Application of Bacillus brevis LYMC-3 in promoting the growth of Masson pine

Abstract

The invention discloses the application of Bacillus pumilus LYMC-3 in promoting the growth of Masson pine, and belongs to the technical field of plant protection. The present application discloses a new use of Bacillus pumilus LYMC-3, that is, the application of Bacillus pumilus LYMC-3 in promoting the growth of Masson pine. The fermentation liquid of Bacillus pumilus LYMC-3 endogenous to Masson pine of the present application can promote the increase of Masson pine biomass; can promote Masson pine to synthesize indoleacetic acid and zeatin, reduce the synthesis of abscisic acid; can improve the root activity of Masson pine; can improve the absorption capacity of Masson pine to nitrogen, phosphorus and potassium; can improve the net photosynthetic rate, intercellular carbon dioxide, transpiration rate and stomatal conductance of Masson pine.

Description

Technical Field

[0001] The invention belongs to the technical field of plant protection, and more specifically relates to application of Bacillus pumilus LYMC-3 in promoting the growth of Masson pine. Background Art

[0002] Masson pine is an important tree species for afforestation and timber production in my country. It is also a pioneer tree species for land reclamation in my country and has both economic and ecological value. At present, the prevention and control of plant diseases mainly relies on drug control, but the long-term use of chemical control has enhanced the resistance of pathogens, and ordinary pesticides cannot kill them, making the disease difficult to control. Therefore, biological control is safe and sustainable, and has gradually become a hot topic in the research on the prevention and control of plant diseases. The use of microorganisms to replace or partially replace chemical fertilizers and pesticides has attracted more and more attention from researchers.

[0003] Anything that can be isolated from a plant tissue that has been detached from the surface, or extracted from the inside of a plant, and if it does not obviously harm the plant, is an endophytic bacterium. Plant endophytes can provide some benefits to the host plant, especially promoting growth and resisting pathogens. The growth-promoting mechanism of endophytic bacteria is mainly manifested in the following two aspects: first, it promotes plant growth through nitrogen-fixing bacteria themselves, or endophytic bacteria produce IAA, which can promote plant growth hormone; second, it indirectly promotes plant growth by inducing plants to produce IAA, inhibiting the synthesis of abscisic acid that originally inhibits the synthesis of IAA, and enhancing the plant's ability to absorb nutrients.

[0004] Bacillus pumilus is a common biocontrol microorganism with multiple functions such as biological control, biodegradation and promotion of plant growth. Bacillus pumilus can prevent and control a variety of plant diseases, such as rice blast, chickpea root rot, cucumber wilt and cotton wilt, etc., which have significant control effects. Therefore, in agricultural production, the use of biological agents of biological bacteria Bacillus to replace chemical pesticides and fungicides has a very broad prospect. It can not only inhibit the occurrence of plant diseases and insect pests, but also maintain the stability of the ecosystem to a large extent, and has little side effects on human health and the environment. LYMC-3 is an endophytic bacterium isolated from Pinus massoniana. It has been found that its fermentation liquid has a strong ability to kill nematodes. However, there is little research on the function of LYMC-3 in promoting plant growth. Summary of the invention

[0005] In view of the above problems existing in the prior art, the technical problem to be solved by the present invention is to provide a new application of Bacillus pumilus LYMC-3 to meet the new demand of Bacillus pumilus LYMC-3 in promoting the growth of Masson pine.

[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0007] Application of Bacillus brevis LYMC-3 in promoting the growth of Masson pine.

[0008] Furthermore, the promoting of Masson pine growth includes one or more of promoting the increase of Masson pine biomass, promoting the synthesis of indoleacetic acid by Masson pine, promoting the synthesis of zeatin by Masson pine, reducing the synthesis of abscisic acid by Masson pine, improving the root activity of Masson pine, improving the absorption capacity of Masson pine for nitrogen, phosphorus and potassium, improving the net photosynthetic rate of Masson pine, increasing the intercellular carbon dioxide of Masson pine, and improving the transpiration rate and stomatal conductance of Masson pine.

[0009] Application of Bacillus brevis LYMC-3 in promoting biomass increase of Masson pine.

[0010] Application of Bacillus pumilus LYMC-3 in promoting the synthesis of indoleacetic acid in Masson pine.

[0011] Application of Bacillus pumilus LYMC-3 in promoting zeatin synthesis in Masson pine.

[0012] Application of Bacillus brevis LYMC-3 in reducing abscisic acid synthesis in Masson pine.

