TWI325891B - - Google Patents

Description

1325891 In view of the various ills of traditional chemical pesticides, the development of bio-rational biological agents (Biocontr.·agent) is the main focus of the current agricultural biotechnology policy promotion in various countries around the world, and the development of various agricultural biological agents is constantly being introduced. Various agricultural biological preparations have been developed with microbial preparations, and related industrialized mass production control technologies are relatively mature and widely applicable, and the effects are easy to appear. Sirepiomyces spp. is a very common Gram-positive bacterium in the soil of nature. It is known to have more than 400 species of Streptomyces, most of which have the ability to produce antibiotics. Has been applied to the control of crop diseases for more than half a century. From the earliest 1943, Wasksman isolated streptomycin from Streptomyces griseus (s. gr/sei/s) and successfully applied it to fruit trees. Prevention and control of bacterial diseases with vegetables So far, similar development success stories 'the most well-known ones are pushing 195〇_7〇 保Blasticidin-S and Kasugamycin in rice rice fever prevention and treatment The application 'and the application of Polyoxjn and Validamycin in the control of rice sheath blight. After 1970, 'the development of relevant applicability is based on the taste of soil-borne disease prevention and control'. The successful examples of the application of prevention and treatment include Rhizoctonia solani (R/?/z〇ciom_aso/am·) Caused by pea root rot, carnation and melon wilt caused by Fuysar/wm; storage disease caused by /\spersf/7/iys of genus Fusarium and including Gibberella yA/iernana spp. of the genus Pseudomonas, culmonum of the genus Fusarium, B〇trytis cinerea, and Pythium sp. 1325891

The inhibition of various important fungal pathogens such as PyM/wm, etc. The application of these known Streptomyces in disease prevention and control, the common characteristics of which are mostly based on the development of antibiotics, and should be attributed to "biochemical preparations" in nature. And the emphasis is on the inhibition or lethal effect of antibiotics on pathogenic bacteria. The application of anti-biomass in agriculture has been controversial in recent years due to the growing problem of important pathogen resistance in human disease treatment; the application is quite limited, and market value and commercial application intention are greatly affected. ~ In view of the questioning about the application of antibiotics in disease prevention and control, in the development and application of microbial biological preparations, the development and application of biologic preparations (B丨〇mass) have received considerable attention in recent years. It was published and confirmed that the actual application can achieve the expected effect of disease prevention and management. Well-known examples are G. foe丨adfum Vfnde (trade name Soil Guard). Trichoderma (7y/c/70de/7?7a spp, trade name p丨ant

Fungal preparations such as helper and the like, and bacterial preparations such as Bacillus subtilis (SWM///S, trade name Kodiak, etc.) and Streptomyces (Sire^〇myces gnsews, trade name Mycostop). The direct application of this kind of biomass is particularly referred to as probiotics or probiotics in the field of animals including aquaculture. Its applicability has been extremely popular in animal breeding in recent years. Its main use is as a feed additive, activation. Intestinal function 'to the utilization of feed and promote animal growth and development (m〇regu|at〇r function). In the application of plant diseases, the above-mentioned bio-living preparations are characterized by the fact that their functions are manifested as multiple mechanisms of action, including the competition of the original nutrition and space, the role of anti-biomass, and the promotion of soil eight: decomposition and nutrition. Absorption of effectiveness, abundance of microbial populations in the soil: the promotion of the degree of dissimilarity and the health of the microbial ecological phase, the promotion of growth, the nature of mites, and the promotion of disease resistance. It is worth noting that its anti-biomass effect is the combined effect of the entire bacterial metabolite' rather than the single anti-biomass effect of traditional applications. Because it is a multiple mechanism of action, 'the common drug resistance problems in traditional pesticide applications will not occur; plus its positive effect on the soil, ecological environment and plant growth, it will ensure the sustainable development of agriculture (Agricu|tura | SUStainabmty) Recommended for plant protection biotechnology applications. The use of natural antagonistic beneficial microorganisms to develop biological rational biological control to reduce or replace the application of chemical pesticides has been an important scientific and technological development theme promoted by China's agricultural administrative units in the past two or three decades. The results, however, are that the existing research work is only limited to the screening and separation of beneficial microorganisms, as well as the laboratory or greenhouse test evaluation of the potential of disease control applications, and its further commercial application attempts are rare and rare, successful industrialization. The application has never been seen before. Reviewing some of the past research work, the inventors found that the reason why there has been no successful industrial application 'mainly lies in the lack of mass production technology; there is a clock to implement these existing research results. Industrialization, the establishment of a platform for mass production and application technology is indeed necessary. Regarding the commercial development and application of microbial live preparations, important key technologies are not necessary. (1) The selected microbial objects must be generally regarded as safe (GRAS), and should not be dangerous. Animal or ecological barrier, (2) I product storage stability performance is the need for commercialization ' (3) easy to integrate into the current cultivation operation system in the field, and (4) product price, smell 'useability, etc. can be accept. As far as crop disease control applications are concerned, the development potential of Streptomyces is quite promising, and there are quite a lot of developers involved. However, as mentioned above, most of the research and development focus is on the development and application of antibiotics. For example, in the United States, where biopesticide development is the most advanced, the Streptomyces living preparations officially registered by the EPA are still only available from Kemjra, Finland.

