CN114806928A - Pepper endophytic Bacillus beleisi PEB23 and application thereof - Google Patents

Abstract

The invention belongs to the field of microorganism and biological control, and particularly relates to bacillus belgii (Bacillus subtilis) with wide antibacterial spectrumBacillus velezensis) And applications thereof. The strain is an endophytic antagonistic bacterium separated from a continuous cropping pepper field for many years, and the preservation number is CCTCCNO: m2021910. The strain has wide antimicrobial spectrum, has remarkable inhibiting effect on sclerotium rolfsii, Ralstonia solanacearum, phytophthora capsici, colletotrichum capsici and fusarium oxysporum f.sp.capsici, and has no bacteriaThe fermentation liquor also has the activity of inhibiting bacteria and fungi, the bacteria can generate siderophores and indole-3-acetic acid (IAA), and also has the capability of dissolving organic phosphorus, a pot experiment shows that the yield of the hot pepper can be increased by 24.7%, the control effect on the southern blight of the hot pepper is 58.8%, and the hot pepper has good disease-resistant and growth-promoting potential.

Description

Pepper endophytic Bacillus beleisi PEB23 and application thereof

Technical Field

The invention belongs to the field of microorganism and biological control, and particularly relates to Bacillus belgii (Bacillus subtilis) with a wider antibacterial spectrumBacillus velezensis) And applications thereof.

Background

The pepper is one of the vegetable crops with the largest planting area in China, the pepper industry is one of the dominant and characteristic industries in Hunan province, and the annual planting area is about 13 ten thousand hm ² . However, with the popularization of greenhouse agriculture such as vegetables and the like and continuous cropping, the soil-borne diseases of the peppers in Hunan province are serious year by year, wherein bacterial wilt and epidemic diseases are two destructive soil-borne diseases of the most serious peppers in the province, pathogenic bacteria of the bacterial wilt and the epidemic disease can survive in soil of a disease area for a long time and are difficult to radically cure, and the whole growth period of the peppers can be damaged, so that serious economic loss is caused. The fumigation of soil by chemical pesticide is an effective method for preventing and treating soil-borne diseases at present, but has high cost, difficult operation, serious damage to soil micro-ecology and environmental pollution. The biological control of soil-borne diseases using antagonistic microbial agents is believed to beThe green and environment-friendly prevention and treatment measures become the hot point of research in recent years. However, it is difficult to obtain an effective antagonistic microbe.

There are many reports on the screening and biological control of antagonistic bacteria of pepper soil-borne diseases (screening of bacillus haustoria, bushua, qiu german. lateral spore a60 and its indoor determination of prevention effect against phytophthora capsici [ J ] biotechnological report, 2017, 33 (9): 160 cake 165.; biological control study of soil-borne diseases such as plum, pepper, and tomato bacterial wilt [ D ]. nanjing: nanjing university of agriculture, 2008.; mein, prunus, zhao, shiyong, etc.. screening and identification of pepper disease antagonistic strains and their prevention effect [ J ] application of ecological report, 2010, 21 (10): 2652.; 201458.; king, liu diming, zhou xin, etc.. screening and identification of capsicum wilt bacteria antagonistic to endophytic bacteria and optimization of fermentation conditions [ J ]. southern agriculture report, 2014, 45 (1781): 1787.: myriad poplar 1787), development of trichoderma biocontrol in pepper blight [ J ] modern agricultural science 2015, (19): 127 and 129; "zhangmin, chendan, yanwanrong", et al, pepper phytophthora blight biological control technology research progress [ J ]. changjiang vegetables, 2013, (8): 1-4.), mainly focuses on the screening of antagonistic bacteria, antagonistic streptomycete and the like in rhizosphere soil, and the screening of endophytes is not common. Compared with soil microorganisms, endophytes can be planted in rhizosphere soil and enter plant bodies through root systems, have more stable living environment than microorganisms exposed to the external environment, and are easier to exert disease-resistant and growth-promoting effects, so that the wide prospect of development of plant endophytes as pollution-free biopesticides for the environment is shown (crystal filling, Chengyxin, Liuyue. plant endophytes and research progress of plant diseases prevention and control thereof [ J ]. ecological bulletin, 2006, 26 (7): 2395. supplement 2401; talent art, Wuyuanhua, Dixiling. plant endophyte research progress and problems existing in [ J ]. journal of ecology, 2004, 23 (2): 86-91.).

