CN113234602B

CN113234602B - Chaetomium globosum, microbial inoculum, seed soaking liquid and application

Info

Publication number: CN113234602B
Application number: CN202110359081.4A
Authority: CN
Inventors: 张颖施; 陈娟; 陈泽怡; 彭锦胜; 梁均钿
Original assignee: Moon Guangzhou Biotech Co ltd
Current assignee: Moon Guangzhou Biotech Co ltd
Priority date: 2021-04-01
Filing date: 2021-04-01
Publication date: 2022-10-18
Anticipated expiration: 2041-04-01
Also published as: CN113234602A

Abstract

The invention discloses chaetomium globosum, a microbial inoculum, a seed soaking solution and application, and relates to the technical field of agricultural biology. Is preserved in Guangdong province microorganism strain preservation center with the preservation number of GDMCC No:61363 under the name Chaetomium globosum. Experiments prove that the strain has broad spectrum for improving the stress resistance of crops, and can obviously improve the drought resistance and the saline-alkali resistance of the crops. Under the stress of drought environment, chaetomium globosum MN110495 can produce extracellular polysaccharide, antioxidant and other substances to resist the drought environment and improve the drought resistance of various plants such as fruits, vegetables, grain crops and the like. Chaetomium globosum also has the function of promoting seed germination and crop growth.

Description

Chaetomium globosum, microbial inoculum, seed soaking liquid and application

Technical Field

The invention relates to the technical field of agricultural biology, and particularly relates to chaetomium globosum, a microbial inoculum, a seed soaking solution and application.

Background

The shortage of water resources is a global problem restricting the agricultural production at present, the global industrialization is rapidly developed, natural disasters are frequent, the precipitation is reduced, about 43 percent of cultivated lands are dry early and semi-dry early areas, the dry early stress seriously influences the growth and development of plants, and the crop yield is reduced. At present, in the agricultural field, seaweed fertilizers, amino acid water-soluble fertilizers and plant growth regulators are mainly used for assisting drought resistance of crops, and humic acid is one of the representatives of the seaweed fertilizers, the amino acid water-soluble fertilizers and the plant growth regulators. Humic acid fertilizer is a kind of chemical fertilizer made up by using peat rich in humic acid and weathered coal as main raw material and making them pass through the processes of ammoniation, nitration and salinization, and adding trace elements and other preparation. Although humic acid has multiple effects of promoting the growth and development of crop roots, enhancing the stress resistance of crops, increasing yield and enhancing efficiency and the like, the humic acid also has a series of problems of high cost, long production period and the like of the traditional fertilizer.

In recent years, researches for promoting crop growth and improving crop stress resistance by using microorganisms are increasing. Patent CN103667132B discloses that bacillus cereus L90 can improve the drought resistance of walnuts. Patent CN110358695A discloses that pseudomonad G3 can improve drought resistance of red date plants. In patent CN105754901B, it is disclosed that a Klebsiella LTGPAF-6F strain can improve the defense ability of wheat seedlings against drought stress. However, the existing patents have the following problems: most strains only have effect on a single certain crop, and cannot generate broad-spectrum synergistic effect on most conventional crops. Therefore, the development of more efficient, broad-spectrum and economic microbial fertilizers is urgently needed to improve the drought resistance and saline-alkali resistance of crops and promote the rapid growth of the crops in adverse environments.

In view of this, the invention is particularly proposed.

Disclosure of Invention

The invention aims to provide chaetomium globosum, a microbial inoculum, a seed soaking solution and application to solve the technical problems.

The invention is realized by the following steps:

the invention provides application of a fungal strain of Chaetomium globosum (Chaetomium globosum) species in preparation of seed soaking liquid, seed soaking powder or a microbial agent.

The chaetomium globosum is preserved in the culture collection of microorganisms in Guangdong province, and the preservation address is as follows: the preservation date of the 5 th building of 59 th building of 100 th college of the Xieli middle Lu of Guangzhou city, guangdong province microbial research institute: 12/10/2020, accession number GDMCC No:61363 under the taxonomic name Chaetomium globosum.

The invention provides an application of a fungal strain of Chaetomium globosum (Chaetomium globosum) species in preparing a crop growth regulator or a seed germination regulator.

The crop growth regulator is suitable for the growth regulation of economic and grain crops in saline-alkali soil, arid land barren soil or fertile soil; the seed germination regulator is suitable for the germination of seeds of economic crops and grain crops;

preferably, the commercial crop is selected from at least one of the following commercial crops: solanaceae, rosaceae, rutaceae, musaceae, cucurbitaceae, papilionaceae, compositae, liliaceae, zingiberaceae, passifloraceae, anacardiaceae, araliaceae and Cactaceae; the food crops are selected from gramineous crops;

preferably, the fungal strain is a strain deposited under the accession number GDMCC No: 61117A deposited Chaetomium globosum or its progeny strain.

The invention provides a chaetomium globosum, which is preserved in the Guangdong province microorganism strain preservation center with the preservation address as follows: the preservation date of the 5 th building of 59 th building of 100 th college of the Xieli Zhonlu of Guangzhou city, guangdong province microbial research institute: 12/10/2020, with a deposit number of GDMCC No:61363 under the name Chaetomium globosum.

In a preferred embodiment of the present invention, the colony culture of chaetomium globosum is characterized by: the hyphae on the fungus culture medium are white initially, light brown later, grey brown to olive brown in the ascocarp shell, medium-sized, scattered or aggregated, and oval.

Optionally, the fungus culture medium is PDA culture medium, SDAY culture medium, liquid Sabouraud culture medium, modified Martin culture medium, malt extract, corn flour agar, high salinity Chaudhur culture medium or Bengal culture medium.

Optionally, the optimal growth temperature of chaetomium globosum is 25-30 ℃.

