CN108441426A - One plant of endophytic bacterial controlled effect that can prevent Plant nematode - Google Patents
One plant of endophytic bacterial controlled effect that can prevent Plant nematode Download PDFInfo
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- CN108441426A CN108441426A CN201710212642.1A CN201710212642A CN108441426A CN 108441426 A CN108441426 A CN 108441426A CN 201710212642 A CN201710212642 A CN 201710212642A CN 108441426 A CN108441426 A CN 108441426A
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Abstract
The present invention relates to the endogenetic fungus that one plant can prevent nematode, microorganism belonging to genus technical field of pesticide.The present invention carries out strain isolation, culture by the method that conventional microbiological detaches from nature water plant, obtain one plant can make plant nematode is lethal in soil aspergillus niger (Aspergillus niger) F25 bacterial strains, F25 bacterial strains are preserved in Wuhan, China China typical culture collection center, preserving number in April, 2016：CCTCC M2016407.The aspergillus niger F25 bacterial strains of the present invention can kill the plant nematode in soil.
Description
Technical field
The water plant endophytic bacterial controlled effect that can prevent plant nematode the present invention relates to one plant, microorganism belonging to genus pesticide technology neck
Domain.
Background technology
Plant nematode is the plant disease generally occurred in a kind of world wide, and only root-knot nematode Known Species just reach
More than 70 kinds, cause harm 3000 various plants, causes huge loss every year.China plant nematode main harm tobacco, flowers,
The industrial crops such as vegetables, cotton, soybean, peanut, Radix Notoginseng, American Ginseng, caused by lose it is very surprising.According to national tobacco pest and disease damage
Prevent information centre incomplete statistics, the only root knot nematode in tobacco onset area of the whole nation in 2000 is up to 500,000 hectares, direct economy
Lose more than 500,000,000 yuan.Only Yunnan Province's onset area is just up to 2.7 ten thousand hectares, and about 100,000,000 yuan of direct economic loss, indirect loss is more tight
Weight.Loss is up to as many as 800,000,000 yuan caused by Heilongjiang Province's only soybean cyst nematode Heterodera glycines are annual.Bursaphelenchus xylophilus is referred to as smokeless Forest Fire
Calamity seriously occurs, the oriented whole nation in Electric Field At Ground In Partial Regions such as Jiangsu, Zhejiang, Guangdong, Shandong, Anhui, Hubei, Shanghai, Taiwan, Hong Kong
The trend stretched.In the vegetables main producing region such as Shandong, Guangdong, Hainan and Yunnan, root knot nematode is in rising trend.Guizhou Province west
American ginseng have to stop production because root-knot nematode seriously occurs.Crop wound is caused after more seriously being infected due to root-knot nematode, is caused
Crop root disease is set seriously to occur.According to incompletely statistics, nematodiasis makes the various crop annual production losses in China reach 10-
15%, the whole world is lost caused by nematode up to 100,000,000,000 dollars every year（Weischer,1994）.
The prevention of nematode at present relies primarily on chemical nematicides, but chemical nematicides are mostly the huge malicious pesticide of natural disposition that goes out,
It not only develops immunity to drugs, more causes environmental pollution, be detrimental to health.Have in succession many disabled or will disable.U.S.'s bromine
Methane was stopped using in, methylene bromide in 2000 in 2005 respectively.Although the iron that largely uses goes out gram to line in production at present
Worm has good preventive effect, but toxicity is high, destroys soil microflora.Chemical nematicides ensure agriculture in prevention eelworm harm
While industry produces, serious negative effect is brought to human health and environment.Due to excessive pesticide residues, China is to outgoing
Tealeaves, Radix Notoginseng, pear, "Hami" melon, fruit juice, flowers, honey and a variety of animal products of mouth repeated commodity and pay compensation for event, directly
Connect the influence export of farm produce.The chemical nematicides that the country uses at present simultaneously are mainly external imported product, and price is excessively high
It is expensive.Therefore, developing nematode biological prevention and control agent is particularly important.
