CN116396906A - Solid state fermentation product of non-pathogenic bacterial wilt and application thereof - Google Patents
Solid state fermentation product of non-pathogenic bacterial wilt and application thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- 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
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
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- A—HUMAN NECESSITIES
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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Abstract
The invention provides a solid state fermentation product of non-pathogenic bacterial wilt and application thereof, belonging to the technical field of microbial fermentation. The solid state fermentation product comprises 150-200 hundred million active bacteria per gram of non-pathogenic bacterial wilt bacteria, 25-29% of organic matter, 0.4-0.7% of total nitrogen, 125-140% of total phosphorus, 0.5-0.7% of total potassium, 12-14% of crude fiber, 17-18% of water and 5.5-6.0% of pH, wherein the contents are all mass percentages. The solid state fermentation product has remarkable prevention effect on solanaceous vegetable bacterial wilt of tomatoes, peppers, eggplants and the like, can solve the problems of complex production process and high production cost in the prior art, can realize the resource utilization of the jujun grass, and has higher commercial value.
Description
Technical Field
The invention belongs to the technical field of microbial fermentation, and particularly relates to a solid-state fermentation product of pathogenic-free bacterial wilt based on huge grass recycling and application of the solid-state fermentation product in solanaceous vegetables.
Background
Crop bacterial wilt caused by ralstonia solanacearum (ralstonia solanacearum for short) is a destructive soil-borne disease, and the disease is particularly serious in the planting process of solanacearum vegetables. Common control strategies include chemical pesticides, soil improvement, disease-resistant varieties, grafting, rotation and the like, but the effects are unstable. So far, few crop varieties with bacterial wilt resistance are available and the resistance is unstable; although the grafting method is successful in preventing and treating tomato bacterial wilt, the cost is too high, the technical difficulty is high, and the popularization and popularization are difficult; rotation can better prevent and treat bacterial wilt to a certain extent, but is influenced by geographical environment and is difficult to popularize in a large area; although the chemical pesticide has a certain prevention effect, with the increasing use amount of the chemical pesticide, the chemical pesticide is easy to generate drug resistance and pesticide residue, the small differentiation type is complex, and an ideal pesticide can prevent and treat bacterial wilt, and the factors limit the application of the chemical pesticide in actual production. The biological control has the advantages of no pollution, being beneficial to human and animal safety, being friendly to the environment, being not easy to generate resistance and the like, so that people gradually turn the eyes to the biological control of the bacterial wilt.
The development of plant vaccines for preventing and treating crop bacterial wilt by utilizing non-pathogenic bacterial wilt has important biocontrol application potential. In the early research and development, a non-pathogenic bacterial wilt strain FJAT-aRS01 (patent number ZL 201510589990.1) with high control efficiency and genetic stability is screened, and the control efficiency on tomato potted seedlings and field cells respectively reaches 100 percent and 85.71 percent. At present, the strain is used for producing pilot-scale products of bacterial wilt plant vaccines, and is applied to the places such as Fujian Pu field, fujian Xiamen, fujian Fuding, zhejianruian, hainan Ling water and the like in an demonstration way, and the prevention effect is over 75 percent. The bacterial wilt plant vaccine is applied to healthy breeding of the seedling of the solanaceae vegetables, so that the disease resistance of the seedling is greatly improved. The plant vaccine is added into products such as seedling culture matrix, cultivation matrix and the like, is used for improving soil, and can promote plant growth and reduce incidence rate of soil-borne diseases.
The biocontrol agent in the market at present mostly adopts a liquid submerged fermentation technology, which has high requirements on equipment and complex production process, while the raw materials adopted by solid fermentation are generally cheap agricultural and sideline products, and the adopted equipment is simpler than liquid fermentation, and the production cost is greatly lower than that of liquid fermentation. At present, the common solid fermentation raw materials are bran, rice bran, bean cake powder and the like, and the problems of increased viscosity, insufficient porosity, small gaps, difficult subsequent inoculation and the like are solved after high-temperature sterilization. In large-scale production, in order to ensure the sustainable production, the selection of fermentation raw materials with wide sources, rich nutrition and low price is a key for reducing the production cost. The jujun grass is a perennial gramineous plant, has strong tillering capability and adaptability, is a plant with highest yield in the gramineous, is known at present, has average annual yield per mu of 20-30t, and is a sustainable development resource. The pennisetum hydridum has high content of crude protein, is rich in lysine, amino acid, flavonoid, saponin, aromatic substances, surfactant and various organic salts, has the characteristics of wide source, large quantity and low price, and is a high-quality biocontrol bacterium solid fermentation raw material.