Disclosure of Invention

The inventor tries to collect plants which are still healthy in the peak period of disease attack from pepper fields which are continuously planted for many years, and separates an endophytic antagonistic bacterium PEB-23 from root systems of the plants, wherein the bacterial strain has obvious inhibiting effects on Ralstonia solanacearum, phytophthora capsici, colletotrichum capsici and fusarium oxysporum of pepper, a strain fermentation culture solution also has obvious antibacterial activity on a plurality of pathogenic fungi and bacteria, the endophytic antagonistic bacterium is preliminarily identified, and growth promoting potential factors of the endophytic antagonistic bacterium are measured. Finally, the present invention has been completed.

The present invention provides a Bacillus belgiiBacillus velezensis) The preservation number is CCTCCNO: m2021910.

Further provides the application of the Bacillus belgii in biological control. In particular, the biological control is for pepper crops. More preferably, the biological control subject is southern blight capsicum, ralstonia solanacearum, phytophthora capsici, colletotrichum capsici and/or fusarium oxysporum capsici.

The invention also provides application of the bacillus beiLeisi in promoting plant growth. In particular, the plant is a pepper crop.

The present invention also provides a biological control agent characterized by containing the Bacillus belgii as an active ingredient. Among them, the cultured Bacillus belgii cells are preferably used as the active ingredient.

The present invention further provides a plant growth promoter characterized by containing the Bacillus belgii as an active ingredient. Preferably, it is added to the fertilizer as a fertilizer additive.

Compared with the reported pepper antagonistic bacteria, the endophytic antagonistic bacterium PEB-23 has a wider antibacterial spectrum, and has obvious inhibition effects on various pepper pathogenic bacteria (pepper ralstonia solanacearum) and pepper pathogenic fungi (pepper southern blight, pepper phytophthora capsici, pepper colletotrichum and pepper fusarium wilt). The strain is identified by 16S rDNA sequencing, and the PEB-23 strain is determined to be Bacillus belgii (Bacillus subtilis)Bacillus velezensis). Besides plants with strong bacteriostatic activity, the bacteria can also produce iron carrier and indole-3-acetic acid (IAA), and have strong dissolved organic phosphorusThe capability of the fertilizer shows good disease-resistant growth-promoting potential, and the fertilizer has the potential of being developed into a biological control microbial agent for soil-borne diseases of the peppers or a disease-resistant growth-promoting microbial fertilizer.

Endogenous antagonistic bacteriaBacillus velezensisPEB23 has been deposited at the China center for type culture Collection (Wuhan university, Wuhan City, China) at 20/7/2021 with the deposition number CCTCC NO: m2021910.

Drawings

FIG. 1 is a colony morphology of the PEB-23 strain.

FIG. 2 spore morphology under a microscope of colonies of the PEB-23 strain.

FIG. 3 antagonistic bacteria PEB-23 against pathogenic bacteria of southern blight of CapsicumSclerotium rolfsii) The bacteriostatic activity of (1). Wherein, the middle of the PDA plate is inoculated with southern blight bacteria, and the upper and lower streaks are respectively inoculated with PEB23 for opposite cultivation; the lower panel is a control inoculated with southern blight bacteria alone).

FIG. 4 antagonizes the bacteriostatic activity of PEB-23 against other pepper pathogens. Wherein, 1. Ralstonia solanacearum (L.) ChiliRalstonia solanacearum) GM 1000; 2. phytophthora capsici (a) ((b))Phytophthora capsici) (ii) a 3. Colletotrichum capsici (C.), (B. cayenne)Colletotrichum capsici) (ii) a 4. Fusarium oxysporum (F.) (Fusarium oxysporum）。

FIG. 5 is a 16S rDNA phylogenetic tree of endogenous antagonist PEB-23.

FIG. 6 shows the control effect of endophyte PEB23 on southern blight of capsicum.

Detailed Description

The invention separates 1 strain of endophytic antagonistic bacterium PEB-23 from a plurality of years of continuous cropping pepper.

Materials and methods

1.1 test materials

Healthy pepper plants are collected from a pepper disease-resistant breeding test field of vegetable research institute of agricultural academy of Hunan province in 2017, the pepper variety is XingShu 105 planted in large area in Hunan, the test field is continuous 3a continuous cropping pepper, and bacterial wilt, epidemic disease and southern blight are serious.

