CN113215000A - Chaetomium gracile, seed soaking agent and application - Google Patents
Chaetomium gracile, seed soaking agent and application Download PDFInfo
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- CN113215000A CN113215000A CN202110360078.4A CN202110360078A CN113215000A CN 113215000 A CN113215000 A CN 113215000A CN 202110360078 A CN202110360078 A CN 202110360078A CN 113215000 A CN113215000 A CN 113215000A
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- chaetomium
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- crops
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Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01C—PLANTING; SOWING; FERTILISING
- A01C1/00—Apparatus, or methods of use thereof, for testing or treating seed, roots, or the like, prior to sowing or planting
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/30—Microbial fungi; Substances produced thereby or obtained therefrom
-
- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05G—MIXTURES OF FERTILISERS COVERED INDIVIDUALLY BY DIFFERENT SUBCLASSES OF CLASS C05; MIXTURES OF ONE OR MORE FERTILISERS WITH MATERIALS NOT HAVING A SPECIFIC FERTILISING ACTIVITY, e.g. PESTICIDES, SOIL-CONDITIONERS, WETTING AGENTS; FERTILISERS CHARACTERISED BY THEIR FORM
- C05G3/00—Mixtures of one or more fertilisers with additives not having a specially fertilising activity
Abstract
The invention discloses chaetomium gracile, a seed soaking agent and application, and relates to the technical field of agricultural biology. Chaetomium gracile with the preservation number of GDMCC No: 61364. the chaetomium gracile has the advantages of improving the drought resistance of crops and promoting the growth of the crops, can be artificially cultured, has high propagation speed, is convenient for large-scale production, and has good development and application prospects.
Description
Technical Field
The invention relates to the technical field of agricultural biology, and particularly relates to chaetomium gracile, a seed soaking agent and application.
Background
At present, seaweed fertilizers, amino acid water-soluble fertilizers and plant growth regulators are mainly used in the agricultural field to assist crops in resisting drought. The rhizosphere microorganisms are a general term for microorganisms distributed in the rhizosphere soil of crops, and microorganisms active in the rhizosphere soil are closely related to the growth state of crops. In order to solve the crop yield reduction caused by drought stress, a new solution is urgently needed to be found.
In view of this, the invention is particularly proposed.
Disclosure of Invention
The invention aims to provide chaetomium gracile, a seed soaking agent 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 tenue species in preparation of seed soaking liquid, seed soaking powder or microbial inoculum.
In a preferred embodiment of the present invention, Chaetomium gracile is deposited in the Guangdong province culture Collection with the following deposition addresses: 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: 61364, strain taxonomic name Chaetomium tenue, biomaterial name Chaetomium MN 113554.
Use of a fungal strain of the species Chaetomium tenue for the preparation of a plant growth regulator;
preferably, the plant growth regulator is a plant drought tolerant growth regulator.
In a preferred embodiment of the present invention, the plant growth regulator is suitable for growth regulation of gramineous crops;
preferably, the gramineous crop is maize, wheat, rice, sorghum or barley;
preferably, the fungal strain is a strain deposited under the accession number GDMCC No: 61364A deposited Chaetomium gracile or progeny strain thereof.
The invention provides a Chaetomium gracile which is preserved in Guangdong province microorganism strain preservation center with the preservation address 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: 61364, strain taxonomic name Chaetomium tenue, biomaterial name Chaetomium MN113554, survived.
In a preferred embodiment of the present invention, the colony culture of chaetomium gracile is characterized by: growing on MEA culture medium, spreading the bacterial colony on the whole plate, culturing later stage to form spherical black spore, and secreting light yellow secretion with smell.
The invention provides a seed soaking agent which comprises the chaetomium gracile.
In a preferred embodiment of the present invention, the seed soaking agent comprises chaetomium gracile as a main active ingredient;
preferably, the seed soaking agent is fermentation liquor of chaetomium gracile or freeze-dried powder of chaetomium gracile.
In a preferred embodiment of the application of the invention, the seed soaking agent is used for seed soaking of crops;
preferably, the crop comprises at least one of a cash crop and a food crop;
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 crop is selected from Gramineae;
preferably, the solanaceae is selected from at least one of the following solanaceae crops: potatoes, peppers and tomatoes; 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 the following Musaceae family crops: bananas; cucurbitaceae family 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;
preferably, the gramineous crop is maize, wheat, rice, sorghum or barley.
The invention also provides an application of chaetomium gracile or a seed soaking agent in preparation of a plant growth regulator;
preferably, the plant growth regulator is a plant drought tolerant growth regulator.