Disclosure of Invention
For this reason, it is necessary to provide a new agricultural byproduct, megaterium residues, as the main medium for solid state fermentation to produce the preventive agent.
In order to achieve the above object, the present inventors provide the following technical solutions:
a solid state fermentation of non-pathogenic bacterial wilt, said solid state fermentation comprising the nutritional ingredients of: the effective viable count of the non-pathogenic bacterial wilt bacteria is 150-200 hundred million/g, the organic matter content is 25-29%, the total nitrogen content is 0.4-0.7%, the total phosphorus content is 125-140%, the total potassium content is 0.5-0.7%, the crude fiber content is 12-14%, the water content is 17-18%, and the pH value is 5.5-6.0, and the above contents are all mass percent.
The preparation method of the solid state fermentation product comprises the following steps:
(1) Preparing seed liquid: inoculating non-pathogenic bacterial wilt into nutrient broth (NutrientBroth, NB) fermentation medium, and shake culturing at 28-32deg.C and 150-200rpm for 20-28 hr to obtain seed solution; the non-pathogenic bacterial wilt (Ralstoniasaranacorus) FJAT-aRS01 is preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) for 4 months and 7 days in 2015, and has an address of Hospital No. 3 of North Chen West Lu in the Korean region of Beijing city and a preservation number of CGMCC No.10693 (patent number ZL 201510589990.1);
(2) Solid state fermentation: adding seed liquid into solid fermentation medium according to 15-20% of inoculation amount, stirring, sealing, and fermenting at 28-32deg.C for 2-3d to obtain solid fermentation product of pathogenic-free ralstonia solanacearum; the preparation method of the solid state fermentation culture medium comprises the steps of adding water into 35-38% of pennisetum sinese residues, 25-30% of corn flour, 25-30% of rice bran and 8-10% of rice husk, uniformly stirring, filling the uniformly mixed materials into a polypropylene sterilization bag according to the mass ratio of 1 (0.7-1), sterilizing at 120-125 ℃ for 15-25min to obtain a sterile fermentation material, and cooling for later use.
Preferably, the solid state fermentation method of the non-pathogenic bacterial wilt comprises the following steps:
(1) Preparing seed liquid: inoculating non-pathogenic bacterial wilt FJAT-aRS01 into a nutrient broth fermentation culture medium, and carrying out shaking culture at 30 ℃ and 170rpm for 24 hours to obtain seed liquid;
(2) Solid state fermentation: weighing 36.36% of pennisetum sinese residues, 27.27% of corn flour, 27.27% of rice bran and 9.09% of rice husk according to the mass ratio, adding water and stirring uniformly, wherein the mass ratio of the water to the material is 1:0.8, filling the uniformly mixed materials into a polypropylene sterilization bag (150 g/bag), and sterilizing for 20min at 121 ℃ to obtain a sterile fermentation material; and (3) cooling the sterile fermentation material, adding seed liquid according to the mass ratio of 15% of the inoculum size, uniformly stirring, sealing by using a sterile edible fungus fruiting lantern ring, and fermenting and culturing at 30 ℃ for 48 hours to obtain the solid fermentation product of the non-pathogenic bacterial wilt FJAT-aRS 01.
Furthermore, the solid state fermentation product of the non-pathogenic bacterial wilt is applied to promoting the germination of the seeds of the solanaceous vegetables.
Furthermore, the solid fermentation product of the non-pathogenic bacterial wilt is applied to the prevention and treatment of the bacterial wilt of the solanaceous vegetables.
Furthermore, the solid state fermentation product of the non-pathogenic bacterial wilt is applied to promoting the growth of solanaceous plants.
