CN111334458B - A kind of biocontrol actinomycetes and its application in the prevention and treatment of ginger stem base rot or soybean blight - Google Patents
A kind of biocontrol actinomycetes and its application in the prevention and treatment of ginger stem base rot or soybean blight Download PDFInfo
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- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/465—Streptomyces
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- 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
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Abstract
The invention relates to a new biocontrol actinomycete and application thereof in the aspect of ginger stem basal rot or soybean epidemic disease prevention and control; the biocontrol actinomycete CGMCC No.18716 (Streptomyces scabies) provided by the invention can effectively prevent and treat soybean epidemic disease and ginger stem basal rot, simultaneously shows broad-spectrum plant disease resistance, is nontoxic to human and livestock, does not pollute the environment, has no residue, is beneficial to sustainable development of ecological environment, and also provides high-quality biological resources for researching and developing new environment-friendly biocontrol actinomycete inoculants.
Description
Technical Field
The invention relates to biocontrol actinomycetes and application thereof in the aspect of ginger stem basal rot or soybean epidemic disease prevention and control, and belongs to the technical field of crop biological prevention and control.
Background
Ginger belongs to the family zingiberaceae and the genus zingiber, belongs to perennial herb perennial root plants, and is a high-yield economic crop. With the continuous expansion of the planting area of ginger, the occurrence of ginger diseases also shows an increasing trend, especially the occurrence of ginger stalk base rot, which is also called as ginger soft rot, ginger pythium mold and the like, is a soil-borne disease caused by pythium infection, and has become one of the main problems in the healthy development of the ginger industry. At present, due to the difference of geographical environment and climatic conditions, pathogenic bacteria in main ginger production areas in northern China are mainly Pythium mycorrhizal fungi (Pythium mycorrhizal um), the main characteristics after the infection of the bacteria are shown in the initial stage of disease, water stain-like scabs with different sizes appear on the stem base part, the scabs are gradually enlarged, the leaves are yellow, the scabs surround the stem base part for a circle in the later stage of disease, the tissue of the stem base part is gradually browned and softened, finally, the stem base part is lodging and rotten and accompanied by malodor, the leaves are yellow and drooping, and the leaves are withered afterwards. When the humidity is high, soil is taken off, and white cotton floccules can be seen in the disease department and the soil 2 cm away from the ground surface.
Soybean is one of the Chinese important food crops, and the soybean epidemic disease caused by phytophthora sojae is a devastating disease which seriously affects the production of soybean. The germ survives in soil by oospores, becomes an initial infection source through winter, is high in humidity or rainy weather, is heavy in soil viscosity and is easy to attack, the yield is generally reduced by 10-30%, the yield is reduced by 50-70% in serious conditions, and some germs even are collected absolutely. The phytophthora sojae may be infected and damaged in any stage of soybean growth, especially the young root and hypocotyl of soybean may be infected most easily to cause rot and seedling damping-off, and the infection in seedling stage may cause root rot or stem rot to result in seedling wilting or death. Under humid conditions, the diseased part is usually in a water stain shape, and the disease is spread quickly, so that the diseased tissue dies quickly.
At present, the chemical prevention and control are mainly performed on soybean epidemic disease and ginger stem basal rot. The chemical agent soil fumigation has good prevention effect on soil-borne diseases, but the method has serious damage to the soil ecosystem and high cost, and is difficult to popularize and apply in a large range. Biological control can protect the virtuous circle of an ecosystem and the environmental safety, thereby reducing the environmental pollution caused by overuse of chemical pesticides and greatly saving the control cost.
The utilization of beneficial environmental microorganisms to control the occurrence of diseases and induce and improve the stress resistance of crops is a hotspot of research in recent years, particularly, the microorganisms have no pollution to the environment, can overcome the defects brought by chemical agent control, and arouse the interest of scientists in controlling diseases and coping with adverse environments by utilizing biological control means; particularly, the method has higher economic benefit and environmental benefit in agricultural production, accords with the development trend of protecting ecological environment and maintaining human health and safety, and can provide wide prospect for national economy of China and sustainable development of future agriculture.
Therefore, those skilled in the art would like to be able to screen out microorganisms that can control ginger stem rot or soybean blight.
