CN117821258A - Endophytic trichoderma viride WJ561 and application thereof in pear rot disease prevention and control - Google Patents
Endophytic trichoderma viride WJ561 and application thereof in pear rot disease prevention and control Download PDFInfo
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- Y—GENERAL 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
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- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The invention belongs to the technical field of microorganism application, and discloses endophytic trichoderma viride WJ561 and application thereof in preventing and treating pear rot, wherein the endophytic trichoderma viride WJ561 is classified and named as trichoderma viride (Trichoderma virens), and the biological preservation number is CGMCC NO.40848. The endophytic trichoderma viride WJ561 obtained by the invention has antagonism on pear rot germs, fusarium oxysporum, fusarium putrescens and the like; the culture filtrate has good antibacterial activity, and can destroy germ hypha cells and inhibit the oxidization resistance of germs by oxidative damage, and the inhibition rate of the growth of the germ hypha of the pear rot germ can reach 100% within 2 days. In the in vitro test of leaves and branches of pear trees, the culture filtrate can effectively inhibit the progress of the disease spots and prevent and treat the rot of pear trees.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to an endophytic trichoderma viride WJ561 and application thereof in preventing and treating pear rot.
Background
Pears are widely planted and consumed worldwide, next to citrus and apples. Pear rot is a destructive mycosis caused by the pear variety of the apple black rot fungi, namely pear black rot fungi (Valsa mali var. Pyri), can lead to pear yield reduction and even death of tree bodies, and has negative influence on growers, consumers and the whole industry. Physical and chemical intervention has been used for the treatment of pear rot in the past decades. Physical methods include pruning and removing diseased, infected branches, but are not effective in eradicating the disease, and in some cases, may further exacerbate the damage to the tree. The chemical bactericide is easy to remain, and has potential toxicity, ecological hazard, pathogen resistance and other problems. Therefore, there is an urgent need for an ideal alternative method for controlling pear rot. Biological control has become a powerful tool in the management of plant diseases in sustainable agricultural practice.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides an endophytic trichoderma viride WJ561 and application thereof in preventing and treating pear rot.
The technical scheme adopted for solving the technical problems is as follows:
trichoderma viride strain WJ561, which is named: WJ561, class name: trichoderma viride (Trichoderma virens), accession number: CGMCC No.40848, date of preservation: 2023, 10, 17, deposit unit: china general microbiological culture Collection center, north Chen Xi Lu No. 1, 3, the Korean region of Beijing, and the China general microbiological culture Collection center.
Further, the trichoderma viride WJ561 strain is separated from the rhizome of ginger in the county of Hebei Ji, and the separation method is as follows: taking ginger rhizome, washing with tap water, sterilizing with 75% alcohol, washing with sterile water for 3 times, placing the rhizome in an ultra-clean bench, properly drying, cutting the rhizome into 0.5cm square pieces with a sterilizing knife, placing on Potato Dextrose Agar (PDA) medium, and culturing at 28deg.C for 6 days; fungus isolates grown from the rootstock were subcultured and purified on the same medium and identified as Trichoderma viride by ITS and RNA polymerase II subunit sequence sequencing and result alignment.
Further, the trichoderma viride WJ561 has the following characteristics:
after 1 day of incubation at 28℃all strains appeared white fluffy on PDA medium, with white back (FIGS. 1a and b); on day 3, light green to dark green spore bands appeared in the center of each medium and gradually extended to the periphery, and the mycelia were densely cotton-like (fig. 1c and d); by day 5, the green spores were spread in concentric circles throughout the dish, exhibiting dark green, greyish green or yellowish green streaks (FIGS. 1e and f), which were initially identified as Trichoderma. WJ561 was identified as Trichoderma viride by ITS, TEF 1-alpha and RPB2 sequence sequencing and result alignment.
The obtained culture filtrate was prepared as described above for Trichoderma viride WJ 561.
The preparation method of the culture filtrate comprises the following specific steps:
placing fresh Trichoderma viride cake of 4-6 days old into potato dextrose water (PDB) culture medium, culturing at 25-28deg.C and 140-160rpm for 2, 4, 6, 10, and 14 days, filtering with 12 layers of sterilizing mirror paper, and filtering with 0.1 μm microporous membrane filter for 2 times to obtain culture filtrate.
Further, the specific steps are as follows:
taking 8 fresh Trichoderma viride cakes which are cultured for 5 days by a 200 microliter sterile gun head, putting the Trichoderma viride cakes into a potato dextrose water (PDB) culture medium, culturing for 4 days at 28 ℃ and 150rpm, filtering the culture filtrate by 12 layers of sterilizing mirror wiping paper, and filtering the culture filtrate by a 0.1 mu m polyvinylidene fluoride microporous filter membrane filter for 2 times.
The application of the culture filtrate in inhibiting the growth of pear rot germs and/or preparing a preparation for inhibiting pear rot germs and/or preventing diseases caused by pear rot germs and/or preparing a preparation for preventing diseases caused by pear rot germs.
Further, the pear rot germ is pear black rot fungus;
alternatively, the formulation is a mixture of trichoderma viride WJ561 culture filtrate and sterile PDB medium;
alternatively, the formulation is a formulation comprising a culture filtrate of trichoderma viride WJ561 at a mass concentration of 0%, 5%, 10% and 15%.