Furthermore, the solid state fermentation product of the non-pathogenic bacterial wilt is applied to the preparation of the disease-resistant growth promoting agent for the solanaceous vegetables.
Further, the solanaceous vegetables include tomatoes, peppers or eggplants.
Furthermore, the application of the solid state fermentation product of the non-pathogenic bacterial wilt in promoting the growth of solanaceous plants is that the solid state fermentation product of the non-pathogenic bacterial wilt with the mass percent less than or equal to 15 percent is added into a culture medium. For tomatoes, the growth promoting effect is best when the addition amount is 5%; for the capsicum and eggplant, the addition amount is 10% and the growth promoting effect is best, and the weight percentages are all.
Furthermore, the application of the solid state fermentation product of the non-pathogenic bacterial wilt in promoting the germination of the solanaceous vegetable seeds is that the seed soaking treatment of the leaching solution of the solid state fermentation product of the non-pathogenic bacterial wilt diluted 50 times is adopted for promoting the germination of the tomato seeds; promoting pepper seed germination, and soaking the pepper seeds in 100 times diluted non-pathogenic bacterial wilt solid fermentation product leaching solution; the eggplant seed germination is promoted by adopting the leaching solution of the non-pathogenic bacterial wilt solid state fermentation product diluted 100 times for seed soaking treatment.
Furthermore, the application of the solid fermentation product of the non-pathogenic bacterial wilt in preparing the disease-resistant growth promoting agent for the solanaceous vegetables is that the solid fermentation product of the non-pathogenic bacterial wilt with the mass percent of 15-25% is added into a cultivation matrix. For tomatoes, the recommended addition of the non-pathogenic bacterial wilt solid state ferment is 15%; for capsicum and eggplant, the recommended addition amount of the non-pathogenic bacterial wilt solid state ferment is 25 percent, and the mass percentages are all.
The invention has the following advantages:
(1) The invention utilizes the agricultural by-product jujun grass as one of the components of the non-pathogenic bacterial wilt FJAT-aRS01 solid fermentation medium, reduces the production cost, simultaneously solves the pollution of the agricultural by-product waste to the environment, and realizes the sustainable development of agriculture;
(2) The non-pathogenic bacterial wilt solid state fermentation product can promote seed germination and plant growth of tomatoes, peppers and eggplants, has obvious bacterial wilt prevention effect on solanaceous vegetables such as tomatoes, peppers and eggplants, has the advantages of no pollution, environmental friendliness, difficult resistance generation and the like, and can be used for green production of the solanaceous vegetables for protecting the driving;
(3) The solid fermentation product of the non-pathogenic bacterial wilt has high viable bacteria content of 150-200 hundred million/g, and the content of the nutrient components exceeds the national standard (NY/T798-2015) of microbial fertilizer.
Drawings
FIG. 1 is a diagram showing the appearance of the solid state fermentation product of non-pathogenic bacterial wilt according to example 2.
FIG. 2 is a graph showing colony morphology of nonpathogenic bacterial wilt FJAT-aRS01 as described in example 2.
Detailed Description
In order to describe the technical content of the technical solution, the achieved objects and effects in detail, the following description is made with reference to the specific embodiments in conjunction with the accompanying drawings. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Example 1: solid state fermentation of avirulent bacterial wilt FJAT-aRS01
1. Preparation of culture medium
Nutrient broth (NutrientBroth, NB) medium: 5g of peptone, 3g of beef extract, 10g of sucrose, and distilled water to a volume of 1L, wherein the pH value is 7.2, and the beef extract is preserved at normal temperature for standby after high-pressure sterilization.
TTC (2, 3, 5-triphenyltetrazolium chloride) medium: glucose 0.5%, peptone 1.0%, hydrolyzed casein 0.1%, TTC 0.05% and agar 1.7%, pH 7.2, and storing at normal temperature after autoclaving.