Nematode biological prevention and control agent has just been listed early in the last century 70's.But after decades of development, to today, it makes
With with promote still not extensively, including China it is many country still be not used.Nematode is shown in many experiments indoors
The effect of the bacterial strain of very strong infection ability in practical applications is undesirable.In field trials, the control effect of biocontrol microorganisms can be by
The influence of various factors.Soil environment (content of organic matter, humidity, temperature etc.) to biocontrol agent survival in the soil, colonize,
Diffusion etc. has important influence.In addition, remaining chemical pesticide in soil, some metal ions present in soil can all influence
Biocontrol agent survival in the soil colonizes and to the control effect of nematode.
Biocontrol bacterial strain is an important prerequisite for studying biocontrol agent.The biological characteristics of different strains, prevent nematode
It is larger to control difference on effect.In nature, the natural enemy of nematode is very much, is mainly had：Fungi, bacterium, mite, predatism nematode etc..
Wherein most is fungi, at present most study and as launch also be mainly fungi.The spy of nematode is infected according to it
Property is broadly divided into：
1, fungus is preyed on：Nematode is captured by the predacious organs that vegetative hyphae specialization is formed；
2, parasitic fungi：Pass through the fungus of the ovum of parasitic nematode, female adult, cyst etc.；
3, toxigenic bacterium object：The nematicide fungus of toxin poison can be generated.
Enter the mainly nematophagous fungi in market, including predation fungus and parasitic fungi as product at present.State so far
The inside and outside nematophagous fungi reported is there are about 400 kinds, and wherein Predacious nematode fungi common are wheel entomophthora there are about 12 categories
Category, Arthrobotrys, Monacrosporium and every spore belong to etc..Endoparasitism nematode fungus is related to be belonged to there are about 13, common are poisonous insect it is mould belong to,
Snag spore category and hair spore etc..About 200 kinds of chance fungus, determining category about 71, study it is more have Arthrobotrys,
Every finger spore category, Metarhizium, paecilomyces, Pukeniya genus and trichoderma etc..Toxigenic bacterium object mainly has the mould category of silk and Pleurotus.
So far, the aquatic Aspergillus niger strain that can kill plant nematode yet there are no document and report for work.
On the one hand effect of the microbial strains to plant nematode is directly made nematode by bacterial strain own growth
With, here it is " generation product " of abbreviation, the main body of effect is microbial cells or cell, without the participation of thalline or cell,
Inhibition or killing effect of the microorganism to nematode are not just shown；The metabolite that another side is generated by strain growth is to plant
Parasitic nematode is had an effect, and here it is " the two generation products " of abbreviation, the main body of effect is the metabolite that microorganism generates, and is passed through
Metabolite inhibits or kills nematode indirectly.Metabolite has the " generation such as environment resistant ability is strong, easy to use, effect is quick
The advantage that product " does not have.By aspergillus niger generate, can act on plant nematode metabolite it is presently found be mostly grass
The organic acids such as acid, citric acid and aspertoxin are mostly inhibiting effect to the effect of plant nematode by oxalic acid, citric acid,
The object of effect is second instar larvae or ovum, while the Aspergillus niger strain metabolite to work to the two is less or effect is not strong,
Ovum, larva and the female adult of nematode can be acted on simultaneously and can effectively kill the Aspergillus niger strain metabolite and bacterial strain of nematode, it is special
It is not the bacterial strain of aquatic environment in addition to the patent that inventor applies yet there are no document report.
Invention content
It is an object of the invention to overcome the deficiency of existing biological and ecological methods to prevent plant disease, pests, and erosion technology bacterial strain, plant root-knot nematode can be killed by providing one plant
Biocontrol bacterial strain.
The present invention bacterial strain be aspergillus niger (Aspergillus niger)-F25, the bacterial strain were protected on July 21st, 2016
Wuhan, China China typical culture collection center is ensconced, address is：Wuhan, China Wuhan University, preserving number：CCTCC M
2016407。
In natural soils environment, culture is more than the bacterium source of the present invention to the killing effect of plant root-knot nematode
60%, 60% is more than to the control effect of plant root-knot nematode.
The bacterial strain of the present invention is different from other bacterial strains of aspergillus niger patent that inventor applies, belongs to of the same race on taxology
But it is different bacterial strain.
The features of the present invention：F25 bacterial strains can have the ovum of nematode, larva, female adult different degrees of lethal effect, cause
Dead rate is better than other bacterial strains.Its lethal ingredient degerming in vitro also have water soluble ingredient, be different from other Aspergillus strains oxalic acid or
Citric acid.
