Some standard content:
This standard is proposed and managed by the Department of Science, Technology and Quality Standards of the Ministry of Agriculture. The drafting unit of this standard: China Agricultural University. The main drafters of this standard: Mei Ruhong, Xu Weimin, Jiang Huimin, 00
1 Scope
Agricultural Industry Standard of the People's Republic of China
Screening methods for yield-increasing bacteria
Screening methods for yield-increasing bacteriaNY336—1998
This standard specifies the screening technical indicators and screening methods for Bacillus sp. yield-increasing bacteria. This standard is applicable to the screening of new strains of Bacillus sp. yield-increasing bacteria. 2 Referenced standards
The provisions contained in the following standards constitute the provisions of this standard through reference in this standard. When this standard is published, the versions shown are valid. All standards will be revised, and parties using this standard should explore the possibility of using the latest versions of the following standards. HG/T3309—1993 Method for determination of fineness of pesticide powder (original HG/T2-896--83) NY337--1998 Method for use of yield-increasing bacteria
3 General
Bacillus of the genus Yield-increasing bacteria is a microecological preparation developed based on the principle of plant microecology. Yield-increasing bacteria adopts the screening method of isolating endophytic symbiotic Bacillus strains from plants. After strain purification, bioassay, strain identification, rejuvenation and activation, the selected strains are selected through two-year two-point field trials and multi-point trials. The strains that can stably increase plant yield, improve quality, enhance stress resistance, and are tested for safety and are non-toxic, harmless to humans, livestock and plants, and non-polluting to the environment are selected for production use. 4 Main method summary and technical indicators
4.1 Method summary
4.1.1 Collect sample plants or tissues of various parts and rinse for 24 hours, strictly disinfect the surface, and then crush them into a suspension and precipitate. 4.1.2 Take the supernatant and culture it on beef juice plate medium for 24 hours after streaking or dilution. 4.1.3 Select representative single colonies for smear, staining and microscopic examination, select single colonies of Bacillus and transfer them to slant medium for numbering, culture and bioassay.
4.1.4 Carry out indoor bioassay respectively, select the best pure strains for bioassay and then conduct field test. 4.1.5 Select strains that have shown good effects of increasing production and preventing diseases through two-year field test or multi-point test, and strains that are non-toxic to humans, livestock and plants and non-polluting to the environment after safety inspection, and then provide them for production use. 4.2 Technical indicators
4.2.1 The collected sample plants should be heated in a water bath at 80℃ for 10~20min to kill the bacteria. 4.2.2 The isolated Bacillus strains should be cultured at 28~35℃ for 18~24h, which is the most suitable environment for the growth of Bacillus.
4.2.3 Place the crops to be bioassayed, including seeds, plants or tubers (roots), in a suspension containing Bacillus at a concentration of 10° and soak for 1 hour.
Approved by the Ministry of Agriculture of the People's Republic of China on June 11, 1998 and implemented on January 1, 1999
5 Screening method
5.1 Collection of samples
5.1.1 Direct sampling method
The following three points should be noted when sampling:
NY 336—1998
a) The sample plants should be consistent with the selected crops for application of yield-increasing bacteria; b) Select plants that have obvious advantages over similar plants in terms of yield, quality or various stress resistance; c) After selecting the sample plants, number and register them, and immediately separate or store them at 4C. 5.1.2 Indirect sampling method
If it is difficult to collect sample plants, you can choose to collect soil from different regions, plant crops indoors, and collect sample plants according to the principles specified in 5.1.1.
5.2 Bacteria separation
5.2.1 Instruments and reagents
Instruments: sterile operating room or clean bench, sterilizing pot, water bath, refrigerator, incubator, sterile mortar and pestle, sterile tube (5mL, 10mI.), magnetic oscillator, sterile plate, sterile test tube, sterile water, alcohol lamp, inoculation loop, microscope, glass slide, sterile glass ball. Reagents: pH test paper, sodium hydroxide, hydrochloric acid, bleaching powder, fuchsin, malachite green and cedar oil, etc. 5.2.2 Culture medium preparation
5.2.2.1 Reagents
Beef slurry (biochemical reagent), protein Chen (biochemical test), agar (biochemical reagent). 5.2.2.2 Culture medium materials
3g beef, 10g egg white·sodium chloride, 20~30g agar, 1000mL distilled water. 5.2.2.3 Culture medium preparation method
Heat the agar to dissolve, add beef juice and egg white, heat again to dissolve, adjust the pH value to 7.0~7.5 with 0.11mol/L hydrochloric acid or 0.1mol/L sodium hydroxide solution, divide into Erlenmeyer flasks or test tubes and place in a sterilizer, sterilize at 0.1MPa pressure for 30min, and make slant culture medium after cooling for use.