In a preferred embodiment of the present invention, the plant growth regulator is suitable for growth regulation of gramineous crops;
preferably, the gramineous crop is maize, wheat, rice, sorghum or barley.
The invention also provides a microbial inoculum which comprises the chaetomium gracile.
The invention also provides a fertilizer which comprises the chaetomium gracile or the microbial inoculum.
In a preferred embodiment of the application of the present invention, the fertilizer is a leaf fertilizer or a root fertilizer.
The invention has the following beneficial effects:
the chaetomium gracile screened by the invention has the advantages of improving the drought resistance of crops and promoting the growth of the crops, can be artificially cultured, has high propagation speed, is convenient for large-scale production, and has good development and application prospects. Through determination, the chaetomium gracile provided by the invention can produce indoleacetic acid, so that the chaetomium gracile acts on the surface area of a plant and promotes the growth and development of the plant. The capability of the plants to absorb nutrients from soil is improved, and the survival rate of the plants under the drought stress condition is obviously improved. Chaetomium elegans can also produce Extracellular Polysaccharide (EPS) to improve the tolerance to drought stress, maintain the stability of soil and water, better adhere to plant roots and increase the available water of the plant roots. When chaetomium gracile acts on plants, the plants can secrete a large amount of enzyme systems capable of clearing active oxygen, antioxidant substances and proline to resist the adverse circumstances, so that the stress resistance of the plants is improved.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed 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 for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 is a phylogenetic tree analysis diagram of Chaetomium gracile;
FIG. 2 is a technical roadmap;
FIG. 3 is a graph of relative germination vigor and seed germination drought resistance index for maize;
FIG. 4 is the average fresh weight of corn at harvest;
FIG. 5 shows the average plant height at the time of corn harvest;
FIG. 6 is the average root surface area at corn harvest;
FIG. 7 is the average dry weight at corn harvest;
FIG. 8 is a diagram of the harvesting of a potted maize plant;
FIG. 9 shows the average plant height at harvest of wheat;
FIG. 10 is the average root surface area at wheat harvest;
FIG. 11 is the average fresh weight of wheat at harvest;
FIG. 12 is the average dry weight of wheat at harvest;
FIG. 13 is the average drought tolerance index after water cut of wheat plants;
FIG. 14 shows the harvest pattern of a potted wheat plant.
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. In fact, 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 invention provides application of a fungal strain of Chaetomium tenue species in preparation of seed soaking liquid, seed soaking powder or microbial inoculum.
In a preferred embodiment of the present invention, Chaetomium gracile is deposited in the Guangdong province culture Collection with the following deposition addresses: 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: 61364, strain taxonomic name Chaetomium tenue, biomaterial name Chaetomium MN 113554. Survival was identified.
Use of a fungal strain of the species Chaetomium tenue for the preparation of a plant growth regulator;
preferably, the plant growth regulator is a plant drought tolerant growth regulator.
In a preferred embodiment of the present invention, the plant growth regulator is suitable for growth regulation of gramineous crops;
preferably, the gramineous crop is maize, wheat, rice, sorghum or barley;
preferably, the fungal strain is a strain deposited under the accession number GDMCC No: 61364A deposited Chaetomium gracile or progeny strain thereof.
The invention provides a Chaetomium gracile which is preserved in Guangdong province microorganism strain preservation center with the preservation address 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: 61364, under the name Chaetomium tenue.
The chaetomium gracile is separated from rhizosphere soil, and the colony culture characteristic is as follows: growing on MEA culture medium, spreading the bacterial colony on the whole plate, culturing later stage to form spherical black spore, and secreting light yellow secretion with smell.
