CN102212483B - Pesticidal Metarhiziumanisopliaevar. anisopliae strain and application thereof - Google Patents
Pesticidal Metarhiziumanisopliaevar. anisopliae strain and application thereof Download PDFInfo
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Abstract
The invention relates to a Metarhiziumanisopliaevar. anisopliae strain CQMa421 of which the CGMCC (China General Microbiological Culture Collection Center) NO is 4609 and an application thereof in the control of rice leaf roller. The production traits of the strain are good and can grow fast and have high spore-producing capability; and the sporulation rate is up to 6.8%, and the strain has high toxicity. The pesticidal fungal agent produced by using the strain can be used to effectively control the number of the rice leaf rollers.
Description
Technical field
The present invention relates to the new bacterial strain of a kind of mikrobe, be specifically related to a kind of desinsection green muscardine fungus bacterial strain and application thereof, belong to field of biological pesticide.
Technical background
Cnaphalocrocis medinali(rice leaf roller) is one of paddy rice primary pest, and domestic Cnaphalocrocis medinali(rice leaf roller) is distributed widely in each rice district, the whole nation, serious is threatening China's grain-production.At present; Mainly use broad spectrum chemical pesticide control Cnaphalocrocis medinali(rice leaf roller); Not only directly influence the safety of HUMAN HEALTH and culture fishery, and, destroy the rice field eubiosis because Cnaphalocrocis medinali(rice leaf roller) develops immunity to drugs and chemical pesticide kills and wounds the Cnaphalocrocis medinali(rice leaf roller) natural enemy in a large number; Seriously undermine the field natural control ability, cause easily Cnaphalocrocis medinali(rice leaf roller) rampant again, be difficult to control.
Insect pathogenic fungus is most important insecticidal microorganism monoid; About under field conditions (factors) 70% insect disease is fungus-caused; Be unique the have characteristic of tagging and epidemic pathogenic micro-organism, its mechanism of causing a disease is complicated, although thereby it be the mikrobe that is applied to pest control the earliest; But not finding yet has resistant insect to produce, and the resistance problem that solves insect is had potentiality.Therefore, fungi receives extensive concern in recent years as important alternative agricultural chemicals; Domestic research to biocontrol fungi starts from the 1950's, is in the first place in the world at aspects such as disinsection fungal liquid-solid two-phase production technology, preparation research and development and large-scale productions at present.Up to now, successively register more than 100 in disinsection fungal agricultural chemicals both at home and abroad.Disinsection fungal has stronger vertical and horizontal proliferation ability; Form prevailing disease easily; The Sustainable Control that helps insect; Control for the migratory pest Cnaphalocrocis medinali(rice leaf roller) is particularly favourable, but utilizes fungi control Cnaphalocrocis medinali(rice leaf roller) not see relevant report as yet, and its one of the main reasons is to lack high virulence disinsection fungal bacterial strain.
Summary of the invention
The object of the present invention is to provide and a kind of Cnaphalocrocis medinali(rice leaf roller) is had good disinsection effect, green muscardine fungus bacterial strain CQMa421 that the production traits is good.
Bacterial strain of the present invention be a strain production traits that filters out through the Cnaphalocrocis medinali(rice leaf roller) bombys batryticatus direct separation of gathering natural infection good, to Cnaphalocrocis medinali(rice leaf roller) have high virulence the little spore mutation of Metarhizium anisopliae (
Metarhizium anisopliaeVar.
Anisopliae) bacterial strain CQMa421; In on 02 25th, 2011 in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) preservation; Address: No. 3, A, DaTun Road, Chaoyang District, BeiJing City, Institute of Microorganism, Academia Sinica, deposit number: CGMCC NO.4609.
Green muscardine fungus bacterial strain CQMa421 of the present invention is seeded on the 1/4SDA flat board, cultivate to observe colony morphology characteristics for 28 ℃, is presented at and cultivates the initial stage bacterium colony flocculence that is white in color, olive-green when producing spore on the 1/4SDA substratum; Mycelia has separation and branch, transparent, and diameter is 1.5-2.0 μ m; Conidium sporophore list is given birth to or is assembled or closely arrangement, is broom shape branch, and diameter is about 2.0 μ m, its terminal doleiform stigma, 8-16 * 1.5-2 μ m of producing; Form the conidium of catena from the end of bottle stalk continuously with basipetal, conidium is unicellular, oblong, big or small 4.8-7 * 2-3 μ m.
