CN100549158C - A kind of campelyco and preparation method thereof - Google Patents
A kind of campelyco and preparation method thereof Download PDFInfo
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- CN100549158C CN100549158C CNB2006101554734A CN200610155473A CN100549158C CN 100549158 C CN100549158 C CN 100549158C CN B2006101554734 A CNB2006101554734 A CN B2006101554734A CN 200610155473 A CN200610155473 A CN 200610155473A CN 100549158 C CN100549158 C CN 100549158C
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Abstract
The invention discloses a kind of campelyco and preparation method thereof, belong to the technical field of utilizing microorganism to prevent and kill off farmland weed.This weedicide is a bacterial classification with curved spore mould (Curvularia Boedijn) Mds0404 bacterial strain CGMCCNO.1827, and the weight percent content of its component and each component is: pure spore 80-95%, chemical herbicide 0-18%, auxiliary 2-5%; Through the preparation of substratum, the activation of bacterial strain Mds0404 and culture of seed liquid, the production of bacterial classification spore cultivate with mixing of collection and each component etc. step be prepared from.Characteristics such as this agent is held concurrently except that weeds such as Herba Setariae Viridis, barnyard grass grass, Herba Eleusines Indicaes to prevent and kill off lady's-grass, has farm crop safety, and especially to monocot crops safety, application adaptability is wide, and is with low cost, pollution-free.Can be applicable in weeds such as lady's-grass harm area.
Description
Technical field
The present invention relates to a kind of technical field of utilizing microorganism to prevent and kill off farmland weed, relate in particular to a kind of preparation method who utilizes fungal bacterial strain to prevent and kill off campelyco and this weedicide of farmland weed, be exclusively used in preventing and kill off of farmland weed lady's-grass, Herba Eleusines Indicae and barnyard grass grass etc.
Background technology
Along with people are residual to chemical weed control, the wideling popularize of understanding, green food and the Organic farming of environmental pollution and resistance and the development of resistance weeds, development campelyco management of weeds has become the active demand in the production, it has less investment, the lead time is short, specificity is strong, environmentally safe, do not produce advantage such as pollution.At present, business-like campelyco kind has Comperico and the Biochon that the DeVine that develops the beginning of the eighties and Collego and the end of the nineties release.These weedicides have played vital role on the relative target weeds of control, can guarantee to use once as the long-lasting of DeVine, and 2-3 is weeding again.Being hopeful business-like campelyco project has tens of, and the campelyco kind of wherein preventing and kill off weeds such as barnyard grass grass, wild water chestnut has very tempting prospect.
Monocotyledon weed lady's-grass (Diditaria sanguinaris) is one of 18 kinds of malignant weeds of generally acknowledging in the world, be distributed widely in the torrid zone, the world and area, temperate zone, it is the malignant weed in autumn maturing Dry crop fields such as corn, Chinese sorghum, soybean, peanut, also very serious to the harm of growth of seedling such as cotton, tobacco, crudefiber crop, sugarcane, also be the advantage weeds in vegetables field, orchard, lawn and garden crop field simultaneously.In China, the hazard area of lady's-grass about about 6,000 ten thousand hectares, accounts for the over half of autumn maturing Dry crop field weed total losses by its crop that causes loss greatly.This weeds fecundity is strong, fast growth, and vegetative period is long, consumes that soil fertility, shading, resistance are strong, artificial, machinery is prevented and kill off the comparison difficulty.At present, though chemical control is effective,, the life-time service of chemical herbicide causes the generation and the serious environmental pollution of resistant weed, causes land deterioration.Therefore develop campelyco and prevent and kill off lady's-grass and not only can effectively prevent and kill off this weeds, and environmentally safe property height.At present, about the research of lady's-grass mycoherbicide, the U.S. has applied for utilizing Curvularia (Curvulariaintermedia Boedijn) bacterial strain, and (international mycology can be collected, and preserving number is 361688 (MT-5), 361689 (CG-L), 375263 (MT-6), 375264 (MT-7)) patent of biological control lady's-grass, the patent No. is 5,635,444 and 5,952,264.But this patent documentation is when proposing its use technology with production method, and is only clearly safer to soybean, cotton, and to the securities of monocot cropss such as corn and paddy rice and use also not mentioned.In addition, domestic Agricultural University Of Nanjing has applied for utilizing hyperbolic spore mould (Nakataea Hara) bacterial strain QZ-2000 (preserving number: the patent of biological control lady's-grass (the ZL patent No. 01134002.9) CGMCC NO.0640), but its single strain preventive effect is undesirable, spray just after needing spore by several individual plants be mixed in proportion and can reach preventive effect preferably, this has just increased the difficulty of producing.Simultaneously, an important feature of mycoherbicide is exactly that it is comparatively harsh to the requirement of envrionment conditions, have only when fully satisfying under the requirement condition of this microbial inoculum to moisture and temperature, could guarantee this bacterial strain effectively infecting to weeds, just can obtain good preventive effect, adapt to and the strange land ecological condition differed surely, easily has certain region, therefore, desired envrionment conditions broad when having only selection that the host is infected for solving this difficult problem, easily the parasitical fungi bacterial strain that can satisfy just has broad popularizing application prospect.
