CN103087924A

CN103087924A - Entomogenous fungus Nomuraearileyi, as well as culture method and application thereof

Info

Publication number: CN103087924A
Application number: CN2012104895577A
Authority: CN
Inventors: 任应党; 崔筱; 张书杰; 刘新涛; 王刚; 刘玉霞; 赵辉; 刘红彦
Original assignee: Henan Academy of Agricultural Sciences
Current assignee: Henan Academy of Agricultural Sciences
Priority date: 2012-11-27
Filing date: 2012-11-27
Publication date: 2013-05-08

Abstract

The invention relates to entomogenous fungus Nomuraearileyi, as well as a culture method and an application thereof. The collection number of the Nomuraearileyi Nr1001 is CGMCCNO. 6387. A microbial agent is formed by mixing a Nomuraearileyi Nr1001 fermentation solution, a surfactant and an anti-corrosion agent. A screened Nr1001 strain has the advantages of broad insecticidal spectrum and good insecticidal effect and stronger dissemination against lepidoptera noctuidae insects, such as larvae of cotton bollworm, asparagus caterpillar, prodenia litura, three-spotted plusia, soybean looper, oriental tobacco budworm and the like, and can prevent the prevalence of field pests and plant diseases, effectively reduce the population density of the pests and realize a better pest control effect.

Description

Entomogenous fungi Nomuraea rileyi and cultural method thereof and application

Technical field

The present invention relates to the biological control field, be specifically related to a kind of disinsection fungal-Nomuraea rileyi and cultural method thereof and application.

Background technology

Compare with other insecticidal microorganism, fungi has its peculiar advantage: tagging property, popularity, environmentally safe and be difficult for developing immunity to drugs; Cultivate and produce simply, industrialization is easy; The control of the subterranean pest-insect that can't prevent and treat at present the Bt insecticides, trunk borer, sucking pest has special efficacy etc., and therefore, the research and development of fungus insecticide have caused that the researchist pays close attention to widely and payes attention to.

The Nomuraea rileyi [ Nomuraea rileyi(Farlow) Samson] be a kind of widely distributed insect pathogenic fungus, parasitic 40 various insects of energy, particularly the virulence to lepidopteran noctuidae pests larva is stronger, can cause the disease popularity of the various pests such as bollworm, prodenia litura, beet armyworm, Anticarsia, soybean noctuid in the field, pest population there is certain control action kou, after the separated evaluation of this bacterium, paid attention to by the various countries scientist gradually, have higher value of exploiting and utilizing.

But not only there is obvious difference in the different Nomuraea rileyi strain of originating at aspects such as colony growth amount, sporulation quantity and spore germination rates, and its insecticidal effect also differs widely, yet sporulation quantity, spore germination rate, insecticidal effect etc. are to affect the important factor that Nomuraea rileyi strain is used in agriculture biological and ecological methods to prevent plant disease, pests, and erosion.

Summary of the invention

The technical problem to be solved in the present invention is to provide a kind of insecticidal effect good entomogenous fungi Nomuraea rileyi, and the Nomuraea rileyi that a kind of sporulation quantity is large and spore germination rate is high cultural method is provided.

For solving the problems of the technologies described above, the technical solution used in the present invention is:

A kind of Nomuraea rileyi ( Nomuraea rileyi) Nr1001, its deposit number is CGMCC No. 6387.

Utilize the living sporogenic method in above-mentioned entomogenous fungi Nomuraea rileyi, comprise the following steps:

(1) test tube strains preparation: the Nr1001 inoculation to test tube SMAY slant medium, is cultivated under 26～28 ℃ and obtained test tube strains in 144 hours;

(2) seed liquor preparation: get slant strains, 0.05% (v/v) Tween-80 of the sterilization of often drawing rinses and makes spore liquid, and being inoculated into 250mL triangular flask to the final concentration that 50mL SMY is housed is 1 * 10 ⁶Spore/mL cultivated 144 hours in 26 ℃, 180 r/min shaking tables, and is standby as liquid seeds liquid;

(3) culture medium preparation :Take a certain amount of substrate material, and enter distilled water by the solid-to-liquid ratio of 2g:1ml in substrate material, cooling after autoclaving, and add the yeast powder of maltose and 1～3% by 4～8% of substrate material weight, culture medium, described substrate material is at least a in wheat, soybean, rice, wheat bran, and described soybean and wheat adding distil water soak and carry out autoclaving at least after 12 hours again and process;

(4) gained matrix of upper step is smashed coagulated lumps, and access wherein step (2) gained seed liquor by the inoculum size of 1ml:12g, and fully shake up, isolate spore after cultivating 14d under following culture condition: 25～30 ℃ of culture temperature, 95% atmospheric moisture, pH6～8,8L illumination.

