CN1225172C - Batch production process of biological herbicide - Google Patents

Abstract

The present invention relates to a method for producing biological herbicide in batches, which belongs to the field of microbial application in the agricultural plant protection. Through a method of liquid and solid combined fermentation, the biological herbicide, colletotrichum gloeosporioides (QZ-97a) of fungi bacterial strains, eupatorium adenophorum alternaria alternata, bacterial strains of crab grass hyperbolic spora, alternaria zinniae pape, etc. can be produced in batches. Liquid fermentation and solid fermentation culture media utilize agricultural and auxiliary foundation products of industrial leftovers, so the raw material resource is rich and the price is low; suitable initial water content is helpful for thalli to obtain nutritive substances and oxygen transfer from the culture media, and the conversion rate is high; the temperature range of the solid fermentation is wide, and the light irradiation condition can be easily satisfied. The present invention has the advantages of simple process of the whole culture, strong operability and no need of special equipment, and is a simple and convenient method for producing biological herbicide in batches.

Description

A method for mass production of biological herbicides

1. Technical field

The invention relates to a method for mass production of biological herbicides, which belongs to the technical field of the application of microorganisms to agricultural plant protection and control of crop weeds, and is specially used for the biological control of weeds in farmland.

2. Technical background

At present, domestic weed control mainly depends on chemical pesticide products, and its usage and area are still expanding. With the integration of global economy and the internationalization of trade, the national pesticide industry is facing severe challenges. The high research and development costs of new varieties of chemical pesticides have hindered the development of the pesticide industry and the needs of agricultural production. On the other hand, the extensive use of chemical pesticides has caused and is causing serious environmental pollution problems, directly endangering the health of humans and animals and affecting the sustainable development of agriculture. Therefore, it is an imminent key to seek new prevention methods and reduce the use of chemical pesticides. The development of biological control technology, especially the replacement of chemical pesticides with biological pesticides, has attracted everyone's attention in order to form a consensus in the world. The research and development of new biological pesticides requires less investment than chemical pesticides, and the cycle is short, which is in line with national conditions. The development and utilization of biological pesticides is becoming a symbolic technology of agricultural modernization and sustainable agriculture. Looking forward to the future, biopesticides will play an increasingly important role as a high-tech in protecting the environment, ensuring human health, and in the sustainable development of agriculture and forestry. At present, as a main component of biopesticides, there is no industrialized bioherbicide in China, but the production of green food and organic food urgently needs non-polluting bioherbicides to solve the weed problem.

Persian mother-in-law (Veronica persica Poir), crabgrass (Digitaria sanguinalis (L.) Scop.), adenophorum (Eupatorium adenophorum Spreng.) and cocklebur (Xanthium occidentale Bertol.) are worldwide malignant weeds, Farmland and the ecological environment have caused extremely serious harm.

Persian gravy is a vicious weed in summer crop fields such as wheat and rapeseed. It also seriously damages the growth of cotton, corn, soybean and other seedlings. It also often occurs in vegetable fields, orchards, lawns and horticultural crop fields. The distribution range of Persian mother-in-law is wide. There are reports of its occurrence and harm in the Yangtze River Basin of my country and North China. In addition, it is also a malignant weed in the farmlands of Europe, West Asia and America. The weed has strong reproductive ability, fast growth rate, long growth period, strong drug resistance, and it is difficult to control artificially, mechanically and chemically.

Crabgrass is a vicious weed in autumn dry crop fields such as corn, cotton, sweet potato, sorghum, soybean, peanut, etc. It is a serious hazard to the growth of seedlings such as cotton, tobacco, hemp, sugarcane, etc. It is also a vegetable field, orchard, lawn and gardening Dominant weeds in crop fields. It is even the main harmful weed in the southern upland rice fields. Crabgrass has a wide distribution range and is widely distributed in tropical and temperate regions of the world. The weed has strong reproductive ability, fast growth, long growth period, soil fertility, shading, and strong drug resistance.

Eupatorium adenophorum invades farmland, grassland, grassland, roadside, houseside, economic woodland and forest, and forms a dominant population quickly due to its heterogeneous effect. The operation caused great losses. Since it was introduced into my country in the 1940s, it has been widely distributed and harmed in Yunnan, Guizhou, Sichuan, Guangxi and Chongqing in the southwest, and it is still spreading eastward and northward with the southwest wind at a speed of about 60 kilometers per year.

