CN112680361A - Trichoderma galnarum and application thereof - Google Patents

Abstract

The invention discloses Trichoderma ganesense, and the strain number TR2122 of Trichoderma ganesense (Trichoderma ghanense) is CGMCC No. 21025. The invention also discloses an organic water-soluble fertilizer which contains the trichoderma gardneri fermentation supernatant produced by trichoderma gardneri fermentation. The invention solves the problem of environmental pollution of the industrial waste water of the trichoderma ganae by a fermentation process, recycles waste and generates double benefits of environment and economy. The organic water-soluble fertilizer prepared by the invention can obviously improve the yield of crops and the quality of the crops, and can improve the soil environment and enhance the stress resistance and disease resistance of plants after long-term use.

Description

Trichoderma galnarum and application thereof

Technical Field

The invention relates to the technical field of microorganisms, and particularly relates to trichoderma californicum and application thereof.

Background

Trichoderma (including Trichoderma "Ganna") is a microorganism in nature which has inhibitory and killing functions against a variety of plant pathogenic bacteria. The excellent characteristics of trichoderma are utilized by human beings to prepare the trichoderma into biological pesticides with different formulations through a specific process technology. The trichoderma has the characteristics of wide distribution, easy separation and culture, capability of generating chlamydospores with strong stress resistance, easy storage, convenient use and the like, and has the following effects: preventing and treating various plant diseases and promoting plant growth.

The water soluble fertilizer is a liquid or solid fertilizer which is dissolved or diluted by water and is used for drip irrigation fertilization, spray irrigation fertilization, foliar fertilization, soilless culture, seed soaking and root dipping and the like. The water-soluble fertilizer has the obvious advantages of instant dissolution, uniform fertilization, high absorption rate, quick response, convenient application and the like, is developed rapidly, and along with the national vigorous popularization of water and fertilizer integration, the market scale and the sales volume of the water-soluble fertilizer are increased year by year. By 6 months and 1 day 2015, 6545 water-soluble fertilizers registered in the Ministry of agriculture comprise 1383 macroelement water-soluble fertilizers, 1668 trace element water-soluble fertilizers, 1630 amino acid-containing water-soluble fertilizers, 1527 humic acid-containing water-soluble fertilizers, 240 medium element water-soluble fertilizers and 97 organic water-soluble fertilizers. The yield of various domestic water-soluble fertilizers is greatly different, wherein 68% of macroelement water-soluble fertilizer, 10% of secondary element water-soluble fertilizer, 12% of humic acid water-soluble fertilizer and 10% of other types. However, the water-soluble fertilizer produced by using the supernatant of the fermentation of the trichoderma gardnerella as a solvent has a certain yield in the current registered fertilizer products in China.

In recent years, with the rapid development of the biopesticide industry in China, trichoderma related products grow vigorously. At present, liquid fermentation technology is mostly adopted by biopesticide enterprises to produce trichoderma preparations in batches. A large amount of fermentation supernatant is produced in the production process of the trichoderma living microbial preparation, 1.5kg of fermentation supernatant is produced per 1kg of trichoderma product, the supernatant contains various trace elements, small molecular active substances such as various amino acids produced in the fermentation process, and various large molecular active substances such as active protease produced in the fermentation process. The active substances are matched with humic acid, so that the growth of the root system of the crop can be promoted, the stress resistance of the crop can be enhanced, the metabolism and the growth of beneficial microorganisms in soil can be promoted, the pH value of the soil can be buffered, the water retention, fertilizer retention and ventilation capability of the soil can be improved, the absorption of the soil on water and fertilizer can be improved, the plant can grow vigorously, the root is deep and the leaves are luxuriant, and the dry weight of the plant can be increased; promoting photosynthesis of leaves, brightening fruits, promoting absorption of medium and trace elements, and increasing fruit weight. However, the normal process of treating fermentation supernatant in the prior trichoderma preparation production factory is that the fermentation supernatant is discharged into the environment after three wastes are treated, so that a simple and rough treatment mode not only causes resource waste to a certain extent, but also has the problem of environmental pollution.