In described step (3), best substrate material is soybean substrate, after immersion, autoclaving, adds maltose by 6% of soybean substrate weight.

Seed liquor is inoculated on soybean substrate can carries out large-scale production; soybean substrate is prepared as follows: the 60g soybean adds the 30ml water soaking and spends the night; smash rear sterilization; add 6% maltose and 2% yeast powder; the above-mentioned seed liquor of access 5ml fully shakes up, and is placed under 25 ℃ and cultivates 14 days; leavened prod normal temperature dries, and namely makes spore powder after pulverizing.

Above-mentioned biotechnological formulation can be used by the method for spraying, can use separately, also can mix use with biotic pesticide, plant modifying agents such as muscardine, green muscardine fungus, Bacillus thuringiensiss.Generally sprayed to 3 ages in the larva hatching.

The present invention has actively useful effect:

Filter out N.rileyi1001 bacterial strain good disinsection effect, indoor Nr1001 bacterial strain reaches more than 70% the insecticidal effect of bollworm third-instar larvae; The effect of the insects such as field control bollworm, beet armyworm reaches more than 90%, compares with other Nomuraea rileyi strains of reporting before, no matter on sporulation quantity or insecticidal effect, certain advantage is arranged; And the insecticidal spectrum of this bacterial strain is wide, and lepidopteran Noctuidae larva such as bollworm, beet armyworm, prodenia litura, Anticarsia, soybean noctuid etc. are all had stronger infection effect, can cause the Field Pests big area popular.This shows, the Nr1001 bacterial strain has insecticidal effect preferably, has suitability for industrialized production prospect preferably.

The preparation method of biotechnological formulation of the present invention is simple, has filtered out industrial large scale culturing substratum with low cost, greatly reduces production cost, is conducive to the application popularization of this biotechnological formulation.

Nomuraea rileyi of the present invention ( Nomuraea rileyi) Nr1001, be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center on July 31st, 2012, it is referred to as CGMCC, the address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, this Nomuraea rileyi ( Nomuraea rileyi) deposit number of Nr1001 is CGMCC NO.6387.

Description of drawings

The affect trend map of Fig. 1 different humidity on the Nr1001 colony growth;

The affect trend map of Fig. 2 different humidity on the Nr1001 spore germination.

Embodiment

Further set forth the present invention below in conjunction with specific embodiment.

Embodiment 1 strain separating

The collection of bacterial strain: 2009-2010, in beach district, Wen County Junxia WANG sample plot, the Wen County institute of agricultural sciences experimental field, Henan Academy of Agricultural Sciences modern agriculture science and technology experiment and demonstration base (is called for short: glutinous rehmannia base, academy of agricultural sciences, Henan), peanut, the Soybean Field collection bombys batryticatus of falling ill.

The separation of bacterial strain: adopt conventional entomogenous fungi method for tissue separation.with flushing with clean water polypide surface, 75% alcohol-pickled 2s, aseptic water washing 2 times, then scalper and the tweezers with sterilization are cut into small pieces polypide, place it in 75% alcohol and soak 1.5min, sterilized water soaks 2 times, each 10s, with aseptic pincet, the polypide piece is received the PDA planar surface again, 28 ℃ of moisturizings are cultivated, when long apparent in view of the mycelia on worm piece surface, the mycelia of provoking the polypide surface with toothpick is transferred on the PDA flat board of the penicillin G (50 μ g/mL) that is added with 100 μ L and Streptomycin sulphate (50 μ g/mL), after 20 d, switching is on the PDA inclined-plane, put 4 ℃ of Refrigerator stores standby.

