CN111944716B - Special compound microbial agent for tobacco seedling culture and preparation method and application thereof - Google Patents

Abstract

The invention belongs to the technical field of agricultural microorganisms, and particularly provides a special compound microbial agent for tobacco seedling culture, a preparation method and application thereofBacillus coagulans) Bacillus tequilensis (B), (B)Bacillus tequila) And Paenibacillus polymyxa: (Paenibacilluspolymyxa) Proportionally mixing. Wherein the bacillus coagulans can promote the growth of tobacco with high IAA yield. The bacillus tequilensis and the paenibacillus polymyxa can effectively inhibit common bacterial wilt and black shank of tobacco, B06 can promote the antibacterial capability of the bacillus tequilensis and the paenibacillus polymyxa, and the three microorganisms are matched for use, so that the growth of the tobacco can be promoted, the yield of the tobacco can be improved, the disease prevention and resistance can be realized, and the quality of the tobacco can be improved.

Description

Special compound microbial agent for tobacco seedling culture and preparation method and application thereof

Technical Field

The invention belongs to the technical field of agricultural microorganisms, and particularly relates to a compound microbial agent special for tobacco seedling culture and a preparation method and application thereof.

Background

Tobacco is an important economic crop in China, mainly focuses on Yunnan, Guizhou, Chongqing, Hubei, Hunan and other provinces, and plays an important role in national economy. In tobacco production, seedling culture is an important link of tobacco leaf production. Currently, the tobacco production adopts a seedling transplanting production mode, namely, tobacco seedlings are intensively cultivated and transplanted to a field after the seedlings are grown. The advantages of this are mainly: the land is economically utilized, and the multiple cropping index is improved; the tobacco seedlings are concentrated in the environment with superior light, temperature and water, and can survive quickly and grow uniformly; overcomes the limitation of frost-free period and prolongs the growing period of tobacco field. The cultivation of strong seedlings is the basis of high-quality tobacco production, so the research and development of tobacco seedling raising technology are very important in the tobacco industry of China all the time, and floating seedling raising, tray seedling raising and conventional seedling raising are mainly used at present.

The cultivation of strong seedlings is the basis of high quality and high yield of tobacco, and the tobacco seedling cultivation work is very important at home and abroad. The excellent microbial strains are inoculated in the seedling stage, so that the nutrition condition of tobacco seedlings can be improved, the physiological metabolism is promoted, the stress resistance is enhanced, the quality of the tobacco seedlings is improved, and strong seedlings are cultivated, thereby being beneficial to improving the yield and the quality of tobacco leaves. The substrate is the environment for the root growth of the tobacco seedling, the moisture and the nutrition of the tobacco seedling are obtained through the substrate, and the components of the substrate directly influence the formation of the mycorrhiza of the tobacco seedling and the growth of the mycorrhiza seedling. Therefore, the development of a special substrate formula for tobacco seedlings and a corresponding microbial inoculation technology are urgent needs for intensive production of flue-cured tobacco seedlings in China

Plant growth-promoting rhizobacteria (PGPR) refers to a microorganism that survives at the root surface and rhizosphere of a plant and can indirectly or directly promote and regulate the growth of the plant. Research shows that rhizosphere growth-promoting bacteria exist widely in plant roots, and more than 20 genera including bacillus and pseudomonas have the potential of promoting growth and preventing diseases. The tobacco growth-promoting rhizobacteria is prepared into the bio-organic fertilizer and applied to tobacco seedling culture, so that the germination rate and the seedling quality of tobacco seedlings in the seedling stage can be effectively improved, and the agricultural property of the tobacco seedlings is promoted. Wu Xiaoping et al research shows that the spraying of photosynthetic bacteria liquid on leaf surface during floating seedling raising of flue-cured tobacco can raise the chlorophyll content and seedling quality of tobacco seedling obviously. 2 growth-promoting strains A03 and B04 are obtained by screening from the rhizosphere of flue-cured tobacco by Matheya and the like, and the PGPR bacterial manure is prepared and used for floating seedling of the flue-cured tobacco, and can obviously improve the quality of the seedling, the germination rate of the seeds and the resistance to tobacco diseases.

At present, some microbial fertilizer products are applied to tobacco seedling raising, but the problems of low product activity, short shelf life, unstable effect and the like exist. According to the invention, microbial strains with growth promoting and disease resisting functions on tobacco seedlings are respectively screened, the prevention and control effects of different microbial agents on tobacco growth and root and stem diseases are evaluated by adopting a matrix bacterium mixing method, an effective tobacco seedling microbial agent combination is obtained, and a foundation is laid for the popularization of a seedling matrix bacterium mixing technology.

Disclosure of Invention

The invention aims to provide a compound microbial agent special for tobacco seedling culture, which comprises the following components in part by weight: the Bacillus coagulans (Bacillus coagulons), the Bacillus tequila (Bacillus tequila) and the paenibacillus polymyxa (paenibacillus polymyxa) have the preservation number of CCTCCNO: m2020066, the preservation number of the Bacillus tequilensis is CCTCC NO: m2020385.

The invention also aims to provide a preparation method of the compound microbial agent.

