CN104073440A - Paecilomyces fumosoroseus and application thereof - Google Patents
Paecilomyces fumosoroseus and application thereof Download PDFInfo
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Abstract
The invention discloses paecilomyces fumosoroseus and application thereof. The paecilomyces fumosoroseus TRCC22001 is collected in the China General Microbiological Culture Collection Center (CGMCC) with the collection number of CGMCC No.7993. The paecilomyces fumosoroseus can be used for preventing and treating piercing-sucking mouthpart pests.
Description
Technical field
The present invention relates to a kind of paecilomyces fumosoroseus (Paecilomyces fumosoroseus), also relate to the preparation method of a kind of this paecilomyces fumosoroseus (Paecilomyces fumosoroseus) simultaneously, and in the application preventing and treating in sucking pest.
Background technology
Along with the foundation that sustainable agriculture is seen, exploring nuisanceless, free of contamination bio-control method has become the important topic in the Plant diseases comprehensive regulation, is day by day subject to the attention of countries in the world.
According to incompletely statistics, the entomogenous fungi approximately 100 of recording in the world belongs to kind more than 800.That has reported in China reaches 405 kinds, wherein 80 kinds of Cordyceps, and 10 kinds of the fungies of parasitic nematode, 215 kinds of the fungal species of parasitic insect, the novel species of report reaches 24 kinds.China since the fifties, approximately 20 kinds of the insect pathogenic fungus of Application and Development, what usable floor area was the widest is muscardine and green muscardine fungus.
Insect pathogenic fungus refer to those under host's normal physiological conditions directly in intrusive body, propagation and cause rapidly dead monoid.They are to absorb nutrient, the decomposition host tissue in hemolymph or to produce toxic metabolite product and kill insects.Causing in the pathogenic microorganism of insect transmission disease, caused that by fungi the situation of insect death is maximum, account for 60%.Meanwhile, insect pathogenic fungus is of a great variety, and population density is played to important regulating effect, so utilize insect pathogenic fungus species resource to carry out the important component part that " controlling worm with bacterium " should be biological control work.The pathogenesis of insect pathogenic fungus: insect pathogenic fungus again produces spore and is generally divided into 10 stages from spore germination to thalline on polypide: (1) Spore adhesion is in Cuticle; (2) spore germination; (3) penetrate epidermis; (4) mycelia is at blood Intracavity; (5) toxin produces; (6) host's death; (7) mycelia is invaded all organs of host; (8) mycelia passes epidermis; (9) produce spore; (10) infect the diffusion of unit.As long as completed front four-stage, invasion and infection to host are just completed; In the time that pathogenic fungi mycelia in vivo breeds in a large number, just can cause host's death.
Summary of the invention
The object of the present invention is to provide a kind of insect pathogenic fungus of preventing and treating sucking pest---paecilomyces fumosoroseus (Paecilomyces fumosoroseus).Its rDNA ITS1-5.8S-ITS2 sequence:
5’-TCTCCGTTGGTGAACCAGCGGAGGGATCATTAACGAGTTTTTTCAACTCCCTAACCCTTTGTGAACATACCTATCGTTGCTTCGGCGGACTCGCCCCGGCGTCCGGACGGCCCTGCGCCGCCCGCGACGGACCCAGGCGGCCGCCGGAGACCCACAAATTCTGTTTCTATCAGTCTTTCTGAATCCGCCGCAAGGCAAAACAAATGAATCAAAACTTTCAACAACGGATCTCTTGGTTCTGGCATCGATGAAGAGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCCGCCAGCATTCTGGCGGGCATGC?CTGTTCGAGCGTCATTTCAACCCTCGACACCTTCGGGGGAGTCGGCGTTGGGGACCGGCAGCATACCGCCGGCCCCGAAATACAGTGGCGGCCCGTCCGCGGCGACCTCTGCGTAGTACTCCAACGCGCACCGGGAACCCGACGCGGCCACGCCGAAACACCCAACTTCTGAACGTTGACCTCGGATCAGGTAGGACTACCCGCTGAACTTAA-3’。
Meanwhile, the present invention also aims to provide spawn culture method and the solid culture method of a kind of paecilomyces fumosoroseus (Paecilomyces fumosoroseus).
