CN109964776A - A kind of method of a variety of tobacco diseases of Synthetical prevention - Google Patents
A kind of method of a variety of tobacco diseases of Synthetical prevention Download PDFInfo
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- CN109964776A CN109964776A CN201910301263.9A CN201910301263A CN109964776A CN 109964776 A CN109964776 A CN 109964776A CN 201910301263 A CN201910301263 A CN 201910301263A CN 109964776 A CN109964776 A CN 109964776A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01B—SOIL WORKING IN AGRICULTURE OR FORESTRY; PARTS, DETAILS, OR ACCESSORIES OF AGRICULTURAL MACHINES OR IMPLEMENTS, IN GENERAL
- A01B79/00—Methods for working soil
- A01B79/02—Methods for working soil combined with other agricultural processing, e.g. fertilising, planting
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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
- 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
- A01G22/45—Tobacco
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- Engineering & Computer Science (AREA)
- Mechanical Engineering (AREA)
- Soil Sciences (AREA)
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- General Health & Medical Sciences (AREA)
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- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention discloses a kind of methods of a variety of tobacco diseases of Synthetical prevention.The present invention effectively reduces disease, insect pest using the beneficial allelopathy of peppermint and flue-cured tobacco, soil physico-chemical property can be not only effectively improved, adjust soil fertility, continuous cropping obstacle can also be weakened, under the premise of not destroying farmland ecological environment and existing plantation function, it can significantly reduce the disease incidence and the extent of injury of tobacco TMV, CMV and PVY disease, the yield and quality of flue-cured tobacco is improved simultaneously, and ecological safety is nontoxic and pollution-free；In addition, making that soil can be fully and rationally used between peppermint and flue-cured tobacco, the total value of crop is improved, increases the income of peasant, the peppermint of plantation can also be used to extract essential oil or as peppermint flavouring etc., have significant social benefit, ecological benefits and economic benefit.This method is easy to operate, economical and practical, and administrative skill is of less demanding, has preferable popularization and application foreground.
Description
Technical field
The invention belongs to tobacco cultivation technical fields.In particular it relates to a kind of method of a variety of tobacco diseases of Synthetical prevention.
Background technique
In recent years, the generation of tobacco virus is in rising trend, seriously affects the quality and yield of tobacco leaf.Take in tobacco grower
It reduces, reduces its enthusiasm for planting tobacco.It is one of the main path that tobacco virus is propagated that vega soil, which carries virus,.Agriculture
Common chemicals sterilization be easy to cause the salinization of soil in industry, aggravates soil hardening.It is developed in production green, environmental protection
The malicious soil restoring technology of band is of great significance.
In human production activity, agricultural land soil is often subject to the pollution of various poisonous and harmful substances, such as: pollution by pesticides,
Crude oil pollution, microbiological contamination etc. not only generate harm to ecological environment, also threaten to human health.In difference
In the soil pollution of type, pathogenic virus has become a big obstacle of agricultural development to the pollution of agricultural land soil.
People are long-standing to viruliferous research is taken in soil.1980, people had found more in soil and waste water
The pathogenic virus of kind of the mankind, and determine soil and waste water be the mankind pathogenic virus major source of infection (Lin Changsheng,
1980).Nineteen ninety-five, researcher explore heliothis armigera nuclear polyhedrosis virus (Heliothis armigera nuclear
Polyhedrosis virus, HaNPv) influence (Zhang Zhong of delay situation and soil pH to HaNPv bioactivity in the soil
Letter, 1995).2006, Zhao Ping Zi (2006) was indicator virus with MS2 and -174, had studied the master that virus migrates in the soil
Want impact factor.Wang Qiuying etc. (2007) has probed into the catharsis of soil to viral absorption behavior and its in the environment, hair
Existing red clay is most strong to the adsorption capacity of virus, and sandy moisture soil is most weak.2016, someone reported cucumber green mottle mosaic virus
Disease plant residuum and disease ground soil can propagate the virus, and have stronger susceptibility, easily cause the sense of succession crop
Disease and morbidity (Cai Meiyan etc., 2016).
Tobacco mosaic virus (TMV) (Tobacco mosaic virus, TMV), cucumber mosaic virus (Cucumber mosaic
Virus, CMV), marmor upsilon (Potato virus Y, PVY) be the main virus for infecting tobacco.The virus that they cause
Disease leads to the huge economic loss of tobacco industry every year, takes in tobacco grower and reduces, and seriously reduces its enthusiasm for planting tobacco.Cigarette
Strain mainly has soil band poison to pass poison, insect biography poison, seedling stage band poison etc. the main reason for virosis occurs for field.Wherein, soil
Passing poison is one of the main path that tobacco virus is propagated.The reason of causing CMV, PVY non-vectored soil transmission mainly CMV, PVY
Survival period in tobacco old complaint is long, and they easily pass through the wound intrusion cigarette strain of root system, can make when field is transplanted seedlings
Root system generates many microtrauma mouths, and the virus in soil invalid body will take advantage of the occasion to invade tobacco seedlings root.Agriculturally common soil disappears
Malicious method is to apply lime and cupric sulphate mixture in the soil.This method can reach to a certain extent inhibits cause of disease in soil
The purpose of object, but can also destroy soil texture simultaneously, lead to arable soil acidification, aggravate soil hardening, it is final to lose agricultural
Cultivation value, the reparation being far from being to soil.Exploitation is directed to the vega soil green recovery technique of TMV, CMV and PVY for subtracting
Less virus primary source of infection, effective prevention and control virosis, land resource protection and sustainable development have important practical significance.
