CN107064435A - The mould leaf spot preventing and treating of handle of crawling and detection method and its application of preventive effect - Google Patents
The mould leaf spot preventing and treating of handle of crawling and detection method and its application of preventive effect Download PDFInfo
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Abstract
The invention discloses the mould leaf spot preventing and treating of handle of crawling and detection method and its application of preventive effect.The mould leaf spot preventing and treating of handle of crawling disclosed by the invention and the detection method of preventive effect, including：Using the mould leaf spot pathogenic bacteria of handle of crawling and chemicals treatment tomato excised leaf, thalline consumption is (8.3 × 106‑1.7×107)cfu/cm2Blade, obtains pathogenic bacteria and medicament coprocessing blade；Determine that medicament is prevented and treated the mould leaf spot of handle of crawling and/or preventive effect according to the incidence of pathogenic bacteria and medicament coprocessing blade；The mould leaf spot pathogenic bacteria of handle of crawling are the strain of eggplant stemphylium botryosum.The method of the present invention can not only detect the mould leaf spot preventing and treating of handle of crawling and preventive effect, it can also screen or assisting sifting preventing and treating prevention is crawled the medicament of the mould leaf spot of handle, this method result is accurate, reliable, repeatability is strong, and method is quick, easy, with very strong practicality.
Description
Technical field
The present invention relates to the detection method of the preventing and treating of handle of in biological technical field, crawling mould leaf spot and preventive effect and its should
With.
Background technology
Handle of crawling, which belongs to fungi (Stemphylium), can infect various crop in global range, can cause various vegetables disease
Evil, main harm vegetables include tomato, lettuce, capsicum, wild cabbage, spinach, garlic, wherein especially the most serious to endanger tomato.It is beautiful
The ground such as state, Israel, New Zealand were reported by the mould caused leaf spot of tomato of handle of crawling, and were caused to local tomato production huge
Economic loss.In recent years, the Tomato gray leaf spot on 500,000 mu of the ground such as China northeast, North China, East China and south China occurs particularly tight
Weight.Wherein especially the most serious with Shandong Shouguang, Haikou, the morbidity of Guangxi Tianyang County, total onset area is more than 100,000 mu, and loss is super
Cross ten million yuan.
Tomato is very important a kind of vegetables in solanaceous vegetables, is the Main Cultivation crop in China's vegetable cultivation industry
One of.At present, plantation, processing and the outlet of Tomato in China are Chinese by the development of more than 20 years all in sustainable growth situation
It is the third-largest production area after the U.S., European Union and as global most important tomato product producing country and exported country
One large outlet state.Major tomato main producing regions occur S. lycopersici leaf spot successively at home since two thousand two, gradually by secondary
Disease rises to Major Diseases, and the development to tomato in China industry brings serious influence.
Sick canopy rate is 43%, and sick canopy rate is 90% within 2003 in 30,000 greenhouses that Shandong Yutai County is planted within 2002,
Prevent and treat not in time, the tomato underproduction 20% or so, the even underproduction 80% having, while causing tamato fruit Quality Down.In Guizhou
The import tomato variety of alpine region plantation shows the high perception to Tomato gray leaf spot, and 2005 in Guiyang white clouds
The ground such as area, Xifeng County, Xiuwen County large area is broken out, because Tomato gray leaf spot is seldom found in the kind planted in the past, agriculture
The people lack experience of prevention and treatment, thus prevent and treat ineffective, and crushing blow is brought to production.Li Baoju etc. is in vegetable disease fact-finding process
It was found that, there is the sick generation on the ground such as Beijing Daxing, Haicheng, Langfang in Hebei Province, Baoding and Shandong Shouguang, but at that time not
Cause production loss (Li Baoju etc., 2009).
Found in the vegetable disease fact-finding process of 2009~2016 years, with being widely applied for antiviral tomato variety,
S. lycopersici leaf spot occurring area expands year by year, in the leading village in Haikou City, Hainan Province Xi Xiu towns, Lingshan Zhen great Chang villages, Shandong
Shouguang Los Angeles Zhen Huangjia Yao Shui villages, old Ma Cun, Shao Jialing villages of Shandong Shouguang Tian Liuzhen, Mao Jia villages, Yan Jia villages village, Guangxi resource
County resource town is with standing grain village duck back of the body village, Huang Bao villages of Ziyuan County, guangxi field township, Guangxi Ziyuan County Malus spectabilis village of township of Dong Tian Miao ethnic group and other places
The serious harm of S. lycopersici leaf spot is had been found that.Especially in Shandong Shouguang old Ma Cun of Tian Liuzhen, tomato is from after being colonized
Soon start the mould leaf spot of handle of sporadicly crawling, run into successive cloudy days high humidity, when temperature is fluctuated, disease Quick Extended,
During to the 6th fringe fruit fruit setting, the incidence of disease reaches more than 90% in canopy, has had a strong impact on the yield of tomato, is brought to peasant huge
Loss.
Because in the market lacks the selectivity bactericide for S. lycopersici leaf spot, the preventing and treating of the current disease is with pre-
Based on anti-.But the effectively generation of control S. lycopersici leaf spot disease, it is very to develop and screen suitable pesticide control
It is necessary.With the increase of environment supervision and pesticide market competitive pressure, the preventing and treating of S. lycopersici leaf spot disease and
The exploitation of novel agrochemical faces huge challenge.Classic flat-plate screening method speed is fast, but the selection result and field efficacy comparison in difference
Greatly, the local bacterial strain concentration of patch bacterium piece method is high, and evaluating drug result is also and field control effect has certain deviation, S. lycopersici
Leaf spot lacks accurately and reliably medicament rapid screening method as serious disease is endangered recently, develops corresponding for the disease
High throughput screening technology there is actual researching value with the successful screening rate for improving novel agrochemical.
