A DX-THS3 bacterial fertilizer of Chaetomium globosum, preparation method and application thereof

Abstract

The present invention belongs to the field of microbial applications, specifically relating to a Chaetomium globosum DX-THS3 bacterial fertilizer, its preparation method, and its application. The present invention finds that Chaetomium globosum DX-THS3 significantly promotes the growth of peppers. Furthermore, it not only significantly inhibits peach brown rot and pythium in the soil, but also improves the soil itself. This significantly broadens the application scope of Chaetomium globosum and provides a new technical means for soil regulation and pepper growth promotion.

Description

Technical Field

[0001] The invention belongs to the field of microbial applications, and particularly relates to a Chaetomium globosum DX-THS3 bacterial fertilizer, a preparation method and application thereof. Background Art

[0002] Chaetomium globosum belongs to the phylum Ascomycota, class Pyrenomycetes, orders Sordariales, family Chaetomiaceae, genus Chaetomium spp. It is recognized as an important family for the production of carbohydrate-active enzymes and antibiotics.

[0003] Research on Chaetomium globosum biofertilizers has primarily focused on the ND35 strain, an endophytic, dominant strain isolated from healthy white poplar (Populus tomentosa). Some research results indicate that ND35 biofertilizer can significantly improve the soil microbial flora in apple cropping soils and increase root vigor and biomass in sweet tea seedlings. It can also enhance the physiological activity of poplar roots and improve the efficiency of light energy utilization in poplar leaves. Furthermore, it significantly promotes the growth and biomass of walnut seedlings. Another strain, Chaetomium globosum D38, isolated from the hairy roots of Salvia miltiorrhiza, has been shown to significantly promote the growth of the plant and increase its production of salvianolic acid and tanshinones. Summary of the Invention

[0004] The present invention discovered a strain of Chaetomium globosum, named Chaetomium globosum DX-THS3, deposited with the China Center for Type Culture Collection on January 4, 2016, with a deposit number of CCTCC NO: M 2016005. A fermentation extract of this strain exhibits significant inhibitory effects against peach brown rot and pythium scabra, with inhibition rates reaching 83.90% and 66.18%, respectively, which are unprecedented in the art. The extract also exhibits inhibitory effects against Phytophthora capsici, rice blast, Sclerotinia sclerotiorum, rice sheath blight, Fusarium oxysporum, Fusarium genus, and Fusarium graminearum.

[0005] In some preferred embodiments, the fermentation extract is produced by inoculating the Chaetomium globosum into a straw solid culture medium and then fermenting in the dark. The specific preparation process is as follows: the Chaetomium globosum is inoculated into PDA culture medium for activation, then inoculated into PDB culture medium, and cultured in the dark at 28°C and 160 rpm for 4-5 days to obtain a seed solution, which is then inoculated into a straw solid culture medium for fermentation in the dark; the product is then extracted and the fermentation extract is obtained.

[0006] The inventors found that when the fermentation product was inoculated into a straw solid culture medium and fermented in the dark for the following specific process, the inhibitory effect on the above-mentioned pathogens was the best, specifically: fermented in the dark at 26°C for 24 days.

[0007] The specific process after fermentation is as follows: After fermentation, the fermented product is stirred with a glass rod to minimize pulverization, and two bottles of fermented product are combined into one bottle. 500 mL of acetone is added to the bottle, and ultrasonic-assisted extraction is performed for 30 minutes, repeated three times. The acetone extract is collected and concentrated under reduced pressure. Then, extraction is performed three times with ethyl acetate in a 1:1 ratio. The ethyl acetate layer is collected and concentrated under reduced pressure to produce an extract.

[0008] Straw is a major agricultural waste, rich in available cellulose resources. However, its dense lignocellulose structure makes it difficult to degrade and recycle. The Chaetomium globosum of the present invention can undergo solid-state fermentation using straw as a substrate (compared to liquid-state fermentation, solid-state fermentation has significant advantages in reducing raw material costs, lowering energy consumption, increasing product concentration, and eliminating wastewater discharge). This not only produces active substances that inhibit the aforementioned pathogens, reducing the use of chemical pesticides to a certain extent, but also enables the recycling of straw waste, which is of great significance for environmental protection and the sustainable development of agriculture.