[0013] Application of Bacillus brevis LYMC-3 in improving root activity of Masson pine.

[0014] Application of Bacillus pumilus LYMC-3 in improving the absorption capacity of Masson pine for nitrogen, phosphorus and potassium.

[0015] Application of Bacillus pumilus LYMC-3 in improving the net photosynthetic rate, intercellular carbon dioxide, transpiration rate and stomatal conductance of Masson pine.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] (1) The fermentation broth of Bacillus pumilus LYMC-3 prepared in the present invention can promote the increase of the biomass of Masson pine.

[0018] (2) The fermentation liquid of Bacillus pumilus LYMC-3 prepared by the present invention can promote the synthesis of indoleacetic acid and zeatin by Masson pine and reduce the synthesis of abscisic acid.

[0019] (3) The fermentation liquid of Bacillus pumilus LYMC-3 prepared by the present invention can improve the root activity of Masson pine.

[0020] (4) The fermentation liquid of Bacillus pumilus LYMC-3 prepared by the present invention can improve the absorption capacity of Masson pine for nitrogen, phosphorus and potassium.

[0021] (5) The fermentation liquid of Bacillus pumilus LYMC-3 prepared by the present invention can increase the net photosynthetic rate, intercellular carbon dioxide, transpiration rate and stomatal conductance of Masson pine. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The effect of LYMC-3 on the growth of Masson pine (A: 50 mL fermentation liquid, B: 100 mL fermentation liquid, C: 200 mL fermentation liquid, D: 300 mL fermentation liquid);

[0023] Figure 2 The figure shows the effect of different inoculation amounts on root growth (A: 50 mL fermentation liquid, B: 100 mL fermentation liquid, C: 200 mL fermentation liquid, D: 300 mL fermentation liquid);

[0024] Figure 3 The standard solution diagrams of indoleacetic acid IAA, abscisic acid ABA, and zeatin ZeatiK (A: IAA standard curve, B: ABA standard curve, C: ZeatiK standard curve);

[0025] Figure 4 This is the effect of strain LYMC-3 on the IAA and ZeatiK content of Masson pine seedlings;

[0026] Figure 5 This is the effect of strain LYMC-3 on ABA content in Masson pine seedlings;

[0027] Figure 6 Mass spectra of IAA of Masson pine seedlings inoculated with strain LYMC-3 (AC: peak area; a: 5 ng / mL IAA standard solution; b: treatment; c: control);

[0028] Figure 7 The mass spectra of ABA in Masson pine seedlings inoculated with strain LYMC-3 (AC: peak area; a: 5 ng / mL IAA standard solution; b: treatment; c: control);

[0029] Figure 8 The mass spectra of ZeatiK in Masson pine seedlings inoculated with strain LYMC-3 (AC: peak area; a: 5 ng / mL IAA standard solution; b: treatment; c: control);

[0030] Fig. 9 is the standard curve of triphenylmethylhydrazone (TTF);

[0031] Fig.10 The effect of different inoculation concentrations on the root activity of Masson pine (A: 50mL fermentation liquid, B: 100mL fermentation liquid, C: 200mL fermentation liquid, D: 300mL fermentation liquid);

[0032] Fig.11The figure shows the effect of different inoculation amounts on the total nitrogen content of Masson pine plants (A: 50mL fermentation liquid, B: 100mL fermentation liquid, C: 200mL fermentation liquid, D: 300mL fermentation liquid);

[0033] Fig.12 The figure shows the effect of different inoculation amounts on the total phosphorus content of Masson pine plants (A: 50mL fermentation liquid, B: 100mL fermentation liquid, C: 200mL fermentation liquid, D: 300mL fermentation liquid);

[0034] Fig.13 The figure shows the effect of different inoculation amounts on the total potassium content of Masson pine plants (A: 50mL fermentation liquid, B: 100mL fermentation liquid, C: 200mL fermentation liquid, D: 300mL fermentation liquid); DETAILED DESCRIPTION

[0035] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described below in conjunction with specific embodiments. In the following embodiments, unless otherwise specified, the technical means used are conventional means well known to those skilled in the art.

[0036] The 2.5-year-old Masson pine seedlings of the present invention are from a nursery base in Shuyang County, Jiangsu Province, and an inoculation experiment is started after the seedlings have been grown for 1 month.

