CN1225172C - Batch production process of biological herbicide - Google Patents
Batch production process of biological herbicide Download PDFInfo
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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
One, technical field
The present invention is a kind of method of producing biological weed killer in enormous quantities, belongs to the technical field that microorganism is applied to agriculture plant protection, prevents and kill off crop weeds, is exclusively used in the biological insect control of farmland weed.
Two, technical background
At present, domestic control crop smothering mainly still depends on the chemical pesticide product, and its usage amount and area are also in continuous expansion.Along with global economic integration and trade internationalization, national pesticide industry is being faced with stern challenge, and the great number of new varieties chemical pesticide is developed the needs that cost has hindered the development and the agricultural production of pesticide industry.On the other hand, a large amount of uses of chemical pesticide and have caused the serious environmental pollution problem, directly jeopardize the sustainable development of human and livestock health and influence agricultural.Therefore, seek new means of prevention, reduce the use of chemical pesticide, become an extremely urgent key.Development biological control technology particularly with biopesticide instead of chemical agricultural chemicals, worldwide to build consensus, has caused everybody concern.The development of biopesticide new product, required less input than chemical pesticide, and the cycle is short, meets national conditions.The development and utilization of biopesticide is just becoming agricultural modernization and the continuable significant technology of agricultural.Look forward to the future, biopesticide will at the protection environment, guarantee human health as a kind of new and high technology, in the agriculture and forestry sustainable development, bring into play more and more important effect.At present, as a chief component of biopesticide, the domestic biological weed killer that does not still have a kind of industrialization, and the free of contamination biological weed killer of the production active demand of pollution-free food and organic food solves the weeds problem.
Persian Veronica (Veronica persica Poir), lady's-grass (Digitaria sanguinalis (L.) Scop.), Eupatorium adenophorum (Eupatorium adenophorum Spreng.) and Siberian cocklebur (Xanthium occidentale Bertol.) are global malignant weeds, and very serious harm has been caused in farmland and ecotope.
The Persian Veronica is the malignant weed in summer crops fields such as wheat class, rape, and growth of seedling such as cotton, corn, soybean are also endangered seriously, and vegetables field, orchard, lawn and horticultural crop Tanaka also often have generation.The distribution of Persian Veronica is extensive, and all there is the report of its generation and harm China Yangtze river basin and North China, and it also is the malignant weed in Europe, West Asia and farmland, America in addition.This weeds fertility is strong, fast growth, and vegetative period is long, and drug resistance is strong, and artificial, machinery and chemical control be difficulty relatively.
Lady's-grass is the malignant weed in autumn maturing Dry crop fields such as corn, cotton, sweet potato, Chinese sorghum, soybean, peanut, and is serious to the harm of growth of seedling such as cotton, tobacco, crudefiber crop, sugarcane, also is the advantage weeds in vegetables field, orchard, lawn and horticultural crop field.Even also be the main harm weeds in southern dryland rice field.The distribution of lady's-grass is extensive, is the torrid zone, the world and area, temperate zone dispersed species.This weeds fertility is strong, fast growth, and vegetative period is long, consumes soil fertility, shading, drug resistance is strong.
Eupatorium adenophorum invasion farmland, meadow, grassland, roadside, other, the economic forest land of residence and forest with the characteristic of its allelopathy effect, comparatively fast form dominant population, cause great loss for farming, woods, animal husbandry production and diversified farming.Since 20th century, imported China the forties into, the Yunnan of southwest, Guizhou, Sichuan, Guangxi and Chongqing extensively distributed and harm, and still with about 60 kilometers speed in every year, spread to propagating eastwards and north with southwester.
Siberian cocklebur is early important harmfulness weeds, and its plant is tall and big, plant growth is caused have a strong impact on.The main distribution occurs in dry crops such as areas such as northeast, North China, Central China, East China, south China and southwest, main harm corn, cotton, soybean, sweet potato, vegetables.
The inventor finds that in the research process of weeds biological control there is the plant of natural occurrence in these four kinds of weeds at natural world, parasitical fungi to these four kinds of weeds carries out screening study, finally be separated to four kinds of bacterial strains, and to these four kinds of culture of strains conditions, production in enormous quantities method, safety and pathogenicly all carried out comparatively detailed research, the result shows, all potential biological weed killer that develops into of these four kinds of bacterial strains.They are respectively glue born of the same parents anthrax-bacilus Veronica specialized form (Colletotrichum gloeosporioides f.sp.veronicae Sheng Qiang et Qing Zeng), eupatorium (Alternaria alternata (Fr.) Keissler), the mould genus of lady's-grass hyperbolic spore (Nakataea Hara) bacterial strain and Alternaria zinniae (Alternaria zinniae Pape) bacterial strain.