Table 1 separates the bacterial strain that obtains

Numbering	Acquisition time	Gather ground	Herborization	The bombys batryticatus kind of separating
					Nr1001	08/20/10	Base, academy of agricultural sciences, Henan	Peanut	The lepidopteran Noctuidae
Nr1002	08/20/10	Base, academy of agricultural sciences, Henan	Peanut	The lepidopteran Noctuidae
					Nr1003	09/25/09	Base, academy of agricultural sciences, Henan	Peanut	The lepidopteran Noctuidae
Nr1004	09/25/09	Base, academy of agricultural sciences, Henan	Peanut	The lepidopteran Noctuidae
					Nr1005	09/25/09	Base, academy of agricultural sciences, Henan	Soybean	The lepidopteran Noctuidae
Nr1006	09/28/09	Base, academy of agricultural sciences, Henan	Soybean	The lepidopteran Noctuidae
					Nr1007	05/09/09	The Wen County institute of agricultural sciences experimental field	Glutinous rehmannia	The lepidopteran Noctuidae
Nr1008	05/09/09	Beach district, Wen County Junxia WANG sample plot	Glutinous rehmannia	The lepidopteran Noctuidae

This example is divided into from the bacterial strain that obtains 8 strain preliminary screening, to produce the spore ability relatively poor due to Nr1006, Nr1007, Nr1008 bacterial strain, so mainly the biological and ecological methods to prevent plant disease, pests, and erosion ability of Nr1001, Nr1002, Nr1003, Nr1004, Nr1005 bacterial strain is measured, the concentration of each spore suspension is about 2 * 10 ⁸Individual spore/mL filters out the Nr1001 bacterial strain that bollworm is had better prevention effect, the results are shown in Table 2.

Table 2 different strains biological and ecological methods to prevent plant disease, pests, and erosion test result

Bacterial strain	For the examination borer population	Infect borer population	Infection rate (%)
				Nr1001	20	5	25
Nr1002	20	4	20
				Nr1003	20	0	0
Nr1004	20	2	10
				Nr1005	20	5	25

By the biological and ecological methods to prevent plant disease, pests, and erosion result as can be known, the infection ability of Nr1001 and Nr1005 bacterial strain is higher, but the Nr1001 bacterial strain is than the product spore Li Genggao of Nr1005, therefore, selected Nr1001 bacterial strain (gathers people: Ren Yingdang, acquisition time: on August 20th, 2010) as the object of after this studying.

Embodiment 2 identification of strains

To the bacterial strain Nr1001 that separates in embodiment 1, by Observation of biological characteristics, carry out further identification of morphology, method is as follows:

(1) form and cultural characteristic: cultivate 10d for 28 ℃ on the PDA substratum, observe its colony characteristics and color etc., with observation by light microscope thalline feature.

(2) ITS sequential analysis: cultured mycelia on scraping PDA flat board, extract according to a conventional method strain gene group DNA, the synthetic universal PC R primer that is used for amplification fungi ITS sequence increases, and the purpose fragment utilizes the method for molecular biology routine to carry out Cloning and sequencing.By BLAST (http: // www. ncbi. nlm. nih.gov/) sequencing result is analyzed, carried out homology relatively with the sequence in the GenBank database, determine the classification position of this bacterial strain.

Above experimental result records as follows:

1. this bacterium of morphological specificity is in cultivating under 28 ℃, and microscopically is observed, conidium ovalize or the round shape of this bacterial strain, one end is point slightly, and an end is slightly blunt, smooth surface, between some spore, connection is arranged, light green, the conidium size is (4.5-5.2) * (2.8-3.5) μ m.Vegetative hyphae is smooth, separates, and is transparent or slightly be with color, and hyphal diameter 2 μ m-3 μ m are without coremium.

2. this bacterial strain of cultural characteristic is on the PDA substratum, bacterium colony is fine and close down shape, and is white at the beginning, colony growthing slow, diameter just reached 0.6～1.8cm in one month, at the colony diameter 2.6cm of SMAY substratum after upper 10 day, approximately produce spore after 5-7 days, after the product spore, bacterium colony is yellow-green colour or green, approximately after 10-15 days, the mycelia that spore can newly be sprouted covers, without musty and exudate, and the colourless or little Huang in the back side.

3.ITS sequential analysis is through order-checking, this bacterial strain ITS sequence length is 619bp, carries out sequence alignment in the GenBank database of NCBI website, and result shows, the bacterial strain that detect and Nomuraea rileyiMAFF 830007(GenBank sequence number: AB268359.1) homology is 99%; With two other Nomuraea rileyiThe sequence of bacterial strain (GenBank sequence number: FJ824809.1, AB100361.1) also only has the difference of 3 bases, judge thus this bacterial strain as the Nomuraea rileyi ( Nomuraea rileyi).