Around allelopathy (allelopathy) refers to a kind of plant or microorganism by itself being synthetically produced and being discharged into
Chemical substance in environment generates direct or indirect repulsion and facilitation to other plant or microorganism.With allelopathy
Substance be allelochemical (allelochemicals).This effect is widely present in nature.By beneficial allelopathy
Being applied to agricultural land soil harmful microbe prevention and control is a kind of very green, environmentally friendly good thinking.By establish reasonable crop rotation,
Between make, interplanting or miscegenation system, beneficial the allelopathy of plant will reduce disease, insect pest, and be effectively improved soil physico-chemical property, adjusting
Soil fertility improves the yield and quality (Zhang Xiaoke, 2004) of crop, can also weaken continuous cropping obstacle (Pal MS, 2006).Fragrant-flowered garlic
Make that banana blight bacteria (the Fusarium oxsporium in the soil of any of several broadleaf plants field can be killed well between dish and banana
f.sp.cubense,FOC).Studies have shown that: making to have centainly balck shank between barley, celery, leek, sweet potato, upland rice and tobacco
Preventive effect (cloth Yun Hong, 2016).But studies have shown that the formation of allelopathy is a complicated process, between crop there is life in kind
The characteristics of object diversity, habitat diversification, genetic diversity, different intercultures are different from the interaction of tobacco, disease resistance
Difference, different community hights, different niches etc. influence also difference to soil harmful microbe, while largely influencing
The growth of tobacco is not to make between carrying out all crop varieties and flue-cured tobacco cultivars arbitrarily, can reach higher yield and product
The preferable resultant effect such as matter.How to explore the reasonable tobacco crop rotation of building, make, interplanting or miscegenation system, effectively reduce disease,
Insect pest and improvement soil physico-chemical property, improve the yield and quality of tobacco, still great challenge.
Real-time fluorescence quantitative PCR (realtime fluoresce quantitative polymerase chain
Reaction, q-PCR) it (is that one kind is sent out based on Standard PCR technology for RT-qPCR) if test object is RNA virus
The new nucleic acid quantification detection technique of one kind of exhibition.Fluorophor is added in PCR reaction system in the technology, tired using fluorescence signal
Product the entire PCR process of real-time monitoring, finally by standard curve to unknown template carry out quantitative analysis method (Li An etc.,
2009).Compared with Standard PCR, the maximum advantage of the technology is that it can carry out quantitative analysis, and its specificity to sample
It is stronger, sensitivity is higher, time saving convenience, high degree of automation, high throughput.In addition, technology experiment operation is whole in addition to experiment
Preparation stage, is entirely stopped pipe operation, and high degree of automation greatly reduces the probability (Tang Yunxia, 2012) of pollution, Er Qie
After the reaction was completed, it is only necessary to which pertinent instruments analyze data, do not need to carry out electrophoresis detection again, can effectively prevent
Only pollution and false positive, while having accomplished that amplification edges frontier inspection is surveyed.
Summary of the invention
The technical problem to be solved by the present invention is to overcome the defect of the above-mentioned prior art and deficiencies, provide a kind of Synthetical prevention
The method of a variety of tobacco diseases.For this method without adding medicament, non-environmental-pollution problem can effectively prevent flue-cured tobacco soil-borne disease
Occur；Implantation methods of the invention simultaneously can simultaneously, efficiently kill TMV, CMV and PVY in vega soil virus, promote flue-cured tobacco
Robust growth improves yield of flue-cured tobacco and quality.
Above-mentioned purpose of the present invention is achieved through the following technical solutions:
A kind of method of a variety of tobacco diseases of Synthetical prevention, by flue-cured tobacco and peppermint intercropping plant.
Flue-cured tobacco cultivation method of the invention carries out intercropping plant using peppermint and flue-cured tobacco with prevention and control flue-cured tobacco soil-borne disease, is one
The completely new biological control method of kind.