The invention also provides seed soaking liquid or seed soaking powder, which comprises the chaetomium globosum.

In a preferred embodiment of the present invention, the chaetomium globosum is an effective component of seed soaking liquid or seed soaking powder.Preferably, the effective amount of chaetomium globosum in the seed soaking solution is 10 ⁴ cfu/mL～10 ⁹ cfu/mL。

Preferably, the effective amount of chaetomium globosum in the seed soaking solution is 10 ⁶ cfu/mL。

Alternatively, the seed soaking liquid or seed soaking powder is suitable for seed soaking of crops.

Optionally, the crop comprises at least one of a cash crop and a food crop.

Optionally, the cash crop is selected from at least one of the following cash crops: solanaceae, rosaceae, rutaceae, musaceae, cucurbitaceae, papilionaceae, compositae, liliaceae, zingiberaceae, passifloraceae, anacardiaceae, araliaceae and Cactaceae; the food crop is selected from Gramineae.

Optionally, the solanaceae is selected from at least one solanaceae crop of: potatoes, peppers and tomatoes; the Rosaceae is selected from at least one of the following Rosaceae crops: strawberry and papaya; the Rutaceae is selected from Rutaceae crops as follows: citrus; the Musaceae family is selected from Musaceae crops as follows: bananas; cucurbitaceae selected from cucumber; the Papilionaceae family is selected from semen glycines, the Compositae family is selected from lettuce, the Liliaceae family is selected from Bulbus Allii, the Zingiberaceae family is selected from rhizoma Zingiberis recens, the Passiflorae family is selected from Passiflora edulis, the Anacardiaceae family is selected from fructus Ananadis Comosi, the Araliaceae family is selected from Notoginseng radix, and the Cactaceae family is selected from fructus Hylocerei.

The invention also provides a microbial inoculum which comprises the chaetomium globosum; preferably, the microbial inoculum takes chaetomium globosum as a main effective component.

The invention also provides application of chaetomium globosum in preparation of seed soaking liquid, seed soaking powder or a microbial inoculum.

The invention also provides application of the chaetomium globosum or the microbial inoculum in preparing a crop growth regulator or a seed germination regulator.

The crop growth regulator is suitable for the growth regulation of economic and grain crops in saline-alkali soil, arid land barren soil or fertile soil; the seed germination regulator is suitable for seed germination of economic crops and grain crops.

Optionally, the cash crop is selected from at least one of the following cash crops: solanaceae, rosaceae, rutaceae, musaceae, cucurbitaceae, papilionaceae, compositae, liliaceae, zingiberaceae, passifloraceae, piperaceae, araliaceae, and Cactaceae; the food crop is selected from Gramineae.

The invention also provides a fertilizer or a pesticide which comprises the chaetomium globosum or the microbial inoculum.

In a preferred embodiment of the present invention, the fertilizer or pesticide is applied to the foliage or roots of the crops.

The invention has the following beneficial effects:

the invention provides Chaetomium globosum (Chaetomium globosum), which is proved by experiments to have broad spectrum for improving the stress resistance of crops and can obviously improve the drought resistance and the saline-alkali resistance of the crops. Under the stress of drought environment, chaetomium globosum MN110495 can produce extracellular polysaccharide, antioxidant and other substances to resist the drought environment and improve the drought resistance of various plants such as fruits, vegetables, grain crops and the like. The chaetomium globosum has the functions of promoting seed germination and crop growth, and can produce indoleacetic acid to resist drought stress, raise the germination speed of seed and the growth speed of crop in drought environment and raise the survival rate of crop. In addition, chaetomium globosum can improve the saline-alkali resistance of crops and promote the growth of the crops in the saline-alkali environment.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and those skilled in the art can also obtain other related drawings based on the drawings without inventive efforts.

FIG. 1 is a phylogenetic tree analysis diagram;

FIG. 2 is a diagram of the harvest of a wheat plant potted in water the next day.

Detailed Description

Reference will now be made in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of explanation, not limitation, of the invention. Indeed, it will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. For instance, features illustrated or described as part of one embodiment, can be used on another embodiment to yield a still further embodiment.

The chaetomium globosum is preserved in the Guangdong province microorganism strain preservation center at the preservation address: the preservation date of the No. 59 building 5 of the No. 100 institute of Pieli Zhonglu, guangzhou province, microorganism research institute: 12/10/2020, accession number GDMCC No:61363 under the name Chaetomium globosum.

preferably, the commercial crop is selected from at least one of the following commercial crops: solanaceae, rosaceae, rutaceae, musaceae, cucurbitaceae, papilionaceae, compositae, liliaceae, zingiberaceae, passifloraceae, piperaceae, araliaceae, and Cactaceae; the food crop is selected from Gramineae;

preferably, the fungal strain is a strain deposited under the accession number GDMCC No:61117 the deposited Chaetomium globosum or its progeny strain.

The invention provides a chaetomium globosum which is preserved in the preservation center of microbial strains in Guangdong province, and the preservation address is as follows: the preservation date of the No. 59 building 5 of the No. 100 college of the Pieli Zhonglu city, guangdong province microbial research institute: 12/10/2020, accession number GDMCC No:61363 under the name Chaetomium globosum.

In a preferred embodiment of the present invention, the colony culture of chaetomium globosum is characterized in that: on the fungus culture medium, the hypha is initially white, and is light brown at the later stage, the ascocarp is grayish brown to olive brown, and is medium-sized, scattered or aggregated, and egg-shaped.

Optionally, the optimal growth temperature of chaetomium globosum is 25-30 ℃.

