CN106967635B - Preparation method of biocontrol microbial inoculum for preventing and treating apple tree fungal diseases - Google Patents

Preparation method of biocontrol microbial inoculum for preventing and treating apple tree fungal diseases Download PDF

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CN106967635B
CN106967635B CN201710191219.8A CN201710191219A CN106967635B CN 106967635 B CN106967635 B CN 106967635B CN 201710191219 A CN201710191219 A CN 201710191219A CN 106967635 B CN106967635 B CN 106967635B
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周天惠
王灵敏
刘辉
孙振娜
李玲娣
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Shaanxi Fengdan Baili Biotechnology Co ltd
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Abstract

The invention discloses a preparation method of a biocontrol microbial inoculum for preventing and treating fungal diseases of apple trees, relates to the technical field of biological microbial inoculants, and particularly relates to a preparation method of a streptomyces cacao FN biocontrol microbial inoculum, wherein the accession number of the streptomyces cacao FN is CGMCC NO.13080, the microbial inoculum has remarkable prevention and treatment effects on apple anthracnose leaf blight and apple alternaria leaf spot, and the microbial inoculum has prevention and treatment functions on common apple tree leaf diseases and other apple tree diseases. The biocontrol microbial inoculum not only can completely replace a chemical control method to eliminate potential food safety hazards, but also has outstanding effects on the effectiveness of controlling common leaf diseases of apple trees and the broad-spectrum bacteriostasis. Meanwhile, the streptomyces inoculant has the characteristics of high fermentation activity, strong heat resistance, broad-spectrum bacteriostasis and the like, and can be used for preventing and treating common diseases of other fruits and vegetables such as cucumber gummy stem blight, cucumber damping-off, tomato early blight and the like.

Description

Preparation method of biocontrol microbial inoculum for preventing and treating apple tree fungal diseases
Technical Field
The invention relates to the technical field of biological agents, in particular to a preparation method of a biological agent for preventing and treating apple tree fungal diseases.
Background
During the growth process of apple trees, common apple tree fungal diseases mainly comprise apple anthracnose leaf blight, apple alternaria leaf spot, apple rot, apple blight and the like. Among them, apple anthracnose leaf blight (glomerilla leaf spot) is a new disease popular in recent years, and it is found by professor caoch chun and its subject group in the pest control research laboratory that fruit growers react with suspected apple anthracnose leaf blight every 7 months since 2009 in our country and the harm is serious. The disease is mainly found in Gala, Jinguan, Qinhuan and Qiannajin varieties. The separation, culture and identification of the pathogen, which is caused by anthrax, are carried out by professor Libaohua university of Qingdao agriculture, and the disease is determined to be called as colletotrichum anthracis (Glomeellacingulata). The disease belongs to the fungal disease of the leaf of an apple tree, the leaf of the apple tree at the early stage of the disease has dark brown near-round scab at the edge, and the leaf can finally turn black, become scorched and fall off when meeting a high-temperature and high-humidity environment. Fruits are also infected with this disease, and near-round necrotic spots form on the fruits.
At present, the prevention and control of the disease are mainly achieved by spraying chemical pesticides before initial infection for protection, only endophytic actinomycete A-1 researched by Zhuming is searched for a biological prevention and control method, the resistance of apple trees to anthracnose leaf blight can be induced, and the prevention effect reaches 70.42%. Because the disease is epidemic in recent years in China, the prevention and treatment technology for the disease is immature, the prevention is mainly performed at present, and the treatment effect is poor.
For example, the article "the prevention and control effect of several medicaments on apple anthracnose leaf blight", songlian, royal snow lotus, bean powder Ting, He Yan Wei, Shaanxi agricultural science 2016, 62(11): 46-48 discloses that 5 medicaments are selected in the test, and the prevention and control of apple anthracnose leaf blight is carried out in a Gala apple orchard, and the result shows that: 1000 times of 25% amisida (azoxystrobin) suspending agent can cause phytotoxicity to apple trees, and is not suitable for use in Gala apple orchard; 400 times of 78% Bordeaux mancozeb (Kebo) wettable powder and 500 times of 80% mancozeb (Dasheng) wettable powder have ideal control effect on anthracnose leaf blight of apples, have good control effect on other leaf droppings, and can be popularized and applied in a large area.
For example, the article, "control effect of different bactericides on apple anthracnose leaf blight", Wangbing, Rongcaixia, Xiangxiangpeng, etc., plant protection, 2014(6): 176-. The pyraclostrobin is used within 72 hours after the pathogen infection, or the prochloraz is used within 24 hours after the pathogen infection, so that the disease spot has a certain treatment effect. The inhibition effect of the Bordeaux mixture on the infection of the colletotrichum anthracnose still reaches 50% 18 days after the Bordeaux mixture is sprayed, the protection effect of 3 medicaments of oxime bacteria, tebuconazole, enoyl, pyrazole ester and azol ether and metiram is still obviously different from that of a contrast at the 11 th day after the spraying, the lasting period reaches 11 days, and the lasting period of 4 protective agents of mancozeb, thiophanate-methyl, copper hydroxide and prochloraz can only be maintained for 6 days. Therefore, the control of the anthracnose leaf blight is mainly based on Bordeaux mixture, only Bordeaux mixture has certain protection effect at 18 days after application, and other 7 medicaments completely lose the protection effect.
The spraying of chemical agent is bound to bring hidden danger to the food safety problem of fruits and vegetables, and because the apple is the food of directly eating, consequently can't avoid chemical agent to remain on the fruit body and chemical agent can permeate into the fruit body along with the growth of plant, causes serious threat to food safety.
Another leaf disease that is very serious and harmful to apple trees is Alternaria alternata, a fungal disease caused by Alternaria alternata apple specialization (Alternaria alternata f.sp.mali), which was found in japan in the early 50 s, and subsequently spread to china in the 70 s, and was also found in both the us and europe in the 90 s. The harm of the alternaria leaf spot of the apple is increased year by year, and the apple is seriously ill in the apple producing areas of Beijing, Shandong, Liaoning, Hebei and the like in China in recent years. The disease mainly infects newly born leaves of apple trees to cause early-stage defoliation, brown round disease spots with the diameter of 2-3mm appear at the early stage of the apple alternaria leaf spot disease, then the disease spots gradually expand into red brown disease spots with the diameter of 5-6mm, the edges are purple brown, concentric ring lines often appear at the center, and the diseased leaves can fall off due to the 3-5 disease spots appearing on the common leaves. The major apple varieties harmed by the disease include red star, marshal, green incense, Fuji and the like, and in recent years, the harm to apple varieties such as golden crown, national light and the like is increased. Apple alternaria leaf spot is a fungal disease which can overwinter on diseased fallen leaves or trees, and conidia can continue to spread with airflow or wind and rain after the spring and rain of the next year, and then infect leaves from stomata.
At present, the alternaria leaf spot of apples is usually prevented and controlled by combining chemical agents, and the commonly used agents mainly comprise 1000-. The mancozeb, the manganese acetate and other medicaments have certain prevention effect on diseases. At present, the research of the biological prevention and control method is mainly focused on Bacillus, for example, Sun Yan and the like prove that the Bacillus subtilis BS-315 has a better inhibiting effect on apple alternaria leaf spot through fruit tree leaf in vitro experiments, a Fourier pool and the like screen Bacillus disease prevention strains B-319(Bacillus brevis) and B-9108(Bacillus cereus) through field disease prevention experiments, and the disease of the apple alternaria leaf spot can be relieved by 60-70% through the indoor experiments and the field experiments.
The invention also discloses a 'synergistic pesticide composition for preventing and treating the defoliation of apple trees' as a patent with application number 201410697783.3, and the synergistic pesticide composition for preventing and treating the defoliation of the apple trees comprises paclobutrazol and tebuconazole in a mass ratio of 100: 1-1: 100. The synergistic pesticide composition for preventing and treating the apple tree defoliation provided by the invention has a certain synergistic effect on preventing and treating the apple tree defoliation, delays the development of resistance and prolongs the service life of a pesticide. The invention discloses a method for preventing and treating alternaria leaf spot of a potted apple tree, which belongs to the field of fruit tree pest control. The control method comprises the following steps: (1) pretreating Chinese herbal medicine raw materials; (2) root soaking treatment of potted apple seedlings; (3) preparing culture soil; (4) transplanting; (5) and (4) watering. The invention has the beneficial technical effects that: the method greatly reduces the alternaria leaf spot of the potted apple trees, and can also reduce the drug resistance degree of alternaria leaf spot germs of the potted apple trees.