2. Seed liquid preparation
Non-pathogenic bacterial wilt (Ralstoniasaranacorus) FJAT-aRS01, offered by the institute of agricultural biological resources, the national academy of agricultural sciences, fujian province. The strain is preserved in China general microbiological culture Collection center (China general microbiological culture Collection center) for 4 months and 7 days in 2015, and has the address of 1 st and 3 rd hospital of North West road in the Korea of Beijing city and the preservation number of 10693 (patent number ZL 201510589990.1).
The FJAT-aRS01 to be tested is activated in a TTC culture medium, cultured for 2 days at 30 ℃, single colony is selected in an NB culture medium, and shake culture is carried out for 24 hours at 30 ℃ in a shaking table at 170r/min, thus obtaining seed liquid of the non-pathogenic bacterial wilt FJAT-aRS 01.
(1) Screening a solid fermentation basic culture medium: mixing corn flour, bran, bean pulp, rice bran and other raw materials with the pennisetum sinese residue and rice husk (90% pennisetum residue and 10% rice husk) respectively according to a mass ratio of 4:6, adding water according to a mass ratio of 1:0.8, uniformly stirring, filling into a polypropylene sterilizing bag, sterilizing at 121 ℃ for 20min, cooling to normal temperature, inoculating seed liquid according to an inoculum size with a mass ratio of 15%, and fermenting for 2d at 30 ℃.10 g of solid state fermentation product is weighed and dissolved in 90mL of sterile water, after 10 times gradient dilution, the solid state fermentation product is coated on a TTC culture medium and cultured for 48 hours at 30 ℃, and the number of viable bacteria in the solid state fermentation product combined by different culture mediums is counted.
As shown in Table 1, the number of viable bacteria of FJAT-aRS01 of the treatment group consisting of pennisetum hydridum residue, rice husk and corn meal was 3.67×10 at the maximum 8 cfu/g; secondly, the live bacterial load of the treatment group FJAT-aRS01 is 2.60 multiplied by 10 8 cfu/g; the viable count of FJAT-aRS01 of the treatment group of the jujun grass slag, the rice hull, the bean pulp, the jujun grass slag, the rice hull and the bran is 1.29 multiplied by 10 respectively 7 cfu/g and 2.17X10 7 cfu/g。
TABLE 1FJAT-aRS01 solid state fermentation basal medium screening
(2) Orthogonal optimization of solid state fermentation media: according to the result of the main matrix, the megaterium grass residues, corn flour, rice bran,Rice husk is used as influencing factor, L9 (3 4 ) Orthogonal table four factor 3 horizontal orthogonal tests are carried out, the factor level setting is shown in table 2, the orthogonal test setting is shown in table 3, the sterilization, inoculation and fermentation methods of the orthogonal optimization culture medium are the same as the above, and the number of FJAT-aRS01 viable bacteria in different orthogonal optimization combination solid state fermented products is counted, so that the method is the same as the above.
TABLE 2 optimization of orthogonal test design factor level for solid fermentation Medium
TABLE 3 optimization of orthogonal test design for solid fermentation media
The results are shown in table 4, treatment level 2: culture medium of 40g of pennisetum sinese residue, 30g of corn meal, 30g of rice bran and 10g of rice husk, and the maximum number of viable bacteria cultivated is 1.01X10 9 cfu/g, treatment differences from the other formulations were very significant (P<0.01 A) is provided; the second is treatment level 1: FJAT-aRS01 viable count cultured by culture medium of 40g of pennisetum sinese residue, 15g of corn meal, 15g of rice bran and 5g of rice husk is 1.37X10 8 cfu/g; FJAT-aRS01 viable count of treatment group 5 was the lowest, and was 1.70X10 7 cfu/g. Therefore, the optimal culture medium formula for FJAT-aRS01 solid state fermentation comprises 40g of pennisetum sinese residue, 30g of corn meal, 30g of rice bran and 10g of rice husk.
TABLE 4 results of optimized orthogonal experiments on solid fermentation Medium
Note that: the same case of different capital letters indicates that the difference is extremely significant (P < 0.01), as follows.
