CN113278532B - Alternaria tenuissima and metabolite and application thereof - Google Patents

Alternaria tenuissima and metabolite and application thereof Download PDF

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CN113278532B
CN113278532B CN202110559463.1A CN202110559463A CN113278532B CN 113278532 B CN113278532 B CN 113278532B CN 202110559463 A CN202110559463 A CN 202110559463A CN 113278532 B CN113278532 B CN 113278532B
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extract
alternaria
alternaria tenuissima
fermentation
virus
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CN113278532A (en
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王红艳
武玉国
姜莉莉
王开运
于颀
王开元
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Shandong Huitaihua Biotechnology Co ltd
Shandong Agricultural University
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Shandong Huitaihua Biotechnology Co ltd
Shandong Agricultural University
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/30—Microbial fungi; Substances produced thereby or obtained therefrom
    • C—CHEMISTRY; METALLURGY
    • C05—FERTILISERS; MANUFACTURE THEREOF
    • C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
    • C05F11/00—Other organic fertilisers
    • C05F11/08—Organic fertilisers containing added bacterial cultures, mycelia or the like
    • C—CHEMISTRY; METALLURGY
    • C05—FERTILISERS; MANUFACTURE THEREOF
    • C05G—MIXTURES OF FERTILISERS COVERED INDIVIDUALLY BY DIFFERENT SUBCLASSES OF CLASS C05; MIXTURES OF ONE OR MORE FERTILISERS WITH MATERIALS NOT HAVING A SPECIFIC FERTILISING ACTIVITY, e.g. PESTICIDES, SOIL-CONDITIONERS, WETTING AGENTS; FERTILISERS CHARACTERISED BY THEIR FORM
    • C05G3/00—Mixtures of one or more fertilisers with additives not having a specially fertilising activity
    • C—CHEMISTRY; METALLURGY
    • C05—FERTILISERS; MANUFACTURE THEREOF
    • C05G—MIXTURES OF FERTILISERS COVERED INDIVIDUALLY BY DIFFERENT SUBCLASSES OF CLASS C05; MIXTURES OF ONE OR MORE FERTILISERS WITH MATERIALS NOT HAVING A SPECIFIC FERTILISING ACTIVITY, e.g. PESTICIDES, SOIL-CONDITIONERS, WETTING AGENTS; FERTILISERS CHARACTERISED BY THEIR FORM
    • C05G3/00—Mixtures of one or more fertilisers with additives not having a specially fertilising activity
    • C05G3/60—Biocides or preservatives, e.g. disinfectants, pesticides or herbicides; Pest repellants or attractants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • 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
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture

Abstract

The invention provides a Alternaria tenuissima (A) and a strainAlternaria alternata) SDTF016, which obtains the fermentation condition and the extraction and separation method of the metabolite telithromycin; the fermentation liquor and the metabolite telithromycin promote plant growth, effectively prevent and control virus diseases on crops such as tomatoes, tobaccos, hot peppers, potatoes and the like, improve the stress resistance and disease resistance of the plants, and improve the yield and quality. The Alternaria tenuissima fermentation liquor and the extract thereof can be diluted by water and then used by seed dressing, seed soaking, root irrigation, spraying and drip irrigation, have high biological activity and broad spectrum of action, can improve the control effect and reduce the pesticide consumption when being mixed with pesticide, have multiple effects of improving the yield and the quality of agricultural products, and are environment-friendly. The fermentation liquor of the alternaria tenuissima Taishan variant metabolite and the Taishan mycin can be widely applied to the field of agricultural production, and have remarkable economic, social and ecological benefits.

Description

Alternaria tenuissima and metabolite and application thereof
Technical Field
The invention belongs to the field of biological pesticides, particularly relates to a functional microorganism, and particularly relates to alternaria tenuissima (A) with a biocontrol effectAlternaria alternata) The variety of Taishan mountain and its metabolite Taishan mountain mycin, and its application in preventing and treating plant diseases.
