CN105265487B - The biocontrol fungi for preventing and treating plant root-knot nematodes mixes microbial inoculum and its application - Google Patents
The biocontrol fungi for preventing and treating plant root-knot nematodes mixes microbial inoculum and its application Download PDFInfo
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Abstract
The present invention relates to the fungies of prevention and treatment plant root-knot nematodes to mix microbial inoculum and its application method.The fungi mixes microbial inoculum and is prepared by two kinds of fungal cultures cultures of pale purple purple spore bacterium (Purpureocillium lilacinum) CGMCC No.9344 and mould (Clonostachys rosea) the CGMCC No.1037 of pink helix poly spore.It can be mixed with seedling medium, organic fertilizer, compound organic and inorganic fertilizer and use or be used alone, handled soil or transplanting front and back cave is applied or pouring root, the prevention and treatment for plant root-knot nematodes.
Description
Technical field
The invention belongs to agriculture, woods and garden crop protection technique fields, in particular to prevent and treat the life of plant root-knot nematodes
Anti-fungal mixes microbial inoculum and its application.
Technical background
Root-knot nematode (Meloidogyne spp.) is a kind of important plant nematode, and host range is very wide, is more than
3000 kinds of plants often result in serious economic loss, can cause nearly 100,000,000,000 dollars of loss every year in world wide.In recent years
With the continuous expansion of greenhouse vegetable cultivated area, its generation also aggravates year by year, and production loss is up to 30%~50%.
Currently, the prevention and treatment of root-knot nematode is mainly based on nematocide, such as chloropicrin, bromomethane, metham-sodium, Aldicarb
Deng.Though nematocide is quick, at high cost, environment is seriously polluted, it is dangerous to people, animal (Yang Wenxiang etc., 2003).Biology
Prevention and treatment can effectively prevent root knot nematode disease, and free from environmental pollution, with very big development potentiality (Tianet al., 2007).
Duddington prevents and treats nematode in nineteen fifty-one proposition earliest biological method, and it is raw that microorganism or microfauna are introduced harmful nematode
Environment living, by mechanism control nematode the effects of competition, predation, parasitism, antagonism.There is the fungi of preventive and therapeutic effect to root-knot nematode
It include: nematode-trapping fungi (the mould category of Arthrobotrys, unit cell, the mould category of little finger of toe spore), endoparasitism nematode fungi (Pochonia
Chlamydosporia, P.lilacinum, Nematophthora gynophila), Toxigenic fungi (picking up the ears) and opportunistic fungus
Four classes such as (Fusarium solani, F.oxysporum)；The bacterium of prevention and treatment root-knot nematode mainly has Pasteurella
(P.penetrans) and rhizosphere bacteria P.fluorescens, B.subtilis, B.sphaericus, Agrobacterium
Radiobacter (Wang Huifang, 2007), B.thuringiensis, B.firmus etc.；Actinomyces such as Avid kyowamycin
Streptomyces avermitilis (Garabedian, 1983).Currently, the plant nematode of registration prevention and treatment both at home and abroad
Preparation mainly have " BioAct WG " and " NemaChek " (active constituent are as follows: Paecilomyces lilacinus Paecilomyces
Lilacinus), " Nematech " (active constituent Pasteuriapenetrans), " (active constituent is TiTera "
Myrothecium verruearia and its metabolin), BioNem and BioSafe (active constituent Bacillusfirmus),
Thick spore Verticillium dahliae (registration number: PD20070381) Gliocladium virens GL-21 (Junaid, 2013), wax gemma bar
Bacterium (registration number: LS20120060) etc..But it is unstable that single biocontrol microorganisms often show preventive effect in Field information.It is used by mixing
Biocontrol microorganisms have an effect to phytopathy original different phase, tool various ways and action site, or can be raw in plant difference
The biocontrol microorganisms that the phase of educating plays a role may be the important channel to solve the above problems.Mendoza and Sikora (2009) are ground