Mycostop ’ and US Natural Industries are registering

Actinovate; the former is composed of a wettable powder prepared from peat soil antagonist & gr/seoWr/des. The number of bacteria per gram is about 108' mainly composed of mycelium and spore. It is used in the cultivation and management of soil-borne diseases. The latter is a water-dispersible granule containing bacteria, and the main control object is also a soil-borne disease. In terms of mass production of Streptomyces living preparations, the conventional techniques are mainly applied to the fermentation of natural grain solids such as wheat grains and brown rice, and the spore preparations, the yield, storage stability, and biological activity can also be achieved. Specific application requirements. As for the liquid fermentation mass production, although the liquid fermentation mass production is known as the most efficient culture method for microbial mass production, it is not possible to grow the streptomyces in the vegetative mycelium state in the liquid. The known spores are stored in the development of metabolites. So far, there has been no technology in the world for the production of a storage-resistant Streptomyces in vivo by liquid fermentation. 1325891 The understanding of the mechanism of action is the basis of applied development and technological improvement. As far as the application of the chain bacillus in disease control is concerned, it is generally believed that the mechanism of action is mainly related to the inhibition or lethal effect of the anti-biomass produced by the pathogen. Related research in recent years has further pointed to the importance of streptomyces including hydrolyzing enzymes such as chitinolytic enzymes and glucan-degrading enzymes; it is worth mentioning that 'there are many existing cognitive observations. The inferences obtained are 'direct evidence support yet to be actually experienced. For the development and application of key mold biologics, liquid fermentation mass production is one of the most widely used, fastest, and most successful mass production methods. However, as far as the know-how is concerned, since the development target is salty and biochemical-based biochemical preparations are the main ones, the technical development focus is mainly on the enhancement of the secreted anti-biomass and quantity, and the development of the bacterial preparation must be emphasized. In the mass production of the spores stored in the female genus, the existing known techniques are attempted by solid fermentation, and the scale is not large, and quantitative production is not easy. As for the development of the storage-resistant spore-forming bacteria and their applicability by liquid fermentation, it has not been seen yet. As far as is known, there have been no successful reports or commercial applications of the production of Streptomyces streptococci in liquid fermentation. The high-density, stable mass production technology that the industrialization of the bacterial preparations should be established is yet to be established. SUMMARY OF THE INVENTION In view of the shortcomings of streptomyces which are difficult to differentiate into reservoir-resistant spores in liquid culture, the present inventors have an understanding of the mechanism of action of pathogenic bacteria against Streptomyces, screening of strain characteristics, preparation of inoculation sources, preparation of medium components, And the regulation of the fermentation conditions of the fermentation process, etc., after many years of attempts to improve 'finally developed successfully through the liquid fermentation culture mass production to the storage of the cubits of the 1328951 main embedded technology platform (technique platform)' and confirmed its Streptomyces strains of different species can also be applied 'and the sample of the produced preparation is applied to cultivated crops through multiple greenhouses and fields. It has been confirmed that it can achieve the prevention of pathogen damage and promote plant growth. The effect. SUMMARY OF THE INVENTION An object of the present invention is to provide a Streptomyces bacterial preparation comprising a reservoir-resistant spore and a liquid medium having chitinolytic activity (C), antibiotic (A) and susceptibility (S). Preferably, the above-mentioned reservoir-resistant spores are obtained by the following steps: providing a pathogenic strain to be tested on the medium; and performing a biochemical screening of the Streptomyces strain and the above-mentioned tested pathogenic strain; measuring the size of the inhibition circle; The above Streptomyces strain capable of forming a large inhibition zone is placed in a medium containing chitin; the size of the permeabilization circle is measured; and the Streptomyces strain having a good sporulation property and a large permeabilization circle is selected, wherein This strain produces resistant reservoir spores. More preferably, the reservoir-resistant spore line of the above strain can be stored at 6. 〇 Up to 1 month. Preferably, the preparation contains at least 101 Qcfu/ml of spores. Preferably, the liquid medium of the above preparation is an oat-containing medium. The present invention further relates to a method for producing a large amount of 10 1325891 reservoir-resistant spores by a liquid mass culture strain, which comprises providing a strain having excellent productivity; The strain is propagated and the spores of the strain are propagated; the spores are cultured in small amounts as the inoculation source; the spores of the inoculation source are put into the liquid medium and fermented in a stirred liquid fermentation tank, the temperature is controlled at 28-37 ° C, and the stirring rate is 80-250 rpm. The ventilation is 0.25-0.75 vvm and the pH is between 5.0-8.0. Preferably, the inoculum source is supplied to the liquid medium at a concentration of 108 cfu/ml. More preferably, the above liquid medium is based on the identification of Kjeldahl in the nutrient solution (Κ2ΗΡ04 0.7 g, ΚΗ2Ρ04 0.5 g, MgS04.7H20 0.5 g, FeS04 7H20 0.01 g, and ZnS04 0.001 g) as the basis of the mineral source' Adding oat, wheat or corn as an organic nutrient substrate. The invention further relates to a liquid medium of a strain of the genus Clematis, wherein the liquid medium comprises a mineral source composition basis for the Kelvin medium, 0.15% to 1% The chitin is a carbon source comprising a group selected from the group consisting of sucrose, mannitol, sorbitol, maltose, starch, cellulose, glucose, pectin and sputum. Preferably, the medium contains a carbon source of from 1 to 3% sucrose and from 0.12% to 0.6% starch. Preferably, the liquid medium promotes the secretion of anti-biomass and chitinolytic enzymes by strains of the genus Streptomyces. More preferably, the liquid medium further comprises from 0 to 12% to 0.60% of 1325891 pectin as a carbon source. More preferably, the liquid medium promotes the secretion of chitinolytic enzymes by strains of the genus Streptomyces. Preferably, the carbon medium of the liquid medium is oats and 1% chitin. Preferably, the carbon medium of the liquid medium is corn and 1 〇 / 〇 chitin 0. Preferably, The liquid medium further comprises a growth factor selected from the group consisting of 0 〇 4% - 1 〇 / 〇 malt extract 'to 1 〇 / 0 yeast extract and up to 1 〇 / 〇 protein Chen. More preferably, the yeast extract in the liquid medium is optimal for promoting mycelial growth. More preferably, the malt extract in the liquid medium is optimal for the transformation of the mycelium into spores. More preferably, the malt extract in the liquid medium maintains the anti-living property of the strain in an elevated state. More preferably, the addition of a growth factor in the liquid medium promotes the secretion of the ruthenium-degrading enzyme. . Further, the present invention relates to a Streptomyces sp. system for controlling plant diseases, which comprises an anti-biomass produced by the strain of the genus Bacillus, and a chitinolytic enzyme. Preferably, the bacterial preparation is for controlling the disease caused by the oomycete. Preferably, the bacterial preparation is for controlling the disease caused by the bacterium of the genus Oomycetes. Preferably, the bacterial system agent is a disease causing disease caused by the pylori pathogen. Preferably, the bacterial cell preparation is better for controlling the Rhizoctonia solani caused by the incomplete bacteria. The bacterial preparation is for controlling the disease caused by the wilting. More preferably, the bacterial preparation is caused by the prevention and control of the bacterium by the genus Gibberella. The method for controlling the disease is to understand the mechanism of the action of the genus Mycobacterium on the anti-αα σ effect of the disease. This month's people use the local separation of the chain · % Pei S. gfnSe〇J{jrtv/7A7ei7s S3 as a test material. S3 liquid sulfhydryl; 5 ice 4 ^ ^ σ nutrient solution and its solid filtrate obtained after separation of solid and liquid, 鳋▲ diluted 100 times, treated by steaming melon young field 'other water H treatment was used as a control group (CK) to compare the control effects against the damage of Pythium. The test results are shown in the first figure. The data shown in the figure is the survival rate of the seedlings under different treatments. The effect of the disease control is better than the best effect of the culture medium. The effect of the culture sputum is the control effect. Relatively worst. It is shown that in terms of the disease control effect, it is mainly the effect of the cells after application, and the existing antibiotics in the culture solution are not critical. f Example 2: The role of the strain of the fungus. The anti-biomass 13 1325891 and the production of hydrolyzed enzymes should be the most important factor in the inhibition or lethal effect on the pathogenic fungi after the application of the bacteria. On the whole, it is apparent that the multiple action mechanism (multiple m〇des 〇f acti〇n)__ includes the competition between the applied antagonistic and pathogenic bacteria and space, the antibiotic action, the super parasitic effect (mycoparasitism), the soil. Medium and large-scale biomolecules include the promotion of decomposition of plant residues remaining in the pathogen, the provision of organic nutrients and the improvement of soil properties, the promotion of crop growth, and the promotion of disease resistance. The combined effect. In order to understand the possible mechanism of action of the test strain culture solution on the pathogen, the inventors of the present invention used the anti-biomass and chitin decomposing enzyme extracted from the S.saracei/C(10)SS31 culture solution as a material, and tested it in vitro (丨n vjtr〇). The effects of pathogens such as Pythium, Rhizoctonia solani (AG4) and Gibberella gerans are added individually or in combination. The anti-biomass (A) and chitin decomposing enzyme (c) obtained by extracting the corn meal of SS31 are prepared by crude purification, and are added to the potato sucrose culture solution alone or mixed (a+c), and tested for Pythium The inhibitory effect of pathogenic fungi such as pathogenic bacteria, Rhizoctonia solani and Gibberella sphaeroides (as measured by dry weight of cultured hyphae for two days). CK is a control treated with water added. As shown in the second figure, the results obtained show that anti-life is clearly the result of the interaction of anti-biomass and chitinolytic enzymes. Source and Streptomyces Growth 牿性方始吐沐# ^ Relationship Between Net J uses liquid synthetic media to further review the nutritional needs of the test strains ‘and the impact of different nutrient sources on the performance of life. In the case of the SS31 strain, the present inventors have confirmed that the anti-biomass and the chitin-decomposing 1335891 production are related to the supply of various multi-audio carbon sources in the growth process, such as various test carbon sources, such as Tables - and Table 2 show that the addition of sucrose, chitin and house powder is most advantageous for chitinolytic enzymes (Table 1) and for the production of antibiotics (Table 2). The test strains in Table 1 were cultured in the carbon source shown in the table, and the sucrose composition in the original K-cell culture solution was replaced by the concentration addition, and the bacteria were placed at 30 after inoculation. 〇, 120 "pm shock culture" · Another treatment without carbon source as a control (CK). The enzyme activity shown in the table is the result of dot blotting, and the enzyme extraction and detection methods are detailed in Ko (2000) paper (Master's thesis of Department of Plant Diseases, Zhongxing University, 2000): No activity detected; +, ++, + + +, + + + + about ι〇·2〇, 1〇〇' 200' 400 units The activity of the chitin decomposing enzymes in /m丨. The test strains in Table 2 were cultured in the carbon source shown in the table, and the treatment methods were the same as those in Table 1 and 2, and the antibiotics shown in the table were bioassay using Pythium. The results of the anti-biomass extraction and detection methods are detailed in κ〇(10). The results of the examples show that 'anti-biomass production is obvious, and the characteristics of chitin-degrading enzymes' are in the middle and late stages. The characteristics of the more obvious performance. The influence of the addition of different carbon sources on the production of S sarace"Ci7S SS31