Test phytopathogen Ralstonia solanacearum (Ralstonia solani) ((R))Ralstonia solanacearum) GM1000 provides for subsidiary professor of Chenwu university in Hunan agricultureSouthern blight of pepper (1)Sclerotium rolfsii) Phytophthora capsici (a), (b), (c) and (c)Phytophthora capsici) Colletotrichum capsici (Fr.), (Colletotrichum capsici) Fusarium oxysporum (F.oxysporum) of Fusarium wiltFusarium oxysporum) Provides a researcher of Zhengjing yuan of vegetable institute of agricultural academy in Hunan province.

The endophytic bacteria are separated by LB culture medium and 1/10 LB culture medium, the conventional bacteria culture adopts LB culture medium, the pathogenic fungus culture medium adopts PDA culture medium, the Ralstonia solanacearum adopts TTC culture medium (Huanhuan, Chengkai, Yangxinging, etc.. pepper bacterial wilt antagonistic bacteria screening and biological control effect [ J]Soil bulletin, 2010, 47 (6): 1225 + 1231.), and the liquid fermentation culture medium is Landy culture medium (Vietnamese, Lvfengxia, Luxiongxin, etc.Bacillus subtilisfmbJIsolation and characterization of lipopeptide antibacterial Agents [ J]Bioengineering, 2006, 22 (4): 644-649.). Except for the PDA culture medium, the pH value of the rest culture media is 7.0-7.2.

LB culture medium: 10g of tryptone, 10g of sodium chloride, 5g of yeast extract, 20g of agar and 1L of distilled water. Landy medium:

PDA culture medium: 200g of potato, 20g of glucose and 15g of agar powder, and adding water to 1000mL

1.2 test methods

1.2.1 isolation and purification of endophytes

Isolation of endophytes minor adjustments were made with reference to the existing literature (Yuan san. isolation of rice endophyte OsiSh-10 and its study of rice blast resistance and plant growth promotion [ D ]. Changsha: university of Hunan, 2015). Cutting off main roots and side roots of hot peppers, cleaning silt with clear water, placing the hot peppers in a superclean workbench for drying, then placing the hot peppers in disodium hydrogen phosphate for ultrasonic treatment for 1 min to further remove surface impurities, taking out roots, sequentially disinfecting the surfaces of the roots with absolute ethyl alcohol, 4% sodium hypochlorite and sodium thiosulfate, washing away residual disinfectants with a large amount of sterile water after disinfection, taking a cleaning solution of the last step, coating an LB (Langmuir-Blodgett) plate for detecting whether disinfection is thorough, and drying the thoroughly disinfected roots in the superclean workbench. And then putting the mixture into a sterilized mortar to be ground into slurry, taking 1, 10 and 50 mu L of juice to coat LB and 1/10 LB plates, culturing the juice in an incubator at 30 ℃ for 3-7 d, and selecting a single colony and streaking the single colony to the LB plate to obtain a pure culture.

Screening of endophyte antagonistic bacteria

Antagonism test of pepper pathogenic fungi adopts a confrontation culture method ^[14] Punching a plurality of holes on the outer edge of the growing hyphae of the pathogenic fungi cultured on the PDA flat plate for 2-3 d by using a puncher, taking out hyphae blocks by using an inoculation needle, placing the hyphae blocks in the center of a new PDA flat plate, marking or dotting the screened bacteria at a position 3 cm away from the center by using an inoculation ring, culturing for 3-5 d at 28 ℃, and observing and measuring the size of a bacteriostatic circle. The antagonism test for the bacterial wilt of hot pepper adopts a double-layer plate method, the bacterial wilt is added into a TTC culture medium, the plate is inverted, screened bacteria are spotted on the plate, the plate is cultured for 2 d, and the size of a bacteriostatic circle is observed and measured.

