CN105039179A - Metarhizium taii MTYJ141025 strain and application thereof - Google Patents
Metarhizium taii MTYJ141025 strain and application thereof Download PDFInfo
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Abstract
The invention provides a Metarhizium taii MTYJ141025 strain and application thereof. The Metarhizium taii MTYJ141025 strain is collected by China General Microbiological Culture Collection Center on December 4th, 2014, and the collection number is CGMCC No.10140. The Metarhizium taii MTYJ141025 strain is separated from Dorysthenes granulosus (Thomson) larvae for the first time, and has the advantages of simple culture process, high growth speed and high toxicity for Dorysthenes granulosus (Thomson) larvae. The conidiophore has the advanages of high pathogenicity for Dorysthenes granulosus (Thomson) larvae, environment friendliness and no pollution, can not easily generate drug resistance, and can be widely used for controlling Dorysthenes granulosus (Thomson).
Description
Technical field:
The invention belongs to microbial technology field, particularly relate to a kind of Metarhizium taii (MetarhiziumtaiiLiang & Liu) MTYJ141025 and application thereof.
Background technology:
Sugarcane root prionid [Dorysthenesgranulosus (Thomson)] is under the jurisdiction of Coleoptera (Coleoptera) Cerambycidae (Cerambycidae), be distributed widely in the Major Sugarcane producing regions such as Guangxi, Guangdong, Hainan and Yunnan, it is the important pests of harm sugarcane, mainly sting food sugarcane rhizome portion and main root with larva, cause the whole strain of sugarcane dead, have a strong impact on sugarcane production.Because sugarcane mostly is stubble cane, add along with the large-area continuous cropping plantation of sugarcane, the occurrence injury of sugarcane root prionid is on the rise, and mainly takes the prophylactico-therapeutic measures based on pesticide control at present.Although chemical pesticide has instant effect, but using of chemical pesticide not only can cause sugarcane pesticide residue and serious environmental pollution, and easily kill the natural enemy of sugarcane root prionid, and target pest is developed immunity to drugs, thus do not meet the sustainability of pests control and the requirement of Ecological Control.And bio-control method has the feature of ecology, Sustainable Control, thus biological control is then that sugarcane root prionid is ecological, the important channel of Sustainable Control.Because it has, nature popularity by force, does not stain environment to insect pathogenic fungus, to people, animal, plant safety, insect seldom or not produces resistance, is conducive to the feature of environment protection, becomes the important measures of biological control of insect pests.Therefore, the biocontrol fungi of research sugarcane root prionid, for the biological control of sugarcane root prionid provides foundation, the resistance also brought for minimizing chemical prevention and environmental pollution provide safeguard.
Metarhizium taii (MetarhiziumtaiiLiang & Liu) belongs to Zygomycotina hyphomycetes hyphomycetales Metarhizium.According to records, the host of Metarhizium taii is a Lepidopterous exigua larvae, and have not been reported infecting of sugarcane root prionid.
Summary of the invention:
The object of the present invention is to provide a kind of Metarhizium taii MTYJ141025 and application thereof, be intended to overcome the deficiency that existing Metarhizium taii does not find the parasitic sugarcane root prionid of energy, by animal nutrition control sugarcane root prionid, avoid or reduce the resistance problems because unreasonable use chemical pesticide brings.
The present invention realizes like this, a kind of Metarhizium taii (MetarhiziumtaiiLiang & Liu) MTYJ141025, China Microbiological preservation management committee's common micro-organisms center is preserved on December 4th, 2014, preserving number is CGMCCNo.10140, 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 taii MTYJ141025 in sugarcane root prionid is prevented and treated.The present invention, in August, 2013, finds Metarhizium taii a large amount of happening and prevelence in sugarcane root prionid when the sugarcane field investigation of Yuanjiang county, causes a large amount of sugarcane root prionid to infect dead.Therefore, this bacterial strain is the parasitic sugarcane root prionid of current domestic Late Cambrian, popular very strong entomogenous fungi, in control sugarcane root prionid, have very large development prospect.
The present invention is separated from sugarcane root prionid larva of falling ill and obtains Metarhizium taii wild strain, the rejuvenation on sugarcane root prionid larva of wild strain tieback is obtained Metarhizium taii bacterial strain, single spore separation is carried out according to a conventional method to the employing of this bacterial strain, cultivation obtains pure, virulent Metarhizium taii.On SDAY substratum, bacterium colony is open and flat, fine hair shape, middle part white its outer be blackish green product spore wheel belt, edge green bean juice is yellow, back side mustard Huang wheel belt.Bottle stalk column, the short sharp 8.4-20 × 1-1.5 μm of tool, is singly born in aerial hyphae or closely takes turns and be born on conidiophore and former bottle stalk, also produce from secondary thecaspore by microcycle conidiation.Conidium column, slightly hang contracting in middle part, two terminal circle or one end attenuate point (4.8-) 7.8 ~ 9.6 (-14) × 2.5 ~ 3 μm, occasionally has 13 × 3 μm, two cell conidium.The conidium formed by microcycle conidiation, its shape is basic identical with the former with size, 7.2-9 × 2.5 μm.
Compared to the shortcoming and defect of prior art, the present invention has following beneficial effect: the present invention is separated from Metarhizium taii body the bacterial strain obtained for the first time, cultivates fairly simple, and fast, sporulation quantity is large in growth.Its conidium is strong to sugarcane root prionid larva virulence, environment friendly and pollution-free, not easily develop immunity to drugs, and can be widely used for the control of sugarcane root prionid.
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
The sugarcane root prionid larva of falling ill after being infected by a kind of entomogenous fungi is collected in Yuanjiang county.