The last purpose of the invention is to provide the application of the microbial agent, and the microbial agent has a disease-resistant function, promotes the root development of tobacco seedlings, and improves the yield and the quality of tobacco.

In order to achieve the purpose, the invention adopts the following technical measures:

the Bacillus coagulans strain is obtained by screening through the following method:

1) collecting multiple tea tree root soil from a tea garden of university of Huazhong agriculture, selecting an IAA high-yield strain from the soil, and identifying the strain as bacillus coagulans through physiological and biochemical processes and a 16S rRNA gene sequence. The strain is used as an initial strain, and then is subjected to normal-pressure room-temperature plasma mutagenesis screening to obtain a bacillus coagulans B06 with high IAA yield.

2) The strain is preserved in China Center for Type Culture Collection (CCTCC) at 4 months and 9 days in 2020, and is classified and named as follows: bacillus coaglulisb 06 with the preservation number of CCTCC NO: m2020066, address: wuhan university in Wuhan, China.

Strain B06 has typical bacillus coagulans characteristics: the strain grows well on an MRS culture medium, forms a circular colony with the diameter of 2-3mm within 48 hours, and is white and glossy, the surface is wet and flat, and the edge is neat. Gram staining and oil lens observation, the shape of the strain B06 is rod-shaped, gram-positive, single, paired or chain-shaped, and spores are grown.

A strain of Bacillus tequila is obtained by screening through the following method:

1) collecting multiple parts of soil from tobacco planting land in Laifeng county of Enshi, Hubei, respectively taking 5g of soil sample to 300ml of YPD liquid culture medium, standing and culturing at 37 ℃ for 3d, then placing the culture solution in 80 ℃ water bath for 15min, diluting by 10 times in gradient, respectively taking 0.1ml of diluent to coat a YPD solid plate, and culturing at 37 ℃. Randomly picking colonies after the colonies grow out, carrying out streaking separation, numbering and storing the purified strains, transferring the strains to a YPD liquid culture medium, culturing at 37 ℃ for 48h, and carrying out a bacteriostasis zone experiment by using fermentation liquor, wherein the bacteriostasis object is pathogenic bacteria Ralstonia solanacearum of tobacco bacterial wilt. And selecting the strain KQ22 with the highest bacteriostatic ability by measuring the size of the bacteriostatic zone.

2) The strain KQ22 is identified through physiological and biochemical processes and 16S rRNA gene sequences, and the strain is determined to be Bacillus tequilensis.

The strain is preserved in China Center for Type Culture Collection (CCTCC) in 7-month and 31-month 2020, and is classified and named as follows: bacillus tequila KQ22 with the preservation number of CCTCC NO: m2020385, address: wuhan university in Wuhan, China.

A compound microbial agent special for tobacco seedling culture comprises: bacillus coagulans (Bacillus coagulons) B06, Bacillus tequilensis (Bacillus tequila) KQ22 and paenibacillus polymyxa; the effective bacteria content ratio of the strain in the composite microbial inoculum is as follows: (1-10) to (1-10); the preservation number of the Bacillus coagulans (Bacillus coagulans) B06 is CCTCC NO: m2020066, accession number of Bacillus tequilensis: CCTCC NO: m2020385, said Paenibacillus polymyxa is a biocontrol strain purchased from commercial sources.

Preferably, the ratio of the viable count of the three bacteria in the composite microbial inoculum is 1:1: 1.

The composition is powder, and the total effective viable count is 20-200 hundred million/g.

According to the conventional mode in the field, the three microorganisms are independently fermented and then compounded according to the proportion of live bacteria, or the three microorganisms are inoculated into a culture medium according to a certain proportion and mixed for culture, so that the composite microbial inoculum can be obtained;

preferably, the preparation method of the complex microbial inoculum comprises the following steps:

the culture medium of the bacillus coagulans B06 is prepared from brown sugar, yeast extract powder, bran, light calcium carbonate and water in a mass ratio of (2-4): (0.5-2): (80-120): (1-3): (80-100), uniformly mixing the culture medium, sterilizing, inoculating 0.5-5% of seed solution (MRS culture medium), standing and culturing at 37-50 ℃ for 48-60h, drying at low temperature and crushing.

The culture medium of the bacillus tequilensis KQ22 is prepared from brown sugar, bran, bean pulp, light calcium carbonate and water in a mass ratio of (2-4): (80-120): (40-60): (0.5-2): (100-140), uniformly mixing culture media, sterilizing, inoculating 3-5% of seed liquid, standing and culturing at 30-40 ℃ for 48-60h, drying at low temperature and crushing.

The wheat bran culture of the bacillus coagulans and the bacillus texatilis and the bacterial powder of the paenibacillus polymyxa are mixed according to the ratio of viable count of (1-10) to obtain the compound microbial agent special for tobacco.

Or prepared according to the following method:

the culture medium is brown sugar, yeast extract powder, bran, light calcium carbonate and water according to a mass ratio (4-6): (0.5-2): (80-120): (1-3): (80-100), uniformly mixing and sterilizing a culture medium, inoculating 0.5-5% of mixed seed solution, standing and culturing at 35-42 ℃ for 48-60h, drying at low temperature and crushing; the mixed seed liquid is prepared from Bacillus coagulans (Bacillus coagulans) B06, Bacillus tequila (Bacillus tequila) KQ22 and paenibacillus polymyxa seed liquid according to the weight ratio of (4-6): (0.5-1): (2-4) in proportion.

