CN101861880A - Microbial agent and application thereof - Google Patents

Abstract

The invention relates to a microbial agent and application thereof, and belongs to the technical field of microbial pesticides. The microbial agent comprises stropharia sp.2052, bacillus nematocidus B16 and diatomite and is prepared by the following steps of: a, respectively preparing 2052 solid culture and B16 bacterial suspension (CFU=2x10<8>) from the stropharia sp.2052 and bacillus nematocidus B16 by a conventional method, and mixing the 2052 solid culture and the B16 bacterial suspension in a mass ratio of 1:1 to obtain a mixture; and b, adding the diatomite in an amount which is 30 to 40 percent of the mass of the mixture to prepare the solid microbial agent by a conventional method, wherein the grain diameter of the diatomite is 4.5 to 5mm. The microbial agent can be used for preparing a preparation for preventing and controlling plant nematodes. The microbial agent has the advantages of high efficiency, low toxicity and no residue, and the effect of preventing and controlling root knot nematode disease of tomatoes in a greenhouse reaches over 90 percent.

Description

A kind of microbial bacterial agent and application thereof

Technical field:

The present invention relates to a kind of microbial bacterial agent and application thereof, the microorganism belonging to genus technical field of pesticide.

Technical background:

Plant nematode is one of disease that generally takes place on the crop, endangers nearly all crop.Only the annual loss that causes in the whole world of root-knot nematode (Meloidogyne spp.) is just up to tens billion of dollars.Lack resistant variety owing to produce to go up, crops cropping index height, the use of chemical nematode killing agent is restricted, and the needs of modern agriculture sustainable development, and the biological control Plant nematode comes into one's own day by day.

Comprise bacterium and fungi in the nematicide microorganism, utilize the inducing action of bacterium, nematode is attracted to around the fungi, utilize unique nematoblast structure of fungi to knock down nematode again, be aided with the enzymolysis of bacterium, reach the purpose of killing nematode nematode.Uniting of this multiple action has potential and actual using value in the biological control of nematode.

Summary of the invention:

The object of the present invention is to provide a kind of microbial bacterial agent and application thereof.

The present invention screens from the nematicide microorganism and seeks the bacterial strain with suitability for industrialized production prospect, and the biological prevention and control agent of a kind of tool better prevention nematode is provided for agricultural production.The present invention specifically is achieved in that

The inventor is in early-stage Study, and screening has obtained to have the basidiomycetes Stropharia rugoso-annulata 2052 (Stropharia sp.2052) and the bacillus B16 (Bacillus nematocidus) of good nematocide function.The B16 bacterial strain is deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center on April 5th, 2004, and preserving number is CGMCC No.1128.2052 bacterial strains have been stored in China Microbial Culture Preservation Commission common micro-organisms center, preserving number: CGMCC No.3399 on November 5th, 2009.

Food nematode bacillus is under the jurisdiction of bacillus (Bacillus), is aerobic bacteria, G ⁺, the long 2.0-3.5 μ of cell m, wide 0.8-1.2 μ m.Growth rate is very fast on the LB flat board, and the endospore of cultivation visible only a few more than 72 hours forms or be discharged into the gemma outside the born of the same parents on the LB medium, and thalline almost generates gemma entirely after 5 days.Gemma is a long column shape, and inferior end is given birth to.The main morphological feature of bacterial strain is: elongated rod shape, and individuality is bigger, and how the blunt circle in two ends exists with discrete form.Bacterium colony is rounded on the LB flat board, rough surface tarnish, similar fine powder shape, lawn white.

