Application of ectomycorrhizal fungus Boletus chrysogenum in the prevention and treatment of larch dieback

Abstract

The invention discloses the application of ectomycorrhizal fungus Boletus thicola in preventing and treating larch dieback, and belongs to the field of microbial technology. The application of the ectomycorrhizal fungus Boletus thicola in preventing and treating larch dieback, potted plant experiments show that the prevention and treatment effect of the Sg strain on larch dieback is 40.00%, and the CAT activity of larch needles treated with the Sg strain is significantly increased, reaching 25.17%. Comprehensive indicators show that the Boletus thicola strain can reduce the damage of larch dieback pathogens to plants, has good disease resistance, and has good practicality.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and more specifically relates to application of ectomycorrhizal fungus Boletus chrysogenum in preventing and treating larch dieback disease. Background Art

[0002] Larix gmelinii (Ruprecht) Kuzeneva has solid wood, strong adaptability, and beautiful appearance. The resin bark extract can be used in the fields of medicine and chemical industry. It is not only a common tree species for landscaping, but also an important economic tree species in Northeast my country, North China, Northwest China and other places. Larix dieback [Neofusicoccum laricinum (Sawada) Y. Hattori & C. Nakash] is a major fungal disease that harms larch, harming the growth and development of larch and affecting the larch industry. The current main method for preventing and controlling larch dieback is to spray chemical agents such as thiophanate-methyl. However, in recent years, due to the large-scale spraying of chemical agents, the pathogens have developed drug resistance, resulting in a decrease in the effectiveness of chemical control methods and causing environmental pollution problems. Therefore, there is an urgent need to find environmentally friendly prevention and control methods.

[0003] Ectomycorrhizal fungi can interact with plants, promote nutrient absorption and enhance plant resistance to stress. Summary of the invention

[0004] In view of the above problems existing in the prior art, the technical problem to be solved by the present invention is to provide an application of the ectomycorrhizal fungus Boletus chrysogenum in preventing and treating larch dieback disease, so as to promote the growth of larch.

[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0006] Application of ectomycorrhizal fungus Boletus chrysogenum in the prevention and control of larch dieback disease.

[0007] The method for preventing and controlling larch dieback disease is to reduce the content of malondialdehyde in larch seedlings.

[0008] The method for preventing and controlling larch dieback disease is to increase the activity of catalase in larch.

[0009] Application of the ectomycorrhizal fungus Boletus chrysogenum in improving catalase activity in larch.

[0010] Application of the slurry of the ectomycorrhizal fungus Boletus chrysogenum in the prevention and control of larch dieback disease.

[0011] The preparation method of the bacterial slurry of the ectomycorrhizal fungus Boletus thunbergii is as follows: the ectomycorrhizal fungus Boletus thunbergii strain is placed in an MMN solid culture medium at 25°C in the dark for more than 14 days, the edge bacterial cake is placed in an MMN liquid culture medium, and is cultured at 25°C and 140 r / min in the dark for 21 days, the mycelial balls are rinsed and then placed in a tissue disruptor for crushing, centrifuged at 5000 r / min for 5 minutes, and the supernatant is discarded; the mycelial tissue is taken and configured into a 100 g / L bacterial solution with sterile water; 150 g of a sterilized mud and sand matrix is ​​taken and mixed evenly with 300 mL of the bacterial solution to obtain the bacterial slurry of the ectomycorrhizal fungus Boletus thunbergii.

[0012] Compared with the prior art, the present invention has the following beneficial effects:

[0013] The ectomycorrhizal fungus Suillus grevillei of the present invention improves the disease resistance of larch. The control effects of the Pisolithus tinctorius (Pt) strain, the Suillus grevillei (Sg) strain and the Suillus bovinus (Sb) strain on larch dieback are 20.01%, 40.00% and 20.01% respectively. The MDA content of the needles of the seedlings treated with mycorrhizal fungi is lower than that of the control, among which the Pt strain treatment and the Sg strain treatment significantly reduce the MDA content, which decreases by 40.41% and 36.09% respectively, and the Sb strain treatment only reduces the MDA content by 17.67%. The CAT activity of the larch needles treated with the Sg strain significantly increases, reaching 25.17%, while the catalase activity of the larch treated with the Pt and Sb strains does not change before and after the onset of the disease. Comprehensive indicators show that the thick-ringed Boletus strain can reduce the damage caused by larch dieback pathogens to plants and has good disease resistance effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is a graph showing the changes in root weight density of larch 4 months after inoculation with three mycorrhizal fungi;