Preliminary identification of endogenous antagonistic bacteria

Extracting endophyte genome by using UNIQ-10 column type bacterial genome DNA extraction kit, and referring to kit instruction. After the endogenous antagonistic bacteria genome is extracted, sampling, carrying out agarose gel electrophoresis to detect the genome extraction quality, and determining the DNA concentration by using a micro-spectrophotometer Nano Drop One. Then, the 16S rDNA of the antagonistic bacteria is subjected to PCR amplification by using the genome DNA of the endogenous antagonistic bacteria as a template and the 16S rDNA 27F/1492R primer which is commonly used by the bacteria, and the amplification product is sent to be sequenced. PCR amplification system, system: PCR Mix 25. mu.L, primer 27F (10. mu. mol/L) 1. mu.L, primer 1492R (10. mu. mol/L) 1. mu.L, template DNA approximately 10 pmoL, and water was added to 50. mu.L. Amplification conditions: 5min at 98 ℃; 35 cycles of 94 ℃ for 35s, 55 ℃ for 35s, and 72 ℃ for 1 min; 10 min at 72 ℃. Adopting a DNA gel recovery kit, recovering a target DNA fragment by agarose gel electrophoresis, and sequencing by Hunan Optimalaceae biotechnology Limited; and performing homology comparison on the sequencing result in GenBank by using BLAST software, performing multi-sequence comparison by using ClustalX, and performing multi-sequence homology analysis by using software Mega 6 to construct a phylogenetic tree.

Preparation of bacterial agent containing endogenetic antagonistic bacteria PEB23 and pot culture test for preventing and treating capsicum sclerotium rolfsii

Activating live bacteria of endogenous antagonistic bacteria PEB23, inoculating to LB liquid culture medium, and culturing at 28 deg.CCulturing at 150rpm at deg.C to logarithmic phase to obtain seed liquid; inoculating the seed liquid into an LB liquid culture medium in an inoculation amount of 2% of the volume, and performing shake culture at 28 ℃ for 40-48h at 150rpm to obtain a fermentation liquid; pouring out the fermentation liquid, counting the number of effective viable bacteria in the final concentration of the fermentation liquid in a flat plate counting mode, centrifuging the bacterial liquid, discarding the supernatant, and diluting the fermentation liquid to 10 degrees by water according to the evaluation result ⁸ cfu/mL of solution for pepper potting experiments. The potting experiment was conducted in two groups, blank (CK) and treatment (PEB 23), each for 15 plants, with potting soil being 2Kg soil per pot, using vegetable field soil taken from the field. Pepper seedlings of a pre-transplanting treatment group (PEB 23) are planted after being treated for 30s by soaking roots with PEB23 bacterial liquid, and then the pepper plants are watered with the prepared microbial inoculum (the concentration of the bacterial liquid is 10) once every 10 days ⁸ cfu/ml), the using amount of the microbial inoculum is 10 ml/strain, and the watering is carried out for 2 times; the blank groups use clear water to replace microbial inoculum for root soaking treatment and irrigation of pepper plants, the management conditions of other water and fertilizers of each treatment group are completely the same, the fertilization is carried out according to the fertilization dosage of farmers, and medicaments for preventing and treating diseases are not used uniformly. After 15 days of planting, all seedlings were inoculated artificially with sclerotium rolfsii pathogen. After 10 days, the growth and the morbidity of the pepper are observed, and the morbidity and the disease index are counted.

Detection of indicator for promoting plant growth of endogenous antagonistic bacteria PEB-23

In order to predict whether the endogenous antagonistic bacteria PEB-23 has the potential of promoting plant growth besides disease-resistant plants, the method of rhizosphere literature (Li Chi Yu, Li Huixin, etc.. screening identification and characteristic research of a peanut rhizosphere growth-promoting bacterium [ J ]. ecological and rural environmental bulletin, 2012, 28 (4): 416-.

