CN102952762A - Paenibacillus polymyxa for controlling plant fungal diseases and production method thereof - Google Patents

Abstract

The invention relates to Paenibacillus polymyxa for controlling plant fungal diseases and a production method thereof and belongs to the technical field of biopesticides. A Paenibacillus polymyxa strain EBL-06 is isolated from wheat leaves and preserved in CGMCC (china general microbiological culture collection center) under the number of CGMCC NO.2377. The Paenibacillus polymyxa strain EBL-06 is capable of controlling various plant fungal diseases to increase crop yield. The Paenibacillus polymyxa is fermented by gourmet powder wastewater to prepare biocontrol agent. The Paenibacillus polymyxa biocontrol agent produced by the production method is capable of effectively controlling various plant fungal diseases, production cost is low, operation is simple, and environment pollution is decreased by effective utilization of the gourmet powder wastewater.

Description

How sticking class bud robe bacillus and the production method thereof of control fungal diseases of plants

The invention belongs to biological pesticide technical field.

Background technology

Can be divided into three classes according to the contained pollutent situation of waste water in the glutamate production process: the one, the high acidity organic waste water of high density is glutamate wastewater; The 2nd, other middle and high concentration organic waste water; The 3rd, need not process the water that cools that directly effluxes.Glutamate wastewater is that it had both contained abundant organic matter, also contained a small amount of inorganic Ao and other trace elements such as N, P, K by behind the ion exchange method extraction L-glutamic acid remaining " waste liquid ", and these materials all can be microorganism growth nutrition is provided.Other middle and high concentration organic waste water comprises: starch wastewater, sugar production wastewater, fermentation are washed and are filled with waste water, refined wastewater etc.Starch wastewater and sugar production wastewater also have some suspended matters except containing certain organic pollution materials; Fermentation washes that to fill with waste water basic identical with the glutamate wastewater ingredient, is that content is lower; It is acid that refined wastewater is sometimes, sometimes is alkalescence, and Organic pollutants material content is higher, and these organic waste waters also are one of more satisfactory source of nutrition of Microbial resources exploitation.But, if these waste water can not get reasonable utilization, not only can cause severe contamination to environment, and resource is wasted.The reported in literature gourmet powder waste water causes than overall situation and pollutes, as pointing out more than 230 of national glutamate production producers in 2005 on the Chinese environmental ecological net, 170,000 tons of year waste discharges are mostly met and are patrolled eedu without administering directly discharging enlightening place orchid. org. and cn/Article/es/envir/ptheory/water/200511/6456. html).Chinese patent (publication number: disclose a kind of method of utilizing gourmet powder waste water to prepare fermention medium and producing the biological pesticide streptomyces griseus CN 1916155A), at present, numerous monosodium glutamate wastewater treatment technology roughly were divided into for two steps, the one, the aminoglutaminic acid thalline single cell protein in the extraction waste water certainly, by this step separating thallus, approximately can remove the COD about 30 %, physical chemistry method commonly used is processed (such as flocculation, the method such as centrifugal); The 2nd, the processing of waste liquid behind the extraction thalline makes it reach emission standard or processing is rear for factory's reuse, and this also is the key issue that numerous monosodium glutamate wastewater treatment technology face, and often adopts bioremediation (such as anaerobically fermenting, biomembrance process, SBR method etc.).[0004 ］ how the full bacillus (Paenibacillus pol minister in ancient times yxa) of sticking class buds is widely distributed at occurring in nature, is a kind of important biocontrol microorganisms, and various plants fungi, Micobial Disease are all had preferably preventive and therapeutic effect.Wang Zhiwen etc. have reported a strain, and how sticking class bud pole bacterium cp one 5316 bacterial strains are to the animals and plants fungi, the bacteriosis original has resistance of wide spectrum, utilize macroporous adsorbent resin to the absorption of the antifungus active substance of many sticking class pole bacterium Cp one 5316 generations, desorption performance, filter out its optimum solution vapor, this active substance is to warm, UV stable, to trypsinase, Proteinase K has certain tolerance, responsive to the chloroform part, stable to acid, to the responsive (Wang Zhiwen of alkali, Yuan Shitao, He Liang etc., the extraction of many sticking class bud pole bacterium cp one 5316 antifungus active substances and some properties research thereof, the agro-environment science journal, 2007,26 (4): 1,464 one 1468).(Granted publication number: report is cultivated cN 11 69942c) Paenibacillus pol minister in ancient times and fermented supernatant fluid thereof can be used for preventing the vegetative bacteria bacterial wilt to Chinese patent, but also can be used to prevent and treat the plant seedling blight, broken sick, and cucumber fusarium axysporum, tomato wilt, wilt of eggplant, root rot, and plant had fairly obvious promotion growth.Chinese patent (Granted publication number: disclose Paenibacillus p river ymyxa cN 1 186441c) and can be used as a kind of composition decomposing straw in the microbe fertilizer microbial inoculum, effectively agricultural waste is too much on the one hand in the solution agriculture production, polluted-water and environment, difficult treatment, chemical fertilizer on the other hand, agricultural chemicals uses too much, soil deterioration

