CN104120084A - Metarhizium flavoviride MFYY090714 and applications thereof - Google Patents
Metarhizium flavoviride MFYY090714 and applications thereof Download PDFInfo
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- CN104120084A CN104120084A CN201410335748.7A CN201410335748A CN104120084A CN 104120084 A CN104120084 A CN 104120084A CN 201410335748 A CN201410335748 A CN 201410335748A CN 104120084 A CN104120084 A CN 104120084A
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Abstract
The invention provides a Metarhizium flavoviride MFYY090714 and applications thereof. The Metarhizium flavoviride MFYY090714 is preserved in China General Microbiological Culture Collection Center on April 8th, 2014. The preservation number is CGMCC No.9020. The bacterium strain is a novel bacterium strain separated from the body of sogatella furcifera. The culture is simple, the growth is rapid, the spore yield is high, and the germination rate of the spores is high. The pathogenicity of the Metarhizium flavoviride spore suspension liquid on sogatella furcifera is high, moreover, the Metarhizium flavoviride spore suspension liquid is environment-friendly and pollution-free, does not generate any drug resistance, and can be widely used to control sogatella furcifera in practice.
Description
Technical field
The invention belongs to microbial pesticide technical field, relate in particular to a kind of yellowish green green muscardine fungus MFYY090714 and application thereof.
Background technology
White backed planthopper is a kind of paddy rice important pests, and normal and brown paddy plant hopper and small brown rice planthopper are referred to as planthopper.White backed planthopper has become Southwestern China area in recent years, especially the sociales of rice in Yunnan district planthopper, because it has that breeding is fast, migrating property and the feature such as resistance is strong, can cause Severe Reduction of Rice while generation greatly, and even total crop failure, is seriously restricting the production of paddy rice.The control of Whitebacked Planthopper is all the important content of pest control research all the time, but mainly rely on for a long time chemical pesticide control, a large amount of chemical pesticides that use threaten to human health and environment, thereby seek eco-friendly white backed planthopper biological control instead of chemical control, the usage quantity that greatly reduces chemical pesticide has become the important topic of white backed planthopper study on prevention.Because white backed planthopper is under the jurisdiction of sucking pest, and the entomogenous fungis such as green muscardine fungus, muscardine are to invade in unique mode, its conidium is mainly sprouted and is invaded insect body wall by epidermis, and this is for utilizing green muscardine fungus and Beauveria treated white backed planthopper to provide the foundation.Yellowish green green muscardine fungus (Metarhizium flavoviride) is as a kind of important insect pathogenic fungus, belong to Deuteromycotina hyphomycetes Metarhizium fungi, its host range comprises that the current Britain of insect such as Orthoptera, Homoptera, Coleoptera, Hemiptera, Diptera, Hymenoptera, lepidopteran have been widely used in the biological control of locust.At present, about the yellowish green Metarhizium Strains of rice field natural infection brown paddy plant hopper and the existing research of Pathogenicity thereof, but the correlative study report of infecting the pathogenic pathogenic of the yellowish green green muscardine fungus of white backed planthopper and Whitebacked Planthopper thereof under natural condition and preventing and treating application aspect yet there are no.
Yellowish green green muscardine fungus genetic stability is better, and people, animal, bird, plant are not all found to poison, and this bacterium have not been reported aspect preventing and treating white backed planthopper.Therefore, in the time of control planthopper, there is again very large development prospect.
Summary of the invention
The object of the present invention is to provide a kind of yellowish green green muscardine fungus (Metarhizium flavoviride) MFYY090714 and application thereof, be intended to, safety efficient, nontoxic with this yellowish green green muscardine fungus, noresidue, easy to use prevent and treat white backed planthopper.
The present invention realizes like this, a kind of yellowish green green muscardine fungus (Metarhizium flavoviride) MFYY090714 has been preserved in Chinese microbial preservation management committee's common micro-organisms center (being called for short CGMCC) on April 8th, 2014, preserving number is No.9020, preservation address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, and postcode is 100101.
