CN105039181A - Metarhizium anisopliae MAYX130921 and application thereof - Google Patents
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Abstract
The invention provides metarhizium anisopliae MAYX130921 and an application thereof. The metarhizium anisopliae MAYX130921 is preserved on Dec 4th, 2014 in China General Microbiological Culture Collection Center with the preservation number of CGMCC No.10139. The metarhizium anisopliae MAYX130921 is a bacterial strain initially separated from holotrichia parallela larvas, is easy to culture and can grow rapidly, sporulation quantity is high, and spore germination rate is high; meanwhile, conidiospores of the metarhizium anisopliae MAYX130921 have strong pathogenicity on the holotrichia parallela larvas, are environment-friendly and hardly produce drug resistance and can be widely applied to prevention and control of holotrichia parallela.
Description
Technical field:
The invention belongs to microbial technology field, particularly relate to a kind of Metarhizium anisopliae (Metarhiziumanisopliae) MAYX130921 and application thereof.
Background technology:
Holotrichia parallela HolotrichiaparallelaMotschulsky is important agriculture subterranean pest-insect, its adult (chafer) takes food damage to crops underground part blade, larva moves in for a long time in soil and takes food harm below rhizome and block root (stem), to cause in soil the infecting of microorganism such as bacterium to cause underground rhizome to rot.To the prevention and controls of Holotrichia parallela larva, once based on chemical prevention, though chemical prevention is to a certain degree reducing the hazard rating of grub, but grub can not be effected a radical cure cause harm, and due to the improper use of chemical pesticide, exacerbate soil and river pollution, the pesticide residue of agricultural-food also exceed standard.These negative impacts make people more pay close attention to biological control in control, the beautiful hook scoliid of arc is the parasitic enemy insect of Holotrichia parallela larva, release arc beautiful hook scoliid control Holotrichia parallela also achieves certain effect, but because arc beautiful hook scoliid is as a kind of natural enemy insect, the Population breeding cycle is longer, add its reactivity comparatively strong, the impact by environment is comparatively large, therefore have impact on large-area applying.Green muscardine fungus is as the important insect pathogenic fungus of a class, and to people, animal, plant safety, insect seldom or not produces resistance, is conducive to environment protection.Therefore, the biological control of research Holotrichia parallela larva, avoids or reduces the resistance problems because unreasonable use chemical pesticide brings, and has become the important selection of the improvement of Holotrichia parallela science and sustainable control.
Metarhizium anisopliae (Metarhiziumanisopliae) MAYX130921 belongs to Deuteromycotina hyphomycetes Metarhizium.According to records, Metarhizium anisopliae var. Anisopliae host is extensive, has important value, and have not been reported infecting of Holotrichia parallela larva in biological control.
Summary of the invention:
The object of the present invention is to provide a kind of Metarhizium anisopliae MAYX130921 and application thereof, be intended to overcome the deficiency that existing Metarhizium anisopliae does not find the parasitic Holotrichia parallela of energy, prevent and treat Holotrichia parallela by animal nutrition, avoid or reduce the resistance problems because unreasonable use chemical pesticide brings.
The present invention realizes like this, a kind of Metarhizium anisopliae (Metarhiziumanisopliae) MAYX130921, China Microbiological preservation management committee's common micro-organisms center is preserved on December 4th, 2014, preserving number is CGMCCNo.10139, and preservation address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City.
Further object of the present invention is to provide the application of above-mentioned Metarhizium anisopliae MAYX130921 in Holotrichia parallela is prevented and treated.The present invention, in September, 2013, finds Metarhizium anisopliae a large amount of happening and prevelence in Holotrichia parallela larva when the field investigation of Yuxi E Shan county of Yunnan Province, causes a large amount of Holotrichia parallela larva to infect dead.Therefore, this bacterial strain is the parasitic Holotrichia parallela of current domestic Late Cambrian, popular very strong entomogenous fungi, in control Holotrichia parallela, have very large development prospect.
The present invention is separated from Holotrichia parallela larva of falling ill and obtains Metarhizium anisopliae wild strain, wild strain tieback is obtained Metarhizium anisopliae bacterial strain in the upper rejuvenation of Holotrichia parallela adult, single spore separation is carried out according to a conventional method to the employing of this bacterial strain, cultivation obtains pure, virulent Metarhizium anisopliae.On SDAY substratum, bacterium colony fine hair shape, to flocculence, is white time initial, is olive-green during product spore.Can see that mycelia tool separates branch at opticmicroscope (400 times), transparent, diameter is 1.5 ~ 2.0 μm.Conidiophore diameter about 2 μm, its end produces doleiform stigma, (2.2) μm, 8 ~ 16 (-25) × 1.5 ~ 2.Form the conidium of long catena continuously with basipetal from bottle stalk end.Conidium is unicellular, and oblong is to cylindric, and blunt section of two ends shape is to blunt circle, and size variation is very large, (4.5) μm, 5 ~ 7 (-9) × 2 ~ 3.Spore is single when existing, and look bright, and olive-green time in heaps, the spore come off often is polymerized to rhombus spore agglomerate or shell-like structure.On SDAY substratum, growth is very fast, and after inoculation, in first 8 days, colony diameter day increases 0.64cm.