Summary of the invention
The present invention seeks at malignant weed-lady's-grass, the disadvantage that the life-time service chemical herbicide is brought, with existing campelyco kind rareness, to the difficult defective that adapts to, uses trouble, poor effect of strange land ecological condition, propose a kind of fungal bacterial strain and be prepared from, to prevent and kill off lady's-grass by the screening acquisition, hold concurrently except that weeds such as Herba Setariae Viridis, barnyard grass grass, Herba Eleusines Indicaes, to farm crop safety, especially to monocot crops safety, application adaptability is wide, with low cost, free of contamination campelyco; Another object of the present invention is the preparation method who proposes this campelyco.
The separation of pathomycete of the present invention, screening and evaluation: collect the lady's-grass blade of falling ill from Yuyao, Zhejiang Province, the sick strong intersection blade of clip, carry out surface sterilization with 6% chlorine bleach liquor, place then on the PSA substratum, in 25 ℃ of thermostat containers, cultivate, carry out single bacterium separation and purification after one week, and then, carrying out single strain produces the spore amount, prevents and kill off monocotyledon weed effect test such as lady's-grass, therefrom filter out high-yield and high-efficiency bacterial strain Mds0404, and the microscopic morphology and the morphological specificity of on the PSA substratum, observing this pathogenic bacteria, identify according to the fungi identification handbook:
Bacterial strain Mds0404: belong to imperfect fungi (Fungi Imperfecti), Moniliales (Moniliales), Moniliaceae (Moniliadeae), Curvularia (Curvularia Boedijn); Well-grown in the PDA substratum that contains sucrose, glucose and saltpetre matrix, bacterium colony are beige or greyish-green, and bacterium colony is a tawny when being matrix with Secondary ammonium phosphate and urea, and bacterium colony is colourless when being matrix with glycerine and trisodium citrate; The differentiation of this bacterium conidiophore is obviously singly given birth to or is grown thickly, and upright or curved, olive colour or black has barrier film, and 48-196 * 4-6um is single or once in a while at the top branch, elongated; Conidium forms continuously, and the top is produced spore part sympodium formula and extend the shape of often going down on one's knees, the shoot top that new spore life is born below previous spore; The spore size is 24-36 * 8-15um, fusiformis or ellipse, 3 barrier films of tool, in every between two parties or placed in the middle substantially, straight or bend towards a side, the little and blunt circle of two terminal cells, colourless or light color, two cells in middle part are big, color depth conidium brown, wide fusiform, 3 barrier films, in every between two parties, not crooked or little curved, zygomorphy, size is 24-36um*10-18um.
This bacterial strain is preserved in the common micro-organisms center C GMCC of China Committee for Culture Collection of Microorganisms, preserving number: No.1827 on September 30th, 2006.
The object of the invention is achieved through the following technical solutions:
A kind of campelyco produces the spore bacterial classification with curved spore mould (Curvularia Boedijn) bacterial strain Mds0404, and the component of this weedicide and the weight percent content of each component are:
Pure spore 80-95%
Chemical herbicide 0-18%
Auxiliary 2-5%.