Similarly, the spraying of chemical agents brings hidden dangers to food safety problems of fruits and vegetables, and poses serious threats to food safety. In addition, the regular use of chemical agents will cause the pathogenic bacteria of crops to generate drug resistance, thereby bringing about the excessive use of chemical agents or the compound use of different chemical agents and further bringing about greater hidden troubles to the food safety problem. Meanwhile, the pollution of chemical agents to the environment such as soil, water source and the like is irreversible.
Compared with a chemical control method, the biological control method has more advantages, besides the easy production and convenient use of biological agents, the mixed microbial inoculum prepared by using the biocontrol bacteria and the metabolites thereof is more environment-friendly, has no pollution and pesticide residue, and meets the strategic requirements of sustainable development in China. And the biological control agent has small sensitivity to environmental changes and stable effect, which is also a great advantage. At present, although the use of chemical agents is intentionally reduced in China, the chemical agents are still main prevention and control means for preventing and controlling apple anthracnose leaf blight and apple alternaria leaf spot, and particularly, no effective biocontrol agent exists for the newly outbreak of apple anthracnose leaf blight in China, so that the development of a biocontrol microbial inoculum for apple leaf diseases is particularly important.
The patent provides a biocontrol microbial inoculum mainly aiming at apple anthracnose leaf blight and apple alternaria leaf spot, overcomes the potential safety hazard of food brought by chemical agents, generates drug resistance to pathogenic bacteria, pollutes the environment and other problems, has remarkable control effect on the two diseases, has remarkable bacteriostatic effect on other diseases of apple trees and pathogenic bacteria of fruit and vegetable diseases, and can be applied to control of fruit and vegetable diseases correspondingly.
Disclosure of Invention
The invention solves the problem of overcoming the defects of the method for preventing and treating the leaf diseases of the apple trees by using chemical agents, provides a biocontrol bacterium, namely Streptomyces cacaoii FN, for preventing and treating the leaf fungal diseases of the apple trees, such as anthracnose and alternaria leaf spot of the apple trees, and other diseases of the apple trees, and the biocontrol bacterium agent for preparing the Streptomyces cacaoii FN is applied to the prevention and treatment of the leaf diseases of the apple trees, can completely replace a chemical prevention and treatment method, eliminates food safety hidden dangers, and obtains outstanding effects on the effectiveness for preventing and treating the leaf diseases of the apple trees and the broad spectrum of bacteriostasis. Meanwhile, the streptomyces has broad-spectrum antibacterial property, and can be used for preventing and treating diseases of other fruits and vegetables such as cucumber gummy stem blight, cucumber damping-off, tomato early blight and the like.
The preparation method of the streptomyces theobromae FN biocontrol microbial inoculum prepared from the streptomyces theobromae FN comprises the following specific steps:
preparing spore suspension: adding sterile water into slant of 2216E solid culture medium containing Streptomyces cacao FNAnd scraping off spores, shaking and uniformly mixing to obtain spore suspension, wherein the thallus concentration of the spore suspension is 108-109cfu/mL。
Seed liquid culture: inoculating the spore suspension into a seed liquid culture medium according to the inoculation amount of 1-3% (v/v), culturing at 28 ℃ and 240r/min for 90-100h by shaking to obtain the seed liquid.
Preferably, the seed liquid culture: inoculating the spore suspension into a seed liquid culture medium according to the inoculation amount of 1-1.5% (v/v), culturing at 28 ℃ for 180-.
More preferably, the seed broth culture: inoculating the spore suspension into a seed liquid culture medium according to the inoculation amount of 1% (v/v), culturing at 28 ℃ at 200r/min for 96h in a shake flask to obtain seed liquid.
Solid fermentation culture, namely inoculating the seed solution into a solid fermentation culture medium with 50 percent of water content according to the inoculation volume mass ratio of 5-15% (v/m), fermenting and culturing for 3-6 days in a curved plate at 28 ℃, air-drying at 20-30 ℃, grinding by using a grinder, collecting a microbial inoculum passing through a 120-mesh sieve, and storing in the shade at room temperature to obtain the streptomyces theobromae FN biocontrol microbial inoculum, wherein the strain activity of the streptomyces theobromae FN biocontrol microbial inoculum is not less than 2 × 1011cfu/g。
Preferably, solid fermentation culture, namely inoculating the seed liquid into a solid fermentation culture medium with 50% of water content according to the inoculation volume mass ratio of 8-12% (v/m), fermenting and culturing for 3-5 days at 28 ℃, starting to finishing from fermentation, maintaining the constant water content of the solid fermentation culture medium at 50%, turning over for 2-3 times during fermentation, air drying at 25-30 ℃ after fermentation is finished, grinding by using a grinder, collecting the microbial inoculum passing through a 120-mesh sieve, and storing in the shade at room temperature to obtain the streptomyces theobromae FN biocontrol microbial inoculum, wherein the strain activity of the streptomyces theobromae FN biocontrol microbial inoculum is not less than 2 × 1011cfu/g。
More preferably, the solid fermentation culture: inoculating the seed liquid into a solid fermentation culture medium with the water content of 50% according to the inoculation volume mass ratio of 10% (v/m), performing fermentation culture for 4 days in a koji tray at the temperature of 28 ℃, and maintaining the constant water content of the solid fermentation culture medium from the beginning to the end of fermentation: 50 percent, turning over for 3 times during the fermentation period and waiting for fermentationAnd (3) finishing air drying at 28 ℃, grinding by using a grinder, collecting the microbial inoculum passing through a 120-mesh sieve, and storing in a shade place at room temperature to obtain the streptomyces theobromae FN biocontrol microbial inoculum, wherein the strain activity of the streptomyces theobromae FN biocontrol microbial inoculum is not less than 2 × 1011cfu/g。
The 2216E solid medium (g/L): bacterial peptone No.25, yeast powder 1, agar 20 and the balance of distilled water, wherein the pH is natural;
the seed liquid culture medium (g/L): 10 parts of corn flour, 10 parts of peptone, 5 parts of yeast powder, 2 parts of dipotassium hydrogen phosphate, 0.3 part of magnesium sulfate and the balance of distilled water, wherein the pH value is natural;
the solid fermentation medium (%) (m/m): 20% of rice hull, 44.7% of bran, 22% of corn flour, 12% of bean cake powder, 0.8% of calcium carbonate, 0.3% of dipotassium hydrogen phosphate, 0.2% of magnesium sulfate and natural pH.
The streptomyces theobromae FN biocontrol microbial inoculum has the capacity of preventing and treating fungal diseases of fruits and vegetables.
Further, the streptomyces theobromae FN biocontrol microbial inoculum product is applied to control of fruit and vegetable fungal diseases.
Further, the fungal diseases of fruits and vegetables are apple tree fungal diseases.
Further, the apple tree fungal diseases comprise apple anthracnose leaf blight, apple alternaria leaf spot, apple rot, apple root rot, apple ring rot and apple blight.
Further, the fungal diseases of the fruits and vegetables are cucumber gummy stem blight, cucumber damping off, tomato early blight, watermelon fusarium wilt, rape sclerotinia rot and Chinese cabbage black spot.
The streptomyces cacao FN in the streptomyces cacao FN biocontrol microbial inoculum is specifically streptomyces cacao (Streptomyces cahooi) FN with the preservation number of CGMCC No. 13080.
The streptomyces theobromae FN is separated from soil of a vegetable garden in the Chaning county of Hebei province. The Streptomyces cacaoensis (Streptomyces cacaoi) FN provided by the invention has been preserved in the China general microbiological culture Collection center (CGMCC) at 10, 12 months and 2016, and the number is CGMCC No.13080, and the location is No. 3 of the Beijing institute of microbiology, West Lu No.1 of the Chaoyang district, Beijing.
The 16S rDNA sequence of the streptomyces theobromae FN is shown in a sequence table SEQ ID No. 1.
