CN114134068A

CN114134068A - Paenibacillus polymyxa microbial agent, preparation method and application thereof

Info

Publication number: CN114134068A
Application number: CN202111250675.8A
Authority: CN
Inventors: 钱晓东; 周桂芬; 袁玉梅; 张晓祥; 李丽琴
Original assignee: Huzhou Central Hospital
Current assignee: Huzhou Central Hospital
Priority date: 2021-10-26
Filing date: 2021-10-26
Publication date: 2022-03-04

Abstract

The invention provides a paenibacillus polymyxa microbial inoculum, a preparation method and application thereof, relating to the technical field of microorganisms. The paenibacillus polymyxa Cs12 can effectively inhibit the growth of fusarium oxysporum, thereby fundamentally reducing the incidence rate of the stigma croci sativi corm rot disease; in addition, the invention also provides application of the paenibacillus polymyxa in preparing the medicine for preventing and treating the saffron corm rot, and a microbial inoculum prepared from the paenibacillus polymyxa can effectively inhibit the growth of fusarium oxysporum, so that the morbidity of the saffron corm rot is reduced, the effect of preventing and treating the saffron corm rot is obvious, and the problems of pesticide residue, environmental pollution and the like are avoided.

Description

Paenibacillus polymyxa microbial agent, preparation method and application thereof

Technical Field

The invention relates to the technical field of microorganisms, and particularly relates to a paenibacillus polymyxa microbial inoculum, and a preparation method and application thereof.

Background

Saffron (Crocus sativus L.) is also called saffron, only three red stigma croci on the upper part of pistil is used as medicine, the yield is low, the medicine is named as red gold seed, and the medicine has the effects of promoting blood circulation to remove blood stasis, cooling blood to remove toxic substances, resolving stagnation and soothing the nerves, is a traditional rare Chinese medicinal material and is also the most rare spice and dye in the world. Saffron is an allotriploid, only blossoms but does not seed, and only can be produced by bulb vegetative propagation, so that the yield and quality of the bulbs directly influence the yield and quality of the saffron. Saffron is originally produced in the Mediterranean region, and the yield of the saffron is over 90 percent of the total world production. China succeeds in introducing seeds to Chongming island in Shanghai in 1986, Zhejiang is the main production area at present, the planting area is about 0.6 ten thousand mu, which accounts for more than 50% of the whole country, and Tibetan safflower is rated as a new Zhe eight-flavor in 2018. But different from the climate of the main producing area, the climate of the middle sea in Jiangzhe area of China is warm and humid in summer, and underground bulbs are easily infected by pathogenic bacteria to grow diseases and rot, so that the new bulbs are dug out in the early 5 months and placed in a room for cultivation for half a year until blooming, and the bulbs are bred in a seed field after the indoor flower picking is finished in 12 months. However, even during indoors, saffron rot still occurs frequently. The annual average reproduction rate of the corms of saffron crocus is only 1.2, the plantation in Zhejiang is about 4000 mu for more than thirty years, and the control of the corm rot of saffron crocus is the bottleneck of the development of the saffron crocus plantation in China.

Thus, the stalk rot has been the most critical destructive factor impeding the development of the saffron industry. The stalk rot disease is mainly caused by that a soil-borne pathogenic fungus fusarium oxysporum (f. oxysporum) infects the stalk from the root by taking soil as a propagation medium, consumes host nutrient substances, causes plant wilting and death, can occur in various cultivation periods, and particularly has the highest morbidity in the late 6 th to late 8 th of high temperature in summer. The incidence of the disease in the Zhejiang Jiande high hair year in the main production area of China can reach 50 percent, and the development of the saffron planting industry in China is seriously hindered, so 80 percent of saffron in China still needs to be imported. At present, chemical pesticides are mainly used by farmers for prevention and treatment, but the effect is poor, and the harmful effects of the chemical substances on the environment and human are fully proved, so that the pesticide residue is serious, the physicochemical properties of soil are changed, the drug resistance of pathogenic bacteria is continuously enhanced, and beneficial microbiota related to plants are also influenced.

Therefore, a new effective method for preventing and treating the crocus sativus corm rot is urgently needed to be found, and the method has important significance for promoting the ecological environment and the healthy development of the crocus sativus industry in China.