15 589 to 5891 ** + + + ++ ++ - + - - . - + + _ ++ +++ +++ ++ +++ +++ ++ +++ +++ + ++ + + ++ Mannitol - Sorbitol - Xylose - Lactose - Maltose - Sucrose + Chitin - Irrigation Powder + CK - Table 2, Addition of Different Carbon Sources to S.saracei/cws SS31 Antiseptic I Pathogen Activity Effect treatment 2 arabinose glucose mannitol sorbitol xylose lactose maltose sucrose chitin starch-QK_ 〇.〇d 0.0d 0.0d 〇.〇d 1.〇〇I. 3b〇〇〇d 1〇〇〇-〇 d II. 1aJA_ I. 3e 〇〇g 00fl 1.0, 2.8C 1- 3ef 2.8C 8.3b2- 2«, II. 2a -15,.

Days after inoculation fg 3 4 5 6 1.0f 〇.〇c 〇.〇d 〇.〇b °09 〇.〇c 〇.〇d 〇.〇b 〇〇g 〇.〇c 〇.〇d 〇.〇 b 〇.〇g 〇.〇c o.od 〇.〇b 1·5ΰβ 〇.〇c 〇.〇d 〇.〇b 1.0f 〇.〇c 〇_〇d 〇.〇b 1-8«, 1.〇c 1.〇c 〇.〇b 1〇7a 7.2a 7.2a 3.0a 1.2ef 1.〇c 1.〇c 〇.〇b 8.3b 6.2b 6.2, 3.2a 2.0, 〇.〇h O .Oh 〇.〇K