Further, the preparation is applied to the cell membrane damage and/or the oxidative damage and/or the inhibition of the generation of antioxidant enzyme and/or the reduction of pathogenicity of pathogenic bacteria to plant leaves and/or the reduction of pathogenicity of pathogenic bacteria to plant branches.
Further, the pear rot germ is pear black rot skin fungus.
Alternatively, the plant is red zodiac pear.
The invention has the advantages and positive effects that:
1. the invention separates an endophytic trichoderma viride (Trichoderma virens) WJ561 from ginger rootstock and has excellent antibacterial property. The strain has obvious antagonism to pear rot disease-causing germ pear black rot skin fungus, the inhibition rate reaches 66.02 percent, and when WJ561 is inoculated for 1 day preferentially, the inhibition rate reaches 92.41 percent; has better antagonism effect on fusarium oxysporum, fusarium putrescens and the like.
2. The culture filtrate of WJ561 of the invention can also inhibit the growth of pathogenic bacteria, the inhibition rate is up to 100% in 2 days, and the culture filtrate with 15% concentration can inhibit the oxidation resistance of pathogenic bacteria, resulting in active oxygen accumulation and serious oxidation damage of bacterial hyphae. In vitro experiments, WJ561 culture filtrate also inhibited the extent of pear rot disease development. In addition, the WJ561 culture filtrate has good environmental stability, and lays a foundation for post production.
Drawings
FIG. 1 is a colony morphology of Trichoderma viride WJ561 on PDA plate according to the present invention; wherein, (a) the front of the plate is incubated for 1 day; (b) culturing the back of the plate for 1 day; (c) incubating the front of the plate for 3 days; (d) culturing the back of the plate for 3 days; (e) incubating the front of the plate for 5 days; (f) culturing the back of the plate for 5 days;
FIG. 2 is a phylogenetic tree of Trichoderma viride WJ561 of the present invention; wherein, (a) a phylogenetic tree of WJ561 constructed based on ITS sequences; (b) Phylogenetic tree of WJ561 constructed based on TEF1- α sequences; (c) phylogenetic tree of WJ561 constructed based on RPB2 sequences;
FIG. 3 is a graph showing a plate-face-up test of Trichoderma viride WJ561 and pear rot pathogen in the present invention; wherein (a) the plates are opposed; (b) A bar chart of the inhibition effect of WJ561 on the growth rate of pear rot germs;
FIG. 4 is a graph showing the plate-face test of Trichoderma viride WJ561 against other 2 pathogenic fungi in accordance with the present invention; wherein, (a) fusarium oxysporum and fusarium putrescens; (b) A bar graph of the inhibition effect of WJ561 on different pathogenic fungi growth rates;
FIG. 5 is a graph showing the growth inhibition of pear rot germs by Trichoderma viride WJ561 culture filtrate at different culture times in the invention; wherein (a) the pathogenic bacteria are grown in 3 days mycelium form on PDA medium containing WJ561 culture filtrate for different culture times: 2 days, 4 days, 6 days, 10 days, 14 days; (b) A bar chart of the inhibition effect of WJ561 culture filtrate with different culture time on the growth rate of pear rot germs;
FIG. 6 is a graph showing the growth test and morphological observation of the culture filtrate of Trichoderma viride WJ561 with different concentrations for inhibiting pear rot germs in the present invention; wherein (a) the pathogenic bacteria grow for 3 days in mycelium morphology on PDB medium containing different concentrations of WJ561 culture filtrate: 0%, 5%, 10%, 15%; (b) A bar chart of the inhibition effect of WJ561 culture filtrate with different concentrations on the growth rate of pear rot germs; (c) A hypha morphological diagram of pear tree rot germs under a scanning electron microscope;
FIG. 7 is a diagram showing the integrity of mycelium membranes of pear rot germs in the present invention; wherein, (a) PI staining; (b) MDA assay; (c) nucleic acid determination; (d) soluble protein assay;
FIG. 8 is a graph showing the measurement of mycelial enzyme activity of Pyricularia pomace and the expression level of the related genes after treatment with different concentrations of Trichoderma viride WJ561 culture filtrate in the present invention; wherein, (a) observing hypha active oxygen accumulation by a laser scanning confocal fluorescence microscope; (b) catalase activity; (c) superoxide dismutase activity; (d) related gene expression levels of catalase; (e) related gene expression levels of the enzyme super-hydrogenase.
FIG. 9 is a graph showing the effect of Trichoderma viride WJ561 culture filtrate on preventing and treating rot of in vitro leaf and branch pear trees; wherein, (a) WJ561 culture filtrate has prevention and treatment effects on in vitro leaf pear rot germs; (b) The WJ561 culture filtrate has the control effect on the in-vitro branch pear rot germs; (c) length of isolated branch lesions after treatment of WJ561 culture filtrate; (d) The inhibition rate of the WJ561 culture filtrate on pear rot of the isolated branches.