The application of the compound microbial agent special for tobacco seedling culture comprises preparing a tobacco root-irrigation agent or an additive of a tobacco seedling culture medium by using the compound microbial agent; or preparing into tobacco microorganism disease-resistant agent and growth promoter;

in the application, the disease-resistant agent is a bacterial wilt-resistant bacterial agent or a black shank-resistant bacterial agent.

Compared with the prior art, the invention has the following advantages:

1) the invention provides a composite microbial inoculum consisting of three microorganisms, wherein bacillus coagulans can promote the growth of tobacco with high IAA yield. The bacillus tequilensis and the paenibacillus polymyxa can effectively inhibit common bacterial wilt and black shank of tobacco, B06 can promote the antibacterial capability of the bacillus tequilensis and the paenibacillus polymyxa, and the three microorganisms are matched for use, so that the growth of the tobacco can be promoted, the yield of the tobacco can be improved, the disease prevention and resistance can be realized, and the quality of the tobacco can be improved.

2) Three microorganisms in the composite microbial inoculum are bacteria capable of producing spores, the stress resistance is strong, and the number of live bacteria after drying is high.

3) The compound microbial inoculum is mainly produced by bran solid state fermentation, and has the advantages of simple process, convenient production, high biomass, low cost and long shelf life.

Detailed Description

The technical schemes of the invention are conventional schemes in the field if not particularly stated; the reagents or materials, if not specifically mentioned, are commercially available.

Example 1:

screening of bacillus coagulans B06:

1) screening of high-IAA-yield wild strain

Culture medium:

MRS liquid medium: 20g/L of glucose, 10g/L of peptone, 5g/L of yeast extract, 10g/L of beef extract powder, 2g/L of diammonium citrate, 5g/L of sodium acetate, 801 g/L of tween, 2g/L of dipotassium phosphate and pH 6.5.

YPD liquid medium: 20g/L of glucose, 20g/L of peptone and 10g/L of yeast extract powder, and the pH is natural.

Solid medium 1.5% agar was added based on liquid medium and all media were sterilized at 115 ℃ for 20 min.

Collecting multiple tea tree root soil from China university tea garden, taking 1g soil sample into MRS culture medium, standing and culturing at 37 deg.C for 7d, water bathing at 80 deg.C for 15min, taking 0.1ml transfer MRS culture medium solid plate, and culturing at 37 deg.C. And randomly picking colonies after the colonies grow out, purifying, numbering and storing. The strain is transferred to YPD liquid culture medium and cultured for 24h at 37 ℃, the content of indoleacetic acid (IAA) in fermentation liquor is measured, and an IAA high-yield strain is selected, wherein the content can reach 96.2 mg/L.

Through physiological and biochemical processes and 16S rRNA gene sequence identification, the similarity of the 16S rRNA gene of the strain and the bacillus coagulans is 99%, and the strain is finally identified as the bacillus coagulans.

2) Carrying out normal-pressure room-temperature plasma mutagenesis breeding on bacillus coagulans B06:

the strain is mutagenized by an Atmospheric Room Temperature Plasma (ARTP) mutagenizing instrument. The working gas used for mutagenesis was helium. Uniformly coating 10 mu L of spore suspension on each metal slide according to the treatment power of 100W, the flow of 12L/min helium, the distance between a sample and an outlet of a plasma generator of 2mm, the operating temperature of 25-35 ℃, treating the spore suspension according to different mutagenesis irradiation times of 30, 60, 90, 120, 150, 180, 210, 240 and 270s, respectively, placing the metal slides in an EP (EP) tube filled with 1mL of sterile physiological saline after mutagenesis, oscillating and suspending, and respectively performing 10 treatment times^-1、10^-2、10^-3Three dilutions were plated, 3 plates were plated for each dilution, counted after 2d incubation at 37 ℃ and the lethality, positive mutation rate calculated by colony-forming units (CFU). Analyzing curve data, taking the time point with the highest positive mutation rate as the optimal mutagenesis time point to carry out subsequent mutagenesis treatment, and determining the IAA content in the fermentation liquor.

After 3 rounds of screening, an IAA high-yield strain Bacillus coagulansB06 is obtained, the content of indoleacetic acid (IAA) in fermentation liquor which is cultured in a YPD liquid culture medium at 37 ℃ for 24 hours can reach 118.5mg/L, and is improved by 23.2 percent compared with the original strain. The strain is subcultured for 5 generations, and the IAA yield is stable. The strain is preserved in China Center for Type Culture Collection (CCTCC) at 4 months and 9 days in 2020, and is classified and named as follows: bacillus coaglulisb 06 with the preservation number of CCTCC NO: m2020066, address: wuhan university in Wuhan, China.

Strain B06 characteristics: the strain grows well on an MRS culture medium, forms a circular colony with the diameter of 2-3mm within 48 hours, and is white and glossy, the surface is wet and flat, and the edge is neat. Gram staining and oil lens observation, the shape of the strain B06 is rod-shaped, gram-positive, single, paired or chain-shaped, and spores are grown.

