CN110301455B - Application of volatile substance generated by streptomyces asjones in plant disease control - Google Patents
Application of volatile substance generated by streptomyces asjones in plant disease control Download PDFInfo
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- CN110301455B CN110301455B CN201910604694.2A CN201910604694A CN110301455B CN 110301455 B CN110301455 B CN 110301455B CN 201910604694 A CN201910604694 A CN 201910604694A CN 110301455 B CN110301455 B CN 110301455B
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- A—HUMAN NECESSITIES
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- 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
- A01N25/00—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests
- A01N25/18—Vapour or smoke emitting compositions with delayed or sustained release
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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
- A01N27/00—Biocides, pest repellants or attractants, or plant growth regulators containing hydrocarbons
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01N31/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic oxygen or sulfur compounds
- A01N31/06—Oxygen or sulfur directly attached to a cycloaliphatic ring system
-
- 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
- A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
- A01N37/10—Aromatic or araliphatic carboxylic acids, or thio analogues thereof; Derivatives thereof
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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
- A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
- A01N43/02—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms
- A01N43/04—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom
- A01N43/20—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom three- or four-membered rings
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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
- A01N55/00—Biocides, pest repellants or attractants, or plant growth regulators, containing organic compounds containing elements other than carbon, hydrogen, halogen, oxygen, nitrogen and sulfur
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01N55/00—Biocides, pest repellants or attractants, or plant growth regulators, containing organic compounds containing elements other than carbon, hydrogen, halogen, oxygen, nitrogen and sulfur
- A01N55/02—Biocides, pest repellants or attractants, or plant growth regulators, containing organic compounds containing elements other than carbon, hydrogen, halogen, oxygen, nitrogen and sulfur containing metal atoms
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- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/02—Oxygen as only ring hetero atoms
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- C12P5/00—Preparation of hydrocarbons or halogenated hydrocarbons
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- C12P5/00—Preparation of hydrocarbons or halogenated hydrocarbons
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- C12P7/00—Preparation of oxygen-containing organic compounds
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Abstract
The invention discloses application of volatile substances generated by Streptomyces askinsonii in plant disease control, and particularly discloses that the volatile substances generated by the Streptomyces askinsonii can inhibit the growth of various pathogenic bacteria such as Phytophthora litchi and the like, and the inhibition rate is 63-100%. The invention further defines 14 compounds with obvious bacteriostatic action in the volatile substances generated by the streptomyces ashsonii, and provides a thought for researching the prevention and treatment medicines of phytopathogen such as peronophythora litchi and the like in the future.
Description
Technical Field
The invention relates to the field of biological control of plant diseases, in particular to application of volatile substances generated by streptomyces asonyssinus in plant disease control.
Background
Volatile substances are a class of low molecular weight (300Da) compounds, non-polar, volatile, with high vapor pressure, which can reach 0.01kPa (Morath et al, 2012) at room temperature. Moore-Landecker & Stotzky reported earlier in 1973 that volatile substances produced by actinomycetes caused conidiophore atrophy of Aspergillus and vacuoles of Penicillium, Fusarium, Trichoderma viride, and the like increased. Strobel et al (2001) reported that volatile substances produced by the endophytic fungus Muscodor albus can control a variety of postharvest diseases (Mercier, 2004; Mercier et al, 2005) and soil-borne diseases by fumigation. In recent years, more and more researchers have been engaged in the study of microbial volatile substances. Wanmingtuo (2008) reported that the Pretz zipper mold can effectively inhibit the occurrence of gray mold of strawberry, rice sheath blight and sclerotinia rot of colza in a closed container. Volatile substances generated by streptomyces globisporus JK-1 cause botrytis cinerea, hypha deformity of penicillium citrinum, cell membrane damage, hypha growth, spore germination and conidiospore formation are inhibited, the penicillium citrinum can be effectively controlled (Li Chaili, 2011), and Zhang Qing (2014) reports that CanR-46 is endophytic fungi of rape, and volatile substances generated by the CanR-46 can inhibit the growth of sclerotinia sclerotiorum.