Cocklebur is an important harmful weed in the early days. Its plants are tall and have a serious impact on crop growth. It is mainly distributed in Northeast China, North China, Central China, East China, South China and Southwest China, and mainly harms dryland crops such as corn, cotton, soybean, sweet potato and vegetables.

During the research process of weed biological control, the inventor found that these four kinds of weeds had naturally occurring plants in nature, screened and studied the parasitic fungi of these four kinds of weeds, finally separated four kinds of bacterial strains, and tested these four kinds of weeds. The culture conditions, mass production methods, safety and pathogenicity of the strains have been studied in detail, and the results show that these four strains have the potential to develop into biological herbicides. They are Colletotrichum gloeosporioides f.sp.veronicae Sheng Qiang et Qing Zeng, Alternaria alternata (Fr.) Keissler, Nakataea Hara) strains and Alternaria zinniae Pape strains.

At present, except that the biological fungus against crabgrass has been reported in the United States (different from the biological control strains of crabgrass referred to in this claim), the biological herbicides of Persia pratense and Eupatorium adenophorum have not been reported yet. And the method for the mass production of these four kinds of biocontrol fungi is rarely reported. In order to realize the industrialization of biological herbicides, the production cost is a crucial factor. Considering economic factors, it is required that the medium components are simple, cheap, and the raw materials can be obtained in large quantities, and the production process is easy to operate, and the labor load is reduced to a minimum. Therefore, research on the mass production process and method of biological herbicides is the core key content in the research of biological herbicides. On the one hand, in order to achieve commercialization, an excellent biocontrol strain must be able to have a relatively complete set of large-scale production Production technology, on the other hand, in agricultural production practice, in order to ensure that the price of agricultural products is competitive, the price of biological herbicides used is required to be cheap.

Most of the fungal herbicides that have been commercialized or used in production are produced by fermentation technology. Solid fermentation, the production cycle is relatively long, so the preferred method of industrial production of bio-herbicides is liquid fermentation, this method does not need to make much improvement to the existing industrial fermentation equipment and can directly use standard fermentation and downstream processing equipment. Of the four bioherbicides commercialized in North America, ^Collego® , ^Devine® and ^BioMal® are produced by submerged liquid fermentation. However, the herbicidal active unit of biological herbicides is often the active part of fungi, such as asexual propagules such as mycelia, conidia, and chlamydospores, and fungal active parts such as conidia produced by liquid fermentation are in the fermentation broth. Preservation, vitality declines faster. For example, the bio-herbicide ^Devine® was first developed as a bio-herbicide, and its infection unit is a fresh suspension of chlamydospores obtained by liquid fermentation, which must be stored like fresh milk and has a shelf life of only six weeks period, causing its commercialization process to be delayed, and it was not commercialized until the early 1980s. In addition, in order to obtain high-concentration bio-herbicides, a series of steps such as filtration and concentration, dehydration and drying are required. The entire process is complicated and requires special equipment, which increases the cost.

Compared with the development speed of chemical herbicides in the same period, the development speed of biological herbicides is undoubtedly slow, and the economical and efficient mass production method has always been the bottleneck factor hindering the development of biological herbicides.

3. Contents of the invention

Technical problem The object of the present invention is a method for producing biological herbicides in large quantities. Aiming at the biology and sporulation characteristics of fungal strains, through the cultivation and fermentation method, the type of medium, pH and water content and cultivation time, light, A series of studies, such as temperature, overcome the bottleneck factors that hinder the development of biological herbicides, and explore the most cost-effective mass production process of biological herbicides.

Technical solution: a method for producing biological herbicides in large quantities, characterized in that a liquid-solid combined fermentation method is adopted, and the specific process flow is as follows:

Inoculate the biological herbicide fungal strains on PDA medium and cultivate them for 5-7 days at 15-30°C to obtain the first-grade inoculum;

Cut out a small piece of the first-grade seed culture, inoculate it into the liquid medium, shake and ferment at 15-35°C for 3-10 days, and the shaking speed is 20-180 rpm. After the mycelium is full, the second-grade Liquid inoculum, that is, bacterial suspension;

The solid medium is composed of wheat bran or rice bran and one, two or three other agricultural by-products, the dry weight of wheat bran or rice bran: the dry weight of other components = 1: 0.1-9, after weighing the dry material of the solid medium in proportion , add 0.3-3.0 times the dry weight of water, mix well and sterilize;

Inoculate the bacterium suspension onto the sterilized solid medium in an aseptic environment, cultivate in aerobic conditions at a temperature of 15-35°C, and produce 3-8 days of solid fermentation under aseptic conditions to obtain large quantities of biological herbicides.