The content of the invention
The technical problems to be solved by the invention are how to determine the crawl mould leaf spot preventing and treating of handle and/or the preventing and treating of prevention medicament
And/or preventive effect, and how to further determine that preventing and treating and/or prevention crawl the mould leaf spot of handle Suitable agents and suitable use
Amount.
In order to solve the above technical problems, present invention firstly provides the mould leaf spot preventing and treating of handle of crawling and/or the inspection of preventive effect
Survey method.
The mould leaf spot preventing and treating of handle of crawling provided by the present invention and/or the detection method of preventive effect, including：It is mould using handle of crawling
Leaf spot pathogenic bacteria and chemicals treatment Vitro Plant blade to be measured, obtain pathogenic bacteria and medicament coprocessing blade；Caused according to described
The incidence of germ and medicament coprocessing blade determines that the medicament is prevented and treated the mould leaf spot of handle of crawling and/or preventive effect.
In the above method, the medicament can be biocontrol microorganisms class medicament or chemical composition class medicament.The medicament is concretely
The mould leaf spot preventing and treating of handle of crawling and/or prevention medicament.
In the above method, it is described treat measuring plants can for it is following 1) or 2) or 3)：
1) dicotyledon；
2) plant of Solanaceae；
3) tomato.
In the above method, the mould leaf spot pathogenic bacteria of handle of crawling can be eggplant stemphylium botryosum strain (Stemphylium
solani)。
In the above method, the big I of the excised leaf is (2-4) cm × (3-5) cm.The size of the excised leaf
Further can be 2cm × (3-5) cm or 3cm × (3-5) cm or 4cm × (3-5) cm, such as 2cm × 4cm, 3cm × 4cm or 4cm ×
4cm。
In the above method, institute can be used by handling the Vitro Plant blade to be measured using the mould leaf spot pathogenic bacteria of the handle of crawling
The bacteria suspension for stating the mould leaf spot pathogenic bacteria of handle of crawling is carried out, and the bacteria suspension can be specifically sprayed to the Vitro Plant blade to be measured
On.The concentration of the bacteria suspension can be (3-6) × 108cfu/mL.The concentration of the bacteria suspension further can for (4-5) ×
108cfu/mL.The thalline consumption for handling the Vitro Plant blade to be measured using the mould leaf spot pathogenic bacteria of the handle of crawling is (8.3
×106-1.7×107)cfu/cm2Blade, concretely (1.1 × 107-1.4×107)cfu/cm2Blade.
In the above method, the medicament can be dissolved in or be suspended using Vitro Plant blade to be measured described in the chemicals treatment
Described treat on the excised leaf of measuring plants is sprayed to after in liquid.
It is described using crawling the mould leaf spot pathogenic bacteria of handle and chemicals treatment Vitro Plant blade to be measured can be in the above method
The blade back of the Vitro Plant blade to be measured is handled.
It is described using crawling the mould leaf spot pathogenic bacteria of handle and chemicals treatment Vitro Plant blade to be measured may include in the above method
Following A 1 or A2：
A1, using the mould leaf spot pathogenic bacteria of the handle of crawling the Vitro Plant blade to be measured is handled, obtain pathogenic bacteria processing
Blade；Then using the blade of pathogenic bacteria processing described in the chemicals treatment, the pathogenic bacteria and medicament coprocessing are obtained
Blade；
A2, using Vitro Plant blade to be measured described in the chemicals treatment, obtain the blade of chemicals treatment；Then institute is utilized
The blade that the mould leaf spot pathogenic bacteria of handle of crawling handle the chemicals treatment is stated, the blade of the pathogenic bacteria and medicament coprocessing is obtained.
In the above method, the mould leaf spot pathogenic bacteria of handle of crawling and the chemicals treatment blade non-concurrent are carried out (i.e. successively
Carry out), the interval time of the two can be 2-6 hours, can be further 3-5 hours, such as 3 hours or 4 hours.
Described air-dry can be carried out or in the mould tikka of handle of crawling before the blade that pathogenic bacteria described in the chemicals treatment are handled
Sick pathogenic bacteria are carried out before handling the blade of the chemicals treatment.
The above method, which may also include, is cultivated the pathogenic bacteria and the blade of medicament coprocessing.The temperature of the culture
Condition can be 26-30 DEG C (such as 28 DEG C).The relative air humidity of the culture can be 70%-85% (such as 80%).
In the above method, the pathogenic bacteria and the incidence of medicament coprocessing blade can be embodied by preventive effect, preventive effect
Calculation formula be：Preventive effect (%)=(control disease index-processing disease index)/control disease index × 100.Wherein, it is right
It is, using the disease index for treating measuring plants described in chemicals treatment, to handle disease index to utilize chemicals treatment institute according to disease index
State the disease index for treating measuring plants.
The calculation formula of disease index is：
Disease index=[∑ (the sick number of sheets × disease index at different levels)/(total number of sheets × highest disease index)] × 100；
Wherein, the determination method of disease index is as follows：
0 grade：Disease-free spot；
1 grade：Lesion area accounts for less than 5% (not including 5% and 0) of whole leaf area；
3 grades：Lesion area account for whole leaf area 5%~25% (including 5%, not including 25%)；
5 grades：Lesion area account for whole leaf area 25%~50% (including 25%, not including 50%)；
7 grades：Lesion area account for whole leaf area 50%~75% (including 50%, not including 75%)；
9 grades：Lesion area accounts for more than 75% (including 75%) of whole leaf area.
In order to solve the above technical problems, present invention also offers screening or assisting sifting preventing and treating and/or preventing the mould leaf of handle of crawling
The method of the medicament of pinta.