Therefore, the research on the control of pathogenic bacteria by volatile substances generated by microorganisms has achieved certain results. However, the research on the Streptomyces avermitilis volatile substances is relatively few, and researchers do not clearly understand the important role of the Streptomyces avermitilis in plant disease control, so that intensive research is urgently needed.
Disclosure of Invention
The invention provides an application of a volatile substance generated by Streptomyces askinsonii in plant disease control, which effectively fills up the technical blank of the Streptomyces askinsonii in plant disease control.
The technical scheme adopted by the invention is as follows:
the invention provides an application of a volatile substance generated by streptomyces asjones in plant disease control, wherein the plant disease is caused by phytophthora litchi, phytophthora capsici, phytophthora taro, pythium aggregatum, alternaria, fusarium graminearum, colletotrichum anthracnose, rice blast, grape canker or common scab. The volatile substance has obvious inhibition effect on phytophthora litchi hypha growth, sporangium yield and oospore yield.
The invention further analyzes by GC-MS, and confirms that the components of volatile substances comprise: tris (trimethylsilyl) arsenite, 2-methylisotrichol, octamethylcyclotetrasiloxane, tridecane, dimethyltetracosane, hexyl benzoate, 2,6, 10-methyldodecane, tetradecane, cis-7, 8-epoxy-2-methyloctadecane, 2,6, 10-methyltridecane, n-octacosane, n-pentadecane, n-hexadecane and longifolene.
The preparation method of the volatile substance comprises the following steps:
s1: performing streak culture on the Streptomyces avermitilis strain on a PDA (PDA) plate for 7d, scraping fresh spores, inoculating the fresh spores into an ISP2 culture medium, placing the fresh spores on a shaking table for shake culture, preparing a spore suspension, and inoculating the spore suspension onto a wheat grain culture medium for culture to obtain a Streptomyces avermitilis wheat grain culture;
s2: the wheat grain culture was placed in a vial, sealed and extracted.
Further, in step S1, the Streptomyces asnyli strain was streaked on PDA plate for 7d, scraped fresh spores were inoculated into a Erlenmeyer flask containing 100mL of ISP2 medium, and then placed on a shaker to be cultured with shaking at 200rpm and 28 ℃ for 3d to prepare a culture having a concentration of 1 × 107Inoculating spore/mL spore suspension onto wheat kernel culture medium at a ratio of 1mL/100g, shaking, and culturing at 25 deg.C for 20 days, wherein the wheat kernel culture medium is shaken every 3 days.
Step S2 is: weighing 20d culture of the wheat grains of the streptomyces askinsus, putting the culture into a bottle, sealing the bottle, standing the bottle at 25 ℃ for 12 hours, and extracting the bottle by using an SPME extraction head.
Compared with the prior art, the invention has the beneficial effects that:
(1) the volatile substances generated by the streptomyces asjones can inhibit the growth of various pathogenic bacteria such as peronophythora litchi and the like, and the inhibition rate reaches 63-100%.
(2) The 14 compounds with obvious bacteriostatic action in the volatile substances generated by the streptomyces asceticus are determined, and the method provides a direction for researching the control drugs of the peronophythora litchi.
(3) The method has the advantages of determining that the volatile substances generated by the streptomyces askinsonii have obvious inhibition effects on the growth of phytophthora litchi hyphae, the yield of sporangium and the yield of oospore.