3) Detection of the directional colonization ability of the bacillus coagulans B06 on tea tree leaves:

(1) induction and culture of rifampicin resistant marker strains: firstly, determining that the B06 strain has the sensitivity concentration of 0.009g/L to rifampicin, then preparing YPD culture media and corresponding YPD plates with rifampicin concentrations of 0.01, 0.02, 0.04, 0.08, 0.16 and 0.32g/L respectively, and then adopting a gradient alternate culture method of YPD liquid culture media containing rifampicin and plate gradients to induce and culture the B06 strain, finally obtaining an anti-rifampicin marker strain B06-r, wherein the strain can normally grow in the YPD culture media of 0.32g/L rifampicin, and the strain has no significant difference from B06 in physiological and biochemical characteristics except rifampicin resistance.

(2) B06-r colonization and quantity detection of tea plant and lettuce leaf surfaces: the amount of viable bacteria is 4.8 × 10⁷CFU/mL of bacterial suspension. B06-r bacterial suspension is uniformly sprayed on leaf surfaces of tea trees and lettuce, sampling and recycling are carried out for 1, 3, 7, 11, 15, 17 and 20 days respectively, sampling is carried out according to a five-point sampling method, then 20 pieces are randomly taken, leaf tissue blocks are taken by using an aseptic puncher with the diameter of 1cm, 10g of the leaf tissue blocks are weighed and put into a triangular flask containing 90mL of sterile water (glass beads are added into the flask), after shaking is carried out for 30min by a 150r/min shaking table, the sterile suspension is subjected to gradient dilution by using the sterile water, and 3 times of treatment are set for each time. The number of bacteria was determined by serial dilution of rifampicin YPD agar plates with 0.32 g/L.

The test is carried out in tea gardens and vegetable test bases in schools of Huazhong agricultural university, after the bacterial liquid containing the rifampicin-resistant marker strain B06-r is sprayed for 15 days, the average daily air temperature is 15-28 ℃, the wind power is 1-2 levels, and no rainfall exists in the period. As is clear from the results in Table 1, the strains 1 to 15d, B06 exhibited good colonization ability on tea leaves, and the number of bacteria was gradually decreased after increasing. Obvious rainfall occurs at 18 days, and the bacterial quantity is greatly reduced to less than 10⁵CFU/g mature leaf. On lettuce leaves, the number of bacteria of the B06 strain continuously and rapidly decreases, and the number of bacteria is less than 10 at 15 days⁴CFU/g mature leaf, probably due to lettuce leaf environment or metabolites not suitable for growth of the strain. In conclusion, the B06 strain can directionally colonize on the leaf surfaces of tea trees, and the colonization time is as long as more than 15 days.

TABLE 1 dynamic of colonization by Strain B06 on plant leaves

4) Fermentation of the bacillus coagulans B06 foliar fertilizer:

bacillus coagulans B06 seed solution grown in YPD liquid medium for 24 hours was inoculated with potato starch wastewater sterilized at 121 ℃ for 30min at an inoculum size of 5% (V/V), cultured at 37 ℃ for 48 hours, adjusted to pH 6.0 with NaOH, and the components thereof were measured as shown in Table 2 below.

TABLE 2 ingredient determination table before and after fermentation of potato starch wastewater

Item	Unit of	Before fermentation	After fermentation	GB 20287 Standard
					pH		6.8	6.0	5.0-8.0
Total solids content	g/L	25	18	--
					Number of viable bacteria	108/mL	0	2.5	--
Protease enzyme	U/mL	0	82	--
					Indoleacetic acid	mg/L	0	16.5	--
COD	mg/L	28065	16438	--
					Total nitrogen	mg/L	2745	1804	--
Total phosphorus	mg/L	86	78	--
					K	mg/L	786	564	--
Fe	mg/L	9.1	7.9	--
					Mg	mg/L	56.5	52.3	--
Zn	mg/L	1.9	1.7	--
					Cu	mg/L	0.52	0.16	--
Cd	mg/L	0.20	0.19	≤10
					As	mg/L	0.26	0.25	≤75
Cr	mg/L	0.25	0.23	≤150
					Pb	mg/L	0.43	0.41	≤100

5) The application of the bacillus coagulans foliar fertilizer on the tea leaves comprises the following steps:

the application test is completed in the tea science test base of Huazhong agriculture university, and the total area of the test tea garden is 1000m²The tea garden is completely flat, the slope is less than 5 degrees, the tea trees grow consistently, and no serious vegetation and mountain shelters are arranged around the tea garden. The tea plant variety is a local group variety (sexual system), the tree age is about 20 years, the group variety is a middle-leaf variety, the sowing time is consistent, and the water and fertilizer management and the picking mode of the test tea garden in the past year (till the planting) are the same.

