CN106577049A - Method of utilizing biomass charcoal to inhibit phytophthora nicotianae - Google Patents
Method of utilizing biomass charcoal to inhibit phytophthora nicotianae Download PDFInfo
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- CN106577049A CN106577049A CN201610918688.0A CN201610918688A CN106577049A CN 106577049 A CN106577049 A CN 106577049A CN 201610918688 A CN201610918688 A CN 201610918688A CN 106577049 A CN106577049 A CN 106577049A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G13/00—Protecting plants
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G22/00—Cultivation of specific crops or plants not otherwise provided for
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
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Abstract
The invention discloses a method of utilizing biomass charcoal to inhibit phytophthora nicotianae. The method includes the following steps: firstly, configuring and adding a biomass charcoal medium; secondly, adjusting pH with the added biomass charcoal medium to be original pH of the medium; and thirdly, configuring bacteria soil. The method is simple, is convenient to use, and has a great effect to inhibit phytophthora nicotianae. Two types of biomass charcoal different in proportion are added separately or added together to an oat medium, the influence of biomass charcoal on phytophthora nicotianae is determined through determination of relevant indexes, and relevant experiments are designed to conduct mechanism verification on the basis of the property of biomass charcoal. Brown soil is selected and rice husk charcoal with different proportions is added in a potted condition to verify an effect of biomass charcoal on phytophthora nicotianae prevention. Therefore, theoretical bases are provided for reasonable applications of biomass charcoal and related research and comprehensive control and treatment of phytophthora nicotianae.
Description
Technical field：
The present invention relates to the suppressing method technical field of tobacco pathogen bacterium, is specifically related to a kind of using biomass carbon suppression tobacco
The method of black shank bacterium.
Background technology：
Black shank is a kind of destructive disease on tobacco leaf production, the serious production for restricting each cigarette district tobacco leaf of China.According to not
Count completely, the economic loss that China is caused because of black shank every year on average is up to more than 100,000,000 yuan.Tobacco black shank bacterium is smokeless
Can survive 5 years or longer in the soil that grass is present, can survive 18 months in wind desiceted soil, after infecting tobacco plant, can be serious
Affect the raising of tobacco production and value amount.For the suppression or preventing and treating of black shank bacterium, generally using chemicals, chemicals is to environment
Pollution is big, and the time is unfavorable for for a long time agricultural production and environmental development, accordingly, it would be desirable to choose a kind of new method to suppress balck shank
Bacterium.
Biomass carbon is that biomass are pyrolyzed at high temperature generation, is a kind of solid matter containing carbon rich（SOHI S P
Et.al, 2010；Yang Pingping, 2012）, it has preferable pore structure and specific surface area, and nutrient enriches, and can improve agriculture
Field environment, is agriculturally widely used.China is tobacco planting big country, and cultivated area occupies first place in the world, and all can be produced every year
Substantial amounts of tobacco waste（Predominantly tabacco straw）, at present for main processing mode be to burn and direct returning to farmland, burn
Element loss and environmental pollution can be caused, and straw-returning is then easily caused serious soil-borne disease.Therefore, by tabacco straw charcoal
Application is carried out after change can give full play to its advantage, reduce the Regional Environmental Pollution that crop straw burning is caused.Note of the prior art
Carry, the utilization of biomass carbon, majority is concentrated in the utilization of charcoal base manure, and for example Chinese patent application CN104292044A is disclosed
A kind of acid soil improvement simultaneously prevents and treats the biomass carbon base of tobacco rhizome disease.（It is by biomass carbon base come conditioned soil
Earth structure, increases water and fertilizer utilization, increases soil fertility, and improves cigarette strain resistance, enriches soil micro-ecosystem, promotes plant growth to send out
Preventing and treating Tobacco Root stem disease is educated and then reached, but has no experimental data to prove its impact to rhizome disease, effect is unknown）
During other are recorded：Applying biomass carbon in the soil-less medium of porous can reduce invading for capsicum and tomato leaf portion fungal disease
Enter（Ellen R.Graber et.al, 2010）.After applying coconut charcoal, the fusarium infection rate of asparagus is substantially reduced
（Lehmann J, 2011）.Compared with the control, in applying some process of charcoal, Ralstonia solanacearum infects substantially reduction to tomato
（Lehmann J, 2011）.Up to the present, found in 6 different pathogenic systems to apply biomass carbon in soil
The occurrence degree of soil-borne disease can be affected（Graber ER et.al, 2014）.By existing document record, it has been reported that pass
It is concentrated mainly in capsicum, tomato, asparagus, Soybean and Other Crops in impact of the biomass carbon to plant disease（Ellen
R.Graber et.al, 2010；Lehmann J, 2011；Jaiswal AK, 2013）, and tobacco and black shank are ground
Study carefully that there is not been reported, and tobacco is one of important industrial crops of China, black shank is affect its yield and quality main
Disease, so it is very necessary that research biomass carbon affects on black shank.In view of above-mentioned prior art, it is contemplated that
There is provided a kind of method simple, it is easy to use, the black shin of tobacco is suppressed using biomass carbon to the good one kind of black shank bacterium inhibition
The method of germ.