The present invention separates and obtains yellowish green green muscardine fungus wild strain MFYY090714 from the white backed planthopper adult of falling ill, wild strain MFYY090714 tieback is obtained to yellowish green Metarhizium Strains in the upper rejuvenation of white backed planthopper adult, this bacterial strain is carried out according to a conventional method monospore separation, cultivates and obtain pure, virulent yellowish green green muscardine fungus.On PDA substratum, bacterium colony is circular launches, yellow-green colour, and aerial hyphae fine hair shape is fine and close in wadding, and the back side is displaing yellow slightly.The conidiophore colyliform branch that loosens, on it raw 1～2 bottle stalk, bottle stalk is shaft-like, become wide angle with sporophore, top acute point (7.2-) 12.0～4.8 (15.6) × 1.8～3.0 μ m, conidium unit cell, wide oval, the end that tool is different, size is 7.2～9.3 × 3.0～4.0 μ m, conidium forms the chain to base on bottle stalk.This yellowish green green muscardine fungus MFYY090714 bacterial strain belongs to fast growth type, and colony diameter has the speed of growth more than 1cm average every day.Product spore is fast, sporulation quantity is large, and cultivate the 5th day on PDA substratum time, sporulation quantity just can reach 1 × 10
8spore/ml.The germination rate of 24 hours spores reaches more than 85%.In the time of 26 DEG C, this bacterium conidium Whitebacked Planthopper adult median lethal concentration(LC&-{50}) (LC
50) be 3.43 × 10
5spore/ml, LT
50it is 4.15 days.
Beneficial effect of the present invention is: this bacterial strain is to separate from white backed planthopper body the new bacterial strain obtaining, and cultivates fairly simplely, and fast, sporulation quantity is large in growth, and spore germination rate is high.Its spore suspension Whitebacked Planthopper adult virulence is better, environment friendly and pollution-free, be difficult for developing immunity to drugs, and can be widely used in the prevention practice of white backed planthopper.
Embodiment
In order to make object of the present invention, technical scheme and advantage clearer, below in conjunction with embodiment, the present invention is further elaborated.Should be appreciated that specific embodiment described herein, only in order to explain the present invention, is not intended to limit the present invention.
The separation of embodiment mono-, pathogenic bacteria, qualification
1, materials and methods
1.1 material
Collect the white backed planthopper Sogatella furcifera that falls ill (the Horv á th) adult after being infected by a kind of entomogenous fungi in Yuanyang terraced fields rice field, Yunnan Province.
Potato glucose (PDA) substratum: peeling potato 200g, be cut into small pieces, 1L adds water, filtered through gauze after 20min, in filtrate, add glucose 20g, agar 15～20g, heating is dissolved, and is dissolved to 1000mL aseptic technique: all vessel and apparatus are all through (121 DEG C, high-temperature sterilization pot, 30min), the operation such as inoculation is all carried out in Bechtop.
Culture condition: be placed in 26 DEG C of illumination (12L:12D) thermostat container and cultivate, after bacterium colony forms, transfer to test tube PDA inclined-plane, cultivate 3～4 days, proceed to 4 DEG C of refrigerator storage.
The separation of 1.2 pathogenic bacterias and purifying
Separate: bacterial isolate bacterium from the white backed planthopper adult corpse of falling ill.By white backed planthopper adult corpse in order successively through 70% alcohol-pickled 30 seconds, soak 3 minutes (thorough disinfection), finally use aseptic water washing three times through 0.1% mercuric chloride solution again, the white backed planthopper adult corpse of handling well is inoculated on PDA substratum.Be to cultivate under 26 DEG C of conditions in temperature, grow and carry out according to a conventional method monospore separation after mycelia, cultivate and obtain pure, virulent yellowish green Metarhizium Strains, transfer on PDA inclined-plane and grow 3～4 days, 4 DEG C of refrigerator storage.
Rejuvenation: the bacterial strain being separated to is cultivated after 7～10 days on PDA substratum, is added containing the sterilized water of 1% tween-80 and collect spore, after transfer in beaker, stir with glass stick, obtain proper concn (10
8spore/ml) spore suspension, be evenly sprayed on white backed planthopper adult body surface (taking polypide surface wettability as degree) with miniaturised nebuliser, keep relative humidity more than 90%.Collect dead worm moisturizing, the adult worm corpse obtaining separates and obtains the bacterial strain that virulence is stronger by above method again.
Purifying: the mycelia on picking substratum on bacterium colony, is seeded on new substratum again.So many cultivation several generations, bacterial strain has just obtained purifying.
The qualification of 1.3 pathogenic bacterias
Identify according to the cultivation proterties of pathogenic bacteria, mycelia and conidial form.At 40 × 10 times of biomicroscopes, microscopy mycelia and conidial form.