Disclosure of Invention

The invention aims to provide trichoderma ganae and application thereof, aiming at the defects in the prior art, not only can reduce wastewater treatment and energy consumption, but also can meet the requirements of high fertility, quick absorption, no environmental pollution and enhancement of stress resistance and disease resistance of plants.

In order to achieve the purpose, the invention adopts the technical scheme that:

the first aspect of the invention provides Trichoderma, and the strain number TR2122 of Trichoderma ghanistan (Trichoderma ghanense) is CGMCC No. 21025.

The Trichoderma ghanistis (Trichoderma ghanense) is preserved in China general microbiological culture collection center (CGMCC), and the preservation address is as follows: xilu No. 1 Hospital No. 3, Beijing, Chaoyang, North; the preservation date is 2020, 12, 21.

The second aspect of the invention provides a preparation method of Trichoderma ghanistense fermentation supernatant, which adopts the Trichoderma ghanistii (Trichoderma ghanense) fermentation, and comprises the following steps:

inoculating the trichoderma strains into a culture medium, and culturing at constant temperature;

step two, transferring the strain cultured in the step one into a culture solution for shake flask culture to obtain a first-level seed solution;

step three, sterilizing the culture solution in a middle-stage fermentation tank at high temperature and high pressure, then pouring the first-stage seed solution obtained in the step two into the middle-stage fermentation tank, pressurizing and introducing sterile air for fermentation to obtain trichoderma fermentation liquor;

and step four, centrifuging the trichoderma fermentation liquor obtained in the step three by using a centrifugal machine to obtain the trichoderma gananense fermentation supernatant.

Further, in the second step and the third step, the culture solution comprises the following components in parts by weight:

further preferably, in the second step and the third step, the culture solution comprises the following components in parts by weight:

further, in the third step, the fermentation conditions are as follows: a temperature of 28-30 ℃, a pressure of 0.4-0.5kg, an air flow of 1500-1800L/H and a rotation speed of 120-150 rpm.

Further, in the third step, the specific conditions of the high-temperature and high-pressure sterilization are as follows: sterilizing at 121 deg.C under 15 pounds pressure for 20 min.

Further, in the third step, before the high-temperature and high-pressure sterilization, a defoaming agent is added into the medium-grade fermentation tank; preferably: 50ml of antifoam was added to the 100L format medium stage fermentor.

Further, in the third step, after the high-temperature and high-pressure sterilization, the medium-grade fermentation tank is kept at positive pressure and cooled to 28-30 ℃.

Further, the medium-sized fermenter means a fermenter of more than 1L and less than 1 ton size, which is common knowledge in the art.

The third aspect of the invention provides an organic water-soluble fertilizer, and the fermentation supernatant of trichoderma gananense prepared by the method comprises the following components in percentage by weight:

the fourth aspect of the invention provides a preparation method of the organic water-soluble fertilizer, which comprises the following steps:

putting the fermentation supernatant of the trichoderma gandrum into a dosing tank according to the proportion, and then starting a stirring device; and adding the mineral potassium humate into the mixing tank, adding the potassium nitrate, the urea, the ammonium polyphosphate, the dipotassium hydrogen phosphate, the molasses powder and the carotene after stirring and completely dissolving, and filtering to obtain the organic water-soluble fertilizer.

By adopting the technical scheme, compared with the prior art, the invention has the following technical effects:

1. the invention solves the problem of environmental pollution of the trichoderma industrial wastewater through a fermentation process, recycles waste and generates double benefits of environment and economy.

2. The organic water-soluble fertilizer prepared by the invention can obviously improve the yield of crops and the quality of the crops, and can improve the soil environment and enhance the stress resistance and disease resistance of plants after long-term use.

Drawings

FIG. 1 shows the conidiophorous morphology of TR2122 of the present invention;

FIG. 2 shows the conidiophorous morphology of TR2122 of the present invention;

FIG. 3 shows the conidiomorphism of TR2122 of the present invention;

FIG. 4 is a photograph of a colony morphology PDA plate of TR2122 of the present invention.

Detailed Description

The invention is further described with reference to the following drawings and specific examples, which are not intended to be limiting. It should be noted that the embodiments and features of the embodiments may be combined with each other without conflict.