FIG. 10 is a graph showing the stability of the culture filtrate of Trichoderma viride WJ561 in the present invention; wherein, (a) the inhibition rate of WJ561 culture filtrate to pear rot germs under different pH environments; (b) The inhibition rate of WJ561 culture filtrate to pear rot germs at different temperatures; (c) The inhibition rate of WJ561 culture filtrate to pear rot germs under different ultraviolet irradiation time periods; (d) The inhibition rate of WJ561 culture filtrate to pear rot germs under different storage time.
Detailed Description
The invention will now be further illustrated by reference to the following examples, which are intended to be illustrative, not limiting, and are not intended to limit the scope of the invention.
The various experimental operations involved in the specific embodiments are conventional in the art, and are not specifically noted herein, and may be implemented by those skilled in the art with reference to various general specifications, technical literature or related specifications, manuals, etc. before the filing date of the present invention. The reagents, consumables and the like used in the examples described below are commercially available unless otherwise specified.
Trichoderma viride strain WJ561, which is named: WJ561, class name: trichoderma viride (Trichoderma virens), accession number: CGMCC No.40848, date of preservation: 2023, 10, 17, deposit unit: china general microbiological culture Collection center, north Chen Xi Lu No. 1, 3, the Korean region of Beijing, and the China general microbiological culture Collection center.
Preferably, the trichoderma viride WJ561 strain is separated from the rhizome of ginger in the county of Hebei Ji, and the separation method is as follows: taking ginger rhizome, washing with tap water, sterilizing with 75% alcohol, washing with sterile water for 3 times, placing the rhizome in an ultra-clean bench, properly drying, cutting the rhizome into 0.5cm square pieces with a sterilizing knife, placing on Potato Dextrose Agar (PDA) medium, and culturing at 28deg.C for 6 days; fungus isolates grown from the rootstock were subcultured and purified on the same medium and identified as Trichoderma viride by ITS and RNA polymerase II subunit sequence sequencing and result alignment.
Preferably, the trichoderma viride WJ561 has the following characteristics:
after 1d of culture at 28 ℃, all strains showed white fluffy shape on PDA medium with white back; when the culture is carried out until the 3 rd day, the center of each culture medium is provided with a light green spore belt to a dark green spore belt, and the spore belts gradually extend to the periphery, and the mycelium is compact cotton-like; by day 5, the green spores were spread in concentric circles throughout the dish, presenting dark green, grayish green or yellowish green carousel; it was initially identified as Trichoderma. WJ561 was identified as Trichoderma viride by ITS, TEF 1-alpha and RPB2 sequence sequencing and result alignment.
The obtained culture filtrate was prepared as described above for Trichoderma viride WJ 561.
The preparation method of the culture filtrate comprises the following specific steps:
placing fresh Trichoderma viride cake of 4-6 days old into potato dextrose water (PDB) culture medium, culturing at 25-28deg.C and 140-160rpm for 2, 4, 6, 10, and 14 days, filtering with 12 layers of sterilizing mirror paper, and filtering with 0.1 μm microporous membrane filter for 2 times to obtain culture filtrate.
Preferably, the specific steps are as follows:
taking 8 fresh Trichoderma viride cakes which are cultured for 5 days by a 200 microliter sterile gun head, putting the Trichoderma viride cakes into a potato dextrose water (PDB) culture medium, culturing for 4 days at 28 ℃ and 150rpm, filtering the culture filtrate by 12 layers of sterilizing mirror wiping paper, and filtering the culture filtrate by a 0.1 mu m polyvinylidene fluoride microporous filter membrane filter for 2 times.
The application of the culture filtrate in inhibiting the growth of pear rot germs and/or preparing a preparation for inhibiting pear rot germs and/or preventing diseases caused by pear rot germs and/or preparing a preparation for preventing diseases caused by pear rot germs.
Preferably, the pear rot germ is pear black rot fungus;
alternatively, the formulation is a mixture of trichoderma viride WJ561 culture filtrate and sterile PDB medium;
alternatively, the formulation is a formulation comprising a culture filtrate of trichoderma viride WJ561 at a mass concentration of 0%, 5%, 10% and 15%.
Preferably, the preparation is applied to the prevention and treatment of the damage of the cell membrane of the pear rot germ and/or the oxidative damage of the cell of the pear rot germ and/or the inhibition of the generation of antioxidant enzyme of the pear rot germ and/or the reduction of the pathogenicity of the germ to plant leaves and/or the reduction of the pathogenicity of the germ to plant branches.
Preferably, the pear rot germ is pear black rot skin fungus.
Alternatively, the plant is red zodiac pear.
Example 1: isolation and identification of strains
1. Isolation and purification of strains
Collecting the rhizomes of 3 ginger plants in the Ji county of Hebei province, washing with tap water, sterilizing the surface with 75% alcohol, and washing with sterile water for 3 times. The rootstock is placed in an ultra clean bench, properly dried, cut into small square blocks of 0.5cm by a sterilizing knife, placed on Potato Dextrose Agar (PDA) culture medium and cultured for 6 days at 28 ℃. Mycelium grown from the rhizome blocks is picked up, cultured and purified for 2 times on PDA culture medium, and the pure fungus is preserved at 4deg.C.
2. Morphological identification of strains
When growing on PDA culture medium, the initial colony is white, expands to four sides, the back of colony is light, the center of the back colony starts to form dark green spores at high speed, hyphae are compact cotton floccules, the surface of the culture medium is green concentric circular patterns with different degrees, the back is dark yellow green, the flat plate can be grown in 5 days, and the trichoderma is primarily identified (figure 1).