Growth-promoting potting test for endophytic antagonist PEB23 strain

Activating the live bacteria of endogenous antagonistic bacteria PEB23, inoculating in LB liquid culture medium,culturing at 28 deg.C and 150rpm to logarithmic phase to obtain seed solution; inoculating the seed liquid into an LB liquid culture medium in an inoculation amount of 2% by volume, performing shake culture at 28 ℃ and 150rpm for 48 hours, sampling and performing microscopic examination, and stopping culture after the spore rate exceeds 90% to obtain the product containing the antagonistic bacteria PEB 23; pouring out the fermentation liquid, counting the number of effective viable bacteria in the final concentration of the fermentation liquid in a flat plate counting mode, centrifuging the bacterial liquid, removing the supernatant, and diluting the fermentation liquid to 10 degrees by water according to the evaluation result ⁸ cfu/mL of solution for pepper potting experiments. The potting experiment was conducted in two groups, namely a blank group (CK) and a treatment group (PEB 23), 20 plants were planted in each group, and potting soil was vegetable field soil taken from the field, and 2Kg of soil was filled in each pot. Pepper seedlings of a pre-transplanting treatment group (PEB 23) are planted after being treated for 30s by soaking roots with PEB23 bacterial liquid, and then the pepper plants are watered with the prepared microbial inoculum (the concentration of the bacterial liquid is 10) once every 10 days ⁸ cfu/ml), the using amount of the microbial inoculum is 10 ml/strain, and the watering is carried out for 3 times; the blank groups use clear water to replace microbial inoculum for root soaking treatment and irrigation of pepper plants, the management conditions of other water and fertilizers of each treatment group are completely the same, the fertilization is carried out according to the fertilization dosage of farmers, and medicaments for preventing and treating diseases are not used uniformly. Observing the growth and disease incidence of the pepper 50 days after planting, and recording plant height and spread index; counting the fresh weight of all mature peppers in the effective harvest period of 50 days from the beginning of pepper plant fruiting, wherein the number of the fruiting is the number of the harvested peppers, and all data are subjected to statistical analysis by SPSS 22.0 software.

2 results and analysis

2.1 isolation and screening results of antagonistic bacteria

132 endophytic bacteria are separated from healthy plant root systems in severely ill fields, and 22 endophytic bacteria with bacteriostatic activity are primarily screened, wherein 1 strain with the number of PEB-23 has the effect of treating the southern blight of the pepper (1)Sclerotium rolfsii) Ralstonia solanacearum (L.) ChiliRalstonia solanacearum) GM1000, Phytophthora capsici (A) and (B)Phytophthora capsici) Colletotrichum capsici (Fr.), (Colletotrichum capsici) Fusarium oxysporum (F.oxysporum) of Fusarium wiltFusarium oxysporum) All have strong antagonistic action, and the average diameters of the inhibition zones are respectively23.8mm, 10.0 mm, 13.8 mm, 26.4 mm, 24.5 mm were reached, showing a broader bacteriostatic spectrum (fig. 4 and table 1).

The colony of PEB-23 is light yellow on LB plate culture medium, with obvious fold (figure 1), the thallus is rod-shaped under microscope, and can form spore (figure 2).

TABLE 1 bacteriostatic diameter of endogenous antagonistic bacterium PEB-23 against 5 pepper pathogens

2.3 16S rDNA sequence of endogenetic antagonistic bacterium and its homology analysis result

In order to further identify the strain species, the genome DNA of the PEB-23 strain mentioned by the kit is adopted, the genome of the PEB-23 strain is taken as a target, a primer 27F/1492R is successfully amplified to a band of about 1.5 kb, the size of a 16S rRNA gene fragment of the endogenous antagonistic bacterium PEB-23 after sequencing is 1427 bp, a phylogenetic tree is constructed by the sequence and a homologous sequence (figure 5), and the PEB-23 and the Bacillus belgii (Bacillus belgii) (a. and a. bacillus) are shown in the figureBacillus velezensis) CBMB205 (accession number: NZ CP 011973.1)) the relationship was recent, and the homology reached 99.65%.

>PEB-23

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Control effect of endophyte PEB23 on capsicum southern blight

Potted plant experiments are carried out, the prevention and control effect of the PEB23 strain on the capsicum sclerotium rolfsii is measured (figure 6), the results in table 2 show that the prevention and control effect of the PEB23 microbial inoculum on the capsicum sclerotium rolfsii can reach 58.8 percent, and the single use of the PEB23 microbial inoculum has better prevention and control effect on the capsicum sclerotium rolfsii.

TABLE 2 potted plant control effect of endophyte PEB23 on southern blight of capsicum

Group of	Number of diseased plants/total number of plants	Incidence (%)	Index of disease condition	Control effect (%)
					PEB23	5/15	33.3	29	58.8
CK	13/15	86.7	68	\

2.5 determination of the plant growth promoting potential of the endogenous antagonist PEB-23

The capability of PEB-23 for producing siderophore, producing indole-3-acetic acid (IAA), dissolving organic phosphorus, dissolving inorganic phosphorus, dissolving potassium, fixing nitrogen and the like is measured.