5.2.3 Isolation and Culture
5.2.3.1 Isolation
Take the root, stem, leaf, flower and fruit, seed or tuber (root) tissue of the sample plants in 5.1.1 and 5.1.2, rinse for 24 hours, strictly disinfect the surface, then take 10g of the tissue, cut it into pieces, put it into a triangular flask containing 40mL sterile water and 10 sterile glass balls, and oscillate at 200-300 rpm for 10 minutes (if the bacteria within the epidermis are isolated, first disinfect the surface with 0.5% bleach solution for 3-5 minutes, grind it with a sterile mortar, oscillate it again, let it stand for 0.5 minutes, and use a sterile pipette to take 5mL of supernatant and inject it into a sterile test tube for use). 5.2.3.2 Culture
Put the test tube containing the supernatant treated in 5.2.3.1 in a water bath at 80℃ for 20 minutes. Then use a sterilized transfer loop to dip the treated liquid in the test tube and streak it on the beef protein culture medium plate. Streaking method: First, streak on half of the culture blood plate in sequence, then turn the culture III 90°, sterilize the transfer loop and start streaking from the second line in sequence (or use an L-shaped or △-shaped sterile coating stick to evenly spread the bacterial solution on the surface of the plate, and use the dilution plate method or single colony method when necessary), incubate at 28~35℃ for 24 hours and then observe. 5.2.3.3 Colony selection
According to the morphology of bacterial colonies formed on the plate culture medium, such as colony size, shape, round or irregular shape, hairy edge, thick or thin bacterial moss, wrinkled, smooth or rough, transparent or dark color, etc., select different types of single colonies (if the colony is impure, it should be purified), and smear, stain and microscopic examination are performed respectively.
5.2.3.4 Smear
Use a sterile inoculation loop to dip a small amount of bacteria on the edge of the selected colony, place it in a test tube containing 10mL sterile water and oscillate to make a bacterial suspension; then use a sterile micropipette or sterile inoculation loop to dip 1 to 3 drops of the suspension, add it to a clean glass slide, and use a sterile inoculation loop to evenly spread the bacterial liquid droplets 502
. After the bacterial liquid dries, it can be stained and observed. 5.2.3.5 Staining
NY 336-1998
Usually, Gram staining is used to stain bacteria, dyeing the bacteria red or purple, and the spores are not colored. Use the following special staining method to dye the bacteria and spores into different colors (the bacteria are red, the spores are green) for easy observation. Reagents: The staining agent is a 5% malachite green aqueous solution, and the staining agent is a 0.5% safranin aqueous solution or a 0.5% alkaline fuchsin aqueous solution. Staining steps: Add stain I to the dry smear, heat it on an alcohol lamp for 1 minute (be careful not to let the staining liquid boil or dry), wash off the excess stain with water, then add stain I for 15 seconds, then wash off the stain with water, dry it with absorbent paper, and observe under a microscope. 5.2.3.6 Microscopic examination
Observe under an oil-immersed microscope, place the smear specimen on the stage of the microscope, align the high-power microscope, and then add a drop of cedar oil to immerse the lens for observation. Note during operation: After adding the oil drop on the glass slide, use the automatic stop device of the microscope to move the oil-immersed lens down into the oil drop until it stops falling. Then use the fine-tuning knob to adjust the focus upward and observe. After use, wipe off the excess oil on the lens and stage with lens paper, then use a cotton ball dipped in a little xylene to gently wipe the oil stain on the lens, and immediately wipe off the xylene with lens paper. Through microscopic examination, any spores that are stained green and bacteria that are stained red can be determined as Bacillus. Number the colonies of Bacillus and transfer them to the slant culture medium, place them in an incubator at 28-35℃ for 24 hours, take them out and store them in the refrigerator for bioassay. 5.3 Indoor bioassay
5.3.1 Instruments and equipment
Flower pots (upper diameter 18cm, height 20cm), tray balance, hemocytometer, microscope, oscillator, test tubes, Erlenmeyer flask, refrigerator, incubator. 5.3.2 Soil
Mix 2 parts of humus soil, 1 part of sandy soil, and 1 part of peat, put them in a small flower pot for later use. 5.3.3 Preparation of bacterial suspension
Transfer the strains to be tested, which are numbered in 5.2.3.6, to the slant, and culture them at 28~35℃ for 24h. Take them out and scrape the bacterial moss into a sterile water test tube or a triangular flask. Oscillate on an oscillator for 2min and mix thoroughly to make a bacterial solution with a spore count of 100/mL (count them under a microscope using a hemocytometer).