[0009] In addition, the inventors have discovered that the above-mentioned Chaetomium globosum can be used as a plant promoter or fertilizer to promote the growth of peppers and increase the yield of peppers, which has not been reported in the prior art. Based on this, the inventors have also proposed a bacterial fertilizer for promoting the growth of peppers, comprising the following raw materials: seed liquid of the above-mentioned Chaetomium globosum, wheat straw, wheat bran, and water. The ratio of the seed liquid of Chaetomium globosum, wheat straw (crushed to 60-100 mesh), wheat bran, and water is 10mL:16g:4g:80mL. The specific preparation process is as follows: the crushed wheat straw, wheat bran, and water are mixed in proportion and sterilized at 121°C for 20 minutes. After cooling, the seed liquid of Chaetomium globosum is inoculated and stirred evenly. The mixture is then allowed to ferment in the dark at 26°C for 24 days to obtain the product.

[0010] After 30 and 120 days of planting pepper seedlings using the bacterial fertilizer of the present invention, the plant fresh weight increased by 98% and 61.88%, respectively, compared to the control; the root fresh weight increased by 61.00% and 73.59%, respectively; the plant height increased by 31.00% and 12.62%, respectively; the stem diameter increased by 78.00% and 23.67%, respectively; and the number of leaves increased by 15.00% and 37.60%, respectively. Furthermore, the application of the bacterial fertilizer of the present invention can significantly improve the yield and quality of pepper fruits. Compared to the control, the number of pepper fruits per plant increased by 60.71%, and the fruit weight increased by 82.49%. The soluble protein content of the pepper fruits increased by 24.42%, the soluble sugar content increased by 7.49%, the organic acid content increased by 34.32%, and the vitamin C content increased by 44.82%.

[0011] The inventors also found that compared with traditional chemical fertilizers, the bacterial fertilizer of the present invention can not only promote the growth of peppers, but also avoid the disadvantages of traditional chemical fertilizers (destruction of the growth environment of microorganisms in the soil, soil compaction, decreased fertility, soil acidification, environmental pollution and other problems), and at the same time has a certain improvement effect on the soil.

[0012] The beneficial effects of the present invention are as follows: the present invention discovered that Chaetomium sphaeroides DX-THS3 has a significant promoting effect on the growth of peppers. At the same time, it not only has a significant inhibitory effect on peach brown rot fungi and sheath rot fungi in the soil, but also has a certain improvement effect on the soil itself; the application scope of Chaetomium sphaeroides is greatly broadened, and a new technical means is provided for soil regulation and pepper growth promotion. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Shown is the result of the inhibition zone experiment of the crude extract of Chaetomium sphaeroides DX-THS3 after the fermentation conditions were optimized;

[0014] Figure 2 Shown is the result of the inhibition zone experiment of the crude extract of Chaetomium sphaeroides DX-THS3 before the fermentation conditions were optimized;

[0015] Figure 3 Shown are the growth appearances of each group in the pot experiment at 30 days;

[0016] Figure 4 Shown are the growth appearances of each group in the pot experiment at 50 days;

[0017] Figure 5 Shown are the growth appearances of each group in the pot experiment for 120 days;

[0018] Figure 6 Shown is the root growth of each group in the pot experiment for 120 days;

[0019] Figure 7Shown are the appearances of pepper fruits in each group at 120 days of the pot experiment. DETAILED DESCRIPTION

[0020] The following will provide a clear and complete description of the concept and technical effects of the present invention in conjunction with the embodiments and drawings to fully understand the purpose, scheme and effects of the present invention.

[0021] Example 1:

[0022] (1) Isolation and preservation of strains

[0023] The Chaetomium globosum DX-THS3 proposed in the present invention is an endophytic fungus isolated and screened from Dongxiang wild rice stems, named Chaetomium globosum DX-THS3, deposited at the China Center for Type Culture Collection on January 4, 2016, and with a deposit number of CCTCC NO: M 2016005.

[0024] (2) Preparation of crude extract from Chaetomium sphaeroides DX-THS3 fermentation

[0025] Inoculate Chaetomium sphaeroides DX-THS3 into PDA medium and incubate in a 28°C incubator for one week to activate the strain. After activation, scrape small amounts of mycelium multiple times and inoculate them into a conical flask containing 150 mL of PDB medium. Incubate in a shaker at 28°C and 160 rpm for 5 days to obtain seed liquid. The seed liquid is then inoculated into straw solid medium and mixed thoroughly. The inoculum volume per flask is 10 mL. The mixture is then allowed to ferment in a 26°C incubator in the dark for 24 days.

[0026] After fermentation is complete, stir the fermented product with a glass rod to break it up as much as possible. Combine the two bottles of fermented product into one. Add 500 mL of acetone to the bottle and perform ultrasonic-assisted extraction for 30 minutes. Repeat three times. Collect the acetone extract and concentrate under reduced pressure. Then, extract three times with ethyl acetate in a 1:1 ratio. Collect the ethyl acetate layer and concentrate under reduced pressure to produce an extract, the crude fermentation extract.

