CN109748735A - A kind of method of the quick decomposition of green plant stalk and prevention and control flue-cured tobacco root knot nematode disease - Google Patents

Abstract

The present invention relates to a kind of quick decomposition of green plant stalk and the methods of prevention and control flue-cured tobacco root knot nematode disease, this method selects green stalk of the oily radish with root-knot nematode resistant gene as green manure, screening has control effect to root knot nematode disease, take into account four kinds of microorganisms of efficient decomposition green stalk, with the suitable concentration and proportion of four kinds of microbial inoculums, decomposition microbial agent is sprayed in full-bloom stage, the green stalk for spraying microbial inoculum is turned on the spot further and is buried decomposition 60 days or more, by promoting green stalk quickly and efficiently decomposition, subtract apply 30% chemical fertilizer under conditions of, the organic matter fast degradation of flue-cured tobacco cultivation rhizosphere soil, N P and K Main Nutrients are sufficient；It is significant to the root knot nematode disease control effect during flue-cured tobacco cultivation；Tobacco leaf Output value significantly improves.The present invention is a kind of to the quick decomposition of green stalk, the short-cut method for effectively increasing root knot nematode disease in the soil organism and lower batch of soil nutrient, prevention and control cultivated crop, improving Crops production and quality.

Description

A kind of method of the quick decomposition of green plant stalk and prevention and control flue-cured tobacco root knot nematode disease

Technical field

The invention belongs to the application fields of plant protection and crop nutrition, and in particular to a kind of green plant stalk passes through hair Yeast-like fungi agent is crop nutrition content in the quick decomposition in field and has the method for control effect to root knot nematode disease.

Background technique

For a long time, usually to apply the yield that chemical fertilizer improves crop, the normal quilt of the quality of crop in the agricultural production in China Ignore.Because of chronic administration chemical fertilizer, the soil texture of cultivated crop is caused to be degenerated, the pollution of area source of water causes lake in agricultural production A series of problems, such as water quality deterioration, drinking water source are sharply reduced (Jin Feng, green science and technology, 2018).In recent years, various circles of society ten Subtract in point concern agricultural production to fertilize with applying organic manure (Lee is proud etc., Agriculture of Anhui science, 2017；Song Jianqun, southern agriculture Industry, 2017), it is hot to improve the quality of crop products, the soil of child care cultivated crop, the moisture holding capacity for enhancing soil and water-saving power etc. Point problem.The approach of traditional applying organic manure has: application fermentation maturity, rich in organic matter and mineral matter element and largely The nutrient utilized can be absorbed by crops；Plantation green manure plant and turning on the spot bury decomposition (thank and set fruit etc., Chinese soil and fertilizer, 2010).The former can greatly improve above-mentioned hot issue using high-quality organic fertilizer, but high-quality organic fertilizer is at high cost, and the latter throws Enter it is at low cost, but it is a small amount of in agricultural production use, to find out its cause, being primarily due to green manure plant decomposition problem, including decomposed Degree is poor and leads to burn seedlings when transplanting crop seedling and cannot be supplied to plant seedlings nutrient in time；Turn over bury decomposition time length then miss It is suitable for the season etc. of plantation.

How suitable green manure plant and quickly battalion by green manure plant decomposition by crop seedling effectively to be absorbed are selected Form point is to hinder one of widely applied critical issue of green manure plant.

Stalk farmyard manure used in traditional agriculture production, belongs to biomass organic fertilizer.Peasant household commonly uses simple stacking mode Organic fertilizer is prepared, is to borrow microorganism present in nature and the self-contained microorganism of stalk, the cause of disease including crop is micro- Biology, wherein more difficult dominant groups as in simple stacking fermentation process of beneficial microbe.Traditional green manure, which turns over, buries decomposition, and Nationality carries out decomposition to green stalk by the microorganism in nature and soil, thus can there are problems that effectively quick decomposition.

Oily radish (Raphanussativus ssp.oleiferus), also referred to as winter sub- dish, eat dish, Oil radish at shore radish, It is Cruciferae radish platymiscium, one kind of Rhaphanus is decided to be in " Yunnan plant will " (Raphanusraphanistroides), it is decided to be in " Chinese Plants will " in the category and cultivates radish (R.sativusL.var.sativus) mutation (R.sativus L.var.raphanistroides).It is called again in Europe Oily radish (Oilradish, R.sativus ssp.Oleiferus).The oily radish in Yunnan Province utilizes mainly as oil crops. In recent years, also have as green manure and animal feeding-stuff utilization.Oily radish has cultivation and distribution in more than 10, Yunnan Province ground, state, city.By Low in the seed oil content of oily radish, the potentiality utilized as oil plant industrial crops are small, therefore in the only a small amount of cultivation of China.But It is that oily radish is resistant to nematode, in the arable farmings such as European beet or rape, usually by oily radish and the economic work of cultivation Work or crop rotation between object, utilize the expression product of nematode resistance gene in oily radish, inhibit the nematode worm of beet (Heterodera Schachtii), the breeding of M hapla (Meloidogyne hapla) etc., to achieve the purpose that prevention and control nematodiasis (H.Peterka et al, TheorAppl Genet, 2004).It has been confirmed that oily radish is resistant to the nematode worm of beet, And by localization of disease resistance genes on D chromosome (H.Peterka et al, TheorAppl Genet, 2004；H.Budan et Al, Euohytica, 2008；H.Budan et al, TheorAppl Genet, 2009；Zhang Shaosong etc., Scientia Agricultura Sinica, 2008).In addition, oily radish also has the character of anti-root-knot nematode.R.B ü nte etc. utilizes wild oily radish (R.sativus Ssp.oleiferus), manually it is pollinated, studies its self progeny to Meloidogyne incognita (M.incognita) and northern root knot The resistance of nematode (M.hapla), the result shows that the resistance category quantitative inheritance type of M hapla in oily radish；And to south The resistance category Dominant gene of square root-knot nematode shows disease-resistant gene segregation ratio (R.B the ü nte, et of 3:1 in offspring al,PlantBreeding,1997).Oily radish goes out notable difference to the Resistant expression of two kinds of root-knot nematodes, shows to two kinds of roots The expression of results of the not same gene of the resistance of tie lines worm, different genes regulation.Further research confirmation, oily radish The resistance of M hapla is not carried by its D chromosome, is not the same base to the resistance of the nematode worm of beet with it Because of the result (H.Peterka, et al, Nematology, 2010) of expression, and oily radish is to Meloidogyne incognita and Java root The resistance of tie lines worm carries (Shaosong Zhang, et al, Nematology, 2014) by its E chromosome.

In addition, carrying out pure-blood ferment using the bacterium and fungi that isolate and purify from nature, decomposition microbial agent is prepared.It is withered Careless bacillus is the bacterium generally used in industrial production, is usually used in producing the enzyme preparations such as protease, amylase, cellulase In industry, display is also had been reported that, bacillus subtilis has the function of prevention and control root knot nematode disease, and (Xia Yan flies etc., Jiangsu's agriculture section It learns, 2014；Zhang Yuqin, northern gardening, 2012)；Bacillus licheniformis is chiefly used in medical production, and relevant report also shows its tool There is prevention and control root knot nematode disease (Feng Haibo etc., Chinese agricultural technology spread, 2016)；Trichoderma viride has been reported to root-knot nematode Disease have control effect (Liu Ting etc., Agricultural University Of Jiangxi's journal, 2007；Zhai Mingjuan etc., China's Vegetable, 2017), and can decompose Lignin, cellulose in plant etc.；The enzyme system of aspergillus niger secretion it is more (tall and big sound etc., Jiangsu's agriculture science, 2017；Have small Juan etc., China's Vegetable, 2017), it is one of common microorganism in food industry, can decompose lignin, cellulose, protein etc. Many plant constitution ingredients.Above-mentioned three kinds of microorganisms with prevention and control root knot nematode disease, relevant report are operationally mostly single Microbial inoculum is applied in crops rhizosphere, since the Tiny ecosystem of rhizosphere soil is complex, rhizosphere microflora and soil enzyme system Multiple action often occurs having biggish difference between indoor application effect and field application effect.

Summary of the invention

The present invention is based on the above problem, the green manure plant suitable from selection is with the efficient decomposition green stalk of screening beneficial to micro- Relevant research and development are carried out in terms of biology and combinations thereof.It is an object of the invention to: slow for field green straw decomposition speed, Rotten degree difference easily leads to transplanting initial stage burn seedlings, cannot effectively be supplied to root-knot nematode disease during seedling nutrient and arable farming The problems such as control effect is poor selects the oily radish of root-knot nematode resistant as the green stalk of green manure, and to root knot nematode disease With control effect, four kinds of microorganisms of rapidly and efficiently decomposition green stalk, the small spring plantation root-knot nematode resistant in field Oily radish, and prepare decomposition microbial agent is sprayed on the green oil radish stalk of full-bloom stage with the suitable concentration and proportion of four kinds of microbial inoculums, It the stalk after microbial inoculum will further be sprayed turns on the spot and bury decomposition 60 days or more, not only promote green stalk quickly and efficiently decomposition, And effective collaboration prevention and control are carried out to the root knot nematode disease during arable farming, and provide crop organic nutrient.