The present invention is accomplished by the following way：
One, the acquisition of bacterial strain：
The bacterial strain of the present invention is coated distribution by the conventional commonsense method that microbe soil detaches, and picking single bacterium colony carries out pure
Change culture to obtain, the culture medium used is cultivated for PDA, is accredited as through fungi ITSAspergillus niger, name as F25；
Form：Mycelia is flourishing, and sprawling is rapid, is just white, after become foresythia until black heavy fleece shape, multi-branched have more
Core, the big spherical shape of top capsule, stigma are double-deck.Conidial head is spherical, and 700~800 μm of diameter, brown-black is radial, conidiophore
It is different in size.Conidium is spherical shape, darkly, dark brown, smooth or coarse.Conidium head such as " chrysanthemum ".
Two, the culture of F25 bacterial strains：
First, the good spore generated is cultivated on sterile collection tablet, and spore inoculating to advance sterilizing and is contained into bead
In liquid PDA culture medium, 28 DEG C of 145rpm/min are cultivated 2-3 days, depending on spare until covering with tiny mycelium pellet in culture medium, this
For liquid seeds.Then, in the liquid PDA culture medium sterile access of the liquid seeds grown to be sterilized in advance, access amount is by new
The 10% of culture medium liquid amount calculates.28 DEG C of 145rpm/min are cultivated 4-7 days, and maximum insecticidal effect is reached after nematodes when being,
The effective cultures of as F25.
Three, F25 measures the control effect of plant root-knot nematode：
It takes out and contains a large amount of egg capsule Tomato Root Systems, gently rinsed with water, carefully remove egg capsule, be placed in 0.5% sodium hypochlorite and disappear
3 min of poison, then with aseptic water washing 3 times, egg capsule be placed in have in the culture dish of a small amount of sterile water, trained in 25 DEG C of insulating boxs
It supports, it is spare to collect the second instar larvae once newly hatched afterwards for 24 hours.
By effective culture be added 32 holes culture plate in, per hole be added the 100 effective cultures of μ l, respectively to processing and it is right
The root-knot nematode second instar larvae newly hatched respectively is added according in, per 50, hole.It is put into 25 DEG C of incubators, is adjusted after 60min, 120min
Rate of knockdown is looked into, nemic death rate is recorded after 12h, calculates corrected mortality.
Advantageous effect of the present invention is：Aspergillus niger (Aspergillus niger) F25 bacterial strains can effectively prevent plant and post
The culture of raw nematode, bacterial strain is more than 60% to the control effect of plant root-knot nematode.
Specific implementation mode
Description of the invention content method of embodiment with Summary.The present invention is made below by embodiment detailed
Explanation.
Example one：
1. with PDA plate culture F25 bacterial strains, when growing black spore on tablet, take out spare；
2. aseptically collecting the black spore on tablet, black spore is accessed into the PDA Liquid Cultures containing bead
In, 28 DEG C of 145rpm/min cultures；
3. culture 2 days, depending on spare until covering with tiny mycelium pellet in culture medium, this is liquid seeds；
4. in the liquid PDA culture medium that the sterile access of the liquid seeds grown is sterilized in advance, access amount fills liquid by new culture medium
The 10% of amount calculates, and 28 DEG C of 145rpm/min are cultivated 3 days；
5. after carrying out raw survey 120min with routine nematodes method, nematode lethality reaches 60%, stops fermentation, as F25
Effective culture.
Example two：
1. with PDA plate culture F25 bacterial strains, when growing black spore on tablet, take out spare；
2. aseptically collecting the black spore on tablet, black spore is accessed into the PDA Liquid Cultures containing bead
In, 28 DEG C of 145rpm/min cultures；
3. culture 3 days, depending on spare until covering with tiny mycelium pellet in culture medium, this is liquid seeds；
4. in the liquid PDA culture medium that the sterile access of the liquid seeds grown is sterilized in advance, access amount fills liquid by new culture medium
The 10% of amount calculates, and 28 DEG C of 145rpm/min are cultivated 5 days；
5. after carrying out raw survey 120min with routine nematodes method, nematode lethality reaches 80%, stops fermentation, as F25
Effective culture.