The content of the invention：
It is an object of the invention to overcome shortcoming present in prior art, there is provided a kind of method is simple, easy to use, to black shin
The method that a kind of good utilization biomass carbon of germ inhibition suppresses tobacco black shank bacterium.
To achieve these goals, the invention provides a kind of utilization biomass carbon suppress tobacco black shank bacterium method,
Wherein, including
Method one, configuration with the addition of the suppression method of biomass carbon culture medium,
Method two, by the pH that with the addition of biomass carbon culture medium the suppression method of the original pH of culture medium is adjusted back,
Method three, configuration bacterium soil suppression method.
Method one comprises the steps：
Configuration oat medium：Oatmeal 33g, agar powder 18g, deionized water 1000ml,
The rice hull carbon and/or tabacco straw charcoal of different proportion is added in the configuration process of oat medium and corresponding pH is determined
Value；
Inoculation balck shank bacterial strain simultaneously determines colony diameter using crossing method, to cultivate the black shin of colony diameter change calculations of 2d
The growth rate and inhibiting rate of germ mycelia.
Method two comprises the steps：
Configuration oat medium：Oatmeal 33g, agar powder 18g, deionized water 1000ml,
The rice hull carbon and/or tabacco straw charcoal of different proportion is added in the configuration process of oat medium and corresponding pH is determined
Value；Then the pH value of change is adjusted back to into initial value with the HCl or NaOH solution of 0.1mol, initial value is to be not added with biomass carbon
PH value corresponding to culture medium,
Carry out connecing bacterium experiment after sterilizing, after culture terminates, picking mycelia is placed on slide, examines under a microscope black shank bacterium
The change of hypha form.
Method three comprises the steps：
Bacterium paddy prepares：Millet is added water and is boiled to half grain in bloom shape, in being fitted into 500ml triangular flasks, 121 DEG C of autoclavings
20 min；Aseptically, the tobacco black shank bacterium bacterium block for taking 1cm × 1cm is transferred in the triangular flask for filling bacterium paddy, 28 DEG C
14d is standby for culture；
Bacterium soil prepares：By the soil and biomass carbon of sterilizing（Rice hull carbon and/or tabacco straw charcoal）By different proportion（0,1%, 2%,
4%, 10%, 20%）Mixing, in being fitted into diameter 9cm, the flowerpot of high 7cm, first fills 3/4 composite soil, is uniformly added into bacterium paddy 0.5g/
Basin, remaining 1/4 composite soil is filled up, and pours permeable, is placed in one month in artificial climate room, is kept hot and humid；
Transplant：The seedling age tobacco seedlings of 60 days or so are transplanted in flowerpot, per one plant of basin.Each processes 10 basins, is repeated 3 times, and is placed in
Phjytotron culture, 28 DEG C of 30 DEG C/night on daytime, illumination 12h/ dark 12h, relative humidity 95% is transplanted after the 5th, 7,9
The morbidity series of its investigation tobacco seedlings, and recorded, calculate disease index and prevention effect.Disease index=∑（Diseased plant numbers at different levels
× series）/（Highest disease series × investigation total strain number）× 100, prevention effect=（Control group disease refers to-treatment group disease refers to）/ control group
Disease refers to × 100%.
Biomass carbon is rice hull carbon and/or tabacco straw charcoal,
Rice hull carbon：Tabacco straw charcoal is 0-20g/L:20-0g/L.
Advantages of the present invention is：Method is simple, easy to use, good to black shank bacterium inhibition, specially：
Impact of the present invention by indoor culture examination observation biomass carbon to tobacco black shank bacterium, from biomass carbon self property point
The possible mechanism of action is analysed, and carries out potted plant checking.Two kinds of lifes of different proportion are individually or collectively added in oat medium
Material charcoal, by being measured to index of correlation, determines impact of the biomass carbon to tobacco black shank bacterium, and from biomass carbon from
Body property is set out, and design related experiment carries out Mechanism Validation；From brunisolic soil, add under condition of pot without ratio rice husk
Charcoal, verifies prevention effect of the biomass carbon to black shank, to for the rationalization utilization and black shank of biomass carbon
Correlative study and the comprehensive regulation provide theoretical foundation.Biomass carbon gradually rises with the increase pathogen growth inhibiting rate of ratio
Height, inhibition is notable.Black shank bacterium colony diameter is as the rising of pH is in the trend being gradually reduced, the adsorptivity of biomass carbon
And toxicant（A&T）Black shank bacterium colony diameter is significantly reduced, and as the increase of ratio is in significantly reduce.To sum up, originally
Invention biomass carbon has significant inhibition to tobacco black shank bacterium.
