Application of Bacillus brevis LYMC-3 in inducing disease resistance in Masson pine

Abstract

The present invention discloses the application of Bacillus pumilus LYMC-3 in inducing disease resistance of Masson pine, and belongs to the technical field of plant protection. The present application discloses a new use of Bacillus pumilus LYMC-3, namely, the application of Bacillus pumilus LYMC-3 in enhancing the resistance of Masson pine. The Bacillus pumilus LYMC-3 described in the present invention can promote the synthesis of H2O2 in plants, increase the CAT activity in plants, increase the POD activity in plants, increase the SOD activity in plants, increase the PPO activity in plants, and increase the PAL activity in plants.

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 inducing disease resistance 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] Under normal circumstances, the active oxygen metabolism in plants is in a dynamic equilibrium state, and the active oxygen content is at a low level. When subjected to environmental stress, active oxygen can accumulate in plants and cause cell damage. Changes in the activity of plant defense enzymes are important physiological and biochemical reactions in which biocontrol bacteria induce plant disease resistance. The main defense enzymes involved include superoxide dismutase (SOD), phenylalanine ammonia lyase (PAL), peroxidase (POD), polyphenol oxidase (PPO) and other defense enzymes.

[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 treat a variety of plant diseases, such as rice blast, chickpea root rot, cucumber wilt and cotton wilt, etc., all have significant control effects. Therefore, in agricultural production, the use of biological agents of biological bacteria Bacillus to replace the effect of chemical pesticides and bactericides has a very broad prospect, which can not only inhibit the occurrence of plant diseases and insect pests, but also maintain the stability of the ecosystem to a great extent, and also have little side effects on human health and the environment. Chinese patent application CN106754586A discloses a strain of pine endophytic bacteria Bacillus pumilus LYMC-3, which has a strong effect of killing pine wood nematodes, and there is no related report of induced disease resistance. 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 application of Bacillus pumilus LYMC-3 in inducing disease resistance 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 inducing disease resistance in Masson pine.

[0008] Furthermore, the inducing disease resistance of Masson pine includes one or more of inducing Masson pine to synthesize H2O2, inducing enhanced CAT activity of Masson pine, inducing enhanced POD activity of Masson pine, inducing enhanced SOD activity of Masson pine, inducing enhanced PPO activity of Masson pine, and inducing enhanced PAL activity of Masson pine.

[0009] Application of Bacillus brevis LYMC-3 in inducing H2O2 synthesis in Masson pine.

[0010] Application of Bacillus brevis LYMC-3 in inducing enhanced CAT activity in Masson pine.

[0011] Application of Bacillus brevis LYMC-3 in inducing enhanced POD activity in Masson pine.

[0012] Application of Bacillus brevis LYMC-3 in inducing enhanced SOD activity in Masson pine.

[0013] Application of Bacillus brevis LYMC-3 in inducing enhanced PPO activity in Masson pine.

[0014] Application of Bacillus brevis LYMC-3 in inducing enhanced PAL activity in Masson pine.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] The Bacillus pumilus LYMC-3 described in the present invention can promote the synthesis of H2O2 in plants, can increase the CAT activity in plants, can increase the POD activity in plants, can increase the SOD activity in plants, can increase the PPO activity in plants, and can increase the PAL activity in plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a graph showing the H2O2 content in the needles of Masson pine seedlings after inoculation with strain LYMC-3 (LYMC-3: inoculated strain, CK1: inoculated sterile culture medium);

[0018] Figure 2 This is a graph of H2O2 content in Masson pine needles after inoculation with pine wood nematode AMA3 (LYMC-3+AMA3: inoculation with strain LYMC-3 followed by inoculation with pine wood nematode AMA3, AMA3: inoculation with sterile culture medium followed by inoculation with pine wood nematode AMA3; CK2: inoculation with sterile water);

[0019] Figure 3 This is a graph of CAT activity in Masson pine needles after inoculation with strain LYMC-3 (LYMC-3: inoculated strain, CK1: inoculated sterile culture medium);

[0020] Figure 4 This is a graph of CAT activity in Masson pine needles after inoculation with pine wood nematode AMA3 (LYMC-3+AMA3: inoculation with strain LYMC-3 followed by inoculation with pine wood nematode AMA3; AMA3: inoculation with sterile culture medium followed by inoculation with pine wood nematode AMA3; CK2: inoculation with sterile water);