Disclosure of Invention

The invention aims to provide paenibacillus polymyxa which can effectively inhibit the growth of fusarium oxysporum so as to fundamentally reduce the incidence rate of the stigma croci corm rot.

The second purpose of the invention is to provide the application of the paenibacillus polymyxa in preparing the medicine for preventing and treating the saffron corm rot. The paenibacillus polymyxa can effectively inhibit the growth of fusarium oxysporum, thereby reducing the morbidity of the stigma croci corm rot, having obvious prevention and treatment effect on the stigma croci corm rot, and avoiding the problems of pesticide residue, environmental pollution and the like.

The third purpose of the invention is to provide a microbial inoculum comprising the paenibacillus polymyxa, which can effectively inhibit the growth of fusarium oxysporum, thereby effectively reducing the incidence rate of corm rot of saffron caused by fusarium oxysporum and further improving the survival rate of saffron.

The fourth purpose of the invention is to provide a using method of the microbial inoculum, which defines the using amount and the using times of the microbial inoculum each time, thereby ensuring that the microbial inoculum can successfully inhibit the growth and development of fusarium oxysporum and play a role in reducing the incidence rate of the corm rot disease of saffron crocus.

The fifth purpose of the invention is to provide a preparation method of the microbial inoculum containing the paenibacillus polymyxa, which is simple and easy to operate and does not cause harm to the environment.

The technical problem to be solved by the invention is realized by adopting the following technical scheme.

The embodiment of the invention provides paenibacillus polymyxa named as paenibacillus polymyxa Cs12Paenibacillus, the preservation number of the paenibacillus polymyxa Cs12 is CGMCC No.22612, the preservation address is the microbial research institute of China academy of sciences No.3 of Beijing West Lu No.1 Hopkins in the area facing the Yang, and the preservation date is 2021, 5 and 26 days. The soil microbial community has important significance on plant health, the healthy growth of plants is closely related to the balance of the rhizosphere soil microbial community, and the imbalance of the rhizosphere soil micro-ecosystem is a main cause of occurrence of soil-borne diseases. Saffron is intercropped in soil where 10-year grapes are planted, developed grape root systems are tightly intertwined with the saffron root systems, and the underground root systems of the grapes are used for forming saffron rhizosphere microecologics with unique intercropping advantages by introducing new microorganisms into the saffron root systems, so that the crocus rhizosphere rot disease prevention and treatment effect is remarkable. The invention utilizes Fusarium oxysporum (Fusarium oxysporum) which is a main pathogenic bacterium of the corm rot disease of saffron to carry out efficient antagonistic separation on the rhizosphere soil microorganisms of saffron intercropped in a grape field, and obtains a bacterium with an obvious effect of antagonizing Fusarium oxysporum, namely the bacillus polymyxa Cs 12. The paenibacillus polymyxa is separated from the soil of grape-saffron intercropping by a high-efficiency 96-pore plate confrontation test and is further separated and purified by a living body disease resistance test and the like, and the paenibacillus polymyxa can effectively inhibit the growth of fusarium oxysporum which is a pathogenic bacterium causing the corm rot of saffron, so that the incidence rate of the corm rot of saffron is fundamentally reduced; and the paenibacillus polymyxa Cs12 can not cause damage and harm to soil and environment, so that the paenibacillus polymyxa Cs12 can be used at ease without worrying about pollution problems. The paenibacillus polymyxa exists in a spore form, has good temperature tolerance and stable quality, treats bacteria with bacteria and has lasting drug effect; the pesticide and fertilizer are both capable of not only being an ideal medicament for preventing and treating soil-borne diseases such as bacterial wilt, fusarium wilt, root rot, soft rot and the like, but also having obvious effects of promoting growth, increasing yield and improving quality; has no any phytotoxicity and residue, and is the first choice microbial preparation for pollution-free and green production.

The embodiment of the invention also provides application of the paenibacillus polymyxa in preparing a medicine for preventing and treating the saffron corm rot. The paenibacillus polymyxa can effectively inhibit the growth of fusarium oxysporum, thereby reducing the morbidity of the stigma croci corm rot, having obvious prevention and treatment effect on the stigma croci corm rot, and avoiding the problems of pesticide residue, environmental pollution and the like.