Description of the drawings：
Impact of Fig. 1 biomass carbons to black shank bacterium mycelial growth rate（A. tabacco straw charcoal；B. rice hull carbon）
The adsorptivity ＆ toxicant of Fig. 2 pH and charcoal（A＆T) to the impact of black shank bacterium mycelial growth rate（A, B, C, D are successively
It is the corresponding pH of tabacco straw charcoal, the A＆T of tabacco straw charcoal, the corresponding pH of rice hull carbon, the A＆T of rice hull carbon to mycelial growth rate
Impact）
Fig. 3 black shank bacterium hypha forms（10 × 40 times）（1. normal hypha form；2. mycelia deformation；3. mycelia branch；4. former
Raw matter is revealed）
Specific embodiment：
The invention provides a kind of method that utilization biomass carbon suppresses tobacco black shank bacterium, wherein, including
Method one, configuration with the addition of the suppression method of biomass carbon culture medium,
Method two, by the pH that with the addition of biomass carbon culture medium the suppression method of the original pH of culture medium is adjusted back,
Method three, configuration bacterium soil suppression method.
Method one comprises the steps：
Configuration oat medium：Oatmeal 33g, agar powder 18g, deionized water 1000ml,
The rice hull carbon and/or tabacco straw charcoal of different proportion is added in the configuration process of oat medium and corresponding pH is determined
Value；
Inoculation balck shank bacterial strain simultaneously determines colony diameter using crossing method, to cultivate the black shin of colony diameter change calculations of 2d
The growth rate and inhibiting rate of germ mycelia.
Method two comprises the steps：
Configuration oat medium：Oatmeal 33g, agar powder 18g, deionized water 1000ml,
The rice hull carbon and/or tabacco straw charcoal of different proportion is added in the configuration process of oat medium and corresponding pH is determined
Value；Then the pH value of change is adjusted back to into initial value with the HCl or NaOH solution of 0.1mol, initial value is to be not added with biomass carbon
PH value corresponding to culture medium,
Carry out connecing bacterium experiment after sterilizing, after culture terminates, picking mycelia is placed on slide, examines under a microscope black shank bacterium
The change of hypha form.
Method three comprises the steps：
Bacterium paddy prepares：Millet is added water and is boiled to half grain in bloom shape, in being fitted into 500ml triangular flasks, 121 DEG C of autoclavings
20 min；Aseptically, the tobacco black shank bacterium bacterium block for taking 1cm × 1cm is transferred in the triangular flask for filling bacterium paddy, 28 DEG C
14d is standby for culture；
Bacterium soil prepares：By the soil and biomass carbon of sterilizing（Rice hull carbon and/or tabacco straw charcoal）By different proportion（0,1%, 2%,
4%, 10%, 20%）Mixing, in being fitted into diameter 9cm, the flowerpot of high 7cm, first fills 3/4 composite soil, is uniformly added into bacterium paddy 0.5g/
Basin, remaining 1/4 composite soil is filled up, and pours permeable, is placed in one month in artificial climate room, is kept hot and humid；
Transplant：The seedling age tobacco seedlings of 60 days or so are transplanted in flowerpot, per one plant of basin.Each processes 10 basins, is repeated 3 times, and is placed in
Phjytotron culture, 28 DEG C of 30 DEG C/night on daytime, illumination 12h/ dark 12h, relative humidity 95% is transplanted after the 5th, 7,9
The morbidity series of its investigation tobacco seedlings, and recorded, calculate disease index and prevention effect.Disease index=∑（Diseased plant numbers at different levels
× series）/（Highest disease series × investigation total strain number）× 100, prevention effect=（Control group disease refers to-treatment group disease refers to）/ control group
Disease refers to × 100%.
Biomass carbon is rice hull carbon and/or tabacco straw charcoal,
Rice hull carbon：Tabacco straw charcoal is 0-20g/L:20-0g/L.
Below in conjunction with the accompanying drawings, the present invention will be described.As Figure 1-3, Fig. 1 biomass carbons are to black shank bacterium mycelial growth
The impact of speed（A. tabacco straw charcoal；B. rice hull carbon）.The adsorptivity ＆ toxicant of Fig. 2 pH and charcoal（A＆T) to black shank bacterium
The impact of mycelial growth rate（A, B, C, D are successively the corresponding pH of tabacco straw charcoal, the A＆T of tabacco straw charcoal, rice hull carbon correspondence
Impact to mycelial growth rate of pH, the A＆T of rice hull carbon）.Fig. 3 black shank bacterium hypha forms（10 × 40 times）（1. normal bacterium
Filament shapes；2. mycelia deformation；3. mycelia branch；4. plasm is revealed）
Concrete example：
1.1 material
For trying tobacco, bacterial strain and biomass carbon：Tobacco bred is little gold 1025, from countries tobacco mid-term storehouse.The black shin of tobacco
Germ Phytophthora parasitica var. Nicotianae（Common black shank bacterium）.Rice hull carbon（S）It is from market purchase
Buy, tabacco straw charcoal（C）,【Biomass carbon（Rice hull carbon or tabacco straw charcoal）It is bio-organic materials（Biomass）In anoxic or
In anoxybiotic environment, the solid product containing carbon rich generated Jing after high temperature pyrolysis.Method is the high temperature at 500 DEG C to 600 DEG C
Under under anaerobic condition, by organic substance pyrolytic.】, basic physical and chemical is shown in Table 1, and biomass carbon is ground before test
0.5mm is sieved.
Reagent：Hydrochloric acid, NaOH, agar powder are traditional Chinese medicines and analyze pure, are purchased from Qingdao Sai Shang scientific ＆ trading Co., Ltd.s；Oatmeal is
The conventional instant oatmeal of supermarket's purchase.
Instrument：NIKON Eclipse E100 binocular microscopes, Shanghai Pu He Electro-optical Technology, INC. (US) 62 Martin Road, Concord, Massachusetts 017；Superclean bench
（Clean bench）, the net cleaning equipment Co., Ltd in Wuxi one；Panasonic high-pressure steam sterilizing pans（MLS-3751L-PC）,
Panasonic Healthcare ＆ Medical Equipment Co., Ltd.；BINDER biochemical cultivation cases, German Binder Co., Ltds；Acidometer（PH meter）, match
Many this scientific instrument of profit（Beijing）Co., Ltd.
1.2 culture mediums are prepared
Oat medium：Oatmeal 33g, agar powder 18g, deionized water 1000ml.
1.3 method
1.3.1 impact of the biomass carbon to black shank bacterium mycelial growth
The charcoal of different proportion is added during oat medium is configured：0,2.5,5.0,10.0,20.0g/L, and determine phase
The pH for answering（It is shown in Table 2）.After the completion of preparation, 121 DEG C of min of autoclaving 20 of Jing pour sterile petri dish into（The cm of diameter 9）In, will
The bacterium colony for activating and growing preferable bacterial strain on oat medium in advance takes side with the sterilization punchers punching of a diameter of 5 mm
Edge bacteria cake, with inoculation shovel plating medium central authorities are inoculated in, and are placed under 28 DEG C of constant temperature dark conditions and are cultivated, and each process sets 5 times
Repeat.Observation measurement daily 1 time, determines colony diameter and makes a record, until mycelia covers with culture dish using crossing method
Till.To cultivate the growth rate of the colony diameter change calculations black shank bacterium mycelia of 2d（Malaysian's victory etc., 2007）And inhibiting rate
（Wang Kun etc., 2009）.Growth rate (cm d-1)=1/2 × (colony diameter-bacteria cake diameter)/incubation time, inhibiting rate %=is (right
According to colony diameter-process colony diameter)/(control colony diameter-bacteria cake diameter) × 100.
1.3.2 impact of the adsorptivity and toxicant of pH and charcoal to black shank bacterium mycelial growth
Impacts of the pH to black shank bacterium mycelial growth：During configuration oat medium, the HCl or NaOH with 0.1mol is molten
Liquid adjusts the acid-base value of culture medium according to table 2, carries out meeting bacterium experiment, same 1.2.1 after sterilizing.
The adsorptivity and toxicant of charcoal（Absorption and Toxins, A＆T）Shadow to black shank bacterium mycelial growth
Ring：The charcoal of different proportion is added during configuration oat medium：0,2.5,5.0,10.0,20.0g/L, with 0.1mol's
The pH value of change is adjusted back to initial value by HCl or NaOH solution（The pH value being not added with corresponding to the culture medium of charcoal）.Carry out after sterilizing
Meet bacterium experiment, same to 1.2.1.
1.3.3 impact of the biomass carbon to black shank bacterium hypha form
After culture terminates, picking mycelia is placed on slide, examines under a microscope the change of black shank bacterium hypha form, and is clapped
According to record.
1.3.4 pot experiment of the biomass carbon to balck shank prevention effect
Bacterium paddy prepares：Millet is added water and is boiled to half grain in bloom shape, in being fitted into 500ml triangular flasks, 121 DEG C of autoclavings
20 min.Aseptically, the tobacco black shank bacterium bacterium block for taking 1cm × 1cm is transferred in the triangular flask for filling bacterium paddy, 28 DEG C
14d is standby for culture.
Bacterium soil prepares：By the soil and biomass carbon of sterilizing（Rice hull carbon and/or tabacco straw charcoal）By different proportion（0,
1%, 2%, 4%, 10%, 20%）Mixing, in being fitted into diameter 9cm, the flowerpot of high 7cm, first fills 3/4 composite soil, is uniformly added into bacterium paddy
0.5g/ basins（Yu Haiqin etc., 2007）, remaining 1/4 composite soil is filled up, pour permeable, it is placed in one month in artificial climate room, protect
Hold hot and humid.
Transplant：The seedling age tobacco seedlings of 60 days or so are transplanted in flowerpot, per one plant of basin.Each processes 10 basins, is repeated 3 times,
It is placed in phjytotron culture, 28 DEG C of 30 DEG C/night on daytime, illumination 12h/ dark 12h, relative humidity 95%, transplant after the 5th,
7th, the morbidity series of 9 days investigation tobacco seedlings, and recorded, calculate disease index and prevention effect.Disease index=∑（Diseases at different levels
Strain number × series）/（Highest disease series × investigation total strain number）× 100, prevention effect=（Control group disease refers to-treatment group disease refers to）/ right
Refer to × 100% according to a group disease.
1.4 data analysis
Test data carries out data statistics and analysis using Excel and the statistical softwares of SAS 9.2, carries out under the level of α=0.05
Duncan ' s Multiple range tests.