Example 1

The embodiment provides Trichoderma californica which is collected and separated from rhizosphere soil of tomato of Suqiao in the new area of Pudong, Shanghai, and the strain number TR2122 of the Trichoderma californica (Trichoderma ghanense) is CGMCC No. 21025.

The Trichoderma ghanistis (Trichoderma ghanense) is preserved in China general microbiological culture collection center (CGMCC), and the preservation address is as follows: xilu No. 1 Hospital No. 3, Beijing, Chaoyang, North; the preservation date is 2020, 12, 21.

Culture characteristics of trichoderma guani strains:

the trichoderma gardnerella strain is cultured in a PDA culture medium for 64 hours at 20-30 ℃ under a dark condition, the radius of a bacterial colony can reach 70mm, and the radius of the bacterial colony can reach 50-60mm after being cultured for 64 hours at 40 ℃. Referring to fig. 4, conidia are uniformly and continuously distributed on colonies, and are dark green; most conidia were formed at 35 ℃ with a gradual decrease in the yield of conidia at temperatures of 30 ℃ and 5 ℃. The aerial hyphae appeared cotton-like (on DMC) at 25 ℃. Culturing at 20 deg.C for 1 hr in dark and 12 hr under cold fluorescent irradiation for 1 week, wherein aerial hyphae on the colony are sparse, and conidia are uniformly distributed in continuous wide band without forming discrete conidium clusters.

Microscopic morphological characteristics of trichoderma gardnerella strains:

referring to FIGS. 1-3, conidiophores of the Trichoderma Gabadensis strain of the invention do not form fertile extensions. Infertile hairy structures are lacking. A typical conidiophores comprise a strong developing medial axis, with the top facing towards the apical direction to give rise to a single phialide, and then from the apical end downwards to give rise to pairs of secondary branches that progressively lengthen. The phialides are directly produced on the secondary branches and are not generally arranged in a vortex shape. The width of the main axis of the conidiophore is (1.8-) 2.5-3.5 (-4.0 μm). The bottle stem is cylindrical or slightly expanded in the middle, straight or bent, the length is (4.5-) 6.0-10.5 (-15.0) mu m, the widest part is (2.0-) 2.5-3.5 (4.5-) mu m, the width of the base part is (1.0-) 1.5-2.5 (-3.5) mu m, the length-width ratio is (1.3-) 2.0-3.0 (-6.8), and the width of the mother cell is (1.8-) 2.5-3.5 (-4.0) mu m. Rarely intercropped phialides. Conidia are green, narrow or thin and oval, the size is (3.5-) 4.5-6.2 (-7.5) × (2.0-) 2.2-3.2 (-4.5) mu m, and the length-width ratio is (1.3-) 1.5-2.5 (-3.2); smooth or warty. The typical strains do not produce chlamydospores, but some strains produce a large amount of chlamydospores, spherical or subspherical, terminal or intergrowth (the character of the mother cell in the process of intergrowth) with the size of (4.5-) 7.0-10.2 (11.7) mu m. No paranoid type was produced.

Molecular biological identification of trichoderma ganassii:

molecular biological identification of trichoderma gardnerella strain TR2122 of the present invention was performed by PCR (Polymerase Chain Reaction).

The sequence segment obtained by using the ITS1-5.8S-ITS2 region sequences amplified by the primers ITS4 and ITS5 is up to 99 percent similar to Trichoderma Gardner by comparing the data in a DNA sequence database established by NCBI Gen Bank.

The ITS sequences are as follows:

TTGAAGGAAAAAAGCGTAACAAGGTCTCCGTTGGTGAaCCaGCGGAGGGAtCATTACCGAGTTTACAACTCCCAAACCCCAATGTGAACGTTACCAATCTGTTGCCTCGGCGGGATCTCTGCCCCGGGCGCGTCGCAGCCCCGGATCCCATGGCGCCCGCCGGAGGACCAACCAAACTCTTTTTTCTCTCCGTCGCGGCTTCCGTCGCGGCTCTGTTTTAACTTTGCTCTGAGCCTTTCTCGGCGACCCTAGCGGGCGTCTCGAAAATGAATCAAAACTTTCAACAACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCCGCCAGTATTCTGGCGGGCATGCCTGTCCGAGCGTCATTTCAACCCTCGAACCCCTCCGGGGGGTCGGCGTTGGGGATCGGCACGCCCTCACACGGGTGCCGGCCCCGAAATCCAGTGGCGGTCTCGCCGCAGCCTCTCCTGCGCAGTAGTTTGCACACTCGCACCGGGAGCGCGGCGCGGCCACAGCCGTAAAACACCCCAAACTTCTGAAATGTtGACCTCGGATCAGGTAGGAATACCCGCTGAACTTAAGCATATCAAA