Drawings
FIG. 1 is a graph showing the effect of different culture times on the bacteriostatic activity of the volatile substance produced by Streptomyces askinsonii TJGA-19; in the figure, a, b, c and d are respectively the treatment of the control and the culture of the streptomyces asjones TJGA-19 cultured for 10d, 20d and 30d on the wheat culture medium;
FIG. 2 is a statistical chart of the results of the effects of different culture times on the bacteriostatic activity of the volatile substances generated by Streptomyces askinsonii TJGA-19;
FIG. 3 is a graph showing the effect of volatile substances produced by different amounts of Streptomyces askinsonii TJGA-19 wheat grain cultures on the growth of phytophthora litchi hyphae; in the figure, a, b, c, d, e, f, g, h and i are respectively the control and the treatment of wheat grain cultures of the streptomyces ashsonii TJGA-19 with 2g/L, 4g/L, 8g/L, 12g/L, 16g/L, 24g/L, 32g/L and 40 g/L;
FIG. 4 is a statistical chart showing the effect of volatile substances produced by different amounts of Streptomyces askinsonii TJGA-19 wheat grain culture on the growth of phytophthora litchi hyphae;
FIG. 5 is a statistical chart showing the effect of volatile substances produced by different amounts of Streptomyces askinsonii TJGA-19 wheat grain culture on the yield of phytophthora litchi sporangia;
FIG. 6 is a graph showing the effect of various amounts of volatile substances produced by the culture of Streptomyces askinsonii TJGA-19 wheat kernels on the yield of oospores; a, b, c, d, e, f, g, h, i, j and k are respectively a reference and 3g/L, 4g/L, 6g/L, 8g/L, 12g/L, 16g/L, 24g/L, 32g/L and 40g/L of the culture of the TJGA-19 wheat kernels of the streptomyces ashsonii;
FIG. 7 is a statistical chart showing the effect of various amounts of volatile substances produced by the culture of Streptomyces askinsonii TJGA-19 wheat kernels on the yield of oospores;
FIG. 8 is a scanning electron microscope graph showing the effect of volatile substances on phytophthora litchi morphology; a, b and c are controls; d, e and f are 100g/L of wheat kernel culture treatment of Streptomyces askinsonii TJGA-19.
FIG. 9 is a diagram of the effect of the observation of the effect of volatile substances on the subcellular structure of phytophthora litchi hyphae by a transmission electron microscope; a, b and c are controls; d, e and f are 100g/L of wheat kernel culture treatment of Streptomyces askinsonii TJGA-19. a and d are longitudinal sections of sporangium; b, e is a transverse section of hypha; c, f is a longitudinal section of hypha; n: cell nucleus; w: a cell wall;
FIG. 10 is a diagram showing the effect of volatile substances on the control of in vitro leaf litchi frost blight; a, b, c, d and e are respectively a control and 8g/L, 16g/L, 24g/L and 32g/L of wheat kernel culture treatment of the streptomyces ashsonii TJGA-19;
FIG. 11 is a statistical chart of the control effect of the volatile substances on in vitro leaf litchi downy blight.
Detailed Description
The present invention will be described in further detail with reference to the following detailed description and accompanying drawings.
The experimental methods used in the examples of the present invention are all conventional methods unless otherwise specified.
The materials, reagents and the like used in the examples of the present invention can be obtained commercially without specific description.
According to the embodiment of the invention, litchi varieties selected from litchi leaves used for experimental inoculation are Huai branches.
The litchi leaves used in the embodiment of the invention are collected from litchi orchards in experimental bases of horticulture academy of agriculture university in south China, the leaves which are tender, consistent in shape, size and leaf age, free of diseases and insect pests and free of mechanical damage are selected, and the leaves are washed clean by running water and dried at room temperature before inoculation.
The Streptomyces arvensis TJGA-19(Streptomyces abikoensis TJGA-19) is separated and stored in a plant disease biological prevention and control laboratory of plant protection college of Hainan university and can be obtained by a conventional method in the prior art.
The phytophthora litchi, phytophthora capsici, phytophthora taro and other pathogenic bacteria in embodiment 8 of the invention are separated and stored in a plant disease biological prevention and control laboratory of plant protection academy of Hainan university, and can be obtained by a conventional method in the prior art.
The wheat grain culture medium of the embodiment of the invention comprises the following components: adding sufficient water into wheat, boiling until the skin cracks, filtering, air drying, packaging, and sterilizing.
