of corn;
1.3 sites of experiment: a fresh corn planting base in Miscanthus sinensis, Delhong, Yunnan province;
2. experimental methods
2.1 preparation of the biocontrol microbial inoculum: the same as 2.2 in example 4.
2.2 preparation of coating agent: dissolving 4g of polyvinyl alcohol in 96mL of distilled water to prepare a 4% polyvinyl alcohol solution; 4g of chitosan is dissolved in 96mL of distilled water to prepare a 4% chitosan solution; 2g of xanthan gum was dissolved in 98mL of distilled water to prepare a 2% xanthan gum solution. According to a weight ratio of 4% polyvinyl alcohol: 4% of chitosan: 2% xanthan gum ═ 4: 1: 1 to prepare a mixed solution, and standing overnight for dissolving.
2.3 coating of the biological seed coating agent: selecting full corn seeds, soaking the corn seeds in clear water for 5-10min, taking out the corn seeds, removing redundant water, and then mixing the corn seeds and the biological seed coating agent according to the volume ratio of 100: 1, uniformly mixing in a container, coating according to the GB15671 standard (the surface area of the medicament-coated seeds is more than or equal to 80 percent), and airing to be completely dry for later use.
2.4 Experimental treatment: fresh corn seeds are coated by using lysobacter enzymogenes BN3-15 microbial inoculum, the shenqinmycin coating treatment is used as a positive control, and the uncoated treatment (CK) of the corn seeds is used as a negative control. The experiment has 3 treatments, each treatment is repeated for 3 times, 9 cells are totally, and 1m protection rows are arranged around the cells. Each cell area is 14m2Planting 4 rows of corns in each cell, wherein the row spacing is 0.45m, the plant spacing is 0.35m, removing redundant seedlings after seedling emergence, and reserving 2 plants in each hole, wherein the number of the plants is about 80.
2.5 data investigation
After the fresh-eating corn is planted, the emergence conditions of the emergence rate, the plant height, the stem thickness and the corn northern leaf blight of each treatment are investigated at the seedling stage and the heading stage respectively. Carrying out yield investigation in the mature period of the corn;
the investigation finds that the main disease of fresh corn in mango market is corn northern leaf blight, and the classification standard is as follows:
level 0: the whole leaf has no disease spots;
level 1: a small amount of disease spots are formed on the whole leaf, and the area of the disease spots is less than or equal to 5 percent;
and 3, level: a small amount of disease spots are formed on the whole leaf, the area of the whole leaf is 6-10%, and sporadic disease spots are formed on the upper leaf of the spike position;
and 5, stage: the disease spots on the whole leaf plant are more, the whole leaf plant occupies 11-30% of the leaf area, and the upper leaf of the spike position has more disease spots;
and 7, stage: the whole plant leaves have a large number of disease spots which are connected and occupy 31-70% of the leaf area, and the lower diseased leaves are withered;
and 9, stage: the whole plant leaves are basically covered by scabs, and the leaves die;
incidence (%) — 100% of diseased plants/total plants;
the disease index is [ ∑ (disease number of all levels of disease levels)/(highest disease value of total number of plants investigated) ] 100%;
the control effect (%) [ (control disease index-treatment disease index)/control disease index ] × 100%.
3. Results of the experiment
TABLE 4 prevention and control effect of fresh corn coated with biocontrol bacterium BN3-15 on corn northern leaf blight
As can be seen from Table 4, the control effect on the northern leaf blight of fresh corn after being coated by the BN3-15 biological type seed coating agent is 40.42 percent, and the effect is better than that of 8.98 percent of that of the shenqinmycin coating treatment. The Control (CK) and shenqinmycin-coated corn disease incidence and disease index were higher than those of the BN3-15 coating agent. The bacterial strain BN3-15 has better disease control effect on the corn northern leaf blight after coating treatment of fresh corn seeds.