Green muscardine fungus bacterial strain CQMa421 of the present invention beneficial effect specific as follows:
The present invention is from the green muscardine fungus bacterial strain CQMa421 of the Cnaphalocrocis medinali(rice leaf roller) bombys batryticatus direct separation of natural infection screening, produce that the spore ability is strong, spore production rate can reach 6.8%, its production traits is good, growth rapidly, strong, the good disinsection effect of insecticidal activity simultaneously.The disinsection fungal preparation that utilizes this bacterial strain to produce has been controlled the Cnaphalocrocis medinali(rice leaf roller) population quantity effectively.
Description of drawings
Fig. 1: CQMa421 bacterial strain conidial fructification and spore shape size;
Fig. 2: the green muscardine fungus genus (
Metarhizium) ITS1-5.8S-ITS2 rDNA zone branch system tree;
Fig. 3: CQMa421 is to Cnaphalocrocis medinali(rice leaf roller) insecticidal activity assay result;
Fig. 4: CQMa421 gives birth to the outdoor control of Cnaphalocrocis medinali(rice leaf roller) and surveys the result.
Embodiment
Through embodiment the present invention is carried out concrete description below; Be necessary to be pointed out that at this following examples only are used for the present invention is further specified; Can not be interpreted as restriction to protection domain of the present invention; Under the situation that does not deviate from the present invention's spirit and essence, modification or replacement to the inventive method, step or condition are done all belong to scope of the present invention.
If do not specialize the conventional means that used technique means is well known to those skilled in the art among the embodiment.
The separation of embodiment 1 bacterial strain
The Cnaphalocrocis medinali(rice leaf roller) bombys batryticatus of the natural infection that collects with 70% alcohol surface sterilization, is put into 28 ℃ of incubators and preserves moisture and cultivated 3-5 days, make its insect surfaces grow fresh spore; Under aseptic condition, on the 1/4SDA culture medium flat plate that contains 100 mcg/ml penbritins, rule with the fresh spore of transfering loop picking trace; The petridish upset that line is good is positioned over cultivates in 28 ℃ of thermostat containers; Select the typical case who grows after 3-4 days and do not have the bacterium colony of assorted bacterium, with moving substratum one fritter that the acicula picking has mycelia, change test tube slant substratum central authorities at colony edge, 28 ℃ constant temperature culture 10-15 days.
The evaluation of embodiment 2 bacterial strains
(1), identification of morphology
The CQMa421 inoculation that is separated on the 1/4SDA flat board, is cultivated for 28 ℃ and observed colony morphology characteristic, conidial fructification and spore shape and size.The result is presented on the 1/4SDA substratum and cultivates the initial stage bacterium colony flocculence that is white in color, olive-green when producing spore.The mycelia tool is separated and branch, and transparent, diameter is 1.5-2.0 μ m.Conidium sporophore list is given birth to or is assembled or closely arrangement, is broom shape branch, and diameter is about 2.0 μ m, its terminal doleiform stigma, 8-16 * 1.5-2 μ m of producing.Form the conidium (referring to the left figure Fig. 1) of catena continuously with basipetal from the end of bottle stalk.Conidium is unicellular, oblong, big or small 4.8-7 * 2-3 μ m (referring to the right figure among Fig. 1).See that from above characteristic bacterial strain CQMa421 should be the little spore mutation of Metarhizium anisopliae
Metarhizium anisopliae var.anisopliae
(2), ITS sequence amplification, sequencing and the molecular classification of CQMa421 bacterial strain
At 1/4SDAY liquid nutrient medium shake-flask culture 3 days (180rpm, 28 ℃), centrifugal (10000 g 20min) collect mycelia, adopt the DNA extraction test kit to extract total DNA of bacterial strain CQMa421 after the liquid nitrogen grinding fragmentation with CQMa421.With total DNA is template, with reference to Curran amplification green muscardine fungus ITS1-5.8S-ITS2 rDNA zone.The primer is TW81 5 '-gtttccgtaggtgaacctgc-3 ' and AB28 5 '-atatgcttaagttcagcgggt-3 ', produces the 547bp amplified band.PCR is reflected at the response procedures on the PCR appearance: 95 ℃ of 4min, 1 circulation; 94 ℃ of 0.5min, 53 ℃ of 0.5min, 72 ℃ of 0.5min, 30 circulations; Last 72 ℃ of 10min.Amplified production is reclaimed test kit with gel reclaim purifying, give birth to the worker by Shanghai and carry out two-way deoxidation method order-checking.CQMa421 ITS1-5.8S-ITS2 sequencing result is seen SEQ ID No.1.
sequence alignment in this sequence and the ncbi database, accord with expectation length does not have the bacterial strain identical with its sequence.This sequence 5 ' end comprises 18S rDNA portion gene sequence (1-34); 3 ' end comprises part 28S rDNA gene order (504-549); Remaining is respectively ITS1 zone (35-167), 5.8S rDNA gene complete sequence (168-326) and ITS2 zone (327-519).Utilize MEGA v4.1 (www.megasoftware.net) software building genealogical tree (1000 repetitions) to confirm: the CQMa421 bacterial strain does
Metarhizium anisopliaeVar.