Peppermint (Mentha haplocalyx Briq.), popular name are Lamiaceae plant, stem is upright, high by 30 " lunar caustic grass "
~60cm is born in by hill wetland river more, and rhizome grows wild underground, is born in 2100m height above sea level, but can also be in 3500m height above sea level more
Upper growth is a kind of fragrant crop for having extraordinary economic value；Complete stool green gas fragrance, is one of Chinese conventional Chinese medicine, Xin Liangxing
Sweating antipyretic medicine, neuralgia, skin can be controlled by controlling diseases, the external applications such as influenza, headache, hot eyes, body heat, throat, gum swelling and pain
Itch, fash and eczema etc., usually with peppermint for tea, clear away heart-fire improving eyesight, and the peppermint of light canopy Greenhouse Fruit Picking is on Spring Festival dining table again
Fresh vegetable, salubrious palatable, temperature, moisture requirement and the tobacco difference planted are little, can be with normal growth in tobacco field.Flue-cured tobacco pair
Required in planting environment more stringent, be one and prefer warm plant, when tobacco leaf picking entirety plant height be 1.5~
1.8m, a height of 30~60cm of peppermint strain, is mutually independent of each other, peppermint and flue-cured tobacco inter-cropping high-efficiency killed TMV in vega soil,
CMV and PVY virus is conducive to the healthy growth for keeping flue-cured tobacco, improves its disease resistance, and better than chemopreventive effects, no
It will appear drug resistance.
Further, in preferred embodiments of the present invention, by flue-cured tobacco succeeding planting, every 1~3 row tobacco planting, 1~2 row
Peppermint.
Further, in preferred embodiments of the present invention, by flue-cured tobacco succeeding planting, 2 rows of every 1 row tobacco planting are thin
Lotus.
Further, in preferred embodiments of the present invention, the spacing of adjacent rows flue-cured tobacco is 1.0~2.0m, and every row is roasting
The spacing of adjacent two plants of flue-cured tobaccos is 0.2~0.7m in cigarette.
Further, in preferred embodiments of the present invention, the spacing of adjacent rows flue-cured tobacco is 1.2~1.5m, every row
The spacing of adjacent two plants of flue-cured tobaccos is 0.5~0.6m in flue-cured tobacco.
Further, in preferred embodiments of the present invention, the spacing of every row peppermint and adjacent a line flue-cured tobacco is 0.2~
0.7m, the spacing of adjacent two plants of peppermints is 20~45cm in peppermint described in every row.
Further, in preferred embodiments of the present invention, the spacing of every row peppermint and adjacent a line flue-cured tobacco is 0.2
~0.4m, the spacing of adjacent two plants of peppermints is 20~30cm in peppermint described in every row.
Further, in preferred embodiments of the present invention, the kind of the peppermint be selected from spearmint peppermint, lavender or
One or more of American mint.
Further, in preferred embodiments of the present invention, the kind of the flue-cured tobacco is selected from dark green No.1, safflower great Jin
One or more of member, K326, cloud 87, Guangdong 97 or NC102.
Further, in preferred embodiments of the present invention, the water content of vega soil is maintained at 65%~75%.
Further, in preferred embodiments of the present invention, flue-cured tobacco top dressing management is conventionally carried out: soil topdressing
(potassium nitrate, potassium sulfate) adds foliage dressing；Peppermint top dressing management: based on decomposed farmyard manure.
Further, in preferred embodiments of the present invention, the control of the planting density of the flue-cured tobacco 1000~1200 plants/
Mu.
Compared with prior art, the invention has the following advantages:
(1) present invention effectively reduces disease, insect pest using the beneficial allelopathy of peppermint and flue-cured tobacco, is effectively improved soil physical chemistry
Property adjusts except soil fertility, continuous cropping obstacle can also be weakened, before not destroying farmland ecological environment and existing plantation function
It puts, can simultaneously, efficiently kill TMV, CMV and PVY virus in vega soil, significantly reduce tobacco TMV, CMV and PVY disease
Disease incidence and the extent of injury, increase the diversity of tobacco field system, keep the ecosystem more stable, resistance is stronger, from root
Vega soil TMV, CMV and PVY virus are killed on source, while improving the yield and quality of flue-cured tobacco, and then improve tobacco leaf
Economic benefit.
(2) flue-cured tobacco-peppermint interplanting technique is applied to green reparation of the tobacco field with pesticide-clay mixture earth for the first time by the present invention, can sufficiently be closed
Reason utilizes soil, improves the total value of crop, increases the income of peasant, and the peppermint of plantation can also be used to extract essential oil or as thin
Lotus flavouring etc. has significant social benefit, ecological benefits and economic benefit, and ecological safety is nontoxic and pollution-free, is a kind of non-
Evergreen color, environmentally friendly agricultural land soil harmful microorganism prevention and control thinking.This method is easy to operate, economical and practical, administrative skill requirement
It is not high, soil physico-chemical property is not destroyed, it is without secondary pollution, there is very much application value in actual production, there is powerful popularization to answer
With prospect, it can be used as the malicious soil remediation method of the novel band of one kind and promoted in tobacco business.