(3) Optimizing solid state fermentation conditions: aiming at 4 influencing factors of inoculation quantity, water mass ratio, fermentation time and bagging quantity, taking the number of the non-pathogenic bacterial wilt FJAT-aRS01 viable bacteria in the fermentation material as an investigation index, adopting L9 (3) 4 ) Orthogonal table four factor 3 horizontal orthogonal test is carried out to optimize FJAT-aRS01 solid state fermentation conditions, the factor horizontal setting is shown in table 5, the orthogonal test setting is shown in table 6, the sterilization, inoculation and fermentation methods of the orthogonal optimization culture medium are the same as the above, and the number of FJAT-aRS01 viable bacteria in different orthogonal optimization combination solid state fermented products is counted, wherein the method is the same as the above.
TABLE 5 horizontal design of orthogonal test factors for optimizing solid state fermentation conditions
TABLE 6 optimization of solid state fermentation conditions orthogonal test design
The results are shown in table 7, treatment level 9: 15% of inoculation amount, 0.8:1 of water-material mass ratio, 150g of bagging amount and 2d of fermentation time, and the maximum number of FJAT-aRS01 viable bacteria is 3.31 multiplied by 10 9 cfu/g, and the treatment levels differed to very significant levels (P<0.01 A) is provided; the second is treatment level 5 and 7, under these 2 fermentation conditions, FJAT-aRS01 viable count is 5.7X10 respectively 8 cfu/g and 5.04×10 8 cfu/g; the treatment level is 6, the FJAT-aRS01 viable count is lowest and is 2.99X10 6 cfu/g. Therefore, the optimal fermentation conditions of FJAT-aRS01 solid state fermentation are 15% of inoculum size, 0.8% of water-material mass ratio, 150g of bagging amount and 2d of fermentation time.
TABLE 7 results of optimization of solid fermentation conditions orthogonal experiments
Example 2: quality detection of non-pathogenic bacterial wilt solid state ferment
1. Sensory index: color, texture, odor, etc.
As shown in FIG. 1, the non-pathogenic bacterial wilt solid state fermentation product is yellow brown, loose in texture, free of caking and has fermentation smell.
2. The technical indexes are as follows: detecting the number of thalli, measuring the germination index of seeds, measuring the bacterial wilt prevention effect, and measuring the plant growth and nutrient components.
(1) Cell number detection: randomly selecting 3 bags of solid state fermentation products, weighing 10g, dissolving in 90mL of sterile water, carrying out 10-time gradient dilution, coating on a TTC (time to temperature) culture medium, culturing at 30 ℃ for 48 hours, and counting the number of viable bacteria of the non-pathogenic bacterial wilt solid state fermentation products.
As shown in FIG. 2, the viable count of the 3-bag random-extracted FJAT-aRS01 solid state fermentation product samples is 2.02X10 respectively 9 cfu/g (FIG. 2A), 1.52X10 9 cfu/g (FIG. 2B) and 1.67×10 9 cfu/g (FIG. 2C).
(2) Seed germination index determination: taking tomatoes, peppers and eggplants as test materials, weighing 100g of non-pathogenic bacterial wilt solid fermentation products, slowly irrigating to saturation by sterile water, standing for 24 hours, wrapping by using 4 layers of gauze, extruding and filtering, and collecting filtrate to obtain leaching liquor. Diluting the leaching solution with sterile water to 5 times, 10 times, 20 times, 50 times, 80 times and 100 times respectively, and preserving at 4deg.C for use. The germination method on paper is adopted, tomato, capsicum and eggplant seeds are rinsed for 2 times with warm water at 55 ℃, fished out and drained, the seeds are soaked for 8 hours with leaching solutions with different dilution factors, and the sterile water soaking seeds are used as a Control (CK). After seed soaking, the seeds are transferred to a 9cm transparent culture dish with two layers of filter paper laid at the bottom, 15 seeds per culture dish are treated in 3 times and 7 times. Placing the culture dish in a 27 ℃ constant temperature artificial climate box, illuminating for 16 hours and darkness for 8 hours, tracking and observing the germination condition of the seeds, recording the germination number until no new germination particles appear in continuous 3 days, and measuring the germination rate, germination index and seed vitality index of the seeds.
Germination percentage (%) = (number of germinated seeds/number of tested seeds on specified days) ×100%
Germination Index (GI) =Σ (Gt/Dt), gt is the number of seed germination on day t, and Dt is the corresponding number of germination days.
Vitality Index (VI) =germination index×radicle length (cm)
The results of the seed germination test show that the germination rates of the tomato seeds treated by 50 times, 80 times and 100 times of the liquid extract of the solid fermentation product of the non-pathogenic bacterial wilt are 79.33 percent, 81.00 percent and 76.17 percent respectively, which are obviously higher than that of the control (72.67 percent) (P < 0.05), and the germination index (18.26) and the vitality index (133.69) of the tomato seeds treated by 50 times of the liquid extract are also obviously higher than that of the control (germination index 12.54 and seed vitality index 109.65). The germination rates of the pepper seeds treated by 80 times and 100 times of the leaching solution of the solid state fermentation product of the non-pathogenic bacterial wilt are 88.80 percent and 93.30 percent respectively, which are significantly higher than that of the control (82.93 percent), and the germination index (60.28) and the seed vitality index (420.67) of the pepper seeds treated by 100 times of the leaching solution are also significantly higher than that of CK (germination index 56.59 and seed vitality index 254.64). The germination rate (97.78%), germination index (14.10) and seed vitality index (59.93) of the eggplant treated with 100-fold dilution of the non-pathogenic bacterial wilt solid state fermentation product extract are significantly higher than those of the control (91.11%), germination index (11.45) and seed vitality index (36.88). Dilution of the non-pathogenic bacterial wilt solid fermentation product extract below 50 times inhibits germination of tomato, capsicum and eggplant seeds, and the germination rate, germination index and seed vitality index are all significantly lower than those of a sterile water control (P < 0.05, table 8).
TABLE 8 influence of non-pathogenic bacterial wilt solid state ferments on seed germination
Note that: the same case of different lower case letters indicates that the difference is significant (P < 0.05), as follows.
(3) And (3) determining the bacterial wilt prevention effect: bacterial wilt strong pathogenic strain FJAT-91 (FJAT-91 was deposited in 2015, 4, 07)The general microbiological center of the national microbiological bacterial conservation management committee, the address is No.1 and No. 3 of North West Lu 1 in the Korean area of Beijing city, the conservation number is CGMCC No. 10692) is activated on a TTC flat plate, transferred into an NB culture medium, cultured for 24 hours at the temperature of 30 ℃ at 170r/min, and the bacterial liquid is diluted to 10 8 CFU/mL. Different amounts of non-pathogenic bacterial wilt solid state fermentation products (0, 5%, 10%, 15%, 20% and 25%) are added into the culture medium, and after fully and uniformly mixing, tomato seedlings, pepper seedlings and eggplants are transplanted into pot bodies (the diameter of the pot bodies is 15cm and the height of the pot bodies is 10 cm) respectively, wherein the pot bodies are 500 g/pot. After 3d transplanting, the root is irrigated with bacterial liquid of FJAT-91, (inoculation concentration is 1 multiplied by 10) 8 CFU/mL), inoculum size was 100 mL/basin, 3 replicates per 15 basin treatment. And observing the morbidity of the plants every day after inoculation, and counting the morbidity and the prevention and treatment effect of the bacterial wilt.
Morbidity (%) = (number of diseased plants/total number of plants) ×100%
Control (%) = (treatment group morbidity-control group morbidity)/control morbidity x 100%
The bacterial wilt prevention test result shows that the tomato, the capsicum and the eggplant control group respectively begin to attack after being inoculated with bacterial wilt strong pathogenic strain FJAT-91, and the attack rate of the plants of 6d, 8d and 12d reaches 100 percent after being inoculated with bacterial wilt strong pathogenic strain FJAT-91, and the attack rate of the plants of 18d, 22d and 28d reaches the same value. In each experimental group of tomatoes, peppers and eggplants, the incidence rate of bacterial wilt in a treatment group with 25% of the addition amount of the non-pathogenic bacterial wilt solid fermentation product is the lowest, and the prevention effect is the best; the additive amount treatment has the bacterial wilt prevention effect on tomatoes, peppers and eggplants of 85.33%, 75.67% and 80.11% respectively (Table 9).