[0037] Indoleacetic acid (IAA) standard sample, abscisic acid (ABA) standard, and zeatin (ZeatiK) standard were purchased from Beijing Century Aoke Biotechnology Co., Ltd., and chromatography-grade methanol was purchased from Nanjing Juxionghua Biological Co., Ltd.

[0038] Nitrogen blowdown instrument, LTQ Orbitrap XL liquid chromatography-mass spectrometer (Thermo Fisher Scientific, USA), desktop high-speed centrifuge, AR5120 electronic balance (AHOMS, USA)

[0039] Example 1: Effect of inoculation with Bacillus pumilus LYMC-3 on growth indicators of Masson pine

[0040] 1. Test strains

[0041] Bacillus pumilus LYMC-3 was isolated and screened from the stems of Pinus massoniana in Luoyang Sui and Tang Botanical Garden. It has been deposited in China Center for Type Culture Collection with the deposit number: CCTCC NO: M 2016775.

[0042] 2. Preparation of Bacillus pumilus LYMC-3 fermentation broth

[0043] The strain LYMC-3 was cultured in bacterial fermentation medium NB at 28°C and 200 r / min for 72 h and then used.

[0044] Bacterial fermentation medium NB: beef extract 5g, peptone 10g, glucose 10g, sodium chloride 5g, pH 8, distilled water 1L;

[0045] 3. Inoculation of Bacillus pumilus LYMC-3

[0046] Root inoculation method: drill holes in the soil around the roots of Masson pine, inject 50mL, 100mL, 200mL, and 300mL of liquid bacterial agent respectively, and inoculate 50mL of sterile culture medium in the control group. Set up 6 replicates for each treatment.

[0047] 4. The results are shown in Table 1. Eight months after inoculation with the LYMC-3 strain, the overall growth-promoting effect was best when the inoculation volume was 200 mL (treatment C), with the seedling height and ground diameter growth rates reaching 40.74% and 36.63%, respectively, and the fresh weight growth rate reaching 68.68%; the effect was second when the inoculation volume was 300 mL (treatment D), with the seedling height and ground diameter growth rates reaching 42.31% and 31.34%, respectively, and there was no significant difference between treatment C and treatment D. The effect was weakest when the inoculation volume was 50 mL (treatment A), with the Masson pine seedling height and ground diameter growth rates only reaching 13.12% and 9.26%, respectively. It can be seen from the comprehensive inoculation results that ( Figure 1 ), inoculation with LYMC-3 had a significant growth-promoting effect on Pinus massoniana, and the growth conditions of Pinus massoniana treated with the four inoculations were significantly better than those of the control.

[0048] There were significant differences in the effects of inoculation with LYMC-3 on the total biomass of Masson pine plants. Among the four inoculation methods, the best effect was achieved when inoculated with 200 mL, with a fresh weight growth rate of 68.68%; the effect of inoculation with 300 mL was second, with a fresh weight growth rate of 63.56% compared with the control, which was not significantly different from treatment C. The effect of inoculation with 50 mL (treatment A) was the worst, with a fresh weight growth rate of only 19.89%. The growth conditions of Masson pine treated with the four inoculation methods were significantly better than those of the uninoculated control.

[0049] Table 1 Effects of different inoculation amounts of LYMC-3 fermentation broth on the growth of Masson pine

[0050]

[0051]

[0052] Different letters in the same column of data indicate significant differences (p < 0.05). The LSD method was used to test the degree of difference between treatments for homogeneity of variance, and the Dunnett's t test was used for heterogeneity of variance. A: 50 mL fermentation broth, B: 100 mL fermentation broth, C: 200 mL fermentation broth, D: 300 mL fermentation broth

[0053] 5. After 8 months, the root systems of different treatments were scanned using a root scanner. Figure 2 As can be seen from Table 2, the inoculated strain LYMC-3 can significantly improve the growth of the root system, and the root system grows vigorously. Among the different treatments, when the inoculation amount is 200 mL of fermentation liquid (treatment C), the root system grows best, and the total root length, total surface area, root volume, total root tip number and branch number are 512.47 cm, 307.97 cm, respectively. 2 、13.24cm 3 , 2228 and 8999, which were 651.73%, 314.29%, 417.80%, 247.83% and 260.99% of the control, respectively. The average diameter of treatment C was smaller than that of the control because treatment C had abundant lateral roots and new roots, and the diameter of lateral roots was smaller, resulting in a smaller average diameter of treatment C.