sequence segments obtained by amplifying a Tef1 alpha sequence by adopting primers Ef728 and Tef1 alpha R are compared through data in a DNA sequence database established by NCBI Gen Bank, and the similarity between the Tef1 alpha sequence and Trichoderma ganesense reaches 99 percent.

The Tef1 α sequence is as follows:

TCATCGAGAAAGTCGAGAAGGTAAGCTTTAGTCCGTTCAGTATCGAGTTTCGAGCCCGATTTTTCCTGCCTCTGTGCCCAACATTTGTCGACCGAATTTCGCTGTCGACGGGATTTTTCCCATTCACCCCGCTTTCTTCTACCCCTCCTTTGAGCGACGCAAAATTTTTTTTGCTACGCCGTTGGTTTTAGCGGGGGTGCATCTCGAGCAACCCCACCATTACTCTCTGGCCGCTCTCTGGTCCTCCAGACAACAGTCAACGCACCCGCATCGTCATTCCTCCAGCAGTTTCTCCAGGATGCTAATCAAATTCCACTCAACAGGAAGCCGCCGAACTCGGCAAGGGTTCCTTCAAGTACGCGTGGGTTCTTGACAAGCTCAAGGCCGAGCGTGAGCGTGGTATCACCATCGACATTGCCCTCTGGAAGTTCGAGACTCCCAAGTACTATGTCACCGTCATTGGTACGTTTGATCCATTGCCTTTTCCGTGCGTCGTTGTCGGCACAAACTAACATGTCCCTCATAGACGCTCCCGGCCACCGTGACTTCATCAAGAACATGATCACTGGTACTTCCCAGGCCGACTGCGCTATCCTCATtATCGCTGCCGgtACTGGTGAGTTCGAGGATGGTATCTCTAAGGGGA

the separated strain can be determined to be the trichoderma ganassium strain by molecular biological strain identification and combining morphological characteristics.

The conidiophore stage of the above-mentioned Trichoderma gardnerella strain is Deuteromycotina (Deuteromycotina), Hyphomycetes (Hyphomycetes), Aphyllophorales (Hyphomycetales), Moniliaceae (Moniliaceae), Trichoderma (Trichoderma), and the active states thereof are Ascomycotina (Ascomycotina), Hypocrea (Hypocrea).

Example 2

The embodiment provides a preparation method of a fermentation supernatant of trichoderma ganae, which comprises the following steps:

inoculating trichoderma gananense separated in the embodiment 1 into a PDA culture medium, and culturing for one week at constant temperature;

step two, transferring the strain cultured in the step one into a culture solution for shake flask culture for 24H to obtain a first-stage seed solution;

step three, sterilizing the culture solution in the middle-stage fermentation tank at the temperature of 121 ℃ and under the pressure of 15 pounds for 20min, keeping positive pressure, cooling to 30 ℃, then pouring the first-stage seed solution obtained in the step two into the middle-stage fermentation tank, introducing sterile air under pressure, and fermenting for 6 days at the temperature of 28-30 ℃, the pressure of 0.4-0.5kg, the flow rate of 1500-plus 1800L/H air and the rotating speed of 120-plus 150rpm to obtain trichoderma fermentation liquor;

and step four, centrifuging the trichoderma fermentation liquor by using a centrifugal machine to obtain the trichoderma gananense fermentation supernatant.