[0015] Figure 2 The mycorrhizal morphology of larch is observed (A is the mycorrhizal morphology observed under a stereomicroscope; B is the root tip; C is the root segment; D is the root hair);

[0016] Figure 3 This is the mycorrhizal infection rate of larch 4 months after inoculation with the three strains;

[0017] Figure 4 The occurrence of larch dieback after inoculation of mycorrhizal fungi (A is not inoculated with mycorrhizal fungi (CK); B is Pt treatment; C is Sg treatment; D is Sb treatment);

[0018] Figure 5This is the effect of three kinds of mycorrhizal fungi on the resistance of larch to dieback disease (A is the disease index of larch; B is the control effect of larch dieback disease);

[0019] Figure 6 This is the change of malondialdehyde (MDA) content in larch needles after different mycorrhizal fungi treatment;

[0020] Figure 7 This is a graph showing the changes in catalase (CAT) activity in larch needles after treatment with three mycorrhizal fungi. DETAILED DESCRIPTION

[0021] 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.

[0022] The ectomycorrhizal fungal strains tested were as follows:

[0023] Pisolithus tinctorius, Suillus grevillei and Suillus bovinus were purchased from Zhili Zhongte Company.

[0024] The pathogenic bacteria strains tested are as follows:

[0025] The pathogen of larch dieback is Neofusicoccum laricinum (Sawada) Y. Hattori & C. Nakash, strain number DHKS 6-3, provided by Jilin Academy of Forestry Sciences, and isolated and identified from larch dieback diseased plants in the larch forest area of ​​Dunhua City, Yanbian Korean Autonomous Prefecture, Jilin Province.

[0026] The tested larch species are as follows:

[0027] The test larch seedlings were 3-year-old Japanese larch, which were purchased from Gaoqiao Town, Hui County, Longnan City, Gansu Province in October 2022 and transplanted into greenhouse pots on October 15, 2022.

[0028] The culture medium used was as follows:

[0029] MMN medium: 14 g MMN medium powder, 1 L distilled water, pH 5.6. Add 20 g / L agar to the solid medium.

[0030] PDA medium: 200g potato, 20g glucose, 1L distilled water, natural pH. For solid medium, add 20g / L agar.

[0031] The experimental instruments are as follows:

[0032] Clean bench (AC2-4S1, Singapore Yisi High-Tech Co., Ltd.), high pressure sterilizer (SX-500, Japan Tomy Digital Biology Co., Ltd.), oven (MOV-212F-PC, Japan Panasonic Healthcare Medical Devices Co., Ltd.), constant temperature incubator (MIR-553, Japan Sanyo Electric Co., Ltd.), shaker (Multitron Standard, China Ifusen Biotechnology Co., Ltd.), water bath (JK-WB-20B, Shanghai Jingxue Scientific Instrument Co., Ltd.), centrifuge (Centrifuge5424R, Germany Eppendorf Company), root scanner (Expression11000XL, EPSON Company), Zeiss stereoscope (SteREO Discovery.V20, ZEISS), Zeiss fluorescence microscope (Imager.M2, ZEISS), UV-visible spectrophotometer (Lambda365, PerkinElmer).

[0033] Trypan blue solution: Dissolve 0.05 g of trypan blue in 100 mL of a mixture of 85% lactic acid, glycerol, and distilled water (volume ratio of 1:1:1).

[0034] Embodiment 1:

[0035] 1. Inoculation of larch with ectomycorrhizal fungi

[0036] The three strains of ectomycorrhizal fungi were placed in MMN solid medium at 25℃ and cultured in the dark for more than 14 days. After the mycelium was fully grown, the edge cake with a diameter of about 6mm was taken with a puncher and placed in MMN liquid medium. Each bottle had 6 cakes and was placed in a shaker at 25℃ and a speed of 140r / min and cultured in the dark for 21 days. The mycelium balls were taken out and rinsed with sterile water, placed in a tissue crusher and broken, centrifuged at 5000r / min for 5min, and the supernatant was poured out. After washing twice with sterile water, the mycelium tissue was weighed and prepared into 100g / L bacterial solution with sterile water. 150g of sterilized mud and sand matrix was mixed evenly with 300mL bacterial solution to make bacterial slurry. The roots of each seedling were dipped in bacterial slurry for 10s and then planted back in the flowerpot. Every month after dipping, 3 small holes were punched in the soil near the roots of the seedlings, and 50mL bacterial solution was applied to each seedling.