The streptomyces cacao FN morphological characteristics are as follows: on 2216E solid culture medium, the hyphae grow well, the hyphae in the medium are yellow, and the aerial hyphae are rat gray; the strain grows slowly on a Gao's synthetic No. I agar culture medium (ISP1), the hyphae in the medium are light yellow, and the aerial hyphae are light gray; well grown on wort yeast extract agar medium (ISP2), with yellow substrate hyphae and rat grey aerial hyphae; in oat flour agar medium (ISP3), the mycelia in the medium are light yellow, and the aerial mycelia are white; in inorganic salt starch agar medium (ISP4), the mycelia in the medium are yellow, and the aerial mycelia are light gray; in the glycerol aspartyl agar culture medium (ISP5), the hypha in the medium is light yellow, and the aerial hypha is white; the spore silk is spiral, and the spore is spherical;
the composition of the Gao's synthetic No. one agar medium (ISP1) is as follows: soluble starch 20g/L, K2HPO40.5g/L，KNO31g/L，MgSO4·7H2O 0.5g/L，FeSO4·7H20.01g/L of O, 20g/L of agar and the balance of distilled water, wherein the pH value is 7.2-7.4;
the wort yeast extract agar culture medium (ISP2) comprises the following components: 4g/L of yeast powder, 10g/L of malt flour, 4g/L of glucose, 20g/L of agar and the balance of distilled water, wherein the pH value is 7.2-7.3;
the oat flour agar medium (ISP3) comprises the following components: 20g/L of oat, 1mL/L of trace salt solution, 20g/L of agar and the balance of distilled water, wherein the pH value is 7.2;
the inorganic salt starch agar medium (ISP4) comprises the following components: soluble starch 10g/L, K2HPO41g/L，MgSO4·7H2O 1g/L，NaCl 1g/L，(NA4)2SO42g/L，CaCO32g/L, 1mL/L of trace salt solution, 20g/L of agar and the balance of distilled water, wherein the pH value is 7.0-7.4;
the glycerol aspartyl agar culture medium (ISP5) comprises the following components: 10g/L of glycerol, 1g/L of L-asparagine and 1m/L of trace salt solution, K2HPO41g/L of Jongqiong20g/L of grease, and the balance of distilled water, wherein the pH is natural;
wherein the trace salt solution comprises: FeSO4·7H2O 0.1g，MnCl2·4H2O 0.1g，ZnSO4·7H2O0.1g, distilled water 100mL, pH natural.
Further, the physiological and biochemical characteristics of the streptomyces theobromae FN are as follows: gelatin liquefaction, strong starch hydrolysis, no milk coagulation, no hydrogen sulfide production, no melanin production, no nitrate reduction, and can utilize alpha-D-lactose, glycogen, D-cellobiose, glycyl-L-glutamic acid, methyl pyruvate, Tween 40, Tween 80, D-mannitol, D, L-alpha-glycerol, N-acetamido-D-glucosamine, and D-aminogluconic acid.
The streptomyces theobromae FN can inhibit pathogenic bacteria causing fungal diseases of fruits and vegetables.
Furthermore, the pathogenic bacteria comprise the growth of various pathogenic bacteria including colletotrichum anthracnose, alternaria alternata apple specialization type, bellopenbergia grape cavity, pythium malorum, fusarium oxysporum, phytophthora infestans, exocarpium citrulli, rhizoctonia solani, alternaria solani, fusarium oxysporum, sclerotinia sclerotiorum, alternaria brassicae and the like.
The streptomyces theobromae FN has the capability of preventing and treating fungal diseases of fruits and vegetables.
Further, the fungal diseases of fruits and vegetables are apple tree fungal diseases.
Further, the apple tree fungal diseases comprise apple tree leaf fungal diseases.
Further, the apple tree leaf fungal diseases comprise apple anthracnose leaf blight and apple alternaria leaf spot.
Further, the apple tree fungal diseases also comprise apple rot, apple root rot, apple ring rot and apple blight.
Further, the fungal diseases of the fruits and vegetables also comprise cucumber gummy stem blight, cucumber damping off, tomato early blight, watermelon fusarium wilt, rape sclerotinia rot and Chinese cabbage black spot.
The streptomyces theobromae FN is applied to prevention and treatment of fruit and vegetable fungal diseases.
Further, the streptomyces theobromae FN is applied to prevention and treatment of apple tree fungal diseases.
Further, the streptomyces cacao FN is applied to prevention and treatment of apple anthracnose leaf blight, apple alternaria leaf spot, apple rot, apple root rot, apple ring rot and apple blight.
Further, the streptomyces theobromae FN is applied to prevention and treatment of cucumber gummy stem blight, cucumber rhizoctonia rot, tomato early blight, watermelon fusarium wilt, rape sclerotinia rot and cabbage black spot.
Has the advantages that:
1. the biocontrol microbial inoculum prepared from the streptomyces theobromae FN has obvious control effects on alternaria leaf spot of apples and anthracnose leaf blight of apples.
The practical field test shows that the streptomyces cacao FN biological control microbial inoculum has the control effects on controlling apple alternaria leaf spot when the dosage is diluted by 1000, 3000 and 5000 times of liquid respectively: 87.10%, 85.58% and 82.74%. On the difference level of 1% and 5%, the control effect of the streptomyces cocoas FN biological control microbial inoculum for controlling apple alternaria leaf spot is superior to that of the streptomyces cocoas FN biological control microbial inoculum when the dosage of the streptomyces cocoas FN biological control microbial inoculum is 1000 times of the diluted liquid, the streptomyces cocoas FN biological control microbial inoculum is 3000 times of the diluted liquid and 5000 times of the diluted liquid, and the control effect of the streptomyces cocoas FN biological control microbial inoculum when the diluted liquid is 1000 times of the diluted liquid is obviously higher than that of the 10% polyoxin wettable powder serving as a control medicament when the dosage of the diluted.
The streptomyces theobromae FN biological control microbial inoculum is used for controlling apple anthracnose and leaf blight, and when the dosage is 1000, 3000 and 5000 times of diluted solution, the control effects are respectively as follows: 82.17%, 77.83%, 73.24%. On the difference level of 1% and 5%, the control effect of the streptomyces cacao FN biological control fungicide on apple anthracnose leaf blight is superior to that of the streptomyces cacao FN biological control fungicide when the dosage is diluted by 1000 times of liquid, namely 3000 times of liquid and 5000 times of liquid. Meanwhile, the control effect of the streptomyces cocoas FN biological control microbial inoculum diluted by 1000 times is obviously higher than that of the control medicament 10% polyoxin wettable powder diluted by 1000 times and that of 80% mancozeb wettable powder diluted by 600 times.
During the test, the occurrence of phytotoxicity phenomenon of the streptomyces cacao FN biocontrol microbial inoculum is not found.
2. The effectiveness and broad spectrum of preventing and treating the apple tree leaf diseases are strong: streptomyces cacaoii (Streptomyces cacaoi) FN CGMCC NO.13080 can also be used for treating other diseases of apple trees: the apple rot, apple root rot, apple ring rot and apple blight also have strong inhibition capability.
3. The effectiveness and broad spectrum of preventing and treating fungal diseases on other fruits and vegetables are strong: streptomyces cococcae FN is used for treating fungal diseases on other fruits and vegetables: cucumber gummy stem blight, cucumber damping off, tomato early blight, watermelon fusarium wilt, rape sclerotinia rot and cabbage black spot have strong inhibition capability.
4. The streptomyces theobromae FN has broad-spectrum antibacterial activity on pathogenic bacteria of fruits and vegetables, and experiments show that the streptomyces theobromae FN has effective inhibiting effect on pathogenic bacteria of fruits and vegetables, and the antibacterial rate of the streptomyces theobromae FN on different pathogenic bacteria is as follows: colletotrichum anthracnose: 86.2 percent of alternaria alternate apple specialization type; 88.9%, Staphylococus Bellengeri: 77.1% and apple Humicola melanosporum: 89.6%, Fusarium oxysporum: 80.9%, phytophthora infestans: 83.6%, watermelon shell two spore: 71.4%, rhizoctonia solani: 74.0%, Alternaria solani: 80.9% and the watermelon fusarium oxysporum: 77.9%, sclerotinia sclerotiorum: 81.4%, alternaria brassicae: 90.1 percent;
5. high genetic stability: after 30 generations of streptomyces theobromae FN, the bacteriostatic rates of the streptomyces theobromae FN on Bacillus anthracis, Alternaria alternata, Alternaria Bellengeri, Humicola malosa, Fusarium oxysporum, phytophthora cactorum, exocarpium citrulli, Rhizoctonia solani, Alternaria solani, Fusarium oxysporum, Sclerotinia sclerotiorum and Alternaria brassicae are respectively 85.3%, 88.0%, 76.5%, 88.5%, 77.1%, 82.5%, 69.3%, 72.5%, 78.6%, 76.1%, 80.2% and 88.6%, and are basically consistent with the bacteriostatic rate of the first generation of the streptomyces theobromae FN, which shows that the streptomyces theobromae FN provided by the invention does not reduce the inhibitory activity on the pathogenic bacteria along with the increase of the generation number of generations, and has higher genetic stability.