A kind of above-mentioned method for producing bio-herbicide in large quantities is characterized in that, the secondary liquid inoculum is preferably pulverized with a pulverizer to obtain a thalline suspension; the condition of solid culture is preferably increased by 20W wavelength 365nm black light lamp, fluorescent lamp or ultraviolet light Intermittent lighting.

The raw materials of agricultural and sideline products of the above-mentioned liquid medium and solid medium are agricultural and sideline basic products of industrial waste, including soybean flour, wheat bran, rice bran, brewer's yeast (wine residue), linseed cake powder, corn flour, sucrose, glucose, Bean dregs, soybean meal, bean cake powder, corn starch, bean cake powder, sweet potato powder, peanut powder, cottonseed cake, peanut shells, corn gluten and various fish meal and other agricultural and sideline products, as well as sawdust from various trees.

Beneficial effects Compared with the prior art, the present invention has the following advantages and positive effects:

1. Compared with pure solid fermentation, the method of the present invention can induce the production of at least 5 times more biological herbicide products, and shorten the solid fermentation time by 2-7 days. During the mass production and development of biological herbicides, the inventors found that some biological herbicides can be produced by liquid fermentation, and some can be obtained by solid fermentation. The bio-herbicide produced by liquid fermentation is stored in the fermentation liquid, and its vitality decays quickly, and it basically loses after six weeks. However, to separate the biological herbicide from the fermentation broth, the technical difficulty is relatively high, a series of additional high-value equipment is required, and a large amount of funds is required. For such a risky bio-herbicide, there is no domestic research technology that has been successfully developed. At the beginning of the development, it is impossible to find sufficient financial support. This will undoubtedly limit the in-depth research of this technology. The production of fungi through solid fermentation can overcome the disadvantage of difficulty in harvesting biological herbicides from the fermentation broth. However, the fermentation process is relatively long, and the growth of fungi in it is extremely uneven, resulting in a great waste of raw materials. Therefore try to utilize the culture method that liquid-solid fermentation combines, and promptly the liquid strain that obtains by liquid fermentation can evenly be inoculated on the solid medium, grows evenly on the medium, can make full use of raw materials, and make the solid fermentation time Shorten 2-7d. The temperature of solid-state fermentation of biological herbicide strains on solid medium is relatively wide, and a large amount of biological herbicides can be obtained when growing under the condition of 15-35°C; Under all dark conditions, they can grow normally to obtain biological herbicides, and can be induced to produce at least 5 times more biological herbicide products under the irradiation of special light sources (black light, ultraviolet light).

2. The nutrition of the selected culture medium of the present invention is comprehensive. The liquid and solid culture medium used in the present invention are agricultural and sideline basic products of industrial leftovers, including soybean flour, wheat bran, rice bran, brewer's yeast (wine residue), linseed cake powder, corn flour, sucrose, glucose, bean dregs, soybean meal , corn starch, bean cake powder, glycerin, sweet potato powder, peanut powder, cottonseed cake, peanut shells, corn gluten and a variety of fish meal and other agricultural and sideline products, as well as sawdust from various trees. These fermentation media are inexpensive, abundant in sources, and highly efficient in being converted into products. The selected medium has comprehensive nutrition and is rich in crude protein, crude fat, crude fiber, nitrogen-free extract, crude ash, calcium, phosphorus and other elements. Reasonable preparation in the preparation of fermentation medium can satisfy the growth and reproduction of strains.

3. Appropriate initial water content makes the medium have a suitable degree of porosity, and there are certain gaps between the particles, which help the bacteria to obtain nutrients and oxygen transfer from the medium, thereby promoting growth and reproduction. Excessively high water content will cause the medium to stick together, reduce the porosity, and affect the transfer of oxygen; if the water content is too low, the expansion degree of the medium will be reduced, and the water activity will be low, thereby inhibiting the growth of bacteria. Fungi grow vegetatively under favorable conditions and then reproduce when conditions are unfavorable. Therefore, when preparing the culture medium, it is necessary to master the amount of water added. If the water is added too little, the humidity is too low, the vegetative growth of the fungus is insufficient, and the reproductive growth is limited. However, when the humidity of the culture medium is too high, it is easy to cause poor ventilation of the culture medium, and it is polluted by bacteria during the fermentation process, and the vegetative growth of fungi is also inhibited, which is also not conducive to the progress of reproductive growth. When water is added properly, the vegetative growth of fungi is sufficient, which will not be conducive to bacterial growth and cause pollution. The study found that the solid fermentation medium weighs the dry material in proportion and mixes it well, and then adds water and mixes it according to the ratio of dry material weight: water weight = 1:0.3-3, and a solid medium with suitable elasticity can be obtained, which is suitable for bacterial strains. grow.