Amplification, sequence analysis and phylogenetic analysis of ITS, TEF 1-alpha and RPB2 fragments
DNA of WJ561 strain was extracted using a fungal genome extraction kit (Probiotics (Shanghai) Co., ltd.), followed by primer pairs ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4
(5'-TCCTCCGCTTATTGATATGC-3') amplifying fragments of IFS, the amplification reaction procedure is as follows: pre-denaturation at 95 ℃ for 5min; denaturation at 94℃for 30s, annealing at 57℃for 30s, elongation at 72℃for 90s, and cycling for 30 times; finally, the extension is carried out for 10min at 72 ℃. By primer pairs EF1-728F (5'-CATCGAGAAGTTCGAGAAGG-3') and TEF1LLErev
(5'-AACTTGCAGGCAATGTGG-3') amplification of fragments of TEF 1-alpha the amplification reaction procedure was as follows: pre-denaturation at 95 ℃ for 5min; denaturation at 95℃for 1min, annealing at 55℃for 90s, extension at 72℃for 90s, and cycling for 30 times; finally, the extension is carried out for 10min at 72 ℃. By primer pair fRPB2-5fl (5 '-GAYGAYMGWGATCAYTTYGG-3') and Frpb2-7cR
(5 '-GAYGAYMGWGATCAYTTYGG-3') amplifying a fragment of RPB2, the amplification reaction procedure being as follows: pre-denaturation at 95℃for 2min; denaturation at 95℃for 1min, annealing at 52℃for 1min (rate 0.2 ℃/s), extension at 72℃for 1min, and cycling for 36 times; finally, the extension is carried out at 72 ℃ for 7min. Primer synthesis and amplified fragment sequencing were all done by biological engineering (Shanghai) Inc. The sequencing results are compared through a national biotechnology information center database, and through MEGA7 software, the sequence analysis is carried out on WJ561 by adopting the neighbor-joining method N-J cluster analysis, and a phylogenetic tree is constructed. The ITS sequence length of the strain WJ561 is 507bp, the sequence length of the TEF 1-alpha is 1230bp, the sequence length of the RPB2 is 988bp, and the result of the constructed phylogenetic tree is shown in figure 2. The sequence of WJ561 can be seen to be clustered with the sequence of Trichoderma virens strain LZ (GenBank ITS accession number MN 533724.1), trichoderma virens strain TRS897 (GenBank TEF 1-. Alpha.accession number KP 008852.1), trichoderma virens isolate YZB-1 (GenBank RPB2 accession number OL 770281.1), combined with the results of morphological analysis, indicating that the WJ561 strain is Trichoderma viride (Trichoderma virens).
Based on the above analysis of the morphology and ITS, TEF 1-alpha and RPB2 sequence homology of the WJ561 strain, the isolated WJ561 strain was identified as Trichoderma viride (Trichoderma virens). And performing biological preservation on the strain, wherein the preservation information is as follows:
strain name: trichoderma viride
Latin name: trichoderma virens
Strain number: WJ561
Preservation mechanism: china general microbiological culture Collection center (China Committee for culture Collection of microorganisms)
The preservation organization is abbreviated as: CGMCC
Address: no. 1 and No. 3 of the north cinquefoil of the morning sun area of beijing city.
Preservation date: 2023, 10, 17
Accession numbers of the preservation center: CGMCC No.40848
Example 2: plate confrontation test
Plate-face test of WJ561 against Pyricularia grisea
The antagonism of the trichoderma viride to the pear rot is clarified by adopting a two-point counter method. A bacterial cake with the diameter of about 6mm is picked from the edge of a fresh-cultured pear rot germ PDA flat plate and placed on one side of the PDA flat plate, and WJ561 bacterial cake is inoculated on the other side 5cm away from the PDA flat plate; in addition, WJ561 bacterial cake was inoculated on one side of the PDA plate, after 1 day of cultivation at 28℃and then pear rot germ bacterial cake was inoculated on the other side 5cm away therefrom. The treatment of placing sterile PDA agar cakes on opposite sides of the pear rot pathogen was used as a control. Culturing at 25deg.C for 3 days, measuring colony diameter growth, and calculating antibacterial rate. The above operation was repeated 3 times.
Equation 1: antibacterial ratio (%) = (control colony diameter-diameter of treated group pathogenic bacteria colony amplified toward trichoderma viride)/control colony diameter) ×100%
Plate challenge experiments showed that WJ561 has excellent antagonism against pathogenic hyphal growth (FIG. 3 a) with inhibition rates as high as 92.41% when WJ561 is preferentially inoculated for 1 day (FIG. 3 b).
Antibacterial spectrum of WJ561
Further, a plate-facing test was used to determine whether the Trichoderma viride has antagonism against other pathogenic bacteria. The selected pathogenic bacteria include Fusarium Jiang Jianbao and Fusarium Jiang Fupi, and the test procedure of reference (1).
Plate facing experiments show that WJ561 also has strong antagonistic effect on Fusarium oxysporum and Fusarium putrescens (figure 4 a), and inhibition rates are 70.82% and 64.40%, respectively (figure 4 b).