Study carefully and shows the nontoxic sickle-like bacteria using induction plant generation system resistance and have the strong of strong lethal effect to root-knot nematode J2
Bacillus can effectively reduce the population density of radopholus similes thorne；Khan etc. (2001) mixes pale purple purple spore bacterium and Trichoderma
The preventive effect of single biocontrol microorganisms is above after use to the preventive effect of tomato root-knot eelworm disease；Frans etc. (1992) is by Verticillum
Chlamydosporium and Pasteuria penetrans is mixed using post-processing soil, and Anastasiadis etc. (2008) will
Pale purple purple spore bacterium and bacillus firmus mixing are used in transplanting pre-treatment soil and apply again in transplanting, can be improved pair
The control efficiency of tomato root-knot eelworm disease；But endogenetic bacteria and actinomyces, which mix, uses (Peng Shuan etc., 2012), F.oxysporum
It is mixed with Rhizobium etli and declines (Martinuz, 2012) using the preventive effect to tomato root-knot eelworm disease.Using compound bacteria
Patent in terms of agent prevention and treatment root knot nematode disease has: Bacillus circulans (Bacillus circulans) bacterium and bacillus subtilis
The composite bacteria agent (Guo Jianhua and Ding Guochun, patent publication No.: CN1568710A) of (Bacillus subtilis), by deinsectization chain
Microorganism formulation made of mould, Paecilomyces lilacinus, Verticillium chlamydosporium and Bacillus sphaericus bacterium solution mixing glycerol (Li Feng, specially
Sharp publication number: CN103039534A), Paecilomyces lilacinus microbial inoculum and Verticillium chlamydosporium microbial inoculum mix microbial inoculum (Wang Xizhuo etc., patent
Publication number: CN102524307A).Obtain new, effect stability root knot nematode disease Biocontrol microorganism combination, to cucumber, tomato,
Capsicum, celery, peanut and other crops root knot nematode disease green prevention and control have important practical significance.
Summary of the invention
The object of the present invention is to provide the biocontrol fungis of prevention and treatment plant root-knot nematodes to mix microbial inoculum, it is characterised in that mixes
Microbial inoculum is mould by pale purple purple spore bacterium (Purpureocillium lilacinum) CGMCC No.9344 and pink helix poly spore
(Clonostachysrosea) culture of CGMCC No.1037 is prepared.
It is a further object of the present invention to provide purposes and its application method that two kinds of biocontrol fungis mix microbial inoculum, can individually make
It mixes and uses with or with the fertilizer such as seedling medium, organic fertilizer, chemical fertilizer, compound organic and inorganic fertilizer, for plant root-knot nematodes
Prevention and treatment.
Technical scheme is as follows:
Root-knot nematode can be survived in soil with ovum or second instar larvae, and wherein second instar larvae, which has plant, infects energy
Power.Pale purple purple spore bacterium (Purpureocillium lilacinum) HS-12 is located away from Hainan Province, the duck pool, Sanya balsam pear root knot
Line eggs have strong parasitic ability to root-knot nematode, are preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms
Center (CGMCC) preservation, deposit number are respectively CGMCC No.9344.Pink mould (the Clonostachys of helix poly spore
Rosea) HLD-1 is located away from sclerotinite (Sclerotinia sclerotiorum), has strong parasitic ability to sclerotinite, can be
It is invaded inside sclerotium in 24 hours, destroys sclerotium tissue, which was stored in Chinese microorganism strain preservation pipe in 2003
The reason committee common micro-organisms center (CGMCC) preservation (deposit number is CGMCC No.1037), and in patent of invention
It is disclosed in CN200310115191 and 201310113676.7.Applicant is directed to two kinds of survival modes of root-knot nematode, further
Pale purple purple spore bacterium HS-12 and the pink mould HLD-1 of helix poly spore are determined to Meloidogyne incognita (Meloidogyne
Incognita) the parasitics of ovum finds that the spore of two plants of bacterium has very high parasitics to ovum, and parasitic rate can reach respectively
51.0% and 44.0%, pale purple purple spore bacterium HS-12 is slightly above the pink mould HLD-1 of helix poly spore to the parasitics of root-knot nematode egg.