At present, both at home and abroad except that the biological fungi (different with this claims indication lady's-grass biocontrol bacterial strain) the U.S. reports to some extent at lady's-grass, the biological weed killer of Persian Veronica and Eupatorium adenophorum does not appear in the newspapers as yet.And the rarely seen especially report of method that relevant these four kinds of biocontrol fungis are produced in enormous quantities.Biological weed killer will be realized industrialization, and production cost is vital factor.Consider from the economic factor aspect, require that nutrient media components is simple, low price, raw material can obtain in a large number, and the production process easy operating, the amount of labour drops to minimum.Thereby, the production in enormous quantities technology and the method for research biological weed killer, it is the core key content in the biological weed killer research, on the one hand, the good biocontrol bacterial strain of a strain must be able to have the comparatively complete complete production in enormous quantities technology of a cover for reaching the commercialization process, on the other hand, in the agricultural production practice, competitive on price in order to guarantee agricultural product, require the biological weed killer low price that uses.
Mostly commercialization now or producing the mycoherbicide that uses is through fermentation technique production.Solid fermentation, the production cycle is longer relatively, thereby the prefered method of biological weed killer suitability for industrialized production is liquid fermentation, and this method does not need that how much existing industrial fermentation equipment done and improves and just can directly utilize the fermentation of standard and downstream device to carry out.In the four kinds of business-like biological weed killers in North America, Collego
, Devine
And BioMal
Produce by deep fermentation.But the activity of weeding unit of biological weed killer is the great-hearted part of fungi often, vegetative propagules such as mycelium, conidium, chlamydospore for example, and fungi activities such as conidium part preservation in zymotic fluid of producing by liquid fermentation, the vigor decline is very fast.Biological weed killer Devine for example
Be that first is used as the biological weed killer development, its unit that infects is the fresh chlamydospore suspending agent that liquid fermentation obtains, must preserve as fresh milk, and the shelf life that had only for six weeks, cause its commercialization process to be delayed, until eighties of last century beginning of the eighties just by commercialization.In addition, in order to obtain the biological weed killer of high concentration, need through filtering and concentrating, series of steps such as dehydrate, the whole process flow complexity needs special equipment, has increased cost.
The development speed of chemical herbicide is compared with the same period, and the development speed of biological weed killer is slow undoubtedly, and the production in enormous quantities method of economical and efficient is to hinder the bottleneck factor of biological weed killer development always.
Three, summary of the invention
Technical problem the objective of the invention is a kind of method of producing biological weed killer in enormous quantities, biology and product spore characteristic at fungal bacterial strain, by to a series of researchs such as kind, pH and the moisture of cultivation and fermentation method, medium and incubation time, illumination, temperature, overcome the bottleneck factor that hinders the biological weed killer development, explore the technology of most economical effective production in enormous quantities biological weed killer.
Technical scheme: a kind of method of producing biological weed killer in enormous quantities is characterized in that adopt the method for liquid-solid combined ferment, its concrete technological process is as follows:
The biological weed killer fungal bacterial strain is inoculated into incubation growth on the PDA medium, under 15-30 ℃ of condition, cultivates 5-7d, behind the acquisition one-level kind bacterium;
Cut one-level kind bacterium culture fritter, be inoculated in the liquid nutrient medium, 15-35 ℃ of oscillation and fermentation cultivation 3-10d, jolting speed is 20-180 commentaries on classics/min, treat that hypha body covers with after, obtain secondary liquid strain bacterium, i.e. 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, and temperature 15-35 ℃, solid fermentation is produced 3-8d under the aseptic condition, obtains large batch of biological weed killer.
Above-mentioned a kind of method of producing biological weed killer in enormous quantities is characterized in that, the most handy cracker of secondary liquid strain bacterium is pulverized and obtained thallus suspension liquid; The condition of solid culture preferably increases 20W wavelength 365nm black light lamp, fluorescent lamp or the irradiation of ultraviolet lamp gap.
The agricultural byproducts raw material of aforesaid liquid medium and solid culture medium is the agricultural and sideline basic product of industrial waste, comprise multiple agricultural byproducts such as analysis for soybean powder, wheat bran, rice bran, brewer's yeast (fermented glutinous rice slag), linseed meal, corn flour, sucrose, glucose, bean dregs, dregs of beans, soybean cake powder, corn starch, soybean cake powder, sweet potato flour, peanut powder, cotton cake, peanut shell, zein and multiple fish meal, and the saw dust of multiple trees.