Background
Tomato virus is one of the most important diseases damaging Tomato production, and the virus has more than 20 virus origins, mainly comprising Tobacco Mosaic Virus (TMV) called Tobacco mosaic virus, Cucumber Mosaic Virus (CMV) called Cucumber mosaic virus, Tobacco Leaf Curl Virus (TLCV) called Tobacco leaf curl virus, Alfalfa Mosaic Virus (AMV) called Alfalfa mosaic virus, Tomato yellow leaf curl virus (TYLV) called Tomato yellow leaf curl virus, Potato Virus Y (PVY) called Potato virus and Whitefly-transmitted geminivirus (WTG) called Tomato bemisia geminivirus, etc. According to the field expression symptoms, the plant can be divided into (1) mosaic type: the leaves are mottled with yellow-green or light-dark interphase or slightly shriveled; (2) fern leaf type: dwarfing the plant, forming linear upper leaves and slightly rolling middle and lower leaves, and increasing the corolla into giant flowers; (3) stripe type: brown spots or cloud spots occur on leaves or brown spots occur on stems and tendrils, and the discolored parts are only positioned in epidermal tissues and do not go deep into the interior; leaf rolling: yellowing of leaf veins, upward curling of leaf edges, distortion and malformation of leaflets, and atrophy or clustering of plants; (4) yellow top type: the leaves at the top are faded or yellowed, the leaves are reduced, the leaves are shriveled, the edges are curled, the plants are dwarfed, and adventitious branches are clustered; (5) necrosis type: part of leaves or the whole leaves are yellowed to generate yellow brown necrotic spots, the diseased spots are irregular and mostly necrotic and dry at the edges, the fruits of the diseased plants are light grayish green, and semitransparent pale spots are penetrated out. Among tomato virus diseases, especially bemisia tabaci virus-transmitted leaf curl virus and yellow leaf curl virus are the most harmful, and because no effective control agent is available, the disease-resistant breeding effect is not ideal enough, and the research and development of efficient, low-toxicity and environment-friendly agents are still in urgent need in production.
Alternaria alternata (Alternaria alternate) Belongs to the fungus doorAlternaria, Aphyllophorales, Hymenochaetaceae, is one of the fungi imperfecti species that is of great economic importance and is distributed in the widest range around the world. Alternaria alternata is one of pathogenic bacteria of plant diseases, and meanwhile, certain metabolites of Alternaria alternata can be utilized by people as biological resources beneficial to human beings, so that Alternaria alternata is currently applied to the aspect of agricultural biological control and has important values in aspects of disease-resistant breeding, disease control, waste treatment, material circulation and the like. Research reports that Sunware (2006) can play an effective prevention and treatment role on tobacco alternaria alternata by spraying nonpathogenic Alternaria alternata spore suspension on tobacco leaves, and the maximum prevention effect of the field can reach 65%. Confirmed from Alternaria longissima (A)Alternaria. Longipes) SRS obtained by separating and screening metabolites2The prepared biocontrol agent is used for preventing and treating alternaria alternate, and can actually increase the tobacco yield and improve the tobacco quality (Dong Han Song, 1995). At home and abroad, secondary metabolites with various activities, such as substances with insecticidal and bactericidal effects, mycotoxins and the like (Montermurro) have been separated from Alternaria alternataet al., 1992). FromA.solaniAlternaric acid (Alternaric acid) separated from the fermentation liquor is a substance with antibacterial activity, has good control effects on sclerotinia rot of colza, gray mold of cucumber and the like, and the control effects can respectively reach 83.3% and 82.7% (plum Zheng et al, 1997; Tian nationality, et al, 1999). Researches report that protein elicitor PeaT1 separated from Alternaria tenuissima by Pengzhi et al (2017) and jinggangmycin are mixed to effectively prevent and treat tobacco virus disease TMV, and the relative prevention effect can reach 73.39%. With the development of times and the advancement of science and technology, more and more beneficial alternaria alternata is well known and utilized, more and more scientists at home and abroad begin to pay attention to the function of the metabolite of the alternaria alternata in the aspect of biological prevention and control, and the metabolite of the alternaria alternata is more and more applied to the actual production through the advancement of technological means.
Disclosure of Invention
Aiming at the problem that biocontrol bacteria for effectively preventing and treating virus diseases are lacked in the prior art, the invention providesFor a strain of Alternaria tenuissima (A) with biocontrol effectAlternaria alternata) The Taishan variant SDTF016 has good antagonistic effect on viruses on tobacco, tomato, eggplant and hot pepper, and can promote plant growth and improve plant stress resistance.
Another object of the present invention is to provide the above-mentioned Alternaria tenuissima (A), (B), (C)Alternaria alternata) A method for producing antibacterial substance by fermenting Taishan mountain variety is provided.
It is still another object of the present invention to provide the above-mentioned Alternaria tenuissima (A), (B), (C)Alternaria alternata) Antibacterial substance produced by fermentation.
In order to achieve the purpose, the invention adopts the following technical scheme.
A strain of Alternaria tenuissima (Alternaria alternata) SDTF016 with preservation number of CCTCC M2021495.
Fermentation liquor and extract of the above Alternaria tenuissima.
Preferably, the fermentation broth may or may not contain biomass. The thallus can be mycelium or spore.
Preferably, the extract is an ethyl acetate extract of the fermentation broth.