By two strain inoculateds in 50mL Cha Shi culture solution (component are as follows: sodium nitrate 3g, dipotassium hydrogen phosphate 1g, magnesium sulfate (MgSO4·7H2O)
0.5g, potassium chloride 0.5g, ferrous sulfate 0.01g sucrose 30g, distilled water 1000mL), 28 DEG C, 180 revs/min of shaken cultivations 2
It, is then inoculated in the fresh Cha Shi culture solution of 500mL with 2% inoculum concentration, and 28 DEG C, 180 revs/min are cultivated 3 days.Fermentation
Liquid is centrifuged 10 minutes in 5000 revs/min, and supernatant is filtered with 0.22 μm of cellulose filter membrane, obtains sterile ferment filtrate.It is sterile
Filtrate has strong lethal effect to the second instar larvae of nematode, wherein the mould 67-1 bacteria-free filtrate of pink helix poly spore is to second instar larvae
Lethal effect is than very fast, and 12 hours after processing, the corrected mortality of second instar larvae reaches 89.9%；And the sterile filter of pale purple purple spore bacterium
Liquid is handled second instar larvae 48 hours, and the corrected mortality of second instar larvae just reaches 91.3%, illustrates the pink mould HLD-1 of helix poly spore
Pale purple purple spore bacterium HS-12 bacterial strain is greater than to the lethal effect of root-knot nematode second instar larvae.
According to the power that HS-12 and HLD-1 acts on root-knot nematode egg and second instar larvae, design uses different mixing bacterium
Agent dosage form.When root-knot nematode is mainly second instar larvae in soil, the liquid bacterial agent of microbial inoculum is mixed using two kinds of fungies；Root in soil
When tie lines worm is mainly ovum, the pulvis of microbial inoculum is mixed using two kinds of fungies.Preparation method: two strain inoculateds are trained in 50mL Cha Shi
Nutrient solution, 28 DEG C, 180 revs/min shaken cultivation 2 days, the fresh Cha Shi culture solution of 500mL is then inoculated with 2% inoculum concentration
In, 28 DEG C, 180 revs/min are cultivated 3 days, and the liquid bacterial agent of two kinds of bacterium is obtained.Liquid bacterial agent deposits in 4 DEG C of refrigerators.Before use,
By the liquid bacterial agent (10 of two kinds of bacterium6~107A viable bacteria/mL) it is mixed with the ratio of 1: 1 (V: V), obtain fungi mixing liquid bacterium
Agent.Liquid bacterial agent is centrifuged 10 minutes in 5000 revs/min, after removing supernatant, is adsorbed with diatomite or turf (W/V=1: 1),
Room temperature is dried, HS-12 and HLD-1 pulvis is obtained.Before use, by the pulvis (10 of two kinds of bacterium6~107A viable bacteria/g) with 1: 1 (W: W)
Ratio mix, obtain mix fungi pulvis.The application method of biocontrol fungi mixing microbial inoculum: microbial inoculum can be added to seedling medium
In, soil can also be handled before transplanting or in transplanting, cave is applied or pouring root.Effective use concentration of biocontrol microorganisms is 106A viable bacteria/
G or mL or more, cave amount of application are the cave 30~50g/.Pouring root dosage is that every young plant pours 100mL.Two kinds of fungies mix microbial inoculum pulvis
63.7% can reach to the preventive effect of root knot nematode disease, higher than the preventive effect of single microbial inoculum；And root-knot nematode was mainly two ages in soil
When larva, the liquid bacterial agent that above two fungi mixes microbial inoculum can reach 43.3-52.4% to the preventive effect of root knot nematode disease.
By literature search, it yet there are no in document at present and above two fungi mixing microbial inoculum be used for grinding for plant root-knot nematode
Study carefully report.Therefore the present invention provides the new biocontrol fungis of prevention and treatment plant root-knot nematodes to mix microbial inoculum, and plants with other
The single biocontrol agent of object root-knot nematode mixes microbial inoculum and compares, the biocontrol fungi mix microbial inoculum be in soil nematode it is main
Existence is higher than single microbial inoculum effect using the different dosage forms for mixing microbial inoculum, control efficiency lasting stability.