6. Strong heat resistance, acid resistance, alkali resistance and high ultraviolet resistance: the streptomyces theobromae FN has strong heat resistance, and experiments show that the streptomyces theobromae FN can keep a good growth state at 50 ℃, and the lethal temperature reaches 95 ℃, so that the streptomyces theobromae FN still has high strain activity under high-temperature stress. The streptomyces theobromae is insensitive to ultraviolet rays, the survival rate of the streptomyces theobromae reaches 77% after the ultraviolet rays are continuously irradiated for 24 hours, and the survival rate of the streptomyces theobromae is still more than 60% after the ultraviolet rays are continuously irradiated for 32 hours.
The streptomyces theobromae FN not only can keep stronger strain activity in the fermentation process, but also can show that the fermentation inoculant can keep remarkable bacteriostatic effect at normal temperature in the prevention and treatment process of pathogenic bacteria, and can still keep good strain activity and bacteriostatic effect in high-temperature weather. And can still maintain good strain activity under outdoor strong light weather.
The streptomyces theobromae FN has acid resistance and extremely strong alkali resistance, still presents a good growth state under the alkali stress of pH10, not only can keep stronger strain activity in the fermentation process, but also can resist different acid-base environments of external sources such as pesticides, rain, snow and the like in the prevention and treatment process of the microbial inoculum applied to pathogenic bacteria, and simultaneously keeps higher bacteriostatic activity.
In addition, the streptomyces theobromae FN has good salt tolerance and can tolerate the salt stress action with the salt ion concentration of 17%.
7. The invention provides the preparation of Streptomyces cacao (cacahooi) FN CGMCC NO.13080 biocontrol microbial inoculum, and the bacterial activity is not less than 2.0 × 10 through liquid fermentation seed liquid and solid fermentation11The cfu/g biocontrol microbial inoculum has very obvious effect on the application of preventing and controlling the leaf diseases of apple trees.
8. The streptomyces cacao FN with antagonism is used for preventing and treating plant diseases, is not easy to generate drug resistance, and is the first choice for biological prevention and treatment. Meanwhile, the streptomyces theobromae FN can avoid hidden dangers to food safety problems of fruits and vegetables and serious threats to human health. The biocontrol microbial inoculum is convenient to produce and use, the mixed microbial inoculum prepared by utilizing the streptomyces theobromae FN and the fermentation metabolites thereof is more environment-friendly, has no pollution and pesticide residue, and meets the strategic requirements of sustainable development in China. And the biological control agent has small sensitivity to environmental changes and stable effect.
It should be noted that the technical effect of the present invention is the result of mutual cooperation and interaction of each process step and parameter, and is not the superposition of simple processes, and the effect produced by the organic combination of each process is far superior to the superposition of each single process function and effect, and has better advancement and practicability.
Drawings
FIG. 1 shows the aerial hypha and spore chain morphology (10X 40 times) of Streptomyces cococcae under FN optical microscope;
FIG. 2 shows normal hypha and spore morphology of apple alternaria leaf spot pathogen and aberrant hypha and spore morphology after Streptomyces cacao FN treatment, wherein 2 a: normal hyphae (10 × 10-fold mirror), 2 b: normal spores (10 × 40 times under the microscope), 2 c: deformed hyphae (10 × 10-fold mirror), 2 d: aberrated spores (under 10 × 40 times of the scope).
Detailed Description
The invention is described below by means of specific embodiments. Unless otherwise specified, the technical means used in the present invention are well known to those skilled in the art. In addition, the embodiments should be considered illustrative, and not restrictive, of the scope of the invention, which is defined solely by the claims. It will be apparent to those skilled in the art that various changes or modifications in the components and amounts of the materials used in these embodiments can be made without departing from the spirit and scope of the invention.
Example 1 isolation and purification of Streptomyces cacao FN and screening
1. Isolation and purification of Streptomyces cacaoi FN
Collecting soil sample from a Changning vegetable garden, filling into a sterile bag, adding 10g into a sterilized triangular flask containing 100mL of distilled water and glass beads, placing in a shaking table at 200rpm, shaking for 30min, and standing for 10min, shaking up, adding 5mL of the solution into a small triangular flask containing 45mL of sterile water by using a pipette, namely diluting the solution by 102Multiple, sequentially carrying out gradient dilution to 10-3、10-4、10-5、10-6Each 100. mu.l of the suspension was pipetted onto a 2216E plate containing antibiotics (50 mg/L potassium dichromate, 15mg/L nalidixic acid, and 50mg/L nystatin) at 3 replicates per concentration, and the plate was incubated at 28 ℃ for 5 to 10 days. Picking single colony of actinomycetes by using a sterilized bamboo stick and placing the single colony on a freshly prepared 2216E plate for separation and purification.
2. Screening of antagonistic bacteria
The method comprises the steps of taking apple anthracnose leaf blight pathogenic bacteria and apple alternaria leaf spot pathogenic bacteria as target bacteria, screening biocontrol strains by a plate confronting culture method, drawing a cross on the back of a plate with the diameter of 90mm, taking a cross point as the center of the plate, respectively inoculating 3 probiotic cakes with the length of 7mm at 3 points of 4 points on the cross line and away from the center of the plate by 25mm, taking the rest 1 point as a control, inoculating the 7mm probiotic cakes of the pathogenic bacteria in the center of a freshly prepared PDA plate, culturing at 28 ℃ for 5-10 days, and screening to obtain the strains with strong bacteriostatic ability on the apple anthracnose leaf blight and the apple alternaria leaf spot, wherein the strains are named as FN.
3. Identification of strains
The FN strain is identified as the streptomyces cacao by combining the morphological characteristics, physiological and biochemical characteristics and 16S rDNA sequence analysis results. The specific identification results are as follows:
streptomyces cocoanut FN morphological characteristics: on 2216E solid culture medium, the hyphae grow well, the hyphae in the medium are yellow, and the aerial hyphae are rat gray; gao's synthetic No. one (ISP1) grows slowly, the substrate hyphae are light yellow, and the aerial hyphae are light gray; wort yeast extract agar (ISP2) grows well, yellow basal hyphae, and gray aerial hyphae; the mycelia in oat flour agar (ISP3) base are light yellow, and the aerial mycelia are white; the mycelia in the base of inorganic salt starch agar (ISP4) are yellow, and the aerial mycelia are light gray; glycerol aspartate agar (ISP5), substrate mycelium is light yellow, and aerial mycelium is white; the spore thread is spiral, and the spore sphere is as shown in figure 1.
Streptomyces cocoas FN physiological and biochemical characteristics: gelatin liquefaction, strong starch hydrolysis, no milk coagulation, no hydrogen sulfide production, no melanin production, no nitrate reduction, and can utilize alpha-D-lactose, glycogen, D-cellobiose, glycyl-L-glutamic acid, methyl pyruvate, Tween 40, Tween 80, D-mannitol, D, L-alpha-glycerol, N-acetamido-D-glucosamine, and D-aminogluconic acid.
Streptomyces cacaoii (Streptomyces cahooi) FN has been preserved in China general microbiological culture Collection center (CGMCC) at 10, 12 and 2016, with the number of CGMCC NO. 13080.
Example 2 growth characteristics of Streptomyces cococcae FN
1. Growth temperature
Subpackaging 2216E liquid culture medium (bactopeptone No. 25g/L, yeast powder 1g/L, and balance distilled water, pH is natural) in transparent test tubes, and performing moist heat sterilization at 121 deg.C for 30min to obtain culture solution without precipitate. Streptomyces cacao FN was inoculated into 2216E liquid medium in shake flask 48 as an inoculum, and inoculated into test tubes containing 2216E liquid medium by 1 ‰, respectively, and control was not inoculated. Culturing in incubator at 0 deg.C, 7 deg.C, 15 deg.C, 20 deg.C, 28 deg.C, 35 deg.C, 40 deg.C, 45 deg.C, 50 deg.C, 55 deg.C, and 60 deg.C for 3d-5d, and observing and recording growth (precipitation) condition.
2. Acid and alkali resistance
The 2216E liquid culture medium is adjusted to different pH values (1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0) by diluted HCl or NaOH, and is packaged into transparent tubes and sterilized by moist heat at 121 ℃ for 30 min. Streptomyces cacao was inoculated into FN to 2216E broth at different pH by 1% o, control was not inoculated, and 3 replicates were set. The culture was incubated at 28 ℃ for 3-5 days and the growth (sedimentation) of the strain was recorded.