Described chemical herbicide be G-30027, alachlor, acetochlor, metolachlor, the third careless amine, isoproturon, chlorotoluron, oxadiazon, Diuron Tech if so or beautiful farming happy etc.;
Described auxiliary is polysorbas20, tween 80, dextran, vegetables oil, Mierocrystalline cellulose, polyether silicone oil etc.;
A kind of method for preparing described campelyco, carry out according to the following steps:
(1) preparation of substratum: bacterial strain Mds0404 can and induce conidial generation with PDA substratum, PSA substratum, bran mass, maize powder medium, starch yeast culture base or the activation of Czapek substratum;
(2) activation culture of bacterial strain Mds0404: this bacterium mycelia piece of a little preservation of picking is to the arbitrary plate substratum of step (1), and 28 ℃ of constant temperature are secretly cultivated, the activation bacterial strain;
(3) bacterial strain culture of seed liquid: the bacterium piece that a little step of picking (2) is cultivated, insert and fill in the triangular flask of the arbitrary nutrient solution of step (1) (not containing agar), 28 ℃, 150rpm enriched mycelium pellet to producing in shaking culture 3-5 days, and is stand-by;
(4) production of bacterial classification spore is cultivated and is collected: can adopt solid or liquid-solid associating cultured method, under aseptic condition, the mycelium pellet of Mds0404 mycelia piece or liquid culture is inoculated in the eggplant bottle that half-mature wheat is housed, placed 25-28 ℃ of dark culturing 6-8 days, after treating that mycelia is covered with solid medium, wheat is smash diffusing, be tiled on the flat board, be controlled to the humidity of 40-65%, interval black lamp illumination in 12 hours 2 days, get final product spore, collect spore with spore sieve shaker or spore collector, standby;
(5) pure spore, chemical herbicide and the auxiliary that will weigh by prescription is mixed into campelyco, packing, sealing, product.
The method that above-mentioned curved spore trichoderma strain Mds0404 weedicide is used for management of weeds is:
Usable concentration is that the pure conidium aqeous suspension of the curved spore trichoderma strain Mds0404 of 3-50g/L carries out foliage-spray or seed treatment to weeds; Maybe with the conidium of this bacterium in addition all kinds of auxiliarys be made into aqeous suspension and use; Or the chemical herbicide mixing water suspension of the mixture of conidium and auxiliary and 5-20% conventional amount used uses; Perhaps with on other weeds separate the pathogenic bacterial strains that obtains and unite use, its method that is used all is the routine techniques that those skilled in the art grasped.
The present invention compared with prior art, its beneficial effect is as follows:
1, bacterial strain Mds0404 is to the seedlings of crops such as wheat, soybean, cotton, cowpea, Sunflower Receptacle, corn, peanut, paddy rice safety, there is part to infect to weeds such as Herba Setariae Viridis, barnyard grass grass, Herba Eleusines Indicaes, and be severe infections to lady's-grass, control effect after one week is more than 85%, the host range of this bacterium is single-minded relatively, is safe (seeing Table 2) to agriculture production; So adopt the curved mould microbial inoculum of spore of lady's-grass of the present invention not only to can be used for preventing and kill off dicotyledonous crops field piece weeds such as soybean, cotton, peanut, also can be used for preventing and kill off monocot crops field piece weeds such as paddy rice, corn and wheat, this routine that obviously is better than almost can not selecting between monocot crops and monocotyledon weed is prevented and kill off the chemical herbicide of monocotyledon weed, therefore, also just be better than United States Patent (USP) of the prior art.
2, be used with relevant auxiliary, this microbial inoculum can reach 85%-100% to the control effect of target weeds lady's-grass, particularly the following plant of 3 leaf ages.