The ITS sequencing result of the strain is shown as the following SEQ ID NO. 1:
ATCATTACAGAGTTGCAAAACTCCCTAAACCATCGTGAACGTTACCTATACCGTTG CTTCGGCGGGCGGCCCCGGGGTTTACCCCCCGGGCGCCCCTGGGCCCCACCGCGGGC GCCCGCCGGAGGTCACCAAACTCTTGATAATTTATGGCCTCTCTGAGTCTTCTGTACTG AATAAGTCAAAACTTTCAACAACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAG CGAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACG CACATTGCGCCCGCCAGTATTCTGGCGGGCATGCCTGTTCGAGCGTCATTTCAACCATC AAGCCCCCGGGCTTGTGTTGGGGACCTGCGGCTGCCGCAGGCCCTGAAAAGCAGTGG CGGGCTCGCTGTCGCACCGAGCGTAGTAGCATACATCTCGCTCTGGTCGCGCCGCGGG TTCCGGCCGTTAAACCACCTTTTAACCCAAGGTTGACCTCGGATCAGGTAGGAAGACC CGCTGAACTTAAGCATATCAATAAGCGGA
the sequencing result of the strain is shown as the following SEQ ID NO. 2:
CCGGGCGGAAGAGCTGGCCGAAGGGGCCGGCACGGACGGCGTCCATGGTGCCG GGCTCCAAGTCGACGAGGACGGCGCG
GGGAACATACTTGTTGCCGGAAGCCTTTATAATACTGTGAGCACGCCATCACTTG GTTGTGCAAAGGTATATGACCCTA
CTAACCTCGTTGAAGTACACGTTCATGCGCTCGAGCTGGAGCTCGGAGGTGCCGT TGTACCTATCGAAGCGGTGAGCGG
GGATTTATCGGTTGGGAACAGTCACGGCCACATACACGCCATTGCTGTCGAGGCC GTGCTCGCCAGAGATGGTCTGCCT
GAAAAGAAGTCAGTCTCGATTGTCATCAGCCACGAGTGTTTGCTTTGCTTGGACG TACCAGAAAGCGGCACCGATTTGG
TTACCCTGGAGAGCTCCATGTCAGTTATCCTGCACCGCTGGTCGGTCGTAATGTTC GATTCGGTCCAACTTACGCACTG
GCCGGTCTGGAGGTGAACCTATAGTAAGAAAAGCAGTCAGCATCATCAAGCTTCG CATCGCCGTCGTGTTGCTATCGGT
CAGGAGCTGTGGAAATGAGGGTCGTCCCGATGGGTGGGGTAGTTCAGGGGCCCA AAAAAAGCTTCCCAGACGCGTCGTA
GCCGTGTGGCTCAGGAATGTGCGGGGCGACTTACAATCTCACCGCATGTTGA
the research of the inventor proves that the separated strain has the functions of plant growth promotion and drought resistance. Can be used for assisting crops in resisting drought and promoting the yield increase of the crops. In addition, the above strains can also be used for preparing seed soaking agents.
Therefore, the invention also provides a seed soaking agent which comprises the chaetomium gracile.
Specifically, the seed soaking agent takes chaetomium gracile as a main effective component; additives such as a preservative, an antioxidant and a coloring agent may be optionally added to the seed soaking agent as required.
Optionally, the seed soaking agent is chaetomium gracilis fermentation liquor or chaetomium gracilis freeze-dried powder.
The dosage form of the seed soaking agent comprises a liquid preparation, a solid microbial inoculum, a pasty microbial inoculum, a suspending agent, an emulsion, a spray or a semisolid microbial inoculum.
In an alternative mode, the chaetomium gracile can be fungus powder prepared by vacuum freeze drying or fungus powder prepared by spray drying. When in use, the bacterial powder is dissolved.
In an alternative mode, the fermentation broth is a fermentation broth obtained by culturing chaetomium gracilis on a fungal culture medium.
In a preferred embodiment of the application of the present invention, the seed soaking agent is used for seed soaking of crops. In application, the seeds are soaked in the seed soaking agent solution. After the seed soaking agent treatment, the relative germination vigor and germination drought-enduring index of the seeds of the crops are obviously improved.
Optionally, the crops include at least one of commercial crops and food 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 crop is selected from Gramineae;
preferably, the solanaceae is selected from at least one of the following solanaceae crops: potatoes, peppers and tomatoes; 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 the following Musaceae family crops: bananas; cucurbitaceae family 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;
preferably, the gramineous crop is maize, wheat, rice, sorghum or barley.
The types of crops described above are only some optional types listed by the inventors, and in other embodiments, the self-adaptive adjustment may be performed as needed, and the types are not limited to the types of crops described above.
The invention also provides an application of chaetomium gracile or a seed soaking agent in preparation of a plant growth regulator.
It should be noted that the above applications include, but are not limited to: promoting the germination of plant seeds, increasing the plant height of seedlings, recovering plant leaves from curling, withering, drooping and the like to a flat shape, increasing the surface area of roots, increasing the fresh weight, increasing the dry weight and the like.
Optionally, the plant growth regulator is a plant drought-enduring growth regulator.
In a preferred embodiment of the present invention, the plant growth regulator is suitable for growth regulation of gramineous crops. In one embodiment, the plant growth regulator may be applied by drip irrigation, spray application or seed soaking.
In one embodiment, the gramineous crop is corn, wheat, rice, sorghum or barley. In other embodiments, the plant growth regulator described above can also be used for growth regulation of other gramineous crops.