The ITS sequencing result of the strain is shown as the following SEQ ID NO. 1:

ACATTACAGAGTTGCAAAACTCCCTAAACCATTGTGAACGTTACCTATACCGTTGC TTCGGCGGGCGGCCCCGGGGTTTACCCCCCGGGCGCCCCTGGGCCCCACCGCGGGCG CCCGCCGGAGGTCACCAAACTCTTGATAATTTATGGCCTCTCTGAGTCTTCTGTACTGA ATAAGTCAAAACTTTCAACAACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGC GAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGC ACATTGCGCCCGCCAGCATTCTGGCGGGCATGCCTGTTCGAGCGTCATTTCAACCATC AAGCCCCCGGGCTTGTGTTGGGGACCTGCGGCTGCCGCAGGCCCTGAAAAGCAGTGG CGGGCTCGCTGTCGCACCGAGCGTAGTAGCATACATCTCGCTCTGGTCGCGCCGCGGG TTCCGGCCGTTAAACCACCTTTTAACCCAAGGTTGACCTCGGATCAGGTAGGAAGACC CGCTGAACTTAAGCATATCA

the sequencing result of the strain is shown as the following SEQ ID NO. 2:

CGGGCGGAAGAGCTGGCCGAAGGGGCCGGCACGGACGGCATCCATGGTGCCGG GCTCCAAGTCGACGAGGACAGCGCGAGGAACATACTTGTTGCCGGAAGCCTGTATAAT ACTATGAGCACGCCATCACTCGGTCGTGTAAAATTATTTGACCCGACTGACCTCGTTGA AGTACACGTTCATACGCTCGAGCTGGAGCTCGGAGGTGCCGTTGTACCTATCGAAGCG GTGAGCGGGGATTTATCGGTTGGGAACTGTCATGCCCACATACACGCCATTGCTGTCG AGGCCGTGCTCGCCAGAGATGGTCTGCCTGAAAAGAAGTCAGTCTCGATTGTCATCA GCCAAGAGTGTTTGCTTTGCTTGGACGTACCAGAAAGCGGCACCGATTTGGTTACCCT AGAGAGCTCCATGTCAGTTATCCTGCGCGGCCGGTCGGTCGTAATGTTCGATTCGGTC CAACTTACGCACTGGCCGGTCTGGAGGTGAACCTATAGTACGAAAAAGCAGTCAGCA TCATCAAGCTTCGCATCATCGTCGTGTTGCTATCCGTCAGGAGCTGTGGAAATGAGGG TCGTCCCGATGGGTGGGGTAGTTCAGGGGCCCAAAAAAAAAGCTCCCAGACGCGTCG TCGCCGTGCGGCTCAGGAATGTGCGGGGTGACTTACA

a phylogenetic tree established based on polygenes (ITS, benA) shows that the strain GDMCC 61363 is Chaetomium globosum.

In a preferred embodiment of the present invention, the chaetomium globosum is an effective component of seed soaking liquid or seed soaking powder. Preferably, the effective amount of chaetomium globosum in the seed soaking solution is 10 ⁴ cfu/mL～10 ⁹ cfu/mL。

The seed culture medium is a culture medium of chaetomium globosum or an aqueous dilution of chaetomium globosum solution.

In other embodiments, the chaetomium globosum can be produced into freeze-dried powder and seed soaking powder, and the seed soaking powder is mixed with water when in use. Optionally, the seed soaking liquid or the seed soaking powder further comprises a preservative, an antioxidant, a stabilizer and other ingredients to improve the stability and activity of the finished product.

In actual use, seeds to be treated can be soaked after being disinfected. The number of spores in the seed soaking liquid can be adjusted by fermentation culture according to the requirements, and optionally, the number of spores in the fermentation liquid is adjusted to 10 ⁴ CFU/mL or more.

Optionally, the crop comprises at least one of a cash crop and a food crop.

Optionally, the commercial crop is selected from at least one of the following commercial crops: solanaceae, rosaceae, rutaceae, musaceae, cucurbitaceae, papilionaceae, compositae, liliaceae, zingiberaceae, passifloraceae, anacardiaceae, araliaceae and Cactaceae; the food crop is selected from Gramineae.

Optionally, the solanaceae is selected from at least one solanaceae crop of: potato, pepper, tobacco, ningxia wolfberry fruit, stramonium, black nightshade and tomato; rosaceae is selected from at least one of the following Rosaceae crops: apple, pear, strawberry and papaya; the Rutaceae is selected from Rutaceae crops as follows: citrus; the Musaceae family is selected from the following Musaceae family crops: bananas; cucurbitaceae is selected from fructus Cucumidis Sativi, cucurbita pepo, pulp Citrulli, fructus Cucurbitae Moschatae, etc.; the Papilionaceae family is selected from semen glycines, semen Viciae Fabae, semen Arachidis Hypogaeae, semen Pisi Sativi, semen Vignae sinensis, semen Phaseoli Radiati or semen Cajani, the Compositae family is selected from lettuce, caulis et folium Lactucae Sativae, herba Xanthii, flos Chrysanthemi or sunflower, the Liliaceae family is selected from Bulbus Allii, rhizoma Polygonati Odorati or rhizoma anemarrhenae, the Zingiberaceae family is selected from rhizoma Zingiberis recens, fructus Amomi or Curcuma rhizome, the Passiflorae family is selected from Passiflora edulis, the Anacardiaceae family is selected from fructus Ananadis Comosi, the Caucaceae family is selected from Notoginseng radix, and the Cactaceae family is selected from Hylochiaceae fructus Pyrolae.

In other embodiments, the seed soaking liquid or the seed soaking powder provided by the present invention may be used for seed soaking of other crops, without being limited to the above-mentioned types of crops.

The invention also provides a microbial inoculum, which comprises the chaetomium globosum; preferably, the microbial inoculum takes chaetomium globosum as a main effective component.