3, the present invention screens the bacterial strain Mds0404 of acquisition, production usefulness as the preparation microbial inoculum is planted, it is to the culture condition wide adaptability, at 10-40 ℃, pH5-10 all can grow, and culture medium raw material is easy to get, and is with low cost, utilize agriculture as the foundation of the economy product commonly used can produce spore output height (2.0 * 10 in enormous quantities
7Individual spore/gram dry weight wheat); And as the susceptible microbial inoculum of management of weeds, its condition broad that causes a disease in the field, 10-40 ℃ of spore all can be sprouted, deactivation temperature is 55 ℃, infect easily and prevent and kill off object, studies show that this bacterium has bigger potential development to become the commercialization campelyco, can be used for most of crops field control weeds lady's-grass.
4, campelyco of the present invention be utilize to live biological itself, come from employed environment, its degraded product is organism capable of circulation, can be because of use causes polluting, therefore environmentally safe, can be used for producing green or organic farm products.
Therefore, the present invention utilizes curved spore trichoderma strain Mds0404 development campelyco control lady's-grass, and this bacterial strain is safe to staple crops, does not infect gramineous crop and broad leaved plant, applied range, thereby this campelyco has very remarkable economical and social benefit.
Embodiment
The present invention is described in further detail by the following examples, but this does not constitute the qualification of going up in all senses to the present invention.
Embodiment 1:(prevents and kill off the screening of the efficient bacterial strain Mds0404 of lady's-grass)
From spontaneous generation in Yuyao, Zhejiang on the geographic open-air lady's-grass disease plant, gather the sick leaf of lady's-grass, carry out the separation and Culture of pathogenic fungi with the PDA substratum, therefrom isolate several fungal bacterial strains, and they pathogenic carried out following test; At the monospore of each bacterial strain of microscopically picking, be seeded in respectively on the PDA substratum, in dark, 28 ℃ of cultivations down, observe the colony characteristics of pathogenic fungi pure culture; Each bacterium colony disk of cut-off footpath 6mm is seeded in the dull and stereotyped central authorities of PDA in addition, measures each colony diameter after 7 days; It is 1 * 10 that each bacterial strain is mixed with concentration
6The spore suspension of individual/ml sprays on 2 leaves, 1 heart lady's-grass plant, handles after 4 days, observes the pathogenic property (seeing Table 1) of each bacterial strain, therefrom selects the strongest Curvularia bacterial strain Mds0404 of death rate of the onset, preserves; Lady's-grass is fallen ill the plant leaf scab just for round point shape, after expand as flat ellipse or the fusiformis scab that differs in size, scab central authorities are brown, be purple or red-purple on every side, be with yellow haloing outward, there is downright bad line at two ends, a plurality of scab associatings when serious cause whole blade to be shrivelled shape shrinkage and curl; The lady's-grass scab central authorities visible black color sporophore that infected by this bacterial strain is done the visible a small amount of conidium identical with culture condition of disease leaf microscopy.
Table 1 lady's-grass pathogenic fungi colony characteristics and to the comparison of lady's-grass pathogenicity rate
The mensuration of embodiment 2:(bacterial strain Mds0404 host range)
Measure clearly through host range, bacterial strain Mds0404 is to crop safeties such as cotton, Sunflower Receptacle, soybean, cowpea, peanut, corn, wheat and paddy rice; Barnyard grass grass, Herba Eleusines Indicae and Herba Setariae Viridis have infection, and lady's-grass is severe infections (seeing Table 2), and the control effect after the week is more than 85%.
The host range of table 2 lady's-grass Curvularia bacterial strain Mds0404 is measured
Annotate: NS represents not have reaction; LS represents that infection is arranged; HS represents severe infections
A kind of campelyco of embodiment 3:(the present invention and preparation method thereof 1)
Take by weighing pure spore 83 grams of bacterial strain Mds0404, G-30027 15 grams, tween 2 grams by prescription, be mixed into campelyco 100 grams.The preparation method of this agent carries out according to the following steps:
(1) preparation of substratum: bacterial strain Mds0404 can and induce conidial generation with following any substratum activation:
PDA substratum: potato 150g, glucose 10g, agar 18g, water 1L;
PSA substratum: potato 150g, sucrose 10g, agar 18g, water 1L;
Bran mass: wheat bran 50g, agar 18g, water 1L;
Maize powder medium: Semen Maydis powder 50g, agar 18g, water 1L;
Starch yeast culture base: starch 10g, yeast powder 2g, agar 18g, water 1L;
Czapek substratum: KNO
32g; K
2HPO
41g; KCl 0.5g; MgSO
4.7H
2O 0.5g;
FeSO
40.01g; Sucrose 30g; Agar 18g; Water 1L.