3. Salt tolerance
And then adding different amounts of NaCl into the 2216E liquid culture medium to prepare 2216E liquid culture medium with salt concentration of 1%, 2%, 5%, 7%, 9%, 11%, 13%, 15%, 17%, 20%, 25% and 30%, subpackaging in transparent test tubes, and performing moist heat sterilization at 121 ℃ for 30 min. Streptomyces cacao FN was inoculated at 1% o into 2216E broth at different salt concentrations, no control was inoculated, and 3 replicates were set. The culture was incubated at 28 ℃ for 3-5 days and the growth (sedimentation) of the strain was recorded.
4. Lethal temperature
Placing Streptomyces cacao FN bacteria liquid into water bath kettle with temperature of 60 deg.C, 65 deg.C, 70 deg.C, 75 deg.C, 80 deg.C, 85 deg.C, 90 deg.C, 95 deg.C and 100 deg.C respectively, timing for 10min, immediately placing into cold water, rapidly cooling, and repeating for 3 times. Mu.l of each was spread on fresh 2216E plates and incubated at 28 ℃ for 3-5 days, and the growth of the strain was recorded.
5. Sensitivity to ultraviolet light
Coating 100 μ l of Streptomyces cacao FN fermentation broth on 2216E plate, irradiating with 30w ultraviolet lamp 40cm for 0h, 0.5h, 1h, 2h, 4h, 8h, 12h, 16h, 20h, 24h, 32h, 40h, and 48h, culturing at 28 deg.C for 3-5 days, and recording strain growth condition.
The growth characteristics of the streptomyces cacao are shown in table 1, the optimal growth temperature is 28 ℃, and the growth temperature range is 15-45 ℃; the optimum growth pH is 7.0, and the growth pH range is 3.0-12.0; tolerance of 15% salt ion concentration. The lethal temperature is 85 ℃ and 10 min. The ultraviolet ray-sensitive antibacterial agent is not sensitive to ultraviolet rays, the survival rate of bacteria reaches 77% after the ultraviolet ray is continuously irradiated for 24 hours, and the survival rate of bacteria is still more than 60% after the ultraviolet ray is continuously irradiated for 32 hours.
TABLE 1 Streptomyces cococcharensis FN growth characteristics
Figure BDA0001256213750000121
Note: + positive; positive; w is weakly positive; negative
As can be seen from Table 1, Streptomyces theobromae FN has strong heat resistance, can keep a good growth state at 50 ℃, and has a death temperature of 95 ℃, thereby indicating that the Streptomyces theobromae FN still has high strain activity under high-temperature stress. The ultraviolet ray-sensitive antibacterial agent is not sensitive to ultraviolet rays, the survival rate of bacteria reaches 77% after the ultraviolet ray is continuously irradiated for 24 hours, and the survival rate of bacteria is still more than 60% after the ultraviolet ray is continuously irradiated for 32 hours.
The streptomyces theobromae FN not only can keep stronger strain activity in the fermentation process, but also can show that the fermentation inoculant can keep remarkable bacteriostatic effect at normal temperature in the prevention and treatment process of pathogenic bacteria, and can still keep good strain activity and bacteriostatic effect in high-temperature weather. And can still maintain good strain activity under outdoor strong light weather.
The streptomyces theobromae FN has acid resistance and extremely strong alkali resistance, still presents a good growth state under the alkali stress of pH10, not only can keep stronger strain activity in the fermentation process, but also can resist different acid-base environments of external sources such as pesticides, rain, snow and the like in the prevention and treatment process of the microbial inoculum applied to pathogenic bacteria, and simultaneously keeps higher bacteriostatic activity.
In addition, the streptomyces theobromae FN has good salt tolerance and can tolerate the salt stress action with the salt ion concentration of 17%.
Example 3 application of Streptomyces cacao FN to prevention and treatment of fungal diseases of fruits and vegetables
Determining the bacteriostasis rate of the streptomyces theobromae FN on pathogenic bacteria of apple anthracnose leaf blight, apple alternaria leaf spot, apple ring spot, apple rot, apple root rot, apple blight, cucumber gummy stem blight, cucumber damping off, tomato early blight, watermelon fusarium wilt, rape sclerotinia rot and cabbage black spot. A straight line is drawn on the back of a PDA flat plate with the diameter of 90mm passing through the center of the flat plate, probiotic cakes with the diameter of 7mm are inoculated on 2 points on the line 25mm away from the center, and a blank PDA culture medium with the diameter of 7mm is inoculated on a contrast flat plate. Inoculating the bacterial cake with 7mm of pathogenic bacteria to the center of a PDA flat plate, culturing at 28 ℃ for 5-10 days, respectively counting the radius of the control pathogenic bacteria and the radius of the treated pathogenic bacteria, and calculating the bacteriostasis rate. The bacteriostasis rate is (control pathogen radius mm-treated pathogen radius mm)/(control pathogen radius mm-pathogen cake radius 3.5mm) × 100%. Subculturing streptomyces theobromae FN, and measuring the bacteriostatic rate of the fungal pathogenic bacteria of fruits and vegetables at the 10 th generation, the 15 th generation, the 25 th generation and the 30 th generation, wherein the experimental method is the same as the above.
TABLE 2 bacteriostasis rate of Streptomyces cococcae FN on pathogenic bacteria of 12 plant diseases in different generations
Figure BDA0001256213750000131
Figure BDA0001256213750000141
As can be seen from Table 2, Streptomyces theobromae FN has strong inhibitory capacity on apple tree diseases (apple anthracnose leaf blight, apple alternaria leaf spot, apple rot, apple root rot, apple ring rot and apple blight), and also has strong inhibitory capacity on fungal diseases (cucumber gummy stem blight, cucumber damping off, tomato early blight, watermelon fusarium wilt, rape sclerotinia rot and cabbage black spot) on other fruits and vegetables.
After 30 generations of streptomyces theobromae FN, the bacteriostatic rates of the streptomyces theobromae FN on Bacillus anthracis, Alternaria alternata, Alternaria Bellengeri, Humicola malosa, Fusarium oxysporum, phytophthora cactorum, exocarpium citrulli, Rhizoctonia solani, Alternaria solani, Fusarium oxysporum, Sclerotinia sclerotiorum and Alternaria brassicae are respectively 85.3%, 88.0%, 76.5%, 88.5%, 77.1%, 82.5%, 69.3%, 72.5%, 78.6%, 76.1%, 80.2% and 88.6%, and are basically consistent with the bacteriostatic rate of the first generation of the streptomyces theobromae FN, which shows that the streptomyces theobromae FN provided by the invention does not reduce the inhibitory activity on the pathogenic bacteria along with the increase of the generation number of generations, and has higher genetic stability.
Example 4 broad-spectrum measurement of the effects of Streptomyces cococcensis FN on pathogenic bacteria hyphae and spores
Take the transformed apple of Alternaria alternata (Alternaria f. spmali) as an example. Inoculating apple alternaria leaf spot pathogenic bacteria to the center of a PDA (PDA) plate, respectively inoculating probiotics on two sides of the position 25mm away from the center of the plate, and growing for 7 days at 28 ℃. Rectangular fungus blocks which are close to probiotics and contain apple alternaria leaf spot pathogenic bacteria are cut from a grown flat plate, a thin layer of the cut fungus blocks is arranged on a glass slide by a small knife, and observation is carried out through an optical microscope. Apple alternaria leaf spot pathogens that were not inoculated with probiotics were treated identically.
The morphological structures of the hyphae and spores of the apple alternaria leaf spot disease pathogenic bacteria are observed through an optical microscope, and the hyphae of the pathogenic bacteria close to the edges of the probiotics are distorted, so that normal spores cannot be formed (figure 2). It is possible that the metabolite produced by the streptomyces theobromae FN is diffused through the PDA plate, the hypha morphology is induced to change after the pathogenic bacteria contact the metabolite, the hypha expands, and normal spores cannot be formed.