ITS sequence 619bp

Figure 2012104895577100002DEST_PATH_IMAGE002

The biological characteristics of embodiment 3 bacterial strains

The bacterial strain of identifying in example 2 is carried out the research of biological characteristics, and method is as follows:

(1) nutritional condition Nomuraea rileyi Nr1001 strain growth and the impact of producing spore are cultivated the solid plate substratum access of different C source, N source, C/N, trace element, VITAMIN on the PDA substratum Nr1001 bacterial strain mycelia piece and (concentration is 1 * 10 with the spore suspension of the 50ml liquid bulk substratum access 100 μ L of different C source, N source, C/N, trace element, VITAMIN ⁷About individual/ml), cultivate after ten days, measure it at the colony diameter under the solid culture condition, sporulation quantity and the mycelia dry weight under the liquid culture condition, with DPS and SPSS software, the experimental result of record is analyzed.

(2) culture condition arranges different temperature, pH, illumination as culture condition to the impact of Nomuraea rileyi Nr1001 strain growth and sporulation quantity, surveys colony diameter and the sporulation quantity of Nr1001 bacterial strain on the SMAY substratum; Different humidity conditions is set, surveys the spore germination rate of Nr1001 bacterial strain on the SMAY substratum; Different PH, inoculum size and shaking speed are set, survey its mycelia dry weight under the liquid culture condition, with DPS and SPSS software, the experimental result of record is analyzed.

Above experimental result records following (seeing Table 3～7):

1. the impact of nutritional condition on Nomuraea rileyi Nr1001 strain growth and product spore

The different carbon sources of table 3 are on Nr1001 strain growth and the impact of producing spore

(through the Duncan multiple comparisons, different capitalization representatives is remarkable at P＜0.01 level error heteropole; Different lowercase representatives is remarkable at P＜0.05 level difference)

The impact of table 4 different nitrogen sources on Nr1001 growth and product spore

The impact of the different carbon-nitrogen ratios of table 5 on Nr1001 growth and product spore

The impact of the different trace elements of table 6 on Nr1001 growth and product spore

Trace element	Colony diameter (mm)	Regression equation	Average sporulation quantity (* 10 ⁶Individual spore/cm ²）	Mycelia weight (g/50mL)
					Iron	35.4±0.68Aa	Y=1.9X＋2.767	0.47±0.09Bb	0.13±0.01Aa
Zinc	34.6±0.24A a	Y=1.84X＋3.48	1.61±0.05Bb	0.18±0.01Aa
					Manganese	34.4±0.68A a	Y=1.78X＋4.34	75.85±20.6Aa	0.13±0.01Aa
Copper	29.0±2.07B b	Y=1.93X＋0.07	0.97±0.24Bb	0.05±0.00Bb
					Contrast	34.6±0.87A a	Y=1.83X＋3.9	3.20±1.68Bb	0.13±0.03Aa

The impact of the different VITAMIN of table 7 on Nr1001 strain growth and product spore

VITAMIN	Colony diameter (mm)	Regression equation	Average sporulation quantity (* 10 ⁶Individual spore/cm ²）	Mycelia weight (g/50mL)
					Pantothenic acid VB ₃	40.0±0.87Aa	Y=1.92X＋2.86	51.00±3.71Aab	0.12±0.01Bbcd
Vitamin H VB ₇	37.8±2.32ABab	Y=2.11X＋3.94	2.80±0.99Ab	0.14±0.00Abc
					Folic acid	36.6±0.93ABCabc	Y=1.97X＋5.2	22.83±4.65Aab	0.14±0.01ABabc
V _C	35.8±0.8ABCabc	Y=1.87X＋4.3	8.07±2.76Ab	0.12±0.02ABbc
					VB ₂	34.6±1.57ABCbcd	Y=1.61X＋5.94	0.00±0.00Ab	0.04±0.00Cd
Nicotinic acid VB ₅	34.4±0.87ABCbcd	Y=2.36X＋1.46	0.22±0.10Ab	0.11±0.02Bbc
					VB ₆	33.0±3.18ABCDbcd	Y=1.69X＋4.12	0.93±0.13Ab	0.11±0.01Bbc
Choline chloride 60	32.2±1.16BCDcd	Y=1.71X＋3.28	0.48±0.13Ab	0.15±0.02ABab
					VB ₁	32.0±0.41BCDcd	Y=1.77X＋3.07	0.48±0.02Ab	0.09±0.01BCc
Thioctic Acid	29.7±0.33CDde	Y=1.64X＋1.94	2.84±0.07Ab	0.19±0.01Aa
					VB ₁₂	27.0±2.51De	Y=1.41X＋3.28	75.42±4.20Aa	0.10±0.00Bbc
Contrast	34.6±0.8ABCbcd	Y=1.83X＋3.9	3.20±1.68Ab	0.13±0.03ABbc

