CN112746024A - Trichoderma composite microbial agent and bio-organic fertilizer prepared from same - Google Patents

Trichoderma composite microbial agent and bio-organic fertilizer prepared from same Download PDF

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CN112746024A
CN112746024A CN202110065741.8A CN202110065741A CN112746024A CN 112746024 A CN112746024 A CN 112746024A CN 202110065741 A CN202110065741 A CN 202110065741A CN 112746024 A CN112746024 A CN 112746024A
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trichoderma
organic fertilizer
bio
salt
halotolerant
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邬海燕
蔡枫
陈巍
沈其荣
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Nanjing Agricultural University
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Nanjing Agricultural University
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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00Microorganisms, 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
    • C12N1/14Fungi; Culture media therefor
    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01NPRESERVATION 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
    • A01N63/00Biocides, 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
    • A01N63/30Microbial fungi; Substances produced thereby or obtained therefrom
    • A01N63/38Trichoderma
    • CCHEMISTRY; METALLURGY
    • C05FERTILISERS; MANUFACTURE THEREOF
    • C05FORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
    • C05F11/00Other organic fertilisers
    • C05F11/08Organic fertilisers containing added bacterial cultures, mycelia or the like
    • CCHEMISTRY; METALLURGY
    • C05FERTILISERS; MANUFACTURE THEREOF
    • C05FORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
    • C05F17/00Preparation of fertilisers characterised by biological or biochemical treatment steps, e.g. composting or fermentation
    • C05F17/20Preparation of fertilisers characterised by biological or biochemical treatment steps, e.g. composting or fermentation using specific microorganisms or substances, e.g. enzymes, for activating or stimulating the treatment
    • CCHEMISTRY; METALLURGY
    • C05FERTILISERS; MANUFACTURE THEREOF
    • C05FORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
    • C05F3/00Fertilisers from human or animal excrements, e.g. manure
    • CCHEMISTRY; METALLURGY
    • C05FERTILISERS; MANUFACTURE THEREOF
    • C05GMIXTURES 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
    • C05G3/00Mixtures of one or more fertilisers with additives not having a specially fertilising activity
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02WCLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
    • Y02W30/00Technologies for solid waste management
    • Y02W30/40Bio-organic fraction processing; Production of fertilisers from the organic fraction of waste or refuse

Abstract

The invention discloses a trichoderma composite bacterial agent and a biological organic fertilizer prepared by the same. A salt-tolerant trichoderma composite microbial inoculum consists of mudflat trichoderma TUCIM10301 spore liquid with the preservation number of CGMCC No.19611 and trichoderma pseudoechinospora TUCIM10320 spore liquid with the preservation number of CGMCC No. 19612. The invention utilizes trichoderma asperellum CGMCC 19612 and trichoderma intertidal CGMCC 19611 to perform secondary fermentation compostingThe total number of the trichoderma in the invention is more than or equal to 3 multiplied by 107cfu g‑1. Experiments show that compared with the treatment of the bio-organic fertilizer prepared by applying single bacteria or other combined strains, the bio-organic fertilizer provided by the invention is applied in saline soil, so that the growth of tomato seedling roots and the growth of plants can be effectively promoted.

Description

Trichoderma composite microbial agent and bio-organic fertilizer prepared from same
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to a trichoderma composite microbial inoculum and a biological organic fertilizer prepared by the trichoderma composite microbial inoculum.
Background
The world saline-alkali soil is about 9.56 hundred million hectares, wherein the saline-alkali soil area of China is 9913.3 ten thousand hectares, and the saline-alkali soil area accounts for about 10 percent of the world saline-alkali soil area. Generally, saline soils with salt contents of 0.2% to 0.5% have a detrimental effect on plant growth. Too much salt can reduce the soil moisture and seriously hinder the growth and development of plants, which becomes a restriction factor for limiting the crop harvest of saline-alkali soil. Therefore, how to improve the salt tolerance of plants becomes a problem to be solved urgently.
Trichoderma spp is soil inhabiting fungus widely existing in nature, has the characteristics of strong decay property, fast growth and reproduction and the like, and can quickly utilize nutrition and occupy space. In recent years, trichoderma has an effect of promoting Plant Growth, is used as one of Plant Growth-promoting Rhizobacteria (PGPR), can interact with other microorganisms in the rhizosphere like other microbial preparations, regulates the ecological balance of the rhizosphere environment, effectively promotes the Plant Growth, and has the characteristics of low production cost, safe use process, stable yield increase effect, good continuous effect, effective guarantee of food safety and the like.
Trichoderma is a saprophytic fungus that grows under a variety of carbon and nitrogen sources. It has many biosynthetic and degradative pathways, and can secrete many degradative enzymes such as macromolecular substances, so it is used as industrial strain for producing enzymes for decomposing various plant materials, including cellulase, hemicellulase, amylase, xylanase, chitinase, etc. Meanwhile, the strong extracellular enzyme of trichoderma can decompose various agricultural organic solid wastes, such as substances with high lignin content, such as coconut shells, and the like, substances with high cellulose content, such as oil cake, straws, bagasse, and the like, fermented livestock and poultry manure, and the like. Therefore, the trichoderma has several abilities which are beneficial to producing the bio-organic fertilizer by decomposing and fermenting the organic fertilizer. At present, the study on salt-tolerant trichoderma is mainly carried out on a single strain, and the screening of a combined strain with a synergistic salt-tolerant effect is rarely reported. The preparation method has the advantages that a large number of different combination compounding and verification tests are required to obtain the compound trichoderma preparation with certain practical significance, so that the research on the combined trichoderma strains is deepened to be significant.
Disclosure of Invention
The invention aims to provide a combined trichoderma preparation with a growth promoting effect superior to that of a single bacterium in saline soil, aiming at the problems that the existing biological organic fertilizer is single in strain, lacks compound bacterium compounding technology and the like.
The invention also aims to provide the application of the combined trichoderma.
The invention also aims to provide a bio-organic fertilizer prepared from the combined trichoderma and application thereof.
The purpose of the invention can be realized by the following technical scheme:
a halotolerant trichoderma complex inoculant consists of mudflat trichoderma (Trichoderma. arenera) TUCIM10301 spore liquid with the preservation number of CGMCC No.19611 and trichoderma asperellum (Trichoderma. asperelloides) TUCIM10320 spore liquid with the preservation number of CGMCC No. 19612.
As a preferable selection of the invention, the volume ratio of two trichoderma spore solutions in the salt-tolerant trichoderma complex inoculant is 1:1, and each spore solution contains more than or equal to 1.5 multiplied by 10 spores8cfu ml-1,pH 8.0。
The salt-tolerant trichoderma combined bacterial agent disclosed by the invention is applied to promotion of plant growth.
3 strains of Trichoderma (Trichoderma asperellum) TUCIM10320 (A), Trichoderma tsunami (Trichoderma arenera) TUCIM10301(B), Trichoderma atroviride T.cf. atroviride 1A51(C)) selected from saline soil and 3 strains of Trichoderma (Trichoderma guizianum T.guizhuense NJAU 4742(D), Trichoderma harzianum CBS 22 (D)) selected from non-saline soil environment were subjected to sand culture conditions containing 0.5% NaCl6.95(E) and T.cf. virens TPL2553 (F)) were grouped in the form of single, double and triple bacteria, respectively, for a total of 14 treatments (A, B, C, AB, AC, BC, ABC, D, E, F, DE, DF, EF, DEF) and 15 treatments with blank not subjected to any treatment as a control. Followed by a suspension of Trichoderma spores (total number of spores 3X 10)8cfu g-1(ii) a The same applies below) were applied to the roots of differently treated crops. Trichoderma was found to reduce the effect of salt on plant growth inhibition relative to the non-inoculated blank control, and the growth promoting effect of the salt tolerant combination trichoderma (i.e., A, B, C and combinations thereof) was generally greater than that of the non-salt tolerant combination trichoderma and the single strain (i.e., D, E, F and combinations thereof).
The biological organic fertilizer is prepared by mixing and fermenting the salt-tolerant trichoderma complex microbial inoculum and a cow dung organic fertilizer.
As a preferable selection of the invention, the salt-tolerant trichoderma complex microbial inoculum is inoculated into cow dung organic fertilizer in an amount of three grams of organic fertilizer per milliliter of spore solution, secondary fermentation is carried out for 7 days, and the final viable count reaches 107cfu g-1And (3) stabilizing the content of the strain to obtain the bio-organic fertilizer.
Preferably, the pH value of the cow dung organic fertilizer is 7.42, and the organic matter content is 457.5g kg-1The total nitrogen, total phosphorus and total potassium contents are respectively 13.25g kg-1、17.78g kg-1、24.34g kg-1
According to the preparation method of the biological organic fertilizer, the salt-tolerant trichoderma complex microbial inoculum is inoculated into the cow dung organic fertilizer according to the ratio of three grams of organic fertilizer per milliliter of spore solution, secondary fermentation is carried out for 7 days, and the final viable count reaches 107cfu g-1And (3) stabilizing the content of the strain to obtain the salt-tolerant trichoderma combined bio-organic fertilizer.
Salinity tolerance testing of the salt tolerant trichoderma combinations described herein.
To screen the most salt tolerant trichoderma combinations, the BIOLOG test (0.5 mol NaCl added) was performed using 3 strains of trichoderma selected from the saline soil (trichoderma asperellum 10320, trichoderma tsunami 10301, trichoderma atroviride 1a51) and their combinations. The results of the study found that the growth of the combination trichoderma intertidal tachii TUCIM10301+ trichoderma asperellum TUCIM10320 (i.e., AB) on three different carbon sources (glucose, sorbitol, sucrose) was best under 0.5% salt stress.
The salt-tolerant trichoderma combination disclosed by the invention is applied to preparation of a biological fertilizer for promoting plant growth.
A salt-tolerant combined trichoderma growth-promoting bio-organic fertilizer is characterized in that: the combined trichoderma growth-promoting bio-organic fertilizer is prepared by mixing and fermenting spore suspensions of trichoderma intertidal TUCIM10301 and trichoderma pseudoasperellum TUCIM10320 and a cow dung organic fertilizer.
In the salt-tolerant composite trichoderma growth-promoting bio-organic fertilizer, the number of spores contained in trichoderma tsugae 10301 is more than or equal to 1.5 multiplied by 108cfu ml-1The Trichoderma asperellum TUCIM10320 contains more than or equal to 1.5 multiplied by 10 spores8cfu ml-1The pH was 8.
The preparation method of the salt-tolerant trichoderma combined bio-organic fertilizer comprises the following steps:
firstly, inoculating Trichoderma tsurui 10301 and Trichoderma pseudoasperellum TUCIM10320 strains on a potato glucose solid culture medium (PDA, BD Difco, Becton, Dickinson and Company), placing the potato glucose solid culture medium in a 25 ℃ illumination incubator for 5 days, taking out the potato glucose solid culture medium, adding about 5mL of sterile aqueous solution on the potato glucose solid culture medium, scraping spores by using a coating rod, and filtering the mixture into a sterile triangular flask wrapped with two layers of sterile gauze to obtain a Trichoderma spore solution. And measuring OD 600 by using a microplate reader to calculate the concentration of the trichoderma spore suspension, and placing the trichoderma spore suspension in a refrigerator at 4 ℃ for later use. Then cow dung organic fertilizer (pH 7.42, organic matter content 457.5g kg) with the weight of about 1 percent of sand is weighed-1The total nitrogen, total phosphorus and total potassium contents are respectively 13.25g kg-1、17.78g kg-1、24.34g kg-1). Two kinds of trichoderma in a volume ratio of 1:1, mixing and inoculating the mixture into cow dung organic fertilizer according to the ratio of spore liquid to spore liquid of three grams, and performing secondary fermentation for 7 days until the final viable count reaches 107cfu g-1And (3) stabilizing the content of the strain to obtain the salt-tolerant trichoderma combined bio-organic fertilizer.
The salt-tolerant trichoderma combination is applied to crop soil in a bio-organic fertilizer mode, and the fact that the trichoderma bio-organic fertilizer is inoculated in a salt environment with 0.5% of NaCl can remarkably promote growth of overground parts of tomato seedlings and root system elongation and lateral root differentiation of underground parts. The bio-organic fertilizer compounded by the trichoderma viride TUCIM10301 and the trichoderma pseudosporangium 10320 has the most obvious growth promoting effect.
Has the advantages that:
the invention mainly utilizes the composite microbial inoculum obtained by mixing two trichoderma to prepare the biological organic fertilizer for application, and can obviously promote the growth of saline soil crops. The composite trichoderma bio-organic fertilizer has obvious growth promotion effect after being applied, has better growth promotion effect than the application of the bio-organic fertilizer combined by single bacteria and other trichoderma, and plays a role in relieving the inhibition of salt on the growth of crops.
Drawings
FIG. 1 shows the effect of the complex microbial inoculum of Trichoderma aureoviride TUCIM10301 and Trichoderma asperellum TUCIM10320 (AB) on promoting the growth of tomato seedlings under salt stress. In the figure, A: TUCIM10320, B: TUCIM10301, C: trichoderma atroviride t.cf. atroviride 1a51, D: trichoderma guizhuense NJAU 4742, E: trichoderma harzianum CBS 226.95, F: trichoderma virens t.cf.virens TPL 2553; the same applies below.
FIG. 2 shows the effect of the complex microbial inoculum of Trichoderma aureoviride TUCIM10301 and Trichoderma asperellum TUCIM10320 (AB) on promoting the growth of tomato seedlings under salt stress.
FIG. 3 shows the salt tolerance (0.5% salt) of the complex microbial inoculum of Trichoderma aureoviride TUCIM10301 and Trichoderma asperellum TUCIM10320 (AB) of the invention.
FIG. 4 shows the promotion effect of the bio-organic fertilizer prepared by the complex microbial inoculum of the Trichoderma tsugae TUCIM10301 and Trichoderma asperellum TUCIM10320 (AB) on the growth of tomato seedling root systems under the salt stress of 0.5%.
Biological material preservation information
TUCIM10320, which is classified and named as Trichoderma asperellides, and is preserved in China general microbiological culture Collection center, wherein the preservation address is the microbiological research institute of China academy of sciences No. 3, West Lu No.1 Hospital, North Cheng, south China, in Beijing, the preservation date is 2020, 4 and 3 days, and the preservation number is CGMCC No. 19612.
TUCIM10301, which is classified and named as Trichoderma, arenerea, and is preserved in China general microbiological culture Collection center, wherein the preservation address is microorganism institute of China academy of sciences No. 3, West Lu No.1 Hospital, North Cheng, south China, and the preservation date is 2020, 4 and 3 days, and the preservation number is CGMCC No. 19611.
Detailed Description
The invention is further illustrated by the following examples.
Example 1: growth promoting effect of salt-tolerant and non-salt-tolerant trichoderma and combination thereof on tomato seedlings
1.1 test materials
1.1.1 test tomato seeds
The tomato variety tested was "Shanghai cooperative 903" purchased from agricultural academy of Nanjing, Jiangsu province. Selecting plump tomato seeds, soaking in warm water for 1-2h, sterilizing with hydrogen peroxide solution (2%, v/v) for 2min, rinsing with sterile distilled water for 6-7 times, placing the seeds in a disposable culture dish paved with two layers of wet filter paper, placing in a 28 ℃ incubator in a dark place for accelerating germination for 24-48h, sowing the tomato seeds germinated in white in a seedling tray, and watering regularly every day until the tomato seeds grow to have two leaves and one core.
1.1.2 test strains
3 strains of salt-tolerant trichoderma (Trichoderma asperellum TUCIM10320, Trichoderma reesei TUCIM10301, Trichoderma atroviride 1A51) and 3 strains of non-salt-tolerant trichoderma (Trichoderma guizhou NJAU 4742, Trichoderma harzianum CBS 226.95, Trichoderma viride TPL 2553)
1.1.3 preparation of Trichoderma spore suspension
Inoculating the trichoderma strains on a potato dextrose solid culture medium (PDA), culturing for 5d in a 25 ℃ illumination incubator, taking out, adding about 5mL of sterile aqueous solution to the culture medium, scraping off spores by using a coating rod, and filtering into a sterile triangular flask wrapped with two layers of sterile gauze to obtain trichoderma spore liquid. And (3) measuring the concentration of the trichoderma spore suspension by using a microplate reader, and storing the trichoderma spore suspension in a refrigerator at 4 ℃ for later use.
1.2 protocol
Respectively combining halotolerant bacteria and non-halotolerant trichoderma into single bacteria, double bacteria and three bacteriaThe resultant patterns were grouped for a total of 14 treatments (A, B, C, AB, AC, BC, ABC, D, E, F, DE, DF, EF, DEF) with no added bacteria treatment as a blank for a total of 15 treatments, 12 basins each containing about 600g of fine sand (0.5-1mm in diameter). And selecting tomato seedlings with consistent growth vigor for transplanting. After one week of seedling transplanting, 0.5% NaCl saline is poured once, and MS nutrient solution is poured once every two weeks. Adding the spore liquid after adding the saline water for the first time, wherein the root irrigation of each seedling is not less than 1070.6ml of spore liquid (i.e. 0.1% of soil weight, 10% of the total volume)4cfu g-1Soil). Measuring the growth condition of tomato plants after the tomato plants grow for four weeks, measuring indexes such as SPAD value (representing photosynthesis intensity), plant height, fresh weight of the plants, dry weight of the plants and the like, and scanning the root systems of the tomato seedlings to analyze the root surface area, the root length and the number of root tips.
1.3 test results
Under the salt stress, the growth vigor of the tomato seedlings inoculated with the trichoderma is obviously superior to that of the tomato seedlings not inoculated with the trichoderma. Under salt stress, tomato seedlings inoculated with salt-tolerant trichoderma generally grow better than tomato seedlings inoculated with non-salt-tolerant trichoderma. The leaves of the tomato seedlings without being inoculated with the fungicide show curling, yellowing, wilting and purple back, and the root system is weak; the leaves of the tomato seedlings inoculated with the trichoderma are emerald green, the leaf surface expansion degree is slightly large, the stems are thick and strong, and the root systems are developed; indexes such as chlorophyll (SPAD), plant and root dry weight of tomato seedlings inoculated with halotolerant bacteria are obviously higher than those of non-halotolerant bacteria (figure 1 and figure 2).
The above shows that under salt stress, the inoculated trichoderma has stronger growth promotion effect on plants compared with the non-inoculated control. The salt tolerant trichoderma exhibits a higher growth promoting capacity than non-salt tolerant trichoderma. Therefore, the growth promoting capability of the trichoderma halodurans and the combined bacteria thereof is explored in a mode of preparing the bio-organic fertilizer.
Example 2: salt tolerance test of trichoderma halodurans and combinations thereof
3 strains of salt-tolerant trichoderma harzianum (trichoderma asperellum) 10320, trichoderma intertidal 10301 and trichoderma atroviride 1A51 are selected for BIOLOG test. Inoculating the three strains to PDA plate, culturing in 28 deg.C incubator 5Around day, the conidiophores are produced in a large amount. Under aseptic conditions, a suitable amount of sterilized 0.5mol NaCl solution was injected into the BIOLOG tube, and the tube was inserted into a turbidimeter to adjust its turbidity to 100% T. The spores were then gently scraped with a cotton swab and inserted into a tube containing saline to adjust the spore concentration to 108cfu ml-1. The three strains are divided into 7 treatments of single strain A (Trichoderma aureoviride TUCIM10301), single strain B (Trichoderma asperellum TUCIM 10320), single strain C (Trichoderma atroviride 1A51), double strain AB, double strain AC, double strain BC and three strain ABC, and each treatment is repeated three times. Each treated salt-containing spore solution was inoculated into 96 wells of FF assay plates containing 96 carbon sources using eight rows of guns, with 90ul of liquid per well. The plates were incubated at 25 ℃ in the dark at constant temperature, and OD (optical density) at 750nm (hyphal growth) was measured and recorded using a microplate reader at 12, 18, 24, 36, 48, 72, 96, 120, 144, and 168 hours, respectively.
Test results show that in trichoderma single-bacterium treatment, the salt tolerance effect of trichoderma asperellum TUCIM10320 and trichoderma atroviride 1A51 is the best; in the treatment of the trichoderma combined double bacteria, the salt tolerance effect of the compound of the trichoderma pseudoasperellum TUCIM10320 and the trichoderma intertidal TUCIM10301 is obviously better than that of the single bacteria, which shows that the mixing of the trichoderma pseudoasperellum TUCIM10320 and the trichoderma intertidal TUCIM10301 can generate a synergistic effect, thereby promoting the improvement of the salt tolerance; similarly, the salt tolerance effect of mixing trichoderma asperellum TUCIM10320 and trichoderma atroviride 1A51 is reduced compared with that of the respective single bacteria, which also indicates that antagonism may exist between trichoderma asperellum TUCIM10320 and trichoderma atroviride 1A51, resulting in reduction of the salt tolerance; in addition, when the three bacteria are mixed together, the salt tolerance effect is worse than that of the three bacteria when the three bacteria are used independently, which indicates that the bacteria can be mutually restrained under the salt stress, so that the growth of the bacteria in the salt environment is not facilitated; under the salt stress, compared with the single use of the trichoderma pseudospora TUCIM10320 and the trichoderma intertidal TUCIM10301(AB), the combined bacterium has the highest utilization rate on glucose, sorbitol and sucrose, and shows that the salt tolerance of the combined bacterium is the strongest (figure 3).