[0027] (3) Preparation of Chaetomium sphaeroides DX-THS3 bacterial fertilizer

[0028] Referring to the fermentation method for preparing crude fermentation extracts, Chaetomium globosum was allowed to ferment in a straw solid culture medium in the dark for 24 days, and then the fermented products were collected and crushed using a grinder to produce bacterial fertilizer.

[0029] Example 2:

[0030] Inhibition zone experiments of the crude fermentation extract of Chaetomium globosum DX-THS3 (prepared in Example 1) against various plant pathogenic fungi:

[0031] The mycelium growth inhibition method was used to determine the antifungal activity of the crude extract. The extract (crude product) was fully dissolved in methanol, and the solution was filtered through a 0.22μm sterile filter and mixed with PDA culture medium at about 50°C to prepare a 1mg / mL drug-containing plate. An equal volume of methanol without extract was used as a blank control. A circular punch with a diameter of 6mm was used to take bacterial cakes of the same size from the edge of the activated plant pathogenic fungus colony and inoculated them in the center of the drug-containing plate. Each treatment was repeated 3 times and cultured in a constant temperature incubator at 28°C in the dark. When the pathogenic fungus in the blank control group grew to near the edge of the culture dish, the mycelial growth diameter was measured by the cross-cross method (excluding the 6mm diameter of the bacterial cake) to calculate the inhibition rate.

[0032] The results are as follows Figure 1 (A~I are blank control pictures of Sclerotinia sclerotiorum, Phytophthora capsici, Rice blast fungus, Pyricularia oryzae, Fusarium oxysporum, Fusarium genus, Pythium graminearum, and Sheath blight of rice; a~i are treatment pictures of crude extracts of each pathogenic fungus). The test results show that the fermentation crude extract of the strain Chaetomium sclerotiorum DX-THS3 of the present invention has an inhibition rate of 100%, 92.86%, 85.94%, 83.90%, 76.12%, 73.02%, 66.18%, 58.96% and 52.99% against Sclerotinia sclerotiorum, Phytophthora capsici, Magnaporthe or yzae, Monilinia fructicola, Fusarium oxysporum, F. culmorum, Sarocladium oryzae, F. graminearum and Rhizoctonia solani, respectively. Compared with the fermentation conditions of Chaetomium sclerotiorum before optimization, the antibacterial activity of the fermentation crude extract is significantly improved (such as Figure 2 , A~I, a~i are the same as above), increasing by 17.72%, 24.86%, 14.05%, 12.60%, 12.88%, 10.86%, 15.68%, 9.60% and 10.13% respectively.

[0033] Example 3:

[0034] Experiment on the effect of Chaetomium sphaeroides DX-THS3 bacterial fertilizer (prepared in Example 1) on pepper growth:

[0035] Select healthy pepper seeds and sterilize them in a 55°C waterbath for 15 minutes. Then soak them in 30°C warm water for 6 hours. Then, place the seeds in a Petri dish lined with moistened filter paper and germinate them at 28°C in a dark, moisturized environment for 4 days. Once the seeds germinate, plant them in seedling trays. Once the peppers have four leaves and a heart, transplant them into plastic pots for potting experiments.

[0036] The five treatments were: ① original soil (no additions); ② soil + 100g of a mixture of wheat straw and wheat bran; ③ soil + 100g of a fertilizer containing Chaetomium sphaeroides; ④ soil + 100g of sterilized fertilizer; and ⑤ soil + 2g of chemical fertilizer. Each treatment was replicated five times, for a total of 25 potted experiments. First, equal amounts of soil were placed in 25 pots. Five pots of soil were left untreated, five pots were supplemented with a mixture of straw and wheat bran, five pots were supplemented with a fertilizer containing Chaetomium sphaeroides, five pots were supplemented with sterilized fertilizer, and five pots were supplemented with chemical fertilizer. Each additive had consistent quality and was thoroughly mixed with the soil. Pepper seedlings with consistent growth were then selected and transplanted into plastic pots at a planting depth of approximately 2.5cm. Two seedlings were grown per pot, with a spacing of approximately 6cm between them. After transplanting, the plastic pots were placed in a greenhouse for cultivation, randomly arranged in order, watered regularly, and photographed weekly to record growth. When the peppers have grown many fruits (about 120 days), the pot experiment can be ended and various indicators of the peppers and soil can be measured.