Beneficial effect the present invention compared with prior art has following advantage and good effect:
1, purer solid fermentation, the inventive method can be induced the biological weed killer product of production more than at least 5 times, and make the solid fermentation time shorten 2-7d.The inventor finds that some biological weed killer can be by liquid fermentation production in the production in enormous quantities development process to biological weed killer, some can obtain by solid fermentation production.Biological weed killer by liquid fermentation production is kept in the zymotic fluid, and the vigor decline is very fast, forfeiture substantially after six weeks.And biological weed killer will be separated from zymotic fluid, the technical difficulty demand is higher, needs a series of extra high value equipment, needs to increase separately very big fund.Bigger for the such risk of biological weed killer, domestic still none succeeds in developing the investigative technique of precedent at present, at the beginning of the development, can not find enough financial supports.This will limit the further investigation of this technology undoubtedly.Fungi can overcome from zymotic fluid the shortcoming of results biological weed killer difficulty by solid fermentation production, and still, fermentation process is longer relatively, and fungi growing way is extremely inhomogeneous therein, cause raw material to waste greatly.Therefore trial utilizes the training method that the liquid-solid fermentation combines, promptly the liquid spawn that obtains by liquid fermentation can evenly be inoculated on the solid culture medium, and growing way is even on medium, can make full use of the raw material thing, and make the solid fermentation time shorten 2-7d.The temperature broad of biological weed killer bacterial classification solid state fermentation on solid culture medium is grown under 15-35 ℃ condition, all can obtain a large amount of biological weed killers; Requirement to illumination is not strict, under natural lighting, full exposure and full dark condition, all can obtain biological weed killer by normal growth, and can induce the biological weed killer product of production more than at least 5 times under special light sources (black light lamp, uviol lamp) irradiation.
2, the comprehensive nutrition of the selected medium of the present invention.Liquid that the present invention is used and solid culture medium are the agricultural and sideline basic product of industrial waste, comprise multiple agricultural byproducts such as analysis for soybean powder, wheat bran, rice bran, brewer's yeast (fermented glutinous rice slag), linseed meal, corn flour, sucrose, glucose, bean dregs, dregs of beans, corn starch, soybean cake powder, glycerine, sweet potato flour, peanut powder, cotton cake, peanut shell, zein and multiple fish meal, and the detritus sawdust of multiple trees.These fermentation mediums are cheap, and the source is sufficient, are converted into the efficient height of product.The comprehensive nutrition of selected medium contains elements such as abundant crude protein, crude fat, raw fiber, nitrogen free extract, coarse ash and calcium, phosphorus.Reasonable preparation can be satisfied growth, breeding when the preparation fermentation medium.
3, Shi Yi initial moisture content makes medium that suitable gas porosity be arranged, and has a fixed gap between particle, helps thalline to obtain the transmission of nutriment and oxygen from medium, thereby promotes growth and breeding.It is agglomerating that too high water content can cause medium to cohere, and porous reduces, and influences the transmission of oxygen; Water content is low excessively, and the medium degrees of expansion is reduced, and the activity of water is low, thereby suppresses thalli growth.Fungi advanced field headquarters health under adapt circumstance is long, carries out reproductive growth then when condition is not suitable for.Therefore, the addition of the good moisture of GPRS when the preparation medium, moisture adds very few, and humidity is too small, and nourishing and growing of fungi is insufficient, and reproductive growth is limited.When but medium humidity is excessive, cause the medium ventilation poor again easily, be subjected to germ contamination during the fermentation, nourishing and growing of fungi is subjected to press down equally, also is unfavorable for the carrying out of reproductive growth.And adding water when suitable, nourishing and growing fully of fungi will be unfavorable for that bacterial growth causes polluting.Discover that the solid fermentation medium takes by weighing siccative in proportion and mixes thoroughly, again in siccative weight: the ratio poach of the weight of water=1: 0.3-3, can access the suitable solid culture medium of degree of tightness, be fit to growth.
4, selected liquid and the solid culture medium of the present invention all can obtain in a large number, and inexpensive, easy to operate, product spore efficient height is beneficial to the control of production cost.The matrix of solid state fermentation test adopts agricultural by products such as wheat bran, rice bran.Wheat bran is the accessory substance of wheat flour processing, contains abundant carbohydrate and nitrogen-containing compound, and contains multivitamin, and quality is loose, is beneficial to ventilation, is the excellent substrates of growth of microorganism breeding; And raw material sources are extensive, and are cheap.