4. Both the liquid and solid culture medium selected by the present invention can be obtained in large quantities, and are cheap, easy to operate, and have high sporulation efficiency, which is beneficial to the control of production costs. The substrate of the solid-state fermentation test uses agricultural by-products such as bran and rice bran. Bran is a by-product of wheat flour processing. It is rich in carbohydrates and nitrogenous compounds, and contains a variety of vitamins. It is loose in texture and good for ventilation. It is an excellent substrate for the growth and reproduction of microorganisms; and the source of raw materials is wide and the price is low.

5. The process is simple and easy to operate. The liquid-solid combined fermentation method combines the advantages of liquid fermentation and solid fermentation, and saves a series of disadvantages of complicated processes and long solid-state fermentation time required for harvesting biological herbicide inoculum from liquid fermentation broth. The fermented seed bacteria are evenly inoculated on the solid medium, which increases the ratio of fermented raw materials into bio-herbicides, reduces the waste of raw materials, and shortens the entire fermentation cycle. Moreover, the produced conidia are adsorbed in the dry solid medium matrix, the post-treatment process is simple and feasible, and the preparation is easy to store.

4. Specific implementation

Example 1 The herbicidal active ingredient of G. anthracnose G. anthracis G. anthracis Popona specialized strain QZ-97a (patent application number: 00112506.0) is the conidia of this fungus. The specific implementation method is as follows: the bacterial strain QZ-97a is cultured on PDA medium for 7 days, inoculated into corn flour, soybean flour and sucrose liquid medium and cultured for 5-7 days, and pulverized by a pulverizer to obtain a bacterial cell suspension. The solid medium is weighed from wheat bran, soybean powder and sweet potato powder according to the dry weight ratio of 7:2:1, then add water 1.5 times the weight of the dry material, mix well and sterilize. Inoculate the bacterial suspension onto the sterilized solid medium in a sterile environment, and culture it aerobically in a self-made incubator for 3-7 days. The temperature of the room is controlled by an air conditioner at 20°C. The amount of spores produced per gram of dry matter It can reach more than 7.8×10 ⁹ spores/g, and after sieving, spore powder with a spore content of 10 ¹⁰ spores or more can be obtained. This amount is sufficient to meet the needs of field use while ensuring that the production cost of the product remains low. The sporulation yield of liquid-solid combined fermentation is at least 5 times higher than that of pure solid fermentation, and the solid fermentation time is shortened by 2-7 days.

Solid culture medium, fermentation cycle, different water content tests and results are as follows in the above-mentioned implementation method:

Prepare 10 groups of solid medium, 1. 50 grams of wheat bran; 2. 25 grams of wheat bran + 25 grams of soybean meal; 3. 25 grams of wheat bran + 25 grams of rice bran; 4. 35 grams of wheat bran + 15 grams of corn flour; 5. 50 grams of rice bran; 6. 15 grams of rice bran + 35 grams of soybean meal; 7. 30 grams of wheat bran + 15 grams of sawdust + 5 grams of fish meal; 8. 15 grams of sawdust + 35 grams of soybean meal; 9. 35 grams of wheat bran + 15 grams of bean dregs; 10 .35 grams of wheat bran + 10 grams of soybean flour + 5 grams of sweet potato flour. Add 1.5 grams of calcium carbonate, 1.5 grams of sucrose, and 40 ml of water to the culture medium of each group, mix and sterilize, and inoculate the secondary liquid inoculum for sporulation. After the sporulation of the solid medium is completed, dry it in an oven at 30°C, take 0.1 g and add 100 ml of water, grind it thoroughly with a mortar, and count the number of spores (Table 1).

Table 1 Effect of solid medium on sporulation of bacterial strain QZ-97a MediaSolid media Conidia yields 10 ⁹ per gram Significance level Significance level 0.05 0.01 Wheat bran + corn flour wheat bran + sawdust + fish meal wheat bran wheat bran + rice bran wheat bran + bean cake flour rice bran + bean cake powder sawdust + bean cake flour wheat bran + bean dregs wheat bran + soybean flour + sweet potato flour 6.67±0.307.41±1.206.99±0.426.76±0.546.38±0.455.79±0.537.24±0.795.46±0.266.12±0.757.80±0.65 babbcdadba BCAABBBBCDADCA

The results in Table 1 show that G. anthracnose QZ-97a can grow a large number of conidia on the 10 kinds of solid medium tested, and the maximum conidia production occurs in the solid medium of wheat bran soybean flour and sweet potato flour. After the fermentation, the amount of spores per gram of dry matter reached 7.8×10 ⁹ .