Compared to the shortcoming and defect of prior art, the present invention has following beneficial effect: the present invention is separated from Holotrichia parallela body the bacterial strain obtained for the first time, cultivates fairly simple, and fast, sporulation quantity is large, and spore germination rate is high in growth.Its conidium is strong to Holotrichia parallela larva virulence, environment friendly and pollution-free, not easily develop immunity to drugs, and can be widely used for the control of Holotrichia parallela.
Embodiment:
In order to make object of the present invention, technical scheme and advantage clearly understand, 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, be not intended to limit the present invention.
The separation of embodiment one, pathogenic bacteria, qualification
1.1 materials and methods
1.1.1 material
Yuxi E Shan county of Yunnan Province collect infected by a kind of entomogenous fungi after fall ill black dull
Gill cockchafer larva.
Sabouraud medium (SDAY): 1% peptone+1% yeast powder+4% glucose+1.5 ~ 2% agar powder+1000ml water.
Aseptic technique: all vessel and apparatus are all through high-temperature sterilization pot (121 DEG C, 30min), and the operations such as inoculation are all carried out in Bechtop.
Culture condition: be placed in 28 DEG C of illumination (12L:12D) thermostat containers and cultivate, after bacterium colony is formed, transfer to test tube SDAY inclined-plane, cultivate 3 ~ 5 days, proceed to 4 DEG C of refrigerator storage.
1.1.2 the abstraction and purification of pathogenic bacteria
Be separated: bacterial isolate bacterium from Holotrichia parallela larva corpse of falling ill.Polypide of being fallen ill by Holotrichia parallela larva takes back laboratory, by Holotrichia parallela larva corpse in order successively through 70% alcohol-pickled 30 seconds, soak 3 minutes (thorough disinfection) through 0.1% mercuric chloride solution, finally use aseptic water washing three times again, then get with inoculating needle the Holotrichia parallela larva tissue handled well and be inoculated on SDAY substratum.Cultivate under temperature is 28 DEG C of conditions, after growing mycelia, carry out single spore separation according to a conventional method, cultivate obtain pure, produce the strong bacterial strain Metarhizium anisopliae of spore ability, transfer on SDAY inclined-plane and grow 3 ~ 4 days, 4 DEG C of refrigerator storage.
Rejuvenation: after the bacterial strain be separated to is cultivated 5 ~ 7 days on SDAY substratum, produce a large amount of conidium, then this conidium is chosen into the sterilized water containing 1% tween-80, stir with glass stick, obtain proper concn (10
8spore/mL) spore suspension, with miniaturised nebuliser be evenly sprayed on Holotrichia parallela larva body surface (with polypide surface wettability for degree), keep relative humidity more than 80%.Collect dead worm and moisturizing, the larva of falling ill obtained is separated by above method and obtains the stronger bacterial strain of virulence.
Purifying: conidial powder on picking substratum, is seeded on new substratum again.In cultivation 2 ~ 3 generation like this, bacterial strain is just purified.
1.1.3 the qualification of pathogenic bacteria
Identify according to the cultural colony of pathogenic bacteria, mycelia and conidial form.Utilize 40 × 10 times of opticmicroscopes, the form of microscopy mycelia, conidium and conidiophore.
1.2 result
Wild strain is obtained from by separation the Holotrichia parallela larva polypide of entomogenous fungi natural infection, in the upper cultivation of Sa Shi agar dextrose culture-medium (SDAY), and tieback obtains a bacterial strain in the rejuvenation of Holotrichia parallela larva, the bacterial strain that single hypha separation obtains purifying is carried out to this bacterial strain, i.e. tortoise green muscardine fungus (Metarhiziumanisopliae) MAYX130921.