5, simple for process.The liquid-solid combined fermentation method combines the strong point of liquid fermentation and two kinds of fermentation process of solid fermentation, a series of required complicated technology of biological weed killer inoculum and long shortcomings of solid state fermentation time from liquid fermentation liquid, gathered in the crops have been save, liquid fermentation kind bacterium evenly is inoculated on the solid culture medium, the ratio that makes fermentation raw material be converted into biological weed killer increases, reduce the waste of raw material, shortened the cycle of whole fermentation.And the conidium of producing is adsorbed in the dry solid culture based substrate, and aftertreatment technology is simple, and preparation is deposited easily.
Four, embodiment
Embodiment 1 colletotrichum gloeosporioides Penz Veronica specialized form bacterial strain QZ-97a (number of patent application is 00112506.0), its herbicidal active component is the conidium of this bacterium.Specific implementation method is as follows: bacterial strain QZ-97a cultivates 7d on the PDA medium, is inoculated in the corn flour analysis for soybean powder liquid sucrose medium and cultivates 5-7d, pulverizes with cracker and obtains thallus suspension liquid.After solid culture medium was taken by weighing than 7: 2: 1 by dry weight by wheat bran, analysis for soybean powder and sweet potato flour, add 1.5 times of heavy water of siccative, mix sterilization thoroughly.Under gnotobasis thallus suspension liquid is inoculated on the solid culture medium of the bacterium of going out, 3-7d is supported in good air culture in homemade incubator, and the temperature in room is controlled 20 ℃ with air-conditioning, and the spore amount of every gram Dry Matter Production can reach 7.8 * 10
9Individual/as more than the gram, after sieving, to obtain the spore amount 10
10Individual/the above conidial powder of gram.This quantity is enough to satisfy when use in the land for growing field crops, guarantees that production cost of products keeps cheap.The purer solid fermentation sporulation quantity of liquid-solid combined ferment has more 5 times at least, and the solid fermentation time shortens 2-7d.
Solid culture medium, fermentation period, the test of different in moisture content and result are as follows in the above-mentioned implementation method:
Prepare 10 groups of solid culture mediums, 1. wheat bran 50 grams; 2. wheat bran 25 gram+dregs of beans 25 restrain; 3. wheat bran 25 gram+rice brans 25 restrain; 4. wheat bran 35 gram+corn flour 15 restrain; 5. rice bran 50 restrains; 6. rice bran 15 gram+dregs of beans 35 restrain; 7. wheat bran 30+ sawdust 15 gram+fish meal 5 restrain; 8. sawdust 15 gram+dregs of beans 35 restrain; 9. wheat bran 35 gram+bean dregs 15 restrain; 10. wheat bran 35 gram+analysis for soybean powder 10 gram+sweet potato flours 5 restrain.Add calcium carbonate 1.5 grams, sucrose 1.5 grams, the sterilization of water 40ml mixing in every group of medium, produce spore behind the inoculation secondary liquid strain bacterium.After solid culture medium product spore finishes, in 30 ℃ of baking ovens, dry, get 0.1g and add 100ml water, after fully grinding with the stone roller alms bowl, counting spore count (table 1).
Table 1 solid culture medium produces the influence of spore to bacterial strain QZ-97a
| Medium Solid media | Sporulation quantity 10 9Individual/gram Conidia yields | 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+beancake powder rice bran rice bran+beancake powder sawdust+beancake powder wheat bran+bean dregs wheat bran+analysis for soybean powder+sweet potato flour | 6.67±0.30 7.41±1.20 6.99±0.42 6.76±0.54 6.38±0.45 5.79±0.53 7.24±0.79 5.46±0.26 6.12±0.75 7.80±0.65 | b a b b c d a d b a | BC A AB B BC D A D C A |
Table 1 is the result show, colletotrichum gloeosporioides Penz QZ-97a growth on 10 kinds of solid culture mediums that supply examination can both obtain a large amount of conidiums, maximum sporulation quantity appears in the wheat bran analysis for soybean powder sweet potato flour solid culture medium, and every gram dry matter spore amount reaches 7.8 * 10 after the fermentation ends
9Individual.
Solid culture medium (wheat bran 35 gram, analysis for soybean powder 10 grams, sweet potato flour 5 grams, calcium carbonate 1.5 grams, sucrose 1.5 grams), inoculate mycelium suspended liquid after, solid is incubation growth 2,3,4,5,6,7,8 days respectively.After the product spore finishes, in 30 ℃ of baking ovens, dry, get 0.1g and add 100ml water, after fully grinding with the stone roller alms bowl, counting spore count (table 2).