The microbial inoculum can be liquid preparation, solid microbial inoculum, pasty microbial inoculum, suspending agent, emulsion, spray or semisolid microbial inoculum.

Besides chaetomium globosum as a main active ingredient, the microbial inoculum can also comprise other addition auxiliary agents.

The invention also provides application of the chaetomium globosum in preparing seed soaking liquid, seed soaking powder or microbial inoculum.

The crop growth regulator is suitable for the growth regulation of economic and grain crops in saline-alkali soil, arid land barren soil or fertile soil; the seed germination regulator is suitable for germination of seeds of economic crops and grain crops.

Optionally, the commercial crop is selected from at least one of the following commercial crops: solanaceae, rosaceae, rutaceae, musaceae, cucurbitaceae, papilionaceae, compositae, liliaceae, zingiberaceae, passifloraceae, piperaceae, araliaceae, and Cactaceae; the food crop is selected from Gramineae.

In a preferred embodiment of the present invention, the fertilizer or pesticide is applied to the foliage or roots of the crops. The leaf fertilizer or root fertilizer can be water-soluble fertilizer or other soluble fertilizer.

The fertilizer can be used as a base fertilizer, and can also be applied by being mixed with other organic fertilizers, inorganic fertilizers or organic-inorganic fertilizers.

In order to make the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are conventional products which are not indicated by manufacturers and are commercially available.

The features and properties of the present invention are described in further detail below with reference to examples.

Preparation of the following reagents

5% sodium hypochlorite: 5ml of sodium hypochlorite and 95ml of sterile water are prepared in situ without sterilization;

75% of ethanol: 26.67ml of sterile water and 73.33ml of 95 percent ethanol are prepared in situ without sterilization;

PDA culture medium: 16.04g of PDA and 400mL of tap water;

PDB culture medium: 9.6g of PDB and 400mL of tap water;

RBM medium: RBM 14.66g, tap water 400mL;

sodium carboxymethylcellulose: 0.7% of CMC suspension, heating and stirring at 655rpm and 100 ℃ for 2h, and sterilizing at 121 ℃ for 20 min;

sterile water: weighing 1000ml of sterile water, sterilizing at 121 ℃ for 20 min.

Hoagland nutrient solution: 1.28g of the calcium nitrate tetrahydrate is weighed into 900mL of distilled water or deionized water, boiled and dissolved, and 0.945g of the calcium nitrate tetrahydrate is also weighed into 100mL of water and dissolved. Mixing the two solutions, and autoclaving at 121 deg.C for 20 min.

AGPS medium: 10g/L of polypeptone, 10g/L of glycerol, 20ml/L of S1, 20ml/L of S2, 20ml/L of S3, 0.1% L-tryptophan (0.1% of the mixed solution) was added before inoculation.

Solution 1: 14g/400mL of anhydrous dipotassium phosphate and 10.8g/400mL of anhydrous potassium dihydrogen phosphate;

solution 2: 50g/L of magnesium sulfate heptahydrate and 25g/L of anhydrous ammonium chloride;

solution 3: 10g/L of calcium chloride dihydrate;

salkowski reagent colorimetric solution: 250ml of H ₂ O，150ml H ₂ SO ₄ And 7.5ml of 5mol/L FeCl ₃ .6H ₂ And storing in an O, 4-degree refrigerator.

EPS special culture medium: yeast extract 2% and K ₂ HPO ₄ 1.5％、MgSO ₄ 0.02％、MnSO ₄ 0.0015％、 FeSO ₄ 0.0015％、CaCl ₂ 0.003%, naCl 0.0015%, agar 1.5%, sucrose 10%, and pH 7.5;

50mM Hoagland saline alkali: naCl 0.59g, naHCO ₃ 7.56g、Na ₂ SO ₄ 12.78g、Na ₂ CO ₃ 1.06g、hoagland 2L。

The culture medium and solution prepared above except for special description are sterilized in vertical pressure steam sterilizer at 121 deg.C for 20 min.

Example 1

This example provides methods for screening, isolating and identifying Chaetomium globosum (Chaetomium globosum) MN110495 strain.

(1) Screening and isolation of strains:

the samples were from root soil collected from cotton plantation in Henan province. Weighing 10g of dry soil, putting the soil into a sterilized mortar, mashing the soil with a pestle, placing the gauze, washing with tap water, attaching clay to the gauze, transferring the gauze to a centrifuge tube, adding 50ml of sterile water, carrying out vortex, centrifuging at 10000rpm for 6min, pouring off supernatant, washing with sterile water, centrifuging again to precipitate particles to the bottom of a conical centrifuge tube, and repeatedly centrifuging and cleaning for 3 times. 1g of the washed soil is diluted with 20mL of sterile sodium carboxymethylcellulose suspension, and the diluted precipitate is coated on an RBM medium and cultured in the dark for 7d. Selecting mycelium, inoculating to PDA culture medium, culturing in dark at room temperature for 7d, and separating to obtain Chaetomium globosum (Chaetomium globosum) MN110495.

(2) Identification of the strains:

hyphae of the colonies on PDA plates appeared initially white and later pale brown. The shell of the ascon ranges from grayish brown to olive brown, of medium size, loose or aggregated, ovoid.

The ITS and BenA sequence determination is respectively carried out on the strain, gene extraction and PCR amplification are finished by adopting a Biospin fungal genome DNA extraction kit, amplification primers and an operation method are shown in table 1, and an amplification product is sequenced by Jinzhi Biotech Limited company.

ITS and BenA sequence sequencing results are shown in SEQ ID NO.1 and SEQ ID NO. 2. The strain was registered to the National Center for Biotechnology Information (NCBI) website, and the sequencing results were subjected to homology search using BLAST search, sequence alignment (https:// BLAST. NCBI. Nlm. Nih. Gov /), phylogenetic tree analysis, and it was determined that the strain was Chaetomium globosum. A phylogenetic tree analysis diagram is shown with reference to figure 1.