Detailed description of the invention
Fig. 1 is the amplification curve that pMD-TMV recombinates positive plasmid；Wherein curve 1-6: being respectively 1.42 × 107–1.42×
102Recombination the positive plasmid DNA, curve 7:ddH of copies/ μ L2O。
Fig. 2 is the standard curve (R that pMD-TMV recombinates positive plasmid2=0.999, E=106.1%).
Fig. 3 is the solubility curve that pMD-TMV recombinates positive plasmid.
Fig. 4 is the amplification curve that pMD-CMV recombinates positive plasmid；Wherein curve 1-6: being respectively 1.31 × 108-1.31×
103Recombination the positive plasmid DNA, curve 7:ddH of copies/ μ L2O。
Fig. 5 is the standard curve (R that pMD-CMV recombinates positive plasmid2=0.986, E=107.9%).
Fig. 6 is the solubility curve that pMD-CMV recombinates positive plasmid.
Fig. 7 is the amplification curve that pMD-PVY recombinates positive plasmid；Wherein curve 1-6: being respectively 1.1 × 108-1.1×
103Recombination the positive plasmid DNA, curve 7:ddH of copies/ μ L2O。
Fig. 8 is the standard curve (R that pMD-PVY recombinates positive plasmid2=0.986, E=108.2%).
Fig. 9 is the solubility curve that pMD-PVY recombinates positive plasmid.
Specific embodiment
To facilitate the understanding of the present invention, below will be to invention is more fully described, there is shown of the invention
Preferred embodiment.But the invention can be realized in many different forms, however it is not limited to embodiment described herein.
Without departing from the spirit and substance of the case in the present invention, to simple modifications or substitutions made by the method for the present invention, step or condition,
It all belongs to the scope of the present invention；Unless otherwise specified, technological means used in embodiment is well known to those skilled in the art
Conventional means.
Unless stated otherwise, following embodiment agents useful for same and material are commercially available.
The method of the present invention wherein a variety of tobacco diseases of embodiment Synthetical prevention, by flue-cured tobacco and peppermint intercropping plant.
The control method of traditional flue-cured tobacco (tobacco) soil virus mainly has cultural control, chemical prevention and biological control.
Cultural control is the disease resistance for passing through the cultivation steps such as Rational Irrigation, the rational application of fertilizer and rational pruning and improving tobacco, mitigates disease
Generation.The chemical prevention for the related disease that studies in China scholar carries out focuses primarily upon indoor and field chemical agent sieve
Choosing, chemical control period etc..And the research of the biological control of vega soil virus still focuses primarily upon beneficial microorganism screening
Etc., it makes slow progress, needs to find new control prece.
In a specific example, by flue-cured tobacco succeeding planting, every 1~3 row tobacco planting, 1~2 row peppermint.It so, it is possible
The propagation for preferably preventing flue-cured tobacco soil virus, forms good isolation barrier.It is appreciated that the concrete mode of intercropping plant is not
It is limited to this, can be adjusted according to the needs of landform.Preferably, every 1 row tobacco planting, 2 row peppermint.
In a specific example, the spacing of adjacent rows flue-cured tobacco is 1.0~2.0m, adjacent two plants of flue-cured tobaccos in every row flue-cured tobacco
Spacing be 0.2~0.7m.Preferably, the spacing of adjacent rows flue-cured tobacco is 1.2~1.5m, adjacent two plants of flue-cured tobaccos in every row flue-cured tobacco
Spacing be 0.5~0.6m, keep the growth of flue-cured tobacco more preferable by the optimization of spacing.
In a specific example, the spacing of every row peppermint and adjacent a line flue-cured tobacco is 0.2~0.7m, in every row peppermint
The spacing of adjacent two plants of peppermints is 20~45cm.Preferably, the spacing of every row peppermint and adjacent a line flue-cured tobacco be 0.2~
0.4m, the spacing of adjacent two plants of peppermints is 20~30cm in every row peppermint.In this way, can either preferably prevent vega soil viral
Propagation, while also can avoid peppermint and flue-cured tobacco and serious Competition for Nutrients occur, the upgrowth situation of peppermint and flue-cured tobacco is preferable.
In a specific example, the kind of peppermint be selected from one of spearmint peppermint, lavender or American mint or
It is several.
In a specific example, the kind of flue-cured tobacco be selected from dark green No.1, Hongda tobacco, K326, cloud 87, Guangdong 97 or
One or more of NC102.
In a specific example, the water content of vega soil is maintained at 65%~75%.In the growth of flue-cured tobacco and peppermint
In the process, need to guarantee suitable moisture, the very few growth for being all unfavorable for flue-cured tobacco and peppermint of excess moisture.
The following are specific embodiments.
A kind of method of the 1 a variety of tobacco diseases of Synthetical prevention of embodiment
It will make between tobacco and peppermint, plant in the high-incidence field of virosis.Use real-time fluorescence quantitative PCR
(realtime fluoresce quantitative polymerase chain reaction, q-PCR) technology is in cigarette strain
The content of TMV, CMV, PVY are detected.Compared to control group (non-work, tobacco nonoculture), analyzes peppermint root exudates pair
Viral killing effect in vega soil.