The invention also provides a microbial inoculum which comprises the chaetomium gracile. It should be noted that the above-mentioned microbial inoculum includes, but is not limited to, liquid preparations, solid microbial inoculants, pasty microbial inoculants, suspending agents, emulsions, sprays or semisolid microbial inoculants.
The invention also provides a fertilizer which comprises the chaetomium gracile or the microbial inoculum.
In a preferred embodiment of the application of the present invention, the fertilizer is a leaf fertilizer or a root fertilizer. 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 clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The features and properties of the present invention are described in further detail below with reference to examples.
The reagents were formulated as follows:
5% of sodium hypochlorite: 5ml of sodium hypochlorite and 95ml of sterile water are prepared on site without sterilization;
75% of ethanol: 26.67ml of sterile water and 73.33ml of 95 percent ethanol are prepared on site without sterilization;
PDA culture medium: PDA 16.04g, 400mL of tap water;
PDB culture medium: 9.6g of PDB and 400mL of tap water;
AGPS medium: 10g/L of polypeptone, 10g/L of glycerol, S120 ml/L, S220 ml/L and S320 ml/L, and 0.1 percent of L-tryptophan (accounting for 0.1 percent of the mixed solution) is added before inoculation;
solution 1 (S1): 14g/400mL of anhydrous dipotassium phosphate and 10.8g/400mL of anhydrous potassium dihydrogen phosphate;
solution 2 (S2): 50g/L of magnesium sulfate heptahydrate and 25g/L of anhydrous ammonium chloride;
solution 3 (S3): 10g/L of calcium chloride dihydrate;
salkowski reagent colorimetric solution: 250ml of H2O，150ml H2SO4And 7.5ml of 5M FeCl3.6H2O；
FeCl3.6H2Otianjin, Baishi chemical Co., Ltd;
polyvalent peptone: kyoto Tokay microbial science and technology Co., Ltd;
glycerol: tianjin, Daimao chemical reagent plant;
l-tryptophan: shanghai Michelin Biochemical technology, Inc.;
anhydrous dipotassium hydrogen phosphate: tianjin, Daimao chemical reagent plant;
anhydrous potassium dihydrogen phosphate: tianjin, Daimao chemical reagent plant;
magnesium sulfate heptahydrate: tianjin, Baishi chemical Co., Ltd;
anhydrous ammonium chloride: tianjin, Daimao chemical reagent plant;
calcium chloride dihydrate: tianjin, Daimao chemical reagent plant;
sterile water: weighing 1000ml of sterile water, sterilizing at 121 ℃ for 20 min.
Improving Hoagland nutrient solution: 1.28g of calcium nitrate tetrahydrate is weighed and dissolved in 900mL of distilled water or deionized water by boiling, and 0.945g of calcium nitrate tetrahydrate is dissolved in 100mL of water. Mixing the two solutions, and autoclaving at 121 deg.C for 20 min.
The culture medium and solution prepared above were sterilized in a vertical pressure steam sterilizer at 121 deg.C for 20min, except for the specific specification.
Example 1
This example provides methods for screening, isolation and identification of the Chaetomium tenue (Chaetomium tenue) MN113554 strain.
The strain was isolated from rhizosphere soil and was named chaetomium gracilis MN 113554.
Extraction of the genome of the strain was accomplished using the Biospin fungal genomic DNA extraction kit and amplified with the primers shown in Table 1 below. The amplified product was sent to Jinzhi Biotechnology Ltd for sequencing.
Table 1 PCR upstream and downstream primers.
ITS and BenA sequence sequencing results are shown in SEQ ID NO.1 and SEQ ID NO. 2.
The sequence sequencing results were aligned for homology by Blast analysis. Phylogenetic tree analysis, and determining the strain as Chaetomium tenue. A phylogenetic tree analysis diagram is shown with reference to figure 1.
Example 2
This example was carried out to prepare a fermentation broth of Chaetomium gracilis strain MN 113554.
The preparation method comprises the following steps: perforating 18 pieces of thallus with sterile perforator, and packaging 18 pieces of thallus in 30mL of PPlacing into 50mL sterile centrifuge tube of DB culture solution, placing in constant temperature shaking incubator, culturing at 28 deg.C under 200rmp for 4d, counting spores with blood counting plate, and adjusting fermentation broth spores to 106CFU/mL。
Example 3
In this example, the effect of chaetomium gracilis MN113554 strain on drought resistance of corn seeds in germination period was studied. The technical route is shown in fig. 2.