Disclosure of Invention
In view of the above-mentioned problems and/or other problems of the related art, the present invention provides, in one aspect, a biocontrol actinomycete, wherein the biocontrol actinomycete is Streptomyces solani (Streptomyces scabies) deposited in the common microorganism center of the China committee for culture collection management of microorganisms with the collection number of CGMCC No. 18716.
In another aspect, the invention provides a bacterial preparation, wherein the bacterial preparation comprises an actinomycete microbial inoculum obtained by fermenting the biocontrol actinomycete.
Preferably, the fungus preparation comprises an actinomycete fungus agent obtained by fermenting the biocontrol actinomycete and an agriculturally and pharmaceutically acceptable auxiliary agent.
The invention further provides a preparation process of the microbial preparation, wherein the strains of the biocontrol actinomycetes are subjected to activation culture on a Gao's first culture medium, the activated strains are subjected to primary fermentation culture to obtain a seed solution, and the seed solution is subjected to secondary fermentation culture to obtain the actinomycetes microbial inoculum.
Preferably, the preparation method of the culture medium adopted by the first fermentation culture comprises the steps of dissolving 9.8-10.2 g of corn flour, 9.8-10.2 g of cane sugar, 1.9-2.1 g of sodium chloride, 2.9-3.1 g of ammonium sulfate and 1.9-2.1 g of calcium carbonate in distilled water for every 1000ml of culture medium, adjusting the pH value to 7.2-7.4, fixing the volume, and then performing high-temperature sterilization; the first fermentation culture adopts the conditions of 25-30 ℃ and the rotation speed of 170-.
Preferably, the preparation method of the culture medium adopted by the second fermentation culture comprises the steps of dissolving 9.8-10.2 g of soybean meal, 14.8-15.2 g of glucose, 1.9-2.1 g of sodium chloride, 2.9-3.1 g of ammonium sulfate and 1.9-2.1 g of calcium carbonate in every 1000ml of culture medium into distilled water, adjusting the pH value to 7.2-7.4, fixing the volume and then sterilizing at high temperature; the second fermentation culture adopts the conditions of 25-30 ℃ and the rotation speed of 170-; and after the second fermentation culture is finished, filtering by using gauze to obtain the actinomycete microbial inoculum.
The invention also provides an application of the biocontrol actinomycete in preventing and treating plant diseases, wherein the plant diseases are caused by phytophthora sojae, pythium nepalense, pythium Stapf, pythium spinulosum, pythium japonicum, pythium forest, pythium ultimum, pythium aphanidermatum, pythium gordonii, phytophthora capsici, phytophthora citri reticulata, fusarium graminearum or fusarium oxysporum.
Preferably, the plant disease is ginger stem rot or soybean epidemic disease.
The invention also provides application of the bacterial preparation in preventing and treating plant diseases, wherein the plant diseases are caused by phytophthora sojae, pythium nepalensis, pythium nodosum, pythium clotocidum, pythium spinulosum, pythium virens, pythium ultimum, pythium aphanidermatum, pythium gordonatum, phytophthora capsici, phytophthora citri reticulata, fusarium graminearum or fusarium oxysporum.
Preferably, the plant disease is ginger stem rot or soybean epidemic disease.
The biocontrol actinomycete CGMCC No.18716 (Streptomyces scabies) provided by the invention can effectively prevent and treat soybean epidemic disease and ginger stem basal rot, simultaneously shows broad-spectrum plant disease resistance, is nontoxic to human and livestock, does not pollute the environment, has no residue, is beneficial to sustainable development of ecological environment, and also provides high-quality biological resources for researching and developing new environment-friendly biocontrol actinomycete inoculants.
Detailed Description
The present invention will be further described with reference to specific embodiments, but the present invention is not limited to these specific embodiments.
Materials, reagents and the like used in the following embodiments are commercially available unless otherwise specified. The specific techniques or conditions are not indicated, and the procedures or conditions are described in the literature in the art (for example, refer to J. SammBruk et al, molecular cloning, A laboratory Manual, third edition, science Press, translated by Huang Petang et al) or in accordance with the product instructions.
First, the operation process of the present inventors for obtaining the biocontrol actinomycete 11X 1 (Streptomyces scabies) CGMCC No.18716 of the present invention by separation and screening from the natural environment will be described below.