1, experimental material
Nicotiana tabacum (flue-cured tobacco) kind K326 is provided by this laboratory.The kind of peppermint is spearmint peppermint, and Labiatae is planted
Object.Purchased from Lv Yong seedling Co., Ltd, the canonical reference " dictionary of medicinal plant " of chosen material (the new medical college in Jiangsu compiles, 1975).
Healthy tobacco seed and peppermint seed are seeded in healthy matrix respectively (through detecting, without virus).Periodically watering, to its hair
Bud seedling (through detecting, without virus).
2, experimental method
It is tested in the field that Agricultural University Of South China's Zengcheng teaching base chooses plantation flue-cured tobacco throughout the year.These fields are all
Virosis district occurred frequently (after testing, all containing TMV, CMV, PVY of high concentration).6 pieces of above-mentioned fields of selection, 1 mu every piece.Respectively
Number is 1,2,3,4,5,6.Wherein 1-3 field is flue-cured tobacco nonoculture area (CK).4-6 field makees peppermint (processing between flue-cured tobacco
Area).
Every year by the end of February by flue-cured tobacco and peppermint seedling replanting to field.By flue-cured tobacco succeeding planting, 2 rows of every 1 row tobacco planting are thin
Lotus.The spacing (line-spacing) of adjacent rows flue-cured tobacco is 1.2m, and the spacing (spacing in the rows) of adjacent two plants of flue-cured tobaccos is 0.5m in every row flue-cured tobacco.Often
The spacing of row peppermint and adjacent a line flue-cured tobacco is 0.3m, and the spacing of adjacent two plants of peppermints is 20cm in every row peppermint.Cigarette strain is raw
Any pesticide is not applied during length, remaining operates equivalent field leaf tobacco production.
Mid-June respectively at two kinds of fields, is randomly selected according to the ratio in 20 plants/1 piece of field and takes cigarette strain blade, utilize q-
PCR detects the content of its TMV, CMV, PVY, takes its average value, compares and analyzes.And count each field TMV, CMV, PVY
Disease incidence and disease index and per mu yield tobacco leaf amount.
Experiment is carried out continuously 2 years.
3, detection method
A, real-time fluorescence quantitative PCR (realtime fluoresce quantitative polymerase chain
Reaction, q-PCR)
(1) total serum IgE extracting and design of primers
With the total serum IgE of RNA isolation kit extracting plant sample.Plant leaf blade RNA extracts kit (TaKaRa MiniBest
Plant RNA E-traction Kit)、Premi-E-TaqTM II kit (TaKaRa) kit, in real time it is glimmering
Light quantification kit One StepPrimeScipt RT-PCR Kit (Perfect Real Time) is purchased from
TaRaKa company.
Design of primers is shown in Table 1, synthesizes in Shanghai Sheng Gong bioengineering Co., Ltd.
The primer that 1 RT-qPCR of table is used
(2) foundation of TMV, CMV, PVY RT--qPCR plasmid standard standard curve
1) by the recombination positive plasmid DNA standard items (offer of this laboratory) of TMV, CMV, PVY for having prepared, determined with absolute
Dilution Easy Dilution is measured using 10 times of concentration gradient serial dilutions 5 times as amplification template, reaction system is prepared, is arranged
Response procedures carry out real-time fluorescence quantitative PCR reaction, while with RNase Free dH2O is the control of template-setup clear water, each
Standard items do 3 parallel repetitions with clear water control respectively.(i.e. according to the logarithm of initial template copy number and the recurring number of reaction
Ct value) linear relationship, building detection TMV, CMV and PVY amplification curve, standard curve, melting curve (Fig. 1 to Fig. 9).Institute
TaRaKa company is purchased from reagent.
2) target gene expands
Target sequence is expanded respectively with above three pairs of primers, one-step method RT-qPCR reaction system is as follows:
3) its response procedures are as follows: 42 DEG C, 5min → 95 DEG C, 10s；
95 DEG C, 5s → 62 DEG C, 30s totally 40 circulations (60 DEG C of collection fluorescence signals)；
95 DEG C, 15s → 60 DEG C, 30s → 95 DEG C, 15s (melting curve: second 95 DEG C of collection fluorescence signal).
(3) interpretation of RT--qPCR result
It is positive findings when the Ct value of sample<35, value>=35 Ct of sample or does not expand, then judging result is feminine gender.
When using the method test sample of absolute quantitation, the Ct value of sample to be tested is substituted into calibration curve equation, acquire-
Value is the starting copy number of sample to be tested.In order to guarantee the consistency of testing result, measured every time using absolute quantification method
Standard curve must be all drawn when sample, and sample to be tested and standard items are put into fluorescence quantitative PCR instrument simultaneously and reacted, instead
The copy number (habit phoenix girl, 2014) of sample to be tested is calculated after answering according to calibration curve equation.