Table 2 fermentation period produces the influence of spore to anthrax-bacilus QZ-97a solid fermentation
| Fermentation period (d) | Sporulation quantity 10 8Individual/ml | Significance level | |
| 0.05 | 0.01 | ||
| 7 9 8 6 5 4 3 2 | 4.35±0.70 4.20±0.19 4.08±0.35 3.99±0.32 3.57±0.44 2.59±0.30 1.18±0.62 0.68±0.25 | a ab ab b c d e f | A A A AB B C D E |
Table 2 is the result show, sporulation quantity is along with time lengthening, and sporulation quantity increases, solid fermentation to the in the time of 7 days sporulation quantity reach maximum.Prolong fermentation time, difference is not remarkable between the sporulation quantity, and therefore, when cultivating, solid culture can finish to cultivate in 7 days, can shorten 3 days solid culture time so at least.
Solid culture medium (wheat bran 35 grams, analysis for soybean powder 10 grams, sweet potato flour 5 grams, calcium carbonate 1.5 grams, sucrose 1.5 grams), add water 5,15,25,35,45,55,65,75,85,95ml respectively, after inoculating mycelium suspended liquid, grow and detected spore count (table 3) in 5 days.
Table 3 different in moisture content produces the influence of spore to solid culture medium
| Moisture (ml) | Sporulation quantity 10 9Individual/gram | Significance level | |
| 0.05 | 0.01 | ||
| 95 85 75 65 55 45 35 25 15 5 | 5.3±0.70 7.2±0.63 7.3±0.80 6.4±0.50 6.3±0.31 6.1±0.61 5.5±0.56 4.7±0.49 4.3±0.41 3.1±0.51 | b ab a b b b c c c d | BC A A AB AB AB BC BC C C |
Moisture content in medium is the important factor that the decision solid culture is produced spore.In the table 3, sporulation quantity increases and increases along with water content, and sporulation quantity reaches maximum when amount of water is 75ml, and when amount of water continued to increase, downward trend appearred in sporulation quantity.Therefore, during solid culture medium, the water content of grasping solid culture medium is most important in preparation, the best when adding heavy 1.5 times of siccative.
Embodiment 2 eupatorium bacterium utilize thick metabolite control weeds, specific implementation method is as follows: alternaric bacteria strain (number of patent application is 00112560.5) was cultivated 7 days on the PDA medium, be inoculated in the analysis for soybean powder corn flour liquid sucrose medium and cultivated 7 days, pulverize with cracker and obtain mycelium suspended liquid.After solid culture medium was taken by weighing than 1: 1 by dry weight by the corn flour soya-bean cake, add 0.33 times of heavy water of siccative, mix sterilization thoroughly.Under gnotobasis, thallus suspension liquid is inoculated on the solid culture medium of the bacterium of going out, after the freshness protection package sealing, suitably punching, temperature control was cultivated 10 days for 25 ℃.Cultivate to finish that after wind is drying to be equipped with thick toxin, the dilution back is with needle point method test organisms activity, produces malicious ability and measures scab diameter (mm) with micro-micrometer and represent.Significantly improve product poison amount by this method cultivation, the pathogenic scab diameter of blade is reached more than the 4.63mm, purer solid fermentation shortening of solid fermentation time 2-7d.
Solid culture medium, fermentation period, the test of different in moisture content and result are as follows in the above-mentioned implementation method:
Use rice bran+wheat bran, corn flour+wheat bran, rice bran+soya-bean cake, corn flour+soya-bean cake, corn flour+analysis for soybean powder, bean dregs+sweet potato flour, peanut shell+peanut powder, fermented glutinous rice slag+cotton cake and the corn starch+linseed meal preparation solid culture medium of 1: 1 mass ratio respectively.The alternaric bacteria filament of bottle shaken cultivation is shaken in inoculation, presses embodiment solid growth under suitable condition and produces poison.After solid culture finished, air-dry by the thick toxin of flow preparation, 5 times (table 4) repeated with needle point method test organisms activity in the dilution back.
Table 4 solid culture medium produces the influence of poison amount to chain lattice spore
| Medium | Scab diameter (mm) | Significance level | |
| 0.05 | 0.01 | ||
| Corn flour+soya-bean cake corn flour+wheat bran rice bran+soya-bean cake rice bran+wheat bran corn flour+analysis for soybean powder bean dregs+sweet potato flour peanut shell+peanut powder fermented glutinous rice slag+cotton cake cornstarch+linseed meal | 2.49±0.23 1.70±0.14 0.51±0.15 0.55±0.10 0.88±0.16 0.65±0.13 1.63±0.12 1.32±0.08 1.25±0.11 | a b d d c d b b b | A B D D C D B B B |
Several solid culture media produce comparison shows that of poison amount to chain lattice spore, and most solid dielectrics can be used for producing poison, but chain lattice spore produces poison in the composite solid culture medium of corn flour and soya-bean cake the highest, can make the blade scab diameter that causes a disease reach 2.49mm.Therefore, select for use the corn flour soya-bean cake to produce the best culture medium of poison as chain lattice spore.