(The data in the table is the inhibition zone (unit is a point) formed by the culture of P. sinensis. The larger the number, the greater the resistance to life. The same column data is lower than the lowercase English letters. # ' indicates that there is more Duncan's. There is no significant difference in the statistics of the variable domain analysis. Based on the convenience and economic considerations of the material on the mass production, the various grains of the temple powder and various easy-to-obtain natural sugar knives are added under the appropriate amount of chitin. The test results of the test strain growth and the activity of chitinolytic enzyme showed that the test was carried out under the condition of 1% chitin 5 stress treatment, including 16 1325891 carbon source species including oat flour, corn flour and potato decoction. The bacteria can grow well and have good chitin-degrading enzyme activity promoting effect (Table 3). The enzyme activity shown in the table is the same as the above-mentioned point-by-point method. The treatment method of the nutrient source for testing and the test The culture method of the bacteria is the same as Table 1. The control treatment medium contains only 1% chitin carbon source. In addition, under the co-addition treatment of 0.1 5% chitin, pectin is found. The addition of ingredients has the most prominent effect on the activity of chitinolytic enzymes (Table 4), while the addition of cellulose and glucose has an inhibitory effect. The enzyme activity shown in the table is the result of the point collapse method. Enzyme extraction and activity detection methods are described in detail in Ko (2000) paper and Table 1. The addition treatment method of the tested nutrient source is the same as that of the test bacteria culture method. The control treatment medium contains only 0.1 5% chitin. Carbon source. Table 3. Different sputum additions at 3% sucrose dosage, combined with 1% chitin, the effect of S.saracei/cws SS31 chitin decomposing enzymes. 2 3 oats++” ++ +++ red beans + + ++ soy + + + sorghum + + ++ corn +++ +++ +++ potato +++ +++ +++ CK (chitin) , 1%) ++ ++ ++ +++ ++ + +++ +++ 4 5 6 + + +++ +++ +++ + + + +++ +++ +++ Table 4 , different carbon sources in different concentrations, individually with 0 · 1 5% chitin added treatment, the impact of S.saracei / 'ct / s SS31 chitin decomposing enzyme 17 1325891 _ days after inoculation _ treatment 123 4 56 Cellulose (0.15%) ^ Cellulose (0.75%) - Glucose (0.12%) - Glucose (0.60%) - Pectin (0.12%) +++ Pectin (0.60%) ++++ Starch (0.12%) - Starch (0.60%) - CK (chitin, 0.15%) ++ + + ++ + +++ ++++ +++ +++ +++ ++ + +++ + +++ ++++ +++ ++ ++ ++ +++ ++++ +++ ++ ++ +++ ++ ++ ++ +++ ++++ +++ + ++ +++ +++ ++ ++ ++ +++ ++++ +++ +++ +++ As for the growth factor requirements, use the Kline medium as the basic medium and use wheat. Different growth factors such as germination (ME), yeast extract (YE) and protein (Pep) were added at 1% (W/V), respectively, and their effects on the growth (dry weight) and anti-life performance of SS31 strain were examined. Please refer to the third figure. The test results confirmed that the addition of the three test growth factors promoted the growth effect. Among them, yeast extract had the best effect on mycelium growth, while protein and malt extract were the best. Divided by. Among them, it is also worth noting that in the growth and treatment group of malt extract, the phenomenon of transforming mycelium into spores is quite obvious. As for the anti-life performance, the effect of adding malt essence is most ideal, and the anti-living property is maintained in a state of obvious improvement during the cultivation process. The addition of both yeast and protein glands significantly reduces the anti-life effect. In addition, the change of the pH value of the culture medium was detected day by day in the external culture process, and it was found that the pH value of the culture liquid was significantly decreased with the increase of the anti-life effect by the addition of the malt essence. On the contrary, the addition of yeast essence and protein chen, the pH value of the culture solution is significantly improved. Then, using the same treatment method, the effects of the addition of malt extract, yeast powder (YP) and egg 1325891 white Chen on the activity of chitin degrading enzymes in the test strain were compared. The test results confirmed that the application of the three additives was The positive effect of the positive effect, among which the addition effect of malt essence is still the most ideal (Table 5), while the addition of yeast powder and protein Chen is divided, and the yeast powder and malt extract are added together (YP + ME). For the positive promotion effect of malt extract, there is a slight inhibition. Based on the above results, it was found that the addition of malt extract to each of the test growth factors has an effect of promoting growth and enhancing the anti-living property (including the participation of chitinolytic enzyme activity). According to this, the effect of different concentrations of malt extract is further tested. As shown in the fourth figure, the concentration is only 0.04%, and the effect is quite obvious. The 0.6% addition has reached the same effect as the 1% addition. . Table 5. Different growth factors such as malt extract (ME), yeast powder (YP) and protein gland were added at 1% (W/V), respectively, to affect the activity of chitinolytic enzyme activity of Sireptomycessaracef/ciysSS31 strain. _ days after inoculation