[0054] Table 2 Effects of different inoculation rates on root growth

[0055]

[0056] Different letters in the same column of data indicate significant differences (p < 0.05). The LSD method was used to test the degree of difference between treatments for homogeneity of variance, and the Dunnett's t test was used for heterogeneity of variance. A: 50 mL fermentation broth, B: 100 mL fermentation broth, C: 200 mL fermentation broth, D: 300 mL fermentation broth

[0057] Example 2: Effect of inoculation with Bacillus pumilus LYMC-3 on endogenous hormones of Masson pine

[0058] 1. Extract hormones:

[0059] Accurately weigh about 1g of fresh plant sample, grind in liquid nitrogen until crushed; add 10mL of isopropanol / hydrochloric acid extraction buffer to the powder, shake at 4℃ for 30min; add 20mL of dichloromethane, shake at 4℃ for 30min; centrifuge at 4℃, 10000r / min for 10min, take the lower organic phase; protect from light, blow dry the organic phase with nitrogen, dissolve with 400μL of methanol (0.1% formic acid); filter through 0.22μm membrane, and perform HPLC-MS / MS detection. The experimental data were statistically analyzed using EXCEL and SPSS 21.0 software.

[0060] 2. Standard curve

[0061] Prepare standard solutions of IAA, ABA, and Zeatin with a gradient of 0.5 ng / mL, 2 ng / mL, 5 ng / mL, 20 ng / mL, 50 ng / mL, and 200 ng / mL using methanol (0.1% formic acid) as solvent. Repeat 3 times for each concentration, and remove points with poor linearity when drawing the standard curve ( Figure 3 ).

[0062] 3. 8 months after vaccination, the hormone levels in the body were detected by liquid chromatography-mass spectrometry. Figure 4 It can be seen that the growth-promoting hormones IAA and ZeatiK in the Masson pine inoculated with LYMC-3 are higher than the control, 0.033ng / g and 0.11ng / g respectively, while the IAA and ZeatiK contents of the control are 0.012ng / g and 0.10ng / g respectively. The IAA in the treatment group is 2.5 times that of the control group, while the ZeatiK is only 1.1 times. It can be seen that the strain LYMC-3 has a greater effect on the synthesis of IAA. Figure 5 It can be seen that the abscisic acid ABA in the treatment with bacteria is significantly lower than that in the control. The treatment is 4.77ng / g, while the control is 5.37ng / g. Abscisic acid ABA is a hormone that inhibits plant growth. The inoculated strain LYMC-3 can significantly reduce the synthesis of abscisic acid ABA, thereby promoting plant growth.

[0063] Depend on Figure 6-8 It can be seen that the mass spectra of the three endogenous hormone standards all have stable peak times and peak areas, indicating that there is no interference from other impurities in the standards. The mass spectra of IAA and ZeatiK of the Masson pine sample have more peaks, and there are more isomers in the sample. The mass spectra of ABA of the Masson pine seedling sample have relatively fewer peaks.

[0064] Table 3 Plant hormone detection results after inoculation of strain LYMC-3

[0065]

[0066] Example 3: Effect of inoculation with Bacillus pumilus LYMC-3 on root activity of Masson pine

[0067] 1. Improved TTC method for determining root activity

[0068] Weigh 0.5g of root tip sample, put it in a weighing dish, add 10mL of equal amount of mixed solution of 0.4% TTC solution and phosphate buffer, immerse the root fully in the solution, keep warm in the dark at 37℃ for 2h, then add 2mL of 1moL / L sulfuric acid to stop the reaction (at the same time, do a blank test, add sulfuric acid first, then add root sample, other operations are the same as above). Take out the root, absorb the water, put it in a 20mL test tube, add 10mL methanol, cover it with a sieve, put it in a dark place and let it stand for several hours until the root turns white, use a spectrophotometer at a wavelength of 485nm, measure the absorbance with the blank test as a reference, check the standard curve, and calculate the tetrazolium reduction amount. The calculation formula is as follows:

[0069] Tetrazolium reduction intensity (mg / g / h) = tetrazolium reduction amount (mg) / root weight (g) / time (h).

[0070] 2. TTC standard curve

[0071] Take 0.2mL of 0.4% TTC solution and put it into a 10mL volumetric flask. Add a little Na2O4S2 and shake well to produce red triphenylmethylhydrazone. Use ethyl acetate to make up to the mark. Take 0.20mL, 0.40mL, 0.80mL, 1.20mL, and 1.60mL of this solution and put them into a 10mL volumetric flask. Use ethyl acetate to make up to the mark to obtain a standard colorimetric series containing 20μg, 40μg, 80μg, 120μg, and 160μg of triphenylmethylhydrazone. Use the blank as a reference, measure the absorbance at 480nm, and draw a standard curve ( Fig. 9 ).