The method of the medicament for the mould leaf spot of handle that screening provided by the present invention or assisting sifting are prevented and treated and/or prevention is crawled, bag
Include：Determine that different agents treat crawling for measuring plants using the detection method of the mould leaf spot preventing and treating of the handle of crawling and/or preventive effect
The preventing and treating of the mould leaf spot of handle and/or preventive effect, determine to prevent and treat and/or prevent described to be measured according to preventing and treating and/or preventive effect
Plant is crawled the medicament of the mould leaf spot of handle.
In the above method, treat that measuring plants are crawled the mould leaf of handle according to preventing and treating and/or preventive effect determine preventing and treating and/or prevention
The medicament of pinta can be carried out according to preventive effect：Preventive effect such as medicament to be measured is more than or equal to 65%, the medicament energy or candidate's energy to be measured
Described treat that measuring plants are crawled the mould leaf spot of handle for preventing and treating and/or preventing；Preventive effect such as medicament to be measured is less than 65%, the medicine to be measured
Agent can not or candidate cannot be used for preventing and treating and/or prevent described to treat that measuring plants are crawled the mould leaf spot of handle.
In order to solve the above technical problems, present invention also offers screening or assisting sifting crawl the preventing and treating of handle mould leaf spot and/or
Prevent the method for dosing.
Screening provided by the present invention or assisting sifting are crawled the method for the preventing and treating of handle mould leaf spot and/or prevention dosing,
Including：Determine to crawl using the detection method of the mould leaf spot preventing and treating of the handle of crawling and/or the preventing and treating of prevention medicament and/or preventive effect
The preventing and treating for treating measuring plants and/or preventive effect of the mould leaf spot preventing and treating of handle and/or prevention medicament under different amounts, according to anti-
Control and/or preventive effect determines the mould leaf spot preventing and treating of the handle of crawling and/or prevents the consumption of medicament.
In the above method, the mould leaf spot preventing and treating of handle of being crawled according to being determined preventing and treating and/or preventive effect and/or prevention medicament
Consumption can be carried out according to preventive effect：As dosing to be measured preventive effect be more than or equal to 65%, the consumption energy of the medicament to be measured or
Candidate can be used to prevent and treat and/or prevent described to treat that measuring plants are crawled the mould leaf spot of handle；Preventive effect such as dosing to be measured is less than 65%,
The consumption of the medicament to be measured can not or candidate cannot be used for preventing and treating and/or prevent described to treat that measuring plants are crawled the mould leaf spot of handle.
The mould leaf spot preventing and treating of handle of crawling of the present invention and/or the detection method of preventive effect and screening or assisting sifting preventing and treating
And/or prevention crawls that not only measurement result is accurate, reliable for the method for the medicament of the mould leaf spot of handle, with field test results complete one
Cause, and this method is quick, easy, meeting bacterium process 3h can complete, preventing and treating and/or preventive effect can be completed in 75 hours
Detection and the screening of medicament, and field test then needs 14-21d, the time saving and energy saving province's seedling of method of the invention；This method blade is easy
, it can meet and use throughout the year, and field test not only needs intact plant, is also limited by season；This method repeatability is strong, and
Field test is easily protected from environmental, causes result repeatability not strong.Compared with pasting bacterium piece method, method of the invention is easy, bacterium
Plant inoculation stable, repeatability is strong, reliably, the prevention effect and field control effect for pasting bacterium piece method test agents exist certain data
Deviation.Bacterium amount and the amount of bactericide are few needed for the method for the present invention, are highly suitable for S. lycopersici leaf spot bactericide
Microscreen, with very strong practicality.
Brief description of the drawings
Fig. 1 is the phenotype of different disease indexes.Wherein, A is 0 grade；B is 1 grade；C is 3 grades；D is 5 grades；E is 7 grades；F is 9
Level.
Embodiment
The present invention is further described in detail with reference to embodiment, the embodiment provided is only for explaining
The bright present invention, the scope being not intended to be limiting of the invention.Experimental method in following embodiments, unless otherwise specified, be
Conventional method.Material, reagent, instrument used etc., unless otherwise specified, are commercially obtained in following embodiments.
Quantitative test in following examples, is respectively provided with three repetition experiments, results averaged.
The calculation formula of disease index and preventive effect in following embodiments is：
Disease index=[∑ (the sick number of sheets × disease index at different levels)/(total number of sheets × highest disease index)] × 100；
Preventive effect (%)=(control disease index-processing disease index)/control disease index × 100.
Wherein, the determination method of disease index is as follows：
0 grade：Disease-free spot；
1 grade：Lesion area accounts for less than 5% (not including 5% and 0) of whole leaf area；
3 grades：Lesion area account for whole leaf area 5%~25% (including 5%, not including 25%)；
5 grades：Lesion area account for whole leaf area 25%~50% (including 25%, not including 50%)；
7 grades：Lesion area account for whole leaf area 50%~75% (including 50%, not including 75%)；
9 grades：Lesion area accounts for more than 75% (including 75%) of whole leaf area.
Eggplant stemphylium botryosum strain (Stemphylium solani) in following embodiments be documented in document (Xie et al.,
First report of Stemphylium solani causing leaf spot on wild eggplant in
China, Can.J.Plant Pathol., 2016, Vol.38, No.4,517-521) in, the strain of eggplant stemphylium botryosum can cause eggplant
Section plant and legume are crawled the mould leaf spot of handle.