Firstly, performing surface disinfection on seeds: taking out semen Maydis (Zhengdan 958) from refrigerator, selecting healthy seed with consistent particle size, sterilizing with 75% ethanol for 1min, 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: the seed was soaked overnight at room temperature using each treatment solution. The fermentation broth of the selected strain prepared in the previous example 2 was used as a treatment group; soaking seeds with commercial seed soaking agent (FA-Pheretima aspergillum) as positive control group; PDB culture broth seed soak was used as a negative control. The blank control group was inoculated with PDB medium and subsequently watered normally in the potting experiment.
(1) Preparing soil: sterilizing the matrix soil at 121 ℃ for 60min in a vertical steam autoclave, fully mixing the Hoagland nutrient solution with the sterilized matrix soil (the volume (mL) to mass (g) ratio is 1:8), and subpackaging the mixture into seedling pots.
Sowing: sowing the soaked seeds into seedling pots, and repeating ten treatments, wherein each treatment is repeated by 3 seeds; blank control group was additionally watered with 50ml of Hoagland nutrient solution per replicate.
(2) Screening experiments for potted plants were performed.
Moisture control during the pot screening test is as follows:
and (3) soil moisture determination: the moisture content of the soil at seven stages was measured using a moisture meter and the statistical moisture% M provided a basis for moisture control scheduling during potting experiments.
Soil is potted and sealed overnight, and the humidity is kept before sowing on the next day;
after sowing, watering for the first time at the humidity of every other day;
humidity of the last day of germination (day 10);
humidity after irrigation on day 14;
humidity before 5-level irrigation is atrophied;
humidity before atrophy 4-stage irrigation;
humidity before harvest.
Moisture content differentiation soil drought grade was graded according to the following parameters: the mild degree is 55-65%, the moderate degree is 40-55%, and the severe degree is 30-40%. The soil humidity of the blank control group potted plant is maintained at a normal irrigation state of 75-85%.
In the seed germination period and seedling growth period (1-13 days after sowing), irrigating 10mL of Hoagland nutrient solution every other day for all treatment groups; in 14 th day, 20mL of Hoagland nutrient solution was poured into the blank control group, and then water was cut off, and 10mL of Hoagland nutrient solution was poured into the blank control group every day.
When the treated plants generally shrink to 4 grades, pouring 20mL of Hoagland nutrient solution to all the treated plants as first rehydration; when treatment groups were universally shrunken to grade 5, all treatments were watered with 20mL Hoagland nutrient solution as a second rehydration.
And after the seed germination period is finished, calculating the drought resistance index of seed germination by counting the number of the seeds germination, and further analyzing the seed germination conditions of different experimental groups in the drought stress environment.
The statistical method of drought tolerance indexes in the seed germination period comprises the following steps:
relative germination potential is 100% of germination number under 4d water stress treatment/4 d normal irrigation treatment.
The drought resistance index of seed germination is the index of seed germination under water stress treatment/the index of control seed germination under normal irrigation treatment.
Seed germination index ═ 1 x nd2+0.75*nd4+0.5*nd6+0.25*nd8
Where nd2、nd4、nd6、nd8The drought resistance index of the seed germination is regarded as a reliable index for evaluating the drought resistance of the seed in the germination period by BOUSLAMA.
Specifically, the relative germination vigor and the drought resistance index of the seed germination of the corn are shown in fig. 3, and as can be obtained from fig. 3, the relative germination vigor of MN113554(Cla.t) at 4 th day is higher than 260% of the negative control of the control group, and the drought resistance index of the seed germination is higher than 132% of the negative control.
(3) And (5) performing water cut-off experiments on the potted plants.
And (5) transplanting the seedlings in each treatment ten repetitions into five seedling pots respectively at 10d after sowing, wherein each pot is provided with three plants, and if the germination rate of 30 seeds sowed in each treatment group in the ten repetitions is lower than 50%, the next pot culture water cut-off experiment is not considered.
Referring to the table 2, the potted soil stress water content is shown in table 2, and it can be seen from table 2 that the test belongs to super severe drought level, and the soil humidity for normal irrigation is 75-85%.
Table 2 potted soil stress water content.
And carrying out experiments according to the water control arrangement, observing the plant drought grade every day, and carrying out twice rehydration arrangement according to the plant drought grade conditions of the treatment group. And (4) counting the plant height, the root surface area, the fresh weight and the dry weight of each treatment group every other day after the second rehydration.
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.
And comprehensively evaluating 7 indexes (relative germination vigor, seed germination drought-tolerant index, root surface area, plant height, fresh weight, dry weight and drought grade) by using a drought-resistant membership function method to obtain the sequence of the drought resistance of the plants.