Solid medium (35 grams of wheat bran, 10 grams of soybean flour, 5 grams of sweet potato flour, 1.5 grams of calcium carbonate, 1.5 grams of sucrose), after inoculating the mycelium suspension, the solids were cultured and grown for 2, 3, 4, 5, 6 , 7, 8 days. After sporulation is complete, dry it in an oven at 30°C, take 0.1 g and add 100 ml of water, grind it thoroughly with a mortar, and count the number of spores (Table 2).

Table 2 Effect of fermentation cycle on spore production of anthracnose bacteria QZ-97a solid fermentation Fermentation cycle (d) Sporulation 10 ⁸ /ml significant level 0.05 0.01 79865432 4.35±0.704.20±0.194.08±0.353.99±0.323.57±0.442.59±0.301.18±0.620.68±0.25 aababbcdef AAAABBBCDE

The results in Table 2 show that the amount of sporulation increases with the prolongation of time, and the amount of sporulation reaches the maximum when the solid fermentation reaches the 7th day. Prolonging the fermentation time, there is no significant difference in the amount of sporulation. Therefore, during cultivation, the cultivation can be completed after 7 days of solid cultivation, which can shorten the solid cultivation time by at least 3 days.

Solid medium (35 grams of wheat bran, 10 grams of soybean flour, 5 grams of sweet potato flour, 1.5 grams of calcium carbonate, 1.5 grams of sucrose), add water 5, 15, 25, 35, 45, 55, 65, 75, 85, 95ml respectively , after inoculating the mycelium suspension, grow for 5 days to detect the number of spores (Table 3).

The impact of table 3 different moisture contents on the sporulation of solid medium Moisture content (ml) Sporulation 10 ⁹ /g significant level 0.05 0.01 9585756555453525155 5.3±0.707.2±0.637.3±0.806.4±0.506.3±0.316.1±0.615.5±0.564.7±0.494.3±0.413.1±0.51 bababbbcccd BCAAAABABABBBCBCCC

The water content of the medium is an important factor to determine the sporulation of solid culture. In Table 3, the amount of sporulation increased with the increase of water content, and reached the maximum value when the amount of water added was 75ml. When the amount of water added continued to increase, the amount of sporulation showed a downward trend. Therefore, when preparing solid medium, it is very important to master the water content of solid medium, and it is best to add 1.5 times the weight of dry material.

Embodiment 2 Alternaria adenophorum utilizes crude metabolites to control weeds, and the specific implementation method is as follows: Alternaria strain (patent application number is 00112560.5) is cultivated on PDA medium for 7 days, and inoculated into soybean flour and corn flour Cultured in sucrose liquid medium for 7 days, crushed with a pulverizer to obtain mycelium suspension. The solid medium is weighed from the cornmeal bean cake according to the dry weight ratio of 1:1, then add water 0.33 times the weight of the dry material, mix well and sterilize. Inoculate the bacterium suspension onto the sterilized solid medium in a sterile environment, seal it with a fresh-keeping bag, punch holes appropriately, and culture at a temperature of 25°C for 10 days. After the cultivation, the crude toxin was prepared by air-drying, and after dilution, the biological activity was tested by acupuncture method, and the toxin-producing ability was expressed by measuring the diameter of the lesion (mm) with a micrometer. Cultivation according to this method significantly increases the toxin production, can make the diameter of the diseased lesion on the leaves reach more than 4.63 mm, and the solid fermentation time is shortened by 2-7 days compared with pure solid fermentation.

Solid culture medium, fermentation cycle, different water content tests and results are as follows in the above-mentioned implementation method:

Use rice bran+wheat bran, corn flour+wheat bran, rice bran+bean cake, corn flour+bean cake, corn flour+soybean flour, bean dregs+sweet potato flour, peanut shells+peanut powder, fermented wine dregs+cotton seeds in a mass ratio of 1:1 Cake and cornstarch + linseed meal to prepare solid medium. Inoculate the Alternaria mycelium of shaking flask shaking culture, and produce toxin by solid growth under suitable conditions according to the embodiment. After solid culture, air-dried to prepare crude toxin according to the procedure, and after dilution, the biological activity was tested by acupuncture method, which was repeated 5 times (Table 4).