By above result as can be known: in the various substratum of different carbon sources, nitrogenous source, carbon-nitrogen ratio, trace element and VITAMIN, Nomuraea rileyi Nr1001 strain growth situation significant difference.Wherein, carbon source is that in the substratum of disaccharides (lactose, sucrose, maltose), colony diameter is larger, and when carbon source was monose or polysaccharide, colony growth was relatively poor; In the substratum of measuring sporulation quantity, produce spore best when carbon source is raffinose, but raffinose is expensive, is not suitable for large-scale production, so choose the maltose that sporulation quantity is only second to raffinose in the relevant mass-produced research of carrying out afterwards.When adding organonitrogen to substratum, can obviously promote the increase of bacterium colony expansion, mycelium growth and sporulation amount, show that organonitrogen is an important factor of this bacteria growing, the organonitrogen substratum is more suitable for the Nomuraea rileyi growth.Add trace element manganese, iron and vitamin V B ₃, VB ₁₂Deng growth and product spore to the Nomuraea rileyi, promoter action is arranged.But research find the Nomuraea rileyi different steps to nutritional condition require inconsistent, the substratum of suitable mycelial growth not necessarily can produce spore by suitable its, be that nourish and grow in the Nomuraea rileyi and reproductive growth is two relatively independent processes, therefore, be according to the different nutritional condition of purpose different choice of research.

2. the impact of culture condition on Nomuraea rileyi Nr1001 strain growth and sporulation quantity

The impact of table 8 differing temps on colony growth and sporulation quantity

Temperature	Colony diameter	The growth regression equation	Growth velocity	Relation conefficient	Sporulation quantity (10 ⁷Individual spore cm ^-1)
						40	6.0± 0.0 0De	-	-	?	0.01±0.00Bb
35	6.0± 0.0 0De	-	-	?	0.01±0.00Bb
						30	24.4± 0.06Aa	Y=2.74+1.36X	1.36	0.997	0.08±0.03Bb
25	24.2± 0.05Aa	Y=4.05+1.24X	1.24	0.999	10.43±0.28Aa
						20	20.2± 0.02Bb	Y=1.32+1.15X	1.15	0.996	0.72±0.32Bb
15	18.2± 0.05Cc	Y=3.67+0.93X	0.93	0.993	0.05±0.05Bb
						10	7.6± 0.02Dd	Y=5.53+0.13X	0.13	0.894	0.03±0.01Bb
5	7.0± 0.0 0Dde	Y=5.37+0.11X	0.11	0.923	0.01±0.00Bb

Annotate: different capitalization representatives is remarkable at P＜0.01 level error heteropole; Different lowercase representatives is at the aobvious significant difference of P＜0.05 level difference

The impact of the different pH of table 9 on colony growth and product spore

pH	Colony diameter	Growth regression equation journey	Growth velocity	Relation conefficient	Sporulation quantity (10 ⁷Individual spore cm ^-1)
						5	11.60±0.02Dd	Y=4.55+0.41X	0.41	0.975	5.50±0.43Bb
6	26.20±0.04Bb	Y=1.08+1.03X	1.03	0.983	55.00±0.38Aa
						7	29.40±0.05Aa	Y=-3.44+1.83X	1.83	0.983	58.17±13.64Aa
8	18.40±0.03Cc	Y=-3.22+1.76X	1.76	0.988	14.50±6.02Bb
						9	7.60±0.03Ee	Y=3.04+0.64X	0.64	0.998	0.30±0.05Bb
10	7.00±0.00Ee	Y=5.41+0.11X	0.11	0.828	0.03±0.01Bb

Annotate: different capitalization representatives is remarkable at P＜0.01 level error heteropole; Different lowercase representatives is remarkable at P＜0.05 level difference