AnisopliaeThe little spore mutation of Metarhizium anisopliae (shown in Figure 2).
Be collected in the ripe spore of the CQMa421 that cultivates on the 1/4SDA flat board, disperse with 0.05% (W/V) tween-80, being configured to final concentration is 1 * 10
8The suspension-s of individual spore/ml.Adopt the topical application rice leaf roller larvae, every examination worm inoculates 30 with micro-sampling device drop medicament 2 μ l, and raise under the condition of humidity 50-70% 28 ℃ of room temperatures the inoculation back, establishes 5 repetitions, is blank with the clear water.Write down dead borer population, calculate survival rate, obtain fungi LT through the analysis of DPS2000 software statistics
50(LT50) and LT
90(terminal hour causes death).
As can beappreciated from fig. 3, green muscardine fungus CQMa421 bacterial strain has tangible insecticidal activity to Cnaphalocrocis medinali(rice leaf roller).5-7 days dead peak periods, inoculation back, the susceptible dead back of rice leaf roller larvae polypide is stiff, and preserving moisture to cultivate after 3 days grows white hypha, and polypide was covered with green spores in 5-7 days.Statistic analysis result shows that its CQMa421 bacterial strain is to the LT of rice leaf roller larvae
50(LT50) and LT
90(terminal hour causes death) was respectively 5.3 days and 9.1 days.
With concentration is 1 * 10
8The CQMa421 spore suspension of individual spore/ml is seeded in (100 μ l/ ware) on the 1/4SDA flat board, differing temps (15 ℃, 20 ℃; 24 ℃; 26 ℃, 28 ℃, 30 ℃; 37 ℃) culture condition cultivate down the sporulation quantity on the analytical unit area after 15 days, confirm that the CQMa421 bacterial strain produces spore the best under 26 ℃ of conditions.Preparing pH respectively is 4.5,5.0,5.5,6.0, and 6.5,7.0,7.5,8.0,8.5 1/4SDA is dull and stereotyped, measures sporulation quantity as stated above, confirms that the CQMa421 bacterial strain is to produce spore the best under 6.5 conditions at pH.
The optimization of embodiment 5 CQMa421 bacterial strain solid fermentation materials
Liquid culture CQMa421 mycelia is as ferment-seeded.Solid fermentation material configuration: the nutritive medium (pH 6.5) or the water that in cultivating solid materials rice or broken corn or wheat, add 15% or 20% or 25% or 30% (V/W) respectively; Nutritive medium is 1/4SDAY or 1/8SDA or 1/6SDA or 1/32SDAY.Pack in the gas-pervious Polypropylene Bag bag in two ends 121 ℃ of high pressure moist heat sterilization 30 min after mixing thoroughly into; Add by ratio (W/V) the inoculation green muscardine fungus CQMa421 bacterium liquid of cultivating material and bacterium liquid=10:1 behind the naturally cooling and mix thoroughly evenly, place 26 ℃ the static fermentation of the proving room drying (30 ℃-34 ℃) that heats up after 15 days, be lower than 5% to material moisture.Randomly draw the material fermented sample, get the sample about 5g behind the mixing, put into the 50ml triangular flask that 20mL 0.05%Tween-80 is housed, with the spore content of blood counting chamber mensuration product, press 200 * 10 behind the abundant mixing
8Individual/g converses the spore production rate of solid fermentation.With the spore production rate is to optimize index, and confirm that at last the solid fermentation material formula of CQMa421 is: rice adds the 1/16SDA nutritive medium (pH 6.5) of 25% (V/W).
The outdoor control effect of embodiment 6 CQMa421 bacterial strains
CQMa421 is seeded in liquid nutrient medium shake-flask culture 3 days (180rpm, 28 ℃) on the 1/4SDAY flat board; Bacterium liquid is seeded on the rice of sterilization, mixes back 26 ℃ thoroughly and cultivated 15 days down; Using salad oil to be mixed with final concentration behind the collection spore is 2 * 10
10Individual spore/ml oil-suspending agent.
Select rice leaf roller larvae that heavier rice field takes place, select 4 10m * 10m sub-district at random, 5 meters at interval.1 observation guard (1m * 2m, spacing is more than 1 meter) is placed in each sub-district, gathers larva and is inoculated in the guard (50-100 head/guard), adopts Mount Taishan-18 type power driven sprayer to carry out ultra-low volume after 24 hours and sprays CQMa421 spore oil suspension agent (2 * 10
13Individual spore/hectare).To spray clear water is blank.Investigate insect pest situation in the guard every day after the dispenser.