[0021] Figure 5 This is a graph of POD activity in Masson pine needles after inoculation with strain LYMC-3 (LYMC-3: inoculated strain, CK1: inoculated sterile culture medium);

[0022] Figure 6 This is a graph of POD activity in Masson pine needles after inoculation with pine wood nematode AMA3 (LYMC-3+AMA3: inoculation with strain LYMC-3 followed by inoculation with pine wood nematode AMA3; AMA3: inoculation with sterile culture medium followed by inoculation with pine wood nematode AMA3; CK2: inoculation with sterile water);

[0023] Figure 7 This is the SOD activity of Masson pine needles after inoculation with strain LYMC-3 (LYMC-3: inoculated strain, CK1: inoculated sterile culture medium);

[0024] Figure 8 This is a graph of SOD activity in Masson pine needles after inoculation with pine wood nematode AMA3 (LYMC-3+AMA3: inoculation with strain LYMC-3 followed by inoculation with pine wood nematode AMA3; AMA3: inoculation with sterile culture medium followed by inoculation with pine wood nematode AMA3; CK2: inoculation with sterile water);

[0025] Fig. 9 This is a graph of PPO activity in Masson pine needles after inoculation with strain LYMC-3 (LYMC-3: inoculated strain, CK1: inoculated sterile culture medium);

[0026] Fig.10 This is a graph of PPO activity in Masson pine needles after inoculation with pine wood nematode AMA3 (LYMC-3+AMA3: inoculation with strain LYMC-3 followed by inoculation with pine wood nematode AMA3; AMA3: inoculation with sterile culture medium followed by inoculation with pine wood nematode AMA3; CK2: inoculation with sterile water);

[0027] Fig.11 This is a graph of PAL activity in Masson pine needles after inoculation with strain LYMC-3 (LYMC-3: inoculated strain, CK1: inoculated sterile culture medium);

[0028] Fig.12This is a graph of PAL activity in Masson pine needles after inoculation with the pine wood nematode AMA3 (LYMC-3+AMA3: inoculation with strain LYMC-3 followed by inoculation with the pine wood nematode AMA3; AMA3: inoculation with sterile culture medium followed by inoculation with the pine wood nematode AMA3; CK2: inoculation with sterile water). DETAILED DESCRIPTION

[0029] 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.

[0030] 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.

[0031] Bacillus pumilus LYMC-3 was isolated and screened from the stems of Pinus massoniana in Luoyang Sui and Tang Botanical Garden.

[0032] Bacillus pumilus LYMC-3, whose classification name is Bacillus pumilus LYMC-3, has been deposited in China Center for Type Culture Collection, with deposit number CCTCC NO: M 2016775, deposit date December 21, 2016, and deposit address: Wuhan University, China.

[0033] Bacterial fermentation medium NB: 5g beef extract, 10g peptone, 10g glucose, 5g sodium chloride, pH 8, 1L distilled water.

[0034] The pine wood nematode strain AMA3 was isolated from Pinus thunbergii in Anhui Province and is currently preserved in the Pathology Laboratory of Nanjing Forestry University.

[0035] Example 1: Effect of inoculation with Bacillus pumilus LYMC-3 on H2O2 content in Masson pine and pine wood nematode AMA3

[0036] 1. Inoculation of Bacillus brevis LYMC-3

[0037] The strain LYMC-3 was cultured in bacterial fermentation medium NB at 28°C and 200 r / min for 72 h and then used.

[0038] Root inoculation method: drill holes in the soil around the roots of Masson pine and inject liquid bacterial agent with an inoculation volume of 200mL. Use 200mL of sterile liquid culture medium as a control, and set up 10 replicates for each treatment.

[0039] 2. Inoculation of pine wood nematodes

[0040] 5 days after the inoculation of strain LYMC-3, 1 mL of nematode solution (containing about 5,000 nematodes) was inoculated into the Masson pine seedlings inoculated with strain LYMC-3 by artificial skin grafting. The Masson pine seedlings without bacteria were inoculated with equal amounts of nematodes as the control (CK1), and the wounds were inoculated with equal amounts of sterile water as the negative control (CK2). Each treatment was repeated three times. Samples were taken every 5 days after inoculation, with 3 replicates each, and the sampling sites between replicates were basically the same.