The embodiment of the invention also provides a microbial inoculum prepared from the paenibacillus polymyxa Cs12, the microbial inoculum prepared from the paenibacillus polymyxa Cs12 can be applied to the roots of saffron crocus infected with fusarium oxysporum in a root irrigation or injection mode, so that the growth of the fusarium oxysporum at the roots of the saffron crocus is inhibited, the incidence of corm rot of the saffron crocus is reduced, and the survival rate of the saffron crocus is improved; the microbial inoculum is pollution-free, does not cause burden to soil and environment, and is convenient for long-term use.

The embodiment of the invention also provides a using method of the microbial inoculum, which comprises the following steps that the microbial inoculum is applied once every 3 days, 5mL of the microbial inoculum is applied every time, and the application lasts for 21-30 days. The using method ensures that the microbial inoculum can effectively exert the effect of inhibiting the fusarium oxysporum by limiting the using amount and the using times of the microbial inoculum each time.

The embodiment of the invention also provides a preparation method of the microbial inoculum, which comprises the following steps of selecting a single colony of Paenibacillus polymyxa Cs12 under an aseptic condition, inoculating the single colony into an NA or PDA culture medium, and carrying out dark culture at 28-37 ℃ and 75% RH for 24-48h to obtain a purified colony; under the aseptic condition, selecting a purified colony, inoculating the colony in an NB or PDB culture medium, and carrying out shaking culture at 28 ℃ and 180rpm for 24 hours to obtain a seed bacterium; inoculating the seed bacteria into NB or PDB culture medium, fermenting at 28 deg.C and 180rpm for 48 hr to form fermentation liquid, and obtaining the microbial inoculum. The method is simple and convenient to operate, single bacterial colony and seed bacteria are obtained by separating and purifying the bacterial strain, and the bacterial strain is obtained by fermenting the seed bacteria of the bacterial colony.

Compared with the prior art, the embodiment of the invention has at least the following advantages or beneficial effects:

the embodiment of the invention provides a paenibacillus polymyxa, which is named as paenibacillus polymyxa Cs12 and is separated from the soil of grape-saffron intercropping, and the paenibacillus polymyxa can effectively inhibit the growth and development of fusarium oxysporum in the soil, thereby reducing the incidence rate of the corm rot disease of saffron crocus.

The embodiment of the invention also provides application of the paenibacillus polymyxa in preparing the medicine for preventing and treating the saffron corm rot, the paenibacillus polymyxa can effectively inhibit the growth of fusarium oxysporum, so that the morbidity of the saffron corm rot is reduced, the effect of preventing and treating the saffron corm rot is obvious, and the problems of pesticide residue, environmental pollution and the like are avoided.

The embodiment of the invention also provides a microbial inoculum, which is prepared from the paenibacillus polymyxa Cs12 and can be absorbed by the roots of saffron so as to play a role in inhibiting the growth of fusarium oxysporum at the roots of the saffron, and because the fusarium oxysporum is a main pathogenic bacterium of the corm rot of the saffron, the incidence rate of the corm rot of the saffron can be reduced by inhibiting the growth of the fusarium oxysporum, so that the microbial inoculum has important significance for the development of the saffron in China.

The embodiment of the invention also provides a using method of the microbial inoculum, and the method ensures that the microbial inoculum can effectively and reasonably inhibit the growth and development of fusarium oxysporum by using a reasonable amount and a reasonable mode, so that the microbial inoculum is ensured to have a remarkable inhibiting effect.

The embodiment of the invention also provides a preparation method of the microbial inoculum, the preparation method comprises the steps of separating and purifying the paenibacillus polymyxa Cs12 to obtain a single colony, subsequently separating and purifying the single colony to obtain seed bacteria, and finally fermenting the seed bacteria to obtain the microbial inoculum.