Treatment of ME protein Chen YP YP+ME CK 1 2 3 I ++1» +4 _ + + _ + + _ + + 4 5 6 ++ ++ ++ ++ ++ Example 4: Suitable for Streptomyces strains CAS three-in-one screening technology for living preparations is the biological live preparation for the antagonism of most pathogens, survival in the natural environment, and ability to compete with other microorganisms, especially pathogenic microorganisms. The key characteristics of success or failure. As for the Streptomyces developed, it is apparent from the above-mentioned mechanism of action that these 1325891 special functions are the result of the interaction of antibiotics with various polysaccharide decomposing enzymes and the biological activity of living hyphae or spores. Based on this consideration, the present invention creates a "CASs integration screening strategy" for the activity of chitinase (C), antibiotic (A) and sporulation (s) in the screening of Streptomyces strains. In terms of its practice, the anti-biotic (A) cotton-selected part is carried out on a PSA (potato, sucrose, and acacia medium) plate by means of a sputum culture method, and the pathogens are detected, including the genus Pythium belonging to the (4) class and belonging to the incomplete bacterium. Rhizoctonia solani (AG4)-based, as shown in Figure 5A, inoculated on the peripheral side of the plate medium for the detection of pathogenic mycelium 'disposed at a central fixed distance (about four centimeters) 〇 _ 5 cm diameter filter paper Disc, and quantitatively inoculate a Streptomyces suspension about 50 microliters, set the colony for 2-5 days in the dark at 28 °c, measure the size of the inhibition circle, and observe the sporulation of Streptomyces. In the lower limit medium containing gelatinous chitin (C〇n〇jdal chitin) [such as removing the sucrose and nitrogen source of the Czpek (s) medium] plate, the spore suspension was inoculated with a filter paper disc. The liquid, as shown in the fifth day of the test, was tested for inoculation with the Streptomyces strain, and the size of the permeabilization circle showing the activity of the chitinolytic enzyme was formed as the colony formed; and the production and holding conditions were examined as described above. Using this "CAS three-in-one comprehensive screening technique", the inventors have confirmed that the biosynthesis of potential bio-pesticide can be rapidly separated and screened from samples of soil or organic materials for rapid development. Separated strains (js〇|ates) with chitinolytic activity and colony morphology similar to Clematis were isolated from 219 root-band soils and natural materials, and confirmed to be resistant to Pythium by sputum culture. There are 72 strains, and those with resistance to Rhizoctonia solani have 彳53 strain; it is worth mentioning that 20 of the 20 strains have resistance to Pythium and Rhizoctonia solani. It is significantly better than the strains that have been commercialized by the COAG technology transfer industry, and most of them have good sporulation characteristics. The Streptomyces strains listed in this application as a development application example are enhanced by this triple-strategy repeated screening and continue to be cultured in a low-medium medium containing chitin to maintain the excellent yield of the selected strains. The guarantee of characteristics. The strains obtained by the three-in-one strategy screening, such as S2, S3, S7, S9 and SS31, have been used to successfully produce the expected stable biological living preparations using the established technology platform. The fermented mass production of the dried plant, the present, and the agricultural application of the Streptomyces sp., the inventor of the present invention has been working for many years, using the separation from Taiwan, the application of crop disease prevention and control. The technology will be transferred to a number of domestic industrialization commercial applications and has been commissioned by the Agricultural Committee of the Provincial Pesticide and Toxicology Laboratory in early 2009 to confirm the safety of SS31 as a model g strain (model iS〇|ate) Through repeated screening of the above-mentioned CAS two-in-one strategy, the offspring of the strains with excellent antagonistic and sporogenic ability were selected, and the high-density and stable bacterial preparations were successfully developed following the improvement of series culture and mass production techniques. Liquid fermentation mass production technology platform. At present, the state of the art, using the traditional stirred fermentation tank, from 5L ~ 75〇i_ mass production scale, mainly the spore form density can reach more than 1 〇 11 ,, and it is refrigerated at 6X for eight months, testing The amount of viable bacteria remained at a stable state above 5X108 Cfu / m丨. 21 1325891 These mass-produced samples stored under different storage time, spin-dried in the greenhouse with cabbage, sweet pepper and other plugs to plant seedlings, combined with artificial inoculation treatment, it has been confirmed that the obvious control of Pythium and Lithium nucleus The effect of pathogen infections such as bacteria. This mass production technique further utilizes the same natively isolated s (S3 strain), S. //nC0, neA?s/s (S7 strain) and S. /7/r^r/aii/s (S9 strain), etc. Three different species, which were also screened by the above-mentioned CAS three-in-one strategy, confirmed that the antagonistic and sporulation abilities were excellent, and the progeny of the Streptomyces strains were tested in a 5-750 L fermentation tank, which has been confirmed to be individual by strain. The characteristics can be changed to a spore state under moderate modification. The spore amount is 107-109 〇~ plus 丨(57,59) or even 1011〇~/yang 丨(33) or more, indicating that the established technology platform is in this class. The liquid fermentation of the bacterial preparations is versatile in green production. The established liquid fermentation mass production technology platform is as follows: 5.1 Preparation of inoculation source The original Streptomyces sp. mold cultured on the slant surface of the test tube is taken out and uniformly coated on the potato plate medium [2 gram per potato, drapes per liter medium) 20 grams of sugar or glucose and 15 grams of agar (Agar); if necessary, add appropriate amount of pickled gelatinous chitin about 20 grams], placed in 30X dark culture 'after sporulation (about 4 -6 days) scrape the spores produced on the medium and fully vortex the suspension with sterile water. Adjust the A62() value to 〇.5 by photoelectric colorimeter to measure the amount of spores to about 1〇10 Cfu/m丨 as the initial inoculation. source. Then, the following solid or liquid culture conditions can be applied to gradually prepare an inoculum source for expanding the fermentation amount. The solid culture inoculation source can be used to quantify the oatmeal, wheat and other tanning medium, and the liquid culture is to use a triangular flask or a small fermentation tank (such as New Brunswick Bioflo III 5L) to make a special preparation for the cultivation of 22 1325891. Containing oat homogeneous liquid 活化5_ 3.5〇/0 (W/v), molasses 0·3_0·5% (ν/ν), and specific sporulation-promoting factors such as malt extract, etc. 30 Χ, smashing rate 200 rpm, aeration 〇 5 vvm 'inoculation about 1X10e cfu / m 丨 the above preparation of the initial inoculation source for cultivation. After 4-6 days of cultivation, the spore-forming differentiation of alfalfa can be used as a source of further enrichment for quantitative growth. 5.2 Preparation of the medium The design of the production culture solution for the production: use water, or match the Krebs

(Czapek's) modified medium (containing κ2ΗΡ〇4 〇.7 g·ΚΗ PO 〇.5g per liter; MgS04. 7H20 0.5g; FeS04 ·7Η20 〇.〇1g; and ZnS〇4 〇.〇〇1g)' Or 1/2 to the full amount of H〇agiand, s culture medium containing the original composition of the original medium, using oats, wheat, corn and other cockroaches as an organic nutrient substrate, adding about 1-3 per liter % (W/VJ. Fresh sputum substrate is firstly treated by low-temperature immersion activation and fine grinding homogenization (small particle size of less than 50μηι), and specific sporulation-promoting factor is added as needed and added with acid-washed gelatinous 20 grams of chitin, molasses and other ingredients 'adjusted pH is about 7_0' can be used for culture after autoclaving. 5.3 Fermentation mass production process parameters are regulated by traditional agitated liquid fermentation tank, and the above-mentioned mass production application medium is subjected to high pressure. After sterilizing and warming up, inoculate the above-mentioned inoculation source made of shake flask or small tank (the concentration of the cells is about 101Qcfu/ml or more); the inoculation concentration is about 1X108 cfu/m丨. At 30 ° C, the stirring rate 80-250rpm, ventilation adjustment 025-0.75 vvm, line batch In the culture of batch culture, the pH of the culture is regulated between 5.0 and 8.0, and the growth of the test bacteria is sampled 23 1325891 daily. 5-4 The results of the mass production test are based on the addition of appropriate amount of pectin to oatmeal. , the production of SS31 strain in 5 liters of the tank' results as shown in the sixth figure, the amount of bacteria in the fourth to fifth day of culture can reach more than 1 〇 10 cfu / ml. Oat flour as the main substrate, followed by 750 liters of trough Mass production of SS31 and S3 strains, the test results are shown in the seventh and eighth figures, the amount of bacteria can reach 1〇11cfu/ml or more on the fifth to sixth day of culture. 5-5 test production of living organisms containing bacteria State and Storage Stability Test Samples of Streptomyces live preparations produced by the above established procedure, with the aromatic odor of the soil characteristic of typical Streptomyces, dark gray to slightly brownish 'liquid slightly viscous' static dark gray The experience gradually settles and is divided into two layers, light and dark. 'Slightly shocked and restored to the original dark state of the suspension state. The culture sample taken from the SS31 strain is placed under the magnification of the optical microscope and can be seen as shown in Figure IX. Show, mainly The contents are tested bacteria that have been transformed into spores, and a very small amount of the filamentous fungus is observed. # · After the same treatment, the scanning electron microscopy is performed at a higher magnification. It was found that the test cells were in a sugar-to-long round shape and scattered in the culture medium (Fig. 9). The spore transformation in the same manner as in the S3 strain culture solution was as shown in Fig. C. The storage ampoules are the key characteristics for the commercial application of microbial live preparations. It is also the main technical bottleneck of liquid fermentation of mold live preparations. During the above-mentioned mass production test, the obtained different formulation products were placed in the liquid state of 24 1325891 and placed in the left side of the mixture. The sample was tested by month to test the survival of the cells. The results are shown in Table 6 and Table 7 below. ^ The majority of the test preparation samples can maintain a viable spore amount of more than 1〇8 per ml after 8-10 months of storage. It is confirmed that the spores contained therein have storage stability. Monthly 1 month 2 months 3 months 4 months 5 months 8 months table,, SS31 strain improved technology platform liquid fermentation mass production products w storage stability test