Stropharia rugoso-annulata (Stropharia) is under the jurisdiction of Basidiomycotina, Hymenomycetes, Agaricales, Stropharia rugoso-annulata section.Its main morphological feature is: the spore of puce to infuscate arranged, be common in places such as the woods, meadow, compost, animal wastes.Fruit body is medium to bigger, bacteria cover diameter 60～160 (200) mm, and flat hemispherical, hemispherical are to flat, and the wine red brown transfers reddish brown brown or cheese look gradually to, and yellow sometimes cheese look also has the claret filament to distribute therebetween.Cap when children taper is to bell, and is bell to open and flat when ripe, level and smooth or cilium shape scale is arranged, and sticking slightly when moistening, there is the velum relic at the initial stage, after polish, the edge is sharp, often inwardly curls.The lamella initial stage is dark brown slightly, and after stain deepens, and dusty blue is closely given birth to and adhesion to atropurpureus, and is close, and broad is not isometric, and the edge is slightly white.Stem white, slightly yellow, thicker sometimes, 70～150 * 15～30mm, cylindrical, base portion slightly expands, and is solid.Collarium is thicker, white.Stem is at smooth, the no squama in the following position of collarium.Bacterial context white is thicker.Green grass fragrance is arranged.The spore print puce, spore is oval to avette, heavy wall, 9.5～12 * 7～9 μ m have germ pore.Singly life or all living creatures are in habitats such as the woods and meadows.The pure culture of nearly all Stropharia fungi can both produce the accessory structure nematoblast, and this structure is considered to a key character of this genus classification of fungi.

Bacillus (Bacillus) is the bacterial strain with eelworm-killing activity that screens from a large amount of soil bacterias.After bacterial strain is screened and improves, obtained stable bacterial strain B16.This bacterium is easy to cultivate, and proterties is stable, is suitable for large-scale production.

Stropharia rugoso-annulata (Stropharia) original strain poor growth.We are separated to different strains from soil, wherein most characteristic that does not possess we required " obvious stable control efficiency, comprehensive proterties is good, is easy to large-scale production ".After bacterial strain is screened and improves, obtained stable bacterial strain 2052.It not only has good control efficiency to root-knot nematode (Meloidogyne spp.), has possessed the basic proterties of suitability for industrialized production simultaneously.

The present invention by solid culture, obtains to comprise the thalline culture of mycelia and mycelia accessory structure with Stropharia rugoso-annulata 2052, and the liquid culture of composite bacterial B16 is made into the solid granula again, the use of spreading manuer in holes.

Microbial bacterial agent of the present invention obtains through following steps:

A. select microbial strains Stropharia rugoso-annulata 2052 (Stropharia sp.2052) and bacillus B16 (Bacillus nematocidus), be prepared into bacteria suspension (CFU=2 * 10 of 2052 solid cultures and B16 according to a conventional method ⁸), obtain mixture in the mixing of 1: 1 ratio of mass ratio;

B. press the diatomite that mass ratio adds the 30-40% of said mixture quality, the diatomite particle diameter is 4.5-5mm, makes solid fungicide according to a conventional method.

Microbial bacterial agent of the present invention is as the application that is used to prevent and treat Plant nematode.

The biological prevention and control agent that this method is made can mix use with other nematicide agricultural chemicals, plant growth regulator, but must guarantee that institute's confection has no side effect to bacillus and the Stropharia rugoso-annulata that the present invention adopts.

Nematode biological prevention and control agent of the present invention is generally used when crop sowing or transplanting.Method is during when sowing and seed are manured into soil jointly, during transplanting this preparation to be applied around the crop rhizosphere.Serious area is taken place nematode can impose in process of crop growth once, and method is that this preparation is applied around the crop rhizosphere.

The present invention compares with the existing plant nematode medicine that kills, and has the advantage of efficient, low toxicity and noresidue.

Embodiment:

The fermentation method for producing of The compounds of this invention (conventional method):

1, the mycelium with 2052 bacterial strains is inoculated on the test tube culture medium slant, and culture medium prescription is the PDA medium, cultivates 4-8 days down, obtains the test tube kind for 20-30 ℃.

2, with the B16 inoculation on the test tube culture medium slant, culture medium prescription is the LB medium, cultivates 1-2 days down, obtains the test tube kind for 35-38 ℃.

3, cultured 2052 solid seeds are inserted in the solid culture medium, the prescription of medium is: 70-80% wheat berry, 10-20% glucose, 5-10% calcium carbonate was cultivated 25-35 days for 22-28 ℃.

4, cultured B16 solid seed is inserted in the liquid LB medium, train 35-38 ℃ and cultivated 1-2 days, be mixed with CFU=2 * 10 ⁸Bacteria suspension.