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 will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

Fig. 1 is a graph showing the inhibitory effect of paenibacillus polymyxa (p.polymyxa) Cs12 on fusarium oxysporum, a pathogen of the corm rot disease of saffron in example 1 of the present invention (a is fusarium oxysporum f. oxysporum, and B is the confrontation of Cs12 on f.oxysporum);

FIG. 2 is a gel electrophoresis diagram of Paenibacillus polymyxa Cs12 amplified based on the 16SrDNA primer in example 1 of the present invention;

FIG. 3 is a phylogenetic tree of Paenibacillus polymyxa Cs12 based on 16SrDNA obtained by the adjacency method in example 2 of the present invention;

FIG. 4 is a colony morphology of Paenibacillus polymyxa (P.polymyxa) on NA solid medium in example 2 of the present invention;

FIG. 5 is a graph showing the growth of bulbs after administration of Fusarium oxysporum in example 2 of the present invention for 28 days (wherein A is a blank control group, B is a model group, and C is a probiotic group).

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict. The present invention will be described in detail below with reference to specific examples.

The embodiment of the invention provides paenibacillus polymyxa named as paenibacillus polymyxa Cs12, wherein the preservation number of the paenibacillus polymyxa Cs12 is CGMCC No.22612, the preservation address is the microbiological research institute of China academy of sciences No.3 of Beijing West Lu No.1 Homeh of the Chaoyang district, and the preservation date is 2021 year, 5 months and 30 days. The paenibacillus polymyxa has strong colonization ability in roots, stems, leaves and other plant bodies, and pathogenic bacteria can be prevented from infecting plants through site competition; meanwhile, broad-spectrum antibacterial substances continuously secreted by the paenibacillus polymyxa can inhibit or kill pathogenic bacteria; in addition, the paenibacillus polymyxa can induce plants to generate disease resistance. The soil microbial community has important significance on plant health, the healthy growth of plants is closely related to the balance of the rhizosphere soil microbial community, and the imbalance of the rhizosphere soil micro-ecosystem is a main cause of occurrence of soil-borne diseases. Saffron is intercropped in soil where 10-year grapes are planted, developed grape root systems are tightly intertwined with the saffron root systems, and the underground root systems of the grapes are used for forming saffron rhizosphere microecologics with unique intercropping advantages by introducing new microorganisms into the saffron root systems, so that the crocus rhizosphere rot disease prevention and treatment effect is remarkable. The invention utilizes fusarium oxysporum, the main pathogenic bacterium of the corm rot of saffron, to carry out efficient antagonistic separation on saffron rhizosphere soil microorganisms intercropped in a grape field, and obtains a bacterium with an obvious fusarium oxysporum resistant effect, namely the paenibacillus polymyxa Cs 12. The paenibacillus polymyxa Cs12 mainly colonizes the roots of saffron, so that the infection of fusarium oxysporum on the corms of saffron is prevented through site competition, the nutrient source of fusarium oxysporum is cut off, the growth of fusarium oxysporum is further inhibited, and the prevalence rate of the fusarium oxysporum rot is reduced; and the paenibacillus polymyxa Cs12 can not cause pollution to soil and environment, thereby being convenient for long-term use.

In some embodiments of the invention, the 16SrDNA sequence of the Paenibacillus polymyxa is shown as SEQ ID NO. 1.

On the other hand, the embodiment of the invention also provides the application of the paenibacillus polymyxa in preparing the medicine for preventing and treating the saffron corm rot disease. Because the paenibacillus polymyxa Cs12 can effectively inhibit the growth of fusarium oxysporum, which is the main pathogenic bacterium of the corm rot of saffron crocus, the medicine prepared by using the strain can effectively reduce the incidence of the corm rot of saffron crocus by inhibiting the growth of fusarium oxysporum.

On the other hand, the embodiment of the invention also provides a microbial inoculum, and the microbial inoculum is prepared from the paenibacillus polymyxa Cs 12. The fungicide prepared by using the paenibacillus polymyxa Cs12 can be applied to the roots of saffron infected with fusarium oxysporum, so that the growth of the fusarium oxysporum at the roots of the saffron is inhibited, the incidence of corm rot of the saffron is reduced, and the survival rate of the saffron is finally improved; the microbial inoculum is pollution-free, does not cause burden to soil and environment, and is convenient for long-term use.

In some embodiments of the present invention, the concentration of the paenibacillus polymyxa Cs12 in the microbial inoculum is 10⁷-10⁸CFU/mL. The paenibacillus polymyxa Cs12 in the concentration range can ensure that the bactericide can effectively exert the prevention and treatment effect and avoid the influence of the paenibacillus polymyxa Cs12 on the growth and development of saffron crocus.