Example 3: growth promotion effect of salt-tolerant trichoderma combined bio-organic fertilizer on tomato seedlings
3.1 test materials
Salt-tolerant trichoderma strains (trichoderma asperellum TuCIM10320, trichoderma intertidal TUCIM10301, trichoderma atroviride 1A 51); tomato seeds (Shanghai cooperative 903).
3.2 test methods
3.2.1 spore suspension preparation
Inoculating the required strain on PDA culture medium, culturing for 7d until spore is completely produced, scraping spore, and uniformly adjusting the concentration of spore suspension to 10 with sterile water8cfu ml-1
3.2.2 growing seedlings
The method comprises the steps of selecting plump tomato seeds, soaking the tomato seeds in warm water for 1-2h, then disinfecting the tomato seeds with hydrogen peroxide solution (2%, v/v) for 2min, then rinsing the tomato seeds with sterile double distilled water for 6-7 times, placing the tomato seeds in an aseptic culture dish paved with two layers of wet filter paper, placing the culture dish in a 28 ℃ culture box in a dark place for accelerating germination for 24-48h, then sowing the tomato seeds with white buds in a seedling culture dish, and watering the tomato seeds regularly every day until the tomato seeds grow to be two leaves and one heart.
3.2.3 preparation of Trichoderma Bio-organic fertilizer
Cow dung organic fertilizer weighing about 1% of sand (PH 7.42, organic matter content 457.5g kg)-1The total nitrogen, total phosphorus and total potassium contents are respectively 13.25g kg-1、17.78g kg-1、24.34g kg-1) Supplied by Junde ecological agriculture science and technology Limited of Jiangsu. Inoculating the spore liquid into cow dung organic fertilizer, fermenting for 7 days until the final viable count reaches 107cfu g-1And (3) the strain content is stable, and the saline-alkali soil growth-promoting bio-organic fertilizer is obtained.
3.2.4 transplanting
The test set eight treatments in total, which are: the method comprises the following steps of applying no fertilizer as a Control (CK), using a Trichoderma koningii TUCIM10301 biological organic fertilizer (A), using a Trichoderma asperellum TUCIM10320 biological organic fertilizer (B), using a Trichoderma atroviride 1A51 biological organic fertilizer (C), using a Trichoderma asperellum TUCIM10320 + Trichoderma asperellum TUCIM10301 composite biological organic fertilizer (AB), using a Trichoderma asperellum TUCIM10301+ Trichoderma atroviride 1A51 composite biological organic fertilizer (AC), using a Trichoderma asperellum TUCIM10320 + Trichoderma atroviride 1A51 composite biological organic fertilizer (BC) and using a Trichoderma asperellum TUCIM10320 + Trichoderma asperellum TUCIM10301+ Trichoderma atroviride 1A51 composite biological organic fertilizer (ABC). 600g of sand is filled in each basin (0.5-1mm), and the trichoderma bio-organic fertilizer which is correspondingly treated is uniformly mixed with the sand for use. Three tomato seedlings were planted per pot, and one treatment was repeated 12 times. After 7d of field planting, 90ml of 0.5% NaCl solution containing 10% Murashige Skoog nutrient solution is poured into each pot, and the pH value is adjusted to 8. Sand and saline water are used for simulating a saline soil environment, and a proper amount of 10% MS nutrient solution is supplemented every other week or so to ensure the supply of plant nutrients.
3.2.5 examination seedling
After 4 weeks of growth, the growth conditions of tomato plants were measured, indexes such as SPAD value (characteristic of photosynthesis intensity), plant height, fresh weight of the plants, dry weight of the plants and the like were measured (Table 1), and the root surface area, root length and root tip number of tomato seedlings were analyzed by scanning the root systems (FIG. 4).
TABLE 10.5% salt concentration stress growth promoting effect of halotolerant trichoderma and its combined bacteria bio-organic fertilizer on tomato seedlings
Figure BDA0002904071880000081
Mean ± standard deviation (n-12) with treatment CK without fertilization as control. There was no significant difference between treatments with the same letter (p ═ 0.05).