More preferably, the preparation method of the extract comprises the following steps:
(1) filtering the fermentation liquor for sterilization and deproteinization to obtain filtrate;
(2) passing the filtrate through silica gel column with ethyl acetate as eluent to obtain effluent;
(3) the eluate is stripped of the leacheate to obtain a solid crude extract.
Further, the above steps also include the process of further purifying the crude extract. Preferably, the method comprises the following steps:
(i) dissolving the crude extract, separating by liquid chromatography, and collecting eluate with absorption peak at 214 nm wavelength;
(ii) removing solvent from the effluent to obtain refined extract.
Preferably, the main effective components in the extract are dibutyl phthalate, 4-methyl-5-thiazole ethanol, 2-methoxy resorcinol, p-hydroxyphenol, 4-coumaric acid and nicotinic acid.
The Alternaria tenuissima and the fermentation liquor and the extract thereof can be used for preparing fertilizers or pesticides.
The control object of the pesticide is plant pathogenic virus. Preferably, the virus is selected from the group consisting of tomato leaf curl virus, cucumber mosaic virus, pepper mild mottle virus, broad bean wilting virus 2, and tobacco mosaic virus.
The fertilizer has the effects of promoting plant growth, improving quality and improving plant stress resistance and disease resistance.
A preparation comprises the above Alternaria tenuissima, fermentation broth or/and extract. Preferably, the formulation is a fertilizer or pesticide formulation. More preferably, the preparation is selected from a microbial agent, a bio-organic fertilizer, a water soluble fertilizer, a water aqua, a water soluble granule or a water soluble powder.
The invention has the following advantages:
the invention provides a strain of Alternaria tenuissima (A)Alternaria alternata) SDTF016 to obtain the fermentation conditions of the Taishan variant of the Alternaria tenuissima; establishing an extraction and separation method of the antibacterial composition of the Taishan mycin; the fermentation liquor of the mutant metabolite of the Alternaria tenuissima and the active ingredient of the Taishan mycin are found to promote the growth of plants, effectively prevent and control virus diseases on crops such as tomatoes, tobaccos, hot peppers, potatoes and the like, can improve the stress resistance and disease resistance of the plants, and have various effects of improving the yield and quality. The Alternaria tenuissima fermentation liquor and the extract thereof can be diluted by adding water and then used by seed dressing, seed soaking, root irrigation, spraying and drip irrigation, have high biological activity and broad spectrum action, can promote the growth of plants, improve the stress resistance and disease resistance of the plants, are used for preventing and treating various virus diseases of vegetables, fruit trees and crops, can improve the prevention effect and reduce the using amount of pesticides when being mixed with the pesticides, have various effects of improving the yield and the quality of agricultural products, and are environment-friendly. The fermentation liquor of the alternaria tenuissima Taishan variant metabolite and the Taishan mycin can be widely applied to the field of agricultural production, and have remarkable economic, social and ecological benefits.
Biological preservation information
Alternaria tenuissima (A), (B), (C)Alternaria alternata) SDTF016, stored in China center for type culture Collection (CGMCC) at 2021, 05 and 06 months, with the preservation address of the preservation center of Wuhan university in Wuhan, China and the preservation number of CCTCC M2021495.
Drawings
FIG. 1 is a transmission electron micrograph of hyphae of strain SDTF016 on a fungus culture medium;
FIG. 2 is a 1% agarose electrophoresis of the ITS sequence PCR product;
FIG. 3 is a high performance liquid chromatogram of crude extract of strain SDTF 016.
Detailed Description
The present invention will be further described with reference to the following examples and drawings, but the present invention is not limited to the following examples.
Example 1 isolation and characterization of Alternaria tenuissima SDTF016
Soil of a farmland near Mount Taishan is collected in Taian City, and fungal strains are isolated therefrom. The specific separation and purification process comprises the following steps: weighing 10 g of sieved soil sample, placing the soil sample into a 250 mL sterilized triangular flask filled with 90 mL of sterile water and a small amount of glass beads, placing the triangular flask on a shaking table with the temperature of 37 ℃ and the speed of 180 rpm for 30 min to enable the triangular flask to be fully suspended, and diluting the suspension to 10 DEG with sterile water-2-10-5And 5 gradients are equal. Respectively sucking 1 mL of each gradient diluent into a 9cm culture dish, pouring 15 mL of improved Martin culture medium (containing 0.033 g/L of Bengal) cooled to about 45 ℃, horizontally shaking and uniformly mixing, repeating each gradient for 3 times, culturing in a constant-temperature incubator at 28 ℃ for 5-7 days, and then picking different single colonies and carrying out plate streaking purification. And screening the purified strains by taking various plant pathogenic bacteria as target bacteria to obtain a target strain with the best bacteriostatic effect, wherein the number of the target strain is SDTF 016. The bacteria are subjected to morphological identification and physiological and biochemical characteristic tests, and the hypha transmission electron microscope photos are shown in figure 1: the hyphae have smooth surfaces, grow in bamboo joints and are in discontinuous segmental states, the tops of the hyphae which just germinate grow outwards in a triangular shape, the strain does not produce spores under proper growth conditions, and the hyphae are flourishing and are distributed in clusters.
Extracting strain genome DNA by using an Ezup column type fungus genome DNA extraction kit (Shanghai biological engineering Co., Ltd.), and amplifying fungus ITS sequences by PCR by using universal primers ITS1 and ITS4 as primers. The PCR product was electrophoresed in 1% agarose as shown in FIG. 2 (M: Marker; 1: amplified band of strain SDTF 016) to obtain a band of about 500 bp in size.
The PCR product was sent to Shanghai Bioengineering Co., Ltd for sequencing. The length of the target gene is 557bp, the NCBI website is logged in, homology comparison analysis is carried out on the sequence obtained by sequencing and the known genome sequence in GenBank by using Blast, complete system evolution analysis is carried out by selecting the genome sequence with similar homology, multiple comparison analysis is carried out on the genome sequence, and then the MEGA 7 software is used for constructing a phylogenetic tree of the strain. According to phylogenetic trees, the sequence consistency of the test strain sequence and the sequence of the alternaria tenuissima is 100 percent; and the phylogenetic tree analysis result shows that the test strain and the alternaria tenuissima mode strain are in the same branch and have the closest genetic distance and affinity relationship. Therefore, according to the observation result of a scanning electron microscope, physiological and biochemical tests and the analysis of ITS sequences, SDTF016 is finally identified as Alternaria tenuissima (A) (B)Alternaria alternata) Abbreviated as Taishan variant.
The strain is preserved in China center for type culture Collection (CGMCC) at 2021, 05 and 06 months, with the preservation number of CCTCC M2021495.
Example 2 obtaining of fermentation broth of Alternaria tenuissima SDTF016 and extraction of fermentation product
Adopting a multi-factor orthogonal test to optimize fermentation conditions of the Taishan mountain variety (the composition and content of a culture medium carbon source, a nitrogen source and inorganic salt, the fermentation time is 12-120h, the fermentation temperature is 20-37 ℃, the rotation speed is 90-150 rpm), and adopting the following optimized conditions to perform fermentation: a punch with the diameter of 7 mm is used for punching a bacterial cake on the edge of a PDA plate bacterial colony of the Alternaria tenuissima SDTF016 strain, and the bacterial cake is inoculated on 150 mL of liquid fungus culture medium (MgSO 4)4·7H2O 0.5 g，K2HPO4 1.0g, peptone 5.0 g, glucose 20 g, yeast powder 2.0 g, water 1.0L, pH = 7.0), inoculating 3 fungus cakes into each bottle, performing shaking culture at constant temperature of 25 deg.C and 120 rpm for 36h to obtain fermentation liquor of Alternaria tenuissima SDTF016 strain, and culturing in parallelFermenting for 5 batches.
And (2) carrying out suction filtration on the fermentation liquor by using a vacuum pump to obtain hypha and degerming fermentation liquor, further passing the degerming fermentation liquor through a 0.2-micron membrane to obtain dark yellow transparent sterile fermentation liquor from which proteins and impurities are removed, adding the dark yellow transparent sterile fermentation liquor into a chromatographic column filled with 200-mesh and 300-mesh chromatographic silica gel, carrying out column chromatography by using ethyl acetate as a solvent, collecting effluent, combining the effluent according to an activity monitoring result, and evaporating the ethyl acetate of the collected transparent liquor to dryness at 40 ℃ by using a rotary evaporator to obtain a light yellow solid, namely a crude extract of a metabolite of the SDTF016 strain of the Alternaria tenuissima. The crude extract was dissolved in acetonitrile and analyzed by Agilent 1200 LC HPLC, and the peak appearance at 214 nm was shown in FIG. 3.
Dissolving the crude extract of metabolite of the Alternaria tenuissima SDTF016 strain with acetonitrile, and performing liquid chromatography on Shimadzu LC-20AP to prepare a sample, wherein the chromatographic column is DALIETER C18Column (packing: sino chrom ODS-BP 1 μm; size: 10.00 mm. times.250 mm), ultraviolet absorption wavelength of 214 nm, mobile phase acetonitrile: water =40:60 (v/v), flow rate 4.0 mL/min, effluent-80 deg.C freeze-drying to obtain pure product of main component of metabolite of Alternaria tenuissima SDTF016, abbreviated as Taishan mycin.
The main components of the telithromycin are 6 of dibutyl phthalate, 4-methyl-5-thiazole ethanol, 2-methoxy resorcinol, p-hydroxyphenol, 4-coumaric acid and nicotinic acid, the weight percentage of the main components in the fermentation liquor is 0.01-0.05 wt%, the average content is 0.02%, and the telithromycin is a mixed active substance with the ultraviolet absorption wavelength of 214 nm.
Example 3 prevention of tomato Virus disease by Alternaria tenuissima SDTF016 fermentation broth and extract
The method comprises the steps of fermenting the Alternaria tenuissima SDTF016 according to the fermentation method in the embodiment 2, extracting the telangin, obtaining bacterium-containing fermentation liquor (the content of the telangin is about 0.02 percent), degerming fermentation liquor (the content of the telangin is about 0.02 percent) and the telangin, and preparing the telangin into a water agent with the content of 0.02 percent by weight by using 0.1 percent of TX-100 aqueous solution. The fermentation broth is obtained by fermenting Alternaria alternate Tsai strain TS001 according to a method of cunning, 2018 (the effect and physiological mechanism of Alternaria alternate Tsai strain metabolite on preventing and treating tomato yellow leaf curl virus). Selecting a greenhouse with tomato leaf curl virus medium-severity occurrence for testing, diluting the fermentation liquor or the aqueous solution by 250 times and 500 times respectively for spray prevention, spraying for 1 time every 7 days in the initial stage of virus disease occurrence, continuously spraying for 3 times, then counting disease indexes 7 days and 14 days after the last application, and calculating prevention effect. For root irrigation prevention and control, root irrigation is carried out according to 1kg and 2kg of dosage per mu at the initial stage of viral disease occurrence, then disease indexes of 14 days and 28 days after pesticide application are counted, and prevention effect is calculated. Each treatment was controlled with clear water.
TABLE 1 Effect of Streptomyces tenuissima SDTF016 fermentation broth and telithromycin spray on prevention and treatment of tomato leaf curl virus disease
Figure DEST_PATH_IMAGE002
The data in the table are counted by a new repeated polarization method, and the data containing the same letters are not obvious in difference.
As shown in the table 1, the tomato leaf curl virus disease is prevented and treated by field spraying of the sterilizing fermentation liquid and the Taishan mycin water aqua, the strain-containing bacteria and the sterilizing fermentation liquid of the Taishan variant of the ultrafine Alternaria alternata are diluted by 250 times in the early stage of the tomato leaf curl virus disease, the whole tomato plant is sprayed for 1 time in 7 days and three times in succession, and the prevention and treatment effects of 7 days and 14 days after the last 1 time of spraying are 91.6%, 92.8%, 85.5% and 85.2% respectively; diluting the bacteria-containing and degerming fermentation liquor by 500 times, carrying out spray treatment on the whole tomato plant, spraying for 1 time in 7 days, continuously spraying for three times, and respectively controlling the control effects of 83.9%, 83.4%, 80.9% and 81.6% 7 days and 14 days after the last 1-time spray treatment; diluting the 0.02% Taishan mycin aqua by 250 times, carrying out spray treatment on the whole tomato plant, spraying for 1 time in 7 days, continuously spraying for three times, and respectively having the control effects of 85.1% and 83.4% 7 days and 14 days after the last 1 time of spray; the 0.02% aqueous solution of the Taishan mycin is diluted by 500 times, the whole tomato plant is sprayed for 1 time in 7 days and continuously sprayed for three times, and the control effects of 7 days and 14 days after the last 1 time of spraying are 81.0% and 80.9% respectively. The prevention effect of the Alternaria tenuissima Taishan variant fermentation broth and the Taishan mycin on tomato leaf curl virus diseases is obviously better than that of Alternaria tenuissima TS001 fermentation broth diluted by 250 times, and the industrialization prospect is better.
TABLE 2 effects of fermentation broth of Alternaria tenuissima and Mount Taishan mycin on preventing and treating tomato leaf curl virus
Figure DEST_PATH_IMAGE004
The data in the table are counted by a new repeated polarization method, and the data containing the same letters are not obvious in difference.
As shown in the table 2, the tomato leaf curl virus disease is prevented and treated by irrigating roots of the field containing the bacterium fermentation liquor and the aqueous solution of the Taishan mycin, the tomato roots are treated by the ultrafine Alternaria alternata Taishan variant fermentation liquor and 0.02% of the Taishan mycin at the initial stage of the tomato leaf curl virus disease, and the prevention and treatment effect can reach 86.0% -98.0% after 14 days and 28 days after the treatment. Compared with spraying, the Alternaria tenuissima Taishan variant fermentation liquid and the Taishan mycin are used for root irrigation, and the prevention and treatment effect on tomato leaf curl virus diseases is better.
Example 4 prevention of Capsicum virens by Alternaria tenuissima SDTF016 fermentation broth and extract
The Alternaria tenuissima SDTF016 is fermented and extracted according to the fermentation method in the embodiment 2, the tebufenomycin is obtained, the sterilized fermentation liquor (the content of the tebufenomycin is about 0.02%) and the tebufenomycin are obtained, and the 0.02wt% aqueous solution of the tebufenomycin is prepared by 0.1% TX-100 aqueous solution. Selecting pepper greenhouses with severe occurrence of virus diseases mainly including Cucumber Mosaic Virus (CMV), pepper mild mottle virus (PMMoV), broad bean wilting virus 2 (BBWV 2) and Tobacco Mosaic Virus (TMV), performing a test, performing spray control on the whole pepper plant, diluting the fermentation liquor or the water agent respectively by 250 times and 500 times, spraying 1 time every 7 days at the initial stage of virus disease occurrence, continuously spraying 3 times, counting disease indexes 7 days and 14 days after the last application, and calculating the control effect.
TABLE 3 prevention of Capsicum virens by Streptomyces tenuis SDTF016 broth and telithromycin
Figure DEST_PATH_IMAGE006
The data in the table are counted by a new repeated polarization method, and the data containing the same letters are not obvious in difference.
As shown in Table 3, the field spraying of the degerming fermentation liquid and the Taishan mycin water aqua can prevent and treat virus diseases mainly comprising CMV, PMMoV, BBWV2 and TMV, the fermentation liquid of the Taishan variant of the ultra-fine alternaria is diluted by 250 times and 500 times in the initial period of occurrence, and the prevention and treatment effects of 7 days and 14 days after the last spraying are respectively more than 80% and 78%; 0.02% of the Taishan mycin AS is respectively diluted by 250 times and 500 times, and the control effect of 7 days and 14 days after the last medicine is respectively more than 77% and 75%. This indicates that the fermentation broth of the mutant of Taishan mountain of the bacterium Alternaria tenuissima and Taishan mycin also have a high effect of preventing and treating viral diseases caused by CMV, PMMoV, BBWV2 and TMV.
Example 5 growth promoting action of fermentation broth and extract of Alternaria tenuissima SDTF016 on plants
The Alternaria tenuissima SDTF016 is fermented and extracted according to the fermentation method in the embodiment 2, the tebufenomycin is obtained, the sterilized fermentation liquor (the content of the tebufenomycin is about 0.02%) and the tebufenomycin are obtained, and the 0.02wt% aqueous solution of the tebufenomycin is prepared by 0.1% TX-100 aqueous solution. The samples were diluted 1000 times and 2000 times respectively to treat cucumber seeds (variety: Jinzhen 11), and 20 seeds per dish were treated for 10 dishes. At 96 hours after the treatment, the number of sprouts, the number of fibrous roots and the length of roots were counted.
TABLE 4 effects of fermentation broth of mutant Taishan of Tenssezia tenuissima and Taishan mycin on germination and growth promotion of cucumber seeds
Figure DEST_PATH_IMAGE008
The data in the table are counted by a new repeated polarization method, and the data containing the same letters are not obvious in difference.
As can be seen from Table 4, the fermentation broth of the mutant of the Alternaria tenuissima Taishan mountain was diluted 1000 times and 2000 times respectively to treat cucumber seeds (variety: Jinzhu 11), and the germination rates were respectively improved by 9.76% and 9.23%, the root lengths were improved by 22.7% and 21.2%, and the number of fibrous roots was increased by 36.2% and 35.6% 96 hours after the treatment; 0.02% of Mount Taishan mycin AS is respectively diluted by 1000 times and 2000 times to process cucumber seeds (variety: Jinzhu 11), the germination percentage is respectively improved by 9.02% and 8.55% compared with the contrast, the root length is improved by 20.9% and 20.4%, and the number of fibrous roots is increased by 31.0% and 27.4% 96 hours after the treatment; has obvious effect of promoting the germination and rooting of the cucumbers.
The method comprises the steps of obtaining fermentation liquor by using alternaria tenuissima SDTF016 according to the method in the embodiment 2, obtaining a solid crude extract, preparing the crude extract into a 500-time solution by using sterile water, soaking cucurbita pepo, tomatoes, hot peppers, eggplants, wheat, corns, cottons and tobaccos, and measuring the influence on the germination rate, wherein the result shows that the germination rate of the seeds can be remarkably improved by 5%. Compared with the clear water control, the complete germination time of each seed is advanced by 1-1.5 days, and the effect of forming the full-length seedlings is achieved.