TABLE 5 biocontrol bacterium BN3-15 coating treatment for promoting growth of fresh corn
As can be seen from Table 5, the rate of emergence was 80.28% for the treatment with BN3-15 coating, which is significantly higher than the treatments with Control (CK) and shenqinmycin coatings. The plant height and stem thickness of the BN3-15 coated seedlings and the plant height and stem thickness of the panicle heading seedlings are higher than those of the other two treatments, which shows that the BN3-15 coated seedlings have a good growth promoting effect on the growth of the corns.
TABLE 6 Effect of biocontrol bacteria BN3-15 coating treatment on field yield of fresh corn
The influence of the biocontrol bacterium BN3-15 coating treatment on the field yield of fresh-eating corns is shown in Table 6, the yield of the field-eating corns is 1098kg per mu by using the lysobacter enzymogenes BN3-15 coating treatment, which is higher than that of the field-eating corns by the Control (CK) and the shenqimycin coating treatment, and the yield of the field-eating corns per mu is increased by 22.71 percent compared with that of the field-eating corns by the control treatment. The yield increasing effect of the zymogenic lysobacter BN3-15 on fresh corn is obvious after coating.
Example 6: growth-promoting field experiment of Lysobacter enzymogenes (Lysobacter enzymogenes) BN3-15 microbial inoculum on pseudo-ginseng
1. Experimental Material
1.1 test reagents: lysobacter enzymogenes (Lysobacter enzymogenes) BN 3-15;
1.2 test article: annual radix Notoginseng;
1.3 sites of experiment: a large river bridge rural big base in Hongdan county of Qudian city, Yunnan province;
2. experimental methods
2.1 preparation of the biocontrol microbial inoculum: the same as 2.2 in example 4;
2.2 Experimental treatment: 3 treatments are set, and the treatments are sequentially BN 3-15: irrigating roots with zymogen lysobacter BN 3-15; backer and super: diluting the agent by 1000 times by using a mountain super agent and irrigating roots; CK: irrigating roots with clear water; the cells were divided into cells 1.5m long by 1.2m wide using disposable chopsticks and string, 3 replicates of each treatment. Irrigating roots once every 14-15 days, and continuously treating for 3 times;
2.3 data investigation: after 90 days of the last root irrigation, 10 panax notoginseng plants (10 × 3 or 30 panax notoginseng plants treated) are taken out from each plot at random and taken back to a laboratory, and indexes such as plant height, root length, fresh weight, dry weight and the like of the panax notoginseng plants in each treatment are measured.
3. Results of the experiment
TABLE 7 post-emergence measurement of physiological indexes of panax notoginseng treated for one year
The results of the physiological indexes of the treated panax notoginseng such as plant height, leaf number, root length, fresh root tuber and the like are shown in table 7. The result shows that the indexes of plant height, leaf number, root length, root tuber fresh weight and the like of the pseudo-ginseng treated by root irrigation with BN3-15 are all higher than those of the pseudo-ginseng treated by Control (CK) and the root-bark treated by Mount-Bac super, which shows that the growth promotion effect of the pseudo-ginseng treated by root irrigation with lysobacter enzymogenes BN3-15 is obvious.
The above description is only for the purpose of illustrating the present invention and the appended claims are not to be construed as limiting the scope of the invention, which is intended to cover all modifications, equivalents and improvements that are within the spirit and scope of the invention as defined by the appended claims.
Claims (9)
1. The lysobacter enzymogenes is characterized in that the lysobacter enzymogenes is a lysobacter enzymogenes BN3-15 strain, and the lysobacter enzymogenes BN3-15 strain has the preservation number of: CGMCC No.21259, deposited in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms.
2. Use of lysobacter enzymogenes BN3-15 as claimed in claim 1 for field disease control including northern leaf blight, pepper anthracnose, crucifer crop clubroot and notoginseng root rot.
3. The use according to claim 2, wherein the lysobacter enzymogenes BN3-15 is used in the form of any one of an aqueous solution, a wettable powder or a seed coating agent.