The preparation corn flour: the soya-bean cake mass ratio is 1: 1 a solid culture medium, and the ratio of dry matter and water was respectively 4: 1,3: 1,2: 1,1: 1, presses the thick toxin of the air-dry back preparation of embodiment inoculated and cultured, dilution lancing test activity.
Table 5 moisture content in medium produces the influence of poison to chain lattice spore
| Dry matter weight: water is heavy | Scab diameter (mm) | Significance level | |
| 0.05 | 0.01 | ||
| 4∶1 3∶1 2∶1 1∶1 | 2.31±0.19 4.63±0.24 3.06±0.25 1.80±0.23 | c a b d | C A B D |
Moisture content in medium has determined the product poison size of chain lattice spore on solid culture medium, at dry matter weight: water is heavy=and 3: 1 o'clock, produce the poison amount and reach 4.63mm, be maximum.Water content continues to increase, and produces the poison amount and descends.
Prepare 1: 1 corn flour of mass ratio+soya-bean cake solid culture medium by embodiment, the sampling after cultivating 5,10,15,20 days respectively under the suitable condition of inoculation back, the thick toxin of air-dry back preparation.Dilution lancing test activity.
Table 6 incubation time produces the influence of poison amount to chain lattice spore
| Incubation time (d) | The scab diameter | Significance level | |
| 0.05 | 0.01 | ||
| 5 10 15 20 | 4.35±0.70 5.57±0.44 5.59±0.30 6.18±0.62 | b a a a | B A A A |
Solid culture 10 days is produced malicious amount and can be reached maximum, further increases incubation time, and the increase effect of contratoxin is not remarkable, grasps this point, is beneficial to the time of getting hold of solid fermentation in practice, and is time saving and energy saving.
The herbicidal active component of the mould genus of embodiment 3 lady's-grass hyperbolic spores is the conidium of this bacterium.Specific implementation method is as follows: bacterial strain QZ-2000 (number of patent application is 011340020.9), on the PDA medium, cultivated 5 days, and be inoculated in the corn flour analysis for soybean powder liquid sucrose medium and cultivated 5-7 days, pulverize with cracker and obtain mycelium suspended liquid.After solid culture medium was taken by weighing than 2: 1 by dry weight by analysis for soybean powder+cotton cake, add 0.6 times of heavy water of siccative, mix sterilization thoroughly.Under gnotobasis, thallus suspension liquid is inoculated on the solid culture medium of the bacterium of going out, good air culture was supported 3-8 days in homemade incubator, place under the 20W black light lamp (wavelength 365nm), fluorescent lamp irradiation 1-3 days or the irradiation of ultraviolet lamp gap amount to 1-10 hour, the temperature in room is controlled at 28 ℃ with air-conditioning.The maximum sporulation quantity of every gram dry matter reaches 8.6 * 10
8Individual/gram, purer solid fermentation sporulation quantity has more 5 times at least, and the solid fermentation time shortens 2-7d.Solid culture medium, illumination and the test of different in moisture content and result are as follows in the above-mentioned implementation method:
Preparation mass ratio 2: 1 solid culture medium (wheat bran list with), selected sawdust timber has Chinese fir, willow, pine tree and robur, according to siccative weight: water weight=1: 0.8 preparation solid culture medium.Press embodiment solid growth under suitable condition and produce spore, treat mycelia confluent cultures base, place to shine after 2 days under the black light lamp and measure sporulation quantity.
Table 7 solid culture medium produces the influence (10 of spore to bacterial strain QZ-2000
8Individual/g
*)
| Medium | The Chinese fir sawdust | The willow sawdust | The pine tree sawdust | Wheat bran | China fir saw+wheat bran | Poplar saw+wheat bran | Pine saw+wheat bran | Oak saw+wheat bran |
| Average sporulation quantity | 2.5c | 3.4c | 3.6c | 6.0ab | 6.6ab | 6.9a | 6.1ab | 5.8b |
| Medium | Analysis for soybean powder+cotton cake | Sweet potato flour+peanut shell | Rice bran+corn flour | Peanut powder+cotton cake | Fish meal+cotton cake | Bean dregs+poplar saw | Corn flour+soybean cake powder | Peanut shell+zein |
| Average sporulation quantity | 7.1a | 5.2b | 6.1b | 4.9b | 4.8b | 6.4ab | 3.6b | 2.66c |
Annotate
*: what data heel difference was alphabetical is significant difference (p＜0.05).