Further, the object of the present invention is to provide the application of a kind of paecilomyces fumosoroseus (Paecilomyces fumosoroseus).To achieve these goals, technical scheme of the present invention has adopted a kind of paecilomyces fumosoroseus (Paecilomyces fumosoroseus), and its preserving number is CGMCC NO.7993.
Meanwhile, technical scheme of the present invention has adopted the spawn culture method of a kind of paecilomyces fumosoroseus (Paecilomyces fumosoroseus), (1) inclined-plane seed culture.Slant strains substratum is Improvement type PDA:20% potato, 2% sucrose, 0.2% peptone, 0.1%K
2hPO
43H2O, 0.3%NaNO
3, 0.05%MgSO
47H2O, 0.05%KCl, 0.01%FeSO
4, 2% agar, water 1000ml.121 degrees Celsius of high pressure steam sterilization 30min.After test tube inoculation, place 26 degrees Celsius of constant temperature culture 10d for subsequent use.(2) liquid seeds is cultivated.Substratum: Improvement type PDA (removing agar).Packed into 500ml triangular flask, every bottled 100ml, 121 degrees Celsius of high pressure steam sterilization 30min.Access test tube slant bacterial classification (every connects two bottles), places 26 degrees Celsius of cultivation 60h of shaking table (180 turn/min) for subsequent use.
Meanwhile, technical scheme of the present invention has adopted the solid culture method of a kind of paecilomyces fumosoroseus (Paecilomyces fumosoroseus): solid medium is 70% wheat bran+30% rice bran.Take raw material by solid medium proportioning, add 1.2 times of water and fully mix thoroughly, the rear 121 degrees Celsius of high pressure steam sterilization 30min that spend the night are for subsequent use.
Further, technical scheme of the present invention has adopted the application of a kind of paecilomyces fumosoroseus (Paecilomyces fumosoroseus) aspect control sucking pest.
Paecilomyces fumosoroseus (Paecilomyces fumosoroseus) involved in the present invention is preventing and treating in process small cabbage moth, paecilomyces fumosoroseus spore powder is 76.2% to the mortality ratio of diamondback moth larvae, paecilomyces fumosoroseus spore suspension is 59.8% to the mortality ratio of diamondback moth larvae, and paecilomyces fumosoroseus spore nutrient solution is 58.5% to the mortality ratio of diamondback moth larvae.
Paecilomyces fumosoroseus (Paecilomyces fumosoroseus) involved in the present invention is preventing and treating in process tea lesser leafhopper, and preventive effect reaches as high as 71.3%.
Bacterial strain of the present invention: paecilomyces fumosoroseus (Paecilomyces fumosoroseus) preservation and China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), preservation address is: China. Beijing. and No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Microbe Inst., Chinese Academy of Sciences, preservation date is on 08 13rd, 2013, and its deposit number is CGMCC No.7993.
Embodiment:
The qualification of embodiment 1TRCC22001 bacterial strain:
Molecular systematics qualification, DNA extraction method: adopt quartzite sand grind CTAB extraction method (Scott et al., 2000).
Genetic marker: rDNA ITS1-5.8S-ITS2, amplimer: ITS5, ITS4 (White et al., 1990).
Pcr amplification reaction condition: first 94 DEG C of heating make template DNA sex change for 3 minutes, then enter following temperature cycle: 94 DEG C of sex change 30 seconds, anneal 30 seconds for 50 DEG C, 72 DEG C are extended 45 seconds, carry out altogether 35 circulations, finally extend 5 minutes (Wang, 2012) at 72 DEG C.
PCR product detects and order-checking: PCR product and 100bp DNA ladder (MBI Fermentas) sepharose (agarose gel) with 2.0% in 0.5 × TBE electrophoretic buffer under 80V voltage electrophoresis 20 minutes, then be placed in the ethidium bromide (EB of 0.5 μ g/ml, ehidium bromide) dye 15 minutes in solution, under the UV-light of wavelength 365 and 254nm, detect product for obvious single band and carry out two-way straight-through order-checking with ABI3700 (Applied Biosystems) after by Qing Ke Bioisystech Co., Ltd purifying.