From amplification figure, (Fig. 1 to Fig. 9) can be seen that linear very good, the line of TMV, CMV and PVY amplification
Property coefficient is all larger than 0.98, and amplification coefficient is 1.06~1.08, between 0.8~1.2, illustrates primed probe and the expansion of design
Increasing system can be very good amplification target gene, not interfere between each amplification.
B, disease index (disease index) and opposite control efficiency count
Disease index=∑ (the diseased plant numbers at different levels × disease grade value)/(investigation total strain number × superlative degree value) × 100.
Wherein, tobacco virus classification and investigation method (GBT23222-2008) are as follows:
Tobacco mosaic viruses disease (TMV), Cucumber Mosaic Virus (CMV), marmor upsilon sick (PVY) are with strain
Unit step investigation.
0 grade: complete stool is disease-free.
L grades: lobus cardiacus veinclearing or slight floral leaf, diseased plant are downgraded without obvious.
3 grades: it is 3/4ths or more of normal plant height that one third blade floral leaf but indeformable or diseased plant, which are downgraded,.
5 grades: one third to half blade floral leaf, or a small number of or deformation or the necrosis of main side arteries and veins or plant are downgraded and are
Normal 3/2nds to four/3rds.
7 grades: half to 2/3rds blade floral leaves, or it is normal that deformation or the necrosis of main side arteries and veins or plant, which are downgraded,
Half is to 2/3rds.
9 grades: complete stool blade floral leaf, severely deformed or downright bad, it is the half of normal plant height or more that diseased plant, which is downgraded,.
C, yield of tobacco counts
By the end of June every year, effective tobacco leaf of every plant of cigarette is collected, its total output is counted.
4, statistical result
A, the area CK and treatment region (make) cigarette strain band poison amount RT--qPCR testing result between flue-cured tobacco-peppermint is shown in Table 2.
The RT--qPCR testing result of 2 cigarette strain band poison amount of table
Judging from the experimental results, the band poison amount of cigarette strain TMV, CMV, PVY are respectively in the check plot CK (flue-cured tobacco nonoculture) field
Treatment region (makees) 402.96 times, 265.90 times, 331.28 times of cigarette strain band poison amount in field between flue-cured tobacco-peppermint.Illustrate that flue-cured tobacco-is thin
Make efficiently kill TMV, CMV, PVY in vega soil between lotus, the present invention is a kind of band pesticide-clay mixture earth of high effective green environmentally friendly
Recovery technique.
B, the area CK and treatment region (make between flue-cured tobacco-peppermint) cigarette strain disease index statistics be shown in Table 3.
Make 3 parallel repetitions and be denoted as CK-1, CK-2, CK-3 respectively in the area CK (flue-cured tobacco nonoculture area)；Area makees 3 between tobacco-peppermint
Secondary parallel repetition is denoted as making to make to make -3 between -2, tobacco-peppermint between -1, tobacco-peppermint between tobacco-peppermint respectively.
3 area CK of table and treatment region (make) cigarette strain field band poison rate and disease index statistical form between flue-cured tobacco-peppermint
As known from Table 3, parallel three times to repeat, treatment region (makees) band of cigarette strain TMV, CMV, PVY in field between flue-cured tobacco-peppermint
Malicious rate is well below the malicious rate of band of cigarette strain in the area CK (flue-cured tobacco nonoculture) field, while treatment region (makees) field between flue-cured tobacco-peppermint
The disease index of middle cigarette strain TMV, CMV, PVY are also significantly lower than the disease index of cigarette strain in the area CK (flue-cured tobacco nonoculture) field, explanation
The disease incidence and the extent of injury that can significantly reduce tobacco between flue-cured tobacco-peppermint, increase the diversity of tobacco field system, make ecosystem
It unites more stable, resistance is stronger, kills vega soil TMV, CMV and PVY virus from the root, reduces tobacco in tobacco
Leaf disease, the propagation of cucumber mosaic virus and potato Y disease, harm, and then improve the economic benefit of tobacco leaf.
C, the area CK and treatment region (make) yield of tobacco statistical result and are shown in Table 4 between flue-cured tobacco-peppermint.
Make 3 parallel repetitions and be denoted as CK-1, CK-2, CK-3 respectively in the area CK (flue-cured tobacco nonoculture area)；Area makees 3 between tobacco-peppermint
Secondary parallel repetition is denoted as making to make to make -3 between -2, tobacco-peppermint between -1, tobacco-peppermint between tobacco-peppermint respectively.
4 area CK of table and treatment region (make) tobacco leaf total output statistical form (kg/ mus) between flue-cured tobacco-peppermint
In summary tobacco can be effectively reduced between tobacco-peppermint it is found that compared with compareing tobacco nonoculture in experimental result
The malicious rate of TMV, CMV, PVY band and disease index in field, greatly improve the per mu yield total amount of tobacco leaf.