[0041] 3. Make a standard curve

[0042] Take 57 μL of 30% analytical pure H2O2 and dissolve it in water; use this solution as the H2O2 standard solution to prepare a standard curve, dilute the standard solution 1, 2, 4, 6, 8, and 10 times, take 1 mL of the diluted standard solution, add 1 mL of PH7.0 potassium phosphate buffer and 1 mL of 1 mmoL / L KI solution, mix thoroughly, and measure the absorbance at 390 nm using a Japanese Hitachi UV-visible spectrophotometer. Perform linear regression with absorbance as the ordinate and H2O2 concentration as the abscissa.

[0043] 4. Determination of H2O2 content

[0044] Accurately weigh 1g of Masson pine leaves, grind them thoroughly under liquid nitrogen, add 10mL of pre-cooled TCA at 1 / 10 (w / v), centrifuge at 10000r / min at 4℃ for 10min, take 2mL of the supernatant, add PH7.2 potassium phosphate buffer and 2mL of 1mmoL / L KI solution, mix thoroughly, and 390 The absorbance was measured at 400 μmol / gFW. The results were expressed as μmol / gFW and the measurement was repeated 3 times to obtain the average value.

[0045] 5. The results are as follows Figure 1 As shown in the figure, the increase of H2O2 content in the Masson pine seedlings after inoculation with strain LYMC-3 showed a trend of first rising and then falling over time. During the entire inoculation time, the H2O2 content in the Masson pine seedlings inoculated with the strain was always higher than that in the control. 10 to 20 days after inoculation, the increase of H2O2 in the Masson pine seedlings treated was large, and the value was the largest at 15 days after inoculation, which was 35.06%. However, the increase of H2O2 was relatively low at 5 and 25 days after inoculation, and the value was the smallest at 25 days after inoculation, which was 18.28%.

[0046] The results are as follows Figure 2As shown in the figure, after inoculation with strain LYMC-3 and then pine wood nematodes, the H2O2 content in the infected Masson pine seedlings was high at the beginning and became low in the later stage. During the entire inoculation time, the H2O2 content in all Masson pine seedlings inoculated with pine wood nematodes was always higher than that in the control. Five days after inoculation with strain LYMC-3 and then pine wood nematodes, the H2O2 content in the Masson pine seedlings was 368.73μmoL / g, while the H2O2 content in the Masson pine seedlings inoculated with pine wood nematodes alone was 343.25μmoL / g. 10 to 25 days after inoculation, the H2O2 content of the inoculated strain was always lower than that of the single inoculated strain, and it decreased significantly 10 days after inoculation. At this time, the H2O2 content of the inoculated strain was 342.76μmoL / g, while the content of the single inoculated strain was as high as 412.57μmoL / g. As time went on, the difference in H2O2 content between the inoculated strain and the strain inoculated with insects alone gradually decreased until 25 days after inoculation, when there was no significant difference between the two.

[0047] Example 2: Effect of inoculation with Bacillus pumilus LYMC-3 on catalase content in Masson pine and pine wood nematode AMA3

[0048] 1. Extract hormones

[0049] Weigh fresh plant materials, a total of 1g of leaves near the inoculation point. Cut the leaves into pieces, add 10mL of 0.1moL / LPH7.0 phosphate buffer, add appropriate amount of quartz sand and polypropylene pyrrolidone in an ice-cold mortar to remove phenolic substances and prevent color interference, grind into a homogenate in an ice bath, then centrifuge at 4℃10000r / min for 10min, pour out the supernatant and store in an ice bath, extract the residue once with 5mL phosphate buffer solution, combine the two supernatants, and the supernatant is the crude enzyme extract for the determination of defense enzyme activity.

[0050] 2. Mix 0.1 mL of enzyme solution with 2.9 mL of buffer (0.1 mol / L pH 7.0 phosphate buffer), place in a 30°C water bath for 10 min to stabilize the temperature, add 1.0 mL of 3% H2O2 and shake well immediately, adjust to zero with a blank tube, and scan with time and absorbance at a wavelength of 240 nm using a Hitachi Type 1 UV-visible spectrophotometer. The decrease of 0.1 in absorbance per minute is regarded as one unit of enzyme activity, and the unit is U / g·min.

[0051] OD 240 is the absorbance at 240 nm, V is the total volume of the extract (mL), a is the amount of enzyme solution used in the measurement (mL), and g is the weighed fresh leaf weight (g).

[0052] 3. The results are as follows Figure 3As shown in the figure, the increase in CAT activity in Masson pine seedlings after inoculation with strain LYMC-3 showed a trend of first rising, then falling, and then rising again. During the entire inoculation time, the CAT activity in Masson pine seedlings inoculated with strains was always higher than that in the control. 10 and 20 days after inoculation, the increase in CAT activity in Masson pine seedlings was large, with the maximum value of 34.12% at 10 days after inoculation and the minimum value of 9.65% at 25 days after inoculation.