In some embodiments of the invention, the above microbial inoculum is administered once every 3 days, 5mL each time, for 21-30 days. The application method of the microbial inoculum ensures that the paenibacillus polymyxa Cs12 can stably inhibit the growth of fusarium oxysporum through the application amount and the application times, and avoids the situation that the paenibacillus polymyxa Cs12 accumulates too much at the roots of saffron to influence the growth and development of the saffron.

In some embodiments of the invention, the method of administration is by drenching or injection. The root of the saffron infected with the crocus coccineus rot is irrigated by using a microbial inoculum or the root of the saffron is directly injected to inhibit the growth of fusarium oxysporum in infected plants, so that the incidence of the crocus coccineus rot is reduced.

On the other hand, the embodiment of the invention also provides a preparation method of the microbial inoculum, which comprises the following steps of selecting a single colony of Paenibacillus polymyxa Cs12 under aseptic conditions, inoculating the single colony into a sterilized NA or PDA culture medium, and carrying out dark culture at 28-37 ℃ and 75% RH for 24-48h to obtain a purified colony; under the aseptic condition, selecting a purified colony, inoculating the colony in a sterilized NB or PDB culture medium, and performing shaking culture at 28 ℃ and 180rpm for 24 hours to obtain a seed bacterium; inoculating the seed bacteria into NB or PDB culture medium, fermenting at 28 deg.C and 180rpm for 48 hr to form fermentation liquid, and obtaining the microbial inoculum. The paenibacillus polymyxa Cs12 is cultured in an NA culture medium for a period of time to obtain a single colony of purified paenibacillus polymyxa Cs12, the single colony is picked and inoculated in an NB or PDB culture medium to obtain a seed bacteria culture medium for subsequent fermentation, and finally the single colony of seed bacteria is inoculated in the NB or PDB culture medium for fermentation to ensure that the paenibacillus polymyxa Cs12 is propagated in the NB culture medium in large quantities to obtain the microbial inoculum.

In some embodiments of the invention, the NA medium comprises peptone 10g, beef extract 3g, sodium chloride 5g, and agar 20g, and the pH of the NA medium is 7.0-7.4; the NB medium comprises 10g of peptone, 3g of beef extract and 5g of sodium chloride, and the pH of the NB medium is 7.0-7.4. PDA culture medium comprises water decoction of potato 200g, glucose 20g and agar 15 g; the PDB culture medium comprises water decoction of potato 200g and glucose 20g, and has pH of 7.0-7.4.

The raw materials are the formulas of the used NA or PDA culture medium and NB or PDB culture medium, so that basic nutrients and raw materials are provided for the growth and metabolism of the paenibacillus polymyxa Cs12, the growth and metabolism of the paenibacillus polymyxa Cs12 can be facilitated, the metabolic rate of the paenibacillus polymyxa Cs12 is at a high level, and the large-scale growth and propagation of the paenibacillus polymyxa Cs12, the formation of seed bacteria and the like are facilitated.

In some embodiments of the invention, the microbial inoculum is inoculated according to the inoculation amount of the NB or PDB culture medium in the volume ratio of 1 (10-100). The seed bacteria in the inoculation amount interval can be greatly proliferated and developed in the fermentation liquor, so that the disease resistance effect of the finally prepared microbial inoculum is improved.

The features and properties of the present invention are described in further detail below with reference to examples.

Example 1

Screening, isolation, purification and identification of paenibacillus polymyxa Cs 12:

(1) collecting healthy saffron rhizosphere soil from grape and saffron intercropping land in modern agriculture demonstration garden of south Taihu lake in Zhejiang lake in 8-15 days in 2020. Weighing 0.5g of soil, adding 2mL of sterile water, oscillating at 180rpm/min for 20min to obtain a suspension, automatically spotting the suspension on a 96-well plate by using a RoToR HDA (British SINGER) colony high-throughput screening workstation, wherein fusarium oxysporum is in the middle, 4 soil suspensions are spotted in a square shape at equal intervals around the fusarium oxysporum, sealing the plate by using a sealing film after spotting is finished, carrying out dark culture at 37 ℃ for 2-3 days, and selecting a colony with an obvious bacteriostasis zone for separation and purification.