Make between 1 flue-cured tobacco of comparative example-leek
It is tested in the field that Agricultural University Of South China's Zengcheng teaching base chooses plantation flue-cured tobacco throughout the year.These fields are all
Virosis district occurred frequently (after testing, all containing TMV, CMV, PVY of high concentration).6 pieces of above-mentioned fields of selection, 1 mu every piece.Respectively
Number is 1,2,3,4,5,6.Wherein 1-3 field is flue-cured tobacco nonoculture area (CK)；4-6 field makees leek (processing between flue-cured tobacco
Area).
By flue-cured tobacco succeeding planting, two row leek are planted between every adjacent rows flue-cured tobacco.Spacing (the row of adjacent rows flue-cured tobacco
Away from) it is 1.0m, the spacing (spacing in the rows) of adjacent two plants of flue-cured tobaccos is 0.5m in every row flue-cured tobacco.Between every row leek and adjacent a line flue-cured tobacco
Away from for 0.3m, the spacing of adjacent two plants of leek is 30cm in every row leek.Cigarette strain growth period does not apply any pesticide, remaining behaviour
Make equivalent field leaf tobacco production.Every year by the end of February by flue-cured tobacco and leek seedling replanting to field.Mid-June respectively at two kinds of fields,
It is randomly selected according to the ratio in 20 plants/1 piece of field and takes cigarette strain blade, the content of its TMV, CMV, PVY are detected using q-PCR, takes it
Average value compares and analyzes.The annual disease incidence for counting each field TMV, CMV, PVY by the end of June and disease index and per mu yield
Tobacco leaf amount.
1, experimental method
It is identical to make related experiment between flue-cured tobacco-peppermint in embodiment 1.
2, experimental result
A, the RT--qPCR testing result of the area CK and treatment region (flue-cured tobacco-leek) cigarette strain band poison amount is shown in Table 5.
The RT--qPCR testing result of 5 cigarette strain band poison amount of table
As shown in Table 5, the band poison amount of cigarette strain TMV, CMV, PVY and treatment region are (roasting in the check plot CK (flue-cured tobacco nonoculture) field
Cigarette-leek) in field cigarette strain band poison amount without marked difference.Illustrate to make effectively to kill in vega soil between flue-cured tobacco-leek
TMV、CMV、PVY。
B, the area CK and treatment region cigarette strain disease index and opposite control efficiency statistics are shown in Table 6.
Make 3 parallel repetitions and be denoted as CK-1, CK-2, CK-3 respectively in the area CK (flue-cured tobacco nonoculture area)；Area makees 3 between tobacco-leek
Secondary parallel repetition is denoted as making to make to make -3 between -2, tobacco-leek between -1, tobacco-leek between tobacco-leek respectively.
6 area CK of table and treatment region (flue-cured tobacco-leek) cigarette strain field band poison rate and disease index statistical form
As shown in Table 6, with the malicious rate of band and disease index of cigarette strain TMV, CMV, PVY in the check plot CK (flue-cured tobacco nonoculture) field
It compares, treatment region (makees) cigarette strain TMV, CMV, PVY in the field malicious rate of band and disease index between flue-cured tobacco-leek is without marked difference.
Illustrate the disease that not can be effectively controlled TMV, CMV, PVY in vega soil between flue-cured tobacco-leek.
C, the area CK and treatment region (flue-cured tobacco-leek) yield of tobacco statistical result are shown in Table 7.
Make 3 parallel repetitions and be denoted as CK-1, CK-2, CK-3 respectively in the area CK (flue-cured tobacco nonoculture area)；Area makees 3 between tobacco-leek
Secondary parallel repetition is denoted as making to make to make -3 between -2, tobacco-leek between -1, tobacco-leek between tobacco-leek respectively.
7 area CK of table and treatment region (flue-cured tobacco-leek) tobacco leaf total output statistical form (kg/ mus)
It can be seen that TMV, CMV, PVY band made between tobacco-leek compared with compareing tobacco nonoculture from upper statistical result
Malicious rate and disease index between tobacco-leek make that tobacco cannot be effectively reduced in the band of TMV, CMV, the PVY in field without marked difference
Malicious rate and disease index can not effectively improve the per mu yield total amount of tobacco leaf.Explanation is not to carry out all crop varieties and flue-cured tobacco
Make between any, can reach better control effect and obtains higher yield and quality.
Embodiment 2
By flue-cured tobacco and peppermint seedling replanting to field.The kind of flue-cured tobacco is dark green No.1.The kind of peppermint is American mint.
By flue-cured tobacco succeeding planting, every 2 row tobacco planting, 2 row peppermint.The spacing (line-spacing) of adjacent rows flue-cured tobacco is 1.5m, in every row flue-cured tobacco
The spacing (spacing in the rows) of adjacent two plants of flue-cured tobaccos is 0.6m.The spacing of every row peppermint and adjacent a line flue-cured tobacco is 0.4m, every row peppermint
In adjacent two plants of peppermints spacing be 25cm.The planting density of flue-cured tobacco is controlled at 1000 plants/acre.Cigarette strain growth period, which is not applied, appoints
What pesticide, the water content of vega soil are maintained at 65%.Remaining operates equivalent field leaf tobacco production.