5.3.4 Seeding of test crops
The crop seeds should be neat and full, with a germination rate of not less than 95%. After the seeds are germinated and whitened, select the seeds with uniform germination and growth, soak them in the test bacterial suspension (5.3.3) of 108/ml for 1h, dry the surface moisture of the seeds, and then sow them in the flower pots in 5.3.2. Sow 10 seeds in each pot, and sow no less than 10 pots for each strain.
5.3.5 Bioassay Crop Management
Water the potted plants treated in 5.3.4 regularly and manage them uniformly to make the growth environment (such as lighting, temperature and humidity, etc.) of each treatment as uniform as possible.
5.3.6 Bioassay Crop Survey Project
After 30 days of crop growth, investigate the total fresh weight of seedlings, root fresh weight and aboveground fresh weight. Repeat the bioassay for each strain 3 to 5 times, and select the strain with significant and stable bioassay effect for field test. 5.4 Field Bioassay (Field Test)
5.4.1 Selection of Test Site
For a certain crop, select the same crop in two different ecological zones, conduct field tests at two points for two years, and clarify the average performance of the yield-increasing performance of the test strain, the stability of the yield-increasing effect and the adaptability of the region. The test plot should be flat and the fertility should be uniform. 5.4.2 Arrangement of experimental plots
Each strain is a treatment, with clean water as the control. The plots are arranged in random blocks or paired blocks. Each treatment is repeated at least 3 times. The plot area is not less than 60m2, preferably 0.0133ha. Protective rows and isolation belts should be set up. 5.4.3 Preparation of microbial agents
The strains obtained from indoor bioassays are fermented and produced into powdered microbial agents for field trials. 5.4.4 Treatment methods
5.4.4.1 Seed dressing
NY 336--1998
Spray or soak the dry seeds, then sprinkle 75-150g/ha of microbial agents on the seeds (see NY337), turn them over several times to make the microbial agents evenly attached to the surface of the seeds, dry the moisture on the surface of the seeds, and then sow them (if the seed rate per hectare exceeds 150kg, the corresponding amount of microbial agents should be increased). bzxZ.net
5.4.4.2 Spray
Dilute the bacterial agent at a dosage of 75-150g/ha with an appropriate amount of water to prepare a suspension (see NY337), stir thoroughly and spray. 5.4.4.3 Dip the roots (seedlings) and seedlings
Dilute the bacterial agent at a dosage of 75-150g/ha with an appropriate amount of water (see NY337), and dip the roots (seedlings) and seedlings so that the bacterial solution is evenly attached to the roots (seedlings). After drying for a while, they can be planted and cut. 5.4.5 Field management
a) The water and fertilizer management of each plot should be consistent;
b) Avoid mutual infection between different strains during spraying, and strictly prohibit water from flowing between plots during irrigation; c) Minimize the harm caused by birds, animals, insects and diseases. 5.4.6 Investigation and Recording
5.4.6.1 Recording Items
The recording items vary with crop types and screening objectives, and their recording contents and required indicators are also different. When the goal is to increase the yield of a certain crop, the traits that affect the yield should be observed and recorded; when the goal is to improve the disease resistance of a certain crop, the main diseases and control levels of the crop should be observed and recorded; when the goal is to improve the quality of a certain crop, the indicators that affect the quality should be recorded. For the same crop and the same screening objective, all observation and recording items must be the same to be comparable. The design can refer to the crop testing method.
5.4.6.2 Sampling units and sampling methods
For crops with tall plant types and fixed plant and row spacing (such as corn and sorghum) and hole-planted crops (such as potatoes and sugar beets), the basic sampling unit is plant or hole; for row-sown and broadcast-sown crops (such as wheat), the basic sampling unit is 1m2 area or 1m long double row. Sampling should be strictly fixed-point, fixed-plant or strictly random to avoid human errors. 5.4.7 Yield determination
The experimental plot is required to conduct full-field yield determination. 5.4.8 Data collation and result analysis
The obtained results are processed according to different block groups, summarized in table form, and then variance analysis is performed to compare the significance of the differences between each treatment, and find the strain with the best effect. If there is multi-point joint test data, the stability of the effect of each strain and the ecological area to which it is adapted can also be clarified. 5.4.8.1 Statistical analysis of paired data The average difference between paired data d is calculated according to formula 1): d = X, - X2
Where X is the treatment average;
X, the control average.