The alternaria tenuissima SDTF016 is used for obtaining sterile fermentation liquor according to the method in the embodiment 2, and the tomatoes, the hot peppers, the tobaccos and the strawberries in the vegetative growth period are subjected to root irrigation treatment according to 50 mL/plant, and the treatment is carried out once every 7 days for 5 times in total. Pot culture results showed an average increase of 3.5% in tomato stem thickness, 5.0% in pepper stem thickness, 9.3% in tobacco ground dry weight and 5.6% in strawberry ground dry weight compared to the clear water control.
The fungus-containing fermentation liquor is obtained by using alternaria tenuissima SDTF016 according to the method in the embodiment 2, root irrigation treatment is carried out on 3-year-old winter jujubes after grafting, the root irrigation treatment is carried out respectively in 3 periods of pre-germination, pre-flowering and fruit expansion period, 5L of each plant is carried out, clear water is adopted for comparison, 10 plants are treated, and the yield is calculated for each plant. Compared with the control, the yield is increased by more than 15%.
Example 6 Effect of fermentation broth and extract of Alternaria tenuissima SDTF016 on crop stress resistance
The Alternaria tenuissima SDTF016 is fermented and extracted according to the fermentation method in the embodiment 2, the tebufenomycin is obtained, the sterilized fermentation liquor (the content of the tebufenomycin is about 0.02%) and the tebufenomycin are obtained, and the 0.02wt% aqueous solution of the tebufenomycin is prepared by 0.1% TX-100 aqueous solution. The samples were sprayed on cherry trees before late spring coldness. And 7d after treatment, counting the freezing injury rate and calculating the anti-freezing effect.
TABLE 5 anti-freeze effect of Alternaria tenuissima Taishan variant fermentation broth and Taishan mycin on cherries
Figure DEST_PATH_IMAGE009
The results in Table 5 show that the Alternaria tenuissima SDTF016 obtained by the method in the example 2 can obtain sterile fermentation liquor and a metabolite telithromycin thereof, has good stress resistance effect on cherry trees, and has obvious antifreezing effect.
The Alternaria tenuissima SDTF016 is fermented to obtain degerming fermentation liquor (the content of the Mount Taishan mycin is about 0.02 percent) to carry out salt tolerance of wheat and herbicide hazard of corn for tests: irrigating root wheat seedlings with 50-500mM sodium chloride solution, spraying and sterilizing the stem and leaf fermentation broth, wherein the growth amount of the wheat seedlings under the concentration of 50mM is not obviously different from that of a control, and the complete growth inhibition concentration of wheat is increased from 400mM to 450 mM; the result shows that the fermentation liquor of the Alternaria tenuissima SDTF016 can relieve the adverse environment damage caused by salt stress. The corn stem and leaf which has slight to moderate phytotoxicity generated by the nicosulfuron, the mesotrione and the acetochlor are sprayed with degerming fermentation liquor respectively, and the results show that the fermentation liquor can reduce the slight to mild phytotoxicity of the nicosulfuron and the mesotrione, and the phytotoxicity is expressed by that the etiolation area or degree of the corn seedling with phytotoxicity is reduced and the heart leaves are greened earlier than that of a control (a culture medium control). The fermentation liquor can reduce moderate acetochlor phytotoxicity, and the leaves can be recovered to be normal earlier; this indicates that the fermentation broth of Alternaria tenuissima SDTF016 can reduce the phytotoxicity of common herbicides on corn.
Example 7 evaluation of environmental impact of fermentation broth and extract of Alternaria tenuissima SDTF016
The method comprises the steps of fermenting the alternaria tenuissima SDTF016 according to the fermentation method in the embodiment 2, extracting the telithromycin, obtaining degerming fermentation liquor (the content of the telithromycin is about 0.02%) and the telithromycin, preparing solutions with different test concentrations by using a 0.1% TX-100 aqueous solution of the telithromycin, and evaluating the acute toxicity of the telithromycin on to environmental organisms such as silkworms, birds and earthworms by using clear water as a control.