Example 3: bacteriostasis test of culture filtrate
1. Preparation of culture filtrate
WJ561 is activated on a PDA plate and then inoculated on a new PDA plate again for 5 days to be cultured, 8 bacterial cakes are taken and put into a 100mLPDB culture medium, after being cultured for 2, 4, 6, 10 and 14 days at 28 ℃ and 150rpm, the bacterial cakes are filtered by 12 layers of sterilizing mirror wiping paper, and then are filtered by a 0.1 mu m polyvinylidene fluoride microporous filter membrane filter for 2 times, so that a culture filtrate is obtained, and the culture filtrate is immediately used or is stored in a refrigerator at the temperature of minus 80 ℃ for a long time.
2. Antibacterial test of WJ561 culture filtrate with different culture time on pear rot germs
5mL of the culture filtrate was mixed with 10mL of warmed PDA, and poured into a petri dish after mixing. After solidification, inoculating a fresh pear rot germ cake which is cultured for 2 days in the center of the plate, and taking the plate added with 5mLPDB liquid culture medium as a control. After 2 days of culture, the colony diameter was measured, the hypha inhibition rate was calculated, and the inhibition rate was calculated as shown in formula 1. The above operation was repeated 3 times.
The results showed that WJ561 culture filtrate can inhibit growth and diffusion of pear rot germ hyphae, and that WJ561 culture filtrate of different culture times has different inhibition effects on pear rot germs (FIG. 5 a). The culture filtrates obtained from WJ561 cultures for 2, 4, 6, 10 and 14 days showed growth inhibition rates of 83.02%, 100%, 76.37%, 36.46% and 35.16% for pear rot pathogen hyphae, respectively (FIG. 5 b). Namely, when WJ561 is cultured for 4 days, the obtained culture filtrate has the best inhibition effect on pear rot germs up to 100% (figure 5 b).
3. Antibacterial test of WJ561 culture filtrate with different concentrations on pear rot germs
The 2-day-old pear rot germ cakes were respectively placed in PDB media containing 0%, 5%, 10% and 15% mass concentrations of 4-day-old WJ561 culture filtrate, and the PDB media without any solution was used as a blank. After incubation at 25℃and 180rpm for 3 days, mycelia were collected and dried and the weight of mycelia was measured. The results showed that the trichoderma viride culture filtrate of the same concentration had a remarkable inhibitory effect on the growth of pear rot germs, and as the content of the filtrate increased, the stronger the inhibitory effect, the smaller the number of mycelium pellets (fig. 6 a). At a trichoderma viride culture filtrate content of 15%, the mycelium dry weight was only 4.6% of the control group (fig. 6 b).
To further clarify the inhibitory effect of the culture filtrate of WJ561 on pear rot pathogens, hyphae were observed with a scanning electron microscope and a laser scanning confocal fluorescence microscope. Hyphae were fixed with 2.5% glutaraldehyde for 24 hours, then dehydrated stepwise with ethanol at a volume concentration of 30%,50%,70% and 90% (v/v), dried, sprayed with gold, observed for the morphology of the hyphae under a scanning electron microscope and photographed. The results showed that the mycelium appearance of the treated group was more curved, rough, wrinkled, shrunken as the culture filtrate concentration increased (fig. 6 c).
In addition, a small amount of mycelia was immersed in 5mM propidium iodide, water-bath was performed at 30℃for 20 minutes, and then washed three times with 0.05M PBS buffer (pH 7.2-7.4) to remove the dye, and then observed under a laser scanning confocal fluorescence microscope. The results showed that the red fluorescence intensity of the mycelia of the treatment group increased as the concentration of the culture filtrate increased. Clearly, a culture filtrate concentration of 10% was sufficient to cause significant damage to the hyphal cell membrane, indicating that the culture filtrate can destroy the hyphal cell membrane (fig. 7 a). Malondialdehyde (MDA) content and cell content leakage assay: a proper amount of mycelium sample treated by the culture filtrate is taken, 1mL of ice-cold 0.05MPBS buffer solution is added for ice bath homogenization, 8000rpm and 4 ℃ are centrifuged for 10min, and the supernatant is taken. MDA content was then determined according to the instructions of the thiobarbituric acid (TBA) commercial kit (Bioroyee Biotechnology). The concentration of soluble nucleic acids and soluble proteins in the mycelium-removed medium was measured with an ultra-micro spectrophotometer. The results showed that as the culture filtrate concentration increased, the MDA content increased (FIG. 7 b) and the nucleic acid and protein concentrations in the medium increased (FIGS. 7c and d). The result shows that the peroxidation damage degree of the mycelium of the pear rot germ increases along with the increase of the concentration of the WJ561 culture filtrate, and the cell content is caused to exude.
Example 4: exploration test of bacteriostasis mechanism of culture filtrate
1. The culture filtrate induces the accumulation of active oxygen (ROS) of hypha of pear rot germ
With 1. Mu.M 2, 7-dichlorofluorescein (H) 2 DCFDA, solarbio) and then flushing the mycelium 3 times with 0.05M PBS buffer, followed by observing ROS accumulation under a laser scanning confocal fluorescence microscope. The results showed that green fluorescence was exhibited in hyphal cells treated with WJ561 culture filtrate, and the fluorescence intensity was concentration-dependent (FIG. 8 a). It was shown that the potential mechanism of WJ561 culture filtrate to induce mycelium damage by Pyricularia is likely to be associated with ROS accumulation.