The average difference between paired data is calculated according to formula (2): Where Ed is the sum of the numbers;
Y is the number of repetitions.
Calculate the standard deviation Sd of the mean of the difference number according to formula (3): ad
Zd2 (Zd)
Sd = -(Y -1)
and the mean of the squared difference number;
(1)
(2)
·(3)
Zd—the sum of the numbers;
NY 336—1998
(Y--1)——degrees of freedom, and check the t value table to determine the significant level of the difference. Calculate the significant level t value according to formula (4):
t = sd
Where: d--.-number of differences;
Sd——standard deviation of the mean of the difference number.
5.4.8.2 Statistics and analysis of randomized block experiments are generally performed using variance analysis (LSR) for statistical analysis. 5.5 Safety Assessment
5.5.1 Preparation of Bacterial Agents
The strains identified through bioassay are fermented into powder or liquid for use. (4)
5.5.2 Safety Test
The bacterial agents in 5.5.1 are subjected to partial safety tests in accordance with the sanitary and epidemic prevention standards. If they are safe for humans and animals, they can be provided for production use. 5.6 Determination of Bacterial Agents
Through the tests in 5.4.8 and 5.5.2, any strain that has obvious advantages in crop yield, quality or various stress resistance traits can be determined as a new strain for promotion and application in production.4.4.2 Spraying
Dilute the bacterial agent at a dosage of 75-150g/ha with an appropriate amount of water to prepare a suspension (see NY337), stir thoroughly and spray. 5.4.4.3 Dip the roots (seedlings) and seedlings
Dilute the bacterial agent at a dosage of 75-150g/ha with an appropriate amount of water (see NY337), and dip the roots (seedlings) and seedlings so that the bacterial solution is evenly attached to the roots (seedlings). After drying for a while, they can be planted and cut. 5.4.5 Field management
a) The water and fertilizer management of each plot should be consistent;
b) Avoid mutual infection between different strains during spraying, and strictly prohibit water from flowing between plots during irrigation; c) Minimize the harm caused by birds, animals, insects, and diseases. 5.4.6 Investigation and Recording
5.4.6.1 Recording Items
The recording items vary with crop types and screening objectives, and their recording contents and required indicators are also different. When the goal is to increase the yield of a certain crop, the traits that affect the yield should be observed and recorded; when the goal is to improve the disease resistance of a certain crop, the main diseases and control levels of the crop should be observed and recorded; when the goal is to improve the quality of a certain crop, the indicators that affect the quality should be recorded. For the same crop and the same screening objective, all observation and recording items must be the same to be comparable. The design can refer to the crop testing method.
5.4.6.2 Sampling units and sampling methods
For crops with tall plant types and fixed plant and row spacing (such as corn and sorghum) and hole-planted crops (such as potatoes and sugar beets), the basic sampling unit is plant or hole; for row-sown and broadcast-sown crops (such as wheat), the basic sampling unit is 1m2 area or 1m long double row. Sampling should be strictly fixed-point, fixed-plant or strictly random to avoid human errors. 5.4.7 Yield determination
The experimental plot is required to conduct full-field yield determination. 5.4.8 Data collation and result analysis
The obtained results are processed according to different block groups, summarized in table form, and then variance analysis is performed to compare the significance of the differences between each treatment, and find the strain with the best effect. If there is multi-point joint test data, the stability of the effect of each strain and the ecological area to which it is adapted can also be clarified. 5.4.8.1 Statistical analysis of paired data The average difference between paired data d is calculated according to formula 1): d = X, - X2
Where X is the treatment average;
X, the control average.
The average difference between paired data is calculated according to formula (2): Where Ed is the sum of the numbers;
Y is the number of repetitions.
Calculate the standard deviation Sd of the mean of the difference number according to formula (3): ad
Zd2 (Zd)
Sd = -(Y -1)
and the mean of the squared difference number;
(1)
(2)
·(3)
Zd—the sum of the numbers;
NY 336—1998
(Y--1)——degrees of freedom, and check the t value table to determine the significant level of the difference. Calculate the significant level t value according to formula (4):
t = sd
Where: d--.-number of differences;
Sd——standard deviation of the mean of the difference number.