The method of the medicament for the mould leaf spot of handle that screening provided by the present invention or assisting sifting are prevented and treated and/or prevention is crawled, bag
Include：According to the detection method of the preventing and treating of handle of crawling mould leaf spot and/or preventive effect determine different agents treat measuring plants handle of crawling it is mould
The preventing and treating of leaf spot and/or preventive effect, determine to prevent and treat according to preventing and treating and/or preventive effect and/or prevention treats that measuring plants are crawled handle
The medicament of mould leaf spot.Wherein, the mould leaf spot preventing and treating of handle of crawling and/or the detection method of preventive effect, including：Utilize the mould leaf of handle of crawling
Pinta pathogenic bacteria and chemicals treatment Vitro Plant blade to be measured, obtain pathogenic bacteria and medicament coprocessing blade；According to pathogenic bacteria with
The scab of medicament coprocessing blade determines that medicament is prevented and treated the mould leaf spot of handle of crawling and/or preventive effect.
Below using tomato as measuring plants are treated, with the 50% Boscalid water of BASF plant protection (Jiangsu) Co., Ltd
Dispersible granule, 50% iprodione wettable powder of Sinon Chemical (China) Co., Ltd., Bayer Cropscience (China) are limited
41.7% fluopyram suspending agent of company, 10% Wettable polyoxin powder of Sinon Chemical (China) Co., Ltd.,
75% Bravo wettable powder of Jiangsu Xinhe Agricultural Chemical Industry Co., Ltd and Hainan Boshiway Agricultural Chemical Co., Ltd.
15% Prochloraz microemulsion is specifically described screening or assisting sifting preventing and treating as medicament to be screened and/or prevents the mould leaf spot of handle of crawling
Medicament method.Tomato excised leaf shoals in morbidity rear blade tissue, and scab is circular brown dot, is evenly distributed one
Cause, morbidity excised leaf and excised leaf of not falling ill distinguish obvious, and distinguishing is strong.
Embodiment 1：S. lycopersici leaf spot pesticide control is screened
The implementation steps of the embodiment are as follows：
A, the processing of tomato excised leaf
Choose the growth aseptic tomato excised leaf of 70-80 days after field planting, with the equal size of sterile scissors clip (2 ×
4cm) healthy leaves；
B, inoculation processing
Bacteria suspension is prepared first：By the strain of eggplant stemphylium botryosum in the PD liquid containing 200g/L potatos Yu 20g/L glucose
In culture medium under conditions of 28 DEG C of temperature with rotating speed 130rpm concussion and cultivate 7 days, obtain eggplant stemphylium botryosum concentration for 6 ×
108Cfu/ml bacteria suspension.
The obtained tomato excised leafs of step A are put on the pallet for being covered with filter paper, are 6 × 10 by 50mL concentration8cfu/ml
Eggplant stemphylium botryosum strain bacteria suspension be equably vacantly sprayed to blade back, under greenhouse natural air drying 2h (i.e. the strain of eggplant stemphylium botryosum and
The interval time of chemicals treatment blade), obtain a kind of tomato excised leaf (i.e. the blade of pathogenic bacteria processing) for being inoculated with pathogen.
Wherein, the inoculum concentration of eggplant stemphylium botryosum strain bacterium is 1.1 × 107cfu/cm2Blade.
C, chemicals treatment
The tomato excised leaf of the obtained inoculation pathogens of step B is divided in moisture preservation box, 1500 will be diluted with clear water
Pharmacy solution (50% pyridine in pharmacy solution of the 50% Boscalid water dispersible granules of times BASF plant protection (Jiangsu) Co., Ltd
The concentration of acyl bacterium aqueous amine dispersible granule is 350 μ g/mL) the tomato excised leaf back side is equably sprayed on, obtain pathogenic bacteria and medicament
Coprocessing blade, the moisturizing culture under conditions of 28 DEG C of temperature and relative humidity 80% by pathogenic bacteria and medicament coprocessing blade；
Then after pathogen 72h described in the tomato Isolated leaf inoculation, disease index (handling disease index) is determined.Utilize
The tomato excised leaf for the inoculation pathogen that amount clear water processing equivalent step B is obtained, as control, obtains compareing disease index.
Preventive effect (prevention effect) is calculated with compareing disease index according to processing disease index.
As a result show, the disease index after pathogen 72h described in tomato Isolated leaf inoculation is 0.25,1500 times of dilution
The prevention effect of 50% Boscalid water dispersible granules is 99%, is shown, this 50% Boscalid water dispersible granules can be used
In preventing and treating S. lycopersici leaf spot.
Embodiment 2：S. lycopersici leaf spot pesticide control is screened
The implementation steps of the embodiment are as follows：
A, the processing of tomato excised leaf
Choose the growth aseptic tomato excised leaf of 70-80 days after field planting, with the equal size of sterile scissors clip (3 ×
5cm) healthy leaves；
B, inoculation processing
Bacteria suspension is prepared first：By the strain of eggplant stemphylium botryosum in the PD liquid containing 200g/L potatos Yu 20g/L glucose
In culture medium under conditions of 26 DEG C of temperature with rotating speed 120rpm concussion and cultivate 6 days, obtain eggplant stemphylium botryosum strain concentration be 4 ×
108Cfu/ml bacteria suspension.
The obtained tomato excised leafs of step A are put on the pallet for being covered with filter paper, are 4 × 10 by 50mL concentration8cfu/ml
Eggplant stemphylium botryosum strain bacteria suspension be equably vacantly sprayed to blade back, under greenhouse natural air drying 3h (i.e. the strain of eggplant stemphylium botryosum and
The interval time of chemicals treatment blade), obtain a kind of tomato excised leaf (i.e. the blade of pathogenic bacteria processing) for being inoculated with pathogen.
Wherein, the inoculum concentration of eggplant stemphylium botryosum strain bacterium is 1.1 × 107cfu/cm2Blade.