The contradiction that cN 10151 9639B specification sheetss are 2 // 3 pages.(Granted publication number: disclose Paenibacillus pol minister in ancient times yxa Jaas cd CN 1325635C) and just entered in the plant materials to foretell in lower concentration, well can field planting in plant materials, breeding and conduction for Chinese patent; Plant is had the growth of promotion simultaneously, improve output, the effect of disease resistance particularly has good prevention effect to soil-borne vascular bundle disease.[0 005 ］ production methods of many sticking class bud pole bacterium biological and ecological methods to prevent plant disease, pests, and erosion agent have multiple, as; (publication number: disclose the production method of a kind of many sticking class bud pole bacterium CN 1844360A), its substratum is bean dregs to Chinese patent.Chinese patent (Granted publication number: use meat substratum in distress as many sticking class bud pole bacteria fermentation culture mediums CN 1 186441C).The method of gluing class bud pole bacterium biological and ecological methods to prevent plant disease, pests, and erosion agent take gourmet powder waste water as substratum production has no bibliographical information more.

Summary of the invention

Of the present invention day provide a kind of can be in plant phyllospheric and rhizosphere field planting, and multiple pathogenic bacteria had the biocontrol microorganisms of antagonistic action-glue class bud pole bacterium EBL one 06 bacterial strains more, a kind of method of utilizing gourmet powder waste water to produce this biocontrol fungicide is provided simultaneously.Biocontrol strain provided by the present invention is that separation screening is out from the blade face of wheat by our laboratory.It is numbered EBL 1, is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, preservation date is 2008 on February 26,, and Classification And Nomenclature is many sticking class bud pole bacterium (Paenibacillus polymyxa).Deposit number is: CGMCC No. 2377.This bacterial strain has following character: this bacterial strain of l colony morphology characteristic well-grown on the PDA substratum, and single bacterium colony is half glass pearl, neat in edge, surperficial thickness is combined closely with substratum, bacterium colony center milk yellow.2 morphological features thalline are shaft-like, and gramstaining is aobvious positive, and bud is embraced end and given birth to, and packing expands.3 utilization of carbon source abilities can be utilized carbon source: Q one cyclodextrin; Glycogen; D one glucose amino acid; Methylene-succinic acid; D one galactosonic acid Y one interior vinegar; D one galacturonic acid; 2 one cyclodextrins; Glycogen; D one glucose amino acid; Methylene-succinic acid; D one galactosonic acid Y one interior vinegar; D one galacturonic acid; 2 one light yl benzoic acids; 4 one light yl benzoic acids; L one arginine; L one Radix Asparagi phthalein amine; L one Serine; Sweet ammonia phthalein one L one L-glutamic acid; D one wood sugar; D one N.F,USP MANNITOL; Phenylethylamine.Can not utilize carbon source: the pyruvic acid first is cruel; D one cellobiose; One glucose former times of p monomethyl one D; I one red liquor-saturated sugar alcohol; N one second phthalein one D glucosamine; D, L one u one phospho-glycerol; U one batanone acid; Y is once butyric acid; L one phenylalanine; L one Threonine; Putrescine.Fungi antagonistic ability bacterial strain is shown the efficient antagonistic effect of grey grape being embraced the former bacterium of pathogenic bacteria mould, scab of cucumber, Trichoderma etc. at the PDA solid medium.5 [0 021 " 2, liquid seeds preparation: in the aobvious liquid nutrient medium of autoclaved soil, the many sticking class bud pole bacterium of inoculation, 11 35oC, ZOOr/min cultivates, and obtains liquid seeds.[002 unreal 4, liquid state fermentation: to the liquid seeds through inoculation 5 % (volume) in the pretreated waste water, in 25 1 35 ℃, logical sterile air was cultivated 24 1 72 hours.[?0?。2315, aftertreatment: cultured fermented liquid is adjusted to 1 08 one 1 mouthfuls after bacterial count.Cell/ml, packing is used.