[0037] The experimental results are as follows Figure 3-7 As shown ( Figure 3 、 4 , 5 (from left to right are treatments ①, ②, ③, ④, ⑤). After 30 days and 120 days of planting pepper seedlings with the bacterial fertilizer of the present invention, the fresh weight of the plants increased by 98% and 61.88% respectively compared with the control (without any addition), the fresh weight of the roots increased by 61.00% and 73.59% respectively, the plant height increased by 31.00% and 12.62% respectively, the stem diameter increased by 78.00% and 23.67% respectively, and the number of leaves increased by 15.00% and 37.60% respectively. In addition, the application of the bacterial fertilizer of the present invention can significantly improve the yield and quality of pepper fruits. Compared with the control, the number of fruits per pepper plant increased by 60.71%, and the fruit weight increased by 82.49%; the soluble protein content of pepper fruits increased by 24.42%, the soluble sugar content increased by 7.49%, the organic acid content increased by 34.32%, and the Vc content increased by 44.82%. The specific data are shown in Tables 1 and 2. It is worth noting that the growth-promoting effect of adding this bacterial fertilizer is equivalent to that of adding chemical fertilizers, and is better than directly adding straw. Moreover, after the bacterial fertilizer is sterilized, its growth-promoting effect is slightly reduced, but the growth-promoting effect is still acceptable and better than directly adding straw.

[0038] Application of a composite endophytic Bacillus fertilizer has been reported to increase pepper plant height by 5.24%, stem diameter by 11.83%, fruit number by 72.22%, fruit weight by 72.55%, fruit soluble sugar content by 28.88%, fruit vitamin C content by 1.29%, and fruit soluble protein content by 7.02%. (Quoted from Effects of Microbial Fertilizers and Soil Remediation Agents on the Growth, Quality, and Yield of Dried Peppers) Therefore, compared to composite endophytic Bacillus fertilizer, the Chaetomium sphaeroides fertilizer of the present invention is significantly more effective in promoting pepper plant growth and increasing pepper fruit yield and quality.

[0039] Table 1

[0040]

[0041] Table 2

[0042]

[0043] At the same time, soil nutrient parameters (pH, moisture content, organic matter, total nitrogen, total phosphorus, total potassium, nitrate nitrogen, ammonium nitrogen, available phosphorus, and available potassium) were monitored at 30 and 120 days using a soil nutrient analyzer. Urease, sucrase, catalase, acid phosphatase, cellulase, and arylsulfatase activities were measured using corresponding kits. Compared to treatment 1 (no addition), the addition of bacterial fertilizer (treatment 4) improved soil fertility and enzyme activities to varying degrees. The specific results are shown in Tables 3-6.

[0044] The microbial fertilizer "Ningdun" is a product whose main component is Bacillus. Application of this fertilizer for 30 days can increase the organic matter, available phosphorus, and available potassium contents in pepper soil by 11%, 21%, and 19%, respectively; increase the activities of soil urease, phosphatase, cellulase, and catalase by 20%, 29%, 100%, and 40%, respectively; but reduce the activity of sucrase (quoted from the effect of application of the microbial fertilizer "Ningdun" on bacterial diversity and soil enzyme activity in the rhizosphere of pepper). After 30 days of application, the fertilizer can increase the organic matter, available nitrogen, available phosphorus, and available potassium contents in the soil by 17.28%, 194.57%, and 59.66%, respectively; and increase the activities of soil urease, phosphatase, cellulase, catalase, and sucrase by 39.23%, 11.33%, 13.38%, 41.3%, and 37.66%, respectively. Therefore, in terms of improving soil fertility, the bacterial fertilizer of the present invention is completely superior to the microbial fertilizer "Ningdun", and is better than the microbial fertilizer "Ningdun" in improving the activities of soil urease, catalase and sucrase.

[0045] Table 3

[0046]

[0047] Table 4

[0048]

[0049] Table 5

[0050]

[0051] Table 6

[0052]

[0053] The above description is merely a preferred embodiment of the present invention. The present invention is not limited to the above-described embodiments. As long as the technical effects of the present invention are achieved by the same means, they shall fall within the scope of protection of the present invention. Within the scope of protection of the present invention, various modifications and variations of the technical solutions and / or implementation methods may be made.

Claims

1. Use of a fermentation extract of Chaetomium globosum in the preparation of a product for inhibiting peach brown rot and / or Pythium scabra, characterized in that: The name of the Chaetomium globosum is Chaetomium globosum DX-THS3, the deposit address is China Center for Type Culture Collection, the deposit date is January 4, 2016, and the deposit number is CCTCC NO: M 2016005; The specific preparation process of the fermentation extract is as follows: Chaetomium globosum is inoculated into PDA culture medium for activation, then inoculated into PDB culture medium, cultured in the dark at 28°C and 160 rpm for 4-5 days to obtain seed liquid, and then inoculated into straw solid culture medium for fermentation in the dark at 26°C for 24 days, and then the product is extracted with acetone and ethyl acetate to obtain the fermentation extract.

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