C, chemicals treatment
The obtained inoculation pathogen excised leafs of step B are divided in moisture preservation box, 1000 times of emerging agricultures will be diluted with clear water
Liquid medicine (the 50% iprodione wettable in liquid medicine of 50% iprodione wettable powder of medicine company (China) Co., Ltd
The concentration of pulvis is 500 μ g/mL) the tomato excised leaf back side is equably sprayed on, pathogenic bacteria and medicament coprocessing blade are obtained, will
Pathogenic bacteria and the moisturizing culture under conditions of 30 DEG C of temperature and relative humidity 70% of medicament coprocessing blade；Then in the tomato
After pathogen 72h described in Isolated leaf inoculation, disease index (handling disease index) is determined.Equivalent is handled using equivalent clear water
The tomato excised leaf for the inoculation pathogen that step B is obtained, as control, obtains compareing disease index.Referred to according to the processing state of an illness
Number calculates preventive effect (prevention effect) with compareing disease index.
As a result show, the disease index after pathogen 72h described in tomato Isolated leaf inoculation is 0.25,1000 times of dilution
The prevention effect of 50% iprodione wettable powder is 99%, is shown, this 50% iprodione wettable powder can be used for preventing
Control S. lycopersici leaf spot.
Embodiment 3：S. lycopersici leaf spot pesticide control is screened
The implementation steps of the embodiment are as follows：
A, the processing of tomato excised leaf
Choose the growth aseptic tomato excised leaf of 70-80 days after field planting, with the equal size of sterile scissors clip (4 ×
4cm) healthy leaves；
B, inoculation processing
Bacteria suspension is prepared first：By the strain of eggplant stemphylium botryosum in the PD liquid containing 200g/L potatos Yu 20g/L glucose
In culture medium under conditions of 30 DEG C of temperature with rotating speed 125rpm concussion and cultivate 5 days, obtain eggplant stemphylium botryosum strain concentration be 3 ×
108Cfu/ml bacteria suspension.
The obtained tomato excised leafs of step A are put on the pallet for being covered with filter paper, are 3 × 10 by 50mL concentration8cfu/ml
Eggplant stemphylium botryosum strain bacteria suspension be equably vacantly sprayed to blade back, under greenhouse natural air drying 4h (i.e. the strain of eggplant stemphylium botryosum and
The interval time of chemicals treatment blade), obtain a kind of tomato excised leaf (i.e. the blade of pathogenic bacteria processing) for being inoculated with pathogen.
Wherein, the inoculum concentration of eggplant stemphylium botryosum strain bacterium is 1.1 × 107cfu/cm2Blade.
C, chemicals treatment
The tomato excised leaf of the obtained inoculation pathogens of step B is divided in moisture preservation box, 2000 will be diluted with clear water
The liquid medicine of 41.7% fluopyram suspending agent of times Bayer Cropscience (China) Co., Ltd is (in liquid medicine
The concentration of 41.7% fluopyram suspending agent is 208.5 μ g/mL) the tomato excised leaf back side is equably sprayed on, caused a disease
Bacterium and medicament coprocessing blade, pathogenic bacteria and medicament coprocessing blade are protected under conditions of 26 DEG C of temperature and relative humidity 85%
Wet culture；Then after pathogen 72h described in the tomato Isolated leaf inoculation, determine that disease index (handles the state of an illness to refer to
Number).The tomato excised leaf for the inoculation pathogen that equivalent step B is obtained is handled using equivalent clear water, as control, is compareed
Disease index.Preventive effect (prevention effect) is calculated with compareing disease index according to processing disease index.
As a result show, the disease index after pathogen 72h described in tomato Isolated leaf inoculation is 0,2000 times of dilution
The prevention effect 100% of 41.7% fluopyram suspending agent, shows, this 41.7% fluopyram suspending agent can be used for
Prevent and treat S. lycopersici leaf spot.
Embodiment 4：S. lycopersici leaf spot pesticide control is screened
The implementation steps of the embodiment are as follows：
A, the processing of tomato excised leaf
Choose the growth aseptic tomato excised leaf of 70-80 days after field planting, with the equal size of sterile scissors clip (3 ×
5cm) healthy leaves；
B, inoculation processing
Bacteria suspension is prepared first：By the strain of eggplant stemphylium botryosum in the PD liquid containing 200g/L potatos Yu 20g/L glucose
In culture medium under conditions of 28 DEG C of temperature with rotating speed 120rpm concussion and cultivate 5.5 days, obtain eggplant stemphylium botryosum strain concentration be 5 ×
108Cfu/ml bacteria suspension.
The obtained tomato excised leafs of step A are put on the pallet for being covered with filter paper, are 5 × 10 by 50mL concentration8cfu/ml
Eggplant stemphylium botryosum strain bacteria suspension be equably vacantly sprayed to blade back, under greenhouse natural air drying 5h (i.e. the strain of eggplant stemphylium botryosum and
The interval time of chemicals treatment blade), obtain a kind of tomato excised leaf (i.e. the blade of pathogenic bacteria processing) for being inoculated with pathogen.
Wherein, the inoculum concentration of eggplant stemphylium botryosum strain bacterium is 1.1 × 107cfu/cm2Blade.
C, chemicals treatment
The tomato excised leaf of the obtained inoculation pathogens of step B is divided in moisture preservation box, 1500 will be diluted with clear water
The liquid medicine of 10% Wettable polyoxin powder of times Sinon Chemical (China) Co., Ltd. (resists more in liquid medicine 10%
The concentration of mycin wettable powder is 70 μ g/mL) the tomato excised leaf back side is equably sprayed on, obtain pathogenic bacteria and coexist with medicament
Blade is managed, the moisturizing culture under conditions of 28 DEG C of temperature and relative humidity 80% by pathogenic bacteria and medicament coprocessing blade；Then
After pathogen 72h described in the tomato Isolated leaf inoculation, disease index (handling disease index) is determined.It is clear using equivalent
The tomato excised leaf for the inoculation pathogen that water process equivalent step B is obtained, as control, obtains compareing disease index.According to
Processing disease index calculates preventive effect (prevention effect) with compareing disease index.