Table 1 PCR upstream and downstream primers.

Example 2

This example was carried out to prepare a fermentation broth of Chaetomium globosum MN110495 strain.

The cells obtained in 18 pieces of the separation and selection in example 1 were punched with a sterile punch, and the cells were filled in a 50mL sterile centrifuge tube containing 30mL PDB medium, incubated in a constant temperature shaking incubator at 28 ℃ under 200rmp for 4 days, and the spores were counted with a hemocytometerCounting, and adjusting the spore number of the fermentation liquor to 10 ⁶ CFU/mL。

Example 3

In this example, the influence of chaetomium globosum MN110495 strain on drought resistance of wheat seeds in germination period was studied. The technical route is shown in fig. 2.

Taking out wheat seeds (Jimai 23) from a refrigerator, selecting healthy seeds with consistent particle size, sterilizing with 75% ethanol for 1 min, washing with sterile water for 3 times, sterilizing with 5% sodium hypochlorite for 5min, and washing with sterile water for 3 times. Then seed soaking treatment is carried out by using the treatment solution under the condition of room temperature. The experimental group and the blank control group were set according to the treatment solutions.

The experimental components comprise an MN110495 treatment group, a negative control group and a positive control group, wherein the MN110495 treatment group uses the strain fermentation liquor in the embodiment 2 to soak seeds overnight; the negative control group is soaked in PDB culture solution; the positive control group was inoculated with a commercially available product (see Table 2). The blank control group was also inoculated with PDB medium.

Then, sowing the seeds soaked in the seed soaking pot into a seedling pot, wherein the seedling pot is filled with a hoagland nutrient solution according to the weight ratio of 1:8 (volume ml/mass g ratio), and repeating ten times, wherein the matrix soil is subjected to sterilization for 60min at 121 ℃ in a vertical steam autoclave before mixing, 3 seeds are sown in each seedling pot, and each group is subjected to sterilization. After sowing, 50mL of hoagland nutrient solution is poured into all seedling pots of the blank control group, and the experimental group is not poured. Then placing the mixture in a greenhouse at 25 ℃, illuminating for 12 hours every day, and carrying out drought treatment.

In the seed germination period (1-10 days after sowing), 10mL of hoagland nutrient solution is irrigated every other day by the experimental group, so that the soil humidity of the experimental group potted plant is maintained in a super severe drought state between 8% and 12%; and the blank control group is watered with 0-20 mL of hoagland nutrient solution every day, so that the soil humidity of the potted plant of the blank control group is maintained in a normal watering state of 75-80%.

After the seed germination period is finished, calculating a seed germination drought resistance index by counting the seed germination number, and further analyzing the seed germination conditions of different experimental groups in the drought stress environment.

The water content is divided into 55-65% of soil drought grade (mild degree, 40-55% of moderate degree and 30-40% of severe degree), and 75-85% of normal irrigation.

And the seed germination drought resistance index = the seed germination index of the experimental group/the seed germination index of the blank control group.

Seed germination index =1 x nd ₂ +0.75*nd ₄ +0.5*nd ₆ +0.25*nd ₈

Where nd ₂ 、nd ₄ 、nd ₆ 、nd ₈ The drought resistance index of the seeds in the germination period is a reliable index for evaluating the drought resistance of the seeds in the germination period.

TABLE 2 list of commercially available products

The results in table 3 show that, although the germination drought resistance index (1.15) of the wheat seeds subjected to seed soaking treatment by the chaetomium globosum MN110495 strain fermentation liquor is lower than that of the chemical fertilizer type commercial product 1 (1.32) with fulvic acid as the main component in the super severe drought state, the germination drought resistance index is obviously superior to that of the similar microbial fertilizers commercial products 2 and 3 (the germination drought resistance indexes are 1.01 and 0.82 respectively), and is improved by 33.7 percent compared with that of a negative control group (0.86). Therefore, the chaetomium globosum MN110495 strain can effectively improve the germination speed of seeds under the drought condition, improve the drought resistance of the seeds and improve the survival rate of crops.

Table 3 comparison table of drought resistance conditions of each group of seeds in germination period.

Example 4 Effect of Chaetomium globosum MN110495 strain on wheat seedling drought resistance.

Transplanting the seedlings with the same growth vigor after the seeds germinate into seedling pots, wherein each seedling pot comprises three seedlings, and each group is provided with five repetitions. 10mL of hoagland nutrient solution is poured into the transplanted seedlings of the experimental group every other day, and 20mL of hoagland nutrient solution is poured into each pot plant of the experimental group on the 4 th day after transplantation, and then water cut-off treatment is started. And the transplanted blank control group seedlings are irrigated with 10mL of hoagland nutrient solution every day. And after 5 days of water cut-off, pouring 20mL of hoagland nutrient solution for rehydration, and counting the average plant height, the root surface area, the fresh weight, the dry weight and the plant drought grade of the plant the next day after rehydration. The results are shown in Table 4.

Evaluation standard of plant drought grade:

a grade 1-9 evaluation system is adopted, with grade 9 being the best and grade 1 being the worst.

Grade 9, plant is intact;

grade 8, the leaves begin to be soft but are not curled, and the color of individual areas of the leaves is lightened;

grade 7, the leaves are softened continuously, the leaf color is lightened continuously, and at least 1 leaf begins to curl;

grade 6, the leaves are softened continuously, the color is lightened, and most of the leaves begin to curl;

grade 5, all leaves start to become soft and curled, even the leaves droop;

grade 4, the lowest leaves begin to dry, the whole plant becomes bad, and the middle leaves are seriously curled;

grade 3, the two lowest leaves wither, and the other leaves begin to dry;

level 2, the middle leaves begin to dry;

grade 1, any more severe than grade 2, is classified as grade 1.