Data processing and statistical analysis: original series is with obtaining sequence accurately after biosoftware Bioedit7.0.9 (Hall, 1999) editor, and the pattern of downloading with GenBank becomes sequence data matrix with authoritative bacterial strain sequence set.For sequence matrix, under Bioedit7.0.9, be necessary human-edited and adjust the new matrix obtaining.By this MEGA5.0 (Tamura et al. for matrix, 2011) carry out neighbours' connection method (neighbor-joining, NJ) analyze, infer phylogenetic tree, and carry out the reliability of 1000 each branches of repeat assessment by the method for bootstrapping (bootstrap).
Bacterial strain phylogenetic tree is shown in accompanying drawing 1, accompanying drawing 2.Result shows that TRCC22001 bacterial strain and (Paecilomyces fumosoroseus) similarity are 98.90%, and its thalli morphology and physiological and biochemical index meet the feature of (Paecilomyces fumosoroseus), so can identify that TRCC82001 bacterial strain is for (Paecilomyces fumosoroseus).By institute of microbiology of the Chinese Academy of Sciences by (Paecilomyces fumosoroseus) called after paecilomyces fumosoroseus.TRCC22001 bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on August 13rd, 2013, and deposit number is: CGMCC No.7993.
Embodiment 2 spawn culture:
(1) inclined-plane seed culture.Slant strains substratum is Improvement type PDA:20% potato, 2% sucrose, 0.2% peptone, 0.1%K
2hPO
43H2O, 0.3%NaNO
3, 0.05%MgSO
47H2O, 0.05%KCl, 0.01%FeSO
4, 2% agar, water 1000ml.121 degrees Celsius of high pressure steam sterilization 30min.After test tube inoculation, place 26 degrees Celsius of constant temperature culture 10d for subsequent use.(2) liquid seeds is cultivated.Substratum: Improvement type PDA (removing agar).Packed into 500ml triangular flask, every bottled 100ml, 121 degrees Celsius of high pressure steam sterilization 30min.Access test tube slant bacterial classification (every connects two bottles), places 26 degrees Celsius of cultivation 60h of shaking table (180 turn/min) for subsequent use.
Embodiment 3 solid culture
(1) Medium Proportion: 70% wheat bran+30% rice bran.Take raw material by proportioning, add 1.2 times of water and fully mix thoroughly, the rear 121 degrees Celsius of high pressure steam sterilization 30min that spend the night are for subsequent use.(2) cultural method and character observation.Substratum is layered on respectively to (thickness is advisable with 2～3cm) on aseptic porcelain dish, respectively, by 10% inoculum size access liquid seeds, mixes cover lid, 26 degrees Celsius of constant temperature culture.7d after stirring 1 time, spray moistening or " pouring water moistening " cultivate, it is moistening or " pouring water moistening " that 15d stirs rear spraying again, cultured continuously 20d, performs upgrowth situation record.Cultivate after 20d, 32 degrees Celsius are dried 40h.(3) sporulation quantity is measured: take 1g, put into triangular flask, add the appropriate distilled water that contains tween-80, break up spore, calculate the spore count of every gram with blood cell counting plate.
The virulence of embodiment 4 paecilomyces fumosoroseus (Paecilomyces fumosoroseus)
1, paecilomyces fumosoroseus (Paecilomyces fumosoroseus) is observed the infection processs of diamondback moth larvae
Small cabbage moth 3 instar larvaes that picking has just been casted off a skin, connect bacterium to small cabbage moth 3 instar larvae body surfaces by cultured paecilomyces fumosoroseus spore powder, connect after bacterium under 20 degrees Celsius, relative humidity 75%～80% condition and raise, and every day, fresh cabbage leaves was changed in timing.Repeat 20 larvas of every repetition for 3 times.After processing, 10h starts sampling, every 2h sampling 1 time, every 4h sampling 1 time after 16h.Sample is fixed by 3% glutaraldehyde, observes, takes pictures after series of processes under scanning electron microscope.The half that spore germination exceedes spore diameter length (non-circular spore is by less diameter) using germ tube length is as sprouting standard.