Embodiment [0.The how sticking class bud pole bacterium biological and ecological methods to prevent plant disease, pests, and erosion agent of 24) being produced by technical solution of the present invention can the multiple fungi plant disease of high-efficiency prevention and control, and nuisanceless, and production cost is low, and has effectively utilized gourmet powder waste water.Below describe enforcement of the present invention in detail by specific embodiment, purpose is to help the reader to understand better spirit of the present invention, but not as to the restriction of the scope of the present invention.[?0?。25 ］ embodiment 1: and how sticking class bud pole bacterium biological pesticide preparation [0026) the seed culture medium preparation: take by weighing 2009 potatoes, chopping was boiled in youngster's Yu tap water 30 minutes, remove by filter residue, add 209 glucose, be settled to IL, namely get the potato substratum.Be distributed in the triangular flask of 5 lL 121oC fire bacterium 20min.[?0?。27) fermention medium preparation: remove the gourmet powder waste water of bacterioprotein through flocculation than the dilution of adding gL tap water, and regulate pH to 6 with sodium hydroxide. 0.[?0?。25 ］ liquid seeds preparation: in the potato liquid nutrient medium, inoculate many sticking class bud pole bacterium, in 30 ℃, shaking flask ZO0r/min, constant-temperature table shaking culture 24 hours obtains liquid seeds.[?0?。29 " leavened prod: in the fermentor tank of 10L, 121 ℃ of skilful min of fiery bacterium are according to the inoculum size inoculation liquid seeds of 5 %; at 30 ℃ of constant temperature, under the condition of logical sterile air, fermented 2 to 3 days; behind the sampling counting packing under the aseptic condition with above-mentioned fermention medium tinning.[?0?。30 ］ embodiment 2: to the antagonistic action of Various Diseases fungal pathogens [0 031 ］ bacterial strain is the pathogenic fungi of 1 cm at the dull and stereotyped center inoculation of PDA solid medium diameter, the good how sticking class bud pole bacterium of distance pathogenic fungi colony edge Zcm place inoculation (point connects) activation, observed the inhibition zone size on the 6th day, find that pathogenic fungi ash grape is embraced the pathogenic bacteria (Cladosporium sp .) of mould (Bot Xiang tis cinerea), scab of cucumber and wooden mould (T richoderma sP .) etc. all has significant antagonistic action.[?0?。32) embodiment 3: to scab of cucumber prevention effect [0.When 33 ］ cucumber seedling in the greenhouse basin alms bowl grows to four true leaves, how sticking class bud pole bacterium bacterium liquid spraying is processed blade once with 107/m1, inoculate the former bacterium of black spot after interval l days, spray l after interval l days again/how sticking class bud pole bacterium bacterium liquid is once for ml, replace the cucumber seedling of many sticking class bud pole bacterium bacterium liquid sprayings as contrast take clear water, the state of an illness of continuous observation cucumber seedling finds that many sticking class bud pole bacterium processing have obvious prevention effect to scab of cucumber.

Claims (4)

1. a strain has the how sticking class bud pole bacterium (Paenibaclllus polymyxa) of controlling plant diseases effect, it is characterized in that: these many sticking class bud pole bacterium are preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, deposit number: CGMCC No. and 2377.

2. described many sticking class bud pole bacterium of claim 1, it is characterized in that: this bacterial strain is well-grown on the POA substratum, single bacterium colony is half glass pearl, neat in edge, surperficial thickness, be combined closely with substratum, bacterium colony center milk yellow, thalline is shaft-like, and gramstaining is aobvious positive, bud is embraced end and is given birth to, the swollen people of packing; Oneself is delivered to the GenBank database partial sequence of 165 rDNA genes of this bacterial strain, and accession number is EF545556.

3. prepare the production method of anti-Plant diseases biological and ecological methods to prevent plant disease, pests, and erosion agent with the above many sticking class bud pole bacterium of claim, it is characterized in that: be the described many sticking class bud pole bacterium of inoculation of medium claim 1 of gourmet powder waste water in main component.

4. the described production method of claim 3 is characterized in that: with 5 one 15 times of gourmet powder waste water dilute with waters, regulating pH is 5 one 7, autoclaving, the described many sticking class bud pole bacterium of inoculation claim 1, in 25 1 35 ℃, logical sterile air was cultivated 24 1 72 hours.