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, cultivates 2-3 days, proceeds to 4 DEG C of refrigerator storage.
1.1.2 the abstraction and purification of pathogenic bacteria
Be separated: bacterial isolate bacterium from sugarcane root prionid larva corpse of falling ill.Polypide of being fallen ill by sugarcane root prionid larva takes back laboratory, to fall ill sugarcane root prionid 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 sugarcane root prionid larva tissue handled well and be inoculated on SDAY substratum.Cultivate under temperature is 28 DEG C of conditions, carry out single spore separation after growing mycelia according to a conventional method, cultivate obtain pure, produce the strong bacterial strain Metarhizium taii MetarhiziumtaiiLiang & Liu 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 2-3 days on SDAY, 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 adult housefly body surface (with polypide surface wettability for degree), keep relative humidity more than 80%.Collect dead worm and moisturizing, the larva worm corpse 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.40 × 10 are utilized to show optical micromirror, the form of microscopy mycelia, conidium and conidiophore.
1.2 result
Wild strain is obtained from being separated by the sugarcane root prionid larva body of entomogenous fungi natural infection, in the upper cultivation of Sa Shi nutrient agar (SDAY), and tieback obtains a bacterial strain in the rejuvenation of sugarcane root prionid larva, the bacterial strain that single hypha separation obtains purifying is carried out to this bacterial strain, i.e. Metarhizium taii MetarhiziumtaiiLiang & LiuMTYJ141025.
On SDAY substratum, bacterium colony is open and flat, fine hair shape, middle part white its outer be blackish green product spore wheel belt, edge green bean juice is yellow, back side mustard Huang wheel belt.Bottle stalk column, the short sharp 8.4-20 × 1-1.5 μm of tool, is singly born in aerial hyphae or closely takes turns and be born on conidiophore and former bottle stalk, also produce from secondary thecaspore by microcycle conidiation.Conidium column, slightly hang contracting in middle part, two terminal circle or one end attenuate point (4.8-) 7.8-9.6 (-14) × 2.5-3 μm, occasionally have 13 × 3 μm, two cell conidium.The conidium formed by microcycle conidiation, its shape is basic identical with the former with size, 7.2-9 × 2.5 μm.
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 the morphological specificity of Metarhizium taii infection symptoms, conidium and mycelia is identified, Metarhizium taii is defined as.
Embodiment two, Metarhizium taii 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 taii is got the punch tool punching of a ware 8mm, be inoculated on another one substratum, do 3 repetitions, constant temperature illumination (12L:12D) 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, then access SDAY media surface, cultivate 7 days in thermostat container, measure every day and a record colony diameter, to measure colony growth situation.
2.2 result
SDAY cultivate under 28 DEG C of conditions well-grown, table 1 result shows, Metarhizium taii MTYJ141025 grows comparatively fast on substratum, when cultivating 1-2d, its bacterium colony mean diameter gathers way lower, and when being cultured to 5d, its bacterium colony mean diameter gathers way as 0.95cm/d, when being cultured to 7d, its bacterium colony mean diameter gathers way 0.75cm/d.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 81.51%.Show that the biological characteristics of this bacterial strain is better, growing state is better.
Table 1 colony growth rate
Table 2 sporulation quantity measurement result
Table 3 conidia germination rate (24 hours)
Embodiment three, Metarhizium taii MTYJ141025 are to the Toxicity Determination of sugarcane root prionid
3.1, materials and methods
3.1.1 for examination worm source
Sugarcane root prionid [Dorysthenesgranulosus (Thomson)] larva, by this laboratory, temperature (28 ± 1) DEG C, in humidity (80 ± 5) %, photoperiod 0L ﹕ 24D illumination box, raises with sugarcane.Secure good health, second instar larvae of the same size as examination worm.
3.1.2 the preparation of spore suspension
Get the purifying Metarhizium taii 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 sugarcane root prionid 2 instar larvae.Being immersed in 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 anistree bottle (4.5cm × 4.5cm × 19.5cm), and at the bottom of anistree bottle, spread thick layer 1cm sterilizing sterilized soil, with sugarcane as feed, with little watering can water spray, moisturizing is carried out to larva, raise in (28 ± 1) DEG C constant incubator.With sugarcane root prionid 2 instar larvae of the sterilized water process of 0.05% tween-80 for contrast, 3 repetitions are established in each process, each repetition 30 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 sugarcane root prionid second instar larvae, Metarhizium taii MTYJ141025 conidium infects the process life history of sugarcane root prionid larva.Experimental result shows: Metarhizium taii MTYJ141025 bacterial strain has higher virulence to sugarcane root prionid 2 instar larvae, after inoculation process, when the 10th day, accumulated correction mortality ratio reaches 86.50%, virulence regression equation is Y=2.25+0.45X (r=0.98), wherein Y is mortality ratio probit value, and X is the logarithmic value of spore concentration.LC
50(spore/mL) is 1.29 × 10
6spore/mL, LT
50=5.69 days.Show that Metarhizium taii MTYJ141025 has the potentiality being developed as the microbial pesticide controlling sugarcane root prionid.
This bacterial strain is that we are first from the Metarhizium taii MTYJ141025 that separation and purification sugarcane root prionid larva obtains, CGMCCNo.10140, 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 sugarcane root prionid larva virulence, can be widely used for the control of sugarcane root prionid 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 sugarcane root prionid 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 taii MTYJ141025, be preserved in China Microbiological preservation management committee's common micro-organisms center on December 4th, 2014, preserving number is CGMCCNo.10140.
2. Metarhizium taii MTYJ141025 according to claim 1 sugarcane root prionid [
dorysthenesgranulosus(Thomson) application of aspect] is prevented and treated.
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