In the second step and the third step, the culture solution comprises the following components in parts by weight:

preferably, the culture solution comprises the following components in weight ratio (table 1):

TABLE 1

Example 3

This example provides an organic water-soluble fertilizer, which respectively corresponds to the fermentation supernatants of trichoderma ganesense obtained by using the culture solutions of groups 1-3 in the above example 2, and respectively includes the following components (table 2) in percentage by weight:

TABLE 2

Components	Example 3-1	Examples 3 to 2	Examples 3 to 3
				Fermentation supernatant of Trichoderma Gardneri	70％	67.11％	62.50％
Mineral potassium humate	3.43％	2.75％	3.25％
				Potassium nitrate	3.72％	8.0％	2.68％
Urea	3.08％	3.25％	8.20％
				Ammonium polyphosphate	2.32％	3.35％	4.82％
Dipotassium hydrogen phosphate	6.25％	6.94％	6.00％
				Honey powder	10.70％	8.50％	12.05％
Carotene	0.5％	0.1％	0.5％

The organic water-soluble fertilizer is prepared by the following method: putting the fermentation supernatant of the trichoderma ganacarii into a dosing tank according to the proportion, and then starting a stirring device; and adding the mineral potassium humate into the mixing tank, adding the potassium nitrate, the urea, the ammonium polyphosphate, the dipotassium hydrogen phosphate, the molasses powder and the carotene after stirring and completely dissolving, filtering after stirring and completely dissolving to obtain the organic water-soluble fertilizer, and storing in a shady and dry place in a dark place.

Application example

And (3) field fertilizer efficiency test: the humic acid-containing water-soluble fertilizer prepared in the above example 3-1 was used to conduct a fertilizer efficiency test for grapes. The test site is carried out in the Wei mountain temple street town of Yunnan province, the grape variety is summer black, and the demonstration area is as follows: 200 mu, demonstration time: 1-6 months in 2020, test method: 12-18L/mu/season. There are 3 treatments in total, which are: the method is characterized in that the method comprises the steps of carrying out detection on the root system, tree vigor, yield, quality and other aspects of the grape 6 months after fertilization without fertilization contrast, humic acid-containing water-soluble fertilizer (Shanghai Wanli Biotech limited) and compound fertilizer (Beijing Zhongkeli agricultural biotechnology research institute).

The test result shows that:

(1) after the humic acid-containing water-soluble fertilizer is treated on grapes, the growth, the color of leaves, the root system and the yield of the grapes are obviously promoted compared with those of a commercially available compound fertilizer and a non-fertilization contrast. Compared with the tree vigor and the fruit bearing condition, the fruit picking time of the grapes is prolonged by using the humic acid-containing water-soluble fertilizer, the tree vigor is more vigorous, the decay time is obviously prolonged, and the color of the fruits is black and bright. From the comparison of root systems, the fibrous roots of the grapes are obviously increased after the humic acid-containing water-soluble fertilizer is used. The test results are shown in table 3:

TABLE 3 growth promotion test results of humic acid-containing water-soluble fertilizer of the present invention on grapes

(2) The sugar content, vitamin C and organic acid of the grapes using the humic acid-containing water-soluble fertilizer are higher than those of the commercial compound fertilizer and the non-fertilization contrast, so that the humic acid-containing water-soluble fertilizer has a very good effect on improving the quality of the grapes. From the color change condition, the color change of the grapes is obviously advanced after the humic acid-containing water-soluble fertilizer is used, and the attached figure 3 is favorable for the grapes to appear on the market in advance and occupy the price market. The test results are shown in table 4:

TABLE 4 quality test results of humic acid-containing water-soluble fertilizers of the present invention on grapes

Detecting items	Humic acid-containing water-soluble fertilizer	Compound fertilizer	No fertilization control
				Vitamin C (mg/100g)	3.26	3.01	2.62
Organic acid (mg/kg)	9.4×103	8.7×103	8.5×103
				Sugar degree (%)	17.8	17.4	17.5

(3) The water-soluble fertilizer containing humic acid is superior to the sold compound fertilizer and the non-fertilization contrast in the improvement aspects of the soil hydrolyzable nitrogen, available phosphorus, quick-acting potassium and the like of the grape field, the porosity, the organic matter content, the cation exchange capacity and the like of the soil are obviously improved, and a large amount of edible wild mushrooms appear in the soil treated by the water-soluble fertilizer containing humic acid. The test results are shown in table 5:

TABLE 5 soil improvement test results of humic acid-containing water-soluble fertilizer of the present invention in the grape field

Detecting items	Humic acid-containing water-soluble fertilizer	Compound fertilizer	No fertilization control
				PH	4.8	4.7	4.3
Organic matter (g/Kg)	32.9	13.2	13.0
				Hydrolyzable Nitrogen (mg/kg)	258	171	182.6
Available phosphorus (mg/kg)	162	151.8	148.8
				Quick-acting potassium (mg/kg)	878	758	738
Cation exchange capacity (cmol/kg (+))	13.38	5.2	4.3
				Volume weight (g/cm)3)	1.32	1.5	1.43

(4) The humic acid-containing water-soluble fertilizer is sprayed on the liquid surface in the growth process of grapes, and has a certain control effect on grape downy mildew. The test results are shown in table 6:

TABLE 6 preventive effect of humic acid-containing water-soluble fertilizer of the invention on grape downy mildew

Treatment of	Dosage of	Index of disease condition	Prevention effect
				Humic acid-containing water-soluble fertilizer	300 times of 500 times	3.85	45.65
Compound fertilizer	300 times of 500 times	5.06	13.89
				No fertilization control	/	5.09	/

While the invention has been described with reference to a preferred embodiment, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the invention.

Claims (10)

1. Trichoderma ghalensis, wherein the strain number TR2122 of Trichoderma ghalensis (Trichoderma ghalensis) is CGMCC No. 21025.

2. A method for preparing a fermentation supernatant of trichoderma ganense, which uses trichoderma ganense as claimed in claim 1, comprising the steps of:

inoculating the trichoderma gananense strain into a culture medium, and culturing at a constant temperature;

step two, transferring the strain cultured in the step one into a culture solution for shake flask culture to obtain a first-level seed solution;

step three, sterilizing the culture solution in a middle-stage fermentation tank at high temperature and high pressure, then pouring the first-stage seed solution obtained in the step two into the middle-stage fermentation tank, pressurizing and introducing sterile air for fermentation to obtain a trichoderma ganense fermentation liquid;

and step four, centrifuging the trichoderma gardnerella fermentation liquor obtained in the step three by using a centrifugal machine to obtain the trichoderma gardnii fermentation supernatant.

3. The method for preparing a fermentation supernatant of trichoderma ganesei according to claim 2, wherein in step two and step three, the culture solution comprises the following components in weight ratio:

4. the method for preparing a fermentation supernatant of trichoderma ganesei according to claim 3, wherein in step two and step three, the culture solution comprises the following components in weight ratio:

5. the method for preparing a fermentation supernatant of trichoderma ganesei according to claim 2, wherein in step three, the fermentation conditions are: a temperature of 28-30 ℃, a pressure of 0.4-0.5kg, an air flow of 1500-1800L/H and a rotation speed of 120-150 rpm.

6. The method for preparing the fermentation supernatant of trichoderma ganense according to claim 2, wherein the specific conditions of the high-temperature and high-pressure sterilization in the third step are as follows: sterilizing at 121 deg.C under 15 pounds pressure for 20 min.

7. The method for preparing a fermentation supernatant of trichoderma gandrum according to claim 2, wherein in step three, before the autoclaving, an antifoaming agent is added to the middle-stage fermenter.

8. The method for preparing a fermentation supernatant of trichoderma ganesei according to claim 2, wherein in step three, after the autoclaving, the medium fermentation tank is kept at positive pressure and cooled to 28-30 ℃.

9. An organic water-soluble fertilizer, wherein the fermentation supernatant of trichoderma ganaraense prepared by the method of any one of claims 2 to 8 comprises the following components in percentage by weight:

10. the preparation method of the organic water-soluble fertilizer as claimed in claim 9, characterized by comprising the following steps:

putting the fermentation supernatant of the trichoderma gandrum into a dosing tank according to the proportion, and then starting a stirring device; and adding the mineral potassium humate into the mixing tank, adding the potassium nitrate, the urea, the ammonium polyphosphate, the dipotassium hydrogen phosphate, the molasses powder and the carotene after stirring and completely dissolving, and filtering to obtain the organic water-soluble fertilizer.

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