[0053] The results are as follows Figure 4 As shown in the figure, after inoculation with strain LYMC-3 and then with pine wood nematodes, the CAT activity in the infected Masson pine seedlings was high at the beginning and continued to decrease in the later stage. Ten days after inoculation, the CAT activity in all Masson pine seedlings inoculated with pine wood nematodes was always higher than that in the control. Five days after inoculation with strain LYMC-3 and then with pine wood nematodes, the CAT activity in the Masson pine seedlings was 89.63U / g, while the CAT activity in the Masson pine seedlings inoculated with pine wood nematodes alone was 84.21U / g. Throughout the inoculation process, the CAT activity of the inoculated strain was always higher than that of the single inoculated strain, and the growth reached the maximum at 10 days after inoculation, which was 33.85%. As time went on, the difference in CAT activity between the inoculated strain and the single inoculated strain gradually decreased, until 25 days after inoculation, when there was no significant difference between the two.

[0054] Example 3: Effect of inoculation with Bacillus pumilus LYMC-3 on the peroxidase content of Masson pine and pine wood nematode AMA3

[0055] 1. Extract hormones

[0056] Same as Example 2.

[0057] 2. Peroxidase (POD) Take 7 clean test tubes, add 2.9mL 0.05moL / L phosphate buffer (pH5.5), 1mL 2% hydrogen peroxide, 1mL 0.05moL / L guaiacol and 0.1mL enzyme solution in turn. Replace the enzyme solution with distilled water in the blank tube and replace the enzyme solution with boiled enzyme solution in the control tube. After adding the enzyme solution to the reaction system, immediately keep it in a 37℃ water bath for 15min, then quickly transfer it to an ice bath, and add 2mL 20% trichloroacetic acid to terminate the reaction. After filtering, dilute appropriately, and measure the absorbance at a wavelength of 470nm. A change of 0.01 in A470 per minute is 1 peroxidase activity unit (U), and the unit is U / (g·min).

[0058] OD470 is the absorbance at 470 nm, V is the total volume of the extract (mL), a is the amount of enzyme solution used in the measurement (mL), and g is the weighed fresh leaf weight (g).

[0059] 3. The results are as follows Figure 5As shown in the results, the inoculated strain LYMC-3 can increase the POD activity of the needles of Masson pine seedlings, and the increase in its content shows a trend of first rising and then falling over time. During the entire inoculation time, the POD activity of the needles of Masson pine seedlings inoculated with the strain was always higher than that of the control. On the 10th day after inoculation, the increase was the largest, at 41.43%, while on the 20th to 25th day after inoculation, the POD increase was not significantly different.

[0060] The results are as follows Figure 6 As shown in the figure, after inoculation with strain LYMC-3 and then pine wood nematodes, the POD activity in the needles of the infected Masson pine seedlings showed a trend of first decreasing and then increasing, while the POD activity in the needles of the Masson pine seedlings inoculated with pine wood nematodes alone continued to decrease. During the entire inoculation period, the POD activity in the needles of all Masson pine seedlings inoculated with pine wood nematodes was always higher than that of the control. On the 15th day after inoculation with pine wood nematodes, the POD activity in the needles of the Masson pine seedlings inoculated with strains was the largest difference from that inoculated with pine wood nematodes alone. The POD activity in the needles of the Masson pine seedlings inoculated with strains was 120.52U / g, while that of the Masson pine seedlings inoculated with pine wood nematodes alone was only 94.66U / g; on the 25th day after inoculation with pine wood nematodes, the POD activity in the leaves of the Masson pine seedlings inoculated with strain LYMC-3 was lower than that of the sterile water treatment, while the Masson pine seedlings inoculated with pine wood nematodes alone began to be lower than that of the sterile water treatment on the 20th day.

[0061] Example 4: Effect of inoculation with Bacillus pumilus LYMC-3 on superoxide dismutase content in Masson pine and pine wood nematode AMA3

[0062] 1. Extract hormones

[0063] Same as Example 2.