Can see that transparent, diameter is 1.5 ~ 2.0 μm at the separation of this bacterial strain mycelia tool, branch at opticmicroscope (400 times).Conidiophore diameter about 2 μm, its end produces doleiform stigma, (2.2) μm, 8 ~ 15 (-25) × 1.5 ~ 2.Form the conidium of long catena continuously with basipetal from bottle stalk end.Conidium is unicellular, and oblong is to cylindric, and blunt section of two ends shape is to blunt circle, and size variation is very large, (4.5) μm, 5 ~ 7 (-9) × 2 ~ 3.Spore is single when existing, and look bright, and olive-green time in heaps, the spore come off often is polymerized to rhombus spore agglomerate or shell-like structure.
According to field infection symptoms, gather the indoor separation observation of polypide of falling ill, and according to " entomomycete ", (Pu bites dragon, Li Zengzhi, 1996) about Metarhizium anisopliae infection symptoms, mycelia and conidial morphological specificity are identified, Metarhizium anisopliae var. Anisopliae is defined as.
Embodiment two, Metarhizium anisopliae purifying Biological Characteristics of Strain
2.1 materials and methods
2.1.1 strains tested
Select to grow after purifying vigorous, grow a uniform ware as strains tested.Get mycelia to be again inoculated on SDAY, cultivate in constant temperature illumination (12L:12D) incubator of 28 DEG C.
2.1.2 the mensuration of colony growth speed and sporulation quantity
Prior cultured Metarhizium anisopliae is got the punch tool punching of a ware 8mm, be inoculated on new substratum, do three repetitions, constant temperature illumination (12D:12L) incubator being placed in 28 DEG C is cultivated, every day, timing measured its diameter and record, until bacterium colony covers with substratum.Be that the punch tool of 8mm gets bacterium cake in the position that substratum is identical with diameter, add 1% tween-80 and 20ml sterilized water, spore is washed down, makes spore suspension, measure sporulation quantity with blood counting chamber.
2.1.3 the mensuration of spore germination rate
By strain culturing 12 ~ 14 days, collect conidium with sterilized water respectively, make suspension, measure spore germination rate with the slide glass of band groove.Directly dropped on sterilized slide glass by spore suspension, 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 process repeats for three times.
2.2 result
As can be seen from Table 1, after cultivating, colony diameter average growth rate is 0.64cm/d in first 8 days, and when the 8th day, rate of growth reaches 0.93cm/d bacterium colony day, belongs to the type of growth fast.And 1-3 days, because of the adaptation of bacterial strain on substratum during just inoculation, therefore poor growth, bacterium colony from the 4th day diameter with the growth rate of 0.73cm/ days.As can be seen from Table 2, from the 6th day, sporulation quantity just can reach 1 × 10
8spore/mL, produce spore speed soon, sporulation quantity is higher.As can be seen from Table 3, the germination rate of 24 hours spores can reach 85.73%.Show that the biological characteristics of this bacterial strain is better, growing state is better.
The speed of growth of table 1 colony diameter
Table 2 sporulation quantity measurement result
Table 3 conidia germination rate (24 hours)
Embodiment three, Metarhizium anisopliae MAYX130921 are to the Toxicity Determination of Holotrichia parallela larva
3.1, materials and methods
3.1.1 for examination worm source
Holotrichia parallela 2 instar larvae, by this laboratory, temperature (25 ± 1) DEG C, in humidity (70 ± 5) %, photoperiod 0L ﹕ 24D illumination box, raises with potato block.Select Holotrichia parallela second instar larvae anosis, of the same size as examination worm.
3.1.2 the preparation of spore suspension
Get the purifying Metarhizium anisopliae sterilized water grown fine conidium is washed down and filters to be configured to 10
8spore/ml conidium bacteria suspension, gets filtrate with aseptic capillary burette one after another drop of on blood cell counting plate, and count spore under the microscope and record data, diluting successively with the sterilized water containing 0.5% tween 80 is 10
8, 10
7, 10
6, 10
5, 10
4the spore suspension of five concentration gradients, with the sterilized water containing 0.5% tween 80 for blank, for toxicity test.
3.1.3 pickling process
Pickling process is adopted to process Holotrichia parallela 2 instar larvae.Immersing concentration respectively by examination worm is 1.0 × 10
8in the spore suspension of spore/mL, shake gently, taken out by examination worm after 30s and dry, the examination worm that vigor of choosing is higher is tested.Examination worm is placed in 24 orifice plates, and with sterilized soil, larva is covered, be cut into small pieces as feed with potato block, with little watering can water spray, moisturizing is carried out to larva, raise in (28 ± 1) DEG C constant incubator.With Holotrichia parallela 2 instar larvae of the sterilized water process of 0.05% tween-80 for contrast, 3 repetitions are established in each process, each repetition 10 examination worm, Continuous Observation 10d.There is the bombys batryticatus quantity of green muscardine spore in the death toll of record examination every day worm and polypide surface, calculates average mortality and bombys batryticatus rate.Data DPS14.0 software carries out linear regression statistical study.