5.4.8.2 Statistics and analysis of randomized block experiments are generally performed using variance analysis (LSR) for statistical analysis. 5.5 Safety Assessment
5.5.1 Preparation of Bacterial Agents
The strains identified through bioassay are fermented into powder or liquid for use. (4)
5.5.2 Safety Test
The bacterial agents in 5.5.1 are subjected to partial safety tests in accordance with the sanitary and epidemic prevention standards. If they are safe for humans and animals, they can be provided for production use. 5.6 Determination of Bacterial Agents
Through the tests in 5.4.8 and 5.5.2, any strain that has obvious advantages in crop yield, quality or various stress resistance traits can be determined as a new strain for promotion and application in production.4.4.2 Spraying
Dilute the bacterial agent at a dosage of 75-150g/ha with an appropriate amount of water to prepare a suspension (see NY337), stir thoroughly and spray. 5.4.4.3 Dip the roots (seedlings) and seedlings
Dilute the bacterial agent at a dosage of 75-150g/ha with an appropriate amount of water (see NY337), and dip the roots (seedlings) and seedlings so that the bacterial solution is evenly attached to the roots (seedlings). After drying for a while, they can be planted and cut. 5.4.5 Field management
a) The water and fertilizer management of each plot should be consistent;
b) Avoid mutual infection between different strains during spraying, and strictly prohibit water from flowing between plots during irrigation; c) Minimize the harm caused by birds, animals, insects, and diseases. 5.4.6 Investigation and Recording
5.4.6.1 Recording Items
The recording items vary with crop types and screening objectives, and their recording contents and required indicators are also different. When the goal is to increase the yield of a certain crop, the traits that affect the yield should be observed and recorded; when the goal is to improve the disease resistance of a certain crop, the main diseases and control levels of the crop should be observed and recorded; when the goal is to improve the quality of a certain crop, the indicators that affect the quality should be recorded. For the same crop and the same screening objective, all observation and recording items must be the same to be comparable. The design can refer to the crop testing method.
5.4.6.2 Sampling units and sampling methods
For crops with tall plant types and fixed plant and row spacing (such as corn and sorghum) and hole-planted crops (such as potatoes and sugar beets), the basic sampling unit is plant or hole; for row-sown and broadcast-sown crops (such as wheat), the basic sampling unit is 1m2 area or 1m long double row. Sampling should be strictly fixed-point, fixed-plant or strictly random to avoid human errors. 5.4.7 Yield determination
The experimental plot is required to conduct full-field yield determination. 5.4.8 Data collation and result analysis
The obtained results are processed according to different block groups, summarized in table form, and then variance analysis is performed to compare the significance of the differences between each treatment, and find the strain with the best effect. If there is multi-point joint test data, the stability of the effect of each strain and the ecological area to which it is adapted can also be clarified. 5.4.8.1 Statistical analysis of paired data The average difference between paired data d is calculated according to formula 1): d = X, - X2
Where X is the treatment average;
X, the control average.
The average difference between paired data is calculated according to formula (2): Where Ed is the sum of the numbers;
Y is the number of repetitions.
Calculate the standard deviation Sd of the mean of the difference number according to formula (3): ad
Zd2 (Zd)
Sd = -(Y -1)
and the mean of the squared difference number;
(1)
(2)
·(3)
Zd—the sum of the numbers;
NY 336—1998
(Y--1)——degrees of freedom, and check the t value table to determine the significant level of the difference. Calculate the significant level t value according to formula (4):
t = sd
Where: d--.-number of differences;
Sd——standard deviation of the mean of the difference number.
5.4.8.2 Statistics and analysis of randomized block experiments are generally performed using variance analysis (LSR) for statistical analysis. 5.5 Safety Assessment
5.5.1 Preparation of Bacterial Agents
The strains identified through bioassay are fermented into powder or liquid for use. (4)
5.5.2 Safety Test
The bacterial agents in 5.5.1 are subjected to partial safety tests in accordance with the sanitary and epidemic prevention standards. If they are safe for humans and animals, they can be provided for production use. 5.6 Identification of Bacterial Agents
Through the tests in 5.4.8 and 5.5.2, any strain that has obvious advantages in crop yield, quality or various stress resistance traits can be identified as a new strain for promotion and application in production.
Tip: This standard content only shows part of the intercepted content of the complete standard. If you need the complete standard, please go to the top to download the complete standard document for free.