[0037] 2. Four months after inoculation, the root volume was measured using a root scanner, and then the root parts of the seedlings were collected and placed in envelopes, dried at 60°C for 48 hours until constant weight, and then weighed using an analytical balance. The root weight density of larch was then calculated, which is the ratio of the root dry weight to the total root volume (g / cm 3 ).

[0038] The results are as follows Figure 1As shown, the root weight density index of larch treated with Pt and Sg strains increased significantly, while there was no significant change in the Sb strain treatment. The root weight density of seedlings treated with Pt strain increased by 20.44%, and that treated with Sg strain increased by 32.43%.

[0039] 3. Mycorrhizal morphology

[0040] Four months after inoculation with ectomycorrhizal fungi, the seedlings were taken out. Three seedlings with average growth status were selected for each strain treatment. Thirty 1-2 cm long mycorrhizal root segments were randomly selected from each seedling and placed in test tubes. They were stained according to the method described in the literature (WM Gill, F. Lapeyrie, T. Gomi, et al. Tricholoma matsutake-an assessment of in situ and invitro infection by observing cleared and stained whole roots [J]. Mycorrhiza, 1999, 9 (4): 227-231.). The specific steps are:

[0041] Add 10% KOH solution into the test tube and boil it at 90℃ for 90 minutes; rinse the mycorrhizae with clean water several times to remove impurities, rinse with rinsing solution (10% H2O2+10% KOH) for 10-30 minutes to make the mycorrhizal tissue transparent; rinse with clean water several times again to remove impurities, acidify with 0.1mol / L hydrochloric acid for 10-20 minutes; pour out the hydrochloric acid, add 0.05% trypan blue solution, and stain in a 90℃ water bath for 90-120 minutes; take out the mycorrhizae and put them into a small bottle, pour in 50% glycerol for 8 hours to decolorize.

[0042] The results are as follows Figure 2 As shown in the figure, under an optical microscope, a large number of hyphae attached to the root surface formed a sheath, while hyphae invading the outer cortex of the root formed a Haugh net. The sheath and Haugh net appeared blue under trypan blue staining. It can be seen that the sheath formed at the root tip can densely wrap the root tip or a local area of ​​the root tip, and the staining is darker ( Figure 2 B), there are also some dense hyphae in the non-root tip area ( Figure 2 C). Most of the extended hyphae on the surface of the mycorrhiza have replaced the original root hairs. Only in the root segment where the hyphae are sparse can the original root hairs of larch be clearly seen ( Figure 2 D).

[0043] 4. Mycorrhizal infection rate

[0044] Thirty root segments with a length of 0.5 cm were randomly selected from each seedling, and the number of mycorrhizae was counted under a stereomicroscope to calculate the mycorrhizal infection rate of the root system of larch seedlings under different strain treatments.

[0045] The results are as follows Figure 2 As shown in A, under a stereomicroscope, there was no obvious difference in morphology between the mycorrhizae formed by the three strains treated with larch, and all of them formed bifurcated or rod-shaped ectomycorrhizae.

[0046] The results are as follows Figure 3 As shown, the mycorrhizal infection rate of larch was significantly increased in the treatments of Pt and Sg strains compared with that in the treatment of Sb strain, reaching 30.00% and 28.89% respectively, while the mycorrhizal infection rate of larch was only 11.11% after the treatment of Sb strain.

[0047] Example 2

[0048] 1. Four months after inoculation of ectomycorrhizal fungi, three seedlings with average growth levels were selected for each treatment to determine relevant growth indicators. Three seedlings with similar growth conditions were selected from the remaining seedlings for each treatment, and inoculated with larch dieback pathogen. The mycelial tissue of the dieback pathogen washed with sterile water was prepared into a 100g / L bacterial solution in the same way, and 50mL of the bacterial solution was sprayed on the branches and leaves of the seedlings. The control group was sprayed with 50mL of sterile water on the branches and leaves of the seedlings. Seven days after inoculation with the pathogen, the symptoms of the pine seedlings were observed, the incidence rate, disease level and disease index were calculated, and the prevention and control effect of the disease was calculated.