1. Isolation of potential biocontrol bacteria present in vitro
1) Preparation of soil suspension: weighing 3g of soil sample (from soybean root soil of a soybean planting field in copper mountain area of Xuzhou city, Jiangsu province), placing the soil sample into a triangular flask (containing sterilized glass beads) filled with 27ml of sterile water, and performing shake culture for 30min at 150rpm of a shaking table;
2) 10-fold gradient dilution: standing for 5min, taking 1ml of soil suspension from triangular flask, placing in test tube containing 9ml of sterile water (diluted by 10 times), shaking, mixing, further diluting in gradient, and making into 10-2、10-3、10-4、10-5、10-6The soil dilution of (1);
3) plating culture: respectively taking 0.1ml from the gradient dilution, uniformly coating on a Gao's No. one culture medium (containing 70mg/ml potassium dichromate), repeating each gradient for 3 times, placing at 28 deg.C, and culturing for about 3 days;
4) selecting a plate with the colony number between 50 and 300, counting, picking all colonies, purifying on a solid Gao's I culture medium, picking a single colony by using an inoculating loop, inoculating the single colony into a test tube containing 5ml of the Gao's I culture medium (liquid), shaking the colony at the speed of 200rpm at the temperature of 28 ℃ for culturing for 24 to 48 hours, uniformly mixing a bacterial suspension with an equal volume of 80 percent glycerol solution, and storing the mixture at the temperature of-70 ℃ for later use (Berg et al, 2000).
2. Enzyme production activity determination for preliminary screening of biocontrol strain
Strains with enzyme-producing activities such as protease, chitinase, glucanase, cellulase and the like are often used as the standard for primary screening of biocontrol bacteria. Selecting a strain in a vigorous growth period, placing the strain on a protease enzyme activity determination culture medium, a culture medium (Chi-adsorbents) taking colloidal chitin as a unique carbon source, a beta-1, 3-glucanase enzyme activity determination culture medium and a cellulase enzyme activity determination culture medium, culturing for 3 days at 28-30 ℃ after inoculation, observing the existence of a transparent ring, and respectively recording the inner diameter and the outer diameter of the transparent ring. Strains having one or more of the above enzyme-producing activities are retained for further screening.
Finally, a strain is separated from the soybean rhizosphere soil and is confirmed to be a potential biocontrol strain through enzyme production activity detection and a flat plate antagonism test, and the strain is named as biocontrol actinomycetes 11 multiplied by 1.
Secondly, the identification process of the biocontrol actinomycete 11X 1 of the invention is as follows:
1. morphological feature observation
Morphological characteristic observation is carried out by using an insertion method, and the method comprises the following specific operations: the sterilized coverslip was inserted obliquely at an angle of 45 degrees into a culture plate of Gao's first culture medium coated with the above-mentioned selected strains, and cultured at a constant temperature of 28 ℃ while observing the culture characteristics and morphological characteristics with an optical microscope.
Morphological characteristics: the single colony is round, and the surface of the single colony is dusted and is opaque; the colony is compact in texture and has mud fishy smell, hyphae are white or grey white, basal hyphae are light yellow, the growth is limited, and pigments are not generated; under an optical microscope, intrabasal hyphae have no transverse septa, aerial hyphae are branched more, spore filaments are mostly long and straight, and parts of spore filaments are bent and have chain spores which are cylindrical or short-rod-shaped.
2. Molecular biological identification
The biocontrol actinomycetes 11 multiplied by 1 obtained by screening are cultured on a Gao's first culture medium at the temperature of 28 ℃ to logarithmic phase, and then the bacteria are collected by centrifugation at 5000r/min for 5 min.
The genome total DNA of the collected thalli is extracted by adopting a genome DNA rapid extraction kit of Shanghai Saibance gene technology Limited company.
Then, a PCR amplification kit of Tiangen Biochemical technology Co., Ltd is adopted to amplify the 16S rRNA sequence of the biocontrol actinomycete 11X 1. The total genomic DNA extracted above is used as a template, and the forward primer sequence adopted by amplification is 5'-AGAGTTTGATCCTGGCTCAG-3' (shown as SEQ No. 1), and the reverse primer sequence is 5'-TACGGCTACCTTGTTACGACTT-3' (shown as SEQ No. 2). The specific PCR amplification procedure was performed according to the kit instructions.
And (3) carrying out 1% agarose gel electrophoresis on the PCR product, observing under an ultraviolet lamp, and recovering the PCR product with a target band. Then, the PCR product is sent to the company of Biotechnology engineering (Shanghai) for sequencing.