Example 5 preparation of Streptomyces cacao FN biocontrol microbial inoculum
Preparing spore suspension: adding sterile water to a slant of 2216E solid culture medium stored with streptomyces cacao FN, further scraping spores, shaking and mixing uniformly to obtain spore suspension with the thallus concentration of 108-109cfu/mL；
Seed liquid culture: inoculating the spore suspension into a seed liquid culture medium according to the inoculation amount of 1% (v/v), culturing at 28 ℃ for 200r/min for 96h in a shake flask to obtain seed liquid;
solid fermentation culture, namely inoculating the seed liquid into a solid fermentation culture medium with 50 percent of water content according to the inoculation volume mass ratio of 10 percent (v/m), fermenting and culturing for 4 days at 28 ℃, maintaining the constant water content of the solid fermentation culture medium at 50 percent from the beginning to the end of fermentation, turning over for 3 times during the fermentation period, air-drying at 28 ℃ after the fermentation is ended, grinding by a grinder, collecting the microbial inoculum passing through a 120-mesh sieve, and storing in the shade at room temperature to obtain the streptomyces theobromae FN biocontrol microbial inoculum, wherein the strain activity of the streptomyces theobromae FN biocontrol microbial inoculum is 3.7 × 1011cfu/g。
The 2216E solid medium (g/L): bacterial peptone No.25, yeast powder 1, agar 20 and the balance of distilled water, wherein the pH is natural;
the seed liquid culture medium (g/L): 10 parts of corn flour, 10 parts of peptone, 5 parts of yeast powder, 2 parts of dipotassium hydrogen phosphate, 0.3 part of magnesium sulfate and the balance of distilled water, wherein the pH value is natural;
the solid fermentation medium (%) (m/m): 20% of rice hull, 44.7% of bran, 22% of corn flour, 12% of bean cake powder, 0.8% of calcium carbonate, 0.3% of dipotassium hydrogen phosphate, 0.2% of magnesium sulfate and natural pH.
The media of examples 6-9 below were all the same as in example 4.
Example 6 preparation of Streptomyces cacao FN biocontrol microbial inoculum
Preparing spore suspension: adding sterile water to a slant of 2216E solid culture medium stored with streptomyces cacao FN, further scraping spores, shaking and mixing uniformly to obtain spore suspension with the thallus concentration of 108-109cfu/mL；
Seed liquid culture: inoculating the spore suspension into a seed liquid culture medium according to the inoculation amount of 1% (v/v), culturing at 28 ℃ for 160r/min for 90h in a shake flask to obtain seed liquid;
solid fermentation culture, namely inoculating the seed solution into a solid fermentation culture medium with the water content of 50% (v/m) according to the inoculation volume mass ratio of 15% (v/m), fermenting and culturing for 3 days at 28 ℃, air-drying at 20 ℃, grinding by using a grinder, collecting a microbial inoculum passing through a 120-mesh sieve, and storing in the shade at room temperature to obtain the streptomyces theobromae FN biocontrol microbial inoculum, wherein the strain activity of the streptomyces theobromae FN biocontrol microbial inoculum is 2.2 × 1011cfu/g。
Example 7 preparation of Streptomyces cacao FN biocontrol microbial inoculum
Preparing spore suspension: adding sterile water to a slant of 2216E solid culture medium stored with streptomyces cacao FN, further scraping spores, shaking and mixing uniformly to obtain spore suspension with the thallus concentration of 108-109cfu/mL；
Seed liquid culture: inoculating the spore suspension into a seed liquid culture medium according to the inoculation amount of 3% (v/v), culturing at 28 ℃ for 240r/min in a shake flask for 100h to obtain a seed liquid;
solid fermentation culture, namely inoculating the seed solution into a solid fermentation culture medium with 50 percent of water content according to the inoculation volume mass ratio of 5 percent (v/m), fermenting and culturing for 6 days at 28 ℃, air-drying at 30 ℃, grinding by using a grinder, collecting a microbial inoculum passing through a 120-mesh sieve, and storing at room temperature and shade to obtain the streptomyces theobromae FN biocontrol microbial inoculum, wherein the strain activity of the streptomyces theobromae FN biocontrol microbial inoculum is 2.3 × 1011cfu/g。
Example 8 preparation of Streptomyces cacao FN biocontrol microbial inoculum
Preparing spore suspension: will be sterileAdding water to the inclined plane of 2216E solid culture medium stored with streptomyces cacao FN, further scraping spores, shaking and mixing uniformly to obtain spore suspension with the thallus concentration of 108-109cfu/mL；
Seed liquid culture: inoculating the spore suspension into a seed liquid culture medium according to the inoculation amount of 1% (v/v), culturing at 28 ℃ for 180r/min in a shake flask for 95h to obtain seed liquid;
solid fermentation culture, namely inoculating the seed liquid into a solid fermentation culture medium with 50 percent of water content according to the inoculation volume mass ratio of 8 percent (v/m), fermenting and culturing for 3 days in a koji tray at the temperature of 28 ℃, maintaining the constant water content of the solid fermentation culture medium at 50 percent from the beginning to the end of fermentation, turning over for 2 times during the fermentation period, air-drying at the temperature of 25 ℃ after the fermentation is ended, grinding by a grinder, collecting a microbial inoculum passing through a 120-mesh sieve, and storing in the shade at room temperature to obtain the streptomyces theobromae FN biocontrol microbial inoculum, wherein the strain activity of the streptomyces theobromae FN biocontrol microbial inoculum is 3.2 × 1011cfu/g。
Example 9 preparation of Streptomyces cacao FN biocontrol microbial inoculum
Preparing spore suspension: adding sterile water to a slant of 2216E solid culture medium stored with streptomyces cacao FN, further scraping spores, shaking and mixing uniformly to obtain spore suspension with the thallus concentration of 108-109cfu/mL；
Seed liquid culture: inoculating the spore suspension into a seed liquid culture medium according to the inoculation amount of 1.5% (v/v), performing shake-flask culture at 28 ℃ for 98h at 220r/min to obtain a seed liquid;
solid fermentation culture, namely inoculating the seed liquid into a solid fermentation culture medium with 50 percent of water content according to the inoculation volume mass ratio of 12 percent (v/m), fermenting and culturing for 5 days in a koji tray at the temperature of 28 ℃, maintaining the constant water content of the solid fermentation culture medium at 50 percent from the beginning to the end of fermentation, turning over for 3 times during the fermentation period, air-drying at the temperature of 30 ℃ after the fermentation is ended, grinding by a grinder, collecting a microbial inoculum passing through a 120-mesh sieve, and storing in the shade at room temperature to obtain the streptomyces theobromae FN biocontrol microbial inoculum, wherein the strain activity of the streptomyces theobromae FN biocontrol microbial inoculum is 3.5 × 1011cfu/g。
Example 10 field test of Streptomyces cacao FN biocontrol microbial inoculum for controlling alternaria leaf spot in apple
Streptomyces cococcharensis FN biocontrol bacterial agent was prepared as described in example 5.
And (3) field test design: 6 treatments are carried out, namely a 1000-time diluent of the streptomycete FN biocontrol microbial inoculum is obtained; 3000 times of dilution of streptomyces theobromae FN biocontrol microbial inoculum; a 5000-time diluent of streptomyces cocoas FN biocontrol microbial inoculum; 1000 times of diluent of 10% polyoxin wettable powder; 80% mancozeb wettable powder 600 times of diluent; the above dilution liquid is clear water, and the control group is clear water. Each treatment was repeated 4 times for a total of 24 plots, 2 apple trees per plot, in a random block arrangement. The test site is carried out in a Bizhen Fujiayuncun apple orchard in Qianyang county of Baoyang province, Baoying, Shaanxi province, the tree species is Fuji, the tree age is 12 years, the plant spacing of the fruit trees is 3m, and the row spacing is 4 m. The streptomyces cocoas FN biocontrol microbial inoculum is prepared by the method in the embodiment 6, diluted by clear water with proper concentration and sprayed.
The total administration time is 6 times, the first time is 5 months and 18 days, the second time is 5 months and 30 days, the third time is 6 months and 12 days, and the fourth time is 7 months and 4 days. 17 days in 7 th month and 3 days in 8 sixth month. Using a Beijing Fengmeng DFH-16A type knapsack manual sprayer, the whole stem and leaf is treated by a uniform spraying method. The liquid spraying amount is equal to that the front and the back of the blade are uniformly sprayed with wet liquid and the liquid medicine drops downwards.
The investigation method comprises the following steps: investigation was conducted before the drug was taken on day 16 at month 5 and after the drug was taken 10-14 days after the end of day 3 at month 8. 2 trees in each plot are investigated, each plant is respectively fixed with 2 new shoots in east, west, south, north and middle 5 directions, all leaves are investigated, and disease index and prevention and treatment effect are calculated. The grading criteria for leaf disease grade are as follows:
level 0: no disease spots;
level 1: the lesion area accounts for less than 10% of the whole leaf area;
and 3, level: the lesion area accounts for 11% -25% of the whole leaf area;
and 5, stage: the lesion area accounts for 26-40% of the whole leaf area;
and 7, stage: the lesion area accounts for 41-65% of the whole leaf area;
and 9, stage: the lesion area accounts for more than 66% of the whole leaf area.