TABLE 4 growth of wheat seedlings in each group under drought conditions

Comparing the blank and negative control groups, it can be seen that the water cut-off treatment is not beneficial to the growth of wheat seedlings. After water supply is cut off, the growth indexes of the wheat seedlings of the negative control group in the experimental group are lower than those of the wheat seedlings of other experimental groups, and therefore the microbial fertilizer and the chemical fertilizer can effectively improve the growth speed of the plant seedlings. The wheat seedlings treated by the commercial product 2 have plant height, root surface area, fresh weight, dry weight and plant drought grade after rehydration which are all higher than those of the commercial product 1, and the chaetomium globosum MN110495 strain shows the treatment effect equivalent to that of the commercial product 1. It is shown that under drought conditions, microbial and chemical fertilizer products exhibit comparable or even better growth promoting effects.

In addition, the MN110495 treated group and the negative control group are independently compared, and the seedling height, the root surface area, the fresh weight and the dry weight of the MN110495 treated group are respectively improved by 12 percent, 15 percent, 14 percent and 11 percent compared with the negative control group in the super severe drought state. The drought grade of the MN110495 treated plant after rehydration is 6.73, which is improved by 15 percent compared with the drought grade of the negative control plant by 5.87 percent. As can be seen from FIG. 2, compared with the negative control group wheat plants, the MN110495 treatment group plants grow greenish, have less withered and yellow parts, and have thicker and longer root systems and more lateral roots. Therefore, the MN110495 treatment group has the effects of promoting the growth of plants and accelerating the revival of the plants in a drought environment state.

Example 5

This example further analyzes the drought-resistant effect of chaetomium globosum MN110495 strain in other crops.

PDB seed soaking is used as a control group, chaetomium globosum MN110495 strain fermentation liquor seed soaking is used as an experimental group, the seed soaking, sowing, seedling transplanting and drought resisting test methods described in the embodiments 3 and 4 are adopted to test the drought resisting effect of more varieties of existing plants in laboratories such as rice, corn, potato and the like, and finally the dry weight conditions of the seedlings of each plant in the next day of water cut-off treatment and rehydration are measured, so that the drought resisting effect of the chaetomium globosum MN110495 strain on the plants is verified.

The chaetomium globosum MN110495 strain acts on the roots of plants and can obviously enhance the growth speed of the roots. This example selects the dry weight of harvested material as the index of drought-resistant effect to illustrate the drought-resistant and growth-promoting effects of Chaetomium globosum MN110495 strain. As can be seen from Table 5, the chaetomium globosum MN110495 strain has broad spectrum in the aspect of improving the drought resistance of crops, can effectively improve the drought resistance of the crops, promotes the growth speed of plants in a drought environment, and obviously improves the dry weight of the plants.

TABLE 5 influence of Chaetomium globosum MN110495 strain on the drought resistance of the plants.

Example 6

This example analyzes the saline and alkaline resistance effect of chaetomium globosum MN110495 strain on corn plants.

The corn seed sterilization, soil preparation and sowing steps were the same as in example 3.

The experimental group and the blank control group were set according to the treatment solutions. The experimental components are that MN110495 treatment group uses the strain fermentation liquor in the embodiment 2 to soak seeds, and a negative control group uses LB culture solution to soak seeds; the positive control group was inoculated with a commercially available product (see Table 6), and the blank control group was also inoculated with LB medium. Measuring OD600, diluting the bacterial liquid concentration with LB until OD600 is 0.8-0.9, and soaking seeds for 5h.

And (3) sowing the seeds after seed soaking into a seedling pot, wherein the seedling pot is filled with matrix soil fully mixed with 50mM saline-alkali Hoagland according to the weight ratio of 1:1 (v/m) (the matrix soil is sterilized for 60min at 121 ℃ by a vertical steam autoclave before being mixed), 3 seeds are sown in each seedling pot, and each group is repeated five times. The blank was treated with an equal amount of normal Hoagland soil. Placing the mixture in a greenhouse at 25 ℃ for 12h each day, and carrying out saline-alkali treatment. After sowing for 1-5 days, pouring 20mL of 100mM saline-alkali Hoagland in each cup of the every-day experimental group; blank treatment 20mL of normal Hoagland was poured per cup. 30mL of 100mM saline alkali Hoagland is poured in an 8-12d alternate-day experimental group; blank treatment 30mL of normal Hoagland was poured per cup. 40mL 100mM haloglan is poured in the 15-22d every other day experimental group; blank treatment 40mL of normal Hoagland was poured per cup. The soil salinity of the pot planting soil of the experimental group is in a severe salinization state, and the blank control group is in a non-salinization state.

After the corn seeds are sown for 23 days, corn plants are harvested to measure the average fresh weight, the surface area, the dry weight and the saline-alkali resistance grade of the plants, and index results are shown in a table 7.

The salinity content distinguishes the soil salinization degree (saturated leachate 0-2 is non-salinization, 2-4 is salinization, 4-8 is medium salt, 8-16 is heavy saline soil, and >16 is extremely heavy saline soil).

The evaluation criteria for saline and alkaline resistance rating are as follows:

a1-5 grade evaluation system is adopted, with grade 5 being the best and grade 1 being the worst.