2. Detection of antioxidant enzyme activity of pear rot germ hypha
Taking a proper amount of mycelium samples treated by the culture filtrate, adding 1mL of ice-cooled 0.05M PBS buffer solution for ice bath homogenization, centrifuging at 8000rpm and 4 ℃ for 10min, and taking the supernatant to obtain a crude enzyme solution. Subsequently, the activities of Catalase (CAT) and superoxide dismutase (SOD) were determined using a commercial kit (Solarbio). The results showed that the activity of both active oxygen scavenging enzymes was inhibited to varying degrees, the activity decrease being most pronounced after 15% culture filtrate treatment (FIGS. 8b and c). To further clarify that WJ561 culture filtrate destroyed the antioxidant capacity of Pyricularia, the gene expression levels encoding these ROS scavenging enzymes were determined by real-time fluorescent quantitative PCR. The results showed that the key genes for CAT and SOD synthesis were significantly down-regulated after WJ561 culture filtrate treatment (fig. 8d and e). The WJ561 culture filtrate is proved to be likely to destroy the antioxidation capability of the pear rot germ silk cells and induce the rapid accumulation of ROS in germ hypha cells, thereby causing damage to germs.
Example 5: ex vivo test
1. In vitro blade test
Taking healthy pear leaves, sterilizing the surfaces of the healthy pear leaves by 75% alcohol, washing the healthy pear leaves by tap water and sterile water, and air-drying the healthy pear leaves at room temperature. Repeatedly smearing 500 mu L of WJ561 culture filtrate with mass concentration of 0%, 5%, 10% and 15% on leaf, and air drying. The leaf blade was pierced with a sterile syringe needle, then a cake of pear rot germ was inoculated on the well, and 3 replicates per treatment were performed with PDA agar cake as control. The leaf is placed on wet sterilized filter paper for moisture preservation, cultured at 25 ℃ and observed for disease spot development. The results showed that the area of lesions caused by pear rot pathogens in the experimental group was smaller than that in the control group after 3 days of treatment, indicating that the WJ561 culture filtrate effectively inhibited the expansion of pathogens and reduced the pathogenic effect of pathogens on pear leaves (FIG. 9 a).
2. In vitro shoot test
Taking a healthy pear branch with the length of about 12cm, sealing two ends by paraffin, wiping and sterilizing the surface by 75% alcohol, washing by sterile water, and air-drying. Scalding the surface of pear branch with hot iron nail cap (diameter about 5 mm), repeatedly smearing the branch with WJ561 culture filtrate with mass concentration of 0%, 5%, 10% and 15%, air drying, and inoculating a pear rot pathogen. Placing the treated branches into a clean tray, wetting sterilized gauze for moisturizing, culturing at 25 ℃ for 5 days, and observing the development of the lesions. The results showed that the WJ561 culture filtrate inhibited lesion expansion on the shoots after 5 days of treatment (fig. 9 b). Compared with the control group (PBD instead of WJ561 CFS), the length of lesions on the branches is significantly smaller than that of the control group after the branches are smeared in advance with the WJ561 culture filtrate with the concentration of 15% (fig. 9c and d). The result shows that the WJ561 culture filtrate effectively inhibits the expansion of germs, reduces the pathogenic effect of germs on pear branches, and lays a foundation for field experiments.
Example 5: stability test of WJ561 culture filtrate
1. Acid-base stability:
the WJ561 culture filtrate was treated with 1mol L -1 HCl and 1mol L -1 NaOH is regulated to pH values of 1, 3, 5, 7, 9, 11 and 13, and after the mixture is placed for 2 hours at room temperature, the pH value of the filtrate is regulated back to the original value. Mixing 5mL of the treated WJ561 culture filtrate with warm PDA culture medium to obtain a PDA plate, setting a bacterial cake of pear rot disease bacteria in the center after solidification, and measuring 3 times of repetition of each treatment by taking the culture filtrate without acid-base treatment as a controlInhibition rate. The results show that after the WJ561 culture filtrate is subjected to acid-base treatment, the antibacterial activity is not affected basically in the pH value range from 1 to 11. Whereas at pH 13 the antifungal activity was significantly reduced (fig. 10 a).
2. Thermal stability
The WJ561 culture filtrate was subjected to temperatures of 4℃at 50℃at 80℃at 100℃and 121℃for 30min, respectively. After the filtrate was cooled to room temperature, 5mL of the treated WJ561 culture filtrate was mixed with a warm PDA medium to prepare a PDA plate, and after solidification, a pear rot pathogen cake was placed in the center thereof, and the inhibition rate was measured with 3 replicates per treatment with the culture filtrate without temperature treatment as a control. The results showed that only 121 ℃ significantly affected the bacteriostatic activity of the WJ561 culture filtrate (fig. 10 b).