Table 7 is the result show, the conidium that the sawdust of multiple tree material and multiple agricultural byproducts can be used for producing QZ-2000, and conversion ratio is higher, wherein uses the sporulation quantity maximum with analysis for soybean powder+cotton cake again, and the maximum sporulation quantity of every gram dry matter reaches 7.1 * 10
8Individual/gram.
The preparation mass ratio is analysis for soybean powder+cotton cake solid culture medium of 2: 1, add water and make that water content is respectively 30%, 40%, 50%, 60% and 70% of a solid culture medium dry weight, according to treating mycelia confluent cultures base after the embodiment inoculation, place under the black light lamp to shine detection spore amount (table 8) 2 days.
Table 8 different moisture content produces the influence (10 of spore to solid culture medium
8Individual/g
*)
| Water content | 30％ | 40％ | 50％ | 60％ | 70％ |
| Average sporulation quantity | 3.19d | 5.25c | 7.25b | 7.64a | 5.75c |
Annotate
*: what data heel difference was alphabetical is significant difference (p＜0.05).
Table 8 is the result show, along with water content increases, sporulation quantity increases.The solid culture medium sporulation quantity maximum of water content 60%, sporulation quantity is 7.64 * 10
8Individual/g.Moisture content in medium increases at 70% o'clock, and sporulation quantity significantly reduces.Therefore, the addition of the good moisture of GPRS when solid fermentation.
The preparation mass ratio is analysis for soybean powder+cotton cake solid culture medium of 2: 1, and water content is 60% of a solid culture medium dry weight.When treating mycelia confluent cultures base after the embodiment inoculation, 1. add a cover and place continuous illumination under black light lamp, uviol lamp, the fluorescent lamp, 12h alternation of light and darkness; Apart from 25cm; 2. go lid to place under above each condition; 3. handle under the continuous darkness condition.Shine in contrast with indoor natural light, room temperature is 25-28 ℃, the quantity (table 9) that the counting spore produces behind the 2d.Each handles 5 repetitions.
The influence of spore is produced in table 9 illumination to bacterial strain QZ-2000
| Handle | Natural lighting | Continuous illumination | The 12h alternation of light and darkness | Dark fully | ||||
| Uviol lamp | Black light lamp | Fluorescent lamp | Uviol lamp | Black light lamp | Fluorescent lamp | |||
| Cover illumination and open wide illumination | 0.48 *H 0.53H | 8.0B 0 | 7.3C 8.6A | 3.4FG 3.9F | 7.9C 0 | 5.4E 6.6D | 2.6G 2.8FG | 0.23H |
* data are spore quantity (* 10 in the table
8/ gram)
Annotate: uppercase expression has utmost point significant difference and significant difference (P=0.01) after the data in row, column
The result of the test of table 9 shows that the later stage illumination of solid culture can greatly promote to produce spore, and greater than the 12h alternation of light and darkness, and three kinds of illumination modes compare to the synergy of producing spore in continuous illumination, again with clarinet light according to the most obvious to the facilitation of producing spore.Maximum sporulation quantity 8.6 * 10
8Individual/gram appears in the group of the unlimited black light lamp continuous illumination of solid culture medium.
Embodiment 4 Alternaria zinniae bacterial strains utilize fungus conidium to kill weeds, the embodiment of producing is as follows: the Alternaria zinniae bacterial strain is (public, the strong victory, Guo Aimin, Bruce A.Auld etc., the technology of producing youth-and-old-age pink mold spore in enormous quantities, 1997. Chinese biological control 13 (4): 169-172) on the PDA medium, cultivated 7 days, be inoculated in the corn flour analysis for soybean powder sucrose calcium carbonate liquid nutrient medium and cultivated 3～4 days, when thalline begins to deposit black pigment, thalline is taken out filtration, and, mycelium is pulverized with cracker with sterile water flush away bacterial metabolism product and residue nutrients.Solid culture medium is with wheat bran or rice bran dry weight: saw dust dry weight=take by weighing at 1: 7, and again in siccative weight: the ratio poach of the weight of water=1: 2.0.Mycelium suspended liquid is inoculated on the solid culture medium, places immediately that the 40cm place carries out 12 hours circulation light photographs under the 20W black light lamp (wavelength 365nm), induce the product spore under the room temperature condition, good air culture was supported 3-8 days in homemade incubator.The spore amount maximum of every gram Dry Matter Production reaches 3.11 * 10
7Individual/gram, purer solid fermentation sporulation quantity has more 5 times at least, and the solid fermentation time shortens 2-7d.