2, Pathogenic Tests
(1) inoculating spores powder: small cabbage moth 3 instar larvaes and the cover glass that scribbles pyroxylin (e) cement are placed in to culture dish lid, then the ware of 25 degrees Celsius, the Paecilomyces Fumosoroseus of SDA culture medium culturing 14d being put to diamondback moth larvae in falling to being inverted in covers, with have gentle hands bat culture dish bottom several, spore powder is fallen on diamondback moth larvae and cover glass, take out that cover glass is counter covers on blood counting chamber, drip two blue liquid of cotton, making spore quantity on cover glass is 6～7 mm
-2.Diamondback moth larvae after treatment is put into and is lined with the culture dish that wet filter paper, diameter are 15cm and raises.Raising condition is 20 degrees Celsius, relative humidity 75%～80%, and every day, fresh cabbage leaves was changed in timing, observed susceptible symptom and recorded dead borer population.Repeat for 3 times, 20 larvas of every repetition, taking the diamondback moth larvae that do not connect bacterium as contrast.Data processing adopts Duncan duncan's new multiple range method.
(2) inoculating spores suspension: get 25 degrees Celsius, the SDA culture medium culturing paecilomyces fumosoroseus conidium of 14 days, make spore suspension (more than magnetic stirrer 10min) with the sterilized water containing 0.1% tween-80, regulating spore suspension concentration is 2 × 10
7individual mL
-1.With atomizer, small cabbage moth 3 instar larvaes and the cover glass that are placed on filter paper are quantitatively sprayed, then take out cover glass microscopy spore quantity, making spore count on cover glass is 6～7 mm
-2.20 larvas of every processing, repeat for 3 times, taking 0.1% tween-80 sterilized water as contrast.Raising condition and data processing are the same.
(3) inoculating spores nutrient solution: get 25 degrees Celsius, the SDA culture medium culturing paecilomyces fumosoroseus conidium of 14 days and be placed in Sharpe liquid nutrient medium, spore concentration is adjusted to 2 × 10
7individual mL
-1, shaking table is cultivated after 8 hours and is connect bacterium processing again, connects bacterium method and raising condition is the same.
3,3 kinds of differences connect the virulence of bacterium method to diamondback moth larvae
It is obviously different that difference connects pathogenic speed and the virulence of bacterium method paecilomyces fumosoroseus to diamondback moth larvae.Paecilomyces fumosoroseus conidium nutrient solution is faster to the pathogenic speed of diamondback moth larvae than spore suspension and spore powder, processes and within latter 44 hours, finds that polypide shows obvious susceptible symptom, within 72 hours, occurs peak mortality, and its median lethal time is 3.18 days; , there is susceptible symptom for 60 hours in the diamondback moth larvae of spore suspension and spore powder processing, than spore nutrient solution late 16 hours, peak mortality appearred in 96h, and the median lethal time is respectively 4.20 days and 4.34 days.
Preparation and the drug effect of embodiment 4 paecilomyces fumosoroseus (Paecilomyces fumosoroseus) preparation
1, spore powder is produced and suspended emulsion agent preparation
The bacterial classification of preservation is activated on SDAY flat board, constant temperature (25e) illumination cultivation is when producing spore, get spore powder to shaking culture in the Sa Shi nutrient solution SDAY of agar (not containing) 24 hours, after switching, continue to cultivate 48 hours then, by the bacterium liquid access containing a large amount of mycelia through soaking and steam sterilizing extremely on the rice of suitable ripe degree, inoculum size is 10%, bacterium liquid spreads out in pallet after fully mixing with rice, under 25 degree, cultivate 10 days, air-dry 1 day 120 mesh sieves after mechanical vibration under 35 degree and collect spore powder, its spore content is greater than 1011g
-1, by quantitative spore powder add formulation optimization can emulsified solvent oil in and be mixed with spore suspended emulsion agent, spore content is 1010ml
-1, this is pure spore suspended emulsion agent, is called for short pure spore agent, and on this basis, with 10% imidacloprid wettable powder, as synergistic agent, adding in pure spore suspended emulsion agent in the ratio of 3% (W/V) (is 3g100ml
-1), obtain thus the spore suspended emulsion agent of mixture, be called for short mixed agent.Select Provado as synergistic agent, the one, because it is to be allowed at present the chemical insecticide that uses in tea place, the 2nd, because fabulous biocompatibility between it and disinsection fungal.