3. Ultraviolet stability
The WJ561 culture filtrate was exposed to UV lamps for 0.5, 1, 2, 3 and 4 hours, respectively. And then 5mL of the treated WJ561 culture filtrate is mixed with a warm PDA culture medium to prepare a PDA plate, a pear rot pathogen cake is placed in the center of the PDA plate after solidification, the culture filtrate which is not subjected to ultraviolet irradiation treatment is used as a control, and the inhibition rate is measured every 3 times of treatment. The results showed that although the bacteriostatic activity of the WJ561 culture filtrate was affected by the irradiation time of ultraviolet rays, the bacteriostatic rate of the culture filtrate was still 60% or more after the irradiation time of ultraviolet rays for 4 hours (FIG. 10 c).
4. Storage stability
After the WJ561 culture filtrate is respectively stored for 4, 8 and 12 months at the temperature of minus 20 ℃, 5mL of the treated WJ561 culture filtrate is mixed with a warm PDA culture medium to prepare a PDA plate, after solidification, a pear rot germ cake is placed in the center of the PDA plate, and the freshly prepared culture filtrate is used as a reference, and the inhibition rate is measured after 3 times of treatment. The results show that the WJ561 culture filtrate has no significant difference in the inhibitory effect on pathogenic bacteria after 4 months of storage at 4 ℃. The observed bacterial inhibition of the culture filtrate at 8 months of storage was slightly below 12 months, which may be due to CFS batch variation. Although antagonism was reduced over time, the inhibition of WJ561 culture filtrate remained 89% after one year of storage (fig. 10 d).
In conclusion, the WJ561 culture filtrate has good environmental stability.
Although embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that: various substitutions, changes and modifications are possible without departing from the spirit and scope of the invention and the appended claims, and therefore the scope of the invention is not limited to the disclosure of the embodiments.
SEQUENCE LISTING
WJ561 strain
Sequence 1
tgtgaacgttaccaaactgttgcctcggcgggatctctgccccgggtgcgtcgcagccccggaccaaggcgcccgccggaggaccaaccaa
aactcttattgtataccccctcgcgggttttttactatctgagccatctcggcgcccctcgtgggcgtttcgaaaatgaatcaaaactttcaacaacg
gatctcttggttctggcatcgatgaagaacgcagcgaaatgcgataagtaatgtgaattgcagaattcagtgaatcatcgaatctttgaacgcacat
tgcgcccgccagtattctggcgggcatgcctgtccgagcgtcatttcaaccctcgaacccctccggggggtcggcgttggggatcggcccttta
cggggccggccccgaaatacagtggcggtctcgccgcagcctctcctgcgcagtagtttgcacactcgcatcgggagcgcggcgcgtccac
agccgttaaacaccccaaacttctgaaatgtgacct
Sequence 2
ctgaccggggtggttcatgacgatgacctgggcggtgaaagaagcggcacccatgggggggtcgttcttggagtcaccggcaacgttaccac
ggcggatttccttgacggaaacgttcttcacgttgaaaccaacgttgtcaccgggaacaccctcaacgagctgctcgtggtgcatctcgacggac
ttgacttcagtggtgacgttggaaggagcgaaggtaacaaccataccgggcttgaggacaccagtctcgatacggccgacgggaaccgttccg
ataccaccgatcttgtacacatcctggaggggaagacggaggggcttgtccgtgggacgcttggggggctcgatggagtcgatggcctcaag
gagggtcttgccggtgaacttgccagccttggtctccttctcccagcccttgtaccaggggcagttggtggagggctggagcatgttgtcaccgtt
gaaaccggagatggggacgaaagcaacagccttggggttgaagccgaccttcttaatgaagttggaggtctccttgatgatttcctggtaacga
gcctcggcccagttggcagtgtccatcttgttgatggcgacgatgagctgcttgacacccagggtgtaggcgagcagagcgtgctcacgggtct
ggccatccttggagataccagcctcgaactcaccagtaccggcggcaatgatgagaatggcgcaatcggcctgggaagtaccagtgatcatgt
tcttgatgaaatcacggtggccgggggcgtctgtgaggtcctggttagcactggtttgcgggggctggatgaatgggacgagacataccaatga
cggtgacatagtacttgggagtctcgaacttccacagggcaatgtcgatggtgataccacgctcacgctcggccttgagcttgtcaagaacccaa
gcgtacttgaaggaacccttgccgagttcggcggcttcctgttgagggaacggtggttagcatcgctgcaaataacgagacacagatcgttgaat
gaggactgggaagtggtagacgatggcaaagagcagcgaggtagtggtggggttgctggtgcacccccactaaaaaccagaggcagcaaa
aaattttgcgtcgctgcaaaggaggggtaatggaacgaaagcggggtgacgaaaaaattgttgcctcgatgattctgaacagaattgtcgagca
caattgaatgtga
Sequence 3
Ttccgtggtatcatgcgaaggatgaacaccgaattggccaattatctgagacgttgcgttgagggcaaccgacacttcaaccttgccgttggtat
caagcccggcacactttcaaacggattgaagtattcgcttgccactggaaactggggtgatcagaagaaggccatgagctcaactgctggtgtg
tctcaggtgcttaatcgttacacgtttgcttcgaccctatcacatttgcgtcgtaccaacacccccattggaagagatggtaagctggcgaagcctc
gacagcttcacaacacgcattggggtttggtgtgtccagccgagacacccgaaggacaggcttgtggtctggtcaaaaacttgtctctgatgtgc
tacgtcagtgtcggttctccctccgaaccactgattgagttcatgatcaacagaggtatggaagtcgtcgaagagtacgagccgctgcgatatcct
catgctaccaagattttcgtgaacggtgtctgggttggagttcaccaagaccctaagcatctggtgaaccaggttctagacactcgtcgcaaatcc
tatctgcagtacgaagtctctctcgtgagagagattcgagaccaggaattcaaaatcttttccgatgcaggccgtgtcatgcgacctgtatttaccgt
tcagcaagaggatgaccctgaaacaggcatcaacaagggccacctggtattaactaaggagctcgtcaacagattggctaaggagcaagctg