Specific embodiment 1: pale purple purple spore bacterium HS-12 and the mould HLD-1 of pink helix poly spore mix pulvis to root knot nematode disease
Control efficiency
Test carries out in Plant Protection institute, Chinese Academy of Agricultral Sciences, base, Langfang.Including HS-12, HLD-1, HS-12+
HLD-1, blank control four processing, 30 young plants of every processing, 3 repetitions, being randomly arranged in area is long 6m × wide 0.7m cell
It is interior.Cucumber transplanting before, five point sampling collect nematode soil, sucrose Centrifugal Flotation method measure cell in nematode density be 300 ovum/
100g dry ground.Single microbial inoculum and mixing microbial inoculum, which are first mixed with a small amount of sandy soil and matrix, (sand: matrix=1: 1), makes biocontrol microorganisms
Final concentration is up to 106Cfu/g, cave is applied when transplanting, every 30~50g of cave.30 plants of cucumber seedlings of each processing, are repeated 3 times.It is every after transplanting
3 plants of statistics cucumber root knot numbers of cucumber seedling were taken at random every 10 days.It is counted referring to Bridge and Page (1980) severity Scaling method (table one)
Calculate disease index, disease index (%)=(strain numbers at different levels × severity series)/(investigation total strain number × highest disease series), prevention and treatment
Effect=(control disease index-processing disease index)/control disease index × 100.
One root knot nematode disease severity Scaling standard of table
The result shows that: after transplanting 50 days, HS-12 and HLD-1 pulvis is mixed to nematode preventive effect up to 63.7%, is significantly higher than
The preventive effect 41.3% and 33.0% (table two) that biocontrol microorganisms HLD-1 and HS-12 are individually handled.
The pale purple purple spore bacterium HS-12 of table two and the mould HLD-1 pulvis of pink helix poly spore mix the field to Cucumber root-knot nematode disease
Between protection effect
The pale purple purple spore bacterium HS-12 of specific embodiment 2. and the mould HLD-1 mixing liquid microbial inoculum of pink helix poly spore are to root knot line
The control efficiency of parasitosis
Test carries out in Chinese Academy of Agricultural Sciences Plant Protection Office Langfang base experimental plot 1 and experimental plot 2.Every piece of experimental plot divides
12 cells (long 6m × wide 0.7m) are divided into HS-12, HLD-1, HS-12+HLD-1 and blank control four processing, every processing three
The repetition of a cell, random alignment.30 young plants are sowed in every processing.Respectively before cucumber is transplanted 15 days, when transplanting and 10 after transplanting
It uses 100mL5 × 106Spore/mL HS-12, HLD-1 and mixing liquid microbial inoculum pouring root (table three).
Three HS-12 and HLD-1 liquid bacterial agent of table and combinations thereof field processing method
First 15 days of transplanting, five point sampling collect the soil in experimental plot respectively, and sucrose Centrifugal Flotation method measures root knot in cell
The density of line eggs and second instar larvae finds that the density of nematode second instar larvae in experimental plot 1 and experimental plot 2 is 60 J2/100 grams
Soil and 84 J2/100 grams of soil.20 days sampling survey incidences after transplanting, it is hereafter primary every 10 days sampling surveys, it investigates altogether
5 times.Investigation method is as follows: 10 plants of every cell random searching, and the soil gently pushed aside around cucumber root investigates root from three directions
The percentage for the total root long degree of root knot number Zhan fastened carries out severity Scaling, and grade scale is the same as table three.Every cell take cucumber plant 3~
5 plants, count root knot number on cucumber root.Calculate disease index and protection effect %.
To two pieces, experimental field the survey showed that, and the liquid bacterial agent mixing processing of HS-12 and HLD-1 can postpone root knot line
The disease time of parasitosis, control efficiency are stablized than single microbial inoculum.In experimental plot 1, when 20 days after cucumber transplanting, three processing it is anti-
Effect is close；At 60 days, HLD-1 liquid bacterial agent and HS-12 and HLD-1 liquid bacterial agent mix processing and prevent Cucumber root-knot nematode disease
Controlling effect is respectively 36.6% and 52.4% (table six), higher than the preventive effect (table four) of HS-12 liquid bacterial agent.In experimental plot 2, transplanting
Afterwards when 20 d, the preventive effect that HS-12 and HLD-1 liquid bacterial agent mixes is 75.8%, is significantly higher than HS-12 and HLD-1 is individually handled
Preventive effect 33.5% and 21.5% (table seven).At 60 days, preventive effect drops to 43.3%, but is higher than the preventive effect (table of single microbial inoculum
Five).