The sequencing results obtained were submitted to the GenBank database for BLAST analysis and alignment. The result shows that the total length of the 16S rRNA sequence of the biocontrol actinomycete 11X 1 is 1490bp, the homology with the Streptomyces sp or Streptomyces solani scabies (Streptomyces scabies) sequence reaches 99.93 percent, and finally the biocontrol actinomycete 11X 1 obtained by screening is identified as the Streptomyces solani scabies (Streptomyces scabies) by combining the results of the morphological characteristics, the culture characteristics and the physiological and biochemical characteristics of the strain.
The strain name of the biocontrol actinomycete 11X 1 is "Streptomyces solani", which may also be called "Streptomyces scabies", and the Latin article thereof is "Streptomyces scabies", which may also be "Streptomyces scabies".
Third, preservation of strains
The biocontrol actinomycete 11 multiplied by 1 of the invention belongs to Streptomyces solani (Streptomyces scabies), is preserved in the common microorganism center of China Committee for culture Collection of microorganisms (address: Beijing Corp. Chaoyang district Beichen Xilu No.1, institute of microbiology of China academy of sciences), has the preservation date of 2019, 10 months and 21 days, and has the preservation number of CGMCC No. 18716.
Fourthly, the invention provides a method for culturing biocontrol actinomycetes 11 multiplied by 1 and the preparation of microbial inoculum
The biocontrol actinomycetes 11 multiplied by 1 of the invention are grown on a Gao's first culture medium (plate streaking), and cultured in a dark environment in a constant temperature incubator at 28 ℃ for 2-4 days until a single colony (an activated actinomycetes strain is obtained). The preparation method of the Gao's No. one culture medium comprises the following steps: 20g of soluble starch, 0.5g of sodium chloride, 1g of potassium nitrate, 0.5g of dipotassium phosphate, 0.5g of magnesium sulfate, 0.01g of ferrous sulfate and 15-20g of agar are used for preparing 1000ml of Gaoshi-one culture medium, the pH is adjusted to 7.2, the volume is fixed to 1000ml by using distilled water, and the culture medium is sterilized for 20min at the temperature of 121 ℃;
selecting a single bacterial colony (activated bacterial strain) and inoculating the single bacterial colony into a 250mL triangular flask filled with 50mL fermentation medium, and carrying out shaking fermentation culture for 120-144 h at the temperature of 28 ℃ and the rotating speed of 180r/min to obtain a seed solution. The preparation method of the fermentation medium used in the step comprises the following steps: using 10g of corn flour, 10g of cane sugar, 2g of sodium chloride, 3g of ammonium sulfate and 2g of calcium carbonate per 1000ml of fermentation medium, adjusting the pH value to 7.2-7.4, using distilled water to contain the mixture to 1000ml, and sterilizing the mixture for 20min at the temperature of 121 ℃;
the seed liquid obtained as described above may be inoculated into a fermentation medium in an inoculum size of 8% for secondary fermentation. The preparation method of the fermentation medium adopted by the secondary fermentation comprises the steps of using 10g of soybean meal, 15g of glucose, 2g of sodium chloride, 3g of ammonium sulfate and 2g of calcium carbonate per 1000ml of the medium, adjusting the pH value to 7.5, using distilled water to fix the volume to 1000ml, sterilizing for 20min at 121 ℃, carrying out shaking fermentation culture for 144-168 h at the rotation speed of 180r/min at the temperature of 28 ℃, filtering with gauze after the fermentation is finished to obtain the actinomycete microbial inoculum, and storing for later use at 4 ℃.
Centrifuging the obtained actinomycete preparation at a rotation speed of 5000r/min, discarding the bottom precipitate, filtering the supernatant fermentation liquid with 0.2 μm bacterial filter to obtain sterile actinomycete preparation (viable bacteria concentration is about 5 × 10)9～1×1010CFU/ml), stored at 4 ℃ until use.
Fifthly, the invention discloses application of biocontrol actinomycetes 11 multiplied by 1
Generally, the sterile actinomycete preparation obtained in the fourth part can be diluted by 100-200 times and subjected to root irrigation on the same day as the crop transplantation; and then, before the crops are attacked, diluting the sterile actinomycete microbial inoculum obtained in the fourth part by 100-200 times according to the growth cycle of the crops every 30-45 days to irrigate roots of the transplanted crops, and simultaneously diluting the sterile actinomycete microbial inoculum obtained in the fourth part by 500-1000 times to spray leaf surfaces.