[0072] 3. Eight months after inoculation, the root activity of Masson pine seedlings inoculated with strain LYMC-3 was measured. The results showed that ( Fig.10 ), different inoculation amounts of LYMC-3 bacterial liquid can improve the vitality of the Masson pine root system. The tetrazolyl reduction strength of Masson pine inoculated with 50mL, 100mL, 200mL, and 300mL fermentation liquid was 111.4mg / g / h, 127.3mg / g / h, 138.6mg / g / h, and 136.5mg / g / h, respectively, all higher than the control of 90.3mg / g / h. When the inoculation amount was 200mL and 300mL, the difference in root activity was not significant.

[0073] Example 4: Effect of inoculation with Bacillus pumilus LYMC-3 on total nitrogen, total phosphorus and total potassium contents of Masson pine

[0074] 1. Take the Masson pine seedlings to be tested, dry them, take 1g of roots, stems and leaves, grind them, pass them through a 0.5mm sieve, weigh 0.050g and place it at the bottom of a Kjeldahl flask, and determine the nitrogen content by the Kjeldahl method.

[0075] Nitrogen determination mixed indicator: weigh 0.5g bromocresol green and 0.1g methyl red and dissolve them in 100mL 95% ethanol. The preparation and calibration of hydrochloric acid standard solution shall be carried out according to GB601.

[0076] The calculation formula for the total nitrogen (N) content (mg / g) of Masson pine plants is as follows:

[0077] Total nitrogen (N) = C (V-V0) × 0.014 × 100 (m × V2 / V1)

[0078] C is the concentration of the acid standard solution (moL / L), V is the acid standard solution used to titrate the sample, V0 is the acid standard solution used to titrate the blank, 0.014 is the millimolar mass of K (g / mmoL), m is the sample size (g), V1 is the volume of the digestion solution (mL), and V2 is the volume of the digestion solution for absorption determination (mL).

[0079] like Fig.11 As shown in the figure, inoculation with LYMC-3 can improve the absorption capacity of Masson pine for N, and the absorption capacity increases with the increase of inoculation volume between 50 and 200 mL. The total nitrogen content of Masson pine inoculated with 50 mL, 100 mL, 200 mL, and 300 mL of fermentation liquid is 6.45 mg / g, 7.23 mg / g, 7.56 mg / g, and 7.49 mg / g, respectively, which are all higher than the control 6.32 mg / g. Among the four different inoculation concentrations, the inoculation of 200 mL (treatment C) has the best effect, and the total nitrogen content in Masson pine seedlings increased by 19.62%; the effect of inoculation with 300 mL (treatment D) is second, but the difference with the former is not significant, and the total N content growth rate reaches 18.51%; while the effect of inoculation with 50 mL (treatment A) is the worst, and the total N content growth rate only reaches 2.06%. It can be seen from the comprehensive inoculation results that the inoculation strain LYMC-3 has a significant effect on improving the absorption of Masson pine N, and the best effect is achieved at 200 mL.

[0080] 2. The method for determining total phosphorus and total potassium in Masson pine seedlings is as follows (Wang Meiqin et al. Growth-promoting effect of walnut endophyte HT3 on tomato and cucumber [J]. Shanxi Agricultural Science, 2013, 41 (2): 130-132.). Take the Masson pine seedlings to be tested, dry them, grind them and pass them through a 0.25 mm sieve, accurately weigh 0.500 g, and place them in a clean conical flask. Add 25 mL of mixed acid (KHO3: HClO4 = 5: 1) to each bottle, place a small funnel at the mouth of the bottle, add glass beads with a diameter slightly larger than the diameter of the funnel, and place them steadily overnight. The next day, place them on an electric hot plate to boil and keep boiling. When a large amount of white smoke disappears and the solution is colorless and transparent, remove the conical flask and dissolve it to 25 mL with deionized water for testing, and make a blank control at the same time. Use a 4300DV inductively coupled plasma emission spectrometer to determine the total phosphorus and total potassium content.