(2) And selecting a colony with an obvious bacteriostatic zone, inoculating the colony to an NA culture medium by adopting a streaking method, and purifying the colony. The purified single colonies were picked and inoculated into 30mL (50mL Erlenmeyer flask) of NB liquid medium and cultured for 12-48h in a constant temperature shaking shaker at 28 ℃ under culture conditions of 180 rpm. Sucking a small amount of microbial inoculum with disposable Pasteur dropper in blood cell counting plate, counting spores under Zeiss positive fluorescence microscope, diluting spore suspension with sterile water to 10⁷-10⁸CFU/mL。

(3) Inoculating fusarium oxysporum (separated from rhizosphere soil of a diseased plant in the same plot) which is a pathogen of the corm rot disease of saffron crocus to a PDA culture medium, culturing for 5-7 days in a dark environment at 26 +/-2 ℃, preparing a bacterial block by using a sterilization puncher with the diameter of 6mm, connecting to the center of a new PDA culture medium, placing 3 sterile filter paper sheets with the diameter of 6mm at equal intervals at a position 2cm away from the center of the new PDA culture medium, and sucking 3 mu L of spore suspension liquid by using a pipette and placing the spore suspension liquid on the filter paper sheets. Equal volumes of NB broth served as negative controls, with 3 replicates per treatment. Placing into an incubator, culturing at 26 + -2 deg.C in dark environment for 5-7d, detecting the opposite culture result, and calculating the relative antibacterial rate according to the result of FIG. 1, as shown in FIG. 1. The relative inhibition rate (%) (control colony radius-treatment colony radius)/(control colony radius-cake radius) × 100%, and strains in which the relative inhibition rate was greater than 85% were selected for molecular identification.

Respectively inoculating the strains with the inhibition rate of more than 85 percent to the fusarium oxysporum to NB liquid culture media, and oscillating the strains in a shaking table at the constant temperature of 30 ℃ for 24-48h at the rotating speed of 180 rpm/min. 2mL of the DNA was aspirated into a centrifuge tube, and the strain DNA was extracted according to the procedure of Tiangen DP302 bacterial genome DNA extraction kit (Beijing Tiangen Biotech Co., Ltd.). Bacterial universal primers were used to amplify the isolated bacterial 16 SrDNA. The sequence of the forward primer 27F is shown as SEQ ID NO.2, and the sequence of the reverse primer 1492R is shown as SEQ ID NO. 3. The amplification conditions were as follows: pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 1min, and 30 cycles; finally, the reaction was terminated by extension at 72 ℃ for 5min, and the amplification sequence of the obtained Paenibacillus polymyxa Cs12 was shown in Table 1. The PCR product detected by agarose gel electrophoresis was sent to Shanghai Bioengineering (Shanghai) Co., Ltd for sequencing, and the gel electrophoresis pattern is shown in FIG. 2. The sequence determined from the PCR product was used as a target sequence, the GenBank database in NCBI was searched for homologous sequences, a reference sequence most similar to the morphotype sequence was downloaded, and the phylogenetic status of the strain to be identified was determined using an adjacent-joining method (NJ) for phylogenetic analysis, and the results are shown in FIG. 3. Through morphological, physiological and biochemical analysis (see table 2, wherein, the + is available or positive, the-is unavailable or negative) and the molecular biological identification of 16SrRNA, the rhizosphere bacterial strain of the invention is determined to be paenibacillus polymyxa Cs12, and the preservation number is CGMCC No. 22612.

TABLE 1 sequence Listing of the 16SrDNA sequence of Paenibacillus polymyxa Cs12 and primers used

TABLE 2 physiological and biochemical characteristics of Paenibacillus polymyxa Cs12

Index (I)	Expression profiles	Index (I)	Expression profiles
				Glucose	+	3% NaCl Hydrogen sulfide	-
Xylose	+	Malonic acid sodium salt	-
				Arabinose	+	Urease	+
Maltose	+	Catalase enzyme	-
				Sorbitol	-	Hydrolysis of gelatin	-
Mannitol	+	MP	-
				Nitrate broth	+	V-P	+

Example 2

Disease resistance test of paenibacillus polymyxa Cs12 microbial inoculum:

(1) taking Fusarium oxysporum strain stored on the inclined plane, picking a little hypha with an inoculating needle under aseptic condition, inoculating to PDA culture medium for activation, and culturing at 26 + -2 deg.C in dark environment for 5-7 d; the bacterial pellets were prepared by using a 6mm diameter sterile punch, and inoculated into PDB liquid medium for culture, and 1 pellet was inoculated per 10ml PDB. Incubate at 30 ℃ and 180rpm/min for 4-5 days. And (3) filtering the cultured fusarium oxysporum fermentation liquid in a super clean bench by using two layers of sterile gauze. Aspirating 100. mu.L spore suspension, counting the number of spores with a hemocytometer, adjusting to F.oxysporum 10⁵-10⁶spores/mL suspension.