The impact of table 4 solid culture medium on Alternaria toxin production culture medium Lesion diameter (mm) significant level 0.05 0.01 Corn flour + bean cake corn flour + wheat bran rice bran + bean cake rice bran + wheat bran corn flour + soybean flour bean dregs + sweet potato flour peanut shell + peanut powder wine residue + cottonseed cake corn starch + linseed cake powder 2.49±0.231.70±0.140.51±0.150.55±0.100.88±0.160.65±0.131.63±0.121.32±0.081.25±0.11 abddcdbbb ABDDCDBBB

The comparison of several solid culture media on Alternaria toxin production shows that most solid media can be used to produce toxins, but Alternaria has the highest toxin production in the solid medium of corn flour and bean cake, which can cause leaf disease Spot diameter reaches 2.49mm. Therefore, cornmeal bean cake was selected as the best culture medium for Alternaria toxin production.

Prepare a solid medium with a mass ratio of corn flour: bean cake of 1:1, and the ratio of dry matter to water is 4:1, 3:1, 2:1, 1:1 respectively, inoculate, culture and air-dry according to the example to prepare crude toxin , diluted needle prick test activity.

The impact of the water content of the medium in table 5 on Alternaria toxin production Dry matter weight: water weight Lesion diameter (mm) significant level 0.05 0.01 4:13:12:11:1 2.31±0.194.63±0.243.06±0.251.80±0.23 cabd CABD

The water content of the medium determines the toxin production of Alternaria on the solid medium. When the dry matter weight:water weight=3:1, the toxin production reaches 4.63 mm, which is the maximum. The water content continued to increase, and the toxin production decreased.

According to the example, a cornmeal+soybean cake solid medium with a mass ratio of 1:1 was prepared. After inoculation, cultured under suitable conditions for 5, 10, 15, and 20 days, samples were taken, and crude toxin was prepared after air-drying. Diluted prick test activity.

The impact of table 6 culture time on Alternaria toxin production Culture time (d) Lesion diameter significant level 0.05 0.01 5101520 4.35±0.705.57±0.445.59±0.306.18±0.62 baaa BAAA

After 10 days of solid culture, the toxin production can reach the maximum value, further increasing the culture time will not have a significant effect on the increase of toxins. Mastering this point will help to grasp the time of solid fermentation in practice, saving time and effort.

Example 3 The herbicidal active ingredient of Bifucus spp. is the conidia of this fungus. The specific implementation method is as follows: bacterial strain QZ-2000 (patent application number is 011340020.9), cultured on PDA medium for 5 days, inoculated into corn flour soybean flour sucrose liquid medium and cultivated for 5-7 days, pulverized with pulverizer to obtain mycelia body suspension. The solid medium is made of soybean flour + cottonseed cake and weighed according to the dry weight ratio of 2:1, then add water 0.6 times the weight of the dry material, mix well and sterilize. Inoculate the bacterium suspension onto the sterilized solid medium in a sterile environment, cultivate it aerobically in a self-made incubator for 3-8 days, place it under a 20W black light (wavelength 365nm), and irradiate it with a fluorescent lamp for 1-3 days. Day, or interval irradiation of ultraviolet light for a total of 1-10 hours, the temperature of the room is controlled at 28°C by air conditioning. The maximum sporulation per gram of dry matter reaches 8.6×10 ⁸ per gram, which is at least 5 times higher than that of pure solid fermentation, and the solid fermentation time is shortened by 2-7 days. Solid culture medium, light and different water content tests and results are as follows in the above-mentioned implementation method:

Prepare a solid medium with a mass ratio of 2:1 (wheat bran is used alone). The selected sawdust woods include fir, poplar, pine and oak, and prepare a solid medium according to dry material weight: water weight=1:0.8. According to the embodiment, solid growth under suitable conditions produces sporulation. After the mycelium is covered with the culture medium, the sporulation is measured after being irradiated under a black light for 2 days.