As a result show, the disease index after pathogen 72h described in tomato Isolated leaf inoculation is 15.80, dilutes 1500 times
10% Wettable polyoxin powder prevention effect 28%, show, this 10% Wettable polyoxin powder is difficult to use
In preventing and treating S. lycopersici leaf spot.
Embodiment 5：S. lycopersici leaf spot pesticide control is screened
The implementation steps of the embodiment are as follows：
A, the processing of tomato excised leaf
Choose the growth aseptic tomato excised leaf of 70-80 days after field planting, with the equal size of sterile scissors clip (3 ×
4cm) healthy leaves；
B, inoculation processing
Bacteria suspension is prepared first：By the strain of eggplant stemphylium botryosum in the PD liquid containing 200g/L potatos Yu 20g/L glucose
In culture medium under conditions of 28 DEG C of temperature with rotating speed 120rpm concussion and cultivate 7 days, obtain eggplant stemphylium botryosum strain concentration be 5 ×
108Cfu/ml bacteria suspension.
The obtained tomato excised leafs of step A are put on the pallet for being covered with filter paper, 50mL concentration upwards, is by vacuum side of blade
5×108Cfu/ml eggplant stemphylium botryosum strain bacteria suspension is equably vacantly sprayed to blade back, natural air drying 6h (the i.e. eggplants under greenhouse
Stemphylium botryosum strain and interval time of chemicals treatment blade), obtain a kind of tomato excised leaf of inoculation pathogen (i.e. pathogenic bacteria
The blade of processing).Wherein, the inoculum concentration of eggplant stemphylium botryosum strain bacterium is 1.1 × 107cfu/cm2Blade.
C, chemicals treatment
The tomato excised leaf of the obtained inoculation pathogens of step B is divided in moisture preservation box, face of blade upwards, will be used
(medicament is molten for the liquid medicine of 75% Bravo wettable powder of 750 times of Jiangsu Xinhe Agricultural Chemical Industry Co., Ltd of clear water dilution
The concentration of 75% Bravo wettable powder is 1mg/mL in liquid) tomato excised leaf front is equably sprayed on, obtain pathogenic bacteria
With medicament coprocessing blade, the moisturizing under conditions of 28 DEG C of temperature and relative humidity 85% by pathogenic bacteria and medicament coprocessing blade
Culture；Then after pathogen 72h described in the tomato Isolated leaf inoculation, disease index (handling disease index) is determined.
The tomato excised leaf for the inoculation pathogen that equivalent step B is obtained is handled using equivalent clear water, as control, obtains compareing the state of an illness
Index.Preventive effect (prevention effect) is calculated with compareing disease index according to processing disease index.
As a result show, the disease index after pathogen 72h described in tomato Isolated leaf inoculation is 7.90,750 times of dilution
The prevention effect 72% of 75% Bravo wettable powder, shows, this 75% Bravo wettable powder can be used for preventing and treating
S. lycopersici leaf spot.
Embodiment 6：S. lycopersici leaf spot prevents Chemicals
The implementation steps of the embodiment are as follows：
A, the processing of tomato excised leaf
Choose the growth aseptic tomato excised leaf of 70-80 days after field planting, with the equal size of sterile scissors clip (3 ×
3cm) healthy leaves；
B, chemicals treatment
The obtained tomato excised leafs of step A are divided in moisture preservation box, 2 layers of white filter paper are covered with box, clear water moisturizing,
By the medicament suspension of the 15% Prochloraz microemulsion of Hainan Boshiway Agricultural Chemical Co., Ltd. that 2500 times are diluted with clear water
(concentration of 15% Prochloraz microemulsion is 60 μ g/mL in medicament suspension) is equably sprayed on the tomato excised leaf back side, in temperature
Natural air drying 6h (i.e. the strain of eggplant stemphylium botryosum and the interval time of chemicals treatment blade), obtains kind that a kind of chemicals treatment is crossed under room
Eggplant excised leaf (i.e. the blade of chemicals treatment).Equivalent tomato excised leaf is handled using equivalent clear water, control blade is used as.
C, inoculation processing
Bacteria suspension is prepared first：By the strain of eggplant stemphylium botryosum in the PD liquid containing 200g/L potatos Yu 20g/L glucose
In culture medium under conditions of 28 DEG C of temperature with rotating speed 120rpm concussion and cultivate 5 days, obtain eggplant stemphylium botryosum strain concentration be 6 ×
108Cfu/ml bacteria suspension, bacteria suspension is equably vacantly sprayed to the tomato tooth in vitro that the chemicals treatment that step B obtains is crossed
The back of the body, obtains pathogenic bacteria and medicament coprocessing blade, by pathogenic bacteria and medicament coprocessing blade in 28 DEG C of temperature and relative humidity
Moisturizing culture under conditions of 85%；Then after pathogen 72h described in the tomato Isolated leaf inoculation, disease index is determined
(handling disease index).Wherein, the inoculum concentration of eggplant stemphylium botryosum strain bacterium is 1.1 × 107cfu/cm2Blade.
According to the method described above, using equivalent bacteria suspension processing equivalent control blade, obtain compareing disease index.
Preventive effect (prevention effect) is calculated with compareing disease index according to processing disease index.As a result show, tomato tooth in vitro
It is 2.47 that piece, which is inoculated with the disease index after the pathogen 72h, 15% Prochloraz microemulsion prevention effect of 2500 times of dilution
91%, show, this 15% Prochloraz microemulsion can be used for preventing and treating S. lycopersici leaf spot.