Example 1 preparation of a volatile substance produced by Streptomyces avermitilis TJGA-19
Streak-culturing strain TJGA-19 on PDA plate for 7 days, scraping fresh spore, inoculating to ISP2 containing 100mL, and culturingA250 mL Erlenmeyer flask of the culture medium was then placed on a shaker at 200rpm and cultured at 28 ℃ for 3 days with shaking to prepare a spore suspension (about 1 × 10)7spores/mL) was inoculated at a ratio of 1mL/100g (V/W) onto a sterile wheat kernel medium. After shaking, the cells were cultured at 25 ℃ for 20 days, during which the wheat grain medium was shaken every 3 days, so that the strain TJGA-19 was grown uniformly.
Example 2 Effect of cultivation time on bacteriostatic Activity of Streptomyces avermitilis TJGA-19 for producing volatile substances
In large culture dishes: (
h is 30mm and the total internal volume is 500mL) are placed at the bottom of four small culture dishes
Wherein 3 small culture dishes contain about 5mL of PDA culture medium, and a piece of Phytophthora litchi strain cake is inoculated in the center of the culture medium
In another small dish, 40g/L of Streptomyces askinsonii TJGA-19 wheat grain culture (cultured at 25 ℃ for 10d, 20d and 30d respectively) was placed, 40g/L of sterile wheat grain was used as a control, and then the large dish was sealed. Culturing at 25 deg.C for 6d, measuring colony diameter and calculating bacteriostasis rate. Each treatment was repeated 3 times and the experiment was repeated 3 times. The results are shown in FIGS. 1 and 2.
Hypha growth inhibition (%) (control colony diameter-treated colony diameter)/control colony diameter × 100
The results show that:
the culture time is different, and the bacteriostatic activity of the volatile substance generated by the streptomyces asjones TJGA-19 is greatly different. The bacteriostatic activity of the volatile substances generated by culturing the streptomyces asjones TJGA-19 on the wheat grain culture medium for 20 days is strongest, the bacteriostatic rate reaches 74.1%, and the bacteriostatic rates of the treatments of culturing 10 days and 30 days are respectively 54.9% and 63.7%, which are obviously lower than that of the treatment of culturing 20 days.
Example 3 collection and identification of volatile substances
Weighing 4g of 20d cultured Streptomyces avermitilis TJGA-19 wheat kernel culture, placing the culture in a 20mL small bottle, sealing the small bottle with a piece of tin foil paper, placing the small bottle at 25 ℃ for 12h, then inserting an SPME extraction head into the bottle, pushing out a fiber head [ metal alloy (PDMS 100 mu m) ] to adsorb for 30min, withdrawing the fiber head, manually injecting the extraction head into a GC-MS (Agilent 7890B American), desorbing the extraction head at 250 ℃ for 3min, and carrying out GC-MS analysis. GC-MS operating conditions were referred to (Strobel, et al 2001). The computer automatically compares the obtained gas composition Mass spectra with data from the international standard database (NIST 14Mass Spectrometry library) to identify the composition of the volatile substance. The control and the culture of wheat grains of Streptomyces askinsonii TJGA-19 were removed by using a sterile blank equal amount of wheat as a control. Each treatment was 3 replicates.
The volatile substances produced by 20d culture of Streptomyces askinsonii TJGA-19 were detected by GC-MS to obtain 14 compounds (Table 1). Most of these compounds belong to alkenes, esters, organic acids, alkanes, etc.
TABLE 1
Example 4 Effect of volatile substances on Peronospora litchi hypha growth and sporangium production
Bottom of 4 small culture dishes
Placed in a large culture dish
In 3 small culture dishes containing about 5mL of PDA culture medium, a cake of Phytophthora litchi was inoculated in the center of the culture medium
The wheat grain culture of Streptomyces askinsonii TJGA-19 of example 1 was placed on another small dish, the large dish was sealed with a sealing film, incubated at 25 ℃ for 6 days, the diameter of the colony was measured, the surface of the colony was washed with sterile water, the hyphae were filtered off, and the sporangium yield was counted. 8 treatments are set in the experiment, the amount of the Streptomyces askinsonii TJGA-19 wheat grain culture is respectively 2g/L, 4g/L, 8g/L, 12g/L, 16g/L, 24g/L, 32g/L and 40g/L, and the same amount of sterile blank wheat grains are used as a control. Each treatment was repeated 3 times and the experiment was repeated 3 times. The results are shown in FIGS. 3-6.