Above-mentioned purpose of the invention is realized by following means:

A kind of method of the quick decomposition of green plant stalk and prevention and control flue-cured tobacco crop root knot nematode disease, it is characterised in that including Following steps:

(1) oily radish plant green stalk is cut off when it grows to full-bloom stage as 30-40cm/ section；

(2) by four kinds of decomposition microbial agent bacillus subtilises (Bacillus subtilis), bacillus licheniformis (Bacillus licheniformis), Trichoderma viride (Trichoderma viride) and aspergillus niger (Aspergillus It niger) is in mass ratio 1-2: 0.5-1.5: 1.5-2: 0.5-2, the ratio of preferably 1:1:1:1 mixes, and the end of every kind of bacterium is dense Degree (spore amount) is allocated as 3 × 10⁹CFU/ml is prepared into suspending agent, and being filtered or being sieved is prepared into mix bacterium agent mother liquor；

(3) the mix bacterium agent mother liquor for obtaining upper step is 1: 20-1: 50 according to the mass ratio of mix bacterium agent mother liquor and water, excellent It is selected as 1: 30 ratio to be uniformly mixed with water, obtains decomposition microbial agent suspending agent, decomposition microbial agent suspending agent miospore concentration is at this time 10⁸CFU/ml；

(4) the decomposition microbial agent suspending agent that upper step is matched is sprayed to the first step to the oily radish green stalk cut down in field On, and the oily radish green stalk for spraying decomposition microbial agent suspending agent is turned on the spot and is buried decomposition 60 days or more；

(5) transplant crop seedling before, turn over plough planting site again, by institute's long-term cropping the smooth milpa of demand and complete its Its farming operations.

The method of the invention is preferable over to be carried out in 2-4 month.

Filtering described in above-mentioned (2) step can be filtered using gauze, and sieving refers to using 20 meshes.

In above-mentioned (4) step, on one mu of oily radish stalk, the amount of spraying of decomposition microbial agent suspending agent is 20-40kg, preferably 25-35kg。

Decomposition microbial agent of the present invention the preparation method comprises the following steps: bacterium using LB culture medium fermentation preparation level-one, second level kind Son and decomposition microbial agent.Culture medium prescription are as follows: yeast extract 0.5%, tryptone 1%, NaCl1%.

Culture medium preparation method are as follows: 60% tap water is added, by the way that 2M NaOH is added, pH value is adjusted to 7.2- 7.4, it adds water to the amount (mass percent) that need to prepare culture medium, packing (40- into the triangular flask of 500mL or 1000mL The culture solution of 50% volume), when preparing solid medium, add 1.8% agar powder, fluid nutrient medium sealing, 121 DEG C of sterilizings 20min.Two kinds of bacteriums of the slant tube or glycerol tube that take cryo-conservation save pipe, aseptically, by the bacterial strain of preservation It is seeded in the solid LB media test tube of preparation respectively on inclined-plane, is placed in 30 DEG C of incubators and cultivates 48hr.

Bacterium decomposition microbial agent the preparation method comprises the following steps: taking the level-one kind (test tube slant) and 500mL triangle of above-mentioned culture respectively Bottle aseptically with oese by sterile working, successively scrapes one ring of lawn on inclined-plane, is inoculated in 500mL respectively In the LB liquid medium of triangular flask, two bottles of culture solutions are placed in constant-temperature table, 30 DEG C, 150rpm by sealing, lasting to cultivate 48hr, the bacterium solution of culture are second level kind.Aseptically, the second level kind bacterium solution of culture is seeded to packing respectively has 40- It in 50% sterilized liquid LB culture medium 1000mL triangular flask, is placed in constant-temperature table, 30 DEG C, 150rpm persistently cultivate 72hr, training Feeding bacterium solution is the decomposition microbial agent of two kinds of bacteriums.

Fungi prepares firsts and seconds seed, culture medium prescription using PDA culture medium are as follows: fresh potato 20%, sucrose 2%.Culture medium preparation method are as follows: fresh potato peeling is added the tap water of 5 times of potato qualities, boils 30min, with double Layer filtered through gauze temperature is down to the potato fruit of room temperature, then dissolves and washes away mashed potato with tap water, filters, the filtrate total amount of collection It is 5 times of potato quality, 20% sucrose and dissolution is added, before dispensing potato culture solution, is added by the 1.8% of dispensed loading amount In triangular flask, culture solution is dispensed into the triangular flask of 500mL or 1000mL (culture solution of 40-50% volume) agar powder, envelope Mouthful, 121 DEG C of sterilizing 20min.

Fungi decomposition microbial agent (second level kind) the preparation method comprises the following steps: taking the slant tube or two kinds of glycerol tube thin of cryo-conservation Bacterium saves pipe, and aseptically, the bacterial strain of preservation is seeded to respectively in the solid PDA medium test tube of preparation on inclined-plane, It is placed in culture 72hr in 25 DEG C of incubators and is prepared into level-one kind (test tube slant).Test tube slant and the sterilizing of above-mentioned culture are taken respectively Aseptically aqua sterilisa is added in water, scrapes the lawn on inclined-plane with inoculation shovel and successively scrapes inclined-plane by sterile working On lawn, two kinds of lawn liquid are collected in respectively in the empty triangular flask of sterilizing, mix spore liquid.The spore of 4mL is drawn with pipette Sub- liquid, the diameter that preparation is added is in the PDA plate of 15cm, and even with coating shovel coating, two kinds of fungies respectively prepare 10 wares, are placed in 25 DEG C 7d is persistently cultivated in incubator, until lawn is covered in plate in PDA culture medium, the complete blackening of the lawn of aspergillus niger, green wood The complete greening of mould lawn (second level kind).

The formula of fungi decomposition microbial agent (three-level kind) culture medium: 1% sucrose, 2% husk, 0.2% (NH₄)₂SO₄.Fungi Decomposition microbial agent (three-level kind) culture medium configuration method: broken corn particulate, diameter 1-2mm, by corn: water=1:1.0- are selected Tap water is added in 1.2 ratio, impregnates r for 24 hours, filters off excessive moisture, then by the ratio addition sucrose of formula, husk and (NH₄)₂SO₄, mix thoroughly, be packaged as 5kg/ bags with polypropylene plastics pocket, 121 DEG C of sterilizing 20min.Fungi decomposition microbial agent (three-level kind) The preparation method comprises the following steps: take the secondary seed of the two kinds of fungies prepared, two kinds of bacterium in plate are respectively charged into PDA culture medium and are gone out It crosses in the vessel of bacterium, it is smashed to pieces respectively with sterilizing spades, the enamel tray of 10 4 × 38 × 52cm is taken, cleans.By the jade of sterilizing Grain of rice culture medium is divided into two parts, claims its weight, and the secondary seed spore and culture medium of above-mentioned preparation are added by 5% inoculum concentration, It mixes thoroughly respectively, the maize culture medium after taking inoculation, is added in enamel tray by the amount of 2kg/ disk, paves, be placed in constant incubator Middle culture when culture substrate temperature is more than 50 DEG C, takes out koji tray, turns over song, keep the mycelia of two kinds of bacterium uniform in maize culture medium Growth, and a large amount of spores are generated, 25 DEG C are persistently cultivated 15 days, and decomposition microbial agent (three-level seed) is prepared into.

Spore (cell) quantity of four kinds of decomposition microbial agent Unit Weights is detected by microscopic counting or dilution-plate method, really Protect the dosage of addition decomposition microbial agent；

The germination percentage of the seed of oil radish strain of the present invention is higher than 60%；Its implantation methods is to enter September, selection The nonirrigated farmland of the crops such as the nonirrigated farmland of optimum plot size or plan next year the first tenday period of a month in late April to May plantation flue-cured tobacco is as oily radish Planting site after having harvested crop in the fall, turn over plough and smooth to nonirrigated farmland, 1-2 days before being rained according to weather forecast selection, into Row oil radish sowing, if after planting without rainwater in the case where, suitable irrigation water can be applied.

The present invention selects oily radish as green manure plant, nutritional ingredient needed for not only having can provide plant after decomposition, but also can prevent The root knot nematode disease in arviculture is controlled, can also improve the soil texture, improve soil water-retaining power and reduce the amount of application of chemical fertilizer Many advantages, such as.

The present invention is directed to oily radish green stalk, is prepared for four kinds of microbial fermentation inoculums, withered grass gemma therein respectively Bacillus, bacillus licheniformis and Trichoderma viride are inhibited to cause of disease root-knot nematode, and generate degraded cellulose abundant, The compound enzyme system such as lignin；Aspergillus niger generates many cellulases, hydrolase etc., and the compound enzyme system that four kinds of bacterium generate, which cooperates with, to be made With the green stalk macromolecular constituent of quickening is degraded to small-molecule substance, the substance of first three bacterium growth metabolism and oily radish The small-molecule substance of stalk is jointly inhibited to cause of disease root-knot nematode.Two kinds of bacteriums belong to bacillus growth and breeding speed Close, consumption nutrient block is spent, equal proportion, promotion thallus abundance balance and metabolic balance are selected.The speed of growth ratio of two kinds of moulds Two kinds of bacteriums are slow, by the growth metabolism of bacterium, promote the temperature of stalk microenvironment, promote the growth of two kinds of fungies, be added etc. The microbial inoculum of ratio promotes the shifting balance Tiny ecosystem of every kind of bacterium growth, is respectively derived between four kinds of bacterium main needed for body Nutrient, but it is nutrient that itself extra ingredient, which is supplied to it to plant bacterium, constitutes the relationship of mutual reciprocity and mutual benefit.Combination microbial inoculum is sprayed on plant Object green stalk, improve turn over bury before four kinds of microorganisms contained by stalk quantity, accelerate green stalk and turn over corruption after burying Speed is solved, has the effect of prevention and control crop root knot nematode disease in the ingredient after promoting oily radish green straw decomposition, and be supplied to Nutritional ingredient needed for plant.

The method of the present invention can be applicable to the plantation of other crops.

" % " of unexplained reference of the present invention is mass percent.

Oily radish of the present invention using anti-root-knot nematode cooperates with work with three kinds of microorganisms with prevention and control root-knot nematode effect With prevention and control root knot nematode disease.The present invention has the advantages that

(1) small spring (2-4 month) is in Yunnan Province and the less area of national rainwater, the especially growing area in water project situation difference Domain such as hillside fields or nonirrigated farmland cannot plant the crop for largely needing water at this season, and oily radish belongs to drought-enduring plant, plants oily radish Later, soil moisture can be consolidated, sand and dust in air is reduced, is beneficial to the ecological environment of Winter-Spring.

(2) operation for planting oily radish is simply extensive, and recruitment is few, at low cost.As described above, because of the hillside fields or mountain in small spring Ground is more arid, and peasant household generally abandons long-term cropping, but after Cotton Varieties by Small Farming Households oil radish, chemical fertilizer amount needed for next season long-term cropping It greatly reduces (30%), therefore the applying quantity of chemical fertilizer in next season can be reduced, to reduce the cost input of peasant household.

(3) after turning over and burying decomposition, the soil organism dramatically increases the oily radish green stalk planted, and improves nitrogen phosphorus in soil Potassium nutrition ingredient and the effect for keeping soil moisture, chesson structure improve the soil texture.

(4) decomposition microbial agent beneficial microbe is added, accelerating green straw decomposition is the nutritional ingredient that can be absorbed by plants Process, decomposed time shortens, rotten degree is high, is conducive to the utilization of plant seedlings efficient absorption, improves surviving for transplanting seedling Rate.

(5) since decomposition microbial agent belongs to beneficial microbe, such quantity of useful microbe in soil is artificially improved, is improved Rhizosphere soil beneficial microorganism ecology, reduces the risk of pest and disease damage generation.

(6) oily radish green stalk, micro- life such as bacillus subtilis, bacillus licheniformis, Trichoderma viride in decomposition microbial agent Object has prevention and control effect to root knot nematode disease, and long-term cropping is effectively reduced and makes because the underproduction caused by root knot nematode disease declines with quality At loss and prevention and control root knot nematode disease needed for pesticide cost, increase the income of peasant household, improve the quality of agricultural product.

Detailed description of the invention

Fig. 1, which builds aqua stalk and sprays different single microbial inoculums and turn over, buries organic matter in processing group phase cigarette strain rhizosphere soil.

Fig. 2 build aqua stalk spray composite bacteria agent various dose turn over bury processing group phase cigarette strain rhizosphere soil in it is organic Matter.

Fig. 3 builds aqua stalk and sprays single microbial inoculum and turn over to bury to handle to be changed in different times soil with organic matter.

Fig. 4 builds aqua stalk and sprays composite bacteria agent various dose and turn over to bury to handle to be become in different times soil with organic matter Change.

The Mengzi Fig. 5 green stalk, which sprays the combination of different microbial inoculums and turns over, buries processing organic matter in group's phase rhizosphere soil.

The Mengzi Fig. 6 green stalk sprays different microbial inoculum combinations and turns over to bury to handle to be changed in different times soil with organic matter.

Fig. 7, which builds aqua stalk and sprays different single microbial inoculums and turn over, buries processing root-knot nematode state of an illness index.

Fig. 8 build aqua stalk spray composite bacteria agent various dose turn over bury processing root-knot nematode state of an illness index.

The Mengzi Fig. 9 green stalk sprays different microbial inoculum combinations and turns over the root-knot nematode state of an illness index for burying processing.

Figure 10 a., which builds aqua stalk and sprays different single microbial inoculums, turns over the per mu yield for burying processing.

Figure 10 b., which builds aqua stalk and sprays different single microbial inoculums, turns over the per mu yield value for burying processing.

Figure 11 a., which builds aqua stalk and sprays composite bacteria agent various dose, turns over the per mu yield for burying processing.

Figure 11 b., which builds aqua stalk and sprays composite bacteria agent various dose, turns over the per mu yield value for burying processing.

The Mengzi Figure 12 a. green stalk sprays different microbial inoculum combinations and turns over the per mu yield for burying processing.

The Mengzi Figure 12 b. green stalk sprays different microbial inoculum combinations and turns over the per mu yield value for burying processing.

Specific embodiment

The method of the present invention is further illustrated with example below.

The preparation of 1: two plant of bacterium decomposition microbial agent of embodiment.

The preparation of (1) two plant of bacteria culture media and the preparation of I and II seed

1) preparation of solid LB media and inoculated and cultured (first order seed)

A. according to the quantity of culture, by weighing as follows: yeast extract 0.5%, tryptone 1%, NaCl1% add The tap water for entering 60% is dissolved and is mixed and (such as prepares the solid medium of 1000mL, be then separately added into: yeast extract 5g, pancreas Peptone 10g, NaCl10g, tap water 600mL), by the way that 2MNaOH is added, pH value is adjusted to about 7.2, adds tap water It is settled to the volume (such as 1000mL) that need to prepare culture medium.

B. it tries to please and accumulates the triangular flask for being 500mL, culture solution is dispensed by 200mL/ bottles of amount, weighs 1.8% agar powder (3.6g) is sub-packed in the triangular flask of 500mL, culture solution 200mL/ bottles is added, beyond the Great Wall cotton plug, with other reagents that need to be sterilized Or vessel 121 DEG C of sterilizing 20min together.

C. prepare the tool plug test tube that 5-10 props up 18 × 180mm, 10mL pipette is packed with old newspaper respectively, with above-mentioned training Feeding base 121 DEG C of sterilizing 20min together.

D. sterilizing finishes, and is down to about 60 DEG C to culture medium temperature, takes out culture medium and mixes, and on superclean bench, uses Culture medium in triangular flask is aseptically drawn about 5mL into sterilizing test tubes by the pipette that sterilizes, and prepares slant medium, standby With.

Two kinds of bacteriums of the slant tube or glycerol tube that E. take cryo-conservation save pipe, aseptically, by preservation Bacterial strain is seeded in the solid LB media test tube of preparation respectively on inclined-plane, is placed in 30 DEG C of incubators and is cultivated 48hr.

2) culture of the preparation of LB liquid medium and secondary seed

A. the formula of fluid nutrient medium is same as described above, is only added without agar powder in preparation process, has adjusted liquid training After the pH value for supporting base, packing, second level kind selects the triangular flask of 500mL, and decomposition microbial agent (three-level kind) selects the triangular flask of 1000mL, It is sealed with 6 layers of gauze, then pricks triangle bottleneck with old newspaper and cotton thread envelope, 121 together with other reagents or vessel that need to be sterilized DEG C sterilizing 20min, it is spare.

B. the LB liquid medium for taking level-one kind (test tube slant) and 500mL triangular flask of above-mentioned culture respectively, ultra-clean On workbench, with oese by sterile working, one ring of lawn on inclined-plane is successively scraped, is inoculated in 500mL triangular flask respectively LB liquid medium in, sealed, two bottles of culture solutions be placed in constant-temperature table, 30 DEG C, 150rpm with six layers of gauze and cotton thread, 48hr is persistently cultivated, it is spare.

(2) preparation of decomposition microbial agent (three-level seed)

A. the quantity that area and decomposition microbial agent are sprayed according to needed for, prepares LB liquid medium, selects the triangle of 1000mL Bottle, every bottle is added LB liquid medium (method is same as above) prepared by 600mL, is sealed with 6 layers of gauze, then is sealed with brown paper and cotton thread Triangle bottleneck is pricked, 121 DEG C of sterilizing 20min are spare.

B. the LB liquid medium for taking the second level kind of above-mentioned culture and the 1000mL triangular flask packing of preparation, in aseptic condition Under, it is inoculated in the LB liquid medium of 600mL by 1% inoculum concentration (6mL bacterium solution), is sealed, set with six layers of gauze and cotton thread In constant-temperature table, 30 DEG C, 150rpm persistently cultivate 72hr.

The measurement of (3) two kinds of bacterium decomposition microbial agent concentration

The clean tube of 6 18 × 180mm is taken, 9mL distilled water is added in every test tube, the bacterial concentration of every kind of bacterium needs 3, respectively by 10 times, 100 times and 1000 times labels of dilution, every kind of bacterium solution 1mL is successively taken, is added in the test tube for diluting 10 times, It mixes；It takes again and mixes the bacterium solution 1mL for diluting 10 times, be added in the test tube for diluting 100 times, mix；Similarly dilute 3 gradients.

A blood counting chamber is taken, the 0.2mL bacterium solution of different microbial inoculums and dilution successively, is respectively added in tally measurement Region, covered are placed under optical microscopy and detect thalline quantity in bacterium solution, and testing result is shown: bacillus subtilis Thallus (gemma or bacterium) concentration be 1.98 × 10⁹CFU/mL, bacillus licheniformis cell concentration be 1.63 × 10⁹CFU/ The bacterial concentration of fermentation preparation is concentrated into 10 by centrifugal concentrating by mL¹⁰CFU/mL or more.

The preparation of 2: two kinds of fungi decomposition microbial agents of embodiment.

The preparation of (1) two fungal strain culture medium and the preparation of I and II seed

The preparation of the preparation of solid PDA medium and inoculated and cultured (first order seed) and secondary seed.

A. it according to the quantity of culture, is prepared in following culture medium prescription ratio (mass percent): peeling fresh potato 20%, small pieces are cut into, 5 times of potato block of distilled water is added and (such as the solid PDA medium of preparation 1000mL, then distinguishes Using: fresh potato 200g, distilled water 1000mL), 30min is boiled in heating, room temperature is down to temperature, with double gauze mistake Filter, then be washed with distilled water the potato block (mud) boiled and filtered with double gauze, filtrate is collected, is mixed, 2% sugarcane is added Sugared (20g) is added water and is settled to 1000mL, dissolve and mix, and the pH value of culture solution is natural.

B. product of trying to please is the triangular flask of 500mL, weighs 1.8% agar powder (3.6g) and is sub-packed in equipped with fluid nutrient medium In 500mL triangular flask, culture solution is added in triangular flask by the amount of every bottle of packing 40-50%, beyond the Great Wall cotton plug, 121 DEG C of sterilizings 20min。

C. prepare the tool plug test tube that 5-10 props up 18 × 180mm, 10mL pipette uses ox-hide paper wrapper, 121 DEG C of sterilizings respectively 20min。

D. sterilizing finishes, and is down to about 60 DEG C to culture medium temperature, takes out culture medium and mixes, and on superclean bench, uses Culture medium in triangular flask is drawn into about 5mL into sterilizing test tubes under sterilizing pipette aseptic condition, prepares slant medium, it is standby With.

E. the test tube slant of cryo-conservation or two kinds of fungies of glycerol tube preservation are taken, aseptically, by the bacterium of preservation Strain is inoculated in the solid PDA medium test tube of preparation respectively on inclined-plane, is placed in 25 DEG C of incubators and is cultivated 7d.

F. taking diameter is the culture dish (about 20 sets) of 15cm, distilled water 200mL/ triangular flask (500mL), the pipette of 10mL 6, and coating shovel packaging sterilizing.The formula of solid PDA medium is same as described above, by 40-50% volume packing triangular flask totally 5 Bottle, is put into 121 DEG C of sterilizing 20min of autoclave.

G. the culture medium of above-mentioned sterilizing is when temperature is down to about 50 DEG C, by culture medium under aseptic condition on superclean bench It pours into the culture dish that diameter is 15cm, 35-40mL/ ware makes it be down to room temperature and solidification naturally, to be seeded.

H. on superclean bench under aseptic condition, with pipette plus sterile purified water culture test tube slant lawn On, 10mL/ pipe scrapes the spore of lawn with inoculation washing-round, two kinds of bacterium are collected in respectively in the empty triangular flask of sterilizing.

I. the spore liquid that 4mL is drawn with pipette, the diameter that above-mentioned preparation is added is in the PDA plate of 15cm, with coating Shovel coating is even, and two kinds of fungies respectively prepare 10 wares, is placed in 25 DEG C of incubators and persistently cultivates 7d, until long in PDA culture medium in plate Full lawn, the complete blackening of the lawn of aspergillus niger, the complete greening of the lawn of Trichoderma viride (secondary seed) are spare.

(2) preparation of decomposition microbial agent (three-level seed)

A. broken corn particulate is selected, diameter 1-2mm such as presses 100 according to the required quantity for spraying area and decomposition microbial agent Mu sprays area, then two kinds of microbial inoculums respectively need about 20kg corn particulate, in corn: water=1:1 ratio, and tap water is added, and impregnates R for 24 hours filters off excessive moisture, and 1% sucrose, 2% husk, 0.2% (NH is added₄)₂SO₄, mix thoroughly, packed with polypropylene plastics pocket It is 5kg/ bags, 121 DEG C of sterilizing 20min are spare.

B. take the secondary seed of two kinds of fungies in the culture dish that diameter is 15cm prepared, by two kinds of bacterium in plate with PDA culture medium is respectively charged into sterilized vessel, is smashed it to pieces respectively with sterilizing spades, spare.

C. the enamel tray of 10 4 × 38 × 52cm is taken, is cleaned.The maize culture medium of sterilizing is divided into two parts, claims its heavy Amount, the secondary seed spore and culture medium of above-mentioned preparation are added by 5% inoculum concentration, is mixed thoroughly respectively, the niblet after taking inoculation Culture medium is added in enamel tray by the amount of 2kg/ disk, paves, be placed in constant incubator and cultivate.During this, supervised with thermometer The temperature of maize culture medium is surveyed, when selecting temperature higher daily (> 50 DEG C), koji tray is taken out, turns over song, the mycelia of two kinds of bacterium is made to exist Maize culture medium homoepitaxial, and a large amount of spores are generated, 25 DEG C are persistently cultivated 15 days, and decomposition microbial agent (three-level kind is prepared into Son).

The measurement of (3) two kinds of decomposition microbial agent concentration

The triangular flask of 2 250mL and the clean tube of 6 18 × 180mm are taken, the distillation of 60mL is added in each triangular flask 9mL distilled water is added in every test tube for water, and the bacterial concentration measurement of every kind of fungi needs 3 test tubes, respectively by dilution 10 times, 100 Again, 1000 times and 10000 times labels, successively weigh every kind of microbial inoculum 10g, are separately added into equipped in 60mL distilled water, sway mixing； It takes again and mixes the bacterium solution 1mL for diluting 10 times, be added in the test tube for diluting 100 times, mix；Similarly dilute 4 gradients.

A blood counting chamber is taken, successively, the 0.2mL bacterium solution of different microbial inoculums and dilution is added dropwise is surveyed in tally respectively Determine region, covered is placed in detection bacterium solution miospore quantity, testing result under optical microscopy and shows: trichoderma viride Concentration be 2.40 × 10¹⁰CFU/g, aspergillus niger spore concentration be 4.35 × 10¹⁰CFU/g reaches every gram 10,000,000,000 or more Spore concentration, the decomposition microbial agent that can be used for spraying.

Embodiment 3: Jianshui County and the sowing of Mengzi city oil radish seed and green stalk plant development.

(1) oily radish seed and enforcement place

A. oily radish seed: oily radish seed is that KUNMING INST OF BOTANY CAS is researched and developed With preservation, expand numerous seed jointly with Honghezhou Branch, Yunnan Tobacco Co., Ltd..

B. enforcement place and area: Jianshui County Linan town village Ming Shao and Mengzi city, dustpan village, the town Ming Jiu, each 5 mu of area.

(2) sowing and Cultivate administration

A. the sowing time of two places is in late September, 2017.Plough first is turned over to milpa before sowing, if the crop of cultivation is roasting The crop that cigarette etc. has been infected by root-knot nematode cause of disease first removes the undesirable root of preceding crop, and smooth milpa, then uses and broadcast sowing Method sowing, spacing in the rows × line-spacing is about 20 × 30cm, and about 0.5kg/ mus of application rate, then milpa after planting is slightly put down with rake It is whole.It preferably selects down sowing before rain or is seeded in smooth rear sprinkling suitable quantity of water, sprout and grow convenient for oily radish seed.

B. oily radish belongs to drought-enduring, the cold-resistant crop with impoverishment tolerant, after preceding stubble long-term cropping, is generally not required to apply fertilizer.If Seeding row spacing is overstocked, can appropriate thinning, reject extra weak seedling.

C. oily radish is crucifer, although winter temperature is low, insect pest is few, if warm in winter climates such as Red River Area plantation, also will appear the insect pest of aphid one kind on oily radish plant, preferably insecticide selected to be prevented and treated.

D. to oily radish growth to full-bloom stage, simultaneously dissection oil radish is cut down in after planting about 100 days, i.e. in early January, 2018 Plant stalk spray microbial inoculum and turn over burying decomposition.The fresh stalk yield of two places is about 3500-4000kg/ mus.

Embodiment 4: the oily radish green stalk of Jianshui County plantation, which turns over, buries decomposition and tobacco planting plot experiment

(1) oily radish green stalk material, enforcement place and area

Oily radish green stalk in September, 2017 sowing plantation, wherein turn over as oily radish green stalk and bury decomposition for 2 mu by choosing And the plot experiment of tobacco planting.Enforcement place is Jianshui County Linan town village Ming Shao.

(2) oily radish green straw harvesting, spray microbial inoculum, turn over and bury and reason moisture in the soil

A. oily radish growth is green by the aerial part oil radish of growth using cutting utensil to full-bloom stage (in January, 2018) Color stalk is cut into 3-4 and breaks (about 30-40cm long).

B. the concentration bacillus subtilis (10 prepared in aforementioned case study on implementation 1¹⁰) and bacillus licheniformis CFU/mL (10¹⁰CFU/mL), the Trichoderma viride (2.40 × 10 prepared in case study on implementation 2¹⁰CFU/g) with aspergillus niger (4.35 × 10¹⁰CFU/ Four kinds of decomposition microbial agents such as g), prepare the mother liquor of composite bacteria agent suspension, and the concentration of every kind of bacterium is 3 × 10⁹CFU/g, with gauze mistake Filter off makes every kind of bacterium spray final concentration of 10 in bacterium solution except big particle by the mother liquor 1kg for applying preparation per acre⁸CFU/ ml；In addition, preparing final concentration of the 3.0 × 10 of four kinds of microbial inoculums respectively⁹The monoxenic mother liquor of CFU/ml.The green straw of Jianshui County It includes two that stalk, which turns over and buries plot experiment: 1. four kinds of single microbial inoculums, which spray respectively and spray and compare with mix bacterium agent, (turns over and buries and sky It is white)；2. 3 kinds of concentration (0.25 × 10 of mix bacterium agent spray liquid⁸CFU/ml、0.50×10⁸CFU/ml and 1.00 × 10⁸CFU/ml And control (turn over and spray clear water when burying and do not plant oily radish and turn over the blank buried)；Mu sprays 2 barrels of dosage, and (sprayer is 15kg/ barrels, the mix bacterium agent of 0.5kg is added, mixes) it is sprayed.

C. it sprays and finishes, the green stalk after spraying is disconnected to be turned over and be earthed that (about 4 ton/mu fresh weights, on January 16th, 2018 are real Apply), keep stalk disconnected decomposition about 60 days natural in the soil.

D. to will plant the plough that turns over before flue-cured tobacco is experimental field transplanted (containing buried and blank control is turned over), and it is normal by flue-cured tobacco Rule planting technology carries out the smooth of planting site, ditching, manages moisture in the soil, opens the preparation before the transplanting of the tobacco seedlings such as the pool.

(3) plot experiment of tobacco planting and transplanting Cultivate administration

A. the cell repetition setting that flue-cured tobacco is planted after burying decomposition is turned over.Build water and turn over that bury test include different single microbial inoculum decompositions effect Fruit (including do not plant oily radish blank control, turn over and bury totally 6 processing of fresh water spraying control and 4 kinds of microbial inoculums) and different bacteria suspension concentrations (do not plant oily radish control including blank, turn over 3 kinds of concentration totally 5 processing for burying fresh water spraying control and mix bacterium agent spray liquid) Two are turned over the test buried, and 3 replicated plots, the area of each replicated plot about 54m is arranged in each processing²。

B. flue-cured tobacco cultivars and tobacco seedlings

The flue-cured tobacco cultivars for building water test point are cloud and mist 87, and tobacco seedlings press conventional seedbed system skill by Jianshui County Linan town flue-cured tobacco seedling-cultivating Art is cultivated and is provided.

C. flue-cured tobacco transplantation of seedlings.After having managed moisture in the soil and having held the pool successfully, carries out compound fertilizer and quantitatively apply fertilizer to the subsoil, do not plant oily radish and turn over The blank control cell for burying processing, by (45g/ plants) application compound fertilizers of 50kg/ mus of amounts of application；It plants oily radish plant and is turned over The test process cell (containing the control for spraying clear water) buried matches by (32g/ plants) of 35kg/ mus of amounts of application and applies compound fertilizer；With cigarette strain Transplanting point is the center of circle, and diameter about 40cm carries out ring and applies.

D. the Cultivate administration after tobacco seedlings transplanting.After flue-cured tobacco transplantation of seedlings, tobacco planting production technology routinely fertilising with It is managed in terms of pest and disease damage prevention and control correlation regulation, but no longer applies the pesticide of prevention and control root knot nematode disease.

Embodiment 5: the oily radish green stalk of Mengzi city plantation, which turns over, buries decomposition and tobacco planting plot experiment

(1) oily radish green stalk material, enforcement place and area

Oily radish green is in September, 2017 sowing plantation, and choosing is wherein turned over as oily radish green stalk and buries decomposition and roasting for 2 mu The plot experiment of cigarette plantation.Enforcement place is Mengzi city, dustpan village, the town Ming Jiu.

(2) oily radish green straw harvesting, spray microbial inoculum, turn over and bury and reason moisture in the soil

A. oily radish growth is green by the oily radish aerial part of growth using cutting utensil to full-bloom stage (in January, 2018) Color is cut into 3-4 and breaks (about 30-40cm long).

B. the bacillus subtilis (3 × 10 prepared in aforementioned case study on implementation 1⁹CFU/mL) with bacillus licheniformis (3 × 10⁹CFU/mL), the Trichoderma viride (2.40 × 10 prepared in case study on implementation 2¹⁰CFU/g) with aspergillus niger (4.35 × 10¹⁰CFU/g) Four kinds of decomposition microbial agents, prepare the mother liquor of composite bacteria agent suspension, and the bulky grain removed with filtered through gauze is prepared by application per acre Mother liquor 1kg, so that every kind of bacterium is being sprayed final concentration of 10 in bacterium solution⁸CFU/ml；In addition, preparing two kinds of four kinds of microbial inoculums respectively Microbial inoculum combination, comprising: bacillus subtilis+Trichoderma viride；Bacillus subtilis+aspergillus niger；Bacillus licheniformis+Trichoderma viride And bacillus licheniformis+aspergillus niger, final concentration of the every kind of bacterium in composite bacterial solution (mother liquor) is 3.0 × 10⁹CFU/ml.Mengzi The green stalk in city, which turns over, buries plot experiment totally 7 processing: a kind of a kind of above-mentioned four kinds of mixing combined respectively by bacterium with fungi Microbial inoculum processing, turns over the blank control for burying and spraying clear water control and not planting oily radish at the processing of four kinds of bacterium composite bacteria agents；Mu sprays The dosage (sprayer is 15kg/ barrels, and the mix bacterium agent of 0.5kg is added, and is mixed) of 2 barrels of microbial inoculums is sprayed.

C. it sprays and finishes, the green stalk after spraying is disconnected to be turned over and be earthed (, make the disconnected decomposition about 60 natural in the soil of stalk It.

D. to will plant the plough that turns over before flue-cured tobacco is experimental field transplanted (containing buried and blank control is turned over), and it is normal by flue-cured tobacco Rule planting technology carries out the smooth of planting site, ditching, manages moisture in the soil, opens the preparation before the transplanting of the tobacco seedlings such as the pool.

(3) plot experiment of tobacco planting and transplanting Cultivate administration

A. the cell setting that flue-cured tobacco is planted after burying decomposition is turned over.Turn over the decomposition effect for burying that test is the combination of different microbial inoculums in Mengzi city The test of (do not plant the control of oily radish including blank, turn over and bury fresh water spraying control and 4 kinds of microbial inoculum groups amount to 7 processing), each 3 replicated plots of processing setting, the area of each replicated plot about 48m²。

B. flue-cured tobacco cultivars and tobacco seedlings

The flue-cured tobacco cultivars of Mengzi testing site are Yun yan85, and tobacco seedlings press conventional seedbed system skill by the Mengzi city town Ming Jiu flue-cured tobacco seedling-cultivating Art is cultivated and is provided.

C. flue-cured tobacco transplantation of seedlings.After having managed moisture in the soil and having held the pool successfully, carries out compound fertilizer and quantitatively apply fertilizer to the subsoil, do not plant oily radish and turn over The blank control cell for burying processing, by (45g/ plants) application compound fertilizers of 50kg/ mus of amounts of application；It plants oily radish plant and is turned over The test process cell (containing the control for spraying clear water) buried matches by (32g/ plants) of 35kg/ mus of amounts of application and applies compound fertilizer；With cigarette strain Transplanting point is the center of circle, and diameter about 40cm carries out ring and applies.

D. the Cultivate administration after tobacco seedlings transplanting.After flue-cured tobacco transplantation of seedlings, tobacco planting production technology routinely and fertilising with Pest and disease damage prevention and control correlation regulation is managed, but no longer applies the pesticide of root knot nematode disease.

Embodiment 6: oily radish green stalk turns over the soil property variation during burying decomposition and flue-cured tobacco cultivation and detects

By pedotheque forward and backward with plantation flue-cured tobacco after the oily radish green stalk of acquisition experimental field middle plantation, and detect The variation of its soil property Main Nutrients detects.

(1) sampling position and sampling time

A. in above-described embodiment, oily radish and with turning over the plot experiment buried and blank control test is planted, in plantation oil Radish turns over bury before, before transplanting tobacco seedlings and after plantation flue-cured tobacco 50 days, acquire soil sample respectively, acquire altogether 3 soil samples (in January, 2018, In April, 2018 and in June, 2018).And pedotheque is sent to Yunnan Yun Nong Science and Technology Ltd. and is detected.

B. the place for acquiring sample, which is respectively as follows: the oily radish of Jianshui County Linan town village Ming Shao plantation and turns over, buries and plants flue-cured tobacco Experimental field；Mengzi city, dustpan village, the town Ming Jiu, which plants oily radish and turns over, buries and plants flue-cured tobacco experimental field.Furthermore it acquires and does not plant oil The neighbour vega soil of radish is as blank control.

(2) acquisition of rhizosphere soil sample is detected with sample presentation

A. the oily radish of kind plant turns over the sample collection method before burying using five point sampling methods；Transplant tobacco seedlings before with plantation flue-cured tobacco 50 Experimental field sampling after it is that each cell acquires 1 part of soil sample.It plants before oily radish turns over and bury, five points is determined, every It is a, the soil of surface layer about 10cm depth is cut out, takes longitudinal soil of about 15cm depth again vertically downward, acquires the soil of about 500g Sample is fitted into valve bag, by the number of the routine operation label sample of acquisition sample.Before transplanting tobacco seedlings, it has been determined that plot experiment Each cell cut out the soil of surface layer about 10cm depth in each cell centre, take the deep longitudinal soil of about 15cm again vertically downward Earth acquires the soil sample of about 500g, is fitted into valve bag, by the number of the routine operation label sample of acquisition sample.Transplant tobacco seedlings After 50 days, soil sample is acquired with method in each cell centre.

B. the sample acquired includes that two of Jianshui County Linan town village Ming Shao turn over and bury test and blank control, Mengzi city ring vulture The soil sample for burying test and blank control is turned in town dustpan village.

C. the above-mentioned pedotheque in different times acquisition turns over and buries cell pedotheque that is rear, acquiring before transplanting tobacco seedlings, will The pedotheque of 3 replicated plots of same processing mixes the sample of (about 600ml) as detection by isometric soil；3rd The cell pedotheque of secondary acquisition, directly as the sample of detection.By pedotheque send to Yunnan Yun Nong Science and Technology Ltd. into Row detection.

D. the soil nutrient project detected in above-mentioned soil sample includes: organic matter, full N, available N, P, K and pH, and method uses The corresponding ministry standard of each detection project is detected.

(3) Analysis of test results of the main soil property project of rhizosphere soil sample

Ministry standard is pressed by Yunnan Yun Nong Science and Technology Ltd., to the main soil property project pH value of pedotheque, full nitrogen, hydrolysis Property nitrogen (available nitrogen), available phosphorus and available potassium are total to five indices and are detected, the results showed that, it builds water test cell and sprays difference It the processing (as shown in Figure 1) of single microbial inoculum and sprays (as shown in Figure 2) test of different microbial inoculum amounts and is subtracting the item for applying 15kg/ mus of compound fertilizers (subtract under part and apply 30%), is planted oily radish and spray microbial inoculum in full-bloom stage and turn over to bury and managed everywhere in decomposition, transplant tobacco seedlings about 50 After it (group's phase), totally four index variations are smaller for the pH value of rhizosphere soil, full nitrogen, rapid available phosphorus and available potassium, and difference is not significant, Show after subtracting the chemical fertilizer for applying 15kg/ mus (30%), four indexs and conventional application chemical fertilizer have similar soil nutrient level, poor It is different not significant；But it plants oily radish and turn in its full-bloom stage and bury decomposition, organic matter and speed in same time tobacco field rhizosphere soil Effect nitrogen nutrient levels are below the control of Zhi Shi compound fertilizer, and whether (F=4.15 > F between different single microbial inoculum processing_0.05 =3.33) or composite bacteria agent various concentration processing between (F=4.45 > F_0.05=3.84) difference is significant, and the content of organic matter is equal Lower than do not plant oily radish with turn over compareing (referring to Fig. 1 and Fig. 2) of burying, theoretically, after planting oily radish green stalk and turning over and bury (about 4 ton/mu of fresh stalk), soil with organic matter content should obviously increase, but reducing instead of showing of test result, the result Show after spraying microbial inoculum, whether single microbial inoculum or composite bacteria agent, degrade to soil with organic matter；From spraying not Organic matter with the processing of single microbial inoculum reduces height analysis, withered grass > trichoderma > clear water > lichens > compound fertilizer CK processing；It is compound from spraying The organic matter of microbial inoculum various dose processing reduces height analysis, at 0.25kg processing > 1.00kg processing > 0.50kg processing > clear water Reason > compound fertilizer CK processing, illustrates that the amount for spraying microbial inoculum can be reduced to 0.25kg/ mus.

By each processing of test being buried and acquiring pedotheques in three different times to building water and spray single microbial inoculum and turn over, detect it Organic matter as the result is shown (referring to Fig. 3), blank control processing the content of organic matter tobacco seedlings transplanting after reduce it is unobvious, but By being added the processing of fermenting agent, the content of organic matter buries processing (clear water) and does not plant oily trailing plants than turning over for fermenting agent is not added Foretell and turn over the blank control buried and be substantially reduced, with three kinds of bacillus subtilis, bacillus licheniformis and Trichoderma viride fermenting agents Reduction amplitude it is maximum, decomposition effect is obvious.

Three kinds of various dose processing of composite bacteria agent are sprayed in the detection of three different times acquisition pedotheques according to water is built As a result (referring to fig. 4), the content of organic matter of blank control processing reduces unobvious in the front and back organic matter of tobacco seedlings transplanting, but passes through The processing of composite fermentation microbial inoculum various dose is added, the content of organic matter buries processing (clear water) and not than turning over for fermenting agent is not added It plants oily radish to be substantially reduced with the blank control buried is turned over, wherein spray the processing that compound bacteria dosage is 1.00kg/ mus, effect The reduction amplitude of organic matter is most prominent, and decomposition effect is most obvious.

It can be seen that plantation green oil radish stalk, sprays 1.00kg/ mus of composite bacteria agent in full-bloom stage and (sprays at 15kg/ barrels The composite bacteria agent of tetra- kinds of bacterium of 0.50kg is added in mist agent, sprays 2 barrels/mu), by effective speed for accelerating green oil radish straw decomposition Degree under conditions of reducing application compound fertilizer 15kg/ mus (30%) in flue-cured tobacco cultivation, fully meets and supports needed for cigarette strain growth Point.

Turning over for Mengzi city, dustpan village, the town Ming Jiu is buried in test, has selected spray clear water, bacillus subtilis+green wood respectively Mould, bacillus subtilis+aspergillus niger, bacillus licheniformis+Trichoderma viride, bacillus licheniformis+aspergillus niger and four kinds of bacterium mixing Microbial inoculum sprays on the oily radish stalk of full-bloom stage, and further turns over and bury decomposition about 60 days, is provided with 6 processing, and will be same Phase does not plant neighbouring one piece of oily radish, and with season vacant land, as blank control, (later testing result shows that the block is not busy with season The soil with organic matter content for setting ground is higher), totally 7 test process.The transplanting tobacco seedlings time is identical, acquisition on June 14 cigarette strain root Border soil, detects its content of organic matter and the results of analysis of variance shows that (referring to figs. 5 and 6), difference is extremely significant between test process (F=7.21 > F_0.01=4.82).Wherein, the content of organic matter is minimum (27.77g/kg) in the rhizosphere soil of blank control, but because In neighbouring cigarette, with belonging to the cigarette of another peasant household, two families have biggish in long-term cropping process, soil organic matter content for experimental plot Difference, generally the piece cigarette the content of organic matter be higher than build water test point.From comparative analysis (April 20 and 6 before and after transplanting tobacco seedlings The respective cell soil sample of the acquisition of the moon 14), after four kinds of microbial inoculums mixing spray, soil with organic matter content reduces maximum, from April 20 The 47.06g/kg of day soil sample is reduced to the 32.22g/kg on June 14, during which reduces 14.84g/kg, followed by sprays clear water pair According to (7.52g/kg) and spray bacillus subtilis+aspergillus niger combination microbial inoculum (6.97g/kg) processing organic matter reduce it is more, The decomposition effect for showing that four kinds of microbial inoculum mixing spray is significant.

In conclusion under conditions of 30% compound fertilizer of Jian Shi, passing through compared with applying compound fertilizer in conventional tillage system reform Plant oily radish and spray microbial inoculum and turn over and bury decomposition processing, can quick decomposition green stalk, effectively improve containing for soil with organic matter Amount, provides the available nutrient of plant growth, improves soil property situation, and provide N P and K needed for cigarette strain growth course and fill Sufficient nutrient.

Embodiment 7: flue-cured tobacco crop adopts the root-knot nematode disease investigation after baking.

(1) oily radish green stalk plantation, spray microbial inoculum, turn over and bury decomposition, tobacco seedlings transplanting, Cultivate administration and adopt flue-cured tobacco etc. It is same as Example 5, implement respectively in Jianshui County Linan town village Ming Shao and Mengzi city, dustpan village, the town Ming Jiu, has carried out single microbial inoculum It sprays, four kinds of compound various concentrations of microbial inoculum spray and two kinds of microbial inoculum combinations spray totally 3 field trials.

(2) after the flue-cured tobacco planted is adopted and baked tobacco leaf, take cigarette strain root system in plot experiment, investigate cigarette strain root system by cause of disease root The hazard rating of nematode infection is tied, cause of disease root-knot nematode endangers cigarette strain rating survey method using " country, the People's Republic of China (PRC) Standard GB/T23222 --- 2008 (classification of tobacco pest and disease damage and investigation methods) " carry out.

(3) according in the experimental plot of investigation cigarette strain by hazard rating, calculate the disease index of each experimental plot, count It is as follows to calculate formula:

Each cell cigarette strain root system disease grade of investigation is calculated to the root-knot nematode state of an illness index of experimental plot by above-mentioned formula, And the variance analysis of disease index is respectively handled in being tested.

(4) disease index and variance analysis of two test process of water are built.

A. root-knot nematode state of an illness index between spraying single microbial inoculum different disposal is built in water test by inquiry, as a result sees Fig. 7 It is shown.It is planted oily radish and sprays microbial inoculum in full-bloom stage and turn over and bury processing (including spraying clear water), make the state of an illness of test process Index is substantially reduced, compared with blank control, relative control effect 36.0-65.8%.Further to the cell disease for testing each processing Feelings index carries out variance analysis, and difference reaches extremely significant (F=25.28 > F between processing_0.01=5.64), show to turn over that bury oily radish green Color stalk has a significant effect to prevention and control flue-cured tobacco root knot nematode disease.Between spraying different single microbial inoculum processing, control effect is successively Are as follows: withered grass > aspergillus niger > lichens > trichoderma > clear water > compound fertilizer CK processing.

B. water oil radish green stalk is built to spray four kinds of mix bacterium agent various dose processing, carry out turning over again and bury decomposition about 60 It, transplanting tobacco seedlings, Cultivate administration and after adopting flue-cured tobacco, uproot and investigate the root-knot nematode state of an illness index of cigarette strain root system, as a result join See Fig. 8.It is planted oily radish and sprays microbial inoculum in full-bloom stage and turn over and bury processing (including spraying clear water), make the state of an illness of test process Index is substantially reduced, compared with blank control, relative control effect 36.0-61.9%.Further to the cell disease for testing each processing Feelings index carries out variance analysis, and difference reaches extremely significant (F=50.08 > F between processing_0.01=7.01), show to turn over that bury oily radish green Color stalk has a significant effect to prevention and control flue-cured tobacco root knot nematode disease.Between the mix bacterium agent processing for spraying various dose, prevention and control effect Fruit is successively are as follows: 1.00kg/ mus > 0.50kg/ mus > 0.25kg/ mus > clear water > compound fertilizer CK processing.

(5) the disease index variance analysis of Mengzi test process.

It turns over and buries the processing for spraying the combination of two kinds of microbial inoculums in test respectively and mixing with four kinds of microbial inoculums, bury decomposition about turning in Mengzi 100 days, transplanting, Cultivate administration and after adopting flue-cured tobacco, uproot and investigate the root-knot nematode state of an illness index of cigarette strain root system, as a result referring to figure 9.Turning over for microbial inoculum combination is sprayed in full-bloom stage and buries processing (including spraying clear water), drops the disease index of test process obviously It is low, compared with blank control, relative control effect 66.6-88.4%.Further the cell disease index for testing each processing is carried out Variance analysis, difference reaches extremely significant (F=96.02 > F between processing_0.01=4.82), show to turn over and bury oily radish green stalk to anti- Control flue-cured tobacco root knot nematode disease has a significant effect.Between the microbial inoculum processing for spraying various combination, control effect is successively are as follows: four kinds of bacterium Agent combination > lichens+aspergillus niger > withered grass+aspergillus niger > withered grass+trichoderma > lichens+trichoderma > clear water > compound fertilizer CK processing.

It is above-mentioned to investigate and divide in cigarette strain root-knot nematode state of an illness index after adopting roasting that Jianshui County and Mengzi city are implemented respectively Analysis the result shows that, by planting oily radish green stalk, spray microbial inoculum and turn over and bury decomposition, during plant flue-cured tobacco, to baking The root knot nematode disease of cigarette has significant control effect, wherein most prominent to spray the control effect of the composite bacteria agent of four kinds of bacterium processing Out.

Embodiment 8: flue-cured tobacco crop yield and the output value are investigated.

(1) oily radish green stalk plantation, spray microbial inoculum, turn over and bury decomposition, tobacco seedlings transplanting, Cultivate administration and adopt flue-cured tobacco etc. It is same as Example 5, implement respectively in Jianshui County Linan town village Ming Shao and Mengzi city, dustpan village, the town Ming Jiu, has carried out single microbial inoculum It sprays, four kinds of compound various doses of microbial inoculum spray and two kinds of microbial inoculum combinations spray totally 3 field trials.

(2) flue-cured tobacco planted routinely adopts flue-cured tobacco, divides bar to adopt as unit of cell roasting, will entirely adopt roasting difference in the process Correspondence is compiled in each cell, is further weighed by tobacco leaf grading standard to roasting cell tobacco leaf grading is adopted, cell tobacco leaf of testing and assessing Yield and the output value and conversion are per mu yield and per mu yield value.

(3) it builds the cell production of two test process of water and the output value is tested and assessed and its variance analysis.

A. by assessment build water turn over bury test in spray cell production and the output value between single microbial inoculum different disposal, further roll over Calculating is per mu yield and per mu yield value, as a result referring to shown in Figure 10 a and Figure 10 b.It plants oily radish and sprays microbial inoculum in full-bloom stage and turn over and bury It handles (including spraying clear water), improves the yield of test process and the output value, compared with blank control, output increased amplitude is 26.9-52.6kg/ mus, it is 686.6-1911.9 member/mu that the output value, which promotes amplitude, further to the conversion per mu yield for testing each processing Variance analysis is carried out respectively with per mu yield value, and difference reaches extremely significant (yield: F=42.49 > F between processing_0.01=5.64；The output value F=14.71 > F_0.01=5.64), show to turn over the promotion for burying oily radish green stalk to flue-cured tobacco per mu yield and per mu yield value with obvious Effect.Output increased and output value promotion are almost the same, successively are as follows: withered grass > lichens > aspergillus niger > trichoderma > clear water > compound fertilizer CK Processing.

B. by assessment build water turn over bury the processing of four kinds of bacterium composite bacteria agent various doses is sprayed in test between cell production and produce Value, further conversion is per mu yield and per mu yield value, as a result referring to shown in Figure 11 a and Figure 11 b.It plants oily radish and is sprayed in full-bloom stage It applies microbial inoculum and turns over and bury processing (including spraying clear water), promoted the yield respectively handled in test and the output value, compared with blank control, Output increased amplitude is 26.7-52.4kg/ mus, and it is 681.3-1501.2 member/mu that the output value, which promotes amplitude, further everywhere to test The conversion per mu yield and per mu yield value of reason carry out variance analysis respectively, and difference reaches the significant (output value: F=5.50 > F between processing_0.05 =3.84) or extremely significant (yield: F=32.37 > F_0.01=7.01), show to turn over after spraying 0.25-1.00kg/ mus of composite bacteria agent Oily radish green stalk is buried, is had a significant effect to flue-cured tobacco per mu yield and the promotion of per mu yield value.From yield angle, spray 1.00kg/ mus of composite bacteria agent is best to effect of increasing production, from output value angle, sprays 0.25kg/ mus of composite bacteria agent and produces to promotion It is best to be worth effect, comprehensive for the control effect of root knot nematode disease, yield and the output value, the effect of three aspects is almost the same, with Spraying 1.00kg/ mus of composite bacteria agent is relatively advisable.

(4) yield of Mengzi test process and output value assessment and its variance analysis.