2 results and analysis
Impact of 2.1 biomass carbons to tobacco black shank bacterium mycelial growth
2.1.1 to the impact of black shank bacterium colony diameter
Add in oat medium after biomass carbon, black shank bacterium colony diameter reduces.With plus charcoal ratio by 2.5 gL-1l
Increase to 20.0 gL-1, colony diameter declines rapidly（Table 3）.
Compared with CK, the black shank bacterium colony diameter that first, second and third day plus different proportion tabacco straw charcoal are processed subtracts respectively
Few 0.57-0.82cm, 0.81-1.54cm, 0.35-1.63cm, add the colony diameter that different proportion rice hull carbon is processed to subtract respectively
Few 0.59-0.76cm, 0.37-1.35cm, 0.29-0.99cm, reduction of two kinds of biomass carbons to black shank bacterium colony diameter is made
With notable.
2.1.2 to the impact of black shank bacterium mycelial growth rate and inhibiting rate
Fig. 1, A are shown in impact of the tabacco straw charcoal to black shank bacterium mycelial growth rate.Compared with CK, plus the mycelial growth that charcoal is processed
Speed has different degrees of decline, with plus charcoal ratio by 2.5g L-1Increase to 20 g L-1, mycelial growth rate reduce amplitude be
15.56%-28.89%.Rice hull carbon is added to reduce the growth rate of mycelia in oat medium（Fig. 1, B, note：Different letters
Represent that Jing Duncan's multiple comparison graphs are verified in P<Significant difference in 0.05 level.Similarly hereinafter.Different letters
indicate significant difference at P<0.05 level by Duncan's Multiple Range
Test. The same below.）.Compared with CK, mycelial growth rate is in the trend being gradually lowered with the increase for adding charcoal ratio,
Reduction amplitude be 11.85%-25.19%, significant difference.Therefore, biomass carbon can reduce black shank bacterium mycelial growth rate, and with
Plus charcoal ratio increase reducing effect is notable.
As shown in Table 4, the tabacco straw charcoal of different proportion is added to have certain suppression to black shank bacterium mycelial growth in the medium
Make and use, the increase inhibition of charcoal ratio is obvious with adding.The inhibiting rate that tabacco straw charcoal grows to black shank bacterium by plus charcoal ratio
The 15.06% of example 5g/L is changed to and adds the 28.62% of charcoal ratio 20g/L, significant difference between process.Likewise, with rice hull carbon ratio
The increase pathogen growth inhibiting rate of example is changed to 25.09% by 11.34%, and inhibition is notable.
Add in the medium after biomass carbon, by black shank bacterium colony diameter, mycelial growth rate, pathogen growth
The analysis of inhibiting rate, can learn that growth of the biomass carbon to black shank bacterium mycelia has an inhibitory action, and this effect is with applying
The increase of charcoal amount is more notable.
With the adsorptivity and toxicant of charcoal（A＆T) to the impact of black shank bacterium mycelial growth
2.2.1 to the impact of black shank bacterium colony diameter
2.2.1.1 impacts of the pH to black shank bacterium colony diameter
In Initial stage of culture, the impact of the tabacco straw charcoal correspondence pH of different proportion to colony diameter is in first reducing becoming of increasing afterwards
Gesture, the colony diameter for adjusting pH process is respectively less than CK process；Cultivate the impact of the 3rd day pH to black stem bacterium colony diameter less,
Difference is not notable.In addition to adding charcoal ratio and having suppression to colony diameter for the corresponding pH of 10g/L, the colony diameter that other are processed is equal
Colony diameter higher than CK.Therefore, the pH for adjusting culture medium has in the early stage certain inhibitory action to the growth of mycelia, but with
The change of incubation time, this inhibition is not obvious, may contact with culture medium with the limited space of culture medium, later stage mycelia
It is less relevant（Table 5）.
As shown in Table 5, the pH of culture medium is adjusted（The corresponding pH of rice hull carbon）There is significant suppression to black shank bacterium colony diameter
Make and use.A few days ago, black shank bacterium colony diameter compared with the control, divides as the rising of pH is in the trend being gradually reduced for culture
Do not reduce 0.39cm, 0.29cm, 0.33cm, 0.37cm and 0.44cm, 0.52cm, 0.57cm, 0.60cm.At the 3rd day, remove
Plus charcoal ratio is 2.5 g L-1With 20 g L-1Outside the corresponding pH of L are significantly inhibited to colony diameter, other process and compare
Without significant difference, mean value is 7.60cm to the colony diameter of process.