Show according to the outdoor result that give birth to survey: after the dispenser 12 days, green muscardine fungus CQMa421 bacterial strain reached (Fig. 4) more than 90% to the control effect of Cnaphalocrocis medinali(rice leaf roller).The one-tenth borer population (/ hundred clumps) of green muscardine fungus CQMa421 bacterial strain treatment group is about 1.5, and the one-tenth borer population of control group (/ hundred clumps) reaches 13.6.
Preparation pH initial value is 6.5 1/16SDA nutritive medium, in the ratio adding rice in 25% (V/W), and in the gas-pervious Polypropylene Bag in two ends of packing into after mixing thoroughly, 121 ℃ of high pressure moist heat sterilization 30 min; (bacterium liquid: rice, the ratio of V/W) inoculation green muscardine fungus CQMa421 bacterium liquid is also mixed thoroughly evenly, places 26 ℃ the static fermentation of the proving room drying (30 ℃-34 ℃) that heats up after 15 days, is lower than 5% to material moisture to press 1:10 behind the naturally cooling.Randomly draw 10 bags of fermentation material, get the sample about 5g behind every bag of mixing behind the mixing, put into the 50ml triangular flask that 20mL 0.05%Tween-80 is housed, with the spore content of blood counting chamber mensuration product, press 200 * 10 behind the abundant mixing
8Individual/g converses the spore production rate of solid fermentation.Replace the 1/16SDA nutritive medium to be made as contrast with clear water.
Table 1 CQMa421 bacterial strain solid fermentation spore production rate
Can find out from table 1: the product spore characteristic of CQMa421 bacterial strain is good, and the spore production rate in the control group is 4.5%, is 6.8% at the spore production rate on the rice material of optimizing, and contrast improves approximately 50%, reaches utmost point level of signification.
Sequence table
< 110>University Of Chongqing
< 120>a kind of desinsection green muscardine fungus bacterial strain and application thereof
<160> 1
<210> 1
<211> 547
<212> DNA
<213>The green muscardine fungus bacterial strain (
MetarhiziumanisopliaeVar.
Anisopliae) CQMa421
<400> 1
tgtttccgta ggtgaacctg cggagggatc attaccgagt tatccaactc ccaacccctg 60
tgaattatac ctttaattgt ?tgcttcggcg ggacttcgcg cccgccgggg acccaaacct?120
tctgaatttt ttaataagta tcttctgagt ggttaaaaaa atgaatcaaa actttcaaca 180
acggatctct tggttctggc atcgatgaag aacgcagcga aatgcgataa gtaatgtgaa?240
ttgcagaatt cagtgaatca tcgaatcttt ?gaacgcacat tgcgcccgtc agtattctgg 300
cgggcatgcc?tgttcgagcg tcattacgcc cctcaagtcc ?cctgtggact ?tggtgttggg?360
gatcggcgag?gctggttttc cagcgcagcc gtccctcaaa ?tcaattggcg gtctcgccgt?420
ggccctcctc?tgcgcagtag taaaacactc gcaacaggag cccggcgcgg tccactgccg480
taaaaccccc caacttttta tagttgacct cgaatcaggt ?aggactaccc gctgaactta?540
agcatat 547
Claims (3)
- The 1. little spore mutation of Metarhizium anisopliae ( Metarhizium anisopliaeVar. Anisopliae) bacterial strain CQMa421, it is characterized in that the deposit number of this bacterial strain is CGMCC NO.4609.
- 2. green muscardine fungus bacterial strain as claimed in claim 1 is characterized in that: the suitable product spore temperature of this bacterial strain is 26 ℃, and suitable product spore pH is 6.5.
- 3. the application of bacterial strain in Cnaphalocrocis medinali(rice leaf roller) is prevented and treated according to claim 1.
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| CN111557307A (en) * | 2020-05-27 | 2020-08-21 | 重庆谷百奥生物研究院有限公司 | Application of metarhizium anisopliae in preventing and treating greenhouse artichoke by using bees as carrier |
| CN111903713A (en) * | 2020-09-16 | 2020-11-10 | 重庆谷百奥生物研究院有限公司 | Tank-mixing auxiliary agent and preparation method thereof |
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| CN113289305A (en) * | 2021-05-24 | 2021-08-24 | 安徽农业大学 | Method for reducing pesticide residue and jointly killing insects by using metarhizium anisopliae |
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