The impact of table 10 different light on colony growth and product spore

Illumination	Colony diameter (mm)	The growth regression equation	Growth velocity (mm/d)	Relation conefficient	Sporulation quantity (10 ⁷Individual spore cm ^-1)
						0L	33.7±0.06Ab	Y=-4.02+2.31X	2.31	0.993	1.48±0.3Dd
4L	26.3±0.08Cd	Y=-2.40+1.82X	1.82	0.994	80.33±13.69Bb
						8L	36.4±0.03Aa	Y=3.09+2.24X	2.24	0.997	96.67±23.51Aa
12L	30.0±0.03Bc	Y=0.11+1.84X	1.84	0.998	8.48±1.28Cc
						16L	24.0±0.03CDe	Y=4.93+1.1X	1.1	0.987	1.10±0.5Dd
20L	23.3±0.03De	Y=4.89+1.17X	1.17	0.996	0.42±0.01Dd
						24L	13.0±0.03Ef	Y=7.96+0.61X	0.61	0.971	0.25±0.3Dd

The impact of table 11 pH on Nr1001 bacterial strain mycelial growth

Figure 2012104895577100002DEST_PATH_IMAGE010

The impact of table 12 inoculum size on the Nr1001 mycelial growth

Figure 2012104895577100002DEST_PATH_IMAGE012

The impact of table 13 shaking speed on the Nr1001 mycelial growth

Figure 2012104895577100002DEST_PATH_IMAGE014

Cultural characters to Nomuraea rileyi Nr1001 bacterial strain studies show that (referring to table 8～13, Fig. 1, Fig. 2), and the optimum growth temperature of this bacterial strain is 25 ~ 30 ℃, and best product spore temperature is 25 ℃, and the highest sporulation quantity reaches 1.04 * 10 ⁸Individual spore mL ^-1, growth hardly under 5 ℃, 10 ℃, 35 ℃, 40 ℃ conditions, the result of observing with state of nature is consistent.This bacterium all can grow under the condition of pH5-9, suitable its growth of neutral slant acidity condition and product spore, and the suitable optimal pH that it produces spore is 7.Long or too short its mycelium growth and sporulation that all affects of light application time, its growth of 8L photo-irradiation treatment optimum and product spore, that sporulation quantity is minimum is 24L, is 0.25 * 10 ⁶Individual spore mL ^-1The colony diameter minimum be 24L, be 13mm, the environment of high humidity is conducive to Nomuraea rileyi Nr1001 strain growth and spore germination, Nomuraea rileyi colony growth under 95% atmospheric moisture is the fastest, spore germination rate is the highest, so the Nomuraea rileyi is suitable its growth and sprouting of spore under high humidity environment.

The indoor virulence test of embodiment 4 Nomuraea rileyi to the bollworm third-instar larvae

(1) for the examination insect

Be bollworm 3 instar larvaes for the examination insect, the worm source comes from the bollworm mature larva that base, Yuanyang, academy of agricultural sciences, Henan Province collects, and indoor feeding turns into adult to pupating, and female male imago pairing is laid eggs, and the access artificial diet were raised to 3 ages after the ovum hatching.

(2) experiment is processed

Under 26 ℃ of conditions, the Nr1001 bacterial strain is cultivated 8 d on the PDA slant medium, with 0.05% aseptic tween-80 aqueous suspension spore, diluted in proportion after producing spore fully, measure through the blood counting plate, the spore suspension of this bacterium is made into concentration is respectively 1.14 * 10 ⁹Individual spore mL ^-1 ,1.14 * 10 ⁸Individual spore mL ^-1 ,1.14 * 10 ⁷Individual spore mL ^-1 ,1.14 * 10 ⁶Individual spore mL ^-1, 1.14 * 10 ⁵Individual spore mL ^-1Adopt the spray method inoculation method that the Nr1001 spore suspension of each concentration of 5ml is sprayed in 3 instar bollworm grub body surfaces with aseptic shower nozzle, allow its 10s that creeps, then it is changed in the aseptic dactylethrae that is added with artificial diet, each dactylethrae connects 1 larva, 20 of every processing, 3 repetitions, 0.05% (volume ratio) tween 80 water aseptic with 5ml replaces bacterium liquid in contrast.