The experimental results show that: the larger the amount of wheat kernel culture, the stronger the bacteriostatic activity. When cultured at 25 ℃ for 6d, the colony diameter of the control is 47.3mm, the colony diameters of the cultures are 46.8mm, 46.2mm and 45.4mm respectively when the culture amount is 2g/L, 4g/L and 8g/L, and the colony diameters are not significantly different from the control, and when the culture amount is 12g/L, 16g/L, 24g/L, 32g/L and 40g/L, the colony diameters are 39.6mm, 33.2mm, 26.3mm, 20.6mm and 1.22mm respectively and are significantly lower than the colony diameter of the control.
The control sporangia yield was 129.2 × 10 when cultured at 25 ℃ for 6 days4The yield of sporangia is 124.7 × 10 when the culture amount is 2g/L and 4g/L4、123.5×104The sporangia yield in the culture of 8g/L, 12g/L, 16g/L and 24g/L treatments was 78 × 104、51.3×104、5.3×104And 2.6 × 104Sporangia/dish, no sporangia formation in the treatment of culture amount of 32g/L, 40 g/L.
Example 5 Effect of volatile substances on Peronospora litchi production
Bottom of 4 small culture dishes
Placed in a large culture dish
In 3 small culture dishes containing about 3mL of CA medium, a cake of Phytophthora litchi was placed in the center of the plate
The Streptomyces askinsonii TJGA-19 wheat grain culture of example 1 was placed on another small dish, the large dish was sealed with a sealing film, incubated at 25 ℃ in the dark for 14 days, and three hypha blocks were randomly cut in an area 10mm from the inoculation site
The resulting mixture was placed in a 10mL centrifuge tube, 3mL of ultrapure water was added, and homogenized with a high-speed homogenizer (6000rpm) for 2min, and then the number of oospores per unit area was determined by counting the number of 50. mu.L of oospores under a microscope (Flier et al, 2001). The experiment sets 10 treatments, the amount of the Streptomyces askinsonii TJGA-19 wheat grain culture is respectively 3g/L, 4g/L, 6g/L, 8g/L, 12g/L, 16g/L, 24g/L, 32g/L and 40g/L, and the same amount of sterile blank wheat grains are used as a control. Each treatment was repeated 3 times and the experiment was repeated 3 times.
Oospore yield per unit area (one/cm)2) Number of oospores × 60/2.36 in 50. mu.L suspension
The experimental results show that: the control group and the 3g/L treatment group both produced a large number of oospores, 2686.4 oospores/cm respectively, when cultured at 25 ℃ in the dark for 14 days22669.5 pieces/cm2(ii) a When the amount of the culture was 4g/L, the oospore yield was 2347.4 spores/cm2Significantly decreased compared to control; when the amount of the culture is 6g/L and 8g/L, the oospore yield is sharply reduced to 1466.1 oospores/cm257.3 pieces/cm2(ii) a When the amount of the culture was increased to 12g/L, 16g/L, 24g/L, 32g/L, 40g/L, no oosporulation occurred (FIG. 7).
Example 6 influence of volatile substances on Peronospora litchi ultrastructure
The phytophthora litchi is inoculated on a CA culture medium, cultured for 3 days at 25 ℃, buckled with a culture dish filled with 100g/L streptomyces asjones TJGA-19 wheat grain culture (prepared by the method of example 1), sealed, continuously cultured for 3 days, cut into about 8mm multiplied by 5mm multiplied by 3mm fungus blocks from the edge of a bacterial colony, cut 3 fungus blocks for each treatment, immediately put into pre-cooled 4% glutaraldehyde fixing solution, and used for preparing samples by a scanning electron microscope at low temperature overnight; the mycelia were gently scraped with a sterile toothpick and pre-fixed in pre-cooled 2.5% glutaraldehyde for transmission electron microscopy sampling. An equal amount of sterile blank wheat grain was used as a control.
