CN113817613B

CN113817613B - Trichoderma asperellum 6S-2 and application thereof in relieving apple continuous cropping obstacle

Info

Publication number: CN113817613B
Application number: CN202111231593.9A
Authority: CN
Inventors: 尹承苗; 王海燕; 毛志泉; 刘鑫; 吕毅; 陈学森; 沈向
Original assignee: Shandong Agricultural University
Current assignee: Shandong Agricultural University
Priority date: 2021-10-22
Filing date: 2021-10-22
Publication date: 2023-05-19
Anticipated expiration: 2041-10-22
Also published as: CN113817613A

Abstract

The invention discloses trichoderma asperellum 6S-2 and application thereof in relieving apple continuous cropping obstacle, and belongs to the technical field of agricultural microorganisms. The biological preservation number of the trichoderma asperellum 6S-2 is as follows: CGMCC No.23275, which is an endophyte and has antagonism to ARD specialization layer fusarium MR5 and various pathogenic bacteria; has protease, cellulase, amylase, laccase and other activities; the iron carrier, the auxin and the ammonia gas are secreted, and the phosphorus dissolving capability is certain; the fermentation extract has antibacterial and growth promoting activities; volatile substances also have bacteriostatic ability. The spore liquid is applied to the soil of the apple orchard, so that the oxidative damage of plants can be reduced, the growth of apple stocks can be promoted, and the apple continuous cropping obstacle can be relieved.

Description

Trichoderma asperellum 6S-2 and application thereof in relieving apple continuous cropping obstacle

Technical Field

The invention relates to the technical field of agricultural microorganisms, in particular to trichoderma asperellum 6S-2 and application thereof in relieving apple continuous cropping obstacle.

Background

Apple continuous cropping obstacle (ARD) is a complex disease caused by a variety of factors (Mazzola and Manici,2012; spath et al, 2015), and it is widely believed that imbalance in soil microflora structure is the main cause of ARD (Mazzola and Manici,2012; manici et al, 2018). Earlier studies showed that Fusarium was the predominant pathogen responsible for ARD in Bay Bohai (Wang et al, 2018), whereas the specialized layer associated with ARD recently screened and identified by the laboratory, fusarium (Fusarium proliferatum) MR5, was highly pathogenic to apple plants. Traditional chemical fumigation and pesticide application are very effective in controlling ARD, but do not follow the concept of ecological development (Lu et al 2020).

Currently, trichoderma, bacillus, pseudomonas and Streptomyces are widely used as biocontrol agents (Harman et al 2004; hu et al 2020; maciag et al 2020), conforming to the concept of "double subtraction". Furthermore, endophytes can colonize plant bodies more effectively than rhizosphere soil microorganisms to adapt to environmental changes, thereby enhancing resistance to disease, promoting plant growth (Bogner et al, 2016), and thus endophytes are also commonly used as biocontrol agents, plant growth promoters (Saad et al, 2019; poveda et al, 2021; poveda,2021 a). For example, endophytic fungi isolated from pelargonium and the like have a rich biocontrol secondary metabolite (Saad et al, 2019). Poveda et al (2021) have also found that endophytes can promote plant growth by increasing nutrient (nitrogen, phosphorus, potassium, zinc, iron) acquisition and production of plant hormones.

Trichoderma is the most traditional beneficial microorganism genus in agricultural production, and shows a strong biocontrol effect on plant diseases. Trichoderma asperellum (Trichoderma asperellum) is an important fungus in the genus Trichoderma, and studies have shown that inoculating onion with Trichoderma asperellum has potential as a synthetic fungicide substitute and can be used to protect onion from infection by pathogenic bacteria (Zapata-Sarminito et al 2020). Trichoderma asperellum can secrete chitinase, glucanase and protease, degrade fungal cell walls, promote fungal parasitism, inhibit fusarium graminearum hyphae by up to 60%, and can produce polyketides, alkanes and other antifungal secondary metabolites to inhibit pathogen growth, the application of which particulate bacterial manure promotes corn growth (Wu et al, 2017; he et al, 2019). The combined action of trichoderma asperellum and mycorrhizal fungi can promote plant defense system and resist black pod disease. Although trichoderma asperellum shows great development potential in green agriculture, no report on the biocontrol effect of fusarium causing ARD specialization has been seen yet.

Disclosure of Invention

The inventor starts from the phenomenon of 'disease-suppressing soil' frequently occurring in continuous cropping orchards through long-term technology and practical exploration, and separates a strain of trichoderma asperellum 6S-2 from healthy apple roots of the continuous cropping orchards, wherein the strain is an endophyte and has antagonism on ARD specialization type fusarium solani MR5 and various pathogenic bacteria; has protease, cellulase, amylase, laccase and other activities; the method secretes siderophores, auxin (IAA) and generates ammonia gas, and has certain phosphorus dissolving capacity; the fermentation extract has antibacterial and growth promoting activities; volatile substances also have bacteriostatic ability. The spore liquid is applied to the soil of the apple orchard, so that the oxidative damage of plants can be reduced, the growth of apple stocks can be promoted, and the apple continuous cropping obstacle can be relieved.

Specifically, the invention relates to the following technical scheme:

in a first aspect of the present invention, a strain of trichoderma asperellum (Trichoderma asperellum) 6S-2, which has been deposited in the China general microbiological culture collection center (CGMCC, address: hospital No. 1, hospital No. 3, beijing, kogyo, and having a biological deposit number of: CGMCC No.23275.

The trichoderma asperellum (Trichoderma asperellum) 6S-2 is separated from the root system of healthy fruit trees in a continuous cropping apple garden and has the following characteristics:

when growing on PDA solid culture medium, the initial mycelium is white wool-like, diffuses to the edge, and the back of colony is colorless; the mid-period gradually turns to light green, spore rings are formed on hyphae which develop earlier, the yellow color is gradually changed to green, aerial hyphae are more luxuriant, and the aerial hyphae are thickened and are cotton-flocculent; the bacterial colony at the later stage can form 3 concentric rings with alternate white and green, dense conidia are attached on the circular ring, the whole bacterial strain is dark green, the darker the color of the part near the center is, the less aerial hypha is, and the flat plate can be grown in 5 days; when growing on the search medium, yellow blister-shaped protruding structures are formed on the initial flat plate, the later stage is gradually matured to become green, and finally yellow-green alternate circular rings are formed, so that the generation and the propagation of spores are facilitated; when the mycelia are thin and thin, the bacterial colony is transparent and radial, the whole culture medium can be covered about 3 days, conidium is in a cluster pad shape, a dark green bullous ring is finally formed, the bullous ring is more polymerized and compact on the CMD culture medium, and in the SNA culture medium, the bullous structure is more dispersed and is in a semi-annular shape or a sheet shape.

When observed under a 60-time common optical microscope, hyphae are long and thin and separated, main branches are dendritic, long and thin conidiophore branches form secondary branches, primary branches generated below the top end of a main shaft are usually opposite, an included angle between the primary branches and the main shaft is approximately 90 degrees, the length of each primary branch is almost equal along with the increase of the distance from the top end, secondary branches formed at the position close to the main shaft are longest, the secondary branches can generate third branches, and the third branches do not further branch. The bottle peduncles on the conidiophores are symmetrically distributed, the bottle peduncles are generated at the top ends of the primary, secondary and tertiary branches, the bottle peduncles are shorter, the bottle peduncles can be sharply contracted and thinned at the tip of the bottle peduncles, are bent and expanded in the middle, are in ampoule bottle shape, are in a set of 3 or 4 wheelsets at the top ends, are arranged in a vortex shape, and can generate conidiophores at the tip of the bottle peduncles after the conidiophores develop and mature. Conidia are green spheres or oval shapes, are gathered or dispersed, have fine thorns on the wall, and can generate chlamydospores.

Spore suspensions, spore powders, fermentation extracts and/or volatile materials of the trichoderma asperellum (Trichoderma asperellum) 6S-2 also fall within the scope of the invention.

Preferably, the spore suspension of trichoderma asperellum (Trichoderma asperellum) 6S-2 is prepared by the following method:

trichoderma asperellum (Trichoderma asperellum) 6S-2 was placed on PDA medium and cultured at 28℃for 7 days, and then the spores were rinsed with sterile water to prepare a spore suspension.