The drug effect calculation method comprises the following steps: disease index is 100 × Σ [ (number of diseased leaves at each stage × relative stage value) ]/(total number of leaves investigated × 9); the prevention and treatment effect (%) is [1- (pre-drug disease index of the control area x post-drug disease index of the treatment area)/(post-drug disease index of the control area x pre-drug disease index of the treatment area) ] x 100%. The data were analyzed using the Duncan's New repolarization test method using the DPS software, and the results are shown in Table 3.
TABLE 3 preventive and therapeutic effects of Streptomyces cacao FN biocontrol microbial inoculum on alternaria leaf spot of apple
Figure BDA0001256213750000181
Note that lower case letters indicate the significance of the difference at the 5% level and upper case letters indicate the significance of the difference at the 1% level.
The streptomyces cacao FN biocontrol microbial inoculum prepared by the method in the embodiment 4 is diluted by 1000 times with clear water, uniformly sprayed on fruit trees until liquid medicine drops on leaves, and sprayed at spring tips for 6 times, and the control effect on apple alternaria leaf spot is 87.10%.
It should be noted that: the streptomyces theobromae FN biological control microbial inoculum prepared in the embodiments 6 to 9 of the invention also has the experimental effect, and the difference between the embodiments and the experimental effect is not great.
Example 11 field test of Streptomyces cacao biocontrol microbial inoculum for controlling apple anthracnose leaf blight
Streptomyces cococcharensis FN biocontrol bacterial agent was prepared as described in example 5.
And (3) field test design: total 6 treatments, 1000 times of dilution of streptomyces theobromae FN biocontrol microbial inoculum; 3000 times of dilution of streptomyces theobromae FN biocontrol microbial inoculum; a 5000-time diluent of streptomyces cocoas FN biocontrol microbial inoculum; 1000 times of diluent of 10% polyoxin wettable powder; 80% mancozeb wettable powder 600 times of diluent; the above dilution liquid is clear water, and the control group is clear water. Each treatment was repeated 4 times for a total of 24 plots, 2 apple trees per plot, in a random block arrangement. The test site is carried out in a Bizhengjia Bay village apple orchard in Qianyang county of Baoyang province, the variety of the test tree is 'golden crown', the tree age is 15 years, the plant spacing of the fruit tree is 3m, and the row spacing is 4 m. The streptomyces cocoas FN biocontrol microbial inoculum is prepared by the method in the embodiment 6, diluted by clear water with proper concentration and sprayed.
The total number of applications is 6, the first time is 5 months and 16 days, the second time is 6 months and 1 day, the third time is 6 months and 13 days, and the fourth time is 7 months and 5 days. 17 days in 7 th month and 7 days in 8 th month. Using a Beijing Fengmeng DFH-16A type knapsack manual sprayer, the whole stem and leaf is treated by a uniform spraying method. The liquid spraying amount is equal to that the front and the back of the blade are uniformly sprayed with wet liquid and the liquid medicine drops downwards.
The investigation method comprises the following steps: anthracnose leaf blight does not occur before the drug is taken for 5, 16 and 16 days. Investigation was carried out 10-14 days after the next dose at the end of 8 months and 7 days. 2 trees in each plot are investigated, each plant is respectively fixed with 2 new shoots in east, west, south, north and middle 5 directions, all leaves are investigated, and disease index and prevention and treatment effect are calculated. The grading criteria for leaf disease grade are as follows:
level 0: no disease spots;
level 1: the lesion area accounts for less than 10% of the whole leaf area;
and 3, level: the lesion area accounts for 11% -25% of the whole leaf area;
and 5, stage: the lesion area accounts for 26-40% of the whole leaf area;
and 7, stage: the lesion area accounts for 41-65% of the whole leaf area;
and 9, stage: the lesion area accounts for more than 66% of the whole leaf area.
The drug effect calculation method comprises the following steps: disease index is 100 × Σ [ (number of diseased leaves at each stage × relative stage value) ]/(total number of leaves investigated × 9); the prevention and treatment effect (%) is [1- (pre-drug disease index of the control area x post-drug disease index of the treatment area)/(post-drug disease index of the control area x pre-drug disease index of the treatment area) ] x 100%. The data were analyzed using the Duncan's New repolarization test method using the DPS software, and the results are shown in Table 4.
TABLE 4 prevention and control effects of Streptomyces cacao FN biocontrol microbial inoculum on apple anthracnose leaf blight
Figure BDA0001256213750000191
Figure BDA0001256213750000201
The streptomyces cacao FN biocontrol microbial inoculum prepared by the method in the embodiment 4 is diluted by 1000 times by clear water, is uniformly sprayed on fruit trees until the liquid medicine drops on leaves, is sprayed at spring tips for 6 times, and has 82.17 percent of control effect on apple anthracnose leaf blight.
The field test results of the embodiment 9 and the embodiment 10 show that the streptomyces cacao FN biological control fungicide for preventing and controlling alternaria leaf spot and anthrax leaf blight of the apple trees is safe and has no phytotoxicity to the apple trees when the dosage is diluted by 1000, 3000 and 5000 times.
The streptomyces theobromae FN biological control microbial inoculum is used for controlling apple alternaria leaf, and when the dosage is diluted by 1000, 3000 and 5000 times of solution, the control effects are respectively as follows: 87.10%, 85.58% and 82.74%. On the difference level of 1% and 5%, the control effect of the streptomyces cocoas FN biological control microbial inoculum for controlling apple alternaria leaf spot is superior to that of the streptomyces cocoas FN biological control microbial inoculum when the dosage of the streptomyces cocoas FN biological control microbial inoculum is 1000 times of the diluted liquid, the streptomyces cocoas FN biological control microbial inoculum is 3000 times of the diluted liquid and 5000 times of the diluted liquid, and the control effect of the streptomyces cocoas FN biological control microbial inoculum when the diluted liquid is 1000 times of the diluted liquid is obviously higher than that of the 10% polyoxin wettable powder serving as a control medicament when the dosage of the diluted.
The streptomyces theobromae FN biological control microbial inoculum is used for controlling apple anthracnose and leaf blight, and when the dosage is 1000, 3000 and 5000 times of diluted solution, the control effects are respectively as follows: 82.17%, 77.83%, 73.24%. On the difference level of 1% and 5%, the control effect of the streptomyces cacao FN biological control fungicide on apple anthracnose leaf blight is superior to that of the streptomyces cacao FN biological control fungicide when the dosage is diluted by 1000 times of liquid, namely 3000 times of liquid and 5000 times of liquid. Meanwhile, the control effect of the streptomyces cocoas FN biological control microbial inoculum diluted by 1000 times is obviously higher than that of the control medicament 10% polyoxin wettable powder diluted by 1000 times and that of 80% mancozeb wettable powder diluted by 600 times.
The 1000-time diluent of the streptomyces cococcae FN biological control microbial inoculum has good control effects on alternaria leaf spot of apples and anthracnose leaf blight of apples, and is superior to 1000-time liquid of 10% polyoxin wettable powder and 600-time liquid of 80% mancozeb wettable powder of a control medicament. During the test, the occurrence of phytotoxicity phenomenon of the streptomyces cacao FN biocontrol microbial inoculum is not found.
It should be noted that: the streptomyces theobromae FN biological control microbial inoculum prepared in the embodiments 6 to 9 of the invention also has the experimental effect, and the difference between the embodiments and the experimental effect is not great.
Example 12 Heat stability of Streptomyces cococcae FN biocontrol microbial Agents
The streptomyces theobromae FN biological control microbial inoculum prepared in the example 5 is respectively treated at the temperature of 50, 70, 90, 100 and 121 ℃ for 30min, and is respectively diluted by 100 times by using sterile water to be tested. Using apple alternaria leaf spot as a test object, inoculating 7mm of pathogenic bacteria cake to the center of a PDA (personal digital assistant) plate, marking through the center of the plate, punching 7mm holes at 2 points on the line 25mm away from the center by using a puncher, respectively adding 100 mu l of liquid to be tested, and adding 100 mu l of sterile water in contrast. Culturing at 28 deg.C for 7-10 days, respectively counting the radius of control pathogenic bacteria and the radius of treated pathogenic bacteria, and calculating the antibacterial rate. The bacteriostasis rate is (control pathogen radius mm-treated pathogen radius mm)/(control pathogen radius mm-pathogen cake radius 3.5mm) × 100%.