4.4χ 1 〇1 1.5Χ101 5.9x10® 6.3x10® 7.〇χ1〇8 6.5x10® 4.1x10® 4.0x1 〇1〇2.2x10® 8.6x1 〇8 9.1 χΐ〇8 4.5><1〇8 4.0 X1ο8 5.〇χ1〇8 3.6χ1010 2.9x1010 1.3x10® 8.9x10® 4.4x10® 4.0x10® 4.5x10® 8.4x10® 3.0x10® 2.9x10® 2.3x10® 3.0x10® 2.0x10® 1.3x10® Table Data Shows the number of viable spores measured after different storage times (cfu/ml)

Table VII, SS31 strain improved technology platform liquid fermentation mass production products 6. 〇

25 1325891 2 months---- 5.5x 1 〇β 3.9χ10β 5.7x109 3 months 4.7x1〇9 1.5χ109 1.8χ109 4 months* ·· «· • • 5 months _ _ * _ · 6 Month 4.〇χ 1 〇9 1.2χ109 1.7χ109 7 months 9·1 χι〇8 7.〇χ1〇8 1.1χ109 8 months 8.7x1〇β 7.2x10® 9.3x10s 9 months 7·9χΐ〇8 7.5 X10® 9·〇χ108 10 months 6·〇Χΐη8 — 4.〇χ108 6.〇χ108 The data in the table shows the number of viable spores measured after storage time of 6_1J without corrosion (CfU/m|) Six: Trial production ίmί » BA ^tl Shiqizhi's applied empirical control of 6.1 kinds of crops, seedlings and dances (Dampjng 〇ff), squatting and other applications in disease control applications (four), using the above established technology to ferment The test samples are applied by conventional application methods such as spraying or watering, which have been proven to be effective in controlling various crops at the seedling stage by Pythium (p ap/^/7/cferma仏) and Rhizoctonia solani (R s The hazards caused by 〇/a;?/AG4); the following examples illustrate their application in disease control. 6.1.1 Quantities

In the greenhouse, 'S1, S3 and SS31 three different Streptomyces strains prepared for live preparation samples for shallow irrigation treatment' test its control effect on the prevention and treatment of Humicpyrosin infection' per-testing acupoints only one milliliter per well Dilute the 26 I liquid-time, and use the money irrigation as the control treatment (CK); the test results can be clearly seen from the seedling emergence rate of the seedlings in the figure, the mass production samples of the three test strains are diluted at 100·200Χ. In the case of flooding, the treatment of strong artificial inoculation of Pythium is extremely obvious (4). 6.1.2 Similarly, in the case of artificially inoculated pathogens, the control effects of the S3 and SS31 strains on the sample production at different dilutions, the control effect of the sweet pepper seedlings caused by Rhizoctonia solani, the test results are as follows: Table (Table 8) does not, from the unearthed rate and average incidence index (average severity index, AS〇 can be clearly seen, the control effect of the two test strains are quite obvious. Table VIII

19.4 ± 2.2 33.3 ± 2.2 37.5 ± 1.6 The treatment was given twice; the first time after the second sowing for two weeks, 1 ml of b was given to each tray for the success of unearthing and standing after standing. Divided into five equals _ homage (1-health plants; 2 = primary root and underground necrosis but the plant can still stand upright; 3 = main sweep magnetic axis sparse & main apical soft odor, and almost no root hair; 4s Death after sowing 'earth unearthed but odorous after sowing; 5 = unearthed or dead seed) 6.1.3 Spore powder preparation of SS31 strain collected by Gu Zhao culture was prepared with corn condensate 27 1325891 powder to 1 gram per gram The powder of sputum spores (SS31+i rice treatment) was mixed in the greenhouse with the infested soil prepared by inoculating the high (four) toxic mud carbon soil, and the effect on the survival density of the pathogenic bacteria in the cultivation medium was tested. 'In the test, corn flour and SS31 suspension were separately added as a comparative treatment' and treated with no bacteria or corn flour at all as a control treatment (CK). During the test, weekly-times#, using the #release plate method combined with the application of selective culture medium, the survival density of pathogenic bacteria in the test medium was detected. Test knot. As shown in the following table (Table 9), the SS31 powder formulated with corn flour, the density of the population of Rhizoctonia solani in the culture medium after treatment was significantly reduced after two weeks of treatment, and treated with SS31 suspension alone. The group of Rhizoctonia solani in the medium also showed a significant decrease after two weeks of treatment. However, the effect was quite different from that of the preparation prepared by corn flour. In addition, the corn flour was compared with the control group and the non-treated control group. The variation of the Rhizoctonia solani population in the medium during the four consecutive weeks of testing is quite limited. The DAT in the table is the number of days after treatment (Days afte "Additional". The data shows the percentage of positive reaction seeds obtained by the Bahia grass (Paspa/i/m noiaitvm Flugge) seed rowing treatment (percentage) The value is positively correlated with the number of surviving propagules of Rhizoctonia solani. Table 9 DAT* CK Maize SS31 SS31 + corn 1 week 100 100 98 82 2 weeks 95 98 87 30 28 1325891 3 weeks 93 96 65 5 4 weeks 90 93 49 4 6.1.4 The main medium of corn and powder is used as the main medium. The live preparation of Streptomyces s (10)s S3 is stored under the condition of 6Χ for 3 to 10 months, and then treated with diluted concentration of 4〇〇χ. With the above method, artificially inoculated with strong pathogens in the greenhouse, the control effect on the infection of the cucumber seedlings by Pythium is detected; the test results are as shown in Figure 11 for the production of the two mediums by 7_1() It is stored in the month, and it has no obvious difference between the control effect and the culture solution which is freshly prepared for two weeks. Among them, the corn culture preparation used in the experiment is worthy of fresh preparation and culture S3 is about 4_5χι〇9 (4) Chanting 7 months and 1 month storage 'the amount of bacteria is 〇.9_1Χ1〇9 and 〇 7_〇8x1〇9cFu/m|, respectively, and its storage stability is quite worthy of recognition. 6.1.5