5, with bacteria suspension (CFU=2 * 10 of cultured 2052 solid cultures and B16 ⁸), mix in 1: 1 ratio, add the diatomite 30-40% of 4.5-5mm, make solid fungicide and use.

Preparation of the present invention is to the efficiency test of root-knot nematode:

1, the cultivation of Meloidogyne incognita (Meloidogyne incognita) statistics

The tomato root that has Meloidogyne incognita of hot-house culture is cleaned with sterile water, the egg capsule of dissecting needle picking root-knot nematode places the KCl solution of 0.4M, use NaClO solution washing and the sterilization of 4ml sterile water (three times), the two anti-solution of the aseptic green grass or young crops-streptomycin of 1ml, 3ml 0.1% in the 5ml centrifuge tube successively, each step is all removed supernatant by of short duration centrifugal (8000 changeed 30 seconds).The worm's ovum that washing is good places the ZnCl of 0.4M ₂Hatching is 48 hours in the solution, and second instar larvae is collected in the 1.5ml centrifuge tube with pipettor, by of short duration centrifugal (5000 changeed 30 seconds) enrichment counting.

2, efficiency test method

(1) test institute's with medicament and processing

A, solid fungicide 10-20%, the disposable crop rhizosphere that imposes on when crop is transplanted;

B, positive control, 0.5% Avermectin is pressed 37.5kg/hm ²Handle;

C, blank be not with microbial inoculum or chemicals treatment.

(2) test method

At diameter is the basin alms bowl adding 500g soil sample (sick soil: sandy soil: humus soil=1: 1: 1), do above several processing of 15cm.Each is handled 5 basins and repeats for 3 times, places 25-28 ℃ greenhouse to cultivate.Transplant the back and watered, make its normal growth every 3 days.Microbial inoculum is handled the back and was investigated the root knot number of each processed group crop root, the worm's ovum number of rhizosphere in 30,60 days respectively, calculates the control efficiency of microbial inoculum to root knot nematode disease.

Embodiment 1: with the sick test of microbial inoculum control tomato root-knot eelworm

Transplant 1 monthly age tomato (4-6 leaf), tomato variety is: improvement hair powder 802, agricultural science and technology development company in Shandong produces.Every basin is used the 50g microbial inoculum, carries out efficiency test by above-mentioned test method.Tabulation is depicted as the preventive effect data.

Table 1 microbial inoculum is to the control efficiency (60d) of tomato root-knot eelworm disease

The result shows that this microbial inoculum has significant preventive and therapeutic effect to the tomato root-knot eelworm disease.

Embodiment 2: the preventive effect of different microbial inoculums relatively

Embodiment 2 methods are with embodiment 1, and difference is: using 2052 microbial inoculums is every basin 50g, and using the B16 microbial inoculum is every basin 100mL.

The synergistic effect of table 2 microbial inoculum

The result shows that microbial inoculum of the present invention improves 10.7% than using 2052 microbial inoculum preventive effects separately, improves 7.3% than using B16 microbial inoculum preventive effect separately.

Claims (3)

1. a microbial bacterial agent is characterized in that microbial bacterial agent comprises bacterial strain Stropharia rugoso-annulata 2052 (Stropharia sp.2052), bacillus B16 (Bacillus nematocidus) and three kinds of materials of diatomite.

2. the described microbial bacterial agent of claim 1 is characterized in that microbial bacterial agent obtains through following steps:

A. select microbial strains Stropharia rugoso-annulata 2052 (Stropharia sp.2052) and bacillus B16 (Bacillus nematocidus), be prepared into CFU=2 * 10 of 2052 solid cultures and B16 according to a conventional method respectively ⁸Bacteria suspension, mix obtaining mixture again in 1: 1 ratio of mass ratio;

B. press the diatomite that mass ratio adds the 30-40% of said mixture quality, the diatomite particle diameter is 4.5-5mm, makes solid fungicide according to a conventional method.

3. the described microbial bacterial agent of claim 1 is characterized in that this microbial bacterial agent is as the application that is used to prevent and treat Plant nematode.