3.2, result
Carried out toxicity test to Holotrichia parallela second instar larvae, Metarhizium anisopliae bacterial strain MAYX130921 conidium infects the process life history of Holotrichia parallela larva.Experimental result shows: Metarhizium anisopliae bacterial strain MAYX130921 bacterial strain has higher virulence to Holotrichia parallela 2 instar larvae, after inoculation process, when the 10th day, accumulated correction mortality ratio reaches 85.90%, virulence regression equation is Y=2.52+0.41X (r=0.97), wherein Y is mortality ratio probit value, and X is the logarithmic value of spore concentration.LC
50(spore/mL) is 1.12 × 10
6spore/mL, LT
50=4.78 days.Show that Metarhizium anisopliae bacterial strain MAYX130921 has the potentiality being developed as the microbial pesticide controlling Holotrichia parallela.
This bacterial strain is that we are first from the Metarhizium anisopliae bacterial strain MAYX130921 that separation and purification Holotrichia parallela larva obtains, CGMCCNo.10139, this bacterial strain temperature be 28 DEG C, relative humidity more than 80% grows fast on SDAY substratum, sporulation quantity is large, and spore germination rate is high.Its spore suspension is better to Holotrichia parallela larva virulence, can be widely used for the control of Holotrichia parallela larva.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 sugarcane root prionid, be typical biological control, the problems such as the anti-medicine that chemical pesticide can be avoided to bring and environmental pollution, will provide foundation for Holotrichia parallela 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 done within the spirit and principles in the present invention, equivalent replacement and improvement etc., all should be included within protection scope of the present invention.
Claims (2)
1. a Metarhizium anisopliae MAYX130921, be preserved in China Microbiological preservation management committee's common micro-organisms center on December 4th, 2014, preserving number is CGMCCNo.10139.
2. the application of Metarhizium anisopliae MAYX130921 according to claim 1 in Holotrichia parallela is prevented and treated.
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| CN106987526A (en) * | 2017-05-25 | 2017-07-28 | 福建省农业科学院植物保护研究所 | One plant of green muscardine fungus FM 03 and its application in preventing and treating mealybug |
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| CN109097290A (en) * | 2018-08-21 | 2018-12-28 | 广西壮族自治区农业科学院 | There is Metarhizium anisopliae bacterial strain and its application of pathogenicity to sugarcane root soil longicorn |
| CN114381380A (en) * | 2022-01-19 | 2022-04-22 | 安徽省农业科学院植物保护与农产品质量安全研究所 | Metarhizium anisopliae rejuvenation method |
| CN115505538A (en) * | 2022-11-17 | 2022-12-23 | 中国农业科学院植物保护研究所 | Metarhizium anisopliae strain CIPPMA0941, application of metarhizium anisopliae strain in preventing and treating red imported fire ants and microbial inoculum |
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| CN105543102A (en) * | 2015-12-09 | 2016-05-04 | 中国热带农业科学院椰子研究所 | Metarhizum anisopliae for preventing and controlling palm pest opisina arenosella walker and application of metarhizum anisopliae |
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| CN107365710A (en) * | 2016-05-12 | 2017-11-21 | 吉林师范大学 | One plant of Metarhizium Strains and its application to hazelnut weevil with High pathogenicity |
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| CN109097290A (en) * | 2018-08-21 | 2018-12-28 | 广西壮族自治区农业科学院 | There is Metarhizium anisopliae bacterial strain and its application of pathogenicity to sugarcane root soil longicorn |
| CN114381380A (en) * | 2022-01-19 | 2022-04-22 | 安徽省农业科学院植物保护与农产品质量安全研究所 | Metarhizium anisopliae rejuvenation method |
| CN114381380B (en) * | 2022-01-19 | 2022-11-22 | 安徽省农业科学院植物保护与农产品质量安全研究所 | Metarhizium anisopliae rejuvenation method |
| CN115505538A (en) * | 2022-11-17 | 2022-12-23 | 中国农业科学院植物保护研究所 | Metarhizium anisopliae strain CIPPMA0941, application of metarhizium anisopliae strain in preventing and treating red imported fire ants and microbial inoculum |
| CN115505538B (en) * | 2022-11-17 | 2023-08-04 | 中国农业科学院植物保护研究所 | Metarhizium anisopliae strain CIPPMA0941, application thereof in preventing and treating solenopsis invicta and microbial inoculum |
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