• Use oats as the main medium to produce the key strains such as 1 bacterium 2 & 2 & stored under 6C - new formula (new formula / -), compared with freshly prepared culture = new formula - 〇) diluted (10) times The control effect of the lower sputum irrigation on the control of the cucumber cucumber Pythium infection was carried out in the experiment. The corn medium (new formula, the new culture solution of the method) and the oat medium were used to improve the front:: freshly prepared culture solution (old formula) As a comparison treatment, water washing was used as a control, and the test results were as shown in Fig. 12. From the investigation of the disease index two weeks after treatment, it can be seen that all the culture liquids were treated with 29 1325891, which significantly reduced the infection effect. The control effect is best in the oatmeal fresh preparation group (new formula - 〇), the incidence index is only about 18%, followed by the freshly prepared culture solution (old formula) according to the old method, the incidence of deduction is about 35%. Left and right, after one year of storage of oatmeal medium preparation (new formula / one)' its control effect is similar to the old method fresh preparation, the incidence index is about 40 〇 /. The prepared culture solution has the worst control effect, and the incidence index is about 60%, while the water-irradiated control treatment has an incidence index of about 76%. 6.1.6 Effect of leaf spray on papaya fruit blight This experiment uses the test samples produced by the laboratory to be tested in the grape center test field of Zhongxing University. The papaya plants with serious damage to the fruiting disease are planted in the lodging field, and the SS31 culture liquid sample produced by the oat medium is diluted by 5 times. After the above-ground spraying, another spraying of phosphorous acid (pH adjusted to 6.5 with KOH) of 1 〇〇〇 ppm was used as a control agent, and water spray was used as a non-application control. The plants were treated weekly. Once, after six times of spraying treatment, the incidence of fruit plague was investigated. After two consecutive years of trial results, the incidence of control fruit plague was 23〇/. 37%, and the spray chain mold treatment group was 7 respectively. % and 3%, as for the phosphorous acid treatment group, 8% and 9% respectively; the control effect of the sample fermentation broth was extremely significant, and it was significantly better than the test chemical 〇 6.1.7 尧 irrigation treatment in citrus rot Application 1325891 The problem of severe degradation of citrus growth has been widespread in the citrus producing areas of the province for many years. Most of the rickets are plants that are more than 10 years old and are full of plant age. The relevant operators have been helpless except for cutting and replanting. , white pomelo, Wendan no-free, is the main limiting factor for the development of the citrus cultivation industry in this province, but its cause has not been able to clarify. The field disease investigation found that the Luo disease plant I see the rot and Huanglong The compound infection of the disease' and its symptoms are related to soil deterioration and root dysplasia; in the application of the prevention and treatment of stalking orange disease, this study has seriously deteriorated the growth of the tree potential in the field, and the degree of leaf month affects Huanglong disease. Symptoms, and a considerable proportion of the stalks of the stalks of the stalks of the stalks and the stalks of the stalks of the stalks of the stalks of the stalks of the stalks of the stalks of the stalks of the stalks of the stalks of the stalks of the stalks of the stalks of the stalks The symptoms of the new branches of Huanglong disease are alleviated or disappeared, and the fruit is normal and the fruit drop is obviously improved. . The relevant test results are as follows: 6.1.1-1 In the field, the stalks at the base of the stem are selected, the growth of the aboveground part is paused, and the leaves begin to show yellowing and deciduous disease. There are six young plants of the annual willow lamp, with SS31 bond mold organisms. The dilution of the preparation was carried out at a concentration of 2% at the base of the stem, and the sacrifice was carried out twice. After the treatment, the control effect on the infection of the wrong rot was examined. It was confirmed that four of them recovered and two died. 6.1.7-2 'Selecting the weak growth, the SS31 and S3 have been mass-produced. In addition, the plants with growth-degrading symptoms began to grow in the orange-brown cultivation field of about 25 years, using 31 1325891 two living biological preparations, diluted 1〇. The test plants of the 〇 times down-watering treatment were selected from three plots, each treatment was 2.3 plants per plot. From 2〇〇2 = April 1st, a total of three perfusion treatments per month, each perfusion water θ was about 60 80 liters, and the control group was replaced by irrigation. The results of the investigation of the incidence of the disease before the monthly treatment are shown in Table 11 below. The improvement effect of the two test strains on the growth of the tree potential is obvious after one month of treatment, and the treatment effect with S3 is more obvious, and the control plants are observed in June and July. It may be because the rainfall is sufficient, and the growth is also short-lived and obviously improved. Compared with the treatment of S3 and SS31, the difference is still very significant. Table 11 Treatments 4/10 5/24 6/18 7/29 Control group 6 2.5+ 0.2 a 2.5+ 0.2 a 1.2+ 0.2a 1.2+ 〇.2a —S3 8 2.8+ 0.2 a 1-9+ 0.1 b 0.4+ 0.2 b 0.1+ 0.1 b SS31 7 2.7+ 0.2 a 2.0± 0.2 ab I 0.4+ 0.2 b 0-6+ 0.2 b The data in the table is the average incidence index, and the letters in the same column are different. The results showed significant differences in Duncan's multivariate domain analysis results (Duncan, s mu丨tjp丨e mn# ratio 4 Ρ = 0·05). The application of Streptomyces microbial resources in the development of biotechnology has its origins, and its importance has been recognized by the academic community. The case stated in this article is the first mass production of stable Streptomyces by liquid fermentation. The technical platform of spore biomass, comprehensive research and development results of the above-mentioned mass production technology and applied development, the established technology platform, the live preparation of the product has storage stability, can be applied by the conventional drug application method, for a variety of important diseases Guardian 32 1325891

The effect can be obviously promoted to the growth and development of the applied crop, and its commercial application value can be confirmed. In terms of application, the established technology platform has been proved to be applicable to different strains of the same species of genus, indicating that it can be applied in the future when developing foreign strains in cooperation with the international community. This trait is of great significance in the development of microbial live preparations. Under the operation of the World Trade Organization (W〇rldTrade〇rganizati〇nw, the liberalization of agricultural trade has become a trend], but it is to avoid foreign pathogens. Invasion, the strengthening of the world's animal and plant epidemic prevention and quarantine jade, the future circulation of microbial living agents in the international market, or will be affected; the corresponding way is to strengthen international cooperation and the acquisition of strains in the field; established in this study The versatility of mass production technology is extremely beneficial to this development. In the application of plant disease prevention and treatment, the characteristics of the above-mentioned bacterial preparations are manifested in the function of multiple effects, including: and pathogen nutrition and '(4) The competition, the role of anti-biomass, the promotion of the decomposition of macromolecules in the soil and the effective absorption of nutrients, the improvement of soil properties, the promotion of crop growth, and the promotion of disease resistance. It is worth noting that its anti-biomass effect is The combined effect of the entire bacterial metabolite, rather than traditional applications