The experiment is totally set to 2 treatments, namely, the unfermented potato starch wastewater in the spraying sterilized potato starch wastewater (step 4) is diluted by 50 times, the viable count in the spraying liquid is 0, the IAA content is 0) and the foliar fertilizer prepared in the spraying foliar fertilizer (step 4) is diluted by 50 times, and the final concentration of the viable count of the bacillus coagulans B06 in the final spraying liquid is 0.5 multiplied by 10⁷CFU/mL, IAA final concentration of 0.33mg/L)1 time. Spraying in a test area in 3 and 2 days in 2019, uniformly spraying sterilized potato starch wastewater or a leaf fertilizer on the front and back surfaces of tea plant leaves by using a fine nozzle sprayer until liquid drops on the leaf surfaces flow down. Each treatment is repeated for 3 times (1 row is used as 1 cell), the treatment is randomly arranged, and protective areas are arranged among the cells at intervals of 2m, and 9 test cells are calculated.

The quality of the buds is checked by adopting a 33cm multiplied by 33cm sample frame, and the sample frame is checked by 3 times in each cell. And weighing and measuring the yield according to a production mode. And (3) deactivating enzymes and fixing the young tea tree shoots by adopting a microwave oven, drying the young tea tree shoots in an oven at 80 ℃, grinding the young tea tree shoots into tea powder, drying and storing the tea powder at a low temperature until analysis and detection are carried out. The total polyphenol content of the tea leaves and the content of the green tea extract are determined by the national standard method.

TABLE 3 Effect of different treatments on the quality of the tea shoots

Group of	1 week after spraying (g)	2 weeks after spraying (g)
			Experimental group	21	28.5
Control group	19	23

As can be seen from Table 3, the weight of the spring tea leaf shutter sprayed with the foliar fertilizer is higher than that of the control group, the weight of the leaf shutter sprayed with the foliar fertilizer is increased by 23.9% for two weeks, and the yield of the spring tea can be effectively increased.

Table 4 effect of different treatments on green tea extract

Group of	1 week after spraying (%)	2 weeks after spraying (%)
			Experimental group	36.5	41.5
Control group	32	37

As can be seen from table 4 and table 5, the contents of the spring tea green tea extract and the tea polyphenol after the foliar fertilizer is sprayed are higher than those of the control group, the content of the green tea extract is increased by 12.2% and the content of the tea polyphenol is increased by 16.6% after the foliar fertilizer is sprayed for two weeks, and the quality of the spring tea can be effectively improved.

TABLE 5 Effect of different treatments on the tea Polyphenol content

Group of	1 week after spraying (%)	2 weeks after spraying (%)
			Experimental group	22.5	24.6
Control group	16.3	21.1

Example 2:

screening of Bacillus terkefir KQ22 against Ralstonia solanacearum

Culture medium:

LB liquid medium: 10g/L of tryptone, 5g/L of yeast extract powder, 10g/L of sodium chloride and natural pH.

YPD liquid medium: 20g/L of glucose, 20g/L of peptone and 10g/L of yeast extract powder, and the pH is natural.

Solid medium 1.5% agar was added based on liquid medium and all media were sterilized at 115 ℃ for 20 min. Collecting multiple parts of soil from tobacco planting land in Laifeng county of Enshi, Hubei, respectively taking 5g of soil sample to 300ml of YPD liquid culture medium, standing and culturing at 37 ℃ for 3d, then placing the culture solution in 80 ℃ water bath for 15min, diluting by 10 times in gradient, respectively taking 0.1ml of diluent to coat a YPD solid plate, and culturing at 37 ℃. Randomly picking colonies after the colonies grow out, carrying out streaking separation, numbering and storing the purified strains, simultaneously transferring the strains to an YPD liquid culture medium, culturing for 48h at 37 ℃, and screening the strains with good disease resistance effect by using a bacteriostatic circle method. Uniformly mixing the cultured Ralstonia solanacearum bacterial liquid with a YPD soft agar culture medium, pouring the mixture on a flat plate, cooling, placing filter paper sheets with the diameter of 1cm soaked with different bacterial strain fermentation liquids on the flat plate, placing 4 filter paper sheets at the position 2cm away from the center of each flat plate, measuring the size of an inhibition zone after culturing for 2 days, and selecting the bacterial strain KQ22 with the highest inhibition capacity, wherein the diameter of the inhibition zone is 25.32 mm.

The strain KQ22 is identified through physiological and biochemical processes and 16S rRNA gene sequences, and the strain is determined to be Bacillus tequilensis. Strain KQ22 characteristics: the strain forms a milky colony which is irregular in edge and dry and wrinkled on the surface on an LB solid plate. The thalli are rod-shaped, arranged singly or in pairs, have no flagellum, are blunt and round at two ends, generate oval spores and are gram-positive. The physiological and biochemical analysis shows that the glucose, xylose and mannose can be utilized, but the D-arabinose can not be utilized, the gelatin, the starch and the casein can be hydrolyzed, and the oxidase reaction, the catalase reaction, the nitrate reduction reaction, the V-P reaction and the indole test are positive.

The strain is preserved in China Center for Type Culture Collection (CCTCC) in 7-month and 31-month 2020, and is classified and named as follows: bacillus tequila KQ22 with the preservation number of CCTCC NO: m2020385, address: wuhan university in Wuhan, China.

Example 3:

the preparation method of the special composite microbial inoculum for tobacco seedling culture comprises the following steps:

the method comprises the steps of respectively culturing and drying bacillus coagulans B06 and bacillus tequilensis KQ22 in respective proper culture media, and then compounding the bacillus coagulans with paenibacillus polymyxa powder according to a certain ratio according to the number of viable bacteria.