[0081] like Fig.12As shown in the results, the inoculation strain LYMC-3 can improve the absorption capacity of Masson pine for P. The total phosphorus content of Masson pine inoculated with 50mL, 100mL, 200mL, and 300mL fermentation liquid was 6.76mg / g, 7.77mg / g, 8.43mg / g, and 8.43mg / g, respectively, which were all higher than the control 6.06mg / g. There were significant differences between different inoculation concentrations when inoculated with 50mL to 200mL. Different inoculation concentrations caused different total phosphorus contents in Masson pine plants. Among the four different inoculation concentrations, the inoculation of 200mL (treatment C) had the best effect, and the total P content in Masson pine seedlings increased by 39.18%; the inoculation of 300mL (treatment D) was the second best, but the difference was not significant, and the total P content growth rate reached 39.09%; while the inoculation of 50mL (treatment A) had the worst effect, and the total P content growth rate only reached 11.63%. The comprehensive inoculation results show that the inoculated strain LYMC-3 has a significant effect on improving the absorption energy of Masson pine P, and the best effect is achieved when the inoculation volume is 200-300 mL.

[0082] like Fig.13 As shown in the results, the inoculated strain LYMC-3 can improve the absorption capacity of Masson pine for K. Between the inoculation amount of 50 and 200 mL, the absorption capacity increases with the increase of the inoculation amount, and the difference is significant. The total potassium content of Masson pine inoculated with 50 mL, 100 mL, 200 mL, and 300 mL of fermentation liquid is 2.23 mg / g, 2.35 mg / g, 2.48 mg / g, and 2.44 mg / g, respectively, which are all higher than the control of 1.98 mg / g. Among the four different inoculation concentrations, the inoculation of 200 mL (treatment C) has the best effect, and the total potassium content in Masson pine seedlings increased by 25.25%; the effect of inoculation of 300 mL (treatment D) is second, but the difference is not significant with the former, and the total K content growth rate reaches 23.23%; while the effect of inoculation of 50 mL (treatment A) is the worst, and the total K content growth rate only reaches 12.63%. From the comprehensive inoculation results, it can be seen that the inoculated strain LYMC-3 has a significant effect on improving the absorption capacity of Masson pine K, and the best effect is achieved when the inoculation volume is 200mL.

[0083] Example 5: Effect of inoculation with Bacillus pumilus LYMC-3 on photosynthesis of Masson pine

[0084] 1. The LI-6400 portable photosynthesis meter from LI-COR of the United States was used to measure the needle clusters at the top of the plants. Each treatment was repeated 3 times. The measurement time was 9 am. The measurement indicators included leaf net photosynthetic rate, transpiration rate, stomatal conductance, and intercellular carbon dioxide concentration.

[0085] 2. After 8 months of inoculation of Masson pine with strain LYMC-3, the results are shown in Table 4. The effects of different inoculation concentrations on the photosynthesis parameters of Masson pine showed an increasing trend within a certain concentration. Among the four concentrations of strain LYMC-3 inoculation, the inoculation of 200mL (treatment C) had the greatest effect on the photosynthesis of Masson pine. The net photosynthetic rate (PK) value, intercellular carbon dioxide (Ci) and transpiration rate (Tr) values ​​of Masson pine increased by 38.91%, 11.51% and 65.20% respectively compared with the control; the effect of the inoculation amount of 300mL (treatment D) was second but the difference was not significant. The values ​​of the first three parameters increased by 38.59%, 9.88% and 63.44% respectively compared with the control. Treatment D had the largest stomatal conductance (GS) value, which was 47.62% higher than the control, and treatment C had a 38.10% higher GS value than the control, and the difference was not significant; the effect of 50 mL inoculation (treatment A) was the worst, with the growth rates of the first four parameters only reaching 15.11%, 14.29%, 2.75% and 19.38%. The intercellular carbon dioxide (Ci) value was not significantly different between different treatments.

[0086] Table 4 Effects of different inoculation amounts on photosynthetic parameters of Masson pine

[0087]

[0088]

[0089] Different letters in the same column of data indicate significant differences (p < 0.05). The LSD method was used to test the degree of difference between treatments when the variance was homogeneous, and the Dunnett's t test was used to test the degree of difference between treatments when the variance was heterogeneous.

Claims

1. Bacillus brevis ( Bacillus pumilus ) The application of LYMC-3 in improving the intercellular carbon dioxide concentration, transpiration rate and stomatal conductance of Masson pine is characterized by: The Bacillus pumilus LYMC-3 is deposited in China Center for Type Culture Collection with the deposit number: CCTCC NO: M 2016775.

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