Nitrogen content is as follows to the test and the result of Alternaria zinniae bacterium sporulation quantity in illumination starting time in the above-mentioned implementation method and the liquid nutrient medium:
Contain corn flour 17g, bean powder 1g, CaCO with every liter
3The liquid fermentation medium of 3g carry out mycelium fermentation gained mycelium after filtration, after washing, pulverizing etc. handle, be seeded in the wheat bran dry weight: saw dust dry weight=1: 7, again in siccative weight: on the solid culture medium of the ratio poach of the weight of water=1: 2.0.Divide 4 groups, 3 plates of every group were induced product spore with black light lamp through 0,3,6,9 hour after the dark respectively.
The light-struck starting time of table 10 near ultraviolet is to Alternaria zinniae bacterium sporulation quantity (10
7/ g
*) influence
| Starting time | 0 | 3 | 6 | 9 |
| Average sporulation quantity | 3.11a | 2.73b | 2.43c | 2.27c |
Annotate: what data heel difference was alphabetical is significant difference (p＜0.05).
Table 10 result shows, adopt immediately after mycelium suspended liquid is inoculated on the solid culture medium spore output that 40cm place under the 20W black light lamp (wavelength 365nm) carries out 12 hours circulation light photographs more at interval 3,6 and the spore output that applied illumination in 9 hours again significantly increase, and blanking time is short more, and sporulation quantity is big more.
Nitrogen content is to the influence of solid culture medium Alternaria zinniae bacterium sporulation quantity in the liquid nutrient medium: in the liquid medium within, bean powder institute nitrogen content accounts for the overwhelming majority of nitrogen content in the medium, and the content of bean powder can be similar to regard as with nitrogen content in the medium and be directly proportional.The medium dry substance mixture is constant keeping, and promptly to add bean powder be 15g/L to corn flour, CaCO
3Content is under the situation of 3g/L, and the content with bean powder becomes 0 (adding corn flour 3g/L), 3,6,9g/L4 kind content respectively.Produce spore, the sporulation quantity of more every at last gram dry matter on the solid culture medium of inoculation by the example preparation.
The Alternaria zinniae bacterium spore output (10 of the different bean powder content of table 11 medium
6/ g
*)
| Bean powder content in the medium | 0g/L | 3g/L | 6g/L | 9g/L |
| Average sporulation quantity | 22.12±1.034a | 17.08±1.560b | 16.12±0.225b | 5.36±0.149c |
Annotate:
*Be every gram dry matter, what data heel difference was alphabetical is significant difference (p＜0.05).
Table 11 is the result show, liquid nutrient medium bean powder content increases, and promptly nitrogen content increases, and the sporulation quantity of solid culture medium significantly descends.Therefore, for increasing the sporulation quantity of solid culture medium, when the obtaining liq medium, to control nitrogen content wherein.
Adopting said method of the present invention to cultivate the other biological weed killer herbicide and can reach same effect as utilizing fungies such as anthrax-bacilus, chain lattice spore, is that those skilled in the art are familiar with.
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.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100426963C (en) * | 2005-01-28 | 2008-10-22 | 南京农业大学 | Use of metabolite of eupatorium in biologically herbicidal |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1312272C (en) * | 2005-06-15 | 2007-04-25 | 四川大学 | Biodegradation method for preventing and eliminating Eupatorium adenophorum spreng |
| CN1928068B (en) * | 2005-09-08 | 2011-03-09 | 上海市农药研究所 | Lilaceous streptomycete and application thereof |
| CN1316898C (en) * | 2005-10-20 | 2007-05-23 | 上海交通大学 | Microbial herbicide for controlling hyacinth, and preparation method |
| CN104237047A (en) * | 2014-08-27 | 2014-12-24 | 广东省农业科学院植物保护研究所 | Method for detecting toxicity of bactericide on bacteria of sigatoka disease of bananas |
| CN105567605A (en) * | 2016-02-19 | 2016-05-11 | 黑龙江省科学院大庆分院 | Lysinibacillus sp. and application thereof, degumming auxiliary containing lysinibacillus sp. and preparation method of degumming auxiliary |
| CN109122727B (en) * | 2018-07-16 | 2021-02-19 | 华南农业大学 | Application of colletotrichum gloeosporioides BWH-1 in preparation of herbicide |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN100426963C (en) * | 2005-01-28 | 2008-10-22 | 南京农业大学 | Use of metabolite of eupatorium in biologically herbicidal |
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