The Paenibacillus polymyxa is purchased from Wuhan Keno Biotech Co., Ltd, the strain is designated by P2 in the invention, and the viable count of the strain powder is 50 hundred million CFU/g.

1) The culture medium of the bacillus coagulans B06 consists of brown sugar, yeast extract powder, bran, light calcium carbonate and water according to the mass ratio of 4:0.5:100:2:80, and the light calcium carbonate, the brown sugar and the yeast extract are dissolved by hot water during preparation and then are uniformly mixed with the bran. Sterilizing at 121 ℃ for 30min, cooling to below 50 ℃, inoculating 5% of seed liquid of bacillus coagulans B06, controlling the culture temperature between 37 and 50 ℃, standing and culturing for 60 hours, turning over once every 20 hours, drying the bran with cold air (below 60 ℃) after the fermentation is finished, crushing the dried bran and sieving with a 20-mesh sieve, and measuring the number of viable bacillus coagulans B06 in the dried bran to be 80 hundred million CFU/g;

the seed solution is MRS liquid culture medium, is inoculated with Bacillus coagulans B06 and is subjected to standing culture at 45 ℃ for 24 hours to obtain the bacillus coagulans strain.

2) The culture medium of the bacillus tequilensis KQ22 is composed of brown sugar, bran, bean pulp, light calcium carbonate and water according to the mass ratio of 2:100:50:1:120, and the light calcium carbonate and the brown sugar are dissolved by hot water and then are uniformly mixed with the bran during preparation. Sterilizing at 121 ℃ for 30min, cooling to below 40 ℃, inoculating 5% seed liquid of Bacillus tequilensis KQ22, controlling the culture temperature between 30-40 ℃, standing for 48h, turning over once after 24h, drying the bran with cold air (below 60 ℃) after fermentation is finished, crushing the dried bran and sieving with a 20-mesh sieve, and measuring the number of viable bacteria of the Bacillus tequilensis KQ22 in the dried bran to be 130 hundred million CFU/g;

the seed liquid is YPD liquid culture medium, inoculated with Bacillus tequilensis KQ22, and cultured at 37 deg.C and 150rpm for 18 h.

The bran cultures of the two microorganisms and the paenibacillus polymyxa powder are mixed according to the viable count ratio of 1:1:1, namely the mass ratio of the three bacteria cultures is 65:40:104, and the compound microbial agent special for tobacco is obtained.

Example 4: disease resistance experiment of potted tobacco

1) The bacillus tequilensis KQ22 and the compound preparation have the disease-resistant effect on the tobacco bacterial wilt:

and setting a pot plant for verification. The total number of the treatment groups is 5, each group comprises 20 pots, each pot is a strain, and the experimental period is 45 days. Transplanting the tobacco seedlings cultured for 30 days into a flowerpot, irrigating the roots of each tobacco seedling with microbial agents according to different treatments (the treatment doses are shown in the table 6), irrigating 5ml of ralstonia solanacearum with the content of 10 hundred million CFU/ml after 5 days, then counting the number of diseases and disease indexes, and counting data such as plant height, dry weight, wet weight and the like. The classification standard of bacterial wilt refers to the tobacco industry standard (GB/T23222-2008):

0 grade, no disease of the whole plant;

1, occasionally chlorosis and scab exist in the stem part, or less than one half of the disease side withers and withers;

grade 3, black streaks exist in the stem part, but the height of the stem part is not more than one half of the height of the stem part, or one half to two thirds of the diseased side leaves are withered;

grade 5, withering the black streak of the stem part which is over one half of the height of the stem but does not reach the top of the stem or more than two thirds of the diseased side;

7, when the black stripe of the stem reaches the top, or the leaves of the diseased plant are completely withered;

and 9, basically withering and dying the diseased plant.

The disease incidence is the number of diseased plants/the total number of plants multiplied by 100%

Disease index ═ Σ (number of diseased plants at each stage × number of corresponding disease stages)/(total number of investigated plants × highest stage) × 100%

The results are shown in table 6, the morbidity of the group without the microbial agent treatment is 100%, the disease condition is serious, the morbidity is reduced to 30% after the bacillus tequilensis KQ22 is added, the disease index is reduced to 3.3, and the bacillus tequilensis KQ22 has a good control effect on bacterial wilt. In addition, after the compound microbial inoculum (prepared in example 3) is treated, the morbidity and disease index are further reduced, the morbidity of the mixed microbial inoculum of bacillus coagulans B06, bacillus tequilensis KQ22 and paenibacillus polymyxa P2 is reduced to 0, the growth condition of the strain is also best, and the leaf number, fresh weight and dry weight are the largest. Therefore, the bacterial wilt resistance effect of the compound bacterial agent is stronger than that of the single use of the Bacillus tequilensis KQ22, and the growth promotion effect on tobacco is also strongest. Note: in the examples of the present invention, the mixed microbial agents in the tables were, as not specifically described, at a volume ratio of 1 between strains.