aacctccggaagacccgagcatgaagattggatgggagggactaatcagggctggtgcggttgaatatctcgacgccgaagaagaggagac
gtcgatgatttgcatgacaccagaggatctcgagctgtatcgtcttcagaaagccggtatctctaccgatgaagacatgggagatgatccgaacaagcgactaaaggacaaagaccaacccaacaacccacatt。
Claims (10)
1. An endophytic trichoderma viride WJ561, characterized in that: the name is as follows: WJ561, class name: trichoderma viride (Trichoderma virens), accession number: CGMCC No.40848, date of preservation: 2023, 10, 17, deposit unit: china general microbiological culture Collection center, north Chen Xi Lu No. 1, 3, the Korean region of Beijing, and the China general microbiological culture Collection center.
2. Trichoderma viride WJ561 according to claim 1, characterized in that: the trichoderma viride WJ561 strain is separated from the rhizome of rhizoma zingiberis of the county of Hebei, and the separation method is as follows: taking ginger rhizome, washing with tap water, sterilizing the surface of the ginger rhizome by 75% alcohol, washing the ginger rhizome with sterile water for 3 times, placing the rhizome in an ultra-clean bench, properly drying the dried rhizome, cutting the rhizome into square small pieces of 0.5cm by using a sterilizing knife, placing the square small pieces on a potato dextrose agar medium, and culturing the potato dextrose agar medium at 28 ℃ for 6 days; fungus isolates grown from the rootstock were subcultured and purified on the same medium and identified as Trichoderma viride by ITS and RNA polymerase II subunit sequence sequencing and result alignment.
3. Trichoderma viride WJ561 according to claim 1, characterized in that: the trichoderma viride WJ561 has the following characteristics:
after 1d of culture at 28 ℃, all strains showed white fluffy shape on PDA medium with white back; when the culture is carried out until the 3 rd day, the center of each culture medium is provided with a light green spore belt to a dark green spore belt, and the spore belts gradually extend to the periphery, and the mycelium is compact cotton-like; by day 5, the green spores were spread in concentric circles throughout the dish, presenting dark green, grayish green or yellowish green carousel; it was initially identified as Trichoderma. WJ561 was identified as Trichoderma viride by ITS, TEF 1-alpha and RPB2 sequence sequencing and result alignment.
4. A culture filtrate obtained by the preparation of trichoderma viride WJ561 as claimed in any one of claims 1 to 3.
5. The method for preparing a culture filtrate according to claim 4, wherein: the method comprises the following specific steps:
placing fresh Trichoderma viride cake of 4-6 days old into potato dextrose water culture medium, culturing at 25-28deg.C and 140-160rpm for 2, 4, 6, 10, and 14 days, filtering with 12 layers of sterilizing mirror cleaning paper, and filtering with 0.1 μm microporous filter membrane for 2 times to obtain culture filtrate.
6. The method for preparing a culture filtrate according to claim 5, wherein: the method comprises the following specific steps:
taking 8 fresh Trichoderma viride cakes which are cultured for 5 days by a 200 microliter sterile gun head, putting the Trichoderma viride cakes into a potato dextrose water culture medium, culturing for 4 days at 28 ℃ and 150rpm, filtering the Trichoderma viride cakes by 12 layers of sterilizing mirror wiping paper, and filtering the Trichoderma viride cakes by a 0.1 mu m polyvinylidene fluoride microporous filter membrane filter for 2 times to obtain a culture filtrate.
7. Use of a culture filtrate according to claim 4 for inhibiting the growth of and/or for the preparation of a formulation for inhibiting and/or for controlling diseases caused by pear rot pathogens.
8. The use according to claim 6, characterized in that: the pear rot germ is pear black rot fungus;
alternatively, the formulation is a mixture of trichoderma viride WJ561 culture filtrate and sterile PDB medium;
alternatively, the formulation is a formulation comprising a culture filtrate of trichoderma viride WJ561 at a mass concentration of 0%, 5%, 10% and 15%.
9. The use according to claim 8, characterized in that: the preparation is applied to the cell membrane damage and/or oxidative damage and/or inhibition of the generation of antioxidant enzyme and/or the reduction of pathogenicity of pathogenic bacteria to plant leaves and/or the reduction of pathogenicity of pathogenic bacteria to plant branches.
10. The use according to claim 8, characterized in that: the pear rot germ is pear black rot fungus;
alternatively, the plant is red zodiac pear.
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