Table 7 Effect of solid medium on sporulation of bacterial strain QZ-2000 (10 ⁸ /g ^* ) culture medium fir tree sawdust poplar sawdust pine sawdust Wheat bran Fir saw+wheat bran Poplar saw+wheat bran Pine saw + wheat bran Oak saw+wheat bran average sporulation 2.5c 3.4c 3.6c 6.0ab 6.6ab 6.9a 6.1ab 5.8b culture medium soybean flour + cottonseed cake Sweet Potato Flour + Peanut Shells Rice Bran + Corn Flour Peanut powder + cottonseed cake Fish Meal + Cottonseed Cake Bean dregs + poplar saw Corn Flour + Bean Cake Flour Peanut Shells + Corn Gluten average sporulation 7.1a 5.2b 6.1b 4.9b 4.8b 6.4ab 3.6b 2.66c

Note ^* : Data followed by different letters means significant difference (p<0.05).

The results in Table 7 show that the sawdust of a variety of trees and a variety of agricultural by-products can be used to produce conidia of QZ-2000, and the conversion rate is relatively high. The maximum sporulation per gram of dry matter reached 7.1×10 ⁸ per gram.

Prepare soybean flour+cotton seed cake solid medium with a mass ratio of 2:1, add water so that the water content is 30%, 40%, 50%, 60% and 70% of the dry weight of the solid medium, and wait for the inoculation according to the embodiment. The mycelium was covered with the culture medium, placed under a black light for 2 days, and the amount of spores was detected (Table 8).

Table 8 Effect of different water contents on sporulation in solid medium (10 ⁸ /g ^* ) water content 30% 40% 50% 60% 70% average sporulation 3.19d 5.25c 7.25b 7.64a 5.75c

Note ^* : Data followed by different letters means significant difference (p<0.05).

The results in Table 8 show that as the water content increases, the amount of sporulation increases. The solid medium with a water content of 60% had the highest sporulation yield, which was 7.64×10 ⁸ /g. When the water content of the medium increased to 70%, the sporulation decreased significantly. Therefore, it is necessary to master the amount of water added during solid fermentation.

Soybean flour+cotton seed cake solid medium with a mass ratio of 2:1 was prepared, and the water content was 60% of the dry weight of the solid medium. After inoculating according to the embodiment, when the mycelium is covered with the culture medium, 1. Cover and place under black light, ultraviolet lamp, fluorescent lamp for continuous light, 12h light and dark alternately; distance 25cm; 2. Remove the cover and place under the above conditions; 3. Handling under continuous dark conditions. Indoor natural light was used as a control, and the room temperature was 25-28° C., and the number of spores produced was counted after 2 days (Table 9). 5 replicates per treatment.

Table 9 Effect of light on sporulation of bacterial strain QZ-2000 deal with natural light continuous light 12h light and dark alternately completely dark UV lamp black light fluorescent lamp UV lamp black light fluorescent lamp cover light open light 0.48 ^* H0.53H 8.0B0 7.3C8.6A 3.4FG3.9F 7.9C0 5.4E6.6D 2.6G2.8FG 0.23H

*The data in the table is the number of spores (×10 ⁸ /g)

Note: There are extremely significant differences and significant differences between the uppercase letters after the data in the rows and columns (P=0.01)

The test results in Table 9 show that the post-lighting of solid culture can greatly promote sporulation, and the synergistic effect of continuous light on sporulation is greater than 12 hours of alternating light and dark. When comparing the three lighting methods, the clarinet light is used to control sporulation. The promotion effect is most obvious. The maximum sporulation amount of 8.6×10 ⁸ per gram appeared in the group exposed to the black light and continuous light on the solid medium.

Embodiment 4 Alternaria zinnia bacterial strain utilizes fungal conidia to kill weeds, and the specific embodiment of production is as follows: Mass production technology of Neurospora zinnia spores, 1997. China Biological Control 13 (4): 169-172) cultured on PDA medium for 7 days, inoculated into corn flour soybean flour sucrose calcium carbonate liquid medium for cultivation After 3 to 4 days, when the thalline begins to deposit black pigment, the thalline is taken out and filtered, and the metabolites and remaining nutrients of the thalline are washed with sterile water, and the mycelium is pulverized with a pulverizer. The solid culture medium is weighed by dry weight of wheat bran or rice bran: dry weight of sawdust = 1: 7, and then mixed with water according to the ratio of dry material weight: water weight = 1: 2.0. Inoculate the mycelium suspension on a solid medium, then place it under a 20W black light lamp (wavelength 365nm) at 40cm for 12 hours of cyclic light, induce sporulation at room temperature, and cultivate it aerobically in a self-made incubator for 3-8 sky. The maximum amount of spores produced per gram of dry matter reaches 3.11×10 ⁷ per gram, which is at least 5 times more than that produced by pure solid fermentation, and the solid fermentation time is shortened by 2-7 days.