Sixthly, the flat plate antagonism test of the biocontrol actinomycetes 11 multiplied by 1 to the plant pathogenic bacteria
The specific test method comprises the following steps:
uniformly mixing the sterile actinomycete inoculum prepared in the fourth part with the cooled V8 solid culture medium according to the ratio of 1:6(V/V) to prepare a culture medium plate containing the biocontrol actinomycete 11X 1 (a control group plate is prepared by mixing the fermentation culture medium containing no biocontrol actinomycete 11X 1 and 10% of V8 solid culture medium in equal proportion).
The above 10% V8 solid medium was cultured in the following ratio of 1: 100(w/V) ratio CaCO added to V8 vegetable juice3Centrifuging at 5000r/min for 10min, collecting supernatant, removing precipitate, adding 9 times of distilled water into the supernatant to obtain 10% V8 vegetable juice culture medium, adding agar in a proportion of 1.5%, sterilizing at 121 deg.C for 20min, and cooling.
Pythium nepalense (Pythium monitum), Pythium aphanidermatum (Pythium aphanidermatum), Pythium gordonii (Pythium sponense), Pythium spinosum (Pythium spinosum), Pythium woodense (Pythium sylvestre), Phytophthora sojae (Phytohthtorasojae), Pythium closterium (Pythium vexans), Pythium ultimum (Pythium uimum) Phytophthora (Phytophthora capsici), Phytophthora citri (Phytophthora citricola), Pythium Oligandrum (Pythium Oligandrum), Fusarium graminearum (Fusarium graminearum), Fusarium oxysporum (Fusarium oxysporum) were each cultured in 10% medium for 1-4 days.
For the plant pathogenic bacteria, respectively taking the parts close to the edges in the bacterial colony, punching a bacterial dish with the diameter of 6mm by using a sterilized puncher, respectively inoculating the hypha surface of the bacterial dish to the center of the culture medium plate containing the biocontrol actinomycetes 11 multiplied by 1, setting each group of treatment for 3 times, carrying out dark culture at 25 ℃ for 1-5 days, measuring the diameter of the bacterial colony by using a cross method, and calculating the inhibition rate of hypha growth by using a formula:
the inhibition rate (%) - (control colony diameter-treated colony diameter)/(control colony diameter-6) × 100%
See table 1 below for results.
TABLE 1
As can be seen from the results in Table 1 above, the biocontrol actinomycete 11X 1 of the present invention has an inhibition rate of more than 86% for phytophthora sojae and pythium aggregatum and has obvious antagonistic activity (inhibition effect).
From this, it is inferred that the biocontrol actinomycete 11X 1 and the microbial inoculum of the present invention can be used for controlling plant diseases caused by phytophthora sojae and pythium aggregatum, such as phytophthora sojae and ginger stem rot.
It can also be seen that the biocontrol actinomycete 11 x 1 of the invention has an inhibition rate of more than 44% for pythium oligandrum, pythium clodinieri, pythium spinulosum, pythium forest, pythium ultimum, pythium aphanidermatum, pythium gordonatum, phytophthora capsici, phytophthora citri-cola, fusarium graminearum and fusarium oxysporum, and can inhibit the growth of these putrescens and fusarium.
From this, it can also be inferred that the biocontrol actinomycetes 11X 1 and the microbial inoculum of the present invention can be used for controlling plant diseases caused by pythium oligandrum, pythium clotocidum, pythium spinulosum, pythium forest, pythium ultimum, pythium aphanidermatum, pythium gordonii, phytophthora capsici, phytophthora citri, fusarium graminearum and fusarium oxysporum.
Seventhly, the biocontrol actinomycete 11 multiplied by 1 of the invention is used for measuring the control effect of the pythium colonization and the phytophthora sojae on the isolated leaves
(1) Culturing actinomycete inoculum for a predetermined time: selecting about 60 pieces of ginger and soybean leaves with the same leaf position, respectively soaking the soybean leaves and the soybean leaves for about 10s by using Tween 20 with the concentration of 0.5%, slightly rinsing by using sterile water, taking out and airing in a super clean bench. Taking the sterile actinomycete agent obtained in the fourth part (viable bacteria concentration is about 5 multiplied by 10)9～1×1010CFU/ml), soaking the soybean leaf and the ginger leaf in a sterile actinomycete agent for 1min respectively (the control group adopts supernatant of fermentation medium without biocontrol actinomycete 11 multiplied by 1), taking out and airing in a clean bench.