4. The use of claim 3, wherein the aqueous solution of lysobacter enzymogenes strain BN3-15 is used by spraying, root irrigation and seed soaking.
5. The use of claim 3, wherein the method for preparing the aqueous solution of lysobacter enzymogenes BN3-15 comprises:
(1) strain culture
Rejuvenation culture: sterilizing an NA culture medium at 121 ℃ for 30min, adding the NA culture medium into a test tube, making an inclined plane, inoculating a strain, and culturing for 24-48 h at 26 ℃ in a constant-temperature incubator;
expansion culture of strains by using a shaking table: inoculating the slant seeds on a slant NA culture medium of an eggplant bottle, and culturing for 24-48 h at 26 ℃ to obtain eggplant bottle seeds;
(2) fermentation of
Preparation of seed suspension: washing the lawn on the slope of the eggplant bottle with sterilized water to obtain a seed suspension;
inoculation: the seed suspension is inoculated to a fermentation tank culture medium by a pressure difference through an inoculation port;
③ fermenting liquid: after inoculation, culturing the fermentation tank culture medium for 36-48 h; the tank temperature of the culture medium of the fermentation tank is 25-32 ℃, and the tank pressure is 0.5kg/cm2The stirring speed in the tank is 200r/min, and the tank pressure is not more than 1.5kg/cm in the process of inoculation pressure increase2。
6. The use of claim 5, wherein the NA medium is formulated and processed by: 0g parts of glucose l, 5 parts of peptone, 3 parts of beef extract, 1 part of yeast extract and 17 parts of agar powder, wherein the volume of the yeast extract is 1000mL, and the pH value is adjusted to 7.0.
7. The use of claim 5, wherein the formulation and processing method of the fermenter medium is: the fermentation medium contained 5% peptone, 10% corn flour, 20% soybean flour, 2% yeast extract, 20% sucrose, 3% dipotassium hydrogen phosphate, and 10% glycerol, the pH was adjusted to 7.0 before sterilization, and sterilization was carried out at 121 ℃ for 30 min.
8. The application of the lysobacter enzymogenes BN3-15 in field disease control, which is disclosed in claim 5, is characterized in that the preparation method of the lysobacter enzymogenes strain BN3-15 coating agent comprises the following steps:
(1) inoculating lysobacter enzymogenes into KB liquid culture solution, performing shake-flask fermentation culture at 28 deg.C and 160r/min for 48h to obtain fermentation solution with initial lysobacter enzymogenes content of not less than 1 × 109cfu/mL；
(2) Mixing polyvinyl alcohol with a chitosan mixture, sodium lignosulfonate, xanthan gum, glycerol and eosin, uniformly stirring, then putting into a grinding machine for grinding for 4-8 hours until the average particle size of the materials is less than 5 micrometers, and preparing into an auxiliary agent;
(3) mixing the zymogenic lysobacter zymogenes obtained in the step (1) and the auxiliary agent obtained in the step (2) according to the volume ratio of 1: 99 to prepare the biological seed coating agent.
9. Use of the lysobacter enzymogenes according to claim 1 as a growth promoter for promoting growth of cruciferous vegetables, corn and pseudo-ginseng.
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| CN117487706B (en) * | 2023-11-02 | 2024-06-04 | 南京农业大学 | Antagonistic bacteria for preventing and controlling root rot of radix angelicae and biological organic fertilizer |
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| CN106434439A (en) * | 2016-09-21 | 2017-02-22 | 云南农业大学 | Lysobacter enzymogenes 1-T-1-4 and application thereof |
| CN107446847A (en) * | 2017-08-14 | 2017-12-08 | 云南农业大学 | One plant of Bei Laisi bacillus GT11 and its application |
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| CN106434439A (en) * | 2016-09-21 | 2017-02-22 | 云南农业大学 | Lysobacter enzymogenes 1-T-1-4 and application thereof |
| CN107446847A (en) * | 2017-08-14 | 2017-12-08 | 云南农业大学 | One plant of Bei Laisi bacillus GT11 and its application |
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