Embodiment 7：Field prevention S. lycopersici leaf spot Chemicals experiment
The implementation steps of the embodiment are as follows：
A, the processing of tomato plants
The tomato plants of 70-80 days of healthy growth after field planting are chosen, without eggplant stemphylium botryosum and other germs
Different agents processing is carried out under environment；
B, chemicals treatment
Different agents solution and pharmacy solution used in embodiment 1-6 is equably sprayed on kind that step A is obtained respectively
On eggplant plants, a kind of liquid medicine or pharmacy solution are each tested, 5 young plants are repeated every time, totally 4 repetitions are periodically carried out clear
Water moisturizing, natural air drying 6h (i.e. the strain of eggplant stemphylium botryosum and the interval time of chemicals treatment plant), obtains a kind of medicine in greenhouse
The treated tomato plants (i.e. the plant of chemicals treatment) of agent.Equivalent tomato plants are handled using equivalent clear water, are made
For adjoining tree.
C, inoculation processing
Bacteria suspension is prepared first：By the strain of eggplant stemphylium botryosum in the PD liquid containing 200g/L potatos Yu 20g/L glucose
In culture medium under conditions of 28 DEG C of temperature with rotating speed 120rpm concussion and cultivate 5 days, obtain eggplant stemphylium botryosum strain concentration be 6 ×
108Cfu/ml bacteria suspension, bacteria suspension is equably vacantly sprayed to the tomato live body that the chemicals treatment that step B obtains crosses and planted
Strain, obtains pathogenic bacteria and medicament coprocessing plants, by pathogenic bacteria with medicament coprocessing plants in 28 DEG C of temperature and phase
To moisturizing culture under conditions of humidity 85%；Then after the tomato plants are inoculated with the pathogen 72h, the state of an illness is determined
Index (handles disease index).Wherein, the inoculum concentration of eggplant stemphylium botryosum strain bacterium is 3.5 × 107cfu/cm2Blade.
According to the method described above, using equivalent bacteria suspension processing equivalent control tomato plants, obtain compareing disease index.
Preventive effect (prevention effect) is calculated with compareing disease index according to processing disease index.As a result show, by chemicals treatment
Tomato plants be inoculated with after the pathogen 72h, it dilutes the preventing and treating of 2000 times of 41.7% fluopyram suspending agent
Effect is 96%；The prevention effect of 50% Boscalid water dispersible granules of 1500 times of dilution is 90%；1000 times of dilution
The prevention effect of 50% iprodione wettable powder is 88%；15% Prochloraz microemulsion prevention effect of 2500 times of dilution
86%；The prevention effect 80% of 75% Bravo wettable powder of 750 times of dilution；10% polyoxin of 1500 times of dilution can
The prevention effect 36% of WP.Show, 41.7% fluopyram suspending agent, 50% Boscalid water dispersible granules,
50% iprodione wettable powder, 15% Prochloraz microemulsion and 75% Bravo wettable powder are to caused by the strain of eggplant stemphylium botryosum
S. lycopersici leaf spot is respectively provided with good prevention effect, can be used for preventing and treating S. lycopersici leaf spot；And resist 10% more
Mycin wettable powder is poor to the prevention effect of S. lycopersici leaf spot caused by the strain of eggplant stemphylium botryosum, it is difficult to for preventing and treating kind
Eggplant is crawled the mould leaf spot of handle, and this is consistent with embodiment 1-6 result.
The comparison of embodiment 8, various pathogenic bacteria inoculation method
I, blade back spray bacterium method：Size identical aseptic tomato excised leaf is put into moisture preservation box, concentration is 1 upwards by blade back
×108Cfu/ml eggplant stemphylium botryosum strain bacteria suspension (its preparation method be the same as Example 1) is equably vacantly sprayed to blade back.It is natural
2.5h is air-dried, is placed into 20 × 40cm moisture preservation boxes, 2 layers of white filter paper are laid in box, clear water drenches, in lower protect of suitably having ready conditions
Wet culture, observes and records incidence.
II, live body spray bacterium method：The healthy tomato plant that 20-30 days are grown after field planting is subjected to live body spray bacterium, is 1 by concentration
×108Cfu/ml eggplant stemphylium botryosum strain bacteria suspension equably sprays completely whole strain blade, places into moisturizing glass cabinet, is suitably there is bar
Moisturizing culture under part, observes and records incidence.
Result of the test shows that excised leaf, which sprays bacterium method and live body spray bacterium method, causes tomato leaf fully to be sent out in a short time
Tomato leaf almost all is fallen ill after disease, 72h.From the point of view of area is infected, bacterium method is sprayed for excised leaf, blade morbidity distribution is big
It is small it is uniform, infect that position is unified, blade occurring degree can be by artificially investigating disease index, time saving and energy saving province's seedling.And live body
Spray bacterium method uneven because blade is bent, the bacteria suspension mobility of contact greatly, causes to infect position disunity, site of pathological change zero
Dissipate, artificial investigation disease index error is larger, and data are inaccurate.
Influence of the cell concentration to result in embodiment 9, bacteria suspension
Using influence of the cell concentration to result in the blade back spray bacterium method detection bacteria suspension of embodiment 8：By size identical
Aseptic tomato excised leaf puts moisture preservation box, and upwards, by the eggplant stemphylium botryosum strain bacteria suspension of various concentrations, (its preparation method is same for blade back
Embodiment 1) to be equably vacantly sprayed in blade back, bacteria suspension used specific concentration be respectively 6 × 108cfu/ml、7×
108cfu/ml、8×108Cfu/ml and 9 × 108Cfu/ml, the inoculum concentration of in vitro tomato leaf is 1.1 × 107cfu/cm2Leaf
Piece.Natural air drying 2.5h, is placed into 20 × 40cm moisture preservation boxes, and 2 layers of white filter paper are laid in box, and clear water drenches, suitably had
Under the conditions of moisturizing culture, observe and record incidence.