(2) Culturing Pythium herding and Phytophthora sojae in 10% V8 solid culture medium for 1-4 days; respectively using a puncher to punch the outer edge of a bacterial colony to prepare bacterial dishes (the diameter is 6mm), then respectively inoculating the bacterial dishes of the pythium colonization with the surfaces facing downwards on ginger blades, respectively inoculating the bacterial dishes of the phytophthora sojae with the surfaces facing downwards on soybean blades, setting 12 repetitions for each treatment, and carrying out dark culture at 25 ℃. The lesion diameters of ginger (cultured for 24h after inoculation) and soybean leaf (cultured for 48h after inoculation) were measured, and the control effect was evaluated. The control effect calculation formula is as follows:
the results of the control (%) control lesion diameter-treatment lesion diameter/control lesion diameter × 100% measurement are shown in table 2 below.
TABLE 2
As can be seen from table 2 above, the biocontrol actinomycete 11 × 1 microbial inoculum (fermentation liquor) of the present invention has an effect of preventing phytophthora sojae on isolated leaves of soybeans of 90%, and an effect of preventing pythium aggregatum on isolated leaves of ginger of 58.3%, which indicates that the biocontrol actinomycete 11 × 1 microbial inoculum (fermentation liquor) of the present invention can be used as a biological agent to be sprayed on ginger and soybean leaves.
Eighthly, determining the potted plant control effect of the biocontrol actinomycete 11 multiplied by 1 on the stem rot of the ginger
(1) Preparation of a suspension of Pythium colonizing zoospores: activating and growing the preserved Pythium colony strain on a 10% V8 culture medium, and culturing in a constant temperature incubator at 25 ℃ in the dark for 1-2 days; cutting the strain into 10mm × 15mm mycelium blocks (10-20 blocks) with scalpel, placing in 15ml sterilized tap water with mycelium side facing upward, changing water every 30min, and repeating for 3 times; finally, adding about 8ml of sterilized tap water, placing and culturing in a constant-temperature incubator at 25 ℃, culturing for about 24 hours to induce the generation of zoospores, and adjusting the zoospore suspension to the concentration of about 1 × 104 spores/ml by using sterile water.
(2) Determination of potted plant control effect: the root of each ginger seedling is filled with 11X 1 microbial inoculum of biocontrol actinomycetes (prepared in the fourth part) by using a ginger seedling cultured by a tissue with the height of 7.5cm, and each ginger seedling is filled with 5ml (the root of the ginger seedling is filled with the equal volume of the ginger seedling of the supernatant of the fermentation culture medium which is not inoculated with the biocontrol actinomycetes 11X 1 is used as a control). And after 3 days of root irrigation, irrigating 2ml of zoospores of pythium colonization to the root of the ginger, repeating 3 groups of treatments for each ginger seedling, and inoculating 36 ginger seedlings in total for each group of 12 seedlings. Culturing in a constant temperature incubator at 25 deg.C, and calculating disease index and preventing and treating effect of the plant 11 days after inoculation. The calculation formula of each parameter is as follows:
disease index is 100 × Σ (number of diseased plants × representative value of each stage)/(total number of investigated plants × representative value of highest stage);
0 ═ the plants remained green and healthy; 1 ═ leaf sheath ring discolours and lower leaves turn yellow; 2-plants survived but leaves were completely yellow or dead; the whole plant died.
The preventing and treating effect (%) is (contrast disease index-treatment disease index)/contrast disease index is multiplied by 100
The results of the measurements are shown in Table 3 below.
TABLE 3
The disease index results in table 3, with significant differences at the 0.01 level.
From the results in table 3, it can be seen that the disease index of ginger seedlings treated by root irrigation with the biocontrol actinomycete 11 × 1 microbial inoculum (fermentation broth) of the present invention is 14.67, the control effect is 85.33%, and ginger seedlings of the control group all die due to ginger stem basal rot, which indicates that the biocontrol actinomycete 11 × 1 microbial inoculum of the present invention can be used for preventing and treating ginger stem basal rot.