Single-anti-biomass effect. Because it is a multiple mechanism of action, the common drug resistance problems in traditional pesticide application will not occur; plus its positive effects on soil, ecological environment and plant growth, it will ensure that the sustainable development of agriculture is recommended. Plant protection biotechnology applications. [Simple description of the diagram] (1) Schematic part, culture filtrate and culture The first figure is the comparative culture solution 33 ^ 3 of the present invention. Schematic diagram of the control effect on the infection of P. glabrata seedlings under the application of both cultures. The figure is the result of the antibiotic activity of the test strain of the present invention as a combination of antibiotics and chitinolytic enzymes. The second graph shows the growth and anti-life effects of SS31 strains with different growth factors. The fourth panel shows the effect of different growth factors on the chitinolytic activity of the SS31 strain. Figure 5A shows the detection of sporulation of 11 strains such as S1-S17 and antagonism against rice fever pathogen (central colony) on PDA plates. Figure 5B shows the activity and sporulation of chitinolytic enzymes at four different strains of S7-S21 and other strains on colloidal chitin medium. The sixth figure is the result of the production of SS31 bacteria in a 5 liter tank using oats and an appropriate amount of pectin as a substrate. The seventh figure is a graph showing the results of mass production of SS31 in a 75 liter liter tank using oats as a substrate. The eighth figure is the result of the production of Lu S3 bacteria in a 750 liter tank using oats as a substrate. Figure IX is the optical microscopy image of the liquid fermentation yield of the SS3i strain. - Figure IX is a scanning electron micrograph of the spores produced by the liquid fermentation of the SS31 strain. Figure IX is a scanning electron micrograph of the spores produced by the liquid fermentation production of the S3 strain. 34 rows of Pantu are the S1, illusion and SS31 strains of the production and production system sword sample surface treatment, the control effect of Pythium on the courgette seedlings. The eleventh figure is the result of detecting the disease control effect of the S3 strain of the present invention after storage for 3 to 1 month under 6<>c. The twelfth figure is a comparison and photograph of the results of the storage of the SS31 strain of the present invention at 6 ° C, the annual preparation of the freshly prepared bacterial liquid. (2) Symbols of components

35 1325891 References: 1. K〇, H.C. 2000. Effect of Nutrient Supplement on Antibiotic and Chitinase Production by Streptomyces saraceticus 31. Master thesis, Department of Plant Pathology, National Chung Hsing University, 90p. (in Chinese with English summary)

36

Claims (1)

1325 milk % Λ 一Γ - .π'Τ.·"3* » -·ί Pickup, I request patent Fangu 1 · A method for screening Streptomyces as a biological pesticide, 兮太土 contains: 乃系 (1) Preparation initial Inoculation source: a strain of streptavidin (Α) to be screened for anti-living sputum. Technical slavery - 蜀 株 strain, the selected strains of the strains of the genus Streptomyces g = t = for anti-life screening, choose to have Anti-living Streptococcus strains (8) Chitin-degrading enzyme activity screening: The anti-living sieves of the <RTI ID=0.0>&&&&&&&&&&&&&&&&&&& The activity of chitin decomposing materials is carried out, 2 can form a larger permeabilization circle; (6) the silk production force _: the anti-living property = the strain obtained by the activity of the butyrozyme (4) And (D) take the stalk as the initial inoculation source; (7) prepare the inoculation source of the batch culture: the initial inoculation source is cultured in a stand or shake flask to obtain the inoculation source, batch of the batch culture. The concentration of the inoculation source is 1 〇 8 cfu/ml or more; the cultivation (3) batch liquid culture: will be obtained The concentration of the old crop is the batch culture inoculation source, and the feed body of the (four) type liquid fermentation tank is expanded for several days. The culture condition is temperature 28·37Χ: the mixing rate is 8〇_, and the aeration rate is 0.25-0.75vvm. The pH value is 5.〇_8 〇; and _ (4) Obtaining the biological pesticide composition: the fermentation product is directly taken from the liquid of the (four) type. The fermentation method is as follows: 2. The method described in the scope of the patent application, wherein the initial method is obtained. The inoculation source is 10丨〇cfu/mh)Ψ 3. As described in the second paragraph of the patent application, in step (1) 37 The 132 strains of the strains obtained by the selection were cultured on a solid medium to obtain an initial inoculation source. (A method as claimed in claim 3, wherein the fermented article contains at least oIGCfu/mi spores. 5. The method described in the patent scope, wherein (4) in step (1) is resistant to life (four) In order to screen the (4) selected Streptococcus (4) strains with or without one or more antagonistic bacteria. The method of claim 5, wherein the antibacterial agent is Pythium, Rhizoctonia solani or a combination thereof. 7' The method of claim i, wherein the step (l) t (A) is resistant to life (four), (B) is a decomposing enzyme activity, and (C) sporulation Screening for force is one or more screenings. 8. The method of claim 7, wherein the antibacterial agent is Pythium, Rhizoctonia solani or a combination thereof. 9. The method according to claim 8, wherein the batch liquid culture medium comprises a chitin modified medium, and a chitin of less than 1%. M%-3% (w/v) one or more selected from the group consisting of mannitol, sorbitol, maltose, house powder, cellulose, glucose 'pectin and scorpion as a carbon source' and at K3% ( w/v) oat, wheat or corn as an organic nutrient substrate. The method of claim 9, wherein the batch liquid culture medium further comprises one or more selected from the group consisting of up to 1% malt extract, up to 1% yeast extract and up to 1% protein. The growth of the ethnic group due to 38 1325891 3. si revised year and month to supplement the year and month?丨日修正 Revision 11. A biological pesticide composition prepared by the method of any one of claims 1 to 10, wherein the antagonistic bacteria is Pythium, Rhizoctonia solani or a combination thereof, and The fermentation product contains at least 10 gfufu/ml bosom. Pick up, pattern: like the next page 39 1325891 jWTTr-f^j — Multi-monthly 曰/曰 Revision Version Wu, Chinese Abstract: The present invention relates to a method for screening Streptomyces as a biological pesticide. The Streptomyces strain is obtained by screening for chitinolytic activity (c), antibiotic resistance Q), and ability to produce and hold (S) to obtain a biological pesticide composition having a disease-preventing hazard and promoting plant growth. Lu and English abstracts: 柒, designated representative map: (1) The representative representative of the case is: Figure IX. (2) A brief description of the symbol of the symbol of the representative figure: None 捌 If the case has a chemical formula, please disclose the chemical formula that best shows the characteristics of the invention: