CN103315005B

CN103315005B - Composite microbial fertilizer bactericide for disease prevention and growth promotion of tobacco and application thereof

Info

Publication number: CN103315005B
Application number: CN201310249962.6A
Authority: CN
Inventors: 顾金刚; 王倩; 李世贵; 张良; 刘好宝; 雷强; 屈健康
Original assignee: Institute of Agricultural Resources and Regional Planning of CAAS
Current assignee: Institute of Agricultural Resources and Regional Planning of CAAS
Priority date: 2013-06-21
Filing date: 2013-06-21
Publication date: 2015-04-29
Anticipated expiration: 2033-06-21
Also published as: CN103315005A

Abstract

The invention discloses a composite microbial fertilizer bactericide for disease prevention and growth promotion of tobacco and an application thereof. The bactericide can be used for promoting tobacco growth and/ or preventing tobacco disease, and comprises active components namely Trichoderma ressei ACCC 30150 and Streptomyces silaceus ACCC 40021. The bactericide is environment-friendly, can inhibit dug resistance development of a disease pathogen, is favor of popularization of green food and organic agriculture, is low in cost and non-pollution, and safe to tobacco crops. By introducing antagonistic microbe in soil, through biological control, fungus and bacterial diseases can be effectively prevented, environment is effectively improved, agroecological system balance is maintained, and a foundation for sustainable development of tobacco can be laid.

Description

A kind of composite microbial fertilizer bactericide for tobacco disease prevention growth-promoting and application thereof

Technical field

The present invention relates to a kind of composite microbial fertilizer bactericide for tobacco disease prevention growth-promoting and application thereof.

Background technology

Tobacco is Chinese important cash crop, and its quality quality is closely related with productivity effect.Tobacco bred, weather condition, cultivation step, soil and Fertilization, complete processing etc. all have material impact to Flue-cured tobacco Quality, and fertilizer practice affects one of most important factor of Flue-cured tobacco Quality.For a long time, in China's cured tobacco production, ubiquity heavy dressing inorganic fertilizer and despises the phenomenon of fertilizer, owing to for a long time using chemical fertilizer in a large number and Rate of Organic Fertilizer reduces year by year, cause that ecological environment of soil worsens, the low and quality of tobacco of utilization rate of fertilizer becomes bad, utilizability reduces, become one of main limiting factor of current quality and efficiency cured tobacco production.In recent years, that produces along with Tobacco develops rapidly, and cultivation technique is constantly reformed, and tobacco planting system is formed gradually, and tobacco nutrient research seems particularly important, and technique of balanced fertilizer becomes the core technology that High Quality Tobacco is produced.For strengthening the Sustainable development of tobacco leaf production, improve constantly quality of tobacco, cured tobacco production increases the popularizing application of fertilizer year by year.Many research shows, fertilizer and inorganic fertilizer compounding application are conducive to growing of flue-cured tobacco.

China's tobacco special microorganism fertilizer developing history is not long, but the use of special fertilizer has played vital role to raising China's leaf tobacco production level and quality of tobacco.Enter the new millennium, China's tobacco industry develops growth rapidly, also increasing to the demand of sound tobacco.Improving Chinese-style cigarette competitive power in the world needs sound tobacco to ensure.Therefore, the production of sound tobacco proposes requirements at the higher level to fertilizer special for tobacco.High-quality compost is good organic fertilizer and soil improvement agent, matured compost is not refer to simply through long-term composting, most of organic matter be decomposed and almost do not have the compost of organic energy and biologic activity, and refer to that labile organic matter is degraded in compost makes, bring disadvantageous effect to after using in farmland soil and plant growth.

Summary of the invention

The object of this invention is to provide and a kind ofly promote tobacco growing and/or the control microbial inoculum of tobacco diseases and microbial organic fertilizer and at control tobacco diseases with promote in tobacco growing application.

The microbial inoculum of promotion tobacco growing provided by the present invention and/or control tobacco diseases, its activeconstituents is made up of Trichodermareesei (Trichoderma reesei) and yellow ocher streptomycete (Streptomyces silaceus); Described Trichodermareesei (Trichoderma reesei) is Trichodermareesei (Trichoderma reesei) ACCC 30150, and described yellow ocher streptomycete (Streptomyces silaceus) is yellow ocher streptomycete (Streptomyces silaceus) ACCC 40021.

In one embodiment of the invention, the colony forming unit number of described Trichodermareesei (Trichoderma reesei) and yellow ocher streptomycete (Streptomyces silaceus) is than being 1:1.

Described microbial inoculum also can comprise carrier, that preferred pesticide field is commonly used and at the carrier being biologically inertia.Described carrier can be solid carrier or liquid vehicle.Described solid carrier is mineral material, vegetable material or macromolecular compound; Described mineral material is at least one in clay, talcum, kaolin, montmorillonite, white carbon, zeolite, silica and diatomite; Described vegetable material is at least one in Semen Maydis powder, bean powder and starch; Described macromolecular compound is polyvinyl alcohol and/or polyglycol.Described liquid vehicle is organic solvent, vegetables oil, mineral oil or water; Described organic solvent is decane and/or dodecane.In described microbial inoculum, described activeconstituents can with by cultivate viable cell, the fermented liquid of viable cell, the filtrate of cell culture or cell and filtrate the form of mixture exist.Described viable cell can be conidium, chlamydospore, mycelia or contains the mycelial form of conidium and mycelia, is preferably conidium or chlamydosporic form.The formulation of described composition can be multiple formulation, as liquor, emulsion, suspension agent, pulvis, granule, wettable powder or water dispersible granules.

As required, tensio-active agent (as polysorbas20, tween 80 etc.), tackiness agent, stablizer (as antioxidant), pH adjusting agent etc. can also be added in described microbial inoculum.

Trichodermareesei (Trichoderma reesei) ACCC 30150 in preparation of the present invention and the spore quantity of yellow ocher streptomycete (Streptomyces silaceus) ACCC 40021 change with formulation and the method used.In solid preparation, conidial quantity is (1 × 10 ⁵)-(1 × 10 ⁸) individual spore/gram, preferred (1 × 10 ⁶)-(1 × 10 ⁷) individual spore/gram.For liquid preparation, in the flushing liquor after dilution, conidial content is generally (2.0 × 10 ⁴)-(5.0 × 10 ⁷) individual spore/mL, preferably 10 ⁷individual spore/mL.

Chemical pesticide that described preparation can be applicable to other with the use of, thus reduce the consumption of chemical pesticide agent, reduce the pollution to environment.

The microbial organic fertilizer of promotion tobacco growing provided by the present invention and/or control tobacco diseases, is made up of above-mentioned promotion tobacco growing and/or the control microbial inoculum of tobacco diseases and fertilizer.

The microbial inoculum of above-mentioned promotion tobacco growing and/or control tobacco diseases or the microbial organic fertilizer containing described microbial inoculum are promoting that the application in tobacco growing and/or control tobacco diseases also belongs to protection scope of the present invention.

In above-mentioned application, during described microbial inoculum can be manured into soil, the consumption of described microbial inoculum is that every square metre of soil applies 3 × 10 ⁹-4.5 × 10 ⁹microbial inoculum described in cfu, as every square metre of soil applies 3 × 10 ⁹microbial inoculum described in cfu.In one embodiment of the invention, the consumption of described microbial inoculum is that every square metre of soil applies 1.5 × 10 ⁹yellow ocher streptomycete described in cfu (Streptomyces silaceus) ACCC 40021 and 1.5 × 10 ⁹cfu Trichodermareesei (Trichoderma reesei) ACCC 30150.

In above-mentioned application, described microbial inoculum can apply in seedling medium, and the consumption of described microbial inoculum is that every gram of seedling medium adds 2 × 10 ⁸-4.5 × 10 ⁸microbial inoculum described in cfu, as every gram of seedling medium adds 3 × 10 ⁸microbial inoculum described in cfu.In one embodiment of the invention, the consumption of described microbial inoculum is that every gram of seedling medium applies 1.5 × 10 ⁸yellow ocher streptomycete described in cfu (Streptomyces silaceus) ACCC 40021 and 1.5 × 10 ⁸cfu Trichodermareesei (Trichoderma reesei) ACCC 30150.

In above-mentioned microbial inoculum, mentioned microorganism fertilizer and above-mentioned application, described promotion tobacco growing can be 7 kinds, 6 kinds, 5 kinds, 4 kinds, 3 kinds, 2 kinds or a kind in a1-a7:

A1, promotion tobacco seedling growth;

The yield of tobacco of a2, raising tobacco;

A3, shorten tobacco time long-term to Moindawang;

The leaf of a4, raising dome phase tobacco top is long;

The leaf of a5, raising dome phase tobacco top is wide;

The leaf of a6, raising dome phase tobacco waist leaf is long;

The leaf of a7, raising dome phase tobacco waist leaf is wide;

Described tobacco diseases can be 3 kinds, 2 kinds or a kind in tobacco bacterial wilt, Alternaria alternate and black shank.

Further, what described promotion tobacco seedling growth can be in following b1-b7 is all or part of:

The growth of b1, promotion tobacco root system;

The growth of b2, promotion cigarette seedling stem;

The growth of b3, promotion cigarette seedling leaf;

B4, raising cigarette seedling cauline leaf weight;

B5, raising tobacco root system weight;

The seedling rate of b6, raising tobacco;

B7, shortening tobacco seedling time.

Further, what the growth of described promotion tobacco root system was presented as in c1-c4 is all or part of:

C1, raising tobacco root system surface-area;

C2, raising tobacco root system volume;

C3, raising tobacco root system mean diameter;

C4, the total root of raising cigarette Miao are long;

The growth of described promotion cigarette seedling stem is presented as and improves the high and/or stem girth of cigarette seedling stem;

The growth of described promotion cigarette seedling leaf is presented as that raising cigarette seedling leaf is long and/or leaf is wide and/or the number of blade.

Above, described tobacco specifically can be cloud and mist 87 or flue-cured tobacco K326.

Above, the microbial organic fertilizer containing described microbial inoculum can be made up of described microbial inoculum and the adsorbing base comprising fertilizer.In one embodiment of the invention, comprise organic fertilizer ground substance material to be mixed by Chicken dung, edible fungi residues and sugar refinery bagasse.

Experiment proves, that the tobacco (T3) of the microbial organic fertilizer process that Trichodermareesei (Trichoderma reesei) ACCC 30150 forms with adsorbing base with the microbial inoculum of yellow ocher streptomycete (Streptomyces silaceus) ACCC 40021 is compared with the tobacco (CK) with identical adsorbing base process with activeconstituents, 11 days in advance long-term to Moindawang, the waist leaf leaf length of T3 is 1.11 times of CK, the waist leaf leaf of T3 is wide is 1.18 times of CK, the top leaf length of T3 is 1.30 times of CK, the top leaf of T3 is wide is 1.33 times of CK, the yield of tobacco of T3 is 1.23 times of CK, T3 is 63.5% to the prevention effect of balck shank, be 86.7% to the prevention effect of red-star like disease, be 100% to the prevention effect of bacterial wilt.The tobacco (T2) of the microbial organic fertilizer process formed with adsorbing base with the microbial inoculum that activeconstituents is yellow ocher streptomycete (Streptomyces silaceus) ACCC 40021 is compared with the tobacco (CK) with identical adsorbing base process, 11 days in advance long-term to Moindawang, the waist leaf leaf length of T2 is 1.07 times of CK, the waist leaf leaf of T2 is wide is 1.14 times of CK, the top leaf length of T2 is 1.30 times of CK, the top leaf of T2 is wide is 1.26 times of CK, the yield of tobacco of T2 is 1.19 times of CK, T2 is 73.0% to the prevention effect of balck shank, be 66.7% to the prevention effect of red-star like disease, be 100% to the prevention effect of bacterial wilt.The microbial inoculum of promotion tobacco growing of the present invention and/or control tobacco diseases is environmentally friendly, can suppress resistance and the development of drug resistance of disease cause of disease, be conducive to the popularization of green food and Organic farming, and cost is low, pollution-free, to tobacco plants safety.In soil, introduce antagonistic microbe, effectively can prevent and treat fungi, bacterial disease can effective environmental protect by biological control, maintain agroecosystem and balance, for walking the backing that tobacco sustainable development road provides powerful.

Accompanying drawing explanation

Fig. 1 is the affinity measurement result of Trichodermareesei (Trichoderma reesei) ACCC30150 and yellow ocher streptomycete (Streptomyces silaceus) ACCC 40021.

A. yellow ocher streptomycete (Streptomyces silaceus) ACCC 40021; B. Trichodermareesei (Trichoderma reesei) ACCC 30150; C. yellow ocher streptomycete (Streptomyces silaceus) ACCC 40021 fermented liquid; Left figure is opposite culture method, and right figure is Odontothrips loti.

Fig. 2 is that tobacco root system SOD, POD, CAT and PAL are active.

Embodiment

Following embodiment is convenient to understand the present invention better, but does not limit the present invention.

Experimental technique in following embodiment, if no special instructions, is ordinary method.

Test materials used in following embodiment, if no special instructions, is and purchases available from routine biochemistry reagent shop.

Quantitative test in following examples, if no special instructions, all arranges and repeats experiment for three times, results averaged.

Biocontrol microorganisms Trichodermareesei (Trichoderma reesei) ACCC 30150 used in following embodiment and yellow ocher streptomycete (Streptomyces silaceus) ACCC 40021 was all concealed in China Committee for Culture Collection of Microorganisms's agricultural microorganism center (being called for short ACCC) before the applying date of the application, the collection day of Trichodermareesei (Trichoderma reesei) ACCC 30150 is on December 1st, 1991, the collection day of yellow ocher streptomycete (Streptomyces silaceus) ACCC 40021 is on December 1st, 1991, from collection, the public can obtain this two bacterial strains from China Committee for Culture Collection of Microorganisms's agricultural microorganism center.

Balck shank pathogenic bacterium used in following embodiment are phytophthora parasitica Oomycete fungal phytophthora parasitica tobacco pathogen mutation (Phytophthora parasitica var.nicotianae), China Committee for Culture Collection of Microorganisms agricultural microorganism center deposit number be ACCC 38065, collection day is on August 10th, 2011, from collection, the public can obtain this bacterial strain, all referred to as black shank bacterium or black shank pathogen in following embodiment from China Committee for Culture Collection of Microorganisms's agricultural microorganism center.

Red-star like disease pathogenic bacterium used in embodiment are rod method (Alternaria alternata), China Committee for Culture Collection of Microorganisms agricultural microorganism center deposit number be ACCC38066, collection day is on August 10th, 2011, from collection, all referred to as Alternaria alternate germ in following embodiment.

Root rot (samping off) pathogenic bacterium used in embodiment are melon and fruit corruption mould (Pythium aphanidermatum), China Committee for Culture Collection of Microorganisms agricultural microorganism center deposit number be ACCC 38064, collection day is on August 10th, 2011, all referred to as Tobacco seedling diseases germ in following embodiment from collection.

Embodiment 1, Trichodermareesei (Trichoderma reesei) ACCC 30150 and yellow ocher streptomycete (Streptomyces silaceus) ACCC 40021 biocontrol microorganisms combine the impact on tobacco seedling growth and disease resistance thereof

The present embodiment take tobacco as test materials, indoor antagonistic effect and Vivo Studies on Screening is adopted to test the method combined, have studied Trichodermareesei (Trichoderma reesei) ACCC 30150 and yellow ocher streptomycete (Streptomyces silaceus) ACCC 40,021 two kinds of affinities that biocontrol microorganisms is composite and antibacterial broad spectrum, and analyze the impact of biocontrol microorganisms combination on tobacco seedling growth, Defense Enzyme Activities and balck shank preventive effect.Result shows: (1) two biocontrol strain is without mutual restraining effect, there is higher affinity, and two bacterial strains itself and meta-bolites thereof all have higher bacteriostatic activity and wider antimicrobial spectrum to the pathogenic bacteria (balck shank, samping off and red-star like disease) under different niches.(2) two strain combinations can affect the morphogenesis of root and overground part, promote the growth of tobacco root system and stem, leaf, significantly can increase the long and root average diameter of underground part root system volume, root system total surface area, root and overground part stem is high, stem girth, leaf are long, leaf is wide and biomass, there is significant growth-promoting usefulness, and the growgh promoting effects of biocontrol microorganisms combination is better than independent inoculation.(3) biocontrol microorganisms combination can significantly improve the Defense Enzyme Activities such as tobacco root system SOD, POD and CAT, reaches 69.3%, have significant diseases prevention usefulness to the prevention effect of black shank.This research shows, Trichodermareesei (Trichoderma reesei) ACCC 30150 and yellow ocher streptomycete (Streptomyces silaceus) ACCC 40021 is the synergistic combination having compared with high-affinity, can synergistic function be played after two bacterial strains are composite in diseases prevention, growth-promoting, more effectively can promote the injury that tobacco seedling growth and control black shank cause.Specific experiment method and experimental result as follows:

1 materials and methods

1.1 test materials

1.1.1 for studying thing

Flue-cured tobacco, kind is K326, and seed is provided by China Tobacco Seed Yuxi Co., Ltd..

1.1.2 strains tested

Biocontrol microorganisms: Trichodermareesei (Trichoderma reesei) ACCC 30150 and yellow ocher streptomycete (Streptomyces silaceus) ACCC 40021.

Tobacco pathogenic bacteria: black shank pathogen, Alternaria alternate germ and Tobacco seedling diseases germ.

Getting respectively culturedly has black shank pathogenic bacteria spore layer culture dish, with transfering loop scraping spore, adds 20mL sterilized water, and adds 1% tween 80 of 1mL, spore suspension impouring is equipped with in the spore dispersion bottle of quartz sand granulated glass sphere, vibration 20-30min.By the filtered through gauze of spore suspension through sterilizing, measure spore count with blood counting chamber, with sterilized water, filtrate is adjusted to 10 ⁵-10 ⁶cfumL ^-1for subsequent use.The preparation of yellow ocher streptomycete (Streptomyces silaceus) ACCC 40021 and Trichodermareesei (Trichoderma reesei) ACCC 30150 spore suspension is the same, and it is 10 that spore liquid concentration is ⁸-10 ⁹cfumL ^-1.Wherein, yellow ocher streptomycete (Streptomyces silaceus) ACCC 40021 substratum used is Gause I substratum (with embodiment 2), and Trichodermareesei (Trichoderma reesei) ACCC 30150 substratum used is PDA substratum (with embodiment 2).

1.1.3 nursery material

Floating seedlings matrix used is commercial matrix, all purchases in Chinese Academy of Agricultural Sciences flowers market, for subsequent use after autoclaving 2h at various nursery material mixing is evenly placed on 121 DEG C.The special nursery fertilizer of tobacco (production of Sichuan gold leaf chemical fertilizer company limited), nutrient content is N:P ₂o ₅: K ₂o=19:10:20, nitric nitrogen accounts for 60% of total nitrogen.Nursery pond specification is that 35.0cm(is long) × 28.0cm(is wide) × 20.0cm(is high), floating disc specification is 26.0cm × 33.0cm × 6.5cm, 70 holes.

1.2 test design

1.2.1 the consistency of bacterial strain measures

Interaction and affinity thereof between two strain biocontrol microorganisms is measured by culture dish mixed culture method and Odontothrips loti.

1.2.2 Antibacterial Activity

Opposite culture method: with reference to (EL-TarabiLy K A such as EL-Tarabily, Nassar A H, Hardy G E.St.J, Sivasithamparam K.Plant growth promotion and biological control of Pythium aphanidermatum, a pathogen of cucumber, by endophytic actinomycetes.Journal of Applied Microbiology, 2009, 106 (1): 13-26) method, after cultivating 3d under each confession examination pathogenic bacteria and biocontrol microorganisms are placed in 25 DEG C of dark conditions, Bechtop buys bacterium cake with aseptic punch tool (5mm) along its colony edge, by Trichodermareesei (Trichoderma reesei) ACCC 30150 respectively with black shank, on on the same straight line in 1.5cm place, PDA substratum Shang Ju center 2 of samping off and red-star like disease pathogenic bacteria pure culture biscuits involvng inoculation, PDA and Gause I equal-volume mixed culture medium choose two long and short dash line inoculation yellow ocher streptomycete (Streptomyces silaceus) ACCC 40021, at distance parallel lines equidistant inoculation tobacco pathogenic bacteria bacterium cake, simultaneously only to connect the substratum of pathogenic bacteria and biocontrol microorganisms in contrast, often process repetition 3 times, be inverted constant temperature culture under 28 DEG C of conditions, observed and recorded after 3d.Mycelial growth inhibition rate (%)=(contrast colony diameter-process colony diameter)/contrast colony diameter × 100.

Odontothrips loti: Trichodermareesei (Trichoderma reesei) ACCC 30150 and yellow ocher streptomycete (Streptomyces silaceus) ACCC 40021 is seeded in respectively in Gause I and PDA liquid nutrient medium (with embodiment 2) and cultivates 2-3d(190rpm, 28 DEG C) to make fermented liquid for subsequent use.It is 1 × 10 that cultured tobacco pathogenic bacteria is made spore concentration ⁶cfumL ^-1bacteria suspension, get pathogenic bacteria bacteria suspension 0.1mL and be applied on PDA flat board, then put 1 Oxford cup in each culture dish centre, add the biocontrol microorganisms fermented liquid of 0.2mL wherein, each process repeats 3 times, 28 DEG C of cultivations, every 24h observed and recorded inhibition zone radius.

1.2.3 Vivo Studies on Screening test

This is tested and carries out at Chinese Academy of Agricultural Sciences's agricultural resource and agricultural regionalization institute heliogreenhouse 2 to May in 2012.5 process are established in test, contrast (CK): only add isopyknic sterilized water in seedling medium, do not inoculate tobacco black shank bacterium, also do not inoculate biocontrol microorganisms; T0: only singly connect tobacco black shank bacterium in seedling medium, do not inoculate biocontrol microorganisms; T1: inoculate tobacco black shank bacterium and Trichodermareesei (Trichoderma reesei) ACCC 30150 in seedling medium; T2: inoculate tobacco black shank bacterium in seedling medium and singly meet yellow ocher streptomycete (Streptomyces silaceus) ACCC40021; T3: inoculate tobacco black shank bacterium and Trichodermareesei (Trichoderma reesei) ACCC 30150 and yellow ocher streptomycete (Streptomyces silaceus) ACCC 40021 in seedling medium.

Biocontrol microorganisms is inoculated: the spore liquid prepared (the same 1.1.2 of preparation method) and appropriate seedling medium are mixed, make seedling medium miospore content be 3 × 10 ⁸cfug ^-1.Wherein, in T1 process, the inoculum size of Trichodermareesei (Trichoderma reesei) ACCC 30150 is 3 × 10 ⁸cfug ^-1, in T2 process, the inoculum size of yellow ocher streptomycete (Streptomyces silaceus) ACCC 40021 is 3 × 10 ⁸cfug ^-1, in T3 process, yellow ocher streptomycete (Streptomyces silaceus) ACCC 40021 is respectively 1.5 × 10 with the inoculum size of Trichodermareesei (Trichoderma reesei) ACCC 30150 ⁸cfug ^-1, contrast and T0 equivalent sterilized water replace biocontrol microorganisms liquid.

Pathogenic bacteria inoculates: adopt leaching root method inoculation tobacco black shank bacterium when cigarette seedling bears the 6th true leaf, inoculum size is every cave 5mL, and spore liquid concentration is 3 × 10 ⁵cfumL ^-1, contrast equivalent sterilized water replaces pathogenic bacteria liquid.Each seedling pan (70 strain) is 1 repetition, often processes repetition 3 times.

1.3 test items and method

1.3.1 the mensuration of Agronomic trait

Seedling rate and the breeding time thereof of each process is instituted an inquiry after sowing 5d; Neat and the representational 10 strain cigarette seedlings of each process random selecting growth after seedling, measure dry weight and the fresh weight of stem high, effective blade number, maximum leaf length, maximum width of blade, stem girth, overground part and underground part respectively, concrete reference tobacco economical character investigation method (YC/T142-1998).Adopt root scanner (Epson Perfection V700 PHOTO) to scan root system sample and obtain digitized image, utilize root system analytical system software (WinRHIZO V2008a, Regent Instrument Inc, Canada) analyze, obtain Root morphology parameter, comprise total root length, root system total surface area, root system volume and root average diameter and diameter and be respectively≤0.2mm(radicula), medium of 0.2-0.6mm() and the thick root of > 0.6mm() root length in scope.

1.3.2 defensive ferment index determining

The representational tobacco root system of random selecting from each processing sample, extracting waste root system 0.5g puts into the mortar of precooling or adds liquid nitrogen grinding, add polyvinylpyrrolidone (PVP) 0.1g therebetween, ground sample is proceeded to 10mL centrifuge tube, add the extracting solution 5mL of 4 DEG C of precoolings, 10, the centrifugal 30min of 000rpm, abandon precipitation, get supernatant for superoxide-dismutase (SOD), peroxidase (POD), phenylalanine ammonia lyase (PAL) and catalase (CAT) determination of activity.Wherein, SOD determination of activity adopts nitroblue tetrazolium(NBT) method [Shanghai Institute of Plant Physiology of Chinese Academy of Science.Shanghai Society for Plant Physiology.Modern Experimental Manual of Plant Physiology.Beijing:Science Press (Science Press), 1999:314-315 (in Chinese)]; POD determination of activity adopts guaiacol colorimetric method [Chen J-X (Chen Jianxun), Wang X-F (Wang Xiaofeng) .Experimental guidance of plantphysiology.Guangzhou:South China University of Technology Press (press of South China Science & Engineering University), 2000:119-122 (in Chinese)]; The mensuration of PAL activity answers method [the Xue Y-L (Xue Yinglong) of dragon and Ou Yangguang with reference to Xue, OU Y-G-C (Ou Yangguangcha), AO S-G (Australia continue root) .Studies on plantphenylalanine ammonia-lyase (PAL) .Journal of PlantPhysiology (plant physiology journal), 1993,19 (3): 301-305 (in Chinese)]; The mensuration of CAT activity is with reference to Cakmak [Cakmak I, Marschner H.Magnesium deficiency and high light intensity enhance activities of superoxide dismutase, ascorbate peroxidase, and glutathione reductase in bean leaves.Plant Physiology, 1992,98:1222-1227] method.

1.3.3 balck shank a situation arises investigation

A situation arises to institute an inquiry balck shank after inoculation black shank bacterium, and disease scale according to tobacco diseases classification investigation method YC/T23222-2008, and calculates sickness rate, disease index and prevention effect.

Sickness rate (%)=(morbidity strain number/investigate total strain number) × 100;

Disease index=[∑ (diseased plant at different levels or the number of sheets × this sick level value)]/(investigating total strain number or the number of sheets × superlative degree value) × 100;

Relative prevention effect (%)=(contrast disease index-process disease index)/contrast disease index × 100.

1.4 data processing

Data, after Excel 2007 arranges, adopt SAS 9.0 to carry out variance analysis, adopt the significance of difference (P<0.05) between Duncan new multipole difference method inspection different treatment, draw to be completed by Excel 2007.

2 results and analysis

The affinity measurement result of 2.1 Trichodermareeseis (Trichoderma reesei) ACCC 30150 and yellow ocher streptomycete (Streptomyces silaceus) ACCC 40021

Adopt mixed culture and Odontothrips loti to measure two bacterial strain consistencies, observe the growth of two strain bacterium after 72h and make situation mutually, result is as Fig. 1.From mixed culture result, there is not mutual antagonistic action in two strain bacterium in whole process of growth.Be embodied in yellow ocher streptomycete (Streptomyces silaceus) ACCC 40021 mycelia normal growth, produce spore; Trichodermareesei (Trichoderma reesei) ACCC 30150 mycelial growth is rapid, and after cultivating 3d, mycelia becomes yellow-green colour from white, starts large volume production spore subsequently; Two strain bacterium contact part mycelia normal growths, and increase to some extent in this part Trichodermareesei (Trichoderma reesei) ACCC 30150 spore amount.From Oxford cup measurement result, in the cup of Oxford, yellow ocher streptomycete (Streptomyces silaceus) ACCC 40021 fermented liquid is to Trichodermareesei around it (Trichoderma reesei) ACCC 30150 unrestraint effect, and Trichodermareesei (Trichoderma reesei) ACCC 30150 sporulation quantity increases to some extent.In a word, measured the growing state of two strain bacterium by comparative analysis mixed culture and fermented liquid, determine two strain bacterium be no matter thalline itself or between fermented liquid and bacterial strain all without antagonistic action, illustrate that two strain biocontrol microorganisms have good affinity, can combine.

The bacteriostatic activity of 2.2 Trichodermareeseis (Trichoderma reesei) ACCC 30150 and yellow ocher streptomycete (Streptomyces silaceus) ACCC 40021 pairs of tobacco pathogenic bacterias

In order to determine two strain bacterium fungistatic effects further, adopting dull and stereotyped opposite culture and Odontothrips loti to measure two strain bacterium and to grow tobacco to 3 the antagonistic action of pathogenic bacteria, the results are shown in Table 1.Result shows, two strain bacterium all can suppress 3 to grow tobacco germ mycelial growth to some extent when opposite culture, it is stronger that Trichodermareesei (Trichoderma reesei) ACCC 30150 passes black shank bacterium bacteriostatic activity to soil, it is comparatively strong that yellow ocher streptomycete (Streptomyces silaceus) ACCC 40021 passes da mping-off fungi bacteriostatic activity to soil, and two strain bacterium are suitable to leaf portion tobacco brown spot pathogen bacteriostatic activity.Odontothrips loti measures and finds, yellow ocher streptomycete (Streptomyces silaceus) ACCC 40021 fermented liquid bacteriostatic activity is totally greater than Trichodermareesei (Trichoderma reesei) ACCC 30150, two strain fermented liquid and shows substantially identical to the bacteriostatic activity Changing Pattern of 3 kinds of pathogenic bacterias and bacteriostatic activity during opposite culture.Illustrate that two bacterial strains itself and meta-bolites thereof all have stronger bacteriostatic activity and wider antimicrobial spectrum to the pathogenic bacteria under soil and leaf portion different niches.

The bacteriostatic activity of table 1 Trichodermareesei (Trichoderma reesei) ACCC 30150 and yellow ocher streptomycete (Streptomyces silaceus) ACCC 40021 pairs of tobacco pathogenic bacterias

Note: in table, data are mean value ± standard errors.

2.3 biocontrol microorganisms are on the impact of seedling rate and growing process

Test the time in after planting starting observed and recorded seedling rate and cigarette seedling and enter each breeding time, the results are shown in Table 2.Result shows, different biocontrol microorganisms process compared with the control seedling rate and growing process differs greatly.As known from Table 2, seedling rate shows as inoculation Trichodermareesei (Trichoderma reesei) ACCC 30150 and significantly reduces seedling rate, other biocontrol microorganisms leaf emergence all significantly increase, and singly connect yellow ocher streptomycete (Streptomyces silaceus) ACCC40021 seedling rate and are significantly higher than combined inoculation again.For cigarette seedling growing process, compared with the control, combined inoculation and singly meet yellow ocher streptomycete (Streptomyces silaceus) ACCC 40021 seeding stage all than contrast 1d in advance, singly connecing Trichodermareesei (Trichoderma reesei) ACCC 30150 seeding stage postpones 2d; Combined inoculation and singly connect the cigarette seedling early growth speed of yellow ocher streptomycete (Streptomyces silaceus) ACCC 40021, early stage breeding time time shorten, along with the continuity of tobacco seedling growth, the difference of time breeding time increases gradually, the seedling time is maximum shortens 6d, and singly meeting Trichodermareesei (Trichoderma reesei) ACCC 30150 owing to affecting the seeding stage, other growth phases are postponed compared with the control all to some extent, last seedling time retardation 2d.It can thus be appreciated that, mainly through suppressing or promoting Seed Germination cigarette seedling seedling rate and seeding stage after biocontrol microorganisms inoculation seedling medium, the seeding stage in advance or delayedly affect again the whole growing process of cigarette seedling further.

The impact of table 2 seedling rate and cigarette seedling growing process

Process	Seedling rate (%)	Sowing time/moon-	Seeding stage/moon-	Cross phase/moon-	The phase of the taking root/moon-	Become seedling stage/moon-
							CK	94.4±0.4c	02-20	02-27	03-08	03-26	04-24
T0	94.3±0.5c	02-20	02-27	03-08	03-26	04-28
							T1	93.3±0.4d	02-20	02-29	03-10	03-28	04-26
T2	96.4±0.4a	02-20	02-26	03-05	03-23	04-20
							T3	95.4±0.4b	02-20	02-26	03-05	03-22	04-18

Note: in table, data are mean value ± standard errors, the different letter representation otherness of same column numerical value reaches 5% conspicuous level.Lower same.

2.4 biocontrol microorganisms are on the impact of cigarette seedling Agronomic trait and biomass

2.4.1 cigarette seedling overground part Agronomic trait

In order to clearly inoculate the impact that biocontrol microorganisms grows cigarette seedling overground part, the stem measuring cigarette seedling when seedling is high, stem girth, leaf long and the Agronomic trait such as the number of blade, the results are shown in Table 3.Result shows, singly connect yellow ocher streptomycete (Streptomyces silaceus) ACCC 40021 and combined inoculation and all have promoter action to Yan Miao stem, the growth of leaf portion and leaf development, but the former is not so good as the latter at effect, and inoculates Trichodermareesei (Trichoderma reesei) ACCC 30150 compared with the control without obvious promoter action.As known from Table 3, compared with the control, the stem height of combined inoculation and stem girth improve 16.0% and 17.0% respectively, and leaf is long, leaf is wide and the number of blade improves 28.0%, 15.0% and 19.0% respectively, and all there is significant difference; Singly connect the every growth indexes of yellow ocher streptomycete (Streptomyces silaceus) ACCC 40021 and be all significantly higher than contrast, singly meet Trichodermareesei (Trichoderma reesei) ACCC 30150 and contrast without significant difference.From the ratio that stem and leaf portion increase, singly meet yellow ocher streptomycete (Streptomyces silaceus) ACCC 40021 and the horizontal and vertical growth balance of combined inoculation cigarette seedling good, after being conducive to cigarette transplantation of seedlings, basal part of stem is sprouted and is formed a large amount of indefinite root system, accelerates differentiation and the development rate of cigarette seedling.It can thus be appreciated that, singly connect raising and nurturing staff that yellow ocher streptomycete (Streptomyces silaceus) ACCC 40021 and combined inoculation are conducive to Yan Miao stem and leaf portion quality.

Table 3 cigarette seedling overground part Agronomic trait

Process

Stem high (cm)

Stem girth (cm)

Leaf long (cm)

Leaf wide (cm)

The number of blade


						CK	10.4±0.3b	1.86±0.08cd	9.7±0.3c	5.3±0.1c	7.5±0.2b
T0	9.5±0.4c	1.75±0.04d	8.9±0.4d	5.2±0.2c	6.8±0.3c
						T1	10.6±0.4b	1.90±0.02bc	9.9±0.4c	5.5±0.2bc	7.7±0.3b
T2	11.5±0.6a	1.99±0.08b	10.8±0.5b	5.7±0.1b	8.5±0.5a
						T3	12.1±0.5a	2.17±0.11a	12.4±0.4a	6.1±0.3a	8.9±0.4a

2.4.2 cigarette seedling underground part Agronomic trait

Cultivate the primary link that healthy and strong cigarette seedling is tobacco leaf production, and the root system of prosperity is one of the standard in strong sprout.During cigarette seedling seedling, measure the Root morphology mathematic(al) parameter of each process cigarette seedling, the results are shown in Table 4, table 5.Result shows, biocontrol microorganisms combination inoculation can promote the root growth of cigarette seedling, and increase total surface area and volume and promotion root system and laterally increase thick and longitudinal tensile strain, inoculation growth-promoting effect is not as combined inoculation separately.As known from Table 4, with regard to tobacco root system volume and total surface area, compared with the control, singly meet yellow ocher streptomycete (Streptomyces silaceus) ACCC 40021 and combined inoculation all significantly increases, and singly meet Trichodermareesei (Trichoderma reesei) ACCC 30150 and contrast without significant difference; Singly meet Trichodermareesei (Trichoderma reesei) ACCC 30150, singly connect that total root of yellow ocher streptomycete (Streptomyces silaceus) ACCC 40021 and combined inoculation is long improves 20.0%, 74.0% and 161.0% respectively, be significantly higher than contrast; From root average diameter, the process of inoculation biocontrol microorganisms is all significantly higher than contrast.Visible, biocontrol microorganisms combination is mainly realized by increase root length and roots oxidizing the growth-promoting functions of cigarette seedling, this also increases total absorption area and the active area of root system of cigarette seedling, optimize the surface property of root system, facilitate growing and morphogenesis of cigarette seedling underground part, the also seedling stage after transplanting will be shortened.

Table 4 cigarette seedling underground part Agronomic trait

Process	Root system surface area (cm ²）	Root system volume (cm ³）	Total root long (cm)	Root average diameter (mm)
					CK	85.2±2.1c	0.5±0.02c	561.7±23.8d	0.31±0.02cd
T0	49.8±1.8d	0.3±0.02d	460.1±20.5e	0.29±0.01d
					T1	91.3±3.3c	0.4±0.02cd	675.3±29.0c	0.34±0.01c
T2	140.5±3.4b	1.7±0.08b	975.8±40.3b	0.42±0.02b
					T3	176.4±5.3a	1.9±0.13a	1465.9±70.2a	0.51±0.02a

The root system of different diameter scope, there is difference in its receptivity, it is generally acknowledged that the receptivity of radicula is better than thick root.Different diameter scope extent of the root system has been carried out analysis in table 5 by this research.Result shows, biocontrol microorganisms combination significantly can increase medium with the length of radicula and in total ratio of following in length; When promoting side root (radicula and medium) to grow, the transverse growth of root system can also be promoted further, thus increase the length of thick root.With regard to the radicula length of diameter≤0.2mm, compared with the control, inoculation biocontrol microorganisms all significantly increases in the radicula length of diameter≤0.2mm, and it is the highest that this part root length accounts for the long ratio of total root; Medium length within the scope of diameter 0.2 ~ 0.6mm, singly connect yellow ocher streptomycete (Streptomyces silaceus) ACCC 40021 and combined inoculation and significantly increase medium length proportion in total root is long, singly meet Trichodermareesei (Trichoderma reesei) ACCC 30150 and contrast without significant difference; Within the scope of combined inoculation diameter ﹥ 0.6mm, medium length significantly increases, but effect is not as singly meeting yellow ocher streptomycete (Streptomyces silaceus) ACCC 40021, and Trichodermareesei (Trichoderma reesei) ACCC 30150 inhibits the growth of thick root.Visible, combined inoculation to increase radicula and medium length obvious, be conducive to the allocation proportion increasing medium, enhancing root to the absorption of nutrient and transportcapacity, is conducive to the growth of bottom and overground part in phase by this.

Root length distribution within the scope of table 5 different diameter

2.4.3 cigarette seedling biomass and root/shoot ratio

The biomass of cigarette seedling and root/shoot ratio weigh the important indicator in strong sprout, in order to more fully assess the growth-promoting effect of biocontrol microorganisms, determining cigarette seedling underground part and overground part cigarette seedling fresh weight and dry weight, calculate root/shoot ratio and fresh and dried ratio, the results are shown in Table 6 when seedling.With regard to fresh weight, combined inoculation significantly increases overground part fresh weight than contrast, and all the other fungi-proofing process cigarette seedling overground part fresh weight differences of singly delivering a child are not obvious, and inoculation biocontrol microorganisms root system fresh weight all exists significant difference with contrasting.In dry-matter accumulation amount, singly meet yellow ocher streptomycete (Streptomyces silaceus) ACCC 40021 and combined inoculation overground part dry weight significantly increases, and singly meet Trichodermareesei (Trichoderma reesei) ACCC 30150 and contrast without significant difference, but inoculation biocontrol microorganisms underground part root system amount of dry matter is all significantly higher than contrast.With regard to root/shoot ratio and fresh and dried ratio, the inoculation fresh and dried ratio of biocontrol microorganisms and root/shoot ratio are all higher than contrast, and root/shoot ratio is all greater than 0.2, meets the standard in tobacco strong sprout.

Table 6 cigarette seedling biomass and root/shoot ratio

Process

Cauline leaf fresh weight (g)

Root system fresh weight (g)

Cauline leaf dry weight (g)

Weight of root system (g)

Fresh and dried ratio

Root/shoot ratio

CK

3.46±0.12b

0.795±0.032b

0.42±0.01b

0.076±0.003d

0.116

0.181

T0

3.14±0.18c

0.571±0.044c

0.38±0.02c

0.054±0.004e

0.116

0.143

T1

3.47±0.08b

0.917±0.044a

0.43±0.02b

0.088±0.004c

0.118

0.205

T2

3.55±0.12b

0.974±0.040a

0.48±0.03a

0.095±0.004b

0.127

0.201

T3

3.98±0.18a

0.924±0.042a

0.51±0.02a

0.103±0.005a

0.124

0.205

2.5 biocontrol microorganisms are on the impact of cigarette seedling disease resistance

2.5.1 cigarette seedling Defense Enzyme Activities

Defense Enzyme Activities is the significant parameter characterizing plant disease resistance power, can reflect the ability of biocontrol microorganisms induction cigarette seedling resistance.When seedling, determine the Defense Enzyme Activities of each process, the results are shown in Figure 2.Result shows, after biocontrol microorganisms combined treatment cigarette seedling, defensive ferment (SOD, POD, CAT and PAL) activity all significantly increases.SOD enzyme activity contrasts without except significant difference except singly connecing actinomycetes with morbidity, and all the other process all exist significant difference, and combined inoculation is than morbidity contrast raising 70%; Combined inoculation improves 40%, 92% and 104.0% respectively than POD, CAT and PAL activity of morbidity contrast, all there is significant difference; Combined inoculation and singly meet Trichodermareesei (Trichoderma reesei) ACCC 30150 PAL and CAT two kinds of enzymic activitys without significant difference, and SOD and POD two kinds of enzymic activitys exist significant difference, but four kinds of enzymic activitys are all significantly higher than the process singly meeting yellow ocher streptomycete (Streptomyces silaceus) ACCC 40021.Visible, biocontrol microorganisms combination can induce the signalling system of cigarette seedling, starts the expression of Defensive Enzyme in plant body, reflect biocontrol microorganisms in the potentiality strengthening cigarette seedling disease resistance, but different biocontrol microorganisms process same enzyme expression amount there are differences.

2.5.2 balck shank prevention effect

Inoculation biocontrol microorganisms after every day observed and recorded cigarette Seedling Diseases a situation arises, the results are shown in Table 7.Result shows, inoculation biocontrol microorganisms significantly reduces black shank sickness rate and disease index, improve prevention effect, the preventive effect of the more single bacterium of biocontrol microorganisms combination all increases significantly, and singly connects Trichodermareesei (Trichoderma reesei) ACCC 30150 preventive effect and is significantly higher than and singly connects actinomycetes.As known from Table 6, in each process, the preventive effect of combined inoculation is best, be 69.3%, singly meet Trichodermareesei (Trichoderma reesei) ACCC 30150 and be better than singly connecing yellow ocher streptomycete (Streptomyces silaceus) ACCC 40021 preventive effect, and there is significant difference.Balck shank sickness rate and the display of disease index calculation result, the sickness rate of biocontrol microorganisms combination and disease index reduce 50.7% and 52.7% than morbidity contrast respectively, and all there is significant difference.By more singly delivering a child, fungi-proofing preventive effect finds, singly connect Trichodermareesei (Trichoderma reesei) ACCC30150 preventive effect to be better than and singly to meet yellow ocher streptomycete (Streptomyces silaceus) ACCC 40021, this may be that the bacterial strain of the different Biocontrol Mechanism of biocontrol microorganisms acts on identical pathogenic bacteria to play Biocontrol Effect strong and weak different.Visible, Trichodermareesei (Trichoderma reesei) ACCC 30150 and yellow ocher streptomycete (Streptomyces silaceus) ACCC 40021 are to preventing and treating the combination that black shank is a synergy.

Table 7 balck shank prevention effect

Process	Sickness rate (%)	Disease index	Prevention effect (%)
				T0	74.3±3.1a	76.1±2.5a	-----
T1	32.7±4.9c	34.3±5.9c	54.9±8.6b
				T2	45.0±5.3b	49.0±6.4b	35.8±7.5c
T3	23.6±2.4d	23.4±2.9d	69.3±3.8a

The research of biocontrol microorganisms combination is mainly concentrated on to the composition research of the aspect such as fungi and fungi, fungus and bacterium, bacterium and yeast both at home and abroad, most research shows that biocontrol microorganisms improves the effect of biological control after composite, but, also there are many reports to point out that mixture preparation does not cause the raising of biological control effect.Wherein, incompatibility between bacterial strain is composite unsuccessful major reason (Saldajeno M G B, Chandanie W A, Kubota M, Hyakumachi M.Effects of interactions of arbuscularmycorrhizal fungi and beneficial saprophytic mycoflora on plant growth and disease protection.Mycorrhizae:Sustainable Agriculture and Forestry, 2008, pp.211-226).This research is composite for research less fungi and actinomycetes, utilizes mixed culture and Odontothrips loti, determines the interaction between two strain biocontrol microorganisms, and determining both is that an affinity combines.Affinity is the composite prerequisite of biocontrol microorganisms, can play the bacteriostasis of synergistic function and bacterial strain self, antimicrobial mechanism and antimicrobial spectrum scope close relation after composite.This research for target, analyzes bacteriostatic activity and the antimicrobial spectrum of two bacterial strains itself and meta-bolites thereof with the multiple pathogenic bacteria from different route of transmission under two kinds, soil and plant leaf portion different niches.Result shows, yellow ocher streptomycete (Streptomyces silaceus) ACCC 40021 fermentation broth on tobacco da mping-off fungi bacteriostatic action is stronger, and Trichodermareesei (Trichoderma reesei) ACCC 30150 mycelia is particularly outstanding to the bacteriostatic action of tobacco black shank bacterium, supposition may produce microbiotic and Trichodermareesei with yellow ocher streptomycete (Streptomyces silaceus) ACCC 40021, and to have multiple antagonism mechanisms relevant.Although these there is different mechanism of action and have affinity biocontrol microorganisms combination can widen antimicrobial spectrum, this does not also mean that the result of combination must be synergy.Under field conditions, many bacterium work in coordination with diseases prevention is not more simple additive effect (Huang B (Huang Bing), Liu N (Liu Ning), Ying H (Huang Ying), et al.Coculture of actinomycetes with Bacillus subtilis and its effecton the bioactive secondary metabolites.Chinese Journal of Biotechnology (biotechnology journal), 2009, 25 (6): 932-940 (in Chinese)), the habitat of crop also must be considered when biocontrol microorganisms is composite, the many factors such as the object of control and route of transmission, the object of biocontrol microorganisms combination synergy could be realized.

This research finds that longitudinally (stem is high, leaf is long, root long) and horizontal (stem girth, Ye Kuan, average root diameter) aspect have growth-promoting and the effect of coordination dispensed materials to inoculation yellow ocher streptomycete (Streptomyces silaceus) ACCC 40021 pairs of cigarette seedlings, especially the increase of tobacco root system and stem mean diameter can ensure its nutritive substance and Water Transportation, thus bottom and overground part growth in phase.

Without mutual antagonistic action when yellow ocher streptomycete (Streptomyces silaceus) ACCC 40021 and Trichodermareesei (Trichoderma reesei) ACCC 30150 mixed culture, show higher affinity; Dull and stereotyped face-off and Odontothrips loti measurement result show that two bacterial strains have antagonistic action in obvious ware to the black shank of under different niches and route of transmission, samping off and brown spot pathogen, and the ferment filtrate of its generation all can suppress the mycelial growth of pathogenic bacteria to some extent.Two strain biocontrol microorganisms lists connect and all can promote cigarette seedling above and below ground some growth to some extent with during hybrid junction, significantly improve the Defense Enzyme Activities such as its SOD, POD, CAT, thus strengthen the resistance against diseases of cigarette seedling, significantly reduce sickness rate and the disease index of cigarette seedling balck shank; The every growth indexes of combined inoculation cigarette seedling and Defense Enzyme Activities are inoculated more separately and are all increased, and singly connecing the every growth indexes of actinomycetes, to be better than single grafting again mould, but the process that the mould Defense Enzyme Activities of single grafting more singly takes over control line bacterium is all significantly increased.Illustrate that yellow ocher streptomycete (Streptomyces silaceus) ACCC 40021 and Trichodermareesei (Trichoderma reesei) ACCC 30150 can not only improve cigarette seedling Defense Enzyme Activities, and growing of cigarette seedling can also be promoted, it is the combination of a synergy after composite, more be conducive to playing complementary advantage and synergy, disease prevention growth-promoting effect is better than single biocontrol microorganisms.

The preparation of embodiment 2, tobacco disease prevention growth-promoting microbial inoculum and microbial organic fertilizer

1, ferment Trichodermareesei (Trichoderma reesei) ACCC 30150

Produce Spore cultivation base: KH ₂pO ₄2.0g/L, NH ₄nO ₃1.0g/L, FeSO ₄.7H ₂o 0.005g/L, MnSO ₄0.0016g/L, CaCl ₂0.3g/L, NaCl 1.0g/L, MgSO ₄.7H ₂o 0.3g/L, corn stalk powder (crossing 40mm sieve) 10g/L.PH 6.0。Sterilizing 20 minutes under 115 DEG C of conditions.

Trichodermareesei (Trichoderma reesei) ACCC 30150 is inoculated in the plate being placed with PDA substratum, cultivate after 5 days, the bacterium cake after getting 10 cultivations is bought with the 0.5cm punch tool of sterilizing, have in being transferred in the triangular flask of the 250ml of 100ml nutrient solution (PDA liquid nutrient medium is housed), put into shaking table 30 DEG C and cultivate 3 days.Then according to 10%(volumn concentration) inoculum size be inoculated in above-mentioned product Spore cultivation base, (shaking speed 250 revs/min) to the content of the fermented liquid miospore of Trichodermareesei (Trichoderma reesei) ACCC 30150 cultivated by 30 DEG C of shaking tables is 4.5 × 10 ⁸cfu/ml, collects the fermented liquid of Trichodermareesei (Trichoderma reesei) ACCC 30150.

PDA substratum: potato (peeling) 200g, glucose 20g, agar 20g, water 1000mL, pH value 7.0-7.2.

Liquid PDA substratum is that the agar in PDA substratum is removed the liquid nutrient medium obtained.

2, ferment yellow ocher streptomycete (Streptomyces silaceus) ACCC 40021

Produce Spore cultivation base: KH ₂pO ₄2.0g/L, NH ₄nO ₃1.0g/L, FeSO ₄.7H ₂o 0.005g/L, MnSO ₄0.0016g/L, CaCl ₂0.3g/L, NaCl 1.0g/L, MgSO ₄.7H ₂o 0.3g/L, corn stalk powder (crossing 40mm sieve) 10g/L.PH 6.0。Sterilizing 20 minutes under 121 DEG C of conditions.

Yellow ocher streptomycete (Streptomyces silaceus) ACCC 40021 is inoculated in the plate that Gause I substratum is housed, cultivate after 5 days, the bacterium cake after getting 10 cultivations is bought with the 0.5cm punch tool of sterilizing, have in being transferred in the triangular flask of the 250ml of 100ml nutrient solution (liquid Gause I substratum), put into shaking table 30 DEG C and cultivate 3 days.Then according to 10%(volumn concentration) inoculum size be inoculated in above-mentioned product Spore cultivation base, (shaking speed 250 revs/min) to the content of the fermented liquid miospore of yellow ocher streptomycete (Streptomyces silaceus) ACCC 40021 cultivated by 30 DEG C of shaking tables is 4.5 × 10 ⁸cfu/ml, collects the fermented liquid of yellow ocher streptomycete (Streptomyces silaceus) ACCC 40021.

Wherein, Gause I substratum: K ₂hPO ₄0.5g, NaCl 0.5g, KNO ₃1.0g, FeSO ₄7H ₂o0.01g, MgSO ₄7H ₂o 0.5g, Zulkovsky starch 20g, agar 20g, water 1000ml; PH 7.2-7.4.121 DEG C of moist heat sterilizations 30 minutes.

Liquid Gause I substratum is that the agar in Gause I substratum is removed the liquid nutrient medium obtained.

3, the preparation of tobacco disease prevention growth-promoting microbial inoculum and microbial organic fertilizer

Prepare following 1 under the same conditions)-3) the solid microbe fertilizer of tobacco disease prevention growth-promoting.Wherein, adsorbing base used is and is mixed by Chicken dung, edible fungi residues and sugar refinery bagasse mass ratio according to 2:4:4, make 100 object powders, 121 DEG C of high-temperature sterilizations cooled after 4 hours, obtain the adsorbing base for the preparation of tobacco disease prevention growth-promoting solid microbe fertilizer, its water content is less than or equal to 10%.

The fermented liquid diatomite adsorption drying of Trichodermareesei (Trichoderma reesei) ACCC 30150 of step 1 is obtained Trichodermareesei (Trichoderma reesei) ACCC 30150 solid fungicide.The fermented liquid diatomite adsorption drying of yellow ocher streptomycete (Streptomyces silaceus) ACCC 40021 of step 2 is obtained yellow ocher streptomycete (Streptomyces silaceus) ACCC 40021 solid fungicide.

1) preparation of the tobacco disease prevention growth-promoting solid microbe fertilizer A of the microbial inoculum be made up of Trichodermareesei (Trichoderma reesei) and yellow ocher streptomycete (Streptomyces silaceus) containing activeconstituents

By the adsorbing base of the above-mentioned solid microbe fertilizer for the preparation of tobacco disease prevention growth-promoting and above-mentioned Trichodermareesei (Trichoderma reesei) ACCC 30150 solid fungicide and above-mentioned yellow ocher streptomycete (Streptomyces silaceus) ACCC 40021 solid fungicide Homogeneous phase mixing, make water content be less than or equal to 10%, and the spore content making Trichodermareesei (Trichoderma reesei) ACCC 30150 is 1.0 × 10 ⁷cfu/g, the spore content of yellow ocher streptomycete (Streptomyces silaceus) ACCC 40021 is 1.0 × 10 ⁷cfu/g, is ground into 100 orders, obtains tobacco disease prevention growth-promoting solid microbe fertilizer A.The spore content of Trichodermareesei (Trichoderma reesei) ACCC 30150 in this tobacco disease prevention growth-promoting solid microbe fertilizer A is 1.0 × 10 ⁷cfu/g, the spore content of yellow ocher streptomycete (Streptomyces silaceus) ACCC 40021 is 1.0 × 10 ⁷cfu/g.

2) containing activeconstituents be the preparation of tobacco disease prevention growth-promoting solid microbe fertilizer B of microbial inoculum of yellow ocher streptomycete (Streptomyces silaceus)

By the adsorbing base of the above-mentioned solid microbe fertilizer for the preparation of tobacco disease prevention growth-promoting and the mixing of above-mentioned yellow ocher streptomycete (Streptomyces silaceus) ACCC 40021 solid fungicide, make water content be less than or equal to 10%, and the spore content making yellow ocher streptomycete (Streptomyces silaceus) ACCC 40021 is 2.0 × 10 ⁷cfu/g, is ground into 100 orders, obtains tobacco disease prevention growth-promoting solid microbe fertilizer B.The spore content of yellow ocher streptomycete (Streptomyces silaceus) ACCC 40021 in this tobacco disease prevention growth-promoting solid microbe fertilizer B is 2.0 × 10 ⁷cfu/g.

3) containing activeconstituents be the preparation of solid microbe fertilizer C of microbial inoculum tobacco disease prevention growth-promoting of Trichodermareesei (Trichoderma reesei)

By the adsorbing base of the above-mentioned solid microbe fertilizer for the preparation of tobacco disease prevention growth-promoting and the mixing of above-mentioned Trichodermareesei (Trichoderma reesei) ACCC 30150 solid fungicide, make water content be less than or equal to 10%, and the spore content making Trichodermareesei (Trichoderma reesei) ACCC 30150 is 2.0 × 10 ⁷cfu/g, is ground into 100 orders, obtains tobacco disease prevention growth-promoting solid microbe fertilizer C.The spore content of Trichodermareesei (Trichoderma reesei) ACCC 30150 in this tobacco disease prevention growth-promoting solid microbe fertilizer C is 2.0 × 10 ⁷cfu/g.

The tobacco disease prevention growth-promoting solid microbe fertilizer of embodiment 3, embodiment 2 promotes tobacco growing

1. materials and methods

1.1 test site

Test arrangement, in spur village, Liangshan State of Sichuan Province Xichang City You Jun town, selects the representational soil such as preceding crop is consistent, the soil texture loose, plot is smooth, soil fertility (table 8) is medium, the row of filling convenience, traffic convenience to layout.Experimental field be there is by a family tobacco grower's management of better plantation experience, take soil testing and fertilizer recommendation scheme simultaneously.

Table 8 Labile soil organic carbon situation records table

Soil fertility	Organic	Alkali-hydrolyzable nitrogen	Rapid available phosphorus	Available potassium	pH
						Medium	18.3g/kg	26.1mg/kg	50mg/kg	59mg/kg	6.95

1.2 test kinds

Experimental cultivar is local main breed cloud and mist 87, unified for planting by company of Sichuan Province.

1.3 test design

Four process are established in experiment, are respectively CK, T1, T2 and T3.

Experiment adopts randomized block design, and arrange three iterons, each iteron arranges four communities at random, is respectively CK treatment zone, T1 treatment zone, T2 treatment zone, T3 treatment zone and T4 treatment zone.The area of each community is 30m ².

1.4 field management

Except transplanting the operation difference of forward direction soil discal patch organic fertilizer, other field management of each process is all identical.

1.4.1 wholely, transplant

At transplanting first 25 days use rotary cultivator real tillages, before transplanting, within 10-15 days, complete ridging work; During transplanting, line-spacing, spacing in the rows all perform according to local sound tobacco production technology scheme; Each iteron completes transplanting within the same day, waters foot and transplant water during transplanting.

Respectively at transplanting forward direction soil discal patch organic fertilizer in each community.Wherein, CK treatment zone (contrast) is according to the adsorbing base for the preparation of tobacco disease prevention growth-promoting solid microbe fertilizer of amount row replacement embodiment 2 step 3 of every square metre of 0.15Kg; T1 treatment zone, according to the tobacco disease prevention growth-promoting solid microbe fertilizer C of amount row replacement embodiment 2 step 3 of every square metre of 0.15Kg, makes every square metre of soil apply 3 × 10 ⁹cfu Trichodermareesei (Trichoderma reesei) ACCC 30150 spore; T2 treatment zone, according to the tobacco disease prevention growth-promoting solid microbe fertilizer B of amount row replacement embodiment 2 step 3 of every square metre of 0.15Kg, makes every square metre of soil apply 3 × 10 ⁹cfu yellow ocher streptomycete (Streptomyces silaceus) ACCC 40021 spore; T3 treatment zone, according to the tobacco disease prevention growth-promoting solid microbe fertilizer A of amount row replacement embodiment 2 step 3 of every square metre of 0.15Kg, makes every square metre of soil apply 1.5 × 10 ⁹cfu Trichodermareesei (Trichoderma reesei) ACCC 30150 spore and 1.5 × 10 ⁹cfu yellow ocher streptomycete (Streptomyces silaceus) ACCC 40021 spore.

1.4.2 fertilizer practice

All carry out according to local conventional fertilizer application standard and fertilizer practice.

1.4.3 field management

The tobacco leaf maturations of link all in strict accordance with local cured tobacco production technical specifications defined such as test nursery, whole, ridging, transplanting, field management, picking and roasting are gathered, science baking (three section of five step formula baking process) strictly operates; The requirement of producing according to locality standardization is carried out, and every farming operation is consistent in time, and same control measures complete within the same day.

Table 9 intertill and clean tillage information slip

Time	Field operates
		May 24	First time imposes saltpetre
June 4	Second time imposes saltpetre
		June 10	Third time imposes saltpetre
July 10	Take off film ridging, in conjunction with intertillage
		July 11	Pinch and smear wooden fork
July 14	Intertill and clean tillage
		July 20	Second time smears wooden fork

The unified anti-governance information slip of table 10

Time	Medicament title	Prevent and treat object
			May 24	Croak venus crystals	Prevent and treat mosaic disease
May 30	Propamocarb	Prevent and treat balck shank
			June 15	Provado	Control cigarette aphid
July 10	Triadimefon	Prevent and treat Powdery Mildew
			July 20	Go out toothpick	Control cigarette aphid

1.5 investigation and analysis projects

1.5.1 the tobacco leaf dome phase investigates the economical character of each process cigarette strain: the leaf of waist leaf and top is long and leaf is wide, effective blade number, pitch, stem girth etc., according to the economical character investigation method (tobacco economical character investigation method, YC/T142-1998) that tobacco industry is promulgated.

1.5.2 the complete tobacco sample 3kg getting B2F, C3F, X2F Three Estate of each process is respectively toasted, for carrying out chemical composition analysis.

1.5.3 the complete cigarette sample 2kg getting B2F, C3F, X2F Three Estate of each process is respectively toasted, smoke panel test for carrying out aesthetic quality, send Dohanykutato Intezet, for tobacco leaf Evaluation on Appearance Quality, physical property mensuration, chemical composition and fragrance matter analysis, smoke panel test.Sample packaging and moisture content are pressed national regulations and are performed.

1.5.4 chemical composition analysis content: starch, total reducing sugar, reducing sugar, protein, nicotine, total nitrogen, total potassium etc.;

1.5.5 sensory evaluating smoking's content: aroma quality, perfume quantity, assorted gas, pungency, strength etc.; Visual appearance qualification, the chemical composition analysis of test sample, roll former cigarette sample, to be concentrated with Dohanykutato Intezet by technique center and carry out former cigarette sensory evaluation.

1.5.6 unified by country's 42 grades of grade scale classifications, price is by local purchasing price statistics.Without each grade ratio, weight, price etc. of the whole former cigarette (comprising sample) of storage after roasting by Dohanykutato Intezet of the Chinese Academy of Agricultural Sciences statistics.Calculate per mu yield respectively, class index, average price, product refer to, per mu yield value, first-class cigarette ratio, upper medium grade cigarette ratio etc.

2 results and analysis

2.1 different treatment are on the impact of flue-cured tobacco primary growth stage

The breeding time of tobacco refers to and is divided into again rice seedling bed (seedbed) breeding time and growing stage the time needed for whole growth and development process that tobacco is gathered in the crops from insemination and emergence to maturation, and rice seedling bed (seedbed) breeding time refers to the number of days from emerging to transplanting.Tobacco roughly can be divided into also seedling stage (surviving from being transplanted to) whole breeding time, growing period of stretching the root phase that (roll into a ball a phase from surviving), prosperous long-term (from group's phase to squaring period) and ripening stage (terminating from squaring period to tobacco leaf picking) 4 maintain close ties with.Squaring period is the date occurring bud.Ripening stage pinches in time and smears wooden fork, and hold ripening degree, timely collecting, Topping Stage is the date of destroying top, and the dome phase is the date of pinching latter 15 days, and the roasting phase of beginning is the date of blade baking of gathering first time, and the roasting phase is the date of blade baking of finally gathering eventually.

The growthdevelopmental stage of table 11 different treatment records table

As can be seen from Table 11, T2, T3 time long-term to Moindawang the earliest, more Zao than CK 11 days, more Zao than T1 10 days.

2.2 different treatment are on the impact of flue-cured tobacco dome phase phase Other Main Agronomic Characters

Table 12 different treatment dome phase economical character records table

As can be seen from Table 12, the waist leaf of T3 and T2 and top leaf area are all significantly higher than CK; The waist leaf leaf length of T3 is 1.11 times of CK, and the waist leaf leaf of T3 is wide is 1.18 times of CK; The waist leaf leaf length of T2 is 1.07 times of CK, and the waist leaf leaf of T2 is wide is 1.14 times of CK.The top leaf length of T3 is 1.30 times of CK, and the top leaf of T3 is wide is 1.33 times of CK; The top leaf length of T2 is 1.30 times of CK, and the top leaf of T2 is wide is 1.26 times of CK.Except T1, all the other blades respectively processed are all more than 20.

2.3 different treatment are on the impact of the yield of flue-cured tobacco output value

Table 13 different treatment affects cartogram to the yield of tobacco output value

The result of table 13 shows, wherein the yield of tobacco of T3 is the highest, is 1.23 times of CK; Wherein the output of T2 is taken second place, and is 1.19 times of CK; That the output value of tobacco leaf is the highest is T3, and being 1.22 times of CK, is secondly T2, and its output value is 1.22 times of CK.

2.4 different treatment are on the impact of flue-cured tobacco on tobacco leaf chemical composition

Table 14 different treatment is on the impact of Chemical Components of Tobacco Leaves content

The result of table 14 shows, total reducing sugar, the reducing sugar content of T3 process are the highest, and promote absorption and the accumulation of potassium ion, the derived values such as sugared alkali ratio, potassium chlorine ratio are coordinated, more all in the optimum range of sound tobacco.

2.5 different treatment are on the impact of flue-cured tobacco on tobacco leaf middle leaf judging parameter

Table 15 different treatment is on the impact of middle leaf tobacco leaf judging parameter

Sensory evaluation has been carried out to test sample.Table 15 result shows: T3 uses has larger impact to tobacco leaf judging parameter, and reveal texture with the processing list of T3 clear, perfume quantity, concentration is higher, and assorted thorn is little.

2.6 different treatment are on the impact of flue-cured tobacco on visual appearance in the middle part of tobacco leaf

Table 16 different treatment is on the impact of tobacco leaf visual appearance

Process

Position

Color

Ripening degree

Blade construction

Identity

Oil content

Colourity

CK

In

Orange

Ripe

Loose

Moderate

Many-

Dense-

T1

In

Orange

Ripe

Loose

Moderate

Many-

Dense-

T2

In

Orange

Ripe

Loose

Moderate

Many-

By force+

T3

In

Orange

Ripe

Loose

Moderate

Many-

By force+

Show from cured tobacco leaf visual appearance: each test process appearance color is orange, and roasting after ripening degree performance is ripe, leaf tissue weave construction, identity and oil content indifference; The tobacco leaf colourity of T2, T3 also shows better, total appraisal best (table 16).

The tobacco disease prevention growth-promoting solid microbe fertilizer control black shank of embodiment 4, embodiment 2 and red-star like disease

1. materials and methods

1.1 test site

Test arrangement is in Gulin County, Luzhou, Sichuan Province.The representational soil such as preceding crop is consistent, the soil texture loose, plot is smooth, soil fertility (table 17) is medium, the row of filling convenience, traffic convenience are selected to layout.Experimental field be there is by a family tobacco grower's management of better plantation experience, take soil testing and fertilizer recommendation scheme simultaneously.

Table 17 Labile soil organic carbon situation records table

Soil fertility	Organic	Alkali-hydrolyzable nitrogen	Rapid available phosphorus	Available potassium	pH
						Medium	18.3g/kg	68.1mg/kg	24mg/kg	78mg/kg	7.2

1.2 test kinds

1.3 test design

Four process are established in experiment, are respectively CK, T1, T2 and T3.

1.4 field management

1.4.1 wholely, transplant

1.4.2 fertilizer practice

1.4.3 field management

Table 18 intertill and clean tillage information slip

Time	Field operates
		May 4	Cigarette transplantation of seedlings
May 11	Impose saltpetre
		June 15	Take off film ridging, in conjunction with intertillage
July 15	Pinch
		July 28	Adopt roasting

The unified anti-governance situation of table 19

Time	Medicament title	Prevent and treat object
			June 15	Insert toothpick	Aphid
July 5	Junkeduke 800 times spraying	Common mosaic
			July 10	Xin Wansheng	Climacteric spot disease

1.5 disease survey

At Topping Stage investigation balck shank and red-star like disease incidence, calculate sickness rate, disease index and prevention effect.

Black shank is generally investigated in units of whole strain, and disease scale standard is as follows:

0 grade: anosis.

1 grade: stem's scab is no more than 1/2 of stem girth, or (with) half with lower blade or top slightly wilting, scab can be there is by bottom minority blade.

2 grades: stem's scab exceedes 1/2 of stem girth, or (with) blade more than half or the above leaf of part waist leaf wilting.

3 grades: stem's scab around stem girth, or (with) 2/3 with blade or lower two canopies wilting with blade.

4 grades: diseased plant is dead.

Black shank disease index=(∑ (sick progression × this grade of diseased plant number)/(investigating total strain number × 4)) × 100;

Black shank prevention effect (%)=((contrast disease index-process disease index)/contrast disease index) × 100%;

Black shank sickness rate=morbidity strain number/investigate total strain number × 100%.

Alternaria alternate is generally investigated in units of blade, and disease scale standard is as follows:

O level: full leaf is anosis;

0.5 grade: lesion area accounts for less than 1% of blade area;

1 grade: the l-5%(that lesion area accounts for blade area does not comprise 1%);

2 grades: the 5.0-10%(that lesion area accounts for blade area does not comprise 5%);

3 grades: the 10.0-20%(that lesion area accounts for blade area does not comprise 10%);

4 grades: lesion area accounts for more than 20% (not comprising 20%) of blade area.

Alternaria alternate disease index=(∑ (sick progression × this grade of sick number of sheets)/(investigating the total number of sheets × 4)) × 100;

Alternaria alternate prevention effect (%)=((contrast disease index-process disease index)/contrast disease index) × 100%;

Alternaria alternate sickness rate=morbidity number of sheets/investigate the total number of sheets × 100%.

According to the Major Diseases of field investigation, a situation arises, balck shank and red-star like disease generation is mainly contained in each process, the prevention effect of T2 and T3 to balck shank and red-star like disease is all better, the prevention effect of T2 and T3 to balck shank is respectively 73.0% and 63.5%, the prevention effect of T2 and T3 to red-star like disease be respectively 66.7% and 86.7%(table 20), illustrate that the tobacco disease prevention growth-promoting solid microbe fertilizer A of embodiment 1 the step 3 and tobacco disease prevention growth-promoting solid microbe fertilizer B of embodiment 1 step 3 all has good prevention effect to black shank and Alternaria alternate.

Table 20 different treatment is to the prevention effect of balck shank and red-star like disease

The tobacco disease prevention growth-promoting solid microbe fertilizer control tobacco bacterial wilt of embodiment 5, embodiment 2

1. materials and methods

1.1 test site

Test arrangement is in Shi Gu village of Hui Long township, Liangshan State of Sichuan Province Mianning County.The representational soil such as preceding crop is consistent, the soil texture loose, plot is smooth, soil fertility (table 14) is medium, the row of filling convenience, traffic convenience are selected to layout.Experimental field be there is by a family tobacco grower's management of better plantation experience, take soil testing and fertilizer recommendation scheme simultaneously.

Table 21 Labile soil organic carbon situation records table

Soil fertility	Organic	Alkali-hydrolyzable nitrogen	Rapid available phosphorus	Available potassium	pH
						Medium	14.8g/kg	67mg/kg	56mg/kg	104mg/kg	6.85

1.2 test kinds

1.3 test design

Four process are established in experiment, are respectively CK, T1, T2 and T3.

1.4 field management

1.4.1 wholely, transplant

Respectively at transplanting forward direction soil discal patch organic fertilizer in each community.Wherein, CK treatment zone (contrast) is according to the adsorbing base for the preparation of tobacco disease prevention growth-promoting solid microbe fertilizer of amount row replacement embodiment 2 step 3 of every square metre of 0.15Kg; T1 treatment zone, according to the tobacco disease prevention growth-promoting solid microbe fertilizer C of amount row replacement embodiment 2 step 3 of every square metre of 0.15Kg, makes every square metre of soil apply 3 × 10 ⁹cfu Trichodermareesei (Trichoderma reesei) ACCC 30150 spore; T2 treatment zone, according to the tobacco disease prevention growth-promoting solid microbe fertilizer B of amount row replacement embodiment 2 step 3 of every square metre of 0.15Kg, makes every square metre of soil apply 3 × 10 ⁹cfu yellow ocher streptomycete (Streptomyces silaceus) ACCC 40021 spore; T3 treatment zone, according to the tobacco disease prevention growth-promoting solid microbe fertilizer A of amount row replacement embodiment 2 step 3 of every square metre of 0.15Kg, makes every square metre of soil apply 0.15 × 10 ⁹cfu Trichodermareesei (Trichoderma reesei) ACCC 30150 spore and 1.5 × 10 ⁹cfu yellow ocher streptomycete (Streptomyces silaceus) ACCC 40021 spore.

1.4.2 fertilizer practice

1.4.3 field management

Table 22 intertill and clean tillage information slip

The unified anti-governance situation of table 23

Time	Medicament title	Prevent and treat object
			June 15	Insert toothpick	Aphid
July 5	Junkeduke 800 times spraying	Common mosaic
			July 10	Xin Wansheng	Weather spot

1.5 disease survey

At Physical property investigation bacterial wilt incidence, calculate sickness rate, disease index and prevention effect.

Tobacco bacterial wilt is generally investigated in units of diseased plant, and disease scale standard is as follows:

0 grade: complete stool is anosis.

1 grade: stem is even chlorisis spot, or is having streak side to have minority blade wilting.

2 grades: there is black streak in stem, but not yet reaches top, or sick side blade more than half is wilting.

3 grades: stem's black streak arrives plant top, or wither with blade tune in sick side 2/3rds.

4 grades: diseased plant is substantially withered.

Tobacco bacterial wilt disease index=(∑ (sick progression × this grade of sick number of sheets)/(investigating the total number of sheets × 4)) × 100;

Tobacco bacterial wilt prevention effect (%)=((contrast disease index-process disease index)/contrast disease index) × 100%;

Tobacco bacterial wilt sickness rate=morbidity number of sheets/investigate the total number of sheets × 100%.

Result shows, a situation arises that difference is comparatively obvious for each process bacterial wilt, T1 and CK process comparatively seriously, sickness rate be respectively 4.2% and 5.2%, T2, T3 without the generation of bacterial wilt.Illustrate that the tobacco disease prevention growth-promoting solid microbe fertilizer B of the tobacco disease prevention growth-promoting solid microbe fertilizer A of embodiment 1 step 3 and embodiment 1 step 3 is to the prevention effect highly significant (table 24) of bacterial wilt.

Table 24 different treatment is to the prevention effect of bacterial wilt

Process

Sickness rate

Disease index

Prevention effect

CK	5.2%	13.35
				T1	4.2%	15.35	-15%
T2	0%	0	100%
				T3	0%	0	100%

The screening of embodiment 6, Trichodermareesei (Trichoderma reesei) ACCC 30150 and yellow ocher streptomycete (Streptomyces silaceus) ACCC 40021

1, plate antagonism screening

Inoculate bacterial strain to be screened at PDA plate center, at radius about 2.5cm circumferentially, equidistant inoculation 3 equivalent various pathogenic bacteria, using every three kinds of different pathogenic bacterias as one group, have three groups, often group establishes three repetitions.Labeled strain numbering is carried out, in order to observed and recorded with marking pen.The culture dish inoculated is put in the incubator of 28 DEG C and cultivates about 7-10d.The next day, takes pictures to done experiment, measures radius R and r that pathogenic bacteria points to bacterium to be screened.Record the morphological specificity between bacterium to be screened and pathogenic bacteria.After germ and bacterium to be screened join, observe pathogenic bacteria to the encirclement of bacterium bacterium colony to be screened, suppression, invade and capture the process of its Vegetation space.

Screen screened bacterial strain, carry out according to following principle: (1) speed of growth, apparently higher than pathogenic bacteria, can fight for nutrition with pathogenic bacteria, thus significantly suppress the growth of pathogenic bacteria; (2) and between pathogenic bacteria, have about 0.2cm space, namely create antibacterial band, illustrate that bacterium to be screened creates certain antibiotics in metabolic process.

Screen from 182 strain trichoderma strains according to above-mentioned screening method and obtain Trichodermareesei (Trichoderma reesei) ACCC 30150(abbreviation Trichoderma 30150), from 45 strain stock streptomycetes, screen yellow ocher streptomycete (Streptomyces silaceus) ACCC 40021(be called for short actinomycetes 40021).

Table 25 Trichoderma 30150 and actinomycetes 40021 and 3 kind of pathogenic bacteria and the two do mutually between plate to stand facing each other record

Strain number	Black shank	Alternaria alternate	Tobacco Root maize ear rot	Actinomycetes 40021	Trichoderma 30150
						Trichoderma 30150	+++	+++	+++	-	/
Actinomycetes 40021	++	++	++	/	-

Note: to pathogenic bacteria and actinomycetic growth-inhibiting effect, by by force to weak, is divided into +++, ++ ,+,-four grades.

2, potted plant Vivo Studies on Screening

2.1 materials and methods

(1) preparation of bacteria suspension

Trichodermareesei (Trichoderma reesei) ACCC 30150 filtered out by plate antagonism and yellow ocher streptomycete (Streptomyces silaceus) ACCC 40021 is re-seeded on the plate of PDA and Gause I substratum after activating respectively, Trichodermareesei (Trichoderma reesei) ACCC 30150 cultivates about 5d, and yellow ocher streptomycete (Streptomyces silaceus) ACCC 40021 cultivates about 7-10d and grows spore.Respectively with spore under writing brush type brush, being made into concentration is separately 10 ⁷the bacteria suspension of cfu/ml.

(2) preparation of fermented liquid

Trichodermareesei (Trichoderma reesei) ACCC 30150 will be filtered out and yellow ocher streptomycete (Streptomyces silaceus) ACCC 40021 is inoculated in the plate of PDA and Gause I substratum respectively, cultivate after 5 days, the bacterium cake after getting 10 cultivations is bought respectively with the 0.5cm punch tool of sterilizing, have in being transferred to separately in the triangular flask of the 250ml of 100ml nutrient solution, put into shaking table 30 DEG C and cultivate 3 days.

(3) growth-promoting Resistance screening

Will for examination Trichodermareesei (Trichoderma reesei) ACCC 30150, yellow ocher streptomycete (Streptomyces silaceus) ACCC 40021 bacteria suspension (being called for short 30150 and 40021 respectively) and the two plastc ring (being called for short 30150+40021) prepare by experimental concentration respectively, Trichodermareesei (Trichoderma reesei) ACCC 30150 bacteria suspension when preventing and treating Tobacco Root stem disease, yellow ocher streptomycete (Streptomyces silaceus) ACCC 40021 bacteria suspension, black shank pathogenic bacterium, the vaccination ways of root rot pathogenic bacterium adopts the inoculation of spore suspension liquid irrigating root, Trichodermareesei (Trichoderma reesei) ACCC 30150 bacteria suspension, yellow ocher streptomycete (Streptomyces silaceus) ACCC 40021 bacterium is hanged and inoculates pathogenic bacterium after the two plastc ring filling root 24h, when preventing and treating tobacco leaf diseases, Trichodermareesei (Trichoderma reesei) ACCC 30150 bacteria suspension, yellow ocher streptomycete (Streptomyces silaceus) ACCC 40021 bacteria suspension, the vaccination ways of red-star like disease pathogenic bacterium all adopts spore suspension spray inoculation method, treat Trichodermareesei (Trichoderma reesei) ACCC 30150 bacteria suspension, pathogenic bacterium are inoculated after yellow ocher streptomycete (Streptomyces silaceus) ACCC 40021 bacteria suspension and the two plastc ring spraying 24h, inoculate rear 28 DEG C of cultivations.

(4) the selection result

Growth-promoting result:

Table 26 Trichodermareesei (Trichoderma reesei) ACCC 30150 and the screening of yellow ocher streptomycete (Streptomyces silaceus) ACCC 40021 growth-promoting

Diseases prevention result:

Table 27 Trichodermareesei (Trichoderma reesei) ACCC 30150 and yellow ocher streptomycete (Streptomyces silaceus) ACCC 40021 disease control screening

Process	Tobacco Root maize ear rot	Alternaria alternate	Black shank
				ACCC 30150	70.327%	72.814%	69.444%
ACCC 40021	55.632%	58.660%	55.422%

30150+40021	71.236%	73.120%	70.145%
				75% m-tetrachlorophthalodinitrile solution	79.897%
70% thiophanate methyl solution	65.153%
				50% dimethomorph solution		94.452%

Claims

1. microbial inoculum or the microbial organic fertilizer containing described microbial inoculum are promoting the application in tobacco growing, and the activeconstituents of described microbial inoculum is made up of Trichodermareesei (Trichoderma reesei) and yellow ocher streptomycete (Streptomyces silaceus); Described Trichodermareesei (Trichoderma reesei) is Trichodermareesei (Trichoderma reesei) ACCC30150, and described yellow ocher streptomycete (Streptomyces silaceus) is yellow ocher streptomycete (Streptomyces silaceus) ACCC 40021.

2. apply as claimed in claim 1, it is characterized in that: the colony forming unit number of described Trichodermareesei (Trichoderma reesei) and yellow ocher streptomycete (Streptomyces silaceus) is than being 1:1.

3. apply as claimed in claim 1 or 2, it is characterized in that: during described microbial inoculum is manured into soil, the consumption of described microbial inoculum is that every square metre of soil applies 3 × 10 ⁹-4.5 × 10 ⁹microbial inoculum described in cfu.

4. apply as claimed in claim 1 or 2, it is characterized in that: described microbial inoculum applies in seedling medium, the consumption of described microbial inoculum is that every gram of seedling medium adds 2 × 10 ⁸-4.5 × 10 ⁸microbial inoculum described in cfu.

5. apply as claimed in claim 1 or 2, it is characterized in that: described promotion tobacco growing is for promoting tobacco seedling growth.

6. apply as claimed in claim 1 or 2, it is characterized in that: described promotion tobacco growing is 6 kinds, 5 kinds, 4 kinds, 3 kinds, 2 kinds or a kind in a2-a7:

The yield of tobacco of a2, raising tobacco;

A3, shorten tobacco time long-term to Moindawang;

The leaf of a4, raising dome phase tobacco top is long;

The leaf of a5, raising dome phase tobacco top is wide;

The leaf of a6, raising dome phase tobacco waist leaf is long;

The leaf of a7, raising dome phase tobacco waist leaf is wide.

7. apply as claimed in claim 5, it is characterized in that: described promotion tobacco seedling growth is all or part of in following b1-b4 and b6-b7:

The growth of b1, promotion tobacco root system;

The growth of b2, promotion cigarette seedling stem;

The growth of b3, promotion cigarette seedling leaf;

B4, raising cigarette seedling cauline leaf weight;

The seedling rate of b6, raising tobacco;

B7, shortening tobacco seedling time.

8. apply as claimed in claim 5, it is characterized in that: described promotion tobacco seedling growth is for improving tobacco root system weight.

9. apply as claimed in claim 7, it is characterized in that: it is all or part of that the growth of described promotion tobacco root system is presented as in c1-c4:

C1, raising tobacco root system surface-area;

C2, raising tobacco root system volume;

C3, raising tobacco root system mean diameter;

C4, the total root of raising cigarette Miao are long;