TABLE 9 prevention and treatment effects of non-pathogenic bacterial wilt solid state fermentation products on tomato, capsicum and eggplant bacterial wilt
(4) Plant growth assay: different amounts of non-pathogenic bacterial wilt solid state fermentation products (0, 5%, 10%, 15%, 20% and 25%) are added into the culture medium, the mixture is fully and uniformly mixed, the mixture is put into a pot (15 cm multiplied by 10 cm), 500 g/pot is put into the pot, tomatoes and seedlings with consistent growth vigor are transplanted into the pot respectively, 4 plants/pot are transplanted into the pot, and 3 repetition is set. The potted seedling is transferred to a artificial climate chamber for culture at the temperature of 30+/-1 ℃ and the relative humidity of 90 percent. After 20d of cultivation, 15 plants were randomly selected to measure the plant height and stem thickness of the plants.
The growth promoting test result shows that the addition amount of the non-pathogenic bacterial wilt solid state ferment is less than or equal to 15 percent, and the growth of tomato, capsicum and eggplant plants can be promoted. For tomatoes, the best growth promoting effect is achieved when the addition amount is 5%, the plant height and the stem thickness of the tomatoes are increased by 29.62% and 25.77% respectively compared with a control, and the addition amount is 25% and the plant growth is obviously inhibited. For capsicum and eggplant, the addition amount of 10% has the best growth promoting effect, the plant height and stem thickness of capsicum plants are respectively increased by 55.15% and 33.66% compared with the control, the plant height and stem thickness of eggplant plants are respectively increased by 20.89% and 13.01% compared with the control, and the addition amount of 20% and 25% can inhibit plant growth, but the difference from the control is not obvious (table 10).
TABLE 10 influence of non-pathogenic bacterial wilt solid state ferments on tomato, capsicum and eggplant plant growth
The control effect and the growth promotion test result are combined, the recommended addition amount of the solid state fermentation product of the non-pathogenic bacterial wilt for tomatoes is 15%, the addition amount can promote the growth of tomato plants, and the control effect on the bacterial wilt of the tomatoes reaches 80.67%; for the capsicum and the eggplant, the recommended addition amount of the non-pathogenic bacterial wilt solid state fermentation product is 25%, the addition amount does not obviously inhibit plant growth, and the control effect on bacterial wilt disease is good and respectively reaches 75.67% and 80.11%.
(5) And (3) nutrient component measurement: randomly selecting 3 parts of non-pathogenic bacterial wilt solid fermentation products, sending the products to a detection qualification unit (institute of agricultural quality standard and detection technology of the national academy of agricultural sciences of Fujian) to measure physicochemical properties and nutritional components, including water (according to GB/T8576-2010 standard), pH (according to NY/T1121.2-2006 standard), organic matters (according to NY/T1121.2-2006 standard), total nitrogen (according to NY/T1121.24-2012 standard), crude fibers (according to GB/T6434-2006 standard), and the like, and counting the average value.
The determination results of the nutritional ingredients and physicochemical properties show that the solid fermentation product of the non-pathogenic bacterial wilt has the average moisture content of 17.8 percent; the average pH value was 5.8, the average organic matter content was 27%, the average total nitrogen content was 0.54%, the average total phosphorus content was 130.33%, the average total potassium content was 0.57%, and the average crude fiber content was 13.15% (Table 11). The nutrient content of the microbial fertilizer exceeds the national standard (NY/T798-2015), namely the total nutrient (N+P) 2 O 5 +K 2 O) is 8.0-25.0%, and the organic matter is more than or equal to 20.0%.