[0064] 2. The reaction system contains 0.05moL / L pH=7.8 phosphate buffer, 130mmoL / L Met, 750μmoL / LNBT, 20μmoL / L riboflavin, and 100μmoL / LEDTA-Na 2. After thorough mixing, place on a transparent test tube rack, place in a light incubator for light reaction for 20 minutes, then stop the reaction in the dark, and quickly measure the A56 value on a UV spectrophotometer. Each treatment is repeated 3 times. The amount of enzyme that inhibits 50% reduction of nitroblue tetrazolium (NBT) under light is 1 enzyme activity unit (μmoL / min)

[0065] ACK is the absorbance value of the control tube at 560 nm, AE is the absorbance value of the sample tube at 560 nm, V is the total volume of the sample solution (mL), VT is the amount of sample used for determination (mL), and W is the fresh weight of the sample (g).

[0066] 3. The results are as follows Figure 7As shown, the inoculated strain LYMC-3 can increase the SOD activity of the needles of Masson pine seedlings, and the increase in its content shows a trend of first decreasing and then increasing over time. During the entire inoculation time, the SOD activity of the needles of Masson pine seedlings inoculated with the strain was always higher than that of the control. Among them, the value was the largest at 24.55% 5 days after inoculation, and the smallest at 2.86% 25 days after inoculation.

[0067] The results are as follows Figure 8 As shown in the figure, after inoculation with strain LYMC-3 and then with pine wood nematodes, the SOD activity in the needles of the infected Masson pine seedlings continued to decrease. The CAT activity in all Masson pine seedlings inoculated with pine wood nematodes was always higher than that in the control during the disease period. Five days after inoculation with strain LYMC-3 and then with pine wood nematodes, the CAT activity in the Masson pine seedlings was 89.63U / g, while the CAT activity in the Masson pine seedlings inoculated with pine wood nematodes alone was 84.21U / g. Throughout the inoculation process, the CAT activity of the inoculated strain was always higher than that of the single inoculated strain, and the growth reached the maximum at 10 days after inoculation, which was 33.85%. As time went on, the difference in CAT activity between the inoculated strain and the single inoculated strain gradually decreased, until 25 days after inoculation, when there was no significant difference between the two.

[0068] Example 5: Effect of inoculation with Bacillus pumilus LYMC-3 on the polyphenol oxidase content of Masson pine and pine wood nematode AMA3

[0069] 1. Extract hormones

[0070] Same as Example 2.

[0071] 2. The reaction system includes pH 7.0 0.1 mol phosphate buffer, catechol solution, and enzyme solution. The inactivated enzyme solution is used as a control, and the reaction is carried out at 30°C for 2 min. The absorbance is measured at 420 nm using a Hitachi UV-visible spectrophotometer. OD 420 Change 01 to one unit of enzyme activity, expressed as U·g -1 Indicates enzyme activity.

[0072] OD420 is the absorbance at 420 nm, V is the total volume of the extract (mL), a is the amount of enzyme solution used in the measurement (mL), and g is the weighed fresh leaf weight (g).

[0073] 3. The results are as follows Fig. 9As shown in the results, the inoculated strain LYMC-3 can increase the PPO activity in the needles of Masson pine seedlings, and the increase in its content shows a trend of first decreasing and then increasing over time. During the entire inoculation time, the PPO activity of the Masson pine seedlings inoculated with the strain was always higher than that of the control. 5 to 15 days after inoculation with pine wood nematodes, the increase in H2O2 in the treated Masson pine seedlings was relatively stable, and 20 days after inoculation, the increase in PPO activity was the smallest, at 11.63%. 25 days after inoculation, the increase was the largest, at 40.54%.

[0074] The results are as follows Fig.10 As shown in the figure, after inoculation with strain LYMC-3 and then pine wood nematodes, the PPO activity in the needles of the infected Masson pine seedlings showed a trend of first increasing and then decreasing. During the entire inoculation time, the PPO activity in the needles of all Masson pine seedlings inoculated with pine wood nematodes was always higher than that of the control. Five days after inoculation with strain LYMC-3 and then pine wood nematodes, the PPO activity in the needles of Masson pine seedlings was 89.54U / g, while the PPO activity in the needles of Masson pine seedlings inoculated with pine wood nematodes alone was 67.22U / g. Ten days after inoculation, the difference between the PPO activity in the needles of Masson pine seedlings inoculated with strains and those inoculated with pine wood nematodes alone was the smallest, at 15.56%. As time went on, the difference in PPO activity between the inoculated strains and the inoculated strains alone increased and tended to be stable.