2, result
From being separated and obtain wild strain by the white backed planthopper adult polypide of entomogenous fungi natural infection, in the upper cultivation of potato dextrose agar (PDA), and tieback obtains a bacterial strain in the rejuvenation of white backed planthopper adult, this bacterial strain is carried out to the bacterial strain of single hypha separation acquisition purifying, be yellow green muscardine fungus (Metarhizium flavoviride) MFYY090714, CGMCC No.9020.
On PDA substratum, bacterium colony is circular launches, yellow-green colour, and aerial hyphae fine hair shape is fine and close in wadding, and the back side is displaing yellow slightly.The conidiophore colyliform branch that loosens, on it raw 1～2 bottle stalk, bottle stalk is shaft-like, become wide angle with sporophore, top acute point (7.2-) 12.0～4.8 (15.6) × 1.8～3.0 μ m, conidium unit cell, wide oval, the end that tool is different, size is 7.2～9.3 × 3.0～4.0 μ m, conidium forms the chain to base on bottle stalk.
The bacterium colony, mycelia and the spore shape that arrive according to the observation, describe with the morphological specificity of the relevant yellowish green green muscardine fungus in " entomomycete " and collection of illustrative plates is analyzed, and is accredited as yellowish green green muscardine fungus.
Embodiment bis-, yellowish green green muscardine fungus purifying Biological Characteristics of Strain
1, materials and methods
1.1 strains testeds are selected to look even an and eugonic ware as strains tested after purifying.Get mycelia and be again inoculated on PDA substratum, in constant temperature illumination (12L:12D) incubator of 26 DEG C, cultivate.
Bacterium is got in the punch tool punching that prior cultured yellowish green green muscardine fungus is got ware 8mm by the mensuration of 1.2 colony growth speed and sporulation quantity, be inoculated on new substratum, do three repetitions, constant temperature illumination (12D:12L) incubator that is placed in 26 DEG C is cultivated, every day, its diameter record were measured in timing, until bacterium colony covers with substratum.The punch tool that is 8mm with diameter is got bacterium cake in the identical position of substratum, adds 1% tween-80 and 20ml sterilized water, and spore is washed down, makes spore suspension, measures sporulation quantity with blood counting chamber.
The mensuration of 1.3 spore germination rates, by strain culturing 5～10 days, is collected conidium with sterilized water respectively, makes suspension, measures spore germination rate with the slide glass with groove.Spore suspension is directly dropped on aseptic slide glass, be placed in the culture dish of end paving filter paper, drip several sterilized waters and keep humidity at 100%RH, cultivate microscopy after 24 hours, each processing repeats for three times.
2, result
As can be seen from Table 1, yellowish green Metarhizium Strains MFYY090714 belongs to the type of Fast Growth.First day poor growth is that bacterium colony was from the 2nd day growth speed because bacterial strain needs for some time to adapt to while rigidly connecting kind.As can be seen from Table 2, on PDA substratum, be cultured to the 5th day sporulation quantity and just can reach 1 × 10
8spore/ml.Sporulation quantity is higher.As can be seen from Table 3, the germination rate of 24 hours spores can reach more than 85%.These all illustrate that this strain growth breeding is very fast, and biological characteristics is good.
The speed of growth of table 1 colony diameter
Table 2 sporulation quantity measurement result
Table 3 conidia germination rate (24 hours)
The indoor Pathogenicity of embodiment tri-, yellowish green green muscardine fungus purifying bacterial strain Whitebacked Planthopper
1, materials and methods
1.1 for examination worm source
Select to sprout wings latter 24 hours same batches, white backed planthopper adult anosis, of the same size as examination worm, will be placed in for examination white backed planthopper adult on the rice seedlings that dependent insect cage plants, under the photoperiod condition of 26 ± 1 DEG C and 12L:12D, raise.
The preparation of 1.2 spore suspensions
Getting the yellowish green green muscardine fungus of the purifying growing fine sterilized water washes down conidium filter and be mixed with 10
8spore/ml conidium bacteria suspension, gets filtrate with aseptic capillary burette one after another drop of on blood cell counting plate, counts under the microscope spores record data, is 10 with diluting successively containing the sterilized water of 0.5% tween 80
8, 10
7, 10
6, 10
5, 10
4the spore suspension of five concentration gradients, taking the sterilized water containing 0.5% tween 80 as blank, for Pathogenic Tests.