Preparing a sample by a scanning electron microscope: samples were incubated at 4% glutaraldehyde fixation (using 0.1 mol. L)-1prepared with phosphate buffer solution with pH of 7.2) at 4 ℃ overnight, and then the mixture is added with 0.1 mol.L-1Rinsing with phosphate buffer solution with pH 7.2 for 3 times, 15min each time, 1% osmic acid fixation for 1h, sequentially dehydrating with 30%, 50%, 70%, 80% and 90% ethanol for 10min each time, then dehydrating with 100% ethanol for 10min twice, finally transiting with isoamyl acetate for 15min twice, drying at critical point, sticking, coating, observing with LEO-1530VP scanning electron microscope, and taking pictures at 5 KV.
Preparing a sample by a transmission electron microscope: samples were fixed at 2.5% glutaraldehyde (using 0.1 mol. L)-1p H phosphate buffer solution of 7.2) at 4 ℃ overnight, 0.1 mol. L-1Rinsing with phosphate buffer solution of pH 7.2 for 3 times, each time for 15min, solidifying after 1% osmic acid, cooling at 4 deg.C overnight, dehydrating with ethanol, infiltrating with epoxy resin, embedding, slicing into 70nm ultrathin sections with an ultrathin microtome (EM UC7, Leica), double staining with uranium acetate and lead citrate, and observing the inner structure of hyphal cells and taking pictures with a transmission electron microscope (Tecnai, FEI) at 100 kV.
Scanning electron microscope results show that phytophthora litchi mycelium was uniform, slender, smooth and full in surface, spherical in sporangium, full and smooth in cell wall in the control treatment (fig. 8a, b and c); when the peronophythora litchi mycelium was exposed to volatile gas produced by 100g/L Streptomyces askinsonii TJGA-19 wheat grain culture, the mycelium collapsed and shriveled, and the sporangium cell wall was rough and depressed (FIG. 8d, e, f). The results of transmission electron microscopy showed that the control sporangia had uniform cytoplasm, aligned organelles, and normal nuclear morphology (FIGS. 9a, b, c); mitochondria and nuclei disappeared, vacuoles increased, and enlarged in 100g/L of wheat cultures (FIG. 9d, e, f).
Example 7 controlling Effect of volatile substances on Perilla Frondosa Frost blight in vitro leaves
In a sterile large petri dish (
h 30mm) is laid with a layer of filter paper
Spraying 8mL of sterile water on the filter paper for keeping moisture, placing a culture dish with the diameter of 90mm at the center of the filter paper, placing different quantities of wheat grain cultures of Streptomyces askinsonii TJGA-19 (prepared by the method of example 1) in the culture dish, then spreading 10 leaves on the outer edge of the filter paper with the leaf back facing upwards, and taking 2 μ L of sporangium suspension (2 × 10)4sporangia/mL) were dropped onto the vein and the large dish was immediately sealed. And culturing at 25 deg.c for 48 hr, and counting the length of the disease spot. 4 treatments were set up in the experiment, and the amount of the Streptomyces askinsonii TJGA-19 wheat grain culture was 8g/L, 16g/L, 24g/L and 32g/L, respectively, with an equivalent amount of sterile blank wheat grains as a control. Each treatment had 30 leaves and each treatment had 3 replicates for a total of 90 leaves, and the experiment was repeated 3 times. The results are shown in FIGS. 10-11.
The results show that: the volatile substance generated by the streptomyces asjones TJGA-19 has good control effect on the frost blight of the litchi leaves in vitro. 48h after inoculation, the diameter of the control lesion is 26.7mm, and when the amount of the Streptomyces askinsonii TJGA-19 wheat kernel culture is increased from 8g/L to 32g/L, the diameter of the lesion is reduced from 21.9mm to 3 mm.