The present embodiment can synchronize under the premise of not destroying farmland ecological environment and existing plantation function, effectively kill tobacco field
TMV, CMV and PVY virus in soil, significantly reduces the disease incidence and the extent of injury of tobacco TMV, CMV and PVY disease, improves
The yield and quality of flue-cured tobacco.
Embodiment 3
By flue-cured tobacco and peppermint seedling replanting to field.The kind of flue-cured tobacco is Hongda tobacco.The kind of peppermint is lavender.
By flue-cured tobacco succeeding planting, every 3 row tobacco planting, 2 row peppermint.The spacing (line-spacing) of adjacent rows flue-cured tobacco is 1.0m, in every row flue-cured tobacco
The spacing (spacing in the rows) of adjacent two plants of flue-cured tobaccos is 0.2m.The spacing of every row peppermint and adjacent a line flue-cured tobacco is 0.2m, every row peppermint
In adjacent two plants of peppermints spacing be 20cm.The planting density of flue-cured tobacco is controlled at 1000 plants/acre.Cigarette strain growth period, which is not applied, appoints
What pesticide, the water content of vega soil are maintained at 70%.Remaining operates equivalent field leaf tobacco production.
The present embodiment can synchronize under the premise of not destroying farmland ecological environment and existing plantation function, effectively kill tobacco field
TMV, CMV and PVY virus in soil, significantly reduces the disease incidence and the extent of injury of tobacco TMV, CMV and PVY disease, improves
The yield and quality of flue-cured tobacco.
Embodiment 4
By flue-cured tobacco and peppermint seedling replanting to field.The kind of flue-cured tobacco is NC102.The kind of peppermint is spearmint peppermint.
By flue-cured tobacco succeeding planting, every 3 row tobacco planting, 1 row peppermint.The spacing (line-spacing) of adjacent rows flue-cured tobacco is 2.0m, in every row flue-cured tobacco
The spacing (spacing in the rows) of adjacent two plants of flue-cured tobaccos is 0.7m.The spacing of every row peppermint and adjacent a line flue-cured tobacco is 0.7m, every row peppermint
In adjacent two plants of peppermints spacing be 45cm.The planting density of flue-cured tobacco is controlled at 1200 plants/acre.Cigarette strain growth period, which is not applied, appoints
What pesticide, the water content of vega soil are maintained at 75%.Remaining operates equivalent field leaf tobacco production.
The present embodiment can synchronize under the premise of not destroying farmland ecological environment and existing plantation function, effectively kill tobacco field
TMV, CMV and PVY virus in soil, significantly reduces the disease incidence and the extent of injury of tobacco TMV, CMV and PVY disease, improves
The yield and quality of flue-cured tobacco.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.
Claims (10)
1. a kind of method of a variety of tobacco diseases of Synthetical prevention, which is characterized in that by flue-cured tobacco and peppermint intercropping plant.
2. the method according to claim 1, wherein by flue-cured tobacco succeeding planting, every 1~3 row tobacco planting 1~2
Row peppermint.
3. according to the method described in claim 2, it is characterized in that, by flue-cured tobacco succeeding planting, every 1 row tobacco planting, 2 row peppermint.
4. method according to claim 1 or 2, which is characterized in that the spacing of adjacent rows flue-cured tobacco is 1.0~2.0m, often
The spacing of adjacent two plants of flue-cured tobaccos is 0.2~0.7m in row flue-cured tobacco.
5. according to the method described in claim 4, every row is roasting it is characterized in that, the spacing of adjacent rows flue-cured tobacco is 1.2~1.5m
The spacing of adjacent two plants of flue-cured tobaccos is 0.5~0.6m in cigarette.
6. method according to claim 1 or 2, which is characterized in that every row peppermint and the spacing of adjacent a line flue-cured tobacco are
0.2~0.7m, the spacing of adjacent two plants of peppermints is 20~45cm in peppermint described in every row.
7. according to the method described in claim 6, it is characterized in that, the spacing of every row peppermint and adjacent a line flue-cured tobacco is 0.2
~0.4m, the spacing of adjacent two plants of peppermints is 20~30cm in peppermint described in every row.
8. the method according to claim 1, wherein the kind of the peppermint is selected from spearmint peppermint, lavender
Or one or more of American mint.
9. the method according to claim 1, wherein the kind of the flue-cured tobacco is selected from dark green No.1, safflower great Jin
One or more of member, K326, cloud 87, Guangdong 97 or NC102.
10. the method according to claim 1, wherein the water content of vega soil is maintained at 65%~75%.
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Application publication date: 20190705 |