The leaves of the whole plant of the 5-grade plant are emerald green or the leaf tips of the plant are slightly withered and yellow

4-grade plant with large withered and yellow leaf area and withered heart leaves

The heart leaves of the 3-grade plants die and the whole leaves of the plants die, and the heart leaves are not unfolded yet and die

The 2-grade plant has severe withering and all leaves have withered and shriveled conditions

All leaves of 1-grade plants die completely

TABLE 6 list of commercially available products

Commercially available product	Number of	Manufacturer of the product	Composition (I)
				Potassium fulvate	Commercially available product	SHANXI HUIYINONG BIOLOGICAL TECHNOLOGY Co.,Ltd.	Bacillus amyloliquefaciens, fulvic acid and the like

TABLE 7 Harvest index of target strains in saline-alkali tolerance experiment

Treatment of	Fresh weight	Root surface area	Salt and alkali resistant grade	Dry weight of
					Blank control group	6.1	59.10	5	0.4
Negative control	3.37	29.64	3.24	0.24
					Positive control	3.61	28.25	4.22	0.26
MN110495 treatment group	3.74	36.44	3.59	0.25

Comparing the blank and negative control groups shows that the growth of corn seedlings is not facilitated in the severe salinization environment. After the severe salinization treatment, the growth indexes of the corn seedlings of the negative control group in the experimental group are lower than those of the corn seedlings of other experimental groups, and the growth speed of the plant seedlings can be effectively increased by microbial fertilizer and chemical fertilizer.

In addition, when the MN110495 treated group and the negative control group are independently compared, the fresh weight, the root surface area, the saline-alkali resistance grade and the dry weight of the seedlings of the MN110495 treated group are respectively improved by 11 percent, 22.9 percent, 10.8 percent and 4.1 percent compared with those of the negative control group under the heavy salinization environment. Therefore, the MN110495 treatment group has the function of promoting the growth of plants under the salinization environment state.

Example 7

This example performed an indoleacetic acid (IAA) production test on the strains.

The strain (spore or hyphae) was inoculated into an AGPS medium containing 0.1% L-tryptophan and cultured at 30 ℃ and 200rpm in a constant temperature shaking incubator for 2 days. mu.L of the strain fermentation solution was dropped on a white ceramic plate, and 200. Mu.L of Salkowski colorimetric solution (1. The white porcelain plate is placed at room temperature and protected from light for 30min, and then the color development result is observed.

And if the color development reaction result is obvious, performing further quantitative test, uniformly mixing the treatment liquid (the strain fermentation supernatant, 50mg/L IAA and AGPS culture medium) with the Salkowski colorimetric solution 1, reacting for 30min at room temperature in a dark environment, and quickly measuring the light absorption value of the mixed solution at the wavelength of 530nm of a spectrophotometer. Calculating the content of the indoleacetic acid according to a standard curve. A standard curve is prepared by pure 3-indoleacetic acid, and the range of the indoleacetic acid is determined to be 5-200 mu g/mL.

TABLE 8 Indolylacetic acid (IAA) production test data for strains

As shown in Table 8, the strain GDMCC 61363 produces IAA and acts on the surface area of the plant to promote the growth of the plant. The capability of plants in absorbing nutrients from soil is improved, and the survival rate of the plants is obviously improved under the drought stress condition.

Example 8

This example carried out EPS exopolysaccharide activity assay on the strains. Dripping 10 μ L of the bacterial solution on a sterile filter paper sheet with the diameter of 5mm in an EPS-producing special culture medium, and culturing for 2d at room temperature. Whether EPS is produced is judged according to the colony size around the filter paper and a transparent ring.

TABLE 9 Exopolysaccharide (EPS) Activity assay data (clear circles for secretion of the strains are indicated by "+")

Test number	Name of strain	Diameter/cm of bacterial colony	Transparent ring for secretion
				GDMCC
61363	Chaetomium globosum	3.27	+

From table 9, it is known that GDMCC 61363 can produce Extracellular Polysaccharide (EPS) to improve tolerance to drought stress, maintain soil and moisture stability, better adhere to plant roots, and increase available moisture of plant roots.

Example 9

In this example, the proline (Pro) content in the leaves of the plant was first determined.

Proline was assayed using a proline kit, and sample pretreatment and assay were performed according to the methods described in the kit instructions. The proline test box manufacturer establishes a bioengineering institute for Nanjing, and has the following goods number: a is 107-1-1, and,

TABLE 10 measurement data of proline (Pro) content in plant leaves

Proline (PRO) test kit

The blank tube is adjusted to zero by sterile water, the optical path is 1cm, and the wavelength is 520nm for color comparison.

Proline (PRO) test formula = ((assay OD value-blank OD value)/(standard OD value-blank OD value))/(standard OD value-blank OD value)/(g tissue wet weight/ml)/(dilution fold before sample test).

In this example, the extraction and activity of antioxidants (SOD, CAT, POD) in plant leaves were further measured.

The SOD activity is measured by adopting a kit for measuring superoxide dismutase by a hydroxylamine method, and sample pretreatment and measurement are carried out according to the method for measuring SOD in plant tissues in the instruction of a test kit.

CAT (EC 1.11.1.6) Activity measurement Using a catalase measurement kit by visible light method, sample pretreatment and measurement were performed according to the method for tissue sample detection described in the kit.

POD (EC 1.11.1.7) activity was measured using a peroxidase test kit, and sample pretreatment and measurement were carried out according to the method described in the specification of the test kit.