Table 6: pot culture experiment of bacillus tequilensis KQ22 for resisting tobacco bacterial wilt

2) The paenibacillus polymyxa P2 and the compound preparation have the disease-resistant effect on the tobacco black shank:

in order to test the disease resistance effect of the paenibacillus polymyxa P2 and the compound preparation on the tobacco black shank, a pot plant is arranged for verification, and the experimental arrangement is the same as that of the pot plant. The tobacco black shank grading standard refers to the tobacco industry standard (GB/T23222-:

0 grade, no disease of the whole plant;

grade 1, withering the leaves of the stem part with scabs not more than one third of the stem circumference or less than one third;

grade 3, the lesion of the stem part slightly withers around one third to one half of the circumference of the stem, or one third to two halves of leaves, or the lesion of a few leaves at the lower part occurs;

grade 5, over one half of the lesion of the stem part but not all surrounding the stem circumference, or one half to two thirds of the leaf withering;

grade 7, the lesion of the stem part surrounds the stem circumference, or more than two thirds of the leaves wither;

and 9, basically withering and dying the diseased plant.

The disease incidence is the number of diseased plants/the total number of plants multiplied by 100%

Disease index ═ Σ (number of diseased plants at each stage × number of corresponding disease stages)/(total number of investigated plants × highest stage) × 100%

The prevention and treatment effect is (contrast disease index-treatment disease index)/contrast disease index multiplied by 100 percent

The results are shown in table 7, the morbidity of the group without the microbial agent treatment is 100%, the disease condition is serious, the morbidity is reduced to 35% after the paenibacillus polymyxa P2 is added, the disease index is reduced from 5.6 to 3.0, and the paenibacillus polymyxa P2 has a good control effect on the phytophthora parasitica. In addition, the morbidity and disease index are further reduced after the composite microbial inoculum is treated, the morbidity of the mixed microbial inoculum of bacillus coagulans B06, bacillus tequilensis KQ22 and paenibacillus polymyxa P2 is reduced to 5%, and the disease index is only 5%; the growth condition of the tobacco strain is the best, the plant height can reach 16.04cm, and the leaf number, fresh weight and dry weight are the largest. Therefore, the black shank resistance effect of the composite microbial inoculum is better than that of the single use of the paenibacillus polymyxa P2, and the growth promotion effect on tobacco is also strongest.

Table 7: paenibacillus polymyxa P2 tobacco black shank resistant potting experiment

Example 5:

a production method of a compound microbial agent special for tobacco comprises the following steps:

besides the method for producing the compound microbial agent described in the embodiment 3, the compound microbial agent can also be prepared by inoculating three kinds of microorganisms into a culture medium according to a certain proportion and mixing and culturing.

The culture medium used for production is composed of brown sugar, yeast extract powder, bran, bean pulp, light calcium carbonate and water according to the mass ratio of 5:0.5:100:50:2:120, and the light calcium carbonate, the yeast extract powder and the brown sugar are dissolved by hot water and then are uniformly mixed with the wheat bran during preparation. Sterilizing at 121 ℃ for 30min, cooling to below 40 ℃, inoculating 5% of mixed seed solution, controlling the culture temperature to be between 35 and 42 ℃, standing and culturing for 60h, turning over once for 20h, drying the bran with cold air (below 60 ℃) after fermentation is finished, crushing the dried bran, and sieving with a 20-mesh sieve to obtain the dried bran with viable bacteria of bacillus coagulans B06, bacillus tequilensis KQ22 and paenibacillus polymyxa P2 of 18 hundred million, 24 hundred million and 12 hundred million CFU/g respectively.

The seed solution is prepared by mixing YPD culture solutions of the three microorganisms according to the ratio of 5:1: 3.

Example 6: tobacco seedling application test with compound microbial agent

Control group: the seedling culture substrate is not added with bacteria.

Experimental groups: one thousandth of compound microbial inoculum is added into the seedling culture substrate, and the production method of the compound microbial inoculum is as described in embodiment 3

The seedling raising is carried out by using the seedling raising plate according to a conventional substrate seedling raising method, the management of an experimental group and a control group is consistent, the seedling emergence rate is measured and calculated after 15 days of sowing, the result is shown in Table 8, the balance average seedling emergence rate of the experimental group is 88.3 percent, the average seedling emergence rate is 24 percent higher than that of the control group by 64 percent, in addition, the seedling emergence time is shortened from 17.3 days to 13.7 days, and the seedling emergence time can be obviously shortened and the seedling emergence rate is improved by adding the composite microbial inoculum.

Table 8: influence of adding complex microbial inoculum on emergence rate and emergence time

After 60 days, sampling and measuring the agronomic characters and biomass of the tobacco seedlings according to tobacco industry standard YC \ T142-2010 (tobacco agronomic character survey method), wherein the statistical result is shown in Table 9, the average plant height of an experimental group is higher than that of a control group, and the number of leaves is more than that of the control group; fresh weight and dry weight of the overground part and the underground part of the experimental group are obviously higher than those of the control group, so that the growth and development of tobacco seedlings can be promoted by adding the composite microbial inoculum, and the biomass of the tobacco seedlings is improved.