2.2.1.2 the adsorptivity and toxicant of charcoal（A＆T) to the impact of black shank bacterium colony diameter
Cultivate a few days ago, compared with CK, the adsorptivity and toxicant of tabacco straw charcoal（A&T）Significantly reduce black shank bacterium
Colony diameter, reduction amplitude be respectively 0.28cm, 0.21cm, 0.32cm, 0.56cm and 0.59cm, 0.63cm, 0.94cm,
1.37cm.Cultivate the 3rd day, compared with CK, in addition to the process of tabacco straw charcoal of 2.5g/L and 5g/L is added, other process equal
Significantly reduce colony diameter.Likewise, the adsorptivity and toxicant of rice hull carbon（A&T）Significantly reduce black shank bacterium bacterium colony straight
Footpath, and as the increase of ratio is in significantly reduced trend.Compared with CK, the culture bacterium colony that a few days ago rice hull carbon A＆T is processed is straight
Footpath reduces respectively 0.37cm, 0.67cm, 0.27cm, 0.49cm and 0.66cm, 0.92cm, 0.49cm, 0.71cm.Plus charcoal ratio
Colony diameter for 2.5g/L reduces amplitude more than process of the charcoal ratio for 20g/L is added, and its reason is probably with external operation not
When or mycelia vigor is very weak relevant when taking bacteria cake.Cultivate the 3rd day, compared to CK, plus charcoal ratio is for 2.5g/L's and 20g/L
Black shank bacterium colony diameter is substantially reduced, and reduces respectively as 0.73cm, 0.50cm（Table 5）.
2.2.2 the impact to black shank bacterium mycelial growth rate
2.2.2.1 impacts of the pH to black shank bacterium mycelial growth rate
Adjust impacts of the pH of culture medium to black shank bacterium mycelial growth rate and see Fig. 2A and C.Compared with CK, the growth speed of mycelia
Rate is reduced with the rising of pH, reduction amplitude is respectively 3.7%, 5.93%, 8.89%, 3.7% and 8.15%, 9.63%, 11.1%,
11.1%, mean value is 5.56% and 10.00%, illustrates that growths of the pH for adjusting culture medium to black shank bacterium mycelia has certain suppression
Make and use.The corresponding pH of tabacco straw charcoal of different proportion is higher than the corresponding pH of rice hull carbon, but adjusts tabacco straw charcoal correspondence
PH to mycelial growth rate reduce amplitude less than rice hull carbon correspondence pH change, illustrate that black shank bacterium is more likely to be appropriate in alkali
Property environment under grow.
2.2.2.2 the adsorptivity and toxicant of charcoal（A＆T) to the impact of black shank bacterium mycelial growth rate
From Fig. 2 B, the A＆T of tabacco straw charcoal reduces the growth rate of black shank bacterium mycelia, and compared with CK, difference adds charcoal
The mycelial growth rate of ratio process reduces 11.11%-25.93%, significant difference.Likewise, from Fig. 2 D, with CK phases
Reduce the growth rate of mycelia than the A＆T of, the rice hull carbon of different proportion, reduction amplitude is respectively 17.04%, 12.04%,
9.63%th, 13.33%, reducing effect is notable.
2.2.3 to the impact of black shank bacterium growth inhibition ratio
2.2.3.1 impacts of the pH to black shank bacterium growth inhibition ratio
The growth of tabacco straw charcoal and the corresponding pH of rice hull carbon to black shank bacterium mycelia is adjusted in the medium produces certain suppression
Make and use.With the rising of medium pH, the amplitude of variation of inhibiting rate is respectively：3.16%-8.74% and 8.18%-11.15%, puts down
Average is 5.25% and 9.90%.As a whole, although inhibiting rate fluctuation is little, still as the rising of medium pH becomes in increase
Gesture（Table 6）.
2.2.3.2 the adsorptivity and toxicant of charcoal（A&T）Impact to black shank bacterium growth inhibition ratio
As shown in Table 6, the adsorptivity and toxicant of tabacco straw charcoal and rice hull carbon（A&T）Growth to black shank bacterium mycelia is produced
Inhibitory action is given birth to.In the A＆T tests of tabacco straw charcoal, inhibiting rate amplitude of variation is 10.97%-25.46%, and mean value is
16.40%.Pathogen growth inhibiting rate with plus the increase of charcoal ratio increased trend is presented, this and normal addition biomass carbon
Experimental rules are consistent.
In the test of rice hull carbon A＆T, with plus charcoal ratio increase, inhibiting rate is respectively 17.10%, 12.27%, 9.11%,
13.20%, mean value 12.29%.Plus rice hull carbon 2.5g/L processes the inhibiting rate to black shank bacterium（17.10%）Higher than normal plus rice
The inhibiting rate of shell charcoal 2.5g/L process（11.34%）, reason may it is improper with external operation or choose bacteria cake vigor it is relevant.
In sum, the change from black shank bacterium colony diameter, mycelial growth rate and pathogen growth inhibiting rate can
Go out, the adsorptivity and toxicant of pH and charcoal（A＆T) the growth to black shank bacterium mycelia has certain inhibitory action.From totality
From the point of view of, this inhibitory action is with pH risings or adds the rising of charcoal ratio more and more significant, therefore, biomass carbon is to balck shank
The inhibitory action of bacterium mycelial growth is mostly derived from two aspects --- the adsorptivity and toxicant of pH and charcoal（A&T).
Microscopy
In incubation, compared with the control, the tobacco black shank bacterium mycelial growth after process is slower.10 × 40 times show
Micro- Microscopic observation finds and normal Hyphal form ratio that height adds the process of charcoal ratio（10g/L and 20g/L）There is deformity, bacterium in mycelia
Silk expands body showed increased, and mycelia branch is a lot, but mostly short and small, and fracture, the phenomenon that plasm leaks occurs in part（Figure
3）.