[0075] Example 6: Effect of inoculation with Bacillus pumilus LYMC-3 on the content of phenylalanine ammonia lyase in Masson pine and pine wood nematode AMA3

[0076] 1. Extract hormones

[0077] Same as Example 2.

[0078] 2. Enzyme activity determination reaction system Take 1mL 0.02moL / L phenylalanine (prepared with 0.1moL / L, pH8.8 boric acid buffer), 2mL 0.05moL / L boric acid buffer, 0.1 enzyme solution, react at 30℃, add 5moL / L HCl. Terminate the reaction, use the mixture without enzyme solution as reference. Use Japan Hitachi Type 1 UV-Vis spectrophotometer to measure OD 290 Value, in OD 290 A change of 0.1 in the value was considered 1 unit of enzyme activity.

[0079] OD 290 is the absorbance at 290 nm, V is the total volume of the extract (mL), a is the amount of enzyme solution used in the measurement (mL), and g is the weighed fresh leaf weight (g).

[0080] 3. The results are as follows Fig.11As shown in the figure, the inoculated strain LYMC-3 can increase the PAL activity in Masson pine seedlings, and the increase in its content shows a trend of first decreasing, then increasing, and then decreasing over time. During the entire inoculation time, the PAL activity in the Masson pine seedlings inoculated with the strain was always higher than that in the control. 15 days after inoculation, the increase in PAL activity in the Masson pine seedlings inoculated with LYMC-3 was the largest, at 20.11%, while 5 to 25 days after inoculation, the increase in PAL was relatively low, and the value was the smallest at 1.88% 25 days after inoculation.

[0081] The results are as follows Fig.12 As shown in the figure, after inoculation with strain LYMC-3 and then pine wood nematodes, the PAL activity in the needles of the infected Masson pine seedlings showed a trend of first rising and then falling. During the entire inoculation time, the PAL activity in the needles of all Masson pine seedlings inoculated with pine wood nematodes was always higher than that of the control. 15 days after inoculation with strain LYMC-3 and then pine wood nematodes, the PAL activity in the needles of Masson pine seedlings reached the maximum, which was 289.77U / g. At this time, the PAL activity in the needles of Masson pine seedlings inoculated with pine wood nematodes alone was 235U / g. 25 days after inoculation, the difference between the PAL activity in the needles of Masson pine seedlings inoculated with strains and Masson pine seedlings inoculated with pine wood nematodes alone was the smallest, only 6.70%, and the PAL activity in the Masson pine seedlings inoculated with pine wood nematodes was lower than that in the sterile water treatment.

Claims

1. Application of Bacillus brevis LYMC-3 in inducing masson pine to synthesize H2O2, wherein the Bacillus brevis LYMC-3 is classified and named as Bacillus brevis ( Bacillus pumilus )LYMC-3, deposited in China Center for Type Culture Collection, with the deposit number: CCTCC NO: M 2016775.

2. Application of Bacillus brevis LYMC-3 in inducing enhanced CAT activity in Masson pine, wherein the Bacillus brevis LYMC-3 is classified and named as Bacillus brevis ( Bacillus pumilus )LYMC-3, deposited in China Center for Type Culture Collection, with the deposit number: CCTCC NO: M 2016775.

3. Application of Bacillus brevis LYMC-3 in inducing enhanced POD activity in Masson pine, wherein the Bacillus brevis LYMC-3 is classified and named as Bacillus brevis ( Bacillus pumilus )LYMC-3, deposited in China Center for Type Culture Collection, with the deposit number: CCTCC NO: M 2016775.

4. Application of Bacillus brevis LYMC-3 in inducing enhanced SOD activity in Masson pine, wherein the Bacillus brevis LYMC-3 is classified and named as Bacillus brevis ( Bacillus pumilus )LYMC-3, deposited in China Center for Type Culture Collection, with the deposit number: CCTCC NO: M 2016775.

5. Application of Bacillus brevis LYMC-3 in inducing enhanced PPO activity in Masson pine, wherein the Bacillus brevis LYMC-3 is classified and named as Bacillus brevis ( Bacillus pumilus )LYMC-3, deposited in China Center for Type Culture Collection, with the deposit number: CCTCC NO: M 2016775.

6. Application of Bacillus brevis LYMC-3 in inducing enhanced PAL activity in Masson pine, wherein the Bacillus brevis LYMC-3 is classified and named as Bacillus brevis ( Bacillus pumilus )LYMC-3, deposited in China Center for Type Culture Collection, with the deposit number: CCTCC NO: M 2016775.

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