3.3 test results
Under the salt stress, the tomato seedlings treated by the trichoderma pseudoasperellum TUCIM10320 and trichoderma intertidal TUCIM10301 composite biological organic fertilizer (AB) have better growth vigor than the tomato seedlings treated by no-fertilizer application and other fertilizer application treatments, and have significant differences in plant height, chlorophyll, fresh and dry weight of overground parts and underground parts and the like, which shows that the combined salt-tolerant trichoderma pseudoasperellum biological organic fertilizer has great effect on improving the salt stress resistance of crops and has obvious growth promotion effect on the plants.

Claims (9)

1. A salt-tolerant trichoderma composite microbial inoculum is characterized by consisting of a trichoderma intertidal 10301 spore liquid with the preservation number of CGMCC No.19611 and a trichoderma asperellum 10320 spore liquid with the preservation number of CGMCC No. 19612.
2. The halotolerant trichoderma complex microbial inoculant according to claim 1, wherein the halotolerant trichoderma complex microbial inoculant is characterized in that the volume ratio of two trichoderma spore solutions in the halotolerant trichoderma complex microbial inoculant is 1:1, and each spore solution contains more than or equal to 1.5 x 10 spores8cfu ml-1
3. The use of the halotolerant trichoderma complex inoculant of claim 1 or 2 for promoting plant growth.
4. The application of the halotolerant trichoderma complex inoculant of claim 1 or 2 in preparation of a bio-organic fertilizer.
5. A bio-organic fertilizer is characterized in that the bio-organic fertilizer is prepared by mixing and fermenting the salt-tolerant trichoderma complex microbial inoculum of claim 1 or 2 and a cow dung organic fertilizer.
6. The biological organic fertilizer as claimed in claim 5, characterized in that the halotolerant trichoderma complex inoculant as claimed in claim 1 or 2 is inoculated into cow dung organic fertilizer in an amount of three grams of organic fertilizer per milliliter of spore liquid, and after secondary fermentation for 7 days, the final viable count reaches 107cfu g-1And (3) stabilizing the content of the strain to obtain the bio-organic fertilizer.
7. The bio-organic fertilizer according to claim 6, characterized in that the pH of the cow dung organic fertilizer is 7.42, and the organic matter content is 457.5g kg-1The total nitrogen, total phosphorus and total potassium contents are respectively 13.25g kg-1、17.78g kg-1、24.34g kg-1
8. The method for preparing the bio-organic fertilizer of claim 5, which is characterized by comprising the following steps: the halotolerant trichoderma complex bacterium of claim 1 or 2The agent is inoculated into cow dung organic fertilizer in an amount of three grams of organic fertilizer per milliliter of spore liquid, secondary fermentation is carried out for 7 days, and the final viable count reaches 107cfu g-1And (3) stabilizing the content of the strain to obtain the salt-tolerant trichoderma combined bio-organic fertilizer.
9. Use of the bioorganic fertilizer of claim 5 for promoting crop growth.
CN202110065741.8A 2021-01-19 2021-01-19 Trichoderma composite microbial agent and bio-organic fertilizer prepared from same Pending CN112746024A (en)

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CN111621424A (en) * 2020-05-07 2020-09-04 南京农业大学 Saline-alkali-resistant neomyces and application thereof
CN114431255A (en) * 2021-10-29 2022-05-06 中国科学院烟台海岸带研究所 Application of trichoderma asperellum and plant growth promoting microbial inoculum
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Publication number Priority date Publication date Assignee Title
CN111394261A (en) * 2020-05-07 2020-07-10 南京农业大学 Saline-alkali-tolerant trichoderma asperellum and application thereof
CN111621424A (en) * 2020-05-07 2020-09-04 南京农业大学 Saline-alkali-resistant neomyces and application thereof
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CN111621424B (en) * 2020-05-07 2022-02-18 南京农业大学 Saline-alkali-resistant neomyces and application thereof
CN114431255A (en) * 2021-10-29 2022-05-06 中国科学院烟台海岸带研究所 Application of trichoderma asperellum and plant growth promoting microbial inoculum
CN114532363A (en) * 2022-02-18 2022-05-27 南京思农生物有机肥研究院有限公司 Application method and application of coating containing trichoderma seed

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