Potted plant prevention effect of 2.4 biomass carbons to black shank
There is obvious disease symptom in the incidence of 5 days investigation tobacco seedlings after transplanting, the tobacco seedlings for not applying charcoal process, with applying charcoal amount
Increase, tobacco seedlings occurring degree significantly reduces successively, and the amplitude that reduces is 5.96% ~ 24.65%.Likewise, compared with the control, transplant
Afterwards the disease of the 7th day and the 9th day investigation tobacco seedlings refers to applying the increase of charcoal amount in reduction trend, reduction amplitude is respectively 4.59% ~
32.38% and 3.15% ~ 33.75%, mitigate disease occurrence degree effect is significant.During investigating, biomass carbon is to black shank
Prevention effect with apply charcoal ratio increase be in increase trend.At the 5th, 7,9 days, the biomass carbon of different proportion was processed to cigarette
The prevention effect of careless balck shank is respectively 14.71% ~ 60.89%, 7.75% ~ 54.73%, 4.35% ~ 45.66%.Show to apply certain
The biomass carbon of amount has preferable prevention effect to black shank, can effectively mitigate the occurrence degree of black shank.
To sum up, many studies have shown that applying biomass carbon in culture medium can reduce the infection rate of fungal diseases of plants.
（Ellen R.Graber et.al, 2010）（Lehmann J, 2011）.（Lehmann J, 2011）.This research is trained by flat board
Support and potted plant efficiency test Design Fundamentals has inquired into impact and the mechanism of action of the biomass carbon to tobacco black shank bacterium.In oat training
Biomass carbon is added to significantly inhibit the growth of black shank bacterium mycelia in foster base, and this inhibitory action can be with the increasing for adding charcoal amount
It is more more and more significant.From the point of view of the property of biomass carbon itself, it is believed that machine of the biomass carbon to black shank bacterium inhibitory action
Reason mainly includes two parts：One side biomass carbon is in alkalescence.In test the biomass carbon used by us is alkalescence（Table
1）, the pH that culture medium is improve in oat medium is added, the growing environment of germ is changed, and then affect the growth of germ.
In the test that pH affects on black shank bacterium mycelial growth, the pH for adjusting oat medium reduces colony diameter, reduces bacterium
The growth rate of silk, the growth to black shank bacterium mycelia produces certain inhibitory action.On the other hand it is the absorption of biomass carbon
Property and toxicant.Because both is difficult to separate, we consider as a factor.There are some researches show biomass carbon
Adsorptivity is several orders of magnitude higher than common soil colloid particle（Graber E. R. et.al, 2014）, this adsorptivity
May absorbing pathogenic bacteria, suppress mycelia growth.Biomass carbon itself contains normal chain alkanoic acid（n-alkanoic acids）, hydroxyl
（hydroxy）, acetoxyl group acid（acetoxy acids）, benzoic acid（benzoic acids）, glycol and three alcohol compounds
（diols and triols）And phenolic compound（phenols）（Ellen R. Graber, 2010）, these organic compounds
Plant growth, induced systemic resistance on the one hand may be stimulated in low concentration on the other hand may to suppress the growth of germ,
So as to mitigate the occurrence degree of disease.In the adsorptivity and toxicant of charcoal are tested, black shank bacterium colony diameter, mycelial growth
Speed is presented significantly reduced trend with the increase for adding charcoal ratio, and pathogen growth inhibiting rate increases with the increase for adding charcoal ratio in notable
Plus trend, show that the growth of the adsorptivity and toxicant of charcoal to black shank bacterium mycelia has inhibitory action.But work as plus charcoal ratio
When example is too high, biomass carbon can not only affect soil quality（Zhang Mingkui, 2012）And the growth of land for growing field crops cigarette strain can be affected, therefore
The amount of application of suitable biomass carbon is selected according to the situation that land used is increased soil fertility, mitigates disease occurrence degree, improve the product of tobacco leaf
Quality.
By more normally adding biomass carbon, adjusting medium pH and the inhibiting rate of the adsorptivity and toxicant test of charcoal
It can be seen that（Table 4 and table 6）, in two kinds of mechanism of action that we are discussed, the adsorptivity and toxicant of pH and charcoal is right
The growth of mycelia has certain inhibitory action, but in the test designed based on tabacco straw charcoal, the adsorptivity and toxicity of charcoal
Material plays a leading role, and rice hull carbon is then that pH and the adsorptivity of charcoal and the action effect proportion of toxicant are similar.This
The reason for planting phenomenon may be relevant with biomass carbon grain class distribution.The commodity rice hull carbon of purchase its grade composition is concentrated mainly on
Between 0.25-0.5mm, account for 80%, and tabacco straw charcoal is ground sieve after, the charcoal of its grade composition less than 0.25mm
Particle accounts for 77%.Grade is less, and its specific surface is bigger, and adsorptivity also can change therewith.