TABLE 11 physicochemical Properties of non-pathogenic bacterial wilt solid state fermentation product
In conclusion, the invention aims at the non-pathogenic bacterial wilt FJAT-aRS01, adopts a solid state fermentation mode, utilizes the pennisetum hydridum residue as one of the components of the solid state fermentation medium, can provide needed nutrients for the fermentation of the bacterial strain, reduces the production cost, and can reduce the pollution of agricultural and sideline product wastes to the environment. The solid state fermentation product of the non-pathogenic bacterial wilt FJAT-aRS01 has the viable count of 150-200 hundred million/g, contains rich nutrient substances, can promote the germination of seeds when used in a seed stage, can promote the growth of plants when used in a plant growth stage, can prevent and treat bacterial wilt, has obvious prevention effect on the bacterial wilt of solanaceous vegetables such as tomatoes, peppers, eggplants and the like, has more than 75 percent, and has important biocontrol potential.
It should be noted that, although the foregoing embodiments have been described herein, the scope of the present invention is not limited thereby. Therefore, based on the innovative concepts of the present invention, alterations and modifications to the embodiments described herein, or equivalent structures or equivalent flow transformations made by the present description and drawings, apply the above technical solution, directly or indirectly, to other relevant technical fields, all of which are included in the scope of the invention.
Claims (9)
1. A solid state fermentation product of non-pathogenic bacterial wilt, wherein the solid state fermentation product comprises the following nutritional components: the effective viable count of the non-pathogenic bacterial wilt bacteria is 150-200 hundred million/g, the organic matter content is 25-29%, the total nitrogen content is 0.4-0.7%, the total phosphorus content is 125-140%, the total potassium content is 0.5-0.7%, the crude fiber content is 12-14%, the water content is 17-18%, and the pH value is 5.5-6.0, and the above contents are all mass percentages.
2. The solid state fermentation product of non-pathogenic bacterial wilt according to claim 1, wherein the preparation method of the solid state fermentation product is as follows:
preparing seed liquid: inoculating non-pathogenic bacterial wilt into nutrient broth fermentation medium, and shake culturing at 28-32deg.C and 150-200rpm for 20-28 hr to obtain seed solution;
solid state fermentation: adding seed liquid into solid fermentation medium according to 15-20% of inoculation amount, stirring, sealing, and fermenting at 28-32deg.C for 2-3d to obtain solid fermentation product of pathogenic-free ralstonia solanacearum; the preparation method of the solid state fermentation culture medium comprises the steps of adding water into 35-38% of pennisetum sinese residues, 25-30% of corn flour, 25-30% of rice bran and 8-10% of rice husk, uniformly stirring, filling the uniformly mixed materials into a polypropylene sterilization bag according to the mass ratio of 1 (0.7-1), sterilizing at 120-125 ℃ for 15-25min to obtain a sterile fermentation material, and cooling for later use.
3. The solid state fermentation product of nonpathogenic bacterial wilt according to claim 1 or 2, wherein the nonpathogenic bacterial wilt is nonpathogenic bacterial wilt (ralstonia acreana) FJAT-aRS01 deposited in the common microorganism center of the chinese microbiological culture collection committee at month 4 of 2015 at the address of north chen west road No.1, no. 3, the region of facing yang in beijing with the deposit number of cgmccno.10693.
4. Use of a solid state fermentation of non-pathogenic bacterial wilt according to any of claims 1-3 for promoting germination of solanaceous vegetable seeds.
5. Use of a solid state fermentation product of non-pathogenic bacterial wilt according to any of claims 1-3 for the control of bacterial wilt of solanaceous vegetables.
6. Use of a solid state fermentation of non-pathogenic bacterial wilt according to any of claims 1-3 for promoting the growth of solanaceous plants.
7. The use of the solid state fermentation product of non-pathogenic bacterial wilt according to claim 6 for promoting the growth of solanaceous plants, wherein the application method is to add the solid state fermentation product of non-pathogenic bacterial wilt with the mass percent less than or equal to 15% into a cultivation substrate.
8. Use of a solid state fermentation product of non-pathogenic bacterial wilt according to any of claims 1-3 for the preparation of a disease resistant growth promoter for solanaceous vegetables.
9. The application of the solid state fermentation product of the non-pathogenic bacterial wilt in preparing the disease-resistant growth promoting agent for the solanaceous vegetables according to claim 8, wherein the application method is to add 15-25% of the solid state fermentation product of the non-pathogenic bacterial wilt in a cultivation matrix.
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