The test and the results of the light start time in the above-mentioned implementation method and the nitrogen content in the liquid medium on the spore production amount of Alternaria zinnia are as follows:

Use a liquid fermentation medium containing 17g of corn flour, 1g of soybean flour, and 3g of CaCO ₃ per liter to carry out mycelium fermentation. Weight = 1: 7, then add water and mix well on the solid medium according to the ratio of dry material weight: water weight = 1: 2.0. Divide into 4 groups, 3 plates in each group, after 0, 3, 6, 9 hours of darkness, induce sporulation with black light lamp.

Table 10 The effect of the start time of near ultraviolet light irradiation on the spore production (10 ⁷ /g ^* ) of Alternaria zinnia start time 0 3 6 9 average sporulation 3.11a 2.73b 2.43c 2.27c

Note: Data followed by different letters means significant difference (p<0.05).

The results in Table 10 show that after the mycelium suspension was inoculated on the solid medium, the spore yield of the 12-hour cycle of light at 40cm under a 20W black light (wavelength 365nm) was compared with that of the spores produced at intervals of 3, 6, and 9 hours, respectively. The spore production increased significantly, and the shorter the interval time, the greater the spore production.

The effect of nitrogen content in liquid medium on sporulation of Alternaria zinnia in solid medium: in liquid medium, the nitrogen content of soybean powder accounts for the vast majority of the nitrogen content in the medium, and the content of soybean powder It can be approximately regarded as directly proportional to the nitrogen content in the medium. Keeping the total dry matter of the medium constant, that is, corn flour plus soybean flour is 15g/L, and the _CaCO content is 3g/L, and the content of soybean flour is changed to 0 (more corn flour 3g/L), 3, 6, 9g/L4 content. Inoculate the solid medium prepared according to the examples to produce sporulation, and finally compare the sporulation per gram of dry matter.

Table 11 Alternaria zinnia spore yield (10 ⁶ /g ^* ) of media with different soybean powder contents Soybean flour content in medium 0g/L 3g/L 6g/L 9g/L average sporulation 22.12±1.034a 17.08±1.560b 16.12±0.225b 5.36±0.149c

Note: ^* is per gram of dry matter, the data followed by different letters means significant difference (p<0.05).

The results in Table 11 show that the amount of sporulation in the solid medium decreases significantly as the content of soybean flour in the liquid medium increases, that is, the nitrogen content increases. Therefore, in order to increase the sporulation of solid medium, the nitrogen content should be controlled when preparing liquid medium.

Adopting the above method of the present invention to cultivate other biological herbicides such as using fungi such as anthracnose and Alternaria can achieve the same effect, which is familiar to those skilled in the art.

Claims (1)

1, a kind of method of producing biological weed killer in enormous quantities is characterized in that, adopts the method for liquid-solid combined ferment, and its concrete technological process is as follows:

With biological weed killer colletotrichum gloeosporioides Penz Veronica specialized form fungal bacterial strain QZ-97a, eupatorium bacterial strain, lady's-grass hyperbolic spore mould genus QZ-2000 or Alternaria zinniae inoculation incubation growth to the PDA medium, under 15-30 ℃ of condition, cultivate 5-7d, behind the acquisition one-level kind bacterium;

Cut one-level kind bacterium culture fritter, be inoculated in corn flour analysis for soybean powder liquid sucrose medium or the corn flour analysis for soybean powder sucrose calcium carbonate liquid nutrient medium, 15 ℃ of-35 ℃ of oscillation and fermentation cultivation 3-10d, jolting speed is 20-180 commentaries on classics/min, after treating that hypha body covers with, obtain secondary liquid strain bacterium, pulverize with cracker and obtain thallus suspension liquid;

Solid culture medium is a kind of by wheat bran or rice bran and other, two or three agricultural byproducts are formed wheat bran or rice bran dry weight: other component dry weight=1: 0.1-9, take by weighing the solid culture medium siccative in proportion after, add 0.3-3.0 times of water of siccative weight, mix sterilization thoroughly;

Under gnotobasis, thallus suspension liquid is inoculated on the solid culture medium of the bacterium of going out, good air culture is supported, 15 ℃-35 ℃ of temperature, solid fermentation is produced 3-8d under the aseptic condition, the condition of solid culture is to increase 20W wavelength 365nm black light lamp, fluorescent lamp or the irradiation of ultraviolet lamp gap, obtains large batch of biological weed killer.