Results from pot experiment test shows that biomass carbon has certain prevention effect to black shank, can effectively mitigate tobacco
The occurrence degree of balck shank.The 9d after transplanting, height applies the process of charcoal amount（20 g L-1）Prevention effect reaches 46.55%, and effect shows
Write.Find that high charcoal is processed by microscopy（10 gL-1 and 20 gL-1）The change of black shank bacterium hypha form can be caused, these changes
Whether change can affect black shank bacterium to need further to study to the infecting potential of cigarette strain.After biomass carbon is manured into soil, can be right
The physicochemical properties of soil produce certain impact, and natural environment is complicated, in addition between soil-charcoal-plant three
Interact and generation common on black shank and spread generation impact.Therefore, in addition to two aspects that we inquire in the text,
About biomass carbon the mechanism of action of fungal disease is also needed further to probe into.
Biomass carbon can be substantially reduced colony diameter, reduce the growth rate of mycelia, suppress the growth of black shank bacterium mycelia,
This inhibitory action is more and more significant with the increase of biomass carbon ratio.From the analysis of biomass carbon self property, this shadow is produced
Loud mechanism mainly includes two parts：The adsorptivity and toxicant (A＆T) of pH and charcoal.Found by microscopy, it is biological at high proportion
The hypha form deformity of matter charcoal process, mycelia expands body showed increased, and branch is a lot, and fracture occurs in part, and plasm leaks.Basin
Under the conditions of cultivation, compared with the control, the biomass carbon for applying different proportion is to the potted plant prevention effect of black shank after 9d
4.35% ~ 46.55%, significant difference.Show that applying a certain amount of biomass carbon has preferable prevention effect, energy to black shank
Effectively mitigate the occurring degree of black shank.
Claims (5)
1. a kind of method that utilization biomass carbon suppresses tobacco black shank bacterium, it is characterised in that include
Method one, configuration with the addition of the suppression method of biomass carbon culture medium,
Method two, by the pH that with the addition of biomass carbon culture medium the suppression method of the original pH of culture medium is adjusted back,
Method three, configuration bacterium soil suppression method.
2. the method for suppressing tobacco black shank bacterium using biomass carbon according to claim 1, it is characterised in that method one is wrapped
Include following steps：
Configuration oat medium：Oatmeal 33g, agar powder 18g, deionized water 1000ml,
The rice hull carbon and/or tabacco straw charcoal of different proportion is added in the configuration process of oat medium and corresponding pH is determined
Value；
Inoculation balck shank bacterial strain simultaneously determines colony diameter using crossing method, to cultivate the black shin of colony diameter change calculations of 2d
The growth rate and inhibiting rate of germ mycelia.
3. according to claim 1 biomass carbon suppress tobacco black shank bacterium method, it is characterised in that method two include as
Lower step：
Configuration oat medium：Oatmeal 33g, agar powder 18g, deionized water 1000ml,
The rice hull carbon and/or tabacco straw charcoal of different proportion is added in the configuration process of oat medium and corresponding pH is determined
Value；Then the pH value of change is adjusted back to into initial value with the HCl or NaOH solution of 0.1mol, initial value is to be not added with biomass carbon
PH value corresponding to culture medium,
Carry out connecing bacterium experiment after sterilizing, after culture terminates, picking mycelia is placed on slide, examines under a microscope black shank bacterium
The change of hypha form.
4. according to claim 1 biomass carbon suppress tobacco black shank bacterium method, it is characterised in that method three include as
Lower step：
Bacterium paddy prepares：Millet is added water and is boiled to half grain in bloom shape, in being fitted into 500ml triangular flasks, 121 DEG C of autoclavings
20 min；Aseptically, the tobacco black shank bacterium bacterium block for taking 1cm × 1cm is transferred in the triangular flask for filling bacterium paddy, 28 DEG C
14d is standby for culture；
Bacterium soil prepares：By the soil and biomass carbon of sterilizing（Rice hull carbon and/or tabacco straw charcoal）By different proportion（0,1%, 2%,
4%, 10%, 20%）Mixing, in being fitted into diameter 9cm, the flowerpot of high 7cm, first fills 3/4 composite soil, is uniformly added into bacterium paddy 0.5g/
Basin, remaining 1/4 composite soil is filled up, and pours permeable, is placed in one month in artificial climate room, is kept hot and humid；
Transplant：The seedling age tobacco seedlings of 60 days or so are transplanted in flowerpot, per one plant of basin；
Each processes 10 basins, is repeated 3 times, and is placed in phjytotron culture, and at 28 DEG C of 30 DEG C/night on daytime, illumination 12h/ is dark
12h, relative humidity 95% transplants the morbidity series after the 5th, 7,9 days investigation tobacco seedlings, and is recorded, calculate disease index and
Prevention effect；
Disease index=∑（Diseased plant number × series at different levels）/（Highest disease series × investigation total strain number）× 100, prevention effect=（It is right
Refer to according to a group disease-treatment group disease refers to）/ control group disease refers to × 100%.
5. the method for suppressing tobacco black shank bacterium using biomass carbon according to claim 1, it is characterised in that biomass carbon
For rice hull carbon and/or tabacco straw charcoal,
Rice hull carbon：Tabacco straw charcoal is 0-20g/L:20-0g/L.
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