Preferably, the spore powder of the trichoderma asperellum (Trichoderma asperellum) 6S-2 is prepared by the following method:

inoculating spore suspension of Trichoderma asperellum (Trichoderma asperellum) 6S-2 into fermentation medium, and culturing at 28 deg.C for 10-14d; air-drying, crushing and sieving after fermentation to obtain spore powder; the fermentation medium is prepared from wheat bran and corn flour according to a volume ratio of 4:1, adding sterile water to make the mass fraction of the sterile water in the fermentation medium 45%.

In one embodiment of the present invention, the preparation method of the spore powder of Trichoderma asperellum (Trichoderma asperellum) 6S-2 is as follows:

activating 6S-2 stored in a test tube, inoculating on a PDA culture medium, growing for 7 days until spores grow, washing the spores with sterile water, and preparing spore liquid; and then adopting a tray fermentation method to propagate the spore powder of 6S-2. 400g of sterilization culture medium (wheat bran and corn flour are mixed according to the volume ratio of 4:1, sterile water is added to ensure that the mass fraction of the sterile water in the fermentation culture medium is 45 percent) is put into a shallow tray with the volume of 30cm multiplied by 20cm multiplied by 5cm, and spore liquid is inoculated according to the proportion of 2 percent (volume fraction) in a liquid inoculation mode to obtain the corn flour ⁵ CFU/ml was placed in a tray, followed by a sterilized double gauze, incubated at 28℃for 2 days with a flip-flop, and kept moist with a humidifier. Fermenting for 12 days, air drying, pulverizing, sieving to obtain spore powder of Trichoderma asperellum 6S-2, and measuring the concentration of spore powder to 1.05X10% ⁹ CFU/g, and storing in a sealed bag.

Preferably, the fermentation extract of trichoderma asperellum (Trichoderma asperellum) 6S-2 is prepared by the following method:

culturing Trichoderma asperellum (Trichoderma asperellum) 6S-2 on PDA culture medium at 28deg.C for 7 days, washing spores with sterile water, and making into spore suspension; adding the spore suspension into PDB culture medium, culturing at 28deg.C for 8 days at 160r/min, filtering, centrifuging at 12000r/min for 5min, and collecting supernatant; the collected supernatant was mixed with ethyl acetate in a volume ratio of 1:1, collecting the extract, concentrating to dry powder, dissolving in methanol solution again, centrifuging, filtering to remove impurities, and making into fermentation extract.

In a second aspect of the invention there is provided the use of a spore suspension, spore powder, fermentation extract and/or volatile material of Trichoderma asperellum (Trichoderma asperellum) 6S-2 or Trichoderma asperellum (Trichoderma asperellum) 6S-2 as described above in at least one of the following (1) - (4):

(1) Inhibiting growth of plant pathogenic bacteria and germination of spores;

(2) Preparing a product for inhibiting plant pathogenic bacteria;

(3) Preventing and treating diseases caused by plant pathogenic bacteria;

(4) A product for controlling diseases caused by plant pathogenic bacteria is prepared.

Preferably, the plant pathogenic bacteria are Fusarium oxysporum (Fusarium proliferatum) MR5, fusarium oxysporum (Fusarium oxysporum), fusarium solani (Fusarium solani), fusarium moniliforme (Fusarium moniliforme), phytophthora (Phytophthora cactorum), pythium aphanidermatum (Pythium aphanidermatum), rhizoctonia solani (Phoma aspargyi), alternaria alternata (Alternaria alternata), rhizoctonia verrucosa (Myrothecium verrucaria), penicillium brazil (Penicillium brasilianum), rhizoctonia solani (Rhizoctonia solani) and/or Aspergillus flavus (Aspergillus flavus) of the ARD specialization layer.

In a third aspect of the invention there is provided the use of trichoderma asperellum (Trichoderma asperellum) 6S-2 in at least one of the following (1) - (4):

(1) Producing protease, cellulase, laccase and/or amylase;

(2) Generating IAA;

(3) Producing a siderophore;

(4) Ammonia gas is generated.

In a fourth aspect of the invention there is provided the use of a fermentation extract or spore suspension of Trichoderma asperellum (Trichoderma asperellum) 6S-2, trichoderma asperellum (Trichoderma asperellum) 6S-2 in any one of (1) to (3) below:

(1) Promoting the generation and elongation of plant lateral roots;

(2) Reducing oxidative damage of plants;

(3) Promote plant growth.

Preferably, the plant is an arabidopsis thaliana or apple rootstock.

In a fifth aspect of the invention there is provided the use of a spore suspension, spore powder and/or fermentation extract of Trichoderma asperellum (Trichoderma asperellum) 6S-2 or Trichoderma asperellum (Trichoderma asperellum) 6S-2 as described above in (1) or (2) as follows:

(1) The apple continuous cropping obstacle is relieved;

(2) Preparing the biocontrol preparation for relieving the continuous cropping obstacle of the apple tree.

In a sixth aspect of the present invention, there is provided a method for alleviating apple continuous cropping obstacle, comprising the steps of:

the spore suspension, spore powder and/or fermentation extract of trichoderma asperellum (Trichoderma asperellum) 6S-2 are applied to apple continuous cropping soil.

The invention has the beneficial effects that:

the invention separates an endophytic trichoderma asperellum (Trichoderma asperellum) 6S-2 from the root system of healthy fruit trees in the continuous cropping apple orchard for the first time, and has potential antagonistic growth promoting capability. The inhibition rate of the fusarium MR5 on the specialization layer causing the apple continuous cropping obstacle is up to 52.41 percent, and the fusarium oxysporum, fusarium solani, fusarium moniliforme, phytophthora wart, humicola citrulline, rhizoctonia solani, alternaria alternata and the like have stronger antagonistic inhibition effects. The strain has the activities of producing protease, cellulase, amylase, laccase and the like; the method secretes siderophores, auxin (IAA) and generates ammonia gas, and has certain phosphorus dissolving capacity. In addition, the fermentation extract of trichoderma asperellum 6S-2 can inhibit the growth of pathogenic bacteria, so that hyphae of the trichoderma asperellum are twisted, shrunken, swelled, ruptured and the content of the trichoderma asperellum is released, and the generation and the extension of lateral roots of arabidopsis thaliana can be promoted to the maximum at the concentration of 50 mg/ml; the volatile substances of the strain also have antibacterial capacity. Under the potting condition, the 6S-2 spore liquid is applied to continuous cropping soil, so that the oxidative damage of plants can be reduced, and the growth of the plants can be promoted; has good effect in relieving apple tree continuous cropping obstacle, and provides theoretical basis for biological control of apple continuous cropping obstacle.

Drawings

Fig. 1:6S-2 strain and MR5 antagonism test chart: MR5 control (a), plate facing front (b), plate facing back (c); 6S-2 tieback re-separation diagram: control (d), 3 days of mycelium morphology (e), 5 days of mycelium morphology (f); after the MR5 and the 6S-2 are co-cultured, a scanning electron microscope is used for observing a hypha morphological image (g-l); MR5 hypha PI staining: MR5 control (m), MR5 co-cultured with 6S-2 (p); root system PAS staining pattern: cross section 100x (n), cross section 400x (o), longitudinal section 100x (q) and longitudinal section 400x (r).

Fig. 2: morphology observation of 6S-2 strain: PDA medium (a), search for medium (b), CMD medium (c), SNA medium (d); 6S-2 morphological analysis: spore morphology (e-g), spore morphology (h) observed under a common light microscope at 60-fold; spore morphology (j-m) under a scanning electron microscope, spore morphology (i); 6S-2 functional analysis: phosphate solubilizing activity (n), amylase activity (o), CAS medium covering siderophore producing activity (p), laccase producing activity (q), cellulase producing activity (r), protease producing activity(s), ammonia producing detection (t): the 6S-2+Nesler reagent (t 1), 6S-2 (t 2), yields IAA activity (u): IAA standard (u 1), 6S-2+Salkowski reagent (u 2), 6S-2 (u 3).

Fig. 3: maximum likelihood phylogenetic tree of the combination of ITS and TEF genes from 44 taxonomic groups of trichoderma. The sequences of Protocrea pallida and Protocrea farinosa were used as outer clusters. The evolutionary tree shows Bootstrap values of > 70% and Bayesian posterior probabilities of > 0.90. The scale bar represents 10 substitutions per nucleotide position. The sequence of the fungal species obtained in the present invention is shown in bold and is 100% identical to the sequence of the known species Trichoderma asperellum SZMC: 24288.