(2) Freezing the Cs12 strain stored in 25% glycerol, coating 100 μ L of the strain on an NA culture medium under aseptic conditions, culturing the strain in the dark at 28-37 ℃ and 75% humidity for 24-48h, and observing the culture characteristics, wherein the obtained results are shown in FIG. 4; selecting a single colony of the activated strain by using an inoculating needle, inoculating the single colony in 25mL NB culture medium, and carrying out shake culture for 24h at 28 ℃ under the condition of 180rpm/min to prepare seed bacteria; inoculating the seed bacteria into a new NB culture medium in an inoculation amount of 1-10%, and fermenting at 28 ℃ and 180rpm for 48h to form the microbial inoculum. The cultured microbial inoculum is diluted with sterile water, and the OD600 of the microbial inoculum is adjusted to 0.4-0.6.

(3) The corm of saffron enters the dormancy stage, and the corm of saffron which has no scab appearance and single weight of 25 +/-1 g is purchased from the professional cooperation of Sandu saffron in Jian de City. After 7 months, when the corms are nearly dormant, removing the outer skin of the corms, sterilizing the surfaces of the corms by a three-step sterilization method (75% ethanol for 60s, 2.5% sodium hypochlorite for 5min and 75% ethanol for 30s), washing the corms with sterile water for multiple times, and drying the corms. Was used for the experiments.

(4) The bulbs were randomly divided into 3 groups (blank Control: Control; model group: fusarium oxysporum + sterile water; probiotic group: Cs12+ fusarium oxysporum), 42 per group, and 3 biological replicates per group. The seedlings were cultivated in a seedling box of 43cm X33 cm X28 cm. The pathogenic bacteria and the probiotics are inoculated by adopting a needle punching method. The Cs12 group was injected 3 days earlier with 10 μ L each of Cs12 at 3 positions at the top of the bulb, and the blank and model groups were inoculated with equal volumes of sterile water. The injection is injected once every 3 days, and in the second inoculation, fusarium oxysporum is inoculated to the model group and the Cs12 group at the same time for 28 days, wherein the growth condition of the bulb after 28 days is shown in figure 5, the lesion area is counted by using AutoCAD2019 software, and the morbidity index is calculated. Grading the disease condition standard: grade 0, no scab; grade 1, the lesion area of the corm is less than 10 percent; grade 2, the lesion area of the corm is 10 to 30 percent; grade 3, the lesion area of the corm is 30 to 50 percent; grade 4, the lesion area of the corm is 50 to 70 percent; grade 5, the lesion area of the corm is more than 70 percent. Disease Index (DI) ([ case-grade plant number × representative value)/total plant number ] × 100. The disease indices of each group are shown in table 3.

TABLE 3 disease index Table for placebo, model and probiotic groups

Group of	Blank control group	Model set	Probiotic group
				Index of disease condition	67±10.2	439±48.3	102±19.2

According to the results in table 3 and the formula, the preventing and treating effect (%) is (model group disease index-probiotic group disease index)/model group disease index x 100%, and the preventing and treating effect of the Cs12 is calculated to be 76.7%, so that the paenibacillus polymyxa Cs12 has an obvious bacteriostatic effect on fusarium oxysporum and can effectively reduce the incidence of the saffron corm rot.