1.3 spray method
The concentration gradient spore suspension of preparation is evenly sprayed on to white backed planthopper adult body surface (taking polypide surface wettability as degree) with miniaturised nebuliser, each dosage repeats for three times, 30 examination worms of each repetition, inoculate as follows: draw the yellowish green muscardine spore suspension of 10ml in miniaturised nebuliser, by spray method to examination worm spray inoculation, after spraying, dependent insect cage is placed in to 26 DEG C, relative humidity and is controlled at more than 85%, the photoperiodic insectary of 12D:12L.Every day, the dead worm amount of examination worm observed in record, and calculate mortality ratio.
2, result
Whitebacked Planthopper adult has carried out toxicity test, and yellowish green green muscardine fungus Metarhizium flavoviride bacterial strain MFYY090714 conidium is infected the process life history of white backed planthopper.Experimental result shows: yellowish green green muscardine fungus Whitebacked Planthopper adult has the effect of significantly causing a disease, and cumulative correction mortality ratio is 88.5%, and the median lethal time (LT50) is 4.15d, median lethal concentration(LC&-{50}) (LC
50) be 3.43 × 10
5spore/ml.Show that yellowish green green muscardine fungus Metarhizium flavoviride bacterial strain MFYY090714 has the potentiality that are developed as the microbial pesticide of controlling paddy rice white backed planthopper.
This bacterial strain is our separation and purification obtains from white backed planthopper first yellowish green green muscardine fungus Metarhizium flavoviride bacterial strain MFYY090714, CGMCC No.9020, this bacterial strain is that more than 80% on PDA substratum, growth is fast for 26 DEG C, relative humidity in temperature, sporulation quantity is large, and spore germination rate is high.Its spore suspension Whitebacked Planthopper adult virulence is better, can be widely used for the control of white backed planthopper.Simultaneously, these strain culturing raw material sources are extensive, low price, cultural method is simple, easily produces in a large number, has larger Application and Development potentiality, with this bacterial strain control white backed planthopper, be typical biological control, the problem such as resistance and environmental pollution that can avoid chemical pesticide to bring, will provide foundation for white backed planthopper green prevention and control.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, all any amendments of doing within the spirit and principles in the present invention, be equal to and replace and improvement etc., within all should being included in protection scope of the present invention.
Claims (2)
1. yellowish green green muscardine fungus (Metarhizium flavoviride) MFYY090714, is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on April 8th, 2014, and deposit number is CGMCC NO.9020.
2. yellowish green green muscardine fungus MFYY090714 claimed in claim 1 application aspect preventing and treating white backed planthopper.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108865896A (en) * | 2018-06-15 | 2018-11-23 | 广东省农业科学院果树研究所 | A kind of method of Fusarium oxysporum Cuba specialized form bacterial strain rejuvenation |
| CN118165841A (en) * | 2024-05-14 | 2024-06-11 | 云南农业大学 | Metarhizium anisopliae Mrysms2308 and method and application thereof in biological control of armyworms |
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| CN1066959A (en) * | 1991-05-23 | 1992-12-16 | 韩国化学技术研究院 | The improvement preparation technology of imbedded microbe insecticide and the insecticide for preparing by this technology |
| CN102212487A (en) * | 2011-05-24 | 2011-10-12 | 重庆大学 | Metarhizium flavoviride bacterial strain and application thereof |
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2014
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1066959A (en) * | 1991-05-23 | 1992-12-16 | 韩国化学技术研究院 | The improvement preparation technology of imbedded microbe insecticide and the insecticide for preparing by this technology |
| CN102212487A (en) * | 2011-05-24 | 2011-10-12 | 重庆大学 | Metarhizium flavoviride bacterial strain and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| 徐阿妹: ""黄绿绿僵菌培养生产技术及其在害虫防治中的应用"", 《中国优秀硕士学位论文全文数据库-农业科技辑》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108865896A (en) * | 2018-06-15 | 2018-11-23 | 广东省农业科学院果树研究所 | A kind of method of Fusarium oxysporum Cuba specialized form bacterial strain rejuvenation |
| CN118165841A (en) * | 2024-05-14 | 2024-06-11 | 云南农业大学 | Metarhizium anisopliae Mrysms2308 and method and application thereof in biological control of armyworms |
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