R(Xi)＝(Xi-Xmin)/(Xmax-Xmin)(1)
R(Xi)＝1-(Xi-Xmin)/(Xmax-Xmin)(2)
Wherein R (xi) is the membership value of each physiological index, and when the calculation result is a negative number, the formula (2) is adopted for calculation;
xi represents an index measurement value; xmax and Xmin represent the maximum and minimum values of the measurement index, respectively.
And accumulating the membership values of all drought-resistant indexes to obtain an average value, wherein the larger the average value is, the stronger the drought resistance is.
In this example, the data of relative germination vigor, germination drought tolerance index, plant height, root surface area, fresh weight, dry weight, drought level at harvest, etc. of corn were statistically analyzed by Excel 2010 and SPSS 19.0.
The average fresh weight, average plant height, average root surface area and average dry weight at the time of corn harvest are shown in FIGS. 4, 5, 6 and 7, respectively. As can be seen from fig. 4, 5, 6 and 7, in the application of the corn plants, the strain MN113554(cla.t) is higher than the negative control by 33%, the root surface area is higher than 84%, the fresh weight is higher than 47% and the dry weight is higher than 5% of the negative control compared with the negative control. The overall experimental results show that: MN113554(Cla.t) has certain growth promoting effect under drought environment state.
The growth indices of maize in the experimental and negative control groups are shown in table 3 below. As can be seen from Table 3, the drought tolerance index of maize was overall better than that of the negative control after MN113554(Cla. t) treatment. (Note: the same English letter after the same number indicates no significant difference at the 0.05 level (DMRT method))
Table 3 maize growth indices for each treatment group.
Referring to fig. 8, it can be seen from fig. 8 that MN113554(cla.t) grows greener, has less withered and yellow parts, and has a thicker, longer and more lateral root part than the negative control in the case of whole corn pot.
Example 4
In this example, the effect of chaetomium gracile MN113554 strain on drought resistance of wheat seeds in germination period was studied.
The experimental procedure was the same as in example 3.
The average plant height during harvest, the average root surface area during harvest, the average fresh weight and the average dry weight of wheat plants are shown with reference to fig. 9, fig. 10, fig. 11 and fig. 12, respectively.
As can be seen from fig. 9 to 12, in the application of wheat plants, compared to the negative control, the wheat plant treated with MN113554(cla.t) was higher than the negative control by 2%, the root surface area was higher than the negative control by 32%, the fresh weight was higher than the negative control by 36%, and the dry weight was higher than the negative control by 11%. The overall experimental results show that: MN113554(Cla.t) has a certain growth promoting effect on wheat under a drought environment state.
As can be seen from Table 4, the drought tolerance index of wheat was overall superior to that of the negative control after treatment of wheat seeds with MN113554 (Cla.t). (Note: the same English letter after the same number indicates no significant difference at the 0.05 level (DMRT method))
Table 4 various growth indicators of wheat under drought stress.
FIG. 13 shows the average drought tolerance index of the wheat plants after water cut off, as shown in FIG. 13, the irrigation of the nutrient solution is stopped from day 14, both MN113554(Cla.t) and the negative control have a relatively obvious decreasing trend, most of the leaves shrink to about 4-5 levels in days 3 and 5 of water cut off, 20ml of the nutrient solution is irrigated, and the leaf revival rate trend of the next day has a significant increase.
From the overall trend, the MN113554(Cla.t) and the negative control are rehydrated in the drought stress resistant environment for a period of time, and have a great trend of rising and falling, and the cyan reviving capability of the MN113554(Cla.t) is stronger than that of the negative control. And at harvest on day 6 of water cut, MN113554(cla. t) still had a drought tolerance index that was 24% better than the negative control.
FIG. 14 shows the harvest of potted wheat plants, and it can be seen from FIG. 12 that compared to the negative control, the whole potted wheat plant showed that MN113554(Cla. t) grew greenish, less withered and yellow parts, thicker root system and more lateral roots.
Example 5
This example further analyzes the drought-resistant effect of chaetomium gracilis strain MN113554 on other crops. The sterilization of the seeds, the soil preparation and the sowing were the same as in example 3. In the seed germination period and seedling growth period (1-7 days after sowing), 20mL of Hoagland nutrient solution is irrigated every other day for all treatments, and the amount of the irrigation improvement/normal nutrient solution is adjusted according to the plant moisture requirement after 8 days.