Fig. 4: counter experiments (PDA medium) of 6S-2 strains with 12 different pathogenic fungi: fusarium oxysporum Fusarium oxysporum (a), fusarium oxysporum Fusarium proliferatum (b), fusarium solani (c), fusarium moniliforme Fusarium moniliforme (d), phytophthora Phytophthora cactorum (e), pythium aphanidermatum Pythium aphanidermatum (f), phoma asparagi (g), alternaria alternata Alternaria alternata (h), verticillium verrucosum Myrothecium verrucaria (i), penicillium Brazil Penicillium brasilianum (j), rhizoctonia solani Rhizoctonia solani (k), aspergillus flavus Aspergillus flavus (l); inhibition bar graph (m) of 6S-2 strain against different pathogenic fungi growth rates.

Fig. 5:6S-2 fermentation extract liquid inhibition pathogenic bacteria growth test: oxford cup test: methanol is on the left side, and sterile water (a) is on the right side; the left side is 6S-2 fermentation extract, and the right side is sterile water (b); inoculating MR5 (c) after inoculating a blank agar block on the cellophane for 7 days; inoculating 6S-2 strain on cellophane for 7 days, and then inoculating MR5 (d); MR5 growth of seven day mycelium morphology on PDA medium containing different concentrations of 6S-2 fermentation extract: 50mg/L (e), 100mg/L (f), 150mg/L (g), 200mg/L (h); under 20 times mirror, the MR5 spore liquid is co-cultured with methanol for 36 hours to obtain spore germination state (i); under 20 times of the mirror, the MR5 spore liquid and the 6S-2 fermentation extract liquid are co-cultured for 36 hours, and then the spore germinates; inoculating a blank agar block on glass paper for 7 days, then inoculating MR5, and scanning the mycelium morphology (k, l) under an electron microscope, wherein the mycelium morphology (o) is 20 times that under a common optical microscope; the 6S-2 strain was inoculated on cellophane for 7 days, and then inoculated with MR5 in 20 times of the mycelium morphology (m, n) under a common optical microscope, and the mycelium morphology (p-w) under a scanning electron microscope.

Fig. 6: bacteriostatic activity of volatile substances of 6S-2 strain: PDA medium double plate assay: the upper layer is MR5, and the lower layer is blank culture medium (a); the upper layer is MR5, and the lower layer is 6S-2 strain (b); the upper layer is MR5, and the lower layer is 6S-2 strain and active carbon (c); PDA medium biplate assay: the top is MR5 control cultured alone, the bottom is MR5 (d) co-cultured with 6S-2 strain; MR5 control mycelium morphology (e, f) under 20-fold normal light microscope; under a 20-fold common optical microscope, the mycelium morphology (g, h) of MR5 after co-culture with 6S-2; comparing MR5 with mycelium morphology (i, j) under a scanning electron microscope; MR5 hyphae morphology (k-n) after co-cultivation with 6S-2 under scanning electron microscope.

Fig. 7: effect of 6S-2 strain volatiles and fermentation extracts on arabidopsis growth: growing Arabidopsis seedlings on 1/2MS medium; arabidopsis seedlings are controlled to be in a growth state on two plates, wherein the left side is 1/2MS culture medium, and the right side is PDA blank culture medium (b); the arabidopsis and the 6S-2 strain are in a growth state when being co-cultured on two plates, wherein the left side is a 1/2MS culture medium, and the right side is a PDA culture medium (c) inoculated with the 6S-2 strain; root length of arabidopsis thaliana on bipartite plate (d); fresh weight of arabidopsis on bipartite plate (e); the number of the primary lateral roots and the length distribution (f) of the lateral roots of the arabidopsis on the bipartite plate; growth status of Arabidopsis thaliana on 1/2MS medium with different concentrations of methanol solution: 10mg/L (g 1), 50mg/L (h 1), 100mg/L (i 1), 150mg/L (j 1), 200mg/L (k 1); growth status of Arabidopsis thaliana on 1/2MS medium containing different concentrations of 6S-2 fermentation extract: 10mg/L (g 2), 50mg/L (h 2), 100mg/L (i 2), 150mg/L (j 2), 200mg/L (k 2); effect of different concentrations of 6S-2 fermentation extract on Arabidopsis root length (l); the effect of the fermentation extracts of different concentrations of 6S-2 on fresh weight of Arabidopsis thaliana (o); effect of fermentation extracts of different concentrations of 6S-2 on primary and lateral root length distribution of Arabidopsis thaliana: 10mg/L (m), 50mg/L (n), 100mg/L (p), 150mg/L (q).

Fig. 8: determination of plant biomass, root system protecting enzyme activity and photosynthetic index under potting conditions: t337 seedlings are planted in continuous cropping soil (a), leaves and root systems NBT are dyed (a 1-a 3), and leaves and root systems DAB are dyed (a 4-a 6); applying 6S-2 spore suspension (b) after T337 seedlings are planted in continuous cropping soil, and staining leaves and root systems NBT (b 1-b 3) and DAB (b 4-b 6); planting Pingyi sweet tea seedlings in continuous cropping soil (c); planting Pingyi sweet tea seedlings in root system scanning patterns (d) in continuous cropping soil; applying 6S-2 spore suspension (e) after the Malus hupehensis seedlings are planted in continuous cropping soil; applying a 6S-2 spore suspension root system scanning pattern (f) after the cognac sweet tea seedlings are planted in continuous cropping soil; the plant height (g) of Malus hupehensis seedlings; crude stem of Pingyi sweet tea seedling (h); fresh weight of the overground part of the Pingyi sweet tea seedling (i); dry weight of the aerial parts of Malus hupehensis seedlings (j); root surface area (k) of Pingyi sweet tea seedling; root system volume (l) of Pingyi sweet tea seedling; root fresh weight (m) of Pingyi sweet tea seedling; root system dry weight of Pingyi sweet tea seedling; intercellular CO ₂ Concentration C _i (o); net photosynthetic rate P _n (p); air hole conductivity G _s (q); transpiration rate T _r (r); SOD enzyme activity(s); POD enzyme Activity (t); CAT enzyme activity (u); MDA content (v).

Fig. 9: soil culturable microbial count map: bacteria (a); fungi (b); actinomycetes (c); bacterial/fungal ratio (d); four fusarium real-time fluorescent quantitative analyses: fusarium oxysporum (e); fusarium layering (f); fusarium solani (g); fusarium moniliforme (h).

Detailed Description

It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the present application. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

As described in the background section, apple continuous cropping obstacles can cause reduced apple fruit yield and quality, aggravated diseases and insect pests, weakened tree vigor and even death, and cause serious economic loss to fruit farmers. At present, researches on trichoderma asperellum in the aspect of preventing and treating apple continuous cropping obstacle are still freshly reported.

Based on the method, the endophytic fungus 6S-2 is separated from the root system of healthy fruit trees in the continuous cropping apple orchard, and the fungus is identified as trichoderma asperellum according to morphological, physiological and biochemical feature detection and phylogenetic analysis based on ITS, tef 1-alpha genes. Unlike available trichoderma asperellum, the trichoderma asperellum 6S-2 is one new kind of biological control bacteria with great development potential, and has the functions of antagonizing bacteria, producing antagonizing enzyme, secreting growth promoting factor, promoting the growth of Arabidopsis and apple stock, relieving continuous cropping obstacle of apple tree, etc. The invention adopts a flat plate opposite method to research the antagonism effect of the fusarium, fusarium oxysporum, fusarium putrescens, fusarium moniliforme, verrucaria verrucosa, phytophthora, pythium citrulli, rhizoctonia solani, penicillium bassinense, aspergillus flavus, alternaria and other 12 pathogenic bacteria on the specialized layer which causes the apple continuous cropping obstacle, the trichoderma aspergilli 6S-2 has stronger antagonism inhibition effect on 12 pathogenic bacteria, and the inhibition rate of the fusarium putrescence MR5 on the ARD specialized layer is up to 52.41 percent. The 6S-2 strain has antagonistic enzyme activities such as protease production, cellulase, amylase, laccase and the like, can secrete siderophores and auxin (IAA) and produce ammonia, and has certain phosphorus dissolving capacity. In addition, the fermentation extract of the 6S-2 strain can inhibit the growth of pathogenic bacteria, so that hyphae of the strain are twisted, shrunken, swelled, ruptured and the content of the strain is released, and the generation and elongation of lateral roots of arabidopsis thaliana can be promoted to the greatest extent at the concentration of 50 mg/ml; the volatile substances of the strain also have antibacterial capacity. Under the potting condition, the 6S-2 spore suspension is applied to continuous cropping soil, so that the degree of oxidative damage of Malus hupehensis seedlings (apple rootstock) can be reduced, the activity of root system protective enzymes is promoted, the MDA content is reduced, the net photosynthetic rate is improved, and meanwhile, the plant growth is promoted, and the 6S-2 strain has good effect in relieving apple continuous cropping obstacle.