Example 3

The field experiment of the fungicide of the paenibacillus polymyxa Cs12 for preventing and treating the saffron corm rot disease comprises the following steps:

selecting a saffron plot planted for 5 years continuously, dividing the saffron plot into three plots with the same size, and setting 2 parallel plots (20m multiplied by 4m) which are respectively a control group and a Cs12 group. The Cs12 group was inoculated with the inoculum of example 2 by root irrigation, 5mL per bulb, using the conventional cultivation management method as a control group. And digging the corms in the corm dormancy stage, counting the corm morbidity, and calculating the prevention and treatment effect. The result shows that the incidence rate of the corm rot disease of the plots applied with the paenibacillus polymyxa Cs12 is below 5 percent and is obviously lower than that of the control group which is not applied, which indicates that the fungicide prepared by the paenibacillus polymyxa Cs12 can effectively reduce the incidence rate of the corm rot disease of saffron crocus.

In summary, the embodiment of the present invention provides a paenibacillus polymyxa and applications thereof, a microbial inoculum comprising the paenibacillus polymyxa and a preparation method thereof:

The embodiment of the invention also provides a microbial inoculum prepared from the paenibacillus polymyxa Cs12, the microbial inoculum can be absorbed by the roots of saffron so as to play a role in inhibiting the growth of fusarium oxysporum at the roots of the saffron, and the fusarium oxysporum is a main pathogenic bacterium of the corm rot of the saffron, so that the incidence rate of the corm rot of the saffron can be reduced by inhibiting the growth of the fusarium oxysporum, and the microbial inoculum has important significance for the development of the saffron in China.

Fourthly, the embodiment of the invention also provides a using method of the microbial inoculum, and the method ensures that the microbial inoculum can effectively and reasonably inhibit the growth and development of fusarium oxysporum in a reasonable using amount and mode, thereby ensuring that the microbial inoculum has excellent inhibition effect.

The embodiments described above are some, but not all embodiments of the invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Claims

1. The paenibacillus polymyxa is characterized in that the paenibacillus polymyxa Cs12 is named, the preservation number of the paenibacillus polymyxa Cs12 is CGMCC No.22612, the preservation address is the microbiological research institute of China academy of sciences No.3 of Beijing West Lu No.1 Homex, Chaoyang, and the preservation date is 2021 year, 5 months and 26 days.

2. The Paenibacillus polymyxa according to claim 1, wherein the 16SrDNA sequence is shown in SEQ ID No. 1.

3. Use of paenibacillus polymyxa according to claim 1 or 2 for the preparation of a medicament for the control of saffron corm rot.

4. A microbial preparation obtained from the Paenibacillus polymyxa Cs12 according to claim 1 or 2.

5. The microbial preparation according to claim 4, wherein the concentration of Paenibacillus polymyxa Cs12 in the microbial preparation is 10⁷-10⁸CFU/mL。

6. The use of the bacterial preparation according to claim 4 or 5, wherein the method comprises the step of administering the bacterial preparation 5mL once every 3 days for 21-30 days.

7. Use according to claim 6, wherein the method of administration is by drenching or injection.

8. A method for preparing the microbial inoculum according to claim 4 or 5, which comprises the following steps of picking a single colony of the Paenibacillus polymyxa Cs12 under aseptic conditions, inoculating the single colony into an NA or PDA culture medium, and carrying out dark culture at 28-37 ℃ and 75% RH for 12-48h to obtain a purified colony;

under the aseptic condition, picking the purified bacterial colony, inoculating the bacterial colony in NB or PDB culture medium, and carrying out shaking culture at 28 ℃ and 180rpm for 24h to obtain seed bacteria;

inoculating the seed bacteria into NB or PDB culture medium, and fermenting at 28 ℃ and 180rpm for 48h to form fermentation liquor, thus obtaining the microbial inoculum.

9. The method for preparing microbial inoculum according to claim 8, wherein the NA culture medium comprises 10g of peptone, 3g of beef extract, 5g of sodium chloride and 20g of agar, and the pH of the NA culture medium is 7.0-7.4; the NB culture medium comprises 10g of peptone, 3g of beef extract and 5g of sodium chloride, and the pH value of the NB culture medium is 7.0-7.4. The PDA culture medium comprises water decoction of 200g of potato, 20g of glucose and 15g of agar; the PDB culture medium comprises 200g of water decoction of potatoes and 20g of glucose, and the pH value of the PDB culture medium is 7.0-7.4.

10. The method for preparing the microbial inoculum according to claim 8, wherein the inoculation is carried out according to the inoculation amount of the seed bacterial liquid to the NB culture medium in the volume ratio of 1 (10-100).