(3) data processing

With LC ₅₀, cumulative mortality, correction cumulative mortality and bombys batryticatus rate be as the index of insect Virulent Analysis.With time (d), bacterial concentration (spore mL ^-1) logarithmic value be X, the probit value of mortality ratio is Y, adopts probit analysis, uses SPSS software, draws virulence regression equation and median lethal concentration(LC﹠-{50}) (LC ₅₀).

(4) experimental result

The Nr1001 spore suspension of different concns has different pathogenic effects (referring to table 14) to bollworm 3 instar larvaes, and spore suspension concentration is higher, and the mortality ratio of insect is higher.Wherein, concentration is 1.14 * 10 ⁹Spore mL ^-1The corrected mortality of processing is the highest, reaches 70.53%, with processing and the contrast of other concentration, utmost point significant difference is arranged, and concentration is 1.14 * 10 ⁸Spore mL ^-1 ,1.14 * 10 ⁷Spore mL ^-1 ,1.14 * 10 ⁶Spore mL ^-1, 1.14 * 10 ⁵Spore mL ^-1The time corrected mortality be respectively 46.4%, 34.21%, 22.19%, 19.12%, but concentration is 1.14 * 10 ⁶Spore mL ^-1With 1.14 * 10 ⁵Spore mL ^-1The time and contrast difference not remarkable.The concentration that therefore, can suitably improve its spore suspension improves its insecticidal effect.

The mortality ratio of getting in 13d from each group of test performs an analysis, and mortality ratio is converted to probit value, and the bacterium amount is converted to logarithmic value.With bacterial concentration (spore mL ^-1) logarithmic value be X, the probit value of mortality ratio is Y, adopts probit analysis, uses SPSS software, draws virulence regression equation, median lethal concentration(LC﹠-{50}) (LC ₅₀).Regression equation is: Y=-3.088+0.383X, LC ₅₀Be 1.163 * 10 ⁸Spore mL ^-1

The pathogenic effect of the Nr1001 spore suspension of table 14 different concns to the bollworm third-instar larvae

^*Different letter representation significant differences (P＜0.05)

*Different?lowercase?alphabet?mean?the?difference?at?0.05?level .

Embodiment 5 Nomuraea rileyi field control effects

(1) experimental field situation and time

Situation experimental field: test is located in Henan Academy of Agricultural Sciences's modern agriculture test and Demonstration Base (Yuanyang) Soybean Field to be carried out, the causing harm seriously of the lepidopteran nocturnal moth class insects such as this piece ground bollworm and beet armyworm.

Experimental period: on July 22nd, 2011

(2) test medicine:

Figure 2012104895577100002DEST_PATH_IMAGE018

Bacillus thuringiensis (800IU/mg), Yixing, Jiangsu Province emerging agriculture heavy chemicals company limited, commercially available; Rynaxypyr (the wide 200g/L of health), Shanghai Dubang Agricultural Chemicals Co., Ltd., commercially available;

Figure 2012104895577100002DEST_PATH_IMAGE022

Worm hydrazides (200g/L), the green prosperous development in science and technology of Beijing North agriculture company limited, commercially available;

Figure 2012104895577100002DEST_PATH_IMAGE024

The Nr1001 spore suspension, the fermentation of this laboratory culture; Chlorpyrifos ec (480 grams/L), Shandong Rupont Chemical Co., Ltd., commercially available;

Figure 2012104895577100002DEST_PATH_IMAGE028

Veratrine (0.5%), the Handan City builds magnificent plant pesticide factory, and is commercially available;

Figure 2012104895577100002DEST_PATH_IMAGE030

The high cryptogam of beauveria bassiana (40,000,000,000 spores/g), Jiangxi Tianren Eco-Industrial Co., Ltd, commercially available;

Figure 2012104895577100002DEST_PATH_IMAGE032

Lambda-cyhalothrin (4.5%), Henan Province's Planck biochemical Industrial Co., Ltd, commercially available;

Figure 2012104895577100002DEST_PATH_IMAGE034

Emamectin-benzoate (2%), Beijing Qi Ke insecticide factory of middle peasant mcroorganism Science and Technology Co., Ltd., commercially available.With fresh water spraying in contrast.