In order to enable those skilled in the art to more clearly understand the technical solutions of the present application, the technical solutions of the present application will be described in detail below with reference to specific embodiments. If experimental details are not specified in the examples, the conditions are generally conventional or recommended by the reagent company; reagents, consumables, etc. used in the examples described below are commercially available unless otherwise specified. Wherein:

PDA medium: peeled potato 200.0g, glucose 20.0g, agar 20.0g, distilled water 1000mL.

Skim milk powder medium: 10.0g of skimmed milk powder, 20.0g of agar, 1000mL of distilled water and natural pH.

Cellulase detection medium: 10.0g of peptone, 10.0g of yeast powder and 10.0g,NaCl 5.0g,KH g of sodium carboxymethylcellulose ₂ PO ₄ 1.0g, agar 20.0g, 1000mL of distilled water, pH 7.0.

Search for medium: KNO (KNO) ₃ 2.0g，K ₂ HPO ₃ 1.0g，KCl 1.0g，MgSO ₄ ·7H ₂ O 0.5g，FeSO ₄ 0.01g, 30.0g of sucrose, 20.0g of agar and 1000mL of distilled water.

CMD culture medium, corn flour 50.0g, glucose 2.0g, agar 15.0g, distilled water 1000mL, pH 6.0.

SNA medium: KH (KH) ₂ PO ₄ 1.0g；KCl 0.5g；KNO ₃ 1.0g；MgSO ₄ 0.5g; glucose 0.2g; sucrose 0.2g; 18.0g of agar; distilled water 1,000 mL.

NBRIP medium: glucose 10.0g, ca ₃ (PO ₄ ) ₂ 10.0g,MgCl ₂ ·6H ₂ O 5.0g,MgSO ₄ ·7H2O 0.25g,KCl 0.2g，(NH ₄ )2SO ₄ 0.1g, agar 20.0g, distilled water 1.000 mL.

Amylase medium: starch 20.0g,KCl 0.5g,NaNO ₃ 2.0g，K ₃ PO ₄ 1.0g，MgSO ₄ ·7H ₂ O0.5 g, naCl 5.0g, agar 18.0g, distilled water 1000mL. After the colony is cultured, iodine solution is dripped on the culture medium.

Siderophore Medium (CAS) Medium CaS 60.5mg, cetyltrimethylammonium bromide 72.9mg, piperazine-1, 4-bis (2-ethanesulfonic acid) 2.4g, 1mM FeC1 was added to 10mM hydrochloric acid and agarose (0.9% w/v) ₃ ·6H ₂ O, 1000mL of distilled water. After culturing the strain on PDA medium for 7 days, the CAS medium was allowed to stand on its surface for 2 hours and then observed.

Laccase medium: peeled potato 200.0g, glucose 20.0g, agar 20.0g, guaiacol 0.4g, distilled water 1000mL.

And (3) detecting ammonia production: the strain was inoculated into a test tube containing 10mL of a 4% peptone broth, cultured at 28.+ -. 0.1 ℃ for 6-7 d, and after culturing, 1mL of Nesler reagent was added to observe the color change.

And (3) auxin detection: the strain was cultured in 10.0ml PDB medium containing tryptophan (1 g/L) at 25.+ -. 0.1 ℃ for 7d. Filtration through Whatman filter paper, 1mL of filtrate with 2mL of Salkowski reagent (2% 0.5M FeCl) ₃ +35% perchloric acid) was mixed in a test tube. The mixture was incubated at room temperature for 20min and the color change was observed.

1/2MS medium: production number: HB8469-6 (Qingdao high technology Industrial park Haibo Biotechnology Co., ltd.).

The ARD specialization layer shows Fusarium (Fusarium proliferatum) MR5, which is also called "Fusarium laminar growth", and MR5 strain is preserved in China general microbiological culture Collection center with the preservation number: CGMCC No.22426, and the preservation time is 2021, 5 and 17. Strain MR5 is pathogenic only to apple rootstock, belonging to the apple specialization type.

Example 1: isolation and identification of strains

1. Isolation and purification of strains:

and (3) adopting an improved root sticking segment method to update and rebuild the 9 old apple orchards (15-25 years old) in the tobacco stand and the Yiyuan area for 4-5 years, and selecting root systems of healthy and vigorous apples to immediately screen endophytic antagonistic fungi. Washing root with tap water for 5min to remove adhered soil particles, soaking in 75% ethanol for 2min for surface sterilization, washing with sterile water for 3 times, soaking in 5% sodium hypochlorite solution for 5min, and washing with sterile water for 5 times. After appropriate drying, the surface sterilized roots were cut into 1cm sections, transferred to PDA medium containing penicillin-streptomycin (100X) to isolate endophytic fungi, four in each dish, and the dishes were kept at 28℃for one week. In addition, sterile water for washing the root system for the last time was spread on the same culture medium and placed in the same environment for cultivation, and whether the root system has been completely disinfected was confirmed. The results showed that no bacteria were produced on the coated plates and that after 3-4 days of incubation of healthy apple root system after surface sterilization, hyphae grew gradually from the cut ends of the root samples. And (5) selecting hyphae to a new PDA plate without antibiotics for continuous culture, and separating 15 endophytic fungi altogether.

2. Screening of strains:

the primary screening of the strain was performed by plate-confrontation with activated ARD specialization layer Fusarium MR5 on PDA plates. Taking fungus blocks at the edges of pathogenic bacteria by using a puncher with the diameter of 5mm, inoculating to the center of a PDA flat plate, inoculating strains separated from root systems on a line 2cm away from the pathogenic bacteria blocks, inoculating pathogenic bacteria only to the center of a culture medium as a blank control, repeating each treatment for 3 times, culturing at 28 ℃, selecting strains with a bacteriostasis ring after 7d, and carrying out re-screening. The antagonistic fungus with the highest antibacterial rate to MR5 is obtained by re-screening, and the width of a bacteriostasis zone is 6mm and is named as 6S-2 (figure 1, b-c). PI staining and scanning electron microscope observation are carried out by picking MR5 strain contacted with 6S-2, and under the illumination of 450-490nm, the mycelium shows red fluorescence (figure 1, p) to indicate that the mycelium structure is damaged, and the control mycelium shows green fluorescence (figure 1, m) to indicate that the mycelium is intact; scanning electron microscopy showed that when MR5 and 6S-2 were grown in opposition, the mycelia of 6S-2 strain began to grow along the mycelia of MR5, then the mycelia of both were gradually cross-wound (FIG. 1, g-h), the mycelia of 6S-2 produced spores on MR5 mycelia, and a large amount of 6S-2 spores were parasitic on MR5 mycelia (FIG. 1, j-k), forming perforations, rupturing MR5 mycelia, releasing the contents, and finally collapsing (FIG. 1, i-l).

To further determine the endophytic nature of the strain, spore suspensions of the 6S-2 strain (3X 10 ⁶ CFU/mL) for 7 days, the M9T337 seedlings were continuously rescreened with the root-adhering stage method for endophytes in the root systems of the M9T337 seedlings, the root systems without inoculating spore suspension were used as a control, and PAS staining was performed. The results showed that 6S-2 strain was detected only in the root system inoculated with the 6S-2 spore suspension, and PAS staining detected oval spores in the root system of M9T337 seedlings (FIG. 1, n-r). In conclusion, it is further demonstrated that 6S-2 is a good antagonist against endophytes.

3. Identification of strains:

(1) Morphological identification:

culturing the separated 6S-2 strain on PDA culture medium, CMD culture medium and SNA culture medium at 28deg.C for 5-7 days, observing mycelium morphology, and observing specific conidiophore structure and conidium of the strain by common microscope observation and scanning electron microscope observation. The 6S-2 strain is inoculated on culture mediums with different functions respectively, and related functions are determined by observing the specific changes of the functional culture mediums.

When growing on PDA solid culture medium, the initial mycelium is white wool-like, diffuses to the edge, and the back of colony is colorless; the mid-period gradually turns to light green, spore rings are formed on hyphae which develop earlier, the yellow color is gradually changed to green, aerial hyphae are more luxuriant, and the aerial hyphae are thickened and are cotton-flocculent; the later colony forms 3 concentric rings with alternate white and green, dense conidia are attached on the circular ring, the whole bacterial strain presents dark green, the more the color of the part close to the center is darker, the aerial hypha is absent, and the flat plate can be grown up in 5 days (figure 2, a). When growing on the culture medium, yellow blister-shaped protruding structures are formed on the initial flat plate, the later stage is mature gradually and turns green, and finally yellow-green alternate circular rings are formed, so that the generation and propagation of spores are facilitated (fig. 2, b). When the mycelia are thin and thin, the bacterial colony is transparent and radial, the whole culture medium can be covered about 3 days, conidia are in a cluster pad shape, dark green bullous rings are finally formed, the bullae are more polymerized and compact on the CMD culture medium, and in the SNA culture medium, the bullae structures are more dispersed and are in a semi-annular shape or a sheet shape (figures 2 and c-d). Optical microscope and scanning electron microscope observations indicate that: the 6S-2 hyphae are long and thin and have a partition, the main branches are dendritic and long, the conidiophore branches form secondary branches, primary branches which are generated below the top end of the main shaft usually form opposite branches, the included angle between the primary branches and the main shaft is approximately 90 degrees, the length of each primary branch is increased along with the increase of the distance from the top end, the length of each pair of branches is almost equal, secondary branches which are formed at the position close to the main shaft are longest, the secondary branches can generate third branches, and the third branches do not further branch. The bottle peduncles on the conidiophores are symmetrically distributed, the bottle peduncles are generated at the top ends of the primary, secondary and tertiary branches, the bottle peduncles are shorter, the bottle peduncles can be sharply contracted and thinned at the tip of the bottle peduncles, are bent and expanded in the middle, are in ampoule bottle shape, are in a set of 3 or 4 wheelsets at the top ends, are arranged in a vortex shape, and can generate conidiophores at the tip of the bottle peduncles after the conidiophores develop and mature. Conidia are green spheres or oval shapes, are clustered or dispersed, have fine thorns on the walls, and have chlamydospores (FIG. 2, e-m).

(2) Amplification of ITS and Tef 1-alpha fragments, sequence analysis and phylogenetic analysis:

DNA of the 6S-2 strain was extracted using a fungal genomic DNA extraction kit (Solarbio cat#D2300), and then fragments of ITS were amplified by primer pair ITS1 (5'-TCCGTAGGTGAACCTGCGG-3'), ITS2 (5'-GCTGCGTTCTTCATCGATGC-3') as follows: pre-denaturation at 94℃for 1min, annealing at 50℃for 1min, extension at 74℃for 90s,30 cycles, and extension at 74℃for 7min; and the tef1 fragment was amplified by primer pairs tef1fw (5'-GTGAGCGTGGTATCACCCATCG-3') and tef1rev (5'-GCCATCCTTGGAGACCAGC-3'), the amplification procedure being as follows: pre-denaturation at 94℃for 1min, annealing at 59℃for 1min, extension at 74℃for 50s,30 cycles, and extension at 74℃for 7min. Primer synthesis and amplified fragment sequencing were all done by Shanghai Biotechnology Inc. BLAST comparison is carried out on sequencing results through a National Center for Biotechnology Information (NCBI) nucleotide database, MEGA7 software is used, sequence analysis is carried out on isolated antagonistic bacteria by adopting an adjacent method N-J cluster analysis, a phylogenetic tree is constructed, a bootstrapping value (bootstrapping value) is repeatedly checked for 1000 times, and then the phylogenetic tree is beautified through iTOL. The ITS sequence length of the strain 6S-2 is 265bp (the sequence is shown as SEQ ID NO.1, genBank accession number MZ 841617), and the Tef 1-alpha sequence length is 262bp (the sequence is shown as SEQ ID NO.2, genBank accession number JQ 040445). Homology alignment was performed through NCBI Blast and phylogenetic tree was established using MEGA 7.0, and the results are shown in FIG. 3. As can be seen, the sequence of 6S-2 was clustered with the sequence of Trichoderma asperellum SZMC 24288 (GenBank ITS accession number MN516477.1, tef 1-alpha accession number N520032.1), and the results of morphological analysis were combined, indicating that the 6S-2 strain was Trichoderma asperellum (Trichoderma asperellum).

Based on the morphological and functional analysis and ITS of the 6S-2 strain and the results of the Tef 1-alpha sequence homology analysis, the isolated 6S-2 strain was identified as Trichoderma asperellum (Trichoderma asperellum). And performing biological preservation on the strain, wherein the preservation information is as follows:

strain name: trichoderma asperellum

Latin name: trichoderma asperellum

Strain number: 6S-2

Preservation mechanism: china general microbiological culture Collection center (China Committee for culture Collection of microorganisms)

The preservation organization is abbreviated as: CGMCC

Address: beijing city, chaoyang area, north Chenxi Lu No. 1 and 3

Preservation date: 2021, 10 and 09 days

Accession numbers of the preservation center: CGMCC No.23275.

Example 2: functional identification of Trichoderma asperellum (Trichoderma asperellum) 6S-2

1. Antagonistic enzyme production:

the 6S-2 strain is respectively inoculated on an NBRIP culture medium, a amylase culture medium, a CAS culture medium, a laccase culture medium, a cellulase culture medium and a protease culture medium by adopting a bacterial cake inoculation mode, and the result shows that the ratio of the phosphate ring diameter (D) of the 6S-2 strain on the NBRIP culture medium to the colony diameter (D) is 0.67, which indicates that the 6S-2 strain has the capability of degrading insoluble phosphate and releasing soluble phosphate (figures 2, n); 6S-2 has strong amylase and protease activities, and plays a role in degrading lipid and protein components of cell walls, thereby enhancing the growth competitiveness (FIG. 2, o, S); the 6S-2 strain also exhibited cellulose degrading ability (FIG. 2, r); after 7d plating of PDA, the colonies were incubated with CAS medium for 2h, the cover medium color changed from blue to orange, indicating that 6S-2 can produce hydroxyl siderophores (FIG. 2, p); and showed a red change on the guaiacol-containing PDA medium, indicating laccase activity (FIG. 2, q). In addition, by inoculating 6S-2 strain into PDB liquid medium containing 1g/L tryptophan, culture was performed at 25.+ -. 0.1 ℃ for 7d. Filtration through Whatman filter paper, mixing 1mL of filtrate with 2mL of Salkowski reagent in a test tube, incubating at room temperature for 20min, and turning pink in color, indicating IAA production (FIG. 2,u); the strain was inoculated into a liquid medium containing 4% peptone broth and cultured at 28.+ -. 0.1 ℃ for 6-7 d, and after 1mL of Nesler reagent was added, the color turned yellow-brown, indicating that the 6S-2 strain had ammonia gas production during growth (FIG. 2, t).

2. Broad-spectrum antibacterial effect:

the broad-spectrum bacteriostatic effect of 6S-2 was evaluated by a plate counter method. And (3) a puncher with the diameter of 5mm is used for punching fungus blocks at the edges of pathogenic bacteria, inoculating the fungus blocks to a position 1cm away from the center of a PDA plate, inoculating 6S-2 fungus blocks on an extension line 2cm away from the pathogenic bacteria blocks, inoculating pathogenic bacteria serving as a control only in the center of a PDA culture medium, measuring the diameter of fungus colonies by a vernier caliper after the control is full of the plates after 7 days, and expressing the size of the bacteriostasis rate by the diameter. The above operation was repeated 3 times.

Antibacterial ratio = [ (control colony diameter-treated colony diameter)/control colony diameter ] ×100%

The opposite test result shows that the 6S-2 strain has extremely strong antagonistic inhibition effect on the growth of 12 pathogenic bacteria hyphae, and the inhibition rate is 31.48% -75.37% (figure 4, m); and produce antagonistic transparent circles of different widths, spores of the parasitic 6S-2 strain are shown on hyphae of some harmful strains, and the edges of the strains are pale yellow (FIG. 4, a-l 2).

Example 3: antibacterial test of liquid fermentation extract

1.6S-2 preparation of fermentation extract:

activating 6S-2 stored in the inclined surface of test tube on PDA plate, inoculating again to new PDA culture, culturing for 7 days until spores are produced, washing with sterile water, and making into 10 ⁶ Spore suspension of CFU/ml was added to PDB medium at a ratio of 1% (volume fraction), 160r/min,28℃for 8 days, filtered through eight layers of gauze, and centrifuged at room temperature for 5min to collect supernatant, which was then mixed with ethyl acetate at a volume ratio of 1:1, carrying out extraction for 3 times, shaking vigorously for 4-5 times during the process to fully mix the two, collecting extract liquid, absorbing excessive water by using anhydrous calcium sulfate, concentrating to dry powder by using a rotary evaporator (36 ℃,80 hPa), collecting solid powder (100 mg of powder is obtained in the experiment) obtained after 6S-2 liquid fermentation extraction, re-dissolving the solid powder in methanol solution (10 ml) to prepare mother solution of 10mg/ml, centrifuging the mother solution by 10000r/min, removing impurities by using a filter membrane (Nylon 66 0.22 mu m), placing the mother solution in a refrigerator of 4 ℃, and storing the mother solution in the refrigerator of-80 ℃ if long-term use is needed.

2. Oxford cup test

Pouring a layer of 2% water agar medium into the bottom of the culture dish, placing two oxford cups 1cm away from the edge, and adding spore liquid (10) of MR5 at a ratio of 1% ⁵ CFU/mL) was mixed with PDA medium cooled to 50 ℃, poured onto the upper layer of water agar medium, after solidification, oxford cup was removed, 20 μl of 6S-2 fermentation extract was added to the left side, and equal volumes of methanol or sterile water were added to the right side, respectively, as controls. The results showed that the 6S-2 strain fermentation extract inhibited the growth and diffusion of MR5 mycelia, forming a transparent zone of inhibition, with sparse mycelia growing in the approximate 1/2 radian range (FIG. 5, b).

3. Cellophane culture antifungal test

The aseptic cellophane film is spread on a plate containing PDA culture medium, a control group is inoculated with a blank agar block in the center of the plate, a treatment group is inoculated with an activated two-generation 6S-2 strain, after 7d of culture, the cellophane and the strain are removed, and the activated two-generation MR5 strain is respectively inoculated on the PDA culture medium. When the control group MR5 hyphae covered the whole plate, the radius of the treatment group hyphae was measured, and the morphology of the treatment group hyphae was observed under a normal optical microscope and under an electron microscope.

The results showed that when the control group was inoculated with MR5 for 7 days, the hyphae grew vigorously and densely (FIG. 5, c), whereas the horizontal growth of MR5 hyphae was almost zero in the treated group (FIG. 5,d), most of them were in a sparse, sporadic state and the vertical growth was evident, indicating that certain metabolites of the 6S-2 strain inhibited the growth of MR5 hyphae. After being dyed by the lactophenol cotton blue, the mycelia of the MR5 strain of the control group are smooth and have no winding fracture, and spores are sickle-shaped, are rich and full and are uniformly and neatly arranged on the mycelia (fig. 5, k-l); in contrast, MR5 mycelia in the control group were uneven in thickness, part of mycelia was bent or even broken, a plurality of mycelia were tangled, spores were small, sparse and wrinkled, and the distribution was uneven (FIG. 5, m-n). The scanning electron microscope results show that the MR5 mycelia of the control group are smooth, obviously separated, uniform in thickness, free from breakage and deformation, and free from overflow of the content (FIG. 5, o). In the treatment group, MR5 mycelia shrink, spores shrink, tips become blunt, sickle-shaped spores deform, growing points of the tips swell, bifurcation is obvious (fig. 5, p), partial mycelia damaged mycelia show swelling, atrophy, bending, folding and uneven arrangement (fig. 5, q), the surfaces of the mycelia gradually become net-shaped, a plurality of mycelia deform, twist and tangle, holes appear on the surfaces of the mycelia, and the severely damaged mycelia have content flowing out (fig. 5,r-w).

3. Antifungal experiments with liquid fermentation extracts of different concentrations

Mixing fermentation extract mother liquor (10 mg/L) of the 6S-2 strain with PDA culture medium cooled to 50 ℃ according to different proportions, diluting to 50mg/L, 100mg/L, 150mg/L and 200mg/L, taking methanol with the same volume as a control, taking PDA culture medium without any solution as a blank control, setting the second generation activated MR5 bacterial block in the center of the culture medium after the culture medium is solidified, measuring the growth radius of the mycelia of different treatments after the MR5 mycelia in the blank control culture medium cover the whole culture dish, and calculating the inhibition rate. The results showed that the growth of MR5 mycelia was inhibited to various degrees after the addition of the 6S-2 fermentation extract (FIG. 5,e-h), and that the extent to which the growth of mycelia was inhibited was increased with the increase in the extract concentration.

4. Spore germination test

To further investigate the bacteriostatic mechanism of the 6S-2 fermentation extract, 30. Mu.L of the extract containing 10 ⁵ The CFU/mL MR5 spore suspension was mixed with an equal volume of 6S-2 fermentation extract as a treatment group, the control group was replaced with an equal volume of methanol, and the culture was performed on a concave slide at 28℃for 36 hours, and the spore germination was observed under a microscope. The results show that after the MR5 spore suspension mixed with methanol is incubated for 36 hours, obvious white hypha exists on the concave glass slide, the spore germination rate reaches 98%, the spore tail is thicker, and the germinated hypha is intertwined (figure 5,i); after 36h incubation with the 6S-2 fermentation extract, there was no apparent turbidity on the concave slide, the spore germination rate was only 15%, the spore tail was relatively slender, the growth was slow, and no apparent hyphae appeared (FIG. 5,j).

Example 4:6S-2 bacterial strain volatile matter bacteriostasis test

The antibacterial effect of the 6S-2 volatile substances is detected by adopting a double-plate buckling and double-plate culture method. Firstly, respectively pouring a PDA culture medium into the bottom of a culture dish and a culture dish cover, inoculating an MR5 bacterial cake into the bottom of the culture dish after the culture medium is solidified, and inoculating a 6S-2 bacterial cake into the dish cover to be used as a treatment group; the control group is replaced by a blank agar block, the blank agar block and the blank agar block are buckled by a liquid paraffin sealing plate to prevent the volatile gas from diffusing, and the blank agar block is sealed by a sealing film, incubated for 5 days at 28 ℃, observed and photographed. The results showed that the growth of MR5 mycelia co-cultured with 6S-2 strain was significantly inhibited (FIG. 6, a), and in order to further confirm the effect of volatile substances, the culture medium on the dish cover was divided into two parts with a sterile scalpel, the left side medium was removed and then covered with activated carbon, only 6S-2 strain was inoculated on the right side, MR5 was inoculated on the bottom of the dish, liquid paraffin was sealed and then wrapped with a sealing film, cultured at 28℃for 5d, observed and photographed, at which time the growth of MR5 mycelia was accelerated and there was no significant difference from the control (FIG. 6, b-c), and it was further clarified that the volatile substances produced by 6S-2 strain had an antibacterial effect, so that the inhibited MR5 mycelia were picked up and observed under an optical microscope and a scanning electron microscope. In the control group, MR5 mycelia were uniformly arranged, smooth and wound without significant twisting, spores were sickle-shaped, uniformly distributed, large and full (FIG. 6,e-f), while MR5 mycelia in the treatment group were uneven in thickness, had significant twisting, breaking and winding phenomena, and spores were small and shrunken (FIG. 6, g-h). Scanning electron microscope observation shows that the MR5 mycelia of the control group are smooth and full, are uniformly arranged, are unbroken, have round and full spore tips (shown in figure 6,i-j), and the mycelia of the treatment group are uneven in thickness, are twisted and wound together, and are arranged in disorder; exhibiting states of atrophy, distortion and deformation; the tip of the mycelium is damaged, and partial epidermis is fallen off; the tips of spores shrink, the bifurcation is obvious, and the spores break off; the hyphae severely damaged were broken and the contents were exuded (FIG. 6,k-n).

Example 5: potential growth promoting effect of 6S-2 strain fermentation extract and volatile matters on arabidopsis thaliana

1. Liquid fermentation extract growth-promoting experiment

In order to rapidly detect whether 6S-2 fermentation extracts have the potential to promote growth. Arabidopsis thaliana was selected as a model plant, seeds thereof were completely sterilized (first placed in a refrigerator at 4℃for 3d, then rinsed with sterile water for 2 times, the floating seeds were removed, placed in 75% alcohol for 3min, rinsed with sterile water for 3 times, then placed in 40% sodium hypochlorite for 1min, finally rinsed with sterile water for 5 times), and then seeds were sown on 1/2MS medium containing 6S-2 strain fermentation extracts of different concentrations (10, 50, 100, 150 and 200 mg/L), with the addition of an equivalent amount of methanol as a control, and with no addition of any substance as a blank control. Five seeds are planted in each culture medium in a straight line, the flat plate is dried on a sterile workbench and placed in a 23 ℃ illumination incubator for 16h/8h culture for 9d, the root length and the lateral root length of the arabidopsis seedlings are measured by a ruler, the fresh weight of the arabidopsis seedlings is weighed by an electronic balance, and the lateral root number is counted. The results showed that the length of main roots of Arabidopsis thaliana was inversely related to the concentration of methanol after adding methanol (10-200 mg/L) to 1/2MS medium compared to the blank, but the number and length of lateral roots were not significantly changed (FIG. 7, g1-k 1). The length of Arabidopsis roots was changed to a different extent after the addition of the 6S-2 fermentation extract compared to the addition of methanol, and Arabidopsis root length, the number of lateral roots and the length of lateral roots were highest at 50mg/L (FIG. 7, g2-k2, L-q). As the concentration of the 6S-2 fermentation extract increased from 50mg/L to 200mg/L, the root length gradually decreased, but the lateral root number and the lateral root length increased to different extents. In the range of 10-100mg/L, fresh weight of Arabidopsis thaliana can be increased by adding the 6S-2 fermentation extract, but at 200mg/L, root growth is obviously inhibited, the whole plant is yellowish-white, and the leaves become thicker and hard (FIG. 7, k 2).

2. Growth-promoting experiment of volatile substance

The volatile substances of the 6S-2 strain are tested by adopting a bipartite plate to determine whether the volatile substances have the potential of promoting the growth of the arabidopsis. The 1/2MS medium was poured on the left side of the bipartite plate and the PDA medium on the right side. Placing 3 sterilized Arabidopsis seeds on 1/2MS culture medium, and uniformly smearing 6S-2 spore solution (10) ⁶ CFU/mL) and with the right side only coated with sterile water as a blank, each treatment was repeated 5 times. Sealing the flat plate with liquid paraffin, wrapping with sealing film, culturing in an illumination incubator for 9d, measuring root length and lateral root length with a meter ruler, weighing fresh weight with an electronic balance, and counting lateral root number. The results showed that when Arabidopsis was grown in 1/2MS medium without any addition of substances for 9d, the root system grew vertically downward and lateral roots were rarely produced (FIG. 7,a). When cultured on a biplate with sterile water applied to the right, the root system grows vertically downward to approximately the same length as the culture medium. Lateral roots are produced in the upper half of the main root, but the whole root system is shorter (fig. 7, b). When Arabidopsis was co-cultured with 6S-2 spore solution, the root system was shorter, the main root was only about 2/3 of the length of the right side control with sterile water applied only, but the lateral root length was three times that of the control (FIGS. 7, c, f), and there was no significant difference in fresh quality between the control and 6S-2 strain treatments (FIG. 7,e). The results show that: the volatile substances of the 6S-2 strain can obviously promote the generation and elongation of the lateral roots of the arabidopsis thaliana.

Example 6: potting test

1. And (3) test design:

potted plant test is carried out in 2021, 3-9 months in apple engineering laboratory center and apple continuous cropping and microorganism laboratory in southern agricultural university, shandong, chinaThe test plants were M9T337 tissue culture seedlings and cognac sweet tea (Malus hupeheusis red.) seedlings. The test soil was obtained from an apple garden of Man Zhuang years old in the Taian Dain area of Shandong province, china, and the soil texture was brown loam. The soil contains 3.22 mg.kg ^-1 Ammonium nitrogen, 6.18 mg.kg ^-1 Nitrate nitrogen, 45.76 mg.kg ^-1 Quick-acting phosphorus, 57.65 mg.kg ^-1 Quick-acting potassium and 8.10 g.kg ^-1 Sieving with 10 mesh sieve, and stirring.

Activating 6S-2 stored in the inclined surface of test tube on PDA plate, inoculating again to new PDA culture, culturing for 7 days until spores are produced, washing with sterile water, and making into 10 ⁶ Spore suspension of CFU/ml.

First, a greenhouse test was performed at 4 months 2021: filling 50 continuous cropping apple orchards with 7.0cm of soil which is sieved and stirred uniformly _* 5.0cm _* Healthy M9T337 (apple stock) tissue culture seedlings of 4-5 leaves are planted in a black plastic basin of 8.5 cm. After 3 days of the seedling stage, 25 pots were used with 1%5×10 ⁸ CFU/mL 6S-2 spore suspension was irrigated and another 25 pots were irrigated with equal amounts of sterile water as a control. After 15 days of culture under the same conditions, functional leaves and roots were taken for NBT and DAB staining and observed under an optical microscope.

After obtaining the greenhouse test results, an outdoor potting test was performed for 2021, 5 years: layering Malus hupehensis seeds at 4 ℃ for about 30 days, and sowing the seeds in a culture pot filled with a culture medium for culturing seedlings after the seeds are exposed to white. Selecting plants with consistent growth vigor and without plant diseases and insect pests when seedlings grow to 6 true leaves, and transplanting to 150cm in 1 day of 5 months _* 90cm _* In a 115cm white plastic pot, at 7 months and 25 days 2021, the index related to photosynthesis was measured, the differences in plant biomass and root protecting enzyme activity between the different treatments were measured using destructive sampling, and the change in the number of soil microorganisms was measured.

2.6 reduction of oxidative damage to M9T337 seedlings by S-2 spore liquid

Nitro Blue Tetrazolium (NBT) staining: the NBT histochemical staining method can rapidly and intuitively observe the accumulation condition of active oxygen in the tissue and qualitatively observe the oxidative damage degree of the plant tissue. Nitroblue tetrazolium (200 mg) powder was added to PBS (100 ml) and incubated at 37℃for 30min and briefly vortexing to dissolve, then 10 functional leaves and roots of differently treated M9T337 seedlings were soaked in NBT (0.2%) staining solution, water bath at 37℃for 2h. Finally, the leaves are decolorized by 80% alcohol aqueous solution at 80 ℃, washed by water, photographed and observed. The results showed that after application of 6S-2 spore liquid, the growth of M9T337 seedlings was improved (FIG. 8, b), the coverage area of blue dye at the edge of the leaf was reduced (FIG. 8, b 1), and the degree of staining was reduced (FIG. 8, b 2). The same results were obtained for NBT staining in roots (FIG. 8, b 3), which initially showed that the administration of 6S-2 spore liquid reduced O in M9T337 tissue ₂ ^- Reducing its oxidative damage.

DAB (3, 3' -diaminobenzidine) staining: the DAB chemical staining method can conveniently and intuitively observe the accumulation condition of hydrogen peroxide in tissues and rapidly detect the active site of peroxidase in tissue cells. The functional leaves and roots of the differently treated M9T337 seedlings were stained using DAB chromogenic kit (20X, DA1010, solarbio): taking 5ml of solution A and 5ml of solution B respectively, diluting to 100ml with 90ml of PBS, preserving in dark, immersing leaves and roots therein, and soaking in water at 37deg.C for 1 hr. Finally, the leaves were decolorized with 80% alcohol in water at 80℃and rinsed with water and observed with photographs. The results showed that in the control treatment, a large amount of peroxide was concentrated on the leaf margin and root system after DAB staining (FIG. 8, a4-a 6), and that the degree of oxidative damage of the leaf and root system was reduced after application of 6S-2 spore liquid (FIG. 8, b4-b 6).

3.6S-2 spore liquid for promoting growth of Malus hupehensis seedling

Determination of the biological amount of Malus hupehensis seedlings: the plant height and stem thickness of Pingyi sweet tea seedlings are measured by a metric ruler and a vernier caliper respectively, and the dry fresh weight is measured by an electronic balance. A Scan Maker i800 plus scanner was used to obtain images of root system scans and a plant image analyzer was used to obtain parameters related to the root system. The results showed that the application of 6S-2 spore suspension promoted the growth of Malus hupehensis seedlings (FIGS. 8,e-f, g-n), which were significantly different from the control.

Photosynthetic property of Malus hupehensis seedlings: the photosynthetic capacity of plants is closely related to plant growth, and the photosynthesis directly affects the yield and quality of crops. 2021Intercellular CO of Pingyi sweet tea seedlings was measured at 10 am on 25 months using CIRAS-3 portable photosynthetic apparatus (PP System, UK) ₂ Concentration (C) _i ) Net photosynthetic rate (P) _n ) Air hole conductivity (G) _s ) And transpiration rate (T) _r ). The results showed that the application of 6S-2 spore liquid can increase the net photosynthesis rate of Malus hupehensis seedlings (FIG. 8, o-r), thereby promoting the increase of plant growth.

Determination of Malus hupehensis seedling root protecting enzyme Activity and MDA content: SOD, POD and CAT activity are the core of the plant cross-protection mechanism, MDA is one of the most important products of membrane lipid peroxidation, and can indirectly measure the degree of membrane lipid peroxidation and plant stress resistance. Fresh white roots of Malus hupehensis seedlings were rinsed and then rapidly frozen in liquid nitrogen to determine the activity of root protecting enzymes, and superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) activity and MDA content were determined by referring to Sun et al. The results indicate that administration of the 6S-2 spore suspension promoted root protecting enzyme activity (FIG. 8,s-u) and reduced MDA accumulation (FIG. 8,v).

4.6 influence of S-2 spore liquid on microbial community structure in continuous cropping soil

10g of fresh soil (preserved at 4 ℃ for no more than 24 hours) is taken, the quantity of bacteria, fungi and actinomycetes which can be cultivated in the soil is counted by adopting a dilution plate method, and the ratio of the bacteria to the fungi is calculated. Bacteria were grown for 24h at 37℃on LB medium, fungi were grown for 48h at 28℃on PDA medium, and actinomycetes were grown for 5d on modified Gao's No. 1 medium. Subsequently, 0.5g of sieved fresh soil was taken and used

DNA was extracted from the soil DNA extraction kit, and the gene copy numbers of four Fusarium species in the soil were determined using CFX96TMthermal Cycler (Bio-Rad). The results show that after application of 6S-2 spore liquid, the number of culturable fungi (in particular the number of copies of fusarium) in the soil is significantly reduced (fig. 9, b, e-h), but the number of culturable bacteria and the number of actinomycetes are increased (fig. 9, a, c), resulting in a significant increase in the ratio of bacteria to fungi (fig. 9,d)。

the foregoing description is only of the preferred embodiments of the present application and is not intended to limit the same, but rather, various modifications and variations may be made by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application should be included in the protection scope of the present application.

SEQUENCE LISTING

<110> Shandong agricultural university

<120> Trichoderma asperellum 6S-2 and application thereof in alleviating apple continuous cropping obstacle

<130> 2021

<160> 6

<170> PatentIn version 3.5

<210> 1

<211> 265

<212> DNA

<213> 6S-2 Strain

<400> 1

cttccgtagg tgaacctgcg gagggatcat taccgagttt acaactccca aacccaatgt 60

gaacgttacc aaactgttgc ctcggcgggg tcacgccccg ggtgcgtcgc agccccggaa 120

ccaggcgccc gccggaggaa ccaaccaaac tctttctgta gtcccctcgc ggacgtattt 180

ctttacagct ctgagcaaaa attcaaaatg aatcaaaact ttcaacaacg gatctcttgg 240

ttctggcatc gatgaagaac gcagc 265

<210> 2

<211> 262

<212> DNA

<213> 6S-2 Strain

<400> 2

tgagcgtggt atcaccatcg acattgccct ctggaagttc gagactccca agtactatgt 60

caccgtcatt ggtatgtttt ggactcttct ctctagctat cgacattcca agtccgccat 120

tctaacatgc tcttcccaca gacgctcccg gtcaccgtga tttcatcaag aacatgatca 180

ctggtacctc ccaggctgac tgcgctatcc tgattatcgc tgccggtact ggtgagttcg 240

aggctggtct ccaaggatgg ca 262

<210> 3

<211> 19

<212> DNA

<213> artificial sequence

<400> 3

tccgtaggtg aacctgcgg 19

<210> 4

<211> 20

<212> DNA

<213> artificial sequence

<400> 4

gctgcgttct tcatcgatgc 20

<210> 5

<211> 22

<212> DNA

<213> artificial sequence

<400> 5

gtgagcgtgg tatcacccat cg 22

<210> 6

<211> 19

<212> DNA

<213> artificial sequence

<400> 6

gccatccttg gagaccagc 19

Claims

1. Trichoderma asperellum strainTrichoderma asperellum) 6S-2, the biological preservation number is: CGMCC No.23275.

2. The trichoderma asperellum of claim 1Trichoderma asperellum) Spore suspension, spore powder and/or fermentation extract of 6S-2;

the trichoderma asperellum is preparedTrichoderma asperellum) The fermentation extract of 6S-2 is prepared by the following method:

the trichoderma asperellum is treatedTrichoderma asperellum) 6S-2 is placed on a PDA culture medium for culturing for 7 days at 28 ℃, and spores are washed by sterile water to prepare spore suspension; adding spore suspension into PDB culture medium at r/min and 28deg.CCulturing for 8 days, filtering, centrifuging for 5min at 12000 r/min, and collecting supernatant; the collected supernatant was mixed with ethyl acetate in a volume ratio of 1:1, collecting the extract, concentrating to dry powder, dissolving in methanol solution again, centrifuging, filtering to remove impurities, and making into fermentation extract.

3. Trichoderma asperellum according to claim 2Trichoderma asperellum) The spore suspension, spore powder and/or fermentation extract of 6S-2 is characterized in that the trichoderma asperellum is Trichoderma asperellum) The spore suspension of 6S-2 is prepared by the following method:

the trichoderma asperellum is treatedTrichoderma asperellum) 6S-2 is placed on a PDA culture medium for culturing for 7 days at 28 ℃, and spores are washed by sterile water to prepare spore suspension;

the trichoderma asperellum is preparedTrichoderma asperellum) The spore powder of 6S-2 is prepared by the following method:

the trichoderma asperellum is treatedTrichoderma asperellum) Inoculating the spore suspension of 6S-2 into a fermentation culture medium, and culturing at 28 ℃ for 10-14 days; and (5) air-drying, crushing and sieving after fermentation to obtain spore powder.

4. The trichoderma asperellum of claim 1Trichoderma asperellum) 6S-2 or Trichoderma asperellum of claim 2 or 3Trichoderma asperellum) Use of a spore suspension and/or spore powder of 6S-2 in at least one of the following (1) - (4):

(1) Inhibiting growth of plant pathogenic bacteria and germination of spores;

(2) Preparing a product for inhibiting plant pathogenic bacteria;

(3) Preventing and treating diseases caused by plant pathogenic bacteria;

(4) Preparing a product for controlling diseases caused by plant pathogenic bacteria;

the plant pathogenic bacteria are ARD specialization Fusarium layeringFusarium proliferatum) MR5 Fusarium oxysporumFusarium oxysporum) Fusarium solani (L.) DCBacteriaFusarium solani) Fusarium moniliforme (Fusarium moniliforme)Fusarium moniliforme) Phytophthora malisPhytophthora cactorum) Humicola aphanidermatum (L.) of melon and fruitPythium aphanidermatum) Pathogenic bacteria of stem blightPhoma asparagi) Alternaria alternata (L.) A. Alternaria alternata) Verrucosa plaque bacteriaMyrothecium verrucaria) Penicillium BrazilPenicillium brasilianum) Rhizoctonia solani (wall.) kuntzeRhizoctonia solani) And/or Aspergillus flavusAspergillus flavus）。

5. The trichoderma asperellum of claim 1Trichoderma asperellum) 6S-2 or Trichoderma asperellum of claim 2Trichoderma asperellum) Use of a spore suspension, spore powder and/or fermentation extract of 6S-2 in (1) or (2) as follows:

(1) The apple continuous cropping obstacle is relieved;

6. A method for alleviating apple continuous cropping obstacle, comprising the steps of:

a method for preparing trichoderma asperellum according to claim 2 or 3Trichoderma asperellum) The spore suspension, spore powder and/or fermentation extract of 6S-2 is applied to apple continuous cropping soil.