In this example, the dry weight of harvested crops was used as an index of drought resistance to demonstrate the drought resistance and growth promotion of Chaetomium gracilis MN 113554. As can be seen from Table 5, the Chaetomium gracile MN113554 strain has broad spectrum in improving the drought resistance of crops, can effectively improve the drought resistance of crops, promotes the growth speed of plants in a drought environment, and obviously improves the dry weight of the plants.
Table 5 effect of chaetomium gracilis MN113554 strain on drought resistance of plants.
Example 6
This example studies the mechanism by which strains assist in drought tolerance in plants.
(1) The growth capacity of the strains was first tested at different water activities.
Media with water activities aw varying from 0.99 to 0.75 were prepared by adding 0%, 10%, 20%, 30%, 40%, 50% glycerol to MEA media, respectively. Chaetomium gracilis MN113554 was inoculated with 10ul spore suspension into the center of MEA media with different water activities, and the colony diameter was recorded on day 7.
The diameter of the colonies at day 7 is shown in Table 6, and it can be seen from Table 6 that the higher the moisture content in the MEA culture medium, the better the colony growth ability. Table 6 shows that in the case of the strain in a water-deficient environment, the growth rate of the strain is reduced along with the reduction of water content, and the growth diameter of the strain is also reduced.
The water activity is expressed as aw and the water activity value is equal to the relative humidity expressed in percentage, which is a number between 0 and 1. Bacteria for aw >0.9, yeast for aw >0.87, and mold for aw > 0.8.
Table 6 strain physiological and biochemical test data.
(2) The strains were tested for indoleacetic acid production (IAA).
Inoculating the strain (spore or hypha) into AGPS medium containing 0.1% L-tryptophan, placing in a constant temperature shaking incubator, and culturing at 30 deg.C and 200rpm for 2 d. mu.L of the strain fermentation solution was dropped on a white ceramic plate, and 200. mu.L of Salkowski colorimetric solution (mixed at a volume ratio of 1: 4) was added thereto, and 50mg/L of IAA and AGPS media were used in place of the strain fermentation solution as a positive control and a negative control, respectively. 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 chromogenic reaction result is obvious, performing further quantitative test, uniformly mixing the treatment solution (the strain fermentation supernatant, 50mg/L IAA and AGPS culture medium) and the Salkowski colorimetric solution according to the volume ratio of 1:1, reacting for 30min in a dark environment at room temperature, and rapidly determining 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 which can be measured by the standard curve is 5-200 mu g/mL.
Table 7 indole acetic acid production data for the strain.
According to the test data of the strain IAA (IAA) of Table 7, the strain MN113554(Cla.t) can produce IAA and can act on the surface of a plant to promote the growth and development of the plant. The capacity of the plants to absorb nutrients from soil is improved, and the survival rate of the plants is obviously improved under the drought stress condition.
(3) This example carried out EPS exopolysaccharide activity assay on the strains.
Mixing 10 μ L of 106The cfu/mL bacterial solution is dripped on a sterile filter paper sheet with the diameter of 5mm in a special EPS-producing culture medium and cultured for 2d at room temperature. And judging whether EPS is produced according to the colony size around the filter paper and a transparent ring.
TABLE 8 Exopolysaccharide (EPS) Activity assay data (clear circles for secretion of the strains are indicated by "+")
| Test number | Bacterial name | Diameter/cm of bacterial colony | Transparent secretion ring |
| MN113554 | Chaetomium tenue | 2.3 | + |
As shown in table 8, MN113554(cla.t) can produce Exopolysaccharide (EPS) to improve the tolerance to drought stress and maintain the stability of soil and water, so that soil and water can be better adhered to plant roots and the available water of the plant roots can be increased.
(4) And (3) measuring the content of proline (Pro) in the wheat leaves.
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: a107-1-1.
Table 9 proline (Pro) assay data in plant leaves.
Proline (PRO) test formula ═ ((assay OD value-blank OD value)/(standard OD value-blank OD value)). standard concentration 5 ug/ml/homogenate concentration (g tissue wet weight/ml). sample dilution factor before test.
The proline (Pro) content in the plant leaves was determined to be 1387.5 ug/tissue weight loss in Table 9.
(5) 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 according to the kit instructions.