TABLE 6 acute toxicity of fermentation broth of the variety Taishan of Tenelian tenziella and 0.02% aqueous solution of Taishan mycin to silkworm (leaf dipping)
Figure DEST_PATH_IMAGE010
TABLE 7 acute oral toxicity of fermentation broth of Taishan variant of Tenonese tenuissima and aqueous solution of 0.02% Taishan mycin on quail
Figure DEST_PATH_IMAGE012
TABLE 8 acute toxicity of fermentation broth of the variety Taishan mountain of the bacterium Tenelix and aqueous solution of 0.02% Taishan mycin to earthworm
Figure DEST_PATH_IMAGE013
The data in tables 6-8 show that the fermentation broth of the tengcia tenuissima and the telithromycin have no acute toxicity expression on the silkworms, the birds (quails) and the earthworms after different dosages of treatment, and are low-toxicity products. This shows that the fermentation broth of the Taishan variant of the Alternaria tenuissima and the Taishan mycin have no adverse effect on the environmental organisms silkworm, bird (quail) and earthworm and are environment-friendly.
Example 8 preparation of an aqueous Fertilizer containing the fermentation product of Alternaria tenuissima SDTF016
The alternaria tenuissima SDTF016 is fermented according to the fermentation method in the embodiment 2 to obtain a degerming fermentation liquid (the content of the telithromycin is about 0.05 percent), then macroelements and microelements are added, the mixture is stirred, then the mixture is stood still and filtered, and the liquid water-soluble fertilizer containing the alternaria tenuissima SDTF016 fermentation product is obtained through filling.
Example 9 bacterial manure preparation containing Alternaria tenuissima SDTF016
Fermenting Alternaria tenuissima SDTF016 according to the fermentation method in example 2 to obtain fermentation liquor, concentrating to 10 times, adsorbing with humic acid, adding organic fertilizer, extruding, granulating, drying, and grading to obtain the product containing Alternaria tenuissima SDTF016 of at least 108cfu/g bacterial manure.
Example 10 preparation of Water-soluble granule of Mount Taishan mycin (SG)
The Alternaria tenuissima SDTF016 is fermented according to the fermentation method in the embodiment 2 to obtain a sterilized fermentation liquid, the sterilized fermentation liquid is concentrated to the content of the telangin of about 10%, 2% of surfactant is added, then urea and cane sugar are used as fillers to obtain wet materials, and then the wet materials are extruded, granulated, dried and granulated to obtain the 1% of telangin water-soluble granules.
EXAMPLE 11 preparation of Mount Taishan mycin Soluble Powder (SP)
The alternaria tenuissima SDTF016 was fermented according to the fermentation method of example 2 to obtain a sterilized fermentation broth, and concentrated to about 1% of the content of the telangin, 2% of a surfactant was added to obtain a spray solution, and then spray-dried to obtain a water-soluble powder of telangin.

Claims (10)

1. A strain of Alternaria tenuissima (Alternaria alternata) SDTF016 with preservation number of CCTCC M2021495.
2. A fermentation broth or extract of alternaria tenuissima according to claim i; the extract is an ethyl acetate extract of fermentation liquor.
3. The extract according to claim 2, wherein the main effective components of the extract are dibutyl phthalate, 4-methyl-5-thiazole ethanol, 2-methoxy resorcinol, p-hydroxyphenol ethanol, 4-coumaric acid and nicotinic acid.
4. The fermentation broth of claim 2, comprising or not comprising bacteria.
5. A process for the preparation of an extract of Alternaria tenuissima as claimed in claim I, comprising the steps of:
(1) filtering the fermentation liquor for sterilization and deproteinization to obtain filtrate;
(2) passing the filtrate through silica gel column with ethyl acetate as eluent to obtain effluent;
(3) the eluate is stripped of the leacheate to obtain a solid crude extract.
6. The method of claim 5, further comprising a process for purifying the crude extract, comprising the steps of:
(i) dissolving the crude extract, separating by liquid chromatography, and collecting eluate with absorption peak at 214 nm wavelength;
(ii) removing solvent from the effluent to obtain refined extract.
7. Use of Alternaria tenuissima according to claim I for the preparation of a fertilizer or a pesticide for controlling phytopathogenic viruses; the plant pathogenic virus is selected from tomato leaf curl virus, cucumber mosaic virus, pepper mild mottle virus, broad bean wilting virus 2 and tobacco mosaic virus;
the fertilizer has the effects of promoting the growth of plants, improving the quality and improving the stress resistance and disease resistance of the plants; the plant is selected from Cucurbita pepo, tomato, capsicum, eggplant, wheat, corn, cotton, tobacco, strawberry or cherry.
8. A formulation comprising alternaria tenuissima according to claim i, a fermentation broth or an extract according to claim 2, characterized in that it is a fertilizer or pesticide formulation.
9. The preparation according to claim 8, wherein the preparation is selected from a microbial agent, a bio-organic fertilizer or a water-soluble fertilizer.
10. The preparation of claim 8, wherein the preparation is selected from water aqua, water soluble granule or water soluble powder.
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