TABLE 11 determination of superoxide dismutase (SOD) content in plant leaves

TABLE 12 determination of Catalase (CAT) content in plant leaves

TABLE 13 determination of Peroxidase (POD) content in plant leaves

From tables 10 to 13, it can be seen that the GDMCC 61363 secretes a large amount of enzyme system and antioxidant substance capable of scavenging active oxygen and proline against stress when acting on plants.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

SEQUENCE LISTING

<110> Muen (Guangzhou) Biotechnology Ltd

<120> chaetomium globosum, microbial inoculum, seed soaking liquid and application

<160> 2

<170> PatentIn version 3.5

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acgccatcac tcggtcgtgt aaaattattt gacccgactg acctcgttga agtacacgtt 180

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ttatcggttg ggaactgtca tgcccacata cacgccattg ctgtcgaggc cgtgctcgcc 300

agagatggtc tgcctgaaaa gaagtcagtc tcgattgtca tcagccaaga gtgtttgctt 360

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Claims

1. Chaetomium globosum (Chaetomium globosum) The application of fungus strains of species in preparing microbial inoculum; the chaetomium globosum is preserved in the Guangdong province microorganism strain preservation center, and the preservation address is as follows: the preservation date of the No. 59 building 5 of the No. 100 college of the Pieli Zhonglu city, guangdong province microbial research institute: 12/10/2020, accession number GDMCC No:61363 under the name of Classification of strainsChaetomium globosum。

2. Chaetomium globosum (Chaetomium globosum) Application of fungus strains of species in preparation of crop growth regulators or seed germination regulators, wherein the fungus strains are selected from the group consisting of the fungus strains with a accession number GDMCC No: 61363A Chaetomium globosum deposited or its progeny strain; the chaetomium globosum is preserved in the Guangdong province microorganism strain preservation center, and the preservation address is as follows: the preservation date of the 5 th building of 59 th building of 100 th college of the Xieli Zhonlu of Guangzhou city, guangdong province microbial research institute: 12/10/2020, accession number GDMCC No:61363 under the name of Classification of strainsChaetomium globosum；

The crop growth regulator is suitable for regulating the growth of economic and grain crops in saline-alkali soil, arid land poor soil or fertile soil;

the seed germination regulator is suitable for germination of seeds of economic crops and food crops, and the economic crops are selected from at least one of the following economic crops: solanaceae, rosaceae, rutaceae, musaceae, cucurbitaceae, papilionaceae, compositae, liliaceae, zingiberaceae, passifloraceae, piperaceae, araliaceae, and Cactaceae;

the solanaceae is selected from at least one of the following solanaceae crops: potato, capsicum, and tomato; the Rosaceae is selected from at least one of the following Rosaceae crops: strawberry and papaya; the Rutaceae is selected from Rutaceae crops as follows: citrus fruit; the Musaceae family is selected from the following Musaceae family crops: bananas; the Cucurbitaceae is selected from cucumber; the Papilionaceae family is selected from soybean, the Compositae family is selected from lettuce, the Liliaceae family is selected from garlic, the Zingiberaceae family is selected from ginger, the Passiflorae family is selected from passion fruit, the Anacardiaceae family is selected from golden pineapple, the Araliaceae family is selected from pseudo-ginseng, the Cactaceae family is selected from dragon fruit;

the grain crop is selected from gramineous crops, and the gramineous crops are selected from rice and corn.

3. The chaetomium globosum is characterized by being preserved in the Guangdong province microorganism strain preservation center at the preservation address: the preservation date of the No. 59 building 5 of the No. 100 college of the Pieli Zhonglu city, guangdong province microbial research institute: 12/10/2020, accession number GDMCC No:61363 under the name of Classification of strainsChaetomium globosum。

4. The chaetomium globosum according to claim 3, characterized in that the colony culture of chaetomium globosum is characterized by: the hypha on the fungus culture medium is white at first, is light brown at later stage, and the ascocarp shell is gray brown to olive brown, medium-sized, scattered or aggregated, and oval; the fungus culture medium is a PDA culture medium.

5. The chaetomium globosum according to claim 4, wherein the optimal growth temperature of chaetomium globosum is 25-30 ℃.

6. A seed soaking liquid or powder comprising chaetomium globosum according to any one of claims 3 to 5.

7. The seed soaking liquid or seed soaking powder according to claim 6, wherein the chaetomium globosum is an effective component of the seed soaking liquid or seed soaking powder.

8. The seed soak solution or seed soak powder of claim 7 wherein the effective amount of Chaetomium globosum in the seed soak solution is 10 ⁴ cfu/mL~10 ⁶ cfu/mL。

9. The seed soak solution or seed soak powder of claim 7 wherein the effective amount of Chaetomium globosum in the seed soak solution is 10 ⁶ cfu/mL。

10. The seed soaking liquid or powder according to claim 7, wherein the seed soaking liquid or powder is suitable for seed soaking of crops including at least one of commercial crops and food crops;

the cash crop is selected from at least one of the following cash crops: solanaceae, rosaceae, rutaceae, musaceae, cucurbitaceae, papilionaceae, compositae, liliaceae, zingiberaceae, passifloraceae, piperaceae, araliaceae, and Cactaceae; the grain crop is selected from gramineous crops; the gramineous crop is selected from rice and maize;

the solanaceae is selected from at least one solanaceae crop of the following plants: potato, capsicum, and tomato; the Rosaceae is selected from at least one of the following Rosaceae crops: strawberry and papaya; the Rutaceae is selected from Rutaceae crops as follows: citrus fruit; the Musaceae family is selected from the following Musaceae family crops: bananas; the Cucurbitaceae is selected from cucumber; the Papilionaceae family is selected from soybean, the Compositae family is selected from lettuce, the Liliaceae family is selected from garlic, the Zingiberaceae family is selected from ginger, the Passiflorae family is selected from passion fruit, the Anacardiaceae family is selected from golden pineapple, the Araliaceae family is selected from pseudo-ginseng, and the Cactaceae family is selected from dragon fruit.

11. A bacterial preparation comprising chaetomium globosum according to any one of claims 3 to 5.

12. The microbial agent according to claim 11, wherein the microbial agent comprises chaetomium globosum as a main active ingredient.

13. A fertilizer, characterized in that it comprises chaetomium globosum according to any one of claims 3 to 5 or a microbial agent according to any one of claims 11 to 12.

14. The fertilizer material of claim 13, wherein said fertilizer material is applied to foliage or roots of a crop.