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 10 determination of superoxide dismutase (SOD) content in plant leaves (optical path 1cm)
TABLE 11 determination of Catalase (CAT) content in plant leaves
TABLE 12 determination of Peroxidase (POD) content in plant leaves
From tables 10 to 12, it can be understood that the strain MN113554(Cla.t) secretes a large amount of enzyme systems and antioxidants capable of scavenging active oxygen and proline against stress when it acts 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 gracile, seed soaking agent and application
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 549
<212> DNA
<213> Artificial sequence
<400> 1
atcattacag agttgcaaaa ctccctaaac catcgtgaac gttacctata ccgttgcttc 60
ggcgggcggc cccggggttt accccccggg cgcccctggg ccccaccgcg ggcgcccgcc 120
ggaggtcacc aaactcttga taatttatgg cctctctgag tcttctgtac tgaataagtc 180
aaaactttca acaacggatc tcttggttct ggcatcgatg aagaacgcag cgaaatgcga 240
taagtaatgt gaattgcaga attcagtgaa tcatcgaatc tttgaacgca cattgcgccc 300
gccagtattc tggcgggcat gcctgttcga gcgtcatttc aaccatcaag cccccgggct 360
tgtgttgggg acctgcggct gccgcaggcc ctgaaaagca gtggcgggct cgctgtcgca 420
ccgagcgtag tagcatacat ctcgctctgg tcgcgccgcg ggttccggcc gttaaaccac 480
cttttaaccc aaggttgacc tcggatcagg taggaagacc cgctgaactt aagcatatca 540
ataagcgga 549
<210> 2
<211> 684
<212> DNA
<213> Artificial sequence
<400> 2
ccgggcggaa gagctggccg aaggggccgg cacggacggc gtccatggtg ccgggctcca 60
agtcgacgag gacggcgcgg ggaacatact tgttgccgga agcctttata atactgtgag 120
cacgccatca cttggttgtg caaaggtata tgaccctact aacctcgttg aagtacacgt 180
tcatgcgctc gagctggagc tcggaggtgc cgttgtacct atcgaagcgg tgagcgggga 240
tttatcggtt gggaacagtc acggccacat acacgccatt gctgtcgagg ccgtgctcgc 300
cagagatggt ctgcctgaaa agaagtcagt ctcgattgtc atcagccacg agtgtttgct 360
ttgcttggac gtaccagaaa gcggcaccga tttggttacc ctggagagct ccatgtcagt 420
tatcctgcac cgctggtcgg tcgtaatgtt cgattcggtc caacttacgc actggccggt 480
ctggaggtga acctatagta agaaaagcag tcagcatcat caagcttcgc atcgccgtcg 540
tgttgctatc ggtcaggagc tgtggaaatg agggtcgtcc cgatgggtgg ggtagttcag 600
gggcccaaaa aaagcttccc agacgcgtcg tagccgtgtg gctcaggaat gtgcggggcg 660
acttacaatc tcaccgcatg ttga 684
Claims (10)
1. Application of fungal strain of Chaetomium tenue (Chaetomium tenue) species in preparation of seed soaking liquid, seed soaking powder or microbial agent is provided.
2. Use according to claim 1, wherein the chaetomium gracile is deposited at the Guangdong province collection of microorganisms at the following deposit 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: 61364, strain taxonomic name Chaetomium tenue, biomaterial name Chaetomium MN 113554.
3. Use of a fungal strain of the species Chaetomium tenue for the preparation of a plant growth regulator;
preferably, the plant growth regulator is a plant drought-tolerant growth regulator.
4. The use according to claim 3, wherein the plant growth regulator is suitable for the growth regulation of gramineous crops;
preferably, the gramineous crop is maize, wheat, rice, sorghum or barley;
preferably, the fungal strain is a strain deposited under the accession number GDMCC No: 61364A deposited Chaetomium gracile or progeny strain thereof.
5. Chaetomium gracile is preserved in 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: 61364, strain taxonomic name Chaetomium tenue, biomaterial name Chaetomium MN 113554.
6. Chaetomium gracile according to claim 5, characterized in that its colony culture characteristics are: growing on MEA culture medium, spreading the bacterial colony on the whole plate, culturing later stage to form spherical black spore, and secreting light yellow secretion with smell.
7. A seed soaking agent comprising the chaetomium gracile of claim 5 or 6.
8. The seed soaking agent according to claim 7, wherein the seed soaking agent comprises chaetomium gracilis as a main effective component;
preferably, the seed soaking agent is the fermentation liquid of chaetomium gracile or the freeze-dried powder of chaetomium gracile.
9. The seed soaking agent according to claim 7 or 8, wherein the seed soaking agent is used for seed soaking of crops;
preferably, the crop comprises at least one of a cash crop and a food crop;
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 grain crop is selected from gramineous crops;
preferably, 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 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;
preferably, the gramineous crop is corn, wheat, rice, sorghum or barley.
10. A fertilizer, characterized by comprising Chaetomium gracile according to claim 5 or 6;
preferably, the fertilizer is a leaf fertilizer or a root fertilizer.
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