Ninth, the invention biocontrol actinomycetes 11X 1 determination of the pot culture control effect on soybean epidemic disease
(1) And (3) phytophthora sojae culture: activating and growing the preserved soybean phytophthora strain on a 10% V8 culture medium, and placing the strain in a constant-temperature incubator at 25 ℃ in the dark for culturing, wherein the culturing time is generally 5-7 days for later use; the strain to be used was cut into a 10mm × 15 mm-sized mycelium block with a scalpel for use.
(2) Determination of potted plant control effect: the roots of soybean seedlings growing for 10 days are filled with 11X 1 microbial inoculum of biocontrol actinomycetes (prepared in the fourth part), and each soybean seedling is filled with 5ml (the soybean seedlings filled with equal volume of fermentation culture medium supernatant without biocontrol actinomycetes 11X 1 at the root position are used as a control). After 3 days of root irrigation, slightly scratching the epidermis of the stem close to the root of the soybean with a scalpel to form a slight wound, enabling the side with hyphae to be tightly attached to the wound, keeping moisture, and then placing the soybean in a constant-temperature illumination incubator at 25 ℃ for growth, wherein the illumination time is 16h illumination/8 h darkness. Each treatment was repeated in 3 groups of 15 seedlings each, and a total of 45 soybean seedlings were inoculated. And calculating the disease index and the prevention and treatment effect of the plants 2 days after inoculation. The calculation formula of each parameter is as follows:
disease index is 100 × Σ (number of diseased plants × representative value of each stage)/(total number of investigated plants × representative value of highest stage);
the preventing and treating effect (%) is (contrast disease index-treatment disease index)/contrast disease index is multiplied by 100
The results of the measurements are shown in Table 4 below.
TABLE 4
The disease index results in table 4, with significant differences at the 0.01 level.
From the results in table 4, it can be seen that the disease index of soybean seedlings treated by root irrigation with the biocontrol actinomycete 11 × 1 microbial inoculum (fermentation broth) of the present invention is 34.32, the control effect is 58.22%, and the soybeans in the control group all die due to soybean blight, which indicates that the biocontrol actinomycete 11 × 1 microbial inoculum of the present invention can be used for controlling soybean blight.
In conclusion, the biocontrol actinomycete 11 multiplied by 1 (Streptomyces scabrosus) can effectively prevent and treat soybean epidemic disease and ginger stem basal rot, simultaneously shows broad-spectrum plant disease resistance, is nontoxic to human and livestock, does not pollute the environment, has no residue, is beneficial to sustainable development of ecological environment, and also provides high-quality biological resources for researching and developing new environment-friendly biocontrol actinomycete microbial inoculum.
After the biocontrol actinomycete 11 x 1 has the control effect on soybean blight and ginger stem basal rot, the microbial agent for controlling soybean blight or ginger stem basal rot can be obtained by combining the microbial agent of the biocontrol actinomycete 11 x 1 with an auxiliary agent acceptable in pesticides.
After the biocontrol actinomycete 11 x 1 has the control effects on soybean blight and ginger stem basal rot, a conventional bactericide (chemical agent) for controlling soybean blight or ginger stem basal rot can be compounded with the bactericide or bacterium powder of the biocontrol actinomycete 11 x 1 to obtain a bacterium-medicine composition.
It should be understood that although the present description refers to embodiments, not every embodiment contains only a single technical solution, and such description is for clarity only, and those skilled in the art should make the description as a whole, and the technical solutions in the embodiments can also be combined appropriately to form other embodiments understood by those skilled in the art.
The above-listed detailed description is only a specific description of a possible embodiment of the present invention, and they are not intended to limit the scope of the present invention, and equivalent embodiments or modifications made without departing from the technical spirit of the present invention should be included in the scope of the present invention.
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| CA2205976A1 (en) * | 1997-05-23 | 1998-11-23 | Universite De Sherbrooke | Biological control of common scab of root and tuber food crops |
| CN102925511A (en) * | 2012-06-19 | 2013-02-13 | 文才艺 | Staurosporine preparation method |
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| CA2205976A1 (en) * | 1997-05-23 | 1998-11-23 | Universite De Sherbrooke | Biological control of common scab of root and tuber food crops |
| CN102925511A (en) * | 2012-06-19 | 2013-02-13 | 文才艺 | Staurosporine preparation method |
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