(2) activation culture of bacterial strain Mds0404: this bacterium mycelia piece of a little preservation of picking is to step (1) PDA plate substratum, and 28 ℃ of constant temperature are secretly cultivated, the activation bacterial strain;
(3) bacterial strain culture of seed liquid: the bacterium piece that a little step of picking (2) is cultivated, insert in the triangular flask that fills step (1) PD nutrient solution (PDA substratum do not add agar), 28 ℃, 150rpm produced to bacterium liquid and to enrich mycelium pellet in shaking culture 3-5 days, and is stand-by;
(4) production of bacterial classification spore is cultivated and is collected: can adopt solid or liquid-solid associating cultured method; Under aseptic condition, the mycelium pellet of Mds0404 mycelia piece or liquid culture is inoculated in the eggplant bottle that half-mature wheat is housed, placed 25-28 ℃ of dark culturing 6-8 days, treat that mycelia is covered with solid medium after, wheat is smash diffusing, be tiled on the flat board, be controlled to the humidity of 40%-65%, 12 hours interval black lamp (20W, vertical luminous range is 25cm) illumination 2 days, get final product spore, spore is collected with spore sieve shaker or spore collector in dry back, and is standby;
(5) pure spore, G-30027, the tween that will take by weighing by prescription is mixed into campelyco, packing, sealing, product.
Embodiment 4:(campelyco of the present invention and preparation method thereof 2)
Take by weighing pure spore 95 grams of bacterial strain Mds0404, vegetables oil 5 grams by prescription, be mixed into campelyco 100 grams; The preparation method of this agent is same as embodiment 3.
Embodiment 5:(campelyco of the present invention and preparation method thereof 3)
Take by weighing pure spore 90 grams of bacterial strain Mds0404, alachlor 7 grams, polyether silicone oil 3 grams by prescription, be mixed into campelyco 100 grams; The preparation method of this agent is same as embodiment 3.
The pure spore of embodiment 6:(is prevented and kill off lady's-grass grass effect test)
At the PDA substratum, 28 ℃, dark culturing brushed surperficial mycelia after 8 days with bacterial strain Mds0404, shine 1 day at interval with black lamp 12 hours after, by filtered through gauze, collect filtrate and microscopy spore concentration (1 * 10 with aseptic water washing
6Individual/as ml), to spray filtrate with manual sprayer, be inoculated on 1 leaf, 1 heart to 3 leaf, the lady's-grass of 1 heart stage, preserved moisture 12 hours, handle after 5 days, the control effect of this grass is reached more than 90%.The pathogenic symptom of this spore inoculating on lady's-grass is: the red-purple scab appears in blade at first, and round point shape is expanded as subcircular or fusiformis brown scab subsequently, and quantity connects in flakes for a long time, and posterior lobe sheet or full wafer are withered.On the immature plant leaf transparent round spot, the very fast death of whole plant when serious.Visible black color sporophore on the sick leaf.Through observing, the spore of sick leaf surface is the same with the Curvularia Mds0404 spore that is sprayed.Disease symptom under the above experiment condition is similar to natural illness.
Embodiment 7:(spore+auxiliary removes lady's-grass grass effect test)
Press embodiment 4 methods, make spore suspension earlier after, add auxiliary tween, vegetables oil, Mierocrystalline cellulose, starch or polyether silicone oil again, handle lady's-grass, the result obviously improves and kills careless speed and efficient.(seeing Table 3).
Table 3 auxiliary is to the influence of Mds0404 effect of inoculation
| Auxiliary agent | Strain preventive effect (%) | Fresh weight preventive effect (%) |
| Adding assistant not | 45.36c | 52.98c |
| The first class Mierocrystalline cellulose | 91.53a | 98.20a |
| TW-20 | 86.79b | 93.12b |
| TW-80 | 93.19a | 97.36a |
| Polyether silicone oil | 94.24a | 99.26a |
Data are shown with significant difference (P≤0.05) with different lowercase alphabets in every row.