Example three：
1. with PDA plate culture F25 bacterial strains, when growing black spore on tablet, take out spare；
2. aseptically collecting the black spore on tablet, black spore is accessed into the PDA Liquid Cultures containing bead
In, 28 DEG C of 145rpm/min cultures；
3. culture 3 days, depending on spare until covering with tiny mycelium pellet in culture medium, this is liquid seeds；
4. in the liquid PDA culture medium that the sterile access of the liquid seeds grown is sterilized in advance, access amount fills liquid by new culture medium
The 10% of amount calculates, and 28 DEG C of 145rpm/min are cultivated 6 days；
5. nematode is routinely after raw survey 120min, nematode lethality reaches 95%, stops fermentation, the effective cultures of as F25.
Example four：
1. with PDA plate culture F25 bacterial strains, when growing black spore on tablet, take out spare；
2. aseptically collecting the black spore on tablet, black spore is accessed into the PDA Liquid Cultures containing bead
In, 28 DEG C of 145rpm/min cultures；
3. culture 3 days, depending on spare until covering with tiny mycelium pellet in culture medium, this is liquid seeds；
4. in the liquid PDA culture medium that the sterile access of the liquid seeds grown is sterilized in advance, access amount fills liquid by new culture medium
The 10% of amount calculates, and 28 DEG C of 145rpm/min are cultivated 7 days；
5. after carrying out raw survey 120min with routine nematodes method, nematode lethality reaches 98%, stops fermentation, as F25
Effective culture.
Example five：
1. with PDA plate culture F25 bacterial strains, when growing black spore on tablet, take out spare；
2. aseptically collecting the black spore on tablet, black spore is accessed into the PDA Liquid Cultures containing bead
In, 28 DEG C of 145rpm/min cultures；
3. culture 2 days, depending on spare until covering with tiny mycelium pellet in culture medium, this is liquid seeds；
4. in the liquid PDA culture medium that the sterile access of the liquid seeds grown is sterilized in advance, access amount fills liquid by new culture medium
The 10% of amount calculates, and 28 DEG C of 145rpm/min are cultivated 7 days；
5. after carrying out raw survey 120min with routine nematodes method, nematode lethality reaches 89%, stops fermentation, as F25
Effective culture.
Example six：Field test results
The effective cultures of F25 are subjected to field trial in Yunnan Province Luliang County greenhouse tomato and Yuxi tobacco planting respectively, are tied
Fruit is as follows：
The effective culture fields F25 use the control effect to root-knot nematode
The result shows that：The effective cultures of F25 act on the diseased plant rate 14% of ensuing crop, root knot percentage 13% is below blank control,
Asafoetide rhzomorph and Furadan, average preventive effect is higher than asafoetide rhzomorph and chemical Furadan.With the present invention in field control crop root knot
Nematode has preferable control effect.
Claims (1)
1. one plant of endogenetic fungus that can prevent Plant nematode, it is characterised in that：The strain isolation is from Yunnan Province's Yuxi Fuxian Lake
Natural, ecological aquatic environment, it is categorized be accredited as aspergillus niger (Aspergillus niger)-F25, bacterial strain April 7 in 2016
Day is deposited in Wuhan, China China typical culture collection center, preserving number：CCTCC M 2016407；The bacterial strain can kill soil
Earth plant nematode.
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| CN201710212642.1A CN108441426A (en) | 2017-04-01 | 2017-04-01 | One plant of endophytic bacterial controlled effect that can prevent Plant nematode |
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| CN201710212642.1A CN108441426A (en) | 2017-04-01 | 2017-04-01 | One plant of endophytic bacterial controlled effect that can prevent Plant nematode |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110724641A (en) * | 2019-10-21 | 2020-01-24 | 北京市植物园 | Rejuvenation method of nematicidal fungi |
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| CN104928194A (en) * | 2015-06-26 | 2015-09-23 | 云南大学 | Aspergillus niger strain and application thereof |
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| CN101445785A (en) * | 2008-12-23 | 2009-06-03 | 北京市农林科学院 | Aspergillus niger fungus capable of poisoning plant parasitic nematodes, preparation method and application thereof |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110724641A (en) * | 2019-10-21 | 2020-01-24 | 北京市植物园 | Rejuvenation method of nematicidal fungi |
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