Control efficiency of the HS-12 and HLD-1 mixing liquid microbial inoculum to Cucumber root-knot nematode disease in four experimental plot 1 of table
Note: different capitalizations indicate to look into test difference using the variable Multiple Poles of Duncan up to the level of signifiance (P < 0.05).
Control efficiency of the HS-12 and 67-1 mixing liquid microbial inoculum to Cucumber root-knot nematode disease in five experimental plot 2 of table
Note: different capitalizations indicate to look into test difference using the variable Multiple Poles of Duncan up to the level of signifiance (P < 0.05).
Claims (3)
1. the biocontrol fungi for preventing and treating plant root-knot nematodes mixes microbial inoculum, which is characterized in that mix microbial inoculum by pale purple purple spore bacterium
(Purpureocillium lilacinum) CGMCC No.9344 and pink helix poly spore are mould (Clonostachys rosea)
The culture of CGMCC No.1037 is prepared；
The biocontrol fungi mix the preparation method of microbial inoculum the following steps are included:
Liquid bacterial agent is mixed after liquid fermentation and culture by two kinds of fungies and is prepared；The viable bacteria of two kinds of bacterium in the liquid bacterial agent
Number is 106~107A/mL；
After the absorption of the adsorbents such as sediment diatomite, turf of the pulvis by liquid bacterial agent after being centrifuged, it is being lower than 30 DEG C of room temperatures
Under dry after mix and be prepared；The viable count of two kinds of bacterium is 10 in the pulvis6~107A/g.
2. biocontrol fungi as described in claim 1 mixes the purposes of microbial inoculum, the prevention and treatment for plant root-knot nematodes.
3. the application method that biocontrol fungi as described in claim 1 mixes microbial inoculum, it is characterised in that exclusive use or and nursery
The mixings such as matrix, organic fertilizer, chemical fertilizer, organic fertilizer and chemical fertilizer use；For soil treatment or seedling stage cave is applied or pouring root.
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| CN106701635B (en) * | 2017-01-25 | 2020-05-05 | 南京农业大学 | Banana endophytic streptomycete with root-knot nematode killing capability and biological seedling culture substrate developed by banana endophytic streptomycete |
| CN106912500A (en) * | 2017-03-23 | 2017-07-04 | 河南省农业科学院园艺研究所 | Pale purple purple spore bacterium and composite bacteria agent and its application of Lactobacillus plantarum |
| CN107410364A (en) * | 2017-06-05 | 2017-12-01 | 陕西省蒲城美尔果农化有限责任公司 | A kind of microbial bacterial agent and its bacterial manure that can be used for preventing and treating root-knot nematode |
| CN109762743B (en) * | 2019-01-30 | 2022-07-29 | 河南省农业科学院植物保护研究所 | Gliocladium roseum, solid fermentation microbial inoculum thereof and application thereof |
| CN110129242B (en) * | 2019-06-13 | 2021-07-13 | 北京市农林科学院 | Continuous cropping resistant composite microbial preparation and preparation method thereof |
| CN111202097A (en) * | 2020-01-14 | 2020-05-29 | 河南农贝得农业科技有限公司 | Agricultural microbial agent for preventing and treating nematode diseases and preparation method thereof |
| CN111849839A (en) * | 2020-08-11 | 2020-10-30 | 江西顺泉生物科技有限公司 | Preparation and application of two composite bacterial liquids for preventing and treating root knot nematode disease |
| CN113956110A (en) * | 2021-09-22 | 2022-01-21 | 江西宝嘉生物发展有限公司 | Bio-organic fertilizer for effectively preventing and controlling weed growth and preparation method and application thereof |
| CN114634878B (en) * | 2022-03-18 | 2024-05-24 | 赤峰市农牧科学研究所 | Composite microbial preparation and application thereof in preventing and controlling root knot nematode |
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| Bio-control activity of Purpureocillium lilacinum strains in managing root-knot disease of tomato caused by Meloidogyne incognita;Singh, S., etc.;《Biocontrol Science & Technology》;20131231;第23卷(第12期);第1469-1489页 * |
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