The chemical herbicide of embodiment 8:(spore+auxiliary+5-20% conventional amount used removes lady's-grass grass effect test)
Press embodiment 3 or 5 methods, make the spore suspension that is added with auxiliary earlier, add again conventional amount used 5-20% G-30027, alachlor, acetochlor, metolachlor, the third careless amine, isoproturon, chlorotoluron, oxadiazon, Diuron Tech if so or beautiful farming happy, evenly spray on the lady's-grass of 2.5-3.5 leaf age phase the two groups of processing of preserving moisture respectively 12 hours and do not preserve moisture; Handled back 4 days, one group the lady's-grass Be Controlled 100% of preserving moisture, one group lady's-grass Be Controlled 70-90% (seeing Table 4) does not preserve moisture.
Table 4 G-30027 and the Mds0404 effect of inoculation that is mixed
The pure spore of embodiment 9:(is prevented and kill off lady's-grass grass seed effect test)
After setting is covered with the culture dish autoclaving of filter paper, put 50 lady's-grass seeds in every ware, will reach (1 * 10 by the spore concentration that embodiment 6 methods are produced
6Individual/ml) spore suspension imposes in the culture dish, and 2 milliliters in every ware repeats 5 times; Other establishes sterilized water and handles in the ware lady's-grass seed in contrast; After 4 days, sprouting of contrast lady's-grass and growing way are all normal, and quilt is infected whole death and the lady's-grass seed that bacterium liquid is handled has just been sprouted promptly.
Claims (3)
1, curved spore mould (Curvularia Boedijn) Mds0404 bacterial strain, its bacterial strain preserving number is CGMCCNO.1827.
2, a kind of campelyco, it is characterized in that this weedicide is a bacterial classification with curved spore mould (Curvularia Boedijn) Mds0404 bacterial strain CGMCC NO.1827, the weight percent content of its component and each component is: pure spore 80-95%, chemical herbicide G-30027 0-18%, auxiliary polysorbas20, tween 80, vegetables oil, Mierocrystalline cellulose or polyether silicone oil 2-5%.
3, a kind of method for preparing the described campelyco of claim 2 is characterized in that this method carries out according to the following steps:
(1) preparation of substratum: bacterial strain Mds0404 can and induce conidial generation with PDA substratum, PSA substratum, bran mass, maize powder medium, starch yeast culture base or the activation of Czapek substratum;
(2) activation culture of bacterial strain Mds0404: this bacterium mycelia piece of a little preservation of picking is to the arbitrary plate substratum of step (1), and 28 ℃ of constant temperature are secretly cultivated, the activation bacterial strain;
(3) bacterial strain culture of seed liquid: the bacterium piece that a little step of picking (2) is cultivated, insert and fill in the arbitrary triangular flask that does not contain the agar nutrient solution of step (1), 28 ℃, 150rpm enriched mycelium pellet to producing in shaking culture 3-5 days, and is stand-by;
(4) production of bacterial classification spore is cultivated and is collected: adopt solid or liquid one solid associating cultured method, under aseptic condition, the mycelium pellet of Mds0404 mycelia piece or liquid culture is inoculated in the eggplant bottle that half-mature wheat is housed, placed 25-28 ℃ of dark culturing 6-8 days, after treating that mycelia is covered with solid medium, wheat is smash diffusing, be tiled on the flat board, be controlled to the humidity of 40-65%, interval black lamp illumination in 12 hours 2 days, get final product spore, collect spore with spore sieve shaker or spore collector, standby;
(5) pure spore, chemical herbicide and the auxiliary that will weigh by prescription is mixed into campelyco, packing, sealing, product.
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Cited By (1)
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| CN109748890B (en) * | 2019-01-23 | 2022-10-11 | 中国科学院新疆生态与地理研究所 | Acetylene compound separated and extracted from curvularia secondary metabolite, preparation method and application thereof |
| CN111436461A (en) * | 2020-03-25 | 2020-07-24 | 福建省农业科学院茶叶研究所 | Biological herbicide |
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