TABLE 5 determination of the thermostability of Streptomyces cococcae FN biocontrol microbial inoculum
Figure BDA0001256213750000211
As can be seen from Table 5, the bacteriostatic ability of the Streptomyces cacao FN biocontrol microbial inoculum is not significantly changed after the Streptomyces cacao FN biocontrol microbial inoculum is treated at high temperature. As can be seen from the bacteriostasis rate, the microbial inoculum after different high-temperature treatments is slightly reduced after being treated for 30min at 100 ℃ and 121 ℃, but still can reach more than 83.7 percent. Therefore, the antibacterial activity of the streptomyces cacao FN biological control microbial inoculum has stronger thermal stability.
It should be noted that: the streptomyces theobromae FN biological control microbial inoculum prepared in the embodiments 6 to 9 of the invention also has the experimental effect, and the difference between the embodiments and the experimental effect is not great.
SEQUENCE LISTING
<110> Shanxi Feng Dan Baili Biotech Co Ltd
<120> preparation method of biocontrol microbial inoculum for preventing and treating apple tree fungal diseases
<130>2017
<160>1
<170>PatentIn version 3.5
<210>1
<211>1490
<212>DNA
<213> Streptomyces cococcensis FN (Streptomyces cahooi) CGMCC No.13080
<400>1
agagtttgat cctggctcag gacgaacgct ggcggcgtgc ttaacacatg caagtcgaac 60
gatgaaccgg tttcggccgg ggattagtgg cgaacgggtg agtaacacgt gggcaatctg 120
ccctgcactc tgggacaagc cctggaaacg gggtctaata ccggatatga ccaccggccg 180
catggtctgg tggtggaaag ctccggcggt gcaggatgag cccgcggcct atcagcttgt 240
tggtggggtg atggcctacc aaggcgacga cgggtagccg gcctgagagg gcgaccggcc 300
acactgggac tgagacacgg cccagactcc tacgggaggc agcagtgggg aatattgcac 360
aatgggcgca agcctgatgc agcgacgccg cgtgagggat gacggccttc gggttgtaaa 420
cctctttcag cagggaagaa gcgcgagtga cggtacctgc agaagaagca ccggctaact 480
acgtgccagc agccgcggta atacgtaggg tgcgagcgtt gtccggaatt attgggcgta 540
aagagctcgt aggcggcctg tcgcgtcgga tgtgaaagcc cggggcttaa ccccgggtct 600
gcgttcgata cgggcaggct agagttcggc aggggagatt ggaattcctg gtgtagcggt 660
gaaatgcgca gatatcagga ggaacaccgg tggcgaaggc ggatctctgg gccgatactg 720
acgctgagga gcgaaagcgt ggggagcgaa caggattaga taccctggta gtccacgccg 780
taaacgttgg gcactaggtg tgggcggcat tccacgtcgt ccgtgccgca gctaacgcat 840
taagtgcccc gcctggggag tacggccgca aggctaaaac tcaaaggaat tgacgggggc 900
ccgcacaagc ggcggagcat gtggcttaat tcgacgcaac gcgaagaacc ttaccaaggc 960
ttgacataca tcggaaaact ctggagacag ggtccccctt tgggtcggtg tacaggtggt 1020
gcatggctgt cgtcagctcg tgtcgtgaga tgttgggtta agtcccgcaa cgagcgcaac 1080
ccttatcctg tgttgccagc atgcctttcg gggtgatggg gactcacggg agactgccgg 1140
ggtcaactcg gaggaaggtg gggacgacgt caagtcatca tgccccttat gtcttgggct 1200
gcacacgtgc tacaatggcc ggtacaatga gctgcgatgc cgtgaggtgg agcgaatctc 1260
aaaaagccgg tctcagttcg gattggggtc tgcaactcga ccccatgaag tcggagtcgc 1320
tagtaatcgc agatcagcat tgctgcggtg aatacgttcc cgggccttgt acacaccgcc 1380
cgtcacgtca cgaaagtcgg taacacccga agccggtggc ccaacccctt gtgggaggga 1440
attgtcgaag gtgggactgg cgattgggac gaagtcgtaa caaggtaacc 1490

Claims (6)

1. The biocontrol microbial inoculum for preventing and treating the fungal diseases of apple trees is characterized by comprising the following specific preparation methods:
preparing spore suspension: adding sterile water to the inclined plane of 2216E solid culture medium stored with streptomyces cacao, further scraping spores, shaking and uniformly mixing to obtain spore suspension, wherein the thallus concentration of the spore suspension is 108-109cfu/mL；
Seed liquid culture: inoculating the spore suspension into a seed liquid culture medium according to the inoculation amount of 1-3%, culturing at 28 ℃ for 160-;
solid hairAnd (2) fermentation culture, namely inoculating the seed solution into a solid fermentation culture medium with the water content of 50% according to the volume mass ratio of the inoculation amount of 5-15%, fermenting and culturing for 3-6 days in a koji tray at the temperature of 28 ℃, air-drying at the temperature of 20-30 ℃, grinding by using a grinder, collecting a microbial inoculum passing through a 120-mesh sieve, and storing in the shade at room temperature to obtain the streptomyces theobromae biocontrol microbial inoculum, wherein the strain activity of the streptomyces theobromae biocontrol microbial inoculum is not less than 1 × 1011cfu /g；
The biocontrol bacterium is streptomyces theobromae (Streptomyces theobromae) (II)Streptomyces cacaoi) FN with preservation number of CGMCC No. 13080.
2. The biocontrol microbial inoculum for controlling apple tree fungal diseases of claim 1, which is characterized in that:
and (3) seed liquid culture: inoculating the spore suspension into a seed liquid culture medium according to the inoculation amount of 1-1.5%, performing shake culture at 28 ℃ for 220r/min for 95-98h to obtain a seed liquid.
3. The biocontrol microbial inoculum for controlling apple tree fungal diseases of claim 2, which is characterized in that:
and (3) seed liquid culture: inoculating the spore suspension into a seed liquid culture medium according to the inoculation amount of 1%, performing shake culture at 28 ℃ at 200r/min for 96h to obtain a seed liquid.
4. The biocontrol microbial inoculum for controlling apple tree fungal diseases of claim 1, which is characterized in that:
and (3) solid fermentation culture: inoculating the seed liquid into a solid fermentation culture medium with the water content of 50% according to the inoculation volume mass ratio of 8-12%, fermenting and culturing for 3-5 days in a koji tray at the temperature of 28 ℃, and maintaining the constant water content of the solid fermentation culture medium from the beginning to the end of fermentation: 50 percent, turning over for 2-3 times during fermentation, air-drying at 25-30 ℃ after fermentation is finished, grinding by a grinder, collecting the microbial inoculum passing through a 120-mesh sieve, and storing in the shade at room temperature to obtain the streptomyces theobromae FN biocontrol microbial inoculum.
5. The biocontrol microbial inoculum for controlling apple tree fungal diseases of claim 4, which is characterized in that:
and (3) solid fermentation culture: inoculating the seed liquid into a solid fermentation culture medium with the water content of 50% according to the inoculation amount, the volume and the mass ratio of 10%, fermenting and culturing for 4 days in a koji tray at the temperature of 28 ℃, and maintaining the constant water content of the solid fermentation culture medium from the beginning to the end of fermentation: and 50 percent, overturning for 3 times during fermentation, after the fermentation is finished, air-drying at 28 ℃, grinding by using a grinder, collecting the microbial inoculum passing through a 120-mesh sieve, and storing in a shade at room temperature to obtain the streptomyces coco FN biocontrol microbial inoculum.
6. The biocontrol microbial inoculum for controlling apple tree fungal diseases of claim 1, which is characterized in that:
the 2216E solid medium comprises the following components in g/L: bacterial peptone No.25, yeast powder 1, agar 20 and the balance of distilled water, wherein the pH is natural;
the seed liquid culture medium comprises the following components in g/L: 10 parts of corn flour, 10 parts of peptone, 5 parts of yeast powder, 2 parts of dipotassium hydrogen phosphate, 0.3 part of magnesium sulfate and the balance of distilled water, wherein the pH value is natural;
the solid fermentation medium comprises the following components in percentage by mass: 20% of rice hull, 44.7% of bran, 22% of corn flour, 12% of bean cake powder, 0.8% of calcium carbonate, 0.3% of dipotassium hydrogen phosphate, 0.2% of magnesium sulfate and natural pH.
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