(3) test is processed: be Su Yunjin with concentration: 120g/15kg water, Rynaxypyr (health is wide): 6ml/15kg water, worm hydrazides: 60g/15kg water, Nr1001:5.5 * 10 ⁷Spore mL ^-1, Chlorpyrifos 94: 0.8ml/15kg water, veratrine: 6ml/15kg water, the high cryptogam of beauveria bassiana: 15g/15kg water, effective cypermethrin: 30/15kg water, emamectin-benzoate: the contrast of 0.8ml/15 kg of water, clear water amounts to 10 processing, every residential quarter spraying liquid amount 5kg.Experimental plot random alignment, the area of each residential quarter are 40m ², 3 repetitions are set, 3m blank (not doing any processing) all is set as the protective belt between repetition and between the residential quarter.

(4) control time and method: time: carry out for the first time insect radix investigation before medication, adopt 5 point samplings to investigate the borer population of 3d, 7d after borer population before each residential quarter dispenser and dispenser, 14d, and calculate prevention effect.Borer population * 100 of living before insect population decline rate (%)=(borer population of living after the borer population-dispenser of living before dispenser)/dispenser, prevention effect (%)=(processing insect population decline rate-check plot insect population decline rate)/(1-check plot insect population decline rate) * 100.

(5) experimental result

9 kinds of chemicals treatment for examination all have certain prevention effect to nocturnal moth class larvas such as Soybean Field bollworm, beet armyworms, referring to table 15.Wherein, in chemical pesticide, the wide prevention effect of health is best, and has long-lasting preferably, after medicine, the preventive effect of 3d is that preventive effect after 63.40%, 14 day can reach 100%, and preventive effect and the persistence of Chlorpyrifos 94 and efficient effective cypermethrin are also better, with the wide otherness of health not significantly but with contrast, utmost point significant difference is arranged, prevention effect and the long-lasting of first dimension salt, veratrine and worm hydrazides are all relatively poor; In biological pesticide, the prevention effect of the Nomuraea rileyi Nr1001 spore suspension of this laboratory culture is best, and long-lasting is also better, after medicine, the preventive effect of 3d is 55.18%, preventive effect after 14 days can reach 98.77%, and long-lasting is far above the high cryptogam of commercially available muscardine and contrast medicament first dimension salt, veratrine and worm hydrazides etc.Generally speaking, though the preventive effect of Nr1001 bacterial strain is wide a little less than the chemical agent health, variance analysis is difference significant difference not with it, this shows, the Nr1001 bacterial strain has insecticidal effect preferably, has development prospect preferably.

Table 15 field test results

Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements, all belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.

Claims

An entomogenous fungi Nomuraea rileyi ( Nomuraea rileyi) the Nr1001 bacterial strain, its deposit number is CGMCC NO.6387.
2. the spore powder of utilizing the described entomogenous fungi of claim 1 Nomuraea rileyi to make.
3. claim 1 described entomogenous fungi Nomuraea rileyi or the described spore powder of claim 2 application in the control of lepidopteran noctuidae pests.
4. utilize the living sporogenic method in the described entomogenous fungi of claim 1 Nomuraea rileyi, comprise the following steps:

(1) test tube strains preparation: the Nr1001 inoculation to test tube SMAY slant medium, is cultivated under 26～28 ℃ and obtained test tube strains in 144 hours;

(2) seed liquor preparation: get slant strains, the volume percent of the sterilization of often drawing is that 0.05% Tween-80 rinses and makes spore liquid, and being inoculated into 250mL triangular flask to the final concentration that 50mL SMY is housed is 1 * 10 ⁶Spore/mL cultivated 144 hours in 26 ℃, 180 r/min shaking tables, and is standby as liquid seeds liquid;

(3) culture medium preparation :Take a certain amount of substrate material, and add distilled water by the solid-to-liquid ratio of 2g:1ml in substrate material, cooling after autoclaving, and add the yeast powder of maltose and 1～3% by 4～8% of substrate material weight, culture medium, described substrate material is at least a in wheat, soybean, rice, wheat bran, and described soybean and wheat adding distil water soak and carry out autoclaving at least after 12 hours again and process;

(4) gained matrix of upper step is smashed coagulated lumps, and access wherein step (2) gained seed liquor by the inoculum size of 1ml:12g, and fully shake up, isolate spore after cultivating 14d under following culture condition: 25～30 ℃ of culture temperature, 95% atmospheric moisture, pH6～8,8L illumination.
5. sporogenic method is given birth in the entomogenous fungi Nomuraea rileyi according to claim 4, it is characterized in that, in described step (3), described substrate material is soybean substrate, after immersion, autoclaving, adds maltose by 6% of soybean substrate weight.