It turns over and buries the processing for spraying the combination of two kinds of microbial inoculums in test respectively and mixing with four kinds of microbial inoculums, bury decomposition about 60 turning in Mengzi It, routinely transplanting, Cultivate administration and after adopting flue-cured tobacco, the yield and the output value for burying each cell in test are turned in assessment, are further rolled over Calculating is per mu yield and per mu yield value, as a result referring to shown in Figure 12 a and Figure 12 b.Turning over for microbial inoculum combination, which is sprayed, in full-bloom stage buries processing (packet Include and spray clear water), improve the yield of test process and the output value, compared with blank control, output increased amplitude is 7.6- 57.9kg/ mus, it is 753.5-1434.5 member/mu that the output value, which promotes amplitude, further to the conversion per mu yield and mu for testing each processing The output value carries out variance analysis respectively, and difference reaches the extremely significant (output value: F=9.96 > F between processing_0.01=4.82；Yield: F= 10.67>F_0.01=4.82), show to spray microbial inoculum combination or composite bacteria agent, turn over and bury decomposition oil radish green stalk, to flue-cured tobacco The promotion of per mu yield and per mu yield value has significant effect.Yield and output value promotion show consistent effect, and display sprays 1.00kg/ mus of composite bacteria agent is best to the effect increased production and increased income.

In conclusion by three plot experiments for building water and Mengzi two places are carried out altogether, according to the knot of 8 embodiments Fruit plants oily radish green stalk, sprays microbial inoculum and turns over and bury decomposition, soil organic matter content can be improved, increases soil nutrient.? It turns over during planting flue-cured tobacco with burying decomposition, under conditions of compound fertilizer's amount of application reduces by 15kg/ mus (30%), soil Green stalk Quick decomposition, npk nutrient meet the needs of cigarette strain growth, and soil property improves.Meanwhile though no longer applying the root knot of prevention and treatment flue-cured tobacco The pesticide of nematodiasis, but control effect is significant, and output increased and the output value are promoted obviously.

Claims (10)

1. a kind of method of quick decomposition of green plant stalk and prevention and control flue-cured tobacco crop root knot nematode disease, which is characterized in that including Following steps:

(1) oily radish plant green stalk is cut off as 30-40cm/ section；

(2) in mass ratio it is 1-2 by four kinds of decomposition microbial agent bacillus subtilises, bacillus licheniformis, Trichoderma viride and aspergillus niger: 0.5-1.5: 1.5-2: 0.5-2 ratio mixes, and the final concentration (spore amount) of every kind of bacterium is allocated as 3 × 10⁹CFU/ml, preparation At suspending agent, being filtered or being sieved is prepared into mix bacterium agent mother liquor；

(3) ratio that the mix bacterium agent mother liquor for obtaining upper step is 1: 20-1: 50 according to the mass ratio of mix bacterium agent mother liquor and water It is uniformly mixed with water, obtains decomposition microbial agent suspending agent, decomposition microbial agent suspending agent miospore concentration is 10 at this time⁸CFU/ml；

(4) the decomposition microbial agent suspending agent that upper step is matched is sprayed to the first step and is cut down on the oily radish green stalk in field, and The oily radish green stalk for spraying decomposition microbial agent suspending agent is turned on the spot and is buried decomposition 60 days or more；

(5) before transplanting crop seedling, plough planting site is turned over again, the smooth milpa of demand and the other agricultures of completion by institute's long-term cropping Thing operation.

2. the method for the quick decomposition of green plant stalk according to claim 1 and prevention and control flue-cured tobacco crop root knot nematode disease, It is characterized in that, the method carries out in 2-4 month.

3. the method for the quick decomposition of green plant stalk according to claim 1 and prevention and control flue-cured tobacco crop root knot nematode disease, It is characterized in that, four kinds of decomposition microbial agent bacillus subtilises, bacillus licheniformis, Trichoderma viride and aspergillus niger are pressed in (2) step The ratio that mass ratio is 1: 1: 1: 1 mixes.

4. the method for the quick decomposition of green plant stalk according to claim 1 and prevention and control flue-cured tobacco crop root knot nematode disease, It is characterized in that, the mass ratio of mix bacterium agent mother liquor and water is 1: 30 in (3) step.

5. the method for the quick decomposition of green plant stalk according to claim 1 and prevention and control flue-cured tobacco crop root knot nematode disease, It is characterized in that, oily radish plant green stalk described in (1) step chooses the stalk of oily radish growth to full-bloom stage.

6. the method for the quick decomposition of green plant stalk according to claim 1 and prevention and control flue-cured tobacco crop root knot nematode disease, It is characterized in that, on one mu of oily radish stalk, the amount of spraying of decomposition microbial agent suspending agent is 20-40kg in (4) step.

7. the method for the quick decomposition of green plant stalk according to claim 1 and prevention and control flue-cured tobacco crop root knot nematode disease, It is characterized in that, the bacterium in four kinds of decomposition microbial agents is using LB culture medium fermentation preparation level-one, secondary seed and decomposition microbial agent, training Support based formulas are as follows: yeast extract 0.5%, tryptone 1%, NaCl 1%.

8. the method for the quick decomposition of green plant stalk according to claim 1 and prevention and control flue-cured tobacco crop root knot nematode disease, It is characterized in that, the fungi in four kinds of decomposition microbial agents prepares firsts and seconds seed, culture medium prescription using PDA culture medium are as follows: Fresh potato 20%, sucrose 2%, culture medium preparation method are as follows: oneself of 5 times of potato qualities is added in fresh potato peeling Water boils 30min, the potato fruit of room temperature is down to double gauze filtration temperature, then dissolve and wash away mashed potato with tap water, Filtering, the filtrate total amount of collection are 5 times of potato quality, and 20% sucrose and dissolution is added, before dispensing potato culture solution, Agar powder is added in triangular flask by the 1.8% of dispensed loading amount, culture solution is dispensed into the triangular flask of 500mL or 1000mL, sealing, 121 DEG C of sterilizing 20min.

9. the method for the quick decomposition of green plant stalk according to claim 8 and prevention and control flue-cured tobacco crop root knot nematode disease, It is characterized in that, fungi decomposition microbial agent the preparation method comprises the following steps: take cryo-conservation slant tube or glycerol tube two kinds of bacteriums protect Pipe is deposited, aseptically, the bacterial strain of preservation is seeded in the solid PDA medium test tube of preparation respectively on inclined-plane, is placed in 72hr is cultivated in 25 DEG C of incubators is prepared into level-one kind；

The test tube slant and aqua sterilisa of above-mentioned culture are taken respectively, aseptically, aqua sterilisa is added, and scrape inclined-plane with inoculation shovel On lawn the lawn on inclined-plane is successively scraped by sterile working, two kinds of lawn liquid are collected in the empty triangular flask of sterilizing respectively In, spore liquid is mixed, the spore liquid of 4mL is drawn with pipette, the diameter that preparation is added is in the PDA plate of 15cm, with coating Shovel coating is even, and two kinds of fungies respectively prepare 10 wares, is placed in 25 DEG C of incubators and persistently cultivates 7d, until long in PDA culture medium in plate Secondary seed is made in full lawn, the complete blackening of the lawn of aspergillus niger, the complete greening of the lawn of Trichoderma viride.

10. the method for the quick decomposition of green plant stalk according to claim 9 and prevention and control flue-cured tobacco crop root knot nematode disease, It is characterized in that, the culture medium prescription of fungi decomposition microbial agent three-level kind are as follows: 1% sucrose, 2% husk, 0.2% (NH₄)₂SO₄； Fungi decomposition microbial agent three-level kind culture medium configuration method are as follows: selection broken corn particulate, diameter 1-2mm, by corn: water=1: Tap water is added in the ratio of 1.0-1.2, impregnates r for 24 hours, filters off excessive moisture, then by formula rate be added sucrose, husk and (NH₄)₂SO₄, mix thoroughly, be packaged as 5kg/ bags with polypropylene plastics pocket, 121 DEG C of sterilizing 20min；

Fungi decomposition microbial agent three-level kind the preparation method comprises the following steps: the secondary seed of the two kinds of fungies prepared is taken, by two kinds in plate Bacterium and PDA culture medium are respectively charged into sterilized vessel, are smashed it to pieces respectively with sterilizing spades, are taken 10 4 × 38 × 52cm Enamel tray, clean, the maize culture medium of sterilizing be divided into two parts, claims its weight, above-mentioned preparation is added by 5% inoculum concentration Secondary seed spore and culture medium, mix thoroughly respectively, take inoculation after maize culture medium, by 2kg/ disk amount be added enamel tray In, it paves, is placed in constant incubator and cultivates, when culture substrate temperature is more than 50 DEG C, takes out koji tray, turn over song, make two kinds of bacterium Mycelia generates a large amount of spores in maize culture medium homoepitaxial, and 25 DEG C are persistently cultivated 15 days, is prepared into decomposition microbial agent three-level Seed.