Table 9: influence of adding composite microbial inoculum on tobacco seedling growth

Example 7: field experiment of tobacco seedling growing with composite microbial inoculum

And (3) respectively selecting the fields which have bacterial wilt and black shank in the previous year for tobacco cultivation experiments, wherein the transplanted tobacco seedlings are the tobacco seedlings cultivated in the embodiment 6. Transplanting tobacco seedlings cultured without the compound microbial inoculum matrix as a control group, transplanting tobacco seedlings cultured with the compound microbial inoculum matrix as an experimental group, wherein the area of each of the experimental group and the control group in a field with bacterial wilt and black shank is 1 mu, and the total area is 4 mu. The cultivation management is the same for each group, and the tobacco is fertilized conventionally: 1000kg of organic fertilizer and 15kg of compound fertilizer special for tobacco are applied to each mu of base fertilizer, and 10kg of compound fertilizer special for tobacco is applied to each mu in the root-building period. The first 100 tobacco plants in 1 ridge of land were randomly selected from each of the experimental group and the control group to perform statistical analysis on plant height, leaf number, yield and disease incidence, and the results are shown in tables 10 and 11.

Table 10: comparison of agricultural characters of different tobacco seedlings

After the tobacco seedlings cultured by adding the composite microbial inoculum matrix are transplanted to a field, the growth conditions of two experimental groups added with the composite microbial inoculum are better than those of a control group, and the results show that the plants grow vigorously, the stalks are thick, the leaves are increased, the tobacco leaves are thick, and the yield and the quality are obviously improved.

Table 11: comparing the disease conditions of different tobacco seedlings

The statistical results of the disease conditions show that after the compound microbial inoculum is added into the seedling culture substrate, the disease resistance of tobacco plants after being transplanted to a field for planting is obviously improved compared with the tobacco without the compound microbial inoculum, the disease incidence of bacterial wilt is reduced from 10.3% to 4.4%, the disease index is also reduced from 5.2 to 2.1, and the disease condition is relieved; the incidence rate of the black shank is reduced from 8.9% to 3.3%, and the disease index is reduced from 4.2 to 2.5.

In conclusion, after the compound microbial agent is added into the tobacco seedling culture medium, tobacco seedlings grow fast, plants are robust, the tobacco seedlings grow vigorously after being transplanted, the plants are robust, the disease resistance is high, and finally harvested tobacco leaves are high in quality and quantity.

Claims (10)

1. A compound microbial agent special for tobacco seedling culture comprises: bacillus coagulans bacterium (A), (B) and (C)Bacillus coagulans) B06, Bacillus tequilensis (B)Bacillus tequila) KQ22 and paenibacillus polymyxa; the Bacillus coagulans strain (A), (B) and (C)Bacillus coagulans) The preservation number of B06 is CCTCC NO: m2020066, the preservation number of the Bacillus tequilensis KQ22 is CCTCC NO: m2020385.

2. The microbial agent according to claim 1, wherein the effective bacteria content of the strain in the complex microbial agent is as follows: (1-10):(1-10):(1-10).

3. The microbial agent according to claim 1, wherein the effective viable count of the microbial agent is 20-200 hundred million/g.

4. The method for preparing the microbial agent of claim 1, comprising:

the culture medium of the bacillus coagulans B06 is prepared from brown sugar, yeast extract powder, bran, light calcium carbonate and water in a mass ratio of (2-4): (0.5-2): (80-120): (1-3): (80-100), uniformly mixing and sterilizing a culture medium, inoculating 0.5-5% of seed liquid, standing and culturing at 37-50 ℃ for 48-60h, and then drying at low temperature and crushing;

the culture medium of the bacillus tequilensis KQ22 is prepared from brown sugar, bran, bean pulp, light calcium carbonate and water in a mass ratio of (2-4): (80-120): (40-60): (0.5-2): (100-140), uniformly mixing and sterilizing a culture medium, inoculating 3-5% of seed liquid, standing and culturing at 30-40 ℃ for 48-60 hours, and then drying at low temperature and crushing;

the wheat bran culture of the bacillus coagulans and the bacillus tequilensis and the bacterial powder of the paenibacillus polymyxa are mixed according to the ratio of viable count of (1-10) to obtain the compound microbial agent special for tobacco.

5. The method for preparing the microbial agent of claim 1, comprising: the culture medium is brown sugar, yeast extract powder, bran, light calcium carbonate and water according to a mass ratio (4-6): (0.5-2): (80-120): (1-3): (80-100), uniformly mixing and sterilizing a culture medium, inoculating 0.5-5% of mixed seed solution, standing and culturing at 35-42 ℃ for 48-60h, drying at low temperature and crushing; the mixed seed liquid is prepared from bacillus coagulans (B.coagulans)Bacillus coagulans) B06, Bacillus tequilensis (B)Bacillus tequila) KQ22 and a paenibacillus polymyxa seed solution according to the ratio of (4-6): (0.5-1): (2-4) in proportion.

6. The use of the complex microbial inoculant of claim 1 in the preparation of a tobacco root-filling agent.

7. The application of the compound microbial agent of claim 1 in preparing tobacco seedling substrate additives.

8. The use of the complex microbial inoculant of claim 1 in the preparation of a tobacco anti-disease agent.

9. The use of claim 8, wherein the anti-disease agent is bacterial wilt-resistant agent or black shank-resistant agent.

10. The use of the complex microbial inoculant defined in claim 1 in the preparation of a tobacco growth promoter.