The bacteria siderophore detection adopts CAS plate, firstly preparing solution I, dissolving 60.5mg Chromium Azure (CAS) in 50ml ultrapure water, and 10ml Fe ³⁺ Mixing the solutions, and adding the mixed solution into a hexadecyl trimethyl ammonium bromide (HDTMA) solution; then, 0.3g of KH solution II was added ₂ PO ₄ 0.5g NaCl, 1.0g NH4Cl, 15g agar powder, 30.24g sodium guazatine diacetate (Pipes), adjusting the pH to 6.8; and preparing other solutions: 20% sucrose solution; hydrolyzing casein with 10% acid; 1mmol/L CaCl2, 10 mmol/L MgSO ₄ . Sterilizing alone, and autoclaving all culture media at 121 deg.C for 30 min. The above solution was melted by heating before placing the CAS plate, cooled to 50 ℃, mixed in proportions and poured onto the plate.

As can be seen from Table 3, endogenous antagonistic bacterium PEB-23 produced an orange-yellow halo on the CAS plate, indicating its siderophore productivity. Also, IAA can be produced by adding tryptophan. In addition, a large transparent hydrolysis loop can be generated on a culture medium with organic phosphorus as the only phosphorus source, but only a small hydrolysis loop can be generated on a culture medium with inorganic phosphorus as the only phosphorus source. The strain can grow well on potassium feldspar and the Artobia sibirica nitrogen-free culture medium, and the potassium decomposing activity and the nitrogen fixing activity are shown.

TABLE 3 determination of growth promotion index of endogenous antagonistic bacteria PEB-23

Growth promotion index

Iron production carrier

Produce IAA

Dissolving organic phosphorus

Dissolving inorganic phosphorus

Decomposing potassium

Fixation of nitrogen

Level of

++

++

+++

+

+

++

Note: "+ + + +" indicates that the item is very active, "+" indicates that the item is less active, and "-" indicates that there is no activity.

Growth promoting effect of endophyte PEB23 on pepper

The growth promoting effect of the PEB23 strain on pepper was measured by pot experiments, and the results are shown in table 4.

TABLE 4 growth promoting Effect of endophyte PEB23 on Pepper

Group of	Number of results (number/strain)	Yield (g/strain)	Plant height (cm)	Stentering (cm)
					PEB23	107±5.7	2013.2±273.4	49.0±2.7	45.0±3.2
CK	82±4.9	1614±123	44.4±2.1	39.2±2.2

As can be seen from the results in Table 4, the pepper plants in the treated group (PEB 23) are more vigorous than those in the control group, and the number of the treated pepper plants, yield, plant height and spread after 50 days of treatment have significant advantages (p is less than 0.05) compared with those in the blank group, wherein the plant height is increased by 10.3%, the spread is increased by 14.8%, and the yield is increased by 24.7%. The siderophore endophyte PEB23 is shown to have obvious growth promoting effect on the pepper and has the potential of being developed into microbial fertilizer.

<110> institute of microorganisms of Hunan province

<120> Bacillus belezii PEB23 growing in capsicum and application thereof

<160> 1

<210> 1

<211> 1336

<212> DNA

<213> Bacillus belgii (Bacillus velezensis)

<400> 1

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Claims (10)

1. Bacillus belgii (B.), (B.), (B.beijerinckii)Bacillus velezensis) The preservation number is CCTCC NO: m2021910.

2. Use of bacillus belgii according to claim 1 for biological control.

3. The use as claimed in claim 2, wherein the biological control is for pepper crops.

4. Use according to claim 2, wherein the biological control subject is southern pepper sclerotinia rot, pepper ralstonia solanacearum, pepper phytophthora capsici, pepper colletotrichum and/or pepper fusarium wilt.

5. Use of bacillus belgii according to claim 1 for promoting plant growth.

6. The use of claim 2, wherein the plant is a pepper crop.

7. A biocontrol agent characterized in that it contains the Bacillus belgii of claim 1 as an active ingredient.

8. The biocontrol agent according to claim 7, characterized in that it is in the form of cultured Bacillus belgii cells as an active ingredient.

9. A plant bio-enhancer, characterized by comprising the Bacillus belgii of claim 1 as an active ingredient.

10. A plant bio-enhancer according to claim 9, which is added to a fertilizer as a fertilizer additive.

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