2, field test
Select representative tea place (tea lesser leafhopper harm is serious and distribute relatively even) to carry out field control effectiveness test, management condition is consistent; This tea place is typical massif tea place, and tea ridge (wide approximately 1～115 meter) is step and distributes, approximately 60 ° of the gradients, and about 40m is risen on Di Zhi slope, slope.Test comprises 4 processing, clear water contrast (CK), mineral oil vehicle contrast (OI) that can humectant containing 3% Provado 10%, pure spore agent (OP) and the mixed agent (OPI) of paecilomyces fumosoroseus.Repeat 3 times every processing community, the about 60m of every community area
2(only ridge that about 60m is long), establishes 1 ridge blank (not doing any processing) as protective belt between community.500 times of sprayings of each microbial inoculum dilute with water, the actual spore concentration that makes hydrojet is 2 × 10
7individual spore ml
-1, the about 5kg of every community spray bacterium liquid, the spouting liquid (CK and OI) of non-microbial inoculum processing is identical, and all processing are arranged by random district group, spray altogether 2 times, and the 12d being sprayed at for the second time after spraying for the first time carries out, and twice use bacterium time is fine day at dusk.
3, insect population investigation
Tea lesser leafhopper happiness cool place and reactivity are strong, and kind jump brings difficulty to field sample survey.Duration of test, sample survey generally morning reveal not dry before when 8:30 (morning before) this worm not too carry out when activity, the cloudy day can whole day carry out.Shi Weiyi ridge, Yin Mei community, therefore adopt the method to tea tree numbering, when investigation, choose at random the tea tree of a numbering as initial sampling strain, then extract 1 strain every 2 strains, every community is looked into 10 strains altogether, every strain 10 tender leafs of selective examination (getting lower second tender leaf of bud), leaf through lightly (bud-leaf being touched is not used in observation), count on the spot and record nymph number and become borer population, finally by processing the every 10 leaf worm numbers of statistics, first with investigation insect population radix before bacterium, then every investigation in 3 or 4 days once, after 2 weeks, do last investigation with bacterium for the second time, local weather data during field test obtains from local weather station.
4, data analysis
Tea lesser leafhopper density (worm amount/10 leaf) to each processing, carries out variance analysis with DPS data handling system software.Each relative control effect (E) of processing calculates as follows:
E=[1-(P
CK0×P
T1)／(P
CK1×P
T0)]×100
In formula, P
t0and P
t1represent to process respectively the average 10 leaf worm amounts of community investigation before and after processing, P
cK0and P
cK1represent respectively the average 10 leaf worm amounts of investigating before and after the spraying of clear water check plot.
5, test-results
(1) duration of test weather conditions: envrionment conditions is the important factor that affects fungus insecticide; The field test of this research is on the occasion of local high temperature season, and from spray bacterium to observing 25 days that finish, range of temperature is 21.2～38.1 degrees Celsius, minimum day 22.7 degrees Celsius of samming, the highest day 31.2 degrees Celsius of samming, 26.6 degrees Celsius of average day sammings.From rainfall situation, have 14 rainfall days, wherein the rainfall of 4 rainfall days is less than 1mm, the light rain day 4d of 1～5mm, at 3 days on the middle rain day of 5～25mm, at 4 days on the large rain day of 25～50mm, total rainfall amount reaches 241.2mm.In a word, test just the midsummer of location Suichang of Zhejiang Province mid-June to mid-July, temperature drift, and rainwater is on the high side, thereby duration of test Changes in weather is larger.
(2) field efficacy: contrast and compare with clear water, 500 times of diluent sprayings of spore suspended emulsion agent of two kinds of fungies all produce certain control effect to tea lesser leafhopper, but be better than pure microbial inoculum with the successful of mixed agent, the highest preventive effect of paecilomyces fumosoroseus is 71.3%.500 times of diluents sprayings of the alone mineral oil vehicle that contains 3% Provado 10% wettable powder also obtain suitable preventive effect, and preventive effect always after using investigation for the first time the highest, then decline to a great extent.To give average and sequence by the preventive effect of all previous investigation after bacterium, result is paecilomyces fumosoroseus mixed agent preventive effect 62.1 ± 12.2%, is secondly mineral oil 50.3 ± 14.3% and the pure microbial inoculum of paecilomyces fumosoroseus containing micro-Provado, only has preventive effect 19.0 ± 23.2%.