[0049] The calculation formulas for the incidence rate, disease index and disease control effect of larch are as follows:

[0050]

[0051]

[0052]

[0053] The results are as follows Figure 4 and Figure 5 As shown, 7 days after inoculation, the larch needles in the control group inoculated with the pathogen developed spots, turned yellow, dried up and fell off ( Figure 4 ), the incidence rate reached 100%, and the disease index was 41.67; although the incidence rate of the three treatment groups inoculated with ectomycorrhizal fungi also reached 100%, the disease index was lower than that of the control group, among which the disease index of Sg treatment was the lowest, which was 25.00, and its control effect was the best, reaching 40.00%, while the control effects of Pt and Sb were both 20.01%.

[0054] 2. Malondialdehyde (MDA) content

[0055] Weigh 1 g of fresh needles of larch for the current year and the control sample, respectively, and homogenize them in 10 mL of 0.1% (w / v) trichloroacetic acid, then centrifuge at 12000 rpm / min for 15 min, take 1 mL of the supernatant and add 4 mL of 0.5% thiobarbituric acid (prepared with 20% trichloroacetic acid), mix well and heat at 95°C

[0056] After cooling in an ice bath for 30 minutes, centrifuge at 12000 rpm / min for 10 minutes, measure the absorbance at 532 nm and 600 nm, and substitute the following formula to calculate the MDA content. The extinction coefficient of MDA is 155 mM -1 cm -1 .

[0057]

[0058] The results are as follows Figure 6 As shown in the data, the malondialdehyde content in the larch seedlings increased significantly after the disease occurred. The malondialdehyde content in the larch seedlings inoculated with three mycorrhizal fungi was lower than that in the control group. Among them, the Pt and Sg strains treatments achieved significant differences, reducing by 40.41% and 36.09% respectively, and the Sb strain treatment only reduced by 17.67%. However, there was no statistically significant difference between the Pt and Sg strains, indicating that the larch seedlings coexisting with the Pt and Sg strains were less affected.

[0059] 3. Defensive enzyme activity

[0060] According to the method in the instructions of the enzyme activity detection kit (Shanghai Sangon Biotechnology Co., Ltd.), the catalase (CAT) activity in the fresh needles of Larix gmelinii treatment group and the control sample was determined.

[0061] The results are as follows Figure 7 As shown in the figure, the catalase activity of larch increased after the Sg strain was treated, and increased significantly after the pathogen was inoculated, reaching 25.17%, while the catalase activity of larch treated with Pt and Sb strains did not change before and after the disease. This shows that the Sg strain can significantly promote the production of catalase in larch and improve the plant's stress resistance.

[0062] Comprehensive indicators show that the thick-ringed Boletus strain (Sg) can reduce the damage caused by larch dieback pathogens to plants and has good disease resistance.

Claims

1. Ectomycorrhizal fungus Boletus edulis ( Suillus grevillei ) in the prevention and treatment of larch dieback; the larch dieback pathogen is Neofusicoccum laricinum .

2. Ectomycorrhizal fungus Boletus edulis ( Suillus grevillei ) in the prevention and treatment of larch dieback disease, wherein the larch dieback disease pathogen is Neofusicoccum laricinum .

3. The ectomycorrhizal fungus Boletus edulis according to claim 2 ( Suillus grevillei ) in the prevention and treatment of larch dieback disease, characterized in that: The preparation method of the bacterial slurry of the ectomycorrhizal fungus Boletus thunbergii is as follows: the ectomycorrhizal fungus Boletus thunbergii strain is placed in an MMN solid culture medium at 25°C in the dark for more than 14 days, the edge bacterial cake is placed in an MMN liquid culture medium, and is cultured at 25°C and 140 r / min in the dark for 21 days, the mycelial balls are rinsed and then placed in a tissue disruptor for crushing, centrifuged at 5000 r / min for 5 minutes, and the supernatant is discarded; the mycelial tissue is taken and configured into a 100 g / L bacterial solution with sterile water; 150 g of a sterilized mud and sand matrix is ​​taken and mixed evenly with 300 mL of the bacterial solution to obtain the bacterial slurry of the ectomycorrhizal fungus Boletus thunbergii.

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