EXAMPLE 8 determination of the bacteriostatic Profile of the volatile substances produced by Streptomyces avermitilis TJGA-19
Inoculating pathogenic bacteria to be tested on PDA culture medium, culturing in 25 deg.C incubator for 6 days, and perforating with hole puncher
Preparing fungus cake at the outer edge of colony, placing in the center of fresh PDA plate, weighing 32g/L Streptomyces asnylensis TJGA-19 wheat grain culture (see example 1 for preparation method)Transferring into a culture dish of 90mm, oppositely buckling the bottoms of the two dishes together, sealing, and making into a double-dish buckling device with pathogenic bacteria on the upper part and a wheat grain culture on the lower part, and using 32g/L sterile wheat grains as a control. The double-dish buckling device is placed in an incubator at 25 ℃ for culture, observation is carried out every day, the diameter of a bacteriostatic circle is measured when pathogenic bacteria contrast colonies grow over the culture dish (a cross method), the inhibition rate is calculated, 3 times of treatment are carried out every time, and the experiment is repeated for 3 times. The results are shown in Table 2.
Hypha growth inhibition (%) - (control colony diameter-treated colony diameter)/control colony diameter × 100%
Table 2: bacteriostatic spectrum of volatile substances generated by streptomyces asjones TJGA-19
Experimental results show that volatile substances generated by the streptomyces asonensis TJGA-19 can inhibit the growth of various plant pathogenic bacteria, wherein the volatile substances have the strongest inhibiting effect on phytophthora litchi, phytophthora capsici, phytophthora taro, pythium aggregatum, alternaria, fusarium graminearum, banana anthracnose, rice blast, grape canker and papaya scab, and the inhibiting rate reaches 63-100%.
The foregoing is a more detailed description of the present invention that is presented in conjunction with specific embodiments, and the practice of the invention is not to be considered limited to those descriptions. It will be apparent to those skilled in the art that a number of simple derivations or substitutions can be made without departing from the inventive concept.
Claims (3)
1. The application of the volatile substances generated by the streptomyces asjones in the prevention and treatment of plant diseases is characterized in that the plant diseases are plant diseases caused by phytophthora capsici or phytophthora taro; the volatile material consists of the following components: tris (trimethylsilyl) arsenite, 2-methylisotrichol, octamethylcyclotetrasiloxane, tridecane, dimethyltetracosane, hexyl benzoate, 2,6, 10-methyldodecane, tetradecane, cis-7, 8-epoxy-2-methyloctadecane, 2,6, 10-methyltridecane, n-octacosane, n-pentadecane, n-hexadecane and longifolene;
the preparation method of the volatile substance comprises the following steps:
s1: performing streak culture on the Streptomyces avermitilis strain on a PDA (PDA) plate for 7d, scraping fresh spores, inoculating the fresh spores into an ISP2 culture medium, placing the fresh spores on a shaking table for shake culture, preparing a spore suspension, and inoculating the spore suspension onto a wheat grain culture medium for culture to obtain a Streptomyces avermitilis wheat grain culture;
s2: the wheat grain culture was placed in a vial, sealed and extracted with a SPME extraction head.
2. The use of the volatile substance produced by Streptomyces avermitilis in the control of plant diseases according to claim 1, wherein in step S1, Streptomyces avermitilis is streaked on PDA plate for 7d, fresh spores are scraped and inoculated into a triangular flask containing 100mL of ISP2 medium, and then the flask is placed on a shaker and cultured with shaking at 200rpm and 28 ℃ for 3d to prepare the volatile substance with concentration of 1 × 107Inoculating spore/mL spore suspension onto wheat kernel culture medium at a ratio of 1mL/100g, shaking, and culturing at 25 deg.C for 20 days, wherein the wheat kernel culture medium is shaken every 3 days.
3. The use of volatile substances produced by streptomyces asjones in the control of plant diseases according to claim 1, characterized in that, step S2: weighing 20d culture of the wheat grains of the streptomyces askinsus, putting the culture into a bottle, sealing the bottle, standing the bottle at 25 ℃ for 12 hours, and extracting the bottle by using an SPME extraction head.
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