Bacteria suspension concentration and inoculation time are listed in table 1 to the result of the test of tomato Isolated leaf inoculation pathogen influential effect
In.
The influence of table 1, bacteria suspension concentration and leaching bacterium time to tomato Isolated leaf inoculation pathogen effect
The result of table 1 clearly illustrates that when the spray bacterium time is identical, the tomato excised leaf incidence of disease is with the drop of bacterial concentration
It is low and reduce, i.e., reduced with the reduction of inoculum concentration.Bacterial concentration is 1 × 10 when being inoculated with 6h9(inoculum concentration is 1.7 to cfu/ml
×105cfu/cm2Blade) and 1 × 108(inoculum concentration is 1.7 × 10 to cfu/ml4cfu/cm2Blade) when can cause tomato in vitro
Blade average attack rate reaches more than 95%, and bacterial concentration is down to 1 × 107(inoculum concentration is 1.7 × 10 to cfu/ml3cfu/cm2
Blade) when, the blade incidence of disease is decreased obviously.At bacterial concentration identical (i.e. inoculum concentration is identical), the tomato excised leaf incidence of disease
As reduction trend is equally presented in the reduction for connecing the bacterium time.Inoculation time is failed to understand for 6h and 3h excised leaf incidence of disease difference
Aobvious, it is only 29.47% to connect the bacterium time 1h maximum concentrations bacteria suspension blade incidence of disease.Comprehensive two Variable Factors, judge tomato from
It is 1 × 10 that body blade, which is most preferably inoculated with bacteria suspension concentration,8(i.e. optimum inoculation amount is 1.7 × 10 to cfu/ml4cfu/cm2Blade), most preferably
Disease time is inoculation 3h, is shown, is formerly connect in the case that bacterium sprays medicament again, medicament can be sprayed by meeting bacterium 3h, formerly be sprayed
In the case that medicament connects bacterium again, meet bacterium 3h and can determine that prevention effect or obtain the result of Chemicals.
Claims (10)
1. the mould leaf spot preventing and treating of handle of crawling and/or the detection method of preventive effect, including：Utilize the mould leaf spot pathogenic bacteria of handle of crawling and medicine
Agent handles Vitro Plant blade to be measured, obtains pathogenic bacteria and medicament coprocessing blade；According to the pathogenic bacteria and medicament coprocessing
The incidence of blade determines that the medicament is prevented and treated the mould leaf spot of handle of crawling and/or preventive effect.
2. according to the method described in claim 1, it is characterised in that：It is described treat measuring plants for it is following 1) or 2) or 3)：
1) dicotyledon；
2) plant of Solanaceae；
3) tomato.
3. method according to claim 1 or 2, it is characterised in that：The mould leaf spot pathogenic bacteria of handle of crawling are that eggplant handle of crawling is mould
Bacterial strain (Stemphylium solani).
4. according to any described method in claim 1-3, it is characterised in that：At the mould leaf spot pathogenic bacteria of the handle of crawling
The thalline consumption for managing the Vitro Plant blade to be measured is (8.3 × 106-1.7×107)cfu/cm2Blade.
5. according to any described method in claim 1-3, it is characterised in that：At the mould leaf spot pathogenic bacteria of the handle of crawling
The bacteria suspension for managing the mould leaf spot pathogenic bacteria of handle of being crawled described in the Vitro Plant blade use to be measured is carried out.
6. method according to claim 5, it is characterised in that：It is described to utilize the crawl mould leaf spot pathogenic bacteria of handle and chemicals treatment
Vitro Plant blade to be measured includes following A 1 or A2：
A1, using the mould leaf spot pathogenic bacteria of the handle of crawling the Vitro Plant blade to be measured is handled, obtain the leaf of pathogenic bacteria processing
Piece；Then using the blade of pathogenic bacteria processing described in the chemicals treatment, the blade of the pathogenic bacteria and medicament coprocessing is obtained；
A2, using Vitro Plant blade to be measured described in the chemicals treatment, obtain the blade of chemicals treatment；Then crawled using described
The mould leaf spot pathogenic bacteria of handle handle the blade of the chemicals treatment, obtain the blade of the pathogenic bacteria and medicament coprocessing.
7. method according to claim 6, it is characterised in that：The mould leaf spot pathogenic bacteria of handle of crawling and the chemicals treatment
The excised leaf for treating measuring plants is successively carried out.
8. according to any described method in claim 1-7, it is characterised in that：Methods described also include by the pathogenic bacteria with
The blade of medicament coprocessing is cultivated.
The method of the medicament of the mould leaf spot of handle 9. screening or assisting sifting preventing and treating and/or prevention are crawled, including：Utilize claim 1-
Any methods described determines that different agents treat preventing and treating and/or the preventive effect of the mould leaf spot of handle of crawling of measuring plants in 8, according to anti-
Control and/or preventive effect determines to treat that measuring plants are crawled the medicament of the mould leaf spot of handle described in preventing and treating and/or prevention.
The method of the mould leaf spot preventing and treating of handle and/or prevention dosing 10. screening or assisting sifting are crawled, including：Will using right
Ask in 1-8 that any methods described determines to crawl the mould leaf spot preventing and treating of handle and/or prevention medicament treats measuring plants under different amounts
Preventing and treating and/or preventive effect, determine that the mould leaf spot of handle of crawling is prevented and treated and/or preventive medicine according to preventing and treating and/or preventive effect
The consumption of agent.
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Application publication date: 20170818 |