It should be noted last that, above example is only illustrative rather than definitive thereof technical scheme of the present invention, although the present invention is had been described in detail with reference to above-described embodiment, those of ordinary skill in the art is to be understood that: still can modify or be equal to replacement the present invention, and not departing from any modification or partial replacement of the spirit and scope of the present invention, it all should be encompassed in the middle of claim scope of the present invention.
Table 1:3 kind difference connects the mortality ratio of the rear diamondback moth larvae of bacterium method processing
| Connect bacterium method | Corrected mortality (%) | Percentage of pupation (%) | Median lethal time |
| Spore powder | 76.2±1.13a | 20.4±1.39a | 4.34 |
| Spore suspension | 59.8±1.36b | 36.2±1.81b | 4.30 |
| Spore nutrient solution | 58.5±2.04b | 38.4±1.56b | 3.18 |
Brief description of the drawings
Fig. 1 is the rDNA ITS1-5.8S-ITS2 sequence of paecilomyces fumosoroseus (Paecilomyces fumosoroseus) TRCC22001CGMCC No.7993.
Fig. 2 is the phylogenetic tree of paecilomyces fumosoroseus (Paecilomyces fumosoroseus) TRCC22001CGMCC No.7993 based on rDNA ITS1-5.8S-ITS2 sequence construct.
Claims (5)
1. Paecilomyces Fumosoroseus strain, it is deposited in the Chinese microorganism strain keeping common micro-organisms center C GMCC of management committee on August 13rd, 2013, and deposit number is 7993.
2. the spawn culture method of paecilomyces fumosoroseus, is characterized in that: (1) inclined-plane seed culture, slant strains substratum is Improvement type PDA:20% potato, 2% sucrose, 0.2% peptone, 0.1%K
2hPO
43H
2o, 0.3%NaNO
3, 0.05%MgSO
47H
2o, 0.05%KCl, 0.01%FeSO
4, 2% agar, 1000 milliliters, water; 121 degrees Celsius of high pressure steam sterilizations 30 minutes; After test tube inoculation, place 26 degrees Celsius of constant temperature culture 10 days for subsequent use; (2) liquid seeds is cultivated, substratum: Improvement type PDA (removing agar); Packed into 500 milliliters of triangular flasks, every bottled 100 milliliters, 121 degrees Celsius of high pressure steam sterilizations 30 minutes; Access test tube slant bacterial classification (every connects two bottles), place shaking table (180 revs/min) 26 degrees Celsius cultivate 60 hours for subsequent use.
3. the solid culture method of paecilomyces fumosoroseus, is characterized in that: take raw material by solid medium proportioning, add 1.2 times of water and fully mix thoroughly, the rear 121 degrees Celsius of high pressure steam sterilizations 30 minutes of spending the night are for subsequent use.
4. the preparation method of paecilomyces fumosoroseus according to claim 3, is characterized in that: solid medium is 70% wheat bran+30% rice bran.
5. Paecilomyces Fumosoroseus strain, is characterized in that the application of described Paecilomyces Fumosoroseus strain in control sucking pest.
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| CN106376600A (en) * | 2016-08-30 | 2017-02-08 | 江西天人生态股份有限公司 | Paecilomyces fumosoroseus wettable powder and preparation method thereof |
| CN106489928A (en) * | 2016-08-31 | 2017-03-15 | 江西天人生态股份有限公司 | A kind of exhibition film oil-suspending agent and its preparation method and application |
| CN106489927A (en) * | 2016-08-31 | 2017-03-15 | 江西天人生态股份有限公司 | A kind of paecilomyces fumosoroseus oil-suspending agent and preparation method thereof |
| CN106417282A (en) * | 2016-09-22 | 2017-02-22 | 江西天祥通用航空股份有限公司 | Paecilomyces fumosoroseus granules and preparing method thereof |
| CN107410367A (en) * | 2017-09-01 | 2017-12-01 | 江西省科学院微生物研究所 | A kind of paecilomyces fumosoroseus finish and preparation method thereof and a kind of insecticide |
| CN107410367B (en) * | 2017-09-01 | 2020-04-17 | 江西省科学院微生物研究所 | Paecilomyces fumosoroseus oil and preparation method thereof and insecticide |
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